Sn2 Lipase Labile Prodrugs and Contact-Facilitated Drug Delivery for

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Chapter 8

Sn2 Lipase Labile Prodrugs and Contact-Facilitated Drug Delivery for Lipid-Encapsulated Nanomedicines D. Pan,1 G. Cui,2 C. T. N. Pham,3 M. H. Tomasson,4 K. N. Weilbaecher,5 and G. M. Lanza*,6 1Departments

of Bioengineering, Materials Science and Engineering and Beckman Institute, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, United States 2Department of Medicine, Division of Cardiology, Washington University Medical School, St. Louis, Missouri 63108, United States 3Department of Medicine, Division of Rheumatology, Washington University Medical School, St. Louis, Missouri 63110, United States 4Department of Internal Medicine, Division of Hematology, Oncology and Blood and Marrow Transplantation, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, United States 5Department of Medicine, Division of Oncology, Washington University Medical School, St. Louis, Missouri 63110, United States 6Department of Medicine, Division of Cardiology, Washington University Medical School, St. Louis, Missouri 63108, United States *E-mail: [email protected].

The concept of achieving Paul Erhlich’s inspired vision of a “magic bullet” to treat disease is now materializing with select monoclonal antibody therapies, but this achievement is not well replicated by current nanomedicine clinical candidates. Nanomedicine technologies have often proven unstable in vivo due to premature release of drug cargoes during circulation resulting in low therapeutic delivery to targeted cells. Compounding this nanoparticle payloads that reach target cells are typically internalized within endosomes, contributing to further drug loss and diminished intracellular drug bioavailability. Historically, size limited extravasation of nanoparticles beyond the circulation followed

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by inhomogeneous and inadequate deep penetration into disease sites has been the major nanoparticle biological barrier. However, nanomedicines can function as excipients and prolonged release systems to favorably alter drug pharmacokinetics and volume of distributions for greater efficacy and lower toxicity. Sn2 phospholipid prodrugs in conjunction with a contact-facilitated drug delivery mechanism have been found to minimize premature drug diffusional loss during circulation and to increase target cell bioavailability. The Sn2 phospholipid prodrug approach has been applied equally well for vascular constrained lipid-encapsulated particles delivering anti-angiogenic therapies, such as fumagillin or docetaxel, and to micelles penetrating through inflamed endothelium into disseminated cancers, such as in multiple myeloma with anti-cMYC payloads. Innovations like Sn2 phospholipid prodrugs in combination with the contact-facilitated drug delivery mechanism are poised to contribute to the translational success of nanomedicines by increasing efficacy and safety for an array of poorly treated diseases.

Introduction Nanomedicine can offer alternative approaches to intractable medical problems by providing probes to detect and characterize disease based on the expression of cell surface biomarkers. Using the same platform technology when appropriate, therapeutic compounds can be more specifically homed to lesions. 1A diverse spectrum of nanotechnologies including solid metal particles, engineered viral systems, polymeric nanosystems, and various lipid-based particles has been researched for medical imaging and drug delivery applications (1). Lipid-based particles have inherently high biocompatibility, a general ease of lipid functionalization, and a “soft” compliant 3D morphology that contributed to early and continued clinical success. The best-known nontargeted translational example of liposomal technology is Doxil™, a pegylated liposomal formulation (2–5). Doxil™ elimination follows a typical bi-exponential curve characterized by a rapid distribution phase with a 2-hour half-life and a much slower beta-elimination rate (~45 hours t1/2) (3). By incorporating doxorubicin into the aqueous phase of liposomes, the systemic drug volume of distribution in patients was decreased from 254 liters to 4 liters. This constrained volume of distribution and extended circulatory half-life, markedly improved the efficacy and tolerance of doxorubicin. The success of Doxil™ and the coincident advent of monoclonal antibody technology helped fuel the current era of targeted particle-based drug delivery. One of the earliest biological barriers recognized in cell culture for ligand-targeted particles was their uptake and internalization within endosomes, which reduced the effectiveness of the compound (6). Innumerable approaches 190 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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to overcome endosomal related losses of therapeutic cargoes of drugs, oligonucleotides, and proteins have been explored including cell-penetrating peptides, such as Tat (transactivator of transcription) (7). However, these cationic peptides interacted with cell-associated glycosaminoglycans and payloads were subsequently internalized by and lost to endocytosis. Arginine-rich cationic peptides coupled directly to peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO) offered improvement at non-cytotoxic doses (7), but this did not hold for cationic nanoparticles (8). In the context of siRNA and gene delivery, effective delivery of the nucleic acid-based therapy from endosomes in the cytosol to the nucleus has achieved some promising successes (9–26).

