Safety evaluation of combination CCNU and continuous toceranib phosphate (Palladia®) in tumour‐bearing dogs: a phase I dose‐finding study

While maintaining a standard toceranib dosage [2.75 mg kg^?1, PO, every other day (EOD)], three dose‐escalating CCNU cohorts up to and including 60 mg m^?2, PO, q3wk, were completed. The dose‐limiting toxicities (DLT) for the combination were neutropenia and the maximum tolerated dose (MTD) for CCNU when given with continuous toceranib was determined to be 50 mg m^?2, q3wk. While activity is not a primary objective of phase I trials, we observed one complete (lymphoma) and four partial responses (lymphoma, sarcoma, undifferentiated carcinoma and prostatic carcinoma) and two dogs experienced stable disease for >6 weeks [gastric adenocarcinoma and metastatic multilobulated osteochondrosarcoma (MLO)] for an objective response rate of 38.4% and a biological response rate of 53.8%. Concurrent continuous toceranib (2.75 mg kg^?1, EOD) and pulse dose CCNU (50 mg m^?2, q3wk) was well tolerated. Phase II effectiveness and phase III prospective randomized trials should further interrogate the potential activity of this combination.     

Investigation of thyroid function in dogs treated with the tyrosine kinase inhibitor toceranib

Tyrosine kinase inhibitors are widely utilized in veterinary oncology for the treatment of mast cell and solid tumours. In man, these drugs are associated with thyroid dysfunction: however, to date only one study has investigated this in dogs. The aim of this study was to prospectively assess thyroid function in a group of dogs with cancer receiving toceranib. Thirty‐four dogs were prospectively enrolled at two referral hospitals into two groups; those receiving toceranib with prednisolone and those receiving toceranib alone. Total thyroxine (TT4) and thyroid stimulating hormone (TSH) was monitored at regular time points during treatment. Follow‐up data was available for 19 dogs. Overall, 12 incidences of elevated TSH occurred but none of these dogs had concurrent low TT4 concentrations. There was a significant difference in median TSH at week six compared with baseline. Hypothyroidism was not diagnosed in any patient during the study period. Patient drop‐out was higher than anticipated which prevented the assessment of longer term toceranib administration on thyroid function. Toceranib therapy was not associated with hypothyroidism in this study but did result in elevations in TSH which confirms what has been previously reported. Toceranib should be considered to cause thyroid dysfunction in dogs and monitoring is advised.     

Safety evaluation of combination carboplatin and toceranib phosphate (Palladia) in tumour‐bearing dogs: A phase I dose finding study

Combining conventional cytotoxic maximum tolerated dose (MTD) chemotherapy with low‐dose metronomic and/or anti‐angiogenic agents is a exciting area of oncologic research. The objective of this study was to establish the MTD, safety and adverse event (AE) profile of 1 such drug combination. This prospective phase I dose‐finding clinical trial assumed an open‐label 3 + 3 cohort design. Client‐owned dogs with 1 or more cytologically and/or histologically confirmed and macroscopically measurable, naive or recurrent, malignant tumours, were enrolled. No preference for tumour histology, grade or stage was expressed. Toceranib was administered at a dose of 2.75 mg kg^?1 by mouth (PO) every other day (EOD), and carboplatin administered intravenously (IV) every 21 days at a starting dose of 200 mg m^?2. A total of 25% dose escalation was proposed for carboplatin, to a maximum of 300 mg m^?2. AEs were graded according to the Veterinary Cooperative Oncology Group's common terminology criteria for AEs (VCOG‐CTCAE). Grade 3 haematologic or gastrointestinal AEs were nominated dose‐limiting. Response to therapy was evaluated according to the VCOG's revised RECIST criteria. Eleven dogs were enrolled. Tumour histologies included sinonasal carcinoma, osteosarcoma, thyroid carcinoma, melanoma and apocrine gland anal sac adenocarcinoma. MTDs of carboplatin and toceranib were identified as 200 mg m^?2 IV every 21 days and approximately 2.75 mg kg^?1 PO EOD, respectively. The dose‐limiting toxicity was neutropenia. Two dogs experienced a partial response, and 6 maintained stable disease. Combination carboplatin and toceranib chemotherapy was well‐tolerated. Clinical benefit was observed in most cases. This protocol warrants further investigation in phase II/III trials.     

