2.1. Patients

Patients with histologically or cytologically confirmed refractory hematologic malignancies, including AML, ALL, chronic myeloid leukemia blast crisis, myelodysplastic syndrome (MDS), and chronic lymphocytic leukemia (CLL), were eligible for this trial. Patients were required to have an Eastern Cooperative Oncology Group performance status of 0-3. There were no limitations on the number of prior therapies, but active toxicities from these prior therapies must have resolved to grade <1 as defined by National Cancer Institute Common Toxicity Criteria version 2.0. Patients were also required to have normal levels of bilirubin, creatinine values less than or equal to 2.0 mg/dL, aspartate aminotransferase (AST) less than or equal to 3.0 × ULN, and alanine aminotransferase (ALT) less than or equal to 3.0 × ULN. All patients were informed of the investigational nature of this protocol in accordance with institutional policies. An Institutional Review Board-approved informed consent form was signed by all patients for both clinical and pharmacology studies; approval was obtained according to the Declaration of Helsinki. Exclusion criteria included heart disease, psychiatric illness that limited compliance with the study, and current enrollment on other standard or investigational therapies for leukemia.

2.2. Study drug

TCN-PM was supplied by Vioquest Pharmaceuticals as lyophilized powder in 50-mg vials. Before use, the drug was reconstituted with 2.5 mL of sterile water for a final concentration of 20 mg/mL per vial with a pH of 6.0 to 7.5. The study drug was then diluted in 500 mL isotonic saline solution and infused intravenously over 1 hour by means of a regulated infusion pump. A cycle of therapy was defined as three doses of TCN-PM administered on days 1, 8, and 15 every 28 days.

2.3. Trial design

TCN-PM was initiated at 15 mg/m² weekly on days 1, 8, and 15 every 28 days based on previously conducted trials [37]. This weekly schedule was chosen based on the long half life of TCN (~89 hours) [26]. The escalation dose for new cohorts was planned as follows: 25, 35, 45, 55, and 65 mg/m². After 65 mg/m², and if no DLT was observed, further increments were to increase by 5 mg/m² per cohort until DLT was observed. All adverse events were graded according to the NCI Common Terminology Criteria for Adverse Events version 3.0. A DLT was defined as any clinically significant grade 3-4 drug-related non-hematologic toxicity with the exception of grade 3 AST, ALT, or amylase/lipase elevations persisting for <7 days. Observations for DLT were performed during cycle 1 for a minimum period of 21 days. Hematologic DLT was defined as grade 3-4 neutropenia and/or thrombocytopenia (secondary to marrow hypoplasia and not leukemic burden) that did not recover to grade ≤ 2 by day 42.

The standard 3 + 3 escalation rule was used for this trial, and the recommended phase II dose was defined as the highest dose level at which no more than 1 of 6 patients experienced a DLT. The maximum tolerated dose (MTD) was defined as the highest dose level in which <2 patients of 6 developed DLT within the first cycle. At the MTD, up to 10 total patients could be accrued to further define the toxicities and response of the agent. All patients were considered to be evaluable for safety and toxicity if they received any dose of TCN-PM. Patients were considered to be evaluable for response if they received at least 1 full cycle of treatment and subsequent bone marrow biopsy or had progressive disease determined prior to completion of 1 full cycle. Patients who were removed from the study prior to day 22 for reasons other than DLT were replaced.

2.4. Response criteria

For acute leukemias and MDS, complete remission (CR) was defined as 5% or fewer leukemia cells in the normocellular or hypercellular marrow with absolute neutrophil count (ANC) equal to or more than 1.0 × 10⁹/L and platelet count equal to or more than 100 × 10⁹/L. A partial response was defined with the same hematologic parameters as for CR, but allowed for abnormal cells of 6% to 25% in the marrow or 50% reduction in marrow blasts. Hematologic improvement was defined as achievement of any of the following parameters: 1) increase in hemoglobin level by ≥ 1 g/dL if ≤ 11 g/dL prior to therapy and either independence or decrease from transfusion requirements by at least 50%; 2) increase in platelet count by 50% if below 100 × 10⁹ prior to therapy, with a net increase ≥10 × 10⁹/L; and 3) increase in ANC by 100% and to greater than 0.5 × 10⁹/L if below this level prior to therapy. For CLL, CR was defined as 1) disappearance of all palpable lymph nodes, spleen, and liver without the appearance of new lesions along with <30% lymphocytes in normocellular marrow; if lymphoid nodules were seen, response was deemed as nodular CR; 2) absolute lymphocyte count (ALC) <4 × 10⁹/L with hemoglobin >11 g/dL, ANC >1.5 × 10⁹/L, and platelet count >100 × 10⁹/L. Partial response was defined as 1) ALC reduced by >50% from pretreatment baseline value, hemoglobin >11 g/dL or 50% improvement over baseline without transfusions, ANC >1.5 × 10⁹/L or 50% improvement over baseline, and platelet count >100 × 10⁹/L or 50% improvement over baseline; and 2) when compared with pretreatment measurements, a reduction of >50% in measurable lesions without the appearance of new lesions.

