Pharmacokinetics of mebendazole in plasma and cerebrospinal fluid following a single oral dose in healthy dogs.

Novel therapies are needed for treatment of gliomas. Mebendazole previously demonstrated anti-neoplastic effects on canine glioma cell lines at in vitro mean inhibitory concentrations (IC50) of 10 ng/mL. Our study aimed to titrate the oral dose of mebendazole necessary to achieve concentrations ≥10 ng/mL in cerebrospinal fluid (CSF) of healthy dogs. We hypothesized that an oral dose up to 200 mg/kg would be necessary. Phase one was a dose titration study using a total of 6 mixed breed dogs that described dose vs. plasma concentrations for 72 h after single oral dosing of either 50 mg/kg (n = 2), 100 mg/kg (n = 2), or 200 mg/kg (n = 2). Based on phase one, phase two dogs (total of 9) received 100 mg/kg (n = 4) or 200 mg/kg (n = 5) orally and blood samples were collected intermittently for 60 h with CSF samples collected intermittently for 24 h. Mebendazole was quantitated in plasma and CSF using high performance liquid chromatography. Median peak plasma concentrations (Cmax) were reached at 7 ± 2 h (100 mg/kg) of 220 ng/mL (81, 283) and at 15 ± 4 h (200 mg/kg) of 147 ng/ml (112, 298). The respective area under the curve (AUC: ng/ml/h) reported as a median was 2,119 (1,876, 3,288) vs. 3,115 (1,559, 4,972). Median plasma concentrations (ng/ml) for 100 vs. 200 mg/kg were 47 (32, 52) vs. 65 (35, 104), respectively. For CSF, the median value for Cmax (at 100 mg/kg vs. 200 mg/kg) was 8 (2, 28) vs. 21 (12, 27) and AUC was 87 (22, 157) vs. 345 (92, 372), respectively. Relative bioavailability in CSF vs. plasma was 4 to 10%. Although several animals demonstrated clinical signs indicative of gastrointestinal upset [i.e., vomiting (n = 2), diarrhea (n = 2), or both (n = 1)], these events were not considered serious. The in vitro IC50 for gliomas can be reached in CSF at 100 mg/kg (n = 1), however a 200 mg/kg dose yielded more consistent concentrations.

Neurologic Causes of Thoracic Limb Lameness.

Although lameness of the thoracic limb typically is due to orthopedic disease, there are several important neurologic conditions that result in lameness. Neurologic diseases cause lameness due to disease of the nerves, nerve roots, spinal cord, or muscles. Common differentials include lateralized intervertebral disc extrusions, caudal cervical spondylomyelopathy (wobbler disease), brachial plexus avulsion, neuritis, and peripheral nerve sheath tumors. Many of these diseases compress or destroy the nerve roots of the cervical intumescence, resulting in non-weight-bearing lameness, or root signature. Advanced diagnostics, such as magnetic resonance imaging, are necessary in these cases to determine the underlying cause.

Pharmacokinetics of multivesicular liposomal encapsulated cytarabine when administered subcutaneously in dogs.

BACKGROUND: Prolonged cytotoxic concentrations of cytarabine (CA) are required for maximum cytotoxicity. DepoCyt is a human liposomal cytarabine (LC) product that lasts longer in plasma and CSF compared with free CA (FC). The use of LC has not been evaluated in dogs. OBJECTIVES: To perform a LC pharmacokinetic (PK) study when administered SC in dogs. ANIMALS: Five healthy female beagles. METHODS: Three-period, 3-treatment, nonblinded, randomized, and crossover design, including a pilot study. LC was administered at 50 mg/m2 SC and FC was administered at 25 and 50 mg/m2 SC and IV. Plasma CA concentrations were measured until 240, 72, and 8 hours after SC LC, SC FC, and IV FC administration, respectively. CA plasma concentrations were quantitated by ultra-high-performance liquid chromatography with mass spectrometry (MS/MS) detection and concentration-time profiles were evaluated by noncompartmental analysis. RESULTS: Subcutaneous LC administration resulted in a maximum plasma concentration of 26.3 to 59.78 ng/mL, time to reach maximum plasma concentration of 2 hours, area under the concentration-time curve to last measurable concentration of 669.3 to 1126 h × ng/mL, and plasma bioavailability (%F) of 19.6% to 31.3%. The PK profiles of FC after SC and IV administration differed when compared with LC. CONCLUSIONS AND CLINICAL IMPORTANCE: In healthy dogs, SC LC administration at 50 mg/m2 results in measurable plasma CA concentrations, is apparently safe and well tolerated, but does not result in prolonged cytotoxic plasma concentrations. Poor absorption of LC prevented establishment of a complete LC PK profile.

Comparative Molecular Life History of Spontaneous Canine and Human Gliomas.

Sporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole-genome, exome, transcriptome, and methylation sequencing of 83 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, TP53 and cell-cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA-methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.

7T MRI Predicts Amelioration of Neurodegeneration in the Brain after AAV Gene Therapy.

GM1 gangliosidosis (GM1) is a fatal neurodegenerative lysosomal storage disease that occurs most commonly in young children, with no effective treatment available. Long-term follow-up of GM1 cats treated by bilateral thalamic and deep cerebellar nuclei (DCN) injection of adeno-associated virus (AAV)-mediated gene therapy has increased lifespan to 8 years of age, compared with an untreated lifespan of ~8 months. Due to risks associated with cerebellar injection in humans, the lateral ventricle was tested as a replacement route to deliver an AAVrh8 vector expressing feline ß-galactosidase (ß-gal), the defective enzyme in GM1. Treatment via the thalamus and lateral ventricle corrected storage, myelination, astrogliosis, and neuronal morphology in areas where ß-gal was effectively delivered. Oligodendrocyte number increased, but only in areas where myelination was corrected. Reduced AAV and ß-gal distribution were noted in the cerebellum with subsequent increases in storage, demyelination, astrogliosis, and neuronal degeneration. These postmortem findings were correlated with endpoint MRI and magnetic resonance spectroscopy (MRS). Compared with the moderate dose with which most cats were treated, a higher AAV dose produced superior survival, currently 6.5 years. Thus, MRI and MRS can predict therapeutic efficacy of AAV gene therapy and non-invasively monitor cellular events within the GM1 brain.

Traumatic atlantoaxial subluxation in dogs: 8 cases (2009-2016).

OBJECTIVE: To demonstrate the utility of advanced imaging in dogs with traumatic atlantoaxial subluxation (TAAS), and to report the presentation, treatment, and outcome for these dogs. DESIGN: Retrospective study (2009-2016). SETTING: University teaching hospital. ANIMALS: Eight dogs diagnosed with TAAS with magnetic resonance imaging (MRI), computed tomography (CT), or both. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Eight dogs met criteria for inclusion. Of these, 6 were male, median age was 4 years (range, 1.5-11 years), and median body weight was 4.9 kg (range, 3.0-25.0 kg). On presentation, 6/8 (75%) dogs were nonambulatory tetraparetic and the most common injury was trauma inflicted by another animal 5/8 (62.5%). Diagnosis of TAAS was made using a combination of imaging modalities including vertebral column radiographs in 7/8 (87.5%) (of which 71.4% were suspicious for TAAS), CT in 7/8 (87.5%), and MRI in 7/8 (87.5%). In 7/8 (87.5%) dogs, CT and/or MRI offered additional information regarding the extent of injuries. Vertebral fractures were identified in 62.5% (5/8) of dogs. The majority of dogs underwent surgical repair (7/8 [87.5%]). The most common complication was aspiration pneumonia (3/8 [37.5%]). All 8 dogs survived to discharge. At the time of discharge, 4/8 (50%) were ambulatory tetraparetic. The 4 dogs that were nonambulatory tetraparetic at discharge progressed to being ambulatory within 2 months of surgery. CONCLUSIONS: Although TAAS is an uncommon occurrence it should be considered a differential for any trauma patient that is presenting with signs of a cranial cervical myelopathy. CT and MRI have been shown to be useful to identify the extent of injuries and to facilitate surgical planning. With appropriate care, these dogs can have an excellent prognosis.

Diagnostic Imaging of Discospondylitis.

Discospondylitis can affect dogs of any age and breed and may be seen in cats. Although radiography remains the gold standard, advanced imaging, such as CT and MRI, has benefits and likely allows earlier diagnosis and identification of concurrent disease. Because discospondylitis may affect multiple disk spaces, imaging of the entire spine should be considered. There is a lengthening list of causative etiologic agents, and successful treatment hinges on correct identification. Image-guided biopsy should be considered in addition to blood and urine cultures and Brucella canis screening and as an alternative to surgical biopsy in some cases.

Adeno-Associated Virus Gene Therapy in a Sheep Model of Tay-Sachs Disease.

Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A (HexA). TSD also occurs in sheep, the only experimental model of TSD that has clinical signs of disease. The natural history of sheep TSD was characterized using serial neurological evaluations, 7 Tesla magnetic resonance imaging, echocardiograms, electrodiagnostics, and cerebrospinal fluid biomarkers. Intracranial gene therapy was also tested using AAVrh8 monocistronic vectors encoding the α-subunit of Hex (TSD α) or a mixture of two vectors encoding both the α and ß subunits separately (TSD α + ß) injected at high (1.3 × 1013 vector genomes) or low (4.2 × 1012 vector genomes) dose. Delay of symptom onset and/or reduction of acquired symptoms were noted in all adeno-associated virus-treated sheep. Postmortem evaluation showed superior HexA and vector genome distribution in the brain of TSD α + ß sheep compared to TSD α sheep, but spinal cord distribution was low in all groups. Isozyme analysis showed superior HexA formation after treatment with both vectors (TSD α + ß), and ganglioside clearance was most widespread in the TSD α + ß high-dose sheep. Microglial activation and proliferation in TSD sheep-most prominent in the cerebrum-were attenuated after gene therapy. This report demonstrates therapeutic efficacy for TSD in the sheep brain, which is on the same order of magnitude as a child's brain.

