• Open Access

An Open-Label, Dose-Escalating Phase I Study of Elsamitrucin (SPI 28090) in Treatment of Malignant Solid Tumors in Dogs

Authors


  • This work was performed at Veterinary Cancer Group, Tustin, CA 92780; Veterinary Cancer Group, Culver City, CA 90232; University of Missouri, Department of Veterinary Medicine and Surgery, Columbia, MO 65211.

Corresponding author: S. C. Fiocchi, DVM, Veterinary Cancer Group, 2887 Edinger Avenue, Tustin, CA 92627; e-mail: sfiocchi@vetcancergroup.com.

Abstract

Background: Elsamitrucin, the most potent topoisomerase II inhibitor available, is unique in that it does not cause neutropenia or cardiotoxicosis. It has antitumor activity in human patients with relapsed or refractory non-Hodgkin's lymphoma.

Objectives: To determine the maximum tolerated dose (MTD), safety, and toxicity of elsamitrucin when administered to tumor-bearing dogs and to evaluate the incidence and severity of adverse events.

Animals: Twenty client-owned dogs with spontaneous malignant solid tumors or lymphoma that were refractory to, or for which the owner declined, conventional therapy were enrolled.

Methods: Prospective, open-label, single-agent study. Escalating doses of elsamitrucin were administered once weekly IV for up to 16 weeks in a modified 3 + 3 Phase I design. The starting dose was 0.06 mg/kg with escalation to 0.08 and 0.09 mg/kg. Dogs that remained on the study were monitored for evidence of toxicoses for at least 4 weeks and for survival every 2 months.

Results: Serious adverse events (SAEs) possibly attributable to elsamitrucin include: 1 dog developed heart failure and another developed hepatotoxicosis manifested by increased alanine aminotransferase, alkaline phosphatase, and total bilirubin (0.06 mg/kg dose); 1 dog developed severe anorexia and diarrhea, another developed severe diarrhea alone, and a 3rd dog went into cardiac arrest (0.09 mg/kg dose). A dose of 0.08 mg/kg was well tolerated with no SAEs.

Conclusions and Clinical Importance: The MTD and recommended dose for Phase II trials of elsamitrucin is 0.08 mg/kg IV weekly. Elsamitrucin might be considered for combination protocols with myelosuppressive chemotherapy agents.

Abbreviations:
AE

adverse event

ALP

alkaline phosphatase

ALT

alanine aminotransferase

BUN

blood (serum or plasma) urea nitrogen

DLT

dose-limiting toxicity

MTD

maximum tolerated dose

RECIST

response evaluation criteria in solid tumors

SAE

serious adverse event

ULN

upper limit of normal

VCOG-CTCAE

Veterinary Co-operative Oncology Group-Common Terminology Criteria for Adverse Events

Elsamitrucin (SPI 28090, BMY-28090, elsamicin A) is an antitumor antibiotic that is more water-soluble and 10–30 times more potent than chartreusin, to which it is structurally related.1 It is the most potent inhibitor of topoisomerase II yet discovered.2

Elsamitrucin exhibits strong inhibitory activity against a variety of murine and human tumor cell lines in vitro1 and has also been evaluated in murine tumors and human tumor xenografts in vivo.3 In preclinical testing, significant survival prolongation, median tumor weight inhibition, or both were observed in elsamitrucin-treated mouse models.

In Phase I trials conducted in human patients, elsamitrucin's dose-limiting toxicity (DLT) was reversible hepatic dysfunction manifested by increased transaminase levels. The increase in transaminase activity was rapid, peaking at day 3, and transient, recovering by day 14. Additionally, there was no evidence of cumulative hepatic dysfunction. Other toxicoses included nausea, vomiting, fatigue, and phlebitis.4 At a dose of 25 mg/m2 (equivalent to 0.65 mg/kg for the average person) IV weekly, the most common toxicoses seen in Phase II studies were nausea, vomiting, or both, lethargy, injection site reaction characterized by redness, swelling, or phlebitis, and less frequently reversible hepatotoxicosis and fatigue.5–7 Elsamitrucin demonstrated modest activity in patients with relapsed or refractory non-Hodgkin's lymphoma. In a Phase II trial of 31 patients with refractory or relapsed NHL, 4 (13%) patients had ≥50% reduction in tumor size (durations of 5, 8, 16, and 30 weeks) and 11 (35%) had stable disease (median duration 8 weeks, range 3–18).5

