Evaluation and Optimization of Blood Micro-Sampling Methods: Serial Sampling in a Cross-Over Design from an Individual Mouse.

PURPOSE: Current practices applied to mouse pharmacokinetic (PK) studies often use large numbers of animals with sporadic or composite sampling that inadequately describe PK profiles.  The purpose of this work was to evaluate and optimize blood microsampling techniques coupled with dried blood spot (DBS) and LC-MS/MS analysis to generate reliable PK data in mice.  In addition, the feasibility of cross-over designs was assessed and recommendations are presented. METHODS: The work describes a comprehensive evaluation of five blood microsampling techniques (tail clip, tail vein with needle hub, submandibular, retro-orbital, and saphenous bleeding) in CD-1 mice.  The feasibility of blood sampling was evaluated based on animal observations, ease of bleeding, and ability to collect serial samples.  Methotrexate, gemfibrozil and glipizide were used as test compounds and were dosed either orally or intravenously, followed by DBS collection and LC-MS/MS analysis to compare PK with various bleeding methods. RESULTS: Submandibular and retro-orbital methods that required non-serial blood collections did not allow for inter-animal variability assessments and resulted in poorly described absorption and distribution kinetics.  The submandibular and tail vein with needle-hub methods were the least favorable from a technical feasibility perspective.  Serial bleeding was possible with cannulated animals or saphenous bleeding in non-cannulated animals. CONCLUSIONS:   Of the methods that allowed serial sampling, the saphenous method when executed as described in this report, was most practical, reproducible and provided for assessment of inter-animal variability.  It enabled the collection of complete exposure profiles from a single mouse and the conduct of an intravenous/oral cross-over study design.  This methodology can be used routinely, it promotes the 3Rs principles by achieving reductions in the number of animals used, decreased restraints and animal stress, and improved the quality of data obtained in mouse PK studies. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Bilateral parotid duct transposition for keratoconjunctivitis sicca in a Connemara stallion.

A 7-year-old Connemara stallion was presented with a 4 month history of blepharospasm, recurrent corneal ulcerations, mucopurulent ocular discharge, and keratoconjunctivitis sicca (KCS) in both eyes unresponsive to medical therapy. Ophthalmic examination revealed lackluster corneas, axial corneal scarring and pigmentation with associated neovascularization, and absolute KCS in both eyes. Computed tomography scan and endoscopic evaluation of the upper airway and guttural pouches revealed no structural abnormalities to indicate neurogenic KCS. The stallion was diagnosed with immune-mediated dacryoadenitis as all other causes of KCS were excluded. Parotid duct transposition (PDT) was performed in the right eye followed by PDT in the left eye 4 weeks later. The right PDT was functional 2 years post-operatively with significant improvement in ocular comfort and reduced corneal fibrosis and neovascularization. The left PDT developed a salivary-cutaneous fistula over the left masseter muscle post-operatively due to avascular necrosis of the distal parotid duct (PD). Surgical reconstruction of the PDT using an expanded-polytetrafluoroethylene (e-PTFE) tube graft, an e-PTFE tube graft to autogenous caudal auricular vein graft, and an autogenous saphenous vein graft were all unsuccessful. Tear production in the left eye improved at 1 year post-surgery as a result of long term lacrostimulant therapy, and a permanent PD-cutaneous fistula was performed on the left PD at the level of the ventral mandible. Bilateral PDT in the horse is effective in resolving clinical signs associated with KCS; however, morbidity associated with avascular necrosis of the transposed PD may be significant and can result in surgical failure.

Complex Feline Disease Mapping Using a Dense Genotyping Array.

The current feline genotyping array of 63 k single nucleotide polymorphisms has proven its utility for mapping within breeds, and its use has led to the identification of variants associated with Mendelian traits in purebred cats. However, compared to single gene disorders, association studies of complex diseases, especially with the inclusion of random bred cats with relatively low linkage disequilibrium, require a denser genotyping array and an increased sample size to provide statistically significant associations. Here, we undertook a multi-breed study of 1,122 cats, most of which were admitted and phenotyped for nine common complex feline diseases at the Cornell University Hospital for Animals. Using a proprietary 340 k single nucleotide polymorphism mapping array, we identified significant genome-wide associations with hyperthyroidism, diabetes mellitus, and eosinophilic keratoconjunctivitis. These results provide genomic locations for variant discovery and candidate gene screening for these important complex feline diseases, which are relevant not only to feline health, but also to the development of disease models for comparative studies.

