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. iii 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. iv 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) v 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.