Animal Safety Studies Research Articles

Risk of Induction of Corneal Neovascularization with Topical Erythropoietin: An Animal Safety Study

Purpose: To evaluate the pro-angiogenic effect of topical erythropoietin on cornea in chemical burn-injured rabbit eyes.
 Methods: The corneal alkali-burn injury was induced in 10 eyes of 10 rabbits using filter paper saturated with 1.0 mol sodium hydroxide. The eyes were categorized into the treatment group (n = 5) that received topical erythropoietin (3000 IU/mL) every 8 hr for one month versus the control group (n = 5) that received normal saline every 8 hr for one month. All eyes were treated with topical ciprofloxacin every 8 hr until corneal re-epithelialization was complete. Corneal epithelial defects, stromal opacity, and neovascularization were evaluated after the injury. At the conclusion of the study, the rabbits were euthanized and their corneas were submitted to histopathological examination.
 Results: Baseline characteristics including the rabbits’ weight and the severity of corneal injury were comparable in two groups. Time to complete corneal re-epithelialization was 37 days in the treatment group and 45 days in the control group (P = 0.83). There was no significant difference between the groups in the rate of epithelial healing or corneal opacification. Clinical and microscopic corneal neovascularization was observed in one eye (20%) in the treatment group and two eyes (40%) in the control group (P = 0.49).
  Conclusion: Recombinant human erythropoietin administered topically did not induce vessel formation in rabbit corneas after chemical burn.

Cardiopulmonary Impact of Electronic Cigarettes and Vaping Products: A Scientific Statement From the American Heart Association.

Vaping and electronic cigarette (e-cigarette) use have grown exponentially in the past decade, particularly among youth and young adults. Cigarette smoking is a risk factor for both cardiovascular and pulmonary disease. Because of their more limited ingredients and the absence of combustion, e-cigarettes and vaping products are often touted as safer alternative and potential tobacco-cessation products. The outbreak of e-cigarette or vaping product use-associated lung injury in the United States in 2019, which led to >2800 hospitalizations, highlighted the risks of e-cigarettes and vaping products. Currently, all e-cigarettes are regulated as tobacco products and thus do not undergo the premarket animal and human safety studies required of a drug product or medical device. Because youth prevalence of e-cigarette and vaping product use was as high as 27.5% in high school students in 2019 in the United States, it is critical to assess the short-term and long-term health effects of these products, as well as the development of interventional and public health efforts to reduce youth use. The objectives of this scientific statement are (1) to describe and discuss e-cigarettes and vaping products use patterns among youth and adults; (2) to identify harmful and potentially harmful constituents in vaping aerosols; (3) to critically assess the molecular, animal, and clinical evidence on the acute and chronic cardiovascular and pulmonary risks of e-cigarette and vaping products use; (4) to describe the current evidence of e-cigarettes and vaping products as potential tobacco-cessation products; and (5) to summarize current public health and regulatory efforts of e-cigarettes and vaping products. It is timely, therefore, to review the short-term and especially the long-term implications of e-cigarettes and vaping products on cardiopulmonary health. Early molecular and clinical evidence suggests various acute physiological effects from electronic nicotine delivery systems, particularly those containing nicotine. Additional clinical and animal-exposure model research is critically needed as the use of these products continues to grow.

0609 Samelisant, Histamine 3 Receptor Agonist for the Potential Treatment of Excessive Daytime Sleepiness in Patients with Narcolepsy

Abstract Introduction Samelisant (SUVN-G3031) is a potent and selective histamine 3 receptor inverse agonist. In orexin knockout mice, samelisant produced wake-promoting and anticataplectic effects suggesting its potential therapeutic utility in the treatment of narcolepsy. Safety and tolerability studies in animals and healthy human volunteers suggested a favorable risk/benefit profile for samelisant. Methods Samelisant is currently being evaluated in a double blind, Phase-2 proof of concept study in USA and Canada for narcolepsy with or without cataplexy (ClinicalTrials.gov Identifier: NCT04072380). The study is recruiting subjects diagnosed with narcolepsy as per ICSD-3 criteria, aged between 18 to 65 years with an Epworth Sleepiness Scale (ESS) score of ≥12 and mean Maintenance of Wakefulness Test (MWT) time of < 12 min. A total of 171 subjects are expected to be randomized into 3 treatment arms (placebo, samelisant 2 mg and samelisant 4 mg) in 1:1:1 ratio. Each subject will receive either placebo or study drug once daily for 2 weeks. The primary efficacy endpoint is change in MWT score from baseline to week 2. Key secondary endpoint is change in ESS from baseline to week 2. Safety will be monitored throughout the study by medical monitor and by data safety monitoring committee. As the study is still recruiting, demographics and baseline characteristic data for the currently enrolled subjects is summarized descriptively. Since the study is blinded, a breakdown of baseline characteristics by treatment group will not be available until after completion. Results At the data cutoff date of Nov 30, 2022, a total of 155 subjects were randomized in the study. The median age of subjects was 30 years (range: 18-57 years) with mean BMI of 28.5 kg/m2 (range: 18.3- 43.9 kg/m2). Overall, 57% subjects were of narcolepsy type-1, 71% were female and 71% were Caucasian. Mean (SD) baseline values of MWT and ESS scores were 6.1 (4.4) and 17.2 (2.8), respectively. Conclusion Baseline characteristics are consistent with the general narcolepsy population. The study is currently enrolling subjects with narcolepsy and the data readout is expected in Q2 2023. Support (if any)  

