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Pharmacokinetic Characterization of Kalata B1 and Related Therapeutics Built on the Cyclotide Scaffold

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Pharmacokinetic Characterization of Kalata B1 and Related Therapeutics Built on the Cyclotide Scaffold Accepted Manuscript Pharmacokinetic characterization of kalata B1 and related therapeutics built on the cyclotide scaffold Aaron G. Poth, Yen-Hua Huang, Thao T. Le, Meng-Wei Kan, David J. Craik PII: S0378-5173(19)30354-0 DOI: https://doi.org/10.1016/j.ijpharm.2019.05.001 Reference: IJP 18331 To appear in: International Journal of Pharmaceutics Received Date: 31 January 2019 Revised Date: 24 April 2019 Accepted Date: 3 May 2019 Please cite this article as: A.G. Poth, Y-H. Huang, T.T. Le, M-W. Kan, D.J. Craik, Pharmacokinetic characterization of kalata B1 and related therapeutics built on the cyclotide scaffold, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Pharmacokinetic characterization of kalata B1 and related therapeutics built on the cyclotide scaffold Aaron G. Potha, Yen-Hua Huanga, Thao T. Lea,b, Meng-Wei Kana, David J. Craika* aInstitute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia Email addresses: Aaron G. Poth: [email protected] Yen-Hua Huang: [email protected] Thao T. Leb: [email protected] Meng-Wei Kan: [email protected] David J. Craik*: [email protected] bPresent address: School of Science, Edith Cowan University, Perth, WA, 6027, Australia *Corresponding author’s address: Professor David J. Craik Institute for Molecular Bioscience The University of Queensland Brisbane, QLD, 4072 Australia Phone: +61 7 3346 2019 Fax: +61 7 3346 2101 Declarations of interest: none 1 Abstract: Oral activity has been described for cyclotide-containing traditional medicines, and demonstrated for reengineered cyclotides bearing grafted therapeutic epitopes, highlighting their potential for translation to the clinic. Here we report preclinical pharmacokinetic parameters for the prototypic cyclotide kalata B1 (kB1) and two orally active grafted analogues, ckb-KAL and ckb-KIN, to provide the first in vivo dose-exposure metrics for cyclotides. Native and grafted kB1 molecules exhibited multiple compartment kinetics and measurable but limited oral bioavailability of similar magnitude to several orally administered peptide drugs in the clinic. Cyclotides are mostly associated with the central compartment, and display small (0.07–0.1 L kg-1 for kB1 and ckb-KIN) to moderate (1 L kg-1 for ckb- KAL) steady state volumes of distribution. This study provides new data essential to the evaluation of cyclotides as therapeutics, validating them as a viable drug design scaffold with tunable pharmacokinetic properties. Keywords: Cyclotide; oral bioavailability; drug scaffold; pharmacokinetics; grafting; knottin Chemical compounds studied in this article: kalata B1 (PubChem CID: 52945815) 2 1. Introduction With the enormous chemical space and extended surface areas accessible by their natural sidechain variations (Khazanov and Carlson, 2013), protein drugs (biologics) can display exquisite selectivity and potency for their targets, typically endowing them with large therapeutic windows compared to traditional small molecule (<500 Da) drugs (Khazanov and Carlson, 2013). However, due to their size (>5000 Da) and physicochemical properties, biologics are often hampered by low membrane permeability, poor oral absorption, and high susceptibility to proteolysis, leading to short biological half-lives. Cyclotides are a family of plant-derived peptides featuring a cyclic backbone cross-braced by a cystine knot in which a trio of disulfide bonds link through one another to stabilize the peptide core (Craik et al., 1999). The traditional uterotonic medicine ‘kalata-kalata’ from which cyclotides were first identified (Craik et al., 1999) is prepared by boiling the leaves of the African herb Oldenlandia affinis, and so reports of its oral bioactivity in ethnomedicinal settings suggest oral and heat stability of the active ingredients (Gran, 1970; Nworu et al., 2017). Subsequent studies on cyclotides isolated from O. affinis have shown them to be impervious to degradation by enzymes in vitro, high temperatures, or low pH environments (Colgrave and Craik, 2004). From the time the ultra-stable cyclic cystine knot (CCK) motif of the cyclotide kalata B1 (kB1) from O. affinis was characterized (Colgrave and Craik, 2004; Saether et al., 1995), considerable effort has been invested in evaluating this motif as a scaffold for drug design. In particular, the ‘grafting’ of bioactive peptide epitopes into the stabilized CCK has been widely