Leukotriene B4 receptor 1 (BLT1) does not mediate disease progression in a mouse model of liver fibrosis.

MASH is a prevalent liver disease that can progress to fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and ultimately death, but there are no approved therapies. Leukotriene B4 (LTB4) is a potent pro-inflammatory chemoattractant that drives macrophage and neutrophil chemotaxis, and genetic loss or inhibition of its high affinity receptor, leukotriene B4 receptor 1 (BLT1), results in improved insulin sensitivity and decreased hepatic steatosis. To validate the therapeutic efficacy of BLT1 inhibition in an inflammatory and pro-fibrotic mouse model of MASH and fibrosis, mice were challenged with a choline-deficient, L-amino acid defined high fat diet and treated with a BLT1 antagonist at 30 or 90 mg/kg for 8 weeks. Liver function, histology, and gene expression were evaluated at the end of the study. Treatment with the BLT1 antagonist significantly reduced plasma lipids and liver steatosis but had no impact on liver injury biomarkers or histological endpoints such as inflammation, ballooning, or fibrosis compared to control. Artificial intelligence-powered digital pathology analysis revealed a significant reduction in steatosis co-localized fibrosis in livers treated with the BLT1 antagonist. Liver RNA-seq and pathway analyses revealed significant changes in fatty acid, arachidonic acid, and eicosanoid metabolic pathways with BLT1 antagonist treatment, however, these changes were not sufficient to impact inflammation and fibrosis endpoints. Targeting this LTB4-BLT1 axis with a small molecule inhibitor in animal models of chronic liver disease should be considered with caution, and additional studies are warranted to understand the mechanistic nuances of BLT1 inhibition in the context of MASH and liver fibrosis.

Discovery of Insulin Receptor Partial Agonists MK-5160 and MK-1092 as Novel Basal Insulins with Potential to Improve Therapeutic Index.

We have identified a series of novel insulin receptor partial agonists (IRPAs) with a potential to mitigate the risk of hypoglycemia associated with the use of insulin as an antidiabetic treatment. These molecules were designed as dimers of native insulin connected via chemical linkers of variable lengths with optional capping groups at the N-terminals of insulin chains. Depending on the structure, the maximal activation level (%Max) varied in the range of ∼20-70% of native insulin, and EC50 values remained in sub-nM range. Studies in minipig and dog demonstrated that IRPAs had sufficient efficacy to normalize plasma glucose levels in diabetes, while providing reduction of hypoglycemia risk. IRPAs had a prolonged duration of action, potentially making them suitable for once-daily dosing. Two lead compounds with %Max values of 30 and 40% relative to native insulin were selected for follow up studies in the clinic.

Functionally selective signaling and broad metabolic benefits by novel insulin receptor partial agonists.

Insulin analogs have been developed to treat diabetes with focus primarily on improving the time action profile without affecting ligand-receptor interaction or functional selectivity. As a result, inherent liabilities (e.g. hypoglycemia) of injectable insulin continue to limit the true therapeutic potential of related agents. Insulin dimers were synthesized to investigate whether partial agonism of the insulin receptor (IR) tyrosine kinase is achievable, and to explore the potential for tissue-selective systemic insulin pharmacology. The insulin dimers induced distinct IR conformational changes compared to native monomeric insulin and substrate phosphorylation assays demonstrated partial agonism. Structurally distinct dimers with differences in conjugation sites and linkers were prepared to deliver desirable IR partial agonist (IRPA). Systemic infusions of a B29-B29 dimer in vivo revealed sharp differences compared to native insulin. Suppression of hepatic glucose production and lipolysis were like that attained with regular insulin, albeit with a distinctly shallower dose-response. In contrast, there was highly attenuated stimulation of glucose uptake into muscle. Mechanistic studies indicated that IRPAs exploit tissue differences in receptor density and have additional distinctions pertaining to drug clearance and distribution. The hepato-adipose selective action of IRPAs is a potentially safer approach for treatment of diabetes.

High Resolution Episcopic Microscopy for Qualitative and Quantitative Data in Phenotyping Altered Embryos and Adult Mice Using the New "Histo3D" System.

