Microglia-Derived Olfactomedin-like 3 Is a Potent Angiogenic Factor in Primary Mouse Brain Endothelial Cells: A Novel Target for Glioblastoma.

Neoangiogenesis, a hallmark feature of all malignancies, is robust in glioblastoma (GBM). Vascular endothelial growth factor (VEGF) has long been regarded as the primary pro-angiogenic molecule in GBM. However, anti-VEGF therapies have had little clinical efficacy, highlighting the need to explore VEGF-independent mechanisms of neoangiogenesis. Olfactomedin-like 3 (OLFML3), a secreted glycoprotein, is an established proangiogenic factor in many cancers, but its role in GBM neoangiogenesis is unknown. To gain insight into the role of OLFML3 in microglia-mediated angiogenesis, we assessed endothelial cell (EC) viability, migration and differentiation following (1) siRNA knockdown targeting endogenous EC Olfml3 and (2) EC exposure to human recombinant OLFML3 (rhOLFML3; 10 ng/mL, 48 h), and conditioned medium (CM) from isogenic control and Olfml3−/− microglia (48 h). Despite a 70% reduction in Olfml3 mRNA levels, EC angiogenic parameters were not affected. However, exposure to both rhOLFML3 and isogenic control microglial CM increased EC viability (p < 0.01), migration (p < 0.05) and differentiation (p < 0.05). Strikingly, these increases were abolished, or markedly attenuated, following exposure to Olfml3−/− microglial CM despite corresponding increased microglial secretion of VEGF-A (p < 0.0001). Consistent with reports in non-CNS malignancies, we have demonstrated that OLFML3, specifically microglia-derived OLFML3, promotes VEGF-independent angiogenesis in primary brain microvascular ECs and may provide a complementary target to mitigate neovascularization in GBM.

Microglia-Derived Olfactomedin-like 3 Promotes Pro-Tumorigenic Microglial Function and Malignant Features of Glioma Cells.

Under the influence of transforming growth factor-beta (TGFß), glioma-associated microglia produce molecules that promote glioma growth and invasion. Olfactomedin-like 3 (Olfml3), a novel, secreted glycoprotein, is known to promote several non-CNS cancers. While it is a direct TGFß1 target gene in microglia, the role of microglia-derived OLFML3 in glioma progression is unknown. Here, we tested the hypotheses that microglial Olfml3 is integral to the pro-tumorigenic glioma-associated microglia phenotype and promotes glioma cell malignancy. Using an Olfml3 knockout microglial cell line (N9), we demonstrated that Olfml3 is a direct target gene of all TGFß isoforms in murine microglia. Moreover, loss of Olfml3 attenuated TGFß-induced restraint on microglial immune function and production of cytokines that are critical in promoting glioma cell malignancy. Importantly, microglia-derived OLFML3 directly contributes to glioma cell malignancy through increased migration and invasion. While exposure to conditioned medium (CM) from isogenic control microglia pre-treated with TGFß increased mouse glioma cell (GL261) migration and invasion, this effect was abolished with exposure to CM from TGFß-treated Olfml3-/- microglia. Taken together, our data suggest that Olfml3 may serve as a gatekeeper for TGFß-induced microglial gene expression, thereby promoting the pro-tumorigenic microglia phenotype and glioma cell malignancy.

The Effects of Mepivacaine Hydrochloride on Antimicrobial Activity and Mechanical Nociceptive Threshold During Amikacin Sulfate Regional Limb Perfusion in the Horse.

OBJECTIVE: To determine the effect of intravenous regional limb perfusion (IVRLP) with a combination of mepivacaine hydrochloride and amikacin sulfate on synovial fluid amikacin sulfate concentration, antimicrobial activity, and mechanical nociceptive threshold (MNT). STUDY DESIGN: Experimental study. ANIMALS: Healthy adult horses (n=9). METHODS: One IVRLP treatment was randomly administered by cephalic vein of each limb: amikacin alone (1 g amikacin in 60 mL saline) or amikacin with mepivacaine (1 g amikacin and 500 mg mepivacaine in 60 mL saline). Opposite treatments were repeated after a 24 hour wash-out period. Amikacin concentration and antimicrobial activity were determined for synovial fluid from middle carpal joints at tourniquet removal and 30 minutes following. Zone of inhibition was determined for Staphylococcus aureus and Escherichia coli. MNT was determined at 3 dorsal metacarpal locations prior to and after sedation, after Esmarch tourniquet application, and 30 minutes after IVRLP prior to and after tourniquet removal. RESULTS: Two limbs from each treatment group were removed because of undetectable amikacin concentrations for a total of 14 data sets analyzed. Synovial fluid amikacin concentrations and zone of inhibition were not significantly different between treatments at any time point. MNT were significantly increased 30 minutes after IVRLP prior to and following tourniquet removal using amikacin and mepivacaine (median, range; 40.0 µg/mL, 38.7-40.0 and 40.0, 25.8-40.0, respectively) compared to amikacin alone (19.5 µg/mL, 18.7-25.6 and 15.3, 13.2-20.5, respectively). CONCLUSION: Addition of mepivacaine to amikacin for IVRLP in the horse as a means of providing analgesia without decreasing antimicrobial activity.

