University of Southern California Dissertations and Theses

Development of an elastin-like polypeptide-based cyclosporine A formulation that improves autoimmune-mediated dry eye characteristic of Sj鰃ren抯 syndrome

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Development of an elastin-like polypeptide-based cyclosporine A formulation that improves autoimmune-mediated dry eye characteristic of Sj鰃ren抯 syndrome

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Content DEVELOPMENT OF AN ELASTIN-LIKE POLYPEPTIDE-BASED CYCLOSPORINE A FORMULATION THAT IMPROVES AUTOIMMUNE-MEDIATED DRY EYE CHARACTERISTIC OF SJÖGREN’S SYNDROME By Hao Guo A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (PHARMACEUTICAL SCIENCES) August 2021 ii Acknowledgements First of all, with great respect, I would like to sincerely thank my advisors, Dr. Andrew MacKay and Dr. Sarah Hamm-Alvarez, for making my graduate school journey such an enlightening and memorable experience. Dr. MacKay has been my mentor throughout my graduate school since 2014. I am truly grateful to him for being so considerate and supportive whenever I am going through challenging situations, both professional and personal. Shortly after in 2016 when joining the PhD program, I was especially fortunate to receive Dr. Hamm-Alvarez’s mentorship. With all her support and encouragement, the style of her mentorship really built my scientific confidence, allowing me to further explore my capabilities. I also want to take this opportunity to express my deep appreciation towards Dr. Xie for his great suggestions, meaningful guidance and generous help in the past a few years. I am also truly grateful to all my previous and current lab members for their generous and selfless help. My special thanks shall go to Dr. Maria Edman, Dr. Zhe Li, Dr. Mihir Shah, Dr. Santosh Peddi, Dr. Yaping Ju, Dr. Changrim Lee, Frances Yarber, Srikanth Reddy Janga, Dr. Anh Truong, Minchang Choi and Jingmei Yu. This Thesis would not be possible without them. I would like to thank Dr. Junji Watanabe from Translational Lab and Dr. Shuxing Li from Nanobiophysics core for training me on various instruments. Finally, I would like to thank my parents and my wife. Their love, support and selfless sacrifices have always been my motivation to keep moving forward. iii Table of Contents Acknowledgements .................................................................................................................... ii List of Tables .............................................................................................................................. v List of Figures ............................................................................................................................ vi Abstract ...................................................................................................................................... ix Introduction ................................................................................................................................. 1 Chapter 1 First generation Elastin-like polypeptides-delivered CsA and its preliminary efficacy against Sjögren’s Syndrome-mediated ocular manifestations. .............................. 4 1.1 Introduction ................................................................................................................... 4 1.2 Materials and Methods ................................................................................................. 8 1.3 Results ........................................................................................................................ 20 1.4 Discussion .................................................................................................................. 41 1.5 Conclusions ................................................................................................................ 46 Chapter 2 Sustained release 2nd generation carriers for Cyclosporine A reduce Th17 mediated autoimmunity in murine model of Sjögren’s syndrome ...................................... 47 2.1 Introduction ................................................................................................................. 47 2.2 Materials and Methods ............................................................................................... 52 2.3 Results ........................................................................................................................ 65 2.4 Discussion .................................................................................................................. 84 2.5 Conclusion .................................................................................................................. 88 Chapter 3 A pharmacokinetics primer for preclinical nanomedicine research ............ 89 3.1 Abstract ....................................................................................................................... 89 3.2 Introduction ................................................................................................................. 90 3.3 Quantifying sample concentrations ........................................................................... 100 3.4 Non-compartmental parameter estimation ............................................................... 106 3.5 Compartmental modeling of parameters .................................................................. 112 iv 3.6 Interpreting the PK of drug carriers ........................................................................... 123 3.7 Structure-function studies to optimize PK parameters .............................................. 126 3.8 Conclusion ................................................................................................................ 138 Conclusion .............................................................................................................................. 140 References .............................................................................................................................. 141 v List of Tables Chapter 1. Table 1. 1: Amino acid sequence and phase behavior of CA192. ................................. 22 Table 1. 2: Comparison of pharmacokinetic parameters observed for dimeric CA192 administered by IV and SC administration to mice ........................................................ 37 Chapter 2. Table 2. 1: Amino acid sequence and thermo-responsive behavior of CAC and CVC. . 70 Table 2. 2: Comparison of pharmacokinetic parameters observed for CAC after IV or SC administration and CVC after SC administration. ........................................................... 78 Chapter 3. Table 3. 1: Size-dependent PK parameters of [ 14 C] sucrose liposomes following IV administration to rats. ................................................................................................... 128 Table 3. 2: Surface charge-dependent PK parameters of gold nanoparticles following IV administration to mice. ................................................................................................. 131 Table 3. 3: Linker-dependent PK parameters of antibody-drug conjugates following IV administration to mice. ................................................................................................. 133 Table 3. 4: PEG-density dependent PK parameters among PRINT nanoparticles following IV administration in mice. .............................................................................. 136 Table 3. 5: Albumin-binding dependent PK parameters for immune-tolerant elastin-like polypeptides (iTEP) administered SC to mice. ............................................................. 138 vi List of Figures Chapter 1: Figure 1. 1: A recombinant elastin-like polypeptide (ELP) fusion protein as a non- covalent drug carrier for cyclosporine A (CsA). .............................................................. 21 Figure 1. 2: Expressed CA192 assembles two species in solution that can be isolated by size exclusion chromatography (SEC). ..................................................................... 24 Figure 1. 3: Isothermal titration calorimetry confirms that dimeric CA192 maintains high binding affinity for CsA. .................................................................................................. 26 Figure 1. 4: CA192 exhibits slow disassociation from CsA under sink dialysis in PBS. . 27 Figure 1. 5: Neither physiological albumin nor whole plasma rapidly displaces CsA from CA192. ........................................................................................................................... 29 Figure 1. 6: CA192 is internalized into Jurkat and Hela cells where it co-localizes with low pH compartments. ................................................................................................... 30 Figure 1. 7: CA192-CsA exhibits comparable IL-2 inhibition efficacy to free CsA. ........ 32 Figure 1. 8: CA192-CsA prevents NFAT1 and NFAT2 dephosphorylation. ................... 33 Figure 1. 9: Pharmacokinetic profile of CA192 dosed via IV or SC. ............................... 36 Figure 1. 10: Subcutaneous administration of CA192-CsA increased basal and stimulated tear production in a mouse model of SS. ...................................................... 40 Figure 1. 11: CA192 delivery suppressed CsA nephrotoxicity relative to free CsA. ...... 41 Chapter 2. Figure 2. 1: IL-17A and IL-2 gene expression is elevated in CD4+ T cells isolated from NOD mouse LG. ............................................................................................................. 68 vii Figure 2. 2: A cyclophilin-based drug-carrier assembles an ELP depot for cyclosporine at physiological temperatures. ....................................................................................... 72 Figure 2. 3: CAC-CsA and CVC-CsA effectively inhibit Th17-like cell differentiation in vitro. ............................................................................................................................... 74 Figure 2. 4: CAC-CsA and CVC-CsA effectively inhibit IL-17A secretion from activated Th17-like cells in vitro. .................................................................................................... 75 Figure 2. 5: The depot-forming CVC-CsA has extended PK profile relative to soluble CAC-CsA. ....................................................................................................................... 77 Figure 2. 6: Two treatments with CVC-CsA significantly reduced Th17.1 and Th2, but not Th1 or Treg, cells in the LG-infiltrating CD4+ population, as well as glandular IL-17A accumulation. ................................................................................................................. 82 Figure 2. 7: One treatment with CVC-CsA over a two-week period effectively altered the LG-infiltrating helper T cell composition. ........................................................................ 83 Figure 2. 8: One treatment with CVC-CsA increased basal tear production. 14-week-old male NOD mice were treated with CAC-CsA or CVC-CsA at a CsA dose of 3.0mg/kg or with PBS once for two weeks via supra-LG injection. .................................................... 84 Chapter 3. Figure 3. 1: Plasma PK profile for a protein-based drug carrier, called CA192, which was administered SC to mice. ........................................................................................ 91 Figure 3. 2: Different ADME processes will have different half-lives. ............................. 95 Figure 3. 3: Fluorescence is a sensitive and adaptable method for studying the PK profiles of nanoparticles. .............................................................................................. 102 Figure 3. 4: Illustration of trapezoidal method. ............................................................. 108 viii Figure 3. 5: One-compartment IV bolus model ............................................................ 115 Figure 3. 6: Two-compartment IV bolus model. ........................................................... 119 Figure 3. 7: One-compartment extravascular bolus model. ......................................... 123 Figure 3. 8: Whole blood PK profile for [ 14 C] sucrose-entrapped into sphingomyelin- phosphocholine liposomes with different sizes, which was administered IV to rats. .... 127 Figure 3. 9: Plasma concentration over time profiles of AuNPs with different surface charges were depicted after injected IV in mice. .......................................................... 130 Figure 3. 10: The plasma concentration versus time profiles of antibodies with different hydrophobicity after IV administration into mice. .......................................................... 132 Figure 3. 11: Blood concentration of nanoparticles with different PEG shielding density over time after IV administration in mice. ..................................................................... 135 Figure 3. 12: Serum concentration of vaccine carriers with or without albumin binding capability over time after SC administration to mice. ................................................... 137 ix Abstract Sjögren’s syndrome (SS) is a multifactorial autoimmune disease with principal symptoms including inflammation and loss of function of lacrimal glands (LG) and salivary glands. As a potent immunosuppressant, topical ophthalmic CsA is approved for SS-mediated dry eye disorders; however, it cannot effectively resolve inflammation due to limited accumulation in the LG. Systemic CsA has dose-limiting side effects that also limit its ability to block LG inflammation. To overcome the limitations of current CsA formulation, in this dissertation, I present a new strategy to carry CsA by fusing its cognate human receptor, cyclophilin A (CypA), to elastin-like polypeptides (ELPs) using recombinant protein expression. Two generations of CsA under this concept were designed and constructed. Their biophysical properties, in vitro/in vivo efficacy and pharmacokinetic performance are systemically evaluated in this dissertation. To summarize and share my experience obtained from my thesis work, general guidelines for performing and interpreting PK studies of nanomedicines are summarized as the 3 rd chapter. 1 Introduction Sjögren’s syndrome (SS) is a chronic systemic autoimmune disease affecting approximately 0.3% to 0.6% of the total population. This infiltration is associated with the principal clinical hallmarks of SS, persistent dry eyes and dry mouth, which leads to severe corneal damage and compromised oral health. Cyclosporine A (CsA), as a potent immunosuppressant, has been shown to be effective in mitigating modulation of inflammatory responses in autoimmune disorders including rheumatoid arthritis and psoriasis (Colombo & Ammirati, 2011). But its systemic use has been associated with dose-dependent toxicities like nephrotoxicity, hepatotoxicity, neurotoxicity and hypertension. When formulated as ophthalmic emulsion and given as eye drops, known as Restasis, CsA is also prescribed to mitigate SS-mediated dry eye symptom. But topical CsA gets effectively drained from ocular surface through nasolacrimal ducts, allowing very limited absorption at the lacrimal glands. Therefore, there is a need to develop a sustained release formulation of CsA capable of maintaining stable drug concentration within its narrow therapeutic window and allowing extended dosing intervals. This formulation could greatly enhance the utility of CsA in the treatment of SS. In order to do that, we explored the feasibility to reformulate CsA with an emerging biocompatible material, named elastin-like polypeptides (ELPs). Derived from human tropoelastin, ELPs are biocompatible, biodegradable and of low immunogenicity (Domb, Kost, & Wiseman, 1998), which makes them prospective candidates for developing new biopharmaceutics. A characteristic property of ELPs is their thermal responsiveness, with an adjustable transition temperature (Tt) controlled by the choice of guest residue, X, and by the number of repetitive units, n, in their pentameric amino acid repeat sequence of 2 (VPGXG)n. When the temperature rises above the Tt, ELPs phase separate from their highly water-soluble form into coacervates (Despanie, Dhandhukia, Hamm-Alvarez, & MacKay, 2016). They revert to the fully water-soluble state again when the temperature falls below the Tt. In Chapter 1, I described the development of the first generation ELP-based CsA carrier for systemic delivery. Through molecular cloning, we have fused the cytosolic sequence of the human receptor of CsA, cyclophilin A (CypA), to a particular ELP, A192, which has the amino acid sequence of G(VPGAG)192Y. This CypA-A192 (CA192) fusion protein was designed to help solubilize CsA and to function as a drug carrier to improve the CsA safety profile when administered systemically. The results reported herein describes the biophysical properties of this protein-based carrier, as well as its high affinity for CsA, which significantly extended the in vitro release profile of CsA and, more importantly, the systemic circulation time upon parenteral administration. Furthermore, this study demonstrates that CsA bound to CA192 exhibits comparable activity in inhibition of IL-2 release in vitro in an activated Jurkat cell system relative to free CsA through the calcineurin-NFAT signaling pathway. Finally, in a proof-of-concept in vivo study, our results show that CsA bound to CA192 injected subcutaneously into male NOD mice with established disease improves tear flow and reduces systemic toxicity, relative to free CsA. Once we proved the feasibility of this drug delivery concept, to extend the concept of ELP-mediated drug delivery based on CypA-CsA binding, the 2nd generation carriers were designed. Unlike CA192 that has only one binding site, one additional cyclophilin was fused to the C-terminus of ELP backbone to improve the drug loading capacity. 3 Different ELPs, A96 or V96, with the amino acid sequences of G(VPGAG)96Y and G(VPGVG)96Y, respectively, were used as the ELP backbone. These two-headed fusion proteins were named as CAC and CVC. Due to the higher hydrophobicity of the guest residue, CVC exhibits much lower transition temperature, below the physiological temperature. Therefore, when injected subcutaneously, in response to physiological temperature, CVC would undergo phase transition and enclose CsA into a drug depot at the injection site, allowing sustained drug release for more than 14 days. When injected subcutaneously to the region overlying LG for enhanced local accumulation, this sustained-release CsA formulation resolves the pathogenic mechanisms of SS by reducing Th17.1 cell infiltration of the LG, and is accompanied by improvement of other manifestations of SS-associated DED. This continued effort was summarized into Chapter 2. The common rationale to develop drug carriers is to increase the therapeutic index (Papahadjopoulos et al., 1991), making drugs more potent and/or safer, by changing the PK profile of the drug to that of the carrier. Therefore, pharmacokinetic evaluation of both ELP carriers and the cargo drug is fundamental in my thesis work. To summarize and share my experience obtained from my research, general guidelines for performing and interpreting PK studies of nanomedicines are summarized as the Chapter 3. 4 Chapter 1 First generation Elastin-like polypeptides-delivered CsA and its preliminary efficacy against Sjögren’s Syndrome-mediated ocular manifestations. 1.1 Introduction Cyclosporine A (CsA) is a well-known lipophilic cyclic immunosuppressant peptide composed of 11 amino acids which works by blocking T-cell proliferation and inhibiting the release of inflammatory cytokines such as interleukin-2 (IL-2) and interferon gamma (IFN-γ) (Stevenson, Chauhan, & Dana, 2012). Through binding to its cognate receptor, cyclophilin A (CypA), CsA can inhibit the calcium-calmodulin activated phosphatase, calcineurin (Gijtenbeek, Van den Bent, & Vecht, 1999), making it a powerful tool in the available arsenal of immunomodulatory therapies. Mechanistically, the nuclear factor of activated T cells (NFAT), the inducible factor that binds the IL-2 promotor in activated T- cells (Shaw et al., 1988), is dephosphorylated by activated calcineurin, which leads to its nuclear translocation and the induction of NFAT-mediated gene transcription of IL-2. When calcineurin phosphatase activity is inhibited, IL-2 gene expression and secretion are markedly reduced, thus generating the principal therapeutic effect of CsA. In addition to IL-2, other pro-inflammatory cytokines, such as IL-3, IL-4, IL-5, TNF-α and IFN-γ can also be downregulated by CsA (Ambroziak et al., 2016). Due to its immunosuppressive effects, CsA has been widely used to prevent rejection after organ transplantation and in modulation of inflammatory responses in autoimmune disorders including rheumatoid arthritis and psoriasis (Colombo & Ammirati, 2011). However, when administered systemically, CsA can lead to a number of serious adverse drug reactions (ADRs) because of its narrow therapeutic window (Mahalati et al., 5 2001). Below the therapeutic window, CsA cannot effectively inhibit T cell proliferation and release of inflammatory cytokines required for its therapeutic actions, while above the therapeutic window, it may elicit severe side effects. In fact, though CsA has potential through modulation of IL-2 and other inflammatory cascades to treat a variety of autoimmune and inflammatory disorders, its clinical usage has been greatly limited due to the side effects including nephrotoxicity (Bennett & Pulliam, 1983), hepatotoxicity (Kassianides, Nussenblatt, Palestine, Mellow, & Hoofnagle, 1990), neurotoxicity (Gijtenbeek et al., 1999), and hypertension (Bellet et al., 1985). In addition, due to its low solubility (27 µg/mL), CsA is usually formulated with polyoxyethylated castor oil (Cremophor EL ® ) for parenteral administration which can cause anaphylactoid reactions. When administrated topically, CsA has also been broadly used to treat dry eye syndrome (DES), a multifactorial disease of the ocular surface associated with decreased tear production and affecting an estimated 5-30% of the population (Cornec et al., 2015; Janine, 2007), presumably by suppressing ocular surface inflammation. Because of its hydrophobic properties, the only commercially-available form for topical administration of CsA is an oil-in-water emulsion, which has led to poor ocular tolerance, low bioavailability and instability (Gupta & Chauhan, 2011). Our goal in this study was to reformulate CsA for systemic delivery in a way that might reduce its systemic toxicity as well as minimize the frequency of administration while retaining its efficacy. We chose an autoimmune dry eye disease, Sjögren’s syndrome (SS), with systemic manifestations in the tear-producing lacrimal gland (LG) to test our reformulated construct. SS is a chronic autoimmune inflammatory disorder characterized by lymphocytic infiltration of exocrine glands, particularly LG and salivary 6 glands (SG), and affecting more than 4 million Americans (Lemp, 2005). The hallmark clinical symptoms of SS are persistent dry eye and dry mouth, which eventually lead to severe corneal damage and compromised oral health. Disease progression is also associated with the development of constitutional symptoms involving pulmonary, neurological, vascular, and renal systems (Manoussakis & Moutsopoulos, 2000). The LG and ocular surface system collectively represent an ideal disease model for evaluation of CsA efficacy, as SS is associated both with autoimmune-mediated LG and systemic inflammation as well as the reduced tear flow characteristic of aqueous-deficient DES, for which topical CsA is prescribed clinically. The well-established murine model for SS that we have used is the male Non-obese Diabetic (NOD) mouse (Shah et al., 2013), which spontaneously develops autoimmune dacryoadenitis (inflammation of the LG) and ocular surface dryness that recapitulates that seen in human SS (Chiorini, Cihakova, Ouellette, & Caturegli, 2009; da Costa et al., 2006). Although SS is more prevalent in women, utilizing the males instead of females in this murine model is based on their different patterns of disease development. Male NOD mice develop an early, profound lymphocytic infiltration of the LG at 8-12 weeks of age but exhibit little SG inflammation (Schenke- Layland et al., 2008). Female NOD mice develop a profound SG lymphocytic infiltration by 16-20 weeks of age, but lesser LG inflammation (Lindqvist et al., 2005; Robinson et al., 1998; Yamano, Atkinson, Baum, & Fox, 1999). In addition to lymphocytic infiltration in the LG and reduced tear secretion, male NOD mice recapitulate other characteristics of human SS including expression of elevated matrix metalloproteinases (MMPs) in LG and in tears (Aluri et al., 2015), and increased LG and tear levels of pro-inflammatory 7 cytokines such as IL-1α, IL-2, IFN-γ, IL-6, and TNF-α (Fox, Kang, Ando, Abrams, & Pisa, 1994). The novel formulation of CsA developed here utilizes elastin-like polypeptides (ELPs). ELPs, derived from human tropoelastin, consist of pentameric repeats of (Val- Pro-Gly-Xaa-Gly)n where Xaa is the guest residue and n is the length of the repetitive units. ELPs have a unique inverse transition behavior. Below their transition temperature (Tt), they are highly water soluble but once the temperature rises above their Tt, ELPs undergo a phase separation process and self-assemble into different kinds of coacervates which can include particles of different sizes (J. Dhandhukia, Weitzhandler, Wang, & MacKay, 2013). This phase separation is a fully reversible process and can be used to effectively purify ELP-conjugated materials (Shah et al., 2013). Phase behavior can be precisely controlled by adjusting the hydrophobicity of guest residue “Xaa” and the number of pentapeptide repeats “n” (Urry, 1997). Here we report that through molecular cloning, we have fused the cytosolic sequence of the human receptor of CsA, cyclophilin A (CypA), to a particular ELP, A192, which has the amino acid sequence of G(VPGAG)192Y. This CypA-A192 (CA192) fusion protein was designed to help solubilize CsA and to function as a drug carrier to improve the CsA safety profile when administered systemically. The results reported herein describes the biophysical properties of this protein-based carrier, as well as its high affinity for CsA, which significantly extended the in vitro release profile of CsA and, more importantly, the systemic circulation time upon parenteral administration. Furthermore, this study demonstrates that CsA bound to CA192 exhibits comparable activity in inhibition of IL-2 release in vitro in an activated Jurkat cell system relative to free CsA 8 through the calcineurin-NFAT signaling pathway. Finally, in a proof-of-concept in vivo study, our results show that CsA bound to CA192 injected subcutaneously into male NOD mice with established disease improves tear flow and reduces systemic toxicity, relative to free CsA. 1.2 Materials and Methods 1.2.1 Reagents NHS-rhodamine (46406), Hoechst 33342 (R37605), Lysotracker green (L7526), RIPA lysis and extraction buffer (89900), Protease and Phosphatase Inhibitor Cocktail (78440), dialysis cassette (20K MWCO, 3 mL) (66003) and Zeba Desalting Chromatography Cartridges (7K MWCO, 5 mL) (89935) were purchased from Thermo Fisher Scientific (Waltham, MA). Fetal Bovine Serum (FBS) (S11150H) was from Atlanta Biologicals, Inc. (Flowery Branch, GA). Penicillin-Streptomycin (10,000 U/mL) (1647113), advanced RPMI 1640 (1897242) and 1M HEPES (15630080) were purchased from Gibco ® (Carlsbad, CA). Dulbecco's Modified Eagle's medium (DMEM) with L-glutamine (30-2002) was from ATCC ® (Manassas, VA). Human serum albumin (A9511), Poly-D- lysine hydrobromide (P0899), phorbol 12-myristate 13-acetate (PMA) (P8139) and ionomycin (I0634) were from Sigma-Aldrich Corporation (St. Louis, MO). NFAT1 primary antibody (4389) and NFAT2 (8032) primary antibody and anti-rabbit IgG, HRP-linked secondary antibody (7074) were generated by Cell Signaling Technology (Danvers, MA). ProSignal Dura components (20-301) were purchased from Genesee Scientific (San Diego, CA). The Zone-Quick™ Diagnostic Threads were from Oassi Medical Inc. (Glendora, CA). Syringe filters (25 mm, 0.2 µM) (4192) were from Pall Corporation (Port Washington, NY). 