In vitro and in vivo effects of toceranib phosphate on canine osteosarcoma cell lines and xenograft orthotopic models

Canine osteosarcoma (OSA) is the most common primary malignant bone tumour in dogs, and it has a high metastatic rate and poor prognosis. Toceranib phosphate (TOC; Palladia, Zoetis) is a veterinary tyrosine kinase inhibitor that selectively inhibits VEGFR‐2, PDGFRs and c‐Kit, but its efficacy is not yet fully understood in the treatment of canine OSA. Here, we evaluated the functional effects of TOC on six OSA cell lines by transwell, wound healing and colony formation assays. Subsequently, two cell lines (Wall and Penny) were selected and were inoculated in mice by intrafemoral injection to develop an orthotopic xenograft model of canine OSA. For each cell line, 30 mice were xenografted; half of them were used as controls, and the other half were treated with TOC at 40 mg/kg body weight for 20 days. TOC inhibited cell growth of all cell lines, but reduced invasion and migration was only observed in Penny and Wall cell lines. In mice engrafted with Penny cells and subjected to TOC treatment, decreased tumour growth was observed, and PDGFRs and c‐Kit mRNA were downregulated. Immunohistochemical analyses demonstrated a significant reduction of Ki67 staining in treated mice when compared to controls. The results obtained here demonstrate that TOC is able to slightly inhibit cell growth in vitro, while its effect is evident only in a Penny cell xenograft model, in which TOC significantly reduced tumour size and the Ki67 index without modifying apoptosis markers.     

Retrospective evaluation of canine heart base tumours treated with toceranib phosphate (Palladia): 2011‐2018

Heart base tumours (HBT) occur commonly in older, brachycephalic dogs. A presumptive diagnosis is made based on location and appearance of the tumour via echocardiogram. Effective treatment options are limited to surgery (when feasible) or radiation therapy. Benefit of medical management is presently unknown. The goal of this retrospective study was to assess the efficacy and tolerability of toceranib phosphate for dogs with HBT. Twenty‐eight dogs with histologically, cytologically confirmed or presumed HBT were evaluated retrospectively. Twenty‐seven dogs were treated with single agent toceranib. One dog received combination therapy with concurrent metronomic chemotherapy. This dog was not included in response or survival analysis. Factors assessed included clinical signs, hematologic/biochemical parameters and response to treatment. For the 27 dogs receiving single agent toceranib, an overall response rate of 10% was found. Overall median survival time was 823 days (range, 68‐1190 days). The overall response rate for the dogs presenting with metastasis was 28.5%, with a median survival time of 532 days (range, 77‐679 days). This was not significantly different than the median survival time of 796 days for dogs who did not present with metastasis. Of the dogs displaying clinical signs at the time of diagnosis, 90% had improvement and 81% had complete resolution of signs after starting toceranib. Toxicity was seen in 54% of dogs with gastrointestinal distress as the most common toxicity but dose reductions were infrequent required. Results demonstrate that toceranib phosphate is a well‐tolerated and effective treatment for inoperable canine heart base tumours including dogs with advanced or metastatic disease.     

Safety evaluation of combination doxorubicin and toceranib phosphate (Palladia®) in tumour bearing dogs: a phase I dose‐finding study

Combination chemotherapy holds promise for improving outcomes in malignancy when compared with single‐agent approaches. Care must be taken to avoid overlapping toxicity and to utilize agents with differing mechanisms of action. A phase I dose‐finding trial was performed to determine the maximally tolerated dose (MTD) of a concurrent toceranib and doxorubicin (DOX) combination protocol where toceranib dose was maintained at or near 2.75 mg kg^?1 by mouth every other day (PO EOD) while escalating DOX dosage. The dose‐limiting toxicity was found to be neutropenia and the MTD of the combination was determined to be 25 mg m^?2 of DOX q 21 days given concurrently with toceranib 2.75 mg kg^?1 PO EOD. This combination was well tolerated with no excessive gastrointestinal toxicity nor novel adverse events (AEs) noted. Anti‐tumour activity was observed in the majority of cases. This combination warrants further investigation in the context of phase II/III clinical trials to characterize efficacy and long‐term AE profiles.     

