Comparison of a microsphere-based platform with a multiplex flow cytometric assay for determination of circulating cytokines in the mouse.

BACKGROUND: Measuring expression profiles of inflammatory biomarkers is important in monitoring the polarization of immune responses; therefore, results should be independent of quantitation methods if they are to be accepted as validated clinical pathology biomarkers. To evaluate effects of differing quantitation methods, the seven major circulating Th1/Th2/Th17 cytokines interleukin 2 (IL-2), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), IL-4, IL-6, IL-10 and IL-17A were quantified in plasma of lipopolysaccharide (LPS)-treated mice with two different multiplex platforms. METHODS: Female C57BL6 mice were treated orally with vehicle or dexamethasone, followed by LPS intravenously. Plasma samples were analyzed 0.5, 1, 2, 4, and 6 h post-LPS challenge with assays at Myriad-RBM and compared to assays performed on a BD Accuri C6 flow cytometer. RESULTS: IL-17A response to LPS was limited but sustained, and the response for the remaining cytokines were early and transient; dexamethasone reduced expression of all 7 cytokines. TNF-α and IL-6 levels were similar across both assays, and IL-4 levels were generally very low. Plasma levels of remaining cytokines were variably lower with BD assays than Myriad-RBM assays. CONCLUSIONS: The present findings demonstrate that quantitation of circulating biomarkers of inflammation can be achieved using multiplexed flow cytometry, but careful consideration must be taken for assay validation when cross-referencing with another multiplexed assay.

Development of a Functional Observational Battery in the Minipig for Regulatory Neurotoxicity Assessments.

A functional observational battery (FOB) is recommended as the first-tier neurotoxicity screening in the preclinical safety pharmacology testing guidelines. Minipigs have increasingly been used in regulatory toxicology studies; however, no current FOB protocol is available for neurotoxicity testing in these species. Hence, a minipig FOB instrument was developed. A complete crossover study with Sinclair minipigs was performed to evaluate physiologic, neurologic, and behavioral effects of amphetamine, ketamine, and diazepam. The treated minipigs were first observed in their home cage, were video-recorded for 10 minutes in an open field, and then went through a complete neurologic examination. Both ketamine and diazepam were shown to reduce the freezing and behavior shifts of treated minipigs, while increasing their exploratory behaviors. Both drugs also caused muscular and gait impairment. The effects of ketamine and diazepam were consistent with their roles as central nervous system (CNS) suppressants. Unique effects were also observed with ketamine and diazepam treatments, which may reflect their unique mechanisms of action. Consistent with its role as a CNS stimulant, amphetamine caused the treated minipigs to be hyperactive and to display increased freezing and behavior shifts and reduced exploring activities. These effects of amphetamine were opposite to those observed with ketamine and diazepam. Amphetamine also increased locomotion in the treated minipigs. The present effects of amphetamine, ketamine, and diazepam are in agreement with observations by others. In conclusion, the minipig is a suitable species for FOB evaluation of pharmaceuticals in preclinical safety pharmacology testing.

The importance of minipigs in dermal safety assessment: an overview.

The use of miniature swine as a non-rodent species in safety assessment has continued to expand for over a decade and their use has become routine, particularly in pharmacology as a model for human integumentary diseases. Translational preclinical swine study data are now favorably compared and contrasted to human data, and miniature swine models provide important information in dermal safety assessment and skin pharmacology. For example, the miniature swine model has been well-accepted for cutaneous absorption and toxicity studies due to swine integument being morphologically and functionally similar to human skin. Subsequently, this model is important to dermal drug development programs, and it is the animal model of choice for assessment of dermal absorption, local tolerance and systemic toxicity following dermal exposures. In conclusion, the miniature swine model has an important role to play in the safety assessment of pharmaceutical products and in multiple aspects of human dermal drug development.

The Miniature Swine as a Model in Experimental and Translational Medicine.