9 1.2.2 Mice Male NOD mice were bred at USC vivarium from breeding pairs obtained from Taconic (Hudson, NY). Male BALB/c mice (Stock No: 000651) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animal use was in compliance with protocols approved by the University of Southern California Institutional Animal Care and Use Committee, and experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 1.2.3 ELP biosynthesis and biophysical characterization The encoding sequence of CypA was designed using Escherichia coli (E. coli) biased codons. The custom encoding sequence comprising the full length human CypA sequence was flanked by restriction recognition sites for NdeI and BamHI at the 5’ and 3’ ends to enable the insertion of the CypA sequence into the pET-25b(+) vector. Another BseRI restriction site was placed immediately ahead of the BamHI restriction site, allowing the ligation of the A192 encoding sequence, which was synthesized by recursive directional ligation in a modified pET-25b(+) vector (S. M. Janib et al., 2014). Thus, the custom encoding sequence indicated below was ordered from Integrated DNA Technologies (IDT): 5’- CATATGGTTAACCCGACCGTTTTCTTCGACATCGCTGTTGACGGTGAACCGCTGGG TCGTGTTTCTTTCGAACTGTTCGCTGACAAAGTTCCGAAAACCGCTGAAAACTTCC GTGCTCTGTCTACCGGTGAAAAAGGTTTCGGTTACAAAGGTTCTTGCTTCCACCGT ATCATCCCGGGTTTCATGTGCCAGGGTGGTGACTTCACCCGTCACAACGGTACCG GTGGTAAATCTATCTACGGTGAAAAATTCGAAGACGAAAACTTCATCCTGAAACAC 10 ACCGGTCCGGGTATCCTGTCTATGGCTAACGCTGGTCCGAACACCAACGGTTCTC AGTTCTTCATCTGCACCGCTAAAACCGAATGGCTGGACGGTAAACACGTTGTTTTC GGTAAAGTTAAAGAAGGTATGAACATCGTTGAAGCTATGGAACGTTTCGGTTCTCG TAACGGTAAAACCTCTAAAAAAATCACCATCGCTGACTGCGGTCAGCTGGAAGGTT ACTGATCTCCTCGGATCC-3’ After verifying the correct sequence through DNA sequencing, the resulting plasmid with the fusion protein sequence was first amplified in TOP10 competent cells and then transfected into BLR competent cells for expression. After expression, the CA192 fusion protein was purified by inverse transition cycling (ITC) (Sun, Hsueh, Janib, Hamm-Alvarez, & MacKay, 2011) (Table 1.1). Briefly, following cell lysis by sonication, the phase transition of CA192 can be triggered by heating to 37°C in the presence of 2 M sodium chloride (NaCl), allowing the collection of CA192 coacervates via centrifugation at 4,500 x g (hot spin). The ELP pellet was then resolubilized in cold PBS and subjected to another centrifugation at 4°C, a temperature below the Tt of CA192, at 16,100 x g (cold spin). This constituted one cycle of ITC. More than 98% purity was obtained by 3 rounds of ITC. The protein yield was around 90 mg/L to 120 mg/L. The concentration of CA192 in phosphate-buffered saline (PBS) was determined by measuring the optical density at 280 nm with a one-centimeter light path using a UV- Vis spectrophotometer (DU800, Beckman Coulter Inc., Brea, CA) after diluting CA192 into 6M guanidine hydrochloride to disrupt any aggregates. The molar extinction coefficient, ε, of CA192 and A192 were estimated to be 9,970 (M -1 cm -1 ) and 1,285 (M - 1 cm -1 ), respectively, based on the following equations (Gill & Von Hippel, 1989): ε = 125 nCysteine + 5500 nTryptophan + 1490 nTyrosine Eq. 1.1 11 and CELP = OD280 / ε Eq. 1.2 The molecular weight of purified fusion protein was verified by SDS-PAGE stained with copper chloride (CuCl2). The phase behavior of CA192, along with the parent ELP, A192, was characterized again using UV-Vis spectrophotometry by measuring the optical density at 350 nm, OD350, where neither fusion protein nor A192 contribute significantly to absorption. ELPs at different concentrations (5 µM to 100 µM) were subjected to a controlled temperature gradient from 25 to 75°C at 1°C/min. The transition temperature (Tt) of each ELP is defined as the temperature at which the first derivative of the optical density with respect to the temperature reaches a maximum. 1.2.4 Size exclusion chromatography (SEC) with multi-angle static light scattering (MALS) To resolve CA192 and estimate the molecular weight of different populations of the protein, 100 μL of 25 μM of CA192 was injected onto a Shodex Protein KW-803 (8.0mm I.D. x 300 mm) (Showa Denko America, New York, NY) column preconditioned and equilibrated with PBS. This was followed by application of an isocratic flow of PBS at 0.5 mL/min was applied to elute CA192. Elution was monitored by three in-line detectors: i) UV 210 nm (SYS-LC-1200, Agilent Technologies, Santa Clara, CA); ii) multi-angle static light scattering (DAWN HELEOS, Wyatt Technology, Santa Barbara, CA); and iii) differential refractometer (OPTILAB rEX, Wyatt Technology, Santa Barbara, CA). ASTRA 6 was used for data analysis and molar mass determination. 12 1.2.5 Dynamic light scattering (DLS) The hydrodynamic radius (Rh) of the ELP fusion proteins was measured via dynamic light scattering (DLS) using a DynaPro Plate Reader II from Wyatt Technology (Santa Barbara, CA) and analyzed by software DYNAMICS V7 (Wyatt Technology, Santa Barbara, CA). Before the DLS measurement, solutions were filtered through syringe filters (25 mm, 0.2 µm). The concentration of each solution was then adjusted to 20 µM. 60 µL from each sample was pipetted into three different wells on a 384 well clear bottomed plate and covered by 15 µL mineral oil in each well to avoid solvent evaporation. Centrifugation was performed to remove air bubbles prior to analysis. 1.2.6 Isothermal titration calorimetry (ITC) ITC (MicroCal PEAQ-ITC, Malvern Instruments Ltd, Worcestershire, United Kingdom) was utilized to study the binding affinity between CsA and CA192. CsA and CA192 were solubilized in the same buffer (2.5% v/v DMSO in PBS) to eliminate the interference by background heat released from buffer disequilibrium. Then, the calorimeter cell was filled with 13 µM CsA and the titration syringe was filled with 150 µM CA192. The titration syringe injected 3 µL of CA192 12 times into the calorimeter cell. When binding occurs, the released heat during gradual titration is measured by the sensitive calorimeter. The MicroCal PEAQ ITC analysis software was then used to fit the resulting isotherm into an “one set of sites” binding model to generate the affinity (Kd), stoichiometry (N) and enthalpy of interaction (ΔH). 1.2.7 Drug encapsulation and reverse-phase high performance liquid chromatography (RP-HPLC) analysis 13 CsA was encapsulated into CA192 based on our previously reported two-phase solvent evaporation method (P. Shi et al., 2013). Briefly, an aqueous phase (PBS containing 300 µM CA192) was mixed with an organic phase (90% hexane/10% ethanol containing 1 mM CsA). Under a nitrogen environment with constant stirring, along with the evaporation of organic solvent, CsA was gradually displaced into the aqueous phase where it was solubilized through binding CA192. This process was followed by high-speed centrifugation at 16,100 x g, ultrafiltration through syringe filters (25 mm, 0.2 µM) and dialysis for 4 hr using a dialysis cassette (20K MWCO, 3 mL) against PBS to remove excess insoluble drug and residual solvent. The loading ratio was determined by RP- HPLC using a C4 column (150 × 4.6 mm, particle size 5 µm, YMC CO., LTD.) The mobile phase was composed of water and methanol, each containing 0.1% trifluoroacetic acid (TFA). The sample was eluted at a flow rate of 1 ml/min with a gradient flow of methanol/water (40:60) to methanol/water (95:5) for the first 5 min and then an isocratic flow of methanol/water (95:5) for another 5 min. The eluate was subject to UV detection at 210 nm. CsA dissolved in methanol at different concentrations: 5 to 50 µM were first analyzed with this method to establish a standard curve. 1.2.8 In vitro drug release assay The in vitro disassociation of CsA from CA192 (120 μM, 3 mL) in aqueous solution was characterized by performing sink dialysis against 1.5 L PBS at 4°C or 37°C. PBS was changed every 48 hr. Samples were collected from the dialysis cassette (20K MWCO, 3 mL) from 2 to 196 hr and analyzed by RP-HPLC. Similarly, to study the free drug release profile, CsA dissolved in DMSO was dialyzed against sink conditions of PBS at 4°C or 37°C, and sampled until 10 hr at 4°C or 5 hr at 37°C. 14 1.2.9 Competitive binding assays To simulate the physiological situation where albumin may displace CsA from CA192, human serum albumin was dissolved into PBS solution with 150 µM CA192-CsA to a final concentration of 1 mM, which is approximately the physiological concentration of albumin in human serum. The mixture was incubated at 37 °C and sampled from 8 to 48 hr. The ELP-mediated phase-separation was then exploited to isolate CA192-CsA from the mixture. Briefly, 5M NaCl solution was added to collected samples to reach a final NaCl concentration of 1 M to induce the phase separation at 37°C. The pellet was obtained by centrifugation at 16,100 x g for 10 min and resuspended for RP-HPLC analysis to measure CsA concentration still retained by CA192. CA192-CsA was also tested against mouse plasma collected from 26-week-old male BALB/c mice. 300 µM CA192-CsA in PBS was diluted 1:1 in mouse plasma to achieve a final concentration of 150 µM. Similarly, the mixture was incubated at 37 °C and sampled from 8 to 48 hr. The phase separation of CA192 was induced with 1M NaCl at 37 °C and the ELP was isolated by centrifugation. The pellet following centrifugation was resuspended for RP-HPLC analysis to measure the remaining CsA bound to CA192. 1.2.10 Cellular uptake of ELPs CA192 was labeled with NHS-rhodamine. The labeling protocol was optimized for CA192 to achieve an approximately 100% labeling efficiency. Briefly, 3.5 mL of 200 µM CA192 was mixed with a 3X molar excess NHS-rhodamine at 4°C for 1.5 hr under constant rotation. Unbound, free dye was removed using Zeba Desalting Chromatography Cartridges (7K MWCO, 5 mL). The absorbance of rhodamine-CA192 (Rho-CA192) at 555 nm (OD555, RhoCA192) was measured using a UV-Vis 15 spectrophotometer. The labeling efficiency was calculated based on the equation below, where the εrhodamine is 80,000 (M -1 cm -1 ) at 555 nm and the concentration of CCA192 was measured before labeling to be 200 µM. 𝐿𝑎𝑏𝑒𝑙𝑖𝑛𝑔 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 = !" !!!,#$%&'( )*+ # , $%-./012 % '( )*+ Eq. 1.3 Jurkat cells, Clone E6-1 (TIB-152™, ATCC ® , Manassas, VA) were first cultured in medium composed of Advanced RPMI 1640 + 10% FBS + 10 mM HEPES + 100 units/mL penicillin + 100 μg/mL streptomycin to a density of 3 × 10 5 /mL. Jurkat cells were then incubated on 35 mm glass bottomed dishes (P35G-0-10-C, MatTek Corporation, Ashland, MA) precoated with Poly-D-lysine for 15 min to enable attachment. Cell density was adjusted to 3 x 10 6 /mL to achieve better adhesion and then diluted back to 3 × 10 5 /mL for further treatment. Following attachment, cells were incubated with 10 µM rhodamine- labeled CA192 for 2 hr. Similarly, HeLa cells (CCL-2™, ATCC ® , Manassas, VA) cultured to 50% confluency in Dulbecco's Modified Eagle's medium (DMEM) with L-glutamine with 10% Fetal Bovine Serum (FBS) and 100 units/mL penicillin + 100 μg/mL streptomycin were incubated with 30 µM rhodamine-labeled CA192 for 2hr. After washing three times with warm PBS to remove residual unbound Rho-CA192, cells were incubated with Hoechst (2 drops/mL culture media) and Lysotracker green at a final concentration of 150 nM for 20 min. Finally, cells were imaged by confocal fluorescence microscopy (ZEISS LSM 800 with Airyscan, Carl Zeiss Microscopy GmbH, Germany). 1.2.11 CsA-mediated suppression of IL-2 secretion from Jurkat cells Jurkat cells, Clone E6-1 (TIB-152™, ATCC ® , Manassas, VA) were cultured in medium composed of Advanced RPMI 1640 + 10% FBS + 10 mM HEPES + 100 units/mL 16 penicillin + 100 μg/mL streptomycin to a density of 3×10 5 /ml. Stimulation of Jurkat cells was with Phorbol 12-myristate 13-acetate (PMA) and ionomycin, each dissolved in DMSO and then added to culture medium to a final concentration of 20 ng/mL for PMA and 1 µg/mL for ionomycin. Immediately after the stimulation, cells were subject to treatment with CA192-CsA or free CsA dissolved in 2.5 % v/v DMSO at CsA concentrations from 10 pM to 100 nM for 6 hr at 37°C. IL-2 concentration in culture medium was assessed using an ELISA kit (EH2IL-2, Thermo Fisher Scientific, Waltham, MA) on a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA) at 450 nm. 1.2.12 NFAT signaling using Western blot analysis Jurkat cells were cultured as above in 6-well-plates to a cell density of 3×10 5 /ml. Two wells were used as negative controls (non-treated) and positive controls (stimulation with PMA and ionomycin at doses listed above), respectively. The remainder of the wells were subjected to pre-incubation with CA192-CsA, CsA dissolved in 2.5% v/v DMSO, CA192, or 2.5 % v/v DMSO for 1 hr at 37 °C, followed by stimulation for another 1 hr at 37 °C. Cells were collected via centrifugation, washed three times with cold PBS, and lysed with RIPA lysis and extraction buffer containing Protease and Phosphatase Inhibitor Cocktail. Electrophoretic transfer was conducted with an iBlot 2 Dry Blotting System (Thermo Fisher Scientific, Waltham, MA) following electrophoretic separation using native PAGE or SDS-PAGE. After 1 hr blocking with 5% milk in Tris-buffered saline with 0.1% Tween 20 at room temperature, the membrane was incubated with primary rabbit antibody to NFAT1 (1:1000 dilution) or NFAT2 (1:1000 dilution) overnight at 4°C. After washing three times for 5 min, the membrane was incubated with anti-rabbit IgG, HRP- linked secondary antibody (1:1000 dilution) for 1 hr prior to washing three times for 5 min. 17 Chemiluminescent substrate (ProSignal Dura components) was mixed 1:1 and placed on the blot with a volume of 0.1 mL/cm 2 for 2 min, followed by draining of the excess reagent. The damp blot was imaged using a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Hercules, CA) to capture the chemiluminescent signal. 1.2.13 Pharmacokinetics and subcutaneous bioavailability of CA192 To study the pharmacokinetic profile of the CA192 carrier, Rho-CA192 (200 µM CA192 concentration; 104% labeling efficiency, 150 µL injection volume/35 g BW) was injected into 12-week male BALB/c mice intravenously (IV) or subcutaneously (SC) for a total dose of 30 nanomoles or 857 nanomoles / kg BW. 20 µL of blood was collected by tail nick at time points from 5 min to 72 hr. The collected blood was immediately added to 80 µL of heparinized PBS at a heparin concentration of 1000 U/ml. Red blood cells were removed by centrifugation at 16,100 x g for 10 min and diluted plasma was collected. CA192 concentration in plasma was calculated based on the fluorescence intensity measured by a SpectraMax iD3 Multi-Mode Microplate Reader (Excitation/Emission: 540/580 nm). Both non-compartmental and compartmental methods were applied to analyze the PK profiles of CA192 after IV or SC administration. Non-compartmental analysis was primarily based on the estimation of body exposure to drug after administration, which is reflected by the area under the plasma concentration-time curve (AUC). The AUC was first computed with the trapezoidal method. Thereafter, the area under the first moment curve (AUMC), mean residence time (MRT) and mean absorption time (MAT) were calculated as follows: 𝑀𝑅𝑇 = 𝐴𝑈𝑀𝐶/𝐴𝑈𝐶 Eq. 1.4 18 𝑀𝐴𝑇 = 𝑀𝑅𝑇 &% −𝑀𝑅𝑇 '( Eq. 1.5 Using these estimates, the SC bioavailability, F, the plasma clearance (CL) were estimated as follows: 𝐶𝐿/𝐹 = 𝐷𝑜𝑠𝑒/𝐴𝑈𝐶 Eq. 1.6 𝐹 = 𝐴𝑈𝐶 &% /𝐴𝑈𝐶 '( Eq. 1.7 Similarly, the terminal half-life, T1/2, Terminal, was best-fit to the log-linear decay observed in each individual over the last three time points. Regarding the compartmental model-based analysis, the volume of distribution of the plasma compartment (Vd), the elimination rate constant (kelimination), the transfer rate constant from plasma to tissue (kplasmaàtissue) and the transfer rate constant from tissue back to plasma (ktissueàplasma) were first solved from the two-compartmental IV model. This IV model was then used to construct the four-compartmental SC analysis under the assumption that Vd and kelimination remain constant from IV to SC. This enabled fitting of additional constants in the SC model including an apparent elimination rate constant from interstitial fluid (ISF) (kISF_elimination), which was used to account for the observed bioavailability, F. If either kISF_elimination or kSC_site->ISF were rate limiting (ie. much greater than the other), than the model would reduce to a one-phase absorption; however, a one- phase absorption model was unable to fit the late peak times observed. This suggested that kISF_elimination and kSC_site->ISF are on the same order of magnitude; therefore, the assumption was made that kISF_elimination = kSC_site->ISF, which enabled good fitting to each mouse. From these best-fit curves generated in SAAM II, the peak concentration, Cmax, and peak time, tmax, were extracted from calculated points of each fit after increasing the 19 Minimum Number of Calculation Intervals to 500. For comparison with the noncompartmental CL, the clearance from the compartmental model was solved by the following equation: 𝐶𝐿 = 𝑘 )*+,-./,0- 𝑉 1 Eq. 1.8 1.2.14 Therapeutic evaluation of CA192/CsA using the male NOD mouse model of autoimmune dacryoadenitis in Sjögren's Syndrome The therapeutic study on male NOD mice was initiated when animals reached 14 weeks of age, when SS-like autoimmune dacryoadenitis was fully established (Hunger, Carnaud, Vogt, & Mueller, 1998). 45 mice were divided into 3 different groups receiving one of the three treatments: 1) CA192-CsA; 2) Free CsA (Sandimmune ® ); and 3) CA192 carrier control. For groups 1 and 3, 400 µL/35g BW of CA192 with a concentration of 300 µM with or without CsA loading, respectively, was injected. The same volume of diluted Sandimmune ® was injected into mice in group 2. Mice in groups 1 and 2 were subject to the same CsA concentration of 2.5 mg/kg. Treatments were given SC every other day for 2 weeks. Basal tear production was measured before and after treatments by performing a thread test (Shah et al., 2017). Briefly, under light anesthesia with isoflurane, a ZoneQuick phenol red-embedded thread was applied in both eyes at the canthus of the ocular surface for 10 sec. Basal tear volume was recorded as the length of thread wetting by basal tears in mm. Stimulated tear collection was conducted under full anesthesia as a terminal procedure. After intraperitoneal injection with a mixture of ketamine/xylazine at concentrations of 100 mg/kg and 10 mg/kg, respectively, mice were subjected to a small bilateral incision on the axis between the outer junction of the eyelid and the ear to expose the LG on both sides. Then 3 µL of 50 µM carbachol (CCh) was applied directly onto the 20 LG to stimulate tear secretion, followed by tear collection from both eyes using 2 µL micro- capillary tubes, which were placed at the tear meniscus in the medial canthus for 5 min. This stimulation and collection procedure was repeated two more times and the volume of collected tears was recorded. Serum chemistry was conducted by ANTECH Diagnostics (Fountain Valley, CA). 1.3 Results 1.3.1 Protein purification and characterization After expression, centrifugation and lysis, the CA192 fusion protein was concentrated and purified by repeatedly inducing ELP phase separation(Sun et al., 2011). The molecular weight of purified fusion protein was verified by SDS-PAGE stained with copper chloride. The parent ELP, A192, served as a control. CypA has a molecular weight of 18.0 kDa. Combined with the 73.6 kDa molecular weight of A192, the molecular weight of CA192 was expected to be around 91.6kDa, which is consistent with the shift seen for CA192 on SDS-PAGE (Figure 1.1B). The phase transition temperature (Tt) of CA192 was reduced with respect to A192 (Figure 1.1C); however, both polymers likely remain soluble at physiological temperatures. At 25 µM, the transition temperature of CA192 was 49.3 °C, significantly lower than that of A192 at 60.9 °C. Consistent with our previous finding (W. Wang et al., 2015), the Tt of CA192 was also found to be a function of concentration: 𝑇 / = 𝑏−𝑚log 23 [𝐶 456 ] Eq. 1.9 where the intercept, b, representing the transition temperature at 1 µM, is equal to 53.1 °C, the slope, m, is the decrease in Celsius for a 10-fold increase in concentration, which 21 equals 2.9 °C, and [CELP] represents the fusion protein concentration (Figure 1.1D, Table 1.1). Figure 1. 1: A recombinant elastin-like polypeptide (ELP) fusion protein as a non-covalent drug carrier for cyclosporine A (CsA). A) A gene was constructed encoding the cyclophilin A protein fused to a high molecular weight ELP, A192. This fusion, called CA192, binds specifically and with high affinity to CsA; B) SDS-PAGE of purified CA192 (91.6 kDa) stained with copper chloride demonstrates a molecular weight shift upon fusing CypA to A192 (73.6 kDa); C) Optical density was determined as a function of temperature (1°C/min) for 25 μM CA192, free A192, and a PBS control. The temperature with the maximum positive slope was defined as the phase transition temperature. D) The concentration-temperature phase diagrams of both CA192 and A192 are plotted, showing that both are expected to remain soluble at physiological temperatures. The 95% confidence interval around each best-fit line is indicated with dashed lines. 22 1.3.2 Particle size determination Size exclusion chromatography with multi-angle light scattering (SEC-MALS) was used to study the aggregation status and molecular weight of CA192. As shown in Figure 1.2A, two different fractions were separated by SEC, suggesting heterogeneity of CA192 in solution. The aggregation and oligomeric state of these two fractions was then evaluated by Multi-angle light scattering, together with a UV detector set at 210 nm and a differential refractometer. Surprisingly, the molecular weight of the second fraction was determined to be 181.0 kDa (± 4.1%), accurately doubling the expected molecular weight of CA192 in its monomeric state and indicating the existence of dimerized CA192. Similarly, fraction 1 was demonstrated to be an aggregated form based on its estimated molecular weight of 8.511 x 10 4 kDa (± 2.5%). Then the dimerized form was collected from SEC and subject to a DLS measurement of hydrodynamic radius (Rh). Native CA192 without SEC isolation was used as a control. As shown in Figure 1.2B, and consistent with data acquired from SEC- Table 1. 1: Amino acid sequence and phase behavior of CA192. Label Amino Acid Sequence *M.W. [kDa] **Slope, m [°C/Log10(μM)] Intercept, b [°C] A192 G(VPGAG)192Y 73.6 8.0 ± 1.7 71.8 ± 2.6 CA192 CypA-(VPGAG)192Y 91.6 2.9 ± 2.2 53.1 ± 3.7 CypA amino acid sequence: MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGFMC QGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKTEWLK HVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE *Expected molecular weight based on the open reading frame for the expressed protein. **The ELP transition temperatures were by Eq. 1.9, yielding an intercept, b, at 1 μM, and a slope, m, representing the change in temperature upon a 10-fold change in concentration. Mean ± 95% CI. 23 MALS, without further separation by SEC, DLS could detect two peaks in the native CA192 with Rh of 7.4 ± 0.7 nm (mean ± SD, n=3) and 113.0 ± 59.1 nm (mean ± SD, n=3). The isolated dimerized form, however, demonstrated a good monodispersity with a Rh of 6.9 ± 0.1 nm (mean ± SD, n=3). 24 Figure 1. 2: Expressed CA192 assembles two species in solution that can be isolated by size exclusion chromatography (SEC). A) SEC-MALS was used to investigate the aggregation and oligomeric state of CA192. The molecular weight of these two fractions were determined to be 8.511 x 10 4 kDa (± 2.5%) and 181.0 kDa (± 4.1%), representing a nano-aggregate and a dimeric species, respectively. The black dashed line represents the molecular weight distribution. B) Purified CA192 dimers were isolated from the nano-aggregate population using SEC, which gave a monodisperse hydrodynamic radius of 6.9 ± 0.1 nm (mean ± SD, n=3). 25 1.3.3 Binding affinity of CA192 to CsA Isothermal titration calorimetry (ITC) was utilized to study the thermodynamics between CA192 and CsA. Two different fractions isolated by SEC were analyzed separately. Interestingly, we discovered that only dimerized CA192 maintains CsA binding capacity (Figure 1.3). Thus, from this point forward, only dimerized CA192 was used in our experiments. The dissociation constant (Kd) was determined to be 189 ± 87 nM (mean ± SD, n = 3) at 37 °C, which is slightly higher than that measured for endogenous CypA and CsA of 35.5 nM (Wear & Walkinshaw, 2006), possibly due to the fusion of CypA to the ELP construct and the presence of 2.5% DMSO in solution. Additionally, as expected, the stoichiometry indicated 1.05 ± 0.06 (mean ± SD, n = 3) consistent with the designed architecture, where one CA192 monomer binds to one CsA molecule. 26 Figure 1. 3: Isothermal titration calorimetry confirms that dimeric CA192 maintains high binding affinity for CsA. A) The heat pulse generated by serial injection of CA192 into a sample cell containing CsA ligand with respect to time. B) The titration curve generated by normalizing the released heat for concentration was fitted to a “one set of sites” binding model to generate the affinity (Kd), stoichiometry (N) and enthalpy of interaction (ΔH). They were determined to be 189.0 ± 87.4 nM, 1.05 ± 0.06 and - 108.2 ± 16.8 kJ/mol, respectively. 27 1.3.4 In vitro drug release profile Binding to CA192 significantly altered the drug release profile of CsA in vitro as evaluated by dialysis under sink conditions at 4°C and 37°C. More than 75% of drug was still recovered in the dialysis cassette when stabilized by CA192 after 8 days at 4°C. As shown in Figure 1.4A and 1.4B, the drug release from CA192 fits a one-phase decay model with a half-life of 954 hr (95% CI: 553 to 3219 hr), of roughly 40 days, at 4°C and a half- life of 52 hr (95% CI: 44 to 61 hr) at 37°C. As a comparison, the free CsA release profile follows a two-phase decay with a burst release due to precipitation along with buffer exchange. The terminal half-life during the second slower decay is 6.3 hr (95% CI: 2.7 to 99.0 hr) at 4°C and 1.1 hr (95% CI: 0.8 to 1.6 hr) at 37°C. Figure 1. 4: CA192 exhibits slow disassociation from CsA under sink dialysis in PBS. In comparison with the rapid release of free CsA, CsA release from CA192 follows a one phase decay model with a half-life of A) 954 hr (95% CI: 553 to 3,219 hr) at 4°C or B) 52 hr (95% CI: 44 to 61 hr) at 37°C. Since loss of CsA loaded on CA192 is much slower than loss of Free CsA from the dialysis cassette, the disassociation kinetics from CA192 appear rate-limiting. Thus, the half-life for CA192-CsA reflects the kinetics of disassociation. Error bar represents mean ± SD from n=3. 