Safety Evaluation of Combination Doxorubicin and Toceranib Phosphate (Palladia®) in Tumour Bearing Dogs: a Phase I Dose-ﬁnding Study

Original Article DOI: 10.1111/vco.12232

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

M. A. Pellin1,∗,R.M.Wouda2,∗,K.Robinson1,K.Tsimbas1,I.D.Kurzman1, B. J. Biller3 andD.M.Vail1,4 1Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA 2Kansas State University, College of Veterinary Medicine, Manhattan, KS, USA 3Flint Animal Cancer Center, Colorado State University, Fort Collins, CO, USA 4Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI, USA

Abstract 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 Keywords with toceranib 2.75 mg kg−1 PO EOD. This combination was well tolerated with no excessive cancer, canine, gastrointestinal toxicity nor novel adverse events (AEs) noted. Anti-tumour activity was observed in chemotherapy, doxorubicin, toceranib, the majority of cases. This combination warrants further investigation in the context of phase II/III Tregs clinical trials to characterize efficacy and long-term AE profiles.

Introduction proven single-agent activity against the tumour A seminal discovery in the evolution of cancer histotype being treated. The antitumour mech- treatment was the advantage of combination anism for each of the drugs should differ and, chemotherapy regimens over single-agent ther- related to this, drugs with considerably similar apy. There now exists ample clinical precedent adverse event (AE) profiles should be avoided. Correspondence address for the use of multi-agent protocols, and almost Finally, the drugs should be able to be administered D. M. Vail School of Veterinary all curative-intent protocols in human oncology at their optimal therapeutic dose and interval. Medicine incorporate a minimum of two or three chemother- Mindful of these criteria, our group has previously University of apeutic agents with additive or synergistic activity. evaluated the combinations of toceranib phos- Wisconsin-Madison ® 2015 Linden Dr. Ideally, such combination chemotherapy protocols phate (Palladia ) with piroxicam, vinblastine, and Madison adhere to several principles, in order to optimize CCNU (lomustine) in dogs.2–5 Further inves- WI 53706 clinical benefit.1 The individual drugs should have tigations of multi-drug chemotherapy protocols USA e-mail: are warranted in veterinary oncology to strive for [email protected] ∗ These authors contributed equally to this work. increased efficacy and improved outcomes in our

© 2016 John Wiley & Sons Ltd 919 920 M. A. Pellin et al. companion animals with spontaneously arising are also seen in humans secondary to RTKI use. tumours. The mechanisms of these toxicities have not been Doxorubicin (DOX) is an anthracycline derived determined but are suggested to occur secondary to from Streptomyces yeast species.6 It has multiple inhibition of VEGF.14 Less frequent toxicities that mechanisms of cellular toxicity, including interca- have been reported in dogs include musculoskele- lation of DNA leading to inhibition of protein syn- tal pain, elevated liver enzymes and azotemia. thesis, free radical formation and topoisomerase Haematopoietic toxicity is uncommonly reported enzyme inhibition. DOX is one of the most com- withtoceranibandistypicallymildifitisnoted monly utilized chemotherapy agents in veterinary at all. Cardiotoxicity including left ventricular medicine and has showed activity against a vari- dysfunction, congestive heart failure and throm- ety of histologies.6,7 This broad spectrum of activ- boembolic disease, is also uncommonly reported ity makes DOX an attractive agent to combine with secondary to RTKI use in human patients.14 To other agents to further enhance antitumour activ- the authors’ knowledge, these effects have not ity. The AE profile of single-agent DOX in dogs is been commonly reported in canine patients. The well established with myelosuppression, in particu- complexity of the RTKIs’ cellular interactions is not lar neutropenia, being the acute dose-limiting AE yet completely appreciated and there is ongoing and myocardial toxicity with cumulative doses over research interrogating the potential roles of the 180–240 mg m−2 being the chronic dose-limiting receptor RTKIs as chemosensitizers, radiation AE.6,8 Other AEs common following DOX include sensitizers, immune modulators and in reversing alopecia and gastrointestinal AEs such as vomit- acquired chemotherapy resistance. Toceranib has ing and diarrhoea, with the latter occasionally being alsobeenshowninonestudytoresultinselective dose-limiting. depletion of circulating regulatory T lymphocytes Toceranib phosphate (Palladia; Zoetis, Florham (Tregs)intumour-bearingdogswhenusedasa Park, NJ) is a receptor tyrosine kinase inhibitor single agent and when combined with metronomic (RTKI) possessing inhibitory activity against cyclophosphamide.15 several members of the receptor tyrosine kinase The hypothesis under investigation in this trial family of transmembrane proteins, including the wasthattoceranibcouldbesafelyadministeredto cell surface receptors for vascular endothelial dogs with naturally occurring malignancies on an growth factor (VEGF), platelet-derived growth every other day (EOD) schedule in combination factor (PDGF), stem cell factor (SCF), cytokine withDOXadministeredevery3weeks,atdosages Flt-3 ligand, and the GDNF-family ligands.9,10 with respective demonstrable single-agent activity. Toceranib’s activity results from competitive block- This study had three objectives. The primary objec- adeoftheATP-bindingsiteofthesetyrosinekinase tive was to employ a conventional 3 + 3phaseI receptors, resulting in impaired phosphorylation study design to determine the maximum tolerated and diminished down-stream signalling.11 In a dose (MTD) of DOX that can be safely admin- phase I dose-finding study of toceranib, clinical istered to tumour-bearing dogs with concurrent responses were observed in a variety of tumour toceranib administered according to a standard types including metastatic carcinomas, sarcomas, dose and schedule.16 The second objective was to multiple myeloma and melanoma.10 Subsequent characterize the AE profile of combination DOX off-label investigation further supported a role for and toceranib in dogs. A third objective was to toceranib in the treatment of numerous canine characterize the effect of the DOX/toceranib com- neoplasms, including osteosarcoma, thyroid car- bination protocol on circulating Tregs owing to cinoma, apocrine gland anal sac adenocarcinoma, toceranib’s previously documented immunomod- nasal carcinoma and head and neck squamous cell ulatory single-agent effect on this lymphocyte carcinomas.12 Toceranib’s most common AEs are subpopulation. Whilst efficacy is never a primary gastrointestinal, especially diarrhoea.10,13 Protein- endpoint of such dose-finding studies, clinical uriaandhypertensionarealsocommonlyseen benefit was also evaluated throughout the study secondary to toceranib use in our institution, and period.

