Preclinical Safety Assessment of a Highly Selective and Potent Dual Small-Molecule Inhibitor of CBP/P300 in Rats and Dogs.

Cyclic adenosine monophosphate-response element (CREB)-binding protein (CBP) and EP300E1A-binding protein (p300) are members of the bromodomain and extraterminal motif (BET) family. These highly homologous proteins have a key role in modulating transcription, including altering the status of chromatin or through interactions with or posttranslational modifications of transcription factors. As CBP and p300 have known roles for stimulating c-Myc oncogenic activity, a small-molecule inhibitor, GNE-781, was developed to selectively and potently inhibit the CBP/p300 bromodomains (BRDs). Genetic models have been challenging to develop due to embryonic lethality arising from germline homozygous mutations in either CBP or P300. Hence, the purpose of this study was to characterize the role of dual inhibition of these proteins in adult rats and dogs. Repeat dose toxicity studies were conducted, and toxicologic and pathologic end points were assessed. GNE-781 was generally tolerated; however, marked effects on thrombopoiesis occurred in both species. Evidence of inhibition of erythroid, granulocytic, and lymphoid cell differentiation was also present, as well as deleterious changes in gastrointestinal and reproductive tissues. These findings are consistent with many preclinical (and clinical) effects reported with BET inhibitors targeting BRD proteins; thus, the current study findings indicate a likely important role for CBP/p300 in stem cell differentiation.

Rapid Development of Glaucoma Via ITV Nonselective ANGPT 1/2 Antibody: A Potential Role for ANGPT/TIE2 Signaling in Primate Aqueous Humor Outflow.

Purpose: Investigate a significant, dose-related increase in IOP, leading to glaucomatous damage to the neuroretina and optic nerve following intravitreal (ITV) administration of a bispecific F(ab')2 [anti-VEGF/Angiopoietins [ANGPT]F(ab')2] molecule in adult monkeys. Methods: ITV ocular tolerability and investigation of anti-VEGF/ANGPT F(ab')2 (blocking both ANGPT1 and ANGPT2) was done in monkeys; mechanistic studies were done in neonatal mice. Results: Following the second ITV dose of anti-VEGF/ANGPT F(ab')2, all 1.5- and 4-mg/eye treated monkeys developed elevated IOP, which eventually was associated with optic disc cupping and thinning of the neuroretinal rim. Histopathologic examination showed nonreversible axonal degeneration in the optic nerves of animals administered 1.5 mg/eye and higher that was considered secondary to high IOP. Anti-ANGPT Fab also caused elevated IOP in monkeys, but anti-VEGF Fab did not contribute to the IOP increase. In addition, an anti-ANGPT2-selective antibody did not change IOP. In mice simultaneous blockade of ANGPT1 and ANGPT2 impaired the expansion and formation of Schlemm's canal (SC) vessels, similar to genetic ablation of Angpt1/Angpt2 and their receptor TIE2. As previously reported, blocking ANGPT2 alone did not affect SC formation in mice. Conclusions: Dual inhibition of ANGPT1/ANGPT2, but not ANGPT2 alone, leads to increased IOP and glaucomatous damage in monkeys. This confirms a role for TIE2/ANGPT signaling in the control of IOP in adults, a finding initially identified in transgenic mice. Dual pharmacologic inhibition of ANGPT1/ANGPT2 may affect aqueous drainage and homeostasis in adult monkeys and may be useful in developing novel models of glaucoma.

Evaluation of the Toxicity of Intravitreally Injected PLGA Microspheres and Rods in Monkeys and Rabbits: Effects of Depot Size on Inflammatory Response.

Purpose: Poly(lactic-co-glycolic) acid (PLGA) inserts have been successfully developed for the treatment of posterior eye disease as a means of reducing injection frequency of intravitreally administered therapeutics. PLGA microspheres are also of interest for the delivery of intravitreal drugs, since they offer the advantage of being easily injected without surgical procedures or large injectors. Methods: In the current study, the toxicity of PLGA microspheres and rods was investigated in nonhuman primates (NHPs) and rabbits. An in vitro assessment of cytokine responses to PLGA in peripheral blood mononuclear cells (PBMCs) and macrophages was also performed. Results: Intravitreal administration of 3, 10, or 12.5 mg/eye of PLGA microspheres in NHPs resulted in a severe immune response characterized by a foreign body response. Follow-up studies in the rabbit confirmed this finding for PLGA microspheres ranging in size from 20 to 100 µm. In contrast, administration of PLGA rod implants with a similar PLGA mass did not elicit a significant immune response. In vitro assays in PBMCs and macrophages confirmed proinflammatory cytokine release upon treatment with PLGA microspheres but not PLGA rods. Conclusions: These data demonstrate a lack of tolerability of PLGA microspheres upon intravitreal injection, and suggest that the size, shape, and/or surface area of PLGA depots are critical attributes in determining ocular toxicity.

Proceedings of the 2015 National Toxicology Program Satellite Symposium.

The 2015 Annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri" was held in Minneapolis, Minnesota, at the American College of Veterinary Pathologists/American Society for Veterinary Clinical Pathology/Society of Toxicologic Pathology combined meeting. The goal of this symposium is to present and discuss diagnostic pathology challenges or nomenclature issues. Because of the combined meeting, both laboratory and domestic animal cases were presented. This article presents summaries of the speakers' talks, including challenging diagnostic cases or nomenclature issues that were presented, along with select images that were used for audience voting and discussion. Some lesions and topics covered during the symposium included hepatocellular lesions, a proposed harmonized diagnostic approach to rat cardiomyopathy, crop milk in a bird, avian feeding accoutrement, heat exchanger in a tuna, metastasis of a tobacco carcinogen-induced pulmonary carcinoma, neurocytoma in a rat, pituicytoma in a rat, rodent mammary gland whole mounts, dog and rat alveolar macrophage ultrastructure, dog and rat pulmonary phospholipidosis, alveolar macrophage aggregation in a dog, degenerating yeast in a cat liver aspirate, myeloid leukemia in lymph node aspirates from a dog, Trypanosoma cruzi in a dog, solanum toxicity in a cow, bovine astrovirus, malignant microglial tumor, and nomenclature challenges from the Special Senses International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Group.

Oligodeoxynucleotide studies in primates: antisense and immune stimulatory indications.

Antisense oligodeoxynucleotide compounds (AS ODN) are being developed as therapeutics for various disease indications. Their safety and pharmacokinetics are most commonly evaluated in rodents and nonhuman primates. Traditional AS ODN are short, single strands of DNA, and they target specific mRNA sequences. Plasma clearance of AS ODN is rapid, broad tissue distribution occurs, and elimination is by nuclease metabolism. Structural modifications to AS ODN have been made to enhance their efficacy and improve their safety. A number of class effects are observed with AS ODN that are unrelated to the specific targeted mRNA sequence. Acute effects include activation of the alternative complement pathway and inhibition of the intrinsic coagulation pathway. In monkeys, rodents, and dogs given repeated doses of AS ODN, accumulation of AS ODN and/or metabolites occurs in the form of basophilic granules in various tissues, including the kidney, lymph nodes and liver. A new potential therapeutic application of ODN is that of immune stimulation. Immunostimulatory ODN (IS ODN) are being investigated for use in treating cancer, infectious disease, and allergy. For the development of both AS and IS ODN, primates will continue to be important for safety assessment.