28 1.3.5 Competitive binding assays Albumin is the most abundant serum protein serving as a carrier for many hydrophobic molecules, which may potentially compete with CA192 to bind CsA during systemic circulation. Here we proposed an in vitro assay where 150 µM CA192-CsA was incubated with albumin at its physiological concentration, 1 mM. As shown in Figure 1.5, over a 48- hr period at 37°C, no significant drug loss from CA192 was observed, consistent with the high binding affinity reported in Figure 1.3. The initial drug loss during the first 8 hr can be explained by incomplete isolation of all of the CA192 from the mixture. Based on the manufacturer’s monograph on Sandimmune ® , instead of albumin, lipoproteins in the plasma, mainly high-(HDL) and low-(LDL) density lipoprotein, predominantly bind to cyclosporine in the plasma. Thus, CA192-CsA was also tested against mouse plasma over a period of 48 hr. Despite an initial drug loss due to incomplete isolation during the hot spin, more than 50% of CsA was maintained as CA192 bound after 48 hr incubation at 37°C (Figure 1.5B). This degree of CsA loss is consistent with the 52 hr half-life of disassociation under sink dialysis at the same temperature (Figure 1.4B). 29 1.3.6 Cell uptake assay Jurkat cells were used to study whether CA192 can be internalized into cells as well to identify its intracellular distribution if internalized. Upon labeling with NHS-rhodamine, the internalization of CA192 was visualized by tracking the fluorescence signal emitted from rhodamine. As shown in Figure 1.6, notable fluorescence accumulation was detected in a punctate pattern observed in cells incubated with CA192 for 2 hr. The intracellular fluorescence was primarily co-localized with lysosomes labeled with Lysotracker Green, suggesting ultimate accumulation in lysosomes after uptake via some mode of endocytosis. Figure 1. 5: Neither physiological albumin nor whole plasma rapidly displaces CsA from CA192. To simulate the physiological situation where purified albumin or lipoproteins may displace CA192 in binding CsA, 150 μM CA192-CsA was incubated with A) 1 mM albumin in PBS or B) heparinized mouse plasma for 2 days at 37°C. CA192 was isolated at different time points by ELP- mediated phase separation. Error bar represents mean ± SD from n=3. 30 1.3.7 In vitro efficacy The in vitro efficacy of CA192-CsA was studied using the Jurkat cell line, an immortalized human T lymphocyte cell line which releases IL-2 in response to stimulation with PMA and ionomycin (Andersson, Nagy, Groth, & Andersson, 1992; Zhu et al., 2010), recapitulating T cell stimulatory responses in vivo. To determine whether the IL-2 release evoked by stimulation was neutralized effectively by CsA either in its free form or bound Figure 1. 6: CA192 is internalized into Jurkat and Hela cells where it co-localizes with low pH compartments. A-C) Jurkat Cells were incubated with Rho-CA192 (10 µM (Jurkat cells) or D-F) Hela cells (30 µM) for 2 hr. Unbound Rho-CA192 was then washed away three times with warmed PBS, and remaining A,D) Rho-CA192 was imaged (red) using confocal laser scanning microscopy. B,E) Lysosomes were stained with lysotracker green. C,F) Nuclei were labeled with Hoechst (blue) in an overlay. Bar represents 20 microns. Areas of strong colocalization is indicated by arrows. 31 to CA192, stimulated cells were incubated with either CA192-CsA or free CsA dissolved in DMSO at serial dilutions indicated for 6 hr. The respective inhibitory effects were quantified through measurement of released IL-2 concentration into the cell culture medium using an ELISA. As shown in Figure 1.7, both CA192-CsA and CsA/DMSO evoked substantial inhibition of IL-2 secretion at sub-nanomolar concentrations. The half maximal inhibitory concentration (IC50) of CA192-CsA was 1.2 ± 0.4 nM (n = 3, mean ± SD), slightly higher than that of CsA in DMSO of 0.5 ± 0.2 nM (n = 3, mean ± SD). To confirm whether this inhibitory effect was achieved through conventional calcineurin and NFAT signaling, we evaluated the effects of CA192-CsA versus free CsA on the phosphorylation state of NFAT1 and NFAT2 in activated Jurkat cells. As shown in Figure 1.8, CA192-CsA, as well as the CsA positive control in DMSO were able to arrest NFATs in their inactive phosphorylated state. The negative controls, CA192 or DMSO, did not reverse the dephosphorylation of NFATs by calcineurin evoked by PMA and ionomycin. The phosphorylated NFATs were differentiated from the dephosphorylated NFATs based on their migration on SDS-PAGE. 32 Figure 1. 7: CA192-CsA exhibits comparable IL-2 inhibition efficacy to free CsA. Upon stimulation, Jurkat cell produce large amounts of IL- 2. CsA can arrest this process by inhibiting calcineurin and the NFAT signaling pathway. The IC50 of CA192-CsA was determined to be 1.2 ± 0.4 nM (mean ± SD from n = 3), slightly higher than free CsA with an IC50 of 0.5 ± 0.2 nM (mean ± SD from n = 3). 33 1.3.8 Pharmacokinetic profile of CA192 The PK profile of CA192 administered intravenously or subcutaneously was investigated in 12-week male BALB/c mice (28.3 ± 1.4 g BW, n=10). The plasma concentration of CA192 was converted from retained fluorescence intensity in the plasma. The plasma concentration of CA192 versus time profile is depicted in Figure 1.9A. Then both non- compartmental and compartmental analyses were utilized to characterize and interpret the PK profile of CA192, with each mouse analyzed individually to obtain statistical reliability. The estimated PK parameters of CA192 are summarized in Table 1.2. Based on non-compartment analysis, the MRT was extended from 7.3 hr to 15.9 hr through Figure 1. 8: CA192-CsA prevents NFAT1 and NFAT2 dephosphorylation. 10 µg of total protein in the supernatant of cell lysate was resolve in each lane by SDS- PAGE. Proteins on gels were transferred to membrane and labeled with anti-NFAT1 or 2 primary rabbit antibodies followed by anti-rabbit IgG, HRP-linked secondary antibody. Jurkat cell pretreatments were with CA192-CsA or with free CsA (dissolved in 2.5% v/v DMSO) at 1 µM CsA concentration, or with vehicle controls, prior to stimulation with 20 ng/mL PMA and 1 µg/mL ionomycin. CA192-CsA and free CsA treatments showed retention of NFAT1 and 2 at their phosphorylated forms with stimulation but neither CA192 vehicle nor DMSO control (2.5%v/v) prevented NFAT dephosphorylation upon stimulation. The cell lysis method used here was not optimized for nuclear protein extraction, which explains why the bands of dephosphorylated NFATs have lower intensity associated with nuclear translocation. The blots shown here are representatives of three independent repeats. 34 switching IV to SC administration, which reflects a mean absorption time (MAT) of 8.6 hr. The bioavailability, F, of SC administration was determined to be 30.9%. The terminal half-life observed was of 29.2, 22.4 hr for IV, SC administration respectively. This long terminal half-life results from the strong biphasic elimination observed as well as the high MW (182 kDa) of dimeric CA192. To better understand the PK for CA192, compartmental models were next developed that fit the observed data, as shown Figure 1.9B, 1.9C. IV CA192 followed a biexponential decay, which was interpreted using a two-compartment pharmacokinetic model. This model assumes the initial distribution of CA192 into an apparent volume of distribution, Vd, from which it distributed to surrounding tissues slowly during eventual elimination from the central plasma compartment. The best estimates for Vd and kelimination from the intravenous two-compartment model were the input into a model for SC administration. When a three-compartment SC model was evaluated for direct absorption from the injection site to the plasma, it was unable to fit the late peak times observed. To accommodate this delay, a four-compartment model was required with a two-phase absorption phase from the subcutaneous injection site to another pool, which may represent the ISF. This second ‘ISF’ pool then drives absorption into the central plasma compartment. In addition, direct elimination from the ‘ISF’ was allowed to account for the observed bioavailability of ~30%, which may reflect degradation of the Rh-CA192 in ISF or elsewhere en route back to the circulatory system. To fit the profiles for each mouse reliably, it was necessary to assume that the absorption rate constants from the SC site to the ISF and from the ISF to the plasma were equal, which reflects that they are on the same order of magnitude. Both of these models are able to accurately fit the observed 35 profiles (Figure. 1.9), and their fit parameters are summarized (Table 1.2). The compartmental model also enables the determination of the peak concentration after SC administration, Cmax = 0.8 µM, which occurred at tmax = 7.1 hrs. Moreover, the clearances derived from noncompartmental, compartmental IV administration of CA192 were 0.49, 0.51 mL/hr respectively, which are in close agreement. 36 Figure 1. 9: Pharmacokinetic profile of CA192 dosed via IV or SC. A) Plasma concentration of CA192 over time following IV or SC administration. Error bars represent mean ± SD from n = 5; B) The compartmental analysis of CA192 administrated via IV injection. A tissue compartment was suggested by its two-phase exponential decay profile; C) The compartmental analysis of CA192 administered via SC injection. This model could be consistent with absorption first into interstitial fluid (ISF) and from there was drained into blood circulation. 37 Table 1. 2: Comparison of pharmacokinetic parameters observed for dimeric CA192 administered by IV and SC administration to mice Parameter (Unit) Route of Administration IV, (n=5) SC, (n=5) CL/F (mL/hr) 0.49 (0.05) 1.66 (0.36) AUC (µM hr) 50.7 (3.8) 15.7 (3.7) AUMC (µM hr 2 ) 371.9 (50.4) 248.9 (60.7) MRT (hr) 7.3 (0.8) 15.9 (1.2) MAT (hr) - 8.6 (1.4) F (%) 100 30.9 (7.3) T1/2, Terminal (hr) 29.2 (15.2) 22.4 (11.7) Model used Two Compartment Four Compartment CL (mL/hr) 0.51 (0.07) *0.51 Vd (mL/g BW) 0.112 (0.013) *0.112 Cmax (µM) 8.6 (1.6) 0.8 (0.3) tmax (hr) 0 7.1 (1.1) kelimination (hr -1 ) 0.16 (0.03) *0.16 ktissueàplasma (hr -1 ) 0.023 (0.019) 0.025 (0.011) kplasmaàtissue (hr -1 ) 0.024 (0.003) 0.072 (0.058) ksiteàISF (hr -1 ) - **0.19 (0.02) kISFàplasma (hr -1 ) - kISF_elimination (hr -1 ) - 0.43 (0.18) AUC, AUMC, MRT, MAT, F, and T1/2, Terminal were calculated using non-compartmental analysis; The remaining parameters are based on compartmental models optimized to fit the data, as shown in Figure 9B and 9C. Values are indicated as the mean (SD). *Compartmental model parameters obtained from the IV analysis were fixed in the SC analysis. **To avoid over-parameterizing the four-compartment SC model both first order absorption rate constants were assumed to equal. 38 1.3.9 Therapeutic effect on the NOD mouse model 2.5 mg/kg of CsA delivered by CA192 was administered SC following the regimen depicted in Figure 1.10A to male NOD mice with established disease aged 14 weeks. Sandimmune (IV), introduced as a free drug positive control, was injected SC at the same dose. CA192 without CsA was used as a negative vehicle control. Since NOD mice have a tendency to develop type 1 diabetes with onset between 4-6 months of age, blood glucose was monitored during the study. Throughout the treatment period, non-fasting blood glucose of every mouse remained below the 250 mg/dL threshold, taken as the onset of diabetes, suggesting that they remained diabetes-free for the duration of the study. Basal tear flow before and after experimental treatments were measured by the thread test and compared. If the basal tear production increased after the treatment, it was interpreted that the mouse benefited from the treatment. Without CsA, the general trend is towards decreased tear production due to progression of inflammation during the study. As shown in Figure 1.10D, most mice treated with Sandimmune or CA192-CsA benefited from treatment, while approximately half of the mice in the control group with CA192 alone showed no benefit. In addition to basal tear production, carbachol- stimulated tear production was compared among groups. Results in Figure 1.10C clearly indicate that Sandimmune and CA192-CsA treated groups exhibited higher stimulated tear production relative to the CA192-treated control group. Notably, CA192-CsA treated mice showed a significantly higher tear production than CA192-treated mice. CA192- CsA did not significantly increase stimulated tear production relative to Sandimmune, which was likely because Sandimmune showed a trend to an increased production that did not rise to statistical significance. To provide insight into the expected plasma levels 39 for CA192 during this study, the optimal compartmental model for SC CA192 (Table 1.2), was then used to predict the plasma concentrations expected during this entire course of treatment (Figure 1.10D). In addition to predicting the CA192 levels at assay days 15 and 16, this prediction shows that a low level of accumulation is expected during the first four days of therapy, after which levels of the carrier follow a steady state profile. The most common clinical side effects of CsA, when administered systemically, are nephrotoxicity and hepatotoxicity(Myers, Ross, Newton, Luetscher, & Perlroth, 1984). Thus, we used blood urea nitrogen (BUN) as a nephrotoxicity biomarker to assess renal function. Interestingly, serum chemistry demonstrated that BUN in the Sandimmune group was approximately 50% higher than in the CA192-CsA and CA192 groups (Figure 1.11A). Similarly, serum alanine transaminase (ALT) was used to assess hepatotoxicity. However, no significant difference among groups was observed (Figure 1.11B). 40 Figure 1. 10: Subcutaneous administration of CA192-CsA increased basal and stimulated tear production in a mouse model of SS. A) The schedule of dosing and acquisition of blood glucose and tear production measurements; B) Basal tear production measure by phenol-red threads suggested that most mice treated with Sandimmune or CA192-CsA benefited from the treatment: only the mean ± 95% CI of CA192 group includes 0; C) On the day of euthanasia, tears were collected after stimulating the LG topically with carbachol as described in the methods. CA192-CsA treated mice exhibited a significant increase in tear volume relative to CA192 control (P = 0.014). Error bars here represent mean ± SD from n=15; D) Using the compartmental model for SC administration (Table 2), the expected concentration profile for CA192 over the duration of the two-week study was estimated. 41 1.4 Discussion SS is a common chronic autoimmune disorder affecting more than 4 million Americans. Persistent DES is one of the clinical hallmarks of SS and can eventually lead to severe corneal damage. One of the few prescribed treatments available for SS- associate DES is an ophthalmic emulsion containing 0.05% CsA (RESTASIS ® ), which is intended to increase tear production as well as manage ocular surface inflammation. However, RESTASIS ® showed disappointingly inadequate clinical efficacy in inflammation-related DES in clinical trials where only 15% of RESTASIS ® treated patients exhibited statistically significant increases in a Schirmer’s wetting test. More importantly, it did not restore LG tear production in SS patients (Dastjerdi, Hamrah, & Dana, 2009; Figure 1. 11: CA192 delivery suppressed CsA nephrotoxicity relative to free CsA. A) Blood urea nitrogen (BUN) is a biomarker of nephrotoxicity. Serum chemistry demonstrated that Sandimmune treated mice had significantly higher BUN than CA192-CsA or CA192 treated mice, indicating higher nephrotoxicity. B) Alanine aminotransferase (ALT), an indicator for hepatotoxicity, was also inspected but no differences in values were observed between groups. Three animals from each group were subject to serum chemistry testing. Error bars represent mean ± SD from n = 3. 42 Hyon, Lee, & Yun, 2007; Zhou & Wei, 2014). For treatment of DES originating with an aqueous tear production deficiency like SS that is principally due to LG deficiency, this is a critical limitation. This inability of CsA to affect the LG may be because the nasolacrimal ducts efficiently drain topically-applied drugs from the ocular surface, allowing only limited systemic absorption at the LG and other sites of inflammation in SS. Topical administration may thus similarly limit other new treatments intended for dry eye, such as the LFA-1 antagonist, lifitegrast (Xiidra TM ) and emerging techniques, including nanowafers or microgels, from being fully effective in SS-associated dry eye (Keating et al., 2006; Shimaoka, Salas, Yang, Weitz-Schmidt, & Springer, 2003). In contrast, the new CsA delivery platform reported here clearly has the ability to restore stimulated tear production from the LG in a mouse model of SS, which topical CsA was unable to clinically achieve. While future studies will investigate in more detail the additional potential therapeutic systemic benefits that this new SC CsA formulation has on exocrine gland inflammation and development of serum autoantibodies, as well as its effects on systemic inflammatory pathways, our proof-of-principle study shows that CA192 shows significant promise in mitigating the efficacy issues associated with use of CsA in SS-associated DES. It does so while reducing dose-limiting toxicities that have been associated with CsA administration for other autoimmune and transplant conditions. As discussed above, topical administration of CsA, despite being noninvasive and accessible, has limited systemic absorption which limits its ability to address many of the underlying mechanisms of SS and other autoimmune diseases. Therefore, several liposome or phospholipid micelle-based CsA carriers for systemic administration have been investigated as replacements for Cremophor EL ® to overcome the poor water 43 solubility and dose-limiting systemic toxicity of CsA, where reduced nephrotoxicity and improved drug disposition in vivo were observed (Aliabadi, Mahmud, Sharifabadi, & Lavasanifar, 2005; Smeesters et al., 1988). However, recognized as foreign substances, liposomes or phospholipid micelles encounter multiple defense systems including reticuloendothelial system (RES), opsonization and immunogenicity (Sercombe et al., 2015). We report here our novel protein-based CsA carrier, CA192, which has the following advantageous properties. First, distinct from conventional liposomes or phospholipid micelles, which are known to be both effective and mildly immunogenic (Forssen & Willis, 1998; Szebeni & Barenholz, 2009), CA192 may be immunologically acceptable and fully biodegradable as both moieties of this fusion protein are initially derived from human self proteins. Second, the hydrodynamic radius of dimeric CA192 was adjusted to approximately 7 nm to allow a favorable SC absorption and biodistribution profile with a particle size exceeding the renal filtration cutoff to permit extended circulation. Third, the high binding affinity of CA192 for CsA (Figure. 1.3) greatly enhances drug solubility and provides a very long duration, one-phase drug release with minimal burst release (Figures. 1.4, 1.5), an improvement not possible with the current liposomal or micelle-based CsA carriers. Finally, since we have shown that CsA can be well maintained in its CA192-bound state without being sequestered by albumin or lipoproteins in the plasma, less drug-drug interaction may be expected when co- administered with other drugs that intensively bind to albumin or lipoproteins, such as methotrexate (R Storb et al., 1986; Rainer Storb et al., 1989) or docetaxel (Malingré et al., 2001; Nakahara et al., 2003), respectively. This strategy may make possible 44 modification with targeting peptides to further improve the enrichment in inflamed tissue while reducing the off-target toxicity of CsA. Regarding the feasibility of advancement from the laboratory scale to a clinical setting, the critical problem we will need to address in the future is to increase the drug loading capacity from the current 0.6% by mass to approximately 5% by mass, comparable to most drug delivery platforms. Although the high yield and ease of purification of ELP-based recombinant fusion proteins makes it possible to scale up from an animal study to clinical studies, where a substantially higher dose is required, subcutaneous injection may need to be replaced by intravenous or intraperitoneal injection. These routes of infusion permit safe administration of higher volumes; however, the high solubility and low burst release of this formulation may also promote local administration, such as intra-lacrimal gland or subconjunctival injection. When CA192 at a higher loading capacity is administered to humans, additional toxicity of CsA may emerge. Our current data demonstrates the efficacy of CA192 to curtail nephrotoxicity, which may extend to related CypA-ELP fusions with higher loading capacity, receptor- mediated targeting ligands, or phase separation at physiological temperatures. Not only being advantageous over traditional drug carriers, the concept of utilizing the cognate receptor small molecule drug conjugated to ELPs through molecular cloning as a drug carrier, reported herein and previously by our lab (J. P. Dhandhukia et al., 2017; Shah et al., 2013; Pu Shi et al., 2013), is potentially superior to other ELP-based small molecule carriers as well. Functional CA192 fusion protein can be directly synthesized and easily purified by exploiting the ELP phase behavior from E. coli with a high yield of 90 -120 mg/L, while other ELP-mediated delivery of small molecules require chemical 45 conjugation for drug attachment (Bhattacharyya et al., 2015; Bidwell III, Fokt, Priebe, & Raucher, 2007; J. A. MacKay et al., 2009), introducing chemical variability and polydispersity, thus, necessitating further chromatographic purification. Another question that we will address in future studies utilizing this carrier is the role of of IL-2 in SS. On one hand, intralesional T cells were demonstrated to predominantly express Th1 cytokines including IL-2 and IFN-γ in SS patients (Boumba, Skopouli, & Moutsopoulos, 1995; Roescher, Tak, & Illei, 2009). Over 40-fold more IL-2 is demonstrated to be produced by CD4+ T cells in SG of SS patients as well (Fox et al., 1994). Thus, an inhibitor effect of CsA on IL-2 should have specific therapeutic potential in the treatment of T cell exocrine gland infiltration in SS. On the other hand, emerging studies have begun to establish a protective role of IL-2 in some autoimmune diseases (Ye, Brand, & Zheng, 2018) due to the key role IL-2 plays in the homeostasis and activation of regulatory T cells (Tregs) (Boyman & Sprent, 2012). However, since Tregs are 7 to 10 times more sensitive to IL-2 than natural killer (NK) cells and other IL-2 responsive CD4+ helper T cells (Tang, 2015), IL-2 levels needs to be constrained to an “ultra-low-dose” range where Tregs can still reliably expand but which has relatively less impact on other IL-2–responsive cells. Therefore, identifying an optimal CsA dose for the specific autoimmune disease of interest, whether it is SS or another, will be critical in future studies. In addition to IL-2 downregulation, the therapeutic efficacy of CsA on SS may be highlighted through its potential inhibitory effects on IL-17 as well (Roescher, Tak, & Illei, 2010; Zhang, Zhang, Yang, & Wu, 2008), whose contribution in immunopathogenesis in various autoimmune disorders, including SS (Espinosa et al., 2009; Katsifis, Rekka, Moutsopoulos, Pillemer, & Wahl, 2009; Nguyen, Hu, Li, Stewart, & 46 Peck, 2008b; Sakai, Sugawara, Kuroishi, Sasano, & Sugawara, 2008), has been newly suggested by mounting evidence. 1.5 Conclusions Here we demonstrate for the first time that the prolyl isomerase protein known as CypA, can be bioengineered into a drug carrier, CA192, for the potent immunosuppressant CsA. Unlike traditional drug encapsulation strategies, this innovative strategy is surfactant-free, does not require the breakage of a covalent linkage, and is instead based on high specificity binding between a drug and its cognate receptor protein. Since the MW of CypA is below the renal filtration cutoff, it was fused to a humanized elastin-like polypeptide to increase its MW by ~80 kDa; furthermore, this fusion assembles a stable, functional, and dimeric species. When bound to CsA this carrier retains drug for extended durations, traffics to low pH compartments in cells, inhibits the NFAT/Calcineurin/IL-2 pathway, enhances the mean residence time following subcutaneous administration, reduces renal drug toxicity, and increases tear production in a non-obese diabetic mouse model of SS. 47 Chapter 2 Sustained release 2nd generation carriers for Cyclosporine A reduce Th17 mediated autoimmunity in murine model of Sjögren’s syndrome 2.1 Introduction Sjögren’s syndrome (SS) is a chronic systemic autoimmune disease affecting approximately 0.3% to 0.6% of the total population. SS is prevalent in 4 million individuals in the United States alone, with the highest female-to-male ratio of 9:1 of any known autoimmune diseases (Delaleu, Jonsson, Appel, & Jonsson, 2008). SS is characterized by lymphocytic infiltration of the exocrine glands, specifically lacrimal gland (LG) and salivary gland (SG) (Nguyen & Peck, 2009; Youinou & Pers, 2011). This infiltration is associated with the principal clinical hallmarks of SS, persistent dry eyes and dry mouth, which leads to severe corneal damage and compromised oral health. SS patients may also experience systemic manifestations including interstitial nephritis (Tu, SHEARN, LEE, & HOPPER JR, 1968), liver disease (Skopouli, Barbatis, & Moutsopoulos, 1994) and obstructive bronchitis (Papiris et al., 1999) in its early stage. Continued development of the disease may lead to B cell lymphoma, resulting in serious morbidity or death (KASSAN et al., 1978). SS-mediated dry eye disease (DED) manifestations and the underlying LG inflammation are the focus of this study. Although B cell dysregulation in SS has been implicated in more severe manifestations in later courses of the disease, initial glandular tissue infiltration is dominated by T lymphocytes, of which 70% are CD4+ helper T (Th) cells (Christodoulou, Kapsogeorgou, & Moutsopoulos, 2010). Historically, Th cells have been categorized into two main subsets, Th1 and Th2, based on their distinct pattern of cytokine secretion (Gor, 48 Rose, & Greenspan, 2003). Th1 cells are characterized by the secretion of IFN-γ and IL- 2, but not IL-4 or IL-5. In contrast, Th2 cells are characterized by the secretion of IL-4, IL- 5 and IL-13, but not IFN-γ or IL-2. This Th1 or Th2 fate is regulated by transcription factors T-bet or GATA3, respectively (Kanhere et al., 2012). The pathogenesis of SS has been considered Th1-mediated, a premise supported by high levels of Th1 cytokines in SG (Wakamatsu et al., 2006) and serum (Hooks et al., 1979) of SS patients. Previous studies have suggested that the balance between Th1/Th2 favors Th1 in the SG of SS patients (Mitsias et al., 2002). The Th1/Th2 ratio, as represented by IFN-γ/IL-4 and TNF-α/IL-4 ratios in saliva, is significantly higher in primary SS patients than in non-SS DED patients (Kang, Lee, Hyon, Yun, & Song, 2011). More recently, the discovery of a distinct subset of helper T cells, which is characterized by increased secretion of IL-17A, has led to a re-examination of infiltrating helper T cells in SS (Singh & Cohen, 2012). This new subset has been named Th17 based on this signature cytokine. As a proinflammatory cytokine, IL-17A overexpression is implicated in the pathogenesis of many autoimmune diseases including multiple sclerosis (Komiyama et al., 2006; Luchtman, Ellwardt, Larochelle, & Zipp, 2014), rheumatoid arthritis (Chabaud et al., 1999; Van Den Berg & Miossec, 2009), inflammatory bowel disease (Fujino et al., 2003), psoriasis (Martin et al., 