Acute toxicity of a new antiseptic for veterinary dentistry

The development steps of new drugs require consistent safety and efficacy studies in animals. This article reflects the research of a new antiseptic for veterinary dentistry containing active ingredients such as chlorhexidine bigluconate and propolis. Acute toxicity studies were carried out following the “Guidelines for the experimental (preclinical) study of new pharmacological substances” (2005), according to GOST 32644-2014 and taking into account the requirements of the Order of the Ministry of Agriculture of the Russian Federation of March 6, 2018 No. 101. rats with an average live weight of 250-280 grams, from which one experimental group of three animals was formed (for the first stage of the study), after receiving the results of the first stage, a second experimental group of three animals was formed for the second stage. The rats of the experimental groups were injected with the studied antiseptic once into the inside of the stomach using a special club-shaped needle at a dose of 2000 mg/kg. As a result of the experiment, it was found that the investigated new antiseptic containing chlorhexidine bigluconate and propolis, intended for the treatment of inflammatory processes in the oral cavity in animals, belongs to substances of hazard class V according to GOST 32644-2014. There were no deaths of animals during the experiment. 

Abstract 2790: Preclinical studies of ZG19018, a novel irreversible covalent inhibitor of KRAS G12C, for the treatment of advanced non-small cell lung cancer and other solid tumors

Abstract Growing evidences of covalent irreversible inhibitors in clinical treatments of solid tumors with KRAS G12C mutation have recently emerged after FDA-approved AMG510 (Sotorasib) for NSCLC chemotherapies. ZG19018 is an irreversible KRAS G12C inhibitor with novel structure that shows promising preclinical results for in vitro molecular and biological studies and in vivo G12C-mutated cancer models and safety studies in animals. For instance, ZG19018 rapidly and selectively forms covalent bonds with G12C-GDP, which accounts for lock-in KRAS protein as the inactive GDP-bound state but inhibits the activation of numerous downstream signaling pathways like RAF/MEK on ERK phosphorylation. ZG19018 showed highly inhibition of a 3D growth panel for human cancer cell lines harboring the KRAS G12C mutation, e.g. H358 (NSCLCs), MIA PaCa-2 (pancreatic) and SW837 (colorectal), etc. For pharmacodynamics, a battery of in vitro and in vivo ADME/DMPK studies indicated a high plasma protein binding (>99.6%) of ZG19018 with over 40% bioavailability in rat and dog via oral administration for the adequate drug levels in various tissues including lung, crossing BBB to reach brain, and accumulation in tumors. For in vivo anti-tumor efficacy, ZG19018 exhibited significant dose-dependent inhibition of tumor growth of G12C-driven subcutaneous models in nude mouse like H358 NSCLC cells but induced further tumor regression in pancreatic cancer MIA PaCa-2 xenograft. ZG19018 also showed an enhanced anti-tumor activities as combined with chemo-reagent Donafinib, a broad tyrosine kinase inhibitor blocking RAF/MEK/ERK signaling pathways of tumor cells, in the same H358 xenograft model. The general toxicology studies in rat under 28 days repeated oral administration (q.d.) showed NOAEL and STD10 values at 75 and 150 mg/kg/day, respectively. The safety pharmacological studies showed no notable effects of ZG19018 on the cardiovascular, respiratory and CNS systems. The genotoxic potential studies indicated ZG19018 without mutagenesis. Collectively, ZG19018 is now in phase I/II clinical trials for the treatments of solid tumors with KRAS G12C mutation in China. Citation Format: Bing Zhu, Dawei Cui, Ruifeng Liu, Weidong Feng, Zelin Sheng, Binhua Lv. Preclinical studies of ZG19018, a novel irreversible covalent inhibitor of KRAS G12C, for the treatment of advanced non-small cell lung cancer and other solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2790.