3D imaging in animal models, during development or in adults, facilitates the identification of structural morphological changes that cannot be achieved with traditional 2D histological staining. Through the reconstruction of whole embryos or a region-of-interest, specific changes are better delimited and can be easily quantified. We focused here on high-resolution episcopic microscopy (HREM), and its potential for visualizing and quantifying the organ systems of normal and genetically altered embryos and adult organisms. Although the technique is based on episcopic images, these are of high resolution and are close to histological quality. The images reflect the tissue structure and densities revealed by histology, albeit in a grayscale color map. HREM technology permits researchers to take advantage of serial 2D aligned stacks of images to perform 3D reconstructions. Three-dimensional visualization allows for an appreciation of topology and morphology that is difficult to achieve with classical histological studies. The nature of the data lends itself to novel forms of computational analysis that permit the accurate quantitation and comparison of individual embryos in a manner that is impossible with histology. Here, we have developed a new HREM prototype consisting of the assembly of a Leica Biosystems Nanocut rotary microtome with optics and a camera. We describe some examples of applications in the prenatal and adult lifestage of the mouse to show the added value of HREM for phenotyping experimental cohorts to compare and quantify structure volumes. At prenatal stages, segmentations and 3D reconstructions allowed the quantification of neural tissue and ventricular system volumes of normal brains at E14.5 and E16.5 stages. 3D representations of normal cranial and peripheric nerves at E15.5 and of the normal urogenital system from stages E11.5 to E14.5 were also performed. We also present a methodology to quantify the volume of the atherosclerotic plaques of ApoEtm1Unc/tm1Unc mutant mice and illustrate a 3D reconstruction of knee ligaments in adult mice.

Discovery of a new class of integrin antibodies for fibrosis.

Lung fibrosis, or the scarring of the lung, is a devastating disease with huge unmet medical need. There are limited treatment options and its prognosis is worse than most types of cancer. We previously discovered that MK-0429 is an equipotent pan-inhibitor of αv integrins that reduces proteinuria and kidney fibrosis in a preclinical model. In the present study, we further demonstrated that MK-0429 significantly inhibits fibrosis progression in a bleomycin-induced lung injury model. In search of newer integrin inhibitors for fibrosis, we characterized monoclonal antibodies discovered using Adimab's yeast display platform. We identified several potent neutralizing integrin antibodies with unique human and mouse cross-reactivity. Among these, Ab-31 blocked the binding of multiple αv integrins to their ligands with IC50s comparable to those of MK-0429. Furthermore, both MK-0429 and Ab-31 suppressed integrin-mediated cell adhesion and latent TGFß activation. In IPF patient lung fibroblasts, TGFß treatment induced profound αSMA expression in phenotypic imaging assays and Ab-31 demonstrated potent in vitro activity at inhibiting αSMA expression, suggesting that the integrin antibody is able to modulate TGFß action though mechanisms beyond the inhibition of latent TGFß activation. Together, our results highlight the potential to develop newer integrin therapeutics for the treatment of fibrotic lung diseases.

Molecular Profiling Reveals a Common Metabolic Signature of Tissue Fibrosis.

Fibrosis, or the accumulation of extracellular matrix, is a common feature of many chronic diseases. To interrogate core molecular pathways underlying fibrosis, we cross-examine human primary cells from various tissues treated with TGF-ß, as well as kidney and liver fibrosis models. Transcriptome analyses reveal that genes involved in fatty acid oxidation are significantly perturbed. Furthermore, mitochondrial dysfunction and acylcarnitine accumulation are found in fibrotic tissues. Substantial downregulation of the PGC1α gene is evident in both in vitro and in vivo fibrosis models, suggesting a common node of metabolic signature for tissue fibrosis. In order to identify suppressors of fibrosis, we carry out a compound library phenotypic screen and identify AMPK and PPAR as highly enriched targets. We further show that pharmacological treatment of MK-8722 (AMPK activator) and MK-4074 (ACC inhibitor) reduce fibrosis in vivo. Altogether, our work demonstrate that metabolic defect is integral to TGF-ß signaling and fibrosis.

In Situ Forming Injectable Thermoresponsive Hydrogels for Controlled Delivery of Biomacromolecules.