Effect of topical naltrexone 0.3% on corneal sensitivity and tear parameters in normal brachycephalic dogs.

OBJECTIVE: To determine the effect of topical naltrexone 0.3% on tear production, corneal sensitivity, and tear film stability in normal brachycephalic dogs. ANIMALS STUDIED: Twenty-two normal brachycephalic dogs. PROCEDURES: Measurements of tear production (Schirmer tear test I and II), intraocular pressure (IOP), central corneal sensitivity (CS), and tear film breakup time (TFBUT) were collected at time 0, 1, and 24 h after administration of either naltrexone (NTX) 0.3% or placebo (SV). Naltrexone or SV was then administered once daily for 1 week, and the above measurements were repeated at 7 days, then again 7 days after discontinuing medication. Owners scored the degree of comfort, redness, rubbing, squinting, and tearing. Serum was collected at time 0, 1, 24 h, and 7 days to determine systemic concentrations. RESULTS: Owners reported no significant change in the degree of comfort, redness, rubbing, squinting, or tearing. Naltrexone was detected in serum of all treated dogs 1-h postadministration (average: 908 pg/mL, range: 319-1570 pg/mL) and in two dogs at the 1-week time point. Naltrexone was not detected at the 24-h time point. There was no significant effect of NTX on STT1, STT2, IOP, CS, or TFBUT. CONCLUSIONS: Naltrexone 0.3% is well tolerated and safe when applied topically to the eye once daily. Naltrexone 0.3% did not show any significant effects on corneal parameters as measured in this study. At once, daily dosing NTX is systemically absorbed; however, the degree of systemic absorption is not likely to be clinically significant.

Cardiac myosin inhibitor, CK-586, minimally reduces systolic function and ameliorates obstruction in feline hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) remains the most common cardiomyopathy in humans and cats with few preclinical pharmacologic interventional studies. Small-molecule sarcomere inhibitors are promising novel therapeutics for the management of obstructive HCM (oHCM) patients and have shown efficacy in left ventricular outflow tract obstruction (LVOTO) relief. The objective of this study was to explore the 6-, 24-, and 48-hour (h) pharmacodynamic effects of the cardiac myosin inhibitor, CK-586, in six purpose-bred cats with naturally occurring oHCM. A blinded, randomized, five-treatment group, crossover preclinical trial was conducted to assess the pharmacodynamic effects of CK-586 in this oHCM model. Dose assessments and select echocardiographic variables were assessed five times over a 48-h period. Treatment with oral CK-586 safely ameliorated LVOTO in oHCM cats. CK-586 treatment dose-dependently eliminated obstruction (reduced LVOTOmaxPG), increased measures of systolic chamber size (LVIDs Sx), and decreased select measures of heart function (LV FS% and LV EF%) in the absence of impact on heart rate. At all tested doses, a single oral CK-586 dose resulted in improved or resolved LVOTO with well-tolerated, dose-dependent, reductions in LV systolic function. The results from this study pave the way for the potential use of CK-586 in both the veterinary and human clinical setting.

Pharmacokinetics of a Single Transdermal Dose of Mirtazapine in Rhesus Macaques (Macaca mulatta).

