LHRH) Analogues

Improving the Bioavailability and Stability of Luteinizing Hormone-Releasing Hormone (LHRH) Analogues

Shayli Varasteh Moradi M.Sc.

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 School of Chemistry and Molecular Biosciences

Abstract

Luteinizing hormone-releasing hormone (p-EHWSYGLRPG, LHRH) is a decapeptide produced by the hypothalamus that stimulates the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland. This neuropeptide plays a crucial role in the regulation of the pituitary/gonadal axis in both males and females. LHRH analogues have extensive therapeutic applications in the treatment of hormone-dependent diseases such as breast and prostate cancers. Chronic administration of LHRH agonists desensitizes the pituitary gland and subsequently suppresses the production of gonadal sex steroids. In addition, LHRH analogues could exert direct growth inhibitory effects on cancer cells which are meditated through the LHRH receptors expressed on the cell’s membrane. All marketed analogues of LHRH are administered parenterally. Given the advantages of the oral administration route such as convenience and patient compliance, developing a practical oral delivery system for LHRH would be more useful and efficient than existing routes of administration. However, the poor bioavailability and rapid enzymatic degradation of the LHRH impede the development of a potent oral pharmaceutical.

The main aim of this research was to develop a carbohydrate-based system for oral delivery of LHRH peptide. This research focused mostly on improving the pharmacokinetic profile of LHRH and enhancing its oral bioavailability using a glycosylation strategy. Conjugation of peptides with carbohydrate moieties has been shown to be a promising strategy in increasing the metabolic stability and changing the physicochemical properties of peptides. A library of LHRH analogue modified by the attachment of different sugar units (glucose, galactose and lactose) to the N- terminus, C-terminus or the middle of the peptide sequence was designed and synthesized. Caco-2 cell monolayers were used as a standard model to evaluate the apparent permeability (Papp) of the newly designed analogues across the cell membranes. A significant improvement was observed in the Papp of all N-terminally glycosylated LHRH derivatives except for the compound-bearing galactose. The greatest Papp value was obtained for the compound bearing a glucose (GS) unit in the middle of the sequence ([GS4][w6]LHRH), and lactose (Lac) at the N-terminus (Lac- 1 6 -7 -7 [Q ][w ]LHRH) with Papp = 58.54×10 cm/s and 38.42×10 cm/s, respectively. The contribution of intestinal active transport systems including the sodium ion-dependent transporter 1 (SGLT1) and a sodium ion-independent facilitative transporter 2 (GLUT2) in the transport of glycosylated LHRH derivatives was also elucidated using the Caco-2 cell monolayer model.

The anti-proliferative activity of the peptide derivatives was also examined in different LHRH receptor-positive prostate cancer cell lines (LNCaP, DU145 and PC3) in this study. The treatment

ii of LNCaP and DU145 cells with glycosylated LHRH derivatives reduced the cell growth significantly (up to 60% decreases in cell viability) after 72 h incubation at 100 µM and 200 µM concentrations. The growth of PC3 cells reduced 20% to 30% after 96 h treatment with glycosylated LHRH analogues (at 100 µM and 200 µM) showing the less inhibitory effect of the glycosylated compounds on PC3 cell growth.

The in vitro metabolic stability of the glycosylated LHRH derivatives was assessed in human plasma, Caco-2 cell and rat kidney and liver homogenates. The plasma half-life of the compounds increased significantly compared to the parent peptide. Lac-[Q1][w6]LHRH and [GS4][w6]LHRH were the most stable derivatives in all these biological matrices. A significant improvement was shown in the metabolic stability of the Lac-[Q1]LHRH analogue in the liver homogenates compared to LHRH (t1/2 of 47 = min vs. 5 min for LHRH).

To evaluate the in vitro efficacy of glycosylated LHRH derivatives, the stimulatory effect of the analogues on LH release was assessed in cultured rat pituitary cells. The secretion of LH was significantly increased following the treatment of the cells with Lac-[Q1][w6]LHRH at 5 nM and 10 nM concentrations. The in vivo efficacy study of Lac-[Q1][w6]LHRH revealed the stimulatory activity of the compound in male rats. A marked increase was observed in the release of LH after oral administration of 20 µg/kg of the lactose-modified derivative (nAUC=11.33± 1.65 ng/24 h) compared to the negative control group (nAUC=4.45± 1.028 ng/24 h).

The oral bioavailability and other pharmacokinetic parameters of Lac-[Q1][w6]LHRH were evaluated in male Sprague Dawley rats following intravenous (2.5 mg/kg) and oral (10 mg/kg) administrations. The absolute oral bioavailability (F%) of the peptide was found to be 14%, which is a remarkable improvement over the poor bioavailability of unmodified peptides (reported to be less than 1%). Maximum serum concentration (Cmax= 0.11 µg/mL) was reached after 2 h (Tmax) following oral administration of the compound at 10 mg/kg with a half-life of 2.6 h.

In conclusion, we improved the pharmacological properties of LHRH peptide through a glycosylation strategy. The attachment of lactose moiety to LHRH had a significant influence on the efficacy and pharmacokinetic parameters of the peptide both in vitro and in vivo. These findings suggest that Lac-[Q1][w6]LHRH is a promising candidate for the development of an orally active peptide with enhanced oral bioavailability.

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Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

Publications during candidature

1. Peer-reviewed articles

Moradi, S.V., F. M. Mansfeld, Toth I., Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH), Bioorg. Med. Chem. , 2013. 21(14): p. 4259-4265. (Incorporated as Chapter 3-section A)

Moradi, S.V, Varamini P., and Toth I., The transport and efflux of glycosylated luteinising hormone-releasing hormone analogues in Caco-2 cell model: contributions of glucose transporters and efflux systems, J. Pharm. Sci., 2014. 103(10): p 3217-22. (Incorporated as Chapter 4)

Moradi, S.V, Varamini P., and Toth I., Evaluation of the biological properties and the enzymatic stability of glycosylated luteinizing hormone releasing hormone analogues, AAPS journal, 2015. P.1-9 (Incorporated as Chapter 3- section B)

Moradi, S.V, Varamini P., Steyn F. and Toth I., In vivo pharmacological evaluation of lactose-conjugated luteinizing hormone releasing hormone analogue, Int. J. Pharm., submitted ( Incorporated as Chapter 5)

2. Conference paper

Moradi, S. V., Varamini, P. and Toth, I. (2014). Glycosylation of luteinizing hormone releasing hormone: towards the development of orally active peptide. In: Dr. Bert L. Schram Young Investigators' Mini Symposium. Abstracts. 33EPS 2014: 33rd European Peptide Symposium, Sofia, Bulgaria, Journal of peptide science (S38-S38). 31 August-5 September, 2014.

3. Conference abstracts

Moradi, S.V., P.Varamini, I. Toth, Biological evaluation of glycosylated derivatives of Luteinizing hormone releasing hormone (LHRH), In 10th SCMB student Symposium, Brisbane, Nov.2014. (Oral)

Moradi, S.V., P.Varamini, I. Toth, Glycosylation of luteinizing hormone releasing hormone: towards the development of orally active peptide. In 33rd European peptide symposium, Sofia, Bulgaria, 31 Aug-5 Sep. 2014. (Oral)

Moradi, S.V., P.Varamini, I. Toth, Biological characterisation of glycosyl conjugates of Luteinizing hormone releasing hormone (LHRH). In NanoBio Australia 2014, Brisbane , 6- 10 July 2014 (Poster)

Moradi, S.V., P.Varamini, I. Toth, Transport of glycosylated Luteinizing hormone releasing hormone (LHRH) analogues in Caco-2 cell model. In 5th FIP Pharmaceutical Science World Congress, Melbourne, Australia, 13-16 April 2014. (Poster)

Moradi, S.V., P.Varamini, I. Toth, Glycosylation as an effective strategy to improve the transport of Luteinizing hormone releasing hormone LHRH across Caco-2 cell monolayer In Brisbane Biological & Organic Chemistry Symposium (BBOCS 2013), Brisbane, Australia, 2 Dec.2013. (Poster)

Moradi, S.V., P.Varamini, I. Toth, Contribution of active transporters in transport of glycosylated Luteinizing hormone releasing hormone (LHRH) analogues across Caco-2 cells. In 9th Annual Research Students Symposium, School of Chemistry and Molecular Biosciences, The University of Queensland,Queensland, Australia, 2013. (Poster)

Moradi, S.V., F. M. Mansfeld, I. Toth, Transport of glycosylated analogues of Luteinizing hormone releasing hormone (LHRH) across Caco-2 cells. In IMB Division of Chemistry and Structural Biology Symposium, Brisbane (QLD, Australia), 2013. (Poster)

Moradi, S.V., F. M. Mansfeld, I. Toth, Toward the development of an oral system for delivery of LHRH analogues. In Drug Discovery & Therapy World Congress , Boston, USA, 2013.(Oral)

Moradi, S.V., 3 Minute thesis presentation competition, Brisbane, Australia, 2013. (Oral)

Moradi, S.V., F. M. Mansfeld, I. Toth, Glycosylation as a promising procedure for oral delivery of LHRH analogues. In Drug Discovery & Therapy World Congress, Boston, USA, 2013. (Poster)

Moradi, S.V., F. M. Mansfeld, I. Toth. Synthesis and in vitro bio evaluation of glycosylated analogues of Luteinizing hormone releasing hormone (LHRH). In The First Biennial SCMB Research Symposium, Brisbane (QLD, Australia), 2013. (Poster)

Moradi, S.V., F. M. Mansfeld, I. Toth, Synthesis and Caco2 Cell Stability Test of Carbohydrate Conjugates of Luteinizing Hormone Releasing Hormone (LHRH). In 6th Annual Meeting of the Australian Chapter of the Controlled Release Society, Parkville (VIC, Australia), 2012. (Poster)

Moradi, S.V., F. M. Mansfeld, I. Toth, In vitro permeability and metabolic stability studies of carbohydrate conjugated luteinizing hormone releasing hormone (LHRH) analogues. In IMB Division of Chemistry and Structural Biology Symposium, Brisbane (QLD, Australia), 2012. (Poster)

Moradi, S.V., F. M. Mansfeld, I. Toth, Synthesis and in vitro bio evaluation of carbohydrate conjugates of Luteinizing hormone releasing hormone (LHRH). In 8th Annual Research Students Symposium, School of Chemistry and Molecular Biosciences, The University of Queensland, (QLD, Australia), 2012. (Poster)

Moradi, S.V., F. M. Mansfeld, I. Toth, Synthesis and in vitro bio evaluation of carbohydrate conjugates of Luteinizing hormone releasing hormone (LHRH). In ECR Poster Symposium, 2012. (Poster)

Publications included in this thesis

Contributor Statement of contribution Author Shayli Varasteh Moradi  Designed experiments (35%) (Candidate)  Synthesis (90%)  In vitro biological study (100%)  Analysis and interpretation of data (80%)  Statistical analysis (100%)  Wrote the paper (100%)  Edited the paper (30%) Author Friederike M. Mansfeld  Designed experiments (25%)  Synthesis (10%)  Analysis and interpretation of data (20%)  Edited the paper (35%) Author Istvan Toth  Designed experiments (40%)  Edited the paper (35%)

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Contributor Statement of contribution Author Shayli Varasteh Moradi  Designed experiments (60 %) (Candidate)  Synthesis (100%)  In vitro biological study (100%)  Analysis and interpretation of data (90%)  Statistical analysis (100%)  Wrote the paper (100%)  Edited the paper (30%) Author Pegah Varamini  Analysis and interpretation of data (10%)  Edited the paper (45%) Author Istvan Toth  Designed experiments (40%)  Edited the paper (25%)

Moradi, S.V, Varamini P., and Toth I., Evaluation of the biological properties and the enzymatic stability of glycosylated luteinizing hormone releasing hormone analogues, AAPS journal,. 2015. P.1-9. (Incorporated as Chapter 3- section B)

Contributor Statement of contribution Author Shayli Varasteh Moradi  Designed experiments (45%) (Candidate)  In vitro biological study (90%)  Analysis and interpretation of data (80%)  Statistical analysis (90%)  Wrote the paper (100%)  Edited the paper (30%) Author Pegah Varamini  Designed experiments (25%)  In vitro biological study (10%)  Analysis and interpretation of data (20%)  Statistical analysis (10%)  Edited the paper (40%)

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Author Istvan Toth  Designed experiments (30%)  Edited the paper (30%)

Moradi, S.V, Varamini P., Steyn F. and Toth I., In vivo pharmacological evaluation of a lactose- conjugated luteinizing hormone releasing hormone analogue, Int. J. Pharm, submitted

Contributor Statement of contribution Author Shayli Varasteh Moradi  Designed experiments (60%) (Candidate)  In vivo biological study (100%)  Analysis and interpretation of data (70%)  Statistical analysis (100%)  Wrote the paper (100%)  Edited the paper (35%) Author Pegah Varamini  Designed experiments (15%)  Analysis and interpretation of data (20%)  Edit the paper (35%) Author Frederik Steyn  Designed experiments (5%)  Analysis and interpretation of data (10%)  Edited the paper (10%) Author Istvan Toth  Designed experiments (20%)  Edited the paper (20%)

Contributions by others to the thesis Dr. Michelle Christie and Dr. Daryn Goodwin provided Galactose succinamic acid and glucose succinamic acid used for the synthesis of the compounds described in chapter 2. Dr. Ken Johnstone assisted in the synthesis of one of the compounds (glucoronic acid conjugated LHRH) in chapter 2. The publications submitted as part of this thesis have had contributions from other authors. The nature and extent of these contributions are detailed above.

Statement of parts of the thesis submitted to qualify for the award of another degree None.

Acknowledgements

I could not have accomplished my dissertation without the care and support of a number wonderful people.

First and foremost, I would like to express my deepest gratitude to my principal supervisor, Professor Istvan Toth for his supervision and continuous encouragement throughout my whole PhD study. I would like to thank him for giving me the chance to work independently and support my ideas, yet providing invaluable comments and guidance, whenever needed.

I would like to acknowledge my co-advisor, Dr. Pegah Varamini for her support and sharing her experiences. She has not been just an advisor, but also a friend. I am also grateful to Dr. Friedrike Mansfeld for her kind mentoring, her time in teaching me lab techniques, peptide synthesis and carbohydrate chemistry during the first year of my study.

I would like to thank all past and present post-docs in our group. In particular, I wish to thank Dr. Mariusz Skwarczynski, Dr. Peter Moyle, Dr. Michelle Christie, Rachel Stephenson and Dr. Pavla Simerska for their valuable ideas and assistance over the last few years. I wish to acknowledge my review panel members, Professor Michael Monteiro (Chair) and Professor Greg Monteith and Dr. Joanne Blanchfield for their thoughtful advice and support during the study period. I also thank Thalia Guerin for all her kind assistance and particularly for her critical review and editing of some of the manuscripts. I would also like to thank all the friendly fellow students in Toth group who always share ideas, assist and support each other; thanks to Abdul, Amirreza, Ashwin, Bita, Fazren, Hugo, Khairul, Nirmal, Nisa, Noushin, Saranya, Sharareh and Vicky for all your help, support and friendship during my study. I wish you the best of luck.

I am grateful to Barbara Arnts for her training and assistance in animal experiments. I would like to thank Dr. Stefanie Henning and Dr. Christina Staaz for their help in analysis of pharmacokinetic parameters. I also thank Certara Inc. for providing pharmacodynamic/ pharmacokinetic analysis software, ‘’Phoenix 1.2’’, which I used for the analysis of my animal study data.

I wish to acknowledge the University of Queensland, the SCMB and my supervisor for their financial support, and the Australian Research Council (ARC) for funding this research.

I would like to specially thank my dearest friend, sister and housemate, Maedeh who I never forget her company, patience and help during the last two years of my phD study. You helped me get through the tough times and you were always there to share the good times. I would also like to thank my kind friend, Siew Ping for listening and offering helpful advice.

Last, but certainly not least, my deepest gratitude goes to my beloved family members for their unconditional love and infinite support throughout my life. To my parents, Minoo and Reza, I am forever grateful for your incredible care, both emotionally and financially. I dedicate this dissertation to you. I thank my brother, Bardia, for his support and encouragement. I love you all and I would not have made it this far without you.

Thank you all!

Keywords: LHRH, glycosylation, peptide, oral delivery, prostate cancer, glucose transporters, efflux pump systems, bioavailability, LH and FSH release

Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 030406 Proteins and Peptides, 30% ANZSRC code: 030499 Medicinal and Biomolecular Chemistry not elsewhere classified, 30% ANZSRC code: 111599, Pharmacology and Pharmaceutical Sciences not elsewhere classified, 40%

Fields of Research (FoR) Classification FoR code: 0304, Medicinal and Biomolecular Chemistry, 60% FoR code: 1115 Pharmacology and Pharmaceutical Sciences, 40%

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Table of Contents

Abstract...... ii Declaration by author...... iv Publications during candidature...... v Publications included in this thesis...... vii Contributions by others to the thesis...... ix Statement of parts of the thesis submitted to qualify for the award of another degree………….....ix Acknowledgements...... x Keywords...... xii Australian and New Zealand Standard Research Classifications (ANZSRC)...... xii Fields of Research (FoR) Classification...... xii Table of Contents...... xiii List of Figures...... xv List of Abbreviations...... xvi Chapter1: Introduction...... 1 1.1 LHRH and its biology...... 2 1.2 Enzymatic degradation of LHRH...... 5 1.3 LHRH analogues and their clinical applications...... 6 1.4 Antiproliferative activity of LHRH and its analogues...... 7 1.5 Strategies to develop orally active peptide...... 8 1.6 Research aim and hypothesis...... 9 1.6.1Hypothesis...... 9 1.6.2 Overall aim...... 9 1.6.3 Specific aims...... 9 1.7 Thesis organisation...... 10 1.8 References...... 12 Chapter 2: Review of Literature...... 15 ‘’Glycosylation, an effective strategy to improve pharmacological properties of therapeutic peptides’’ Abstract…………………………...... ……………………………………….16 2.1 Introduction...... 16 2.2 Barriers limiting oral delivery of peptides...... 17 2.2.1 Mucus and unstirred water layer...... 17 2.2.2 Epithelial cell membrane...... 18

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2.2.2.1 Paracellular pathway...... 18 2.2.2.2 Transcellular pathway...... 19 2.2.3 Efflux drug transporters...... 20 2.2.4 Metabolic enzymes...... 21 2.3 Barriers limiting brain delivery...... 22 2.4 Role of glucose transporters in the transport of carbohydrates across biological membranes.....22 2.5 Glycosylation strategy for peptide delivery...... 25 2.5.1 Glycopeptide synthesis...... 26 2.5.2 Impact of glycosylation in physicochemical properties...... 29 2.5.3 Impact of glycosylation in pharmacological characteristics of peptides...... 29 2.6 Development of therapeutic peptides using glycosylation strategy...... 32 2.6.1 Neuropeptide therapeutics...... 32 2.6.2 Radiopharmaceuticals...... 32 2.6.3 Targeted delivery...... 33 2.7 Conclusion...... 34 2.8 References...... 35 Chapter 3: Glycosylated LHRH analogues and their biological properties…………………...... 43 Section A: ...... 44 ‘’Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH)’’...... 44 3.1 Introduction to this publication...... 44 3.2 Reprint of this peer-reviewed publication...... 44 Section B...... 52 ‘’Evaluation of the biological properties and the enzymatic stability of glycosylated Luteinizing hormone releasing hormone (LHRH) analogues’’...... 52 3.3 Introduction to this publication ...... 52 3.4 reprint of the peer-reviewed article...... ……………...... ………………………....52 Chapter 4: Roles of transporters in the vectorial transport of the glycosylated LHRH analogues....64 ‘’The Transport and Efflux of Glycosylated Luteinising Hormone-Releasing Hormone Analogues in Caco-2 Cell Model: Contributions of Glucose Transporters and Efflux Systems’’ 4.1 Introduction to this publication…………………...... ……………………………………...... 64 4.2 Reprint of this peer-reviewed publication...... 64 Chapter 5: Lactose derivative of LHRH with enhanced pharmacological profile……...... 77 ‘’In vivo pharmacological evaluation of lactose-conjugated luteinizing hormone-releasing hormone analogue’’ xiv

5.1 Introduction to this chapter……………………………………………………………...... 77 5.2 Prepared manuscript to be submitted to the journal of ‘’ pharmaceutical research’’...... 77 Chapter 6: Conclusion and future directions………………………………………………...... 92 6.1 Conclusion...... 93 6.2 Future directions...... 95

List of Figures Figure1.1: The hypothalamic–pituitary–gonadal axis...... 3 Figure 1.2. Molecular structure of natural LHRH...... 3 Figure 1.3. Schematic structure of LHRH in folded conformation in which it is bound to its receptor...... 5 Figure 1.4. Cleavage sites within the LHRH peptide...... 6 Figure 2.1. Barriers in oral delivery of peptide and proteins...... 21 Figure 2.2. Localisation of glucose transporters on the membrane of enterocytes...... 24 Figure.2 3 Localization of GLUT transporter isoforms in the brain...... 25 Figure 2.4: A) Examples of O-linked and N-linked glycosylated amino acids, B) Direct and convergent strategies for glycopeptide synthesis...... 27 Figure 2.5. The attachment of galactose residue to the N-terminal of A) NAPamide and B) LHRH peptides...... 28

List of abbreviations ABC ATP-binding cassette ACE Angiotensin-converting enzyme AUC Area under the curve BBB Blood brain barrier BSA Bovine serum albumin Caco-2 cells Heterogeneous line of human epithelial colorectal adenocarcinoma cells

Cmax Maximum plasma concentration Cl Clearance cLogP Water-octanol partition coefficient CNS Central nervous system CV Coefficient of variation Da Dalton DCM Dichloromethane DIPEA N,N’-diisopropylethylamine DMEM Dubelcco’s Modified Eagle Medium DMF N,N’-dimethylformamide ELISA Enzyme linked immunosorbent assay F% Bioavailability FBS Foetal bovine serum Fmoc 9-Fluorenylmethoxycarbonyl FSH Follicle-stimulating hormone GIT Gastrointestinal tract GalNAc N-acetylgalactosamine Glc Glucoronic acid GlcNAc N-acetylglucosamine GLUT Glucose transporter GnRH Gonadotropin releasing hormone GPCR G-coupled protein receptor GS Glucose HBSS Hank’s balanced buffer solution HBTU 1-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HEPES N-2-hydroxyethylpiperazine- xvi

N’-2-ethanesulfonic acid IM Intramuscular IS Internal standard IV Intravenous LAA Lipoamino acid Lac Lactose LC-MS Liquid chromatography- mass spectrometry LH Luteinizing hormone LHRH Luteinizing hormone releasing hormone LHRHR LHRH receptors LLOQ Lower limit of quantitation MeOH Methanol MeCN Acetonitrile MRP Multidrug resistance associated protein MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide nAUC Normalised area under curves NMR Nucleic magnetic resonance

Papp Apparent permeability PBMC Peripheral blood mononuclear cell PBS Phosphate buffer saline P-gp P-glycoprotein PE Prolyl endopeptidase PEPT Peptide transporters PO Per oral QC Quality control RP-HPLC Reverse phase- high performance liquid chromatography S.C Subcutaneous SD Standard deviation SDS Sodium dodecyl sulphate SLC Solute carrier families SGLT Sodium glucose transporter SPPS Solid-peptide phase synthesis

Tmax Time to reach maximum plasma concentration TEER Trans epithelial electrical resistance xvii

TFA Trifluoroacetic acid TIPS Triisopropylsilane UWL Unstirred water layer

Vd Apparent volume of distribution

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Chapter 1: Introduction

1 1.1 LHRH and its biology

Luteinizing hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone (GnRH), plays a crucial role in the control of the reproductive system. LHRH was first isolated and synthesized by Schally et al. in 1971 (1). The prohormone contains 92 amino acids in its sequence which is produced by the neurosecretory cells of the hypothalamus. Endopeptidases process the precursor peptide and convert it to the active form. The active decapeptide is released from the hypothalamus in a pulsatile manner and exerts its function by binding to its receptor on the anterior pituitary gland where it stimulates the secretion of the gonadotropin hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH). Subsequently, the production of LH and FSH controls the function of testes and ovaries, by stimulating the secretion of sex steroids including testosterone in males, and oestrogens and progesterone in females (2). Generally, sex steroids regulate the hypothalamic–pituitary–gonadal axis through a negative feedback loop (Fig. 1.1). When the level of sex steroids reaches a threshold, it reduces the secretion of LHRH via two ways, either by direct suppression of the LHRH receptor in pituitary cells or by blocking the LH secretion and gonadotropin expression (3).

The amino acid sequence of LHRH is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (Fig. 1.2). To date, more than 23 isoforms of this hormone have been identified in both mammals and non-mammalian vertebrates. There are highly-conserved amino acids in both the N-terminus and the C-terminus of the LHRH peptide in all vertebrates indicating that these residues are critically important for receptor-binding and hormone activity (4). In addition to their regulatory function in the neuroendocrine system, LHRH peptides act as neurotransmitters and neuromodulators in the central nervous system or have autocrine/paracrine roles in several peripheral tissues (5). The autocrine activity of LHRH is mediated through the locally expressed LHRH receptors on the peripheral tissues and may have diverse functions including the facilitation of steroidogenesis, cell proliferation, apoptosis, fertilization, and cell migration (6). It has also been demonstrated that LHRH plays an important role in regulating tumour growth including ovarian and prostate cancers via an autocrine/paracrine function (6, 7).

Figure 1.1. The hypothalamic–pituitary–gonadal axis. Pulsatile secretion of LHRH controls the synthesis of FSH and LH in the pituitary gland. The production of LH and FSH in turn, regulates the secretion of sex hormones from the testes and ovaries that act as feedback hormones in the regulation of LHRH secretion.

Figure 1.2. Molecular structure of natural LHRH. The N-terminus of the peptide contains pyroglutamic acid (pGlu) and the C-terminus is amidated.

3 LHRH receptors (LHRHRs) belong to the family of rhodopsin-like G-coupled protein receptors (GPCR) located on the surface of the pituitary gonadotrope cells. The amino acid sequence of the LHRH receptor was first identified in 1992 by cDNA clones from rat and human pituitaries (8). LHRHR protein contains an extracellular N-terminal region and seven α-helical transmembrane (7TM) domains with specific sites for ligand binding (4, 9). There are also three hydrophobic loops in both extracellular and cytoplasmic sides which connect 7TM domains to each other.

Furthermore, the intracellular loops have specific domains for coupling to Gq/11 mediator protein, which is involved in the activation of the intracellular signalling pathway (9, 10). Unlike the other GPCRs, the intracellular region of LHRHR does not contain a C-terminal tail required for the internalisation of the receptor complex. This region is important for desensitisation of the receptor (10, 11). The receptor has to be stimulated continuously in order to be desensitized and down- regulated, which is the mechanism of LHRH agonists on the cognate receptor (12). Identification of individual amino acid residues involved in receptor-binding and receptor activation of LHRH is critical for designing biologically active LHRH analogues. Studies have shown that His2 and Trp3 play essential roles in the biological activity of the hormone and any modification in these positions can decrease the activity of the peptide. Gly6 in the middle of the sequence facilitates the folding of the peptide. Substitution of position six with a D-amino acid generates potent agonist derivatives of LHRH. This modification has been shown to enhance the binding affinity to the receptor due to a change in folding conformation. In addition, D-amino acid substitution at position six improves the metabolic stability of the modified analogue. The N- terminal residues in the peptide contribute to the receptor activation predominantly. Replacement of the first three N-terminal amino acids with D-amino acids leads to the development of analogues with antagonist activity (Fig.1.3) (13, 14).

Figure 1.3. Schematic structure of LHRH in folded conformation in which it is bound to its receptor. The amino-terminal residues (green) are associated with receptor activation and receptor-binding. Gly residue at position six is responsible for a specific folding conformation and it is required for high-affinity interaction with mammalian receptors. The last three amino acids at the carboxy-terminus (red) are necessary for specificity and high-affinity binding to the LHRH receptor.

1.2 Enzymatic degradation of LHRH

Native LHRH has a short half-life of 2-5 min in human plasma (15). It has been shown that the degradation of LHRH in the brain and peripheral tissues is caused mainly by two enzymes: 1) prolyl endopeptidase (PE) and 2) zinc metalloendopeptidase EC 3.4.24.15 (EP24.15). LHRH is initially cleaved by PE resulting in the production of nonapeptide (LHRH-(1-9)). The produced nonapeptide is a suitable substrate for EP24.15. In the second step, EP24.15 cleaves the peptide bond of LHRH- (1-9) at position five and six (Tyr5-Gly6) and forms LHRH-(1-5) (Fig. 1.4). The resulting pentapeptide (pGlu1-His2-Trp3-Ser4-Tyr5) is biologically active and functionally distinct from the parent peptide, since it can regulate the gene expression of LHRH and may also stimulate cell proliferation in peripheral tissues. A high level of these hydrolysing enzymes has been found in the tissues regulating the production of LHRH, such as testes (6). It is also believed that pyroglutamyl peptidase (also known as pyrolidone carboxyl peptidase) which eliminates pGlu residue from peptides, hydrolyses LHRH at position one (16, 17). Moreover, this enzyme has been detected in mammalian serum that might account for plasma degradation of the hormone (18).