The use of the miniature swine as a nonrodent species in research has continued to expand for over a decade, and they are becoming routinely used both in experimental pharmacology and as a therapeutic model for human diseases. Miniature swine models are regularly used for studies designed to assess efficacy and safety of new therapeutic compounds given through different routes of exposure and are used as an alternative model to rodents, canines, or nonhuman primates. Translational preclinical swine study data presented here support the current understanding that miniature swine are the animal model of choice for the assessment of drugs targeting endocrine, dermal, and ocular disorders. Because research investigators need to be familiar with some of the important features of the models developed in the miniature swine in order to place clinical and experimental findings in their proper perspective, relevant references and data from these models will be presented, compared, and partially illustrated.

Miniature Swine Breeds in Toxicology and Drug Safety Assessments: What to Expect during Clinical and Pathology Evaluations.

The use of miniature swine as a nonrodent species in safety assessment has continued to expand for over a decade, and they are becoming routinely used in toxicology and in pharmacology as well as a model for human diseases. Miniature swine models are regularly used for regulatory toxicity studies designed to assess safety of new therapeutic compounds given through different routes of exposure and are used as an alternative model to the canine or the nonhuman primate. Translational preclinical swine study data presented support the current finding that miniature swine are the animal model of choice for assessment of drug absorption, tolerance, and systemic toxicity following systemic exposures. Because research investigators need to be familiar with important anatomic and histopathologic features of the miniature swine in order to place toxicopathologic findings in their proper perspective, clinical and anatomic pathology data from a large number of Sinclair, Hanford, Yucatan, and Göttingen breeds from control groups from a wide variety of studies performed between 2004 and 2014 will be presented, compared, and partially illustrated.

Background Pathological Changes in Minipigs: A Comparison of the Incidence and Nature among Different Breeds and Populations of Minipigs.

Swine, especially the miniature swine or minipigs, are increasingly being used in preclinical safety assessment of small molecules, biopharmaceutical agents, and medical devices as an alternate nonrodent species. Although swine have been used extensively in biomedical research, there is a paucity of information in the current literature detailing the incidence of background lesions and differences in incidence between commonly used breeds. This article is a collaborative effort between multiple organizations to define and document lesions found in the common breeds of minipigs used for toxicological risk assessment in North America (NA) and the European Union (EU). We retrospectively assessed 10 years of historical control data from several institutions located in NA and EU, covering the period of 2004-2015. Here we report the background lesions with consideration of breed and geographical location. To our knowledge, this is the first report documenting spontaneous background lesions in commonly used breeds of swine in both NA and EU. This report serves as a resource to pathologists and will aid in interpretation of findings and differentiation of background from test article-related changes.

Pigs in Toxicology: Breed Differences in Metabolism and Background Findings.

Both a rodent and a nonrodent species are required for evaluation in nonclinical safety studies conducted to support human clinical trials. Historically, dogs and nonhuman primates have been the nonrodent species of choice. Swine, especially the miniature swine or minipigs, are increasingly being used in preclinical safety as an alternate nonrodent species. The pig is an appropriate option for these toxicology studies based on metabolic pathways utilized in xenobiotic biotransformation. Both similarities and differences exist in phase I and phase II biotransformation pathways between humans and pigs. There are numerous breeds of pigs, yet only a few of these breeds are characterized with regard to both xenobiotic-metabolizing enzymes and background pathology findings. Some specific differences in these enzymes based on breed and sex are known. Although swine have been used extensively in biomedical research, there is also a paucity of information in the current literature detailing the incidence of background lesions and differences between commonly used breeds. Here, the xenobiotic-metabolizing enzymes are compared between humans and pigs, and minipig background pathology changes are reviewed with emphasis on breed differences.

Tolerable Levels of Nonclinical Vehicles and Formulations Used in Studies by Multiple Routes in Multiple Species With Notes on Methods to Improve Utility.

