The Embrace Hydrogel Embolic System: Evaluation Of Its Safety And Efficacy in A Rabbit Kidney Model

for more information:ali.ma@wecistanche.com

Suvranu Ganguli, MD, FSIR, Raymond Lareau, MS, Tim Jarrett, BA, and Michael C. Soulen, MD, FSIR

ABSTRACT

Purpose: To evaluate a novel aqueous-based liquid embolic (Embrace Hydrogel Embolic System, [HES]) that has been developed to embolize hypervascular tumors by filling the tumor vascular bed and solidifying it into a hydrogel. HES(Embrace Hydrogel Embolic System) was evaluated for embolization safety and efficacy relative to microspheres in a preclinical rabbit kidney model.

Materials and Methods: A renal embolization model in New Zealand white rabbits was utilized. Twenty-four rabbits underwent unilateral kidney embolization via the main renal artery with either HES(Embrace Hydrogel Embolic System) or 40-μm microspheres. Twenty-two rabbits survived the procedure and were monitored for 2, 12, 17.5, or 26 weeks before sacrifice. All rabbits underwent a repeat renal angiogram before necropsy. HES(Embrace Hydrogel Embolic System) was evaluated for nontarget embolization, safety, and embolization effectiveness as measured by recanalization and viability of embolized tissue.

Results: Both embolization materials were found to be safe, with targeted tissue necrosis and the absence of nontarget embolization. Prenecropsy angiograms found vascular recanalization in 0/14 (0%) HES(Embrace Hydrogel Embolic System)-embolized kidneys and in 3/8 (38%) microsphere-embolized kidneys (P¼ .036). Viable kidney tissue was observed in 2/14 (14%) kidneys embolized with HES(Embrace Hydrogel Embolic System) and 5/8 (63%) kidneys embolized with microspheres (P ¼ .052). All kidneys embolized with microspheres that showed vascular recanalization had viable tissue on histological sections. HES(Embrace Hydrogel Embolic System) was observed in vessels as small as 10 μm in diameter in histological analysis.

Conclusions: HES(Embrace Hydrogel Embolic System) provided deep, durable vascular bed embolization that resulted in less recanalization and, on average, less viable target tissue compared with 40-μm microspheres. No systemic effects or nontarget tissue embolization were identified.

Click to cistanche powder health benefits for kidney disease

INTRODUCTION

Current commercially available liquid-based embolic are limited by their inability to provide consistent and controlled embolization due to challenges in delivery technique. An optimal liquid embolic product should be easy to use, have reproducible and controllable deep penetration for tumor and organ applications, be safe from nontarget embolization, and be compatible with commercially available aqueous contrasts, drugs, and microcatheters.

The Embrace Hydrogel Embolic System (HES; Instylla, Bedford, Massachusetts), commercially unavailable at the time of this publication, is a biocompatible, hydrophilic hydrogel designed to offer controlled, safe, and targeted embolization(1). While HES(Embrace Hydrogel Embolic System) is being developed for the diffuse embolization of hypervascular tumors, the modulation of delivery rate allows for targeted proximal embolization in the setting of hemorrhage control. HES(Embrace Hydrogel Embolic System) is formed from the precursors of functionalized polyethylene glycol and an initiator that rapidly self-reacts to form a hydrogel in situ. The injected HES(Embrace Hydrogel Embolic System) precursors are designed to be dilution-sensitive, which reduces hydrogel embolic formation when diverted into high-flow shunts or with reflux, thus limiting non-target embolization. This report describes the preclinical application of HES and subsequent evaluation of nontarget embolization, embolization effectiveness, and embolization durability relative to commercially available microspheres in a rabbit kidney model. Micro-spheres were selected as the control agent since this study focused on bland oncologic embolization and not bleeding Control.

MATERIALS AND METHODS

Water-Based Liquid Embolic

The polyethylene glycol-based liquid embolic HES(Embrace Hydrogel Embolic System) consists of 2 low viscosity liquid precursors(polymer and initiator)that, when mixed together in the bloodstream, solidify into a soft hydrogel embolic. Before use, these precursors are mixed with commercially available iodinated contrast media for radiopacity, Upon simultaneous injection. the precursors flow deep into the vascular bed where they mix, causing the polyethylene glycol molecules to crosslink and form a soft, resorbable, water-based hydrogel cast of the distal vasculature. The hydrogel contains no solvents, the reaction produces no measurable temperature change, and the resulting vascular occlusion is rapid and coagulation independent. Over a period of approximately 6 months, the hydrogel embolic liquifies via hydrolysis, and the hydrolysis products are absorbed and cleared via renal filtration.