Fungal Infections of the Central Nervous System in Small Animals: Clinical Features, Diagnosis, and Management.

Small animal mycoses vary geographically. Different clinical presentations are seen in animals with infection of the central nervous system (CNS), including multifocal meningoencephalomyelitis, intracranial lesions that accompany sinonasal lesions, rapidly progressive ventriculitis, or solitary granuloma of the brain or spinal cord. Systemic, nasal, or extraneural clinical signs are common but, especially in granuloma cases, do not always occur. Surgery may have a diagnostic and therapeutic role in CNS granuloma. There have been recent advancements in serology. Fluconazole, voriconazole, and posaconazole cross the blood-brain barrier, but voriconazole is neurotoxic to cats. Liposomal and lipid-encapsulated formulations of amphotericin B are preferred.

Lipidomic Evaluation of Feline Neurologic Disease after AAV Gene Therapy.

GM1 gangliosidosis is a fatal lysosomal disorder, for which there is no effective treatment. Adeno-associated virus (AAV) gene therapy in GM1 cats has resulted in a greater than 6-fold increase in lifespan, with many cats remaining alive at >5.7 years of age, with minimal clinical signs. Glycolipids are the principal storage product in GM1 gangliosidosis whose pathogenic mechanism is not completely understood. Targeted lipidomics analysis was performed to better define disease mechanisms and identify markers of disease progression for upcoming clinical trials in humans. 36 sphingolipids and subspecies associated with ganglioside biosynthesis were tested in the cerebrospinal fluid of untreated GM1 cats at a humane endpoint (∼8 months), AAV-treated GM1 cats (∼5 years old), and normal adult controls. In untreated GM1 cats, significant alterations were noted in 16 sphingolipid species, including gangliosides (GM1 and GM3), lactosylceramides, ceramides, sphingomyelins, monohexosylceramides, and sulfatides. Variable degrees of correction in many lipid metabolites reflected the efficacy of AAV gene therapy. Sphingolipid levels were highly predictive of neurologic disease progression, with 11 metabolites having a coefficient of determination (R2) > 0.75. Also, a specific detergent additive significantly increased the recovery of certain lipid species in cerebrospinal fluid samples. This report demonstrates the methodology and utility of targeted lipidomics to examine the pathophysiology of lipid storage disorders.

Novel Biomarkers of Human GM1 Gangliosidosis Reflect the Clinical Efficacy of Gene Therapy in a Feline Model.

GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers using blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, 7 T MRI, and magnetic resonance spectroscopy in GM1 cats-either untreated or AAV treated for more than 5 years-and compared them to markers in human GM1 patients where possible. Significant alterations were noted in CSF and blood of GM1 humans and cats, with partial or full normalization after gene therapy in cats. Gene therapy improved the rhythmic slowing of electroencephalograms (EEGs) in GM1 cats, a phenomenon present also in GM1 patients, but nonetheless the epileptiform activity persisted. After gene therapy, MR-based analyses revealed remarkable preservation of brain architecture and correction of brain metabolites associated with microgliosis, neuroaxonal loss, and demyelination. Therapeutic benefit of AAV gene therapy in GM1 cats, many of which maintain near-normal function >5 years post-treatment, supports the strong consideration of human clinical trials, for which the biomarkers described herein will be essential for outcome assessment.

IMAGING DIAGNOSIS-MAGNETIC RESONANCE IMAGING FEATURES OF A MULTIFOCAL OLIGODENDROGLIOMA IN THE SPINAL CORD AND BRAIN OF A DOG.

An 8-year-old neutered male Toy Poodle was presented with chronic, progressive tetraparesis, and possible seizures. Magnetic resonance images demonstrated an extensive, T1 and T2 hyperintense contrast enhancing mass in the cervical spinal cord. Three nodules were present on the surface of the thalamus, with enhancement most evident on delayed images. A diagnosis of high-grade oligodendroglioma was confirmed with postmortem histopathology and immunohistochemical labeling. Oligodendroglioma should be considered as a differential for T1 hyperintense intraaxial or intramedullary lesions with contrast enhancement. If enhancement is not visualized on postcontrast images, delayed images may be beneficial.

Use of a frameless computed tomography-guided stereotactic biopsy system for nasal biopsy in five dogs.

