| Blood. 2006 December 1; 108(12): 3674–3681. Prepublished online 2006 August 15. doi: 10.1182/blood-2006-02-005702. | PMCID: PMC1895460 |
The success of BCR-ABL kinase inhibition by imatinib mesylate in the treatment of patients with chronic myelogenous leukemia1-5 has provided a stimulus to the development of other kinase inhibitors as potential therapies for hematologic malignancies. FMS-like tyrosine kinase 3 (FLT3) is a transmembrane protein that belongs to the type III receptor tyrosine kinase family. Other members of this family include platelet-derived growth factor receptor (α/β-PDGFR), KIT, and CSF1R. Activating mutations of FLT3 are found in 20% to 30% of patients with newly diagnosed acute myelogenous leukemia (AML), the majority of these taking the form of an internal tandem duplication (ITD) in the juxtamembrane region of the receptor.6-12 Activating point mutations in the kinase activation loop of the receptor also occur but with lower frequency (5%-10% of patients newly diagnosed).9-14 Both ITD and activation loop mutations appear to have a negative effect on prognosis: patients with these mutations relapse sooner following initial induction chemotherapy and have inferior survival compared with patients with only the wild-type receptor.6-8,10,12,14-17
Tandutinib is a piperazinyl quinazoline compound that resulted from screening of chemical libraries and subsequent optimization.18,19 In cell-based assays tandutinib inhibited FLT3, β-PDGFR, and KIT with IC50 values of 95 to 122 ng/mL but had no significant effect against a broad range of other kinases.18,19 In Ba/F3 cells expressing various FLT3-ITD mutants, tandutinib inhibited IL-3–independent growth and FLT3-ITD autophosphorylation with IC50 values of 6 to 17 ng/mL.19 Tandutinib also inhibited in vitro proliferation of human leukemia cell lines containing FLT3-ITD mutations with IC50 values of approximately 6 ng/mL.19 Given twice daily by oral gavage, tandutinib increased survival of nude mice with leukemia or lymphoma arising from Ba/F3 cells expressing FLT3-ITD mutations and increased survival of mice with myeloproliferative disease arising from transfection of hematopoietic progenitor cells with such mutations.18,19
Tandutinib has a very limited spectrum of activity outside the type III receptor kinase family. However, in a broad in vitro general pharmacology screen that included various receptor and enzyme assays, tandutinib yielded IC50 values less than 500 ng/mL against the muscarinic nonselective central nervous system acetylcholine receptor (434 ng/mL) and the muscle-type nicotinic acetylcholine receptor (483 ng/mL) (Millennium Pharmaceuticals, data on file). In a competitive human ether-a-go-go related gene (hERG) binding assay, tandutinib had a Ki of 216 ng/mL and an IC50 of 550 ng/mL. In a whole-cell variant of the patch-clamp assay using cells transfected with cloned human cardiac K+ channel hERG, tandutinib had a tail current IC50 of 1742 ng/mL (Millennium Pharmaceuticals, data on file).
Evaluation of tandutinib in rats, dogs, and monkeys showed it to be orally bioavailable, metabolically stable, and most likely eliminated by biliary excretion without biotransformation. Acute administration of high oral doses of tandutinib in dogs produced symptoms suggestive of central nervous system or neuromuscular toxicity, such as lack of coordination and tremors. However, under conditions of chronic oral dosing tandutinib was generally well tolerated in both rats and dogs. The principal toxicologic findings at high chronic doses in both species were (1) mild and reversible hypocellularity of the bone marrow with associated anemia and leukopenia and (2) reversible inflammatory infiltrates in hepatic portal triads, associated with reversible increases in liver function tests (Millennium Pharmaceuticals, data on file).
On the basis of preclinical models showing activity against leukemia cells driven by mutant FLT3 and given tandutinib's relatively favorable in vivo pharmacology and toxicology, a phase 1 clinical trial was initiated to evaluate tandutinib in patients with AML or high-risk myelodysplastic syndrome (MDS).