Methods the DOX dose increasing with each cohort and the toceranib dose remaining constant. The ini- Patient selection tial cohort received DOX at a dose of 20 mg m−2 Client-owned dogs with one or more cytologically administered intravenously (IV) every 21 days for a and/or histologically diagnosed malignant tumours total of four doses. DOX was escalated by 5 mgm−2 of any histology (excluding mast cell tumours), foreachcohortuntilMTDorthestandarddoseof gradeandstagewereconsideredforenrollment. 30 mg m−2 was reached, whichever came first. The Dogs with mast cell tumours were excluded given toceranib dose was maintained at approximately the investigators’ concern regarding the paraneo- 2.75 mg kg−1 by mouth (PO) EOD throughout the plastic gastrointestinal effects observed with this trial period and extending for a minimum of 7 days histology, which could confound interpretation following the fourth DOX dose. Toceranib dosing of gastrointestinal AEs secondary to proposed was necessarily approximate to accommodate the therapy, especially in this smaller study population. drug’s commercially available tablet sizes. Owners Prior surgery was allowed, hence, measurable were given the option of having their dog receive macroscopic disease was not an inclusion criteria. a fifth dose of DOX and/or continuing toceranib Prior radiation therapy, chemotherapy (other than after trial conclusion. DOX or toceranib) and corticosteroid therapy Three dogs were enrolled in each cohort and were also allowed, with adherence to the prede- then observed for dose-limiting toxicities (DLTs). termined minimum washout periods of 3 weeks ADLTwasdefinedasanygrade3orhigherAE for both radiation and chemotherapy, and 72 h for (VCOG-CTCAE v1.117) with the exception of corticosteroid therapy. Concurrent non-steroidal haematopoietic AEs (i.e. neutropenia, thrombo- anti-inflammatory therapy was permitted if it was cytopenia) for which a grade 4 AE was defined instituted at least 30 days prior to enrollment. as dose-limiting. Furthermore, any grade AE that A complete physical examination, haematologic wasrefractorytosupportivecareorpersisting analysis [complete blood count (CBC)], serum beyond 7 days was also considered dose-limiting. biochemistry profile, urinalysis and blood pres- If no AEs were observed within the first cohort sure measurement were performed prior to initial of dogs following 3 weeks of treatment, a second treatment. Additional inclusion criteria included a cohort was treated with the DOX dose escalated minimumbodyweightof10kg,haematologicand by 5 mg m−2.IfaDLTwasobservedinoneofthe biochemical parameters deemed adequate for the three dogs, the cohort was expanded to a total of safe administration of chemotherapy, the absence six dogs. If two or more DLTs were noted in any of severe or uncontrolled concurrent disease(s), cohort, it was considered that the MTD had been and a Veterinary Cooperative Oncology Group exceeded. Therefore, the MTD was defined as the (VCOG) common terminology criteria for AEs highest dose level at which no more than one of six (VCOG-CTCAE) v1.1 performance status of 0 dogs developed a DLT. (fully active, able to perform at pre-disease level) or 1 (activity less than pre-disease level, but able to Assessment of AEs function as an acceptable pet).17 The clinical trial protocol was approved by the Animal Care and Use All dogs were evaluated at the University of Wis- Committee of the School of Veterinary Medicine, consin Veterinary Medical Teaching Hospital University of Wisconsin-Madison, and written (UW-VMTH) prior to trial enrollment. Physical informed consent was obtained from owners prior examination, haematologic analysis (CBC), serum to clinical trial commencement. biochemistry profile, urinalysis and blood pressure measurement were performed at baseline, then every 21 days immediately prior to each DOX Study design and treatment protocol dose. The urine protein:creatinine ratio (UPC) A conventional open-label phase I dose-cohort wascalculatediftheurinalysisand/orbloodpres- 3 + 3escalationstudydesignwasemployed16 with sure measurement were suggestive of pathologic