Fit-for-purpose validation of a drug-tolerant immunogenicity assay for a human mAb drug in animal safety studies

A modified biotin-drug extraction and acid dissociation (BEAD) immunogenicity assay was developed to detect anti-drug-antibodies (ADA) against the human anti-FXIIa monoclonal antibody (mAb) drug, Garadacimab (previously called CSL312). Multiple strategies were tested to optimize the signal-to-background (S/B) ratio, assay sensitivity and the drug tolerance. The modified BEAD assay was found to be highly drug tolerant (>500 μg/ml) with a sensitivity of 100 ng/ml, in line with current FDA regulatory guidelines. The assay was validated for use in a repeat-dose animal safety study and showed an acceptable intra-assay precision and robustness but a lower inter-assay precision. In-study sample analysis confirmed that the assay was fit-for-purpose (FFP) for the context-of-use (COU) in the nonclinical study and the results obtained were deemed meaningful.

2022 Monkeypox Case Report: Beyond Borders and Expectations

Monkeypox is a zoonotic virus that is in the same family as smallpox. It is primarily spread through various forms of close contact with an infected individual, including direct contact with an infected person’s bodily fluids or surfaces and fabrics that have been contaminated. Its symptoms are generally characterized by rashes or lesions that are firm or supple, restricted to its area, and raised in its center. Vaccines for this disease were actually developed for smallpox. There are two main types of vaccinations: ACAM2000 and 0Jynneos. ACAM2000 uses a live vaccine virus while Jynneos uses a non-replicating virus. Due to the similarity between smallpox and monkeypox, Tpoxx is being researched as a potential treatment. FDA approved Tpoxx – or Tecovirimat – in 2018 as an effective treatment for smallpox after extensive animal trials and safety studies. Currently, the drug is not FDA-approved for monkeypox; however, under an expanded access protocol, it is clinically available. There are two cases presented in this article. The first case was a heterosexual male in his 30s who contracted monkeypox while having multiple sexual encounters with women while moving from tent to tent as an unhoused resident. He also shared pipes with others for recreational marijuana use. This case illustrates that monkeypox is a virus that isn’t restricted to sexuality, especially that of the LGBTQ+ community. To clarify, this virus is not an STI/STD, it’s spread by skin-to-skin contact, contact with an object that was in contact with an infected individual, or contact with respiratory secretions. However, there are fears of singling out the LGBTQ+ community and using the monkeypox virus to stigmatize these communities, much like the AIDS pandemic in the 1980s. In this case, a large proportion of those infected identifies as non-heterosexual, which may just be a result of the nature of a viral illness. Tightly knit groups and communities that spend a lot of time with each other create many opportunities for contact and facilitate disease spread. Second case is a homosexual male in his 60s who received childhood vaccination for smallpox, and yet contracted the monkeyvirus during his travels to Germany and Switzerland, where he had encountered multiple sex partners. This case presents how previous vaccination for smallpox may not be effective against monkeypox virus. Thus, prior smallpox vaccines in the distant past offered no protection against monkeypox and are capable of spreading to people outside of the borders of LGBTQ+ community.

LOCL-07 LOCO-REGIONAL INFUSION OF GB-13 (IL13.E13K-PE4E) AS A POTENTIALLY PROMISING TREATMENT FOR RECURRENT HIGH-GRADE GLIOMA

INTRODUCTIONHigh grade gliomas (HGG) are devastating diseases with largely unchanged survival outcomes despite decades of research. Recent studies suggest the interleukin 13 receptor subunit alpha 2 (IL-13Rα2) is selectively upregulated in up to 80% of HGG, including glioblastoma (GBM) and diffuse midline gliomas (DMG) harboring H3K27 alterations. Immunotoxins targeting IL-13Rα2 have been demonstrated as safe and have shown some benefit for patients with HGG in previous phase I/II and III clinical trials. We hypothesized that by using GB-13 (IL13.E13K-PE4E), a novel peptide-toxin that binds IL-13Rα2 with high specificity and possesses a Pseudomonas exotoxin moiety, we would enhance the anti-tumor effects of this immunotherapy for HGG in vitro and in vivo while decreasing off-target toxicity. METHODSWe examined the pharmacological effects of GB-13 in multiple patient-derived cell lines and rodent models of HGG. GBM and DMG lines were used to confirm IL-13Rα2 expression and sensitivity towards GB-13. Tumor naïve rats were evaluated for toxicity, and orthotopic PDX mice were used to monitor tumor size and survival following loco-regional infusion of GB-13. RESULTSGB-13 induced a potent cytotoxic response strongly predicated on IL-13Rα2 expression in vitro. No treatment-related adverse effects were noted after 7-day continuous intracranial infusion of GB-13 in tumor naïve rats. Further, in IL-13Rα2-upregulated orthotopic PDX mice, direct intratumoral administration of GB-13 via convection-enhanced delivery abrogated tumor growth and prolonged survival. CONCLUSIONSGiven these promising results as well as the critical need for novel therapies in CNS malignancies, we are progressing to human trials using GB-13 targeting recurrent HGG. Ongoing safety studies in tumor-bearing animals will be able to define dose levels for the initial adult study-arm and the following pediatric study-arm. In this Phase 1 clinical trial, we hypothesize that loco-regional infusion of GB-13 will safely enhance tumor clearance by causing selective killing of IL-13Rα2-upregulated HGG cells.