Figure 1.4. Cleavage sites within the LHRH peptide. Prolyl endopeptidase (PE) and EP24.15 are involved in the hydrolysis of LHRH. First, PE cleaves the peptide at the ninth position and the resulting nonapeptide is cleaved by EP24.15. The hydrolysis products of the latter enzyme are LHRH-(1-5) and LHRH-(6-9), of which LHRH-(1-5) is biologically active (6).

1.3 LHRH analogues and their clinical applications

Elucidation of the LHRH structure and its pivotal role in the reproductive system prompted the development of several LHRH agonists and antagonists. These analogues are widely used for the treatment of hormone-dependent malignancies including breast, ovary, prostate and endometrial cancers, as well as endocrine disorders such as precocious puberty and infertility (19, 20). LHRH analogues have been found to be effective for the treatment of Alzheimer disease to decrease the level of gonadotropins elevated in patients with dementia (21-23). The ultimate effect of both agonists and antagonists of LHRH is to suppress the production of sex steroids in males and females via different mechanisms. In agonist therapy, the continuous administration of LHRH agonists results in an initial rise in the levels of LH and FSH hormones (so-called “flare effect”) followed by the down-regulation of LHRH receptors and the consequent decrease of sex steroids (24). For the treatment of prostate cancer in androgen deprivation therapy, using LHRH agonists has several advantages including good efficacy with a low level of adverse effects and psychological issues compared to the conventional therapies (surgical castration) (25).

6 This is because the medical castration achieved by LHRH agonists is reversible and therefore makes it preferable as a first-line of therapy (26). To date, a number of different LHRH analogues (both agonists and antagonists) have been synthesized by substitution of amino acids in the middle of the peptide sequence at position six, and also by the alteration of the N-terminus and C-terminus of the peptide. These modifications can protect the hormone against enzymatic degradation and improve their potency (19, 27). [D-Trp6]LHRH (triptorelin) is one of the agonists in the market with a higher biological activity than the native peptide due to the presence of D-Trp at position six which enhances the enzymatic stability of the peptide and its potency. The introduction of a hydrophobic group in the side chain of the amino acid residues in the peptide’s sequence can also reduce the sensitivity to proteases and protects it from digestion (28). [D-Leu6, Pro9-NHEt] LHRH (leuprolide), [D-Ser(But)6, Pro9- NHEt]LHRH (buserelin) and [D-Ser (But)6, Aza-Gly10]LHRH (goserelin) are other currently available LHRH agonists in the market with improved metabolic stability compared to the parent peptide (27, 29).

1.4 Antiproliferative activity of LHRH and its analogues

It has been shown that LHRH analogues not only suppress the pituitary-gonadal axis but also exert a direct growth-inhibitory effect on the tumour growth (30). In addition to pituitary expression, LHRH and its receptor are slightly expressed in the peripheral tissues but overexpressed in numerous tumours such as prostate and breast cancers (31, 32). It has been shown that LHRH receptors in malignant tumours mediate the anti-tumour activity of the LHRH derivatives (33, 34). A number of studies demonstrated the different impact of LHRH agonists on the growth rate of the cancer cells, e.g. prostate cancer cell lines (35-37). It has been suggested that LHRH agonists could exert a biphasic impact on the cell growth; they inhibit cell proliferation at high concentrations whereas they stimulate the growth at low concentrations in cultured cancer cells (38, 39). Medium- to-high-affinity binding sites were reported for LHRH agonists in LHRH-receptor positive prostate cancer cell lines including LNCaP, DU145 and PC3 cells (40). These analogues exert their growth inhibitory effect in a time- and dose-dependent manner (41). Studies showed that the growth inhibitory effect of LHRH analogues in PC3 is lower than the other cancer cell lines, which could be due to the lower rate of LHRH receptors expressed in PC3 cells (30). The inhibitory mechanism of LHRH analogues is through a different signalling transduction pathway from that in the pituitary cells (42, 43). The antiproliferative activity of LHRH analogues is mediated through the plasminogen activator system by reducing the activity of the enzyme in

7 prostate cancer cells which subsequently decreases the migration and invasiveness of cancer cells (43).

1.5 Strategies to develop orally active peptide

All LHRH analogues are administered through parenteral routes including subcutaneous (SC), intramuscular (IM) and intravenous (IV) (44). Due to the advantages of oral administration including convenience, high patient compliance and avoidance of infection risk caused by injection, the development of an orally active LHRH analogue is highly desirable. LHRH peptide has a poor oral bioavailability because of its relatively high molecular weight (limits the absorption through intestinal cells) and sensitivity to proteolytic enzymes that prevent its uptake by gastrointestinal cells (45). In order to improve the bioavailability of peptides, it is necessary to increase their intestinal absorption and protect them from enzymatic degradation. Chemical modifications of the peptide’s sequence using glycosylation and lipidation are known to be effective strategies to facilitate the oral peptide delivery. Various lipoamino acid (LAA) conjugates of LHRH have been synthesized by our group and the enhanced in vitro and in vivo stability and pharmacokinetic profiles of the lipidated analogues have been illustrated(46-48). The half-lives of LAA-conjugated peptides were increased significantly in Caco-2 cell homogenate in comparison with the parent peptide(46). The in vivo intestinal uptake of the lipid-modified LHRH analogues was also improved after oral administration (48). Conjugation of carbohydrate units to the peptide drugs is a useful strategy to improve their pharmacological properties including the bioavailability and stability. Carbohydrate moieties can alter the physiochemical properties of peptides and improve their enzymatic stability while retaining the biological activity. In addition, the attachment of sugar units to peptides facilitates the transport across the biological cell membranes via carrier-mediated transport systems and subsequently increases the intestinal absorption of the sugar-conjugated peptides (49, 50). The favourable impact of glycosylation on the membrane permeability, metabolic stability and the biological activity of glycosylated peptides has been shown in a number of studies (51-55). For instance, the glycosylation of somatostatin resulted in a ten-fold increase in the oral bioavailability of the peptide without changing its bioactivity to inhibit the release of growth hormone (54).

8 1.6 Research aim and hypothesis

1.6.1 Hypothesis

We hypothesized that conjugation of carbohydrate moieties to LHRH enhances the stability of the complex as well as increasing the uptake through biological membranes.

1.6.2 Overall aim

The main objective of this project was to enhance the oral bioavailability of LHRH analogues and develop an orally active peptide. To achieve this goal, we applied a glycosylation strategy to improve the metabolic stability of the LHRH peptide and enhance its intestinal absorption. This could consequently lead to improved pharmacokinetic properties and increased oral bioavailability of LHRH analogues, while maintaining their efficacy.

1.6.3 Specific aims:

Aim 1: Design and synthesis of a small library of LHRH analogues comprised of peptides conjugated with carbohydrate moieties and compounds modified by both carbohydrate moieties and amino acid substitution. Aim 2: Biological evaluation of all synthesized LHRH analogues in vitro including to:

 examine their enzymatic degradation in Caco-2 cell homogenate to evaluate the stability of peptide drug in vitro  investigate the apparent permeability of all peptide derivatives in Caco-2 cell monolayers  measure the half-lives of glycosylated derivatives in human plasma, rat liver and kidney membrane homogenates  investigate the role of transporters on the vectorial transport of glycosylated LHRH  evaluate the in vitro efficacy of the analogues on release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from cultured pituitary cells.

Aim 3: Perform an in vivo test for the lead compound (lactose-[Q1][w6]LHRH) in order to:  measure the change in the level of LH in rat plasma following oral administration of the compound  determine the oral bioavailability of lactose-[Q1][w6]LHRH in Sprague Dawley rats by measuring the plasma concentration of the compound after oral and IV administration and evaluating the pharmacokinetic (PK) parameters of the compound in rat serum

9 1.7 Thesis organization

In addition to Chapter 1, there are five more chapters included in this thesis. Chapter 2-5 are presented in the form of published or prepared manuscripts. Specifically, Chapter 2 is a review of literature related to the thesis topic. All the original research data are covered in Chapters 3-5. The last chapter consists of the conclusion to the thesis and future prospects of the project. Chapter 2 describes the factors limiting the oral and brain delivery of peptides and introduces the glycosylation strategy as an effective system for the delivery of peptide-based therapeutics. Several methods of glycoconjugate synthesis and recent progress in the development of glycosylated peptides therapeutics have been also explained in this section. Moreover, the impact of glycosylation on changing the physicochemical properties and pharmacological profiles of peptides has been highlighted in this chapter. Chapter 3 is divided into two sections and describes the glycosylation of LHRH peptide and in vitro biological activity of the glycosyl-modified analogues. The first section is a published research article in the journal of ‘Bioorganic and Medicinal Chemistry’ and describes the design and synthesis of a library of LHRH analogues by the attachment of different sugar moieties such as glucose, galactose and lactose to the peptide sequence. This article also reports the in vitro biological evaluation of the glycosylated LHRH analogues including their metabolic stability in Caco-2 cell homogenate and apparent permeability across the Caco-2 cell monolayer model. The second section of Chapter 3 is an accepted manuscript in the journal of the ‘American Association of Pharmaceutical Sciences’. In this section, the metabolic stability of glycosylated LHRH analogues in different tissue homogenates including human plasma, rat kidney membrane and liver homogenates has been discussed. Their anti-proliferative effect on a number of tumour cell lines along with the toxic effect on normal peripheral blood mononuclear cells (PBMC) is also reported in this study. Additionally, the impact of selected analogues on stimulating the release of LH and FSH in the cultured rat pituitary cells is highlighted in the article. Chapter 4 includes a published research article in the journal of ‘Pharmaceutical Sciences’. This paper focuses on the role of glucose transporters (GLUT-2 and SGLT1) in the transport of the glycosylated LHRH derivatives. The role of efflux transport systems (P-gp and MRP2) in the extrusion of LHRH derivatives to the extracellular environment has been also described in this article. Chapter 5 describes the pharmacological evaluation of the lead compound, lactose-modified LHRH derivative, based on the preliminary studies. This article focuses on the PK parameters of this compound including bioavailability, half-life, and the maximum concentration (Cmax) and time to reach Cmax. The stimulatory effect of the lactose-modified analogue on the release of LH

10 hormone was also reported following oral administration to rats. This chapter was prepared as a research article which was submitted to the journal of ‘Pharmaceutical Research’.

11 1.8 References 1. Schally A, Arimura A, Baba Y, Nair R, Matsuo H, Redding T, et al. Isolation and properties of the FSH and LH-releasing hormone. Biochem Biophys Res Commun. 1971;43(2):393-9. 2. Conn PM, Marian J, Mcmillian M, Stern J, Rogers D, Hamby M, et al. Gonadotropin- releasing hormone action in the pituitary: a three step mechanism. Endocr Rev. 1981;2(2):174-85. 3. Clayton RN, Catt KJ. Gonadotropin-releasing hormone receptors: characterization, physiological regulation, and relationship to reproductive function. Endocr Rev. 1981;2(2):186-209. 4. Millar RP, Lu ZL, Pawson AJ, Flanagan CA, Morgan K, Maudsley SR. Gonadotropin- releasing hormone receptors. Endocr Rev. 2004;25(2):235-75. 5. Limonta P, Moretti RM, Marelli MM, Motta M. The biology of gonadotropin hormone- releasing hormone: role in the control of tumor growth and progression in humans. Front Neuroendocrinol. 2003;24(4):279-95. 6. Walters K, Wegorzewska IN, Chin YP, Parikh MG, Wu T. Luteinizing hormone-releasing hormone I (LHRH-I) and its metabolite in peripheral tissues. Exp Biol Med. 2008;233(2):123-30. 7. Kang S, Choi K, Yang H, Leung P. Potential role of gonadotrophin-releasing hormone (GnRH)-I and GnRH-II in the ovary and ovarian cancer. Endocr Relat Cancer. 2003;10(2):169-77. 8. Flanagan CA, Millar RP, Illing N. Advances in understanding gonadotrophin-releasing hormone receptor structure and ligand interactions. Rev Reprod. 1997;2(2):113-20. 9. Schneider F, Tomek W, Gründker C. Gonadotropin-releasing hormone (GnRH) and its natural analogues: A review. Theriogenology. 2006;66(4):691-709. 10. Harrison G, Wierman M, Nett T, Glode L. Gonadotropin-releasing hormone and its receptor in normal and malignant cells. Endocr Relat Cancer. 2004;11(4):725-48. 11. Kaiser UB, Conn PM, Chin WW. Studies of gonadotropin-releasing hormone (GnRH) action using GnRH receptor-expressing pituitary cell lines. Endocr Rev. 1997;18(1):46-70. 12. McArdle C, Franklin J, Green L, Hislop J. Signalling, cycling and desensitisation of gonadotrophin-releasing hormone receptors. J Endocrinol. 2002;173(1):1-11. 13. Sealfon SC, Weinstein H, Millar RP. Molecular mechanisms of ligand interaction with the gonadotropin-releasing hormone receptor. Endocr Rev. 1997;18(2):180-205. 14. Millar RP, Newton CL. Current and future applications of GnRH, kisspeptin and neurokinin B analogues. Nature Reviews Endocrinology. 2013;9(8):451-66. 15. Redding T, Kastin A, Gonzalez-Barcena D, Coy D, Coy E, Schalch D, et al. The half-life, metabolism and excretion of tritiated luteinizing hormone-releasing hormone (LH-RH) in man. J Clin Endocrinol Metab. 1973;37(4):626-31. 16. Mendez M, Cruz C, Joseph-Bravo P, Wilk S, Charli JL. Evaluation of the role of prolyl endopeptidase and pyroglutamyl peptidase I in the metabolism of LHRH and TRH in brain. Neuropeptides. 1990;17(2):55-62. 17. Dando PM, Fortunato M, Strand GB, Smith TS, Barrett AJ. Pyroglutamyl-peptidase I: cloning, sequencing, and characterisation of the recombinant human enzyme. Protein Expression and Purification. 2003;28(1):111-9. 18. Cummins PM, O’Connor B. Pyroglutamyl peptidase: an overview of the three known enzymatic forms. Biochim Biophys Acta. 1998;1429(1):1-17. 19. Tarlatzis BC, Bili H. Safety of GnRH agonists and antagonists. Expert Opinion on Drug Safety. 2004;3(1):39-46. 20. Hackshaw A. Luteinizing hormone-releasing hormone (LHRH) agonists in the treatment of breast cancer. Expert Opin Pharmacother. 2009;10(16):2633-9. 21. Wilson AC, Vadakkadath Meethal S, Bowen RL, Atwood CS. Leuprolide acetate: a drug of diverse clinical applications. Expert Opin Investig Drugs. 2007;16(11):1851-63. 22. Short RA, O'Brien PC, Graff-Radford NR, Bowen RL. Elevated gonadotropin levels in patients with Alzheimer disease. Mayo Clin Proc. 2001;76(9):906-9. 23. Meethal SV, Smith MA, Bowen RL, Atwood CS. The gonadotropin connection in Alzheimer’s disease. Endocrine. 2005;26(3):317-25.

12 24. Thompson IM. Flare associated with LHRH-agonist therapy. Rev Urol. 2001;3(Suppl 3):S10. 25. Jan de Jong I, Eaton A, Bladou F. LHRH agonists in prostate cancer: frequency of treatment, serum testosterone measurement and castrate level: consensus opinion from a roundtable discussion. Curr Med Res Opin. 2007;23(5):1077-80. 26. Andrew V S. Luteinizing hormone-releasing hormone analogs: their impact on the control of tumorigenesis. Peptides. 1999;20(10):1247-62. 27. Moreau J-P, Delavault P, Blumberg J. Luteinizing hormone-releasing hormone agonists in the treatment of prostate cancer: a review of their discovery, development, and place in therapy. Clin Ther. 2006;28(10):1485-508. 28. Coy DH, Vilchez-Martinez JA, Coy EJ, Schally AV. Analogs of luteinizing hormone- releasing hormone with increased biological activity produced by D-amino acid substitutions in position 6. J Med Chem. 1976;19(3):423-5. 29. Werle M, Bernkop-Schnürch A. Strategies to improve plasma half life time of peptide and protein drugs. Amino Acids. 2006;30(4):351-67. 30. Kraus S, Naor Z, Seger R. Gonadotropin-releasing hormone in apoptosis of prostate cancer cells. Cancer Lett. 2006;234(2):109-23. 31. Moretti R, Marelli MM, Van Groeninghen J, Motta M, Limonta P. Inhibitory activity of luteinizing hormone-releasing hormone on tumor growth and progression. Endocr Relat Cancer. 2003;10(2):161-7. 32. Aguilar-Rojas A, Huerta-Reyes M. Human gonadotropin-releasing hormone receptor- activated cellular functions and signaling pathways in extra-pituitary tissues and cancer cells (Review). Oncol Rep. 2009;22(5):981-90. 33. Schally AV, Nagy A. New approaches to treatment of various cancers based on cytotoxic analogs of LHRH, somatostatin and bombesin. Life Sci. 2003;72(21):2305-20. 34. Mezo G, Manea M, Szabo I, Vincze B, Kovacs M. New derivatives of GnRH as potential anticancer therapeutic agents. Curr Med Chem. 2008;15(23):2366-79. 35. Ravenna L, Salvatori L, Morrone S, Lubrano C, Cardillo M, Sciarra F, et al. Effects of Triptorelin, a Gonadotropin‐Releasing Hormone Agonist, on the Human Prostatic Cell Lines PC3 and LNCaP. J Androl. 2000;21(4):549-57. 36. Morgan K, Stewart AJ, Miller N, Mullen P, Muir M, Dodds M, et al. Gonadotropin- releasing hormone receptor levels and cell context affect tumor cell responses to agonist in vitro and in vivo. Cancer Res. 2008;68(15):6331-40. 37. Dondi D, Limonta P, Moretti RM, Marelli MM, Garattini E, Motta M. Antiproliferative effects of luteinizing hormone-releasing hormone (LHRH) agonists on human androgen- independent prostate cancer cell line DU 145: evidence for an autocrine-inhibitory LHRH loop. Cancer Res. 1994;54(15):4091-5. 38. Tolkach Y, Joniau S, Van Poppel H. Luteinizing hormone‐releasing hormone (LHRH) receptor agonists vs antagonists: a matter of the receptors? BJU international. 2013;111(7):1021-30. 39. Cheung LW, Wong AS. Gonadotropin‐releasing hormone: GnRH receptor signaling in extrapituitary tissues. FEBS J. 2008;275(22):5479-95. 40. Halmos G, Arencibia JM, Schally AV, Davis R, Bostwick DG. High incidence of receptors for luteinizing hormone-releasing hormone (LHRH) and LHRH receptor gene expression in human prostate cancers. J Urol. 2000;163(2):623-9. 41. Pappa EV, Zompra AA, Spyranti Z, Diamantopoulou Z, Pairas G, Lamari FN, et al. Enzymatic stability, solution structure, and antiproliferative effect on prostate cancer cells of leuprolide and new gonadotropin‐releasing hormone peptide analogs. pep sci. 2011;96(3):260-72. 42. Millar RP, Pawson AJ, Morgan K, Rissman EF, Lu Z-L. Diversity of actions of GnRHs mediated by ligand-induced selective signaling. Front Neuroendocrinol. 2008;29(1):17-35. 43. Dondi D, Festuccia C, Piccolella M, Bologna M, Motta M. GnRH agonists and antagonists decrease the metastatic progression of human prostate cancer cell lines by inhibiting the plasminogen activator system. Oncol Rep. 2006;15(2):393-400.

13 44. Padula AM. GnRH analogues—agonists and antagonists. Anim Reprod Sci. 2005 8//;88(1– 2):115-26. 45. Nakpheng T, Sawatdee S. Stabilization of luteinizing hormone-releasing hormone in a dry powder formulation and its bioactivity. Asian Biomed. 2011;5(2):225. 46. Toth I, Flinn N, Hillery A, Gibbons WA, Artursson P. Lipidic conjugates of luteinizing hormone releasing hormone LHRH and thyrotropin releasing hormone TRH that release and protect the native hormones in homogenates of human intestinal epithelial (Caco-2) cells. Int J Pharm. 1994;105(3):241-7. 47. Blanchfield JT, Lew RA, Smith AI, Toth I. The stability of lipidic analogues of GnRH in plasma and kidney preparations: the stereoselective release of the parent peptide. Bioorg Med Chem Lett. 2005;15(6):1609-12. 48. Flinn N, Coppard S, Toth I. Oral absorption studies of lipidic conjugates of thyrotropin releasing hormone (TRH)1 and luteinizing hormone-releasing hormone (LHRH). Int J Pharm. 1996;137(1):33-9. 49. Patel M, Barot M-I, Renukuntla JW, Mitra AK. Protein and peptide drug delivery. Adv Drug Deliv. 2013:241-5. 50. Simerska P, Moyle PM, Toth I. Modern lipid , carbohydrate , and peptide based delivery systems for peptide, vaccine, and gene products. Med Res Rev. 2009;31(4):520-47. 51. Ueda T, Tomita K, Notsu Y, Ito T, Fumoto M, Takakura T, et al. Chemoenzymatic synthesis of glycosylated glucagon-like peptide 1: effect of glycosylation on proteolytic resistance and in vivo blood glucose-lowering activity. J Am Chem Soc. 2009;131(17):6237-45. 52. O’Harte FPM, Mooney MH, Lawlor A, Flatt PR. N-terminally modified glucagon-like peptide-1(7–36) amide exhibits resistance to enzymatic degradation while maintaining its antihyperglycaemic activity in vivo. Biochim Biophys Acta. 2000;1474(1):13-22. 53. Varamini P, Mansfeld FM, Blanchfield JT, Wyse BD, Smith MT, Toth I. Synthesis and biological evaluation of an orally active glycosylated endomorphin-1. J Med Chem. 2012;55 (12):5859–67. 54. Albert R, Marbach P, Bauer W, Briner U, Fricker G, Brums C, et al. SDZ CO 611: a highly potent glycated analog of somatostatin with improved oral activity. Life Sci. 1993;53(6):517-25. 55. Nomoto M, Yamada K, Haga M, Hayashi M. Improvement of intestinal absorption of peptide drugs by glycosylation: Transport of tetrapeptide by the sodium ion‐dependent D‐glucose transporter. J Pharm Sci. 1998;87(3):326-32.

14 Chapter 2: Review of literature

“Glycosylation, an effective strategy to improve the pharmacological properties of therapeutic peptides”

15 Glycosylation, an effective strategy to improve the pharmacological properties of therapeutic peptides

Abstract Given the advantages of peptides over conventional drugs, there is an increased interest in the development of peptides as therapeutic agents for the treatment of various diseases. However, several drawbacks such as undesirable physicochemical properties, short metabolic stability and low bioavailability hamper their clinical applications and successful development. Biological barriers are also formidable obstacles that impede the delivery of peptide drugs to the target site. Recent advances in the understanding of glycobiology together with synthetic methods in carbohydrate chemistry have had a great impact on the development of therapeutic peptides and proteins. Glycosylation of peptides is one of the promising strategies to modulate the physicochemical properties of peptide drugs and improve their absorption through biological membranes. This review summarises the biological barriers restricting oral and brain delivery of peptides. The effective impact of peptides’ glycosylation on overcoming the barriers is discussed. Various methods of glycoconjugate synthesis and the recent progress in the development of glycosylated peptide therapeutics are also described herein.

Keywords: peptides/proteins, glycosylation, oral delivery, vaccine development, bioavailability, glycoconjugate

2.1 Introduction

Peptides represent a promising therapeutic potential in the treatment of several types of disease with the low incidence of adverse effects (1). They offer several advantages over conventional therapeutic drugs including high activity, higher target specificity, low toxicity, and minimal non- specific and drug-drug interactions (2, 3). Development of peptide-based therapeutics is one of the major goals of the pharmaceutical industry due to the various therapeutic potentials of these molecules for the treatment of diseases. Therefore, numerous attempts have been made to improve the pharmacological properties of peptide drugs and deliver them efficiently to the target sites particularly through non-parenteral routes (4-6). The poor physicochemical and pharmacological properties of peptides impede their efficient delivery. More importantly, peptide delivery through the oral route is the most persistent challenge due to the biological conditions such as variable pH, presence of proteases and physical barriers resulting in unfavourable conditions for successful

16 delivery (7, 8). The phospholipid bilayer in the biological membranes is a common barrier that limits the adequate penetration of peptide drugs inside the intestinal cells. Furthermore, inadequate absorption and rapid degradation by proteolytic enzymes are the other obstacles resulting in the low oral bioavailability of peptides (less than 1-2%) (8-10). Chemical strategies have been explored to overcome these obstacles and can be classified into two major groups: 1) chemical modification of peptides’ structure and 2) peptides’ formulation together with other components (absorption enhancers and enzyme inhibitors) in order to alter their unfavourable physicochemical characteristics (3, 11). Lipidation, glycosylation and cyclisation are some of the chemical approaches to improve the pharmacological profile of the therapeutic peptides (12-14). Among chemical modification strategies, incorporation of carbohydrate units into the peptides’ structure has become one of the most effective strategies of overcoming the related challenges (4). Attachment of glycosyl units to peptides causes several changes in their features including their conformational structures, their chemical, physical, and biochemical properties, as well as their functions (15, 16). This review provides insights into the barriers limiting the oral and brain delivery of peptides. The glycosylation strategy and the synthetic methods of glycoconjugate production have been described. The impact of glycosylation as an effective strategy on peptide delivery and the applications of this strategy towards the development of therapeutic peptides have also been discussed.

2.2 Barriers limiting oral delivery of peptides

Physical and biochemical barriers are major issues restricting the bioavailability of oral peptide drugs. Physical barriers include cell membranes and tight junctions between the epithelial cells and mucosa that hamper the diffusion of peptides into intestinal cells. Enzymatic digestion and first-pass metabolism are considered as biochemical barriers limiting the proper absorption of peptides. Enzymatic digestion of peptides results in a short half-life and the inability of the molecules to reach the site of action in adequate concentrations and produce the desired pharmacological effect.

2.2.1 Mucus and unstirred water layer

The whole area of intestine is coated with a stable layer containing mucus, water and glycocalyx. Mucus acts as a lubricant and protective barrier which is composed of a high molecular weight of a glycoprotein component called mucin (10). Mucus is a negatively charged layer which can interact with positively charged peptides and inhibit diffusion through the layer. Unstirred water layer (UWL) is a stringent aqueous layer which is a potential barrier to the absorption of drugs and

17 is considered as a diffusion barrier (12, 17, 18). The thickness of this layer is estimated to be about 25µm in human jejunum (19). Early studies demonstrated that the absorption rate of the drug affected by UWL depends on the concentration of the solute and in higher concentrations, the permeation rate is not reduced (20). However, further studies showed that in vivo permeability of drugs is affected by this aqueous layer particularly for hydrophilic compounds (18, 21). In absorption of compounds like peptides and proteins, the mucus layer and glycocolyx are known as the first barrier (22) by which they retard the penetration of peptides through the intestinal membrane (23, 24).

2.2.2 Epithelial cell membrane

The membrane of the enterocyte in the gastrointestinal tract (GIT) is another formidable physical barrier in drug delivery research fields and the transport of peptides across the intestine is restricted by this membrane. Permeation of molecules across the intestinal epithelial cells (enterocytes) proceeds naturally by paracellular and/or transcellular pathways (8, 25). These pathways contribute to the penetration of drugs based on the physicochemical features of molecules. Generally, the paracellular pathway which comprises aqueous channels is involved in the transport of small hydrophilic components, whereas the hydrophobic molecules are typically transported through transcellular systems. Both pathways are size-dependent and limit the penetration of large hydrophilic molecules.

2.2.2.1 Paracellular pathway This is an aqueous extracellular route between adjacent cells in the epithelia (Fig. 2.1-a). In this pathway, drug molecules pass through intercellular spaces where tight junctions exist and influence the permeation of passing molecules (26). The paracellular route has some advantages in the delivery of peptides because it provides an aqueous route and has no proteolytic activity (26, 27). However, the peptide transport through this route is restricted because of the small size of the aqueous channel. The channel pore size is estimated to be between 0.3 to 1 nm in humans (28) and it is just permeable to the peptides with a molecular weight of less than 100-200 dalton (Da) (26, 29). The permeability of the solutes depends on influential factors such as the molecular weight and molecular size of the passing molecules (30). Tight junctions also act as charge (cation) selective channels to pass positively charged molecules through the spaces; however, the molecular size is dominant over the charge and the molecules larger than 200 Da cannot pass through tight junctions (31-33).

