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Supplementary Figure 12 shows the ELISA assay with the dose-dependent C1q binding to plate-adsorbed sasanlimab and the ADCC assay of sasanlimab performed with activated T cells (target cells) and stimulated PBMCs (effector cells).

Supplementary Figure 6 shows the rCGE profile for sasanlimab with two predominant peaks, consistent with L chain and H chain

Supplementary Figure 10 shows the selectivity of sasanlimab binding to human and cynomolgus monkey PD-1 using HEK-293T transiently transfected cells and flow cytometry

Table S2 shows IC50 values for CDK4/6 inhibitors in human and rat bone marrow.

Table S3 shows A: Most significantly impacted pathways. B: Most significantly impacted upstream regulators.

Table S4 shows expression change in solute carrier family genes.

Supplementary Figure 5 shows the analysis of sasanlimab by SE-HPLC with UV detection at 280 nm

Supplementary Figure 11 shows sasanlimab binding to human and cynomolgus monkey Fcγ receptor and FcRn

Supplementary Figure S1. Anti-4-1BB and anti-PD-1 synergized to suppress B16F10 tumor growth. Supplementary Figure S2. The flow cytometric gating strategy to analyze immune cells. Supplementary Figure S3. Elevated expression of genes associated with anti-tumor immune response in MC38 colon carcinomas. Supplementary Figure S4. Accumulation of CD8+ T cell and memory phenotype differentiation in MC38 model. Supplementary Figure S5. Serum liver enzyme levels and hematology alterations in response to 4-1BB activation therapy in B16F10 tumor-bearing mice.

Supplementary Figure 3 shows the analysis of the primary structure of sasanlimab by LC-MS peptide mapping.

Supplementary Figure 9 shows the SPR analysis of sasanlimab binding to PD-1 from various species

Supplementary Figure 16 shows the functional blockade of PD-1 receptors in cynomolgus monkeys treated with sasanlimab.

Supplementary Figure 14 shows that sasanlimab blocks the binding of human PD-1 to its ligands, PD-L1 and PD-L2, by SPR analysis.

Development of antagonistic mAbs that specifically target the immune checkpoint receptor, programmed cell death protein-1 (PD-1), is of great interest for cancer immunotherapy. Here, we report the biophysical characteristics and nonclinical antagonistic activities of sasanlimab (PF-06801591), a humanized anti-PD-1 antibody of IgG4 isotype. We show that sasanlimab binds selectively and with similar high potency to human and cynomolgus monkey PD-1 receptor and blocks its interaction with PD-L1 and PD-L2, with no detectable Fc-dependent effector function. The binding of sasanlimab to human and cynomolgus PD-1 is associated with the formation of a stable complex, which is likely to be the main driver of this high-affinity interaction. In vitro, sasanlimab significantly augmented T-cell proliferation and cytokine production in mixed lymphocyte reaction and superantigen stimulation assays. In vivo, sasanlimab accelerated the incidence of GvHD by enhancing T-cell proliferation and cytokine secretion in a xenogeneic model of acute GvHD and halted the growth of MC-38 colon adenocarcinoma tumors in human PD-1 knock-in mice. Pharmacokinetic and toxicokinetic findings from cynomolgus monkey showed that sasanlimab was active and well-tolerated. Taken together, the data presented here support the clinical development of sasanlimab for the treatment of patients with advanced cancers as a single agent or in combination with other immunotherapies.

Immunotherapies targeting the programmed death 1 (PD-1) coinhibitory receptor have shown great promise for a subset of patients with cancer. However, robust and safe combination therapies are still needed to bring the benefit of cancer immunotherapy to broader patient populations. To search for an optimal strategy of combinatorial immunotherapy, we have compared the antitumor activity of the anti–4-1BB/anti–PD-1 combination with that of the anti–PD-1/anti–LAG-3 combination in the poorly immunogenic B16F10 melanoma model. Pronounced tumor inhibition occurred only in animals receiving anti–PD-1 and anti–4-1BB concomitantly, while combining anti–PD-1 with anti–LAG-3 led to a modest degree of tumor suppression. The activity of the anti–4-1BB/anti–PD-1 combination was dependent on IFNγ and CD8+ T cells. Both 4-1BB and PD-1 proteins were elevated on the surface of CD8+ T cells by anti–4-1BB/anti–PD-1 cotreatment. In the tumor microenvironment, an effective antitumor immune response was induced as indicated by the increased CD8+/Treg ratio and the enrichment of genes such as Cd3e, Cd8a, Ifng, and Eomes. In the spleen, the combination treatment shaped the immune system to an effector/memory phenotype and increased the overall activity of tumor-specific CD8+ CTLs, reflecting a long-lasting systemic antitumor response. Furthermore, combination treatment in C57BL/6 mice showed no additional safety signals, and only minimally increased severity of the known toxicity relative to 4-1BB agonist alone. Therefore, in the absence of any cancer vaccine, anti–4-1BB/anti–PD-1 combination therapy is sufficient to elicit a robust antitumor effector/memory T-cell response in an aggressive tumor model and is therefore a candidate for combination trials in patients. Cancer Immunol Res; 3(2); 149–60. ©2014 AACR.

