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.

Inflammation and immunogenicity limit the utility of the rabbit as a nonclinical species for ocular biologic therapeutics.

The nonclinical safety evaluation of therapeutic drug candidates is commonly conducted in two species (rodent and non-rodent) in keeping with international health authority guidance. Biologic drugs typically have restricted species cross-reactivity, necessitating the evaluation of safety in non-human primates and thus limiting the utility of lower order species. Safety studies of cross-reactive ocular biologic drug candidates have been conducted in rabbits as a second toxicology species, despite the fact that rabbits are not a rodent species. Such studies are often confounded by the development of anti-drug antibodies and severe ocular inflammation, the latter requiring studies to be terminated prematurely for animal welfare reasons. Notably, these confounding factors preclude the interpretation of safety. Nonclinical toxicology programs should be designed with consideration of ethical animal use and 3Rs principles (Replacement, Reduction and Refinement). The experience of several pharmaceutical sponsors, demonstrating that toxicology studies of ocular (intravitreal and topical ocular) biologic drug candidates in the rabbit are of limited interpretive value, calls into question the utility of such studies in this species and indicates that such studies should not be conducted.

Pharmacologic and pharmacokinetic profile of repifermin (KGF-2) in monkeys and comparative pharmacokinetics in humans.

Repifermin (truncated, recombinant human keratinocyte growth factor-2, KGF-2) was evaluated in cynomolgus monkeys and healthy humans during a phase 1 trial. Monkeys received vehicle or repifermin at 20, 75, or 200 microg/kg IV or 750 microg/kg subcutaneous (SC) daily for 29 days. Clinical observations were made during the entire dosing period. Gross and microscopic changes were assessed at necropsy. Pharmacokinetic parameters and immunogenicity were evaluated in these monkeys and in humans, following a single or 7 daily IV bolus injections of 1, 5, 25, or 50 microg/kg repifermin. In monkeys, repifermin was well tolerated, and histologic evaluation demonstrated dose-dependent, reversible thickening of the mucosa throughout the alimentary tract, except for the stomach. In the alimentary tract tissues, nonepithelial tissues were not affected, indicating a specificity of repifermin for epithelial cells. Pharmacokinetics in both monkeys and humans were dose proportional, showed lack of drug accumulation with repeated daily dosing, and were characterized by high volumes of distribution and clearance rates, indicating substantial tissue binding and metabolism. Repifermin was not markedly immunogenic following multiple daily IV injections in either species. Serum repifermin concentrations in humans were comparable to those attained in monkeys that produced significant pharmacological effects on epithelial cells in the alimentary tract. These findings provide additional support for the ongoing clinical development of repifermin for diseases involving epithelial injury.