© 2016 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 15, 3, 919–931 922 M. A. Pellin et al. proteinuria and/or hypertension. No specific car- progressivedisease(PD)wasdefinedasatleast diac monitoring was performed unless dictated a 20% increase in the sum of the target lesion(s’) by changes (such as new murmur, arrhythmia) longest diameter(s) or the appearance of new noted on physical examination. As our protocol lesions; stable disease (SD) was defined as neither would result in cumulative doses of less than 180 CR,PRnorPDforatleast6weeks.Dogsthat mg m−2, and previous evidence has demonstrated developedPDwerewithdrawnfromthetrialand that routine echocardiogram or electrocardiogram owners were offered alternative treatment options. (ECG) monitoring is insensitive for detection of Progression-free survival (PFS) was defined as the DOX-induced cardiotoxicity in non-symptomatic time (days) from the start of treatment to docu- patients, this was not deemed necessary for patient mented progression (local and/or distant) or death assessment.18 Physical examination and CBC were from any cause, for cases with macroscopic disease, also performed by clinicians at the UW-VMTH or or time to documented recurrence, metastasis the referring veterinarian 1 week following each or death from any cause for cases with micro- DOX dose. scopic disease at treatment initiation. Patients were All AEs were characterized and graded accord- censored if still alive at last assessment prior to ing to VCOG-CTCAE v1.1.17 Disease progression manuscript preparation. or clinical signs that were attributed to the underlying disease were not considered AEs. Permissible Peripheral blood mononuclear cell (PBMC) supportive care for gastrointestinal AEs included sample preparation a 7-day toceranib drug holiday, anti-diarrhoeal medications, such as metronidazole and tylosin, In all dogs in the 25 mg m−2 and higher DOX antiemetics, such as maropitant, ondansetron and cohorts, an additional 15 mL of ethylenediaminete- metoclopramide, and gastric protectants, such as traacetic acid (EDTA) whole blood was collected famotidine, ranitidine and omeprazole. Hyperten- immediatelypriortotheinitialDOXdoseandthen sion was managed with angiotensin converting every 21 days, immediately prior to each subse- enzyme (ACE) inhibitors, either benazepril or quentDOXdose,forperipheralbloodmononuclear enalapril, and/or amlodipine as recommended by cell (PBMC) enumeration and lymphocyte subset the UW-VMTH cardiology service. flow-cytometric analysis. PBMCs were isolated from the EDTA blood using density gradient separation. Briefly, whole blood was diluted 1:1 in Antitumour response assessment Hanks’ balanced salt solution (HBSS) followed by Tumour response was assessed for those dogs 10 mL of lymphocyte separation medium (Cellgro, with macroscopically measurable disease on study Manassas, VA, USA). Following centrifugation at days 0, 21, 42, 63 and 84. Follow-up after day 84 830 × g × 18 min, PBMCs were removed from the varied because not all patients continued therapy gradient and washed in HBSS. Cells were then or monitoring. Tumour response was assessed centrifuged at 340 g ××7 min and red blood cells according to either VCOG response evaluation were lysed by adding ACK red cell lysis buffer criteria for solid tumours (RECIST) in dogs v1.019 (Lonza, Walkersville, MD, USA). Cells were then or VCOG v1.020 response evaluation criteria for washed in HBSS, centrifuged and re-suspended in peripheral nodal lymphoma as was case applicable. lymphocyte freezing medium [90% foetal bovine Tumour measurement was performed as indicated serum (FBS)/10% demethyl sulfoxide (DMSO)] at using callipers, thoracic radiographs, ultrasonog- a concentration of ∼5.0 × 106 cells mL−1.Vialscon- raphy and/or computerized tomography (CT) with taining the cells were placed at −80∘ Candfrozen the associated imaging programme. A complete at a rate of cooling of −1o Cmin−1 in a Mr. Frosty response (CR) was defined as complete regression freezing container (Thermo Scientific, Waltham, of all measurable disease; partial response (PR) MA, USA). After 24 h, cells were transferred to was defined as at least a 30% reduction in the liquid nitrogen and stored until shipment. Samples sum of the target lesion(s’) longest diameter(s); were shipped in batch on dry ice to the laboratory

