A Third MLPH Variant Causing Coat Color Dilution in Dogs.

Altered melanosome transport in melanocytes, resulting from variants in the melanophilin (MLPH) gene, are associated with inherited forms of coat color dilution in many species. In dogs, the MLPH gene corresponds to the D locus and two variants, c.-22G > A (d1) and c.705G > C (d2), leading to the dilution of coat color, as described. Here, we describe the independent investigations of dogs whose coat color dilution could not be explained by known variants, and who report a third MLPH variant, (c.667_668insC) (d3), which leads to a frameshift and premature stop codon (p.His223Profs*41). The d3 allele is found at low frequency in multiple dog breeds, as well as in wolves, wolf-dog hybrids, and indigenous dogs. Canids in which the d3 allele contributed to the grey (dilute) phenotype were d1/d3 compound heterozygotes or d3 homozygotes, and all non-dilute related dogs had one or two D alleles, consistent with a recessive inheritance. Similar to other loci responsible for coat colors in dogs, this, alongside likely additional allelic heterogeneity at the D locus, or other loci, must be considered when performing and interpreting genetic testing.

Phase I study of PF­04895162, a Kv7 channel opener, reveals unexpected hepatotoxicity in healthy subjects, but not rats or monkeys: clinical evidence of disrupted bile acid homeostasis.

During a randomized Phase 1 clinical trial the drug candidate, PF-04895162 (ICA-105665), caused transaminase elevations (≥grade 1) in six of eight healthy subjects treated at 300 mg twice daily for 2-weeks (NCT01691274). This was unexpected since studies in rats (<6 months) and cynomolgus monkeys (<9 months) treated up to 100 mg/kg/day did not identify the liver as a target organ. Mechanistic studies showed PF-04895162 had low cytotoxic potential in human hepatocytes, but inhibited liver mitochondrial function and bile salt export protein (BSEP) transport. Clinical relevance of these postulated mechanisms of liver injury was explored in three treated subjects that consented to analysis of residual pharmacokinetic plasma samples. Compared to a nonresponder, two subjects with transaminase elevations displayed higher levels of miRNA122 and total/conjugated bile acid species, whereas one demonstrated impaired postprandial clearance of systemic bile acids. Elevated taurine and glycine conjugated to unconjugated bile acid ratios were observed in two subjects, one before the onset of elevated transaminases. Based on the affinity of conjugated bile acid species for transport by BSEP, the profile of plasma conjugated/unconjugated bile acid species was consistent with inhibition of BSEP. These data collectively suggest that the human liver injury by PF-04895162 was due to alterations in bile acid handling driven by dual BSEP/mitochondrial inhibition, two important risk factors associated with drug-induced liver injury in humans. Alterations in systemic bile acid composition were more important than total bile acids in the manifestation of clinical liver injury and may be a very early biomarker of BSEP inhibition.

Current Practices and Challenges in Method Validation.

Method validation is a cornerstone on which biomarker development and utilization rest. However, given the abundance of biomarker candidates that are being identified and characterized, validation of these entities for the use in nonclinical studies can be complex. The objective of this continuing education course was to review current practices and challenges encountered during the validation of methods for the analysis of novel biomarkers. Additionally, the importance of biological validation and correlation with pathology end points for biomarker candidates was discussed. This article is a summary of the materials presented at the 36th Annual Symposium of the Society of Toxicologic Pathology for a continuing education course titled "Current Practices and Challenges in Method Validation." The speakers were subject-matter experts in the validation of quantitative mass spectrometry, multiplex binding assays, biological biomarkers, and immunophenotyping and anatomic and clinical pathology considerations in biomarker qualification.

Assessment of serum bile acid profiles as biomarkers of liver injury and liver disease in humans.