2013; Raychaudhuri, 2013), systemic lupus erythematosus (Crispín & Tsokos, 2010) and others. In pSS patients, abnormal expression of IL-17A was observed in SG lesions as well as in plasma (Katsifis et al., 2009; Nguyen, Hu, Li, Stewart, & Peck, 2008a). When immunized with SG protein as an autoantigen to induce experimental SS, IL-17A knockout (KO) mice were resistant to induction of SS-associated changes in the SG. Moreover, infusion with IL-17A- 49 expressing Th17 cells expedited the onset of autoimmune sialoadenitis in these IL-17A KO mice, causing markedly reduced salivary secretion and profound SG inflammation (Lin et al., 2015). Notably, IL-17A production has also been implicated in SS development in CD25 knockout mice (De Paiva et al., 2010) and in a non-obese diabetic (NOD)-derived SS disease model (Coursey et al., 2017). Initially, T helper cell type 17 (Th17) cells were reported as the only IL-17A secreting lineage in CD4+ T cells (Harrington et al., 2005; H. Park et al., 2005a, 2005b). Later, IL- 17A-producing CD4+ T cells were recovered from synovial fluid of patients with juvenile idiopathic arthritis and analyzed for their cytokine and surface marker expression. Relative to Th17 cells, these synovial IL-17A-producing cells exhibited uniform Chemokine Receptor 6 (CCR6) expression but distinct CCR4 expression. Some of these cells with an intermediate phenotype between Th1 and Th17 expressed both IL-17A and IFN-γ (Nistala et al., 2008). These IFN-γ-expressing Th17 cells, or Th17.1 (also called Th17/1, Th1Th17 or Th1/Th17) were also identified in the gut of patients with Crohn's disease (Annunziato et al., 2007) and in the central nervous system during experimental autoimmune encephalomyelitis (Abromson-Leeman, Bronson, & Dorf, 2009; Ivanov et al., 2006). The majority of IL-17A-secreting cells in the joints of arthritis patients were also found to express cytokines characteristic of both Th17 and Th1 cells (Nistala et al., 2010). TGF-β may be a key regulator responsible for this developmental plasticity of Th17 cells. Th17 cells require TGF-β for sustained expression of IL-17F and IL-17A, two prominent members of the IL-17 family. The expression of IFN-γ is enhanced in the absence of TGF- β, leading to the Th17.1 phenotype (Y. K. Lee et al., 2009). The phenotypic conversion 50 from Th17 to Th17.1 cells has been demonstrated both in vitro (Nistala et al., 2010) and in vivo (Bending et al., 2009). Cyclosporine A (CsA) exerts pharmacological activity upon binding to its cytoplasmic cognate receptor, cyclophilin. This complex interferes with the phosphatase activity of calcineurin, impeding the dephosphorylation of nuclear factor of activated T cells (NFAT). This event interrupts proinflammatory cytokine release, in particular of IL-2, and reduces lymphocyte proliferation (Matsuda & Koyasu, 2000). Although Th1 cells are often thought to be its primary target, CsA is reported to inhibit IL-17A production (Chi et al., 2010; Cho et al., 2007; Holan et al., 2011) and to attenuate Th17 cells (Schewitz-Bowers et al., 2015), likely via inhibition of T cell activation (Zhang et al., 2008), supporting its potential use to modulate Th17 and related cells and reduce local IL-17 treatment for SS-associated DED. This possibility is explored in this study. However, a necessary additional step to test the potential of CsA to target LG-specific Th17 and related cells is the development of a formulation that allows CsA accumulation in the LG. Derived from human tropoelastin, elastin-like polypeptides (ELPs) are biocompatible, biodegradable and of low immunogenicity (Domb et al., 1998), which makes them prospective candidates for developing new biopharmaceutics. A characteristic property of ELPs is their thermal responsiveness, with an adjustable transition temperature (Tt) controlled by the choice of guest residue, X, and by the number of repetitive units, n, in their pentameric amino acid repeat sequence of (VPGXG)n. When the temperature rises above the Tt, ELPs phase separate from their highly water-soluble form into coacervates (Despanie et al., 2016). They revert to the fully water-soluble state again when the temperature falls below the Tt. Previously, we reported the development of a construct for 51 systemic delivery of CsA with ELPs (H. Guo et al., 2018). Cyclophilin A (CypA), the cytosolic receptor of CsA, was fused to the N-terminus of an ELP called A192 with the amino acid sequence of G(VPGAG)192Y. The resultant fusion protein, CA192, maintained nanomolar binding affinity to CsA. CA192-CsA exhibited a strong pharmacological efficacy both in vitro by inhibiting IL-2 secretion from activated Jurkat cells and in vivo by increasing stimulated tear production in NOD mice when given subcutaneously (H. Guo et al., 2018). To extend the concept of ELP-mediated drug delivery based on CypA-CsA binding, the current report makes two significant enhancements: 1) improved drug loading capacity; and 2) sustained drug release through phase separation following supra-LG injection in response to physiological temperature. The male NOD mouse is a well-established murine model of autoimmune dry eye disease (DED). This model recapitulates the lacrimal component of human SS by spontaneously developing a progressive autoimmune dacryoadenitis (inflammation of LG) at 8-12 weeks in age, which parallels reduction in tear flow and production of tear proteins characteristic of SS-associated DED (Chiorini et al., 2009; da Costa et al., 2006; Ju et al., 2018; Lindqvist et al., 2005; Yamano et al., 1999). In the current study, using this established model of SS-associated DED, we have identified IL-17A as a proinflammatory cytokine that is markedly and selectively increased in the LG in parallel with disease development. We further show that sustained release of CsA via the supra-LG injection of a novel ELP depot reduces Th17.1 cell infiltration of the LG and is accompanied by improvement of other manifestations of SS-associated DED. 52 2.2 Materials and Methods 2.2.1 Murine model Both NOD/ShiLtJ mice (Stock No: 001976) and BALB/cJ mice (Stock No: 000651) were purchased from The Jackson Laboratory (Bar Harbor, ME). Animal use was in compliance with protocols approved by the University of Southern California Institutional Animal Care and Use Committee an in accordance with the Guide for the Care and Use of Laboratory Animals 8 th edition (Council, 2011). 2.2.2 Lymphocyte isolation from LG, spleen and lymph nodes. This isolation method was revised from methods in a previous report (Z. Guo et al., 2000). Sterile filtered solutions (0.2 µm) were prepared as follows: 1) s-Ham’s, which is Ham’s F12 medium (11765054, Gibco, Grand Island, NY), supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL) (15070063, Gibco, Grand Island, NY), L-glutamine (2 mM) (25030081, Gibco, Grand Island, NY), n-butyric acid (2 mM) (B2503, Sigma- Aldrich, St. Louis, MO), linoleic acid (0.3 µM) (L1376, Sigma-Aldrich, St. Louis, MO), soybean trypsin inhibitor (50 µg/mL) (T9003, Sigma-Aldrich, St. Louis, MO), bovine serum albumin (BSA) (5 mg/mL) (A3912, Sigma-Aldrich, St. Louis, MO), and 2-[4-(2- hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (10 mM) (11344041, Gibco, Grand Island, NY) adjusted to pH 7.6 with 1 M NaOH (221465, Sigma-Aldrich, St. Louis, MO); 2) CHD-Ham’s, which is s-Ham’s further supplemented with 350 U/mL collagenase (17100017, Gibco, Grand Island, NY), 300 U/mL hyaluronidase (LS02592, Worthington Biochemical Corp, Lakewood, NJ), and 40 kU/mL DNase (07469, STEMCELL Technology, Cambridge, MA); 3) s-Hank’s, which is Hank’s balanced salt powder (9.5g/L) (H4891, Sigma-Aldrich, St. Louis, MO), Ethylenediaminetetraacetic acid (EDTA) (2 mM) 53 (E5124, Sigma-Aldrich, St. Louis, MO) and HEPES (10 mM) adjusted to pH 7.6 with 1 M NaOH in a volume of 1 L; 4) and negative selection buffer, which is 1X Dulbecco’s Phosphate Buffered Saline (PBS) (14190144, Gibco, Grand Island, NY), supplemented with 0.5% (w/v) BSA and 2 mM EDTA. To isolate lymphocytes from tissue samples, freshly isolated LGs were minced into 1 mm 3 pieces and sequentially incubated in s- Hank’s and CHD-Ham’s in a shaking water bath set at 37°C for 10 min each. 3mL of each solution was used to digest a pair of LGs from one mouse. CHD-Ham’s incubation was repeated once to allow adequate enzymatic digestion. The supernatant was collected after every incubation. The pooled supernatant was filtered through a 10 µm cell strainer (43-50010-03, pluriSelect, El Cajon, CA) to enrich lymphocytes in the filtrate. After washing twice in PBS, enriched lymphocytes were resuspended either in negative selection buffer (480017, BioLegend, San Diego, CA) at a density of 10 8 cells/mL for negative selection detailed in section 2.3 or in PBS at a density of 10 7 cells/mL for flow cytometry analysis detailed in section 2.13. For preparation of lymphocytes from lymph node or spleen for negative selection, freshly collected spleen or lymph nodes were placed in a 70 µm cell strainer pre-wetted with PBS on top of 50 mL centrifuge tube. The thumb side of a 10 ml syringe plunger was then used to smash the spleen and lymph nodes, while constantly adding up to 5 mL PBS. The isolated cells were washed twice with PBS and resuspended in negative selection buffer at a density of 10 8 cell/mL. 2.2.3 Negative selection of CD4+ T cells. CD4+ T cells were negatively selected from LG-derived lymphocyte and lymphoid tissues, such as spleen and lymph nodes, using the Mouse CD4+ T Cell Isolation Kit 54 (480033, BioLegend Inc, San Diego, CA). Lymphocytes isolated as described above were first adjusted to £10 8 cells/mL in negative selection buffer. A biotin-antibody cocktail composed of biotin-labeled anti-CD8a, CD11b, CD11c, CD19, CD24, CD45R/B220, CD49b, CD105, I-A/I-E (MHC II), TER-119/Erythroid, and TCR-γδ was diluted 1:10 into cell suspension. After 15 min incubation on ice, an equal volume of freshly vortexed streptavidin magnetic nanobeads was added into the cell suspension, followed by another 15 min incubation on ice. The cell suspension was then transferred to 12 x 75 mm round bottom polystyrene tubes, in which the total volume of cell suspension was increased to 2.5 mL with negative selection buffer. The tube was placed in the magnet (MAG-4902-10, ThermoFisher Scientific Inc., Waltham, MA) for 5 min for magnetic separation, prior to collection of fluid containing unbound cells. This process was repeated with another 2.5 mL of negative selection buffer. The unbound fractions, which are enriched CD4+ T cells, were pooled. 2.2.4 mRNA purification and quantitative real-time PCR mRNA was extracted from negatively isolated CD4+ T cells using the RNeasy Mini kit (74104, Qiagen, Germantown, MD), followed by reverse transcription using TaqMan Reverse transcription Reagents (N8080234, ThermoFisher Scientific Inc., Waltham, MA). Gene expression levels were measured using TaqMan gene expression assays on a QuantStudio 6K Flex Real-time PCR system (ThermoFisher Scientific Inc., Waltham, MA) with the following primers: IL-17A (Mm00439618_m1), IL-2 (Mm00434256_m1), aquaporin 5 (Mm00437578_m1) and GAPDH (Mm99999915_g1). GAPDH was used as an endogenous control. The following equations were used to calculate relative quantification (RQ): 55 ∆𝐶 / = 𝐶 / (𝑡𝑎𝑟𝑔𝑒𝑡 𝑔𝑒𝑛𝑒)−𝐶 / (𝐺𝐴𝑃𝐷𝐻) Eq.2.1 ∆∆𝐶 / = ∆𝐶 / (𝑒𝑥𝑝𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑔𝑟𝑜𝑢𝑝)−∆𝐶 / (𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝) Eq.2.2 𝑅𝑄(𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑔𝑟𝑜𝑢𝑝 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝 ⁄ ) = 2 7∆∆% 3 Eq.2.3 A serial of nonparametric Kruskal-Wallis tests was conducted to capture significant difference in gene expression levels in CD4+ T cells isolated from the same tissue but different strains of mice and from different tissues in the same group of mice. Dunn’s multiple comparison test was then used for multiple comparison. 2.2.5 In vitro differentiation of Th17-like cells Tissue culture-treated plates were first coated with 5 µg/mL anti-mouse CD3ε antibody (100314, BioLegend, San Diego, CA), at 37°C for 4 hr. Lymphocytes were harvested from mouse spleen through a 70 µm cell strainer as described above and resuspended in PBS. Then, these CD4+ T-cells were negatively isolated from splenocytes as described above. The isolated CD4+ T cells were resuspended into culture media (RPMI 1640 (11875093, ThermoFisher Scientific Inc., Waltham, MA) + 10% FBS (26140079, ThermoFisher Scientific Inc., Waltham, MA) + 10 mM HEPES + 1X Antibiotic-Antimycotic (15240062, ThermoFisher Scientific Inc., Waltham, MA) supplemented with 5 μg/ml anti-mouse CD28 antibody (102112, BioLegend, San Diego, CA), 50 ng/ml recombinant mouse IL-6 (575704, BioLegend, San Diego, CA), 5 ng/ml recombinant human TGF-β1 (580702, BioLegend, San Diego, CA), 10 ng/mL recombinant mouse IL-23 (589002, BioLegend, San Diego, CA), 10 µg/mL anti-mouse IL-4 antibody (504108, BioLegend, San Diego, CA), and 10 µg/mL anti-mouse IFN-γ antibody (505812, BioLegend, San Diego, CA) to a cell density of 0.8 - 1 x 10 6 /mL and incubated at 37°C with 5% CO2 for 2 days. Fresh medium with the same supplements 56 was added into each well to increase the initial volume by 50% and incubation was continued for another 2 days. 2.2.6 Biosynthesis and biophysical characterization of CAC and CVC A96 and V96 were previously synthesized by recursive directional ligation in a modified pET-25b(+) vector (McDaniel, MacKay, Quiroz, & Chilkoti, 2010). E. coli biased codon-modified encoding sequence of CypA were flanked by restriction recognition sites of NdeI and BamHI at the 5’ and 3’ termini, allowing its insertion into the pET-25b(+) vector. A BseRI restriction site placed ahead of a BamHI restriction site to allow further ligation with the ELP encoding sequence. Both CypA-pET-25b(+) vector and A96 or V96- modified pET-25b(+) vectors were digested with BseRI and BssHII to allow CypA insertion to the 5’ end of the ELP encoding sequence. CypA was slightly modified to enable C- terminal ligation. A glycine-encoding codon, GGT, was inserted into the 5’ terminus of the CypA encoding sequence. The BseRI restriction site at the 3’ terminus was also removed. This modified C-term CypA encoding sequence was first inserted to the modified pET- 25b(+) vector using NdeI and BamHI, as described above. To finish the construction of CAC and CVC, the CypA-A96/V96-modified pET-25b(+) vectors and the C-term CypA- modified pET-25b(+) vector were digested with BssHII and AcuI, and BseRI and BssHII, respectively. The custom coding sequence of CypA and C-term CypA indicated below were ordered from Integrated DNA Technologies Inc. (IDT) (Coralville, IA): CypA encoding sequence: 5’-CATATGGTTAACCCGACCGTTTTCTTCGACATCGCTGTTGACGGTGAACC GCTGGGTCGTGTTTCTTTCGAACTGTTCGCTGACAAAGTTCCGAAAACCGCT GAAAACTTCCGTGCTCTGTCTACCGGTGAAAAAGGTTTCGGTTACAAAGGTT 57 CTTGCTTCCACCGTATCATCCCGGGTTTCATGTGCCAGGGTGGTGACTTCA CCCGTCACAACGGTACCGGTGGTAAATCTATCTACGGTGAAAAATTCGAAG ACGAAAACTTCATCCTGAAACACACCGGTCCGGGTATCCTGTCTATGGCTAA CGCTGGTCCGAACACCAACGGTTCTCAGTTCTTCATCTGCACCGCTAAAAC CGAATGGCTGGACGGTAAACACGTTGTTTTCGGTAAAGTTAAAGAAGGTAT GAACATCGTTGAAGCTATGGAACGTTTCGGTTCTCGTAACGGTAAAACCTCT AAAAAAATCACCATCGCTGACTGCGGTCAGCTGGAAGGTTACTGATCTCCT CGGATCC-3’ C-term CypA encoding sequence: 5’-CATATGGGTATGGTTAACCCGACCGTTTTCTTCGACATCGCTGTTGACGG TGAACCGCTGGGTCGTGTTTCTTTCGAACTGTTCGCTGACAAAGTTCCGAAA ACCGCTGAAAACTTCCGTGCTCTGTCTACCGGTGAAAAAGGTTTCGGTTACA AAGGTTCTTGCTTCCACCGTATCATCCCGGGTTTCATGTGCCAGGGTGGTG ACTTCACCCGTCACAACGGTACCGGTGGTAAATCTATCTACGGTGAAAAATT CGAAGACGAAAACTTCATCCTGAAACACACCGGTCCGGGTATCCTGTCTAT GGCTAACGCTGGTCCGAACACCAACGGTTCTCAGTTCTTCATCTGCACCGC TAAAACCGAATGGCTGGACGGTAAACACGTTGTTTTCGGTAAAGTTAAAGAA GGTATGAACATCGTTGAAGCTATGGAACGTTTCGGTTCTCGTAACGGTAAAA CCTCTAAAAAAATCACCATCGCTGACTGCGGTCAGCTGGAAGGTTGATAAT GATCTTCAGGATCC-3’ For protein expression, ClearColi BL21(DE3) electrocompetent cells (60810, Lucigen, Middleton, WI) were transformed by CAC or CVC encoding plasmids for recombinant expression following the manufacturer’s protocol (M. Park et al., 2020). Expressed CAC and CVC were purified from cell lysates using inverse transition cycling (Sun et al., 2011). More than 98% purity can be obtained by 3 rounds of ELP collection by cycling above and below the phase transition temperature of the fusion protein. For concentration measurements, purified ELPs (CAC and CVC) were diluted 1X with 8M 58 guanidine-hydrochloride and assayed for absorbance at 280 nm using a UV-Vis spectrophotometer with a one-centimeter light path (DU800, Beckman Coulter Inc.). CELP= OD280 / ε Eq. 2.4 The molar extinction coefficient, ε, of CAC and CVC, was estimated to be 17,960 (M -1 cm - 1 ) (Pace, Vajdos, Fee, Grimsley, & Gray, 1995) based on the following equation: ε = 125 nCysteine + 5500 nTryptophan + 1490 nTyrosine Eq. 2.5 SDS-PAGE was used to resolve proteins, which were then stained with Bio-Safe TM Coomassie Stain (1610786, Bio-Rad Laboratories) to verify the molecular weight and determine the purity of the fusion proteins. Their transition behavior was characterized by measuring optical density at 350 nm through a controlled temperature gradient from 25 to 75°C at 1°C/min using DU800 UV/Visible Spectrophotometer (Beckman Coulter, Brea, CA) The transition temperature (Tt) was defined as the temperature at which the maximum first derivative of the optical density with respect to the temperature was reached. 2.2.7 Characterization of ELP monomeric/oligomeric state and drug binding affinity To obtain and characterize mono-dispersed materials for functional investigations, CAC and CVC isolated by ELP-mediated phase separation were further purified by size exclusion chromatography (SEC) to separate different oligomeric states included in these ELP solutions. For characterization purpose, 100 µL of CAC or CVC at 25 µM was first resolved with a size exclusion HPLC column (Shodex Protein KW-803, Showa Denko America, Inc, New York, NY) at a 0.5 mL/min isocratic flow of PBS. Elution was subject to analysis by 59 three in-line detectors: 1) a variable wavelength detector (SYS-LC-1200, Agilent, Santa Clara, CA) at 210 nm; 2) a multiangle light scattering detector (MALS) (DAWN HELEOS, Wyatt Technology Corporation, Santa Barbara, CA); and 3) differential Refractive Index (dRI) detector (OPTILAB rEX, Wyatt Technology Corporation). ASTRA 6 software was used for data analysis and molar mass estimation. For preparative scale chromatography, a BioLogic Duo-Flow system (Bio-Rad Laboratories, Inc., Hercules, CA) and HiLoad ® 26/600 Superdex ® 200 pg SEC column (28989336, Cytiva, Marlborough, MA) was used to purify mono-disperse material. In detail, 5mL of CAC or CVC with no more than 300µM concentration were resolved at 2.0mL/min isocratic flow of PBS. The drug binding kinetics of CAC and CVC were studied using isothermal titration calorimetry (ITC) (MicroCal PEAQ-ITC, Malvern Instruments Ltd, Northampton, MA). Both constructs were diluted to 25 µM to avoid phase separation at room temperature, which was the temperature used for the assay. Both CsA and ELP constructs were equilibrated in the same buffer (3% v/v DMSO in PBS) to minimize the background heat released by buffer mismatch. CAC or CVC was titrated 12 times, in 3 µL aliquots, into 280 µL of a 5 µM CsA solution. Each injection generates a heat pulse that is integrated with respect to time and normalized for concentration to generate a titration curve of kcal/mol vs molar ratio (ligand/sample). The resulting isotherm is fitted to an “one set of sites” binding model to generate the affinity (KD), stoichiometry (n) and enthalpy of interaction (ΔH) using MicroCal ITC analysis software (Malvern Instruments Ltd, Northampton, MA). 2.2.8 Drug encapsulation and loading efficiency measurements Nine volume parts of CAC or CVC solution in PBS was mixed with one volume part of CsA in ethanol (T038181000, ThermoFisher Scientific Inc., Waltham, MA), at three 60 times molar excess, dissolved in ethanol. The mixture was stirred for one hour at 4°C to maximize drug loading, followed by high-speed centrifugation at 16,100 x g and ultrafiltration through syringe filters (25 mm, 0.2 µm) (4612, Pall Corporation, Port Washington, NY) to remove unbound, insoluble drug. Ethanol was removed by dialysis against PBS (~5 mL against 1 L incubated at 4 °C with 2-4 changes of buffer over 1-2 days). The loading efficiency was determined by RP-HPLC (1260 Infinity II, Agilent, Santa Clara, CA). The binary mobile phase was composed of water and methanol, each containing 0.1% trifluoroacetic acid (TFA) (1081780050, Sigma-Aldrich, St. Louis, MO). ELPs and ELP-bound CsA was resolved by a C4 column (BU12S05-1546WT, YMC CO., Devens, MA), eluted by a gradient flow of methanol (A452, ThermoFisher Scientific Inc., Waltham, MA) /water (40:60) to methanol/water (95:5) for the first 5 min and then an isocratic flow of methanol/water (95:5) for another 5 min at a flow rate of 1 mL/min, and detected at 210 nm. 2.2.9 Endotoxin removal Drug-loaded CAC and CVC were subject to endotoxin removal by filtration through Acrodisc ® Mustang E syringe filters (MSTG25E3, Pall Corporation, Port Washington, NY) at 4 °C, prior to any efficacy and pharmacokinetic evaluation. 0.2 µm pore size of these filters also ensured sterility suitable for in vivo injection. Per recommendation of United States Pharmacopeia, the remaining endotoxin burden was measured with chromogenic Limulus Amebocyte Lysate (LAL) assay (C1500, Associates of Cape Cod, Inc., East Falmouth, MA) following manufacturer’s protocol. 61 2.2.10 CsA mediated inhibition of IL-17A secretion from Th17 cells In vitro differentiated Th17-like cells were stimulated with 20 ng/mL PMA and 1 µg/mL ionomycin, immediately following treatment with free CsA dissolved in DMSO, CAC-CsA or CVC-CsA at CsA concentrations from 10 pM to 1 µM for 5 hr at 37°C. IL- 17A concentration in culture medium was measured using ELISA (432504, BioLegend, San Diego, CA) on a SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA) at 450 nm. 2.2.11 Pharmacokinetic study CsA-loaded CAC and CVC were fluorescently labeled with rhodamine. 3 times molar excess of NHS-Rhodamine (46406, ThermoFisher Scientific Inc., Waltham, MA) dissolved in DMSO was mixed with 200-300 µM CAC-CsA or CVC-CsA in PBS. The mixture was protected from light and incubated with constant rotation for 1.5 hr at 4°C. Unbound free dye was then removed by Zeba desalting column (89893, ThermoFisher Scientific Inc., Waltham, MA) and sink condition dialysis against PBS at 4°C. As reported previously (H. Guo et al., 2018), the labeling efficiency was determined to be 177% for CAC-CsA 137% for CVC-CsA. To compare the pharmacokinetic profiles of CAC and CVC, rhodamine-labeled CsA- loaded CAC was injected into 12-week-old male BALB/c mice intravenously (IV), or subcutaneously (SC) to an area of skin overlaying the LG (supra-LG injection) CsA- loaded CVC was also injected supra-LG SC. The injected dose was 1095 nanomoles of drug-loaded protein/ kg BW. 20 μL of blood was collected from the tail vein by tail nicking at various time points up to 120 hrs. The collected blood was immediately added to 80 μL of heparinized PBS at a heparin concentration of 1000 U/mL. Diluted plasma was 62 collected after spinning down blood cells at 1,000 x g for 10 min. Fluorescence intensity (Excitation/Emission: 540/580 nm) was measured by a SpectraMax iD3 Multi-Mode Microplate Reader to estimate plasma concentration of CAC and CVC. The area under the plasma concentration-time curve (AUC) was first calculated with the trapezoidal method to reflect total body exposure to drugs administered, i.e., CAC IV, CAC SC or CVC SC, which subsequently enabled non-compartmental computation of the area under the first moment curve (AUMC), mean residence time (MRT), mean absorption time (MAT) after SC injection, the SC bioavailability, F, the plasma clearance (CL) with following equations. 𝑀𝑅𝑇 = 𝐴𝑈𝑀𝐶/𝐴𝑈𝐶 Eq. 2.6 𝑀𝐴𝑇 = 𝑀𝑅𝑇 &% −𝑀𝑅𝑇 '( Eq. 2.7 𝐶𝐿/𝐹 = 𝐷𝑜𝑠𝑒/𝐴𝑈𝐶 Eq. 2.8 𝐹 = 𝐴𝑈𝐶 &% /𝐴𝑈𝐶 '( Eq. 2.9 Due to the phase transition behavior of CVC at physiological temperature, CVC IV was not explored, as that was expected to form micron-sized droplets in the bloodstream. The MRTIV and AUVIV of CVC were assumed to be equal to those of CAC. The last two time points of each profile were fit to log-linear decay to estimate the terminal half-life, t 1/2, terminal . Besides circulating concentration measurements, the local retention after supra- LG injection was monitored using an in vivo imaging system (IVIS) (Lumina Series III, Perkin Elmer, Waltham, MA). Epifluorescence images of mice treated with SC CAC or CVC were captured at different points from 4 hr to 14 days. The total fluorescence intensity within the region of interest (ROI) against the time profile was fit to a two-phase 63 decay model. The terminal half-lives of both constructs after supra-LG injection were estimated and compared. The depot-forming construct, CVC, was expected to extend the duration of drug retention at SC injection site. To confirm this hypothesis, CAC-CsA or CVC-CsA were injected into BALB/c mice SC via supra-LG injection with n=6 at a drug dose of 2.7mg/kg. Both LG and whole blood were collected from three animals in each group on day 7 and from the remaining animals on day 14 for LC-MS analysis to determine CsA concentration. 2.2.12 Therapeutic study Male NOD mice typically develop a SS-like autoimmune dacryoadenitis from 8-12 weeks, which is well-established by 14 weeks of age (H. Guo et al., 2018). In the first therapeutic study, four groups of 14-week-old male NOD mice with n=10/group were treated with either CAC-CsA, CVC-CsA, or A192 + Sandimmune ® at a CsA dose of 2.0 mg/kg or with PBS once a week for two weeks, via supra-LG SC injection. The Sandimmune ® Injection formulation of CsA was used as a free drug control; however, it was supplemented with additional free ELP to control for the presence of ELPs in the CAC treatment. A192, which has the sequence G(VPGAG)192Y, was used, which shares a similar MW as CAC and CVC, but lacks the drug-binding specificity. A different dosing regimen was used in the second therapeutic study. Male NOD mice with n=15/group were treated with CAC-CsA, or CVC-CsA at a CsA dose of 3.0mg/kg or with PBS once via supra-LG injection. Two weeks after the first injection, lymphocytes were prepared from LGs under the conditions in each therapeutic study using the methods described above and subjected to analysis with flow cytometry. 64 Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey’s posthoc test using Prism 9 (GraphPad Software, La Jolla, CA). A p- value < 0.05 was considered statistically significant. 