2.5. Cell lines and TCN pharmacology

The ML-1 myeloid leukemia cell line was a gift from Dr. M. J. Kastan (Memphis, TN). OCI-AML3 cells were kindly provided by Dr. M. Minden (Ontario Cancer Institute, Toronto, ON, Canada). All cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and 2 mM L-glutamine and maintained at 37°C in 5% CO₂ in a fully humidified incubator. The cell doubling time was 24 hours under these conditions. Cells were incubated with various concentrations of TCN (Vioquest Pharmaceuticals) for 3 hours before being harvested to quantitate the cellular accumulation of TCN-PM by high-performance liquid chromatography (HPLC). In other experiments, cells were exposed to 1 and 10 μM TCN before being harvested to assess the levels of pAkt and cellular viability.

2.6. Samples for clinical pharmacology

Blood samples were collected at time 0 (prior to infusion) and as post-infusion samples of TCN-PM at 2 and 24 hours after completion of day 1 of cycle 1. Each blood sample (10 mL) was collected in a Vacutainer green top tube (heparin) and placed in an ice-water bath until transport to the laboratory.

2.7. Pharmacokinetics of TCN-PM

Blood samples were diluted with phosphate-buffered saline, and leukemia cells were isolated by Ficoll-Hypaque density gradient step-gradient centrifugation procedures. A Coulter channelyzer (Coulter Electronics, Hialeah, FL) was used to determine cell number and the mean cell volume. We used 1-2 × 10⁷ cells to extract nucleotides including TCN-PM, after which nucleotides were separated and quantitated with HPLC using UV detection. The results were analyzed for the accumulation and retention of TCN-PM in leukemia blasts during therapy.

2.8. Western blotting

Cellular lysates were harvested at 0 and 24 hours after infusion of TCN-PM and immunoblotted with antibodies against pSer⁴⁷³Akt, pSer¹¹²Bad, total AKT and Bad (Cell Signaling Technology), or GAPDH (Upstate Biochemicals). Proteins were quantitated using the densitometry function on the Odessey-LiCor and expressed as a ratio of pAkt/Akt or pBad/Bad.

3.1. Toxicity

The toxicity profile of TCN-PM is detailed in Table 2. Overall, TCN-PM was well-tolerated. DLTs included mucositis (1 patient at the 35 mg/m² cohort), lipase elevation (2 patients at the 65 mg/m² cohort), and triglyceride elevation (1 patient at the 65 mg/m² cohort). DLTs were reversible in all patients following discontinuation of TCN-PM. Common treatment-emergent non-hematologic toxicities included pain, infection/febrile neutropenia, nausea, bleeding, mucositis, and diarrhea, with the large majority being grade 1-2. Due to concerns for metabolic toxicity, the MTD was declared at 55 mg/m².

3.2. Clinical response

Of the 41 treated patients, 32 were considered evaluable for response based on the criteria described above. Overall, there were no complete or partial responses. Of the 32 evaluable patients, 15 had progressive disease and 17 had stable disease following 1 cycle of treatment. Of the patients with stable disease, 3 patients with AML achieved ≥50% bone marrow blast reduction and a fourth patient with CMML had marked spleen reduction and resolution of leukocytosis. These patients are highlighted in Table 3. Eight patients received at least 2 cycles of TCN-PM.