18 2.2.2.2 Transcellular pathway

In this pathway, molecules cross the cells by means of passive diffusion, transcytosis and carrier-mediated transport (Fig. 2.1-b) (8, 12). The passive transcellular transport is non-saturable and non-sensitive to the stereochemistry of a drug, because there is no specific binding site in the lipid bilayer of the membranes (34). The transport occurs according to the concentration gradient of the solute from the higher to the lower concentration. This diffusion is suitable for the absorption of lipophilic drugs due to their affinity for the phospholipid bilayer. The size, charge and water-octanol partition coefficient (cLogP) of the molecules are also essential factors that affect the transport of the molecules through passive diffusion (34). Therefore, the Lipinski ‘rule of 5’ (Ro5) can be used to predict the ability of the drug to be diffused by passive transcellular transport (35). It was believed that this is the dominant route of the transport of drugs in the biological membrane (34). However, different carrier proteins are also involved in drug transport that could explain the possible permeation route of drug-like properties. It is believed that this mechanism contributes to the transport of biologically active peptides, the hydrophilic properties and large molecular size of which do not allow their transport through the lipid bilayer. Large size molecules can cross the epithelial membrane via a facilitative mechanism called transcytosis in which the macromolecules are taken up by vesicles. Transcytosis is an energy dependent mechanism and occurs vectorially in polarized cells like enterocytes. Receptor-mediated transcytosis and adsorptive transcytosis are two types of this system in which the ligands are internalized the cells through carrying vesicles (36, 37). The specifically bound ligands are transported through receptor-mediated pathways whereas non-specifically adsorbed ligands are internalized into the cells through adsorptive transcytosis. The transport of oligopeptides through the adsorptive mechanism has been reported in several studies (37, 38). The carrier-mediated transport system involves the transport of molecules through an active transport and facilitated diffusion mechanism by intervention of transmembrane proteins called transporters or carriers. The poorly absorbed molecules with hydrophilic property are mainly transported through this transport system. The system is divided into two groups of active transport and facilitated diffusion depending on the consumption of ATP as an energy source. Unlike passive diffusion, the carrier-mediated pathway is saturable and occurs when the concentration of the substrate is higher than the Michaelis constant (Km) (34). In the facilitated transport pathway, no energy source is required and the compounds pass through the membrane according to the concentration gradient; whereas, the active transport is energy-dependent and can transport the solutes against the concentration. There are more than 400 proteins classified into two major families of transporters: the ATP- binding cassette (ABC) and the solute carrier (SLC) families (39, 40). The ABC transporter family

19 is an example of active transporters and requires the hydrolysis of ATP (primary active transport) to mediate the transport of the solutes (40). Many SLC transporters are involved in the uptake of various substrates including oligopeptides, glucose and other sugars, water-soluble vitamins, inorganic cations and anions (41, 42). SLC transporters can carry the substrate by different mechanisms including passive (uniport), coupled (symport) or exchange (anti-port) (43). Glucose transporters (GLUT) are an example of a uniport system. They transport sugars across the membrane according to their concentration gradient. Sodium sugar co-transporters (SGLT) and peptide transporters (PEPT1 and PEPT2) are among the coupled transporters. They carry substrates against the concentration gradient using the energy stored in the concentration gradient of the driving ion (Na+ or H+) (41). GLUT and SGLT are involved in the transport of carbohydrates and PEPT1 contributes to the transport of diverse peptide and peptidomimetic across intestinal membranes substrates (44, 45). Overall, the optimum physico-chemical properties of the drugs are required to take advantage of the transcelluar route. It has been reported that both passive diffusion and carrier- mediated transport cooperate in the permeation of drugs across biological membranes particularly through intestinal cells (34).

2.2.3 Efflux drug transporters Efflux transport systems are classified in the family of ABC proteins and have an important impact on the disposition of orally administered drugs including peptides. These systems consist of several proteins that affect the intestinal absorption in combination with intracellular metabolising enzymes (e.g. cytochrome P450). Thus, they can change the pharmacokinetic features of therapeutic agents leading to their poor oral bioavailability (46). Multidrug resistance proteins (MRP) and P- glycoprotein (P-gp) transporters are examples of these systems that are broadly studied. The main role of P-gp and MRP proteins is to extrude xenobiotics out of the cell leading to a restriction of the transcellular flux of drug molecules. P-gp and MRP2 are localized in the apical membrane of enterocytes whereas MRP1 are located on the basolateral side of the intestinal cells (46-49). It is believed that MRP1 is responsible for the extrusion of substrates into the blood. P-gp transporter is expressed not only in normal tissues such as the liver, kidney, brain endothelial cells and intestinal lumen, but also in cancerous cells. Therefore, it plays a pivotal role in resistance to anti-cancer therapeutic peptides (50). It has been illustrated that inhibition of efflux systems improved the permeability of peptides across biological membranes significantly (51, 52). This supports the contribution of the efflux system in limiting the absorption of peptides through cell membranes. Cyclic peptides were also shown to be the substrates of the P-gp transporter and have a higher binding affinity to P-gp than

20 linear ones. Cyclosporine is an example of a cyclic peptide that is a proper substrate of efflux transporters (53). The hydrophobic residues in the peptides’ structure interact with P-gp and have a great impact on increasing the binding affinity to the transporter in both linear and cyclic constructs (54, 55).

Figure 2.1. Barriers in oral delivery of peptide and proteins: A) Physical barriers limiting peptide transport through the intestinal membrane via: a) paracellular, and b) transcellular pathways, B) biochemical barriers comprised of metabolic digestion by brush border and/or intracellular metabolism and apical polarized efflux systems(10). P= peptide, M=metabolite

2.2.4 Metabolic enzymes Enzymatic digestion is the other reason for the low bioavailability of orally administered peptides. Most compounds are affected by the enzymatic hydrolysis that occurs at the lumen, brush border membrane and cytosol. GIT contains various digestive enzymes including cytochromes P450 (CYP450), transferases, peptidases and proteases that are involved in the digestion of therapeutic agents (56). Peptides are degraded in the lumen mainly by the proteolytic enzymes, trypsin, chymotrypsin, carboxypeptidase A and elastase secreted by pancreas (57). Elastase (serine

21 endopeptidase), trypsin and chymotrypsin are the examples of endopeptidases that hydrolyse the bond in the middle of a peptide sequence (58). The exopeptidases such as aminopeptidase A and dipeptidyl aminopeptidase are the enzymes in the brush border and the cytosol of enterocytes involved in peptide metabolism. These exopeptidases cleave the peptide bond at the NH2– or COOH–terminal of the peptide chain (58, 59). Furthermore, lysosomal peptidases such as leukocyte elastase, cathepsins B and D are involved in the degradation of peptides endocytosed by epithelial or endothelial cells (60).

2.3 Barriers limiting brain delivery

For delivery of peptides to the central nervous system (CNS), it is necessary to overcome the blood brain barrier (BBB) which is a formidable obstacle to the passage of therapeutics into brain tissue. Similar to GIT, the BBB acts as both a physical and biochemical barrier towards the delivery of peptides to the brain and consequently restricts the absorption of the target drug. Endothelial cells in the brain contain proteolytic enzymes along with cytosolic enzymes and polarized efflux systems that protect the brain from the entry of undesirable substances (61, 62). Moreover, brain capillaries are not only surrounded by endothelial cells, but are also covered by pericytes and astrocytic perivascular end-feet, which make a thick membrane around the capillary and restrict the transport of the molecules. On the other hand, the tight junctions between the endothelial cells of the brain have no fenestration which impedes the transport of the solute via the paracellular pathway (63, 64). The transport of drugs across the BBB occurs via two general pathways: 1) passive diffusion across the cell membrane and, 2) passage through specific transport systems. Drug molecules with a molecular size of smaller than 400 Da and bearing less than 8 hydrogen bonds may cross the BBB through passive diffusion. However, these properties are lacking in the majority of drug molecules (65). Due to relatively large and hydrophilic characteristics, peptides do not cross the BBB by passive diffusion and they require a particular transport mechanism to penetrate into the cells (66). The transport mechanisms including carrier-mediated transport, fluid phase endocytosis, transcytosis, adsorptive and receptor-mediated endocytosis are involved in the passage of the nutrients and small molecules to the brain (62, 66, 67). It has been shown that adsorptive mediated endocytosis and transcytosis mainly contribute to the penetration of the opioid peptide’s analogues across the BBB (68, 69). In addition, the BBB possesses proteolytic enzymes acting as metabolic barriers and causes the rapid degradation of the endogenous peptides (70).

2.4 Role of glucose transporters in the transport of carbohydrates across biological membranes

22 Transport of hexoses across cell membranes is mediated by two different types of membrane protein families (GLUT and SGLT transporter families) in most mammalian cells (71). Glucose transporters are expressed in tissues and involved in the control of glucose homeostasis, i.e. muscle, adipose tissue, liver, intestine and brain (72). GLUT are Na+-independent facilitative transporters and they move the hexoses across the cell membranes according to their concentration gradient (73). GLUT transporters consist of 14 identified isoforms and they are classified in the SLC2 transporters’ family. The distribution of the isoforms of these transporters differs in tissues and each transporter shows different affinities to hexoses. Based on the molecular structures, the isoforms are divided into three subclasses: class I comprises the classical transporters GLUT1–4 and GLUT14; the ‘‘odd’’ isoforms of GLUT5, 7, 9, and 11 belong to class II and class III contains the isoform GLUT6, 8, 10, 12 and the proton-driven myo-inositol transporter HMIT (GLUT13) (74). In addition to glucose, GLUT transporters mediate the transport of other monosaccharides including galactose, mannose, and fructose across cell membranes (72, 74). The other group of glucose transporters known as SGLT are Na+-dependent transporters and they are classified as the members of the SLC5 transporter family. SGLT transporters carry hexoses actively through cell membranes against the sugar concentration gradient by utilisation of the energy provided by Na+- K+ ATPase pumps (40, 45, 75). These transporters also contribute in the transport of amino acids, vitamins, osmolytes, and some ions across the intestinal brush border and renal tubules (76). Among all six identified isoforms, SGLT1 is the most known member involved in the transport of glucose across the intestinal brush border. SGLT1 has the same affinity for both glucose and galactose. SGLT2 is a high-capacity and low-affinity transporter located in the membrane of proximal renal tubules (77). It is responsible for a vast amount of D-glucose reabsorption in kidney (76, 78). SGLT3 is expressed in cholinergic neurons and skeletal muscles. Unlike the other members of this family, SGLT3 is not a glucose transporter and it seems to have a role as a glucose sensor (79). The functions of SGLT4, SGLT5, and SGLT6 are poorly understood (78). The detailed information concerning the structure and function of all GLUT isoforms has been extensively reviewed elsewhere (74, 80). The transport of carbohydrates in the intestine is mediated by both active transporters (SGLT) and facilitative transporters (GLUT) located on the epithelial cell membranes (73). In the intestine, SGLT1 and GLUT5 are located mainly on the apical side of the brush border of epithelial cells. GLUT2 is the dominant isoform of the transporter with a low affinity to glucose and resides in the basolateral side of enterocytes (Fig. 2.2) (40, 41). In response to high concentrations of sugar in the lumen, GLUT2 is transiently expressed on the apical surface of entrocytes and provides a chief pathway for carbohydrate absorption (41, 42). GLUT2 and SGLT1 transporters show different affinities for D-glucose and D-galactose. At low concentrations, glucose is transported against a

23 concentration gradient by SGLT1 (81), whereas, GLUT2 contributes to the glucose uptake at high concentrations of the sugar (82, 83). GLUT2 is also expressed in the basolateral surface of kidney absorptive cells and liver sinusoidal membrane where it is involved in blood glucose uptake (72). These transporters are useful targets to transport therapeutic peptides through biological membranes. For instance, Nomoto et al. showed that SGLT1 had an important role in the transport of glycosylated tetrapeptide (Gly-Gly-Tyr-Arg) which is not transported by di- or tripeptide transporters (84). The presence of D-fructose increases the level of GLUT5 mRNA confirming the role of this transporter in the intestinal absorption of fructose.(85) GLUT7, is another isoform of hexose transporters expressed in the apical membrane of intestinal cells and responsible for the transport of glucose and fructose (42).

Figure 2.2. Localisation of glucose transporters on the membrane of enterocytes (86): GLUT-2 is located on the basolateral side of the cells and mediates the transport of monosaccharides down their concentration gradient. SGLT-1 is located on the luminal (apical) side of the intestinal cells. Monosaccharides are co-transported with Na+ against their concentration gradient by contribution of this membrane transporter.

In the brain, GLUT1 and GLUT3 are the main glucose transporters expressed in endothelial cells and neuronal cells, respectively. GLUT1 is the first identified isoform of the GLUT family and has been extensively studied. Two molecular forms of 45 (kilo dalton) kDa and 55 kDa exist in the brain in which they are localized at the perivascular end-feet of astrocytes and at the membranes of the endothelial cells, respectively (Fig. 2.3) (87). GLUT3 is localized at the neuronal cell membrane and mediates glucose transport from extracellular space into neuronal cells (88). It has been

24 suggested that the GLUT1 transporter is responsible for the improved uptake of glycosylated opioid peptide into the brain (43, 89).

Figure 2.3 Localization of GLUT transporter isoforms in the brain: The 55-kDa form of GLUT1 is found in the endothelial cells, whereas the lower molecular weight of the 45- kDa form is expressed in astrocytes. The GLUT3 isoform is located in neuronal cells and it is responsible for the transport of glucose from extracellular spaces into the cells (87).

It has been demonstrated that the isoforms of GLUT transporters such as GLUT1 and GLUT3 are overexpressed in various cancer cells that can be used as a target for anticancer therapy and immunodiagnostic markers (90-93). It has been found that the overexpression of GLUT1 is associated with tumour progression and the reduced expression of GLUT1 suppresses the tumour growth in vitro and in vivo (92, 94, 95). SGLTs are also expressed on the tumour cell that can be targeted to deliver chemotherapeutic agents into the cancer cells (96).

2.5 Glycosylation strategy for peptide delivery

Carbohydrate moieties are used to change the physiological properties of peptides and improve their bioavailability. Some advantages of glycosylation include: 1) targeting specific organs and enhancing biodistribution in tissues (66), 2) improving penetration through biological membranes, 3) increasing metabolic stability and lowering the rate of clearance (97), 4) improving receptor- binding (98), 5) protecting amino acid’s side chain from oxidation, and 5) maintaining and stabilizing the physical properties of peptides such as precipitation, aggregation, thermal and kinetic denaturation (4, 99, 100). Conjugation of sugars with peptides can also facilitate the active transport of modified compounds across cell membranes by targeting glucose transporters on the surface of

25 biological membranes (101). The favourable impact of glycosylation on pharmacokinetic/pharmacodynamics properties of the indigenous peptides leads to an increase in their oral absorption and bioavailability. Glycosylated somatostatin is one pioneering example with potent biological activity through the oral route. The oral bioavailability of the modified peptide improved markedly which corresponded to the enhanced inhibitory effect on the release of growth hormone (102).

2.5.1 Glycopeptide synthesis

The synthesis of glycoconjugates is a challenging process due to the presence of several functional groups (hydroxyl groups) in their structure. Therefore, the functional groups of the sugar moiety should be masked by permanent or temporary protecting groups to protect them from reacting with the reagents during synthesis (103). Benzyl ether and benzoyl ester are permanent protecting groups removed by reduction reaction at the end of the synthesis. Temporary groups such as trityl ethers, acetate and fluorenylmethyloxycarbonyl are removed during the intermediate steps of the synthesis. They are easier to cleave but stable during the synthesis (103, 104). Regio- specificity (when only a single hydroxyl group is glycosylated) and streo-selectivity (when the β- anomer is produced selectively) are two important factors in glycosylation reaction resulting in the production of higher yield of final products with reduction of producing side products. The type of protecting groups for masking the hydroxyl groups controls the regio-selectivity of the glycosides and determines their stereo-selectivity (103, 105). N- and O-linked glycosylation are naturally occurring processes in which carbohydrates are attached to polypeptide chains. This attachment can be through co-translational or post-translational modifications via either N- or O-glycosidic linkages (106). N-glycosylation occurs through the amine group of asparagine (Asn) residue resulting in the formation of an amide bond. In O-linked glycopeptide, the oxygen atom in the side chain of serine or threonine residues binds to the carbohydrate moiety through an ether bond (Fig. 2.4 A) (106-108). Synthetic glycopeptides are produced through a combination of the techniques used in carbohydrate and peptide chemistry. Generally, chemical and chemo-enzymatic methods are the reliable approaches for the synthesis of glycopeptides and glycoproteins. Direct and convergent syntheses are two common chemical strategies for N- or O-linked glycopeptides (Fig. 2.4B). In the direct method, the pre-synthesized glycosylated amino acid is coupled to the elongating peptide through solid phase peptide synthesis (SPPS) in a stepwise fashion (108). However, the synthesis of long peptides with more than 50 residues is difficult by this method due to the incomplete couplings and epimerization. This leads to the formation of side

26 products and a low yield of final product (105). Therefore, convergent (fragment–condensation) synthesis is applied as an alternative strategy to overcome this problem. The convergent approach is particularly used for N-linked glycopeptide synthesis as O-glycosylation is not achievable by this method (109). In the convergent method, the glycosylamine unit is conjugated to an aspartic acid containing peptide through condensation of the amino acid (110). This strategy is hampered due to the peptide’s racemization at the C-terminus and formation of aspartimides. To overcome the drawback, a modified convergent method has been employed in which pseudoproline motif is incorporated at Thr residues to avoid aspartimide formation (111).

Figure 2.4: A) Examples of O-linked and N-linked glycosylated amino acids, B) Direct and convergent strategies for glycopeptide synthesis.

In addition to O- and N-linked glycosylation approaches, several chemical methods have been established for the attachment carbohydrate units to different amino acid residues at N-terminus of peptide’s sequence. Conjugation of galactose unit to the N-terminus of α-melanocyte-stimulating hormone octapeptide analogue (NAPamide) is one of the examples in which the anomeric carbon of the carbohydrate was modified by ethanoic acid and attached to the N-terminus of NAPamide via the SPPS strategy (Fig. 2.5A) N-terminus modification of peptides is also achievable by conjugation of carbohydrate units to peptide through a succinamic linker. In this method, the azide

27 derivative of sugar moiety is replaced by succinamic acid at anomeric carbon and coupled to the N- terminus of the peptide through a peptide bond (Fig. 2.5B).

(A)

(B) ( B)

Figure 2.5. The attachment of galactose residue to the N-terminal of A) NAPamide and B) LHRH peptides. A: Galactose was conjugated to the peptide through anomeric carbon modified by ethanoic acid (112); B: Sugar was attached to the peptide through a succinamic acid linker (113).

Chemo-enzymatic approaches are powerful tools that combine the flexibility of chemical synthesis and high regio- and stereo-selectivity of enzyme-catalysed reactions to achieve highly efficient synthesis of complex carbohydrates. Particularly, these techniques are ideal choices for complex chemical synthesis like sialic acid-containing molecules or attachment of oligosaccharides to polypeptides (114). To date, several enzymes have been found with transglycosylation activity that can attach glycosyl moieties to peptides. Endo-β-N-acetylglucosaminidases, Endo-A (form

Arthrobacter) and Endo-M (from Mucor hiemalis) are common endoglycosidases with distinct substrate activity (115). These enzymes are able to couple an intact oligosaccharide to N- acetylglucosamine (GlcNAc)-containing peptide or protein in a single step (116, 117). β-(1,3)-N-

28 acetylglucosaminyltransferase (EC 2.4.1.56; LgtA) is an enzyme isolated from Neisseria meningitides and was used for the conjugation of GlcNAc residue to glycosylated endomorphin-1 and lactose-enkephalin derivatives (118-120). Lipopolysaccharyl α-1,4-galactosyltransferase (EC 2.4.1.; LgtC) is another glycosyltransferase derived from Neisseria meningitides and is applied for attachment of the galactose unit to the terminal lactose residue in lipooligosachharide (121). This enzyme was used to attach galactose residue to a glycosylated enkephalin to improve the metabolic stability of the peptide and target the ASGPR receptor (122). Using glycosyltransferases for glycosyl unit synthesis has some advantages including high yield and regio- and stereo-specificity without the need for protecting groups. Advances in the field of glycoconjugate synthesis will further improve the medical applications of these important and diverse biologically active molecules.

2.5.2 Impact of glycosylation on physicochemical properties

Physicochemical properties of peptide drugs play an important role in their pharmacokinetic profile and metabolic fate in the human body. Attachment of carbohydrate units to the peptides improves the physicochemical properties of the construct. Glycosylation can enhance the molecular stability and change the conformation of the peptide backbone (123-125). Lin et al. showed that the modification of hamster prion peptide with different sugar entities such as mannose, galactose, and N-acetylgalactosamine (GalNAc) exerts diverse impacts on the conformational properties of the polypeptide chain. Mannosylation of the prion has an inhibitory impact on the formation of amyloid fibril (a special type of aggregation) implying the anti-aggregation function of this sugar entity on the prion peptide (125). It has been shown that the position of the glycosyl unit in the peptide’s structure is an important factor in changing the conformation of the peptide backbone and may affect the biological properties of the modified peptide. For instance, attachment of N-acetyl galactose amine (GalNac) to Thr6 and Thr21 in calcitonin peptide broke the helical structure of the intact peptide resulted in a reduction in receptor-binding affinity and loss of its bioactivity (126).

2.5.3 Impact of glycosylation on pharmacological characteristics of peptides

One of the main obstacles in the development of therapeutic peptides is their poor pharmacological properties. Glycosylation of peptides can modulate their pharmacokinetic/pharmacodynamic properties and improve their therapeutic efficacy. Several factors such as position, type and the number of carbohydrates are crucial to enhance the

29 pharmacological properties of the manipulated peptides and influence their biological functions (127). The position of the glycosyl unit attached to the peptide can influence the peptide-receptor interactions, biodistribution and pharmacological activity of the glycosylated peptides (70, 112, 128). The structure-activity studies with enkephalin-based glycopeptides demonstrated that the position of the glycosyl units attached to the opioid peptides had different effects on binding affinity and the potency of the glycopeptide. The addition of β-D-glucose to the cyclized region of the opioid peptide Met-enkephalin decreased the binding to the receptor and eliminated its in vivo activity. Whereas, the glycosylation at position six of the linear peptide improved its analgesic activity significantly with retained receptor-binding affinity (129). If carbohydrate units are attached to peptides at the proper position, they preserve the structure-activity relations (SAR) of native peptide with the target receptor and enhance their biological activity (112). The site- dependent effect of glycosylation was also investigated for O-glycosylated calcitonin analogues and it was shown that glycosylation affects both the conformation and biological activity of calcitonin in a site-dependent manner (130). The type and number of carbohydrate (i.e. mono, di, and tri-saccharide) conjugated to the peptide can also influence its biological activity. Elmagbari et al. reported that the type of monosaccharide including α-mannose, β-xylose and β-glucose exerted different impacts on the opioid receptor-binding affinity of the hexapeptide (Tyr-D-Thr-Gly-Phe-Leu-Ser-CONH2). The best binding affinity was observed for the hexapeptide bearing β-glucose. Furthermore, conjugation of disaccharide to the hexapeptide caused a higher antinociceptive activity than the monosaccharide-bearing analogue after intravenous administration (131). The impact of carbohydrate type (glucose, galactose and mannose) on renal delivery of arg-vasopressin (AVP) peptide was studied in rats. The glucose and mannose derivatives of AVP peptide exhibited higher renal uptake than the galactose-modified analogue. It was also shown that the glucosyl and mannosyl conjugates that was bound specifically to the kidney microsomal membrane in vitro resulted in high renal uptake (132). The attachment of tri-saccharide (galactose-lactose) to enkephalin resulted in a higher binding affinity to the asialoglycoprotein receptor compared to the galactose-modified analogue. The enzymatic stability of the trisaccharide (galactose-lactose) conjugated enkephalin in human plasma and Caco-2 cell homogenate improved significantly compared to the peptide alone (133). The higher number of sugar units is not always accompanied by an improvement in the biological properties of the modified peptides. A single GlcNAc unit attached to calcitonin had the best hypocalcemic effect with improved biodistribution of the peptide; whereas, increasing the number

30 of carbohydrate moieties (multiple copies of mannose and GlcNAc) decreased the activity of calcitonin (130). Improving the metabolic stability of peptide drugs is another advantage of glycosylation leading to enhance their in vivo bioavailability. Typically, endogenous peptides have short half-lives in the biological environment due to the enzymatic digestion. Incorporation of carbohydrate units to peptides can protect them from proteolysis without changing their bioactivities (113, 134-139). Different strategies of glycosylation (N-linked, O-linked or N-terminal glycosylation) have been applied for improving the metabolic stability of modified peptides both in vivo and in vitro. Introduction of O-β-glucosylated serine glucose (Ser(Glc)) to the analgesic compound TY027 (Tyr-

D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3´,5´-Bzl(CF3)2) at position six (O-linked glycosylation) enhanced its metabolic stability significantly (128). The longer serum half-life was reported for Major-Histocompatibility-Complex (MHC)-binding peptides (MHC receptor inhibitors) following glycosylation. Substitution of valine with N-acetyl glucosamine-modified Asn in (MHC)-binding peptide (N-linked glycosylation) stabilized the modified peptide against serum peptidases significantly compared to the unmodified analogue (134). N-terminal modification of glucagon-like peptide 1 (GLP-1) with glucitol residue improved the resistance of the compound to enzymatic degradation after intraperitoneal administration to Wistar rats (135). In another study, the attachment of sialyl N-acetyllactosamine (lacNAc) to Asn residue of GLP-1 peptide via N-linked glycosylation improved the in vivo stability of the modified peptide and prolonged its antihyperglycaemic activity (136). N-terminal attachment of the glycosyl unit to endomorphine-1 via the succinamic acid linker improved the metabolic stability of the peptide in human serum significantly (137). The same strategy was applied for N-terminal modification of LHRH which resulted in significant enhancement in the metabolic stability of the modified peptides in Caco-2 cell homogenate (containing a series of proteolytic enzymes) (113). Improving the penetration of peptides across biological membranes is another advantage of glycosylation. Glycosylation of endomorphine-1 resulted in a 700-fold increase in membrane permeability across the Caco-2 cell monolayer. It was suggested that this analogue was transported across membranes through a lactose-selective transporter (137). N-terminally modified LHRH with sugar moieties including glucose, galactose and lactose had a significant impact on improving the permeability of the analogues through the Caco-2 cell monolayer (113). It was reported that GLUT2 and SGLT1 contributed to the transport of the glycosylated LHRH analogues across Caco-2 cell membranes. It was also shown that the efflux pumps (P-gp and MRP2 transporters) did not affect the apparent permeability of any of the glycosylated analogues except for the galactose derivative (52).

2.6 Development of therapeutic peptides using glycosylation strategy 2.6.1 Neuropeptide therapeutics Neuropeptides have therapeutic potential for the treatment of neurological disorders. However, the successful delivery of these peptides to the CNS has been hampered due to formidable obstacles like the BBB, enzymatic digestion and liver clearance limiting the efficient delivery of peptides to the brain (140, 141). Glycosylation has been shown to be one effective strategy to improve brain delivery of therapeutic peptides. This approach promotes the penetration of opioid peptides including enkephalins, endorphins and dynorphins into the brain and increases their pharmacological activity. Therefore, the glycosylated analogues of opioid peptides have shown improved analgesic activity (129, 137, 142, 143). Enkephalin is a pentapeptide involved in antinociception with a short half-life in blood and an inability to pass the BBB. Attachment of

Ser(Glc) residue to Lue-enkephalin amide (Tyr-D-Thr-Gly-Phe-Leu-Ser-NH2) improved the permeability of the opioid peptide across the BBB in mice. The glycosylated analogue produced a similar antinociceptive effect to morphine (144). The improved permeability and higher metabolic stability of the glycosylated neuropeptides results in a significant increase in their bioavailability that might account for the enhanced analgesic effect of the glycopeptides (64, 143). The decreased renal clearance of glycosylated analogue of Met-enkephalin (conjugated β-D glucose) resulted in significant improvement in the bioavailability and analgesic effect of the peptide in rats (142). Conjugation of lactose succinamic acid to endomorphine-1 produced a significant analgesic activity after oral administration in a chronic pain model in rats and the modified peptide showed a significant change in binding-affinity to μ-opioid receptor (137). It is postulated that glycopeptides penetrate the BBB through adsorptive endocytosis (145). However, the exact mechanism involved in improving the permeation of the glycosylated peptides through biological barriers is yet to be elucidated.

2.6.2 Radiopharmaceuticals Glycosylation is a promising strategy for improving the biodistribution and poor pharmacological profiles of the radiolabeled peptides for diagnostic and therapeutic purposes. The radiolabeled derivatives of bombesin peptide have potential applications in cancer cell imaging and peptide receptor radiotherapy. However, they possess unfavourable pharmacokinetic properties such as hepatic accumulation and hepatobiliary excretion (146). Conjugation of radiolabeled bombesin analogues with glucose moiety (through a traizole group) reduced the abdominal accumulation and increased the tumour uptake without affecting the cell internalization of the modified peptides (147). Glycosylation was applied to increase the hydrophilic property of

32 radiolabeled Tyr3-octreotide peptide and overcome the drawbacks restricting its diagnostic application in cancer radiotherapy. Carbohydrate modifications of the peptide using glucose, maltose and maltotriose resulted in a higher renal clearance and subsequently less accumulation of the peptide in the liver and abdominal region. This modification made Tyr3-octreotide analogues (particularly maltose and glucose-conjugated peptides) suitable for targeted imaging and radiotherapy of somatostatin receptor-expressing tumours (148). In another study, the radiolabeled arginine–glycine–aspartic acid (RGD)-containing pentapeptide (cyclysed (Arg-Gly-Asp-D-Tyr-Lys) was glycosylated to improve the pharmacokinetic profiles of the modified analogues. These peptides are antiangiogenetic agents and act as an antagonist of an integrin glycoprotein (αVβ3) expressed in malignant tumour cells. It was observed that conjugation of GlcNAc to Lys residue of the peptide decreased its lipophilicity and reduced the hepatic uptake of the modified peptide leading to a significant increase in tumour uptake (149).