Tandutinib dose escalation. The dose selected for initial evaluation was 50 mg twice daily This represents approximately a sixth of the highest nontoxic dose (200 mg/m2 twice daily) in a 28-day toxicology study in dogs (Millennium Pharmaceuticals, data on file). Separate groups of 3 to 6 patients were enrolled to successively higher doses of tandutinib using a modified Fibonacci dose escalation scheme. No dose escalation within individual patients was allowed. New patients could not be enrolled to the next higher dose level of tandutinib until at least 3 patients had been treated at the preceding dose level, had received at least 14 days of treatment with tandutinib, and had not experienced DLT, defined as any grade 3 or 4 nonhematologic toxicity or grade 2 neurologic toxicity. Toxicities were graded according to the National Cancer Institute's Common Toxicity Criteria, version 2.0.
Denaturing wave high-performance liquid chromatography (D-HPLC). Aliquots (5-20 μL) of each PCR reaction were assessed for FLT3 mutations using a Transgenomic WAVE HPLC system (Transgenomic, Omaha, NE). Samples were run at 50°C to distinguish fragments of different lengths in exon 14 and at 56.9°C (exon 14) and 59.1°C (exon 20) to detect point mutations. Amplimers with abnormal D-HPLC profiles were bidirectionally sequenced on an ABI 310 Sequencer using the BigDye Terminator Kit (Applied Biosystems, Foster City, CA). Mutation gene dosage was determined by integration of the mutant-specific HPLC peak area and comparison of the calculated percentage mutant allele versus blast percentage. Samples for which the ratio of calculated percentage mutant allele to blast percentage greater than 0.75 were judged to be homozygous or hemizygous for the FLT3-ITD mutation.
The pharmacokinetics of tandutinib were evaluated with nonlinear mixed effects modeling, using NONMEM (version V level 1.1)21 and Wings for NONMEM,22 running under Compaq Visual Fortran (version 6.6c). The first-order conditional estimation (FOCE) method with interaction was applied to all analyses. Model acceptance was based on successful minimization, significant reduction in objective function values, diagnostic plots, and a posterior predictive check procedure.
Immunoprecipitation. Frozen cell pellets were lysed in 500 μL freshly made lysis buffer, and total FLT3 was immunoprecipitated from cell lysate supernatants by addition of 3 μg anti-FLT3 polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; catalog no. SC-480) followed by protein-A agarose beads (Pierce, Rockford, IL; catalog no. 20333). Beads were washed twice with 1 mL cold lysis buffer before the addition of 40 μL/tube of sodium dodecyl sulfate (SDS) protein loading buffer. Tubes were boiled for 5 minutes, and supernatants were loaded onto 12% to 20% Tris-glycine SDS-polyacrylamide gel for electrophoresis. Proteins were transferred to nitrocellulose membranes for 1.5 hours at 125 V.
Phosphorylated FLT3 (pFLT3) detection. Blots were incubated in blocking buffer (5% nonfat dry milk in 1X Tris-buffered saline [TBS] containing 0.5% Tween 20) for 1 hour at ambient temperature, washed 3 times in washing buffer (1X TBS containing 0.5% Tween 20), and incubated overnight with mouse anti-pFLT3 monoclonal antibodies (diluted 1:1000 in blocking buffer). After washing, blots were incubated with horseradish peroxidase (HRP)–conjugated goat anti–mouse IgG (Biosource International, Camarillo, CA; catalog no. AMI4404) for 1 hour at ambient temperature. Blots were washed again prior to incubation with enhanced chemiluminescence (ECL) substrate (Amersham Bioscience, Piscataway, NJ; catalog no. RPN2106) for 1 minute at ambient temperature and were rapidly exposed to X-ray films (Hyperfilm; Amersham; catalog no. RPN3114K).
Total FLT3 detection. Blots were stripped of antibodies by incubation in Western Stripping buffer (Pierce; catalog no. 21059) for 30 minutes at room temperature. After washing, blots were incubated for 30 minutes in blocking buffer. Total FLT3 was detected by anti-FLT3 polyclonal antibodies (Santa Cruz Biotechnology; catalog no. SC-480) for 1 hour at ambient temperature, followed by sheep anti–rabbit IgG-HRP (Chemicon International, Temecula, CA; catalog no. AP304P). Blots were washed, incubated with ECL substrate, and exposed to X-ray film, as described in “Phosphorylated FLT3 (pFLT3) detection.”
