Abstract
Background/Aim: BAT-90 is an innovative active implantable device designed for the irradiation of unresectable tumors (e.g., liver cancer) or surgical tumor beds, based on the combination of Yttrium-90 beta-emitting microspheres and a tissue adhesive hydrogel, currently used in cardio-vascular surgery. The rationale behind BAT-90 is to localize the Yttrium-90 activity on the administration site, while minimizing its body dispersion. Materials and Methods: The effective induction of necrosis in the target injection area was tested in a pig liver model, whereas the safety of BAT-90 was assessed and demonstrated in biocompatibility tests for acute systemic toxicity, intracutaneous reactivity, delayed hypersensitivity and subcutaneous implantation. Results: BAT-90 administration induced necrosis into the target site, while the safety experiments in the treated animals highlighted results very similar to the controls. Conclusion: BAT-90 could be considered as a safe and innovative treatment option for inoperable solid tumors of the liver.
Surgical excision is the treatment for resectable primary and secondary hepatic malignancy, offering the best long-term outcome (1). However, many liver tumors are inoperable, either due to infiltration, adherence, or too close location to other organs and/or vascular structures, or to their too advanced stage, which makes surgery unsafe and inadvisable. In most resectable cases, adjuvant therapy is indicated after surgical excision to eradicate potential residual cancer cells and prevent tumor recurrence (1).
Selective internal radiation therapy (SIRT), based on intra-arterial embolization of Yttrium-90 (90Y)-coated microspheres, is a treatment option that has been developed in recent years for the treatment of primary or metastatic liver cancer (2). 90Y is a pure β-radiation emitter, with a mean decay energy emission of 2.28 MeV which, when delivered to a tumor, causes cellular breakdown and tumor necrosis. The isotope’s tissue penetration is between 1 to 11 mm (average: 2.5 mm), which then limits exposure to the surrounding tissues; its half-life is approximately 64 h, with a complete decay after 21 days (3).
The efficacy of SIRT depends on the preferential arterial vascularization of liver tumors, given its administration via hepatic artery cannulation. It is however limited, in many cases, by the presence of significant vascular shunts to the surrounding organs (mostly the lungs and stomach), that can lead to actinic inflammation (2). Intra-arterial injection of radiation particles has advantages over external beam radiation of the liver, since it limits the exposure of normal parenchyma to radiation, placing the highest possible dose of radiation into the liver segment harboring the tumor (4). Restricting 90Y activity even further to the target tumor could minimize the potential side effects of the treatment, as well as the number of 90Y-charged microspheres needed, thus providing an additional safety advantage to the patients and the medical staff.
This goal led to the development of BAT-90, a novel medical device that combines the activity of 90Y microspheres with the retention capability of a surgical adhesive hydrogel composed of bovine serum albumin and glutaraldehyde (4:1 ratio), widely used for achieving hemostasis during cardiac and vascular procedures (5), as well as for treating lung alveolar air leaks in thoracic surgery (6), bronchopleural fistulae (6) or for the repair of traumatic liver lacerations (7). Both BAT-90 individual components have been marketed as medical devices world-wide for several years, with a very well-known safety profile.
The aim of the current study was to investigate in-vivo the performance of BAT-90 to induce 90Y-dependent tumor lysis on the target injection area, as well as its safety characteristics. This data will pave the way to clinical studies on the potential of BAT-90 as a new internal electron radiotherapy (IERT) tool, with the overall objective of improving the treatment of unresectable tumors in the liver and other organs, as well as of irradiating surgical beds immediately after tumor removal and therefore cleaning them from microscopic foci of cancer cells.
Materials and Methods
Animal care. All experiments were carried out with the approval of the local Animal Research Ethics Committee and the Italian Ministry of Health, as appropriate; the professional staff of the Biotechnology Research Center of the “A. Cardarelli” Hospital (Naples, Italy: pig study; local Animal Research Ethics Committee approval on February 28th 2019) and of the Eurofins Biolab (Vimodrone, Milan, Italy: biocompatibility studies with CD-1 mice, albino rabbit or guinea pig; Certification N. 2017/16 released by the Italian Ministry of Health on May 11th 2017) supervised the housing and care of the animals.
BAT-90 administration to induce necrosis into pig liver. Eight adult female large white pigs (75-100 kg, mean 83.7±9.9 Kg), 6-month-old, free of symptoms of disease and with current immunizations were enrolled in this study. All animals were routinely examined by a veterinarian in order to assess their well-being and the absence of any symptoms.
