Distribution,metabolism, and excretion of toceranib phosphate (Palladia™, SU11654), a novel tyrosine kinase inhibitor,in dogs

Yancey, M. F., Merritt, D. A., White, J. A., Marsh, S. A., Locuson, C. W. Distribution, metabolism, and excretion of toceranib phosphate (Palladia?, SU11654), a novel tyrosine kinase inhibitor, in dogs. J. vet. Pharmacol. Therap. 33 , 154–161. Toceranib phosphate (Palladia?, SU11654), a multireceptor tyrosine kinase inhibitor with anti‐tumor and anti‐angiogenic activity, has been developed for the treatment of mast cell tumors in dogs. An overview of the distribution, metabolism, and excretion of toceranib phosphate in dogs is presented. When [¹⁴C]‐toceranib was orally administered to dogs, the majority of the radioactivity (92%) was excreted in feces and only a small portion (7%) was excreted in urine. Seven days after a single 3.25 mg/kg oral dose, radioactivity was the highest in bile and liver, with measurable concentrations in lymph nodes, colon, adrenals, bone marrow, kidneys, lungs, spleen, pancreas, and skin. Plasma protein binding of toceranib in fresh plasma ranged from 90.8% to 92.8% at concentrations between 20 ng/mL and 500 ng/mL and was independent of concentration. Microsomal and hepatocyte incubations resulted in the formation of a single metabolite. Spectrometric analysis of the metabolite was consistent with the formation of an alicyclic N‐oxide of toceranib. The combination of the high rate of fecal excretion and the long elimination half‐life of toceranib indicate enterohepatic recirculation of the parent compound and/or the N‐oxide metabolite.     

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.     

Effect of tyrosine kinase inhibition by imatinib mesylate on mast cell tumors in dogs

BACKGROUND: Imatinib mesylate is a small molecule targeted at dysregulated protein-tyrosine kinase. Mutation of c-kit exon 11, which induces constitutive phosphorylation of KIT, is one of the mechanisms for the development or progression of mast cell tumor (MCT) in dogs. The purpose of this study was to examine the therapeutic potential of imatinib mesylate in canine MCT. HYPOTHESIS: Imatinib mesylate has activity against MCT in dogs, and response to treatment can be correlated to presence of mutation within exon 11 of c-kit. ANIMALS: Twenty-one dogs with MCT with gross tumor burden and median tumor size of 7.2 cm (range, 1.0-25.3 cm) before treatment. METHODS: Tumors were analyzed for mutation of c-kit exon 11. Imatinib mesylate was administered PO to the dogs at a dose of 10 mg/kg daily for 1-9 weeks. RESULTS: Ten of 21 dogs (48%) had some beneficial response to imatinib mesylate treatment within 14 days of treatment initiation. All 5 dogs with a demonstrable c-kit mutation in exon 11 responded to the drug (1 complete remission, 4 partial remission). CONCLUSIONS AND CLINICAL IMPORTANCE: Imatinib mesylate has clinical activity against MCT in dogs. Response could not be predicted based on presence of absence of a mutation in exon 11 of c-kit.     

Phosphorylated KIT as a predictor of outcome in canine mast cell tumours treated with toceranib phosphate or vinblastine

Canine cutaneous mast cell tumour (MCT) is the most common malignant skin tumour in dogs and can exhibit variable biologic behaviour. Dysregulated signalling through the receptor tyrosine kinase (RTK) KIT can promote cell proliferation and survival, and assessment of its dysregulation via detection of activating c‐kit gene mutations or assessment of KIT protein localization is associated with multiple features of malignancy. The aim of the current study was to use a previously validated immunohistochemical (IHC) assay to directly measure phosphorylated KIT (pKIT) in order to investigate its association with other established prognostic markers, response to therapy, progression free interval (PFI) and overall survival time (OST) in dogs treated medically for measurable MCT. Tumour tissue from 74 dogs enrolled in a prospective study comparing toceranib and vinblastine for MCT treatment were evaluated for pKIT immunoreactivity. pKIT was variably expressed, with some degree of positivity observed in 49/74 cases (66%). pKIT immunoreactivity was significantly associated with aberrant KIT localization, high mitotic index and high histologic grade. On univariate analysis, pKIT immunoreactivity predicted shorter PFI and OST in the entire patient population as well as shorter PFI in the toceranib treated group, and was the sole predictive factor for OST upon multivariate analysis, while mitotic index was the sole independent predictive factor for PFI. These results demonstrate that IHC detection of pKIT correlates with several features of aggressive behaviour, and may confer information that is complementary to other prognostic factors. However, the role of pKIT in predicting outcome needs to be studied further before recommendations can be made for its routine use.     