HES(Embrace Hydrogel Embolic System) is delivered via a microcatheter coaxially inserted within another microcatheter to form a dual-lumen system. The outer catheter can be any commercially available microcatheter having an inside diameter ≥0.027 inches, whereas the inner catheter is a 1.7-F microcatheter(Instyle). Following the navigation of the outer catheter to the target site over a commercially available guidewire, the guidewire is removed and replaced with the 1.7-F inner microcatheter, positioned with its tip just beyond the outer catheter tip. The 2 precursor lumens are primed with precursors, and the 2 precursor syringes are placed in a dual-syringe holder, ensuring simultaneous precursor injection (Fig 1). This coaxial catheter configuration ensures that the precursors do not mix before entering the bloodstream, thus preventing catheter plugging.

Rabbit Kidney Embolization Model

This study was performed with Institutional Animal Care and Use Committee approval in an independent Association for Assessment and Accreditation of Laboratory Animal Care International-accredited research laboratory compliant with all regulations governing the care and use of laboratory animals. The chronic vascular response can only be assessed in a whole-body system, and the rabbit renal artery embolization model is an established model emulating hypervascular tumor embolization (2). Twenty-four New Zealand white rabbits underwent a unilateral renal embolization procedure on day 0. The study size was selected to balance responsible animal use with adequate assessment of safety and efficacy in vivo. During the procedure, vital signs (electrocardiogram, heart rate, respiratory rate, oxygen saturation, and temperature) were monitored and documented at regular intervals. Target arteries were accessed via right common femoral artery cutdown and systemic heparin (1,000 IU, intravenous)was administered. Using fluoroscopic guidance and through an angiographic introducer sheath, a guide catheter (5F ConcierGE Judkins Right Guide Catheter; Merit Medical, South Jordan, Utah) was advanced into the main renal artery, followed by the introduction of the microcatheter (Renegade HIL-FLO 2.8 F × 135 cm; Boston Scientific, Marlborough, Massachusetts) to be used for embolic delivery. Animals were randomized to which single kidney was embolized(left or right) and embolization with either HES(Embrace Hydrogel Embolic System) or microspheres. Microspheres（40-μm Embozene Microspheres; Varian, Palo Alto, California)from the manufacturer were supplied as 2 mL of micro-spheres in approximately 5 mL of transport solution. Before delivery,5 mL of non-ionic contrast solution was added to the microsphere syringe and the suspension was agitated until homogenous, resulting in a final concentration of 2-mL beads in approximately 10 mL transport solution (50%contrast agent by volume). Embolization in all treated kid-neys was performed until complete stasis. The angiograms of the treatment site were performed before, during, and after the embolization procedures. Embolization time and amount of embolic required to obtain stasis for each treatment were noted. All procedures were performed by an interventionalist with >8 years of experience.

Animals were recovered, and clinical observations were performed at least once daily for the first 14 postoperative days and at least once per calendar week thereafter through the day of necropsy. Bodyweight/body condition scores were collected before surgery, within 14 days following surgery, monthly thereafter, and on the day of necropsy. Blood chemistry samples for hematology, coagulation, and serum chemistry were taken at 0,14, 30,60, and 90 days, and pre-necropsy.

Figure 1. Simultaneous injection of both Embrace HES(Embrace Hydrogel Embolic System) pre-cursors is achieved using a coaxial delivery system: standard high-flow microcatheter (>0.027-inch ID) with the Instylla 1.7-F inner catheter, ID = inner diameter.

Survival timepoints of2(n =4 HES,2 microspheres), 12 (4 HES,2 microspheres),17.5 (2 HES,2 microspheres), and 26(4 HES,2 microspheres)weeks were selected to focus on sub-acute,sub-chronic, and chronic safety and efficacy of the HES(Embrace Hydrogel Embolic System) compared with the control microspheres (Fig 2). Before necropsy, all animals underwent a repeat renal angiography, which was compared with the renal angiogram after embolization from day 0 and assessed for any vascular recanalization. Recanalization was defined as perfusion in a previously embolized arterial vasculature. This was followed by a comprehensive necropsy, including the examination of all external surfaces and orifices and the cranial, thoracic, and abdominal cavities and contents by a veterinary pathologist. The macroscopic observations noted at necropsy were correlated with the microscopic observations of collected tissues. All animals underwent gross examination of tissue and organ weights along with tissue collection/histological examination of the treated kidneys. The veterinary pathologist, with >10 years of experience, also assessed the presence or absence of viable kidney tissue in HES(Embrace Hydrogel Embolic System) and microsphere-treated kidney histology sections and the depth of embolic penetration in the HES(Embrace Hydrogel Embolic System) and microsphere-treated kidneys. Hydrogel in histology sections was visualized by staining slides with hematoxylin and eosin, periodic acid-Schiff, and Lugol iodine with fast green counterstain.