© 2016 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 15, 3, 919–931 Safety of concurrent doxorubicin and toceranib combination therapy 923 of one author (B. J. B.) for lymphocyte subset atoraboveDOXMTDwerecomparedusinga analysis. non-parametric repeated measures analysis of vari- ance (ANOVA) analysis (Friedman’s) and Dunn’s Multiple Comparison post hoc Test for comparison Flow cytometric lymphocyte subset analysis of individual time points. All statistical analyses Cryopreserved PBMC samples were batch analysed were performed with a commercial software pack- forTregsubsetanalysisinthelaboratoryofDr.Barb age (Prism v5.0, GraphPad Software, LaJolla, CA, Biller at Colorado State University using previously USA). P values less than 0.05 were considered published and validated techniques.15,21–23 Briefly, statistically significant. sampleswerethawedandthePBMCswashed, then added to a 96-well plate at a concentration Results of 5 × 105 perwell.Allsamplesweretreatedfor 5 min with a mixture of normal canine serum, Patient demographics human IgG and anti-mouse CD16/32 antibod- A total of 21 dogs were enrolled between January ies to block non-specific binding. Samples were 2014 and February 2015. Two dogs were not evalu- then immunostained for surface expression of able because they received only a single DOX dose CD4, CD8 and CD25 with the following reagents with no follow-up blood work as a result of dis- respectively: Pacific Blue conjugated anti-canine ease progression and euthanasia (n = 1) or owner CD4 (clone YKIX302.9, Serotec [Raleigh, NC, withdrawal (n = 1); these patients never received USA], Raleigh, NC, USA), Alexa-647 conjugated toceranib. Three additional dogs were considered anti-canine CD8, clone YCATE 55.9, Serotec), and only minimally evaluable because they received FITC conjugated anti-dog CD25 (clone P4A10, only the initial DOX dose and very short-term eBioscience, San Diego, CA, USA). Following toceranib before being withdrawn from the study surface staining, the cells were washed then fixed because of disease progression. Whilst these three and permeabilized using the Foxp3/Transcription dogs did have a CBC performed 7-days post-DOX factor staining buffer set (eBioscience) according with no dose-limiting AEs, no further monitor- to the manufacturer’s instructions. Samples were ing was performed. A total of 16 dogs were, there- then stained intracellularly with PE-conjugated fore, evaluable. Their demographic characteristics anti-mouse/rat Foxp3 (clone FJK-16s, eBioscience). are presented in Table 1. Of these, 12 dogs had Flow cytometry was performed using a Cyan ADP the diagnosis of neoplasia made via histopathology, flow cytometer (Beckman Coulter, Miami, FL, whereas four had the diagnosis made via cytology. USA) and the data analysed using FlowJo anal- Four dogs had received previous chemotherapy. ysis software (FlowJo, Ashland, OR, USA). Live lymphocytesweregatedbasedonforwardand Dose and safety evaluation side-scatter properties, then analysed for CD4 and CD8 expression. The percentage of Tregs was deter- The number of dogs treated in each cohort and mined based on percentage of CD4+ Tcellsthat number of DLTs per cohort are presented in expressed both Foxp3 and CD25. The percentages Table 2. AEs per cohort and grade are presented in of CD4+ and CD8+ Tcellswerealsodetermined. Table3.Nodeathsoccurredastheresultofcom- Absolute numbers of Tregs in peripheral blood bination therapy. The mean and median dosages were then calculated based on the total lymphocyte of toceranib received were 2.67 mg kg−1 PO EOD countdeterminedfromaCBCobtainedfromthe and 2.7 mg kg−1 PO EOD, respectively (range: sameblooddrawaspartoftheclinicalmonitoring. 2.25–2.81 mg kg−1 PO EOD). A total of 14 patients (87.5%) received the intended four DOX doses. One dog received only one dose and another dog Statistical analyses received three doses before disease progression was Changes in total lymphocytes and Tregs over time noted and trial participation ceased. The patient from dogs receiving the combination protocol receiving only one DOX dose did receive 21 days

Table 1. Patient demographics

Patient demographics and outcome

Age (years) Median 9.2 Range 6.7–14.8 Weight (kg) Median 28.2 Range 13.3-53.5 Sex MN 5 MI 2 FS 9 Breed Labrador retriever 5 Pit bull terrier 3 Golden retriever 2 Beagle, Brittany, GSD, Samoyed, Alaskan Malamute, mixed breed 1 each Tumour types Hemangiosarcoma 8 PFS (days) Splenic stage I 1 197 Splenic stage II 2 112, 225 Splenic stage III 2 122, 176 Right auricular 1 146 Intramuscular 1 199 Retroperitoneal 1 92 Multicentric lymphoma 3 189, 159, 64 Anal sac adenocarcinoma 2 228, 232 Prostatic carcinomaa 2 281, 135 Cutaneous lymphoma 1 22

GSD, German Shepherd Dog; MN, male neutered; MI, male intact; FD, female spayed. aThis patient also had a splenic sarcoma. of toceranib and did have appropriate monitoring doses had been well tolerated and a CBC 8 days at day 21 for AEs. The dose-limiting toxicity of after the fourth DOX dose was within normal lim- combination DOX and toceranib was determined its.Thispatientwasconcurrentlydiagnosedwith to be myelosuppression, specifically neutropenia. a urinary tract infection and it is unknown if this In the 30 mg m−2 DOX cohort, two out of five dogs contributed to the febrile neutropenic episode or developed a febrile grade 4 neutropenia 1 week was a consequence of the neutropenia. This patient after the first or second DOX dose, and one dog responded well to supportive care of hospitalization developed a febrile grade 3 neutropenia 2 weeks with intravenous fluids and antibiotics. This was the after receiving DOX. One of the patients in the only patient in the 25 mg m−2 cohort experiencing 30 mg m−2 DOX cohort who developed febrile DLT,andasaresult,theMTDofDOX,whengiven neutropenia did not have further neutropenic concurrently with toceranib at ∼2.75 mg kg−1 PO episodes with a 20% dose reduction and went on to EOD, was established as 25 mg m−2 IV every 21 receive two additional DOX doses (a total of four days. Owing to availability of funds and drugs, two doses). The other patient continued to have grade additional dogs were subsequently treated at the 25 4 febrile neutropenia episodes despite 20% dose mg m−2 cohort bringing the total number treated reductions; it should also be noted that this patient in this cohort to 8. had other febrile neutropenia episodes after other Gastrointestinal AEs (vomiting, diarrhoea, col- chemotherapy agents that were received prior to itis, hyporexia or anorexia) were also observed being enrolled in the clinical trial. Within other with the combination of DOX and toceranib. cohorts, neutropenia without fever (grade 3) and Most of these AEs were mild and self-limiting thrombocytopenia (grade 3) were also seen. One and responded to supportive care. A total of dog who received DOX at a dose of 25 mg m−2 three dogs received a 7-day toceranib drug hol- developedagrade4febrileneutropenia2weeks iday for treatment of gastrointestinal side effects following the fourth DOX dose. Previous DOX (colitis, diarrhoea and anorexia). All three dogs had