Effect of MK‐467 on organ blood flow parameters detected by contrast‐enhanced ultrasound in dogs treated with dexmedetomidine

ObjectiveTo evaluate the dexmedetomidine‐induced reduction in organ blood flow with quantitative contrast‐enhanced ultrasound (CEUS) method and to observe the influence of MK‐467 on such reduction.Study designRandomized cross‐over study.AnimalsSix adult purpose‐bred laboratory beagle dogs (mean body weight 15.3 ± 1.9 kg).MethodsContrast‐enhanced ultrasound was performed on six conscious healthy laboratory beagles. The animals on separate occasions underwent three treatments: awake without any medication (CTRL), dexmedetomidine 10 μg kg^?1 (DEX) and DEX + MK‐467 500 μg kg^?1 (DMK) intravenously (IV). The kidney (10–15 minutes post‐treatment), spleen (25–30 minutes post‐treatment), small intestine (40–45 minutes post‐treatment) and liver (50–55 minutes post‐treatment) were examined with CEUS. A time curve was generated and the following perfusion parameters were analysed: arrival time (AT), time to peak from injection (TTPinj), peak intensity (PI) and wash‐in rate (Wi). In addition to CEUS, renal glomerular filtration rate was indirectly estimated by the rate of iohexol elimination.ResultsAT and TTPinj were significantly higher for DEX than for CTRL in all studied organs. The same parameters were significantly higher for DEX than for DMK in the kidney, spleen and small intestine. PI was significantly lower for DEX than for CTRL or DMK in the kidney. Wi was significantly lower for DEX than for CTRL or DMK in the kidney and significantly lower than for CTRL only in the small intestine. Plasma concentration of iohexol was significantly higher after DEX than CTRL administration.ConclusionsContrast‐enhanced ultrasound was effective in detecting DEX‐induced changes in blood flow. MK‐467 attenuated these changes.Clinical relevanceClinicians should consider the effects of the sedation protocol when performing CEUS. Addition of MK‐467 might beneficially impact the haemodynamic function of sedation with alpha‐2 adrenoceptor agonists.     

Sentinel lymph node mapping by near‐infrared fluorescence imaging and contrast‐enhanced ultrasound in healthy dogs

Sentinel lymph node (SLN) mapping is a valuable and crucial diagnostic procedure in staging malignancies. We compared two non‐invasive techniques, near‐infrared (NIR) fluorescence imaging and contrast‐enhanced ultrasound (CEUS), to identify the SLNs in three superficial anatomical regions in an animal model. Six healthy laboratory dogs were included in a proof‐of‐concept trial. A NIR fluorescent dye (Indocyanine Green) and microbubbles (Sonovue) were consecutively injected subdermally in the Inguinal, axillary and popliteal region to map the SLNs. Transcutaneous NIR fluorescence imaging identified SLNs in 17 out of a total of 18 occasions. CEUS identified SLNs in all regions (18/18). Whereas NIR fluorescence imaging performed better in the visualization of the afferent lymphatic tract, CEUS demonstrated different filling patterns of the SLNs, a feature potentially critical for the concept of SLN mapping in cancer patients. Both NIR fluorescence imaging and CEUS are safe, non‐invasive, practical and accurate methods to perform real‐time transcutaneous SLN mapping with potential in a clinical setting.     

Novel contrast agent Visphere™ is feasible for contrast‐enhanced ultrasonography in dogs

Contrast‐enhanced ultrasonography provides a more functional diagnostic image than conventional ultrasonography. This prospective exploratory study compared the novel contrast agent, Visphere^?, with commercial contrast agents in five healthy Beagle dogs. Visphere^? has the smallest diameter and highest concentration compared with Sonazoid^® and SonoVue^®. Each dog received an intravenous injection of Visphere^?, Sonazoid^®, or SonoVue^®. Images were recorded for 300, 600, and 60 s in the heart, liver, and left kidney, respectively. The mean pixel values of the regions of interest for each organ were expressed as time intensity curves (TIC). The agents all improved the visualization of left ventricular endocardial border delineation in the heart, and had similar TICs and clinical useful durations. In contrast, Visphere^? expressed the highest mean pixel value in the liver parenchyma at an early observation time and maintained the intensity until 600 s, like Sonazoid^®. The renal evaluation results indicated there were no statistically significant differences in time‐to‐peak for the renal cortex or medulla among the agents. Compared with the other two agents, SonoVue^® had the lowest peak enhancement for the renal cortex and medulla. No dogs had any adverse reactions during or after the study. All three agents provided adequate results for left ventricular endocardial border delineation, and Visphere^? may have the same potential as Sonazoid^® to detect and characterize hepatic lesions. Visphere^? and Sonazoid^® may offer better visualization quality to evaluate renal function. In conclusion, the novel contrast agent, Visphere^?, is comparable with commercial agents and could be applied in different major organs in dogs.     