Figure 2. Study flow diagram.

Statistic

The Mann-Whitney rank-sum test was used to compare the time required for embolic delivery. Fisher exact test was used to compare the incidence of pre-necropsy recanalization and viable tissue findings on pathology between the groups. Student t-test was performed using Microsoft Excel 2012 (Microsoft Corporation, Redmond, Washington) to compare HES(Embrace Hydrogel Embolic System) and microsphere-embolized kidney weights at each necropsy timepoint. GraphPad Prism version 6(GraphPad Software, San Diego, California) was used for all other tests. A P value >,05 was considered statistically insignificant.

RESULTS

A total of 24 rabbits underwent an embolization procedure, with 22 surviving to the scheduled time point and used for analysis. Two animals developed hind limb paralysis secondary to baseline interventional access complications and were sacrificed at that time. Both HES(Embrace Hydrogel Embolic System) and microspheres were successfully delivered to all targeted kidneys (100%) with stasis per angiographic assessment and no angiographic evidence of nontarget embolization. Stasis was achieved with 2.3 mL ± 1.7 and 1.1 mL ± 0.4 of HES(Embrace Hydrogel Embolic System) and microsphere suspension delivered in procedures that took 3.1 minutes ±2.0 and 2.9 minutes +1.8 (P=.826), respectively

No significant differences in hematologic and chemical parameters were noted between the HES(Embrace Hydrogel Embolic System) and microsphere groups. The treatments with HES(Embrace Hydrogel Embolic System) and microspheres had a comparable nephrotoxic response, with no differences in urea nitrogen and creatinine values over time. Only minor fluctuations in clinical laboratory parameters including prothrombin time and activated partial thromboplastin time were observed, with no differences between the groups. Weight gain and body condition were no different between the HES(Embrace Hydrogel Embolic System) and microsphere groups.

Figure 3. Representative renal angiograms at days 0 and 26 weeks showed recanalization, renal artery flow, and kidney perfusion in a microsphere-embolized rabbit, with no visibility or recanalization of the renal artery or kidney in a rabbit embolized with HES.

Renal angiograms performed immediately before necropsy found renal artery blood flow in 0/14(0%) of HES(Embrace Hydrogel Embolic System)-embolized kidneys and in 3/8 (38%) of microsphere-embolized kidneys (P =.036). Microsphere-embolized kidney recanalization was observed in l kidney at 12 weeks and in 2 kidneys at 26 weeks(Fig 3). Macroscopic observations noted at necropsy and the correlative microscopic observations were consistent with anticipated renal necrosis due to vascular obstruction by both HES(Embrace Hydrogel Embolic System) and microspheres. All other tissues evaluated were macroscopically normal. Nontarget embolization was not observed in any animals.

The microsphere-embolized kidney average weights were greater than those of the HES(Embrace Hydrogel Embolic System)-embolized kidneys at 12 (4.7g±0.1 vs3.2g±0.2, P=.017),17.5(3.2g±0.9vs 2.5g±0.9, P=.646), and 26(4.5g±2.3 vs 2.3g±0.6, P=.261)weeks (mean± SEM, as shown in Figure 4. At 26 weeks, the microsphere-embolized kidneys had 93%more mass compared with the HES(Embrace Hydrogel Embolic System)-embolized kidneys. Minor differences in the weights of other organs were within the expected variation of the animal model, and no specific trends were identified.

Figure 4. Average microsphere and HES-embolized kidney weights at 12,17.5, and 26 weeks.SEM = standard error of the mean.

Histological sections from the embolized kidneys exhibited some level of viable kidney tissue in 5/8(63%)treated with microspheres and 2/14(14%)treated with HES(Embrace Hydrogel Embolic System)(P = .052). All microsphere-embolized kidneys with recanalization had viable tissue in histological sections. Further evaluation of the hematoxylin and eosin-stained histology slides in the 2-week group found HES(Embrace Hydrogel Embolic System) present in the vessels down to 10 um (Fig 5).In contrast, microspheres were not found in the vessels of <40 um diameter in control animals. In histological sections, HES(Embrace Hydrogel Embolic System) was noted to undergo gradual absorption, with a wispy, translucent to faintly basophilic appearance noted at 26 weeks. Upon HES(Embrace Hydrogel Embolic System) absorption, the vessels did not recanalize but remained occluded with fibrosis.