Decreased appetite is a common clinical problem in captive rhesus macaques (Macaca mulatta). Mirtazapine, a tetracyclic antidepressant originally developed for humans, has shown promise as a safe and effective promoter of weight gain and appetite in several veterinary species including rhesus and cynomolgus macaques. Although mirtazapine is available as oral formulations, transdermal delivery in macaques with reduced appetite would allow quick, painless, topical application. Here we describe the pharmacokinetics of a single application of a widely available veterinary transdermal mirtazapine formulation in 6 rhesus macaques. A dose of 0.5 mg/kg of transdermal mirtazapine ointment that has proven to be effective in rhesus was applied to the caudal pinnae of 3 female and 3 male young adult macaques. Serum was collected at 0, 0.5, 1, 3, 6, 8, 12, 24, 36, 48, and 72 h after administration. Our data indicate transdermal mirtazapine is absorbed at a lower level in rhesus as compared with published values in domestic cats (rhesus peak serum concentration: 1.2 ± 0.3 ng/mL), while drug half-life is longer than that reported in cats (rhesus: 33 ± 7 h). Mirtazapine reaches peak plasma concentrations in rhesus at 16 ± 10 h after administration; our model indicates that up to 5 d of serial dosing may be necessary to reach steady state. Our preliminary data also suggest that sex differences may contribute to efficacy and/or indicate sex-based differences, as male macaques reached Tmax more quickly than females (19 ± 2 h in females and 8 ± 3 h in males) and showed higher variation in half-life (33 ± 4 h in females and 34 ± 11 h in males). While previous work indicates clinical efficacy of the 0.5-mg/kg dosage in macaques, further investigation is warranted to determine if rhesus may benefit from higher recommended doses than companion animal species.

Vinblastine pharmacokinetics in mouse, dog, and human in the context of a physiologically based model incorporating tissue-specific drug binding, transport, and metabolism.

Vinblastine (VBL) is a vinca alkaloid-class cytotoxic chemotherapeutic that causes microtubule disruption and is typically used to treat hematologic malignancies. VBL is characterized by a narrow therapeutic index, with key dose-limiting toxicities being myelosuppression and neurotoxicity. Pharmacokinetics (PK) of VBL is primarily driven by ABCB1-mediated efflux and CYP3A4 metabolism, creating potential for drug-drug interaction. To characterize sources of variability in VBL PK, we developed a physiologically based pharmacokinetic (PBPK) model in Mdr1a/b(-/-) knockout and wild-type mice by incorporating key drivers of PK, including ABCB1 efflux, CYP3A4 metabolism, and tissue-specific tubulin binding, and scaled this model to accurately simulate VBL PK in humans and pet dogs. To investigate the capability of the model to capture interindividual variability in clinical data, virtual populations of humans and pet dogs were generated through Monte Carlo simulation of physiologic and biochemical parameters and compared to the clinical PK data. This model provides a foundation for predictive modeling of VBL PK. The base PBPK model can be further improved with supplemental experimental data identifying drug-drug interactions, ABCB1 polymorphisms and expression, and other sources of physiologic or metabolic variability.

A systems biology approach to identify molecular pathways altered by HDAC inhibition in osteosarcoma.

Osteosarcoma (OS) is the most common primary tumor in humans and dogs affecting the skeleton, and spontaneously occurring OS in dogs serves as an extremely useful model. Unacceptable toxicities using current treatment protocols prevent further dose-intensification from being a viable option to improve patient survival and thus, novel treatment strategies must be developed. Histone deacetylase inhibitors (HDACi) have recently emerged as a promising class of therapeutics demonstrating an ability to enhance the anti-tumor activity of traditional chemotherapeutics. To date, gene expression analysis of OS cell lines treated with HDACi has not been reported, and evaluation of the resultant gene expression changes may provide insight into the mechanisms that lead to success of HDACi. Canine OS cells, treated with a clinically relevant concentration of the HDACi valproic acid (VPA), were used for expression analysis on the Affymetrix canine v2.0 genechip. Differentially expressed genes were grouped into pathways based upon functional annotation; pathway analysis was performed with MetaCore and Ingenuity Pathways Analysis software. Validation of microarray results was performed by a combination of qRT-PCR and functional/biochemical assays revealing oxidative phosphorylation, cytoskeleton remodeling, cell cycle, and ubiquitin-proteasome among those pathways most affected by HDACi. The mitomycin C-bioactivating enzyme NQ01 also demonstrated upregulation following VPA treatment, leading to synergistic reductions in cell viability. These results provide a better understanding of the mechanisms by which HDACi exert their effect in OS, and have the potential to identify biomarkers that may serve as novel targets and/or predictors of response to HDACi-containing combination therapies in OS.

Pharmacokinetics of isavuconazole in healthy cats after oral and intravenous administration.