To assess the potential of individual bile acids (IBA) and their profiles as mechanistic biomarkers of liver injury for humans in real world situations, we interrogated samples collected under minimum controlled conditions (ie subjects were not fasted). Total bile acids (TBA) have been considered to be biomarkers of liver injury for decades, and more recently, monitoring of IBA has been proposed for differentiation of variety of etiologies of liver injury. We established a LC-MS/MS methodology to analyze nine IBA, generated reference ranges, and examined effects of age, gender, and ethnicity for each IBA. Furthermore, we evaluated the ability of IBA and their profiles to detect hepatic injury in subjects with a broad range of liver impairments. To date, our study utilized the largest total cohort of samples (N = 645) that were divided into 2 groups, healthy or liver impaired, to evaluate IBA as biomarkers. The TBA serum levels in the Asian ethnic group trended higher when compared to other ethnic groups, and the serum concentrations of IBA, such as glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), chenodeoxycholic acid (CDCA), and taurochenoxycholic acid (TCDCA) were significantly increased. To our knowledge, this report is the first to describe ethnic differences in serum concentrations of IBAs. In patients with hepatic impairments, with the exception of deoxycholic acid (DCA), the concentrations of IBAs were significantly elevated when compared with healthy subjects. The conjugated bile acids displayed greater differences between healthy subjects and subjects with hepatic impairments than non-conjugated bile acids. Furthermore, the subjects with hepatic impairments exhibited distinct profiles (signatures) of IBAs that clustered subjects according the nature of their liver impairments. Although additional studies are needed, our data suggested that the analysis of IBA has the potential to become useful for differentiation of various forms of liver injury.

The Validation of Quantitative Mass Spectrometry Assays for Clinical Chemistry Assessments in Animal Models.

Mass spectrometry (MS) has become a key platform in the clinical pathology laboratory and is being used more frequently for clinical pathology assessments in preclinical species for drug development studies. MS assays are being utilized for some traditional clinical pathology end points as well as novel biomarker analyses. For effective deployment in drug development toxicology studies, assays must be validated for use, and these validations are not very different from other bioanalytical platforms commonly found in the clinical pathology laboratory. Validations for MS assays include accuracy and precision assessments, analyte stability evaluations, carryover determinations, and recovery measures. The MS platform does present some unique challenges that should be considered, including ion suppression and availability of reference standards with MS data. Understanding the caveats of the MS platform is important for thorough validations and effective deployment.

Therapeutic Effects of FGF23 c-tail Fc in a Murine Preclinical Model of X-Linked Hypophosphatemia Via the Selective Modulation of Phosphate Reabsorption.

Fibroblast growth factor 23 (FGF23) is the causative factor of X-linked hypophosphatemia (XLH), a genetic disorder effecting 1:20,000 that is characterized by excessive phosphate excretion, elevated FGF23 levels and a rickets/osteomalacia phenotype. FGF23 inhibits phosphate reabsorption and suppresses 1α,25-dihydroxyvitamin D (1,25D) biosynthesis, analytes that differentially contribute to bone integrity and deleterious soft-tissue mineralization. As inhibition of ligand broadly modulates downstream targets, balancing efficacy and unwanted toxicity is difficult when targeting the FGF23 pathway. We demonstrate that a FGF23 c-tail-Fc fusion molecule selectively modulates the phosphate pathway in vivo by competitive antagonism of FGF23 binding to the FGFR/α klotho receptor complex. Repeated injection of FGF23 c-tail Fc in Hyp mice, a preclinical model of XLH, increases cell surface abundance of kidney NaPi transporters, normalizes phosphate excretion, and significantly improves bone architecture in the absence of soft-tissue mineralization. Repeated injection does not modulate either 1,25D or calcium in a physiologically relevant manner in either a wild-type or disease setting. These data suggest that bone integrity can be improved in models of XLH via the exclusive modulation of phosphate. We posit that the selective modulation of the phosphate pathway will increase the window between efficacy and safety risks, allowing increased efficacy to be achieved in the treatment of this chronic disease. © 2017 American Society for Bone and Mineral Research.

