Ultrasonography of Osteosarcoma in Dogs
Sonography Atlas of Dogs

Satraplatin (JM216) is the first PO-delivered platinum chemotherapy drug used in human patients for cancer treatment, developed initially in the mid-1990s.¹ As a platinum (IV) compound, the drug is more lipophilic and stable than other platinum agents, allowing oral delivery and bioavailability. In addition to the oral formulation, satraplatin is unique as compared with other platinum agents in that it causes less neuro- and nephrotoxicity.^2–4 The major metabolite of JM216 is JM118, which itself has significant anticancer activity.⁵ The dose-limiting toxicity (DLT) seen in people and rodents to date is myelosuppression, with lowest neutrophil and platelet counts both occurring at 2–4 weeks. Other adverse effects include nausea and diarrhea in approximately one-third of patients.⁶ Pharmacokinetics (PKs) and physical properties are known in people.^7–9 As a result of its lipophilic nature and reactivity, satraplatin tends to accumulate in tissues, maintaining detectable concentrations for up to 2 weeks after cessation of therapy.⁶ The ideal dosing scheme is once daily for 5 days and repeated every 3–5 weeks.⁹ This schedule has been used in people, and a decrease in tumor size has been seen in human patients with ovarian cancer, among others, as well as palliation of tumor-related pain even in the absence of objective tumor response.¹⁰

Although resistance to other platinum drugs such as cisplatin is a frequent cause of treatment failure, satraplatin creates bulkier adducts in DNA that may be more difficult for cellular machinery to repair.¹¹ Accordingly, in vitro evidence demonstrates satraplatin efficacy in cisplatin-resistant cell lines.^5,12 Satraplatin enters the cell primarily by passive diffusion, likely because of its enhanced lipophilicity.¹³ Satraplatin also has been shown to inhibit cytochrome P450,¹⁴ and resistance to satraplatin may occur by glutathione conjugation.¹⁵

While in vitro investigations have included both chemosensitive and chemoresistant cell lines of human prostate, colon, breast, leukemia, and ovarian cancer,⁵ no published reports exist of in vitro activity of satraplatin or its metabolites against nonhuman cancer. Identification of efficacy in vitro at concentrations that can be achieved in the plasma based on PK evaluation provides preclinical support for the use of a novel chemotherapy agent in vivo.

Satraplatin has not been evaluated in animals with spontaneously occurring neoplasia. Oral chemotherapy agents offer many advantages for treatment of dogs with cancer. The purpose of this study was to determine the toxicity and the maximally tolerated dose (MTD) of satraplatin in dogs with cancer. PK data were collected to allow determination of the optimum dose and dosing interval for future clinical studies in order to evaluate efficacy in a prospective manner. Our hypothesis was that nephro- and neurotoxicity would not be dose limiting for satraplatin in dogs and that a dosage resulting in acceptable toxicity would be established. Based on available human data, we hypothesized that gastrointestinal toxicity or myelosuppression would be dose limiting for this drug in dogs.

Dogs with spontaneously occurring neoplasia were recruited from the oncology service at the University of Missouri Veterinary Medical Teaching Hospital. Enrollment criteria included cytologic or histologic diagnosis of cancer, adequate performance status, life expectancy of at least 16 weeks, satraplatin administered as the sole anticancer therapy, adequate renal and bone marrow function as determined by chemistry profile and CBC, and informed owner consent. At the University of Missouri, IACUC approval is not required for clinical trials in animals if owner consent is obtained and the protocol could be performed by general practitioners. Informed owner consent was obtained in all cases.

CBC, chemistry profile, and urinalysis were performed on all dogs as well as 3-view thoracic radiographs and additional staging tests as appropriate for the tumor type such as abdominal ultrasound examination or nuclear scintigraphy. For chemotherapy, normal renal function, a neutrophil count of at least 3,000/μL, and a platelet count of at least 150,000/μL were required within 24 hours of the 1st dose.