Table 2. Scheduled treatment cohorts

Toceranib dose Number of dogs Cohort (mg kg−1 EOD) DOX (mg m−2,q21days) Dogstreated experiencing DLT

1 2.75 20 3 0 2 2.75 25 8 1 3 2.75 30 5 3

Table 3. AEs by grade and cohort dog did have toceranib discontinued after 2 weeks because of persistent anorexia. This dog developed Cohort PDandonlyreceivedoneDOXdose.Another AE classiﬁcation Grade 1 (n = 3) 2 (n = 8) 3 (n = 5) dog who experienced anorexia was concurrently Neutropenia 1 1 1 1 receiving enalapril and amlodipine for proteinuria 21 3 and hypertension, and this dog’s appetite improved 312 412when these drugs were discontinued. Thrombocytopenia 1 1 2 Proteinuria was pre-existing in two dogs at 2131enrollment. Proteinuria remained stable in one dog 32for the duration of therapy. The other dog had pro- Anaemia 1 6 2 211gression of proteinuria; it is unknown whether this Anorexia 1 1 2 was due to therapy or progression of underlying 22disease. Two dogs developed proteinuria during Weight loss 1 1 the trial. An ACE inhibitor was commenced in 21 −1 Lethargy 1 both cases (enalapril 0.5 mg kg ), and proteinuria 21improved on subsequent evaluations. Vomiting 1 1 Hypertension was noted in seven patients, Diarrhoea 1 1 1 2 23although four of these patients were hypertensive at Colitis 2 3 baseline. Two patients who developed hypertension Elevated ALT 1 1 3 1 during the clinical trial had concurrent disease that 21 1 can also be associated with hypertension (diabetes Elevated AST 1 1 5 2 Elevated ALP 1 2 3 mellitus, pre-existing proteinuria) and therefore 21 it is difficult to determine the true contribution Hypertension 1 1 1 of toceranib to these AEs. All patients were able 222to have their hypertension controlled with oral 31a Elevated BUN 2 1 medical therapy (ACE inhibitors, amlodipine); Elevated creatinine 1 1 1 1 two patients did receive both medications for dual Proteinuria 1 1 control of proteinuria and hypertension. One addi- 211 tional patient received both medications for control Arrhythmia 2 1 Left ventricular 21of hypertension but this patient was hypertensive diastolic at baseline prior to receiving DOX or toceranib. dysfunction Elevated liver enzymes (ALT, ALP and AST) and ALT, alanine aminotransferase; AST, aspartate aminotrans- renal values (creatinine, BUN) were noted in a total ferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen. of 25 patients. These elevations were of low grade a Patient was hypertensive at baseline and managed with and transient. Grade 1 aspartate aminotransferase amlodipine and enalapril. (AST) elevation was most common amongst these elevations and occasionally occurred without ele- resolution of their clinical signs during the drug vations of other enzymes. These elevations have holiday and toceranib therapy was reinstituted been previously reported with toceranib and may without recrudescence of the clinical signs. One be related to the idiopathic musculoskeletal pain