BAT-90 was prepared in sterile conditions in the radio-pharmacy of the G. Pascale Foundation (IRCCS), National Cancer Institute Naples, Italy. The total dose to be administered to animals consisted of 24 ml of surgical adhesive (BioGlue®, Cryolife Inc., Kennesaw, GA, USA) containing 60/80 mCi of 90Y microspheres (SIR-Spheres®, Sirtex Medical Ltd, St. Leonards, NSW, Australia), divided into three separate doses (see below for further details).
Following an 8-h fasting, the animals underwent a median laparotomy and liver exposure under general anesthesia. BAT-90 was administered under ultrasonographic guidance, targeting a liver ablation area of about 3.5 square centimeters, in three different liver segments: left lobe, right lobe and lower right para-caval lobe. After each injection, 1-2 min were waited for the hydrogel matrix to solidify.
After treatment, all pigs were transferred to their dedicated boxes and checked daily. The housing conditions were in compliance with current legislation; all the animals were free to move and to feed “ad libitum”. The animals were divided into four groups (2 animals each): they were sacrificed under general anesthesia 7, 14, 21, and 28 days after BAT-90 administration, respectively, and their livers harvested.
The explanted organs were immediately submitted to PET/CT investigations, in order to assess the presence of a radioactive signal in the injection site, and then provided to the pathology lab for a macroscopic and microscopic evaluation of BAT-90-induced necrosis.
Blood samples from the jugular vein were drawn before surgery (T0), after surgery and before awakening (T1) and before liver explantation (T2). Complete blood counts (CBC) and liver enzymes [alanine aminotransferase (ALT), aspartate transferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase, bilirubin] were obtained from each sample.
Acute systemic toxicity test. The test was performed according to ISO 10993-11:2017 and ISO 10993-12, in Good Laboratory Practice (GLP) conditions using CD-1 mice (male; Weight: 19.7-24 g). Two extracts of the solidified test sample of BAT-90 (300 Me/cc), one in a non-polar vehicle, cottonseed oil, and one in a polar vehicle, sodium chloride injection (0,9%), were prepared by dipping the sample in the relative solvent in order to reach a weight/volume ratio of 0.2 g/ml and incubating at (50±2)°C for (72±2 h) in dynamic conditions (orbital stiller).
The test sample obtained diluting the 50 ml/kg of the test sample extract in sodium chloride injection was intravenously injected in one group of 5 CD-1 mice (treated group), while 50 ml/kg of the test sample extract in cottonseed oil was intraperitoneally injected in another group of 5 CD-1 mice (treated group). Sodium chloride injection and cottonseed oil were injected with the same modality in two other groups used as control. The rate of injection didn’t exceed 2 ml/min.
The animals were observed immediately after the injection and after 4, 24, 48, and 72 h. Clinical signs, systemic effects and mortality (if present) were recorded.
To evaluate the toxicity of injected test sample or control vehicle this scale was used: 0=no symptoms; 1=tremors; 2=hair brising; 3=diarrhea; 4=abdominal pain; 5=sialorrhea; 6=depression state of sensorium; 7=state of excitement; 8=polypnea; 9=hypopnea; 10=tachycardia; 11=cyanosis; 12=ataxia; 13=convulsions; 14=nose-bleeding; M=death).
Intracutaneous reactivity test. The test was performed according to ISO 10993-10:2010 and ISO 10993-12, in GLP conditions using albino rabbit (male; strain: New Zealand; young adult; weight: 3,325-3,355 g). Two extracts of the solidified test sample of BAT-90 (300 Mq/cc), one in non-polar vehicle, cottonseed oil, and one in polar vehicle, sodium chloride injection (0,9%), were prepared by dipping the sample in the relative solvent in order to reach a weight/volume ratio of 0.2 g/ml and incubating at (50±2)°C for (72±2h) in dynamic conditions (orbital stiller).
0.2 ml of the two extracts and 0.2 ml of the respective solvents were intracutaneously injected in five sites of 3 albino rabbits, and macroscopic skin reactions, such as erythema, edema and eschar formation were evaluated (24, 48, and 72 h after injections).
Injection sites were examined for evidence of any tissue reaction: erythema (0=no erythema; 1=very slight erythema; 2=well defined erythema; 3=moderate erythema; 4=severe erythema) and oedema (0=no oedema; 1=very slight oedema; 2=well defined oedema; 3=moderate oedema; 4=severe oedema).