2.2.13 Flow cytometry To monitor in vitro Th17-like cell differentiation, Th17-like cells were stimulated with 50 µg/mL phorbol 12-myristate 13-acetate (PMA) (P8139, Sigma-Aldrich, St. Louis, MO) and 1 mg/mL ionomycin (I0634, Sigma-Aldrich, St. Louis, MO) in the presence of 5 µg/mL Brefeldin A (BFA) (420601, BioLegend, San Diego, CA), the protein transport inhibitor, for 5 hr at 37°C to allow intracellular accumulation of IL-17A. Cells collected from each well of 12-well plate were collected, washed once with 1mL PBS and resuspended in 1mL fresh PBS, into which 1 µL of reconstituted Aqua dead cell stain (L34957, ThermoFisher Scientific, Waltham, MA) was added. This cell suspension was briefly vortexed and incubated on ice for 30 minutes in the dark. Cells were then washed 1X with 1 mL PBS, followed by surface antigen staining. Cells were resuspended in 100 µL PBS, and mixed well with 0.25 µg PerCP anti-mouse CD45 antibody (103129, BioLegend, San Diego, CA) before incubating on ice for 30 min in the dark. Cells were then washed 1X with 1mL PBS and fixed with 500 µL of 4% paraformaldehyde at room temperature for 20 min in the dark. The cell membrane was permeabilized by washing cells 1X with an intracellular staining permeabilization wash buffer (421002, BioLegend, San Diego, CA). Cell were then resuspended in 100 µL permeabilization buffer, mixed well with 0.25 µg FITC-labeled anti- mouse IL-17A antibody (506908, BioLegend, San Diego, CA) and incubated on ice for 30 min in the dark. Finally, cells were washed 1X with permeabilization buffer and resuspended in 500 uL PBS before being acquired on a Fortessa X-20 Cell Analyzer (BD 65 Bioscience, San Jose, CA). CD45+ cells with positive intracellular IL-17A staining were considered as Th17-like cells. FlowJo TM V10 software (BD Biosciences, San Jose, CA) was used for data analysis. To evaluate cells obtained from the therapeutic studies, helper T cell subtypes were distinguished based on their surface markers. LG-derived lymphocytes were first subject to Aqua staining as described above, following by surface staining with serial antibodies purchased from BioLegend (San Diego, CA): Brilliant Violet 421™ anti-mouse CD196 (CCR6) (129817), PerCP/Cy7 anti-mouse CD194 (CCR4) (132213), PE anti- mouse CD183 (CXCR3) (126505), APC anti-mouse CD25 (102011), BV605 anti-mouse CD127 (IL-7Rα) (135025), Alexa Fluor® 700 anti-mouse CD3 (152316), FITC anti-mouse CD4 (100406), APC/Cyanine7 anti-mouse CD8a (100713) and PerCP anti-mouse CD45 antibodies (103129) at the manufacturer’s recommended concentration. Cells were acquired on a Fortessa X-20 Cell Analyzer and data analyzed with FlowJo TM V10 software. 2.2.14 Basal thread test of tear production Tear production was assessed as described (Shah et al., 2017), A ZoneQuick phenol red-embedded thread was applied in both eyes at the canthus of the ocular surface for 10 sec, while the animal was under light anesthesia with isoflurane. Basal tear volume was recorded as the length of thread wetting by basal tears in mm. 2.3 Results 2.3.1 Cytokine expression by CD4+ T cells present in healthy LG versus LG with autoimmune dacryoadenitis. To better understand the spectrum of immune cells infiltrating the LG upon establishment of autoimmune dacryoadenitis, the infiltrating lymphocytes and glandular 66 epithelial cells were prepared from LG from age-matched male NOD and BALB/c mice via enzymatic digestion. Lymphocytes were subsequently enriched by filtration through a 10 µm cell strainer, from which the CD4+ T cell population was negatively selected with 81.6 ± 9.7% (mean ± SD) purity. The CD4+ T cell population was then negatively selected from total lymphocytes. Similarly, CD4+ T cells were also isolated from the LG-draining cervical lymph nodes (LN) and spleen (SP). qRT-PCR was conducted to measure gene expression levels of two pro-inflammatory cytokines of interest, IL-2 and IL-17A, the signature cytokines of Th1 and Th17 cells, from these purified CD4+ T cells. As shown in Figure 2.1A, a considerably higher IL-17A gene expression was observed in CD4+ T cells isolated from NOD mouse LG relative to LG from age-matched BALB/c mice. Due to the lack of lymphocytic infiltration, the relative abundance of CD4+ T cells to glandular epithelial cells was markedly lower in BALB/c mice. CD4+ T cells derived from BALB/c mouse LG were thus subject to higher epithelial cell contamination, as exhibited by higher expression of the membrane water channel, Aquaporin 5 (AQP5) (Figure 2.1C). Unlike CD4+ T cells isolated from the LG, IL-17A gene expression levels were similar in age- matched NOD and BALB/c mice in LN and SP. Notably, when comparing IL-17A gene expression among different organs, CD4+ T cells from NOD mice also exhibited dramatically higher IL-17A gene expression in LG than LN and SP, which was not observed for BALB/c mice (Figure 2.1A). The expression profiles of IL-2 and IL-17A in tissue-infiltrating CD4+ T-cells were distinct. As shown in Figure 2.1B, LG IL-2 expression in these cells are comparable in NOD and BALB/c at all time points measured, while IL-2 expression showed a trend to an increase in CD4+ T-cells from LN and SP from BALB/c versus NOD mice. Although 67 IL-2 expression from infiltrating CD4+ T-cells was still much higher than in LN and SP in older NOD mice, the fold increase of 2-3 in this tissue was much less than the ~100-fold increase for IL-17A in the same cell population. Since the elevation of IL-17A is much more prominent than IL-2, we decided to focus on IL-17 and IL-17 secreting cells in the rest of this study. 68 2.3.2 Characterization of recombinant CAC and CVC, two protein-based carriers of CsA. As depicted in Figure 2.2A, by fusing CypA to both the N and C-termini of an ELP backbone with the same number of pentameric repeats but differing hydrophobicity, CAC ss Figure 2. 1: IL-17A and IL-2 gene expression is elevated in CD4+ T cells isolated from NOD mouse LG. mRNA was extracted from CD4+ T cells, negatively isolated from LG, LN or SP, and used to quantify A) IL-17A, B) IL-2 gene expression and C) epithelial cell contamination in the LG samples quantified by the expression of the water channel protein, AQP5. BALB/c mice were analyzed at 11 and 22 weeks (B11, B22). NOD mice were analyzed at these timepoints as well as at 7 and 15 weeks (N7, N11, N15, N22) to better characterize the development of disease. A) IL-17A was significantly higher in LGs of NOD mice older than 7 weeks in comparison with age-matched BALB/c mice. In contrast to LG, IL-17A was expressed to the same extent in CD4+ T cells from LN and SP for both strains. B) CD4+ T cells from LG also showed notably higher IL-2 expression than cells from LN and SP in older NOD mice. C) The expression of the epithelial-cell-specific membrane water channel protein, AQP5, was used to evaluate the relative contamination of isolated T cells with epithelial cells. Due to the limited abundance of T cells in LGs with low lymphocyte infiltration (B11,B22,N7), the epithelial cell contamination is greater for LG preparations from BALB/c mice at 11 and 22 weeks compared to NOD mice at 22 weeks (N22). N=7. Error bar represents SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. 69 and CVC were cloned and expressed from E. coli. As reported earlier (Sun et al., 2011), CAC and CVC were purified from cell lysates by repeatedly inducing ELP-dependent phase separation. The molecular weights of the purified constructs were resolved by SDS-PAGE and staining with Coomassie Blue (Figure 2.2B). The absolute MW of CAC and CVC were confirmed by MALDI-TOF mass spectroscopy to be 72.8 kDa and 75.4 kDa, respectively (Figure 2.2C, Table 2.1). Consistent with our previous finding (H. Guo et al., 2018), the Tt of both constructs were a log-linear function of concentration as follows (Figure 2.2D): 𝑇 / = 𝑏−𝑚log 23 [𝐶 456 ] Eq. 2.10 where the intercept, b, represents the transition temperature at 1 μM, the slope, m, is the decrease in Celsius for a 10-fold increase in concentration and [CELP] represents the fusion protein concentration. Values for b and m as summarized in Table 2.1 were used to estimate the transition temperature (Tt) of the in vivo injection formulation, which are 44.5, 21.8 ºC for CAC, CVC respectively. These results show that CAC is expected to remain soluble at the physiological temperatures, while CVC is expected to phase separate. 70 Previously, we reported that two populations of CA192 with different oligomeric status could co-exist in solution: a nano-aggregate and a dimeric species, as determined by SEC-MALS, while only the dimeric form bound to CsA (H. Guo et al., 2018). Similar to CA192, as shown in Figure 2.2E, CAC also exhibited two oligomeric states, despite a much smaller calculated nano-aggregate fraction. Unlike CA192, the 2 nd low MW fraction of CAC retained by SEC was monomeric in solution, with an absolute MW estimated to be 77.6 kDa. This is very close to its expected MW, which was confirmed by mass spectrometry (Table 2.1). In contrast to CAC, a nano-aggregate fraction eluting in the void volume was not observed during SEC of CVC; however, CVC also existed in solution as a homogeneous monomer with an absolute MW of 75.6 kDa (Figure 2.2F). Additionally, the binding kinetics between CAC/CVC and CsA were analyzed by isothermal titration calorimetry (ITC) at 25°C, the temperature at which both constructs remain soluble (Figure 2.2G). The equilibrium dissociation constant (Kd) was determined to be 207 ± 133 nM and 46 ± 35 nM for CAC and CVC, respectively, comparable with that Table 2. 1: Amino acid sequence and thermo-responsive behavior of CAC and CVC. Label Amino Acid Sequence 2 M.W. [kDa] 3 Est. M.W. by MALS [kDa] 4 Est. M.W. by MS [kDa] 5 Slope, m [°C/Log10(μM)] 6 Intercept, b [°C] 7 Est. Tt at 300µM [°C] CAC 1 CypA-(VPGAG)96-CypA 72.9 77.6 72.8 4.3 ± 0.8 55.2 ± 1.2 44.5 CVC CypA-(VPGVG)96-CypA 75.6 75.6 75.4 7.3 ± 1.6 39.9 ± 2.4 21.8 1 CypA amino acid sequence: MVNPTVFFDIAVDGEPLGRVSFELFADKVPKTAENFRALSTGEKGFGYKGSCFHRIIPGFMC QGGDFTRHNGTGGKSIYGEKFEDENFILKHTGPGILSMANAGPNTNGSQFFICTAKTEWLK HVVFGKVKEGMNIVEAMERFGSRNGKTSKKITIADCGQLE 2 Expected molecular weight based on the open reading frame for the expressed protein. 3 Estimated molecular weight by MALS, data shown in Figure 2.2E, 2.2F. 4 Estimated molecular wight by MALDI-TOF mass spectrometry, data shown in Figure 2.2C 5 The transition temperatures of both constructs follow Eq. 2.10, yielding an intercept, b, at 1 μM, and a slope, m, representing the change in temperature upon a 10-fold change in concentration. Mean ± 95% CI. 6 The estimated transition temperature at the injection concentration used in in vivo therapeutic evaluation described in section 2.5, based on Eq. 2.10. 71 of CA192 of 189 ± 87 nM (H. Guo et al., 2018). The stoichiometry of CAC and CVC was determined to be 0.48 ± 0.04 and 0.33 ± 0.02, respectively, consistent with the designed architecture, by which each CAC or CVC molecule can maximally bind two CsA molecules. When binding with CsA, the binding enthalpy of CAC was determined to be - 236 ± 39.2 kJ/mol, stronger than that of CVC of -109 ± 11.9 kJ/mol. 72 Figure 2. 2: A cyclophilin-based drug-carrier assembles an ELP depot for cyclosporine at physiological temperatures. A) Cartoon depicting the ELP fusion proteins, CAC or CVC, which specifically bind CsA and behave differently in response to physiological temperature when injected subcutaneously; B) Compared to a MW ladder (lane 1), SDS-PAGE of purified CAC (lane 2) and CVC (lane 3) confirmed their expected MW and high purity; C) The absolute MW of both CAC and CVC was confirmed by MALDI-TOF mass spectroscopy to be 72.8 kDa and 75.4 kDa, respectively, very close their expected MW. D) ELP phase separation was monitored using optical density as a function of temperature and concentration and plotted as a phase diagram. Fusions assemble coacervates in solution above a log-linear (Eq. 2.10) fit line. Dashed lines represent the 95% confidence interval. At relevant injection concentrations (~100 µM), CAC, but not CVC, remains soluble at physiological temperature. E, F) SEC- MALS was used to analyze the oligomeric state of both constructs in solution. Two populations of CAC were separated with the 2 nd fraction being the major population, with an estimated MW of 77.6 kDa. Unlike CAC, CVC appeared homogeneous, with an estimated MW of 75.6 kDa. By comparing the observed and expected (Table 1) MW, both constructs were concluded to be primarily monomeric in solution. G) The binding thermodynamics between CAC or CVC and CsA were analyzed by ITC. The Kd of CsA was 207 ± 133 nM for CAC and 46 ± 35 nM for CVC. The binding stoichiometry of CsA was 0.48 ± 0.04 to CAC and 0.33 ± 0.02 to CVC, which confirmed the availability of two binding sites per fusion protein. The binding enthalpy between CsA was -236 ± 39.2 kJ/mol for CAC and -109 ± 11.9 kJ/mol for CVC. 73 Finally, prior to efficacy and pharmacokinetic evaluation, CAC-CsA and CVC-CsA were first subject to endotoxin removal. The remaining endotoxin level at 300µM protein concentration was determined to be 40 EU/mL for CAC-CsA and 43 EU/mL for CVC-CsA. 2.3.3 In vitro efficacy of CAC-CsA and CVC-CsA against Th17-like T cells. IL-17A producing Th17-like cells can be differentiated and polarized from CD4+ T cells isolated from NOD mouse splenocytes when incubated with a cocktail of cytokines and antibodies. After a 4-day incubation, cells with positive intracellular staining for IL- 17A were identified by flow cytometry. As shown in Figure 2.3, about 5% of these helper T-cells were polarized into Th17-like cells. This frequency was significantly reduced to below 1% when co-incubated with 10 nM CsA, either as free drug, or delivered by CAC or CVC. 74 These in vitro differentiated Th17-like cells, when activated by ionomycin and phorbol myristate acetate (PMA), secrete large amounts of IL-17A in the absence of CsA. The cytokine release from activated Th17-like cells differentiated as described above was also observed to be effectively inhibited by CAC-CsA and CVC-CsA in a dose-dependent manner as shown in Figure 2.4. The half maximal inhibitory concentrations (IC50) of CAC- CsA and CVC-CsA were determined as 28.5 ± 9.1 nM and 9.1 ± 0.6 nM, respectively, slightly higher than that of free CsA which was 1.0 ± 0.4 nM. Figure 2. 3: CAC-CsA and CVC-CsA effectively inhibit Th17-like cell differentiation in vitro. CD4+ T-cells obtained by negative selection from NOD mouse splenocytes can be differentiated into Th17-like cells in vitro when treated with a cocktail of cytokines and antibodies (IL-6, TGF-β1, IL-23, anti-mouse CD28, anti-mouse IL-4, and anti-mouse IFN- γ) on anti-mouse CD3ε antibody-coated plate. As CD4+ T cells were pre-selected prior to the differentiation, pan-leukocyte marker CD45 was used here in place of CD4. A subset of these helper T cell population, Th17-like cells were captured by flow cytometry based on their intracellular accumulation of IL-17A upon activation. Normally, 4.9 ± 1.6% (mean ± SD from n=3) of CD4+ T-cells can be differentiated into Th17-like cells. However, Th17-like cell abundance was significantly reduced to 0.6 ± 0.1%, 0.8 ± 0.2 and 0.9 ± 0.1 when co-incubated with 10 nM CsA, delivered by CAC, CVC or in its free form, respectively. **p<0.01. 75 2.3.4 Pharmacokinetic profiles of CAC and CVC in mice. To enhance accumulation in the target tissue, the LG, and to more effectively control the local elevation of IL-17A expression in LG associated with IL-17A expressing immune cells, we developed a new route of administration, supra-LG injection, which consists of a subcutaneous (SC) injection to the region overlaying the LG (Figure 2.5A). The pharmacokinetic (PK) profiles of CAC and CVC after supra-LG injection and CAC after IV injection were studied in healthy BALB/c mice. Plasma concentrations of both constructs versus time profiles are depicted in Figure 2.5B. Non-compartmental analysis was conducted under the assumption that the PK profile of CVC given IV is roughly the same as that of CAC given IV, considering their similar architecture and MW. Figure 2. 4: CAC-CsA and CVC-CsA effectively inhibit IL-17A secretion from activated Th17-like cells in vitro. Upon activation with PMA and ionomycin, Th17-like cells produce a large amount of IL-17A, which is secreted into the culture medium. CsA, regardless of its delivery vehicle, inhibits this IL-17A secretion in a dose- dependent manner. The IC50 of free CsA, CAC-CsA and CVC-CsA were estimated to be 1.0 ± 0.4 nM, 28.5 ± 9.1 nM and 9.1 ± 0.6 nM (mean ± SD from n=3), respectively. 76 CVC was not given IV since the coacervation may disrupt venous circulation. As summarized in Table 2.2, the depot-forming construct, CVC, exhibited a 4-times longer mean residence time (MRT) of 52 hr relative to its soluble counterpart, CAC, of 12.6 hr when given supra-LG. The slower absorption phase of CVC is expected to be the rate- limiting step, supported by the mean absorption time (MAT) extension from 1.6 hr to 40.9 hr. The bioavailability, F, after supra-LG SC injection was calculated to be 40.9% and 24.4% for CAC and CVC, respectively. Additionally, the depot-forming CVC demonstrated a terminal half-life of 56.2 hr, significantly longer than the 15.2-hr terminal half-life of CAC after supra-LG SC administration. 77 Figure 2. 5: The depot-forming CVC-CsA has extended PK profile relative to soluble CAC-CsA. A) The cartoon illustrates supra-LG injection (subcutaneous injection to the region overlaying the LG). This new route of administration was developed to maximize the local effect of CsA; B) Using a rhodamine-labeled approach, the plasma concentration of CAC was determined after IV administration and compared to CAC or CVC after supra-LG injection. Supra-LG administration of CVC gave the lowest relative bioavailability and the longest terminal half-life (15.2 hr for CAC SC and 56.2 hr for CVC SC) (Table 2). Error bars represent mean ± SD from n = 5 BALB/c mice; C) Whole animal IVIS imaging was used to compare the local retention of CAC (left) and CVC (right) on day 5 post supra-LG injection. CAC was completely eliminated by day 5, while CVC remained easily detectable. D) Image analysis was used to quantify absorption of CAC and CVC from the supra-LG injection site, which follows a two- phase decay model with a terminal half-life of 28 hr for CAC and 92 hr for CVC. E, F) HPLC-MS was used to quantify CsA drug concentrations in plasma and LG at 7 and 14 days after supra-LG administration. CVC-CsA maintained a stable CsA concentration in whole blood (panel E) and a much higher level in the LG lysate (panel F) for at least 14 days, the longest time measured. In contrast, by these timepoints CsA in the CAC-CsA formulation was undetectable. 78 Use of an in vivo imaging system (IVIS) confirmed the longer retention of CVC at the injection site. Fluorescently labeled CAC-CsA and CVC-CsA were administered via supra-LG injection. As shown in Figure 2.5C, by day 5 after injection, CAC is no longer detectable at the injection site, while CVC is still easily detectable. Based on the remaining fluorescence intensity retained at injection, absorption of CAC and CVC from the injection site follows a two-phase decay model with a terminal half-life of 28 hr (CAC) and 92 hr (CVC) (Figure 2.5D). More importantly, the depot-forming CVC achieved sustained drug release and retained much higher CsA both in whole blood (Figure 2.5E) and LG lysate (Figure 2.5F). 2.3.5 Therapeutic effect of CAC versus CVC on disease pathogenesis in the NOD mouse model of autoimmune dacryoadenitis Initially, 14-week-old male NOD mice were divided into four treatment groups: PBS, A192 + Sandimmune ® (US-FDA approved IV formulation for CsA), CAC-CsA and CVC- CsA, with n = 10. The PBS group served as a negative control, while the A192 + Sandimmune group served both as an ELP carrier control and a free drug control. Mice received supra-LG injection once a week for two weeks at a CsA dose of 2.0 mg/kg. Two Table 2. 2: Comparison of pharmacokinetic parameters observed for CAC after IV or SC administration and CVC after SC administration. Parameters (Unit) CAC IV CAC SC CVC SC CL/F (mL/hr) 0.3 (0.04) 0.76 (0.12) 1.34 (0.23) AUC (µM hr) 108.6 (13.8) 44.4 (3.4) 26.5 (3.9) AUMC (µM hr 2 ) 1196.7 (180.9) 559.8 (67.7) 1366.4 (213.7) MRT (hr) 11.0 (1.2) 12.6 (0.9) 52.0 (7.5) MAT (hr) - 1.6 (0.9) 40.9 (7.5) F (%) 100 40.9 (3.1) 24.4 (3.6) V/F (mL/30g BW) 0.99 (0.27) *0.99 (0.27) *0.99 (0.27) T1/2, Terminal (hr) 41.0 (4.3) 15.2 (2.7) 56.2 (5.4) Values are indicated as mean (SD) *Volume distribution estimated from IV analysis were assumed in SC analysis. 79 weeks after the first injection, infiltrating lymphocytes were prepared from LG and subsequently analyzed by flow cytometry to understand how different treatments affected helper T cell composition. Surprisingly, based on our gating strategy for different helper T cell subsets illustrated in Figure 2.6A, we observed that Th17 cells, traditionally defined as CD3+ CD4+ CCR6+ CCR4+ CXCR3-, were fairly rare. Instead, the CCR6+ helper T-cell population was predominantly CCR4-. Therefore, our focus became the CCR6+ CCR4- CXCR3+ Th17.1 cell population (also called Th1Th17 cells) as the likely origin of the IL17A expressed by CD4+ T cells in the LG in disease (Figure 2.1). As shown in Figure 2.6B, relative to PBS controls and an equivalent CsA dose given as free Sandimmune ® plus ELP, only CVC-CsA was able to significantly reduce Th17.1 cells. CAC-CsA given supra-LG did not exert a therapeutic effect. It was not surprising that CAC-CsA or Sandimmune ® , even at an equivalent CsA dose, did not significantly reduce Th17.1 cell frequency. Supra-LG injection was intended to maximize the CsA accumulation in LG. Unlike the depot-forming CVC-CsA, CAC-CsA remained fully soluble after being injected. As illustrated in Figure 2.5C and D, absorption of CAC- CsA from the injection site was much faster than CVC-CsA, making it incapable of exerting a sustained effect on the LG. In addition to being quickly absorbed from the injection site, free CsA in Sandimmune ® is also subject to rapid renal filtration, making it even less bioavailable to the LG than CAC-CsA. With respect to their yield from treated LG in the experimental groups, we also investigated other T cell subtypes including Th1, Th2 and regulatory T cells (Treg) which is known to suppress immune response and maintain self-tolerance. No significant 80 differences in the frequencies of Th1 cells (Figure 2.6E) and Treg cells (Figure 2.6F) were seen. However, as summarized in Figure 2.6D, the anti-inflammatory Th2 cells were increased in the CVC-CsA group relative to the PBS and A192 + Sandimmune ® treated groups. The CAC-CsA treated group also increased this Th2 cell population relative to the PBS group. No significant difference between the CAC-CsA and A192 + Sandimmune ® groups were observed. As shown in Figure 2.5, CVC-CsA administration resulted in high CsA concentration both locally in LGs and systemically in the blood for more than 14 days, potentially allowing an even longer dosing interval. Moving forward, we further tested the in vivo efficacy of CVC-CsA when given as a single supra-LG injection over a two-week period. Three treatment groups with n=15 per group were included in this study: PBS, CAC-CsA given supra-LG SC and CVC-CsA given supra-LG SC. At 14 days after injection, CD4+ T cell composition was examined by flow cytometry. As shown in Figure 2.7A, CVC-CsA significantly outperformed CAC-CsA, as well as PBS, by reducing the frequency of Th17.1 cells recovered in the LG-infiltrating CD4+ T cell population. However, IL-17A accumulation was not significantly reduced by either treatment, possibly due to less drug exposure than when a weekly dose was given (Figure 2.7B). Consistent with the weekly dose treatment findings in Figure 2.6, both CAC-CsA and CVC-CsA increased the prevalence of the pro-repair Th2 cell population (Figure 2.6D) while not eliciting obvious effects on Th1 cells (Figure 2.6E) or Treg cells (Figure 2.6F). In addition to modulating Th17.1 and Th2 cell levels in LG, we measured functional recovery of tear flow in response to therapeutic treatments using a basal thread test. As shown in Figure 2.8, 81 CVC-CsA given supra-LG SC significantly increased tear production in these mice, but CAC-CsA did not improve tear production. 82 Figure 2. 6: Two treatments with CVC-CsA significantly reduced Th17.1 and Th2, but not Th1 or Treg, cells in the LG-infiltrating CD4+ population, as well as glandular IL-17A accumulation. 14-week-old male NOD mice were treated twice (weekly) by supra-LG administration and compared to controls. In addition to PBS, a combination of ELP (A192) that lacks the ability to bind CsA combined with an approved formulation of CsA known as Sandimmune ® was also used as a control. A) Different CD4+ T cell subsets were gated based on surface antigen expression. With prior gates set on CD3+ CD4+, Th17 cells were defined as CCR6+ CCR4+ CXCR3-, Th17.1 as CCR6+ CCR4- CXCR3+, Th1 as CCR6- CCR4- CXCR3+, Th2 as CCR6- CCR4+ CXCR3- and Treg as CD25+ CD127-. B) Relative to control groups treated with PBS or A192 + Sandimmune ® , only CVC-CsA treatment reduced the frequency of Th17.1 cells obtained from the diseased NOD mouse LG. CAC-CsA treatment was ineffective in reducing the Th17.1 cell population. C) As an alternative to isolation and flow cytometry, the total IL-17A content of LG lysates was measured by ELISA. Compared with the PBS group, only the CVC-CsA group showed significantly lower IL-17A concentration in LG lysate. CVC-CsA treatment also increased the frequency of D) Th2 cells in LG-infiltrating CD4+ T cell population but didn’t effectively alter the frequency of E) Th1 or F) Treg. Error bars represent mean ± SD from n=10. Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by Tukey’s posthoc test. *p<0.05; **p<0.01. 83 Figure 2. 7: One treatment with CVC-CsA over a two-week period effectively altered the LG-infiltrating helper T cell composition. A) CVC-CsA appeared to be more effective than CAC-CsA in reducing the abundance of Th17.1 cells; B) IL-17A was not reduced by either CAC-CsA or CVC-CsA, likely due to reduced drug exposure from the weekly dose treatment. Consistent with findings in Figure 6, CVC-CsA also increased C) Th2 cell abundance but didn’t alter the frequency of D) Th1 or E) Treg in NOD mice LGs. Error bars represent mean ± SD from n=15. **p<0.01. 84 2.4 Discussion The therapeutic approach reported here was inspired by initial findings of increased expression of the proinflammatory cytokines, IL-17A and IL-2, which are characteristic of Th17 and Th1 cells, in diseased NOD mice LG-derived CD4+ T cells. Overexpression of IL-17A in particular has been strongly correlated with pathogenesis of several autoimmune diseases (Chabaud et al., 1999; Crispín & Tsokos, 2010; Fujino et al., 2003; Komiyama et al., 2006; Luchtman et al., 2014; Martin et al., 2013; Raychaudhuri, 2013; Van Den Berg & Miossec, 2009). Male NOD mice have been previously reported to develop lymphocytic infiltration of the LG as early as 8 weeks of age and fully established autoimmune dacryoadenitis by 12 to 14 weeks of age (Ju et al., 2018). Our findings from the analysis of gene expression levels of IL-17A in LG-infiltrating CD4+ T Figure 2. 8: One treatment with CVC-CsA increased basal tear production. 14- week-old male NOD mice were treated with CAC-CsA or CVC-CsA at a CsA dose of 3.0mg/kg or with PBS once for two weeks via supra-LG injection. 14-week-old male NOD mice were treated with PBS, CAC-CsA or CVC-CsA as a single injection over a two-week period. The injection dose of CsA in CAC-CsA and CVC-CsA groups was 3mg/kg. The basal tear production in both eyes was measured by thread test before and after the treatment. No significant difference was observed in (A) PBS group and (B) CAC-CsA group. Significant increase in tear production was only observed in (C) CVC-CsA group. Error bars represent mean ± SD from n=30 (both eyes of 15 animals). **p<0.01. 