Pharmacokinetic comparisons of tail-bleeding with cannula- or retro-orbital bleeding techniques in rats using six marketed drugs.

INTRODUCTION: The evaluation of drug disposition properties of chemical entities in drug discovery research typically involves the conduct of pharmacokinetic studies in rodents that requires blood sampling over several time points, preferably without disrupting the physiological status of the animals. Several blood withdrawal methods have been employed throughout the industry, yet these methods have not been comprehensively evaluated with regard to their effects on pharmacokinetic profiles of the drug investigated to recommend best practices. METHODS: In this paper, the pharmacokinetics of six marketed drugs from four distinct therapeutic classes were compared using tail-vein, femoral-artery cannula-, and retro-orbital sinus bleeding techniques. The marketed drugs used in these studies were pentoxifylline, gemfibrozil, glipizide, methotrexate, clonidine, and fluoxetine. RESULTS: Following oral administration, peak plasma concentration (C(max)), and area under the curve (AUC(0-24)) values for all compounds were not significantly different with the tail-vein method when compared to cannula- or retro-orbital sinus bleeding, except for fluoxetine and gemfibrozil for which minor, but statistically significant differences were observed. The effect of arterial versus venous tail-bleeding on the pharmacokinetics of pentoxifylline indicated no statistical differences in either C(max) or AUC(0-24) values. However, for fluoxetine, higher exposures were observed with tail arterial than venous sampling (2-fold with respect to C(max) and 1.7-fold with respect to AUC(0-24), p<0.05). DISCUSSION: The observed differences with fluoxetine may be due to its pharmacological effects on thermoregulatory responses that influence tail blood flow, a hypothesis that remains to be tested. Based on these observations, we recommend the tail-bleeding technique for pharmacology or toxicology exposure and F% studies, particularly in early discovery work. Retro-orbital bleeding is controversial and is no longer considered a humane method. Cannula-bleeding, especially coupled with automated blood-collection techniques, has become the most efficient way for pharmaceutical industry to perform rat bioavailability studies.

Assessment of personal protective equipment used for facial mucocutaneous exposure protection in nonhuman primate areas.

Animal caretakers working in NHP areas must wear facial PPE to protect themselves from the zoonotic hazards related to splash exposures, but PPE that is uncomfortable may present its own risks. The authors evaluated the level of protection offered by several types of facial PPE against a variety of simulated facial mucocutaneous exposures of the sort that could occur during typical procedures in Old World NHP facilities and determined that less restrictive PPE can be used without compromising safety.

Longitudinal studies of cardiac troponin I concentrations in serum from male cynomolgus monkeys: resting values and effects of oral and intravenous dosing on biologic variability.

BACKGROUND: There is a paucity of information regarding cardiac troponin (cTn) concentrations in peripheral blood of nonhuman primates (NHP). Even less is known regarding cTn concentrations in monkeys that are restrained for oral or intravenous (iv) dosing. OBJECTIVES: The objectives of these studies were to (1) determine cardiac troponin I (cTnI) concentration in resting Cynomolgus monkeys and investigate biologic variability in cTnI concentration over time, (2) determine cTnI changes in restrained monkeys given sham oral dosing, and (3) determine cTnI changes in restrained NHP given a sham intravenous dosing. METHODS: The Research Use Only Erenna cTnI ultrasensitive immunoassay based on single molecule counting technology was used to determine serum cTnI concentration in longitudinal studies of male Cynomolgus monkeys at rest, and after sham oral and intravenous dosing. Animals were catheterized prestudy, and blood samples were collected by an automated sampling device to limit disturbance of the animals during studies. RESULTS: In resting monkeys cTnI concentrations were relatively low and constant and ranged from 0.2 to 9.6 pg/mL (mean = 2.5 pg/mL), with minimal variability during a 24-hour period. Animals given sham oral dosing also had low cTnI concentration with little variability similar to the resting values. Several animals restrained for intravenous dosing had a small transient increase in cTnI concentration (~5-25 pg/mL) that resolved quickly within one to 3 hours postinjection. CONCLUSIONS: Results of this longitudinal study provide information that may be important in differentiating effects of animal handling from those associated with compound-related effects in preclinical toxicology studies of drugs in development.