Figure 5. Histology section of HES(Embrace Hydrogel Embolic System)-treated kidney 2 weeks after implant. Hematoxylin and eosin stain (a), periodic acid-Schiff stain (b), and Lugol iodine with fast green counterstain (c). Differential staining of HES(Embrace Hydrogel Embolic System) is represented within the cortical arteries by “*,” afferent glomerular arterioles by “arrows,” and glomerular capillary tufts by “G.” HES(Embrace Hydrogel Embolic System) embolic essentially filled the renal vascular bed from the renal artery to the fine distal vasculature.

DISCUSSION

A limitation common to all solid embolic is that the vascular occlusion occurs proximal to the target tumor vasculature(3). Even with complete angiographic stasis of the feeding artery, the intratumoral vessels remain patent and are perfused at the microvascular level(4). Therefore, therapeutic payloads on drug-eluting embolic are often delivered into normal tissue outside of the target tumor, with randomized trials showing no significant improvement in oncologic outcomes compared with non-drug-loaded microspheres(5,6)with greater hepatobiliary injury(7,8).In addition, vessel recanalization following transarterial embolization is a common occurrence, with I study found no changes in the embolized hepatic arterial tree in 83% of patients despite an average of 6 embolizations over 4 years (9). The continued perfusion of an ischemic tumor following incomplete embolization may upregulate hypoxia-inducible factor-1 u and vascular endothelial growth factor, enabling tumor progression (10.11). Therefore, an embolic that deeply penetrates and comprehensively occludes tumor vessels with no recanalization could reduce the expression of vasogenic hormones, potentially reducing the rates of tumor progression.

Currently available liquid embolic are not deeply penetrating, water-based, absorbable, or approved for permanent implantation or peripheral use in the United States. N-butyl cyanoacrylate(TRUFILL; Johnson & Johnson, New Bruns-wick, New Jersey) is a free-flowing liquid that exothermically polymerizes upon body fluid contact. Due to the rapid polymerization, the penetration depth is limited and requires custom mixing with Lipiodol (Guerbet LLC, Princeton, New Jersey), an oil-based radio-opaque contrast before use. De-livery control is also difficult and catheter entrapment is a real concern(12). Another commercially available liquid embolic consists of ethylene-vinyl alcohol copolymer and tantalum powder in dimethyl sulfoxide(DMSO) solvent(Onyx; Medtronic, Minneapolis, Minnesota). Injected as a viscous polymer suspension, DMSO diffusion solidifies the injected embolic in the vessel. DMSO-compatible catheters are required due to solvent incompatibility with some commercially available microcatheters. The slow injection required to prevent vasospasm and DMSO flush vascular toxicity coupled with the material viscosity limits the embolic penetration depth (12).

While liquid embolic currently used in peripheral applications are limited, there is potential for a liquid embolic to be effective in hypervascular tumor applications. In this study, HES(Embrace Hydrogel Embolic System) was comparable with microspheres in safety, with comparable application times and improved embolization durability. The coaxial catheter lumen prevented catheter occlusion and the non-adhesive nature of the hydrogel embolic prevented catheter plugging by the embolic or catheter entrapment. Both the HES(Embrace Hydrogel Embolic System) and micro-spheres exhibited similar safety in this model, with animals from both the groups having normal body conditions and similar weight gain throughout the study, similar hemato-logic, and chemical parameters and organ weights(except for the treated kidneys).

While safety between the groups was equivalent, there were notable differences in the response of the treated kids-neys between the 2 embolic evaluated. Kidneys embolized with HES(Embrace Hydrogel Embolic System) exhibited no recanalization, greater organ shrinkage, and less viable tissue than the microsphere-embolized kidneys. These data suggest that the mode of HES(Embrace Hydrogel Embolic System) embolization (complete vessel filling with capillary level penetration)is fundamentally different than that of microsphere embolization (incomplete vessel filling and limited penetration depth)(Fig 6). This results in a more complete and persistent flow stasis with the HES(Embrace Hydrogel Embolic System). Additionally, the absence of vessel recanalization through 6 months suggests permanent occlusion despite eventual HES absorption.