In a single-dose dose ranging study performed in normal dogs,a overt clinical toxicosis was observed only in dogs given more than 0.08 mg/kg of elsamitrucin. Dogs given doses of ≥0.16 mg/kg rapidly developed emesis, pallor, or both with labored respirations and generally died within 3 days of dosing. Severe hemorrhage and necrosis of the gastrointestinal tract were observed, as were hydrothorax, congestion of the mucosa of the stomach and small intestine, pulmonary edema, and hepatic and renal congestion. Transient decreases in food intake and weight loss were seen at the 0.094 mg/kg dose level, and higher doses caused clinicopathologic changes including neutrophilic leukocytosis, lymphopenia, and hemoconcentration. No consistent pattern of abnormalities was observed in serum chemistries, liver function, or renal function.

The objectives of the following Phase I study were (1) to determine the maximum tolerated dose (MTD), safety, and toxicoses of elsamitrucin when administered weekly to tumor-bearing dogs that had failed or declined standard therapy and (2) to evaluate the incidence and severity of adverse events (AEs) related to elsamitrucin administration. As such, no formal hypothesis testing was performed.

Methods and Materials

Animals

Elsamitrucinb was administered to client-owned dogs that had histologically or cytologically confirmed spontaneous malignancies that were refractory to, or for which the owner declined, conventional therapy. Inclusion criteria included a neutrophil count >2,000 cells/μL, platelet count ≥100,000 cells/μL, bilirubin ≤2.0 mg/dL, alanine aminotransferase (ALT) ≤3 × the upper limit of normal (ULN), creatinine ≤1.25 × ULN, serum or plasma urea nitrogen (BUN) <45 mg/dL, and an expected minimum survival ≥6 weeks without treatment. Exclusion criteria included administration of steroids or nonsteroidal anti-inflammatory medications within 7 days of entry, chemotherapy or radiation therapy within the preceding 3 weeks, uncontrolled hypertension, or any complicating comorbidities. Additionally, neither the pet nor the owner could be pregnant or lactating. The study was a prospective, bi-institutional, Phase I clinical trial carried out at the Veterinary Cancer Group in Tustin, CA and Culver City, CA and the University of Missouri-Columbia Veterinary Medical Teaching Hospital. Written informed consent was obtained from all clients before dogs entered the study.

Study Design

This study was a modified Phase I dose-escalating clinical trial with 3 dogs per dosing cohort. Elsamitrucin was administered weekly for at least 4 weeks, and 3 additional treatment cycles were allowed in dogs that had no evidence of tumor progression and did not experience a DLT. The maximum total treatment time allowed was 16 weeks.

Drug doses used were 0.06, 0.08, and 0.09 mg/kg body weight, administered as a 10-minute IV infusion via the side port of free-flowing D5W. The starting dose of 0.06 mg/kg was chosen after a dog in a field study received 0.05–0.06 mg/kg weekly for several weeks without any adverse effects (unpublished, personal communication with Spectrum Pharmaceuticals). Dosage was escalated in decreasing increments as is standard for a Phase I trial, although the authors did not follow a modified Fibonacci escalation design since, as a result of preclinical studies, it was believed that the MTD would be within the planned dosing range. Additionally, we felt that fewer numbers of decimals would decrease the risk of calculation errors.

Three dogs were treated per dosing cohort and escalation to the next cohort was not permitted until at least 1 dog had completed 4 weeks of treatment in any given cohort level. When 1 of 3 dogs experienced a DLT (grade 3 or higher), another 3 dogs were treated at that level before further dose escalation. When 2 dogs experienced a DLT, the previous dose was considered to be the MTD and 3 additional dogs were treated at that dose. A modification to the standard 3 + 3 design was made in the 0.06 mg/kg dosing cohort because AEs (n = 3 of the first 6 dogs treated) were difficult to attribute to elsamitrucin. AEs in which attribution was difficult are described below. An additional cohort of 3 dogs was enrolled in the 0.06 mg/kg dosing cohort for a total of 9 dogs treated at that level. In the final 0.06 mg/kg cohort, 1 of the 3 dogs had serious adverse events (SAEs) that were attributed to disease (vomiting, melena, hypotension, anorexia) as they were consistent with mast cell tumor degranulation (either because of effects of drug on the tumor, or due to the tumor itself). Given that the AEs were difficult to clearly attribute to elsamitrucin, and given that in the dose-ranging study no AEs were seen at doses <0.08 mg/kg, escalation was permitted to the next cohort.