Assessment of endogenous 25-hydroxyvitamin D serum concentrations by liquid chromatography-tandem mass spectrometry in various animal species.

BACKGROUND: Mass spectrometry (MS) has become the preferential method for the analysis of vitamin D in the clinic, yet no single platform is utilized for preclinical species in drug development studies. For vitamin D, the MS platform can provide certain benefits such as applicability of a single assay for multiple species, low cost, and high specificity. OBJECTIVES: A quantitative liquid chromatography-tandem MS (LC-MS/MS) assay for 25-hydroxyvitamin D3 (25OHD3 ) and D2 (25OHD2 ) was validated for rat, dog, mouse, and monkey, and suitability for drug development studies was assessed. METHODS: Standards were used to determine intra- and inter-assay accuracy and precision for LC-MS/MS. Extraction recovery and carryover due to instrumentation were determined. Repeat analyses of pooled serum samples from rat, dog, mouse, and monkey were assessed for precision, and other serum samples were used to determine the normal range in each species and detect biologically relevant changes. RESULTS: For both 25OHD3 and 25OHD2 , inaccuracy was ≤ 6%, and imprecision was ≤ 13%. Extraction recovery was 75% for 25OHD3 and 72% for 25OHD2 , and carryover was ≤ 0.1%. Measurable concentrations of 25OHD3 were recorded in serum samples from all species tested, but no 25OHD2 as diets were only fortified with 25OHD3 . This dataset provides preliminary information for the determination of RIs for 25OHD3 in rat, dog, mouse, and monkey with the LC-MS/MS platform. CONCLUSIONS: The LC-MS/MS assay was accurate and precise for determination of endogenous concentrations of 25OHD3 in serum samples from drug development studies in rat, dog, mouse, and monkey.

Inflammatory Cell Findings in the Female Rabbit Heart and Stress-associated Exacerbation with Handling and Procedures Used in Nonclinical Studies.

Despite the use of rabbits in biomedical research, including regulatory toxicology and cardiovascular studies, little data exist on heart findings in this species. This study was designed to document myocardial findings in female rabbits and the impact of study-related procedures typical for vaccine toxicology studies. One hundred and forty 6- to 8-month-old female New Zealand White rabbits were divided equally into 2 groups, high and low study procedure groups (group 1 and group 2, respectively). All animals received intramuscular (IM) injections of sterile saline every 2 weeks for 5 times and were necropsied 2 days after the final IM injection. Clinical chemistry, hematology, and urinalysis were evaluated. Blood for stress biomarkers (norepinephrine, epinephrine, cortisol, and corticosterone), C-reactive protein, cardiac troponin I, and creatine kinase were collected at time 0 (just before dose administration) and then at 4, 24, and 48 hr after dose administration in group 1 only. Hearts were assessed histologically. Focal to multifocal minimal inflammatory cell infiltrates were common (∼80%), particularly in the left ventricle and interventricular septum, and were similar to the types of infiltrates identified in other laboratory animal species. Additionally, study-related procedures elevated serum stress biomarkers and exacerbated the frequency and severity of myocardial inflammatory cell infiltrates.

Evaluation of serum bile acid profiles as biomarkers of liver injury in rodents.

Bile acids (BAs) have been studied as potential biomarkers of drug-induced liver injury. However, the relationship between levels of individual BAs and specific forms of liver injury remains to be fully understood. Thus, we set out to evaluate cholic acid (CA), glycocholic acid (GCA), and taurocholic acid (TCA) as potential biomarkers of liver injury in rodent toxicity studies. We have developed a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) assay applicable to rat and mouse serum and evaluated levels of the individual BAs in comparison with the classical biomarkers of hepatotoxicity (alanine aminotransferase [ALT], aspartate aminotransferase [AST], glutamate dehydrogenase (GLDH), alkaline phosphatase, total bilirubin, gamma-glutamyl transferase, and total BAs) and histopathology findings in animals treated with model toxicants. The pattern of changes in the individual BAs varied with different forms of liver injury. Animals with histopathologic signs of hepatocellular necrosis showed increases in all 3 BAs tested, as well as increases in ALT, AST, GLDH, and total BAs. Animals with histopathologic signs of bile duct hyperplasia (BDH) displayed increases in only conjugated BAs (GCA and TCA), a pattern not observed with the other toxicants. Because BDH is detectable only via histopathology, our results indicate the potential diagnostic value of examining individual BAs levels in serum as biomarkers capable of differentiating specific forms of liver injury in rodent toxicity studies.