The limitations of this study include limited sample size, a safety assessment in animals rather than humans, and anatomical differences between rabbit kidneys and hyper-vascular tumors. While safety assessment in animals has limitations, it allows for exaggerated doses. HES(Embrace Hydrogel Embolic System) randomized animals in this study received over 13 times the expected human HES(Embrace Hydrogel Embolic System) dose, making the safety findings more relevant, Additionally, even though rabbit kidneys are an imperfect model for hypervascular tumors such as hepatocellular carcinoma, they do provide for a highly branched and vascularized parenchymal tissue. This model can be studied closely and in a controlled manner for the comparison of an investigational device to an established treatment. An additional limitation was the use of only I control material （40-μm microspheres）. Other liquid embolic products were not included as they are not used routinely in hyper-vascular tumors. HES exhibited effective and permanent blood flow cessation in this study, suggesting potential efficacy for use in hypervascular tumors such as hepatocellular carcinoma. Effective, deep, complete, and durable vascular stasis following embolization may lead to more effective tumor response and potentially improved local tumor progression outcomes. As HES is water-based. there is considerable interest in the potential for drug delivery. Work in this area is underway and will be the focus of future publications.

In conclusion, both HES(Embrace Hydrogel Embolic System) and microspheres safely embolized targeted structures in this study. However, the deep, distal HES(Embrace Hydrogel Embolic System) vascular penetration suggests a different mechanism of embolization than microspheres, resulting in improved outcomes with less recanalization, more target tissue shrinkage, and reduced amounts of viable tissue. Clinical studies are required to determine if the HES(Embrace Hydrogel Embolic System) mechanism of embolization results in improved outcomes in hepatocellular carcinoma patients.

REFERENCES

1. Gangui S, Weintraub J, DiBartholomeo T, LareauR, Clesson H, Bean R. A novel aqueous Liquid hydrogel embolic for peripheral vascular applications. J Vasc Interv Radiol 2019; 30.S170.

2.Kwak BK Shim HJ, Han SM Park ES. Chitin-based embolic materials in the renal artery of rabbit; pathological evaluation of an absorbable particulate agent.Radiology 2005; 236;151-158.

3. Devcic Z, Toskich BB.Embolic locoregional therapy for the treatment of hepatocellular carcinoma: A general survey of embolization agents, characteristics, data, and applications. Endovascular Today 2020;19:54-59.

4. Johnson CG.Shamu VK, Lew EB, et aL Mcrovascularperfusion changes following transarterial hepatic tumor embolization. J Vasc Interv Radiol 2016;27:133-141.e3.

5. Mdoari K. Pomoni M.Kelekis Aet al. Prospective randomized comparison of chemoembolization with doxorubicin-eluting beads and bland embolization with BeadBlock for hepatocellular carcinoma.Cardovasc Intervent Radiol 2010;33:541-551.

6. Brown KT, Do RK Gonen M, et alL Randomized tri of hepatic artery embolization for hepatocellular carcinoma using doxorubicin eluting microspheres compared with embolization with microspheres alone.J Cin Oncol 2016;34:2046-2053.

7. Joskin JL de Bocre T, Aupein A et aL Predisposing factors of Fever n- crosis after transcatheter arterial chemoembolization in liver metastases from neuroendocrine tumors. Cardiovasc Intervent Radiol 2015;38:372-380.

8. Mornler A. Guu B. Duran R.et al. Liver and bi Eary damages following transarterial chemp embonation of hepatocellular carcinoma; comparison between deep-Altina heads and Fniornl emulsion Fur Badol 2017:27 1431-1439.

9.Erirjeri JP, Salhab HM, Covey AM,Getrajdman Gl, Brown KT.Arterial patency after repeated hepatic artery bland particle embolization. J Vasc Interv Radiol 2010:21:522-526.

10. Yana W Liz. Cin R. et a. WYIpromoes endo the lalcek depend on tumor angiogenesis in hepatocellular carcinoma by transcriptionally activating VEGFA. Front Oncol 2019; 9:1187.

11. Nahm JH, Rhee H, Kim H, et a. increased expression of stemness markers and altered tumor stroma in hepatocellular carcinoma under TACE-induced hypoxia: a biopsy and resection matched study.Ono0-target 2017; 8:99359-9e371

12. Hu J, Abadawi H, Chong BW, et al.Advances in biomaterials and technologies for vascular embolization. Adv Mater 2019; 31:e1901071.