2.6.3 Targeted delivery Carbohydrate-mediated delivery also termed as glycotargeting is a strategy that employs cell surface recognition in order to target specific organs. Carbohydrates are useful candidates for receptor-targeted peptide delivery as their receptors, known as lectin receptors, are expressed in the membrane of different cells such as liver, tumour cells, and kidney. Therefore, the therapeutic agents conjugated with carbohydrate units can be recognized by those receptors and internalized into the cells (150). Asialoglycoprotein receptor (ASGPR) is a lectin receptor expressed on the surface of liver hepatocytes that recognizes the galactose and the galactosyl residue of the glycoproteins (150, 151). ASGPR can be targeted for peptide delivery to the hepatocytes. Reports indicated that kidney and brain targeting of peptides are also achievable through a glycosylation strategy. Suzuki et al. showed that the mannose and glucose derivatives of arg-vasopressin had high in vivo renal uptake (132). Glucose transporters such as GLUT1 and GLUT3 are overexpressed in various cancer cells that can be targeted for anticancer therapy and immunodiagnostic markers (90-93). It has been found that the overexpression of GLUT1 is associated with tumour progression and the reduced expression of GLUT1 suppresses the tumour growth in vitro and in vivo (92, 94, 95). Glycopeptides can also target specific cancer cells. The impact of galactose and glucose matotriose on the pharmacokinetic properties of α-melanocyte-stimulating hormone (α-MSH) was evaluated to target melanoma cancer. It was shown that the glycosylated analogues exhibited excellent binding affinities (in nanomolar and subnanomolar ranges) to the specific receptor (melanocortin receptor 1 (MC1R) overexpressed in melanoma cells) in vitro. Among all glycopeptides, the analogue-bearing galactose unit at the N-terminus of the peptide had a favourable

33 pharmacokinetic profile (higher tumour uptake with a lower kidney uptake) for melanoma targeting (112).

2.7 Conclusion

The successful development of peptide-based therapeutics requires the optimization of their pharmacological profiles. Glycosylation can be applied to enhance the therapeutic behaviour of peptide drugs by optimizing their pharmacokinetic and pharmacodynamic properties. Incorporation of carbohydrate moieties into the peptides’ sequence can change their physicochemical properties leading to increasing the membrane permeability across biological membranes and improving their proteolytic stability against digestive enzymes. The significant therapeutic potential of glycosylated peptides accounted for the establishment of several techniques for the synthesis of glycoconjugates which had an important impact on the development of carbohydrate-based peptide drugs. Further advancements in understanding the fundamental impact of glycosylation on the pharmacological properties of peptides are still required for the rational design of glycopeptides with enhanced biological activity.

34 2.8 References

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Chapter 3: Glycosylated LHRH analogues and

their biological properties

43 Section A “Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH)’’

3.1 Introduction to this publication

This section was published as a research article in the journal of ‘’Bioorganic and Medicinal Chemistry’’. In this article, the synthesis of the glycosylated LHRH derivatives by the attachment of different sugar moieties including lactose, galactose and glucose was described. The in vitro biological properties of the glycosyl conjugates of LHRH including their metabolic stability in Caco-2 cell homogenate and their membrane permeability across Caco-2 cell monolayer were investigated and reported in this publication.

Moradi SV, Mansfeld FM, Toth I. Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH). Bioorg Med Chem. 2013;21(14):4259-65.

3.2 Reprint of this peer-reviewed publication

44 Bioorganic & Medicinal Chemistry 21 (2013) 4259–4265

Contents lists available at SciVerse ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH) ⇑ Shayli Varasteh Moradi a, Friederike M. Mansfeld a, Istvan Toth a,b, a School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD 4072, Australia b School of Pharmacy, The University of Queensland, Woolloongabba, QLD 4102, Australia article info abstract

Article history: Luteinizing hormone-releasing hormone (LHRH) analogues are used extensively for the treatment of var- Received 14 February 2013 ious hormone-dependent diseases. However, none of the currently marketed derivatives can be admin- Revised 18 April 2013 istered orally. Modification of peptide sequences by attachment of carbohydrate moieties is a promising Accepted 26 April 2013 strategy that may increase the metabolic stability of the target peptide and enhance its transport across Available online 6 May 2013 cell membranes, subsequently improving peptide bioavailability. In this study, either the N- or C-terminus of the LHRH peptide was altered by attachment of carbohydrate moieties. Caco-2 cells were chosen as Keywords: an in vitro model to investigate both the permeability and stability of the new LHRH analogues. Our find- Luteinizing hormone-releasing hormone ings show that conjugating sugar moieties to the N-terminus of the LHRH peptide significantly increased Carbohydrate moieties Peptide modification both permeability and metabolic stability of most of the modified LHRH derivatives. Permeability Ó 2013 Elsevier Ltd. All rights reserved. Metabolic stability

1. Introduction compliance and ease of treatment. Therefore, orally available LHRH analogues are highly desirable. However, the development of orally Luteinizing hormone-releasing hormone (LHRH), also known as active LHRH derivatives has been hampered by poor stability and gonadotropin releasing hormone (GnRH), is a neuropeptide that bioavailability in vivo. Hence, for oral delivery, it is necessary to plays an important role in regulating the reproductive system.1 It improve the pharmacokinetic proﬁle of LHRH to enable transport is produced by neurosecretory cells in the hypothalamus and to the target site in an adequate concentration. mediates its effect by binding to the cognate receptor on gonado- Physical and biochemical barriers are major issues restricting trope cells in the pituitary gland, where it stimulates the secretion the oral bioavailability of peptide drugs.12 Physical barriers, includ- of luteinizing hormone (LH) and follicle-stimulating hormone ing the mucosa, cell membranes and tight junctions between epi- (FSH). The release of FSH and LH controls the secretion of sex ste- thelial cells, impede the absorption of peptides from the roid hormones in the gonads.1,2 gastrointestinal (GI) tract.13 Digestive enzymes in the GI tract have LHRH is a decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro- an important impact on peptide drugs, decreasing the half-life and

Gly-NH2, Fig. 1) and has a short half-life of 3–4 min in human plas- affecting their ability to reach the site of action in an adequate con- ma due to the rapid enzymatic degradation.3,4 It is mainly cleaved centration after oral administration.12,14 The intact LHRH peptide by a metalloendopeptidase called EP24.15 at position six (Tyr5– passes the blood brain barrier (BBB) via a saturable transport sys- Gly6), prolyl endopeptidase (PE) at position nine (Pro9–Gly10) and tem.15–17 The principal factors impeding drug absorption in the pyroglutamyl peptidase that cleaves pGlu residue at position one central nervous system (CNS) include proteolytic enzymes in brain (pGlu1–His2) of the peptide sequence.5–7 endothelial cells, and an apically polarised efﬂux system that Elucidation of the structure of LHRH by Schally et al. in 19718 pumps the solute back to the blood circulation.18,19 prompted the development of several LHRH analogues, which have To date, several strategies have been explored to overcome the a variety of applications in the treatment of hormone-dependent challenges presented by delivering peptides orally. Among them, diseases such as prostate cancer, precocious puberty, endometri- chemical modiﬁcation of peptide sequences (e.g. attachment of li- osis and breast cancer. All clinically approved analogues currently pid and carbohydrate moieties or amino acid substitution) is con- on the market are administered daily, either parenterally or intran- sidered to be an effective approach, enhancing bio-distribution of asaly.9–11 Oral administration has advantages in terms of patient the target peptide by changing its pharmacodynamic and pharma- 20,21 cokinetic properties. Substitution of L-amino acids with ⇑ Corresponding author. Tel.: +61733469892; fax: +61733654273. D-amino acids is a common method of chemical alteration which E-mail address: [email protected] (I. Toth). protects peptides from proteolysis and can stabilise the desired

HN NH2

NH NH

HO O N O O O H H H N N N N HN N N N O H H H N H O O O O O HN NH

OH H2N

Figure 1. Structure of LHRH. conformation.22 Attaching carbohydrates to C-terminus, N-termi- tor binding affinity due to the stabilisation of a conformation nus or the middle of the peptide sequence is another method that important for receptor binding.9,32 Compound 11 was synthesised can increase the metabolic stability of the peptide, and improve its by the replacement of Gly with D-Trp at position six and conjuga- absorption across biological barriers.23–26 tion of the succinamic acid derivative of lactose at its N-terminus. The purpose of this study was to examine the role of carbohy- Compound 10 (the commercially available analogue-triptorelin) drate moieties in improving the metabolic stability and in vitro was synthesised by substitution of Gly with D-Trp at position six permeability of LHRH. Therefore, we synthesized a small library for comparison with compound 11. of LHRH derivatives by conjugating carbohydrate moieties to either All of the peptides were synthesised on Rink amide MBHA resin the N- or C-terminus of the peptide sequence. We also synthesized using Fmoc chemistry via standard solid phase peptide synthesis.33 an analogue which was modified by substituting the amino acid The crude peptides were then purified using preparative HPLC, sequence in addition to the attachment of a sugar moiety to its characterised by electrospray ionisation mass spectrometry (ES- N-terminus. The analogues were analysed using established MS). in vitro assays to assess their stability against enzymatic metabolism (exposure to Caco-2 cell homogenate), and their permeability across biological barriers (Caco-2 cell monolayer permeability). 2.2. Caco-2 cell monolayer permeability assay

2. Results and discussion Due to similarities with human intestinal epithelium, Caco-2 cell monolayers are routinely used in pharmaceutical research 2.1. Design and synthesis of glycosylated derivatives of LHRH as a useful model to predict the transport of drug molecules across biological barriers.34,35 The apparent permeability of LHRH The glycosylated LHRH analogues that were synthesized using analogues was examined using Caco-2 (Fig. 3) cell monolayer at solid phase peptide synthesis (SPPS) are shown in Table 1. Com- pH 7.4 and 37 °C to mimic conditions similar to those of the pound 1, native LHRH, was used as a control. In compound 2, pyro- physiological environment. Freshly prepared Hank’s balanced glutamic acid (pGlu) at position one was replaced with glutamic buffer solution (HBSS) in 25 mM of N-2-hydroxyethylpiperazine- acid (Glu). Carbohydrate units were made compatible with SPPS N0-2-ethanesulfonic acid (HEPES) buffer was used to keep the by modification with a succinamic acid linker (for N-terminus pH within this range during the test. Furthermore, the compound attachment) or an azide (for C-terminus attachment) at the ano- solutions were prepared in HBSS/25 mM HEPES. The integrity of meric carbon in four synthetic steps. For the synthesis of com- tight junctions within the monolayer was evaluated using trans- pounds 3–5, pyroglutamic acid (pGlu) was substituted with epithelial electrical resistance (TEER) values before and 24 h after glutamic acid (Glu) at position one, to provide a free amine group the experiment. Values obtained after performing the test were at the N-terminus to allow attachment of lactose (3), galactose (4) within 15% of those measured before the start of the experiment. or glucose (5). The synthesis of compound 6 was done by addition This confirmed that the cells were stayed quite intact and none of of glucoronic acid to C-terminus of LHRH. Two main changes were the compounds were toxic and damaging to the cell. Additionally, performed for the synthesis of compounds 7–9. Instead of glutamic [14C]mannitol was used as a paracellular flux marker to show acid, glutamine was added in position one, then the N-termini of that the cell monolayer remained intact. This assay was those compounds were modified by attachment of glycosyl units. repeated twice independently and in triplicate for each tested The reason for replacement of glutamic acid with glutamine at po- compounds. sition one was because of faster cyclisation of glutamine compared The parent peptide (compound 1) had very low permeability of to glutamic acid at physiological pH.27,28 Furthermore, the natural 3.98 107 cm/s (Fig. 2). Among all tested compounds, the appar- sequence of the LHRH prohormone contains glutamine instead of ent permeability of compounds 3, 5 and 9 increased significantly glutamic acid which is cyclised when the hormone converts to (One-way ANOVA, p <0.05) by approximately 6–7-fold compared the biologically active peptide.29 with the native peptide. The permeability of compound 8 As described in the introduction, Tyr5–Gly6 is the main cleavage (16.2 ± 2.23 107 cm/s) was also higher than that of the native site of LHRH.5,7 It is well-known that substitution of glycine in po- peptide (1). The apparent permeability of analogue 4 was about sition six with a D-amino acid results in increased metabolic stabil- three times higher than LHRH, however this increase was not sta- 30,31 ity and lipophilicity. Moreover, placing D-amino acid residue at tistically significant. The permeation rate of analogue 7 did not position six generates agonist derivatives of LHRH with high recep- change notably compared with peptide 1. 46 S. V. Moradi et al. / Bioorg. Med. Chem. 21 (2013) 4259–4265 4261

Table 1 Structure of LHRH analogues Compound Structure 1 2

The Caco-2 cell monolayer permeability assay has known limi- gation to prepare the soluble fraction of the cell homogenates. tations. For example the underestimation of carrier-mediated Among all derivatives of [E1]LHRH, compound 3 (modified with 36,37 transport due to low expression of transport proteins and over- lactose at the N-terminus) had the highest stability (t1/ 38 expression of the efflux transporter P-glycoprotein. Nevertheless, 2 = 77 min). Compound 6 showed the lowest half-life at t1/ we observed a considerable increase in the transport of most su- 2 = 28.4 min, which was even lower than that of the native peptide. gar-conjugated LHRH analogues across the Caco-2 cell monolayer. Our results are consistent with previous studies that indicated that the stability of peptides with C-terminal carbohydrate modifica- 2.3. Caco-2 cell homogenate stability assay tions is quite similar to the parent peptide.40,41 We also observed a significant increase in the half-life of all su- The in vitro stability of carbohydrate conjugated LHRH was gar conjugated analogues of [Q1]LHRH. In particular, (Table 2) ana- tested using Caco-2 cell homogenates. Well-differentiated Caco-2 logue 7 was the most stable compound (t1/2 = 133 min) in Caco-2 cells express a series of proteolytic enzymes that may be a good cell homogenate. Although compound 11 was less stable than model for investigating the metabolic profile of potential therapeu- compound 10, it exhibited a significant improvement in stability tic peptides.39 In this assay, the stability of all compounds were of approximately fourfold when compared with LHRH. The half-life tested in quadruplicate and repeated three times independently. of each analogue is presented in Table 3. The half-life of endoge- Caco-2 cells were lysed and separated from cell debris by centrifu- nous LHRH in Caco-2 cell homogenates was found to be 32.7 min 47 4262 S. V. Moradi et al. / Bioorg. Med. Chem. 21 (2013) 4259–4265

possible that the Caco-2 cell homogenates lack the enzymes neces- 4.0×10-05 sary to cleave the derivative to liberate the LHRH peptide. 2.0×10-05 3. Conclusions

-06 *** We successfully synthesised and puriﬁed a small library of car- 4.0×10 *** *** -06 bohydrate conjugated LHRH analogues. Based on the results of

Papp (cm/s) Papp 3.0×10 -06 ** in vitro assays performed, we conclude that modification of the 2.0×10 ns ns N-terminus of LHRH with carbohydrate improved its in vitro bio- 1.0×10-06 availability both by stabilizing the peptide towards enzymatic 0 digestion and increasing its permeation across the Caco-2 cell 3 4 5 7 8 9 monolayers. Coupling carbohydrate to the N-terminus of the peptide (e.g. 3) decreased its degradation by endonucleases in Caco-2 Mannitol 1 (control) propranolol cell homogenates. It was also observed that all the derivatives modified with lactose succinamic acid (Compounds 3, 7 and 11) Figure 2. Apparent permeability of sugar-conjugated LHRH in Caco-2 cell mono- exhibited higher stability compared with analogues conjugated layer. Statistical analysis was performed using one-way ANOVA analysis followed by Dunnet post-test. Each value represents mean ± SEM (n = 2 independent to other sugar moieties. Data analysis of the permeability assay experiments in triplicate for each compound). ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001, and ns also indicated a significant enhancement in absorption of N-termi- p > 0.05, analogues vs LHRH. nal glycosylated LHRH peptides with the exception of compound 7, which exhibited low permeability. The in vitro–in vivo correlation studies show that the transport which is higher than the half-life measured in the human body (t1/ 3 rate of the compounds with high permeability coefficient of 2 = 3–4 min). It has previously been shown that this cell line lacks 6 several metabolic enzymes.42,43 Additionally, it is possible that (Papp >1 10 cm/s) is twofold to fourfold less than the rate in 34 in vivo a large proportion of LHRH is cleared from systemic circu- human jejunum in situ. As the permeability co-efficient of the lation by renal or biliary excretion.3,43,44 most of our derivatives was higher than the mentioned rate, we anticipate a significant increase in oral absorption when these ana- 2.4. Release pattern of the parent peptide by LHRH analogues logues are tested in vivo.

In addition to investigating the stability of the tested com- 4. Experimental pounds, the level of parent LHRH peptide released from the analogues was also measured in order to investigate whether any of 4.1. General the modiﬁed compounds could act as a prodrug. Minimal levels of native peptide were detected for any of the analogues (Fig. 4). Fmoc-protected amino acids and Rink amide MBHA resin It is possible that the rate of LHRH release is too slow to be de- (100–200 mesh, 0.59 mmol/g) were purchased from Novabiochem tected during the limited time of the experiment (2 h). It is also (Melbourne, Australia) or Mimotopes (Clayton, VIC, Australia).

A B 150 1 150 1 3 125 7 4 125 8 100 5 100 9 6 75 75

50 50

25 25 Peptide concentration %

0 peptide concentration % 0 0 20 40 60 80 100 120 140 0 20406080100120140 Time Time

C 150

125 1 100 10 11 75

Peptide concentration % 0 0 20 40 60 80 100 120 140 Time

Figure 3. Degradation proﬁle of LHRH analogues in Caco-2 cell homogenate: (A) percentage of [E1]LHRH derivatives remaining in Caco-2 cell homogenate, (B) percentage of [Q1]LHRH derivatives remaining in cell homogenate, (C) percentage of 10 and 11 remaining in cell homogenate. Data is presented as the mean ± SD (n = 3, the number of independent experiment) and analysed by one phase decay analysis using Graphpad Prism 5. 48 S. V. Moradi et al. / Bioorg. Med. Chem. 21 (2013) 4259–4265 4263

Table 2 Characterisation of glycosylated LHRH analogues

Compound Pure ﬁnal yield (%) HR-MS Retention time (tR) on RP-HPLC (min) Calculated Found 1 48 [M+H]+ 1182.5803 1182.5805 13.8 2 45 [M+2H]+ 600.7791 600.8008 13.6 3 31 [M+2H]+ 812.3679 812.3672 13.6 4 36 [M+2H]+ 731.3415 731.3450 13.5 5 39 [M+2H]+ 731.3415 731.3431 13.5 6 22 [M+2H]+ 688.3231 688.3258 13.2 7 26 [M+2H]+ 811.8759 811.8789 13.2 8 34 [M+2H]+ 730.8495 730.8530 13.3 9 27 [M+2H]+ 730.8495 730.8530 13.2 10 68 [M+H]+ 1311.6382 1311.6406 15.7 11 17 [M+H]+ 1751.8012 1751.8012 15.6

acetonitrile was purchased from RCI Labscan Ltd, (Bangkok, Table 3 The half-life of glycosylated LHRH analogues in Caco-2 cell homogenate and their Thailand). All media and supplements for cell culture work were permeability across the Caco-2 cell monolayer. Data of stability assay was obtained by purchased either from Sigma–Aldrich or Life Technologies (VIC, LC–MS analysis and values are mean ± SD (n = 3). Each sample was tested in 4 Australia). Caco-2 cells were obtained from American Type Culture replicates and the experiment was independently repeated three times. ND, not Collection (Rockville, USA). determined. 1H nuclear magnetic resonance (1H NMR) spectra were re- 7 Compound Peptide derivatives Half-life (min) Papp (10 cm/s) corded on Bruker Avance 300 MHz spectrometer (Bruker Biospin, 1 [pE1]LHRH 32.6 3.98 ± 0.96 Germany). Peptides were characterised by electrospray ionisation 2 [E1]LHRH 30.4 ND mass spectrometry (ES-MS) on a PE Sciex API3000 triple quadru- 3 Lactose-[E1]LHRH 77.0 28.7 ± 0.56 pole mass spectrometer using a mixture of solvent A1 (0.1% acetic 4 Galactose-[E1]LHRH 51.0 10.8 ± 1.23 1 acid in water) and B (0.1% acetic acid in 9:1 acetonitrile/water) at 5 Glucose-[E ]LHRH 56.7 26.5 ± 3.27 1 6 [E1]LHRH-Glucoronic acid 28.4 ND 0.05 mL/min. The Varian Cary 50 Bio UV/vis spectrophotometer 7 Lactose-[Q1]LHRH 133.0 7.6 ± 1.73 (k = 570 nm) was used for absorbance measurements. LC–MS was 8 Galactose-[Q1]LHRH 92.2 16.2 ± 2.23 carried out using solvents A and B at a flow rate of 0.5 mL/min, 1 1 1 9 Glucose-[Q ]LHRH 73.5 23.1 ± 1.29 and a splitter after the Phenomenex Luna C18 column (5 lm, 10 [pE1][w6]LHRH 185.7 ND 11 Lactose-[Q1][w6]LHRH 132.8 ND 50 2.0 mm) was used to achieve a flow rate of 0.05 mL/min in the ion source of the mass spectrometer. Analytical HPLC was performed on an Agilent 1100 system fitted with a binary pump, autosampler, and a UV detector set to a wavelength of 214 nm. Dimethylformamide (DMF), trifluoroacetic acid (TFA), N,N-diiso- Preparative RP-HPLC was performed on a Shimadzu instrument propylethylamine (DIPEA) and piperidine of peptide synthesis (Kyoto, Japan; LC-20AT, SIL-10A, CBM-20A, SPD-20AV, FRC-10A) grade were obtained from Merck Biosciences (Kilsyth, VIC, Austra- in linear gradient mode using 20 mL/min flow rate, with detection lia) or Sigma–Aldrich (VIC, Australia) and O-benzotriazole- at 230 nm. Separations were performed with solvent A2 (0.1% TFA 0 0 N,N,N ,N -tetramethyl-uronium-hexafluoro-phosphate (HBTU) was in water) and solvent B2 (0.1% TFA in 9:1 acetonitrile/water) on Vy- purchased from Mimotopes (Melbourne, Australia). HPLC-grade dac preparative C18 column (218TP1022; 10 lm, 22 250 mm).

150 150 4 150 5 3 1

1 % 125 125 125 1 100 100 100

75 75 75

50 50 50

25 25 25 Peptide concentration % Peptide concentration Peptide concentration % 0 0 0 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 Time Ti me Time

150 150 150 7 125 125 8 125 9 1 1 100 100 100 1

75 75 75

50 50 50

25 25 25 Peptide concentration % concentrationPeptide % Peptide concentration % 0 0 0 0 20 40 60 80 100 120 140 0 25 50 75 100 125 150 0 20 40 60 80 100 120 140 Time Time Time

Figure 4. Level of native LHRH released from glycosylated analogues of LHRH in Caco-2 cell homogenate. The carbohydrate moieties were not cleaved from the LHRH peptide in any of the tested compounds. Data is presented as the mean ± SD (n = 3, independent experiment) using Graphpad Prism 5. 49 4264 S. V. Moradi et al. / Bioorg. Med. Chem. 21 (2013) 4259–4265

4.2. Synthesis of glycosyl units 4.4. In vitro experiments

Succinamic derivatives of the carbohydrates were synthesized 4.4.1. Cell culture in four steps according to published methods.45–49 The azide deriv- Cells were grown in T75 ﬂasks containing Dulbecco’s Modiﬁed ative of glucoronic acid was synthesized according to the protocol Eagle’s Medium (DMEM) media supplemented with 10% FBS and described in the literature.46 1% nonessential amino acids. The monolayers were incubated at

37 °C with 95% humidity in a 5% CO2 atmosphere. The medium 4.2.1. 4-Oxo-4-[(2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl)- was changed every other day. Cells were subcultured using 0.25% amino]-butanoic acid trypsin–EDTA when 80% conﬂuency was reached. Passage numbers

C18H25O12N, 447.40; melting point: 152–155 °C; ES-MS, m/z: 38–43 were used in these experiments. 448.3 [M+H]+, 470.1 [M+Na]+, 895.5 [2M+H]+, 917.7 [2M+Na]+. NMR data correspond to those reported in literature.48 4.4.2. Caco-2 cell homogenate stability assay The medium was removed from a ﬂask containing Caco-2 cells 4.2.2. 4-Oxo-4-[(2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl)- at 80% conﬂuence. The cells were washed once with EDTA 0.02% amino]-butanoic acid and three times with 25 mM HBSS/HEPES buffer and the cells were

C18H25O12N, 447.40; melting point: 142–143 °C; ES-MS, m/z: detached from the ﬂask using a cell scraper. The resulting cell sus- 448.2 [M+H]+, 470.2 [M+Na]+, 895.5 [2M+H]+, 917.7 [2M+Na]+. pension was lysed using two 10 sec pulses on a Sonics Vibracell NMR data correspond to those reported in literature.45 ultrasonic processor with 40% amplitude in 130 W mode. Cell debris was precipitated by centrifuging at 2000 rpm for 10 min at 4 °C. 4.2.3. 4-Oxo-4-[(2,3,6-tetra-O-acetyl-4-O-20,30,40,60-tetra-O- The protein content was measured using the Bio-Rad assay and the acetyl-b-D galactopyranosyl-b-D-glucopyranosyl)amino] ﬁnal protein concentration was adjusted to 0.5–0.8 mg/mL in butanoic acid HBSS/HEPES buffer. Test compound solutions (200 lM) were pre-

C30H41NO20; melting point: 97–98 °C. ES-MS, m/z: 736.3 pared in HBSS buffer containing 25 mM of HEPES buffer. Cell [M+H]+, 758.3 [M+Na]+, 781.6 [M+2Na]+ 1472.2 [2M+H]+. 1H homogenate (100 lL) was mixed with 100 lL of test compound

NMR (300 MHz, CDCl3): d = 6.44 (d, 1H, JNH,1 = 9.3 Hz, NH), 5.35 solution in 96-well plates and incubated at 37 °C. At selected time 0 (dd, 1H, J40,50 = 1.0 Hz, J30,40 = 3.5 Hz, H-4 ), 5.29 (dd, 1H, intervals (0, 5, 10, 15, 20, 30, 40, 60, 90 and 120 min), 10 lL of mix- J3,4 = 8.9 Hz, J2,3 = 9.6 Hz, H-3), 5.22 (dd, 1H, JNH,1 = 9.3 Hz, ture was taken and transferred to another 96-well plate, containing 0 J1,2 = 9.3 Hz, H-1), 5.10 (dd, 1H, J10,20 = 7.9 Hz, J20,30 = 10.4 Hz, H-2 ), a mixture of TFA (5 lL), water (75 lL) and DMSO (10 lL). Samples 0 4.95 (dd, 1H, J30,40 = 3.5 Hz, J20,30 = 10.4 Hz, H-3 ), 4.84 (dd, 1H, were then analysed using LC–MS. 0 J1,2 = 9.6 Hz, J2,3 = 9.6 Hz, H-2), 4.47 (d, 1H, J10,20 = 7.9 Hz, H-1 ), 4.43 (m, 1H, H-6a), 4.14 (m, 2H, H-6b,H-6a0), 4.07 (m, 1H, H-6b0), 4.4.3. Caco-2 cell monolayer permeability assay 0 3.87 (m, 1H, H-5 ), 3.76 (dd, 1H, J3,4 = 8.8 Hz, J4,5 = 8.8 Hz, H-4), Caco-2 cells were cultured in semi-permeable polycarbonate Ò 3.73 (m, 1H, H-5), 2.72 (m, 1H, CH2CO), 2.63 (m, 1H, CH2CO), Transwell plates with the following characteristic: 6.5 mm inserts 2.47 (m, 2H, CH2CO), 2.15 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.07 (s, diameter, and polycarbonate membrane pore size of 0.4 lm. The 3H, OAc), 2.05 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.04 (s, 3H, OAc), cell suspension was adjusted to a concentration of 1 106 cells/ 1.96 (s, 3H, OAc). mL and 100 lL of the cell suspension was seeded into the inserts. Both apical (0.1 mL) and basolateral (0.6 mL) chambers were filled 4.3. Peptide synthesis with cell culture medium containing DMEM with 10% FBS, 1% nonessential amino acids and 1% penicillin/streptomycin (10,000 units 4.3.1. Solid phase peptide synthesis (SPPS) of penicillin sodium salt/mL and 10 mg streptomycin sulphate/mL All peptide derivatives were synthesised on a scale of in 0.85% saline). The media in both chambers was changed every 0.025 mmol using Fmoc SPPS. Protected amino acids (4.2 equiv) other day. For formation of well-differentiated monolayers of suit- were activated using 4 equiv of HBTU and 6 equiv of DIPEA. Each able confluence the cells were grown for 21–28 days in the plates. coupled amino acid was deprotected using 20% piperidine in Before the start of the experiment, the media was removed from DMF before addition of the next amino acid. The coupling effi- both chambers, washed three times with preheated 25 mM of ciency of each amino acid was quantified by nihydrin test,50 with HBSS/HEPES (pH 7.4), then refilled with the same buffer. After the exception of proline which was tested using the chloranil equilibration for 30 min at 37 °C, TEER values were measured with test.51 Couplings were repeated until a coupling efficiency of a MillicellÒ-ERS epithelial volt ohmmeter instrument (Millipore >99% was achieved. Coupling of glucoronic acid to the C-terminus Company) to evaluate the integrity of tight junctions. Values rang- of the peptide was achieved using the protocol described in the lit- ing from 0.9 to 1.4 k O cm2 were deemed acceptable for use in the erature.47,52 Attachment of carbohydrate succinamic acid deriva- experiment. tives to the N-terminus of the peptides was achieved by Solutions of test compounds (200 lM) were prepared in HBSS/ overnight coupling. After completion of the sugar coupling, the car- 25 mM HEPES. The compound solutions (200 lL) were added to bohydrate protection groups were removed by treatment with 75% the apical side of the monolayer and incubated at 37 °C. At prede- of hydrazine hydrate in methanol twice (15 min and 30 min, termined time points (30, 90, 120, and 150 min) samples (400 lL) respectively). The completed peptides were cleaved from the resin were taken from the basolateral chamber and replaced with fresh using a mixture containing 95% TFA, 2.5% water and 2.5% triisopro- HBSS/HEPES buffer. At the end of the experiment, a 50 lL of aliquot pyl silane. was collected from apical chamber of each well. All samples were analysed by LC–MS. 4.3.2. Peptide purification Radiolabelled mannitol and propranolol were used as negative All crude peptides were purified by preparative RP-HPLC using a and positive controls respectively. The radioactivity of 14C-manni- Vydac C18 column (22 250 mm) with a gradient of 10–50% sol- tol was quantified by liquid scintillation counting (Liquid Scintilla- vent B over 72 min at a flow rate of 20 ml/min. The purity of the tion Systems, Beckman LS3801) after addition of scintillation collected fractions was checked by ES-MS and analytical RP-HPLC. cocktail (Perkin–Elmer-Optiphase). The apparent permeability of Pure fractions were combined, lyophilized, and stored at 20 °C. test compounds was calculated using the following equation: 50 S. V. Moradi et al. / Bioorg. Med. Chem. 21 (2013) 4259–4265 4265

Papp ¼ V r ðdCÞ=ðdtÞ1=AC0 ð1Þ 18. Deeken, J. F.; Löscher, W. Clin. Cancer Res. 2007, 13(6), 1663. 19. Scherrmann, J. M. Vasc. Pharmacol. 2002, 38(6), 349. 3 Vr is the volume of the receiver chamber (cm ); dC/dt is the steady 20. Pauletti, G. M.; Gangwar, S.; Knipp, G. T.; Nerurkar, M. M.; Okumu, F. W.; state rate of the change in the chemical concentration (M/s); A is the Tamura, K.; Siahaan, T. J.; Borchardt, R. T. J. Controlled Release 1996, 41(1–2), 3. 2 21. Morishita, M.; Peppas, N. A. Drug Disc. Today 2006, 11(19–20), 905. surface area of the cell monolayer (cm ) and C0 is the initial concen- 22. Werle, M.; Bernkop-Schnürch, A. Amino Acids 2006, 30(4), 351. tration in the donor chamber (M). 23. Witczak, Z. J. Carbohydrate Therapeutics: New developments and Strategies In ; ACS Publications, 2006; Vol. 932,; pp 25–46 24. Blanchfield, J.; Toth, I. Curr. Med. Chem. 2004, 11(17), 2375. 4.4.4. LC–MS analysis 25. Witt, K. A.; Gillespie, T. J.; Huber, J. D.; Egleton, R. D.; Davis, T. P. 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D.; Smith, M. T.; Toth, I. 14. Woodley, J. F. Crit. Rev. Ther. Drug Carrier Syst. 1994, 11(2–3), 61. J. Med. Chem. 2012, 55(12), 5859. 15. Barrera, C. M.; Banks, W. A.; Fasold, M. B.; Kastin, A. J. Pharmacol. Biochem. 50. Sarin, V. K.; Kent, S. B. H.; Tam, J. P.; Merrifield, R. B. Anal. Biochem. 1981, 117(1), Behav. 1992, 41(1), 255. 147. 16. Begley, D. J. Ann. N.Y. Acad. Sci. 1994, 739(1), 89. 51. Vojkovsky, T. Peptide Res. 1995, 8(4), 236. 17. Barrera, C. M.; Kastin, A. J.; Fasold, M. B.; Banks, W. A. Am. J. Physiol. Endocrinol. 52. Malkinson, J. P.; Falconer, R. A.; Toth, I. J. Org. Chem. 2000, 65(17), 5249. Metab. 1991, 261(3), E312.