AEs were recorded and graded by the treating clinician according to the Veterinary Co-operative Oncology Group-Common Terminology Criteria for Adverse Events (VCOG-CTCAE) v1.0.8 An AE was attributed either to elsamitrucin, to disease, or to an unrelated cause. Using timing and expected AEs, attribution was assigned (1) by the treating clinician at the time of clinical evaluation and (2) by the Principal Investigator after case review. Attribution standards were divided into 5 categories: unrelated, unlikely, possibly, probably, or definitely related (personal communication, VCOG-CTCAE Consensus Committee).

Treatment was stopped if there was progressive disease, life-threatening toxicoses, a treatment delay >14 days, or study exit initiated by the owner or clinician for noncompliance or any other reason. After trial discharge, dogs were to be followed for toxicoses for at least 4 weeks and for survival every 2 months until 1 year after study enrollment. The study remained open until all dogs had progressive disease, had withdrawn from the study or had completed 1 year of follow-up. Dogs that were lost to follow-up or those that were still alive at the time of writing were censored.

Prestudy Evaluation

At the time of study enrollment a complete history was obtained and a physical examination, baseline CBC, serum biochemistry panel, urinalysis, and indirect blood pressure measurement were performed. Depending on the diagnosis, additional staging tests were also completed to allow determination of the extent of disease as this might impact drug pharmacokinetics and toxicity attribution.

Toxicity Assessment

Owners completed a weekly diary in which they graded appetite, attitude, and activity level as excellent, good, decreased, or poor. Dogs were evaluated weekly by review of the diary, physical examination, CBC, and liver function tests (SGPT/ALT and alkaline phosphatase [ALP]); urinalysis and chemistry profile were reevaluated every 4 weeks. AEs were recorded and were graded according to VCOG-CTCAE v1.0.8

Tumor Response Assessment

Target lesions were selected based on accessibility and ability to reproduce measurements; a minimum diameter of 20 mm was required. Lesions were assessed by response evaluation criteria in solid tumors (RECIST).9 The measurement used for response evaluation was the sum total of the lengths of all target lesions, recorded as the sum longest diameter. Nontarget lesions were recorded at the clinician's discretion, and if smaller than 20 mm were monitored by volume (length × width × height/depth ×π/6). Tumor measurements were taken via calipers or ultrasound, before initiation of treatment and every 4 weeks thereafter. Efficacy was evaluated after the first 4 weeks of treatment (ie, dogs must have maintained an objective response for a minimum of 4 weeks to consider the response durable). Responses were assessed by the investigator on the basis of RECIST criteria.

Results

Dog Population

Baseline information for the 20 dogs enrolled into the study is as follows. Median age was 7 (4.3–11) years. There were 11 spayed females, 8 castrated males, and 1 intact male. Tumor types included multicentric lymphoma (n = 9), mast cell tumor (n = 4), apocrine gland anal sac adenocarcinoma (n = 2), soft tissue sarcoma (n = 2), epitheliotropic lymphoma (n = 1), SC hemangiosarcoma (n = 1), and malignant melanoma of the digital pad (n = 1). Prior treatments included surgery in 8 dogs and chemotherapy in 9 dogs. None of the dogs had received radiation therapy before study enrollment. A total of 9 dogs were treated at 0.06 mg/kg, 6 dogs at 0.08 mg/kg, and 5 dogs at 0.09 mg/kg. Only 2 dogs in the study received the maximum allowed 16 doses of elsamitrucin: one, in the 0.08 mg/kg cohort, withdrew from the study after receiving the maximum allowed 16 doses of elsamitrucin to pursue other chemotherapy for multiple mast cell tumors (119 days after entering the study); the other, in the 0.06 mg/kg cohort, remained in the study (pursued no additional treatment after 16 weeks of elsamitrucin therapy) and was euthanized because of progressive disease 195 days after entering the study. Although 3 dogs (1 MCT, 2 lymphoma) lived longer than 1 year beyond study initiation, those dogs had been withdrawn from the study to pursue other therapeutic options. None of the dogs remained on the study for 1 year. Reasons for discontinuation included disease progression (n = 13), SAE (n = 5), and study exit by the pet owner (n = 2).