BACKGROUND: Isavuconazole is a triazole antifungal drug that has shown good efficacy in human patients. Absorption and pharmacokinetics have not been evaluated in cats. OBJECTIVES: To determine the pharmacokinetics of isavuconazole in cats given a single IV or PO dose. ANIMALS: Eight healthy, adult research cats. METHODS: Four cats received 100 mg capsules of isavuconazole PO. Four cats received 5 mg/kg isavuconazole solution IV. Serum was collected at predetermined intervals for analysis using ultra-high performance liquid chromatography-tandem mass spectrometry. Data were analyzed using a 2-compartment uniform weighting pharmacokinetic analysis with lag time for PO administration and a 2 compartment, 1/y2 weighting for IV administration. Predicted 24 and 48-hour dosing intervals of 100 mg isavuconazole administered PO were modeled and in vitro plasma protein binding was assessed. RESULTS: Both PO and IV drug administration resulted in high serum concentrations. Intravenous and PO formulations of isavuconazole appear to be able to be used interchangeably. Peak serum isavuconazole concentrations occurred 5 ± 3.8 hours after PO administration with an elimination rate half-life of 66.2 ± 55.3 hours. Intersubject variability was apparent in both the PO and IV groups. Two cats vomited 6 to 8 hours after PO administration. No adverse effects were observed in the IV group. Oral bioavailability was estimated to be approximately 88%. Serum protein binding was calculated to be approximately 99.0% ± 0.03%. CONCLUSIONS AND CLINICAL IMPORTANCE: Isavuconazole might prove to be useful in cats with fungal disease given its favorable pharmacokinetics. Additional studies on safety, efficacy, and tolerability of long-term isavuconazole use are needed.

Population pharmacokinetics identifies rapid gastrointestinal absorption and plasma clearance of oral chlorambucil administered to cats with indolent lymphoproliferative malignancies.

OBJECTIVE: To establish the pharmacokinetics of a single 2-mg oral dose of chlorambucil in cats with indolent lymphoproliferative malignancies. ANIMALS: 24 client-owned cats. PROCEDURES: Cats were assigned to 1 of 4 groups, with each group having a total of 3 sample collection time points over 12 hours after receiving a single 2-mg oral dose of chlorambucil. Each time point combined to generate 6 full patient plasma chlorambucil concentration-time curves from the 24 cats. Chlorambucil treatment was continued every other day and a single, variably timed sample collection was obtained on day 14. Population parameter estimates were obtained by nonlinear mixed-effects modeling. Covariates investigated included age, sex, baseline serum cobalamin, study location, weight, and body condition score. RESULTS: Chlorambucil administered orally to cats was found to have a peak plasma concentration of approximately 170 ng/mL (SE, 31.1 ng/mL), percent coefficient of variation (%CV) of 18.4% within 15 minutes, and a terminal half-life of 1.8 hours (SE, 0.21 hour; %CV, 12.4). At the 4-hour mark, a smaller secondary peak in plasma chlorambucil was found. Day 14 samples were similar to those of the initial dose. No covariates showed a significant effect in the population model. CLINICAL RELEVANCE: In these cats, chlorambucil at a 2-mg dose administered every other day undergoes rapid gastrointestinal absorption and plasma clearance with no drug accumulation between doses. These data are critical to inform future work investigating the association of chlorambucil drug exposure with adverse events and outcome of cats with lymphoproliferative diseases.

Prevalence and Clinicopathologic Features of Canine Metastatic Melanoma Involving the Central Nervous System: A Retrospective Analysis and Comparative Review.