The tyrosine kinase inhibitor imatinib [STI571] induces regression of xenografted canine mast cell tumors in SCID mice

Canine mast cell tumors (MCTs) are the most common cutaneous tumors in the dog. They have a wide range of behaviour, which can make these tumors challenging to treat. Recently, mutations in c-kit proto-oncogene have been identified in several canine MCTs. Imatinib is the first member of a new class of agents that act by inhibiting particular tyrosin kinase enzymes, including KIT which is a product of the c-kit. In this study the efficacy of imatinib to reduce or abolish canine MCT [CMC-1] using xenografted MCT in severe combined immunodeficient [SCID] mice was evaluated. Imatinib was administered at doses of 200 mg/kg and 100 mg/kg once a day for one week. The antitumor responses in SCID mice with CMC-1 xenografts following treatment with imatinib were observed. Significant tumor regression occurred with 100 mg/kg on days 7, 10, 14 and 21, and 200 mg/kg on all days. Our results indicate that imatinib is effective against canine mast cell tumor in mouse xenograft models. Canine MCTs might be a potential target for imatinib therapy.     

CT features of subcutaneous,intermuscular, and intramuscular mast cell tumors in dogs

Surgical removal is the treatment of choice for subcutaneous (SC), intermuscular (InterM), and intramuscular (IntraM) mast cell tumors (MCTs). Advanced imaging (CT or MRI) is frequently used for presurgical planning, but InterM and IntraM MCTs can be difficult to identify and delineate on CT. Aims of the current retrospective, diagnostic accuracy, observer agreement study were to describe the imaging features of SC, InterM, and IntraM MCTs on CT and to assess the limitation of CT to identify the full local extent of the MCT. Inclusion criteria for the study were dogs with a cytologically or histologically diagnosed MCTs determined to be SC, InterM, or IntraM MCT based on histology and/or a CT scan performed in the gross disease setting. Two board-certified veterinary radiologists reviewed the CT images and recorded location, contrast enhancement pattern, and delineation between the normal and abnormal tissue. Sensitivity and specificity of CT for determining location (SC/InterM versus IntraM) was 85.71% and 55.56%, respectively, when compared to consensus location based on surgical pathology report/CT/MRI review. There was a low inter-rater agreement for delineation (kappa: 0.150 (−0.070 to 0.370) and measurement had a low/moderate correlation (rho: 0.4667 to 0.5792). Upon review by a surgical oncologist, CT findings were deemed insufficient for curative surgical planning in 13 of 16 due to inadequate definition of tumor depth, compartment boundary (fascial plane) or MCT margins. The use of CT for presurgical planning of SC/InterM/IntraM MCT dogs has limitations, especially when differentiating MCT from the adjacent muscle.     

Calcitriol (1,25‐dihydroxycholecalciferol) enhances mast cell tumour chemotherapy and receptor tyrosine kinase inhibitor activity in vitro and has single‐agent activity against spontaneously occurring canine mast cell tumours*

Calcitriol potentiates the effect of multiple chemotherapy agents in a variety of tumour models. In this study, we examine whether calcitriol increases chemotherapy or tyrosine kinase inhibitor in vitro cytotoxicity in canine mastocytoma C2 cells. We also evaluate the in vivo effect of DN101, a highly concentrated oral formulation of calcitriol designed specifically for cancer therapy, as a single‐agent therapy in dogs with mast cell tumours (MCTs). Calcitriol exhibits synergistic, antiproliferative activity when used in combination with CCNU, vinblastine, imatinib or toceranib in vitro. The concentrations required for 50% growth inhibition were generally two‐ to six‐fold lower when the drugs were used in combination than when used individually. High‐dose oral calcitriol induced remission in 4 of 10 dogs (one complete remission, three partial remissions), although the majority experienced toxicity, necessitating discontinuation of the trial. Further evaluation of calcitriol in combination therapy for dogs with MCTs is warranted.     