51 Section B ‘’Evaluation of the biological properties and the enzymatic stability of glycosylated Luteinizing hormone releasing hormone (LHRH) analogues’’

3.3 Introduction to this publication

This section was accepted in ‘’AAPS journal’’ as an original article. In this article, the metabolic stability of the glycosyl derivatives of LHRH has been evaluated in different tissue homogenates including human plasma, ray liver and kidney membrane homogenates. The metabolites produced by the digestive enzymes in kidney membrane enzymes were characterised. The antiproliferative effect of the glycosylated compounds in a number of prostate cancer cell lines including LNCaP, DU145 and PC3 cell were investigated. Moreover, the stimulatory effect of selected LHRH analogues on the secretion of LH and FSH hormones from cultured rat pituitary cells was evaluated.

3.1 Reprint of the peer-reviewed publication

52 The AAPS Journal ( # 2015) DOI: 10.1208/s12248-015-9769-x

Research Article

Evaluation of the Biological Properties and the Enzymatic Stability of Glycosylated Luteinizing Hormone-Releasing Hormone Analogs

Shayli Varasteh Moradi,1 Pegah Varamini,1 and Istvan Toth1,2,3

Received 12 March 2015; accepted 6 April 2015

Abstract. The enzymatic stability, antitumor activity, and gonadotropin stimulatory effects of glycosylated luteinizing hormone-releasing hormone (LHRH) analogs were investigated in this study. Conjugation of carbohydrate units, including lactose (Lac), glucose (GS), and galactose (Gal) to LHRH peptide protected the peptide from proteolytic degradation and increased the peptides’ half-lives in human plasma, rat kidney membrane enzymes, and liver homogenate markedly. Among all seven modified analogs, compound 1 (Lac-[Q1][w6]LHRH) and compound 6 (GS4-[w6]LHRH) were stable in human plasma during 4 h of experiment. The half-lives of compounds 1 and 6 improved significantly in kidney membrane enzymes (from 3 min for LHRH to 68 and 103 min, respectively). The major cleavage sites for most of the glycosylated compounds were found to be at Trp3-Ser4 and Ser4-Tyr5 in compounds 1–5. Compound 6 was hydrolyzed at Ser4-Tyr5 and the sugar conjugation site. The antiproliferative activity of the glycopeptides was evaluated on LHRH receptor-positive prostate cancer cells. The glycosylated LHRH derivatives had a significant growth inhibitory effect on the LNCaP cells after a 48-h treatment. It was demonstrated that compound 1 significantly increased the release of luteinizing hormone (LH) at 5 and 10 nM concentrations and compound 5 (GS-[Q1]LHRH) stimulated the release of follicle-stimulating hormone (FSH) at 5 nM concentration in dispersed rat pituitary cells (p<0.05). In our studies, compound 1-bearing lactose and D-Trp was the most stable and active and is a promising candidate for future preclinical investigations in terms of in vitro biological activity and metabolic stability. KEY WORDS: antiproliferative activity; carbohydrate conjugation; LH and FSH release; LHRH; peptide.

INTRODUCTION 3). There is no oral analog of LHRH in the clinic due to its poor pharmacokinetic profiles and low oral bioavailability. Luteinizing hormone-releasing hormone (LHRH) is a The rapid clearance of peptides from the human body is one hypothalamic decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu- of the main issues in the development of therapeutic Arg-Pro-Gly-NH2) with a regulatory function in the repro- compounds. They have very low stability in digestive enzymes ductive system. The secretion of this hormone from the in the physiological environments. Therefore, in order to hypothalamus stimulates its cognate receptor in the pituitary develop an orally active LHRH derivative, the stability of the gland to produce gonadotropins including luteinizing hor- peptide should be improved. Several strategies have been mone (LH) and follicle-stimulating hormone (FSH). Subse- explored to reduce the enzymatic cleavage of peptide in quently, the release of gonadotropins regulates the secretion physiological media. Conjugation of peptides with carbohy- of sex steroids in both males and females (1). A substantial drate moieties has been shown to be a useful approach in number of LHRH derivatives have been developed and are improving stability and permeability of the modified analogs administered parenterally for the treatment of various through biological membranes (4–6). Furthermore, a number hormone-dependent diseases such as breast cancer, prostate of studies demonstrated that the efficacy of the sugar- cancer, endometriosis, infertility, and precocious puberty (2, modified peptides is preserved in different animal models (7–9). To develop a potent drug, it is necessary to fully Electronic supplementary material The online version of this article characterize the designed compounds for biological and (doi:10.1208/s12248-015-9769-x) contains supplementary material, physicochemical properties at the early stage of the discovery which is available to authorized users. process. The promising drug candidate selected from preliminary characterization should possess desirable characteristics 1 School of Chemistry and Molecular Biosciences, The University of such as high potency, long half-life, and an appropriate Queensland, St. Lucia, Queensland 4072, Australia. fi 2 School of Pharmacy, The University of Queensland, St. Lucia, physicochemical pro le. Understanding the metabolic path- Woolloongabba, Queensland 4102, Australia. way of the target peptide is an important factor in evaluating 3 To whom correspondence should be addressed. (e-mail: the therapeutic potential of the peptide drug candidates (10). [email protected]) The blood, liver, and kidney are the main compartments of

1550-7416/15/0000-0001/0 # 2015 American Association of Pharmaceutical Scientists 53 Moradi et al. enzymatic digestion of peptides resulting in a reduction in the MATERIALS AND METHODS peptide’s half-life (11). It has been reported that LHRH is metabolized by proteolytic enzymes within a short period General of time post administration (12). The main cleavage sites of this peptide are Trp3-Ser4 and Tyr5-Gly6 amide bonds High-performance liquid chromatography (HPLC) grade (12, 13). Typically, substitution of Gly at position 6 with a acetonitrile (MeCN) was purchased from Labscan (Bangkok, fl D-amino acid in LHRH increases its metabolic stability Thailand) and tri uoroacetic acid (TFA) was obtained from and the binding affinity to LHRH receptor (14, 15). Merck Biosciences (Kilsyth, VIC, Australia). Reversed- 6 Triptorelin is one of the LHRH super agonists bearing D-Trp phase HPLC (RP-HPLC) was performed using Shimadzu and it is more stable to enzymatic degradation. Triptorelin is Instrumentation (Kyoto, Japan) (LabSolutions software, administered parenterally for the treatment of prostate cancer SIL-20AC HT auto-sampler, LC-20AB pump, SPD-M10A and hormone- dependent diseases (16). detector, DGU-20A5 degasser). The analysis was achieved It has been shown that LHRH analogs do not only usingalinear0–100 or 20–60% gradient of solvent B suppress the pituitary-gonadal axis but also exert a direct (solvent A, 0.1% TFA in H2O; solvent B, 90% fl growth inhibitory effect on the tumor growth (17). LHRH ACN:H2O:0.1% TFA) for 30 min with a 1-mL/min ow receptors are overexpressed in malignant tumors such as rate and detection at 214 nm. The crude peptides were prostate, breast, and ovarian cancers, and they can then purified by preparative HPLC with a linear 20–45% mediate the antitumor activity of LHRH derivatives (18, gradient of solvent B to ≥95% purity. Analytical separa- 19). Various studies showed that LHRH analogs induce a tionswereperformedusingaC8column(Vydac 2 significant decrease in the growth rate of the cancer cells, 208TP5205; 5: m, 2.1×50 mm , Columbia, MD, USA) or 2 e.g., prostate cancer cell lines such as LNCaP and DU145 a C18 column (Vydac 218TP5405; 5: m, 4.6×50 mm , (20–22). LNCaP cells are androgen-dependent and are Columbia, MD, USA). Electrospray ionization mass derived from a lymph node metastasis of prostatic spectrometry was performed on a Perkin-Elmer Sciex carcinoma with a high level of LHRH receptor expression API 3000 operating in positive ion mode. A Varian Cary (20). PC3 and DU145 cells are androgen-independent and 50 Bio UV–vis spectrophotometer was used for absor- are derived from bone and brain metastasis of prostate bance measurement. cancer, respectively (20, 22). Medium-to-high affinity LNCaP (androgen-sensitive prostate adenocarcinoma) binding sites were also reported for LHRH agonists in and DU145 (androgen-independent human carcinoma) hu- LHRH receptor-positive prostate cancer cells (23). The man cell lines were used and were kindly provided by agonists exhibited their antiproliferative effect in a dose- Professor Judith Clements at the Translational Research and time-dependent manner (24). LHRH agonists and Institute, Queensland University of Technology. PC3 (steroid antagonists were shown to reduce the enzymatic activity hormone-independent prostate adenocarcinoma) cells were of the plasminogen activator system in prostate cancer kindly donated by Professor Rodney Minchin, School of cells leading to a decrease in the migration and invasive- Biomedical Sciences, The University of Queensland; ness of cancer cells (25). The mechanism of their growth Dulbecco’smodified Eagle’smedium(DMEM); inhibitory effects is through a different signaling transduc- penicillin/streptomycin; fetal bovine serum (FBS); and tion pathway from that in the pituitary cells (25, 26). phosphate-buffered saline (PBS) were obtained from Life Treatment of cultured pituitary cells with LHRH ago- Technologies Australia (Mulgrave, VIC, Australia). Tris nists has been shown to alter the secretion level of LH and (hydroxymethyl) aminomethane hydrochloride (Tris–HCl) FSH from gonadotrophs. This occurs when the agonist was purchased from ICN Biomedical Inc. (OH, USA). binds to the receptor and the hormone-receptor complex MgCl2·6H2O was obtained from Sigma-Aldrich (Castle is internalized (27, 28). The long-time exposure of LHRH Hill,NSW,Australia). receptors to the high concentrations of LHRH agonists resulted in receptor desensitization and the lower doses of Peptide Synthesis the agonists increased the number of LHRH receptors followed by stimulation of LH release (29, 30). All LHRH analogs were synthesized using Fmoc solid In the present study, we designed LHRH derivatives by phase peptide synthesis (SPPS) based on the previously replacement of Gly residue with D-Trp and attachment of a published methods (Fig. 1)(31). glycosyl unit to either N-terminus or middle sequence of the peptide. The enzymatic stability of the designed compounds In Vitro Metabolic Stability Assay (1–6) was then evaluated in human plasma and rat tissue homogenates. The metabolites produced by the digestive Human Plasma Stability Assay activity of kidney membrane enzymes were characterized, followed by the examination of the degradation profile of The test was performed on fresh human plasma of the analogs. The direct antiproliferative potency of consenting and healthy volunteers (ethics approval number: glycosylated LHRH analogs was examined in prostate 2006000950). Plasma was separated from red blood cells by a cancer cells including LNCaP, DU 145, and PC3. We also 15-min centrifugation at 1500×g and diluted to 80% by adding investigated the stimulatory activity of the selected glyco- 1× PBS. The compound’s solution was prepared in PBS at sylated LHRH derivatives (compounds 1, 2, 5, and 6) at 600 μM. Plasma (300 μL) was spiked with the peptide low concentrations to release LH and FSH in the cultured solutions at 1:1 ratio (incubated at 37°C). During the time pituitary cells. course of the experiment (4 h), samples were collected and

54 Biological Properties of Glycosylated LHRH Analogs

Fig. 1. Chemical structure of the glycosylated LHRH analogs mixed with acetonitrile for quenching the reaction. Finally, samples were then homogenized with the ice-cold buffer in a the protein mixture was centrifuged at 7400×g for 10 min and Teflon homogenizer using 4–6 pestle strokes. The homogenate the supernatant was separated from the mixture and analyzed was centrifuged at 3000×g for 15 min at 4°C and the supernatant by RP-HPLC. A calibration curve of each compound was was decanted. The total protein count was determined using plotted (peak area of serial dilutions versus the concentra- Bradford assay and the protein concentration was adjusted to tions) to calculate the concentration of the peptide in the 2.5 mg/mL. The kidney membrane homogenate was prepared sample’s solutions. according to the procedure described by Vergote et al. with minor modifications (10). In brief, rat kidneys were washed with Rat Tissue Preparation ice-cold 0.9% sodium chloride and transferred into the Tris–HCl buffer (2 mM containing 10 mM mannitol, pH 7.3). After cutting Male Sprague–Dawley rats (180±20 g were obtained from into pieces, the tissue was homogenized by a Teflon homoge- the Animal Resource Centre (ARC). All experimental proce- nizer followed by centrifugation of the homogenate suspended dures were approved by The University of Queensland Animal in ice-cold 10 mM MgCl2·6H2O and 2 mM Tris–HCl buffer Ethics Committee (AEC#SCMB/005/11/ARC) and performed (1500×g, 15 min at 5°C). The supernatant was removed and the according to NHMRC animal handling guidelines. Animals pellet was re-suspended in the ice-cold buffer and centrifuged at were euthanized and their kidneys and livers were removed to 15,000×g for 15 min at 5°C. The supernatant was discarded again prepare tissue homogenates. The rat liver homogenate, S9 and the pellet was re-suspended in the buffer and centrifuged at (containing both cytosolic and microsomal enzymes) was 2200×g for 15 min at 5°C. After discarding the supernatant, the prepared according to the previously published methods (32, suspended pellet was again centrifuged at 15,000×g for 15 min at 33). Briefly, the fresh rat liver was weighed and washed with ice- 5°C. The supernatant was decanted and the final pellet was re- cold 0.9% sodium chloride solution. The tissue was cut into suspended in the same Tris–HCl buffer mix. The total protein small pieces followed by mixing with 3 mL of 20 mM Tris–HCl content of the suspended pellet was measured by Bradford assay buffer (pH 7.4), containing 0.25 M sucrose per 1 g of tissue. The and adjusted to 2.5 mg/mL.

55 Moradi et al.

Incubation of the Peptide Analogs with Homogenates volume of RPMI. Diluted cell suspension was layered over 4 mL Ficoll and centrifuged at 400×g for 30 min. The white The homogenates were added (100 μL) into each well of cell interface including mononuclear cells was aspirated and the 96-well plates. Prior to the start of the experiment, the washed three times with RPMI 1640. Cells were re-suspended homogenates were pre-warmed for 15 min at 37°C. LHRH in 10% FBS:RPMI and seeded in a 96-well flat bottom plate compounds were dissolved in PBS and added to the (TPP) at the density of 1×106 cells/mL. Cells were then homogenates to give a final concentration of 100 μM. The activated by adding 10 μg/mL of phytohemagglutinin and reaction was initiated by incubating the plates at 37°C and incubated at 37°C in a 5% CO2 atmosphere. After 1-h shaking at 50 rpm (Thermo Scientific MaxQ 4000 Benchtop incubation, 10 μL of LHRH derivative was added to each shaker, USA). Samples of 50 μL were collected from each well at 50 and 200 μM. An MTT assay was performed after well at pre-determined time intervals (0, 5, 10, 15, 20, 30, 40, 48-h incubation using the same method as described for the 60, 90, 120, 180, and 240 min) and added to the 50 μL of 80% cell proliferation assay. acetonitrile containing 0.1% formic acid to stop the enzymatic activity. Samples were finally centrifuged at 3000×g for LH and FSH Release Assays 15 min; the supernatants were collected and analyzed using HPLC on a C8 column. Rat Pituitary Cell Preparation

Identification of Metabolites Pituitary cell dispersion was performed as described elsewhere with some modifications (35). Briefly, anterior The metabolites formed by the degradation of the pituitaries were removed immediately after euthanizing rats by compounds in the kidney membrane homogenate and human CO2 inhalation and rinsed with Hanks’ balanced salt solution plasma were characterized using HPLC and ESI-mass (HBSS) containing 25 mM N-2-hydroxyethylpiperazine-N-2- spectrometry. The peaks from the HPLC were collected and ethane sulfonic acid (HEPES) buffer (pH 7.2). Tissues were the corresponding mass was identified using mass spectrom- minced with a razor blade into small pieces. The buffer was etry (PerkinElmer-Sciex API3000). removed and replaced by a collagenase enzyme solution (1 mg/ mL dissolved in 1% bovine serum albumin (BSA)/HBSS). The In Vitro Cell Proliferation Assay pituitary fragments were incubated with the enzyme for 1 h at 37°C to become dissociated. After a gentle trituration, cells were LNCaP and DU145 cell lines were grown in 75 cm2 passed through a cell strainer (Costar) to remove clumps and culture flasks containing RPMI-1640 medium supplemented centrifuged at 400×g for 10 min. The supernatant was decanted with 10% fetal bovine serum (FBS) and 1% non-essential and the cells were suspended in DMEM media supplemented amino acids in a humidified atmosphere of 5% CO2. with 10% FBS (growth media). Afterwards, cells were plated in Dulbecco’s modified Eagle’s medium (DMEM) was used to 96-well plates at the density of 30,000 cells/well and incubated grow the PC3 cell line. The cell media were changed every for 72 h at 37°C. 2 days. The cell proliferation was evaluated by assessing the LH and FSH Measurement mitochondrial reduction of MTT using the established procedure (34). Tumor cells including LNCaP, DU145, and Plated pituitary cells were spun down at 1200×g for PC3 were seeded at the density of 2.0×104 cells/well and 10 min. Before the addition of LHRH compounds, pituitary allowed to attach for 4 h. LHRH compounds were added to cells were washed and replaced by challenging media the plates at different concentrations (10, 50, 100, and containing DMEM with 0.1% BSA. Plates were then 200 μM) in triplicate. Cells were incubated for 48, 72, and incubated with 10 μL test solutions at 37°C for 2 h. 96 h at 37°C. Fresh compound solution was added to the cells Compounds were used at 1, 10, and 50 nM for the LH every other day. After the end of the treatment, 10 μL MTT release assay and at 0.5, 5, and 10 nM (concentrations (5 mg/mL) was added to each well. Plates were incubated for were chosen based on some preliminary experiments) for a further 4 h. The medium was aspirated and 200 μL acidified the FSH release experiment. The level of LH and FSH isopropanol (0.1 N HCl) was added to the wells to dissolve was quantified with the commercial ELISA kit (USCN formazan crystals. The absorption of each well was measured Life Science Inc., Wuhan, China) according to the using a Spectramax 250 microplate reader at the wavelength manufacturer’s instructions. of 570 nm. The percentage of cell viability for each compound was calculated by comparing the absorbance of PBS added RESULTS samples (as a negative control). Sodium dodecyl sulfate (SDS) was used as a positive control. Each experiment was Designed LHRH Analogs repeated twice. Peptide derivatives were synthesized and purified to 95– Isolation of Peripheral Blood Mononuclear Cells (PBMCs) 99% purity according to previously published methods (6, 31). The pure peptides were used for the biological evalua- Assay was performed with the approval from the tions. Figure 1 shows the chemical structure of the glycosyl- University of Queensland Ethics Committee (ethical approval ated LHRH analogs. In all compounds, the carbohydrate number: 2009000661). A blood sample (4 mL) was collected units were attached to the N-terminus of the peptide via a from a healthy adult volunteer and diluted with an equal succinamic acid linker except for compound 6 in which the

56 Biological Properties of Glycosylated LHRH Analogs glucose unit was coupled to the serine residue at position 4 chromatographic separation using a gradient solvent system. through O-glycosylation. The analysis of the fragments illustrated indicated that the peptide bonds Trp3-Ser4 and Ser4-Tyr5 were the most In Vitro Metabolic Stability of LHRH Derivatives susceptible positions in compounds 1–5, which were cleaved after 10- and 15-min incubation with the homogenate, To assess the metabolic stability of LHRH derivatives in respectively. Moreover, the cleavage of the Tyr5-Gly6 bond human plasma, compounds were incubated with plasma for was detected in compounds 1–5. The lactose unit was cleaved 4 h and the half-life of each LHRH derivative was calculated from the LHRH peptide in compound 1 after 40 min, while after quantifying the collected samples using HPLC. The the cleavage between the glucose residue and the peptide in results showed that compounds 1 (bearing Lac at N-terminus compounds 4 and 5 was observed after 20 min. 6 4 6 and D-Trp ) and 6 (bearing GS and D-Trp ) were stable Compound 6 was stable in the kidney homogenate, and during the time course of the experiment. A significant no metabolites were detected in the samples until 180 min. increase was also observed in the plasma half-lives of the After 180 min, a metabolite was detected by the cleavage of compounds 2–5 compared to the native peptide (from the glucose from the peptide. Another degradation site was 4 5 t1/2=6 min to more than 120 min). found for compound 6 after 240 min at Ser -Tyr . The enzymatic stability of the glycosylated conjugates of LHRH was also studied in rat liver and kidney membrane In Vitro Antiproliferative Study homogenates at pH 7.3 (Table I). The half-lives of compounds 1 and 2 in liver homogenate were 117 and 42 min, The growth inhibitory effect of glycosylated conjugates respectively, which were enhanced significantly compared to of LHRH was investigated in three LHRH receptor-positive the parent peptide (t1/2=5 min). The metabolic stability of prostate cancer cell lines (LNCaP, DU145, and PC3). Cells compounds 1 and 2 was also improved between 7- and 22-fold were treated with the different doses of LHRH compounds in kidney membrane homogenate. Compound 6 was stable in and their effects on cell growth were assessed during four liver homogenate for 4 h and showed the highest half-life in consecutive days. The cell growth in LNCaP cells decreased kidney membrane homogenate (t1/2=103 min) compared to between 35 and 53% after 48-h treatment with compounds 1– the other derivatives. The shortest half-life in both homoge- 6 (at 100 and 200 μM concentrations). Similar inhibitory nates was obtained for those compounds that had glucose and effects on cell growth were observed after the treatment of galactose units in the N-terminal of their structures (com- LNCaP cells for 72 and 96 h (30 to 55% growth reduction) pounds 3–5). It was demonstrated that the glycosylated (Fig. 2a, S1-A). It was shown that LHRH compounds induced LHRH derivatives were more rapidly hydrolyzed in the the same or even higher levels of growth inhibition in LNCaP kidney membrane homogenates than the liver homogenates. cells compared to triptorelin at 200 μM concentration. This Among the tested LHRH derivatives, compound 1 and effect was in a concentration-dependent manner. compound 6 were the most stable analogs, whereas com- The growth rate of DU145 cells was not changed pound 3 had the least metabolic stability in all three matrices. significantly when cells were treated with glycosylated LHRH derivatives for 48 h. However, the cell growth was inhibited Characterization of the Metabolites evidently (40% reduction) after 72 h treatment of the cells with the compounds at 50, 100, and 200 μM concentrations The metabolites generated by the enzymatic digestion of (Fig. S1-B). Compounds 1, 2, 4, and 6 reduced the viability of LHRH analogs were identified in kidney membrane and DU145 cells to less than 50% after a 4-day treatment human plasma stability experiments. No degraded products (Fig. 2b). Similar to the LNCaP cell line, the cell growth were found in the samples incubated with plasma; however, inhibitory effect of the sugar-modified compounds was several metabolites were identified upon the incubation of the concentration-dependent in DU145 cells. analogs in the kidney membrane homogenate. All fragments No significant change was observed in the growth of PC3 were eluted from HPLC earlier than the native peptide in the cells after 48 and 72 h of treatments (Fig. S1-C). However, the

Table I. Half-Lives of Glycosylated LHRH Derivatives in Human Plasma and Tissue Homogenates

Half-life (min)

Compound number Peptide derivatives Rat liver homogenate Rat kidney membranes Human plasma

– [pGlu1]LHRH (native) 5 3 6 1 Lac-[Q1][w6] LHRH 117 68 Stablea 2 Lac-[Q1]LHRH 42 22 187 3 Gal-[Q1]LHRH 17 8 124 4 GS-[E1]LHRH 17 8 138 5 GS-[Q1]LHRH 22 8 146 6GS4-[w6]LHRH Stablea 103 Stablea

All metabolic stability assays were performed in triplicate in two independent repetitions. The half-life values are reported as the mean of repeated experiments LHRH luteinizing hormone-releasing hormone, Lac lactose, Gal galactose, GS glucose a Compounds were stable during the time course of the experiment (4 h)

57 Moradi et al.

Fig 2. Antiproliferative effects of glycosylated LHRH analogs in cancer cell lines. a LNCaP, b DU145, and c PC3. Cancer cells were treated with LHRH compounds for 48, 72, and 96 h at 10, 50, 100, and 200 μM. In this figure, the growth inhibitory effect of compounds is shown after 96-h treatment at 100 and 200 μM. All other relevant graphs are presented in supplementary information. d In normal PBMCs, the growth inhibitory effects of LHRH derivatives at 50 and 200 μM was determined following 48-h incubation with cells. Each column represents the mean±SD of the data obtained from experiments performed in triplicate. Statistical analysis was performed using a one-way ANOVA followed by the Dunnett’s post hoc test and compared to PBS group (*p<0.05, **p<0.01, ***p<0.01) incubation of PC3 cells with LHRH derivatives elicited up to selected based on our preliminary results) with the cultured 37% growth inhibition after a 4-day incubation at 100 and pituitary cells. Compound 1 at 10 nM concentration stimulated 200 μM concentrations (Fig. 2c). All tested compounds the release of LH significantly from 47 ng/mL in the negative showed a superior inhibitory effect on the growth of LNCaP control samples to 117 ng/mL (Fig. 3). The concentration of and DU145 cell lines than PC3 cells. LNCaP cells were kept 1 nM caused a 2-fold increase in LH release, although it was not for an additional 5 days without any treatment with com- statistically significant. However, a higher concentration of the pounds. The cells resumed growth after 5 days and their compound 1 (50 nM) did not affect the LH level. Compound 6 viability reached the level of the negative control wells. inhibited the LH release significantly at 50 nM. Compounds 2 and 5 did not show significant effects in altering the release of Cell Toxicity Against Non-cancerous Peripheral Blood LH at any concentration (Fig. 3). Compound 5 had a significant Mononuclear Cells (PBMCs) effect in increasing the secretion of FSH at 5 nM. The FSH level was not significantly altered when the cells were treated by The toxicity of compounds 1–6 was evaluated in PBMCs compounds 1 and 2 at any concentration. isolated from the whole blood. None of the compounds showed any toxic effect on these cells at 50 and 200 μM DISCUSSION concentrations (Fig. 2d). The therapeutic benefits of LHRH derivatives have been In vitro LH and FSH Release proven in the treatment of hormone-dependent diseases (36, 37). However, all agonists and antagonists of LHRH are The level of released LH and FSH was measured after 2-h characterized by poor pharmacokinetic properties when they incubation of compounds 1, 2, 5, and 6 (lead compounds are orally administered. The development of orally active

