Improving drug delivery to the kidney using renal-targeted therapeutics is a promising but underdeveloped area. We aimed to develop a kidney-targeting construct for renal-specific drug delivery. Elastin-like polypeptides (ELPs) are nonimmunogenic protein-based carriers that can stabilize attached small-molecule and peptide therapeutics. We modified ELP at its NH-terminus with a cyclic, seven-amino acid kidney-targeting peptide (KTP) and at its COOH-terminus with a cysteine residue for tracer conjugation. Comparative in vivo pharmacokinetics and biodistribution in rat and swine models and in vitro cell binding studies using human renal cells were performed. KTP-ELP had a longer plasma half-life than ELP in both animal models and was similarly accumulated in kidneys at levels fivefold higher than untargeted ELP, showing renal levels 15- to over 150-fold higher than in other major organs. Renal fluorescence histology demonstrated high accumulation of KTP-ELP in proximal tubules and vascular endothelium. Furthermore, a 14-day infusion of a high dose of ELP or KTP-ELP did not affect body weight, glomerular filtration rate, or albuminuria, or induce renal tissue damage compared with saline-treated controls. In vitro experiments showed higher binding of KTP-ELP to human podocytes, proximal tubule epithelial, and glomerular microvascular endothelial cells than untargeted ELP. These results show the high renal selectivity of KTP-ELP, support the notion that the construct is not species specific, and demonstrate that it does not induce acute renal toxicity. The plasticity of ELP for attachment of any class of therapeutics unlocks the possibility of applying ELP technology for targeted treatment of renal disease in future studies.

Introduction. Fusion of therapeutic agents to Elastin-like Polypeptide (ELP) is a novel drug delivery strategy for prevention of placental drug transfer. Previous studies have used a 60 kDa ELP tag for this purpose. However, placental transfer of ELP may be size dependent. The goal of this study was to measure the effects of ELP polymer size on pharmacokinetics, biodistribution, and placental transfer of ELP. Methods. Three ELPs ranging from 25 to 86 kDa (4.1 to 6.8 nm hydrodynamic radius) were fluorescently labeled and administered by i.v. bolus to pregnant Sprague Dawley rats on gestational day 14. Plasma levels were monitored for 4h, organ levels and placental transfer determined by ex vivo fluorescence imaging, and placental localization determined by confocal microscopy. Results. Increasing ELP size resulted in slower plasma clearance and increased deposition in all major maternal organs, except in the kidneys where an opposite effect was observed. Placental levels increased with an increase in size, while in the pups, little to no ELP was detected. Discussion. Pharmacokinetics and biodistribution of ELPs during pregnancy are size dependent, but all ELPs tested were too large to traverse the placental barrier. These studies verify that ELP fusion is a powerful method of modulating half-life and preventing placental transfer of cargo molecules. The tunable nature of the ELP sequence makes it ideal for drug delivery applications during pregnancy, where it can be used to target drugs to the mother while preventing fetal drug exposure.

Preeclampsia is characterized by the development of elevated blood pressure during the second and third trimesters of pregnancy that is accompanied by end organ dysfunction. The pathogenesis of preeclampsia is multifactorial but is commonly characterized by endothelial dysfunction and the overproduction of antiangiogenic factors, including the soluble VEGF (vascular endothelial growth factor) receptor sFlt-1 (soluble Fms-like tyrosine kinase receptor 1). Previously, administration of exogenous VEGF-A, bound to a carrier protein called ELP (elastin-like polypeptide), significantly reduced free sFlt-1 levels and attenuated the hypertensive response in a rodent model of preeclampsia. However, VEGF-A administration induces multifactorial effects mediated through its direct activation of the Flk-1 receptor. In response to this, we developed a therapeutic chimera using ELP bound to VEGF-B, a VEGF isoform that binds to sFlt-1 but not to Flk-1. The purpose of this study was to evaluate the in vitro activity and pharmacological properties of ELP-VEGF-B and to test its efficacy in the reduced uterine perfusion pressure rat model of placental ischemia. ELP-VEGF-B was less potent than ELP-VEGF-A in stimulation of endothelial cell proliferation and matrix invasion, indicating that it is a weaker angiogenic driver. However, after repeated subcutaneous administration in pregnant rats, ELP-VEGF-B was maternally sequestered and reduced blood pressure when compared with saline treated animals following induction of placental ischemia (123.38±11.4 versus 139.98±10.56 mm Hg, P =0.0129). Blood pressure reduction was associated with a restoration of the angiogenic capacity of plasma from rats treated with ELP-VEGF-B. ELP-VEGF-B is a nonangiogenic, maternally sequestered protein with potential efficacy for treatment of preeclampsia.