Assessment of tumor enhancement by contrast‐enhanced CT in solid tumor‐bearing dogs treated with toceranib phosphate

In humans, contrast‐enhanced CT (CECT) has been used to indirectly assess the antiangiogenic effects demonstrated by a number of tyrosine kinase inhibitors. This retrospective, cross‐sectional study aimed to quantitatively evaluate changes in tumor contrast‐enhancement (CE) using CECT in solid tumor‐bearing dogs treated with toceranib phosphate (TOC). The changes in tumor size and CE were measured using the Hounsfield unit (HU) scale in CECT images before TOC treatment and between 30 and 90 days after initiating the treatment. Among the 36 dogs treated with TOC, eight (22.2%) showed a partial response, 22 (61.1%) showed stable disease, and six (16.7%) showed progressive disease. Thirty (83.3%) of 36 dogs showed a decrease in tumor CE (median: ?20%, range: ?1% to ?48%) after initiating the treatment. The results indicated that tumor CE and size changes were observed in tumor‐bearing dogs that were treated with TOC; however, tumor CE was not significantly correlated with tumor regression. We suggest that these results could serve as pilot data to evaluate the antiangiogenic effects associated with TOC.     

Successful treatment of a metastatic,gastrointestinal stromal tumour in a dog with toceranib phosphate (Palladia)

A ten‐year‐old, female‐entire English springer spaniel presented with a large intra‐abdominal mass but no other clinical signs. Gastrointestinal stromal tumour of the caecum with widespread abdominal metastasis was confirmed. Treatment with toceranib phosphate resulted in complete response, despite the absence of exon‐8 or exon‐11 c‐kit mutation. There was no clinical evidence of tumour recurrence nine months after diagnosis.     

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.     

Relationship of disease progression and plasma histamine concentrations in 11 dogs with mast cell tumors

Plasma histamine concentrations (PHCs) were measured serially over 9 months or until death in 11 dogs with mast cell tumors (MCTs). Eight dogs had grossly visible disease and the other 3 dogs had microscopic disease. Initial PHCs in the dogs with gross disease were significantly higher than PHCs in healthy dogs (median, 0.73 ng/mL and 0.19 ng/mL respectively; P < .009), whereas initial PHCs in dogs with microscopic disease showed no difference from controls. Seven dogs subsequently had progressive increases in PHC, and developed hyperhistaminemia (median, 14.0 ng/mL; range, 5.11-30.1 ng/nL). These 7 dogs died from MCTs, and 1 had general weakness with rapid lysis of a large tumor burden after radiation therapy. PHCs of the other 4 dogs were less than 1 ng/mL during the study. These 4 dogs were still alive with adequate control of the tumor at the conclusion of the study. Four of the 11 dogs initially had gastrointestinal (G1) signs, which abated soon after administration of histamine-2 (H-2) blockers. No significant difference was found between PHCs in dogs with GI signs and those without GI signs (median, 0.86 ng/mL and 0.35 ng/mL. respectively). Thereafter, 7 dogs had serious GI complications for which H-2 blocker therapy was ineffective. PHCs in these 7 dogs were extremely high (median, 12.2 ng/mL; range, 3.42-30.1 ng/nL). Results of this study demonsrated that PHC was one factor related to disease progression, and indicated that marked hyperhistaminemia was associated with the GI signs refractory to H-2 blocker therapy in dogs with MCTs.     