Tolerability of graded levels of tall oil fatty acids as a nutritional additive for broiler chickens: a 45-day target animal safety study

ABSTRACT 1. The following study was conducted to evaluate the tolerability of tall oil fatty acid (TOFA) to broiler chickens, at three graded levels as a nutritional additive in complete feed. 2. 256 one-day-old female and male Cobb 500 broiler chickens were assigned to four dietary treatment groups with TOFA at 0 (control), 1.0, 3.0, or 5.0 g/kg within a complete feed for 45 d. 3. Birds were weighed individually on days 0, 16, 31 and 45, and the feed intake, bird weight gain, and feed conversion ratio were calculated for the respective starter, grower and finisher phases and over the whole study. On day 45, blood samples were drawn from each bird for haematology and blood chemistry measurements. Two birds per pen were subjected to gross pathological examination and sampling of several tissues for histopathology, including weighing the liver. 4. The dietary treatments did not affect zootechnical performance variables or mortality over the whole study period. Bird performance was typical for the breed. 5. Haematology, clinical chemistry and histopathology did not reveal any changes associated with dietary TOFA dosing. However, the 5.0 g/kg dose level increased the relative weight of the liver, as a percentage of final body weight, compared to the control group, but there was lack of corresponding histopathology findings. 6. In conclusion, the study indicated that oral administration of TOFA for 45 d in feed was well tolerated by the birds at dietary levels of up to 5.0 g/kg.

Development and characterization of anti-SARS-CoV-2 intravenous immunoglobulin from COVID-19 convalescent plasma.

Background: The authors describe the developmental process of intravenous anti-COVID-19 hyperimmune immunoglobulin from anti-SARS-CoV-2 neutralizing antibody-containing plasma. Furthermore, the authors investigated its safety and protective activity in animal models. Materials & methods: The manufacturing process included standard ethanol fractionation, chromatographic purification steps and virus removal or inactivation. Results: The authors produced pure and safe immunoglobulin for intravenous administration, with 98.1 ± 6.5 mg/ml protein content, of which 97.6 ± 0.7% was IgG. The concentration factor of SARS-CoV-2 neutralizing antibodies was 9.4 ± 1.4-times. Safety studies in animals showed no signs of acute/chronic toxicity or allergenic or thrombogenic properties. Intravenous anti-COVID-19 hyperimmune immunoglobulin protected immunosuppressed hamsters against SARS-Cov-2. Conclusion: The obtained results can allow the start of clinical trials to study the safety and efficacy in healthy adults.

0624 Samelisant (SUVN-G3031), Baseline Characteristics from a Phase-2 Study Evaluating Efficacy and Safety in Patients with Narcolepsy

Abstract Introduction Histamine H3 receptor (H3R) antagonists/inverse agonists increase histaminergic neurotransmission and offer a therapeutic option for the treatment of narcolepsy. Samelisant (SUVN-G3031) is a potent H3R inverse agonist and exhibits very high selectivity over other targets. In orexin knockout mice, samelisant produced wake-promoting and anticataplectic effects suggesting its potential therapeutic utility in the treatment of excessive daytime sleepiness and cataplexy associated with narcolepsy. It showed dose-dependent H3R occupancy at efficacy doses. Safety and tolerability studies in animals and healthy human volunteers suggested a favorable risk/benefit profile for samelisant.Samelisant is being evaluated in a Phase-2, multicenter, double-blind, placebo controlled, parallel-group study in patients with narcolepsy with or without cataplexy (ClinicalTrials.gov Identifier: NCT04072380). Methods Eligibility criteria for the study include subjects diagnosed with narcolepsy as per ICSD-3, aged between 18 to 50 years with an Epworth Sleepiness Scale (ESS) score of ≥12 and mean maintenance of Wakefulness Test (MWT) time of <12 min. A total of 171 subjects will be randomized into 3 treatment arms (placebo, samelisant 2 mg and samelisant 4 mg) in a fixed 1:1:1. Further, the randomization will be stratified according to the type of narcolepsy (Type-1 or Type-2). Each subject will receive either placebo or study drug once daily for 2 weeks. The primary efficacy endpoint is change in MWT score from baseline to week 2. Secondary endpoints are change in ESS and Clinical Global Impressions of Severity (CGI-S) from baseline to week 2. Safety will be monitored throughout the study by medical monitor and by an independent data safety monitoring committee. Baseline clinical and demographic data for the currently enrolled study is summarized descriptively. Since the study is blinded, a breakdown of baseline characteristics by treatment group will not be available until after completion. Results At the data cutoff date of Nov30, 2021, a total of 108 subjects were randomized in the study. The median age of subjects was 30 years (range: 18-50 years) with mean BMI of 26.4 (range: 18.3- 43.1 kg/m2). Overall, 58% subjects were of narcolepsy type-1, 69% were female and 74% were Caucasian. Mean (SD) baseline values of MWT and ESS were 6.20 (4.53) and 17.17 (2.93), respectively. Conclusion Baseline characteristics are consistent with the general narcolepsy population. The study is currently enrolling subjects with narcolepsy and the data readout is expected in Q3 2022. Support (If Any) None

Preclinical studies using CAR-T cells.

