Liver Microvascular Injury and Thrombocytopenia of Antibody-Calicheamicin Conjugates in Cynomolgus Monkeys-Mechanism and Monitoring.

Purpose: Adverse reactions reported in patients treated with antibody-calicheamicin conjugates such as gemtuzumab ozogamicin (Mylotarg) and inotuzumab ozogamicin include thrombocytopenia and sinusoidal obstruction syndrome (SOS). The objective of this experimental work was to investigate the mechanism for thrombocytopenia, characterize the liver injury, and identify potential safety biomarkers.Experimental Design: Cynomolgus monkeys were dosed intravenously at 6 mg/m2/dose once every 3 weeks with a nonbinding antibody-calicheamicin conjugate (PF-0259) containing the same linker-payload as gemtuzumab ozogamicin and inotuzumab ozogamicin. Monkeys were necropsied 48 hours after the first administration (day 3) or 3 weeks after the third administration (day 63).Results: PF-0259 induced acute thrombocytopenia (up to 86% platelet reduction) with nadirs on days 3 to 4. There was no indication of effects on megakaryocytes in bone marrow or activation of platelets in peripheral blood. Microscopic evaluation of liver from animals necropsied on day 3 demonstrated midzonal degeneration and loss of sinusoidal endothelial cells (SECs) associated with marked platelet accumulation in sinusoids. Liver histopathology on day 63 showed variable endothelial recovery and progression to a combination of sinusoidal capillarization and sinusoidal dilation/hepatocellular atrophy, consistent with early SOS. Among biomarkers evaluated, there were early and sustained increases in serum hyaluronic acid (HA) that correlated well with serum aspartate aminotransferase and liver microscopic changes, suggesting that HA may be a sensitive diagnostic marker of the liver microvascular injury.Conclusions: These data support the conclusion that target-independent damage to liver SECs may be responsible for acute thrombocytopenia (through platelet sequestration in liver sinusoids) and development of SOS. Clin Cancer Res; 23(7); 1760-70. ©2016 AACR.

Contribution of membrane trafficking perturbation to retinal toxicity.

The retina is a highly structured tissue that is formed by layers containing 7 different cell types. The photoreceptor cell is a specialized type of neuron in the retina that is capable of absorbing and converting light into electrophysiological signals. There is a constant renewal process for photoreceptors consisting of intermittent shedding of the distal tips of the photosensitive outer segment and subsequent phagocytosis (uptake, degradation and recycling) by retinal pigmented epithelial (RPE) cells. This rebuilding process is essential for vision and the survival of photoreceptors and RPE cells. Drugs with a basic moiety have the potential to accumulate in the lysosome and impair its functions including the phagocytosis process, which could hinder clearance of outer segments and ultimately induce retinopathy. To determine the prevalence of this cellular mechanism in retinal toxicity, a collection of proprietary compounds associated with retinal toxicity were subjected to a battery of in vitro tests using the human adult retinal pigmented epithelium cell line, ARPE-19. The tests included a phagocytosis assay, and lysosomal and autophagosomal staining. The compounds that induced retinopathy clustered in the basic and lipophilic region, which drives lysosomal sequestration. This accumulation coincided with phagocytosis inhibition and an increase in autophagosome staining, suggesting a blockage of the membrane trafficking process. A correlation between the physicochemical properties and in vitro lysosomal pathway effects was established. These data reveal the importance of physicochemical properties of compounds and lysosome accumulation as a potential mechanism for drug-induced retinopathy and demonstrate the usefulness of in vitro screening in predicting this liability.

Comparative nonclinical assessments of the proposed biosimilar PF-05280586 and rituximab (MabThera®).