A phase II clinical trial of vinorelbine in dogs with cutaneous mast cell tumors

Background: Few effective drugs are available to treat dogs with locally aggressive or metastatic mast cell disease.
Hypothesis: Vinorelbine, a semisynthetic derivative of vinblastine, is an effective drug for the treatment of canine mast cell tumors (MCT).
Animals: Twenty-four dogs with cutaneous MCT.
Methods: Dogs with at least 1 measurable, cytologically confirmed, and previously untreated cutaneous MCT received a single treatment with vinorelbine at the previously established dosage of 15 mg/m² IV. Tumor measurements and CBC were evaluated before and 7 days after treatment. Adverse events were graded according to Veterinary Cooperative Oncology Group (VCOG) guidelines.
Statistics: Data were accrued in accordance with a Simon's 2-stage design with a noninteresting response rate of .05, a target response of .25, and α and β values of .10.
Results: Three of 24 dogs (13%) had a response to treatment, including 1 measurable complete response and 1 measurable partial response. The 3rd dog had microscopic complete response to treatment with stable measurable disease. Twenty other dogs (83%) had stable disease and 1 dog (4%) had progressive disease. Neutropenia occurred in 13 dogs (54%) (grade 1, n = 4; grade 3, n = 6; grade 4, n = 3). Gastrointestinal toxicity occurred in 11 dogs (46%) (anorexia: grade 1, n = 3; grade 2, n = 1; grade 3, n = 1; diarrhea: grade 1, n = 2; grade 3, n = 1; vomiting: grade 1, n = 5; grade 3, n = 1).
Conclusions and Clinical Importance: Vinorelbine was associated with an overall response rate of 13% and a high prevalence of neutropenia. Additional studies are indicated to determine if repeated dosing of vinorelbine or combination of vinorelbine with other drugs increases the observed biologic activity against canine MCT.     

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.     

Long-term postsurgical outcomes of mast cell tumors resected with a margin proportional to the tumor diameter in 23 dogs

We evaluated the postsurgical outcomes of cutaneous or subcutaneous mast cell tumors (MCTs, n=25) in 23 dogs, resected with lateral surgical margins proportional to the widest tumor diameter, including at least one facial plane. The tumor diameter range was 0.3–2.6 cm (median: 0.9 cm), and all were histologically diagnosed as Kiupel’s low-grade MCT. Resection was histologically complete in 20, close (deep margin) in three, and incomplete (deep margin) in two. No dogs developed local recurrence at the site of initial surgery during follow-up of 161–2,219 days (median: 976 days). These results suggest that resection of low-grade, relatively small MCTs with surgical margins proportional to the tumor diameter is a practical procedure with high success rates.     

Outcomes of dogs with grade 3 mast cell tumors: 43 cases (1997-2007)

This study reports the outcomes of dogs with grade 3 mast cell tumors (MCTs). Clinical and histopathological data were available for 43 dogs. Median progression-free survival (PFS) and overall survival (OS) were 133 and 257 days, respectively. Tumor size, lymph node (LN) status, and mitotic index (MI) significantly influenced PFS in univariate analysis. Tumor size and LN status remained significant in the multivariate analysis. Lymph node status, local tumor control, LN treatment, and MI significantly influenced OS in univariate analysis but only LN status remained significant in multivariate analysis. These results confirm that locoregional control improves outcomes in patients with grade 3 MCTs.     

The tyrosine kinase inhibitor sorafenib decreases cell number and induces apoptosis in a canine osteosarcoma cell line

Canine osteosarcoma, an aggressive cancer with early distant metastasis, shows still despite good chemotherapy protocols poor long term survival. The aim of our study was to determine whether sorafenib, a novel multikinase inhibitor, has any effect on D-17 canine osteosarcoma cells.A cell proliferation kit was used for detecting surviving cells after treatment for 72 h with sorafenib or carboplatin or their combination. A significant decrease of neoplastic cells was observed after incubation with 0.5-16 μM sorafenib or with 80-640 μM carboplatin. Using immunocytochemistry for activated caspase 3 to evaluate apoptosis, we found significantly more positive cells in the sorafenib treated groups. Paradoxically, expression of the nuclear proliferation marker Ki-67 was also significantly higher in sorafenib treated cells. The drug sorafenib showed potent antitumour activity against D-17 canine osteosarcoma cells in vitro, suggesting a potential as a therapeutic tool in the treatment of bone cancer in dogs.     