A dose‐escalation study of combretastatin A4‐phosphate in healthy dogs

Combretastatin A4 ‐Phosphate (CA4P ) is a vascular disrupting agent revealing promising results in cancer treatments for humans. The aim of this study was to investigate the safety and adverse events of CA4P in healthy dogs as a prerequisite to application of CA4P in dogs with cancer. Ten healthy dogs were included. The effects of escalating doses of CA4P on physical, haematological and biochemical parameters, systolic arterial blood pressure, electrocardiogram, echocardiographic variables and general wellbeing were characterised. Three different doses were tested: 50, 75 and 100 mg m^?2. At all 3 CA4P doses, nausea, abdominal discomfort as well as diarrhoea were observed for several hours following administration. Likewise, a low‐grade neutropenia was observed in all dogs. Doses of 75 and 100 mg m^?2 additionally induced vomiting and elevation of serum cardiac troponine I levels. At 100 mg m^?2, low‐grade hypertension and high‐grade neurotoxicity were also observed. In healthy dogs, doses up to 75 mg m^?2 seem to be well tolerated. The severity of the neurotoxicity observed at 100 mg m^?2, although transient, does not invite to use this dose in canine oncology patients.     

Renal perfusion parameters measured by contrast‐enhanced ultrasound in healthy dogs demonstrate a wide range of variability in the long‐term

Contrast‐enhanced ultrasound may be helpful for detecting early renal microvascular damage and dysfunction in dogs. However, before this noninvasive imaging method can be tested as an early‐stage screening tool in clinical patients, an improved understanding of long‐term variation in healthy animals is needed. In this prospective, secondary, longitudinal, serial measurements study, variability of contrast‐enhanced ultrasound renal perfusion parameters was described for eight healthy dogs, using seven time points and a period of 83 weeks. Dogs were sedated with butorphanol (0.4 mg/kg), and contrast‐enhanced ultrasound of each kidney was performed after an intravenous bolus injection of a microbubble contrast agent (0.04 mL/kg). Time‐intensity curves were created from regions‐of‐interest drawn in the renal cortex and medulla. Intensity‐related parameters representing blood volume and time‐related parameters representing blood velocity were determined. A random‐effects model using restricted maximum likelihood was used to estimate variance components. Within‐dog coefficient of variation was defined as the ratio of the standard deviation over the mean. Time‐related parameters such as time‐to‐peak, rise and fall time had lowest within‐dog variability. Intensity‐related parameters such as peak enhancement, wash‐in and wash‐out area under the curve, total area under the curve, and wash‐in and washout rates had high within‐dog variability (coefficient of variation > 45%). Authors therefore recommend the use of time‐related parameters for future studies of renal perfusion. Within‐dog variability for bilateral kidney measurements was extremely low, therefore contrast‐enhanced ultrasound may be particularly useful for detecting unilateral changes in renal perfusion. Future studies are needed to compare contrast‐enhanced ultrasound findings in healthy dogs versus dogs with renal disease.     

Development of an ELISA to detect circulating anti‐asparaginase antibodies in dogs with lymphoid neoplasia treated with Escherichia coli l‐asparaginase

Resistance to Escherichia coli l ‐asparaginase in canine lymphoma occurs frequently with repeated administration, a phenomenon often attributed, without substantiation, to the induction of neutralizing antibodies. To test the hypothesis that treated dogs develop antibodies against the drug, we created an enzyme‐linked immunosorbent assay (ELISA) to measure plasma anti‐asparaginase immunoglobulin G responses. Using samples from dogs that had received multiple doses, specific reactivity against l ‐asparaginase was demonstrated, while naïve patients' samples were negative. The optimized ELISA appeared sensitive, with endpoint titers >1 600 000 in positive control dogs. Intra‐ and inter‐assay coefficients of variation were 3.6 and 14.5%. The assay was supported by the observation that ELISA‐positive plasma could immunoprecipitate asparaginase activity. When clinical patients were evaluated, 3/10 dogs developed titers after a single injection; with repeated administration, 4/7 dogs were positive. l ‐asparaginase antibodies showed reduced binding to the PEGylated drug formulation. The ELISA should prove useful in investigating the potential correlation of antibody responses with resistance.