Delayed hypersensitivity test. To evaluate the BAT-90 potential sensitizing effect (see above for its dose), a Guinea Pig Maximization (GPMT) test was performed (using male albino guinea pigs; strain: Dunkin-Hartley; Weight: 300-500 g). This study was modelled after the work of Magnusson et al. (8), and complied with the standards of GLP. It was performed according to the ISO 10993-10:2010 and ISO 10993-12.
In GPMT, two extracts of the solidified test sample, one in non-polar vehicle, cottonseed oil, and one in polar vehicle, sodium chloride injection (0.9%), were prepared by dipping the sample in the relative solvent in order to reach a weight/volume ratio of 0.2 g/ml and incubated at 50±2°C for 72±2 h. in dynamic conditions (orbital stiller). For each extract, 15 guinea pigs were used: 10 treated with the extract and 5 treated with the solvent. The total number of animals was 30 (two test item extracts). The guinea pigs were treated with 3 (0.1 ml each one) double intradermal injections (induction phase): i) injected with 50:50 (v:v) stable emulsion of Freund’s complete adjuvant mixed with the solvent; ii) injected with undiluted extract; the control animals injected with the solvent alone; iii) injected with undiluted extract, emulsified in a 50:50 (v:v) stable emulsion of Freund’s complete adjuvant and the solvent (50%); the control animals injected with an emulsion of the solvent with adjuvant. After 7 days the extracts of test sample (or only extraction solvent for the control animal) were applied (0.5 ml/animal) to the skin of the animals for a period of 48 h through an occlusive patch (Topic application phase). Fourteen days after topical induction the challenge phase was performed applying the extracts of test sample (at a dose of 0.5 ml/animal for 24 h) on the right flank of each animal and about 0.5 ml of the solvent on the left flank.
In the GPMT, a score of 1 or greater for redness was considered a positive reaction (0=no visible change; 1=discrete or patchy erythema; 2= moderate and confluent erythema; 3=intense erythema and swelling). Sensitization potential was determined based on the percentage of animals showing a positive response.
Subcutaneous implantation test. In order to evaluate the potential local and systemic effect after an exhausted BAT-90 subcutaneous implantation (see above for its dose) for a 26- and 52-week exposure, a subcutaneous implantation test was performed as per ISO 10993-6:2016 and ISO 10993-11:2017, in GLP conditions.
For each test, 20 26- or 52-weeks male albino rats were used (male; strain: SD; weight: 195-212 g); 10 were implanted with the prepared test sample and 10 with a negative control (USP low density polyethylene).
The explant time was scheduled 26 or 52 weeks after the implantation. Animals were daily submitted to general objective examination with the purpose to detect possible local and systemic effects due to the sample. At the explant day, before the sacrifice, the blood of the animals was harvested. The analyzed hematological parameters were: ALT, AST, creatinine, glucose, total protein, urea, albumin, calcium, sodium, and potassium. A full necroscopy was performed. Implantation sites of each animal and the following organs were harvested and weighted: heart, liver, adrenal gland, kidney, skin, spleen, muscle, brain, testis, lung, femur, bone marrow, mesenteric lymph node and local lymph node. They were preserved in formalin 10% buffer solution and used for histological evaluation. The evaluation of organ slides (thickness 3-5 um) was performed after hematoxylin-eosin staining according to the following parameters, observations and corresponding scores: architecture (normal=0, minimal changed=1, mild changed=2, moderate changed=3 or totally changed=4); cell hypertrophy (absent=0, minimal=1, mild=2, moderate=3 or marked=4); necrosis (absent=0, minimal=1, mild=2, moderate=3 or marked=4); inflammatory population (absent=0, minimal=1, mild=2, moderate=3 or marked=4); atrophy (absent=0, minimal=1, mild=2, moderate=3 or marked=4); oedema (absent=0, minimal=1, mild=2, moderate=3 or marked=4); hyperemia (absent=0, minimal=1, mild=2, moderate=3 or marked=4); fibrosis (absent=0, minimal=1, mild=2, moderate=3 or marked=4); vascular congestion (absent=0, minimal=1, mild=2, moderate=3 or marked=4). The group total score was calculated as the sum of the total score of each animal belonging to the group. The final index of total relative reaction was calculated as the difference between total group score of the treated group and total group score of the control group. A negative difference was recorded as zero. Mean Final Index of reaction of each group was calculated dividing the total group score for each group by the number of animals included into that group (10 animals/group).