The therapy with genetically modified T cells to express chimeric antigen receptors (CAR) is a promising strategy for immunotherapy against cancer. CAR-T cells can specifically recognize antigens on the surface of tumor cells and then effectively kill those cells. Several researchers have presented the development of CAR-T cells for various hematological targets and the treatment of solid tumors. Quality control and preclinical evaluation of these products are essential to demonstrate their safety and efficacy and allow development to the clinical trial phase. This chapter will present relevant guidelines regarding pre-clinical research of CAR-T cell products.
 Preclinical research on cell therapy products should include in vitro and in vivo pharmacodynamics studies (antitumor activity), pharmacokinetics (proliferation, distribution, and persistence of CAR-T cells in vivo), and animal safety studies.

Ferric citrate is a safe and digestible source of iron in broilers and piglets.

BackgroundIron (Fe) is traditionally supplemented in poultry and swine diets using inorganic forms (e.g. sulfates, oxides). However, research suggests that organic sources are more beneficial due to greater bioavailability. In this paper, we present results from four studies aimed at assessing ferric citrate (CI-FER™, Akeso Biomedical Inc., Burlington, MA, USA) as a safe and effective source of Fe for broilers and piglets.MethodsA total of four studies were performed in Germany following standard farming practices for each species. One study in day-old broiler chicks and one study in weaned piglets were designed as target animal safety studies where animals were randomly allocated to one of three treatment groups: a negative control group, the proposed dose group and a multifold dose group. Broilers and pigs were fed the experimental diets for 35 and 42 days, respectively. In each study, average daily feed intake, average daily weight gain and feed conversion ratio were measured, and blood samples were taken at study end for routine biochemistry and haematology. The other two studies were designed to evaluate different sources of dietary Fe for weaned piglets bred and managed under standard farm conditions. All piglets received routine Fe injections (200 mg Fe dextran, intramuscular) on day 3 of age, as well as the experimental diets for 42 days. In both studies, performance parameters were measured. In one study, Fe digestibility and serum Fe, superoxide dismutase and haptoglobin were also measured. In all studies, the general health status of the animals was monitored daily and all culls and mortality recorded. Each study followed a complete randomised block design.ResultsIn broilers, ferric citrate was well tolerated up to 2,000 mg/kg feed (×10 the recommended inclusion rate) and no adverse effects on growth, blood parameters or mortality were observed. In piglets, ferric citrate was well tolerated up to 5,000 mg/kg feed (×10 the recommended inclusion rate) with no adverse effects on growth, blood parameters or mortality. In addition, piglets fed ferric citrate performed significantly better than animals fed the negative control diet (containing only endogenous Fe) and those fed inorganic forms of Fe. Moreover, piglets fed ferric citrate demonstrated improved Fe digestibility and improved oxidative status. Altogether, these findings show that ferric citrate is a safe and easily digestible source of dietary Fe for broilers and piglets.

Development and Characterization of Hyperimmune Anti-SARS-CoV-2 Intravenous Immunoglobulin From COVID-19 Convalescent Plasma