Table S1 shows plasma exposure in humans or rats.

Figure S2 shows A) significantly perturbed genes, B) Venn diagram of changed genes, C) hierarchical clustering of changed genes, D) volcano plot of all genes.

Supplementary Figure 13 shows the structure of sasanlimab bound to human PD-1: Final data collection and refinement statistics and exemplar electron density shown for the reported structure displaying the interface between sasanlimab light chain and hu-PD-1.

Table S5 shows normalized intensity of GSK3b, CDK4 and CDK6 in bone marrow and intestine of control rats.

Supplementary Figure 15 shows no body weight changes in response to sasanlimab treatment in mice.

Figure S3 shows Axin2 expression normalized to vehicle.

Supplementary Figure 8 shows the solution binding affinity of sasanlimab to human and cynomolgus monkey PD-1 using the KinExA

Supplementary Figure Legends

Supplementary Figure 2 shows the confirmation of the primary structure and multi-chain architecture of intact sasanlimab and the identification of the major and minor product isoforms using ESI MS

Supplementary Figure 1 shows the complete, confirmed amino acid sequence of sasanlimab

Supplementary Figure 7 shows the binding of sasanlimab to PD-1 by ELISA

In the fight against cancer, immunotherapies are one of the largest growing therapeutics in development. Immunotherapeutics are designed to boost or harness the power of the immune system to prevent, control, or eliminate cancer; while many immune therapies have been found to be safe others have induced severe toxicities. For instance, even compounds that target the same molecule/antigen can have dramatically differing safety profiles. Current preclinical models evaluating these therapies are underequipped to assess the safety of these compounds: in vitro assays fail to predict systemic responses and traditional animal models often fail to correlate with human responses. To better meet the needs of assessing preclinical toxicity we developed a PBMC-humanized mouse model to test a variety of therapeutics, including both monoclonal and bispecific antibodies and induce human cytokine release responses which can manifest within hours or days later resulting in tissue damage and lethality of the mice. To date we have tested a variety of therapeutics, including Blinatumomab, Rituximab, EGFRxCD3 BiTE, CAR-T, and others in our platform while evaluating the ability of the therapeutic to induce human cytokines, bodyweight loss, clinical symptom assessment, and survival in the context of toxicity alone or along with the evaluation with efficacy. We found that many of the therapeutics tested in our platform showed similarities to clinical data in humans. For example, urelumab and utomilumab are both fully humanized monoclonal antibodies against 4-1BB (CD137). However, during clinical trials, urelumab was shown to induce severe liver toxicities while utomilumab was well tolerated. In our huPBMC mouse model, we likewise showed that huPBMC mice dosed with 10 mpk of urelumab experienced body weight loss, showed liver necrosis, and met the clinical criteria for early euthanasia compared to mice treated with 10 mpk utomilumab and PBS treated controls. Serum levels of enzymes associated with liver damage: AST, ALT and GLDH were significantly higher in urelumab treated mice and terminal serum cytokine analysis revealed similarities with those found to be increased in urelumab clinical trials, including elevated IFNγ, IP-10, MIG, and MIP-1α and MIP-1β. Further, HuPBMC mice are also capable of detecting variability among donors. We have screened well over 60 human PBMC donors in huPBMC mice treated with OKT3 and αCD28 and while we always see an increase in cytokines such as IFNγ - the range of induction varies greatly among donors. Further, we see PBMC-donor variability in body weight loss and survival rate after OKT3 and αCD28 treatments. We demonstrate that the PBMC humanized mouse model shows clinical relevance. The use of these models for preclinical safety assessments has the potential to become an important part of novel immunotherapeutic development for patient safety and reducing drug development costs. Citation Format: Destanie Rose, Won Lee, Guoxiang Yang, Jiwon Yang, Mingshan Cheng, Wenqian He, Bernard Buetow, Allison Vitsky, Maggie Liu, Bart Jessen, James Keck. The use of PBMC humanized mice to test the efficacy and safety of antibody and cell-based cancer immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB349.