Mean Final Index of reaction (treated group versus control group) was assessed according to the following response category: from 0.0 to 2.9, minimal or no reaction; from 3.0 to 8.9, slight reaction; from 9 to 15, moderate reaction; from 15.1 and above, severe reaction.
Statistical analysis. Descriptive analysis of the data was performed with Microsoft Excel 14.1.0 (Microsoft, Redmond, WA, USA) and GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) and plotted or reported in tables as mean±SD. Where appropriate, statistical significance was checked using unpaired Mann-Whitney test.
Results
BAT-90 application is able to induce necrosis into the liver injection site. To verify the capability of BAT-90 administration to induce necrosis into the target site, it was administered into 3 different liver segments of 8 adult female pigs, under ultrasonographic guidance: left lobe, right lobe and lower right para-caval lobe. Two animals were euthanized at each time point (1 week, 2 weeks, 3 weeks and 4 weeks) and the anatomopathological evaluation of the explanted livers was performed to quantify the dimensions of necrosis area inside the injection sites (Figure 1). The analysis demonstrated a stable ablation area at each evaluated time point. The necrotic mean±SD area (cm2), as assessed at histo-pathology, was 18.8±3.7, 20.5±2.8, 19.2±2.1 and 20.5±2.0, after 1, 2, 3, and 4 weeks from BAT-90 injection, respectively.
BAT-90-induced necrosis. The graph represents the extent of necrosis in pig livers after 1, 2, 3 and 4 weeks from BAT-90 injection. The data are reported as mean area (cm2)±SD. The mean area for each time point was calculated from necrosis evaluations following BAT-90 administration in 3 different hepatic segments of two animals (Data did not show any significant difference: Mann-Whitney test). Two representative photographs of the necrosis area induced by BAT-90 after 1 and 4 weeks are also shown.
The blood samples collected at the same time points did not show any significant alteration of the hemo-coagulation parameters (data not shown).
BAT-90 was safe in biocompatibility tests. Acute systemic toxicity, intracutaneous reactivity and delayed hypersensitivity tests were performed to verify the safety of BAT-90 administration to experimental animals. The results observed in the treated animals were very similar to the controls: no symptoms were detected in the acute systemic toxicity test (all animals injected with treatment or control vehicle showed a score=0, meaning no symptoms, at all evaluated time points); no erythema nor slight erythema were detected in the intracutaneous reactivity test (all animals injected with treatment or control showed a score=0 for both erythema and oedema, meaning that no erythema and no oedema were observed when sodium was used as administration liquid; a score=1 for erythema and a score=0 were observed in all animals injected with treatment or control when cottonseed oil was used as administration liquid) and no visible changes were observed in the delayed hypersensitivity test (all animals treated with BAT-90 or the control presented a score = 0 meaning “no visible change”).
Moreover, considering the partial non-bioabsorbable nature of BAT-90 and the 90Y decay time, the local and systemic effects of subcutaneous BAT-90 implantation in male albino rats were assessed after 26 and 52 weeks from implantation. Considering the local effects, no abnormality was detected after macroscopic evaluation and necropsy in all implanted sites (Table I). Comparing the treated and control animals, the hematological parameters from the samples collected at the same time points did not document any significant alteration (Table II). The histological evaluation allowed to calculate a Mean Final Index of reaction for the treated and control groups, which were 1.5 and 1.8, respectively, at 26 weeks (values from 0.0 to 2.9 mean minimal or no reaction), 3.5 and 4.2, respectively, at 52 weeks (values from 3.0 to 8.9 mean slight reaction).
Summary of the local and systemic effects of BAT-90 and control after subcutaneous implantation, verified at 26 and 52 weeks. The tests were performed according to the ISO 10993-6:2016 and ISO 10993-11:2017 standards for the biological evaluation of medical devices.
Hematological parameters at explantation day after 26 weeks and 52 weeks of the subcutaneous implantation (Comparison between treated and control animals did not show any significant difference: Mann-Whitney test).
As far as the systemic effects, BAT-90 did not cause significantly different effects, when compared to the controls (Figure 2 and Figure 3), when the following parameters were evaluated: tissue architecture; cell hypertrophy; necrosis; inflammatory population; atrophy; oedema; hyperemia; fibrosis; vascular congestion.