© 2016 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 15, 3, 919–931 926 M. A. Pellin et al. and lameness that has been noted with use of the one intramuscular hemangiosarcoma, PFS = drug.13,24 No dogs developed musculoskeletal pain 199 days), three SD (two prostatic carcinoma, or lameness, or evidence of liver dysfunction during PFS = 281 and 135 days; 1 stage III splenic this clinical trial. Two dogs which developed ele- hemangiosarcoma, PFS = 122 days) and one PD vated liver enzymes were concomitantly diagnosed (cutaneous lymphoma) were observed. with progressive lymphoma and this could also have Eight dogs had surgical removal of all mea- contributed to the elevations. One of these dogs was surablediseaseandcommencedthetrialinthe also concurrently receiving prednisone therapy at microscopic disease setting: four splenic heman- the time of day 21 blood work due to poor appetite, giosarcoma (PFS = 112, 176, 197, 225 days), two despite this being a contraindication for the clinical anal sac adenocarcinoma (PFS = 228 and 232 days) trial. It is probable that prednisone also contributed and one each of right auricular hemangiosarcoma to elevated ALT and ALP. (PFS = 146 days) and retroperitoneal hemangiosar- One patient in the 25 mg m−2 cohort was diag- coma (PFS = 92 days). The two patients with anal nosed with supraventricular tachycardia on ECG sac adenocarcinoma were still alive and censored at after an elevated heart rate was detected on phys- time of last evaluation. The previously noted patient ical examination prior to the third DOX dose. died secondary to complications of diabetes melli- Thispatientwastreatedwithdiltiazemwhichcon- tuswithstabledisease.AllotherpatientshadPDor verted the arrhythmia to normal sinus rhythm. died due to their disease. This patient went on to have two subsequent DOX doses. This patient had progression of disease (ini- Effect of simultaneous combination treatment tially stage I splenic hemangiosarcoma) noted after on peripherally circulating lymphocytes 197daysandwassubsequentlytreatedwithmulti- and Tregs ple other chemotherapy agents (cyclophosphamide, In dogs receiving the combination of toceranib vincristine, carboplatin). Six months after receiving ∼2.75 mg kg−1 POEODandDOX25or30mgm−2 the last DOX the patient was noted to have a new IV q 21 days, no statistically significant declines arrhythmia and an echocardiogram was performed were observed in circulating total lymphocytes showing mild left ventricular systolic dysfunction. or in absolute or relative (percent) Treg lympho- This was treated with mexiletine and pimobendan. cyte numbers, when measured at 21 day intervals This was believed to be secondary to DOX; no right (Figure 1). PBMCs and therefore Tregs were not atrial mass was seen. The patient was ultimately collected in patients receiving the lowest DOX euthanized due to PD. cohort 20 mg m−2. The patient in the 30 mg m−2 cohort who experienced multiple episodes of febrile neutropenia Discussion despite DOX dose reductions was diagnosed with severe left ventricular systolic dysfunction and mild The MTD for the combination protocol was deter- congestive heart failure after presenting for cough mined to be DOX administered intravenously at 25 and syncopal episodes 2 months after the fourth mg m−2 every 21 days and toceranib administered DOX dose. This was believed to be secondary to PO at 2.75 mg kg−1 EOD. The DLT of the combi- DOX. The patient was successfully treated for heart nation of DOX and toceranib was myelosuppres- failure and died due to complications from diabetes sion,specificallyneutropenia.NoAEswerenoted mellitus. with this combination that would not have been predicted based upon the known AE profiles for either drug. It should be noted that some dogs did Response and outcome assessment tolerate the higher (30 mg m−2)DOXdoseincom- Eight dogs had macroscopic measurable disease at bination with toceranib and therefore, as with what the time of enrollment. Of these, one CR (multi- should be standard oncology practice, DOX dose centric lymphoma, PFS = 189 days), three PR (two increasesshouldbeconsideredinthosepatientstol- multicentric lymphoma, PFS = 159 and 64 days; erating 25 mg m−1 when a lack of appropriate nadir

A B C Total Lymphocytes Absolute Tregs % Tregs P = 0.29 P = 0.69 P = 0.96 4000 200 15

3000 150 10 2000 100

% Tregs 5 1000 50

0 0 0 Lymphocytes (cells/uL) Absolute Tregs (cells/uL) pre 21 42 63 84 pre 21 42 63 84 pre 21 42 63 84 Days Post-Treatment Days Post-Treatment Days Post-Treatment

Figure 1. Box and traditional Tukey whiskers plots of total lymphocytes and Treg numbers and proportion following simultaneous combination of DOX and toceranib in tumour-bearing dogs. The medians, 75 percentile (box) and whiskers (1.5 times the interquartile distance or the highest or lowest point, whichever is shorter) are depicted. (A) Total circulating lymphocytes did not change significantly over time after treatment (P = 0.29). (B) Absolute Tregs did not change significantly over time after treatmentP ( = 0.69). (C) Relative (percent) Tregs did not change significantly over time after treatment (P = 0.96). is observed in order to ensure maximum potential masitinib have specifically been shown to inhibit efficacy. MDR systems.32–36 As DOX is a known MDR The DLTs of neutropenia observed in this study substrate of both ABCB1 and ABCG237 this could, in dogs in the 30 mg m−2 DOX cohort along with in part, explain the enhanced myelosuppression 2.75 mg kg−1 toceranib was more marked than is seen in combination with toceranib. Theoretically, known to occur with either agent alone at those thismayalsoleadtodecreasedincidenceofDOX dosages. Previous trials performed by our group resistance and increased tumour cytotoxicity when and others have also showed apparent sensitiza- used in combination, although no supporting tion of the myeloid compartment by toceranib as evidence for this theory is nor would be evident evidenced by enhanced myelosuppression noted inasmallphaseIstudysuchasthis.Whilemost when toceranib is used in combination with episodes of neutropenia or thrombocytopenia CCNU and vinblastine.2–4 In human clinical tri- occurred at the expected nadir for DOX (day 7), als, similar findings have been observed with the two patients did appear to have a more delayed combination of cytotoxic agents and RTKIs includ- nadir. This can occasionally be seen secondary to ing gefitinib/gemcitabine, gefitinib/vinorelbine, DOX administration, but it is possible that the erlotinib/vinorelbine and vinca alkaloids/EGFR toceranib administration could have affected this inhibitor combination protocols.25 –28 One sug- as well. gested mechanism for this increased haematologic When used in a single agent setting both of these toxicity is that the RTKIs, DOX and vinca alkaloids drugs can lead to gastrointestinal toxicity, there- are all metabolized by CYP3A4 enzymes, result- fore potentiation of these toxicities was a concern ing in prolonged systemic exposure to the active with combination. While gastrointestinal toxicities metabolites due to saturation of hepatic metabolism were seen in a number of patients, they were mild when these agents are combined.29,30 Another and transient and were successfully managed with potential explanation is that toceranib and similar symptomatic care and supportive medications. drugs inhibit colony stimulating factor-1 receptor, Cardiotoxicity is an established cumulative which could exacerbate the level of neutropenia toxicity of DOX and has been reported in human seen when combined with cytotoxic drugs.24 In patients with RTKI use.14 Possible DOX-related addition, several RTKIs have been shown to inhibit cardiotoxicity was seen in two patients in this ATP-binding cassette (ABC) multidrug resistant study. Neither patient succumbed to cardiac dis- transporters (MDR-ABC proteins).31 Sunitinib, ease. Post-mortem exam was not performed in a RTKI that targets identical RTKI receptors either patient, so it is difficult to truly establish as toceranib, as well as imatinib mesylate and whether this was secondary to DOX or other