Vascular Endothelial Growth Factor (VEGF), a key mediator of angiogenesis and vascular repair, is reduced in chronic ischemic renal diseases, leading to microvascular rarefaction and deterioration of renal function. We developed a chimeric fusion of human VEGF-A121 with the carrier protein Elastin-like Polypeptide (ELP-VEGF) to induce therapeutic angiogenesis via targeted renal VEGF therapy. We previously showed that ELP-VEGF improves renal vascular density, renal fibrosis, and renal function in swine models of chronic renal diseases. However, VEGF is a potent cytokine that induces angiogenesis and increases vascular permeability, which could cause undesired off-target effects or be deleterious in a patient with a solid tumor. Therefore, the current study aims to define the toxicological profile of ELP-VEGF and assess its risk for exacerbating tumor progression and vascularity using rodent models. A dose escalating toxicology assessment of ELP-VEGF was performed by administering a bolus intravenous injection at doses ranging from 0.1 to 200 mg/kg in Sprague Dawley (SD) rats. Blood pressure, body weight, and glomerular filtration rate (GFR) were quantified longitudinally, and terminal blood sampling and renal vascular density measurements were made 14 days after treatment. Additionally, the effects of a single administration of ELP-VEGF (0.1–10 mg/kg) on tumor growth rate, mass, and vascular density were examined in a mouse model of breast cancer. At doses up to 200 mg/kg, ELP-VEGF had no effect on body weight, caused no changes in plasma or urinary markers of renal injury, and did not induce renal fibrosis or other histopathological findings in SD rats. At the highest doses (100–200 mg/kg), ELP-VEGF caused an acute, transient hypotension (30 min), increased GFR, and reduced renal microvascular density 14 days after injection. In a mouse tumor model, ELP-VEGF did not affect tumor growth rate or tumor mass, but analysis of tumor vascular density by micro-computed tomography (μCT) revealed significant, dose dependent increases in tumor vascularity after ELP-VEGF administration. ELP-VEGF did not induce toxicity in the therapeutic dosing range, and doses one hundred times higher than the expected maximum therapeutic dose were needed to observe any adverse signs in rats. In breast tumor—bearing mice, ELP-VEGF therapy induced a dose-dependent increase in tumor vascularity, demanding caution for potential use in a patient suffering from kidney disease but with known or suspected malignancy.

Pregnancy has been a limiting factor in drug development, and pregnant females are often excluded from clinical trials. Developmental and reproductive toxicology studies are often done in mice, rats and rabbits during the drug development process for a novel therapeutic, however very little drug development occurs for disorders specific to pregnancy due to the risk of deleterious effects on the developing fetus. Nevertheless, there is still a need for drug administration during pregnancy and for development of treatments for pregnancy – specific conditions. We have approached this problem by developing a novel drug delivery system that prevents placental drug transfer. In the emerging field of biologics, the effects during pregnancy are variable. While some protein therapeutics are too large to cross the placental barrier, many biologics are monoclonal antibodies or Fc domain fusion proteins that are substrates for the placental antibody transport machinery, and these agents can be actively shuttled into the fetal circulation. Our lab has developed a drug delivery system based on a biopolymer called elastin‐like polypeptide (ELP) that does not cross the placental barrier. ELPs consist