The first 3 dogs enrolled had CBCs collected Monday and Thursday of each week for the first 3 weeks to determine neutrophil and platelet nadirs. Such frequent blood sampling is impractical in client-owned animals so we chose not to evaluate the nadir in all subjects. The nadir should be at the same time point regardless of dose, although more profound with higher doses. CBCs were performed at the predicted nadir in subsequent dogs. Biochemical evaluation with urinalysis was performed at baseline and at the time of the third or (most often) the 4th cycle of chemotherapy. Sixteen dogs had at least 1 biochemical profile after receiving satraplatin, 2 died unexpectedly before the 2nd cycle of treatment, 2 dogs exited the study without biochemical evaluation because of disease progression early in the course of treatment, and 3 dogs either were not evaluated because of lack of owner compliance or because the information was not available from other veterinary clinics. Once dogs exited the study, they were not routinely monitored except to recommend thoracic radiographs every 3 months and to request postmortem examination at the time of their death.

Although not a primary objective of this study, clinical response was noted for the purpose of justifying a future phase II trial. Complete response (CR) was defined as resolution or disappearance of all clinically detectable disease, partial response (PR) was defined as >50% reduction in tumor volume (length × width × height ×π/6), stable disease (SD) was defined as <25% increase and <50% decrease in tumor volume, and progressive disease (PD) was defined as >25% increase in tumor volume or the detection of new lesions. Response assessment was made at 3 months after enrollment and objective responses were required to be maintained for at least this period of time. Outcome of patients treated in the adjuvant setting was evaluated with progression-free survival (PFS), defined as the date of enrollment to the date of detectable new lesions. Overall survival (OS) was defined as the date of enrollment to the date of death.

Adverse events were graded according to the Common Terminology Criteria for Adverse Events as published by the Veterinary Cooperative Oncology Group (VCOG-CTCAE v.1).¹⁶ For most adverse events, grade 3 or higher toxicity was considered dose-limiting for that individual. However, afebrile, asymptomatic grade 3 neutropenia or thrombocytopenia was not considered dose-limiting, whereas febrile grade 3 neutropenia or grade 4 myelosuppression of any kind constituted a DLT. If a dog experienced DLT, the next lowest dose was used subsequently for that dog so that chronic toxicity still could be evaluated. If a dog experienced only nausea as a DLT, that dog was allowed to receive an antiemetic (mirtazapine) before each treatment for subsequent doses at the same dosage. If grade 3 or higher gastrointestinal toxicity was encountered despite the addition of antinausea medication, the next lowest dose was used for subsequent treatments. Owners were provided with a diary to record any changes in thirst, urination, appetite, stool, or other physical variables at home.

Groups of 3–6 dogs were treated at each dose level until the MTD was reached in a standard 3 + 3 design as detailed below. All dogs meeting the eligibility criteria and receiving at least 1 dose of satraplatin were considered evaluable for toxicity. Dogs were dosed initially at 50 mg/m² PO once daily for 5 days. This dosage was selected based upon data from a preliminary study (unpublished data) in which healthy Beagle dogs were treated at various dosages daily for 5 days repeated every 28 days for a total of 7 cycles, and was the dose at which no clinically relevant toxicity was seen. Dosage initially was increased or decreased by 10 mg/m²/d increments until DLT was encountered, then further titrated at 5 mg/m²/d increments. Using 10 and 50 mg capsules, dosing was rounded to the nearest 10 mg, without exceeding the calculated dose by more than 10%. The dose was administered to each dog after at least a 5-hour fast, although a small amount of food was allowed if needed to facilitate administration. After dosing, dogs were encouraged to drink, and food was withheld for an additional hour to allow absorption. Because effects of motility-modifying drugs on satraplatin absorption were unknown, metoclopramide was avoided and mirtazapine was used as the initial antiemetic and appetite stimulant, if needed. During the course of the trial, an NK1 receptor antagonist (maropitant) became available and was used as an initial or secondary antiemetic when needed.

