Kirstin F. Barnhart

ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics

Reference intervals (RI) are an integral component of laboratory diagnostic testing and clinical decision-making and represent estimated distributions of reference values (RV) from healthy populations of comparable individuals. Because decisions to pursue diagnoses or initiate treatment are often based on values falling outside RI, the collection and analysis of RV should be approached with diligence. This report is a condensation of the ASVCP 2011 consensus guidelines for determination of de novo RI in veterinary species, which mirror the 2008 Clinical Laboratory and Standards Institute (CLSI) recommendations, but with language and examples specific to veterinary species. Newer topics include robust methods for calculating RI from small sample sizes and procedures for outlier detection adapted to data quality. Because collecting sufficient reference samples is challenging, this document also provides recommendations for determining multicenter RI and for transference and validation of RI from other sources (eg, manufacturers). Advice for use and interpretation of subject-based RI is included, as these RI are an alternative to population-based RI when sample size or inter-individual variation is high. Finally, generation of decision limits, which distinguish between populations according to a predefined query (eg, diseased or non-diseased), is described. Adoption of these guidelines by the entire veterinary community will improve communication and dissemination of expected clinical laboratory values in a variety of animal species and will provide a template for publications on RI. This and other reports from the Quality Assurance and Laboratory Standards (QALS) committee are intended to promote quality laboratory practices in laboratories serving both clinical and research veterinarians.

A Peptidomimetic Targeting White Fat Causes Weight Loss and Improved Insulin Resistance in Obese Monkeys

A peptide-based drug that targets the vasculature of adipose tissue induces weight loss and improves metabolic function in spontaneously obese monkeys. Fat Monkeys Get Trim The rapidly increasing rate of obesity worldwide is one of the biggest health challenges facing society today. Unlike related threats such as cancer, cardiovascular disease, and diabetes, very few approved drugs are available to treat obesity despite some promising early-stage candidates. In a new study, Barnhart and colleagues take a fresh approach to treating obesity by developing a peptide-like molecule that targets the blood vessels that feed fat tissue. They test their peptidomimetic called adipotide in obese monkeys and show that it both reduces fat tissue and decreases resistance to insulin. Adipotide is a short peptide-based agent that selectively targets a receptor expressed by the vascular endothelial cells that comprise the blood vessels that support subcutaneous and visceral fat. This peptidomimetic carries a molecule that, once internalized by the endothelial cells, causes them to undergo programmed cell death, thereby inducing gradual elimination of excess fat. In placebo-controlled experiments, spontaneously obese rhesus monkeys treated with adipotide for 28 days showed a 7 to 15% weight loss as well as improved insulin resistance. Two forms of imaging revealed that the weight loss occurred primarily through a reduction in fat tissue and did not reflect fluid loss or muscle wasting. Monkeys treated with adipotide displayed a 38% reduction in total body fat and a 27% reduction in abdominal fat compared to pretreatment baseline values. Early weight loss drug candidates are typically screened in rodent models of obesity. However, the central nervous system control and metabolic regulation of food intake and fat storage in rodents is quite different from that of monkeys and humans. Spontaneously obese monkeys are a more accurate model of obesity in humans and provide a valuable setting for testing anti-obesity drug candidates. Adipotide therapy resulted in a reduction in body mass, an improvement in insulin resistance, and a decrease in abdominal circumference, key predictors of diabetes in humans. These encouraging results support the further development of adipotide as a potential new prototype drug to combat obesity in humans. Obesity, defined as body mass index greater than 30, is a leading cause of morbidity and mortality and a financial burden worldwide. Despite significant efforts in the past decade, very few drugs have been successfully developed for the treatment of obese patients. Biological differences between rodents and primates are a major hurdle for translation of anti-obesity strategies either discovered or developed in rodents into effective human therapeutics. Here, we evaluate the ligand-directed peptidomimetic CKGGRAKDC-GG-D(KLAKLAK)2 (henceforth termed adipotide) in obese Old World monkeys. Treatment with adipotide induced targeted apoptosis within blood vessels of white adipose tissue and resulted in rapid weight loss and improved insulin resistance in obese monkeys. Magnetic resonance imaging and dual-energy x-ray absorptiometry confirmed a marked reduction in white adipose tissue. At experimentally determined optimal doses, monkeys from three different species displayed predictable and reversible changes in renal proximal tubule function. Together, these data in primates establish adipotide as a prototype in a new class of candidate drugs that may be useful for treating obesity in humans.

Targeting the interleukin‐11 receptor α in metastatic prostate cancer: A first‐in‐man study

Receptors in tumor blood vessels are attractive targets for ligand‐directed drug discovery and development. The authors have worked systematically to map human endothelial receptors (“vascular zip codes”) within tumors through direct peptide library selection in cancer patients. Previously, they selected a ligand‐binding motif to the interleukin‐11 receptor alpha (IL‐11Rα) in the human vasculature.