I. Contact-Facilitated Drug Delivery i. Mechanism In contradistinction to investigators seeking to internalize particles into cells for drug delivery, a novel approach called contact-facilitated drug delivery (CFDD) was developed (27). CFDD is a slow second order process dependent upon the persistent interaction of the phospholipid-encapsulated nanoparticle with target membrane surfaces. Figure 1 illustrates the transfer of rhodamine-coupled phosphatidylethanolamine, a particle membrane marker, into the outer cell membrane and then inner membranes of a C32 melanoma cell in culture (28). Close microscopic examination (inset) revealed streaming of the fluorescent phospholipid from the particle into cell membrane.

Figure 1. Contact facilitated drug delivery illustrated with rhodamine perfluorooctylbromide (PFOB) nanoparticle bound to C32 melanoma cell (transfected with Rab GFP endocytic markers). Reproduced with permission from reference (28). Copyright (2008) Elsevier Ltd. (see color insert) Electron microscopic examination captured the hemifusion complexation of the monolayer of a perfluorocarbon (PFC) nanoparticle (~250nm) with the bilayer of target cell membrane (29). Unlike ligand-receptor interactions that occur in equilibrium, the hemifusion of the particle with the cell membrane is irreversible and the surfactant entrapped drug payload is delivered directly into 191 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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the outer membrane. Translation of the phospholipid-drug complex into the outer membrane can occur at 4°C, independent of ATP, but the translation of the phospholipid-prodrug from the outer to the inner membrane leaflet requires ATP. Since the inner cell membranes, excluding mitochondrial membranes, are contiguous, drug is transported throughout the cytosol. CFDD delivers the “kiss of death” while circumventing endosomal particle internalization and drug payload losses. (Figure 2)

Figure 2. SEM showing perfluorooctylbromide (PFOB) nanoparticle hemifusion complex to C32 melanoma cell. Reproduced with permission from reference (29). Copyright (2008) American Chemical Society. (see color insert) ii. Preclinical Examples of Drug Delivery For some very hydrophobic drugs, such as fumagillin, the contact facilitated drug delivery mechanism proved effective in vivo, particularly for anti-angiogenic therapy, which was often pursued in combination with MR neovascular molecular imaging. Fumagillin is an anti-angiogenic agent, isolated from Aspergillus fumigatus, specific for proliferating endothelial cells through inhibition methionine aminopeptidase 2. Its water-soluble clinical analogue, TNP-470, was produced semi-synthetically, and effective in rodents. In patients, it possessed only anecdotal effectiveness even at high doses, which often were complicated by numerous toxicities, including neurocognitive dysfunction (30–32). The effectiveness of fumagillin as a nanomedicine was first shown In the Vx2 syngeneic rabbit tumor model using αvβ3-targeted perfluorocarbon (PFC) nanoparticles. Native fumagillin (0.049mg/kg) incorporated into the phospholipid surfactant was given in 3 serial minute doses to the animals, reducing tumor development and angiogenesis (33). This constituted a greater than 10,000-fold reduction of drug versus the water-soluble TNP-470 analogue used in clinical 192 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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studies (33). Figure 3a Similar results were obtained in hyperlipidemic NZW rabbits with early aortic atherosclerosis in which αvβ3-targeted fumagillin PFC nanoparticles provided an MR neovascular estimate of plaque progression, delivered effective fumagillin anti-angiogenic therapy, and provided quantitative follow-up of treatment response (34). Figure 3b In the serum transfer K/BxN mouse model of inflammatory arthritis, which expresses the T-cell receptor transgene KRN and the MHC class II molecule A(g7), serial αvβ3-targeted fumagillin PFC particles decreased arthritic score, ankle thickness, inflammation, proteoglycan depletion, and angiogenesis (35). Figure 3c In each example, cumulative fumagillin dosage was well below the high levels of TNP-470 (30mg to 60 mg/kg/dose) used in cancer patients with neurocognitive deficits (30, 36, 37).