The qualitative assessment of FLT3 phosphorylation was done visually. Because the amount of protein loaded onto the gels was not standardized for every time point, the intensity of each pFLT3 band was normalized to that of the corresponding total FLT3 band at each time point. Inhibition of FLT3 phosphorylation was then assessed with reference to the intensity of the bands in the baseline sample. Given the semiquantitative nature of the assay and the qualitative analysis, results were categorized as “no inhibition,” “inhibition,” or “not assessable” (eg, no detectable band in the predose sample or samples without detectable total FLT3).
 | Table 1. Tandutinib phase 1 experience: patient characteristics |
| Table 2. Patients with FLT3 mutations |
| Table 3. Summary of tandutinib dose escalation |
At the 525-mg twice daily dose level, 1 of the first 3 patients enrolled terminated dosing on day 10 because of grade 3 generalized muscular weakness and fatigue. The patient had a preexisting benign intention tremor that worsened, and she also developed hyperreflexia with clonus. There was no change in mental status, speech, or affect, and there were no focal abnormalities on neurologic examination. At the time of these observations the tandutinib plasma concentration was 1060 ng/mL. The patient's weakness improved to grade 1 to 2 within 72 hours of stopping tandutinib therapy. Complete resolution of all abnormal findings could not be documented because the patient was subsequently transferred to hospice care with progressive leukemia. In light of this DLT, 3 additional patients were enrolled to the 525-mg twicedaily dose level. None of these additional patients experienced DLT or weakness or fatigue related to tandutinib.
Three patients were enrolled to the 700-mg twice-daily dose level. Two of these patients terminated tandutinib therapy on days 9 and 16, respectively, because of grade 3 and 4 generalized muscular weakness. The corresponding trough plasma concentrations of tandutinib in these 2 patients were 1390 and 2220 ng/mL. No focal neurologic deficits were demonstrable in either patient, and neither patient had any change in mental status, speech, or affect. In contrast to the patient experiencing dose-limiting weakness at 525 mg twice daily, neither of these patients exhibited hyperreflexia or tremor. The patient with grade 3 weakness fully recovered within 24 hours of stopping tandutinib, and the patient with grade 4 weakness recovered after 3 days.
In the 3 cases of tandutinib-related muscular weakness, the tandutinib plasma concentration determined 12 hours or more after the last dose exceeded 1000 ng/mL. Only 1 other patient, treated at 400 mg twice daily, had a similarly high plasma concentration. On day 14 of treatment this patient's predose tandutinib plasma concentration was 1010 ng/mL. A review of this patient's dosing history indicated that the day 14 sample was taken before that morning's dose of tandutinib. However, unlike the patients who developed dose-limiting muscular weakness, the preceding trough tandutinib plasma concentrations in this patient were significantly lower (577 ng/mL on day 10), and, as tandutinib therapy continued, plasma concentrations remained well below 1000 ng/mL (432 ng/mL on day 17).
| Table 4. Tandutinib phase 1 experience: selected drug-related adverse events by CTC grade |
Although mild myelosuppression was observed in preclinical toxicology studies with the chronic administration of tandutinib at high doses, no evidence of hematologic toxicity was observed in this study. Decreases in peripheral blood cell counts were always accompanied by increases in bone marrow blast counts. Four patients without FLT3-ITD mutations maintained stable peripheral blood counts and bone marrow blast counts for relatively long periods of time, ranging from 154 to 190 days, at tandutinib doses ranging from 100 to 525 mg twice daily.