Two Hypomorphic Alleles of Mouse Ass1 as a New Animal Model of Citrullinemia Type I and Other Hyperammonemic Syndromes

Citrullinemia type I (CTLN1, OMIM# 215700) is an inherited urea cycle disorder that is caused by an argininosuccinate synthetase (ASS) enzyme deficiency. In this report, we describe two spontaneous hypomorphic alleles of the mouse Ass1 gene that serve as an animal model of CTLN1. These two independent mouse mutant alleles, also described in patients affected with CTLN1, interact to produce a range of phenotypes. While some mutant mice died within the first week after birth, others survived but showed severe retardation during postnatal development as well as alopecia, lethargy, and ataxia. Notable pathological findings were similar to findings in human CTLN1 patients and included citrullinemia and hyperammonemia along with delayed cerebellar development, epidermal hyperkeratosis, and follicular dystrophy. Standard treatments for CTLN1 were effective in rescuing the phenotype of these mutant mice. Based on our studies, we propose that defective cerebellar granule cell migration secondary to disorganization of Bergmann glial cell fibers cause cerebellar developmental delay in the hyperammonemic and citrullinemic brain, pointing to a possible role for nitric oxide in these processes. These mouse mutations constitute a suitable model for both mechanistic and preclinical studies of CTLN1 and other hyperammonemic encephalopathies and, at the same time, underscore the importance of complementing knockout mutations with hypomorphic mutations for the generation of animal models of human genetic diseases.

Methicillin-resistant Staphylococcus aureus prevalence among captive chimpanzees, Texas, USA, 2012

Methicillin-resistant Staphylococcus aureus (MRSA) infection in humans and animals is concerning. In 2012, our evaluation of a captive chimpanzee colony in Texas revealed MRSA prevalence of 69%. Animal care staff should be aware of possible zoonotic MRSA transmission resulting from high prevalence among captive chimpanzees.

Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

Preclinical Mammalian Safety Studies of EPHARNA (DOPC Nanoliposomal EphA2-Targeted siRNA)

To address the need for efficient and biocompatible delivery systems for systemic siRNA delivery, we developed 1,2-Dioleoyl-sn-Glycero-3-Phosphatidylcholine (DOPC) nanoliposomal EphA2-targeted therapeutic (EPHARNA). Here, we performed safety studies of EPHARNA in murine and primate models. Single dosing of EPHARNA was tested at 5 concentrations in mice (N = 15 per group) and groups were sacrificed on days 1, 14, and 28 for evaluation of clinical pathology and organ toxicity. Multiple dosing of EPHARNA was tested in mice and Rhesus macaques twice weekly at two dose levels in each model. Possible effects on hematologic parameters, serum chemistry, coagulation, and organ toxicity were assessed. Following single-dose EPHARNA administration to mice, no gross pathologic or dose-related microscopic findings were observed in either the acute (24 hours) or recovery (14 and 28 days) phases. The no-observed-adverse-effect level (NOAEL) for EPHARNA is considered >225 μg/kg when administered as a single injection intravenously in CD-1 mice. With twice weekly injection, EPHARNA appeared to stimulate a mild to moderate inflammatory response in a dose-related fashion. There appeared to be a mild hemolytic reaction in the female mice. In Rhesus macaques, minimal to moderate infiltration of mononuclear cells was found in some organs including the gastrointestinal tract, heart, and kidney. No differences attributed to EPHARNA were observed. These results demonstrate that EPHARNA is well tolerated at all doses tested. These data, combined with previously published in vivo validation studies, have led to an ongoing first-in-human phase I clinical trial (NCT01591356). Mol Cancer Ther; 16(6); 1114–23. ©2017 AACR.

Response to Comment on “A Peptidomimetic Targeting White Fat Causes Weight Loss and Improved Insulin Resistance in Obese Monkeys”

Several lines of evidence indicate that the effects of adipotide on weight loss and food consumption in obese monkeys are not due to a toxic effect, but rather result from the targeting mechanism of adipotide. Several lines of evidence indicate that the effects of adipotide on weight loss and food consumption in obese monkeys are not due to a toxic effect, but rather result from the targeting mechanism of adipotide.

MLH1-rheMac hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques.