of a repetitive five amino acid motif. ELPs can be easily modified to attach therapeutic proteins or peptides, and small drugs can be chemically attached to them. Previous studies have showed that little to no ELP crosses the placental barrier, and ELPs can be safely used as carriers to prevent placental drug transfer. However, all previous work in pregnancy models has been done with a single ELP molecule of 60 kDa. Recently, we have studied the effect of molecular weight (MW) on the pharmacokinetics, biodistribution and renal deposition of ELPs in mice. However, given that pharmacokinetics could change during pregnancy and that placental transfer may be size dependent, the goal of this study was to measure the effects of polymer size on pharmacokinetics, biodistribution, and placental transfer of ELP in a rodent pregnancy model. Three ELP proteins, 25, 50 and 86 kDa, were fluorescently labeled and administered via bolus intravenous injection to timed pregnant Sprague Dawley rats on gestational day 14. Plasma clearance was determined to define the plasma pharmacokinetics. The biodistribution of each ELP construct was determined by whole‐organ ex vivo fluorescence imaging. Placental distribution was determined in feto‐amnio‐placental units by quantitative fluorescence and histological analysis. Increasing MW resulted in an increase in organ ELP levels, except in the kidneys where an opposite effect was observed. Kidney levels were 6.7, 5, and 1.7 μM for 25, 50 and 86 kDa ELP following an intravenous dose of 1.5 μmol/kg. Placental levels increased with an increase in MW from 0.05, 0.26 to 0.5 μM for 25, 50 and 86 kDa ELP, while in the pups, little to no ELP was detected. Plasma clearance rate was inversely related to MW, and terminal half‐life increased with an increase in MW. Pharmacokinetics and biodistribution of ELPs are size dependent, but they do not affect placental transfer.Support or Funding InformationSupported by NIH NHLBI Grant R01HL121527 (GLB).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Placenta ischemia, the initiating event in preeclampsia (PE), is associated with fetal growth restriction. Inhibition of the agonistic autoantibody against the angiotensin type 1 receptor, AT1-AA, using an epitope-binding inhibitory peptide ('n7AAc') attenuates increased blood pressure at gestational day (G)19 in the clinically relevant reduced uterine perfusion pressure (RUPP) model of PE. Thus, we tested the hypothesis that maternal administration of 'n7aac' does not transfer to the fetus, improves uterine blood flow and fetal growth, and attenuates elevated placental expression of miRNAs implicated in PE and FGR. Sham or RUPP surgery was performed at G14 with vehicle or 'n7aac' (144µg/day) administered via osmotic pump from G14 to G20. Maternal plasma levels of the peptide on G20 were 16.28 ± 4.4 nM, and fetal plasma levels were significantly lower at 1.15 ± 1.7 nM (p=0.0007). Uterine artery resistance index was significantly elevated in RUPP (P<0.0001), but was not increased in 'n7aac'-RUPP or 'n7aac'-Sham versus Sham. The significant reduction in fetal weight at G20 in RUPP (P=0.003) was not observed in 'n7aac'-RUPP. Yet, percent survival was reduced in RUPP (P=0.0007) and 'n7aac-RUPP (P <0.0002). Correlation analysis indicated the reduction in percent survival during gestation was specific to the RUPP (r=0.5342, p=0.043) and independent of 'n7aac'. Placental miR-155 (P=0.0091) and miR-181a (P=0.0384) expression were upregulated in RUPP at G20 but were not elevated in 'n7aac'-RUPP. Collectively, our results suggest that maternal administration of 'n7aac' does not alter fetal growth in the RUPP implicating its potential as a therapeutic for the treatment of PE.