85 cells suggests that increased IL-17A is established as early as 7 weeks of age, suggesting it as one of the earliest events in the onset of LG pathogenesis. Additionally, we observed that CCR6+ CXCR3+ Th17.1 cells, the “alternative” Th17 cells, are more abundant within the LG-infiltrating CD4+ T cell population, and are likely the principal source of the IL-17A accumulated in the LG. These “alternative” Th17 cells have also been shown to be enriched in the target organs of several other autoimmune diseases (Bettelli et al., 2004; Neurath et al., 2002), likely being particularly pathogenic in tissue inflammation and autoimmunity (Peters, Lee, & Kuchroo, 2011). In addition to Th1, Th2 and Th17, another independent CD4+ T cell lineage, follicular helper T (Tfh) cells, has also been identified, defined by surface expression of C-X-C chemokine receptor type 5 (CXCR5) and the induced costimulatory molecule (ICOS). Through the production of IL-21, Tfh cells exhibit strong effects in stimulating the differentiation of B cells into antibody-producing cells (King, Tangye, & Mackay, 2008). It is interesting to note that Th17 cells share several features, including the expression of IL-21 (Bauquet et al., 2009) and ICOS, triggering B-cell proliferation and promote the formation of germinal centers (Mitsdoerffer et al., 2010). We speculate that Th17.1 cells may share these properties and function as strong B-cell helpers in the LG of SS. Additionally, T cells may also express B-cell activating factor (BAFF) in inflamed glandular tissue of patients with SS (Lavie et al., 2004). BAFF is essential for B cell maturation and survival (F. Mackay & Browning, 2002) and found to be elevated in serum and SGs of SS patients. Taken together, we speculate that Th17 cells, both classical and alternative, may be responsible for the dysregulation of B cells and B cell expansion in autoimmune lesions in SS. 86 Patients with SS-associated dry eye disease (DED) represent 11% (Akpek et al., 2009) of the total DED patients, as defined by symptoms alone. This includes patients with many different forms of the disease (non-autoimmune-mediated aqueous deficient dry eye, evaporative dry eye, etc). DED is highly prevalent in the population, affecting as many as 17% of women and 11.1% of men in the United States (Moss, Klein, & Klein, 2000); however, DED is multifactorial and patients with different etiologies are often not distinguished in clinical trials. The few US- FDA approved medications for DED such as topical cyclosporine A (Restasis ® ) and lifitegrast (Ziidra ® ) provide relief for some patients with SS-associated DED by suppressing ocular surface inflammation. However, they have not been shown to suppress the lymphocytic inflammation of the LG, the principal driver of SS-associated DED. Thus, topical medications treat the symptoms and not the major cause of SS-associated DED. There are currently no US-FDA approved treatment options for SS-associated DED that suppress the underlying lymphocytic infiltration of the LG. Our injectable sustained release construct represents a first step in addressing this limitation. As an immunosuppressant, CsA has been widely explored for use in various autoimmune indications, such as psoriasis (Griffiths et al., 2004), rheumatoid arthritis (Gremese & Ferraccioli, 2004), atopic dermatitis (Bunikowski et al., 2001), uveitis (Song, 2003), systemic lupus erythematosus (Wallace, 2002) and others. Its systemic use is has been correlated with dose-dependent systemic side effects including nephrotoxicity (Bennett & Pulliam, 1983), hepatotoxicity (Kassianides et al., 1990), neurotoxicity (Gijtenbeek et al., 1999) and hypertension (Bellet et al., 1985). Organic additives such as polyoxyethylated castor oil (Cremophor EL) are often required for CsA formulation as 87 solubilizers and emulsifiers to enable parenteral administration which may also cause anaphylactoid reactions. Previously, using ELPs as a drug delivery platform, we successfully constructed a first generation ELP-based CsA carrier, named CA192. CA192-CsA, the drug with this carrier, administered every other day at a drug dose of 2.5 mg/kg, significantly increased stimulated tear production and mitigated nephrotoxicity compared (H. Guo et al., 2018). The second generation ELP-based CsA carriers, CAC and CVC, reported here retained an additional CypA fused to the C-terminus of the ELP backbones, A96 and V96, exhibiting a higher drug loading capacity. More importantly, the characteristic thermo-responsiveness of ELPs was utilized to achieve sustained drug release. Specifically, the temperature sensitive construct, CVC, undergoes temperature- dependent phase transition at the injection site at physiological temperature upon SC injection, exhibiting sustained drug release for more than 2 weeks, and functioning much like a depot implant. This 2-week duration of effect was not exhibited by its temperature- insensitive counterpart CAC. In this proof-of-concept study, we have administered our CsA sustained release formulation via “supra-LG injection”, SC injection to the region overlaying the LG, which may be feasible in human patients at least for a significant part of the gland. This may be further resolved by incorporating a targeting moiety to our drug carriers. Previously in our lab, we have successfully exercised this concept and enhanced inflamed LG accumulation of another ELP-based drug carrier with ICAM-binding peptide (Ju et al., 2019). The abundance of IL-23 and TGF-β have been reported to be skewing factors deciding whether naïve T cells differentiate to “classical” Th17 cells or “alternative” Th17.1 88 cells. Both cytokines were present during in vitro differentiation of Th17 cells, likely leading to the co-existence of two phenotypes. The incomplete inhibition of both effector cell differentiation and cytokine secretion that was observed in Figures 2.3 and 2.4 suggest that CsA may inhibit the development of a specific Th17-expressing cell phenotype. Taken together with in vivo findings that percentage of Th17.1 cell in CD4+ T cells was reliably reduced by CVC-CsA, we conclude that this susceptible population may be the Th17.1 cells, a finding which will be confirmed in future studies. 2.5 Conclusion Here, using the male NOD mouse as a model of SS-associated dry eye disease, we identified increased IL-17A expression by LG-infiltrating CD4+ T cells as an early potential pathogenic factor in development of disease. ELPs in fusion with cyclophilin can deliver CsA to exert an inhibitory effect on the principal cell type responsible for IL-17 expression, the Th17.1 cell, impeding their cellular development and cytokine secretion. More importantly, in response to physiological temperature, ELP-mediated depot formation allows the sustained release of CsA, enhancing its in vivo efficacy against SS-mediated ocular surface manifestations. 89 Chapter 3 A pharmacokinetics primer for preclinical nanomedicine research 3.1 Abstract As for any drug, nanomedicines must achieve a pharmacokinetic (PK) profile that is acceptable in the clinic. To do so, PK parameters should be evaluated through rigorous structure-function studies during preclinical development. The purpose of this chapter is to provide experts in nanoparticle design with general guidelines for performing and interpreting PK studies of potential nanoparticles. With a focus on issues applicable to nanoparticles, this chapter includes functional definitions of concepts, brief summaries of experimental strategies, equations for use in calculating parameters, and guidance on interpretation using compartmental and non-compartmental analyses. Real examples of structure-function studies are presented, which can guide the design of future nanoparticle PK studies. Key Words: Pharmacokinetics, half-life, bioavailability, blood, plasma, compartmental modeling, non- compartmental analysis, clearance, volume of distribution. Variables studied in this chapter: Half-life t1/2, Terminal half-life t1/2,terminal, maximum blood concentration Cmax, time to achieve maximum blood concentration tmax, absorption half-life t1/2, abs, elimination half-life t1/2, elim, distribution half-life t1/2, dist, clearance CL, volume of distribution V, volume of distribution at steady state Vss, area under the curve AUC, area under the moment curve AUMC, mean residence time MRT, mean absorption time MAT, bioavailability F. 90 3.2 Introduction As suggested by its linguistic roots in Greek, pharmacokinetics (PK) involves the characterization of the use of drugs ‘pharmakeia’ and their motion ‘kinetikos’ in the body. PK developed because pharmacists realized that timing the appearance and disappearance of drugs in the body would determine their dose, frequency, and route of administration. Thus, scholars and practitioners of pharmacy began collecting blood- based samples as a function of time after dose and standardizing methods for their interpretation. A primary endpoint for most first-in-humans phase I clinical trials is to produce a PK profile after a single, safe dose(Olmos et al., 2010; Wilson et al., 2010). With estimates of human PK parameters, it becomes possible to evaluate therapeutically relevant, multi-dose regimens during subsequent clinical trials. A reasonable PK profile can mean several things. For example, the half-life must be sufficient that the patient can take it at reasonable intervals without trough concentrations too low to maintain therapy. During peak concentrations following dosing, the drug concentration must remain below pharmacodynamically acceptable limits for acute and chronic toxicity. Alternatively, the extent of absorption for an extravascular dose (including oral and subcutaneous administration), should be high enough to limit dose-to-dose variability in blood levels and toxicity. Unless efficacy is of remarkable benefit to the patient, drug candidates that fail to have a clinically-relevant half-life and oral bioavailability usually do not succeed. While there remains deep PK expertise within major pharmaceutical companies, better dissemination of PK expertise is required to academic groups developing early-stage nanoparticles, like those shown in Fig 3.1. 91 Throughout the 1970s to 2000s, a number of attempts were made to develop drug carriers, which by their nature would change the PK profile of a drug to that of the carrier. A common rationale was that the drug carrier could increase the therapeutic index(Papahadjopoulos et al., 1991), making drugs more potent and/or safer. The most successful example are liposomes, into which more than 10 different small molecules have been entrapped and approved by the United States Food and Drug Administration(T. M. Allen & Cullis, 2013). Optimized liposomes do a good job of retaining and then releasing active drugs; furthermore, they force encapsulated/engrafted drugs to adopt the PK of the liposomes. Since liposomes cannot typically be administered orally, it was important that their dose frequency remain on the order of once a week. This requires that liposomes have an ~2 day half-life in humans(Gabizon, Shmeeda, & Barenholz, 2003), which they achieved. It took twenty years of research and development to achieve Figure 3. 1: Plasma PK profile for a protein-based drug carrier, called CA192, which was administered SC to mice. A) After a single dose, the formulation exhibited both an absorption and distribution phase before settling into an elimination phase with a terminal half-life of ~24 hr. B) A modeling approach was used to estimate how plasma concentrations would appear over time during multiple dosing at 48 hr intervals (tau). Cmax and Ctrough are indicated. Data extracted from (H. Guo et al., 2018). 92 this in liposomes, which was facilitated by grafting an optimal polymer (polyethylene glycol), at the optimal molecular weight (~2,000 Da), at the optimal surface grafting density (~5% by mol lipid)(T. M. Allen & Cullis, 2013). During the 2000s, the conviction spread that many other materials and architectures would make capable drug carriers, and ‘nanomedicine’ entered the lexicon. During this period drug delivery technologies expanded well beyond Schools of Pharmacy and major pharmaceutical companies, where traditional expertise in PK resided. Biologists, bioengineers, chemists, chemical engineers, and electrical engineers have developed numerous technologies, as detailed in this book and elsewhere. These range from implantable digitally-operated pumps, bioerodable wafers(Voskerician et al., 2003), PEGylated proteins(Veronese, 2001), sterically-shielded liposomes(T. M. Allen & Cullis, 2013), antibody-drug conjugates(Wu & Senter, 2005), and other nanoparticles(Giljohann et al., 2010). The purpose of this chapter is to quickly orient scholars investigating preclinical nanoparticles to the design and analysis of PK studies. Based on personal observations, reputable academic journals commonly peer-review or even publish PK studies that fail to collect adequate time-points, to calculate basic PK parameters, or to use simple models of drug disposition. It is common to see PK studies reduced to the observation that the concentrations for one formulation remain higher than another, which essentially removes kinetics from the analysis. Why does this matter? Unlike the non-linear, indirect connections between drug regimen and its pharmacodynamic effect, the tools to relate drug regimen to blood concentrations of nanoparticles over time are very effective. When those profiles are used to estimate established PK parameters, structure-function studies will reveal significant strengths and weaknesses of emerging nanoparticles. Such studies 93 should be used to interpret and optimize the nanoparticle physico-chemistry (size, shape, surface chemistry, rate of drug release, ligand grafting density, etc.) to reach dosing regimens sufficient for therapeutic endpoints. In this way scholars of early-stage nanoparticles can maximize the likelihood of developing approved nanomedicines. 3.2.1 Inputs to a PK study The inputs to a PK study are concentration-time profiles, which represent nanoparticle concentration in the plasma, serum, or whole blood. Whole blood contains ~42 % hematocrit, which typically excludes nanoparticles. When blood samples are allowed to clot, blood cells and clotting factors can be removed by centrifugation, and the supernatant is defined as serum. Nanoparticles that bind blood cells or clotting factors may thus have lower concentrations than in whole blood. When blood is prevented from clotting, usually through the chelation of divalent cations and heparin, the blood cells can be removed by centrifugation, and the supernatant is defined as plasma. Thus, plasma contains albumin, immunoglobins, lipoproteins, clotting factors, and complement. All of the above factors may interact to different extents with nanoparticles. As these interactions raise or lower the observed sample concentrations, they will also affect estimates of PK parameters. For this reason, it is important to consider and identify the method for collecting blood samples. To establish reliable PK parameter estimates, samples must be collected at sufficiently placed timepoints. In the absence of prior information about a nanoparticle, timepoints may be spread at factors of 2 (e.g. 0, 0.5, 1, 2, 4, 8, 16, 32, 64 hrs) to capture both absorption/distribution phases with a short half-life and terminal phases with a longer half-life. In subsequent studies, timepoints can be removed or added such that each log- 94 linear phase is covered by 3-4 timepoints ranging over about 3 half-lives. While the planning and collection of this data is critical, these concentration-time profiles only constitute an input to a PK study. Having determined these concentration-time profiles, the prudent investigator must next estimate output parameters, statistically compare these between formulations, and place these in the context of approved drugs and scientific literature. 3.2.2 Half-life One potential output of a PK study is half-life, which is a familiar concept to many scholars. Half-life is a useful way to characterize first-order processes throughout the chemical, physical, electrical, biological, and even economic worlds. First-order processes can be easily identified by semi-log plots as a function of time, whereby log- linear regions can be fit by an exponential growth or decay to estimate the half-life. PK processes often reveal half-lives, which can predict how a dose regimen will affect nanoparticle levels in the body. Dose too high, and side effects dominate. Dose too infrequently, and periods pass with subtherapeutic drug concentrations. Yet, calculation of blood half-life alone is inadequate to compare formulations. Observed PK profiles in the blood reflect multiple half-lives related to Absorption, Distribution, Metabolism, and Excretion (ADME) (Figure 3.2A). Absorption includes transfer of nanoparticles at a site of introduction to its detection in blood samples. Distribution includes the reversible transfer of nanoparticles from the blood into other tissues, which fill and release over time like a reservoir. Metabolism includes processes that irreversibly modify the nanoparticle from one species to another, keeping in mind that nanoparticle metabolites exhibit their own PK profiles in the body. Excretion includes irreversible transfer of nanoparticles from the 95 body into urine or feces. Each of these processes takes time. If the dose were directly introduced into any one of these compartments, its concentration at that site often exhibits a constant, unique half-life (Fig. 3.2B). In contrast, the PK profile in the blood reflects contributions from all ADME processes. Even the terminal blood half-life following IV administration depends on processes of distribution and elimination (metabolism and/or excretion). A reasonable goal of a PK study might be to study a parameter specific to one ADME process, and its structure-activity relationship with the nanoparticle. Since half-life is neither specific for, nor solely dependent on any particular ADME process, other PK parameters are recommended to evaluate structure-function studies of a nanoparticle. 3.2.3 Clearance Arguably the most important PK parameter for any nanoparticle (or cargo) is clearance, Cl. Like half-life, clearance is a constant that is independent of the timepoints selected for analysis. While observed half-lives are inter-related functions of all four ADME processes, clearance only quantifies processes that irreversibly eliminate the Figure 3. 2: Different ADME processes will have different half-lives. A) Summary of important sites of mass transfer involved in absorption, distribution, metabolism, and excretion (ADME) processes after different routes of administration. The PK profile observed in systemic circulation (blood) results from mass balances around all ADME processes relevant to the site of administration. B) For first-order ADME processes, the nanoparticle concentration driving each isolated flux falls by a constant half-life over time. Following a log-linear decay, this representative profile has a half-life of 3 hr. 96 nanoparticle from the blood. Since clearance isolates the effects of distribution (and absorption) from those of elimination (metabolism and excretion), PK studies are well- served by estimating this parameter. This chapter describes multiple ways to estimate clearance; however, its specific mathematical definition is as follows: Rate of drug elimination from the body = 𝐶𝑙 𝐶(𝑡) Eq. 3.1 This essentially states that the loss of the nanoparticle (or its cargo) in the body with respect to time is proportional to the measured concentration in a blood sample, C(t). An important assumption is that the capacity to clear the nanoparticle is a first-order, non- saturable process. For a first-order process, no matter how high the concentration, the rate of drug elimination can always increase. Though there are exceptions, this is a reasonable assumption for many small molecule drugs and nanoparticles near their intended doses. A nanoparticle that results in the same clearance at different doses is said to follow linear PK. Inspection of Eq. 3.1 reveals that Cl has units of volume per time. Clearance scales with the size of the clearance organs, and therefore with the size of the organism. As such, it is common to normalize clearance to body weight or surface area. This can be useful in comparing subjects of different size, age, or species. Clearance estimates are immediately useful because they allow the calculation of a maintenance dose, MD, which will maintain an average target concentration, Ctarget over multiple doses at steady state: 𝑀𝐷 = /.9 %* % 3.,423 : Eq. 3.2 Where the bioavailability, F, represents the total absorbed dose (F=1 for an IV bolus), tau represents the time interval between doses. When designing a dosing regimen 97 for a novel nanoparticle, calculation of a maintenance dose can help achieve target blood concentrations, which may be guided by in vitro cell-based or biochemical assays. When nanoparticle clearance is determined only from the blood and without inspecting measures of flux into particular organs, this is more accurately described as total clearance. The major two clearance organs are the kidney and the liver, and one or both play a major role in the clearance of nanoparticles. Many small molecule drugs have sufficient permeability or specificity to transport into hepatocytes, where they may be metabolized and/or excreted into the bile. Due to larger size, nanoparticles generally have low membrane permeability, and instead become substrates for active internalization into liver cells through receptor-mediated endocytosis, macropinocytosis, or phagocytosis. Nanoparticles in the ~10-100 nm size range, can diffuse through fenestrations of the basement membrane supporting the liver sinusoidal endothelial cells(Braet & Wisse, 2002). This slows transport to hepatocytes due to the size-dependence of a Fickian diffusion constant and to a reduction in ‘permeable’ surface area through fenestrations. Another significant complication, nanoparticles undergo opsonization by factors in blood that form a protein corona(Owens III & Peppas, 2006). This corona tags them for capture by the reticuloendothelial system (RES). Distributed through various organs of the body, the RES is composed of circulating monocytes and resident tissue macrophages capable of engulfing particulates. In the liver, these are the Kupffer cells. Thus, within liver, multiple mechanisms are capable of clearing nanoparticles and their cargo. Total clearance does not identify the organ, system, or physiological mechanism responsible for clearing the nanoparticle. The total clearance is the sum of all routes of nanoparticle elimination. Thus, if one estimates the contribution of a specific clearance 98 mechanism, it is possible to estimate how significant each clearance mechanism is to the overall PK as follows: 𝐶𝑙 = 𝐶𝑙 ;)-.* +𝐶𝑙 <)=./,> +𝐶𝑙 ?4&,-0-<)=./,> +𝐶𝑙 0/<); Eq. 3.3 For particles larger than the glomerular filtration cutoff (~5-10 nm), the renal clearance, Clrenal, is typically low, such that hepatic clearance dominates. In contrast, small proteins, polymers, micelles, and inorganic nanoparticles may have very significant renal clearance. As suggested, C hepatic, can represent accumulation in hepatocytes, direct capture by Kupffer cells, or binding to other cell types. Other tissues within the RES may also accumulate/retain/degrade nanoparticles through ClRES,nonhepatic. Having identified the total nanoparticle clearance, it may be possible to determine the significance of individual clearance mechanisms. By identifying which mechanism of elimination dominates clearance for a particular nanoparticle, effort can be focused on blocking that specific mechanism. This could more systematically reduce the nanoparticle clearance, increase the observed half-life, increase intervals between doses, and enhance therapeutic effects. 3.2.4 Volume of distribution As a nanoparticle moves around the body, there are a number of places it may go before it is irreversibly cleared. It may bind tissues or blood cells, making its apparent concentration in the plasma lower. It may associate with plasma proteins or lipoproteins, while remaining concentrated in the plasma. Many drugs partition rapidly away from the plasma, such that observed plasma concentrations are much lower than would be expected by dilution into the available plasma volume, which is approximately 0.05 mL/g body weight for a rodent(H. Lee & Blaufox, 1985). In addition, many drugs partition slowly 99 enough to observe a distribution phase in their PK profile before they follow a log-linear terminal phase. In either case, distribution processes decrease plasma concentrations which decreases the driving-force to engage a pharmacological target. This also decreases concentrations passing through clearance organs, which lowers rate of drug clearance (Eq. 3.1) and can result in a longer terminal half-life. So, a nanoparticle with a significant distribution component could have a very long half-life, albeit concentrations too low to support efficacy. To account for distribution processes, as distinct from elimination processes, it is possible to calculate apparent volumes, V, into which the drug distributes: 𝑉 = total amount of drug in the body apparent drug concentration in blood Eq. 3.4 Depending on the behavior of the drug, there are multiple further definitions of the volume of distribution. For example, immediately after a bolus dose to the vascular compartment, the drug concentration is measurable and in rapid equilibrium. By either taking a concentration immediately after dosing or by extrapolating back to time zero, it is possible to estimate V. Many drugs and nanoparticles have slower distribution process, which causes concentrations at later times to fall to lower levels then captured by the initial volume of distribution. For these drugs, it is common to calculate an apparent volume at steady state, Vss. Volume of distribution estimates are immediately useful because they allow the calculation of a loading dose, LD, which can immediately bring a drug to a target concentration, Ctarget: 𝐿𝐷 = ( % 3.,423 : Eq. 3.5 100 Where the bioavailability, F, represents the total absorbed dose. Estimation of a LD may be necessary to optimize therapies using nanoparticles with long half-lives, because the PK concentration profiles will not rise to steady-state levels for approximately 4 half-lives. Thus, nanoparticles with a long-half life like an antibody-drug conjugate may benefit from an initial loading dose. 3.3 Quantifying sample concentrations Across assayed PK timepoints, the concentration of the nanoparticle and/or any critical cargo should be assessed. The analytical technique must be sensitive and accurate enough to estimate reliable concentrations in biological solutions. Concentrations below the limit of detection of an assay should be excluded from log-linear fitting because baseline signal will not decrease over time. Fitting data beyond the limit of detection will artifactually increase the nanoparticle half-life in the terminal phase. In addition, for any label attached to a nanoparticle, it is both important that the free label be completely removed prior to administration and it is also important that biological factors do not lead to premature separation from the label. The following techniques are commonly useful to evaluate nanoparticle PK. 3.3.1 Fluorescence Fluorescence is a sensitive and adaptable method for studying the PK profiles of nanoparticles as illustrated in Fig 3.3. Fluorescence is the emission of light when excited electrons transition back to the ground state and can be detected by microplate assays in concentrations down to ~10 nM. Fluorophores span a range of sizes from small molecules, to recombinant proteins, to Quantum Dot (Qdots) nanoparticles. Among these, wavelengths of excitation and emission can vary from the ultraviolet (UV) to the infrared 101 (IV) range of the electromagnetic spectrum(Gonçalves, 2008). While whole blood contains significant hemoglobin, diluted plasma has low absorption of excitation wavelengths or background from auto fluorescent species that contribute to emission of fluorescent samples. Chemically-conjugated fluorophores can be used as reporters enabling the detection and quantification of polymers, proteins, micelles, liposomes, and other nanoparticles of interest. Organic fluorophores, such as rhodamine and fluorescein, have generally useful properties in nanoparticle PK. More so than for small molecule drugs, nanoparticles are large enough that low levels of fluorophore modification are less likely to significantly alter the PK profile. Protein-fluorophores (Green Fluorescent Protein) are perhaps less useful due to their lack of stability and higher molecular weight. Nanoparticle labels such as quantum dots (Qdots) have excellent fluorescent properties; however, their sizes rival the size of many potential nanomedicines, which increases the likelihood that unless they are entirely masked by the nanoparticle their properties may affect the observed PK profile (Toseland, 2013). For all fluorophores, the coupling of the labels to the target proteins could compromise their plasma stability as well as the biochemical functions of peptides and proteins. 