Significance The discovery of MLH1-rheMac hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques (MLH1-rheMac HNPCC), which is an orthologue of Lynch syndrome in humans, is highly significant in the field of oncology. The hereditary nature of this disease should allow for planned cross-breeding of rhesus macaques to assess the effects of homozygous versus heterozygous MLH1 gene mutations, as well as other comutations and environmental factors that may affect the development of colon cancers. Also, the MLH1-rheMac HNPCC syndrome in rhesus macaques can serve as an important model for development of novel approaches to diagnosis and therapy of Lynch syndrome in human patients. Over the past two decades, 33 cases of colonic adenocarcinomas have been diagnosed in rhesus macaques (Macaca mulatta) at the nonhuman primate colony of the Keeling Center for Comparative Medicine and Research at The University of Texas MD Anderson Cancer Center. The distinctive feature in these cases, based on PET/computed tomography (CT) imaging, was the presence of two or three tumor lesions in different locations, including proximal to the ileocecal juncture, proximal to the hepatic flexure, and/or in the sigmoid colon. These colon carcinoma lesions selectively accumulated [18F]fluorodeoxyglucose ([18F]FDG) and [18F]fluoroacetate ([18F]FACE) at high levels, reflecting elevated carbohydrate and fatty acid metabolism in these tumors. In contrast, the accumulation of [18F]fluorothymidine ([18F]FLT) was less significant, reflecting slow proliferative activity in these tumors. The diagnoses of colon carcinomas were confirmed by endoscopy. The expression of MLH1, MSH2, and MSH6 proteins and the degree of microsatellite instability (MSI) was assessed in colon carcinomas. The loss of MLH1 protein expression was observed in all tumors and was associated with a deletion mutation in the MLH1 promoter region and/or multiple single-nucleotide polymorphism (SNP) mutations in the MLH1 gene. All tumors exhibited various degrees of MSI. The pedigree analysis of this rhesus macaque population revealed several clusters of affected animals related to each other over several generations, suggesting an autosomal dominant transmission of susceptibility for colon cancer. The newly discovered hereditary nonpolyposis colorectal cancer syndrome in rhesus macaques, termed MLH1-rheMac, may serve as a model for development of novel approaches to diagnosis and therapy of Lynch syndrome in humans.

Abstract 2094: Translating EPHARNA (DOPC nanoliposomal EphA2-targeted siRNA) to the clinic

Introduction: To address the need for efficient and biocompatible delivery systems for systemic siRNA delivery, we have developed a 1,2-Dioleoyl-sn-Glycero-3-Phosphatidylcholine (DOPC) nanoliposomal EphA2-targeted therapeutic (EPHARNA). We have previously demonstrated the efficacy of EPHARNA in multiple tumor models. Here, following FDA guidance, we performed safety studies of EPHARNA in murine and primate models. Methods: Single dosing of EPHARNA was tested at 5 concentrations in mice (N = 15 per group) and groups of animals were sacrificed on days 1, 14, and 28 for evaluation of clinical pathology and organ toxicity. Multiple dosing of EPHARNA was tested in mice twice weekly for 1 month (N = 10 per group) and in Rhesus macaques weekly (N = 4 per group) at two dose levels in each model. Possible effects on hematologic parameters, serum chemistry, coagulation, and organ toxicity were assessed in all animals. Results: Following single dose EPHARNA administration to mice, no gross pathological or dose-related microscopic findings were observed in either the acute (24 hrs) or recovery (14 and 28 days) phases of this study. The no-observed-adverse-effect level (NOAEL) for EPHARNA is considered > 225 μg/kg when administered as a single injection intravenously in CD-1 male and female mice. With twice weekly injection, EPHARNA appeared to stimulate a mild to moderate inflammatory response in a dose-related fashion. In addition, there appeared to be a mild hemolytic reaction in the female mice. Systemic toxicity related to both the DOPC formulation as well as EphA2 silencing was assessed in Rhesus macaques due to the near 100% homology between Rhesus and human EphA2. One animal developed signs consistent with an infusion reaction on its first administration, and was dosed with a slow infusion over 15-30 minutes with subsequent infusions. Minimal to moderate infiltration of mononuclear cells was found in some organs including the GI tract, heart, and kidney in all Rhesus macaques in all groups. The significance of these infiltrates to the general health of the animals is unknown. One female in the control group and one in the high dose group had ovarian mineralization, which was considered incidental in this study. No differences attributed to EPHARNA were observed in the hematologic or anatomic parameters assessed. Conclusion: These results demonstrate that EPHARNA is well tolerated at all doses tested without significant complications. These data, combined with previously published validation studies demonstrating siRNA as an effective therapeutic in vivo, led to the development of a first in human Phase I clinical trial that is currently underway (NCT01591356). Citation Format: Michael J. Wagner, Rahul Mitra, Wallace Baze, Kirstin Barnhart, Robert L. Coleman, Gabriel Lopez-Berestein, Anil K. Sood. Translating EPHARNA (DOPC nanoliposomal EphA2-targeted siRNA) to the clinic. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2094.