© 2016 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 15, 3, 919–931 928 M. A. Pellin et al. underlying disease. Given the short follow-up total lymphocyte and absolute Treg subpopula- period inherent in phase I trials, it is not possible to tions, did not selectively decrease Treg numbers adequately access whether the combination of DOX and therefore, did not result in a decrease in the and toceranib alters the severity or incidence of percent of Treg populations as is reported to occur DOX associated cumulative cardiac changes; such following metronomic cyclophosphamide.21 Here, a characterization will await further evaluations in it was hypothesized that the addition of MTD-DOX a phase II/III trial context. resulted in indiscriminant lymphocyte depletion While efficacy is not a primary endpoint of phase which overshadowed the selective Treg depletions I clinical trials, antitumour activity was observed in observed when metronomic cyclophosphamide is the majority of cases treated in the macroscopic dis- used alone.21 Thisfinding,however,wasincontrast ease setting with this combination. Current dogma to another report by Mitchell et al.22 who found instructs that antitumour activity may be compro- that in a small number of dogs with lymphoma, mised if dose reductions are required to safely com- asingletreatmentofMTD–DOXresultedina bine active agents into a combination protocol. This selective decrease in peripherally circulating Tregs is of concern as in all trials investigating the concur- at day 7 post-treatment. The lack of observable rent combination of toceranib with standard cyto- declines in total lymphocytes, as well as absolute toxic agents (e.g. vinblastine, CCNU).2–4 enhanced and relative (percent) Treg numbers, in this study myelosuppression has been observed, resulting in could be the result of some as of yet unknown inter- anMTDforthecytotoxicagentthatisapproxi- action between the two agents used including the mately 20% less than that when the agent was used possibility that the Treg subset of lymphocytes is as a single-agent. However, this concern may be more sensitive to storage and processing. Any com- overstated owing to the response rates observed in parisons between this study and those of Mitchell these trials despite the dose reduction, and the inhi- andextrapolatedconclusionsare,however,inher- bition of MDR systems that may result in enhanced ently flawed because of the varied tumour types activity when RTKIs are used in combination with included, small population numbers, and the fact MDR substrate chemotherapeutics. Further explo- that in this study lymphocyte subpopulations were ration of efficacy with these combinations in the quantified 21 days after treatment initiation, rather context of phases II and III clinical trials will be nec- than at 14 days as in the Mitchell study.15 essary to determine if improved response rates and Thelimitationsofthisstudyaresimilartothose durations result. This study, as with any phase I trial inherent in all phase I dose-finding clinical tri- with small patient numbers, a variety of tumour als. These include small patient numbers and short types, and short follow-up time, is not designed to follow-up periods. Phases II and III clinical trials show improved survival times. are needed to more accurately assess response rates No significant declines in absolute or relative and durability, as well as the combination protocol’s (percent) Treg lymphocytes were observed in dogs long-term AE profile. undergoing combination therapy in this study. Pre- viously, Mitchell reported statistically significant Conclusions decreases in both absolute and relative (percent) Tregs in dogs receiving single-agent toceranib at In general, the combination of DOX and toceranib similar doses to those utilized in this study when waswelltolerated.TheMTDandscheduleforcom- measured at 14 days post-initiation of toceranib bination was determined to be DOX 25 mg m−2 therapy.15 Thisdeclinewasmaintainedforupto every 21 days and toceranib 2.75 mg kg−1 EOD. This 56 days, but interpretation of subsequent mea- concurrent combination shows an approximately surements were complicated by the addition of 17% dose reduction for DOX when compared metronomic cyclophosphamide beginning at day with its MTD as a single-agent. Despite this dose 14. Rasmussen et al.38 reported that the addi- reduction, responses to the protocol were seen and tion of MTD-DOX treatment to metronomic enhanced myelosuppression was noted, indirectly cyclophosphamide, while resulting in decreased suggesting activity. Prospective randomized phase