Introduction Preeclampsia is clinically characterized by the development of elevated blood pressure combined with proteinuria or other symptoms during the second and third trimester of pregnancy. sFlt‐1, a soluble form of the extracellular domain of the Flt‐1 receptor, is up‐regulated in the blood of preeclamptic women and functions as a trap for vascular endothelial growth factor (VEGF), preventing healthy VEGF signaling to endothelial cells and contributing to endothelial dysfunction. Previously, our lab has shown that administration of a VEGF isoform, VEGF‐A, bound to a carrier protein called elastin‐like polypeptide (ELP), significantly reduced free s‐Flt1 levels and normalized blood pressure in a rodent model of preeclampsia. However, VEGF‐A administration has a host of side effects mediated thorough its activation of the Flk‐1 receptor. In response to this, we have developed a separate therapeutic chimera which utilizes ELP bound to a different isoform of VEGF, VEGF‐B, that has no known interaction with the Flk‐1 receptor but still possess the potential to bind to sFlt‐1. In this study we have conducted pharmacokinetic, biodistribution, and efficacy experiments to test our hypothesis that a biopolymer chimera of VEGF‐B (ELP‐VEGF‐B) would function as a sequestrant of sFlt‐1 in order to lower blood pressure in the reduced uterine perfusion pressure (RUPP) model of preeclampsia. Methods Pharmacokinetics were determined by injecting pregnant rats with labeled protein and measuring clearance by repeated tail vein blood draws. Biodistribution and placental transfer of ELP‐VEGF‐B was determined by ex vivo imaging of organs, placentae, and pups of animals injected with labeled protein using an IVIS Spectrum In Vivo Imaging System (IVIS). Efficacy studies were conducted in pregnant SD rats from day 13 to day 18 of gestation. Pregnant SD rats received either the RUPP or sham procedure and were subsequently administered ELP‐VEGF‐B (50mg/kg) or sterile saline, subcutaneously, on days 13, 15, and 17 of gestation. Body weights were monitored daily, and blood pressure was measured, through carotid catheters, on day 18 of gestation. Animals were then sacrificed and harvested on day 18 of gestation. Results Plasma clearance of ELP‐VEGF‐B fit well into a two‐compartment pharmacokinetic model, resulting in half lives of 48.4hr(terminal) and 0.22hr (distribution). ELP‐VEGF‐B accumulated primarily in the maternal kidney, liver, and placenta, with undetectable amounts in the pups. ELP‐VEGF‐B administration had no significant effect on maternal body weight, pup weight, or placental weight amongst any of the treatment or control groups. However, administration of ELP‐VEGF‐B in a rodent model of placental ischemia did achieve a significant reduction in blood pressure when compared to saline treated animals following induction of placental ischemia (123.38 ± 11.4 versus 139.98 ± 10.56 mmHg, p=0.0129). Conclusions The biopolymer fusion peptide, ELP‐VEGF‐B, has a pharmacokinetic profile similar to our previously tested therapeutic, EL...

Placenta ischemia, the initiating factor in preeclampsia (PE), is associated with intrauterine growth restriction (IUGR) and increased blood pressure (BP) in offspring. Yet, the only treatment for PE is delivery of the baby and placenta. The Reduced Uterine Perfusion Pressure (RUPP) rat model induced by placental ischemia at gestational day 14 (G14) mimics many facets of human PE including pregnancy-specific hypertension, an increase in the agonistic ANG II Type 1 receptor autoantibody (AT1-AA), IUGR and increased BP in the offspring. Inhibition of AT1-AA using an epitope-binding inhibitory peptide ('n7AAc') attenuates increased BP at gestational day 19 in the RUPP. Yet, whether use of ‘n7aac’ improves fetal growth and mitigates increased BP in the offspring is unknown. Thus, we tested the hypothesis that maternal administration of ‘n7aac’ improves fetal growth by attenuating reduced uterine blood flow and impaired placental remodeling. Sham or RUPP surgery was performed at G14 with administration of vehicle or ‘n7aac’ (144μg/day) via mini osmotic pump until gestational day 20 (G20). At G20 uterine artery resistance index was significantly elevated in vehicle RUPP (0.69±0.02 mm/s n=10) compared to vehicle Sham (0.48±0.02 mm/s n=8) (P<0.0001) and not increased in treated RUPP (0.49±0.02 mm/s n=10) or treated Sham (0.48±0.02 mm/s n=9). Fetal weight was significantly reduced in vehicle RUPP (3.24±0.2 g) compared to vehicle Sham (3.92±0.05 g) (P=0.013) and not decreased in treated RUPP (3.70±0.04 g) or Sham (3.98±0.10 g). Litter size of viable pups at G20 was only reduced in treated RUPP (5.3±1.4) compared to vehicle Sham (11.56±0.7) (P=0.003). Importantly, using in vivo imaging, little to no auto fluorescence of rhodamine-labeled peptide (480 μg/kg/day, n=4) was detectable in the pups at G20. Thus, our results demonstrate that maternal treatment with ‘n7aac’ in the RUPP rat model of PE improve UARI, which is associated with improved fetal weight at G20 in response to placental ischemia. Whether this benefit continues to birth and mitigates increased BP in IUGR offspring is unknown but is the focus of future studies. In conclusion, inhibition of the AT1AA during PE may not only provide benefit to the mother, but may also be associated with benefit in the offspring.