Use of the dTAG system in vivo to degrade CDK2 and CDK5 in adult mice and explore potential safety liabilities.

The degradation tag (dTAG) system for target protein degradation can remove proteins from biological systems without the drawbacks of some genetic methods, such as slow kinetics, lack of reversibility, low specificity, and the inability to titrate dosage. These drawbacks can make it difficult to compare toxicity resulting from genetic and pharmacological interventions, especially in vivo. Because the dTAG system has not been studied extensively in vivo, we explored the use of this system to study the physiological sequalae resulting from CDK2 or CDK5 degradation in adult mice. Mice with homozygous knock-in of the dTAG sequence onto CDK2 and CDK5 were born at Mendelian ratios despite decreased CDK2 or CDK5 protein levels in comparison with wild-type mice. In bone marrow cells and duodenum organoids derived from these mice, treatment with the dTAG degrader dTAG-13 resulted in rapid and robust protein degradation but caused no appreciable change in viability or the transcriptome. Repeated delivery of dTAG-13 in vivo for toxicity studies proved challenging; we explored multiple formulations in an effort to maximize degradation while minimizing formulation-related toxicity. Degradation of CDK2 or CDK5 in all organs except the brain, where dTAG-13 likely did not cross the blood brain barrier, only caused microscopic changes in the testis of CDK2dTAG mice. These findings were corroborated with conditional CDK2 knockout in adult mice. Our results suggest that the dTAG system can provide robust protein degradation in vivo and that loss of CDK2 or CDK5 in adult mice causes no previously unknown phenotypes.

Editor's Highlight: Plasma miR-183/96/182 Cluster and miR-124 are Promising Biomarkers of Rat Retinal Toxicity.

Retinal toxicity is one of the leading causes of attrition in drug development, and drug-induced retinal toxicity remains an issue in both drug discovery and postmarketed drugs. Derisking strategies to help with early identification of retinal injury utilizing a predictive retinal miRNA biomarker would greatly benefit decision-making in drug discovery programs, ultimately reducing attrition due to retinal toxicity. Our previous work demonstrated elevation of circulating retina-enriched miRNAs in a retinal toxicity model. To further validate our previous observation, 2 additional rat retinal injury models were utilized in this study: NaIO3-induced retinal injury and laser-induced choroidal neovascularization (CNV) injury model. Following induction of retina tissue injuries, circulating miR-183/96/182 cluster (miR-183 cluster), and miR-124 was investigated, as well as evaluations using an electroretinogram (ERG) and histopathology analysis. Data revealed that circulating miR-183/96/182 cluster was significantly increased (2- to 15-fold) compared with baseline/control in both laser-induced CNV and NaIO3-induced retinal injury models. Moreover, the severity of the retinal injury evaluated by ERG and histopathology correlated highly with elevation of these retina-enriched miRNAs in plasma. MiR-124 was also significantly increased in comparison with baseline/control by ∼25-fold postrepeat-doses of 30 mg/kg NaIO3 treatment. Increased level of these plasma miRNA biomarkers appeared to be dose- and time-dependent upon NaIO3 or laser treatment. The results suggest that the retina-enriched miRNAs (miR-183/96/182 cluster and miR-124) could serve as convenient and predictive biomarkers of retinal toxicity in drug development.

Application of electroretinography (ERG) in early drug development for assessing retinal toxicity in rats.

Retinal ocular toxicity is among the leading causes of drug development attrition in the pharmaceutical industry. Electroretinography (ERG) is a non-invasive functional assay used to assess neuro-retinal physiological integrity by measuring the electrical responses. To directly assess the utility of ERG, a series of studies was conducted following intravitreal and/or iv administration of pan-cyclin-dependent kinase inhibitors: AG-012,986 and AG-024,322 in rats. Both compounds have previously shown to induce retinal toxicity. Retinal injury was evaluated by ERG, histopathology and TUNEL staining. Intravitreal injection of AG-012,986 at ≥ 10 µg/eye resulted in decreases (60%) in ERG b-wave and microscopic changes of mild to moderate retinal degeneration, and at 30 µg/eye led to additional ophthalmic findings. Intravenous administration of AG-012,986 daily at ≥ 5 mg/kg resulted in dose-related decreases (25 to 40%) in b-wave and sporadic to intense positive TUNEL staining. Intravitreal injection of AG-024,322 at 30 µg/eye also resulted in decreases (50 to 60%) in b-wave, mild to marked retinal degeneration and mild vitreous debris. These experiments demonstrate that ERG can be used as a sensitive and reliable functional tool to evaluate retinal toxicity induced by test compounds in rats complementing other classical ocular safety measurements.

An assessment of the ocular safety of excipient maleic acid following intravitreal injection in rabbits.