Figure 3. A) 3D MR angiogenesis maps of control and integrin-targeted fumagillin NP in Vx2 model. B) Angiogenesis contrast before and 1 week after a fumagillin or control NPs in hyperlipidemic rabbits C). Decreased arthritic score and ankle thickness following targeted fumagillin in the serum transfer K/BxN model of inflammatory arthritis. Reproduced with permission from reference (33). Copyright (2008) Federation of American Societies for Experimental Biology, from reference (34). Copyright (2006) Wolters Kluwer Health/Lippincott Williams & Wilkins and from reference (35). Copyright (2009) Federation of American Societies for Experimental Biology. (see color insert) iii. Lipid-Dissolved Drug Instability Similar, to fumagillin, other hydrophobic drugs, such as paclitaxel, dissolved readily into the phospholipid membrane component of PFC nanoparticles and were retained during in vitro dissolution studies. However, when given systemically, these compounds were rapidly and prematurely lost from the PFC nanoparticle surfactant (38). Simultaneous pharmacokinetic analysis of the phospholipid-anchored homing ligands and gadolinium chelates, dissolved hydrophobic drugs, and perfluorocarbon core indicated that the functionalized lipid components were stably integrated into the particle membrane and tracked with the PFC core (unpublished), but the free chemotherapeutic agents 193 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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were prematurely leaked from the particles into blood. As a theranostic, the molecular imaging functions were outstanding, but systemic drug retention was compromised. Closer examination of fumagillin dissolved into the nanoparticle surfactant also revealed that despite its efficacy in preclinical models, it diffused into the circulation during particle transit (39). Perhaps only 10 to 20% of the initial fumagillin drug load was retained and delivered to targeted neovasculature, but the remaining low dose of this highly potent anti-angiogenic was effective in vivo. Although fumagillin has negligible off-target toxicity due to its biochemical specificity for proliferating endothelial cells, the general translational benefit for fumagillin and similar hydrophobic compounds was diminished. iv. Phospholipid Sn2 Prodrugs Following these results, prodrugs coupling the active ingredients to phosphatidyl ethanolamine were synthesized. This prodrug motif proved ineffective because the compounds were too large for the ATP dependent transfer of compound from the outer to inner cell membranes. This was a benefit for the rapid bioelimination of phospholipid anchored gadolinium chelates but problematic for drug delivery. A new series of phospholipid compounds were synthesized with the drug tethered to phosphatidyl ethanolamine through various short spacers with enzymatically labile regions, such as esters. These were generally ineffective due to low drug bioavailability caused low release rates from the particle surface or low uptake of the liberated drug into the target cells when compared with free drug alone. Consequently, a different approach to a phospholipid prodrug was imagined wherein the active pharmaceutical ingredient (API) is coupled to the Sn2 acyl position (i.e., stereospecific numbered hydroxyl group of the second carbon of glycerol) (39, 40). This design retains the advantages of contact facilitated