Preclinical evaluation of tandutinib suggested that it may have the potential for prolongation of the QT interval. However, it was not possible in this complex, often acutely ill patient population to rigorously assess the effect of tandutinib on the QT interval. Nevertheless, a regression analysis (Figure 1) was performed to explore the relation between tandutinib dose and change in QTc from baseline, measured after 28 days of tandutinib administration. The day 28 time point was chosen because at earlier time points tandutinib plasma concentrations were generally lower. Linear regression analysis showed the slope of the line to be 0.064, suggesting that for each 100-mg increase in the dose of tandutinib there was a 6.4-msec increase in QTc compared with baseline. However, although the slope of the line is positive, it is not statistically different from zero (P = .240, t test). Further analysis revealed that 1 patient treated at 525 mg twice daily, who had a 270-msec increase in QTc on day 28, is responsible for the positive slope of the linear regression line. Without the inclusion of this patient the slope is slightly negative (–0.007), although not statistically different from zero (P = .812, t test). Interestingly, this patient's profound QT interval prolongation on day 28 escaped initial clinical detection, and he continued therapy with tandutinib for a total of 162 days. His QT interval returned to within normal limits as dosing continued. In addition, this patient's tandutinib plasma concentrations were much below the population average throughout his course of treatment: on day 28 his predose tandutinib plasma concentration was only 54 ng/mL, with a maximum of 156 ng/mL measured 1 hour after dosing.
 | Figure 1. Change in day-28 rate-corrected QT interval (QTc) from baseline versus tandutinib dose. |
 | Figure 2. Tandutinib plasma concentration versus time profile for a patient receiving 525 mg twice daily for 28 days. (A) Plasma concentration versus time profile during days 0 to 30. (B) Plasma concentration versus time profile following completion of dosing. (more ...) |
The tandutinib plasma concentration versus time curve following the day 28 dose shows 2 phases of plasma concentration versus time decay. This observation was subsequently corroborated by modeling, whereby a 2-compartment open linear model with first-order absorption best described the pharmacokinetic data. Estimates of absolute pharmacokinetic parameter values could not be made because intravenous tandutinib administration data were not available. Therefore, the pharmacokinetic parameters were calculated relative to absolute bioavailability (F). The parameters determined were relative total body clearance (CL/F), intercompartmental clearance (Q2/F), apparent central volume of distribution (Vc/F), apparent peripheral volume of distribution (Vp/F), mean residence time (MRT), absorption half-life (t1/2 abs), and lag time (Tlag) (Table 5).
| Table 5. Summary of model-based pharmacokinetic parameters |
The large value for CL/F (148 L/h/70 kg) implies that the extent of tandutinib systemic uptake is incomplete, perhaps because of incomplete absorption; antitransport; or first-pass, extrahepatic elimination, or both. If tandutinib is distributed equally in plasma and red cells and liver blood flow is 90 L/h, then an estimate of the extent of uptake into the systemic circulation would be 21%. The 2-compartment model population parameters predict that, on average, 90% and 95% of steady-state plasma concentrations are achieved after 8.6 and 11.4 days of treatment, respectively.
The patient with wild-type FLT3 received tandutinib at 150 mg twice daily. Total FLT3 levels in this patient were comparable to levels in patients with ITD mutations. However, the level of pFLT3 prior to the first dose of tandutinib was very low and did not change through day 3 (last data point available), making assessment of inhibition difficult. The tandutinib plasma concentrations for this patient were 23.3, 6.4, and 13.3 ng/mL at 2 and 8 hours, day 1, and day 3, respectively, all well below the concentration predicted necessary for inhibition of receptor phosphorylation.
The patients with FLT3-ITD mutations received tandutinib at 300 mg (1 patient) and 525 mg (2 patients) twice daily. Figure 3 depicts the total FLT3 and pFLT3 status for 1 of the patients treated at 525 mg twice daily, prior to tandutinib administration, 2 and 8 hours after the first dose, and prior to the morning dose of tandutinib on day 3. Prior to tandutinib administration, both total FLT3 and pFLT3 can be readily detected. Subsequently, with associated tandutinib plasma concentrations of 219, 177, and 128 ng/mL, total FLT3 is unchanged to increased, whereas pFLT3 is clearly reduced. Reduction in pFLT3 compared with prior to dosing was also observed at different time points in the presence of tandutinib in the other patients with FLT3-ITD mutations, showing that tandutinib has an inhibitory effect on FLT3 phosphorylation (Table 6).
