Optimization of the In Vivo Potency of Pyrazolopyrimidine MALT1 Protease Inhibitors by Reducing Metabolism and Increasing Potency in Whole Blood.

The paracaspase MALT1 has gained increasing interest as a target for the treatment of subsets of lymphomas as well as autoimmune diseases, and there is a need for suitable compounds to explore the therapeutic potential of this target. Here, we report the optimization of the in vivo potency of pyrazolopyrimidines, a class of highly selective allosteric MALT1 inhibitors. High doses of the initial lead compound led to tumor stasis in an activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) xenograft model, but this compound suffered from a short in vivo half-life and suboptimal potency in whole blood. Guided by metabolism studies, we identified compounds with reduced metabolic clearance and increased in vivo half-life. In the second optimization step, masking one of the hydrogen-bond donors of the central urea moiety through an intramolecular interaction led to improved potency in whole blood. This was associated with improved in vivo potency in a mechanistic model of B cell activation. The optimized compound led to tumor regression in a CARD11 mutant ABC-DLBCL lymphoma xenograft model.

Structure-activity relationship study of a series of caspase inhibitors containing γ-amino acid moiety for treatment of cholestatic liver disease.

A series of caspase inhibitors containing γ-amino acid moiety have been synthesized. A systemic study on their structure-activity relationship of anti-apoptotic cellular activity is presented. These efforts led to the discovery of compound 20o as a potent caspase inhibitor, which demonstrated preclinical ameliorating total bilirubin efficacy with a significantly improved pharmacokinetic profile.

Rational Drug Design of Topically Administered Caspase 1 Inhibitors for the Treatment of Inflammatory Acne.

The use of an interleukin ß antibody is currently being investigated in the clinic for the treatment of acne, a dermatological disorder affecting 650M persons globally. Inhibiting the protease responsible for the cleavage of inactive pro-IL1ß into active IL-1ß, caspase-1, could be an alternative small molecule approach. This report describes the discovery of uracil 20, a potent (38 nM in THP1 cells assay) caspase-1 inhibitor for the topical treatment of inflammatory acne. The uracil series was designed according to a published caspase-1 pharmacophore model involving a reactive warhead in P1 for covalent reversible inhibition and an aryl moiety in P4 for selectivity against the apoptotic caspases. Reversibility was assessed in an enzymatic dilution assay or by using different substrate concentrations. In addition to classical structure-activity-relationship exploration, topical administration challenges such as phototoxicity, organic and aqueous solubility, chemical stability in solution, and skin metabolic stability are discussed and successfully resolved.

In Vivo-Near Infrared Imaging of Neurodegeneration.

In vivo near-infrared (NIR) imaging of molecular processes at the preclinical stage promises to provide more valuable mechanistic information about pathological pathways involved in neurodegeneration. NIR imaging has the potential to improve in vivo therapeutic screening protocols by enabling noninvasive monitoring of presymptomatic responses to treatment. We have developed new NIR fluorescent contrast agents conjugated to markers of cell death, and using these agents we have identified molecular pathways associated with prion-induced neurodegeneration and determined the optimal window for meaningful therapeutic intervention in prion disease. This chapter provides a description of the synthesis and purification of our NIR cell Death (NIRD) contrast agent and the application of in vivo NIRD (iNIRD) imaging to a prion model of neurodegeneration.

In Vivo Evaluation of Radiofluorinated Caspase-3/7 Inhibitors as Radiotracers for Apoptosis Imaging and Comparison with [18F]ML-10 in a Stroke Model in the Rat.

PURPOSE: The first biological evaluation of two potent fluorine-18 radiolabelled inhibitors of caspase-3/7 was achieved in a cerebral stroke rat model to visualize apoptosis. PROCEDURES: In vivo characteristics of isatins [(18)F]-2 and [(18)F]-3 were studied and compared by µPET to previously described 1-[4-(2-[(18)F]fluoroethyl)benzyl]-5-(2-methoxymethylpyrrolidin-1-ylsulfonyl)isatin ([(18)F]-1) and to 2-(5-[(18)F]fluoropentyl)-2-methyl-malonic acid ([(18)F]ML-10) used as a reference radiotracer in a rat stroke model. RESULTS: [(18)F]-2 and [(18)F]-3 were radiolabelled with high radiochemical purity and high specific radioactivity. Radioactivity uptakes in ischemic and contralateral brain regions were weak for the three radiolabelled isatins and lower for [(18)F]ML-10. In µPET, time activity curves showed significant uptake differences between both regions of interest for [(18)F]-1 after 45 min. No differences were observed for [(18)F]ML-10. CONCLUSIONS: Radiolabelled isatins are more promising radiotracers to image apoptosis than [(18)F]ML-10 in this stroke animal model without craniectomy. In particular, [(18)F]-1 presented significant uptake in apoptotic area 45 min after administration.