Comparative nonclinical studies were conducted with the proposed biosimilar PF-05280586 and rituximab-EU (MabThera®). In side-by-side analyses, peptide maps and complement-dependent cytotoxicity assay results were similar. Sexually-mature cynomolgus monkeys were administered PF-05280586 or rituximab-EU as a single dose of 0, 2, 10, or 20 mg/kg on day 1 and observed for 92 days (single-dose study) or as 5 weekly injections of 0 or 20 mg/kg and necropsied on day 30, the day after the 5th dose, or on day 121 (repeat-dose study). The pharmacokinetic and pharmacodynamic profiles for both molecules were similar. Marked depletion of peripheral blood B cells 4 days after dosing was followed by near or complete repletion (single-dose study) or partial repletion (repeat-dose study). In the single-dose study, anti-drug antibodies (ADA) were detected by day 29 in all animals administered PF-05280586 or rituximab-EU and persisted through day 85, the last day tested. In the repeat-dose study, ADA were detected on day 121 in 50% of animals administered PF-05280586 or rituximab-EU. Both molecules were well tolerated at all doses. In all endpoints evaluated, PF-05280586 exhibited similarity to rituximab-EU.

Hemodynamic correlates of drug-induced vascular injury in the rat using high-frequency ultrasound imaging.

Several classes of drugs have been shown to cause drug-induced vascular injury (DIVI) in preclinical toxicity studies. Measurement of blood flow and vessel diameter in numerous vessels and across various tissues by ultrasound imaging has the potential to be a noninvasive translatable biomarker of DIVI. Our objective was to demonstrate the utility of high-frequency ultrasound imaging for measuring changes in vascular function by evaluating blood flow and vessel diameter in the superior mesenteric arteries (SMA) of rats treated with compounds that are known to cause DIVI and are known vasodilators in rat: fenoldopam, CI-1044, and SK&F 95654. Blood flow, vessel diameter, and other parameters were measured in the SMA at 4, 8, and 24 hr after dosing. Mild to moderate perivascular accumulations of mononuclear cells, neutrophils in tunica adventitia, and superficial tunica media as well as multifocal hemorrhage and necrosis in the tunica media were found in animals 24 hr after treatment with fenoldopam and SK&F 95654. Each compound caused marked increases in blood flow and shear stress as early as 4 hr after dosing. These results suggest that ultrasound imaging may constitute a functional correlate for the microscopic finding of DIVI in the rat.

Mechanistic investigations of test article-induced pancreatic toxicity at the endocrine-exocrine interface in the rat.

Pancreatic toxicity commonly affects the endocrine or exocrine pancreas. However, it can also occur at the endocrine-exocrine interface (EEI), where the capillary network of the islet merges with the capillaries of the surrounding acinar tissue, that is, the insulo-acinar portal system. The goal of this article is to describe a novel, test article-induced pancreatic toxicity that originated at the EEI and to summarize investigations into the mechanistic basis of the injury. This injury was initially characterized by light microscopy in 7/14 day-toxicity studies in Sprague-Dawley (Crl: CD®[SD]) rats with undisclosed test articles. Microvascular injury at the interface resulted in peri-islet serum exudation, fibrin deposition, hemorrhage, inflammation, and secondary degeneration/necrosis of surrounding exocrine tissue. More chronic injury presented as islet fibrosis and lobular atrophy. Direct cytotoxicity affecting the capillary endothelium at the EEI was confirmed ultrastructurally on day 4. Endothelial microparticle and blood flow studies further confirmed endothelial involvement. Similar lesions occurred less frequently in 2 other rat strains and not in the mouse, dog, or cynomolgus macaque. In summary, in vivo and investigative study data confirmed primary endothelial cytotoxicity in the pathogenesis of this lesion and suggested that the lesion may be rat/rat strain-specific and of uncertain relevance for human safety risk assessment.

Effects of age on noninvasive assessments of vascular function in nonhuman primates: implications for translational drug discovery.