Toxicity

AEs were recorded, graded, and assigned attribution as described above. AEs, based on dose, are listed in Table 1; Table 2 lists only SAEs. Those AEs that are listed in Table 1 but not in Table 2 were mild to moderate (grades 1 or 2). Specific AEs are described below by system. The total number of treatments with elsamitrucin by dosing cohort was 46 doses in the 0.06 mg/kg group, 37 doses in the 0.08 mg/kg group, and 25 doses in the 0.09 mg/kg group. In no case was a delayed toxicosis (more than 4 weeks after treatment with elsamitrucin) reported. A dose of 0.08 mg/kg was well tolerated in all dogs treated at that dose.

Table 1.   Adverse events (grades 1–5) attributed to elsamitrucin by dose.
Toxicosis TypeSign/ParameterDose Level (mg/kg)
0.06 (n = 9)0.08 (n = 6)0.09 (n = 5)
  1. n, the number of dogs in each dose level; %, the percent of dogs that experienced the specified adverse event at each dose level.

GastrointestinalAnorexia2 (22%)3 (50%)1 (20%)
Vomiting1 (11%)2 (33%)1 (20%)
Diarrhea01 (17%)2 (40%)
HepaticIncreased ALT3 (33%)1 (17%)0
Increased ALP2 (22%)00
Increased bilirubin1 (11%)00
RenalIncreased creatinine02 (33%)0
Increased BUN2 (22%)3 (50%)0
CardiopulmonaryCardiopulmonary arrest001 (20%)
Cardiac failure1 (11%)00
Hypotension1 (11%)00
HematologicAnemia1 (11%)00
Other laboratoryHyperkalemia1 (11%)00
Other signsWeight loss1 (11%)00
Injection site edema1 (11%)00
Behavior change001 (20%)
Table 2.   Serious adverse events (grades 3–5) attributed to elsamitrucin by dose.
Toxicosis TypeSign/ParameterDose Level (mg/kg)
0.06 (n = 9)0.08 (n = 6)0.09 (n = 5)
  1. n, the number of dogs in each dose level; %, the percent of dogs that experienced the specified adverse event at each dose level.

GastrointestinalAnorexia001 (20%)
Diarrhea002 (40%)
HepaticElevated ALP1 (11%)00
Elevated bilirubin1 (11%)00
CardiopulmonaryCardiopulmonary arrest001 (20%)
Cardiac failure1 (11%)00
Hypotension1 (11%)00

Hepatic. Eight days after receiving a single dose of elsamitrucin, a dog with lymphoma developed grade 4 ALP, grade 3 bilirubin, and grade 2 ALT increases in addition to grade 1 hypoalbuminemia. The dog did not have sonographic evidence of lymphoma in the liver and hence the increases were deemed possibly related to the study drug. Another dog had a grade 3 ALP increase that was not attributed to elsamitrucin because it was pre-existing. Both dogs were in the 0.06 mg/kg dosing cohort, and no SAEs related to the liver were reported in the 0.08 and 0.09 mg/kg dosing cohorts. Other changes in liver values (n = 1) that were attributed to elsamitrucin were grades 1–2.