Recently three different cyclin-dependent kinase 4 and 6 (CDK4/6) dual inhibitors were approved for the treatment of breast cancer (palbociclib, ribociclib, and abemaciclib), all of which offer comparable therapeutic benefits. Their safety profiles, however, are different. For example, neutropenia is observed at varying incidences in patients treated with these drugs; however, it is the most common adverse event for palbociclib and ribociclib, whereas diarrhea is the most common adverse event observed in patients treated with abemaciclib. To understand the mechanism of diarrhea observed with these drugs and in an effort to guide the development of safer drugs, we compared the effects of oral administration of palbociclib, ribociclib, and abemaciclib on the gastrointestinal tract of rats using doses intended to produce comparable CDK4/6 inhibition. Rats administered abemaciclib, but not palbociclib or ribociclib, had fecal alterations, unique histopathologic findings, and distinctive changes in intestinal gene expression. Morphologic changes in the intestine were characterized by proliferation of crypt cells, loss of goblet cells, poorly differentiated and degenerating enterocytes with loss of microvilli, and mucosal inflammation. In the jejunum of abemaciclib-treated rats, downregulation of enterocyte membrane transporters and upregulation of genes associated with cell proliferation were observed, consistent with activation of the Wnt pathway and downstream transcriptional regulation. Among these CDK4/6 inhibitors, intestinal toxicity was unique to rats treated with abemaciclib, suggesting a mechanism of toxicity not due to primary pharmacology (CDK4/6 inhibition), but to activity at secondary pharmacologic targets.

Microphysiological systems (MPS) are making advances to provide more standardized and predictive physiologically relevant responses to test articles in living tissues and organ systems. The excitement surrounding the potential of MPS to better predict human responses to medicines and improving clinical translation is overshadowed by their relatively slow adoption by the pharmaceutical industry and regulators. Collaboration between multiorganizational consortia and regulators is necessary to build an understanding of the strengths and limitations of MPS models and closing the current gaps. Here, we review some of the advances in MPS research, focusing on liver, intestine, vascular system, kidney and lung and present examples highlighting the context of use for these systems. For MPS to gain a foothold in drug development, they must have added value over existing approaches. Ideally, the application of MPS will augment in vivo studies and reduce the use of animals via tiered screening with less reliance on exploratory toxicology studies to screen compounds. Because MPS support multiple cell types (e.g. primary or stem-cell derived cells) and organ systems, identifying when MPS are more appropriate than simple 2D in vitro models for understanding physiological responses to test articles is necessary. Once identified, MPS models require qualification for that specific context of use and must be reproducible to allow future validation. Ultimately, the challenges of balancing complexity with reproducibility will inform the promise of advancing the MPS field and are critical for realization of the goal to reduce, refine and replace (3Rs) the use of animals in nonclinical research.

e14602 Background: Immunotherapy is proven to be powerful and effective in treating cancer. One of the strategies for the therapy is to boost a patient’s immune system by stimulating tumor necrosis factor receptors via the activation of 4-1BB (CD137) costimulatory molecule. Urelumab was the first 4-1BB agonist investigated in clinical trials and showed clinical promise but doses were limited by severe liver toxicity. Preclinical assessment of toxicity failed to predict the clinical safety outcome. Methods: To address the critical needs of in vivo evaluation of toxicity in a preclinical setting, we have developed an animal model using PBMC humanized mice to assess the safety of immunotherapy. In this model, agents targeting human immune system including monoclonal and bispecific antibodies can induce an acute response of cytokine release within hours of treatment and with some therapies a systemic response can manifest in tissue damage and lethality of mice days later. Results: The potential clinical relevance of the PBMC humanized mouse model was demonstrated using 4-1BB agonists urelumab and utomilumab. The antibody-treated PBMC humanized mice were evaluated daily for clinical score and assessed for clinical chemistry and liver histopathology at study terminus. Animals dosed with urelumab at 10 mg/kg exhibited body weight loss and underwent early euthanasia, while animals dosed with utomilumab survived to scheduled euthanasia with minimum body weight loss. Urelumab showed marked liver toxicity relative to utomilumab and controls with more necrosis in the tissue and higher mean ALT, AST and GLDH activities, and the tolerability of urelumab was increased by lowering the dose to 1 mg/kg. The differences captured by the humanized mouse model presented a safety profile similar to the findings about urelumab and utomilumab from the clinical trials. Utomilumab was well tolerated at the dose that caused severe toxicity for urelumab in patients, and the toxicity of urelumab could be reduced by lowering the dose. Conclusions: Our data suggest that the PBMC humanized mouse model could identify human 4-1BB agonists that caused potentially severe liver toxicity. Preclinical safety assessment using humanized mouse models as used for these studies could be an important step in the course of the development of novel immunotherapy for the safety of patients as well as mitigating drug development cost.