Background: Central nervous system (CNS) involvement is the leading cause of death in malignant melanoma. Rodent models, while vital to mechanistic investigation, have had limited success identifying effective therapies for melanoma brain metastases. The companion dog with de novo melanoma is a promising complementary model for developmental therapeutic investigation, as these tumors occur in an immunologically outbred host that has shared environmental exposures with humans. However, relatively little is known regarding the prevalence and clinicopathological features of canine melanoma metastasis to the CNS. To further validate the dog as an appropriate model for human metastatic melanoma, the aims of this study were to determine the rate of CNS metastasis and associated clinicopathologic features in canine malignant melanoma. Methods: Medical records of dogs diagnosed with malignant melanoma from 1985-2019 at the University of California Davis Veterinary Medical Teaching Hospital were assessed retrospectively. Clinicopathologic features were compared between dogs with CNS metastasis (CNS+) and dogs without CNS metastasis (CNS-). Site of CNS involvement and associated neurological signs were analyzed via Wilcoxon-Mann-Whitney rank sum and Fisher's exact tests. Survival data were analyzed via Kaplan-Meier estimates. Results: CNS metastasis was identified in 38% of dogs in this study (20/53). The oral cavity was the most common site of primary melanoma in both groups [CNS+: n=12 (60%) vs. CNS-: n=22 (67%); p>0.99]. The total burden of metastatic disease was higher in the CNS+ group (CNS+: 4, 95% CI 3-5 vs. CNS-: 3, 95% CI 1-3; p<0.001). The cerebrum was the most common site of CNS metastasis (n=15, 75%) and seizures were the most observed neurological sign (n=9, 64%). There was no difference in overall survival between CNS+ and CNS- groups. However, the median survival time following onset of neurological signs was 9.5 days (95% CI 1-43), with 5 dogs euthanized within 24 hours of the onset of neurological signs. Conclusions: Canine and human MM patients share similar rates of CNS metastasis and clinical presentation. This study will guide clinical management of canines with malignant melanoma and inform future studies using dogs with spontaneously occurring melanoma as a preclinical model for human melanoma brain metastases.

Effect of standard-dose and high-dose pimobendan on select indices of renal and cardiac function in dogs with American College of Veterinary Internal Medicine stage B2 myxomatous mitral valve disease.

BACKGROUND: Pimobendan might have favorable effects on renal function but this has not been well-studied in dogs with myxomatous mitral valve disease (MMVD). OBJECTIVES: Determine the effects of standard-dose (SD_pimo) and high-dose pimobendan (HD_pimo) on glomerular filtration rate (GFR) and cardiac size and function in dogs with preclinical MMVD. ANIMALS: Thirty nonazotemic dogs with stage B2 MMVD. METHODS: Prospective, randomized, double-blinded, placebo-controlled clinical study. Dogs had an echocardiographic examination, assessment of GFR (iohexol clearance), N-terminal probrain natriuretic peptide (NT-proBNP), and quality of life (QOL) score at baseline and 7 to 10 days after placebo (n = 6), SD_pimo 0.2 to 0.3 mg/kg q12 (n = 12), or HD_pimo 0.5 to 0.6 mg/kg q12h (n = 12). RESULTS: No significant differences in GFR or QOL scores were detected between groups (P ≥ .07). After HD_pimo, the mean [SD] percent change of NT_proBNP (-46.1 [20.2]%), left atrial volume (LAV; -27.1 [16.9]%), left ventricular end-diastolic volume (EDV; -21.8 [15.0]%), and end-systolic volume (ESV; -55.0 [20.7]%) were significantly different (P ≤ .004) from placebo (0.5 [19.9]%, 1.3 [15.6]%, -0.2 [8.2]%, -7.3 [35.6]%, respectively) but not the percent change after SD_pimo (-36.6 [16.1]%, -22.7 [14.9]%, -16.7 [12.5]%, -41.6 [14.8]%, respectively; P > .05). After SD_pimo, percent change of NT_proBNP, LAV, EDV, and ESV were significantly different from placebo (P < .05). CONCLUSIONS AND CLINICAL IMPORTANCE: Results suggest that pimobendan (SD_pimo or HD_pimo) might not affect renal function in nonazotemic dogs with stage B2 MMVD. High-dose pimobendan did not demonstrate advantages over SD_pimo within the constraints of our study.

Serum concentrations of gabapentin in cats with chronic kidney disease.

OBJECTIVES: The purpose of this study was to assess serum concentrations of gabapentin in cats with chronic kidney disease (CKD) vs clinically healthy cats. METHODS: Five healthy cats were enrolled in a pharmacokinetic study. A single 20 mg/kg dose of gabapentin was administered orally and blood was obtained at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 8, 12, 24 and 36 h via a jugular catheter. Serum gabapentin concentrations were measured using liquid chromatography coupled to tandem mass spectrometry. Non-compartmental pharmacokinetic analysis was performed. The same five healthy cats plus 25 cats with stable International Renal Interest Society stage 2 (n = 14) and 3 (n = 11) CKD were enrolled in a limited sampling study. Cats in both groups received a single 10 mg/kg dose of gabapentin, and serum gabapentin concentrations and compliance scores were obtained 3 and 8 h post-administration. RESULTS: Cats with CKD had significantly higher dose-normalized serum gabapentin concentrations than normal cats at 3 h (P = 0.0012 CKD vs normal 10 mg/kg; P = 0.008 CKD vs normal 20 mg/kg) and 8 h (P <0.0001 CKD vs normal 10 mg/kg; P <0.0001 CKD vs normal 20 mg/kg). Both 3 and 8 h dose-normalized serum gabapentin concentrations were significantly correlated with serum creatinine (3 h: P = 0.03, r = 0.39; 8 h: P = 0.001, r = 0.57) and symmetric dimethylarginine (3 h: P = 0.03, r = 0.41; 8 h: P = 0.007, r = 0.48). There was a significant correlation between 3 h serum gabapentin concentrations and compliance scores (P = 0.0002, r = 0.68). CONCLUSIONS AND RELEVANCE: Cats with CKD that received 10 mg/kg of gabapentin had significantly higher dose-normalized serum concentrations than normal cats that received 20 mg/kg, supporting the need to dose-reduce in this patient population.