Formulation of nonclinical evaluations is a challenge, with the fundamental need to achieve multiples of the clinical exposure complicated by differences in species and routes of administration-specific tolerances, depending on concentrations, volumes, dosing regimen, duration of each administration, and study duration. Current practice to approach these differences is based on individual experience and scattered literature with no comprehensive data source (the most notable exception being our 2006 publication on this same subject). Lack of formulation tolerance data results in excessive animal use, unplanned delays in the evaluation and development of drugs, and vehicle-dependent results. A consulting firm, a chemical company, and 4 contract research organizations conducted a rigorous data mining operation of vehicle data from studies dating from 1991 to 2015, enhancing the data from this author's 2006 publication (3 of the six 2015 contributors were also 2006 contributors). Additional data were found in the published literature. The results identified 108 single-component vehicles (and 305 combination formulations) used in more than 1,040 studies across multiple species (dog, primate, rat, mouse, rabbit, guinea pig, minipig, pig, chick embryo, and cat) by multiple routes for a wide range of study durations. The tabulated data include maximum tolerated use levels by species, route, duration of study, dose-limiting toxicity where reported, review of the available literature on each vehicle, guidance on syringe selection, volume and pH limits by route with basic guidance on nonclinical formulation development, and guidance on factors to be considered in nonclinical route selection.

Investigation of 4-piperidinols as novel H3 antagonists.

Compounds containing a substituted 4-piperidinol core have been found to be potent antagonists of the human H(3) receptor. The compounds exhibited up to a 60-fold preference for inhibiting the human H(3) receptor over the mouse and showed a low binding affinity for the hERG channel.

3-Indolyl sultams as selective CRTh2 antagonists.

CRTh2 (DP(2)) is a prostaglandin D(2) receptor implicated in the recruitment of eosinophils and basophils within the asthmatic lung. Here we report the discovery of a novel series of 3-indolyl sultam antagonists with low nM affinity for CRTh2. These compounds proved to be selective over the other primary prostaglandin D(2) receptor (DP1) as well as the thromboxane A(2) receptor (TP).

Measurement of glomerular filtration rate in anesthetized and conscious rhesus monkeys (Macaca mulatta).

OBJECTIVE: To validate a method to assess glomerular filtration rate (GFR) in conscious monkeys via transcutaneous radiation detection after IV injection of technetium Tc 99m pentatate (99mTc-DTPA). ANIMALS: 4 healthy rhesus monkeys. PROCEDURES: On day 1, each monkey was anesthetized, lothalamate sodium I 125 (125l-iothalamate) was administered via continuous rate infusion (0.0037 MBq/min); blood and urine samples were obtained for determination of 125l-iothalamate plasma clearance variables and estimation of GFR. One dose of 99mTc-DTPA (74 MBq/kg, IV) was also administered during the 125l-iothalamate plasma clearance test, and transcutaneous measurements of technetium 99m-emitted radiation were obtained by use of an ambulatory renal monitor (ARM) applied to a brachium of each monkey. Determination of GFR by use of the ARM was repeated on days 8 and 45 in the same monkeys without anesthesia. RESULTS: Sensitivity, accuracy, and precision of the 2 methods were similar. By use of the ARM, GFR determined by use of the renal rate constant (κGFR) was calculated; the value obtained on day 1 under anesthesia was similar to values determined via 125l-iothalamate plasma clearance testing on the same day, but was 16% to 23% less than that measured on days 8 and 45 in conscious monkeys. CONCLUSIONS AND CLINICAL RELEVANCE: The ARM method for assessment of GFR was less invasive, faster, and more convenient than the standard clearance method, but yielded comparable results. The need to train animals and size restrictions of the device may limit the use of this technique in other nonhuman animals.

PTP1B regulates leptin signal transduction in vivo.