102 3.3.2 Radiotracers Radiolabeling enables accurate quantitative determination of very low amount of target macromolecules, making it a reliable method to trace biotherapeutics throughout the body during a PK study. The most commonly used radionuclides for peptide and protein studies are 3 H, 125 I, 14 C, 35 S and 32 P (Patel & Matthewson, 1998). Radioactive labels can be introduced into the macromolecules during biosynthesis, i.e. recombinant protein expression, or after biosynthesis through chemical modification of amino acids side chains. In addition to high sensitivity, a major advantage of radiolabeling is that it need not modify the original structure of the labeled groups, which can make the nanoparticle chemically identical to their unlabeled analogues(Holtzhauer, 2006). From a research perspective, exposure to radiation is more highly regulated, which necessities specialized facilities and personnel trained in PK studies using radioisotopes. Despite the fact that radiotracers are the gold-standard for following nanoparticles over the widest dynamic range, their need for added protective measures remains a limitation to their broader use in nanoparticle PK. Figure 3. 3: Fluorescence is a sensitive and adaptable method for studying the PK profiles of nanoparticles. Fluorescent tags can be chemically-conjugated to nanoparticles that absorb/emit lower energy photons, which can be quantified accurately over a range of low concentrations. Using validated microplate assays, plasma samples may be assayed to estimate nanoparticle concentration at different timepoints. 103 3.3.3 Enzyme-Linked Immuno-Sorbent Assay (ELISA) ELISA is another widely used biochemistry technique for quantification of blood concentrations. Target molecules are first immobilized on a solid surface by passive binding or capture through antibody recognition. Detection is accomplished by measuring the activity of enzyme which is pre-conjugated to a highly specific detection antibody through chromogenic reaction. Two commonly used enzyme labels are horseradish peroxidase (HRP) and alkaline phosphatase (AP). ELISA assay is known for its high specificity and sensitivity. However, performing an ELISA involves at least one antibody with specificity for a particular antigen, restricting the use of ELISA in PK studies, given that novel nanoparticles may not have validated antibodies. A recognition sequence may be genetically or chemically conjugated to the biotherapeutic under investigation to enable the use of commercially available antibodies; however, like other labeling approaches this may alter their original activity and performance. 3.3.4 Mass Spectrometry Liquid chromatography-Mass spectrometry (LC-MS) is another quantitative analytical technique broadly applied in PK studies, combining the resolving power of liquid chromatography (LC) with the detection specificity of mass spectrometry (MS). LC-MS has the ability to quantify multiple analytes simultaneously with high selectivity and sensitivity. Just like other quantification methods, LC-MS has its own disadvantages. In addition to high operational cost, LC-MS has significant interlaboratory variability (Christians et al., 2015) due to the absence of standardized LC-MS method validation guidelines and documents (Clarke, Rhea, & Molinaro, 2013). LC-MS is especially 104 powerful at detecting the PK for drugs which may be cargo on nanoparticles as well as protein and polymeric nanoparticles. 3.3.5 Obtaining samples from blood There are multiple methods for collecting blood samples, which largely depend on the volume and sensitivity of the assay for the nanoparticle and cargo. Of course, it is generally better to develop a full PK profile within each subject. For studies in mice with relatively small blood volumes, this may not be possible. For this reason, rats are often the species of choice for evaluating PK studies. Cardiac puncture is a terminal procedure which can be applied to both mice and rats under deep anesthesia, allowing the collection of a single, good quality and large volume of blood (up to 1 mL from mouse and 15 mL from rat) from the experimental animals(Adeghe & Cohen, 1986). This method be performed through a ventral, left lateral, or open approach. For studies in mice, this approach may be necessary for the evaluation of larger volumes using LC-MS. Tail venipuncture can be performed multiple times to collect small volume of blood (20 µL to 0.2 mL from mice and 0.1-2 mL from rats) from incision with sterile scalpel blades of the tail vein(Omaye, Skala, Gretz, Schaus, & Wade, 1987). To avoid bruising and damage to the tail, unless otherwise approved by Institutional Animal Care and Use Committee (IACUC), normally no more than two blood samples from mice or eight blood samples from rats should be taken within 24 hours. In particular, when using this approach with mice, it is helpful to immediately dilute the whole blood into a volume of heparinized saline. Upon centrifugation, this yields plasma that can be corrected for its dilution factor. This approach works well for fluorescence and radiotracers. 105 Retro-orbital sampling can be performed in both rats and mice by penetrating a capillary into the retro-orbital sinus to collect medium to large volume of blood (up to 0.5mL). Retro-orbital bleeding should only be used under general anesthesia or as a terminal procedure because it causes “more than minimal or transient pain and distress” according to Animal Research Advisory Committee (ARAC)(Health, 2012). The use of topical ophthalmic anesthetic before bleeding is also recommended. Repeated blood sampling via this route is not recommended. Indwelling catheter. The use of temporary or surgical cannulation methods should be considered when continuous and multiple sampling in the experimental animal is required(Parasuraman, Raveendran, & Kesavan, 2010). Temporary cannulation is usually made in the tail vein through a nonsurgical procedure, which can only be used for a few hours. Surgical cannulation, on the other hand, is usually done in the femoral artery, femoral vein, carotid artery, jugular vein, vena cava and dorsal aorta, requiring proper anesthesia and analgesia to minimize the pain. After cannulation, animals should be single housed in a large and spacious cage. Blood sample may be collected over 24 hours at the volume of 0.1 to 0.2 ml/sample from surgical cannula. The surgical cannula needs to be flushed with anticoagulant after every blood withdraw. For preclinical PK it is possible to purchase rats that already have been surgically catheterized. 3.3.6 Molecular imaging The miniaturized version of positron-emitting tracers (PET), named as microPET, has been widely used in preclinical pharmacokinetic study with small animals to non- invasively acquire quantitative and repetitive images of radiolabeled biopharmaceutics and depict their spatial distribution, which is then aligned with 3D anatomic images 106 obtained by computed tomography (CT) scanner. The use of microPET-CT is restricted by the difficult handling of radioactive materials; however, for calibrated studies the accuracy of quantification rivals that of gamma counting plasma samples. Thus, by fitting the signal from a tissue that mostly represents the blood volume, such as the heart, it is possible to obtain useful PK data related to both blood half-lives and also the clearance to the liver and kidneys(S Mohd Janib et al., 2012). Alternatively, optical imaging modalities are available to track the distribution of biomaterials pre-labeled with bioluminescent and fluorescent reporters; however, their quantification is more challenging due to scattering and attenuation of signal through even shallow tissues. 3D tomography also is available in more recent optical imaging modalities. Similar to microPET, when used in combination with other tomographic technologies such as magnetic resonance imaging (MRI) or CT, the optimal images can also be correlated with anatomical context. 3.4 Non-compartmental parameter estimation Compartmental and non-compartmental analyses are two common approaches to understand the PK properties of nanoparticles. Compartmental modeling assumes the body is composed of a certain number of kinetically homogeneous interconnected compartments. The established model can be used to predict drug concentrations at any time points and, therefore, optimize the dosing regimen(H. Guo et al., 2018). On the other hand, non-compartmental analysis is model-independent. In comparison with compartment modeling, non-compartmental analysis is more robust, especially when estimating the PK parameters using fewer timepoints. In addition to providing key parameters to compare between formulations, the clearance and volume of distribution 107 estimates can be used to develop maintenance doses and loading doses respectively. The remainder of section 3.4 introduces equations used to determine non-compartmental parameters. 3.4.1 Terminal half-life Terminal half-life is defined as the time required to reduce the blood sample concentration by half after absorption and distribution phases have ended. If sufficient timepoints are collected, the PK profile will adopt a log-linear decay, and this fraction of the dataset Cz(t) can be fit using nonlinear regression to the following: 𝐶 A (𝑡) = 𝐶 A (0) 𝑒 7B 5 / = 𝐶 A (0) 𝑒 7 67(+) 3 3 )/+ Eq. 3.6 In the above equations, λz is the apparent terminal rate constant and t1/2 is the respective terminal half-life. Cz(0) represents the y-intercept of this curve, which may be significantly lower than the observed initial concentration following the IV dose of a nanoparticle. 3.4.2 Area under the curve Area under the curve (AUC) is the definite integral in the nanoparticle blood concentration vs. time profile, representing the total drug exposure across time. AUC can be used to determine a number of parameters, most importantly clearance, bioavailability, and mean residence time. First, the AUC extending from the last timepoint obtained, n, until infinity can be estimated assuming the profile will continue to follow a mono-exponential decline at the terminal half-life estimated above as follows: 𝐴𝑈𝐶 -7C = % 1 B 5 Eq. 3.7 108 Where Cn is the last concentration determined at the last timepoint. Second, to determine the AUC from the time of the dose until the end of the collected timepoints, the ‘trapezoid method’ can be used. The idea is to divide the plasma drug concentration over time profile into trapezoids between each timepoint. Summing up these areas allows an estimate the AUC. Based on trapezoidal method and Eq. 3.7, the total AUC can thus be calculated as below: 𝐴𝑈𝐶 37C = ∑ T (% 0 E% 0<) )(/ 0<) 7/ 0 ) G U ,H- ,H3 + % 1 l 5 Eq. 3.8 Summation of all of the individual trapezoids and terminal contribution, thus allows estimation of the AUC (Figure 3.4). Figure 3. 4: Illustration of trapezoidal method. The complete AUC for any PK profile can be estimated using the trapezoid rule between each data point added to the AUC following the last timepoint (Eq. 3.8). For example, the ‘tail’ contribution, AUC7-¥, can be estimated as C7/lz (Eq. 3.7), where lz is estimated by fitting the terminal log-linear data points to Eq. 3.6. The remaining areas between each data point can be estimated using the trapezoid rule. For example, trapezoid #5 has an area of (C5+C6)(t6-t5)÷2. 109 3.4.3 Bioavailability Due to incomplete absorption from an extravascular dose as well as first-pass metabolism from oral administration, only a certain fraction of a dose may reach circulation. For small molecule drugs, oral and/or subcutaneous bioavailability can be high (F ~ 1). Many nanoparticles have low bioavailability, which is due to their low permeability and slow diffusion constant. Bioavailability can be estimated as the ratio of the AUC following an extravascular dose, DEV, to an intravenous dose, DIV: 𝐹 = IJ% => " => " ?> IJ% ?> Eq. 3.9 This bioavailability represents the total absorbed dose with respect to an IV dose. For low bioavailability nanoparticles it may be necessary to use higher extravascular doses to reach similar concentration levels in blood samples. This definition can be used to estimate any extravascular bioavailability, including intramuscular injection (IM), oral administration (PO), and subcutaneous administration (SC). 3.4.4 Clearance As the most fundamentally important PK parameter measuring the body’s capacity to clear drug from the blood, clearance accounts for elimination by metabolism or excretion. Under all routes of administration, clearance can be calculated as follows: %* : = " IJ% Eq. 3.10 For IV administration the bioavailability, F, is 1 or 100%. As the dose, D, is known by the investigator, estimation of total clearance for an IV bolus of a nanoparticle requires only an accurate estimate of the AUC as described above (Eq 3.8). 110 3.4.5 Area under the moment curve If the concentration versus time data are again multiplied by time, the area under the ‘first statistical moment’ (AUMC) is obtained. The first moment at each timepoint can be calculated by multiplying the time by the blood sample concentration. Making use of the trapezoidal method again and adding the contribution to AUMC following the last observed timepoint, the AUMC can be estimated as follows: 𝐴𝑈𝑀𝐶 37C = ∑ T (/ 0 % 0 E/ 0<) % 0<) )(/ 0<) 7/ 0 ) G U ,H- ,H3 + / 1 % 1 l 5 + % 1 B 5 + Eq. 3.11 This parameter is useful to estimate several additional PK parameters, including the mean residence time and the volume of distribution at steady state. 3.4.6 Mean residence time Upon administration, drug molecules distribute throughout the body and reside in the body for various periods. Mean residence time (MRT) is the average amount of time a nanoparticle will reside in the body: 𝑀𝑅𝑇 = IJK% IJ% Eq. 3.12 The MRT can be directly estimated from any nanoparticle PK profile simply by dividing the AUMC by the AUC. 3.4.7 Mean absorption time All extravascular doses (IM, PO, SC, etc.) have an absorption phase before the nanoparticle enters the circulatory system. Mean absorption time (MAT) is the average amount of time drug molecules take to appear in blood samples. It can be calculated by subtracting the MRT of IV dose from the MRT of an extravascular dose as follows: 111 𝑀𝐴𝑇 = 𝑀𝑅𝑇 4( −𝑀𝑅𝑇 '( Eq. 3.13 The MAT is thus a useful measure of how long it takes for a nanoparticle formulation to be absorbed. In contrast, bioavailability (Eq. 3.9) measures the total extent to which a nanoparticle is absorbed. 3.4.8 Volume of distribution The extent of drug distribution is quantified by the apparent volume of distribution V, which as described above is the amount of drug in the body divided by the concentration of drug observed in a blood sample. One way to estimate V is based on the initial concentration observed after administering a known dose, DIV, of drug by IV administration: 𝑉 = " ?> % @ Eq. 3.14 C0 is the drug concentration immediately after dosing. Notably, V is an apparent volume, which for many small molecules greatly exceeds the physical volume of blood or even the body itself. In contrast, due to their high hydrodynamic radius and steric stabilization, many nanoparticles dilute only into the pool of available plasma. Thus, nanoparticles often have very low volumes of distribution compared to many small molecule drugs. The lower bound on V is the total volume of blood or plasma in the subject. 3.4.9 Volume at steady-state The volume of distribution at steady-state (Vss) is defined as the volume of distribution when pseudo-equilibrium between blood circulation and tissue is reached, which can be estimated as follows: 112 ( AA : = 𝐶𝑙 𝑀𝑅𝑇 = " IJK% IJ% + Eq. 3.15 Vss is always equal to or greater than V; therefore, if a dose, D, is needed to reach a target concentration (Eq. 3.5), the volume at steady-state will always yield a higher loading dose than the initial volume of distribution. This elevates blood concentrations to a greater extent immediately after the first dose, which may be problematic if a nanoparticle exhibits a dose-limiting acute toxicity in the central pool of blood. Thus, using the initial volume of distribution to estimate a loading dose yields more conservative peak nanoparticle levels, but may require a longer delay to achieve steady-state effects in pharmacological target tissues. 3.5 Compartmental modeling of parameters While non-compartmental analysis described above is an excellent strategy for quantifying and comparing relevant PK parameters for nanoparticles, it cannot predict the expected blood levels under different doses, dose intervals, routes of administration, or make other helpful predictions. An alternative is to apply a model based on mass balances around one or more ‘compartments.’ One of these compartments will represent the location of the sample, and the other compartments may represent sites of drug absorption, target tissues, clearance organs, or unspecified tissues. To make solutions tractable, the assumption is usually made that fluxes between compartments are either first or zero-order. The solution of these models requires either analytically solving or numerically integrating a system of linear differential equations, which is beyond the scope of this chapter. Compartmental PK modeling can estimate many of the same parameters as non-compartmental analysis. In addition, compartmental models can estimate concentrations at timepoints not assayed. Through the principle of 113 ‘superposition,’ the solution for single dose can be added sequentially together in time to estimate the profile for a multi-dose regimen. There are a number of analytical solutions for common PK cases; however, compartmental modeling can also make use of computational packages that do not require an analytical solution to fit or predict PK profiles. In the remainder of this section, three very common compartmental models for a bolus dose will be examined: i) elimination of an IV dose for a drug with one-compartment PK; ii) distribution and elimination of an IV dose for a drug exhibiting two-compartment PK; and iii) the absorption and elimination of an extravascular dose for a drug exhibiting one-compartment PK. 3.5.1 One-compartment IV bolus For the simplest case after an IV dose, DIV, some drugs and nanoparticles behave as if they were introduced into a single, well-mixed compartment. For example, due to its rapid decline below detectable conditions, the non-PEGylated hydrogel nanoparticle discussed in section 3.7.4 fits well into a one-compartment model(Perry et al., 2012). The model assumes the bolus mixes instantaneously into a single compartment, that observed concentrations reflect an apparent volume of distribution, V, and that a single flux accounts for total drug clearance, via a first-order rate constant, kelim (Fig. 3.5A). Due to the additivity of clearance (Eq. 3.3), this flux is the sum of multiple mechanisms of clearance. Based on these assumptions, a mass balance integrates into the following solution: 𝐶(𝑡) = 𝐶 3 𝑒 7L 2B0/ / = " ?> ( 𝑒 7L 2B0/ / Eq. 3.16 114 When PK data is plotted on a semi-log scale and fit to the above equation, the y-intercept gives an estimate of C0, from which V can be estimated (Eq. 3.14). As a first-order process, the flux arrow leaving the blood compartment has the definition: rate of loss from compartment = 𝑘 )*,+ 𝑋(𝑡) = 𝑘 )*,+ 𝑉 𝐶(𝑡) Eq. 3.17 Recognizing the rate of loss from the compartment must equal the defined rate of clearance from the body (Eq. 3.1) leads to the useful relationship: 𝐶𝑙 = 𝑘 )*,+ 𝑉 Eq. 3.18 The above holds for all compartmental models with a single, first-order flux representing clearance from a blood-compartment with an apparent volume of distribution, V. It should be noted that direct integration of Eq. 3.16 from time zero until infinity results in an AUC as follows: 𝐴𝑈𝐶 = ∫ 𝐶 3 𝑒 7L 2B0/ / /HC /H3 𝑑𝑡 = % @ L 2B0/ Eq. 3.19 Which using Eq. 3.10 estimates the same Cl as Eq. 3.18. Finally, it is useful to relate the half-life to the kelim, CL, and V, which can be obtained by substituting the relationship of a half-life into Eq. 3.18: 𝑡 2/G, )*,+ = NO (G) L 2B0/ = NO(G) ( %* Eq. 3.20 This relationship shows that even under constant clearance, an increase in the volume of distribution has the direct effect of increasing the half-life. Thus, when comparing related nanoparticles, it is only possible to state that an increase in observed half-life means that 115 clearance has decreased if the volume of distribution remains unchanged. This is why it is important to calculate clearance and volume of distribution, in addition to the half-life. 3.5.2 Two-compartment IV bolus While some drugs and optimized nanoparticles fit the one-compartment model quite well, this is not generally the case. Specific examples of structure-function studies for nanoparticles that fail to fit one-compartment models are found below in sections 3.7.1, 3.7.3 and 3.7.4, such as liposomes, antibodies, and PEGylated hydrogel nanoparticles(T. Allen & Everest, 1983; Lyon et al., 2015; Perry et al., 2012). To visualize deviations from the one-compartment behavior, PK profiles should always be plotted with the Y-axis concentration on a log scale. Semi-log plotting makes it easy to determine if the nanoparticle follows a single log-linear decay (Eq. 3.16), or if another model is required. If the semi-log plot clearly shows a rapid early decline in concentration, followed by a Figure 3. 5: One-compartment IV bolus model A) Model depiction for an IV bolus with one-compartment behavior; B) A representative PK profile of a one-compartment model. On a semi-log scale, the blood concentration decreases by a straight line, which indicates it is well-fit to a single exponential decay with a slope of -kelim. 116 slower, log-linear elimination phase then the PK profile would be better described by two- compartment model (Fig. 3.6A). In addition to the assumptions of the one-compartment model, the two-compartment model assumes that there is a second ‘tissue’ compartment that serves as a reservoir for material being transferred from the blood compartment. This tissue compartment is filled and drained by first-order fluxes with constants, k12, and k21 respectively. Based on these assumptions, a mass balance integrates into a solution of the following form: 𝐶(𝑡) = 𝐴𝑒 7P/ +𝐵𝑒 7Q/ Eq. 3.21 Where ‘macroconstants’ A, B, a, and b, can be fit with nonlinear graphing software (Fig. 3.6B). If defined such that a is greater than b, then the distribution half-life can be determined as: 𝑡 2/G,1,R/ = NO (G) P Eq. 3.22 and the observable elimination half-life as: 𝑡 2/G,)*,+ = NO (G) Q Eq. 3.23 Direct integration of Eq. 3.21 from time zero until infinity results in an AUC as follows: 𝐴𝑈𝐶 = ∫ Z𝐴𝑒 7P/ +𝐵𝑒 7Q/ [ /HC /H3 𝑑𝑡 = I P + S Q Eq. 3.24 Which using Eq. 3.10 can be used to estimate Cl. At time zero, Eq. 3.21 simplifies to: 117 𝐶 3 = 𝐴+𝐵 Eq. 3.25 Using this relationship and Eq. 3.14 allows the estimation of the apparent volume of distribution specific to the blood sample compartment, V1: 𝑉 2 = " ?> IES Eq. 3.26 Eq. 3.18, which also applies to the two-compartment model, can be rearranged as follows: 𝑘 )*,+ = %* ( ) Eq. 3.27 Estimates for Cl and V1, can be used to estimate kelim, which is equivalent to the single elimination flux constant described in the one-compartment model above. Based on their mathematical relationship, a, b, and kelim cannot equal to one another. Thus, the observed half-life of elimination does not have the same value as the estimated half-life of the flux associated with clearance. While its derivation is beyond the scope of this chapter, the relationship between macroconstants (a, b) and the ‘microconstants’ (kelim, k12, k21) is defined so that they simultaneously satisfy both of the following relationships: 𝛼+𝛽 = 𝑘 )*,+ +𝑘 2G +𝑘 G2 and 𝛼𝛽 = 𝑘 )*,+ 𝑘 G2 Eq. 3.28 Rearranging the above equations, it is possible to first estimate: 𝑘 G2 = PQ L 2B0/ Eq. 3.29 and then: 𝑘 2G = 𝛼+𝛽−𝑘 )*,+ −𝑘 G2 Eq. 3.30 118 Thus, it is possible to estimate all of the microconstants for the two-compartment model by first fitting the PK profile to the bi-exponential (Eq. 3.21) The remaining macroconstants, A and B, are compound parameters, which depend on a number of other parameters as follows: 𝐴 = " ?> (P7L +) ) ( ) (P7Q) Eq. 3.31 and: 𝐵 = " ?> (L +) 7Q) ( ) (P7Q) Eq. 3.32 For a one-compartment nanoparticle, the entire PK profile may be accurately fit by just 4 or 5 accurate time-points spaced over 2-3 half-lives. In contrast, for a two- compartment nanoparticle with both distribution and elimination phases, it is important to collect at least 3-4 timepoints centered around the distribution half-life as well as 3-4 timepoints more broadly centered around the elimination half-life. Additional timepoints are essential, because the bi-exponential equation has 4 independent parameters (Eq. 3.21), which for 4 or fewer timepoints leaves no degrees of freedom to fit the model. Having accurately estimated all 4 macroconstants, it is then possible to estimate the microconstants, the clearance, and the volume of distribution stepwise in the order presented above. With these parameters, investigators can ask how changing the nanoparticle’s physicochemical properties might specifically affect the PK profile. A striking feature of many nanoparticle PK profiles is how they often have volumes of distribution nearly as low as the blood or plasma volume(T. Allen & Everest, 1983; Arvizo et al., 2011; Banskota, Yousefpour, Kirmani, Li, & Chilkoti, 2019; C. Lee et al., 2019; Lyon et al., 2015; Perry et al., 2012; P. Wang et al., 2018). However, even those formulations 119 with significant two-compartment behavior are associated with a decrease in AUC, and increase in clearance, and overall lower blood concentrations. Thus, adopting specific modifications to the nanoparticle that resist either clearance or extensive two- compartment distribution may be needed to achieve therapeutic endpoints in preclinical models. 3.5.3 One-compartment extravascular (EV) bolus Many preclinical PK studies of nanoparticles explore only IV administration. There are excellent reasons for this, as a clear understanding of the IV disposition of the nanoparticle should precede any other route of administration. There are excellent Figure 3. 