Inhaled recombinant human IL-15 in dogs with naturally occurring pulmonary metastases from osteosarcoma or melanoma: a phase 1 study of clinical activity and correlates of response.

PURPOSE: Although recombinant human interleukin-15 (rhIL-15) has generated much excitement as an immunotherapeutic agent for cancer, activity in human clinical trials has been modest to date, in part due to the risks of toxicity with significant dose escalation. Since pulmonary metastases are a major site of distant failure in human and dog cancers, we sought to investigate inhaled rhIL-15 in dogs with naturally occurring lung metastases from osteosarcoma (OSA) or melanoma. We hypothesized a favorable benefit/risk profile given the concentrated delivery to the lungs with decreased systemic exposure. EXPERIMENTAL DESIGN: We performed a phase I trial of inhaled rhIL-15 in dogs with gross pulmonary metastases using a traditional 3+3 cohort design. A starting dose of 10 µg twice daily × 14 days was used based on human, non-human primate, and murine studies. Safety, dose-limiting toxicities (DLT), and maximum tolerated dose (MTD) were the primary objectives, while response rates, progression-free and overall survival (OS), and pharmacokinetic and immune correlative analyses were secondary. RESULTS: From October 2018 to December 2020, we enrolled 21 dogs with 18 dogs reaching the 28-day response assessment to be evaluable. At dose level 5 (70 µg), we observed two DLTs, thereby establishing 50 µg twice daily × 14 days as the MTD and recommended phase 2 dose. Among 18 evaluable dogs, we observed one complete response >1 year, one partial response with resolution of multiple target lesions, and five stable disease for an overall clinical benefit rate of 39%. Plasma rhIL-15 quantitation revealed detectable and sustained rhIL-15 concentrations between 1-hour and 6 hour postnebulization. Decreased pretreatment lymphocyte counts were significantly associated with clinical benefit. Cytotoxicity assays of banked peripheral blood mononuclear cells revealed significant increases in peak cytotoxicity against canine melanoma and OSA targets that correlated with OS. CONCLUSIONS: In this first-in-dog clinical trial of inhaled rhIL-15 in dogs with advanced metastatic disease, we observed promising clinical activity when administered as a monotherapy for only 14 days. These data have significant clinical and biological implications for both dogs and humans with refractory lung metastases and support exploration of combinatorial therapies using inhaled rhIL-15.

The interaction between RUNX2 and core binding factor beta as a potential therapeutic target in canine osteosarcoma.

Osteosarcoma remains the most common primary bone tumour in dogs with half of affected dogs unable to survive 1 year beyond diagnosis. New therapeutic options are needed to improve outcomes for this disease. Recent investigations into potential therapeutic targets have focused on cell surface molecules with little clear therapeutic benefit. Transcription factors and protein interactions represent underdeveloped areas of therapeutic drug development. We have utilized allosteric inhibitors of the core binding factor transcriptional complex, comprised of core binding factor beta (CBFß) and RUNX2, in four canine osteosarcoma cell lines Active inhibitor compounds demonstrate anti-tumour activities with concentrations demonstrated to be achievable in vivo while an inactive, structural analogue has no activity. We show that CBFß inhibitors are capable of inducing apoptosis, inhibiting clonogenic cell growth, altering cell cycle progression and impeding migration and invasion in a cell line-dependent manner. These effects coincide with a reduced interaction between RUNX2 and CBFß and alterations in expression of RUNX2 target genes. We also show that addition of CBFß inhibitors to the commonly used cytotoxic chemotherapeutic drugs doxorubicin and carboplatin leads to additive and/or synergistic anti-proliferative effects in canine osteosarcoma cell lines. Taken together, we have identified the interaction between components of the core binding factor transcriptional complex, RUNX2 and CBFß, as a potential novel therapeutic target in canine osteosarcoma and provide justification for further investigations into the anti-tumour activities we describe here.