 | Figure 3. Relation between inhibition of FLT3 phosphorylation in peripheral blasts and plasma concentration of tandutinib in a single patient receiving 525 mg twice daily. D1 indicates day 1; D3, day 3. |
| Table 6. Inhibition of FLT3 phosphorylation versus tandutinib plasma concentration in assessable patients with FLT3-ITD mutations by dose level and time point |
 | Figure 4. Single-patient hematologic data for 2 patients treated with tandutinib. The patients received (A) tandutinib 525 mg twice daily or (B) tandutinib 700 mg twice daily. WBCs indicate, white blood cells; ANC, absolute neutrophil count; APB, absolute peripheral (more ...) |
Among the remaining 6 patients with FLT3-ITD mutations, a number of factors potentially precluded or confounded the observation of an antileukemic effect. One patient was treated at 150 mg twice daily and did not achieve tandutinib plasma concentrations expected to consistently inhibit receptor activation. A patient treated at 300 mg twice daily developed overwhelming sepsis on day 13 of treatment, prompting cessation of tandutinib dosing. Although this patient's absolute peripheral blast count was decreasing when tandutinib dosing was stopped, the patient was also receiving therapy with hydroxyurea. As described earlier, another patient with a FLT3-ITD mutation was given a single 400-mg dose of tandutinib and within hours developed atrial fibrillation and hypoxemia considered to be unrelated to study therapy; the decision was made to not continue protocol therapy. Two patients, 1 treated at 525 mg twice daily and the other treated at 700 mg twice daily, stopped therapy on days 10 and 9, respectively, because of tandutinib-induced weakness. Neither of these patients resumed treatment with tandutinib, thereby precluding response evaluation. An additional patient treated at 525 mg twice daily withdrew from the study on day 32 and, although evaluable for response, there was no evidence of an antileukemic effect.
One patient had a D835Y point mutation in the activation loop of FLT3. After 31 days of tandutinib therapy at 400 mg twice daily, this patient's leukemia had progressed.
Although preclinical toxicology data suggested that myelosuppression and hepatic inflammation would be the main limitations of tandutinib therapy, the dose-limiting toxicity of tandutinib proved to be generalized muscular weakness, fatigue, or both. The patient who developed weakness at the 525-mg twice-daily dose level had generalized hyperreflexia with clonus, which suggested a centrally mediated effect. However, no other signs or symptoms of central nervous system toxicity were manifest in this patient, and none were manifest in either of the 2 patients treated at 700 mg twice daily who developed generalized muscular weakness. The current hypothesis is that this toxicity may result from an effect of tandutinib at the neuromuscular junction, as suggested by preclinical data showing that tandutinib has the capacity to bind to a muscle-type nicotinic receptor. This toxicity does not appear to be related to tandutinib's inhibition of FLT3, KIT, or PDGFR, given that muscular weakness has not been reported with other FLT3 antagonists in clinical development.23-25
Despite the slow elimination of tandutinib, tandutinib-induced muscular weakness proved to be rapidly reversible. The time course required for resolution of this toxicity is likely explained by tandutinib's biphasic pharmacokinetic profile: following oral dosing and achievement of maximum plasma concentration there is an initially rapid decline in tandutinib plasma concentration, reflecting drug distribution, followed by a much slower phase of drug elimination (Figure 2B). All 3 patients presenting with muscular weakness were found to have trough tandutinib plasma concentrations greater than 1000 ng/mL. They undoubtedly had higher plasma concentrations immediately after tandutinib dosing. Only one other patient in this trial exhibited a trough concentration greater than 1000 ng/mL, but this concentration (1010 ng/mL) was achieved only transiently. These observations have led to the provisional hypothesis that the general muscular weakness associated with tandutinib is related to its plasma concentration, and that trough concentrations of 1000 ng/mL or greater should be avoided. However, the limited data regarding the ability of tandutinib to inhibit the activation (phosphorylation) of either wild-type or ITD-mutated FLT3 are consistent with preclinical data, suggesting that the in vivo IC90 is 150 ng/mL or greater.18,19 Therefore, assuming that the goal of therapy is to continuously maintain plasma concentrations of IC90 or greater, the therapeutic index of tandutinib is 1000/150 = 6.7.