Background: Polyclonal immunoglobulin G (IgG) medications have successfully treated different bacterial and viral infections for several decades. Herein, we describe the development process of hyperimmune anti-Coronavirus disease 2019 (COVID-19) intravenous immunoglobulin (COVID-globulin) from anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibody (nAb)-containing plasma. Furthermore, we investigate the safety and protective activity of the COVID-globulin in animal models.Methods: COVID-19 convalescent plasma samples collected from Russian blood service organizations with known anti-SARS-CoV-2 nAb titers were used for COVID-globulin production. The manufacturing process included standard ethanol fractionation, chromatographic purification steps, and virus removal or inactivation. Immunoglobulin batches were characterized based on Russian and EU Pharmacopeia guidelines, with the anti-SARS-CoV-2 nAb titers determined using a virus neutralization test (performed in Vero E6 cells) and enzyme-linked immunosorbent assay (ELISA). COVID-globulin safety was assessed by evaluating its toxicity and thrombogenic potential. COVID-globulin protective efficacy was evaluated in immunosuppressed Syrian hamsters via the intraperitoneal administration of COVID-globulin or a placebo Changes in animal body weight and health status were observed daily. Lungs were subjected to macroscopic, histological, and virological studies. The lung virus load was determined using titration of 10% lung tissue homogenate in Vero E6 cells, and the cytopathic effect was visually evaluated.Findings: We produced pure and safe immunoglobulin for intravenous administration, with 98·1±6·5 mg/mL protein content.. The concentration factor of SARS-CoV-2 nAbs in ten COVID-globulin lots was 9·4±1·4 times. Safety studies in animals showed no signs of acute/chronic toxicity or allergenic or thrombogenic properties. COVID-globulin demonstrated protective effects against SARS-CoV-2 in immunosuppressed hamsters. The survival rate in the COVID-globulin groups was 66·6–100% compared with total mortality in the control groups.Interpretation: COVID-globulin, manufactured in accordance with the Ph. Eur. requirements, protected immunosuppressed hamsters against SARS-Cov-2.Funding Information: This work was sponsored and performed by JSC Nacimbio.Declaration of Interests: We declare no competing interests.Ethics Approval Statement: Studies were performed at The National Research Center for epidemiology and Microbiology named after Honorary Academician N.F. Gamaleya of the Ministry of Health of the Russian Federation, after receiving bioethical approval №26 from 08.10.2020 and №28 from 16.11.2020.

Intervention with selution SLR™ Agent Balloon for Endovascular Latent Limus therapy for failing AV Fistulas (ISABELLA) Trial: Protocol for a pilot clinical study and pre-clinical results.

The aim of this pilot clinical study is to evaluate the safety and efficacy of the Selution Sustained Limus Release (SLR)™ sirolimus-coated balloon (M.A. MedAlliance SA, Nyon, Switzerland) for improving the patency of failing arterio-venous fistulas (AVF) in hemodialysis patients. We also present herein a pre-clinical pharmacokinetic and safety evaluation of Selution™ to justify its first use in hemodialysis patients for endovascular access salvage. This is an investigator-initiated prospective single-center, non-blinded single-arm trial. Forty patients with clinically significant de novo or recurrent stenoses in a mature AVF circuit will be recruited. All stenotic lesions will be prepared with high pressure non-compliant conventional balloon angioplasty (CBA) prior to deployment of the Sustained-Release Selution™ sirolimus drug-eluting balloon. The primary efficacy endpoint is 6-month target lesion primary patency and the primary safety endpoint is freedom from localized or systemic serious adverse events through 30 days. Secondary endpoints of interest include technical and clinical success rates and circuit access patency at 3 and 6 months. Follow-up will occur for 2 years for those patients whose AVFs remain patent. Pharmacokinetic and histological animal safety studies performed with the Selution™ coating formulation showed prolonged arterial tissue retention of sirolimus with therapeutic levels up to 60 days and non-toxic and rapidly declining blood levels. Histological results in animal models demonstrated safety, freedom from intraluminal thrombus, reduction in restenosis by sirolimus elution compared to CBA, and no evidence of embolic phenomena indicative of adverse particulate effects. Long release sirolimus coated balloons may serve as a promising novel alternative therapy to paclitaxel-based technology for treating conduit stenosis secondary to neointimal hyperplasia. Pre-clinical pharmacokinetic and histological animal data are encouraging and provide suggestion of safety and efficacy in this setting. This single-center trial will provide a first step toward demonstration of efficacy and safety of this device for treatment of stenotic fistulas.

Long-term safety evaluation of Daxocox\xae tablets (enflicoxib) in dogs after weekly oral administrations for seven months

BackgroundDaxocox® [Ecuphar/Animalcare Group] contains the selective COX-2 inhibitor enflicoxib, approved in the EU for the treatment of pain and inflammation associated with osteoarthritis in dogs. The safety of Daxocox® was evaluated in a target animal safety study: Groups of 4 dogs per sex each were treated once weekly with placebo or Daxocox tablets at 1-, 3- and 5-times (1X, 3X and 5X) the maximum recommended therapeutic dose of enflicoxib (0, 4, 12 or 20 mg/kg, respectively). After an initial loading dose, dogs in the placebo control, 1X and 3X groups were administered for 32 weeks, and those in the 5X group were administered for 13 weeks. Dogs were subjected to daily food consumption measurements and clinical and dose observations. Body weight measurements, physical examinations, clinical pathology, urinalysis, faecal occult blood (FOB) and electrocardiographic (ECG) and blood pressure measurements, buccal mucosal bleeding time (BMBT), ophthalmology and gastroduodenal endoscopy examinations were conducted throughout the study. At study completion, all dogs were subjected to gross necropsy. Histopathology was performed on selected tissues from all animals in all groups. ResultsNo clinical signs were noted, and no toxicologically relevant dose-associated effects were observed.ConclusionsResults show that Daxocox® is well-tolerated and has a broad safety margin when administered as directed in dogs.