Due to their relatively large molecular sizes and delicate nature, biologic drugs such as peptides, proteins, and antibodies often require high and repeated dosing, which can cause undesired side effects and physical discomfort in patients and render many therapies inordinately expensive. To enhance the efficacy of biologic drugs, they could be encapsulated into polymeric hydrogel formulations to preserve their stability and help tune their release in the body to their most favorable profile of action for a given therapy. In this study, a series of injectable, thermoresponsive hydrogel formulations were evaluated as controlled delivery systems for various peptides and proteins, including insulin, Merck proprietary peptides (glucagon-like peptide analogue and modified insulin analogue), bovine serum albumin, and immunoglobulin G. These hydrogels were prepared using concentrated solutions of poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PLGA-PEG-PLGA), which can undergo temperature-induced sol-gel transitions and spontaneously solidify into hydrogels near the body temperature, serving as an in situ depot for sustained drug release. The thermoresponsiveness and gelation properties of these triblock copolymers were characterized by dynamic light scattering (DLS) and oscillatory rheology, respectively. The impact of different hydrogel-forming polymers on release kinetics was systematically investigated based on their hydrophobicity (LA/GA ratios), polymer concentrations (20, 25, and 30%), and phase stability. These hydrogels were able to release active peptides and proteins in a controlled manner from 4 to 35 days, depending on the polymer concentration, solubility nature, and molecular sizes of the cargoes. Biophysical studies via size exclusion chromatography (SEC) and circular dichroism (CD) indicated that the encapsulation and release did not adversely affect the protein conformation and stability. Finally, a selected PLGA-PEG-PLGA hydrogel system was further investigated by the encapsulation of a therapeutic glucagon-like peptide analogue and a modified insulin peptide analogue in diabetic mouse and minipig models for studies of glucose-lowering efficacy and pharmacokinetics, where superior sustained peptide release profiles and long-lasting glucose-lowering effects were observed in vivo without any significant tolerability issues compared to peptide solution controls. These results suggest the promise of developing injectable thermoresponsive hydrogel formulations for the tunable release of protein therapeutics to improve patient's comfort, convenience, and compliance.

Spatial and temporal studies of metabolic activity: contrasting biochemical kinetics in tissues and pathways during fasted and fed states.

The regulation of nutrient homeostasis, i.e., the ability to transition between fasted and fed states, is fundamental in maintaining health. Since food is typically consumed over limited (anabolic) periods, dietary components must be processed and stored to counterbalance the catabolic stress that occurs between meals. Herein, we contrast tissue- and pathway-specific metabolic activity in fasted and fed states. We demonstrate that knowledge of biochemical kinetics that is obtained from opposite ends of the energetic spectrum can allow mechanism-based differentiation of healthy and disease phenotypes. Rat models of type 1 and type 2 diabetes serve as case studies for probing spatial and temporal patterns of metabolic activity via [2H]water labeling. Experimental designs that capture integrative whole body metabolism, including meal-induced substrate partitioning, can support an array of research surrounding metabolic disease; the relative simplicity of the approach that is discussed here should enable routine applications in preclinical models.

Engineering Glucose Responsiveness Into Insulin.

Insulin has a narrow therapeutic index, reflected in a small margin between a dose that achieves good glycemic control and one that causes hypoglycemia. Once injected, the clearance of exogenous insulin is invariant regardless of blood glucose, aggravating the potential to cause hypoglycemia. We sought to create a "smart" insulin, one that can alter insulin clearance and hence insulin action in response to blood glucose, mitigating risk for hypoglycemia. The approach added saccharide units to insulin to create insulin analogs with affinity for both the insulin receptor (IR) and mannose receptor C-type 1 (MR), which functions to clear endogenous mannosylated proteins, a principle used to endow insulin analogs with glucose responsivity. Iteration of these efforts culminated in the discovery of MK-2640, and its in vitro and in vivo preclinical properties are detailed in this report. In glucose clamp experiments conducted in healthy dogs, as plasma glucose was lowered stepwise from 280 mg/dL to 80 mg/dL, progressively more MK-2640 was cleared via MR, reducing by ∼30% its availability for binding to the IR. In dose escalations studies in diabetic minipigs, a higher therapeutic index for MK-2640 (threefold) was observed versus regular insulin (1.3-fold).

Measurement of catecholamines in rat and mini-pig plasma and urine by liquid chromatography-tandem mass spectrometry coupled with solid phase extraction.

A tandem mass spectrometry method combined with an ion-pair chromatographic separation after weak cation exchange solid phase sample extraction for epinephrine (E), norepinephrine (NE) and dopamine (DA) has been developed. Two surrogate matrixes for plasma and urine as well as stable isotope labeled internal standards were utilized for quantitation. The observed dynamic range of E, NE and DA was 0.025-100ng/ml for plasma, and 0.25-1000ng/ml for urine with a r(2) regression coefficient >0.99. Extraction recoveries were greater than 60% and the lower limit of quantitation was 25pg/ml for all three analytes in plasma. This method provided excellent sensitivity and selectivity for use with small sample volumes (≤25uL), enabling high-throughput pharmacodynamic animal model development and screening of adverse effects.

