International consortium for innovation and quality: An industry perspective on the nonclinical and early clinical development of intravitreal drugs.

The eye, which is under constant exposure to environmental pathogens, has evolved various anatomic and immunological barriers critical to the protection of tissues lacking regenerative capacity, and the maintenance of a clear optic pathway essential to vision. By bypassing the ocular barriers, intravitreal (IVT) injection has become the mainstay for the delivery of drugs to treat conditions that affect the back of the eye. Both small molecules and biotherapeutics have been successfully administered intravitreally, and several drugs have been approved for the treatment of (wet) age-related macular degeneration and diabetic macular edema. However, IVT injection is an invasive procedure, which requires sufficient technical expertise from the healthcare professional administering the drug. Potential side effects include bleeding, retinal tear, cataracts, infection, uveitis, loss of vision, and increased ocular pressure. Pharmaceutical companies often differ in their drug development plan, including drug administration techniques, collection of ocular tissues and fluids, ophthalmology monitoring, and overall conduct of nonclinical and clinical studies. The present effort, under the aegis of the Innovation & Quality Ophthalmic Working Group, aims at understanding these differences, identifying pros and cons of the various approaches, determining the gaps in knowledge, and suggesting feasible good practices for nonclinical and early clinical IVT drug development.

Ketamine/Xylazine Anesthesia-Related Corneal Lesions in Rats With Surgically Implanted Venous Catheters Utilized in Nonclinical Intravenous Studies.

Nonclinical rodent studies with repeat slow intravenous dosing, such as safety assessments of anticancer therapeutics, often require the use of animals with surgically implanted catheters. Catheterization is a relatively short surgical procedure but requires use of anesthesia. Ketamine/xylazine injectable anesthesia is typically used because it has advantages over inhalation anesthesia including ease of administration, safety and predictability of effects, and relatively low cost. However, ketamine/xylazine anesthesia in rodents can also be associated with the development of undesirable corneal lesions of uncertain mechanism such as mineralization of Bowman's membrane or stroma, erosion/ulceration, inflammation, fibroplasia, and neovascularization. Such findings have the potential to confound study interpretation in programs for which the cornea is a potential target tissue. This case report describes the occurrence of ketamine/xylazine-related corneal lesions observed in surgically catheterized rats in a 16-day toxicity study for an oncology compound.

From the Cover: Inhibitors of Nicotinamide Phosphoribosyltransferase Cause Retinal Damage in Larval Zebrafish.

Nicotinamide phosphoribosyltransferase (NAMPT) has been investigated as a target for oncology because it catalyzes a rate-limiting step in cellular energy metabolism to produce nicotinamide adenine dinucleotide. Small molecule inhibitors of NAMPT have been promising drug candidates but preclinical development has been hindered due to associated retinal toxicity. Here we demonstrate that larval zebrafish can predict retinal toxicity associated with this mechanism revealing an attractive alternative method for identifying such toxicities. Zebrafish permit higher throughput testing while using far lower quantities of test article compared with mammalian systems. NAMPT inhibitor-associated toxicity manifested in zebrafish as a loss of response to visual cues compared with auditory cues. Zebrafish retinal damage associated with NAMPT inhibitor treatment was confirmed through histopathology. Ranking 6 NAMPT inhibitors according to their impact on zebrafish vision revealed a positive correlation with their in vitro potencies on human tumor cells. This correlation indicates translatable pharmacodynamics between zebrafish and human NAMPT and is consistent with on-target activity as the cause of retinal toxicity associated with NAMPT inhibition. Together, these data illustrate the utility of zebrafish for identifying compounds that may cause ocular toxicity in mammals, and, likewise, for accelerating development of compounds with improved safety margins.

C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice.

The expansion of a hexanucleotide (GGGGCC) repeat in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both the function of C9ORF72 and the mechanism by which the repeat expansion drives neuropathology are unknown. To examine whether C9ORF72 haploinsufficiency induces neurological disease, we created a C9orf72-deficient mouse line. Null mice developed a robust immune phenotype characterized by myeloid expansion, T cell activation, and increased plasma cells. Mice also presented with elevated autoantibodies and evidence of immune-mediated glomerulonephropathy. Collectively, our data suggest that C9orf72 regulates immune homeostasis and an autoimmune response reminiscent of systemic lupus erythematosus (SLE) occurs in its absence. We further imply that haploinsufficiency is unlikely to be the causative factor in C9ALS/FTD pathology.

Hormonal profiles of captive Galapagos tortoises (Chelonoidis nigra).

Monthly blood samples, daily mating observations from Galapagos tortoises (Chelonoidis nigra), and local rainfall and temperature were collected at the Honolulu Zoo as part of a fertility evaluation. Testosterone concentrations were measured for males (n = 6), two of which were seen copulating and were considered sexually active. Estrone sulfate and progesterone concentrations were measured for female tortoises (n = 9), two of which nested and only one had laid eggs. Testosterone profiles were similar for both sexually active and sexually inactive males, both of which were positively correlated with temperature but not rainfall. Peak testosterone concentrations (12.0 ± 1.4 ng/ml sexually active animals vs. 14.4 ± 2.4 ng/ml sexually inactive animals) occurred at the end of the nesting season, from April to July. Estrone sulfate concentrations were similar for nesting (n = 2) and non-nesting (n = 7) female tortoises, rising from non-detectable concentrations (September), and increasing to peak concentrations during the nesting season. Progesterone concentrations remained low and spiked (9.44 ng/ml) only for the female that nested and laid eggs. Testosterone was negatively correlated with mating behavior, and the male tortoises were likely capable of spermatogenesis even though only two of them engaged in mating behavior. The female tortoises were not senescent, as the estrone sulfate concentrations likely reflected waves of ovarian follicular activity. Endocrine parameters were not in synchrony with rainfall, and a disconnect between the timing of reproductive events and the environmental milieu may help to explain the poor fertility of these tortoises. Zoo Biol. 35:237-245, 2016. © 2016 Wiley Periodicals, Inc.

Isoproterenol effects evaluated in heart slices of human and rat in comparison to rat heart in vivo.

Human response to isoproterenol induced cardiac injury was evaluated by gene and protein pathway changes in human heart slices, and compared to rat heart slices and rat heart in vivo. Isoproterenol (10 and 100µM) altered human and rat heart slice markers of oxidative stress (ATP and GSH) at 24h. In this in vivo rat study (0.5mg/kg), serum troponin concentrations increased with lesion severity, minimal to mild necrosis at 24 and 48h. In the rat and the human heart, isoproterenol altered pathways for apoptosis/necrosis, stress/energy, inflammation, and remodeling/fibrosis. The rat and human heart slices were in an apoptotic phase, while the in vivo rat heart exhibited necrosis histologically and further progression of tissue remodeling. In human heart slices genes for several heat shock 70kD members were altered, indicative of stress to mitigate apoptosis. The stress response included alterations in energy utilization, fatty acid processing, and the up-regulation of inducible nitric oxide synthase, a marker of increased oxidative stress in both species. Inflammation markers linked with remodeling included IL-1α, Il-1ß, IL-6 and TNFα in both species. Tissue remodeling changes in both species included increases in the TIMP proteins, inhibitors of matrix degradation, the gene/protein of IL-4 linked with cardiac fibrosis, and the gene Ccl7 a chemokine that induces collagen synthesis, and Reg3b a growth factor for cardiac repair. This study demonstrates that the initial human heart slice response to isoproterenol cardiac injury results in apoptosis, stress/energy status, inflammation and tissue remodeling at concentrations similar to that in rat heart slices.