Simultaneous absolute quantitation of ATP-binding cassette transporters in normal dog tissues by signature peptide analysis using a LC/MS/MS method.

Membrane transport proteins are fundamental components of blood-tissue barriers and affect the absorption, distribution and elimination, and interactions of many of the drugs commonly used in veterinary medicine. A quantitative, simultaneous measurement of these proteins across dog tissues is not currently available, nor is it possible with current immune-based assays such as western blot. In the present study, we aimed to develop a sensitive and specific liquid chromatography tandem-mass spectrometry (LC/MS/MS) based quantitation method that can simultaneously quantitate 14 ATP-binding cassette transporters. We applied this method to a panel of normal canine tissues and compared the LC/MS/MS results with relative messenger RNA (mRNA) abundance using quantitative real-time polymerase chain reaction (qRT-PCR). Our LC/MS/MS method is sensitive, with lower limits of quantitation ranging from 5 to 10 fmol/µg of protein. We were able to detect and/or quantitate each of the 14 transporters in at least one normal dog tissue. Relative protein and mRNA abundance within tissues did not demonstrate a significant correlation in all cases. The results presented here will provide for more accurate predictions of drug movement in dogs through incorporation into physiologically based pharmacokinetic (PBPK) models; the method described here has wide applicability to the quantitation of virtually any proteins of interest in biologic samples where validated canine antibodies do not exist.

Doxorubicin area under the curve is an important predictor of neutropenia in dogs with naturally occurring cancers.

Doxorubicin (DOX) area-under-the-curve (AUC) was calculated for 40 dogs with spontaneously occurring cancers using a previously validated limited-sampling approach. All dogs were administered a dose of 30 mg/m2 by intravenous infusion and serum samples were collected at 5, 45 and 60 minutes post-infusion. DOX and its major metabolite, doxorubicinol (doxol), were quantified in serum samples using high-performance liquid chromatography tandem-mass spectrometry. Wide interpatient variability was observed in the predicted DOX AUC with a coefficient of variation of 34%. A significant relationship was found between DOX AUC and absolute white blood cell count (P = 0.003), absolute neutrophil count (ANC; P = 0.002) and surviving fraction of neutrophils (P = 0.03) approximately 1 week after dosing (nadir). No changes in other hematologic parameters (red blood cells, platelets, lymphocytes, haemoglobin) were found to correlate with DOX AUC. The absolute dose (mg) and the dose per unit body weight (mg/kg) were not significantly correlated with nadir ANC. No relationships were found between maximum serum doxol concentration and myelosuppression. Baseline ANC was also significantly correlated to nadir ANC and a model was constructed using baseline ANC and DOX AUC that significantly described the nadir ANC. These findings demonstrate the important relationship between systemic DOX exposure and degree of neutropenia in dogs, and suggest a potential for individualized, pharmacokinetically-guided DOX dosing in dogs.

Pharmacokinetics of cannabidiol administered by 3 delivery methods at 2 different dosages to healthy dogs.

The purpose of this study was to determine the pharmacokinetics of cannabidiol (CBD) in healthy dogs. Thirty, healthy research dogs were assigned to receive 1 of 3 formulations (oral microencapsulated oil beads, oral CBD-infused oil, or CBD-infused transdermal cream), at a dose of 75 mg or 150 mg q12h for 6 wk. Serial cannabidiol plasma concentrations were measured over the first 12 h and repeated at 2, 4, and 6 wk. Higher systemic exposures were observed with the oral CBD-infused oil formulation and the half-life after a 75-mg and 150-mg dose was 199.7 ± 55.9 and 127.5 ± 32.2 min, respectively. Exposure is dose-proportional and the oral CBD-infused oil provides the most favorable pharmacokinetic profile.