Carcinogenicity assessment of the pan-caspase inhibitor, emricasan, in Tg.rasH2 mice.

Emricasan, formerly IDN-6556, is a small molecule currently being evaluated in clinical trials to reduce hepatic injury and liver fibrosis. Since emricasan is an irreversible pan-caspase inhibitor that potently inhibits caspase-mediated apoptosis and inflammation, its carcinogenic potential was evaluated in a humanized mouse model. Tg.rasH2 mice received LabDiet formulated with 0, 10, 25, and 75mg/kg/day of emricasan, for 26weeks. At terminal sacrifice, blood was collected for clinical pathology analysis and tissues were collected, processed, and evaluated microscopically. There were no treatment related deaths or overt signs of toxicity for the duration of the study. There was no evidence of a carcinogenic effect in the peripheral blood leukocyte counts. Liver microgranulomas, which are background lesions, were slightly increased, especially in males. Increases in the incidence of the activated germinal centers were seen in the spleens and mesenteric lymph nodes of males and females, and in the mandibular lymph nodes of male mice. Atrophy of ovaries and testicular degeneration were also seen in emricasan treated animals. Although several non-neoplastic lesions were observed, there was no evidence of emricasan-related tumor formation in any tissue. In addition, the non-neoplastic lesions were not considered pre-neoplastic. Thus, emricasan is not considered carcinogenic.

A peptide-based positron emission tomography probe for in vivo detection of caspase activity in apoptotic cells.

PURPOSE: Apoptosis, or programmed cell death, can be leveraged as a surrogate measure of response to therapeutic interventions in medicine. Cysteine aspartic acid-specific proteases, or caspases, are essential determinants of apoptosis signaling cascades and represent promising targets for molecular imaging. Here, we report development and in vivo validation of [(18)F]4-fluorobenzylcarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone ([(18)F]FB-VAD-FMK), a novel peptide-based molecular probe suitable for quantification of caspase activity in vivo using positron emission tomography (PET). EXPERIMENTAL DESIGN: Supported by molecular modeling studies and subsequent in vitro assays suggesting probe feasibility, the labeled pan-caspase inhibitory peptide, [(18)F]FB-VAD-FMK, was produced in high radiochemical yield and purity using a simple two-step, radiofluorination. The biodistribution of [(18)F]FB-VAD-FMK in normal tissue and its efficacy to predict response to molecularly targeted therapy in tumors was evaluated using microPET imaging of mouse models of human colorectal cancer. RESULTS: Accumulation of [(18)F]FB-VAD-FMK was found to agree with elevated caspase-3 activity in response to Aurora B kinase inhibition as well as a multidrug regimen that combined an inhibitor of mutant BRAF and a dual PI3K/mTOR inhibitor in (V600E)BRAF colon cancer. In the latter setting, [(18)F]FB-VAD-FMK PET was also elevated in the tumors of cohorts that exhibited reduction in size. CONCLUSIONS: These studies illuminate [(18)F]FB-VAD-FMK as a promising PET imaging probe to detect apoptosis in tumors and as a novel, potentially translatable biomarker for predicting response to personalized medicine.

Dogs are more sensitive to antagonists of inhibitor of apoptosis proteins than rats and humans: a translational toxicokinetic/toxicodynamic analysis.

Inhibitor of apoptosis (IAP) proteins suppress apoptosis and are overexpressed in a variety of cancers. GDC-0152 is a potent and selective IAP antagonist being developed as an anticancer agent. In preclinical safety studies, dogs were particularly sensitive to GDC-0152 showing adverse signs of a tumor necrosis factor alpha (TNF-α) driven systemic inflammatory response, related to cellular IAP degradation and activation of NFκB signaling, at lower exposures compared with rat. In addition, downstream increases in systemic levels of cytokines and chemokines, such as monocyte chemotactic protein-1 (MCP-1), were observed. A semimechanistic population toxicokinetic/toxicodynamic (TK/TD) model incorporating transit compartments was used to fit MCP-1 plasma concentrations from rats or dogs given iv GDC-0152 doses. Estimated TD parameters inferred that lower GDC-0152 plasma concentrations triggered more severe increases in plasma MCP-1 in dogs compared with rats. Human simulations performed using dog TD parameters and human pharmacokinetics predicted 300-2400% increases of MCP-1 in humans at iv doses from 0.76 to 1.48mg/kg. Similar simulations using rat TD parameters suggest little or no change. Patients given iv doses of GDC-0152 up to 1.48mg/kg iv showed no substantial increases in systemic MCP-1 or signs of a severe TNF-α driven systemic inflammatory response. Emerging clinical data reported for other IAP antagonists are consistent with our observations. Taken together, the data suggest dogs are more sensitive to IAP antagonists compared with humans and rats. This study illustrates how TK/TD analysis can be utilized to quantitatively translate and context an identified preclinical safety risk in dogs to humans.