6: Two-compartment IV bolus model. A) Model depiction for an IV bolus with two-compartment behavior; B) A representative PK profile of a two-compartment model. The blood concentration follows two-phase exponential decay with both distribution and elimination phases. Following an IV bolus, material in the blood compartment begins to distribute to the tissue compartment. After the flux from blood to tissue and tissue to blood equalize, the profile enters an elimination phase, which can be recognized as a straight line on semi-log plot. A best-fit line with intercept B and slope b can be fit to the elimination phase. Subtraction of the elimination phase contribution during the distribution phase, generates residuals, which can then be plotted as a ‘residual’ line representing with Y- intercept of A and slope a. 120 reasons to study extravascular delivery. For example, oral administration could allow patients to easily take nanomedicines by pill. Even if oral administration is not feasible, the avoidance of IV administration would advance nanomedicines. Subcutaneous administration offers easier patient self-administration, nanomedicine access to the lymphatics, and extension of the MRT in the blood compartment through extended release. As in prior sections, simple compartmental models can be derived for an extravascular bolus of a drug that exhibits one-compartment PK (Fig. 3.7A). A compartment representing the dose site has been included, which could represent a subcutaneous pocket, the GI tract, or the peritoneal cavity. The key assumptions of this model are that the extravascular bolus, DEV, appears instantaneously in the dose site, that drug is absorbed by a first-order process with constant kabs, that only a fraction of the dose, F, reaches detection in the blood sample compartment, that the concentrations observed in the blood sample compartment reflect an apparent volume of distribution, V, and that total clearance is accounted for by a first-order process with rate constant, kelim. Another important assumption of this model is that kelim is not equal to kabs. Based on these assumptions, mass balances can be solved as follows: 𝐶(𝑡) = : " => L .CA ((L .CA 7L 2B0/ ) (𝑒 7L 2B0/ / −𝑒 7L .CA / ) Eq. 3.33 To fit this equation to an absorption PK profile, the following form can be used: 𝐶(𝑡) = 𝐵(𝑒 7Q/ −𝑒 7P/ ) Eq. 3.34 Integration of the above equation from zero to infinity allows estimation of the AUC as follows: 121 𝐴𝑈𝐶 = ∫ Z𝐵𝑒 7Q/ −𝐵𝑒 7P/ [ /HC /H3 𝑑𝑡 = S Q − S P Eq. 3.35 There are two ways to interpret data fit to Eq. 3.33. Since kelim and kabs cannot be equal for this solution, one of the two processes must have a longer half-life. Thus, the process with the longer half-life remains a significant contribution at the terminal phase of the PK profile. Conversely, the process with the shorter half-life will decay nearly to zero by the terminal phase. From an extravascular PK profile alone, it is impossible to determine whether the terminal half-life reflects either kelim or kabs. If an IV one- compartment PK profile for the same nanomedicine has already been quantified, then the kelim and V will be known. Based on this knowledge, it may be possible to interpret Eq. 3.34 by one of two approximations. First, if absorption is faster than elimination (kabs >> kelim) then: 𝑘 .TR = 𝛼 and 𝑘 )*,+ = 𝛽 Eq. 3.36 Using the fit parameter B from Eq. 3.34, the volume of distribution divided by bioavailability can be estimated: ( : = " => L .CA S(L .CA 7L 2B0/ ) Eq. 3.37 Alternatively, if absorption is slower than elimination (kabs << kelim) then: 𝑘 .TR = 𝛽 and 𝑘 )*,+ = 𝛼 Eq. 3.38 Using the fit parameter B, and the volume of distribution divided by bioavailability can be estimated: ( : = " => L .CA S(L 2B0/ 7L .CA ) Eq. 3.39 122 For either approximation, substituting the dose DEV into Eq. 3.10 then allows estimation of the clearance divided by bioavailability: %* : = " => IJ% Eq. 3.40 The absorption half-life can be determined as: 𝑡 2/G,.TR = NO (G) L .CA Eq. 3.41 The elimination half-life can be estimated as: 𝑡 2/G,)*,+ = NO (G) L 2B0/ Eq. 3.42 By solving the time where the first derivative of the above Eq. 3.33 becomes equal to zero, it is possible to calculate the time until the peak concentration is reached: 𝑡 +.U = NO( D .CA D 2B0/ ) (L .CA 7L 2B0/ ) Eq. 3.43 Note that the time until the maximum concentration is independent of the dose, volume of distribution, or bioavailability. It only depends on the absorption and elimination rate constants. To estimate the peak concentration, Cmax, one only needs to substitute the tmax into Eq. 3.33. Lastly, it is important to note that analysis of an extravascular PK profile only allows the determination of V/F and Cl/F. It is impossible to determine the absolute V or Cl unless the bioavailability is known. Bioavailability would need to be determined by comparison with an IV dataset for the same nanoparticle. 123 3.6 Interpreting the PK of drug carriers Drug carriers are sometimes designed to prolong the blood circulation and improve PK properties of small molecule drugs, which might enhance potency while diminishing systemic toxicity. The modulatory effect of drug carrier needs to be validated by comparing the drug and carrier PK profiles. Through monitoring carrier and drug PK simultaneously post-administration, actionable information regarding drug release kinetics and mechanism can be obtained. 3.6.1 Renal filtration cutoff The glomerular capillary wall is composed of three layers: endothelium, glomerular basement membrane (GBM) and podocyte extensions of glomerular epithelial Figure 3. 7: One-compartment extravascular bolus model. A) Model depiction for an extravascular bolus for a drug with one-compartment behavior; B) A representative PK profile of the one-compartment extravascular bolus model. Following an extravascular dose, material in the absorption site starts entering the blood compartment. After the absorption phase ends, the profile enters an elimination phase, which can be recognized as a straight line on semi-log plot. A best-fit line with intercept B and slope b can be fit to the terminal ‘elimination’ phase. Subtraction of the early absorption phase data from the contribution of the elimination phase, generates residuals that plot as a log-linear line with Y-intercept of B and slope a. If elimination is faster than absorption, then kelim = a and kabs = b; however, if absorption is faster than elimination, then kelim=b and kabs=a. This has consequences for interpreting the PK for oral and SC doses of nanoparticles. 124 cells(Longmire, Choyke, & Kobayashi, 2008). The endothelium is fenestrated, allowing free filtration. The spaces between podocyte extensions constitute the primary size barrier with a physiologic pore size of 4.5-5 nm. Consistent with this size range, the molecular weight cutoff for macromolecules is thought to be 30-50 kDa. Nanoparticles < 6 nm are usually small enough to undergo free glomerular filtration; however, nanoparticles > 8 nm do not filter through the GBM due to the size limit. Drug carriers exceeding that limit have significantly lower renal elimination and thus longer circulation half-life. Also, the highly negatively charged GBM makes it easier for positively charged molecules to filter relative to those negatively charged ones, which are repelled by the GBM due to charge-charge repulsion. Charge-charge interaction is especially important for those macromolecules with sizes around the renal filtration size cutoff. These observations inspired various nanoparticle architectures that decrease both renal and total clearance. 3.6.2 Absorption from EV administration SC administration outperforms IV administration in several aspects, including ease of administration, high patient compliance, low risk of systemic infection and, in some cases, prolonged drug exposure(McDonald, Zepeda, Tomlinson, Bee, & Ivens, 2010). However, the bioavailability of SC administered therapeutics is limited by incomplete absorption. In addition, biotherapeutics with different MW reach the systemic circulation via different routes. Compounds with MW less than or equal to 16 kDa usually enter the systemic circulation through blood capillaries, while biotherapeutics with higher MW have to be transported through the interstitial fluid. They are taken up into lymphatic capillaries draining the injection site and eventually are channeled back into the circulatory system (Richter, Bhansali, & Morris, 2012). In addition, extracellular matrix (ECM) maintains a 125 negatively charged environment for interstitial fluid, which may delay the absorption of positively charged macromolecules in comparison with negatively charged macromolecules with similar MW. Oral dosage, due to its convenience, cost-effectiveness, and high patient compliance, is the most preferred route of administration. Orally administered drugs need to be absorbed by the small intestinal tract before entering the systemic circulation. The incomplete absorption and first-pass elimination greatly constrain the bioavailability of orally administered nanoparticles, often ranging from 1-5%. Strategies to increase the magnitude and reproducibility for the oral bioavailability of nanoparticles would overcome major barriers to their use. Incidentally, were orally bioavailable nanoparticles developed that could be taken daily, they might not require a 2-day half-life in humans, which was necessary to enable parenteral/IV administration of clinically approved liposomes(Gabizon et al., 2003). 3.6.3 Opsonization and capture by the reticuloendothelial system Opsonization is the process through which nanocarriers or foreign organisms are tagged with opsonin proteins, thereby making them easily recognizable to phagocytes. Nanoparticles have been shown to be readily opsonized upon entering the blood circulation and rapidly taken up by the reticuloendothelial system (RES) consisting of phagocytic cells such as monocytes and macrophages. But neutrally charged particles, including zwitterionic species, have much lower opsonization rate than charged nanoparticles(Roser, Fischer, & Kissel, 1998). To reduce opsonization, different shielding groups blocking electrostatic and hydrophobic interactions, such as polysaccharides (dextrans) and polyethylene glycols (PEGs), have been broadly investigated(Nie, 2010). 126 Strategies to reduce opsonization will remain essential to maintain the PK properties of nanoparticles. 3.7 Structure-function studies to optimize PK parameters 3.7.1 The effect of particle size distribution Liposomes composed of bovine brain sphingomyelin-phosphatidylcholine (SM- PC), 4:1 molar ratio, were used to deliver [ 14 C] sucrose, as a hydrophilic tracer (T. Allen & Everest, 1983). Three different methods were used to formulate liposomes, which generate different distributions of particle sizes. The small unilamellar vesicles (SUV) are the smallest and least polydisperse. The reverse phase evaporation vesicles (REV) are intermediate in size. In contrast, the multi-lamellar vesicles (MLV) are large and polydisperse. Their PK profiles were compared in rats and replotted here in terms of concentration vs. time (Fig 3.8). All three liposomes significantly reduce the clearance compared to free sucrose. Despite having identical surface properties, the small size of the SUV promotes the highest blood concentrations, highest AUC, lowest clearance, and lowest volume at steady-state (Table 3.1). All liposome diameters are well above the renal filtration cutoff, which prevents rapid filtration. However, the larger MLV, being more subject to opsonization and RES clearance, exhibit substantially higher clearance. For the MLV, nearly 88% of their bi-exponential fit follows a fast-distribution half-life of 0.3 hr. Accumulation in organs of the RES is partially supported by higher liver and spleen accumulation. In contrast, all three liposome formulations maintain similar elimination half-lives. This data demonstrates the critical importance of reaching the window of 10- 100 nm to develop clinically relevant nanoparticles. 127 Figure 3. 8: Whole blood PK profile for [ 14 C] sucrose-entrapped into sphingomyelin-phosphocholine liposomes with different sizes, which was administered IV to rats. Small Unilamellar Liposomes (SUV) lack a distribution phase, have the highest AUC, and the longest terminal half-life. Reverse phase Evaporation Vesicles (REV) have a more significant distribution phase, which reduces their AUC even though they have a similar terminal half-life. Multi-Lamellar Liposomes (MLV) have a very significant distribution phase, which greatly reduces their AUC; however, they too have a similar terminal half-life. Free sucrose has a 3 hour half-life; therefore, the majority of signal remaining in the liposome samples is a good estimate of liposome-entrapped label. Data extracted from (T. Allen & Everest, 1983). 128 Table 3. 1: Size-dependent PK parameters of [ 14 C] sucrose liposomes following IV administration to rats. Parameters (Unit) Small Unilamellar Vesicles (SUV) Reverse-phase Evaporation Vesicles (REV) Multi-Lamellar Vesicles (MLV) Free sucrose Hydrodynamic diameter (nm) 20-100 100-400 200-3000 N/A AUC (µmol/mL hr) 5.1 3.6 0.9 0.2 AUMC (µmol/mL hr 2 ) 43.9 49.6 5.1 0.07 MRT (hr) 8.5 13.9 5.5 0.4 t1/2, terminal (hr) 6.3 11.2 6.6 1.5 Vss (mL) 13.2 31.2 47.6 17.8 Cl (mL/hr) 1.6 2.2 8.7 46.6 Liver/spleen at 0.5 hrs (% of Injected Dose) 8.6 ±3.2 29.6 ± 3.2 50.5 ± 11.2 N/A Model used Two-compartment IV bolus N/A A (µmol/mL) 0.13 0.32 0.50 B (µmol/mL) 0.45 0.25 0.07 a(hr -1 ) 1.93 2.17 2.10 b(hr -1 ) 0.07 0.09 0.13 t1/2, dist (hr) 0.4 0.3 0.3 t1/2, elim (hr) 8.1 8.0 5.5 AUC (µmol/mL hr) 5.3 3.0 0.8 V1 (mL) 14.0 14.0 14.0 Cl (mL/hr) 1.5 2.6 10.4 kelim (hr -1 ) 0.11 0.19 0.74 k12 (hr -1 ) 1.53 1.00 0.36 k21 (hr -1 ) 0.38 1.07 1.12 Dose: 8 mmol of phosphocholine administered to 200 g rats. Blood volume, V1, estimated 7% of body weight. Mean±SD. Data interpreted from (T. Allen & Everest, 1983) 129 3.7.2 Surface charge Surface charge is another important consideration in the optimization of nanoparticle PK. In this case(Arvizo et al., 2011), through chemical modification authors generated four types of gold nanoparticles (AuNPs) with different surface charges, albeit consistent hydrodynamic radii of 9-10 nm: neutral (TEGOH), positive (TTMA), negative (TCOOH) and zwitterionic (TZwit). The PK profiles of these four nanoparticles were determined after IV administration (Fig. 3.9). PK parameters are summarized in Table 3.2. Some parameters were calculated in the original publication; however, the remainder have been estimated based on extracted data. Zwitterionic and neutral AuNPs clearly have the optimal PK profiles relative to charged particles, with higher drug exposure, longer MRTs, and slower elimination. Charged particles, on the other hand, get eliminated rapidly. Being more subject to opsonization, charged particles, especially positively charged particles, are more likely to be eliminated rapidly by RES clearance(Roser et al., 1998). Also, the positively charged (TTMA) particles exhibit a greater distribution phase and volume of distribution at steady state, which may reflect their adsorption to the negatively charged glycocalyx. Clearly, the PK of these nanoparticles benefits greatly from maintaining charge neutrality. 130 Figure 3. 9: Plasma concentration over time profiles of AuNPs with different surface charges were depicted after injected IV in mice. Highly charged TTMA and TCOOH exhibit the most severe distribution phase upon administration and short terminal half-life, which leads to the lowest AUC. Negatively charged TCOOH slightly outperforms positively charged TTMA due to slower redistribution. Zwitterionic TZwit and neutral TEGOH nanoparticles demonstrate more optimal PK profiles with milder distribution phases, longer terminal half-lives, and thus significantly higher AUC. Data extracted from (Arvizo et al., 2011). 131 3.7.3 Hydrophobicity Antibody drug conjugates (ADCs), which combine the specificity of an IgG antibody and the potency of small molecule therapeutics attached via chemical linker, can greatly improve the PK profile of small molecule drugs due to their ability recycle back out of hepatocytes via the neonatal Fc receptor (FcRn). Even so, excessive hydrophobicity contributed by the linker appears to induce a PK penalty. In this study different ADCs with the same antibody backbone (h1F6) were compared with different linkers (linker 1, 2 and 3) (Lyon et al., 2015). Using hydrophobic interaction chromatography, the hydrophobicity of different ADCs was sequenced as h1F6-1> h1F6-2> h1F6-3> h1F6. Then a PK study was performed as shown in Fig 3.10. Based on this data, the PK parameters have been summarized in Table 3.3. In comparison to other nanoparticles, antibodies are exceptional drug carriers with clearance (0.01 – 0.05 mL/hr) a magnitude lower than Table 3. 2: Surface charge-dependent PK parameters of gold nanoparticles following IV administration to mice. Parameters (Unit) Surface charge TEGOH TZwit TTMA TCOOH Zeta Potential (mV) -1.1 -2.0 +24.4 -37.9 AUC (µg/mL hr) 436,080 486,819 10,318 45,682 AUMC (µg/mL hr 2 ) 2,449,506 2,240,577 8,551 19,111 MRT (hr) 5.6 4.6 0.8 0.4 t1/2, terminal (hr) 6.0 3.4 0.7 0.2 V (mL) 1.9 1.5 21.1 2.3 Vss (mL) 1.8 1.5 14.0 2.1 Cl (mL/hr) 0.4 0.3 16.9 4.9 Dose: TEGOH 160 mg, TTMA 174 mg, TCOOH 224 mg, TZwit 167 mg to mice. Data estimated from (Arvizo et al., 2011) 132 observed for other optimized nanoparticles (0.1-0.5 mL/hr) in mice. Their avoidance of renal filtration and RES clearance are critical. Even so, the evidence suggests that the linker chemistry can play a large role in harnessing the innate ability of IgG antibodies to recycle back out of hepatocytes and avoid clearance. Figure 3. 10: The plasma concentration versus time profiles of antibodies with different hydrophobicity after IV administration into mice. Unconjugated h1F6 and least hydrophobic conjugate h1F6-3 have the highest AUC, due to milder distribution phases and longer terminal half- lives. Along with the increment in hydrophobicity (h1F6-3àh1F6- 2àh1F6-1), the distribution phase gets intensified and the terminal half- life gets shortened, causing an AUC reduction. Data extracted from (Lyon et al., 2015). 133 Table 3. 3: Linker-dependent PK parameters of antibody-drug conjugates following IV administration to mice. Parameters (Unit) ADCs with different linker hydrophobicity H1F6 H1F6-3 H1F6-2 H1F-1 AUC (µg/mL hr) 4,546 8,723 1,482 873 AUMC (µg/mL hr 2 ) 556,854 4,529,965 193,531 53,294 MRT (hr) 122 519 131 61 t1/2, terminal (hr) 66 375 103 66 V (mL) 0.9 1.3 1.2 1.8 Vss (mL) 1.2 2.7 4.0 3.1 Cl (mL/hr) 0.01 0.005 0.03 0.05 Model used Two-compartment IV bolus A (µmol/mL) 24.3 18.8 28.0 24.8 B (µmol/mL) 24.3 16.6 9.5 1.8 a(hr -1 ) 0.214 0.114 0.277 0.059 b(hr -1 ) 0.005 0.002 0.007 0.004 t1/2, dist (hr) 3.2 6.1 2.5 11.7 t1/2, elim (hr) 153 322 99 195 AUC (µmol/mL hr) 5,478 7,866 1,462 926 V1 (mL) 0.9 1.3 1.2 1.7 Cl (mL/hr) 0.008 0.006 0.031 0.049 kelim (hr -1 ) 0.009 0.005 0.026 0.029 k12 (hr -1 ) 0.109 0.055 0.075 0.007 k21 (hr -1 ) 0.101 0.057 0.183 0.027 Dose: 45 µg of antibody. The calculation is based on the assumption that 4-6wk old female BALB/c mice have an average body weight of 15g. Data interpreted from (Lyon et al., 2015) 134 3.7.4 Density and thickness of shielding layer Blocking the electrostatic and hydrophobic interactions has the potential to help nanoparticles evade the uptake by RES and thus prolong circulation time. The mostly investigated shielding group is poly(ethylene glycol) (PEG). Better stealth has been reported by increasing the surface density of PEG. In this case, PRINT hydrogel nanoparticles were coated with PEG into two different conformations: brush and mushroom, by high-density PEG and low-density PEG, respectively(Perry et al., 2012). As shown in Fig 3.11, the PK properties of these two confirmations were compared, with non-PEGylated nanoparticles as a negative control. Authors utilized two-compartmental modeling to calculate PK parameters, as summarized in Table 3.4. PEGylation significantly decreased the clearance, prolonged the circulation period and increased the total exposure than non-PEGylated nanoparticles. Notably, the high-density brush confirmation has superior shielding effect than low-density mushroom confirmation, yielding better PK performance. 135 Figure 3. 11: Blood concentration of nanoparticles with different PEG shielding density over time after IV administration in mice. PEGylated nanoparticles, in comparison with the non-PEGylated one, have notably milder distribution phases, longer terminal half-lives and thus significantly higher AUC. But high-density PEG appears to have better shielding effect, which results in lower distribution to ‘tissue’ compartment. Therefore, PEG brush has higher AUC than PEG mushroom. Data extracted from (Perry et al., 2012). 136 3.7.5 Serum albumin binding In addition to IgG, serum albumin can also undergo FcRn-mediated recycling, avoiding lysosome degradation and achieving longer circulation half-life. Another case reported that introducing an albumin-binding domain (ABD) to a vaccine carrier, termed as iTEP, significantly improved its PK profile(P. Wang et al., 2018). The serum concentration over time profiles of iTEP and ABD-iTEP administered subcutaneously were compared and depicted in Fig 3.12. As summarized in Table 3.5, fusing to ABD Table 3. 4: PEG-density dependent PK parameters among PRINT nanoparticles following IV administration in mice. Shielding density Parameters (Unit) PEG brush PEG mushroom Non-PEGylated PEG5k density (number/nm) 0.083 ± 0.006 0.028 ± 0.002 0 AUC (µg/mL hr) 2,863 1,726 8.3 AUMC (µg/mL hr 2 ) 73,189 33,544 4.5 MRT (hr) 25.6 19.4 0.5 t1/2, terminal (hr) 19.0 14.9 0.5 V (mL) 1.5 1.4 4.8 Vss (mL) 2.2 2.4 14.3 Cl (mL/hr) 0.087 0.126 26.2 Model used Two-compartment One- compartment A (µg/mL) 65.756 103.323 N/A B (µg/mL) 100.044 74.177 N/A a(hr -1 ) 1.503 5.009 N/A b(hr -1 ) 0.036 0.044 N/A t1/2, dist (hr) 0.46 0.14 N/A t1/2, elim (hr) 19.2 15.7 0.2 AUC (µg/mL hr) 2810 1703 10 V1 (mL) 1.5 1.2 4.8 Cl (mL/hr) 0.088 0.127 21.85 kelim (hr -1 ) 0.059 0.104 4.524 k12 (hr -1 ) 0.921 2.119 N/A k21 (hr -1 ) 0.559 2.830 N/A Dose: non-PEGylated 218.5 µg, PEG mushroom 217 µg, PEG brush 248.6 µg Data interpreted from (Perry et al., 2012) 137 significantly reduced the clearance of iTEP by 50%, yielded 4-fold higher AUC. Thus, strategies to hijack natural biological mechanisms used by proteins abundant in the blood appear to be excellent candidates for enhancing the PK of nanoparticles. Figure 3. 12: Serum concentration of vaccine carriers with or without albumin binding capability over time after SC administration to mice. Terminal half-life was only slightly altered with the addition of albumin-binding domain (ABD), which likely results from delayed absorption from the site of injection. Due to this, ABD-iTEP has clearly higher AUC than iTEP. This can be explained by lower tissue distribution (lower Vss) and reduced clearance achieved by the albumin-binding capability. In the absence of IV data, it is not clear if the ABD had an effect on bioavailability, which may also be responsible for the elevated levels observed. Concentration at time 0 was assumed to be 3 ug/mL to be presented within this log10- scale Y axis. Data extracted from (P. Wang et al., 2018). 138 3.8 Conclusion A clinically-relevant PK profile is essential to develop therapeutic nanoparticles. This should be achieved through structure-function optimization of PK parameters discussed above. The most important PK parameters include clearance, volume of distribution, half-life, and bioavailability in cases of extravascular administration. These parameters can be estimated based on blood-based concentrations versus time as inputs, which must be collected at the correct time and assayed using accurate and precise methods. Nanoparticle and/or cargo concentration in biological solutions can then be quantified by fluorescence, radioactivity, ELISA, and mass spectrometry. Optimal PK studies of nanoparticles can often be recognized because nanoparticles usually have an Table 3. 5: Albumin-binding dependent PK parameters for immune-tolerant elastin- like polypeptides (iTEP) administered SC to mice. Parameters (unit) ABD-iTEP iTEP AUC (µg/mL hr) 2,685 897 AUMC (µg/mL hr 2 ) 214,659 113,659 MRT (hr) 80 127 t1/2, terminal (hr) 54 88 Vss/F (mL) 2.6 10.4 Cl/F (mL/hr) 0.03 0.08 Model used One-compartment extravascular bolus B (µg/mL) 49.7 8.7 kabs (hr -1 ) 0.209 0.559 kelim (hr -1 ) 0.022 0.013 t1/2, abs (hr) 3 1 t1/2, elim (hr) 32 51 AUC (µg/mL hr) 2021 654 V/F(mL) 2.0 8.7 Cl/F (mL/hr) 0.044 0.113 tmax (hr) 12 6.9 Cmax (µg/mL) 34.1 7.8 Dose: 89.1 µg of ABD-iTEP and 73.8 µg of iTEP per mouse. The compartmental model fit was interpreted assuming that kabs<<kelim. Data interpreted from (P. Wang et al., 2018) 139 initial volume of distribution similar to the total blood or plasma volume. Two different analytical strategies are presented: i) non-compartmental analysis and ii) compartmental modeling. Non-compartmental analysis is based on the calculation of AUC using the trapezoidal method. Being model-independent, non-compartmental analysis is sometimes considered more robust when the timing, number, and reproducibility of concentration may be insufficient to fit an appropriate model. Compartmental modeling, on the other hand, can predict nanoparticle concentrations, which may be useful to design multi-dose regimens. For nanoparticles, unsatisfactory PK properties can arise from low hydrodynamic radius, which leads to increased renal clearance and larger distribution effects. Optimal nanoparticles are typically charge neutral, because either positive or negative surfaces increase clearance. Optimal nanoparticles also benefit from steric shielding, which reduces opsonization and clearance by the RES. Examples of nanoparticle structure-function studies evaluating several of these variables are presented and reanalyzed using the approaches described. 140 Conclusion First, I demonstrate in this Thesis that the prolyl isomerase protein known as CypA, can be bioengineered into drug carriers for a potent immunosuppressant CsA. Unlike traditional drug encapsulation strategies, this innovative strategy is surfactant- free, does not require the breakage of a covalent linkage, and is instead based on high specificity binding between a drug and its cognate receptor protein. When bound to CsA this carrier retains drug for extended durations, traffics to low pH compartments in cells, inhibits the NFAT/Calcineurin/IL-2 pathway, enhances the mean residence time following subcutaneous administration, reduces renal drug toxicity, and increases tear production in a non-obese diabetic mouse model of SS. Second, using the male NOD mouse as a model of SS-associated dry eye disease, we have identified increased IL-17A expression by LG-infiltrating CD4+ T cells as an early potential pathogenic factor in development of disease. ELP delivered CsA exert strong inhibitory effect on the principal cell type responsible for IL-17 expression, the Th17.1 cell, impeding their cellular development and cytokine secretion. More importantly, in response to physiological temperature, ELP-mediated depot formation allows the sustained release of CsA, enhancing its in vivo efficacy against SS-mediated ocular surface manifestations. 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Creator Guo, Hao (author)

Core Title Development of an elastin-like polypeptide-based cyclosporine A formulation that improves autoimmune-mediated dry eye characteristic of Sj鰃ren抯 syndrome

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Degree Conferral Date 2021-08

Publication Date 07/17/2022

Defense Date 04/02/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag autoimmune disease,cyclosporine A,dry eye,elastin-like polypeptide,interleukin-17,OAI-PMH Harvest,Sj?ren syndrome,T-cell

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Language English

Contributor Electronically uploaded by the author (provenance)

Advisor MacKay, Andrew (committee chair), Hamm-Alvarez, Sarah (committee member), Xie, Jianming (committee member)

Creator Email guohao@usc.edu,guohaoxmu@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC15613661

Unique identifier UC15613661

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