Le but de la présente étude était de déterminer la pharmacocinétique du cannbidiol (CBD) chez des chiens en santé. Trente chiens de recherche en santé ont été assignés à recevoir une des trois formulations (de l'huile micro-encapsulé dans des billes par voie orale, de l'huile infusé de CBD par voie orale, ou une crème infusée de CBD par voie transdermique), à une dose de 75 mg ou 150 mg q12h pendant 6 semaines. Les concentrations plasmatiques de cannabidiol ont été mesurées pendant les 12 premières heures et répétées après 2, 4 et 6 semaines. Les expositions systémiques les plus élevées ont été observées avec la formulation d'huile infusé de CBD administrée par voie orale et la demi-vie après une dose de 75 mg et de 150 mg était de 199,7 ± 55,9 et 127,5 ± 32,2 min, respectivement. L'exposition est proportionnelle à la dose et l'huile infusée de CBD par voie orale fournie le profile pharmacocinétique le plus favorable.(Traduit par Docteur Serge Messier).

A genetically engineered microRNA-34a prodrug demonstrates anti-tumor activity in a canine model of osteosarcoma.

Osteosarcoma (OSA) represents the most common primary bone tumor in humans and pet dogs. Little progress has been made with regard to viable treatment options in the past three decades and patients presenting with metastatic disease continue to have a poor prognosis. Recent mouse studies have suggested that microRNA-34a (miR-34a) may have anti-tumor activities in human OSA models. Due to the conservation of microRNA across species, we hypothesized that a bioengineered miR-34a prodrug (tRNA/miR-34a) would have similar effects in canine OSA, providing a valuable preclinical model for development of this therapeutic modality. Using a panel of canine OSA cell lines, we found that tRNA/miR-34a reduced viability, clonogenic growth, and migration and invasion while increasing tumor cell apoptosis. Furthermore, canine OSA cells successfully process the tRNA/miR-34a into mature miR-34a which reduces expression of target proteins such as platelet derived growth factor receptor alpha (PDGFRα), Notch1 and vascular endothelial growth factor (VEGF). Additionally, our subcutaneous OSA xenograft model demonstrated in vivo tumor growth delay, increased necrosis and apoptosis by tRNA/miR-34a, and decreased cellular proliferation ability. Taken together, these data support that this novel microRNA-based therapy may possess clinical utility in a spontaneously-occurring large animal model of OSA, which can then serve to inform the clinical development of this therapy for human OSA patients.

Preliminary pharmacokinetics of intravenous and subcutaneous dolasetron and pharmacodynamics of subcutaneous dolasetron in healthy cats.

Objectives The objectives were to evaluate the pharmacokinetics (PK) of subcutaneous (SC) and intravenous (IV) dolasetron and the pharmacodynamics (PD) of SC dolasetron in healthy cats. Methods Five cats with unremarkable complete blood count, serum biochemistry and urinalyses were utilized. In the PK study, cats received 0.8 mg/kg SC and IV dolasetron in a crossover format. Serum samples were obtained via a jugular catheter at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36 and 48 h after the administration of dolasetron. Dolasetron and the active metabolite hydrodolasetron were measured using liquid chromatography/tandem mass spectrometry. Non-compartmental PK analysis was performed. In the PD study, SC dolasetron (0.8 mg/kg and 1.0 mg/kg) and saline were administered 30 mins prior to administration of 0.44 mg/kg intramuscular xylazine in a randomized three-way crossover. Number of emetic events, lip licks, time to onset of emesis and visual nausea score were scored by a blinded observer. Results In the PK study, dolasetron was quickly metabolized to the active metabolite hydrodolasetron, limiting assessment of dolasetron PK parameters. Median (range) PK parameters for IV hydrodolasetron were as follows: maximum serum concentration (Cmax) 116 ng/ml (69-316 ng/ml), time to maximum concentration (Tmax) 0.5 h (0.3-0.5 h), half-life 3.3 h (2.9-7.2 h) and area under the curve until the last measurable concentration (AUClast) 323 h/ng/ml (138-454 h/ng/ml). Median (range) PK parameters for SC hydrodolasetron were as follows: Cmax 67.9 ng/ml (60.4-117 ng/ml), Tmax 0.5 h (0.5-1.0 h), half-life 3.8 h (2.9-5.3 h) and AUClast 437 h/ng/ml (221.5-621.8 h/ng/ml). There was no significant difference in exposure to hydrodolasetron between the routes of administration. With regard to PD, when dolasetron was administered prior to xylazine, there was no significant difference in the mean number of emetic events, lip licks, time to onset of emesis or visual nausea score when compared with saline. Conclusions and relevance Administration of 0.8 mg/kg dolasetron does not maintain serum concentrations of active metabolite for 24 h. Administration of dolasetron at 0.8 mg/kg and 1 mg/kg did not prevent xylazine-induced vomiting. Additional feline dose studies are needed to determine if a higher dose is efficacious.