Cyclin-dependent kinases (CDKs) are serine/threonine kinases that regulate cell cycle and have been vigorously pursued as druggable targets for cancer. There are over 20 members of the CDK family. Given their structural similarity, selective inhibition by small molecules has been elusive. In addition, collateral damage to highly proliferative normal cells by CDK inhibitors remains a safety concern. Intestinal epithelial cells are highly proliferative and the impact of individual CDK inhibition on intestinal cell proliferation has not been well studied. Using the rat intestinal epithelial (IEC6) cells as an in vitro model, we found that the selective CDK4/6 inhibitor palbociclib lacked potent anti-proliferative activity in IEC6 relative to the breast cancer cell line MCF7, indicating the absence of intestinal cell reliance on CDK4/6 for cell cycle progression. To further illustrate the role of CDKs in intestinal cells, we chose common targets of CDK inhibitors (CDK 1, 2, 4, 6, and 9) for targeted gene knockdown to evaluate phenotypes. Surprisingly, only CDK1 and CDK9 knockdown demonstrated profound cell death or had moderate growth effects, respectively. CDK2, 4, or 6 knockdowns, whether single, double, or triple combinations, did not have substantial impact. Studies evaluating CDK1 knockdown under various cell seeding densities indicate direct effects on viability independent of proliferation state and imply a potential noncanonical role for CDK1 in intestinal epithelial biology. This research supports the concept that CDK1 and CDK9, but not CDKs 2, 4, or 6, are essential for intestinal cell cycle progression and provides safety confidence for interphase CDK inhibition.

Development of antagonistic mAbs that specifically target the immune checkpoint receptor, programmed cell death protein-1 (PD-1), is of great interest for cancer immunotherapy. Here, we report the biophysical characteristics and nonclinical antagonistic activities of sasanlimab (PF-06801591), a humanized anti-PD-1 antibody of IgG4 isotype. We show that sasanlimab binds selectively and with similar high potency to human and cynomolgus monkey PD-1 receptor and blocks its interaction with PD-L1 and PD-L2, with no detectable Fc-dependent effector function. The binding of sasanlimab to human and cynomolgus PD-1 is associated with the formation of a stable complex, which is likely to be the main driver of this high-affinity interaction. In vitro, sasanlimab significantly augmented T-cell proliferation and cytokine production in mixed lymphocyte reaction and superantigen stimulation assays. In vivo, sasanlimab accelerated the incidence of GvHD by enhancing T-cell proliferation and cytokine secretion in a xenogeneic model of acute GvHD and halted the growth of MC-38 colon adenocarcinoma tumors in human PD-1 knock-in mice. Pharmacokinetic and toxicokinetic findings from cynomolgus monkey showed that sasanlimab was active and well-tolerated. Taken together, the data presented here support the clinical development of sasanlimab for the treatment of patients with advanced cancers as a single agent or in combination with other immunotherapies.

Purpose: Palbociclib (PD-0332991) is the first selective cyclin-dependent kinase (CDK) 4/6 inhibitor approved for metastatic breast cancer. Hematologic effects, especially neutropenia, are dose-limiting adverse events for palbociclib in humans. Experimental Design: Reversible hematologic effects and bone marrow hypocellularity have been identified in toxicology studies in rats and dogs after palbociclib treatment. To understand the mechanism by which the hematologic toxicity occurs, and to further differentiate it from the myelotoxicity caused by cytotoxic chemotherapeutic agents, an in vitro assay using human bone marrow mononuclear cells (hBMNC) was utilized. Results: This work demonstrated that palbociclib-induced bone marrow suppression occurred through cell-cycle arrest, with no apoptosis at clinically relevant concentrations, was not lineage-specific, and was reversible upon palbociclib withdrawal. In contrast, treatment with chemotherapeutic agents (paclitaxel and doxorubicin) resulted in DNA damage and apoptotic cell death in hBMNCs. In the presence or absence of the antiestrogen, palbociclib-treated hBMNCs did not become senescent and resumed proliferation following palbociclib withdrawal, consistent with pharmacologic quiescence. The breast cancer cells, MCF-7, conversely, became senescent following palbociclib or antiestrogen treatment with additive effects in combination and remained arrested in the presence of antiestrogen. Conclusions: Palbociclib causes reversible bone marrow suppression, clearly differentiating it from apoptotic cell death caused by cytotoxic chemotherapeutic agents. This study also distinguished the cell-cycle arresting action of palbociclib on normal bone marrow cells from the senescent effects observed in breast cancer cells. These results shed light on the mechanism and support risk management of palbociclib-induced bone marrow toxicity in the clinic. Clin Cancer Res; 22(8); 2000–8. ©2015 AACR.