drug delivery previously established and stably incorporated the phospholipid prodrug into surfactant membrane of nanoparticles during self-assembly (27). Moreover, with the active pharmaceutical ingredient (API) nestled within the protective hydrophobic environment, exposure to the surrounding media during circulatory transit was minimized. Upon docking to a cell surface receptor, contact mediated streaming of the Sn2 phospholipid prodrug into the outer leaflet of the target cell membrane was facilitated by hemifusion. Figures 1 and 2 ATP dependent translation to the inner lipid leaflet followed by rapid distribution throughout the intracellular membranes was achieved (29). In the cytosol, numerous lipases are present with the capacity to effectively liberate drug from the membrane, although specific enzymes and their trigger for activity have yet to be defined (39, 40). v. Background for Phospholipid Sn2 Prodrugs Although the concept of Sn2 phospholipid prodrugs had never been considered for targeted drug delivery in association with CFDD as pursued herein, precedent for the formation of Sn2 phospholipid prodrugs was reported by David Thompson et al (41) in the context of triggered release mechanisms. Later, 194 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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Thomas Lars Andresen and colleagues (42–52) pursued this approach to deliver chemotherapeutics as Sn2 prodrugs via untargeted liposomes. They hypothesized that increased liberation of phospholipases into blood by cancers could trigger the local release of the Sn2 linked compounds enriching drug bioavailability. Ultimately this strategy, which was the basis for a small European biotechnology start-up company, failed. These investigators noted that non-pegylated liposomes were resistant to phospholipase A2 (PLA2) drug liberation as compared to stealthy pegylated liposomes, which retained drug poorly. Unfortunately, in vivo simple liposomes were highly susceptible to intravascular destruction and bioelimination, and unsuitable for clinical translation. Physical chemical modelling suggested that pegylation undesirably enhanced water hydration around the particle and increased enzyme access to the Sn2 ester bonds leading to premature drug release by PLA2. The same lipid prodrugs incorporated into natural lipid membranes were resistant to water and enzyme penetration, protecting against Sn2 hydrolysis and premature drug loss (42–52). Targeted PFC nanoparticles, nanoemulsions and other lipid micelle particles subsequently discussed were adequately stable for rapid targeting in vivo without pegylation. In the case of the PFC particles, which are vascular-constrained by size (~200-250 nm), target saturation as evidenced by MRI molecular imaging of angiogenesis in cancer was achieved in less than 3 hours (53). Moreover, robust serial repeatability of this homing within the same animal over time allowed construction of precise Vx2 tumor 3D neovascular maps illustrating the progression of neovascular expansion (54). Protection of the drug within the outer membrane reduced water accessibility to the Sn2 ester and prevented premature drug loss until ligand-mediated binding and CFDD ensued. Via the membrane hemifusion process, the entire phospholipid prodrug is translocated into the outer membrane of the target cell in minutes and then into the internal cell membrane system.