Gastrointestinal. Nausea and vomiting were observed in dogs at each dosing level but were dose-limiting only at 0.09 mg/kg. One dog developed diarrhea after 3 of his 5 doses of elsamitrucin and 1 of those episodes was serious (grade 3). The dog also developed low-grade anorexia which became more serious as his disease (hypercalcemic lymphoma) progressed. The diarrhea and anorexia were deemed possibly related to the study drug. Another dog was euthanized 3 days after a single dose of elsamitrucin because of anorexia and severe weakness. On necropsy the dog had gastrointestinal hemorrhage (the inner lumen of duodenum was grossly hemorrhagic) despite having had no clinical sign of gastrointestinal hemorrhage. The dog's disease was melanoma of the digital pad with pulmonary metastasis. The gastrointestinal hemorrhage was attributed to elsamitrucin, although the anorexia and weakness were attributed to disease because they were pre-existing, which the owner disclosed only after euthanasia. In the 0.06 mg/kg dosing cohort, a dog that had a recurrent, metastatic mast cell tumor developed hypotension, and clinical signs associated with grade 2 gastrointestinal hemorrhage (melena, vomitus with coffee ground appearance, anorexia, anemia) and was euthanized 3 days after receiving a single dose of elsamitrucin. On necropsy the dog had a splenic hemangiosarcoma in addition to mildly congested lungs, a mildly edematous myocardium, and no significant microscopic changes in sections of liver, kidney, or intestine. Because the clinical signs were typical of a dog with mast cell tumor degranulation, the AEs were deemed unlikely related to the study drug.

Six dogs developed mild, transient increases in BUN (3 in the 0.06 mg/kg cohort and 3 in the 0.08 mg/kg cohort), 3 of which did not have concurrent increases in creatinine. Because these changes were mild and transient, they did not determine whether to advance to the subsequent dosing level.

Cardiopulmonary. Two SAEs related to the cardiopulmonary system were difficult to attribute to elsamitrucin. In the 0.06 mg/kg dosing cohort, a dog with lymphoma had previously received a total cumulative doxorubicin dose of 240 mg/m2 IV and had a grade 4 of 6 heart murmur at the initiation of the study. The heart murmur did not change after receiving the 1st and 2nd doses of elsamitrucin, but the day after receiving the 3rd dose, the dog was examined for coughing. Heart failure with hypotension was diagnosed, treatment was initiated, and the dog lived an additional 4 months. The progression to heart failure was deemed possibly related to elsamitrucin but it could have resulted from progression of the dog's pre-existing cardiac disease. In the 0.09 mg/kg dosing cohort a dog with a recurrent, metastatic mast cell tumor, and no history or prior clinical signs of cardiac disease presented to his local emergency clinic 3 days after receiving a single dose of elsamitrucin, with a <24-hour history of dyspnea. Upon presentation to the emergency clinic the dog was severely dyspneic and pale. There was no indication of a heart murmur and the heart rate was 130 bpm. The dog's breathing became agonal shortly after presentation and it soon went into cardiac and respiratory arrest. Attempts at resuscitation were unsuccessful. The emergency clinician was not made aware of the study's requirement for necropsy and no necropsy was performed. Nine days prior, thoracic radiographs had shown a narrowing of the dog's trachea consistent with upper airway collapse. This had been diagnosed on previous radiographs and was consistent with changes that are typical of this breed (Pug); no cardiovascular abnormalities had been noted. The dog's electrocardiogram and blood pressure at study entry did not reveal abnormalities. The dog had received previously vinblastine, lomustine, cyclophosphamide, and chlorambucil to treat the mast cell tumor but none of those had been given within 5 months of his death. The cause of the cardiopulmonary arrest was unclear. Mast cell degranulation, elsamitrucin toxicosis, and the dog's pre-existing airway disease were all considered as possible causes of the arrest. Considered less likely but still possible for sudden death are fatal arrhythmia and thromboembolic event.

Renal. There was no SAE related to the renal system; however, 5 dogs developed mild, transient increases in BUN, and 3 dogs demonstrated mild increases in creatinine. The BUN increases might have been unrelated to the renal system, as gastrointestinal hemorrhage can also result in an increased BUN.

Hematologic. Hematologic toxicoses were minimal. It is concluded that elsamitrucin is not myelosuppressive at the doses tested.

Other. One dog experienced grade 2 injection site edema which did not result in permanent tissue necrosis. Elsamitrucin is a vesicant and the edema was probably related to elsamitrucin administration. AEs that were attributed to something other than elsamitrucin were uncommon and include a fatal seizure, a urinary tract infection, polyuria/polydipsia, lethargy, weakness, fever, inflamed skin not limited to the injection site, hyphema, and glaucoma. Most of these events were attributed to disease progression. Specifically, the seizure occurred in a dog with lymphoma in the 0.06 mg/kg dosing cohort, 4 days after its 7th weekly dose of elsamitrucin. Seizure is an unexpected toxicity and was thought to possibly represent progressive lymphoma. Even if related to elsamitrucin, this AE was so unique and unexpected that it might not define MTD.