Mice lacking the protein-tyrosine phosphatase PTP1B are hypersensitive to insulin and resistant to obesity. However, the molecular basis for resistance to obesity has been unclear. Here we show that PTP1B regulates leptin signaling. In transfection studies, PTP1B dephosphorylates the leptin receptor-associated kinase, Jak2. PTP1B is expressed in hypothalamic regions harboring leptin-responsive neurons. Compared to wild-type littermates, PTP1B(-/-) mice have decreased leptin/body fat ratios, leptin hypersensitivity, and enhanced leptin-induced hypothalamic Stat3 tyrosyl phosphorylation. Gold thioglucose treatment, which ablates leptin-responsive hypothalamic neurons, partially overcomes resistance to obesity in PTP1B(-/-) mice. Our data indicate that PTP1B regulates leptin signaling in vivo, likely by targeting Jak2. PTP1B may be a novel target to treat leptin resistance in obesity.

Inactivation of fatty acid transport protein 1 prevents fat-induced insulin resistance in skeletal muscle.

Insulin resistance in skeletal muscle plays a major role in the development of type 2 diabetes and may be causally associated with increases in intramuscular fatty acid metabolites. Fatty acid transport protein 1 (FATP1) is an acyl-CoA synthetase highly expressed in skeletal muscle and modulates fatty acid uptake and metabolism by converting fatty acids into fatty acyl-CoA. To investigate the role of FATP1 in glucose homeostasis and in the pathogenesis of insulin resistance, we examined the effect of acute lipid infusion or chronic high-fat feeding on insulin action in FATP1 KO mice. Whole-body adiposity, adipose tissue expression of adiponectin, intramuscular fatty acid metabolites, and insulin sensitivity were not altered in FATP1 KO mice fed a regular chow diet. In contrast, FATP1 deletion protected the KO mice from fat-induced insulin resistance and intramuscular accumulation of fatty acyl-CoA without alteration in whole-body adiposity. These findings demonstrate an important role of intramuscular fatty acid metabolites in causing insulin resistance and suggest that FATP1 may be a novel therapeutic target for the treatment of insulin resistance and type 2 diabetes.

Evaluation of functional and binding assays in cells expressing either recombinant or endogenous hERG channel.

INTRODUCTION: The hERG (human ether-a-go-go related gene) potassium channel is required for normal cardiac repolarization, is susceptible to inhibition by a wide variety of compounds, and its blockage can lead to cardiac QT interval prolongation and life threatening arrhythmias. The present report examines the ability of hERG binding and functional assays to identify compounds with potential cardiovascular liabilities at the earliest stages of drug discovery. METHODS: Competitive binding assays were developed using (3)H-dofetilide and membranes from HEK293EBNA cells stably expressing recombinant hERG (HEK293-hERG) and IMR-32 cells expressing hERG endogenously. hERG functional assays were also developed using membrane potential indicator dye and rubidium efflux. The ability of these assays to identify compounds with potential adverse cardiac effects was examined using drugs with known cardiac effects ranging from those with no known adverse effects to drugs that were withdrawn from the market due to increased risk of sudden death associated with Torsades de Points. RESULTS: Binding assays using HEK293-hERG membranes and (3)H-dofetilide were robust (Z'=0.69+/-0.015, mean+/-S.E.M.), highly reproducible (test-retest slope=1.04, r(2)=0.98), and correlated well with IC(50) values obtained by patch clamp (slope=0.98, r(2)=0.89). Binding assays using IMR-32 membranes were less sensitive (Z'=0.4+/-0.03, mean+/-S.E.M., false negative rate=0.4) but still correlated well with patch clamp data (slope=1.06, r(2)=0.83). The hERG membrane potential assay could detect potent hERG inhibitors (defined by hERG patch clamp IC(50)<0.1 muM) using HEK293-hERG cells, but were prone to generate false-negative results with less potent inhibitors (false negative rate=0.5). Finally, the rubidium efflux assay gave highly reproducible results (Z'=0.80+/-0.02, mean+/-S.E.M.) that correlated with patch clamp IC(50) values (slope=0.87, r(2)=0.73). DISCUSSION: The hERG binding and rubidium efflux assays are robust, predictive of patch clamp results, and can be used at the earliest stages of drug discovery.