3.3. Pharmacokinetics of TCN-P in AML blasts

We initially determined the feasibility and reproducibility of detecting TCN-P by HPLC. To this end, a quality control standard was generated by adding a known amount of purified TCN-PM to an acid-soluble extract from AML blast cells. The standard curve indicated a well-defined TCN-P peak (Figure 1A). Next, we evaluated whether TCN-P could be detected in the leukemic cells from AML patients undergoing therapy with this Akt inhibitor. Our data demonstrate that TCN-P was absent in the leukemic blasts obtained before therapy (Figure 1B) but accumulated a discrete TCN-P peak 24 hours after TCN-PM infusion (Figure 1C).

We then determined the pharmacokinetics of TCN-P in two leukemia cell lines (ML-1 and OCI-AML3), which were exposed to 0, 0.05, 0.1, 0.2. 0.5, 1, 3, and 10 μM TCN for 3 hours (TCN is metabolically converted to its active form TCN-P in cells) [26]. Our data demonstrate a dose-dependent accumulation of TCN-P in both leukemia cell lines. Cells exposed to <0.05 μM TCN accumulated between 20 and 40 μM of TCN-P, whereas exposure to 10 μM TCN resulted in the accumulation of over 500 μM of TCN-P (Figure 1D).

Next, leukemic blasts from 8 patients obtained at 0, 2, and 24 hours after start of TCN-PM therapy (day 1, cycle 1) were evaluated for the accumulation of TCN-P. Similar to the results obtained in the AML cell lines, primary AML blasts also demonstrated a dose-dependent accumulation of TCN-P, albeit to much lower concentrations (Figure 1E). In AML blasts, the peak level of TCN-P was achieved within 2 hours after the end of the infusion in the cohorts that received 15 (n=1), 25 (n=3), and 35 mg/m² (n=2). AML blasts accumulated a median peak concentration of 4 μM (range of 2.1-7.5 μM) (Figure 1E). In patients who received 65 mg/m² (n=2), the peak TCN-P levels occurred 24 hours after infusion (Figure 1E). Collectively, 4 of 6 samples in these cohorts retained at least 50% of their intracellular TCN-P after 24 hours (Figure 1E).

3.4. Pharmacodynamics of TCN-P in AML cell lines and primary leukemic blasts

Both AML cell lines and AML blasts are known to express robust levels of Akt that is phosphorylated on Ser⁴⁷³ and Thr³⁰⁸ [38]. It is also established that exposure to chemotherapy is associated with decreased levels of pSer⁴⁷³ and pThr³⁰⁸ and increased levels of cell death in vitro and in primary AML blasts during therapy [38]. Because TCN inhibits Akt phosphorylation, we first evaluated the response of OCI-AML3 cells exposed to 1 or 10 μM TCN for 0, 12, 24, 36, and 48 hours. Our data show that the levels of pSer⁴⁷³Akt decreased in a time-dependent manner (Figure 2, A and B). Loss of pSer⁴⁷³Akt was associated with a reciprocal increase in the numbers of apoptotic cells, as measured by the appearance of annexin V positivity (Figure 2C).

Because TCN-PM has been recently shown to inhibit Akt phosphorylation in tumor biopsies following treatment of patients whose tumors had high levels of activated Akt [37], we first compared the levels of pSer⁴⁷³Akt in the lymphocytes of normal donors and AML blasts from eight patients before therapy. Our data indicate that normal lymphocytes (n=5) express low levels of pSer⁴⁷³Akt, whereas AML blasts displayed variable levels of pSer⁴⁷³Akt (Figure 3A). Five samples expressed levels that were similar or 2-fold higher than the levels in normal lymphocytes, whereas three samples displayed between 30- and 200-fold higher expression of pSer⁴⁷³Akt, suggesting that these three patients were likely to have an activated Akt pro-survival signaling (Figure 3A). We then determined the levels of phosphorylation of Akt (pSer⁴⁷³) and its substrate Bad (pSer¹¹²) in AML blasts obtained before and 24 hours after TCN-PM infusion. Figure 3B shows that the pSer⁴⁷³Akt/Akt levels in the 5 patients whose tumors had low basal levels of phosphorylated Akt (1 treated with 15 mg/m², 2 treated at 25 mg/m², and 2 treated with 65 mg/m²) were little affected by TCN-PM treatment. In contrast, the three patients who had high basal levels of pAkt (2 treated at 35 mg/m² and 1 at 65 mg/m²) demonstrated significant decreases in pSer⁴⁷³Akt/Akt levels. Similar results were observed with pSer¹¹²Bad/Bad levels, consistent with an inhibition of this survival pathway and the activity of TCN-P in this patient population (Figure 3C).