Enrollment was staggered to ensure safety. Dogs were enrolled in a new cohort concurrently, but at least 3 dogs must have completed the 1st cycle and have been evaluated for toxicity before dose escalation could occur. The standard 3 + 3 design involves dose escalation in the absence of DLT, and cohort expansion by an additional 3 dogs if 1 DLT is seen. If 2 or more DLTs are found at a given dosage, then the MTD has been exceeded. Once the MTD was determined, a minimum of 6 additional dogs were planned to be enrolled at this level to further characterize the safety profile at the MTD dose level. This also would allow preliminary justification for a phase II trial if at least 1 dog showed evidence of efficacy. These dogs did not have PK samples collected. De-escalation was permitted in the event of life-threatening adverse events, even if a cohort was not complete.

During the dose escalation phase, all dogs had PK evaluation performed at each dose level with blood drawn to determine concentrations of total and free platinum. Sampling times were as follows: day 1: 0 (predose), 0.5, 1, 1.5, 2, 3, 4, 6, 12, and 24 hours after administration; day 5: 0 (predose), 0.5, 1, 1.5, 2, 3, 4, 6, 12, 24, 48, and 72 hours after administration. Prechilled tubes and a precooled centrifuge (4°C) were used to collect EDTA plasma from blood samples at each time point. One milliliter of plasma was transferred into a cryotube and immediately frozen at −80°C. Aliquots of plasma were ultrafiltered with Centrifree 30,000 D ultrafiltration devices^a in a precooled fixed angle rotor centrifuge (4°C) for 20 minute at 2,000 ×g. Aliquots (approximately 500 μL total) of the plasma ultrafiltrate (PUF) were immediately frozen at −80°C. The use of these special filters allowed the separation of free platinum because it is considered the biologically relevant portion. However, because the relative value of free and total platinum has been debated, both fractions were evaluated. Accumulation of drug was determined by dividing day 5 PK values by day 1 PK values for maximal plasma concentration (C_max) and area under concentration (AUC) time curves.

The PK parameters of total platinum (Pt_TOT) and ultrafiltered platinum (PUF) were calculated by noncompartmental methods and equations by Microsoft Excel software.¹⁷C_max and time to maximal plasma concentration (T_max) were determined from the plasma concentration-time profiles. AUC time curves and area under the moment curve (AUMC) were calculated by linear trapezoidal summation. Clearance was calculated as dose/AUC time curves and MRT as AUMC/AUC time curves.

Before collection of clinical samples, the effect of various serum conditions was evaluated. Serum from unaffected, healthy animals that was lipemic, hemolyzed, or both, was obtained and spiked with satraplatin. Samples were processed and evaluated by ANOVA for differences among expected results. Additionally, acid balancing was evaluated with 0.9% HCl with 2 different concentrations of HNO₃ (0.7 and 7%).

The working standard solutions were prepared every 7 days. These stock standard solutions were prepared by diluting the 1,000 μg/mL platinum standard solution with 0.24 M HCl and stocked at room temperature. The standard solutions had concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 20 ng/mL platinum and contained 10 ng/mL iridium as internal standard.

For metal analysis of PUF, a more economical digestion method, heated sonic water bath digestion, was used. The samples were prepared by heating at 60°C with sonication for 30 minutes. One hundred microliters of aliquots of PUF were gravimetrically transferred into 15 mL polypropylene Eppendorf centrifuge tubes. Each sample was mixed with 300 μL of aliquot Optima grade HNO₃ (69%) and 100 μL of aliquot Fluka TraceSelect grade H₂O₂ (30%) and then digested by water bath with sonication. Digested samples then were analyzed by ICP-MS.