Electrolyte abnormalities consisting of a minor deviation from the reference range were not considered to be reportable AEs; hypokalemia and hypercalcemia were seen in 1 dog each and were not attributed to the study drug. Although minor and not considered a significant AE, mild hyperchloridemia (1–4 U above the reference range) was noted 13 times in 6 dogs. Although additional minor laboratory abnormalities were noted in some dogs, none were the cause of morbidity or poor performance status, and all were pre-existing and did not change following study enrollment.

Responses

Although evaluation of tumor response was not an objective of this study, dogs were evaluated for response according to the RECIST criteria.9 There were no complete or partial responses. Stable disease was seen in 6 dogs with the following neoplastic diseases: lymphoma (n = 2), mast cell tumor (n = 2), apocrine gland anal sac adenocarcinoma (n = 1), and epitheliotropic lymphoma (n = 1). Progressive disease was recorded in 11 dogs. Responses were not evaluable in 3 dogs because of death (n = 2) or withdrawal from the study (n = 1) <4 weeks after study enrollment.

Discussion

Elsamitrucin is a novel chemotherapy agent that exhibits in vitro cytotoxicity and in vivo antitumor activity. In this Phase I study, dogs treated at a dose of 0.08 mg/kg given weekly did not develop SAEs. The most common toxicoses in this study included vomiting, anorexia, and diarrhea. Less frequently, increases in ALT, ALP, bilirubin, BUN, and creatinine were noted. Heart failure, respiratory compromise, and seizure were each seen in 1 dog. As has been reported previously, elsamitrucin did not cause myelosuppression.

Gastrointestinal toxicosis is a known AE of DNA intercalating agents and was the most common toxicosis noted in this study. Most gastrointestinal effects were mild and transient; however, gastrointestinal hemorrhage was seen in 2 dogs 3–4 days after receiving a single dose of elsamitrucin. In the dog with metastatic mast cell tumor in the 0.06 mg/kg dosing cohort that had melena, vomitus with coffee ground appearance, anorexia, anemia, azotemia, and hypotension, the clinical signs were deemed unlikely related to the study drug because they were typical of a dog with mast cell tumor degranulation. Mast cell tumor degranulation might have been caused by the effect of elsamitrucin on the tumor, or it might have been unrelated. Because gastrointestinal toxicosis is a known AE of the study drug, relatedness of these SAEs to the study drug could not be entirely ruled out.

In a Phase I trial in human patients, the DLT was hepatic dysfunction manifested by transient transaminase (ALT and AST) increases.4 In single-dose toxicology studies performed in Beagle dogs, although significant increases in transaminases were generally not observed, at necropsy a marked, diffuse hepatocellular swelling was seen in the livers of dogs that received doses ≥0.08 mg/kg.a In our study, 1 dog had biochemical evidence of hepatic dysfunction (increased ALP, ALT, and bilirubin) 1 week after receiving a single dose of elsamitrucin; he was withdrawn from the study because of progressive disease (hyphema, lymphocytosis) and died the following day. At the time of study withdrawal, the liver was sonographically unchanged compared with the week prior (when the liver enzymes were within reference range) and was not further examined. The hepatic dysfunction was deemed possibly related to elsamitrucin and no other hepatic SAEs were attributed to elsamitrucin.

Heart failure and hypotension are not reported sequelae of elsamitrucin administration; cardiotoxicosis is a known risk of anthracyclines. The dog in this study that had previously received 240 mg/m2 of doxorubicin and had a grade 4 of 6 heart murmur at the initiation of the study developed congestive heart failure after receiving 3 doses of 0.06 mg/kg elsamitrucin. In a pharmacodynamic safety assessment in dogs, elsamitrucin administered by single IV injection to anesthetized dogs at 0.1 mg/kg did not appear to induce alterations in cardiovascular functions based on cardiac and hemodynamic system function tests including blood pressure, heart rate, ECG, cardiac output, total peripheral resistance, stroke volume, left ventricular work, and left ventricular contractility and relaxation parameters.a Given the lack of prior reports of cardiovascular toxicosis in multiple species, in addition to the high-cumulative dose of doxorubicin that the dog had received before entering the study, the heart failure was difficult to attribute to the study drug. The SAE was deemed possibly related, considering that elsamitrucin may have in some way caused the heart to decompensate.

