As the development of 3-AP has been limited by toxicity, our primary purpose was to determine if toxicity was associated with pharmacokinetic parameters. We identified a relationship between CL and the number cycles the patient received, where a significantly reduced clearance was associated with patients who stopped therapy after receiving only one cycle of chemotherapy. This 32% decrease in CL is a population-based finding and was found irrespective of which starting dose to which a patient was randomized. Overall, eight patients went off study after only one cycle of therapy. Of these eight patients, four experienced dose limiting toxicities and three others discontinued therapy because of toxicity that was not defined as dose limiting. Only one patient discontinued therapy for progressive disease, which was based on physician and patient judgement and did not meet RECIST criteria. It is important to point out that this evaluation provides an association and does not determine a cause-and-effect relationship. It is unlikely that stopping therapy after one cycle causes a decrease in clearance. Nonetheless, if a patient had to terminate therapy after one cycle, they tended to have lower 3-AP clearance. The mechanism of reduced clearance is unknown. A polymorphism in ABCB1 is not likely the explanation as these polymorphisms did not contribute to pharmacokinetic variability. However, a larger controlled clinical trial powered to detect an appropriate difference would be necessary to demonstrate that finding. Reduced renal and hepatic function are also not explanations, as normal and renal and hepatic function were required for study entry. A potential explanation may be variable intracellular binding of 3-AP to ribonucleotide reductase and changes in intracellular clearance (22
Prior investigations have demonstrated that ABCB1 genotype was predictive of both outcome (20
) and toxicity (20
), in patients treated with 3-AP and an in vitro evaluation suggested 3-AP was a substrate for p-glycoprotein (15
). We therefore hypothesized individuals with variant genotypes had altered pharmacokinetics and evaluated this relationship in the current study, showing the ABCB1 genotype did not influence 3-AP pharmacokinetic parameters. While some studies have shown a pharmacokinetic relationship between ABCB1 genotype and plasma concentrations, a number of studies have failed to demonstrate a relationship (23
). This may be explained by the wide and variable distribution of ABCB1 including the blood brain barrier, GI tract, liver and tumor cells, all of which may distribute 3-AP back to a central compartment in a variable manner, resulting in no consistent PK relationship between ABCB1 genotype and 3-AP plasma concentrations.
BSA was not identified as a significant covariate for any 3-AP PK parameter. While BSA based dosing is widely used in oncology and generally believed to reduce inter-individual pharmacokinetic variability, comparisons between flat dosing strategies and BSA based dosing have generally shown them to be equivalently ineffective in decreasing inter-individual variability (24
). Since gender did influence V2, a gender specific, flat dose could be considered in future clinical trials.
The relationship between toxicity and exposure was further evaluated by assessing grade III–IV myelosuppression and gastrointestinal toxicity. However, nearly universal myelosuppression was observed in both clinical studies, even with lower doses (25–45mg/m2) of 3-AP used in combination with doxorubicin. In addition, only five patients experienced gastrointestinal toxicity, all of which were observed in the study with concurrent irinotecan. Since gastrointestinal toxicity with irinotecan is a well known dose limiting toxicity of irinotecan and no toxicities were noted in the doxorubicin arm, this finding suggests 3-AP contributed little to the observed GI toxicity.
When considering grade III–IV hypoxia, only three patients experienced episodes of hypoxia. All of these occurred at the highest dose levels of 3-AP administered in this study of 60–85mg/m2. This is consistent with other trials of 3-AP where G6PD deficient patients were excluded, methemoglobinemia has still been reported at doses ranging from 96–105mg/m2 in 5–25% of subjects. A potential explanation for this toxicity may be preferential distribution or accumulation of 3-AP into erythrocytes. Our data is best fit by a three compartment model, with V2, sampled from the erythrocyte compartment, having a 3–6 fold increased volume when compared to V1, sampled from the plasma compartment. This suggests extensive intracellular distribution of 3-AP in the erythrocyte compartment. We also noted a significantly decreased V2 in female subjects, compared to males. This may have a protective effect, as all hypoxia episodes occurred in male subjects, even though 50% (8 male, 8 female) of the subjects treated at the highest doses of 3-AP were female.