Tandutinib therapy was associated with other toxicities that, although not dose limiting, are clinically important. In the majority of patients, tandutinib-related nausea, vomiting, and diarrhea were grade 1 in severity and could be managed successfully with standard supportive therapies such as 5-HT3 antagonists and loperamide. However, tandutinib tended to exacerbate preexisting nausea, vomiting, or diarrhea and resulted in one instance of dose-limiting diarrhea. The periorbital and peripheral edema associated with tandutinib therapy were mild and manageable and are mainly of interest because similar edema is observed with imatinib mesylate.2-5 The edema associated with imatinib mesylate has been attributed to PDGFR inhibition, and tandutinib is a potent inhibitor of PDGFR.19 Although no relation could be found between tandutinib dose and change from baseline in length of the QTc interval, this analysis should not be viewed as conclusive. Definitive conclusions about the possible effect of tandutinib on the QT interval will require dedicated studies in more stable patients or in healthy subjects.
This phase 1 trial was limited in its ability to assess the antileukemic activity of tandutinib. The majority of patients were treated at doses not expected to be effective, and only 8 patients in the study had AML with FLT3-ITD mutations. Only 1 patient with an activating point mutation in FLT3 was treated in this study, and in this patient there was no evidence of an antileukemic effect. Although no conclusions can be drawn from this experience, tandutinib is known to have lower potency against activating point mutations in FLT3 than against ITD mutations.26 Even among the patients with FLT3-ITD mutations who were treated at potentially effective doses, response evaluation was often not possible because of rapid disease progression, sudden disease-related clinical deterioration, or tandutinib-related toxicity. Nevertheless, evidence of antileukemic activity was observed in 2 patients treated at 525 and 700 mg twice daily, respectively. This activity did not fulfill the traditional, protocol-specified definition of a partial or complete remission, which perhaps is not surprising given the patient population and the complexity of this disease. However, combined with the evidence that tandutinib inhibits the activation (phosphorylation) of FLT3 in patients' leukemic blasts, the observed activity provides hope that phase 2 testing of tandutinib 525 mg twice daily in patients with AML and FLT3-ITD mutations may confirm the therapeutic activity of this agent.27
D.J.D. designed and performed research, analyzed data, and assisted in the final preparation of the manuscript; R.M.S. and M.A.C. designed and performed research; M.L.H. and R.B.K. designed and performed research and analyzed data; S.D.N. designed and performed research and edited the paper; R.L.P. and P.T.C. performed research; M.R.C. was medical monitor for the trial, analyzed the clinical data, and wrote initial drafts of the manuscript; J.-M.L. performed research, analyzed data, and wrote a section of the paper; M.D.K. performed the pharmacokinetic analysis, contributed to the pharmacokinetic results, and reviewed the paper; S.S. was the biostatistician for trial, analyzed and reviewed data, and reviewed the paper; N.H. performed the pharmacokinetic analysis and contributed details of pharmacokinetic methods and results for the manuscript; B.J.D. performed research and analyzed data; M.C.H. designed and performed research, contributed vital new reagents or analytical tools, analyzed data, and wrote a section of the paper.
D.J.D., R.M.S., M.L.H., S.D.N., R.L.P., R.B.K., M.A.C., P.T.C., and B.J.D. have no commercial interests to disclose. M.R.C., J.-M.L., M.D.K., and S.S. are employed by Millennium Pharmaceuticals Inc, whose product was studied in the present work. N.H. and M.C.H. are consultants to Millennium Pharmaceuticals Inc, whose product was studied in the present work. M.C.H. is also a consultant to Novartis Pharmaceuticals.
We thank Laura McGreevey and Tina Harrell for assisting with the FLT3 mutational analyses, Ilene Galinksy (D.J.D. and R.M.S.), and Jeffrey Gardner for technical assistance (M.L.H.). We also thank Steve Hill (medical writer) and Rachel Higgins (editor with Gardiner-Caldwell London) for their support in drafting the manuscript.
This work was supported in part by research funding from Millennium Pharmaceuticals (grant POI CA66996-06A1), by a Veterans Administration (VA) Merit Review Grant (M.C.H.), aLeukemia and Lymphoma Society Specialized Center of Research (LLS SCOR) grant (S.D.N.), and a Doris Duke Charitable Foundation Distinguished Clinical Scientist Development Award (B.J.D.).
References
