European Polymer Journal 120 (2019) 109241 Contents lists available at ScienceDirect European Polymer Journal journal homepage: www.elsevier.com/locate/europolj Tuning drug release from polyoxazoline-drug conjugates T ⁎ J. Milton Harrisa, ,1, Michael D. Bentleya, Randall W. Moreaditha, Tacey X. Viegasa,1, Zhihao Fanga, Kunsang Yoona, Rebecca Weimera, Bekir Dizmanb, Lars Nordstiernac a Serina Therapeutics, Inc., 601 Genome Way, Suite 2001, Huntsville, AL 35806, USA2 b Sabanci University, Faculty of Engineering and Natural Sciences, Tuzla, 34956 İstanbul, Turkey2 c Department of Chemistry and Chemical Engineering, Chalmers University of Technology, SE-412 96 Göteborg, Sweden ARTICLE INFO ABSTRACT Keywords: Poly(2-oxazoline)-drug conjugates with drugs attached via releasable linkages are being developed for drug Poly(2-oxazoline) or POZ delivery. Such conjugates with pendent ester linkages that covalently bind drugs to the polymer backbone ex- Poly(2-ethyl-2-oxazoline) or PEOZ hibit significantly slower hydrolytic release rates in plasma than the corresponding PEG- and dextran-drug Pendent drugs conjugates. The slow drug release rates in-vitro of these POZ-drug conjugates contribute to extended in-vivo Degradable ester linkages pharmacokinetic profiles. In some instances, the release kinetics may be relatively sustained and ideal foronce-a- Pharmacokinetics week subcutaneous injection, whereas the native drug by itself may only have an in-vivo half-life of a few hours. Drug delivery Phenolic drugs The origin of this unusual kinetic and pharmacokinetic behavior is proposed here to involve folding of the POZ conjugate such that the relatively hydrophobic drug forms a central core, and the relatively hydrophilic polymer wraps around the core and slows enzymatic attack on the drug-polymer chemical linkage. Here we present evidence supporting this hypothesis and demonstrate how the hypothesis can be used to tune hydrolytic release rates and pharmacokinetics. Evidence for the folding hypothesis is taken from hydrolysis kinetics of a range of drugs in plasma, pharmacokinetics of a range of drugs following subcutaneous injection in laboratory animals, and nuclear magnetic resonance (NMR) studies showing folding of the POZ-rotigotine molecule. The drugs included in this study to test the hypothesis are: rotigotine, buprenorphine, dexanabinol, cannabidiol (CBD), Δ9- tetrahydrocannabinol (THC) and cannabigerol (CBG). 1. Introduction This surprisingly slow release of rotigotine from the POZ conjugate in plasma is reflected in a near linear pharmacokinetic (PK) profile for Use of polyoxazolines (POZ) for drug modification and drug de- once-a-week subcutaneous injection of POZ-rotigotine in humans [4]. livery has attracted increasing research activity in recent years [1–6]. We have now observed similar behavior in other pendent POZ-drug One aspect of this research involves attachment of drugs as hydro- conjugates. The purpose of this publication is to provide an explanation lytically releasable pendent groups along the polymer backbone. of the unusual and useful slow hydrolysis rates and extended PK profiles Structure 1 in Fig. 1 shows such an example in which the drug roti- for pendent POZ-drug conjugates. gotine is attached to POZ 20 kDa via a triazole-ester linkage and in Our operating hypothesis for the unusually slow pendent POZ-drug which a is random, as opposed to block, m is typically 190, o is typically release kinetics has been that the conjugate folds loosely such that the 10, and p can be varied to change the hydrolysis rate of the ester. In one relatively hydrophobic pendent drugs form a “core” surrounded by the of our recent publications we showed that rotigotine attached via relatively hydrophilic polymer backbone “shell”. In this “core-shell” triazole-ester linkages to branched PEGs, dendrimer PEGs, and mod- hypothesis, the surrounding polymer shell is flexible and mobile and ified dextran with pendant functional groups was released inplasma interferes with but does not prevent esterase approach to the core. This much more rapidly than rotigotine attached to POZ through the same interference to esterase approach and binding slows ester hydrolysis linkages; this data is summarized in Table 1 [7]. In contrast, reactive and drug release. Here we describe several experiments designed to test groups attached to the terminus of a linear POZ show very similar this core-shell hypothesis. These experiments are: (1) NMR studies of hydrolysis rates to the corresponding PEG derivative [1]. POZ-rotigotine (Structure 1) and POZ-cholesterol using correlation ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (J.M. Harris). 1 These authors contributed equally to writing this manuscript. 