II/III trials are needed to further characterize WithrowandDMVailEds.,Philadelphia,WB activity and the chronic/cumulative AE profile of Saunders, 2013: 157–172. the combination relative to single-agent protocols. 7. Ogilvie GK, Reynolds HA, Richardson RC, et al. Phase II evaluation of doxorubicin for treatment of various canine neoplasms. Journal of the American Acknowledgements Veterinary Medical Association 1989; 195: 1580–1583. ThisstudywassupportedbyaZoetisAnimal 8. Ogilvie GK, Richardson RC, Curtis CR, et al. Acute Health Canine Oncology Resident Research Grant and short-term toxicoses associated with the and the Barbara A. Suran Comparative Oncology administration of doxorubicin to dogs with Research endowment at the University of Wiscon- malignant tumors. Journal of the American sin, School of Veterinary Medicine. The authors Veterinary Medical Association 1989; 195: 1584–1587. further acknowledge Jonathan Coy, whose techni- 9.LiaoAT,ChienMB,ShenoyN,MendelDB, cal expertise in Treg staining and flow-cytometric McMahon G, Cherrington JM, London CA. analysis was greatly appreciated. The funding agen- Inhibition of constitutively active forms of mutant ciesplayednoroleinstudydesign,caseaccrual, kit by multitargeted indolinone tyrosine kinase data collection and analysis, the decision to publish, inhibitors. Blood 2002; 100: 585–593. or preparation of the manuscript. doi:10.1182/blood-2001-12-0350. 10. London CA, Hannah AL, Zadovoskaya R, Chien MB, Kollias-Baker C, Rosenberg M, et al. Phase I References dose-escalating study of SU11654, a small molecule 1. DeVita VT and Chu E. Principles of medical receptor tyrosine kinase inhibitor, in dogs with oncology. In: Cancer: Principles and Practice of spontaneous malignancies. Clinical Cancer Research Oncology.8th edn., VT DeVita and SA Rosenberg 2003; 9: 2755–2768. Eds., Philadelphia, Lippincott-Raven Publishers, 11. Lemmon MA and Schlessinger J. Cell signaling by 2008: 340–342. receptor tyrosine kinases. Cell 2010; 141: 2. Pan X, Tsimbas K, Kurzman ID and Vail DM. Safety 1117–1134. doi:10.1016/j.cell.2010.06.011. evaluation of combination CCNU and continuous 12. London C, Mathie T, Stingle N, Clifford C, Haney S, toceranib phosphate (Palladia®)intumour-bearing Klein MK, et al. Preliminary evidence for biologic dogs: a phase I dose-finding study. Veterinary and activity of toceranib phosphate (Palladia®)insolid Comparative Oncology 2014. doi:10.1111/vco.12091. tumours. Veterinary and Comparative Oncology [Epub ahead of print] PMID: 24735385 2012; 10: 194–205. 3. Robat C, London C, Bunting L, McCartan L, Stingle doi:10.1111/j.1476-5829.2011.00275.x. N, Selting K, et al. Safety evaluation of combination 13.BernabeLF,PortelaR,NguyenS,KisseberthWC, vinblastine and toceranib phosphate (Palladia®)in Pennell M, Yancey MF, and London CA. Evaluation dogs: a phase I dose-finding study. Veterinary and of the adverse event profile and pharmacodynamics Comparative Oncology 2012; 10: 174–183. of toceranib phosphate administered to dogs with doi:10.1111/j.1476-5829.2011.00261.x. solid tumors at doses below the maximum tolerated 4. Burton JH, Venable RO, Vail DM, et al. dose. BMC Veterinary Research 2013; 9: 190. Pulse-administered toceranib phosphate plus doi:10.1186/1746-6148-9-190. lomustine for treatment of unresectable mast cell 14. Bronte G, Bronte E, Novo G, Pernice G, Lo Vullo F, tumors in dogs. Journal of Veterinary Internal Musso E, et al. Conquests and perspectives of Medicine 2015; 29: 1098–1104. cardio-oncology in the field of tumor doi:10.1111/jvim.13573. angiogenesis-targeting tyrosine kinase 5. Chon E, McCartan L, Kubicek LN and Vail DM. inhibitor-based therapy. Expert Opinion on Drug Safety evaluation of combination toceranib Safety 2015; 14: 253–267. phosphate (Palladia®)andpiroxicamin 15. Mitchell L, Thamm DH and Biller BJ. Clinical and tumour-bearing dogs (excluding mast cell immunomodulatory effects of toceranib combined tumours): a phase I dose-finding study. Veterinary with low-dose cyclophosphamide in dogs with and Comparative Oncology 2012; 10: 184–193. cancer. Journal of Veterinary Internal Medicine 2012; doi:10.1111/j.1476-5829.2011.00265. 26: 355–362. doi:10.1111/j.1939-1676.2011.00883.x. 6. Gustafson DL and Page RL. Cancer chemotherapy. 16. Thamm DT and Vail DM. Veterinary oncology In: Small Animal Clinical Oncology.5th edn., SJ clinical trials: design and implementation.

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