BACKGROUND: Endothelium-dependent flow mediated dilation (FMD) and pulse-wave velocity (PWV), are used as measures of vascular health and predictors of cardiovascular risk in clinical studies, and both are age-dependent. Numbers of circulating endothelial microparticles (EMPs) and endothelial progenitor cells (EPCs) are also associated with cardiovascular risk, but independent of age in humans. The use of these measurements for pre-clinical assessment of drug cardiovascular safety and efficacy in non-human primates (NHPs) may promote the translation of drug-induced effects on vascular function to clinic outcomes. However, in NHPs, the age effects on the non-invasive measurements of FMD and PWV and the relationship of EMPs/EPCs with FMD are unknown. METHODS: A non-invasive, clinically-relevant approach to assess FMD and PWV was used to examine their relationship with age and with EMPs/EPCs in NHPs. The effects on FMD of nicotine and rosiglitazone were evaluated in senescent primates in an effort to validate our FMD method for pre-clinical assessment of vascular function. RESULTS: FMD and PWV methods were established in a colony (n = 25) of metabolically healthy, cynomolgus monkeys ranging in age from 6 to 26 years. FMD, defined as the percent change, at 1 min of cuff release, from baseline vascular diameter (0.15 ± 0.03 cm), had a strong, negative correlation with age (r = -0.892, p < 0.0001), ranging from 6% to 33%. PWV positively correlated with age (r = 0.622, p < 0.002) in the same healthy monkeys. Nicotine and rosiglitazone, were evaluated in subsets of senescent primates (mean age 16.3 ± 1.5[SEM] years). Rosiglitazone significantly improved FMD (21.0 ± 1.6% vs. vehicle 16.3 ± 1.6%, p < 0.01) without changing baseline diameters, and coincided with a significant increase in circulating numbers of endothelial progenitor cells (CD45-CD31 + CD34 + VEGFR2+ 7.1 ± 1.3 vs. 4.8 ± 1.1 counts/µl) and a decrease in endothelial microparticles (CD45-CD42a-CD54+ 26.7 ± 11.1 vs. 62.2 ± 9.8 counts/µl)(p < 0.05). Conversely, FMD was significantly reduced with nicotine (8.7 ± 1.4% vs. vehicle 20.1 ± 2.2%, p < 0.05). CONCLUSIONS: Adult NHPs demonstrate the characteristic linear relationship between age and vascular function using the non-invasive clinically-related measurements of FMD and PWV. However, numbers of circulating EMPs and EPCs did not correlate with age. Endothelial function assessed with FMD, together with EMPs/EPCs assessment, may serve as a novel approach for translational research and therapeutic discovery. Age should be considered in the study design or data analyses when FMD or PWV is used in NHPs.

Pregabalin induces hepatic hypoxia and increases endothelial cell proliferation in mice, a process inhibited by dietary vitamin E supplementation.

The preceding article identified key components of pregabalin's mode of action on nongenotoxic hemangiosarcoma formation in mice, including increased serum bicarbonate leading to decreased respiratory rate, increased blood pH, increased venous oxygen saturation, increased vascular endothelial growth factor and basic fibroblast growth factor expression, increased hepatic vascular endothelial growth factor receptor 2 expression, and increased iron-laden macrophages. Increased platelet count and platelet activation were early, species-specific biomarkers in mice. Dysregulated erythropoiesis, macrophage activation, and elevations of tissue growth factors were consistent with the unified mode of action for nongenotoxic hemangiosarcoma recently proposed at an international hemangiosarcoma workshop (Cohen, S. M., Storer, R. D., Criswell, K. A., Doerrer, N. G., Dellarco, V. L., Pegg, D. G., Wojcinski, Z. W., Malarkey, D. E., Jacobs, A. C., Klaunig, J. E., et al. (2009). Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18). In this article, we present evidence that pregabalin induces hypoxia and increases endothelial cell (EC) proliferation in a species-specific manner. Dietary administration of pregabalin produced a significant 35% increase in an immunohistochemical stain for hypoxia (Hypoxyprobe) in livers from pregabalin-treated mice. Increased Hypoxyprobe staining was not observed in the liver, bone marrow, or spleen of rats, supporting the hypothesis that pregabalin produces local tissue hypoxia in a species-specific manner. Transcriptional analysis supports that rats, unlike mice, adapt to pregabalin-induced hypoxia. Using a dual-label method, increased EC proliferation was observed as early as 2 weeks in mouse liver and 12 weeks in bone marrow following pregabalin administration. These same assays showed decreased EC proliferation in hepatic ECs of rats, further supporting species specificity. Dietary supplementation with vitamin E, which is known to have antioxidant and antiangiogenic activity, inhibited pregabalin-induced increases in mouse hepatic EC proliferation, providing confirmatory evidence for the proposed mode of action and its species-specific response.