Pharmacological inhibition of diacylglycerol acyltransferase 1 reduces body weight and modulates gut peptide release--potential insight into mechanism of action.

OBJECTIVE: Investigation was conducted to understand the mechanism of action of diacylglycerol acyltransferase 1 (DGAT1) using small molecules DGAT1 inhibitors, compounds K and L. DESIGN AND METHODS: Biochemical and stable-label tracer approaches were applied to interrogate the functional activities of compounds K and L on TG synthesis and changes of carbon flow. Energy homeostasis and gut peptide release upon DGAT1 inhibition was conducted in mouse and dog models. RESULTS: Compounds K and L, dose-dependently inhibits post-prandial TG excursion in mouse and dog models. Weight loss studies in WT and Dgat1(-/-) mice, confirmed that the effects of compound K on body weight loss is mechanism-based. Compounds K and L altered incretin peptide release following oral fat challenge. Immunohistochemical studies with intestinal tissues demonstrate lack of detectable DGAT1 immunoreactivity in enteroendocrine cells. Furthermore, (13) C-fatty acid tracing studies indicate that compound K inhibition of DGAT1 increased the production of phosphatidyl choline (PC). CONCLUSION: Treatment with DGAT1 inhibitors improves lipid metabolism and body weight. DGAT1 inhibition leads to enhanced PC production via alternative carbon channeling. Immunohistological studies suggest that DGAT1 inhibitor's effects on plasma gut peptide levels are likely via an indirect mechanism. Overall these data indicate a translational potential towards the clinic.

Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis.

The use of nicotinic acid to treat dyslipidemia is limited by induction of a "flushing" response, mediated in part by the interaction of prostaglandin D(2) (PGD(2)) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr(-/-)ApoE(-/-) mice versus ApoE(-/-) mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr(-/-)ApoE(-/-) mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE(-/-) mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr(-/-) mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.

(1aR,5aR)1a,3,5,5a-Tetrahydro-1H-2,3-diaza-cyclopropa[a]pentalene-4-carboxylic acid (MK-1903): a potent GPR109a agonist that lowers free fatty acids in humans.

G-protein coupled receptor (GPCR) GPR109a is a molecular target for nicotinic acid and is expressed in adipocytes, spleen, and immune cells. Nicotinic acid has long been used for the treatment of dyslipidemia due to its capacity to positively affect serum lipids to a greater extent than other currently marketed drugs. We report a series of tricyclic pyrazole carboxylic acids that are potent and selective agonists of GPR109a. Compound R,R-19a (MK-1903) was advanced through preclinical studies, was well tolerated, and presented no apparent safety concerns. Compound R,R-19a was advanced into a phase 1 clinical trial and produced a robust decrease in plasma free fatty acids. On the basis of these results, R,R-19a was evaluated in a phase 2 study in humans. Because R,R-19a produced only a weak effect on serum lipids as compared with niacin, we conclude that the beneficial effects of niacin are most likely the result of an undefined GPR109a independent pathway.

Reconstituted HDL elicits marked changes in plasma lipids following single-dose injection in C57Bl/6 mice.

High-density lipoprotein (HDL)-targeting therapies, including reconstituted HDL (rHDL), are attractive agents for treating dyslipidemia and atherosclerosis, as they may increase HDL levels and enhance therapeutic activities associated with HDL, including reverse cholesterol transport (RCT). Using CSL-111, a rHDL consisting of native human apolipoprotein AI (hApoAI) and phospholipids, we characterized the acute effects of rHDL administration in C57Bl/6 mice to (i) further our understanding of the mechanism of action of rHDL, and (ii) evaluate the usefulness of the mouse as a preclinical model for HDL-targeting therapies. After a single injection of CSL-111, there was a dose- and time-dependent increase of hApoAI, human pre-ß HDL, total cholesterol, and triglycerides in serum, consistent with the effects of CSL-111 in humans. However, unlike in humans, there was no measurable increase in cholesteryl esters. Evaluated ex vivo, the ATP binding cassette A1 (ABCA1)- and scavenger receptor type BI (SR-BI)-dependent cholesterol efflux capacity of serum from CSL-111-treated mice was increased compared with serum from vehicle-treated animals. Fractionation by size exclusion chromatography of lipoproteins in serum from treated mice revealed hApoAI in particles the size of endogenous HDL and slightly larger, cholesterol-enriched particles of all sizes, including sizes distinct from endogenous HDL or CSL-111 itself, and triglyceride-enriched particles the size of very-low-density lipoprotein (VLDL). These results suggest that in mouse blood CSL-111 is remodeled and generates enhanced cholesterol efflux capacity which increases mobilization of free cholesterol from peripheral tissues. Our findings complement the previous reports on CSL-111 in human participants and provide data with which to evaluate the potential utility of mouse models in mechanistic studies of HDL-targeting therapies.