A descriptive analysis for breed, sex, age, weight, plasma albumin concentration, target dose, actual dose, dosage, measurable disease, and diagnosis was performed. To evaluate the relationship between PK and hematologic toxicity, regression analysis was conducted between the t_1/2, T_max, C_max, and AUC time curves (for Pt_TOT and PUF), and the nadir neutrophil and platelet counts. In addition, regression analysis between the actual dose administered and the nadir neutrophil and platelet counts also was performed. Kaplan-Meier analysis was calculated for the PFS and survival time points. A Kaplan-Meier log-rank test was used to calculate the difference in survival between the measurable and nonmeasurable disease osteosarcoma groups. All parameters were considered significant if the P value was <.05.

Results

In Vitro Studies

We determined the MTD of orally administered satraplatin in tumor-bearing dogs to be 35 mg/m²/d for 5 days. Because additional DLTs occurred in an extended cohort treated at this dosage, it is reasonable to consider a range of 30–35 mg/m²/d for 5 days for clinical use. Our in vitro studies showed biologic activity at concentrations that are achievable in vivo as shown by the PK data. Regarding PK, we also demonstrated that nadir neutrophil count was inversely correlated with the AUC_{0–24 h}. Dosage also impacted some PK parameters, as would be expected. During optimization, we were able to show that acid balance is important for accurate determination of platinum concentrations after satraplatin administration.

The platelet nadir occurred before the neutrophil nadir in most cases. This is unusual because neutrophils are shorter lived (6–8 hours) than platelets (6 days) and thus typically are depleted more rapidly. It is unlikely that an early neutrophil nadir was missed because blood counts were assessed twice weekly in the first 3 dogs. Failure to identify the nadir was possible, however, given the wide variability in time to nadir. Possible explanations include selective effects on different stages of precursors for the 2 cell lines, but further studies are required to confirm this hypothesis. Apart from myelotoxicity, the drug was well tolerated with minimal gastrointestinal and no nephro- or neurotoxicity. The 1 dog that developed a perforating gastric ulcer had 2 other risk factors for ulceration, either of which could have caused ulceration alone. Whether satraplatin contributed to the ulcer is unknown but considered unlikely because the dog had only received 3 doses of the first 5-day course. In people, the DLT also is myelosuppression with platelet and neutrophil nadirs at 17 and 21 days, respectively, with recovery by day 28, and a large variability in days to nadir which is very similar to the dogs treated here.³

Preliminary efficacy was suggested in this group of dogs. Of note, dogs with appendicular osteosarcoma treated in the adjuvant setting experienced a median survival time (577 days) comparable or superior to other published outcomes with platinum-based chemotherapy regimens (cisplatin or carboplatin alone or in combination with doxorubicin, median survival 10–14 months).^18–21 Although we studied a small group of dogs, based on previous studies very few dogs with osteosarcoma treated in the adjuvant setting would be expected to live beyond a year if the drug was ineffective, yet 4 of 6 did so in the present study. Orally administered satraplatin may offer an alternative to the costly and time-consuming fluid diuresis required for cisplatin administration in dogs, and to the expense of IV carboplatin.

Satraplatin use in people has produced few objective responses, but has resulted in notable palliation of cancer-related clinical signs in patients with a variety of tumors such as nonsmall cell lung cancer, mesothelioma, and hormone-refractory prostate cancer.^6,10 However, only patients with advanced or refractory disease are evaluated in early phase trials, and satraplatin may be most effective in the minimal disease setting, as is the case for most cytotoxic chemotherapy. Additionally, the palliation of pain and cancer symptoms is valuable even in patients with advanced disease.

In conclusion, orally administered satraplatin was well tolerated in this group of tumor-bearing dogs. Preliminary efficacy in an extended treatment group warrants a larger scale phase II trial.

^aCentrifree YM-30, Millipore, Billerica, MA

^bPersonal communication, Agennix Inc, Princeton, NJ

This trial was supported in part by a University of Missouri College of Veterinary Medicine Committee on Research Clinician-Scientist grant, NSEI Reactor Utilization grant from the University of Missouri, Phi Zeta grant, and the Scott Endowed Program in Veterinary Oncology. Satraplatin was generously provided by Agennix Inc.