Cyclin-dependent kinases (CDKs) are serine/threonine kinases that bind to cyclins and together act as regulators of cell cycle progression. CDK inhibitors have various therapeutic potentials including cancer (e.g. CDK4/6 inhibitors). Intestinal epithelial cells are highly proliferative; however, the impact of individual CDK inhibition on intestinal cell proliferation has not been well studied. The IEC6 cell line, a non-transformed rat small intestinal epithelial cell line with characteristics of crypt epithelial cells, was utilized in the current study to understand the role of CDKs in the proliferation and survival of intestinal cells. In the initial experiment CDK4/6 inhibitor palbociclib treatment demonstrated a lack of potent toxicity in the IEC6 in comparison with MCF7 (breast cancer cell line), indicating the absence of intestinal cell reliance on CDK4 or CDK6 for cell cycle progression. To further illustrate the role of CDKs in intestinal cells, targeted gene knockdown experiments using CDK1, 2, 4, and 6 siRNA and lipofectamine-mediated transfection were conducted. Surprisingly, only CDK1 knockdown causes profound cell death. CDK2, 4, or 6 knockdowns, whether single, double or triple combinations, did not have significant impact on IEC6 cells, as measured by multiple endpoints including impedance, ATP viability, and caspase activity. To further understand the role of CDK1 in cell cycle, various seeding densities of IEC6 cells were used with CDK1 siRNA. Although the impact of CDK1 knockdown on IEC6 cells decreased at the initial proliferation phase with the higher seeding density, there was still a decrease of viability observed at the later time point (when cells reached proliferation plateau), indicating a non-canonical role of CDK1 that is not related to cell cycle. This research supports the concept that CDK1, but not CDKs 2, 4, or 6, is essential for intestinal cell cycle progression and explains the lack of GI toxicity observed with palbociclib. Citation Format: Shuyan Lu, Wenyue Hu, Tae Sung, Brad Hirakawa, Marina Amaro, Bart Jessen. Phenotypic characterization of knockdown of CDKs in the intestinal epithelial cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 99.

Latanoprost is used for the treatment of an increased intraocular pressure (IOP) to prevent the progression of glaucoma. Since the lack of compliance with topical ocular dosing may compromise efficacy, alternate methods of delivery are being sought. A 9-month study was conducted to assess the safety and tolerability of latanoprost-containing subconjunctivally implanted devices.Dutch-belted rabbits were implanted subconjunctivally with up to 5 placebo or drug-loaded devices containing from 50 to 180 μg of latanoprost per device. Study assessment consisted of irritation scoring, clinical signs, ophthalmic exams, IOP, electroretinography (ERG), ocular histology of cohorts at 3 and 9 months postimplantation, and systemic exposure to latanoprost acid.The implants were well tolerated, with minimal-to-mild clinical and microscopic ocular findings attributable to either the placebo or drug-loaded devices. Mild conjunctival congestion persisted through week 13 of the study and tended to correlate with the number of devices and presence of drug. Ophthalmic examinations revealed no effects beyond conjunctival surface hyperemia. No effects on the IOP, corneal thickness, or ERG parameters were observed. The lack of changes in the IOP was expected due to the known lack of the IOP-lowering effects in rabbits from latanoprost. Microscopically, implants at the 3-month necropsy were associated with subconjunctival tissue cavities (containing the implants), fibrous encapsulation, and an infiltrate of lymphocytes and macrophages, sometimes as multinucleate cells, into the subconjunctival implant cavity. The drug-containing implants were often associated with inflammatory cell infiltrates, including heterophils (neutrophils), within the implant subconjunctival cavities and adjacent to the implant sites. At the 9-month necropsy, heterophils were no longer common among the inflammatory cell infiltrates; macrophages and lymphocytes persisted; most of the biodegradable implants were fragmented and disintegrating; and fibrovascular proliferation was present within implant luminal remnants. None of the findings were considered adverse. Systemic exposures were above the limit of quantification (0.1 ng/mL) for up to 96 h in the higher-dose groups, consistent with the initial burst phase of compound release.Overall, the study supports the safety of the latanoprost-containing subconjunctival device as a means of extended delivery of the antiglaucoma medication. Latanoprost-containing subconjunctival implants were well tolerated by Dutch-belted rabbits for up to 9 months. Such devices may improve patient compliance and serve as a means of extended delivery of antiglaucoma medications.

PF-06653157 is a bifunctional antagonist monoclonal antibody (mAb) that targets human VEGF-A ligand and PDGF-Rβ. With the advent of PF-06653157 as an angiogenesis inhibitor and potential treatment for angiogenesis deregulation diseases, a relevant toxicology species is needed for toxicity and efficacy studies. Investigative studies were conducted to validate the mAb dual antagonist properties in a human system and determine its cross-reactive pharmacology in nonhuman cells.Sequence alignment was used to determine percent sequence identity of VEGF and PDGF receptors and ligands; qualitative reverse transcription polymerase chain reaction (qRT-PCR) was used to determine the presence of PDGF-Rβ on cells of interest. The functional activity of PF-06653157 antibody was assessed in human, dog, porcine, rabbit, rat, mouse, and cynomolgus monkey cells treated with VEGF and PDGF ligands through cell proliferation assays and western blot analysis of AKT and p44/p42 (ERK1/2) protein phosphorylation and enzyme-linked immunosorbent assay.PF-06653157 attenuated phosphorylation of AKT and p44/p42 proteins in human and cynomolgus monkey cells. The antibody did not attenuate AKT nor p44/p42 phosphorylation in any other species tested. PDGFR signaling could not be activated with human PDGF ligand in the porcine cells, so PF-06653157 activity in porcine remains inconclusive.The PF-06653157 mAb cross-reacts with cynomolgus monkey cells in a similar manner to human cells. Therefore, cynomolgus monkeys are considered the appropriate species for efficacy and regulatory toxicology studies in PF-06653157 development.