58 Biological Properties of Glycosylated LHRH Analogs

Fig. 3. Effect of glycosylated LHRH derivatives on the release of a LH and b FSH in rat pituitary cells. Statistical analysis was performed using a one-way ANOVA followed by the Dunnett’s post hoc test. ##p<0.05, decrease in the LH level when compared to the PBS group (*p<0.05, **p<0.01, increase in the LH level when compared to the PBS group) peptide drugs has been one of the major goals in the homogenate. Compound 6 with the glucose unit attached to the pharmaceutical industry. Enzymatic digestion is one of the middle of the sequence showed higher metabolic stability than main limiting factors in the oral delivery of peptides leading other derivatives. No hydrolyzed metabolites were formed until to poor absorption from the gastrointestinal tract into the 180-min incubation with the homogenate. After 180 min, a 5 systemic circulation. Several approaches have been employed glucose-free fragment and after 240 min, a hexapeptide (Tyr -D- 6 7 8 9 10 to overcome these challenges. We applied the glycosylation Trp -Leu -Arg -Pro -Gly -NH2) was detected. It was found approach and amino acid substitution with D-isoform to that the kidney endopeptidases cleaved the peptide bond at overcome the poor oral bioavailability of native LHRH positions 3 and 4 in compounds 1–5. This caused the formation 5 6 6 7 8 9 10 peptide. We previously reported the enhanced stability of of a hexapetide (Tyr -Gly [or D-Trp ]-Leu -Arg -Pro -Gly - 4 5 6 6 7 8 the LHRH glycosylated derivatives in the Caco-2 cell NH2) and heptapeptide (Ser -Tyr -Gly [or D-Trp ]-Leu -Arg - 9 10 5 6 homogenates (31). Caco-2 cells are derived from human Pro -Gly -NH2) in the homogenate. The Tyr -Gly bond was colon adenocarcinoma, and they express the typical enzymes also degraded in compounds bearing Gly at position 6 of intestinal cells such as peptidases in high levels. Therefore, (compounds 2–5). This digestion pattern was reported for they are applied as a useful model for drug metabolism LHRH, and its analogs in other studies in which the stability studies (38). In addition to intestinal enzymes, the liver and of the peptide’s derivatives was examined in different tissue kidney are the two main organs responsible for metabolizing homogenates and organs (41–43). It has been shown that hydrophilic peptides using different types of proteases. In this metalloendopeptidase EP24.15, EP24.11, and the angiotensin- study, the metabolic stability of the designed LHRH analogs converting enzyme (ACE) are mainly responsible for digestion was evaluated in rat liver and kidney membrane homoge- of the LHRH peptide. The endopeptidase E24.15 cleaves the nates. The results indicated that the metabolic stability of all peptide bonds at Tyr5-Gly6 and His2-Trp3 whereas ACE glycosylated analogs improved significantly in the liver degrades the bond at Try3-Ser4 (44–46). The cleavage of the homogenate compared to the native peptide and the com- sugar entities was found to be processed in the later stage of the 6 pounds bearing glucose and lactose with D-Trp amino acid in proteolytic reaction. It was also observed that pyroglutamyl the sequence (compounds 1 and 6) were the most resistant residue (pGlu) was cleaved after the removal of the sugar analogs against enzymatic digestion. The glycosylated LHRH entities from the N-terminus of the peptides’ sequence. The compounds were shown to be less stable against metabolizing cleavage of pGlu from the amino terminus of the peptides is enzymes in the kidney compared to that of the liver. This was instigated by the enzymatic activity of pyroglutamyl peptidase in line with the previous studies showing that the kidney plays distributed in different mammalian tissues (47). an important role in the metabolism of LHRH derivatives In addition to the suppression of gonadal steroids, a (39, 40). The native LHRH is degraded rapidly in blood and number of studies reported the direct antiproliferative activity has a short half-life of 3–4min(36). We showed that of LHRH analogs in different tumor cell lines (17, 48, 49). compounds 1 and 6 remained intact in the human plasma However, the mechanism of the action of LHRH analogs on during the 4-h incubation. The half-lives of compounds 2–5 tumor cells is mediated through a different signal transduc- increased 20- to 30-fold compared to the LHRH peptide. tion pathway from that in the anterior pituitary (20, 50). We Overall, it was found that the introduction of a D-amino acid tested the growth inhibitory effect of the LHRH derivatives and a lactose unit to the structure of LHRH peptide had a in three prostate cancer cell lines including LNCaP, DU145, significant impact in improving the metabolic stability of the and PC3. We found that all tested peptide analogs markedly modified analogs. Furthermore, the attachment of a glucose inhibited the growth of LNCaP and DU145 cells after 48- and unit at position 4 in compound 6 was shown to be the most 72-h treatment, respectively. There was no significant differ- effective approach in the current study to protect the peptide ence between the antiproliferative effects of compound 6 against enzymatic degradation. bearing a sugar unit at position 6 and the other glycosylated The production of major metabolites of the compounds 1–6 analogs in which the sugar entities were attached to the N- was examined upon incubation in the kidney membrane terminus of the peptide. There are reports in the literature on

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Mechanism of LH release in 35. Pelletier JC, Chengalvala M, Cottom J, Feingold I, Garrick L, cultured rat pituitary cells. Endocrinol Jpn. 1989;36(5):739–46. Green D, et al. 2-Phenyl-4-piperazinylbenzimidazoles: orally 53. Levine JE, Duffy MT. Simultaneous measurement of luteinizing active inhibitors of the gonadotropin releasing hormone hormone (LH)-releasing hormone, LH, and follicle-stimulating (GnRH) receptor. Bioorg Med Chem. 2008;16(13):6617–40. hormone release in intact and short-term castrate rats*. Endo- 36. Moreau J-P, Delavault P, Blumberg J. Luteinizing hormone- crinology. 1988;122(5):2211–21. releasing hormone agonists in the treatment of prostate cancer: a 54. Drouin J, Fernand L. Selective effect of androgens on LH and review of their discovery, development, and place in therapy. FSH release in anterior pituitary cells in culture. Endocrinology. Clin Ther. 2006;28(10):1485–508. 1976;98(6):1528–34.

61 Supporting information

Evaluation of the biological properties and the enzymatic stability of glycosylated luteinizing hormone releasing hormone (LHRH) analogues

Shayli Varasteh Moradi 1, Pegah Varamini1 and Istvan Toth1,2

Figure S1 : Antiproliferative effects of glycosylated LHRH analogues in A) LNCaP and B) DU145 C) PC3 after 48,72 and 96h treatment with glycosylated LHRH analogue

Chapter 4: Roles of transporters in the vectorial transport of the glycosylated LHRH analogues

‘’The Transport and Efflux of Glycosylated Luteinising Hormone-Releasing Hormone Analogues in Caco-2 Cell Model: Contributions of Glucose Transporters and Efflux Systems’’

Moradi SV, Varamini P, Toth I. The Transport and Efflux of Glycosylated Luteinising Hormone‐ Releasing Hormone Analogues in Caco‐2 Cell Model: Contributions of Glucose Transporters and Efflux Systems. J Pharm Sci. 2014;103(10):3217-24.

4.1 Introduction to this publication

This chapter was published in ‘’journal of pharmaceutical sciences’’ as an original article. In this paper, the role of sugar transporters including GLUT2 and SGLT1 on transport of glycosylated LHRH analogues was investigated in Caco-2 cell monolayer. The efflux ratio (ER) and the role of efflux transport systems including P-gp and MRP2 in pumping back the glycosylated LHRH derivatives was also examined in Caco-2 cell monolayer.

4.2 Reprint of this peer-reviewed publication

64 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

The Transport and Efﬂux of Glycosylated Luteinising Hormone-Releasing Hormone Analogues in Caco-2 Cell Model: Contributions of Glucose Transporters and Efﬂux Systems

SHAYLI VARASTEH MORADI,1 PEGAH VARAMINI,1 ISTVAN TOTH1,2

1School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland 4072, Australia 2School of Pharmacy, The University of Queensland, Woolloongabba, Queensland 4102, Australia

Received 12 May 2014; revised 14 July 2014; accepted 14 July 2014 Published online 29 August 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24120

ABSTRACT: Luteinising hormone-releasing hormone (LHRH) analogues have wide therapeutic applications in the treatment of prostate cancers and endocrine disorders. The structure of LHRH was modified using a glycosylation strategy to increase the permeability of the peptide across biological membranes. Lactose, galactose and glucose units were coupled to LHRH peptide, and the impact of glucose transporters, GLUT2 and SGLT1, was investigated in the transport of the analogues. Results showed the contribution of both transporters in the transport of all LHRH analogues. In the presence of glucose transporter inhibitors, reduction in the apparent permeability (Papp) was greatest for compound 6, which contains a glucose unit in the middle of the sequence (Papp = 58.54 ± 4.72 cm/s decreased to Papp = 1.6 ± 0.345 cm/s). The basolateral to apical flux of the glycosylated derivatives and the impact of two efflux pumps was also examined in Caco-2 cell monolayers. The efflux ratios (ERs) of all LHRH analogues in Caco-2 cells were in the range of 0.06–0.2 except for compound 4(galactose modified, ER = 8.03). We demonstrated that the transport of the glycosylated peptides was facilitated through glucose transporters. The proportion of glucose and lactose derivatives pumped out by efflux pumps did not affect the Papp values of the analogues. C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:3217–3224, 2014 Keywords: LHRH; SGLT1; GLUT2; transport; Caco-2 cell; peptides; MRP2; P-glycoprotein; glycosylation; efflux pumps

INTRODUCTION moieties and active components can assist the carrier-mediated active transport across biological membranes. Glycosylation Luteinising hormone-releasing hormone (LHRH) is a neuropep- can also modify the physicochemical properties, such as po- tide produced by the hypothalamus that stimulates the ac- larity, solubility and stability of the conjugated peptide.7 We tivation of LHRH receptors in the pituitary to release LH have previously shown that glycosylation of LHRH enhanced and follicle-stimulating hormone. The secretion of these two both metabolic stability and membrane permeability of this hormones results in controlling the production of sex steroid peptide.8 In the present study, we investigated the role of car- hormones in both male and female reproductive systems.1 bohydrate transporters in the transport of LHRH-glycosylated LHRH analogues are currently applied for treatments of var- analogues across human colorectal adenocarcinoma (Caco-2) ious types of diseases including hormone-dependant cancers cell monolayers. This cell line has been extensively used in (i.e., prostate, breast and ovarian cancers), infertility and pre- pharmaceutical research, especially in intestinal drug absorp- cocious puberty.2,3 Because there is no oral derivative of this tion studies as a model for transport across the intestinal hormone, developing an analogue with improved oral bioavail- mucosa.9 Generally, a carrier-mediated system is required for ability is highly desirable. the penetration of hydrophilic constructs through phospholipid Oral administration is the most preferred and accepted route membranes.10 Hexoses including glucose, galactose and fruc- by patients. However, the poor metabolic stability and low per- tose are examples of hydrophilic components, which are not meability of peptides across biological barriers are one of the able to pass through cell membranes by passive transport. major challenges for the pharmaceutical industry in the de- Active and facilitative transport systems are responsible for velopment of oral peptide therapeutics.4 Research in peptide their transfer through intestinal cell membranes.11 Two differ- delivery has demonstrated that improving oral absorption of ent types of membrane protein families mediate the transport peptides is achievable through different strategies such as cycli- of compounds containing carbohydrate entities in most mam- sation, lipidation and glycosylation.5,6 Among these, incorpora- malian cells.11 Facilitative glucose transporters (GLUT) are tion of carbohydrate residues into the structure of peptides has + Na -independent transporters, which transport sugars across proven to be effective in enhancing metabolic stability against the membrane according to their concentration gradient.11 The enzymatic degradation and penetration of peptides through + other group known as Na -dependent transporters (sodium– membrane barriers. Generally, conjugation of carbohydrate glucose-linked transporter or SGLT) transport hexoses actively through cell membranes against the sugar concentration gra- Correspondence to: Istvan Toth (Telephone: +614-6689-7933; Fax: +617- dient by using the energy obtained from the electrochemical 3365-4273; E-mail: [email protected]) sodium ion gradient.12 In intestinal cells, both SGLT1 and This article contains supplementary material available from the authors upon request or via the Internet at http://onlinelibrary.wiley.com/. GLUT5 are monosaccharide transporters mainly residing on Journal of Pharmaceutical Sciences, Vol. 103, 3217–3224 (2014) the apical side of the brush border, whereas GLUT2 is the dom- C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association inant isoform of the transporter located on the basolateral side 65 Moradi, Varamini, and Toth, JOURNAL OF PHARMACEUTICAL SCIENCES 103:3217–3224, 2014 3217 3218 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology of the enterocytes.12 GLUT2 is also transiently expressed on Synthesis and Purification of LHRH Derivatives the apical surface of enterocytes in response to high concentra- The method for synthesis and purification of compounds 1–5 tions of sugar in the lumen; hence, it provides a major path- followed those previously published by our group.8,21 Briefly, way for carbohydrate absorption.13 GLUT2 and SGLT1 trans- glycosyl units were first synthesised from the D isomer of glu- porters show different affinities for D-glucose and D-galactose. cose, galactose and lactose in four steps followed by purifica- At low concentrations, glucose is transported against a concen- tion using flash or dry column chromatography. Then, peptide tration gradient by SGLT1, whereas, at higher concentrations derivatives were synthesised using the standard Fmoc solid of glucose, GLUT2 is mainly involved in the transport of this phase peptide synthesis procedure on MBHA resin and 4.2 eq hexose.11,14 These transporters are proposed as useful targets to N"-Fmoc amino acids activated with 4 eq. HATU and 6 eq. transport therapeutic peptides through biological membranes. DIPEA. For synthesis of compound 6, Fmoc glycosyl serine was Drug efflux systems also affect oral absorption of drug synthesised separately according to the protocol described in molecules. P-glycoproteins (P-gp) and multidrug resistance- the following section and then coupled to the elongating pep- associated protein (MRP) are efflux pumps present in the in- tide by an overnight reaction using a proportion of 1.5 eq. The testinal epithelium. They belong to a superfamily of membrane acetyl group of the glycosyl residues was de-protected on a solid proteins known as ATP-binding cassette (ABC) proteins.15,16 support using a solution of 75% (v/v) hydrazine hydrate in P-gp is predominantly located at the apical domain of entero- DMF twice for 15 and 20 min, respectively. Cleavage of pep- cytes and is encoded by multidrug resistance (MDR1) genes tide from the resin was accomplished using a cocktail of TFA, in humans.4 Among the MRP family, MRP2 is found in the triisopropylsilane and water (95:2.5:2.5). Crude products were apical membrane of enterocytes and accounts for secretion of characterised by analytical HPLC on a C18 column and mass compounds into the lumen.17 Several studies have revealed spectrometry. Finally, they were purified by preparative HPLC that peptides and glycosides are both appropriate substrates on a C18 column. Pure fractions were lyophilised and kept at for P-gp efflux pumps, which can contribute in their low oral −20◦C for the tests. absorption.18–20

In the current study, we investigated the role of GLUT2 and SGLT1 in the transport of glycosylated LHRH derivatives using 4-1-1 Synthesis of N -(9-Fluorenylmethoxycarbonyl)-3-O-(2,3,4,6- Caco-2 cell membranes. The efflux ratio (ER) and the impact of Tetra-Oacetyl-D-Glucopyranosyl)-L-Serine ABC transporters located in Caco-2 cell membranes were also This compound was synthesised according to the protocol examined. published by Kihlberg et al.22 Briefly, glucosepentaacetate (600 mg, 1.5 mmol) and Fmoc-L-Ser-OH (660 mg, 2 mmol) were dissolved in CH2Cl2 (30 mL), and then BF3·Et2O complex MATERIALS AND METHODS (2 mL) was added drop-wise under N2 atmosphere and stirred General overnight at room temperature. The reaction mixture was then diluted with CH2Cl2 and the organic phase was extracted with Trifluoroacetic acid (TFA), N,N-dimethylformamide (DMF), 2- 1% HCl (3 × 30 mL) and H2O(2× 30 mL). The organic phase (1H-7-Azabenzotriazol-1-yl)–1,1,3,3-tetramethyl uronium hex- was dried with MgSO4 and concentrated by rotary evapora- afluorophosphate (HATU) and acetic acid were purchased tion. The crude product was purified by preparative HPLC on from Merck Biosciences (Kilsyth, Victoria, Australia). High- a C8 column (10 m, 250 × 21.20 mm2; Phenomenex, Torrance, performance liquid chromatography (HPLC) grade acetonitrile California) running with a gradient of 30%–60% solvent B over (MeCN) was purchased from Labscan ((Bangkok, Thailand). 60 min (solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in Fmoc-protected amino acids and Rink amide MBHA resin 90% acetonitrile/water) at a flow rate of 10 mL/min. The pure (100–200 mesh, 0.4–0.8 mmol/g loading) were obtained from fractions were subsequently combined and lyophilised to give a Nova-Biochem (Melbourne, Victoria, Australia) or Mimotopes white powdery substance, which was characterised using NMR (Clayton, Victoria, Australia). All media and supplements for spectroscopy and ESI–MS (Fig. 1a; Supplementary Figs. S1 and cell culture work were purchased from Life Technologies and S2). Pure yield: 32%. the human epithelial colorectal adenocarcinoma Caco-2 cell 1 H-NMR (300 MHz, CDCl3) * 1.90, 1.95, 1.98, 2.0 (4s, 3H, line was obtained from the American Type Culture Collection Ac), 3.92–3.95 (dd, 1H, J = 4.08 and 6.48 Hz, H-$), 4.10–4.17 (Rockville, Maryland). (2d, 1H each, J = 5.44 and 6.16 Hz, H-6,6), 4.20–4.25 (m, 1H, Analytical reverse-phase HPLC (RP-HPLC) was performed H-5, H-$), 4.35–4.39 (t, 1H, 7.2 Hz, Fmoc CHAr), 4.40 (m, 1H, using the Agilent 1100 series system equipped with an au- Fmoc OCH2), 4.42–4.47 (m, 1H, H-") 4.72–4.74 (d, 1H, J = tosampler, UV detector, and fraction collector with a 1 mL/min 7.08 Hz, H-1), 5.08–5.15 (m, 2H, H-2.3), 5.36 (dd, 1H, J = 2Hz flow rate and detection at 214 nm. Electrospray ionisation– and 1.9 Hz, H-4), 6.32 (d,1H, J = 8.12 Hz, NH). mass spectrometry (ESI–MS) was performed on a PerkinElmer- ESI–MS (M+H)+ calcd 658.2136, obsd 658.2140. Sciex API3000 instrument using Analyst 1.4 (Applied Biosys- tems/MDS Sciex, Toronto, Canada) software. A flow of a 1:1 Cell Culture mixture of solvent A (0.1% acetic acid in water) and B (0.1% acetic acid in acetonitrile/water 9:1) at a rate of 0.05 mL/min Caco-2 cells (passage 28–39) were cultured in Dulbecco’s modi- was used as the mobile phase. Separation was achieved using fied Eagle’s medium (Invitrogen, Carlsbad, California) contain- a Shimadzu system equipped with a CBM-20A controller, LC- ing 10% foetal bovine serum and 1% nonessential amino acids. 20AT pump, SIL-10A autosampler, SPD-20A UV/Vis detector Cells were grown in 5% CO2 with 95% relative humidity at (230 nm) and a FRC-10A fraction collector on either an analyt- 37◦C. The media were changed every 3 days. Cells were sub- ical C8 column (Vydac 208TP5205; 5 :m, 2.1 × 50 mm2)ora cultured using 2 mL of trypsin–EDTA 0.05% after reaching C18 column (Vydac 218TP5405; 5 :m, 4.6 × 50 mm2). more than 80% confluence. 66 Moradi, Varamini, and Toth, JOURNAL OF PHARMACEUTICAL SCIENCES 103:3217–3224, 2014 DOI 10.1002/jps.24120 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology 3219

Figure 1. (a) O-glycosylation of Fmoc–serine reaction using peracetylated glucose (yield = 32%). (b) Structure of O-linked glycosylated LHRH.

Hexose Transport Experiment donor side (apical chamber in the A–B direction, absorptive or basolateral in the B–A direction secretory) was removed and When Caco-2 cells reached more than 80% confluence, 1 × 106 replaced with 200 :M of compound solution (pH 7.4) without cells/well were seeded in inserts of 12-transwell plates (Corning inhibitors. During the time course of the experiment, the plates Costar, Cambridge, Massachusetts). The plate media were ◦ were incubated at 37 C and shaken at 50 rpm. Samples of changed every other day and after 21–28 days; the integrity of 400 :L were collected from the acceptor chamber at pre- the cells was checked by measuring their trans-epithelial elec- determined time points over 150 min (30, 90, 120 and 150 min) trical resistance (TEER) value. Cells exhibiting a TEER of 0.85– from the basolateral side or 80 :L from the apical side), and the 1.3 K cm2 were chosen for the experiment. Initially, cells were same volume of buffer was replaced after each sampling. Dur- washed three times with pre-warmed Hanks’ balanced salt so- ing the time course of the experiment, the pH was kept at 7.4 to lution (HBSS) containing 25 mM N-2-hydroxyethylpiperazine- avoid any possible influence of this factor on the permeability N-2-ethane sulfonic acid (HEPES) buffer (pH 7.2) and incu- across the cells. Collected samples were subsequently analysed bated for 30 min at 37◦C. Afterwards, the buffer was removed by LC–MS. The ER of the compounds was calculated according and the wells refilled with 50 :L of transporter inhibitors, ei- to the equation shown in the analysis section. ther phloretin or phlorizin, and incubated for 30 min at 37◦C. : According to the literature, a concentration of 100 Mofeach Efflux Transporter Inhibition Study inhibitor in phosphate-buffered saline has been shown to provide the best inhibitory effect.23,24 Compounds 1–6 (50 :L) at fi- For the inhibition study, verapamil was used as a specific in- nal concentrations of 200 :M in HEPES/HBSS were then added hibitor of P-gp transporter, whereas MK-571 was used as a to each apical (A) chamber. At pre-determined time intervals selective inhibitor to block MRP2. To give the maximum in- (30, 90, 120 and 150 min), 400 :L samples were collected from hibitory effect, a concentration of 100 :M of verapamil and the basolateral (B) chamber, which were refilled by adding fresh 50 :M of MK-571 was added to both chambers. Results of this buffer after each sampling. A mixture of phloretin and phlorizin study were then compared with the bidirectional transport of and (100 :M each) was also used to give an optimum inhibi- tested compounds in the absence of inhibitors. tion. Radio-labelled mannitol [14C] and propranolol were used as negative and positive controls, respectively. Finally, sam- Data Analysis ples were quantified by liquid chromatography–mass spectrom- Results are expressed as mean (±SEM) values performed in etry (LC–MS). The radioactivity of the [14C]-D-mannitol sam- triplicate. The apparent permeability (Papp) of both tests (un- ples was quantified by liquid scintillation counting (LS6500; der sink conditions) was calculated according to the following Beckman Coulter, Fullerton, California, USA). To determine equation: the reproducibility of the test, this experiment was repeated twice separately and in triplicate for each sample. Papp = dC/dt × Vr/A × C0

Bidirectional Transport Experiment where dC/dt is the steady-state rate of the change in the chemi- Preparation of Caco-2 cells was performed as previously de- cal concentration (M/s), Vr is the volume of the receiver chamber 3 2 scribed in an earlier section. After 30 min incubation of cells (cm ), A is the surface area of the cell monolayer (cm )andC0 with the transport buffer (HEPES/HBSS), the buffer from the is the initial concentration in the donor chamber (M). 67 DOI 10.1002/jps.24120 Moradi, Varamini, and Toth, JOURNAL OF PHARMACEUTICAL SCIENCES 103:3217–3224, 2014 3220 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Table 1. The Sequence of the Glycosylated LHRH Analogues

Compound Peptide Pure Number Derivative Sequence Yield (%)

1 Native LHRH EHWSYGLRPG 76 2Laca- (Lac-)QHWSYwLRPG 33 [Q1][w6]LHRH 3Laca-[E1]LHRH (Lac-)EHWSYGLRPG 39 4Galb-[Q1]LHRH (Gal-)QHWSYGLRPG 47 5 Glcc-[Q1]LHRH (Glc-)QHWSYGLRPG 58 6 Glc4-[Q1][w6] EHW(Glc)SYwLRPG 26 LHRH Figure 2. Changes in Papp of glycosylated LHRH derivatives in the aLactose. presence or absence of glucose transporter inhibitors. Both phloretin bGalactose. and phlorizin signiﬁcantly reduced the permeation rate of glycosyl c Glucose. derivatives 2–6. The statistical analysis was performed using one-way ANOVA followed by Dunnett’s post-hoc test. Each value represents ± = < Under non-sink conditions, the following equation25 was mean SEM (n 3 from three independent experiments). **p 0.01 and ***p < 0.001 analogues versus untreated samples. used to calculate the Papp of the compounds:

− / + / = / + + − / + PappA(1 VD 1 VR)t CR(t) [M (VD VR)] (CR,0 [M (VD VR)])e these compounds was calculated based on non-sink conditions. We found that the Papp of derivatives 2–6 dropped significantly where CR,0 is the drug concentration in the receiver chamber in the presence of phloretin and phlorizin. This confirmed that at time t; M is the total amount of drug in both chambers; VR both SGLT1 and GLUT2 transporters are involved in the trans- and VD are the volumes of receiver and donor chambers, respec- port of these LHRH analogues (Figs. 2 and 3). The inhibition of tively; C is the drug concentration in the receiver chamber at R(t) GLUT2 resulted in a sixfold to 10-fold decrease in the Papp of a previous time; f is the sample replacement dilution factor; A compounds 2–6 compared with the negative control. The trans- is the surface area of the monolayer and t is the time interval. port of analogues 2–6 was significantly reduced in the presence The statistical significance of differences among the groups of phlorizin (twofold to fourfold) compared with the negative was evaluated using one-way ANOVA and Dunnett’s post-hoc control (Supplementary Table S1). Specifically, the transport of < test. The differences were considered significant when p 0.01 compound 6 was the most affected. < and p 0.001. The concurrent addition of both phloretin and phlorizin in- The ER was calculated using the following equation: hibitors to the cells also resulted in a meaningful reduction in the transport of all glycosylated LHRH compounds through the = / ER Papp,BA Papp,AB Caco-2 cell membranes. where Papp,BA is the permeability value from basolateral to api- Evaluation of the Integrity of Caco-2 Cell Monolayers cal side and P is the permeability value from apical to app,AB [14C]-D-mannitol (negative control) was also used to investi- basolateral side. 14 gate the monolayers’ integrity. The Papp value of the [ C]-D- mannitol was obtained at 0.77 × 10−7 cm/s, indicating that the RESULTS monolayers’ integrity was acceptable for use. There was less than 10% reduction in TEER values at 2, 16 and 24 h after Synthesis of Glycosylated LHRH Derivatives the experiments, as compared with the initial values, which > The synthesis and purification of compounds 1–5 were per- was not statistically significant (p 0.05). This indicated that formed by previously published methods.8 The glycosyl units the monolayers’ integrity was not affected by any LHRH ana- (lactose, galactose and glucose) were attached to the N- logues. In the presence of phloretin and phlorizin, an initial 40% terminus of LHRH peptide via a succinamic acid linker to decline in the TEER values was observed; however, the cohe- achieve compounds 1–5 (Table 1). In compound 2, a glycine sion of the monolayer was retrieved after 18 h (Supplementary residue was replaced by D-tryptophan. A glucose unit was at- Fig. S4). tached to a serine residue in the middle of the peptide sequence through an O-glycosidic linkage to achieve compound 6 (Fig. 1b; ER Measurement Supplementary Fig. S3). To determine the ER of LHRH derivatives, the transport of the compounds was measured in both the secretory (B–A) and the Hexose Transport Assay absorptive direction (A–B) in Caco-2 cell monolayers (Table 2). Phloretin, a GLUT2 inhibitor,26 and phlorizin, a specific and The native peptide 1 showed a high transport rate in the se- competitive SGLT1 inhibitor,26,27 were used to study the con- cretory direction that resulted in an ER value of 8.75. Among tribution of sugar transporters to permeation of glycosylated the glycosylated LHRH derivatives (2–5), the transport of ana- LHRH compounds. Sink conditions were achieved for the lac- logue 4 was significantly higher in the B–A direction (Papp = −7 tose derivative of LHRH. However, the experiment for the 87 × 10 cm/s) than in the A–B direction (Papp = 11 × transport of compounds 4 and 5 was not maintained under sink 10−7 cm/s). This resulted in a higher ER (8.03) than for the conditions because of the back diffusion of compounds from the other analogues. The lowest ER belonged to compound 5 (0.06), receiver chambers. Therefore, the permeability coefficient of which contained glucose in its sequence. 68 Moradi, Varamini, and Toth, JOURNAL OF PHARMACEUTICAL SCIENCES 103:3217–3224, 2014 DOI 10.1002/jps.24120 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology 3221

Figure 3. The concentration of the compounds transported across Caco-2 cell monolayers over time: the amount of each LHRH derivative transported over time is presented separately. Compounds (200 :M) were incubated with cells in the presence and absence of the inhibitors. Six sequential samples were taken at different time points (0, 30, 60, 90, 120 and 150 min) and were analysed by LC–MS. Data are shown as the mean ± SEM (n = 3).