Pregabalin induces hepatic hypoxia and increases endothelial cell proliferation in mice, a process inhibited by dietary vitamin E supplementation.

The preceding article identified key components of pregabalin's mode of action on nongenotoxic hemangiosarcoma formation in mice, including increased serum bicarbonate leading to decreased respiratory rate, increased blood pH, increased venous oxygen saturation, increased vascular endothelial growth factor and basic fibroblast growth factor expression, increased hepatic vascular endothelial growth factor receptor 2 expression, and increased iron-laden macrophages. Increased platelet count and platelet activation were early, species-specific biomarkers in mice. Dysregulated erythropoiesis, macrophage activation, and elevations of tissue growth factors were consistent with the unified mode of action for nongenotoxic hemangiosarcoma recently proposed at an international hemangiosarcoma workshop (Cohen, S. M., Storer, R. D., Criswell, K. A., Doerrer, N. G., Dellarco, V. L., Pegg, D. G., Wojcinski, Z. W., Malarkey, D. E., Jacobs, A. C., Klaunig, J. E., et al. (2009). Hemangiosarcoma in rodents: Mode-of-action evaluation and human relevance. Toxicol. Sci. 111, 4-18). In this article, we present evidence that pregabalin induces hypoxia and increases endothelial cell (EC) proliferation in a species-specific manner. Dietary administration of pregabalin produced a significant 35% increase in an immunohistochemical stain for hypoxia (Hypoxyprobe) in livers from pregabalin-treated mice. Increased Hypoxyprobe staining was not observed in the liver, bone marrow, or spleen of rats, supporting the hypothesis that pregabalin produces local tissue hypoxia in a species-specific manner. Transcriptional analysis supports that rats, unlike mice, adapt to pregabalin-induced hypoxia. Using a dual-label method, increased EC proliferation was observed as early as 2 weeks in mouse liver and 12 weeks in bone marrow following pregabalin administration. These same assays showed decreased EC proliferation in hepatic ECs of rats, further supporting species specificity. Dietary supplementation with vitamin E, which is known to have antioxidant and antiangiogenic activity, inhibited pregabalin-induced increases in mouse hepatic EC proliferation, providing confirmatory evidence for the proposed mode of action and its species-specific response.

Studies on the glycosylation of wild-type and mutant forms of Aspergillus niger pectin methylesterase.

Pectin methylesterase (PME) is one of a number of enzymes released by the fungus Aspergillus niger that are involved in the degradation of specific plant cell-wall structures. PME is a glycoprotein with three potential sites for N-linked glycosylation. The glycosylation may affect the hydrolytic activity or the substrate specificity of PME. In this work, we investigate first the structures and the attachment sites of the glycans present on recombinant wild-type PME. Further, a series of PME mutants was created in which the three potential N-linked glycosylation sites were eliminated in all possible combinations. The glycosylation of the mutants and their activities were then studied. Mass spectrometric techniques tailored for carbohydrate analysis were applied to both characterize the glycan structures and to determine the specific sites of attachment. High mannose structures with variable numbers of mannose were found on the wild-type, as well as the mutant forms. Studies using the mutants suggest that glycosylation does not strongly influence the activity. Whether it may affect the substrate specify of the enzyme is unknown, and that aspect will be explored in future work.