PF-04254644 is a selective kinase inhibitor of mesenchymal epithelial transition factor/hepatocyte growth factor receptor with known off-target inhibitory activity against the phosphodiesterase (PDE) family. Rats given repeated oral doses of PF-04254644 developed a mild to moderate myocardial degeneration accompanied by sustained increase in heart rate and contractility. Investigative studies were conducted to delineate the mechanisms of toxicity. Microarray analysis of Sprague–Dawley rat hearts in a 6 day repeat dose study with PF-04254644 or milrinone, a selective PDE3 inhibitor, revealed similar perturbation of the cyclic adenosine monophosphate (c-AMP) pathway. PDE inhibition and activation of c-AMP were further substantiated using PDE3B immunofluorescence staining and through a c-AMP response element reporter gene assay. The intracellular calcium and oxidative stress signaling pathways were more perturbed by treatment with PF-04254644 than milrinone. The rat cardiomyocytes calcium assay found a dose-dependent increase in intracellular calcium with PF-04254644 treatment. These data suggest that cardiotoxicity of PF-04254644 was probably due to activation of c-AMP signaling, and possibly subsequent disruption of intracellular calcium and oxidative stress signaling pathways. The greater response with PF-04254644 as compared with milrinone in gene expression and micro- and ultrastructural changes is probably due to the broader panel of PDEs inhibition. Copyright © 2012 John Wiley & Sons, Ltd.

Attrition due to safety reasons remains a serious problem for the pharmaceutical industry. This has prompted efforts to develop early predictive in vitro screens that can assist in selecting compounds with a more desirable safety profile early on in the drug discovery process. Here we examined the relationship between physicochemical properties, such as partition coefficient (clogP), topological polar surface area (TPSA), acid dissociation constant (p K a ), and in vitro mechanistic endpoints generated using a high content imaging approach. We demonstrate in our initial analysis that compounds with clogP > 2 and p K a > 5.5 flagged more endpoints than compounds with clogP ⩽ 2 and p K a ⩽ 5.5. In contrast, TPSA did not stand on its own in predicting cytotoxicity. When this knowledge was applied to eight different mechanistic cytotoxicity endpoints (cell loss, apoptosis, ER stress, DNA fragmentation, mitochondrial potential, nuclear size, neutral lipids/steatosis and lysosomal mass), we found that compounds with such properties preferentially flagged in the lysosomal endpoint. We also saw a slight enrichment of such compounds in the endpoints cell loss, DNA fragmentation and nuclear size. We demonstrate that lysosomal compound accumulation is a potential contributor to cell death and possibly organ toxicity.

The Bcr-abl tyrosine kinase inhibitor imatinib mesylate is the frontline therapy for chronic myeloid leukemia. Imatinib has been reported to cause congestive heart failure and left ventricular contractile dysfunction in patients and cardiomyopathy in rodents, findings proposed to be associated with its pharmacological activity. To investigate the specific role of Abelson oncogene 1 (c-Abl) in imatinib-induced cardiac toxicity, we performed targeted gene inhibition of c-Abl by RNA interference in neonatal cardiomyocytes (NCMs). Suppression of c-Abl did not lead to cytotoxicity or induction of endoplasmic reticulum (ER) stress. To further dis associate c-Abl from imatinib-induced cardiac toxicity, we designed imatinib structural analogs that do not have appreciable c-Abl inhibition in NCMs. The c-Abl inactive analogs induced cytotoxicity and ER stress, at similar or greater potencies and magnitudes as imatinib. Furthermore, combining c-Abl gene silencing with imatinib and analogs treatment did not significantly shift the cytotoxicity dose response curves. Imatinib and analogs were shown to accumulate in lysosomes, likely due to their physicochemical properties, and disrupt autophagy. The toxicity induced by imatinib and analogs can be rescued by bafilomycin A pretreatment, demonstrating the involvement of lysosomal accumulation in cardiac toxicity. The results from our studies strongly suggest that imatinib induces cardiomyocyte dysfunction through disruption of autophagy and induction of ER stress, independent of c-Abl inhibition.