Long-term and acute safety of a novel orally administered combination drug product containing milbemycin oxime and lotilaner (Credelio\xae Plus) in juvenile and adult dogs

BackgroundThe combination of milbemycin oxime (MO) and lotilaner (Credelio® Plus) is a novel systemic endectocide that provides month-long effectiveness in dogs after a single oral treatment. The safety of Credelio® Plus flavored chewable tablets was investigated in three target animal safety studies. Two studies (one in juveniles and one in adults) evaluated the long-term safety, and one study evaluated the acute safety of the product when administered orally at the upper end of the recommended dose range (0.75–1.53 mg/kg MO and 20–41 mg/kg lotilaner) and multiples of this dose.MethodsThe objectives of these studies were to determine the long-term and acute safety of MO and lotilaner flavored chewable tablets in healthy dogs. All three studies were randomized, blinded, parallel-group design studies in healthy Beagle dogs. In each of the two long-term studies, 32 dogs were randomized among four groups to untreated controls or to treated groups at target doses of 1X, 3X, or 5X. Treatment was administered on seven (adult dogs) or nine (juvenile dogs) occasions with dosing every 4 weeks. In the acute study, 48 dogs were randomized among four groups to untreated controls or to treated groups at 1X, 3X, or 6X. In all three studies, the control group was administered placebo tablets. All dogs were fed 30 to 45 min prior to treatment and the assessment of safety was based on health observations, complete physical/neurological examinations, and food consumption. For the long-term safety studies, safety assessments also included clinical pathology evaluations (hematology, clinical chemistry and urinalysis), body weight, pharmacokinetic blood collections, and macroscopic and microscopic examinations of collected tissues.ResultsMO and lotilaner did not induce any treatment-related adverse effects based on health observations, physical/neurological examinations, or food consumption in the long-term or acute studies. Additionally, in the long-term studies, MO and lotilaner did not induce any treatment-related effects on clinical pathology, body weight, and macroscopic and microscopic examinations.ConclusionsThese three studies demonstrate that Credelio® Plus has a wide safety margin when administered at monthly intervals to puppies and dogs at the high end of the commercial dose band.Graphical

506 Samelisant: Baseline Characteristics from a Phase 2 Study Evaluating Efficacy and Safety in Patients with Narcolepsy

Abstract Introduction histamine H3 receptor (H3R) antagonists/ inverse agonists increase histaminergic neurotransmission and offer a therapeutic option for the treatment of narcolepsy. Samelisant (SUVN-G3031) is a potent and selective H3R inverse agonist exhibited selectivity over 70 other targets. Samelisant showed wake-promoting and anticataplectic effects in orexin knockout mice suggesting its potential therapeutic utility in the treatment of EDS and cataplexy associated with narcolepsy. Safety and tolerability studies in animals and healthy humans suggested a favorable risk/benefit profile. Methods The current study is a 2 week treatment, multicenter, double-blind, placebo controlled, parallel-group study in patients with Narcolepsy with or without Cataplexy. Eligibility criteria include age between 18 to 50 years old, an ESS score of ≥ 12; and mean MWT time of < 12 minutes and a confirm diagnosis of narcolepsy as per ICSD-3. Further, the randomization will be stratified according to type of narcolepsy (Type-1 or Type-2). Each subject will receive either placebo or study drug once daily for 2 weeks in a fixed ratio of 1:1:1. The primary efficacy endpoint is change in maintenance of wakefulness test (MWT) score from baseline to week 2. Key secondary endpoints include change from baseline to week 2 in ESS and an improvement in CGI-S scores. Safety will be monitored by medical monitor and by an independent data safety monitoring committee. Baseline clinical and demographic data for the currently enrolled study is summarized descriptively. Since the study is blinded, a breakdown of baseline characteristics by treatment group will not be available until after completion. Results As of data cutoff date of Dec 20, 2020, a total of 54 subjects were completed in the study. The median age of subjects was 30 years (range: 18 - 50 years) with mean BMI of 28.6 (range: 18.3 - 43.1 kg/m2). Overall, 74% of subjects were female and 83% were Caucasian. Mean (SD) baseline values of MWT and ESS are 5.65 (3.5) and 16.7 (2.5), respectively. Conclusion Baseline characteristics are consistent with the general narcolepsy population. The study is currently enrolling the subjects with Narcolepsy with or without Cataplexy, and the Data readout is expected in the second half of 2021. Support (if any):

The food additive EDTA aggravates colitis and colon carcinogenesis in mouse models