Plasma lipid profiling across species for the identification of optimal animal models of human dyslipidemia.

In an attempt to understand the applicability of various animal models to dyslipidemia in humans and to identify improved preclinical models for target discovery and validation for dyslipidemia, we measured comprehensive plasma lipid profiles in 24 models. These included five mouse strains, six other nonprimate species, and four nonhuman primate (NHP) species, and both healthy animals and animals with metabolic disorders. Dyslipidemic humans were assessed by the same measures. Plasma lipoprotein profiles, eight major plasma lipid fractions, and FA compositions within these lipid fractions were compared both qualitatively and quantitatively across the species. Given the importance of statins in decreasing plasma low-density lipoprotein cholesterol for treatment of dyslipidemia in humans, the responses of these measures to simvastatin treatment were also assessed for each species and compared with dyslipidemic humans. NHPs, followed by dog, were the models that demonstrated closest overall match to dyslipidemic humans. For the subset of the dyslipidemic population with high plasma triglyceride levels, the data also pointed to hamster and db/db mouse as representative models for practical use in target validation. Most traditional models, including rabbit, Zucker diabetic fatty rat, and the majority of mouse models, did not demonstrate overall similarity to dyslipidemic humans in this study.

ApoA-I mimetic peptides promote pre-ß HDL formation in vivo causing remodeling of HDL and triglyceride accumulation at higher dose.

Reverse cholesterol transport promoted by HDL-apoA-I is an important mechanism of protection against atherosclerosis. We have previously identified apoA-I mimetic peptides by synthesizing analogs of the 22 amino acid apoA-I consensus sequence (apoA-I(cons)) containing non-natural aliphatic amino acids. Here we examined the effect of different aliphatic non-natural amino acids on the structure-activity relationship (SAR) of apoA-I mimetic peptides. These novel apoA-I mimetics, with long hydrocarbon chain (C(5-8)) amino acids incorporated in the amphipathic α helix of the apoA-I(cons), have the following properties: (i) they stimulate in vitro cholesterol efflux from macrophages via ABCA1; (ii) they associate with HDL and cause formation of pre-ß HDL particles when incubated with human and mouse plasma; (iii) they associate with HDL and induce pre-ß HDL formation in vivo, with a corresponding increase in ABCA1-dependent cholesterol efflux capacity ex vivo; (iv) at high dose they associate with VLDL and induce hypertriglyceridemia in mice. These results suggest our peptide design confers activities that are potentially anti-atherogenic. However a dosing regimen which maximizes their therapeutic properties while minimizing adverse effects needs to be established.

Altered lipoprotein metabolism in P2Y(13) knockout mice.

The purinergic receptor P2Y(13) has been shown to play a role in the uptake of holo-HDL particles in in vitro hepatocyte experiments. In order to determine the role of P2Y(13) in lipoprotein metabolism in vivo, we ablated the expression of this gene in mice. Here we show that P2Y(13) knockout mice have lower fecal concentrations of neutral sterols (-27%±2.1% in males) as well as small decreases in plasma HDL (-13.1%±3.2% in males; -17.5%±4.0% in females) levels. In addition, significant decreases were detected in serum levels of fatty acids and glycerol in female P2Y(13) knockout mice. Hepatic mRNA profiling analyses showed increased expression of SREBP-regulated cholesterol and fatty acid biosynthesis genes, while fatty acid ß-oxidation genes were significantly decreased. Liver gene signatures also identified changes in PPARα-regulated transcript levels. With the exception of a small increase in bone area, P2Y(13) knockout mice do not show any additional major abnormalities, and display normal body weight, fat mass and lean body mass. No changes in insulin sensitivity and oral glucose tolerance could be detected. Taken together, our experiments assess a role for the purinergic receptor P2Y(13) in the regulation of lipoprotein metabolism and demonstrate that modulating its activity could be of benefit to the treatment of dyslipidemia in people.

GPR109a agonists. Part 2: pyrazole-acids as agonists of the human orphan G-protein coupled receptor GPR109a.

5-Alkyl and aryl-pyrazole-acids have been identified as a new class of selective, small-molecule, agonists of the human orphan G-protein-coupled receptor GPR109a, a high affinity receptor for the HDL-raising drug nicotinic acid.