AG-012986 is a pan-CDK (cyclin-dependent kinase) inhibitor that has in vitro and in vivo antitumor properties but was stopped in development due in part to rapid bone-marrow-independent white blood cell toxicity in preclinical studies and the potential for acute and delayed immunosuppression in humans. Because peripheral lymphocytes are largely nonproliferating, it was hypothesized the toxicity of AG-012986 was due to an off-target mechanism and not driven by the intended pharmacology. We show the toxicity mechanism in primary human immune cells is caspase driven. T-cells treated with AG-012986 and acutely stimulated through the T-cell receptor exhibited decreased toxicity while still maintaining cell division inhibition. This indicated that the pharmacology of AG-012986 functioned as expected but the toxicity had now been decoupled through activation. Induced phosphorylation of p38 and IL-2 production was impaired with AG-012986. Thus, AG-012986 could cause apoptosis of T-cells by targeting upstream kinases in the p38 Mitogen-activated protein kinase (MAPK) pathway and impairing cellular survival. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:101–108 2012; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.20415

Aim To determine if there is an association between the use of latanoprost ophthalmic solution and malignant melanoma and to assess the evidence of a plausible biological mechanism. Methods Two safety databases were reviewed: one representing all latanoprost (n=24) and fixed-combination latanoprost/timolol (n=16) clinical trials conducted from November 1992 through November 2007 and a global safety database of all spontaneous non-trial-related clinical reports spanning 13 and 9 years for latanoprost and for latanoprost/timolol, respectively. A systematic PubMed search for studies evaluating potential mechanisms was conducted. Results Amongst 12 880 latanoprost-treated subjects in clinical trials, no reported cases of ocular melanoma and three cases of cutaneous melanoma were identified. Of 19 940 cases recorded in the global safety database, 22 reports of ocular/cutaneous neoplasms were identified. Of these neoplasms, 11 were ocular and six were cutaneous melanomas. Possible association with latanoprost use could not be excluded in three ocular and one periorbital report. In vitro and in vivo data were consistent with a mechanism whereby the increased iris pigmentation results from stimulation of melanin synthesis by induction of tyrosinase transcription without increasing mitotic activity. Conclusion There is no evidence at present that establishes a link between latanoprost use and either ocular or cutaneous melanoma.

Lysosomes are acidic organelles that are essential for the degradation of old organelles and engulfed microbes. Furthermore, lysosomes play a key role in cell death. Lipophilic or amphiphilic compounds with a basic moiety can become protonated and trapped within lysosomes, causing lysosomal dysfunction. Therefore, high-throughput screens to detect lysosomotropism, the accumulation of compounds in lysosomes, are desirable. Hence, we developed a 96-well format, high content screening assay that measures lysosomotropism and cytotoxicity by quantitative image analysis. Forty drugs, including antidepressants, antipsychotics, antiarrhythmics and anticancer agents, were tested for their effects on lysosomotropism and cytotoxicity in H9c2 cells. The assay correctly identified drugs known to cause lysosomotropism and revealed novel information showing that the anticancer drugs, gefitinib, lapatinib, and dasatinib, caused lysosomotropism. Although structurally and pharmacologically diverse, drugs that were lysosomotropic shared certain physicochemical properties, possessing a ClogP>2 and a basic pKa between 6.5 and 11. In contrast, drugs which did not lie in this physicochemical property space were not lysosomotropic. The assay is a robust, rapid screen that can be used to identify lysosomotropic, as well as, cytotoxic compounds, and can be positioned within a screening paradigm to understand the role of lysosomotropism as a contributor to drug-induced toxicity.

Preservatives are used in ocular medications to prevent microbial contamination. The use of benzalkonium chloride (BAC), the most widely used preservative in ocular medications, has been scrutinized with a number of studies indicating its toxicity to monolayer cultures of corneal and conjunctival epithelial cells. The purpose of this study was to evaluate and compare the toxicity of BAC and other preservatives and common components of ocular formulations on monolayer and stratified air-lifted cultures of Chang conjunctival cells. Air-lifting Chang cells grown on transwell filters increased stratification as assessed by transepithelial electrical resistance and histology. Unlike monolayer cultures in which ocular medications containing BAC caused near complete loss of cell viability, stratified, air-lifted cultures were not affected by the presence of BAC in ocular medications with up to 30-min exposures. Stratification shifted the dose-response curve to the right for benzalkonium chloride, thimerosal, chlorhexidine digluconate, potassium sorbate and EDTA. These results demonstrate that stratification significantly affects cell viability of Chang conjunctival cells in response to preservatives and additives of ophthalmic preparations.

