Rapamycin pharmacokinetic and pharmacodynamic relationships in osteosarcoma: a comparative oncology study in dogs

Abstract

Background: Signaling through the mTOR pathway contributes to growth, progression and chemoresistance of several cancers. Accordingly, inhibitors have been developed as potentially valuable therapeutics. Their optimal development requires consideration of dose, regimen, biomarkers and a rationale for their use in combination with other agents. Using the infrastructure of the Comparative Oncology Trials Consortium many of these complex questions were asked within a relevant population of dogs with osteosarcoma to inform the development of mTOR inhibitors for future use in pediatric osteosarcoma patients.

Methodology/principal findings: This prospective dose escalation study of a parenteral formulation of rapamycin sought to define a safe, pharmacokinetically relevant, and pharmacodynamically active dose of rapamycin in dogs with appendicular osteosarcoma. Dogs entered into dose cohorts consisting of 3 dogs/cohort. Dogs underwent a pre-treatment tumor biopsy and collection of baseline PBMC. Dogs received a single intramuscular dose of rapamycin and underwent 48-hour whole blood pharmacokinetic sampling. Additionally, daily intramuscular doses of rapamycin were administered for 7 days with blood rapamycin trough levels collected on Day 8, 9 and 15. At Day 8 post-treatment collection of tumor and PBMC were obtained. No maximally tolerated dose of rapamycin was attained through escalation to the maximal planned dose of 0.08 mg/kg (2.5 mg/30 kg dog). Pharmacokinetic analysis revealed a dose-dependent exposure. In all cohorts modulation of the mTOR pathway in tumor and PBMC (pS6RP/S6RP) was demonstrated. No change in pAKT/AKT was seen in tumor samples following rapamycin therapy.

Conclusions/significance: Rapamycin may be safely administered to dogs and can yield therapeutic exposures. Modulation pS6RP/S6RP in tumor tissue and PBMCs was not dependent on dose. Results from this study confirm that the dog may be included in the translational development of rapamycin and potentially other mTOR inhibitors. Ongoing studies of rapamycin in dogs will define optimal schedules for their use in cancer and evaluate the role of rapamycin use in the setting of minimal residual disease.

Figure 1. The mTOR pathway is integral in cell metabolism and protein translation in cancer.

The mTOR pathway is the “nutrient sensor” of the cell and proximate targets of the pathway are responsible for both TOP and cap-dependent translation of proteins. Many of these proteins have been shown to be important in cancer progression, angiogenesis, autophagy and anti-apoptotic mechanisms. Rapamycin inhibits mTOR (via TORC1) following the formation of a complex with FKBP-12.

Figure 2. Rapamycin exposure in dogs with osteosarcoma is dose dependent.

Serial rapamycin whole blood concentrations (ng/ml) were measured by HPLC with MS/MS detection for all dogs that completed study (n = 19). After a single parenteral dose of rapamycin, 7-point PK analysis (samples collected at 0, 30 minutes, 1,2, 6, 24 and 48 hours) was performed. Over the dose range studied, A. average concentration – time curves for each dose level, and B. rapamycin exposure (AUC_0–48h) increased proportionally to dose.

Figure 3. Translationally relevant exposures of rapamycin were achieved in dogs with cancer.

In this study, dogs in the 4^th (0.06 mg/kg, approx 2.1 mg) and 5^th (0.08 mg/kg, approx 2.8 mg) dose cohorts had measurable trough levels ≥10 ng/ml. Trough concentrations in dogs with osteosarcoma after 7 and 8 days (C_D8, C_D9, ng/ml) of rapamycin treatment are similar to those intended for human cancer patients. Translationally relevant exposures of rapamycin are achievable in dogs with cancer, and support the use of the comparative approach in rapalog development.

Figure 4. Rapamycin therapy inhibits tumoral and PBMC downstream targets of mTOR in a clinical setting.

Modulation of mTOR pathway targets were evaluated in matched tumor (A.) and PBMC samples (B.) to compare pS6RP pre- and post-rapamycin therapy. Electrochemiluminescence (ECL) was utilized to accurately quantify phospho-protein status in tumor and PBMC. Quality control assessments defined 10 tumor and 8 PBMC samples eligible for evaluation. A. The red bars below the x-axis indicate patient dosing cohorts. Pre-treatment bars (purple) represent p-S6RP tumor levels prior to rapamycin dosing and post-treatment bars (blue) represent Day 8 levels at tumor surgical excision. Rapamycin led to >2-fold inhibition of tumoral p-S6RP in 8/10 dogs (A, p<0.0001). B. PBMC phosphorylation of S6RP was significantly inhibited in 8/8 dogs evaluated at Day 8 after rapamycin therapy and was maintained through Day 15 (7 days after the cessation of rapamycin therapy) (B, p<0.0001). Matched PBMC and tumor sample data were concordant. Marked post-treatment mTOR pathway inhibition was seen in dogs from all dose cohorts, including the lowest dose cohorts (0.01–0.02 mg/kg), proving that p-S6RP is a very sensitive biomarker of rapamycin administration.

Figure 5. Tumoral AKT phosphorylation is unchanged after short-term exposure to rapamycin in dogs with osteosarcoma.

AKT is an important pro-survival pathway in a variety of tumor types. In 9 tumor samples that passed quality control standards there was no significant (p = 0.069) post treatment up-regulation of pAKT (measured by ECL) after 8 days of exposure to rapamycin.