explored. The CCK scaffold is especially suitable for this application and has been used to stabilize a range of peptide epitopes with potential applications in the treatment of cancer (Aboye et al., 2016; Chan et al., 2015; Dsouza et al., 2015; Getz et al., 2013; Huang et al., 2015; Quimbar et al., 2013), obesity (Eliasen et al., 2012), pain (Wong et al., 2012), cardiovascular disease (Getz et al., 2011), multiple sclerosis (Thell et al., 2016) and inflammation (Thongyoo et al., 2009). Acyclic knottins also feature a stable cystine knot motif (Colgrave and Craik, 2004; Werle et al., 2006) and several of them have been investigated for their capacity to increase the stability of contrast agents for tumor imaging (Kimura et al., 2012; Moore et al., 2013) or to modulate therapeutic targets (Krause et al., 2007). 3 Recently, several grafted knottins and cyclotides have been shown to exhibit oral activity and have been evaluated from in vitro concentration-response or in vivo dose-response perspectives. Wong et al. (Wong et al., 2012) reported the oral activity of two engineered cyclotides bearing therapeutic grafts (ckb-KAL and ckb-KIN) targeting bradykinin B1 receptors in a mouse model of inflammatory pain. These grafted cyclotides inhibited pain pathways in peripheral sensory ganglia, indicating their systemic distribution (Wong et al., 2012). In another study, the cyclotide MCoTI-I was shown to undergo cellular uptake in vitro and interact with intracellular targets, thus opening up new avenues for peptide therapeutics (Contreras et al., 2011). In terms of biodistribution in rats for intravenously administered MCoTI-II, the intact peptide was primarily detected in the serum and kidneys indicating renal clearance, although no uptake into brain tissues was observed (Wang et al., 2016). Furthermore, fluorescently tagged [T20K] kB1 was observed to associate particularly with the kidneys following oral administration to rats, and detectable amounts of unlabeled [T20K] kB1 were observed in plasma (although not quantitated) (Thell et al., 2016). In combination, these studies indicate promise in linking in vitro dose-effect with in vivo dose-responses, but a critical part of evaluating the clinical translatability of any therapeutic is gaining an understanding of its dose-exposure metrics. Preclinical pharmacokinetic analyses of investigational therapeutics remain a prerequisite for progression to first-in- human clinical studies, whereby allometric scaling allows reasonable prediction of the primary clinical pharmacokinetic parameters (Caldwell et al., 2004). A key metric for assessing the clinical translatability of cyclotides is oral bioavailability (F%) as drugs with a low F% tend to exhibit higher variability in drug exposure among recipients, leading to higher risks of administering sub-therapeutic or toxic doses, particularly when administering drugs with a narrow therapeutic index (Hellriegel et al., 1996). Notwithstanding the variable predictive correlation between human and animal drug bioavailability (Musther et al., 2014), at present there remains a dearth of information regarding the in vivo pharmacokinetics of cyclotides, which have been only minimally explored quantitatively in animal studies (Colgrave et al., 2005; Melander et al., 2016). In the original study of grafted cyclotides ckb-KAL and ckb-KIN, the authors suggested that the variability in absolute oral activity between them could be due to differences in their oral bioavailability, potentiated by the presence of two charged amino acids and a proline in the grafted loop- the presence of which might favor membrane 4 interaction (Wong et al., 2012). Thus, the aim of the present work was to investigate the pharmacokinetic parameters of the prototypic cyclotide kB1 alongside grafted cyclotides ckb-KAL and ckb-KIN (Figure 1) to uncover quantitative bases for their differences in activity that could be used to improve the future design of grafted cyclotides. In this study, we compared the plasma protein binding, blood component distribution, distribution and elimination kinetics, and oral bioavailability of cyclotides and evaluate their strengths, weaknesses, addressable knowledge gaps, and overall potential for translation as clinical therapeutics. 2. Materials and methods 2.1 Chemicals and peptides All chemicals were of analytical grade and sourced from Sigma-Aldrich. The native cyclotide (kB1) was extracted and purified from O. affinis as previously described (Plan et al., 2007). Grafted kalata peptides ckb-KAL and ckb- KIN were synthesized via solid phase peptide synthesis using established methods (Daly
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