Immunohistochemical detection of proliferating cell nuclear antigen and Ki-67 in mast cell tumors from dogs

OBJECTIVE: To determine whether immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and Ki-67 correlated with prognosis for dogs with cutaneous mast cell tumors (MCT). DESIGN: Case series. ANIMALS: 120 dogs with solitary cutaneous MCT that were excised. PROCEDURE: Information on signalment, history, and outcome was obtained by sending a questionnaire to referring veterinarians. Tumors were graded histologically, and immunohistochemical staining for Ki-67 and PCNA was performed. RESULTS: Survival rates 12, 18, and 24 months after surgery were significantly different among groups when dogs were grouped on the basis of histologic grade. Although mean number of PCNA-positive nuclei/1,000 tumor nuclei was significantly higher for dogs that died of MCT than for those that survived, there was great overlap in values. Mean number of Ki-67-positive nuclei/1,000 tumor nuclei was significantly higher for dogs that died of MCT than for those that survived, without any overlap in values between groups, and number of Ki-67-positive nuclei/1,000 tumor nuclei was significantly different among groups when tumors were grouped on the basis of histologic grades. For dogs with grade-II tumors, number of Ki-67-positive nuclei/1,000 tumor nuclei (< 93 vs > or = 93) was significantly associated with outcome (survived vs died). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that for dogs with solitary cutaneous MCT, determining number of Ki-67-positive nuclei may be useful in predicting prognosis, particularly for dogs with grade-II tumors.     

Correlation of DNA ploidy to tumor histologic grade, clinical variables, and survival in dogs with mast cell tumors.

By using flow cytometry, a retrospective analysis of the DNA content of 40 primary canine mast cell tumors and seven lymph nodes that contained metastatic mast cell tumor from 44 dogs of various breed, sex, and age was performed on formalin-fixed, paraffin-embedded samples of the tumors and nodes. These samples were chosen according to the following criteria: samples contained sufficient well-preserved tumor tissue in the paraffin block for processing, sufficient patient history data were available, clean and homogeneous cell suspensions were obtained after processing, and interpretable DNA histograms were produced on analysis. The ploidy data obtained were compared with the histopathologic grade, the anatomical site of occurrence, the clinical stage of the tumors, and the survival of the dogs. Over 70% (29/40) of the mast cell tumors were diploid. Three metastatic mast cell tumors in lymph nodes had the same ploidy status as their corresponding primary tumors. In five dogs, mast cell tumors from multiple sites in each dog displayed similar ploidy status. Of 26 dogs evaluated for survival times, 69% (18/26) had diploid tumors and 31% (8/26) had aneuploid tumors. When numbers of diploid versus aneuploid tumors were compared, no significant difference was found between any two grades, clinical stages, or anatomic sites. A significant difference (P = 0.02) was found, however, between aneuploid and diploid tumors when comparing Stage I and non-Stage I disease. The Kaplan-Meier survival plot indicated a tendency towards an increased survival within the first year in dogs with diploid versus aneuploid tumors (P = 0.06).     

Frequency and severity of mastocytemia in dogs with and without mast cell tumors: 120 cases (1995-1997).

OBJECTIVE: To elucidate frequency of detection on blood smears and severity on quantitative buffy coat evaluation of mastocytemia between dogs without mast cell tumors (MCT) and dogs that had MCT, and to expand the list of diseases associated with mastocytemia in dogs without MCT. DESIGN: Retrospective study. ANIMALS: 94 dogs without MCT and 26 dogs with MCT. PROCEDURE: Medical records of all dogs with mast cells detected on blood or buffy coat smears during a 2-year period were reviewed. Dogs with mastocytemia were grouped by disease into dogs with MCT and dogs without MCT. Twenty-five of the dogs without MCT that had mast cells detected on blood smears also had evaluations of buffy coat smears. Quantitative buffy coat results of the 25 dogs without MCT were compared with those of the 26 dogs with MCT. RESULTS: 95.5% of blood smears with mast cells detected during CBC determination were from dogs without MCT. For these dogs, diagnoses included inflammatory disease (28.2%), regenerative anemia (27%), neoplasia other than MCT (25.9%), and trauma (11.8%). Dogs with MCT had a mean of 71.4 mast cells/buffy coat smear, whereas dogs without MCT had a mean of 276.2 mast cells/buffy coat smear. The 2 highest counts of mast cells/buffy coat smear were for dogs without MCT. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of results of quantitative buffy coat evaluations, severity of mastocytemia in dogs without MCT often exceeds that detected during tumor staging in dogs with MCT. Random detection of mast cells in blood smears during CBC determination in dogs is usually not secondary to MCT.