CASE DESCRIPTION: 5 dogs (median age, 9 years; median body weight, 31 kg [68.2 lb]) with undefined nasal masses were examined after undergoing CT of the head and nasal biopsy via a rostral rhinoscopic or unaided (blind) approach because histologic results for collected biopsy specimens (inflammatory, necrotic, or hemorrhagic disease) suggested the specimens were nonrepresentative of the underlying disease process identified via CT (aggressive or malignant disease). CLINICAL FINDINGS: Clinical signs at the time dogs were evaluated included open-mouth breathing, sneezing, or unilateral epistaxis. Histologic findings pertaining to the original biopsy specimens were suggestive of benign processes such as inflammation. In an attempt to obtain better representative specimens, a frameless CT-guided stereotactic biopsy system (CTSBS) was used to collect additional biopsy specimens from masses within the nasal and sinus passages of the dogs. The second set of biopsy specimens was histologically evaluated. TREATMENT AND OUTCOME: Histologic evaluation of biopsy specimens collected via the CTSBS revealed results suggestive of malignant neoplasia (specifically, chondrosarcoma, hemangiopericytoma, or undifferentiated sarcoma) for 3 dogs, mild mixed-cell inflammation for 1 dog, and hamartoma for 1 dog. No complications were reported. These findings resulted in a change in treatment recommendations for 3 dogs and confirmed that no additional treatment was required for 1 dog (with hamartoma). For the remaining dog, in which CT findings and clinical history were strongly suggestive of neoplasia, the final diagnosis was rhinitis. CLINICAL RELEVANCE: Biopsy specimens were safely collected from masses within the nasal and sinus passages of dogs by use of a frameless CTSBS, allowing a definitive diagnosis that was unachievable with other biopsy approaches.

Cerebrospinal fluid inflammatory cytokines and chemokines in naturally occurring canine spinal cord injury.

Canine intervertebral disk herniation (IVDH) is a common, naturally occurring form of spinal cord injury (SCI) that is increasingly being used in pre-clinical evaluation of therapies. Although IVDH bears critical similarities to human SCI with respect to lesion morphology, imaging features, and post-SCI treatment, limited data are available concerning secondary injury mechanisms. Here, we characterized cerebrospinal fluid (CSF) cytokines, and chemokines in dogs with acute, surgically treated, thoracolumbar IVDH (n=39) and healthy control dogs (n=21) to investigate early inflammatory events after SCI. A bioplex system was used to measure interleukin (IL)-2, -6, -7, -8, -10, -15, and -18, granulocyte macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), keratinocyte chemoattractant (KC)-like protein, IFN-γ-inducible protein-10, monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor alpha. Cytokine and chemokine concentrations in the CSF of healthy and SCI dogs were compared and, in SCI dogs, were correlated to the duration of SCI, behavioral measures of injury severity at the time of sampling, and neurological outcome 42 days post-SCI as determined by a validated ordinal score. IL-8 concentration was significantly higher in SCI cases than healthy controls (p=0.0013) and was negatively correlated with the duration of SCI (p=0.042). CSF MCP-1 and KC-like protein were positively correlated with CSF microprotein concentration in dogs with SCI (p<0.0001 and p=0.004). CSF MCP-1 concentration was negatively associated with 42-day postinjury outcome (p<0.0001). Taken together, these data indicate that cytokines and chemokines present after SCI in humans and rodent models are associated with SCI pathogenesis in canine IVDH.

Application and machine accuracy of a new frameless computed tomography-guided stereotactic brain biopsy system in dogs.

The purpose of this study was to describe application and machine accuracy for a new computed tomography (CT) guided, frameless, stereotactic brain biopsy system in dogs. Heads from ten canine cadavers were secured to a bite-plate with six attached fiducial markers and imaged using CT. Fiducialized CT images were imported into stereotactic software and spherical phantom lesions between 3.9 and 5.5 mm in diameter were created in six locations. Infrared cameras and reflective markers were used to register fiducials to the reconstructed image set. Coordinates in the X, Y, and Z planes were identified for each lesion center. Iohexol (1.5 µl of 240 mgI/ml) was injected into the center of each lesion and CT scans were repeated. Pre- and postinjection CT images for each cadaver were fused using the system software. Application accuracy was calculated using the center of each phantom lesion and the center of each injected contrast material location. Machine accuracy was calculated using a phantom with known distances between four fixed points in the X, Y, and Z planes. Mean application accuracy in the first 5 cadavers was 4.3 mm (95% confidence interval [CI] 2.9-4.3 mm) and in the second 5 cadavers was 2.9 mm (95% CI 2-3.9 mm). The more superficial lesions were targeted significantly less accurately than the deeper lesions (P = 0.0183). Median machine accuracy was 0.1 mm and the range was 0.1-0.2 mm. Findings supported use of the new biopsy system for canine brain lesions >3.9 mm in diameter.