Respiratory compromise is also not a reported AE with elsamitrucin. In this study, a dog with a metastatic mast cell tumor became dyspneic and within 24 hours went into cardiopulmonary arrest after receiving a single dose of elsamitrucin. The dog had no history of cardiac disease or severe respiratory distress before elsamitrucin administration. Elsamitrucin was considered a possible etiology of the clinical signs as was mast cell degranulation, related or unrelated to the study drug. Mast cell granules contain histamine, which is a vasoactive protein that results in vasodilation and constriction of smooth muscle in the bronchi. Mast cell degranulation may have exacerbated the dog's pre-existing airway narrowing/collapse or caused severe hypotension. If mast cell tumor degranulation were caused by the effect of elsamitrucin on this dog's disease, these SAEs would not necessarily be used to define MTD.

Seizure was a unique AE seen in 1 dog that on necropsy had stage V lymphoma (in the lungs, lymph nodes, spleen, and liver); the central nervous system was not evaluated. Based on preclinical data, seizure was not an expected adverse effect from the study drug, and given the extensive disease involvement in addition to the duration between the dog's last treatment and the AE (4 days), the seizure was attributed to the lymphoma. Even if attributed to drug, this was an AE that was not seen in any other dog in the study and would not be used to define MTD.

A weakness of this study that is inherent to a Phase I trial is related to the challenge of determining whether an AE is related to the tested agent. While clinician bias influences these decisions, determining attribution for AEs can be challenging and is a function of what is expected because of drug versus expected because of disease.10,11 Observed toxicoses that are expected from neither drug nor disease should be attributed to drug in the most conservative evaluation for safety reasons, but if AEs are not repeatable or are very discrepant, an investigator may feel there is enough doubt so to not use a given AEs to define the DLT, even if it may be attributable to the drug. Other factors that help an investigator determine AE attribution include timing of an AE relative to drug administration and histopathological evaluation of tissues. Unfortunately, necropsies were not obtained in all cases, despite owners' willingness to commit to this at the time of enrollment. This is an unfortunate complication of clinical trials with client-owned animals. We were able to use preclinical dataa to determine which AEs were expected because of drug, and used our knowledge of cancer to determine which were expected because of disease. The modifications we made in the 3 + 3 design allowed for a more thorough evaluation of this drug than if escalation had not been allowed.

There were no complete or partial responses in this study; however, as with most Phase I trials, the study population included dogs with advanced cancer or refractory disease at enrollment. Lack of response could be attributed in part to study dog selection and should not preclude advancing to a Phase II study. Because the lack of myelosuppression allowed continuous weekly administration, it is also possible that the stable disease responses that were seen constituted a response to the metronomic-like administration schedule.

In conclusion, the recommended dose for Phase II trials of elsamitrucin is 0.08 mg/kg IV weekly. This is consistent with a previous dose ranging study in which overt clinical toxicosis was only seen in dogs given elsamitrucin doses >0.08 mg/kg. Depending on attribution of AEs, the MTD could be considered either 0.06 or 0.08 mg/kg, however, we feel that the lack of toxicosis at 0.08 mg/kg supports the latter as the MTD. For cytotoxic chemotherapy, the therapeutic margin is narrow and it is important to treat at the true MTD to evaluate efficacy. The most common toxicoses are low-grade anorexia and vomiting. Given the lack of myelosuppression, elsamitrucin may be considered for use in combination chemotherapy protocols.

Footnotes

a Elsamitrucin Investigator Brochure, Spectrum Pharmaceuticals Inc, Irvine, CA

b Spectrum Pharmaceuticals Inc

Acknowledgments

The authors thank Spectrum Pharmaceuticals for providing the study drug and sponsoring this study.

Ancillary