Identification of novel and improved antimitotic agents derived from noscapine.

Analogues of the natural product noscapine were synthesized and their potential as antitumor agents evaluated. The discovery of a novel regioselective O-demethylation facilitated the synthesis of the potent aniline 6, which arrests mammalian cells in the G2/M phase of the cell cycle at 0.1 microM and also affects tubulin polymerization. Aniline 6 is orally bioavailable and is 250-fold more potent than noscapine in reducing cell proliferation in rapidly dividing cells.

The prolactin-releasing peptide receptor (GPR10) regulates body weight homeostasis in mice.

To identify new drug targets for the treatment of obesity, we employed a degenerate reverse transcriptasepolymerase chain reaction technique to isolate novel members of the G-protein coupled receptor superfamily from mouse hypothalamus. One of our clones was found to encode a protein with 90% amino acid identity to human GPR10, which was previously identified as the receptor for prolactin-releasing peptide (PrRP) and has been implicated in lactation, the regulation of food intake and other physiological functions. To investigate the role of GPR10 in food intake and body weight homeostasis, we generated mice carrying a targeted deletion of the GPR10 gene. First, using these knockout animals, we confirmed that GPR10 is the principle receptor for PrRP in the mouse hypothalamus because deletion of GPR10 completely abolished PrRP binding to isolated hypothalamic cell membranes. Second, we investigated the effect of normal and high-fat diets on energy intake, body weight, and glucose homeostasis in wild-type and GPR10 knockout mice. After fasting and refeeding, food intake in knockout animals was unchanged relative to control littermates. However, beginning at 16 wk of age on a normal diet, knockout mice became hyperphagic, obese, and showed significant increases in body fat and the levels of leptin and insulin, as well as decreased glucose tolerance. This metabolic profile was similar to the effect of a high-fat diet on wild-type animals. Our findings provide direct evidence that GPR10 is the receptor for PrRP and that it is involved in the regulation of energy balance in mice. GPR10 knockout mice will also prove useful for investigating other proposed activities for PrRP.

Orexins/hypocretins in the ob/ob mouse: hypothalamic gene expression, peptide content and metabolic effects.

Orexins (forms A and B) belong to a new family of peptides that, as neuropeptide Y (NPY), stimulate food intake when centrally injected. The ob/ob mouse is a well-characterized model of hyperphagia and obesity associated with strong metabolic disturbances and a central dysregulation of peptides involved in the control of feeding. In the present report, we investigated the hypocretin (Hcrt)/orexin (OX) peptide pathway in lean and ob/ob mice. Prepro-Hcrt/OX mRNA expression, measured by in situ hybridization was restricted to the lateral hypothalamus area. It was significantly decreased in ob/ob mice (-18%; p<0.01). When estimated by real time RT-PCR in the whole hypothalamus, this decrease amounted to 65% (p<0.001). Hcrt-1/OX-A peptide concentrations, measured by RIA in microdissected hypothalamic nuclei were high in the lateral hypothalamus (LH) and lower in the arcuate (ARC) and paraventricular nuclei (PVN). In ob/ob mice, OX-A levels were significantly lower than in lean mice in the LH (-34%; p<0.02) and in the PVN (-72%; p<0.005). Acute intracerebroventricular injection of Hcrt-1/OX-A (1-10 nmol) stimulated feeding in lean, but not in ob/ob mice, whereas Hcrt-2/OX-B (1-10 nmol) had the opposite effect. Acute third ventricle (i3vt) injections of Hcrt/OX peptides in ob/ob mice transiently increased their metabolic rate and stimulated lipid substrate utilization. These findings provide direct evidence that Hcrt/OX peptides are down-regulated in the hypothalamus of ob/ob mice, contrary to the NPY system. The present data argues that Hcrt/OX peptides are not primarily responsible for the metabolic syndrome of the ob/ob mice. The diminution in the OX tone might participate in a counterregulatory system necessary to limit the adverse effects of NPY on food intake and body weight.