Inflammatory bowel disease is a group of conditions with rising incidence caused by genetic and environmental factors including diet. The chelator ethylenediaminetetraacetate (EDTA) is widely used by the food and pharmaceutical industry among numerous other applications, leading to a considerable environmental exposure. Numerous safety studies in healthy animals have revealed no relevant toxicity by EDTA. Here we show that, in the presence of intestinal inflammation, EDTA is surprisingly capable of massively exacerbating inflammation and even inducing colorectal carcinogenesis at doses that are presumed to be safe. This toxicity is evident in two biologically different mouse models of inflammatory bowel disease, the AOM/DSS and the IL10−/− model. The mechanism of this effect may be attributed to disruption of intercellular contacts as demonstrated by in vivo confocal endomicroscopy, electron microscopy and cell culture studies. Our findings add EDTA to the list of food additives that might be detrimental in the presence of intestinal inflammation, but the toxicity of which may have been missed by regulatory safety testing procedures that utilize only healthy models. We conclude that the current use of EDTA especially in food and pharmaceuticals should be reconsidered. Moreover, we suggest that intestinal inflammatory models should be implemented in the testing of food additives to account for the exposure of this primary organ to environmental and dietary stress.

Abstract PS8-13: Silicone implant based sustained localized drug delivery of fulvestrant to prevent breast cancer

Abstract Background: Hereditary breast cancer is common. With enhanced awareness and the recent introduction in affordable multi-gene germ line testing, an estimated 0.5-1 million women will learn that they carry a considerable risk to develop breast cancer. Thus, a rapidly increasing number of women, many of them very young, will be in need of effective strategies for breast cancer prevention. Current options to prevent breast cancer in women at high risk include bilateral mastectomies or systemic anti-estrogen therapy. Both options, while effective, may have a detrimental impact on the physical and emotional well-being of the patient. Localized delivery of an established anti-estrogen to breast tissue only may thus offer an attractive alternative for cancer prevention and may replace systemic therapy for ductal carcinoma in situ and early stage breast cancer with minimal risk for metastases. Methods: As such, we have sought to develop a silastic-silicone device, which when placed under breast tissue, will deliver the anti-estrogen fulvestrant directly to the target tissue. Sustained slow release of fulvestrant from a silastic-silicone device directly into the mammary tissue will provide the risk reducing benefits of systemic hormonal therapy while minimizing systemic exposure and the resulting poor compliance due to adverse effects. Results: Using a combination of in vitro and in vivo studies, we show that fulvestrant can be delivered through a silastic-silicone device. Implanted adjacent to mammary tissue, this drug delivery device provides sustained high levels of fulvestrant to inhibit estrogen receptor signaling and breast cancer cell proliferation. In a MCF-7 breast cancer xenograft model we have shown that silastic-silicone delivers fulvestrant selectively to mouse mammary tissue for more than 1 year with anti-tumor effects similar to those achieved with systemic fulvestrant exposure. Using the Sprague-Dawley rat DMBA spontaneous breast cancer induction model, we further demonstrate that fulvestrant delivered by silastic-silicone devices implanted adjacent to mammary tissue significantly delays time to first tumor compared to control animals (n=90, HR = 0.42, 95% CI 0.21-0.83) with minimal systemic exposure (plasma average 1.1 ng/mL, SD ±1.5 ng/mL). Initial large animal safety and toxicity studies in female sheep (ewes, n=2) support the surgical strategy to place the device between breast tissue (i.e. udder) and the chest wall (abdominal wall in ewes). Following 1 month of fulvestrant release, gross and histopathological analysis found no adverse effects or implant related toxicity. Bioanalytical analysis of mammary tissues suggested that drug was found highest near the implant (max 341 ng/g, udder average 58.4 ng/g, SD ±17.1 ng/g), with diminishing levels distal from the implant. Systemic exposure was low, with plasma levels 1.2 ng/mL, SD ±0.22 ng/mL, comparing favorably to clinical plasma levels achieved by intramuscular fulvestrant of 500 mg: 28.0 ng/mL, SD ±27.9 ng/mL (FDA package insert). Subsequent large animal studies will focus on the safety and pharmacokinetics of device delivered fulvestrant in longer studies (e.g. 3 to 6 months). Conclusions: The greater awareness and genetic identification of individuals at risk for breast cancer brings about an increased need for novel approaches to breast cancer prevention. The development of a silastic-silicone based device for sustained and localized drug delivery with an approved and effective anti-estrogen should allow rapid transition into clinical testing. This strategy will provide an alternative option to mastectomies and allow breast conservation for women identified to have a more than 40% lifetime chance of developing breast cancer, as well as provide an alternative to systemic hormonal therapy in women with ductal carcinoma in situ or stage I breast cancer. Citation Format: Pamela N Munster, Pujan K Desai, Nela Pawlowska, Elysia Roche, Romain Pacaud, Maxim A Daud, Emily Hsu, David Gingrich, Amelia Deitchman, Fran Aweeka, Scott Thomas. Silicone implant based sustained localized drug delivery of fulvestrant to prevent breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-13.