Histological images at 26 weeks. Representative images of distant organs after 26 weeks of subcutaneous BAT-90 or control implantation.
Histological images at 52 weeks. Representative images of distant organs evaluated after 52 weeks of subcutaneous BAT-90 or control implantation.
Discussion
Despite improvements in its treatment, liver cancer remains one of the most difficult cancers to treat (9). Moreover, liver metastases are significantly more common than primary liver cancer, and patient long-term survival after radical surgical treatment is approximately 50% (10).
Selective internal radiation therapy using 90Y microspheres has been recognized as a potential therapy for unresectable primary and metastatic liver cancers (11). However, reducing the 90Y off-target radiation exposure could increase the safety of this treatment, while decreasing the dose of 90Y microspheres required to induce tumor control in the injection site. With this goal in mind BAT-90, an innovative medical device was developed combining the known activity of 90Y microspheres with a surgical adhesive hydrogel, acting as a carrier of the 90Y microspheres. The hydrogel is contained in a dual chamber syringe, with BSA and glutaraldehyde in a 4:1 ratio, and is administered to the target tissues through a mixing tip attached to the syringe itself, designed to optimize the mixing of its components during the injection procedure.
Although there are some differences between the human and pig liver, this animal model is recognized to be very useful in experimental hepatic surgery, since it has a similar number of segments as the human liver, shows a common vascular structure and has a similar stiffness to normal and pathologic liver tissue (12, 13).
To test the efficacy of BAT-90 in delivering the β-radiation energy to the liver injection site, we measured the necrosis area (cm2) induced by the injection of 8 ml of BAT-90 into each of three different liver segments (left lobe, right lobe and lower right para-caval lobe) of eight adult female pigs. There was no difference in necrotic area among the three different observations, performed at four evaluation times; this is indicative of an effective and early ablation of the target liver tissue, of a size which is comparable to what is achieved by other hepatic loco-regional treatments (14). The stability over time of the BAT-90-induced necrosis further supports the retention effect of the hydrogel matrix over the 90Y microspheres (Figure 1).
Blood samples collected from the test animals at the different timepoints did not document any significant abnormalities of the blood and liver parameters; no radioactivity effects were detectable on the complete blood counts after BAT-90 administration.
To further verify the safety of BAT-90 after subcutaneous implantation, an acute systemic toxicity test, an intracutaneous reactivity test, a delayed hypersensitivity test and a subcutaneous implantation test were performed according to current ISO standards for biocompatibility testing of medical devices. All the tests showed similar results between the BAT-90 and the control groups, supporting the safety of the new medical device.
Taken collectively, all these results represent the first evidence that BAT-90 could be considered as an innovative treatment option for inoperable solid tumors of the liver, by combining the known efficacy of 90Y-released β-radiation particles on tumor growth with the capacity of the polymerized hydrogel to keep the 90Y-coated microspheres strictly within the compound administration site.
Additional experiments in other target organs are warranted to confirm size and characteristics of BAT-90-induced tumor necrosis and the effective retention of 90Y microspheres in the injection site; they will further support BAT-90’s potential for treating unresectable tumors of different origin, or for ablating residual tumor cells after cancer resection procedures.
Acknowledgements
The Authors are grateful to Prof. Francesco Izzo, MD, Secondo Lastoria, MD, Sabrina Bimonte, MD and their extended team at Fondazione G. Pascale (IRCCS), National Cancer Institute, Naples (Italy) for the design and conduct of the pig study.
This work was made possible by a research agreement between FIC Srl and Cardarelli Biotechnology Institute, Naples (Italy); the results were later acquired by BetaGlue Technologies SpA, Milan (Italy).
Footnotes
Authors’ Contributions
A. Amato and PL. Carriero contributed to the design, analysis and reporting of the safety tests included in the manuscript; they also contributed to the analysis and reporting of the pig model. R. Cianni, G. Ettorre, G. Paganelli and G. McVie contributed to the review and critical appraisal of the manuscript.
Conflicts of Interest
A. Amato and PL. Carriero are employees of BetaGlue Technologies SpA; R. Cianni, G. Ettorre, G. Paganelli and G. McVie are members of the Scientific Advisory Board of BetaGlue Technologies SpA.
- Received June 30, 2022.
- Revision received July 19, 2022.
- Accepted July 20, 2022.
- Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
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