Feeding response to a potent prolactin-releasing peptide agonist in lean and obese Zucker rats.

Prolactin (PRL)-releasing peptide (PrRP) is a new peptide present in the hypothalamus and in the circulation that may be involved in the regulation of feeding behavior. In the present experiment, we measured it in a well-known model of obesity, the Zucker rat. We also measured the reactivity of this animal in terms of food intake after the intraperitoneal (I.P.) or central injection of PrRP-13, a potent PrRP agonist. Plasma PrRP levels were 35% lower in obese fa/fa than in the lean rats (p<0.005). I.P. injections of PrRP-13 (10 mg/kg) stimulated food intake in lean and had no effect in obese rats (p<0.001). Intracerebral injections of PrRP-13 had no effects in both genotypes. Altogether, these results do not support a role for PrRP in the hyperphagia and obesity syndrome of the Zucker rat.

Ghrelin and body weight regulation in the obese Zucker rat in relation to feeding state and dark/light cycle.

Ghrelin is a new orexigenic peptide primarily produced by the stomach but also present in the hypothalamus. It has adipogenic effects when it is chronically injected in rodents but in obese humans, its plasma concentration is decreased. It can reverse the anorectic effects of leptin when it is co-injected with this peptide in the brain ventricles. The Zucker fa/fa rat is a genetic model of obesity related to a default in the leptin receptor. It is characterized by a large dysregulation of numerous hypothalamic peptides but the ghrelin status of this rat has not yet been determined. Through several experiments, we determine in lean and obese Zucker rats its circulating form in the plasma, its tissue levels and/or expression, and studied the influence of different feeding conditions and its light/dark variations. Ghrelin expression was higher in the obese stomach and hypothalamus (P < 0.05 and P < 0.02, respectively). The ratio of [Octanoyl-Ser3]-ghrelin (active form) to [Des-Octanoyl-Ser3]-ghrelin (inactive form) was approximately 1:1 in the stomach and 2:1 in the plasma in lean and obese rats (no differences). After fasting, plasma ghrelin concentrations increased significantly in lean (+ 64%; P < 0.001) and obese (+ 60%; P < 0.02) rats. After 24 hours of refeeding, they returned to their initial ad lib levels. Ghrelin concentrations were higher in obese rats by 69% (P < 0.005), 65% (P < 0.02), and 73% (P < 0.005) in the ad libitum, fast, and refed states respectively. These results indicate that the obese Zucker rat is characterized by increases in the stomach mRNA expression and in peptide release in the circulation. They clearly support a role for ghrelin in the development of obesity in the absence of leptin signaling.

Feeding response to ghrelin agonist and antagonist in lean and obese Zucker rats.

Ghrelin is a new orexigenic and adipogenic peptide primarily produced by the stomach and the hypothalamus. In the present experiment, we determined the circulating ghrelin levels in 60-week old fa/fa Zucker rats with a well-established obesity (n = 12) and in their lean (FA/FA) counterparts (n = 12). We also tested the feeding response of both groups to intra-peritoneal (I.P.) injection of ghrelin agonist and antagonist. Obese rats ate significantly more than the lean rats (21.7 +/- 1.1 vs. 18.3 +/- 0.3 g/day; p < 0.01). Their plasma ghrelin concentration was 35% higher than that in the lean homozygous rats (p < 0.025). GHRP-6 (1 mg/kg I.P, a GHS-R agonist) stimulated food intake in lean but not in obese rats (p < 0.01), whereas [D-Lys)]-GHRP-6 (12 mg/kg I.P., a GHS-R antagonist) decreased food intake in both groups (p < 0.0001). These results indicate that the obese Zucker rat is characterized by an increase in plasma ghrelin concentrations and by an attenuated response to a GHS-R agonist. They support a role for ghrelin in the development of obesity in the absence of leptin signaling.