Table 2. Apparent Permeability of Glycosylated LHRH Derivatives Efflux Transporter Inhibition Experiment in Absorptive (A–B) and Secretory (B–A) Direction Across Caco-2 Cell Monolayers The role of P-gp and MRP2 transporters in the efflux of LHRH analogues was investigated by comparing the P (B–A) in the P (A–B)a P (B–A)b app app app presence and absence of verapamil and MK-571 (Fig. 4). The for- Compound ×10−7 (cm/s) Efflux Ratio (ER) mer is a selective inhibitor of P-gp and the latter blocks MRP2 transporters. The efflux of glycosylated LHRH derivatives was 1 1.02 ± 3.34 8.93 ± 2.45 8.75 not significantly altered by verapamil in Caco-2 cell monolay- ± ± 2 38.42 3.55 5.47 0.54 0.14 ers (p > 0.05). However, co-incubation with MK-571 exhibited 3 36.76 ± 6.65 8.70 ± 0.96 0.22 a significant decrease in P (B–A) of compound 2, 3 and 4 (p < 4 16.04 ± 2.46 86.8 ± 2.15 8.03 app 5 34.61 ± 3.25 1.67 ± 0.72 0.06 0.001). Compound 4 was affected the most by MK-571 showing −7 a Papp (B–A) value at 86.8 × 10 cm/s post-treatment as com- a −7 The apparent permeability from apical to basolateral direction pared with a Papp value at 7.2 × 10 cm/s in untreated cells. (absorptive). bThe apparent permeability from basolateral to apical direction (secretory). No significant change was observed in Papp (B–A) of compound Data are shown as the mean ± SEM (n = 3). 5 in the presence of the inhibitors. Native peptide 1 showed an

69 DOI 10.1002/jps.24120 Moradi, Varamini, and Toth, JOURNAL OF PHARMACEUTICAL SCIENCES 103:3217–3224, 2014 3222 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

Glycosyl units are attached to the N-terminus of peptides through a linker such as succinamic acid. They are also coupled to the serine or threonine residues in the middle of the sequence.22 In this study, sugar units were attached to the N- terminus of the peptide derivatives 2–5. In compound 6,the glycosyl unit was attached to the side chain hydroxyl group of serine residue through a process known as O-linked glycosylation. This compound (6) was designed and synthesised to allow comparison between the transport rates of LHRH analogues with glycosyl units at different positions. In a comparison between the Papp of compounds 5 and 6, twice the transport rate was observed for compound 6. As the only difference between the structures of 5 and 6 was the position of glucose unit, it is plausible that the higher permeability of 6 might be because of Figure 4. Effect of MK-571 and verapamil on the Papp (B–A) of the the conjugation of the sugar in the middle of the sequence. glycosylated LHRH derivatives in Caco-2 cell monolayers. MK-571 There is a proper correlation between the permeability of caused a significant decrease in P from basolateral to apical (B– app drugs in Caco-2 cells and the absorption process in intesti- A) of compounds 2–4 in which verapamil resulted in a drop in its Papp. The statistical analysis was performed using one-way ANOVA analy- nal cells that results in a reliable prediction of intestinal drug 30,31 sis followed by Dunnett’s post-hoc test. Each value represents mean absorption. In our previous study, we observed a signifi- ± SEM (n = 3, from two independent experiments). *p < 0.05 and cant increase in the transport of some of the glycosylated ana- ***p < 0.001 inhibitor-treated versus control samples. logues of LHRH across Caco-2 cells.8 Here, we reported the impact of a carrier-mediated transport system, where glycosylated LHRH analogues penetrated through the intestinal cells. Ac- tively growing Caco-2 cell monolayers means they are differentiated and have become polarised resulting in the formation of distinguishable apical and basolateral domains on the surface of the semipermeable membrane.32 This results in the expression of SGLT1 and GLUT2 transporters at the same position as in human intestinal cells.33,34 A comparison study between the localisation of SGLT1 in Caco-2 cell membranes and the human jejunum showed the same distribution pattern in both models.35 SGLT1 transporters are located on the apical side of the brush borders and are mostly responsible for the transport of hexoses into cells. GLUT2 transporters are localised on the basolateral and apical sides of epithelial cells and mediate the absorp- Figure 5. Mean Papp values of compound 4 from apical to basolateral tion of carbohydrates from the intestinal lumen into the blood (A–B) and basolateral to apical (B–A) direction in Caco-2 cell mono- circulation.36,37 In Caco-2 cell monolayers, SGLT1 is predomi- layers in the presence or absence of 50 :M of MK-571 (n = 3, two nantly situated on the apical side, whereas GLUT2 is mostly independent experiments). found on the basolateral side of the monolayer.36 In the presence of carbohydrates, GLUT2 proteins are also distributed on the apical side of the monolayer.36 We described that inhibition 86% reduction in the P (B–A) after treatment with verapamil app of GLUT2 and SGLT1 transporters by phloretin and phlorizin, (Supplementary Table S2). respectively, caused a significant reduction in the P of com- The P (A–B) of compound 4 was measured when MRP2 app app pounds 2−5. However, these data indicated that the transport was blocked by MK-571. This inhibition caused more than a of the compounds was mostly facilitated through the GLUT2 43% increase in the transport of compound 4 from apical to the transporter system. In the brush border membrane, the trans- basolateral direction (Fig. 5). port of glucose by GLUT2 is three times greater than that of SGLT1.38 This is in agreement with the results we obtained for all sugar-modified analogues including compounds comprising DISCUSSION lactose and galactose in their structure. Several transporters are involved in the transport of drug can- We also reported the impact of efflux pump systems in didates across intestinal cells. We examined the impact of glu- pumping back the glycosylated LHRH from the Caco-2 cells. cose transporters and efflux systems on vectorial transport of P-gp and MRP2 serve as ATP-dependent efflux pumps, which glycosyl derivatives of LHRH peptide. The efficacy of glycosy- are localised in the apical membranes of polarised cells and lation in improving the drug-like properties of peptides has play an important role in drug disposition. They are involved been confirmed in several studies. In a study carried out by in the extrusion of xenobiotics to the extracellular environ- our group, oral administration of a glycosylated derivative of ment. These proteins are also functionally expressed in Caco- endomorphine-1 produced significant pain relief in a rat model 2 cells.39,40 We observed that the permeability of analogues of neuropathic pain.28 It was also reported that the glycosy- 2–5 was variably influenced by the efflux transport systems. lation of serine or threonine in enkephaline opioid peptide Galactose-conjugated LHRH (4) showed the highest efflux rate not only increased the binding affinity of the compound to compared with the other analogues. This explains why the per- opioid receptors but also enhanced anti-nociceptive potency.29 meation rate of this compound is lower than that of glucose- and 70 Moradi, Varamini, and Toth, JOURNAL OF PHARMACEUTICAL SCIENCES 103:3217–3224, 2014 DOI 10.1002/jps.24120 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology 3223

lactose-coupled analogues. The native LHRH peptide is also an 6. Patel M, Barot M-I, Renukuntla JW, Mitra AK. 2013. Protein and appropriate substrate for efflux pumps. In Caco-2 cell studies, peptide drug delivery. Adv Drug Deliv:241. the drug candidates with ER > 2 are considered as appropriate 7. Yamamoto T, Nair P, Jacobsen NE, Vagner J, Kulkarni V, Davis P, substrates for efflux transporters.41–43 In this study, the ER of Ma S, Navratilova E, Yamamura HI, Vanderah TW. 2009. Improving all glycosylated compounds was less than one except for com- metabolic stability by glycosylation: Bifunctional peptide derivatives that are opioid receptor agonists and neurokinin 1 receptor antagonists. pound 4 and the native peptide (ER > 2). J Med Chem 52(16):5164–5175. Several modulators are competitive inhibitors of P-gp and 8. Moradi SV, Mansfeld FM, Toth I. 2013. Synthesis and in vitro evalu- MRP2 transporters. Among them, verapamil, which is com- ation of glycosyl derivatives of luteinizing hormone-releasing hormone monly used for inhibition of P-gp and MK-571, has been proven (LHRH). Bioorg Med Chem 21(14):4259–4265. to be a selective inhibitor of MRP2.44,45 The ERs of glycosy- 9. Shah P, Jogani V, Bagchi T, Misra A. 2008. Role of Caco-2 cell lated compounds in the presence of MRP2 and P-gp inhibitors monolayers in prediction of intestinal drug absorption. Biotechnol Prog indicated that the compounds were predominantly effluxed by 22(1):186–198. MRP2. Thus, in comparison to MRP2, the contribution of P- 10. Cai Z, Huang J, Luo H, Lei X, Yang Z, Mai Y, Liu Z. 2013. Role gp was less significant. A more than 12-fold decrease in the of glucose transporters in the intestinal absorption of gastrodin, a highly water-soluble drug with good oral bioavailability. J Drug Tar- Papp (B–A) of compound 4 in the presence of MRP2 inhibitor get 21(6):574–580. proved the role of this efflux transporter in extrusion of that 11. Drozdowski LA, Thomson A. 2006. Intestinal sugar transport. compound. Furthermore, the inhibition of MRP2 enhanced the World J Gastroenterol 12(11):1657–1670. apical to basolateral transport of compound 4 to more than 12. Tamai I, Tsuji A. 1996. Carrier-mediated approaches for oral drug 43%; this finding was in line with a previous study performed delivery. Adv Drug Deliv Rev 20(1):5–32. 46 by Chen et al. In our study, the Papp (B–A)offlavonoidgluco- 13. Cheeseman C, Harley B. 1991. Adaptation of glucose transport sides was not altered by inhibition of P-gp, whereas the contri- across rat enterocyte basolateral membrane in response to altered di- bution of MRP2 in the efflux of those compounds was confirmed etary carbohydrate intake. J Physiol 437(1):563–575. in their research.46 It has been demonstrated that MRP2 trans- 14. Kellett GL, Brot-Laroche E, Mace OJ, Leturque A. 2008. Sugar porter is involved in the efflux of other flavonoid glucosides like absorption in the intestine: The role of GLUT2. Annu Rev Nutr 28:35– quercetin.47 54. 15. Gutmann H, Fricker G, Tor¨ ok¨ M, Michael S, Beglinger C, Drewe J. 1999. Evidence for different ABC-transporters in Caco-2 cells modulat- ing drug uptake. Pharm Res 16(3):402–407. CONCLUSIONS 16. Schinkel AH, Jonker JW. 2003. Mammalian drug efflux trans- We found that the incorporation of glycoside moieties including porters of the ATP binding cassette (ABC) family: An overview. Adv glucose and lactose into LHRH peptide enhanced the penetra- Drug Deliv Rev 55(1):3–29. 17. Chan LMS, Lowes S, Hirst BH. 2004. The ABCs of drug transport tion of the modified analogues in Caco-2 cell monolayer. GLUT2 in intestine and liver: Efflux proteins limiting drug absorption and and SGLT1 transporters contributed to the transport of sugar- bioavailability. Eur J Pharm Sci 21(1):25–51. modified LHRH derivatives across cells. The proportion of glu- 18. Burton PS, Conradi RA, Hilgers AR, Ho NFH. 1993. Evidence for a cose and lactose derivatives of LHRH pumped out of the cells polarized efflux system for peptides in the apical membrane of Caco-2 through MRP2 transporter did not affect the absorption rate of cells. Biochem Biophys Res Commun 190(3):760–766. the analogues significantly. These results indicated that lactose 19. Tang F, Borchardt RT. 2002. Characterization of the efflux trans- and glucose sugar moieties are promising absorption enhancers porter (s) responsible for restricting intestinal mucosa permeation of for the transport of LHRH peptide across biological membranes. the coumarinic acid-based cyclic prodrug of the opioid peptide DADLE. Pharm Res 19(6):787–793. 20. Day AJ, Gee JM, DuPont MS, Johnson IT, Williamson G. 2003. Ab- ACKNOWLEDGMENTS sorption of quercetin-3-glucoside and quercetin-4 -glucoside in the rat small intestine: The role of lactase phlorizin hydrolase and the sodium- This study was funded by Australian Research Council project dependent glucose transporter. Biochem Pharmacol 65(7):1199– grant (DP110100212) and Shayli Varasteh Moradi was sup- 1206. ported by a UQ International (UQI) Scholarship. 21. Blanchfield JT, Toth I. 2005. Modification of peptides and other drugs using lipoamino acids and sugars. In Peptide synthesis and applications. Springer, pp 45–61. REFERENCES 22. Kihlberg J, Elofsson M, Salvador LA. 1997. [11] Direct synthesis of glycosylated amino acids from carbohydrate peracetates and Fmoc 1. Clayton RN, Catt KJ. 1981. Gonadotropin-releasing hormone recep- amino acids: Solid-phase synthesis of biomedicinally interesting gly- tors: Characterization, physiological regulation, and relationship to re- copeptides. Methods Enzymol 289:221–245. productive function. Endocr Rev 2(2):186. 23. Nistor Baldea LA, Martineau LC, Benhaddou-Andaloussi A, 2. Hackshaw A. 2009. Luteinizing hormone-releasing hormone (LHRH) Arnason JT, Levy´ E,´ Haddad PS. 2010. Inhibition of intestinal glucose agonists in the treatment of breast cancer. Expert Opin Pharmacother absorption by anti-diabetic medicinal plants derived from the James 10(16):2633–2639. Bay Cree traditional pharmacopeia. J Ethnopharmacol 132(2):473– 3. Jan de Jong I, Eaton A, Bladou F. 2007. LHRH agonists in prostate 482. cancer: Frequency of treatment, serum testosterone measurement and 24. Martell R, Slapak C, Levy S. 1997. Effect of glucose trans- castrate level: Consensus opinion from a roundtable discussion. Curr port inhibitors on vincristine efflux in multidrug-resistant murine Med Res Opin 23(5):1077–1080. erythroleukaemia cells overexpressing the multidrug resistance- 4. Hamman JH, Enslin GM, Kotze AF. 2005. Oral delivery of peptide associated protein (MRP) and two glucose transport proteins, GLUT1 drugs—Barriers and developments. BioDrugs 19(3):165–177. and GLUT3. Br J Cancer 75(2):161. 5. Simerska P, Moyle PM, Toth I. 2011. Modern lipid-, carbohydrate-, 25. Hubatsch I, Ragnarsson EG, Artursson P. 2007. Determination of and peptide-based delivery systems for peptide, vaccine, and gene prod- drug permeability and prediction of drug absorption in Caco-2 mono- ucts. Med Res Rev 31(4):520–547. layers. Nat Protoc 2(9):2111–2119. 71 DOI 10.1002/jps.24120 Moradi, Varamini, and Toth, JOURNAL OF PHARMACEUTICAL SCIENCES 103:3217–3224, 2014 3224 RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology

26. Zheng Y, Scow JS, Duenes JA, Sarr MG. 2012. Mechanisms of glu- 37. Play B, Salvini S, Haikal Z, Charbonnier M, Harbis A, Roussel M, cose uptake in intestinal cell lines: Role of GLUT2. Surgery 151(1):13– Lairon D, Jourdheuil-Rahmani D. 2003. Glucose and galactose regulate 25. intestinal absorption of cholesterol. Biochem Biophys Res Commun 27. Masumoto S, Akimoto Y, Oike H, Kobori M. 2009. Dietary phlo- 310(2):446–451. ridzin reduces blood glucose levels and reverses Sglt1 expression in the 38. Kellett GL HP. 2000. The diffusive component of intestinal glucose small intestine in streptozotocin-induced diabetic mice. J Agric Food absorption is mediated by the glucose-induced recruitment of GLUT2 Chem 57(11):4651–4656. to the brush-board membrane. Biochem J 350:155–162. 28. Varamini P, Mansfeld FM, Blanchfield JT, Wyse BD, Smith 39. Anderle P, Niederer E, Rubas W, Hilgendorf C, Spahn-Langguth H, MT, Toth I. 2012. Synthesis and biological evaluation of an Wunderli-Allenspach H, Merkle HP, Langguth P. 1998. P-glycoprotein orally active glycosylated endomorphin-1. J Med Chem 55(12):5859– (P-gp) mediated efflux in Caco-2 cell monolayers: The influence of cul- 5867. turing conditions and drug exposure on P-gp expression levels. J Pharm 29. Elmagbari NO, Egleton RD, Palian MM, Lowery JJ, Schmid WR, Sci 87(6):757–762. Davis P, Navratilova E, Dhanasekaran M, Keyari CM, Yamamura 40. Alluri RV, Ward P, Kunta JR, Ferslew BC, Thakker DR, Dallas S. HI. 2004. Antinociceptive structure–activity studies with enkephalin- 2014. In vitro characterization of intestinal and hepatic transporters: based opioid glycopeptides. J Pharmacol Exp Ther 311(1):290– MRP2. In Optimization in drug discovery. Springer, pp 369–404. 297. 41. US Food and Drug Administration, US Department of Health and 30. Artursson P, Palm K, Luthman K. 2001. Caco-2 monolayers in ex- Human Services, Center for Drug Evaluation and Research, Center for perimental and theoretical predictions of drug transport. Adv Drug Biologics Evaluation and Research(CBER). 2012. Guidance for indus- Deliv Rev 46(1–3):27–43. try: Drug interaction studies—Study design, data analysis, and impli- 31. Artursson P, Palm K, Luthman K. 2012. Caco-2 monolayers in ex- cations for dosing and labeling. Silver Spring, Maryland: US FDA. perimental and theoretical predictions of drug transport. Adv Drug 42. European Medicines Agency. 2010. Guideline on the investigation Deliv Rev 64(Supplement 0):280–289. of drug interactions. 32. Borlak J, Zwadlo C. 2003. Expression of drug-metabolizing en- 43. Giacomini KM, Huang SM, Tweedie DJ, Benet LZ, Brouwer KLR, zymes, nuclear transcription factors and ABC transporters in Caco-2 Chu XY, Dahlin A, Evers R, Fischer V, Hillgren KM, Hoffmaster KA, cells. Xenobiotica 33(9):927–943. Ishikawa T, Keppler D, Kim RB, Lee CA, Niemi M, Polli JW, Sugiyama 33. Harris DS, Slot JW, Geuze HJ, James DE. 1992. Polarized distri- Y, Swaan PW, Ware JA, Wright SH, Yee SW, Zamek-Gliszczynski MJ, bution of glucose transporter isoforms in Caco-2 cells. Proc Natl Acad Zhang L, International T. 2010. Membrane transporters in drug devel- Sci USA 89(16):7556–7560. opment. Nat Rev Drug Discov 9(3):215–236. 34. Mahraoui L, Rodolosse A, Barbat A, Dussaulx E, Zweibaum 44. Jedlitschky G, Leier I, Buchholz U, Center M, Keppler D. 1994. A, Rousset M, Brot-Laroche E. 1994. Presence and differen- ATP-dependent transport of glutathione S-conjugates by the multidrug tial expression of SGLT1, GLUT1, GLUT2, GLUT3 and GLUT5 resistance-associated protein. Cancer Res 54(18):4833–4836. hexose-transporter mRNAs in Caco-2 cell clones in relation 45. Sandstrom¨ R, Karlsson A, Lennernas¨ H. 1998. The absence of stere- to cell growth and glucose consumption. Biochem J 298:629– oselective P-glycoprotein-mediated transport of R/S verapamil across 633. the rat jejunum. J Pharm Pharmacol 50(7):729–735. 35. Khoursandi S, Scharlau D, Herter P, Kuhnen C, Martin D, Kinne 46. Chen Z, Ma T, Huang C, Zhang L, Zhong J, Han J, Hu T, Li J. 2014. RK, Kipp H. 2004. Different modes of sodium-D-glucose cotransporter- Efficiency of transcellular transport and efflux of flavonoids with dif- mediated D-glucose uptake regulation in Caco-2 cells. Am J Physiol ferent glycosidic units from flavonoids of Litsea coreana L. in a MDCK Cell Physiol 287(4):C1041–C1047. epithelial cell monolayer model. Eur J Pharm Sci 53(0):69–76. 36. Grefner N, Gromova L, Gruzdkov A, Komissarchik YY. 2010. Com- 47. Walle T, Walle UK. 2003. The $-D-glucoside and sodium-dependent parative analysis of SGLT1 and GLUT2 transporters distribution in rat glucose transporter 1 (SGLT1)-inhibitor phloridzin is transported by small-intestine enterocytes and Caco-2 cells during hexose absorption. both SGLT1 and multidrug resistance-associated proteins 1/2. Drug Cell Tissue Biol 4(4):354–361. Metab Dispos 31(11):1288–1291.

72 Moradi, Varamini, and Toth, JOURNAL OF PHARMACEUTICAL SCIENCES 103:3217–3224, 2014 DOI 10.1002/jps.24120 Supporting information

The transport and efflux of glycosylated Luteinizing hormone releasing hormone (LHRH) analogues in Caco-2 cell model: contributions of glucose transporters and efflux systems

Shayli Varasteh Moradi1, Pegah Varamini1, Istvan Toth1,2,*

Additional figures:

+Q1: 4.708 to 5.409 min from Sample 1 (TuneSampleID) of MT20130715164007.wiff (Ion Spray) Max. 2.8e5 cps.

658.1 2.8e5 (2) (1) 2.6e5 331.4 2.4e5

2.2e5

2.0e5

1.8e5

1.6e5

1.4e5

1.2e5 Intensity, cps

1.0e5

8.0e4

6.0e4

4.0e4 327.1 2.0e4 675.4 436.2 459.2 696.1 313.0 355.0 371.2 500.1 509.5 577.4 589.6 598.2 663.5 736.4 773.3 821.3828.9 891.2 904.1 976.4 988.0 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 m/z, amu

Figure S1: Electrospray ionization mass spectrometry (ESI-MS) and analytical HPLC spectra of GS-Fmoc Serine: The compound was characterized using mass spectrometry and analytical HPLC. The purification of the glycosylated serine was performed on Vydac C8 column (22

73 x 250 mm) at a flow rate of 20 mL/min with a 60 minute gradient of 30 to 70% solvent B (0.1% TFA in acetonitrile/water 9:1; solvent A: 0.1%TFA in water). The purity of the fractions was confirmed by analytical HPLC on a Vydac C8 column (4.6 x 250 mm, 5 µm) at a flow rate of 1 mL/min and a gradient of 0 to 100% B over 42 minutes.

Figure S2: A 400 MHz 1H NMR spectrum of Glucose-FMOC-Serine: compound was dissolved in CDCl3.

74 +Q1: 2.755 to 3.055 min from Sample 1 (TuneSampleID) of MT20130811182648.wiff (Ion Spray) Max. 3.3e6 cps.

737.6 3.3e6 (3) (2) (1) 3.2e6

3.0e6

2.8e6

2.6e6

2.4e6

2.2e6

2.0e6

1.8e6

1.6e6

Intensity,1.4e6 cps

1.2e6

1.0e6

8.0e5 1474.5

6.0e5

4.0e5 557.7 983.5 2.0e5 492.4 586.2 857.1 1106.5 922.7 1312.3 1477.9 1793.0 1871.5 2003.4 2109.6 2411.5 2470.6 2694.1 2947.7 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 m/z, amu

Figure S3: ESI-MS and analytical HPLC spectra of C6: Characterization of compound was done using mass spectrometry and analytical HPLC. The crude peptide was purified by preparative HPLC on Vydac C18 column (22 x 250 mm) at a flow rate of 20 mL/min with a 60 minute gradient of 30 to 60% solvent B. The purity of the fractions was confirmed by analytical HPLC on a Vydac C8 column (4.6 x 250 mm, 5 µm) at a flow rate of 1 mL/min and a gradient of 0 to 100% B over 42 minutes.

75 (A) (B)

Figure S4. Trans-epithelial electrical resistance (TEER) value of Caco-2 cell monolayers (A) effect of phloretin and phlorizin (B) and glycosylated derivatives of LHRH on the integrity of the monolayers.

Fig S5. The concentration of the compounds transported across Caco-2 cell monolayers in basolateral to apical direction as a function of time.

Additional table:

Table S1: The apparent permeability values of LHRH analogues in Caco-2 cell monolayers

P (A-B) P (A-B) P (A-B) app app app Compound P (A-B) -7 -7 -7 app -7 (×10 cm/s± SEM) (×10 cm/s± SEM) (×10 cm/s± SEM) (×10 cm/s± SEM) Inhibitor: Inhibitor: Inhibitor: phloretin phlorizin phloretin+phlorizin 1 1.02±3.34 - - - 2 38.42±3.55 5.41±0.137 11.8±2.06 1.2±0.48 3 36.76±6.65 3.62±0.248 17.2±2.73 0.84±0.11 4 17.04 ±2.46 3.35±0.12 6.9±2.67 1.00±0.365 5 34.61±3.25 4.5±0.285 11.4±2.46 1.45±0.474 6 58.54±4.72 5.3±2.4 13.7±3.04 1.6±0.345

Table S2: Basolateral to apical permeability of glycosylated LHRH analogues in the presence of verapamil (P-gp inhibitor) and MK571 (MRP2 inhibitor) separately. Data are presented as the mean ± SEM (n=3)

-7 -7 Compound P (×10 ,cm/s) P (×10 ,cm/s) Number app app Inhibitor: Inhibitor: Verapamil MK571 C1 1.22±0.43 5.05±0.85 C2 3.79±0.73 0.62±0.14 C3 6.10±1.03 1.14±0.10 C4 72.00±4.26 7.17±1.03 C5 1.89±0.81 1.21±0.56

76 Chapter 5: Lactose derivative of LHRH with enhanced pharmacological profile

‘’In vivo pharmacological evaluation of a lactose-conjugated luteinizing hormone releasing hormone analogue’’

Shayli Varasteh Moradi, Pegah Varamini, Frederik Steyn and Istvan Toth

5.1 Introduction to this publication

This chapter was submitted to ‘’International Journal of Pharmaceutics’’ journal. In this manuscript, the pharmacokinetic parameters of lactose derivative of LHRH as a lead glycosylated analogue (based on in vitro studies) were evaluated following oral administration to rats. The stimulatory effect of this compound on the secretion of LH was also investigated in vivo.

5.2 Reprint of the submitted manuscript to the journal of ‘’ International Journal of Pharmaceutics’’

77 In vivo pharmacological evaluation of a lactose-conjugated luteinizing hormone releasing hormone analogue

Shayli Varasteh Moradi 1, Pegah Varamini1, Frederik Steyn2 and Istvan Toth1,3

1School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Queensland 4072, Australia; 2 The University of Queensland Centre for Clinical Research and the School of Biomedical Sciences, The University of Queensland, St. Lucia, Queensland 4072, Australia; 3School of Pharmacy, The University of Queensland, Woolloongabba, Queensland 4102, Australia

Abstract:

In the current study, the efficacy and pharmacokinetic profile of lactose-conjugated luteinizing hormone releasing hormone (LHRH) was examined following oral administration in male rats. A rapid and sensitive liquid chromatography/mass spectrometry technique was developed and applied for measuring the concentration of lactose[Q1][w6]LHRH (compound 1) in rat plasma in order to allow measurement of pharmacokinetic parameters. LH release was evaluated using a sandwich

ELISA. Maximum serum concentration (Cmax= 0.11 µg/ml) was reached at 2 h (Tmax) following oral administration of the compound at 10 mg/kg. The half-life was determined to be 2.6 h. The absolute bioavailability of the orally administered compound was found to be 14%, which was a remarkable improvement compared to zero-to-low oral bioavailability of the native peptide. Compound 1 was effective in stimulating LH release at 20 mg/kg after oral administration. The method was validated at a linear range of 0.01–20.0 µg/ml and a correlation coefficient of r2 ≥0.999. The accuracy and precision values showed the reliability and reproducibility of the method for evaluation of the pharmacokinetic parameters. These findings showed that the lactose derivative of LHRH has a therapeutic potential to be further developed as an orally active therapeutics for the treatment of hormone-dependent diseases.