Cyclin-dependent kinases (CDKs) play a significant role in the control of cell-cycle progression and exhibit aberrant regulation in various neoplastic diseases. AG-024322 is a potent inhibitor of CDK1, CDK2, and CDK4 that produces cell-cycle arrest and antitumor activity in preclinical models. This study evaluated the toxicity of AG-024322 when given by intravenous (IV) infusion to cynomolgus monkeys, including reversibility of effects.Male and female monkeys received AG-024322 by 30-min IV infusion once daily for 5 days at doses of 2, 6, and 10 mg/kg (24, 72, and 120 mg/m(2), respectively). Controls received vehicle alone which was aqueous 5% dextrose, pH 3.8. Three animals/sex/group were necropsied on day 6, and two animals/sex/group at 6 and 10 mg/kg were necropsied on day 22 (reversal cohort). Doses were based upon the results of a dose range-finding study in monkeys; decreased white blood cells occurred ator =3 mg/kg and 12 mg/kg produced central nervous system effects and was above the maximum-tolerated dose.No deaths occurred and clinical signs of toxicity, including swelling at the IV administration site, were seen ator =6 mg/kg. AG-024322 ator =6 mg/kg produced pancytic bone marrow hypocellularity, lymphoid depletion, and vascular injury at the injection site. Renal tubular degeneration occurred at 10 mg/kg. These changes were either reversible or in a process of repair following the 17-day recovery period. Hematology changes included decreases in reticulocytes and/or granulocytes ator =6 mg/kg, which were reversible and consistent with changes in the bone marrow. Lymphoid and bone marrow depletion are consistent with pharmacologic inhibition of CDKs by AG-024322 and were expected findings. On day 22, vacuolar degeneration of pancreatic acinar cells with increased serum amylase and lipase levels occurred in one female at 10 mg/kg. Neither sex-related differences in toxicokinetics nor plasma accumulation over 5 days of dosing were seen. Terminal phase overall mean half-life on day 5 ranged from 6.69 to 8.87 h (across dose levels) and was not dose dependent.The no-adverse-effect dose of AG-024322 was 2 mg/kg and associated with overall mean plasma AUC(0-24.5) of 2.11 microg h/mL.

AG-012986 is a multitargeted cyclin-dependent kinase (CDK) inhibitor active against CDK1, CDK2, CDK4/6, CDK5, and CDK9, with selectivity over a diverse panel of non-CDK kinases. Here, we report the potent antitumor efficacies of AG-012986 against multiple tumor lines in vitro and in vivo. AG-012986 showed antiproliferative activities in vitro with IC50s of 83.1%) in 10 of 11 human xenograft tumor models when administered at or near the maximum tolerated dose for 8 or 12 days. AG-012986 caused dose-dependent hypophosphorylation at Ser795 of the retinoblastoma protein, cell cycle arrest, and apoptosis in vitro. Colony-forming assays indicated that the potency of AG-012986 substantially decreased with treatment time of

The inhibition of 11betahydroxysteroid dehydrogenase 1 (11betaHSD1), an enzyme that catalyzes the conversion of inactive cortisone to active cortisol, is an attractive target to treat diabetes by suppressing hepatic gluconeogenesis. To test this hypothesis, we developed a novel glucocorticoid-induced diabetic KK mouse model and used 11betaHSD1 antisense oligonucleotide (ASO) as an inhibitory tool. KK mice were treated with 25 or 50mg/kg/day of 11betaHSD1 ASO for 28 days. On day 25, cortisone pellets were surgically implanted to induce diabetes. In the ASO-treated mice, plasma blood glucose levels were significantly reduced by up to 54%. In parallel, cortisol and other diabetes endpoints were also significantly reduced. Hepatic 11betaHSD1 mRNA was suppressed by up to 84% with a concomitant respective decrease of up to 49% in the expression of PEPCK. The results suggest that inhibition of 11betaHSD1 activity reduces the availability of cortisol to activate the glucocorticoid receptor, down regulates gluconeogenesis and thus reduces plasma glucose levels in cortisone-induced diabetic KK mice.

Cyclin-dependent kinases (CDKs) have been pursued for more than a decade for the treatment of cancer. CDK inhibitors are expected to slow the rate of cell division and potentially increase the apoptotic fraction of rapidly dividing cells. Although CDK activity is often increased in tumors, normal dividing tissues are also susceptible to the cytostatic and cytotoxic effects of CDK inhibitor action. Therefore the typical toxicity profile associated with cytotoxic anti-cancer therapy, bone marrow suppression and gastrointestinal toxicity, is expected with CDK inhibitors. Bone marrow toxicity and the ensuing delayed peripheral leukocyte suppression often limit the therapeutic application of cytotoxic anticancer drugs. Here we characterize an unusual bone marrow-independent acute toxicity toward leukocytes from broad spectrum CDK inhibitors in monkeys and rodents. The potential combination of both acute and delayed immunosuppression would likely further restrict the application of these particular compounds. Since the cells targeted were non-proliferating, it was assumed that the toxicity was not driven by the intended pharmacological mechanism thereby facilitating the development of a testing strategy to identify compounds with a reduced potential for acute leukocyte toxicity. This testing strategy resulted in a CDK inhibitor void of bone marrow-independent leukocyte toxicity that is currently undergoing clinical testing.

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.