Targeting of 4-1BB by monoclonal antibody PF-05082566 enhances T-cell function and promotes anti-tumor activity.

4-1BB (CD137, TNFRSF9) is a costimulatory receptor expressed on several subsets of activated immune cells. Numerous studies of mouse and human T cells indicate that 4-1BB promotes cellular proliferation, survival, and cytokine production. 4-1BB agonist mAbs have demonstrated efficacy in prophylactic and therapeutic settings in both monotherapy and combination therapy tumor models and have established durable anti-tumor protective T-cell memory responses. PF-05082566 is a fully human IgG2 that binds to the extracellular domain of human 4-1BB with high affinity and specificity. In preclinical studies, this agonist antibody demonstrated its ability to activate NF-κB and induce downstream cytokine production, promote leukocyte proliferation, and inhibit tumor growth in a human PBMC xenograft tumor model. The mechanism of action and robust anti-tumor efficacy of PF-05082566 support its clinical development for the treatment of a broad spectrum of human malignancies.

Diamide amino-imidazoles: a novel series of γ-secretase inhibitors for the treatment of Alzheimer's disease.

The synthesis and structure-activity relationship (SAR) of a novel series of di-substituted imidazoles, derived from modification of DAPT, are described. Subsequent optimization led to identification of a highly potent series of inhibitors that contain a ß-amine in the imidazole side-chain resulting in a robust in vivo reduction of plasma and brain Aß in guinea pigs. The therapeutic index between Aß reductions and changes in B-cell populations were studied for compound 10 h.

Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: discovery of PF-3084014.

A novel series of tetralin containing amino imidazoles, derived from modification of the corresponding phenyl acetic acid derivatives is described. Replacement of the amide led to identification of a potent series of tetralin-amino imidazoles with robust central efficacy. The reduction of brain Aß in guinea pigs in the absence of changes in B-cells suggested a potential therapeutic index with respect to APP processing compared with biomarkers of notch related toxicity. Optimization of the FTOC to plasma concentrations at the brain Aß EC(50) lead to the identification of compound 14f (PF-3084014) which was selected for clinical development.

Pharmacodynamics and pharmacokinetics of the gamma-secretase inhibitor PF-3084014.

PF-3084014 [(S)-2-((S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-3-ylamino)-N-(1-(2-methyl-1-(neopentylamino)propan-2-yl)-1H-imidazol-4-yl)pentanamide] is a novel gamma-secretase inhibitor that reduces amyloid-beta (Abeta) production with an in vitro IC(50) of 1.2 nM (whole-cell assay) to 6.2 nM (cell-free assay). This compound inhibits Notch-related T- and B-cell maturation in an in vitro thymocyte assay with an EC(50) of 2.1 microM. A single acute dose showed dose-dependent reduction in brain, cerebrospinal fluid (CSF), and plasma Abeta in Tg2576 mice as measured by enzyme-linked immunosorbent assay and immunoprecipitation (IP)/mass spectrometry (MS). Guinea pigs were dosed with PF-3084014 for 5 days via osmotic minipump at 0.03 to 3 mg/kg/day and exhibited dose-dependent reduction in brain, CSF, and plasma Abeta. To further characterize Abeta dynamics in brain, CSF, and plasma in relation to drug exposure and Notch-related toxicities, guinea pigs were dosed with 0.03 to 10 mg/kg PF-3084014, and tissues were collected at regular intervals from 0.75 to 30 h after dose. Brain, CSF, and plasma all exhibited dose-dependent reductions in Abeta, and the magnitude and duration of Abeta lowering exceeded those of the reductions in B-cell endpoints. Other gamma-secretase inhibitors have shown high potency at elevating Abeta in the conditioned media of whole cells and the plasma of multiple animal models and humans. Such potentiation was not observed with PF-3084014. IP/MS analysis, however, revealed dose-dependent increases in Abeta11-40 and Abeta1-43 at doses that potently inhibited Abeta1-40 and Abeta1-42. PF-3084014, like previously described gamma-secretase inhibitors, preferentially reduced Abeta1-40 relative to Abeta1-42. Potency at Abeta relative to Notch-related endpoints in vitro and in vivo suggests that a therapeutic index can be achieved with this compound.