Keywords: LHRH, LC/MS, LH release, bioavailability, pharmacokinetic

1. Introduction

LHRH is a neuroendocrine decapeptide (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) secreted by hypothalamus in a pulsatile manner, which stimulates its cognate receptor in the pituitary gland to release gonadotropins including luteinising hormone (LH) and follicle-stimulating hormone (FSH). Subsequently, the production of gonadotropins regulates the secretion of sex

78 hormones in both males and females (Clayton and Catt, 1981). Due to the biological importance, various LHRH analogues have been designed and developed after LHRH was sequenced in 1971 (Matsuo et al., 1971; Schally et al., 1971). LHRH analogues are used clinically for the treatment of various hormone dependent diseases including prostate and breast cancers, endometriosis, fertility disorders and precocious puberty (Huirne and Lambalk, 2001). The continuous administration of LHRH or its agonists leads to an initial surge in the release LH, FSH and sex hormones subsequently, followed by down-regulation of the LHRH receptors and suppression of gonadotropin secretion (Millar et al., 2004). Endogenous LHRH has a short half-life of 4-8 min in human plasma (Barron et al., 1982; Redding et al., 1973). It is also rapidly degraded by organs including liver, kidneys, anterior pituitary, posterior pituitary, and hypothalamus (Müller et al., 1997). Due to poor stability, LHRH is not able to provide a long-term stimulation of the pituitary gland and exert a strong and long-acting agonist effect. Replacement with D-amino acids at cleavage sites of native peptides improves their stability against enzymatic digestion and thereby prolongs the biological half-life. Substitution of Gly6 with D-amino acid in endogenous LHRH enhances the enzymatic resistance and also receptor binding affinity of the peptide, which leads to a better agonist activity (Coy et al., 1976; Schally AV, 2003). Triptorelin is a synthetic super agonist of LHRH containing D-Trp at position 6 with a longer half-life (19 min) than the native peptide (Barron et al., 1982) and is parenterally administered for prostate cancer treatment (Heyns, 2005). Although LHRH agonists have shown improved metabolic stability compared to the native peptide, they are still not orally effective. Poor membrane permeability and susceptibility to digestion by gastrointestinal enzymes give rise to a low oral bioavailability of LHRH agonists (less than 1%) (Iqbal et al., 2012). These analogues are distributed into the extracellular space and metabolized by digestive enzymes and kidney. It is believed that LHRH analogues are predominantly cleared by the kidney due to rapid and extensive renal uptake following IV administration (Handelsman and Swerdloff, 1986). All commercial analogues of LHRH are administered through parenteral routes including subcutaneous and intramuscular (Beyer et al., 2011; Padula, 2005). As the oral route is the preferred route of administration by patients, development of an oral delivery system for therapeutic peptides like LHRH is highly desirable. Manipulation of peptide structures using glycosylation strategy is known to be an effective approach to improve the metabolic stability and membrane permeability of modified analogues (Christie et al., 2014; Moradi et al., 2014; Powell et al., 1993). Glycosylation has been shown to improve the efficacy of some peptides in vitro and in vivo (Egleton et al., 2000; Wu et al., 2006; Yamamoto et al., 2009).

79 Liquid chromatography/mass spectrometry (LC/MS) is a highly selective automated tool for quantitative sample analysis (Niessen, 2003). In addition to high sensitivity and selectivity, LC/MS has the advantage of providing data that are easy to interpret compared to other applicable techniques (Gillespie and Winger, 2011). Immunoassays are other routine techniques used for screening and quantification of peptides. However, these assays have some drawbacks such as higher incidence of false positive results and the requirement for specific antibodies. Therefore, developing an LC/MS-based method can provide highly accurate and reliable peptide quantification in biological samples (Sofianos et al., 2008). In our previous study, we showed that conjugation of glycosyl units to LHRH peptide enhanced the in vitro stability and permeability of the modified peptides across Caco-2 cell monolayers (as an intestinal model). Among all glycosylated analogues, lactose conjugated LHRH demonstrated the best in vitro stability and membrane permeability (Moradi et al., 2013). In the current study, we measured the bioavailability and pharmacokinetic parameters of the lactose- modified LHRH as the lead compound in rats. An LC/MS-based method was developed for quantitative sample analysis followed by compound extraction from serum using an optimized method of extraction. The stimulatory effect of the orally administered compound on LH release in rats was also examined over 24 hours.

2. Experimental 2.1 Materials and apparatus Lactose[Q1][w6]LHRH (compound 1) and lactose[Q1]LHRH were synthesized according to the published methods (Moradi et al., 2013). Lactose[Q1]LHRH was used as the internal standard (IS). HPLC-grade acetonitrile (MeCN) was purchased from RCI Labscan Ltd. (Bangkok, Thailand). Methanol (MeOH) in HPLC grade was purchased from Merck biosciences (VIC, Australia) and Formic acid (analytical grade, 99%) from Univar, Australia. Phosphate buffer saline (PBS) was purchased from Invitrogen Life Technologies.

2.2 Animals Male Sprague Dawley rats weighing between 140–170 g were purchased from UQ Animal Resource Centre (ARC) and were kept for one week acclimatization period prior to initiation of experimental procedures. Rats were housed in groups of two or three with ad libitum access to food and water, in a room with controlled temperature (22.2 ± 0.2 ◦C) and humidity (51–65%) on a photo period of 12 h light/12 h dark. For experiments, rats were divided into 3 groups of 5 animals; negative control, intravenously (IV) and orally (PO) dosed groups. All experimental procedures

80 were approved by The University of Queensland Animal Ethics Committee (AEC#SCMB/005/11/ARC).

2.3 Sample preparation The rat plasma samples were thawed at room temperature and spiked with 100 µl of IS solution (to a final concentration of 1 µg/ml). Analytes were extracted from plasma using a liquid-liquid extraction method. Different concentrations of acidified MeCN (95%, 80% and 60%) and MeOH (95%, 80% and 60%) with 0.1% formic acid were used to optimize the analyte extraction method. Plasma proteins were precipitated by the addition of 500 µl extraction solutions and were then vortexed and centrifuged at 14000 × g, 15 min. The supernatant was collected and evaporated under a slow stream of nitrogen gas using a five-valve glass manifold. Extracted samples were reconstituted in 40 µl of water-acetonitrile-formic acid (90:10:0.1) and injected into the LC/MS system for analysis.

2.4 Preparation of calibration standards and quality control samples Stock solutions of compound 1 (100 µg/ml) and IS (10 µg/ml) were prepared in water: acetonitrile 90:10 containing 0.1% formic acid. Serial dilutions of the compound (60-0.2 µg/ml) were then prepared from the stock solution to be used as calibration samples. Calibration standards were freshly prepared by spiking dilutions into blank rat plasma to yield final plasma concentrations ranging from 0.05 to 30.0 µg/ml of compound 1 and 1µg/ml of IS solution. Quality control (QC) samples at the lower limit of quantification (LLOQ), low, middle and high concentrations (0.01, 2, 10 and 20 µg/ml) were made daily from separately prepared stock solutions. The selected dilutions were added to 100 µL of plasma spiked with 20 µl of IS solution at a final concentration of 1 µg/ml). Similar extraction method was then used for the preparation of QC samples. Calibration curves were plotted as the peak area ratio (Compound 1:IS) vs concentration.

2.5 LC–MS analysis and quantification LC/MS was carried out on a Shimadzu HPLC (LC-10AT) system coupled to a PE Sciex (AB Sciex) API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Concord, Ontario, Canada). The instrument was operated in positive ion mode under the following conditions: Ion-Spray voltage, 5000 V; source temperature, 550 °C; curtain gas (nitrogen) at 8; collision gas (nitrogen) at 5; declustring potential at 50; focusing potential at 220 and entrance potential at 10. Chromatographic separation was performed on a Phenomenex luna C18 column (5 μm, 50 mm × 2.0 mm) with a gradient mobile phase of solvent A (0.01% acetic acid in water) and solvent B (90% acetonitrile, 10% water and 0.01% acetic acid). The compound was eluted with a

81 20-35% solvent B over 5 min at a flow rate of 0.5 ml/min. Total injection volume was 15 µL. The extracted ion chromatogram with m/z 877.1 at 3.3–3.4 min and m/z 812.2 at 3.9–4.1 min was detected for compound 1 and IS, respectively.

2.6 Method validation The accuracy and precision, calibration curve performance and recovery of the method were evaluated. The calibration curve was plotted in the range of 0.05-20 µg/ml using 1/x weighting regression model. Calibration curves were evaluated using three separately prepared batches. For testing the accuracy and precision of the method, intra- and inter-day assays were performed for all QC samples. The intra-day assay was performed within one day by analysing triplicate of each concentration of QC samples. The inter-day assay was carried out on four separate days for QC samples (each concentration in triplicate) and repeated twice (in two separate weeks). The recovery efficiency of the extraction procedure was performed at three concentrations of compound 1 (0.01, 10, and 20.0 µg/ml) and IS (1 µg/ml) from rat plasma.

2.7 In vivo pharmacokinetic study Rats were administered compound 1 at 2.5 mg/kg IV, 10 and 20 mg/kg PO. About 300 µL of blood sample was collected from each rat by tail bleed prior to the start of the experiment and at selected time points (0.5, 1, 2, 4, 6, 8, 12 and 24 h). Samples were allowed to clot for 2 h at room temperature, followed by centrifugation at 1000×g for 20 min. Serum aliquots were stored at −80◦C.

2.8 LH release assay Serum concentration of LH was measured by a sandwich ELISA, strictly adhering to methodology as published previously (Steyn et al., 2013). Briefly, a 96-well high-affinity binding microplate was coated with monoclonal antibody (anti-bovine LH beta subunit) and incubated overnight at 4°C. A standard curve was generated using a 2-fold serial dilution of mouse LH in 0.2% (w/v) BSA- 1×PBS-T (PBS with 0.05% Tween 20). The LH standards and plasma samples were incubated with 50 µL of detection antibody (polyclonal antibody, rabbit LH antiserum) for 1.5 h followed by the addition of 50 µL horseradish peroxidase-conjugated antibody (polyclonal goat anti-rabbit antibody) and 1.5 h incubation at room temperature. O-phenylenediamine, substrate containing

0.1% H2O2 was added to each well and left at RT for 30 minutes. The reaction was stopped using 3 M Hydrochloric acid. The absorbance of each well was read at a wavelength of 490 nm (Sunrise; Tecan Group). The concentration of LH in whole blood samples was determined by interpolating the OD values of unknowns against a nonlinear regression of the LH standard curve. LH secretory

82 responses were expressed as the area under the curve (AUC) after normalizing data to the baseline value. The within and between assay coefficient of variation of LH assays were below 5%.

2.9 Data analysis and statistical evaluation The pharmacokinetic profiles, including area under the plasma concentration vs. time curve (AUC), half-life (t1/2) and clearance (Cl) of each rat were analyzed by non-compartmental analysis (Phoenix

WinNonlin 1.2; Certara Inc., Princeton, NJ, USA). The maximum plasma concentration (Cmax) and the time the Cmax is reached (Tmax) were directly computed from the plasma concentration vs. time graph. The oral bioavailability (F%) was calculated by the following equation:

퐷표푠푒 퐼푉 × 퐴푈퐶 푃푂 푭% = 100 × 퐷표푠푒 푃푂 × 퐴푈퐶 퐼푉

All the data are expressed as mean ± standard deviation. Statistical analysis of pharmacokinetic parameters was calculated using ANOVA followed by the Dunnett’s post hoc test.

3. Results 3.1 Method validation and quantification of compound 1 in rat plasma The chemical structures of compound 1 and compound 2 (IS) are shown in Fig. 1. Lactose[Q1]LHRH was used as IS due to the structural similarity to compound 1. The extraction of compound from rat plasma was performed using different concentrations of acidified MeOH and MeCN solutions. Among all extraction solutions tested, the best recovery of compound 1 and IS from rat plasma was obtained using 95% of MeCN in water. The recovery (%) was found to be above 80% for 0.05, 2 and 10 µg/ml of compound 1 (83.7%, 89.8%, and 91.3%, respectively). The calibration curve showed an acceptable linearity at r2=0.999 for the range of concentration used, from 0.05 to 20 µg/ml (Fig. S1). The lower limit of quantification (LLOQ) was 0.01 µg/ml. The reproducibility of the method was confirmed by intra- and inter-day assays which were determined by analysing the LLOQ (0.01 µg/ml), low QC (2 µg/ml, n=3), medium QC (10 µg/ml, n=3) and high QC (20 µg/ml, n=3) on four separate runs. The inter-day precision did not exceed 12% for the four concentrations of QC samples and the intra-day precision of the assay was between 2% and 15%. The accuracy of intra- and inter-day assays ranged from 95% to 105% for the analyte and IS (Table S. I). The method selectivity was determined by analysing the plasma samples spiked with the highest concentration of compound 1 without IS. No signal was detected for IS showing that there was no interference of the IS signal with the compound’s peak. Analysis of four sources of blank

83 plasma prepared from different rats showed no signal for compound 1 and IS indicating that source of plasma did not affect the signals obtained from compound 1 and IS.

(A)

(B )

Figure 1: Chemical structures of (A) lactose [Q1][w6]LHRH (compound 1) and (B) lactose [Q1]LHRH (Compound 2; IS)

3.2 Pharmacokinetic studies Pharmacokinetic parameters were analysed by non-compartmental analysis. The results are summarised in Table I as mean±SD of measurements from five animals per group. Following IV and PO administrations, the plasma concentration of the compound at various time points (during 24 h) was measured using the established LC/MS method (Fig. 2). The AUCs were obtained at 1.27±0.76 µg/ml*h and 0.709±0.24 µg/ml*h for IV and PO doses, respectively. The mean volume

of distribution (Vd) and clearance of compound 1 were found to be 36.49 ml/kg and 1.96±0.52 ml/h/kg, respectively, after IV administration. The peak plasma concentration of the compound was

achieved after 2 h (Cmax= 0.11±0.03 µg/ml). The half-life of compound 1 in plasma was 2.6 h after PO dose, which was a significant improvement compared to the short half-life of native LHRH as reported in literature (4 min) (Redding et al., 1973). The oral bioavailability of the compound was measured to be 14%.

Figure 2: Plasma levels of compound 1 (mean ± SD, n=5) following 2.5 mg/kg IV bolus (A) and 10 mg/kg oral gavage (B) in intact male rats measured by LC/MS.

Table 1: Pharmacokinetic parameters of compound 1 in rats after administration at 2.5 mg/kg IV and 10 mg/kg PO. Data are presented as mean ± SD (n = 5). Parameters Value (Mean+SD) Value (Mean+SD)

IV PO

Body weight (g) 171±25.08 164.4±18.40

AUC0-∞ (µg/ml*h) 1.27±0.76 0.709±0.24

T1/2 (h) 2.9±0.63 2.6±0.84

Cl (ml/h/kg) 1.96±0.52 -

Vd (ml/kg) 36.49 -

Cmax (µg/ml) - 0.11±0.03

Tmax (h) - 2

F (%) - 14

T1/2: half-life; Cl: clearance; Vd: volume of distribution; Cmax: maximum concentration; Tmax: time to reach Cmax.

3.3 Efficacy of compound 1 in the release of LH

To determine the changes in LH level after oral administration of compound 1, the normalised area under curves (nAUC) of LH released was obtained over 24 h. A marked increase in the level of LH was observed after oral administration of 20 mg/kg of compound 1 compared to the control (PBS)

85 group (from nAUC=4.45± 1.028 ng/24h in control group to nAUC=11.33± 1.65 ng/24h). However, the oral dose of 10 mg/kg did not stimulate the release of LH significantly over 24 h (Fig. 3).

Figure 3: Changes in the plasma level of LH calculated from a nAUC over 24 h following oral administration of compound 1 at 10 and 20 mg/kg. Each column represents the Mean ± SD (n=5). Statistical analysis was performed using a one-way ANOVA followed by the Dunnett’s post hoc test and compared to PBS group (***, p<0.01).

4. Discussion In the present study, the pharmacological properties of lactose-[Q1][w6]LHRH was evaluated after PO and IV administration to rats. Due to the poor oral bioavailability, all commercial derivatives of LHRH are administered parenterally which is inconvenient for patients. The main objective of this research was to improve the bioavailability of LHRH following oral administration. Modification of therapeutic peptides by substitution of D-isoform of amino acids is known to be the effective strategy to improve their pharmacological properties and metabolic stability (Seitz, 2000; Werle and Bernkop-Schnürch, 2006). Glycosylation is another useful approach to improve the metabolic stability of peptides in physiological environments and increase their biological activity (Simerska et al., 2009; Ueda et al., 2009). We applied D-amino acid substitution together with glycosylation strategy to address the associated challenges and enhance the bioavailability of LHRH peptide. In our preliminary in vitro studies, we showed that glycosylation significantly enhanced the metabolic stability (up to 4-fold) and apparent permeability (7 to 15-fold) of LHRH across intestinal cell membranes (Moradi et al., 2013; Moradi et al., 2014). Based on those results, we selected

86 compound 1 as the most promising LHRH glycosylated derivative for oral administration and investigated the pharmacological profile of this analogue followed by oral administration to rats. For pharmacokinetic evaluation of compound 1, an accurate analytical tool was required to determine the peptide’s plasma concentration. Therefore, an LC/MS method was developed and validated for quantitation purposes. LC/MS is an automated technique with broad applications in quantification of pharmaceutical compounds and their metabolites in biological matrices. The selectivity, sensitivity and cost effectiveness of MS based methods make them a preferred analytical technique in pharmaceutical industry (Lee, 2003). The developed method in this study was capable of detecting the compound over a range of 0.01 to 20 µg/ml with a detection limit of 0.01 µg/ml. The precision and accuracy of the method was assessed by performing intra- and inter-day assays The precision value obtained was below 12% for the four QC levels which was in acceptable limit based on FDA guidelines (the precision value should not exceed 15%) (Rower et al., 2010). The high accuracy (98.2% and 97.9% for intra-day and inter-day, respectively) was also obtained showing the reliability of the developed method. Consequently, the validation of the method showed good reproducibility, accuracy, precision and linearity for quantification of compound 1 in rat plasma. A quick and easy extraction protocol was performed for precipitation of plasma proteins using liquid-liquid extraction method. It yielded over 80% recovery for three selected concentration of the compound. The high and consistent extraction recovery showed a negligible loss of the analyte during the sample preparation process, and did not vary from sample to sample. A significant improvement in the bioavailability (14%) and half-life of compound 1 (2.6 h) was observed after oral administration of compound 1 compared to the endogenous peptide (Iqbal et al., 2012; Redding et al., 1973). The increased half-life of the compound may account for the enhancement of its bioavailability. These findings demonstrate that the attachment of lactose to LHRH analogue significantly improved the pharmacological properties of native LHRH. This is in agreement with other studies we previously published showing that the modification of opioid peptide endomorphin-1 by a lactose moiety resulted in significant analgesic activity of the peptide following oral administration to rats (Varamini et al., 2012). To the best of our knowledge, this is the first report of an orally administered LHRH analogue with a remarkable half-life and bioavailability. Acute administration of LHRH agonists stimulates the pituitary gland to release LH, whereas the chronic administration results in suppression of the pituitary-gonadal axis and blockade of LH secretion (Pinski et al., 1996). Following single-dose oral administration (acute administration) of compound 1 to rats, their plasma LH level increased significantly compared to the negative control group. This finding demonstrated that compound 1 was able to produce

87 stimulatory effect in vivo following acute administration. Taken together, we improved the oral bioavailability of LHRH by conjugation of a lactose moiety and D-Tyr6 substitution while maintaining the activity.

5. Conclusions In the present study, we applied glycosylation strategy together with D-amino acid substitution to develop an orally active LHRH analogue. A sensitive and specific LC/MS quantification method was developed and validated to evaluate the pharmacological activity of lactose-[Q1][w6]LHRH in vivo. The method was reproducible and reliable to be used for quantitative analysis of the compound in rat plasma. We showed that the conjugation of lactose residue to LHRH peptide increased its half-life and oral bioavailability in rats significantly. The newly designed analogue could also stimulate LH secretion upon oral administration. The improved pharmacological properties of lactose-[Q1][w6]LHRH render this analogue a promising candidate towards the development of an orally available LHRH therapeutics.

Acknowledgments We would like to acknowledge the Australian Research Council for their support of this work with the Discovery Project Grant and Professorial Research Fellowship to Istvan Toth (DP110100212). Shayli Varasteh Moradi was supported by UQ International (UQI) Scholarship. The pharmacokinetic analysis was performed using Phoenix 1.2 supplied by Certara Inc. We would also thank Dr. Stefanie Henning and Dr. Christina Staaz for their assistance in analysis of pharmacokinetic parameters.

88 References:

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90 Supporting information

In vivo pharmacological evaluation of lactose-conjugated luteinizing hormone releasing hormone analogue

Shayli Varasteh Moradi 1, Pegah Varamini1, Frederik Steyn2 and Istvan Toth1,3

1School of Chemistry and Molecular Biosciences, the University of Queensland, St. Lucia, Queensland 4072, Australia, 2School of Biomedical Sciences, the University of Queensland, St. Lucia, Queensland 4072, Australia, 3School of Pharmacy, the University of Queensland, Woolloongabba, Queensland 4102, Australia

Calibration curve C1.rdb (C1): "Linear" Regression ("1 / x" weighting): y = 2.59 x + 0.0674 (r = 0.9990)

15 A nalyte A rea / IS A rea

0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Analyte Conc. / IS Conc. Figure S1. A representative calibration curve for compound 1 quantification in rat plasma. Analyte concentration was in the range of 0.01-20 µg/mL and IS concentration was 1 µg/mL.

Table S. I: Intra- and inter-day precision and accuracy values for the developed method Nominal Intra-day (n=8) Inter-day (n=6) concentration Accuracy Precision Accuracy Precision (µg/mL) (mean±SD,%) (CV,%) (mean±SD,%) (CV,%) QC1: 0.01 101.04± 0.58 2.40±1.70 101.11±0.67 1.15±0.16

QC2: 2 99.67±1.39 4.17±2.27 102.33±0.58 3.17±2.35

QC3: 10 103.47±1.98 10.74±3.51 103.89± 2.55 8.38±1.61

QC4:20 104.75±2.16 14.75 ±5.15 95.56±1.18 12.17±1.26 SD: standard deviation; CV: coefficient of variation

91 Chapter 6: Conclusion and future directions

92 6.1 Conclusion LHRH analogues have extensive therapeutic applications for the treatment of hormone dependent diseases such as infertility, endometriosis, breast and prostate cancers. Due to the poor oral bioavailability, all LHRH derivatives have to be administered through parenteral routes. This study focused on developing an effective system to improve the pharmacological properties and oral bioavailability of LHRH peptide.

Chemical modification of peptides using glycosylation has been proven to be an effective approach in oral peptide delivery. We successfully designed and synthesized a library of LHRH derivatives by conjugation of different sugar units including glucose, galactose or lactose to the N- terminus (via succinamic acid linker) or to Ser4 of LHRH sequence (through O-glycosylation) using SPPS approach. In addition to carbohydrate modification, two derivatives were synthesised with D- Trp at position six instead of the Gly residue. The substitution of D-amino acids to LHRH at position six leads to 10- to 200- fold increase in the potency and enhancement of the enzymatic stability of the peptide. We aimed to address the poor pharmacokinetic profiles of the native LHRH by combining these two approaches (D-amino acid substitution and glycosylation).

The impact of glycosylation on the apparent permeability of LHRH across cell membrane was investigated in Caco-2 cell monolayer as a well-established in vitro model of intestinal epithelium. The apparent permeability of the compounds bearing glucose and lactose at the N-terminus was improved significantly by approximately 6-7 folds compared to the native LHRH. The highest permeability value was observed for the compound bearing glucose at Ser4 (15 folds increase compared to native LHRH) and the galactose modified derivatives exhibited the lowest permeability. The position and the type of glycosyl units were shown to be influential to the permeation of LHRH through Caco-2 cell monolayer. We showed that glucose and lactose units could serve as promising absorption enhancers for improving the penetration of LHRH through Caco-2 cell monolayer. Moreover, the conjugation of glucose to Ser4 residue had the best impact on increasing the penetration of LHRH across Caco-2 cell monolayer.

The impact of certain glucose transporters including GLUT2 and SGLT1 on the transport of glycosylated LHRH derivatives across Caco-2 cell monolayer was also investigated. It was confirmed that both transporters were involved in facilitating the transport of glycosylated peptides across Caco-2 cell monolayer.

The contribution of efflux transporters in the extrusion of glycosylated LHRH derivatives from Caco-2 cells was examined and reported in this thesis. Efflux transport systems are considered as formidable biological barriers to oral drug delivery. They reduce the drug absorption by pumping back the drugs into the extracellular spaces. It was found that the permeability of the glycosyl

93 conjugates of LHRH was not significantly affected by the efflux pumps except for the galactose modified compound. This analogue had the highest efflux ratio showing that it was a proper substrate for one or more efflux transport systems. The possible role of MRP2 and p-gp transporters (as highly expressed isoforms of efflux transporters in Caco-2 cell membrane) was also investigated. Our studies showed that MRP2 was mainly involved in the efflux of glycosylated LHRH analogues (particularly galactose modified compound) whereas, p-gp contributed to the extrusion of the native LHRH.

Therapeutic peptides are susceptible to the proteolytic enzymes located in the gastrointestinal tract. These digestive enzymes degrade the peptides before they reach to the target sites. The glycosylation of LHRH appeared to confer an increase in the enzymatic stability of the peptide. The glycosylated LHRH derivatives exhibited enhanced half-lives in different tissue homogenates (Caco-2 cell, rat liver and kidney membrane homogenates) and human plasma. In addition to stability data obtained for LHRH derivatives, it was found that kidney had a significant metabolic effect in the hydrolysis of the glycosylated LHRH analogues compared to the other matrices tested here. Among all tested analogues, GS4-[w6]LHRH and Lac-[Q1][w6]LHRH were the most stable derivatives in all studied biological matrices.

The efficacy of glycosyl conjugates of LHRH was also studied by evaluating the stimulatory effect of the compounds in the release of LH and FSH from pituitary gland in vitro. Among tested analogues, Lac-[Q1][w6]LHRH demonstrated the best stimulatory effect in the secretion of LH hormone from the rat pituitary cells. The analogue bearing glucose at Ser4 residue exhibited an inhibitory effect on LH release in the treated pituitary cells. Moreover, GS-[Q1]LHRH was capable of stimulating the release of FSH in dispersed rat pituitary cells. These findings provided useful insight into the stimulatory activity of the carbohydrate modified LHRH derivatives in the release of gonadotropins.

The anti-proliferative activity of the glycosylated LHRH analogues was investigated in three LHRH-receptor positive cancer cells, PC-3, LNCaP and DU145. A meaningful reduction was observed in the viability of LNCaP and DU145 cells following treatments by glycosylated compounds in concentration-dependent manner. The treatment of PBMCs at the same concentrations did not show any growth inhibitory effect indicating that glycosylated LHRH analogues are non-toxic to normal cells like PBMCs.

Based on the data obtain from in vitro studies, Lac-[Q1][w6]LHRH was chosen as the lead candidates for in vivo investigations. The oral bioavailability of this compound was improved 14- fold which was remarkable compared to the poor bioavailability of native peptide. Moreover, a significant increase was observed in the half-life of this analogue following oral administration

94 which may account for the improved bioavailability of the lactose bearing LHRH analogue compared to the endogenous peptide. The in vivo efficacy of Lac-[Q1][w6]LHRH was also evaluated in rats at 20 mg/kg following oral administration. An increase was observed in the level of LH secreted into the blood compared to the control group (measured over 24 hours). According to these results, Lac-[Q1][w6]LHRH possessed promising pharmacological properties to be considered as an orally active analogue.

Taken together, the promising results of these in vitro and in vivo studies demonstrated that the conjugation of carbohydrate units to LHRH had a pronounced effect in improving the pharmacokinetic profiles of the modified peptides. Moreover, the lactose and glucose bearing analogues exhibited direct antitumor activity in prostate cancer cells and stimulated LH release form pituitary gland. The pharmacokinetic advantages and maintained efficacy of the lactose and glucose derivatives of LHRH rendered these analogues promising candidates for the development of orally active LHRH pharmaceuticals.

6.2 Future directions This thesis provided some useful information on the improved pharmacokinetic profiles of glycosyl conjugates of LHRH to be orally administered. The further investigations could help to improve the current knowledge concerning the designed system for oral delivery of LHRH peptide. In this project, we showed the efficacy of lactose and glucose modified LHRH analogues to stimulate the LH and FSH release from pituitary gland. Receptor binding affinity and understanding the mechanism of action of LHRH glycosyl conjugates would be the future in vitro studies that could give useful knowledge on the activity and specific binding of the glycosylated analogues to the cognate receptor.

One of the major finding of this project was the improved pharmacological properties of Lac- [Q1][w6]LHRH. Further investigation of whether the increase in the administered dose will enhance the bioavailability and efficacy of this compound would be desirable. Evaluating the long-term effect of this analogue on the suppression of LH production (four to seven weeks of consecutive treatment) through oral route would be also worth of study. Another important study is to investigate the biodistribution of this compound following oral administration to rats which will provide pivotal information on the accumulation of this analogue in different organs including kidney, liver, stomach and brain. As described in this thesis, Lac-[Q1][w6]LHRH exerted a significant anti-proliferative activity on prostate cancer cells. Further studies could be performed to assess the in vivo anti-tumour activity of this analogue in a prostate cancer model and evaluate its

95 long-term effect on the tumour size. Histological observation by monitoring the morphological changes in the prostate organ is clearly beneficial.

Based on the in vitro data provided in this thesis, GS4-[w6]LHRH is another promising analogue with improved metabolic stability and membrane permeability to be further investigated for in vivo biological properties. The comparison studies between the pharmacological profiles of GS4-[w6]LHRH and Lac-[Q1][w6]LHRH will also offer informative data to find out the impact of the position and type of the glycosylation on biological properties of the glycosylated LHRH analogues.