Maleic acid was formulated in 0.7% saline and injected intravitreally in rabbits in order to evaluate ocular safety and tolerability. Maleic acid was formulated within a narrow pH range (2-3), administered in a fixed volume (100 µl), and concentrations ranged from 0.00 to 2.00 mg/eye (0.00 to 12.30 mM vitreous). Ocular evaluations were conducted at 2, 4, and 8 days post injection. Ocular irritation responses were observed at doses from 0.50 mg/eye (3.07 mM vitreous) to 2.00 mg/eye (12.30 mM vitreous) and included conjunctival redness and scleral swelling. Chemosis was observed at 2.00 mg/eye (12.30 mM vitreous). Funduscopic evaluations revealed enlarged retinal blood vessels and optic disk swelling at doses ≥1.50 mg/eye (9.22 mM vitreous), retinal folds and retinal discoloration at 2.00 mg/eye (12.30 mM vitreous). Histopathologic evaluations on days 4 and 8 post injection revealed retinal degeneration at doses ≥1.0 mg/eye (6.15 mM vitreous), conjunctival inflammation at doses ≥1.5 mg/eye (9.22 mM vitreous), and retinal pigment epithelial hypertrophy, optic nerve demyelination, anterior chamber fluid, and conjunctival fibrosis at 2.00 mg/eye (12.30 mM vitreous) maleic acid. The data suggest that maleic acid formulations at ≥1.00 mg/eye (6.15 mM vitreous) were not suitable for intraocular indications.

Cardiovascular effects in rats following exposure to a receptor tyrosine kinase inhibitor.

The receptor tyrosine kinase receptor (RTK) signaling pathway, mesenchymal-epithelial transition factor (c-Met)/hepatocyte growth factor receptor (HGFR), has been implicated in oncogenesis and is a target of interest in cancer therapy. PF-04254644 is a potent and selective inhibitor of c-Met/HGFR. Wide ligand binding profiling of PF-04254644 revealed a potentially significant interaction with phosphodiesterase (PDE) 3, and follow-up PDE enzyme activity assays confirmed PF-04254644 as a potent inhibitor of PDE3 as well as other PDEs (1, 2, 5, 10, and 11). Clinical observations, laboratory, and echocardiography parameters were recorded in Sprague-Dawley (SD) rats that received PF-04254644 oral dosing for up to seven consecutive days. Toxicological evaluations revealed myocardial degeneration as an adverse event at all tested doses. Echocardiographic evaluations revealed an increase in heart rate (HR) and contractility after the first dose with PF-04254644 and myocardial fibrosis correlated with decreased cardiac function after repeat dosing. A study in telemetry-instrumented rats substantiated that PF-04254644 induced a sustained increased HR and decreased contractility after six days of treatment. Data suggest that the decreased cardiac function and cardiotoxicity are likely due to inhibition of multiple PDEs by PF-04254644.

PD0325901, a mitogen-activated protein kinase kinase inhibitor, produces ocular toxicity in a rabbit animal model of retinal vein occlusion.

OBJECTIVE: PD0325901, a selective inhibitor of mitogen-activated protein kinase kinase (MEK), was associated with the occurrence of ocular retinal vein occlusion (RVO) during clinical trials in patients with solid tumors. As previous animal safety studies in rats and dogs did not identify the eye as a target organ of toxicity, this work was conducted to develop a rabbit model of ocular toxicity with PD0325901. METHODS: Dutch-Belted rabbits were administered a single intravitreal injection of PD0325901 (0.5 or 1 mg/eye) or saline control, and ophthalmic examinations and retinal angiography were conducted over a 2-week period post-dose. In addition, mechanism of ocular toxicity was further explored in rat with microarray analysis. RESULTS: PD0325901 treatment produced RVO with retinal vasculature leakage and hemorrhage within 48-h postinjection in Dutch-Belted rabbits. Subsequent retinal detachment and degeneration were also detected on day 8 postinjection. To evaluate the potential mechanism(s) of PD0325901-mediated RVO, male Brown Norway rats were orally administered PD0325901 (45 mg/kg/day) up to 5 days and retinal tissue was collected for gene array analysis. Although PD0325901 did not produce clinical evidence of RVO in rats, retinal gene expression suggested an increased oxidative stress and inflammatory response, endothelium and blood-retinal barrier damage, and prothrombotic effects. Moreover, soluble endothelial protein C receptor (sEPCR), a biomarker for RVO, was elevated in human umbilical vascular endothelial cells (HUVECs) cultured with PD0325901. CONCLUSIONS: This work has developed a rabbit model of PD0325901-induced RVO that may be used to characterize the cellular and molecular mechanisms of this effect in humans.