2 All coauthors except Nordstierna are stockholders of Serina Therapeutics and have a financial interest in Serina Therapeutics. https://doi.org/10.1016/j.eurpolymj.2019.109241 Received 3 July 2019; Received in revised form 3 September 2019; Accepted 5 September 2019 Available online 11 September 2019 0014-3057/ © 2019 Elsevier Ltd. All rights reserved. J.M. Harris, et al. European Polymer Journal 120 (2019) 109241 conjugates with less hydrophobic drugs, assuming other factors such as steric and electronic effects are similar. Third, loading more ofahy- drophobic drug onto a POZ backbone should lead to a tighter, more- compact core, and this in turn should give a slower hydrolysis rate. Fourth, adding inert, hydrophilic molecules in pendent positions would be expected to attract water and swell and “loosen” the core, which would lead to increased enzymatic access to the core, and an increase in hydrolysis rates of attached drugs. Inert hydrophobic pendants would be expected to have the opposite effect, and such work will be the subject of future publication. In the following sections we explore the above four experiments. Finally, we show how combination of the various controlling factors (ester linkage, drug loading, polymer MW) can be used to tune the pharmacokinetics of POZ-drug conjugates. 2. Experimental 2.1. Materials The ethyl oxazoline monomer was purchased from Polymer Chemistry Innovation, Tucson, Arizona. The functional pentynyl Fig. 1. Structure 1 of POZ-rotigotine conjugate using a degradable ester linkage monomer was prepared at Serina Therapeutics. The solvents used in the where p can be 1–3. A branched alkyl linker can also be used. synthesis and extraction of the polymer and polymer conjugates were ACS anhydrous grade or better and were acquired from EMD Chemicals. The initiators, reagents and catalysts used in the synthesis of Table 1 the polymer and polymer conjugates were acquired from Sigma-Aldrich Effect of polymer on hydrolytic release rate of rotigotine from roti- in St. Louis, MO. Samples of the active molecules used in the POZ gotine 3-propionate ester conjugates of POZ, PEG, and modified conjugation were rotigotine (from Sai Chemicals, Hyderabad, India), dextran in female rat plasma at 37 °C [7]. cannabidiol, Δ9-THC, cannabigerol and buprenorphine (from Noramco, Polymer Plasma half-life (t1/2) Wilmington, DE), and dexanabinol (from Cayman Chemical, Ann Arbor, MI). Polyethylene glycol (PEG) reagents with 4-arm and 8-arm chains POZ 10 pendent 11.9 h PEG 4 arm 8 min were obtained from Creative PEGWorks, Chapel Hill, NC and the dex- PEG 8 arm dendrimer 11 min tran 6-arm polymer was synthesized at Serina Therapeutics. Dextran 6 pendent < 2 min 2.2. Chemical synthesis of POZ and POZ conjugates a. POZ is MW 20 kDa, acid terminus, 10 pendants, each pendant ter- minated with triazole-CH CH -COO-rotigotine. 2 2 Synthesis of POZ with pendent functional groups and pendent, re- b. PEG 4 arm has propargyl core, MW 10 kDa, each arm terminated leasable drugs (typically with ester linkers to phenolic drugs) via the with triazole-CH2CH2-COO-rotigotine. c. PEG dendrimer, 8 arms, MW 26 kDa, each arm terminated with route shown in the reaction scheme below (Fig. 2) has been described in triazole-CH2CH2-COO-rotigotine. several of our publications and patents [4,7]. d. Dextran 6 pendent MW 20 kDa [8], each pendant terminated with POZ conjugates of the following phenolic drugs were prepared by triazole-CH2CH2-COO-rotigotine [2]. this procedure (Fig. 3). An azido alkyl carboxylic acid linker was first coupled to the phenolic –OH of the small molecule drug to make a spectroscopy and diffusometry; (2) effect of drug hydrophobicity on mono-ester. Drugs such as cannabidiol and cannabigerol have two po- plasma release kinetics; (3) effect of drug loading on plasma release tential phenolic –OH groups, and the diesters formed were removed by kinetics; and (4) effect of hydrophilic pendent groups on rate of release preparative chromatography. The azido alkyl esters of these drugs were of hydrophobic drugs. then “clicked” to the pentynyl pendants on the POZ polymer as shown The reasoning behind choice of these experiments is as follows. in Fig. 2. Examples of alkyl carboxylic acid linkers are acetic, 2-pro- First, NMR spectroscopy is capable of showing chemical shifts corre- pionic and 3-propionic acid. The number of drug molecules loaded can lations consistent with nuclei in close spatial proximity, and thus can vary and will depend on the number of equivalents (eq) of the Drug- reveal polymer conformation. Moreover, phase-sensitive 1H–1H NOESY Linker-Azide used in the click reaction. When 10 eq are used the con- pulse sequence NMR provides molecular self-diffusivity which depends jugate is found to be fully clicked on all 10 pendants of the POZ polymer on polymer conformation, size and aggregation. The concept of col- chain. Conjugates with lower number of drug loading were also pre- lapsing a macromolecule into itself using different polymers such as pared using less than 10 eq of the Drug-Linker-Azide compound; i.e. 8 polystyrene and poly(methyl methacrylate) in solvents such as toluene and 6 eq. The percentage (% w/w) of drug loaded was chromato- and tetrachloroethane has been previously studied [9]. Some have de- graphically assayed then calculated using the molecular weight of the monstrated that pulsed-gradient spin-echo NMR (PGSE-NMR) and small molecule drug.