II. Sn2 Phospholipid Prodrugs and Contact-Facilitated Drug Delivery i. Fumagillin as an Anti-Angiogenic Sn2 Prodrug In addition to premature drug loss and despite the promising preclinical nanomedicine results across different pathologies, the “druggability” of native fumagillin was inherently compromised by photochemical instability related to the conjugated decatetraenedioic tail and to reactive epoxide rings at the active site. To address these problems, an Sn2 fumagillin prodrug (Fum-PD) was envisioned and the compound was synthesized in a straightforward way in two steps involving saponification of fumagillin dicyclohexylamine salt to fumagillol and a subsequent esterification with oxidized lipid 1-palmitoyl-2-azelaoyl-sn glycero-3-phosphocholine (PAzPC) (39). (Figure 4)

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Figure 4. Structure of fumagillin with sources of instability indicated. Synthetic strategy for the preparation of sn-2 fumagillin prodrug and development of site-targeted nanoparticles: saponification of fumagillin with MeOH : water (1:1), 35% NaOH; esterification with PAzPC, DCC/DMAP; preparation of lipid thin film from a phospholipids mixture of 98.7 mole% lecithin PC, 0.15 mole% of ανβ3-ligand conjugated lipid and 1.12 mole% of fumagillin prodrug; self-assembly by brief sonication and microfluidization, perfluorocarbon, glycerin, pH 6.5, at 20,000 psi for 4 minutes. Reproduced with permission from reference (39). Copyright (2012) Future Medicine Ltd. (see color insert) The anti-angiogenesis efficacy of αvβ3-Fum-PD nanoparticles was visualized with photoacoustic microscopic imaging (PA) of neovasculature in a subcutaneous Matrigel® rodent model (55). Mice implanted with Matrigel™ 18 days previously received either ανβ3-copper oleate in oil nanoparticle (ανβ3-CuNPs), nontargeted CuNPs, or ανβ3-CuNP preceded by 10 minutes with a competitive dose ανβ3-oil only NPs (1:1). As seen in Figure 5, at 0 min, forming vascular tubules were observed by the inherent PA contrast imparted by erythrocyte hemoglobin. Following ανβ3-CuNP injection, numerous incomplete vascular sprout offshoots largely devoid of erythrocytes were noted. These sprouts were previously characterized to be nonpolarized immature endothelial cells (ανβ3+, Tie-2-, CD31+) as opposed to formed microvessels with polarized endothelium, which were ανβ3-, Tie-2+, CD31+ (56). Animals given nontargeted-CuNPs had very little change in PA signal with minimal passive accumulation and slight blood pool enhancement. In the competition group, very little change in the PA signal from 196 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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the vascular tubules or sprouts was observed. Pretreatment with ανβ3-targeted NPs comprising oil core (no Cu oleate) blocked the receptors to ανβ3-CuNP binding and even precluded significant passive blood pool accumulation.

Figure 5. In vivo PA images of the MatrigelTM plug area implanted in 4 groups of mice at 18 days using αvβ3-CuNP. (A)-(B) Targeted CuNP group. The enhanced neovasculature by Cu oleate NPs are marked by arrows in B. (C)-(D): Nontargeted CuNP group. (E)-(F): Competition group: mice received a competitive dose αvβ3-oil only NP (1:1) 10 min before αvβ3-CuNP. (G)-(H): Fum-PD group: mice received αvβ3-CuNP with Fum-PD 11 and 15 days after the MatrigelTM implantation then αvβ3-CuNP w/o Fum-PD on day 18 for PA imaging. For all PA images, laser wavelength = 767 nm. Reproduced with permission from reference (55). Copyright (2015) Ivyspring International Publisher. (see color insert) In follow up, ανβ3-CuNP incorporating Fum-PD within the phospholipid membrane were administered on days 11 and 15 post Matrigel™ implant. Again on day 18, the amount of neovasculature observed by PA imaging was sparse and similar for all groups. Following ανβ3-CuNP injection control animals that received either drug free ανβ3-CuNP or nontargeted ανβ3-CuNP with Fum-PD had marked neovascular expansion. However, animals pre-treated with ανβ3-CuNP incorporating Fum-PD had negligible neovascular sprouting, only microtubules noted at baseline were appreciated after contrast injection. Dr. Rakesh Jain first suggested “Pruning” of neovasculature in a visionary manner and in this study the concept was visually apparent with PA microscopic imaging (57–59). ii. αvβ3-Fum-PD Nanoparticles Suppress Inflammation The K/BxN model of inflammatory arthritis is a polyarticular inflammatory arthritis resembling rheumatoid arthritis (RA) (60) . Serial i.v. injection of αvβ3-Fum-PD NP after K/BxN serum transfer in mice with established arthritis 197 Ilies; Control of Amphiphile Self-Assembling at the Molecular Level: Supra-Molecular Assemblies with Tuned ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

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attenuated disease progression at 8-fold lower doses than previously reported using native fumagillin (0.3mg/kg versus 2.4mg/kg) (35, 61). By comparison, inflammation progressed unabated in mice that received αvβ3-Ctrl NP. Figure 6. Clinical scores and histologic examination of arthritic paws revealed that αvβ3-Fum-PD NP limited inflammatory leukocyte recruitment into the inflamed paws, protected against bone erosions, and minimized cartilage damage.

Figure 6. αvβ3-Fum-PD NP suppressed inflammatory arthritis in the KRN model. αvβ3-targeted particle with or without drug were administered on days 2, 3, and 4 (arrows). Clinical changes in ankle thickness (A), arthritic score (B), and body weight (C) were monitored daily. Histology on day 7 for inflammatory cell number per high power field (HPF, D, E, J), erosions (F,G,K) proteoglycan depletion (H, I, L) *p