Choosing Antifungals for the Midostaurin-Treated Patient: Does CYP3A4 Outweigh Recommendations? A Brief Insight from Real Life.

INTRODUCTION: Patients treated with midostaurin and chemotherapy are at risk of invasive fungal disease. Prophylactic posaconazole is recommended for these patients, but posaconazole strongly inhibits the CYP3A4 isozyme that metabolizes midostaurin. Posaconazole therefore introduces a risk of patient's overexposure to midostaurin. METHODS: Blood samples were obtained from 4 patients treated with midostaurin for newly diagnosed FLT3-mutAML. Patients had received a concomitant treatment with posaconazole, isavuconazole, or micafungin, respectively. All blood samples were drawn before daily dose administration of midostaurin. RESULTS: Posaconazole caused a ≥8-fold increase of midostaurin plasma levels at through, which was accompanied by a decreased plasma exposure to O-demethylated or hydroxylated midostaurin metabolites. We also show that hematologists react to risk perception by replacing posaco-nazole with antifungals like micafungin or isavuconazole, which lack a strong inhibition of CYP3A4 and fail to modify midostaurin pharmacokinetics but are not formally recommended in these settings. DISCUSSION: In real-life scenarios, concerns about CYP3A4 inhibition may outweigh compliance with recommendations. Large studies are needed to survey the risk:benefit of hematologist's decision to replace posaconazole with other antifungals.

Drug-drug interactions of newly approved small molecule inhibitors for acute myeloid leukemia.

Several small molecule inhibitors (SMIs) have been recently approved for AML patients. These targeted therapies could be more tolerable than classical antineoplastics, but potential drug-drug interactions (DDI) are relatively frequent. Underestimation or lack of appropriate awareness and management of DDIs with SMIs can jeopardize therapeutic success in AML patients, which often require multiple concomitant medications in the context of prior comorbidities or for the prevention and treatment of infectious and other complications. In this systematic review, we analyze DDIs of glasdegib, venetoclax, midostaurin, quizartinib, gilteritinib, enasidenib, and ivosidenib. CYP3A4 is the main enzyme responsible for SMIs metabolism, and strong CYP3A4 inhibitors, such azoles, could increase drug exposure and toxicity; therefore dose adjustments (venetoclax, quizartinib, and ivosidenib) or alternative therapies or close monitoring (glasdegib, midostaurin, and gilteritinib) are recommended. Besides, coadministration of strong CYP3A4 inducers with SMIs should be avoided due to potential decrease of efficacy. Regarding tolerability, QTc prolongation is frequently observed for most of approved SMIs, and drugs with a potential to prolong the QTc interval and CYP3A4 inhibitors should be avoided and replaced by alternative treatments. In this study, we critically assess the DDIs of SMIs, and we summarize best management options for these new drugs and concomitant medications.

Midostaurin, a Novel Protein Kinase Inhibitor for the Treatment of Acute Myelogenous Leukemia: Insights from Human Absorption, Metabolism, and Excretion Studies of a BDDCS II Drug.

The absorption, metabolism, and excretion of midostaurin, a potent class III tyrosine protein kinase inhibitor for acute myelogenous leukemia, were evaluated in healthy subjects. A microemulsion formulation was chosen to optimize absorption. After a 50-mg [14C]midostaurin dose, oral absorption was high (>90%) and relatively rapid. In plasma, the major circulating components were midostaurin (22%), CGP52421 (32.7%), and CGP62221 (27.7%). Long plasma half-lives were observed for midostaurin (20.3 hours), CGP52421 (495 hours), and CGP62221 (33.4 hours). Through careful mass-balance study design, the recovery achieved was good (81.6%), despite the long radioactivity half-lives. Most of the radioactive dose was recovered in feces (77.6%) mainly as metabolites, because only 3.43% was unchanged, suggesting mainly hepatic metabolism. Renal elimination was minor (4%). Midostaurin metabolism pathways involved hydroxylation, O-demethylation, amide hydrolysis, and N-demethylation. High plasma CGP52421 and CGP62221 exposures in humans, along with relatively potent cell-based IC50 for FMS-like tyrosine kinase 3-internal tandem duplications inhibition, suggested that the antileukemic activity in AML patients may also be maintained by the metabolites. Very high plasma protein binding (>99%) required equilibrium gel filtration to identify differences between humans and animals. Because midostaurin, CGP52421, and CGP62221 are metabolized mainly by CYP3A4 and are inhibitors/inducers for CYP3A, potential drug-drug interactions with mainly CYP3A4 modulators/CYP3A substrates could be expected. Given its low aqueous solubility, high oral absorption and extensive metabolism (>90%), midostaurin is a Biopharmaceutics Classification System/Biopharmaceutics Drug Disposition Classification System (BDDCS) class II drug in human, consistent with rat BDDCS in vivo data showing high absorption (>90%) and extensive metabolism (>90%).

Comparison of two endogenous biomarkers of CYP3A4 activity in a drug-drug interaction study between midostaurin and rifampicin.

PURPOSE: Midostaurin, a multitargeted tyrosine kinase inhibitor, is primarily metabolized by CYP3A4. This midostaurin drug-drug interaction study assessed the dynamic response and clinical usefulness of urinary 6ß-hydroxycortisol to cortisol ratio (6ßCR) and plasma 4ß-hydroxycholesterol (4ßHC) for monitoring CYP3A4 activity in the presence or absence of rifampicin, a strong CYP3A4 inducer. METHODS: Forty healthy adults were randomized into groups for either placebo or treatment with rifampicin 600 mg QD for 14 days. All participants received midostaurin 50 mg on day 9. Midostaurin plasma pharmacokinetic parameters were assessed. Urinary 6ßCR and plasma 4ßHC levels were measured on days 1, 9, 11, and 15. RESULTS: Both markers remained stable over time in the control group and increased significantly in the rifampicin group. In the rifampicin group, the median increases (vs day 1) on days 9, 11, and 15 were 4.1-, 5.2-, and 4.7-fold, respectively, for 6ßCR and 3.4-, 4.1-, and 4.7-fold, respectively, for 4ßHC. Inter- and intrasubject variabilities in the control group were 45.6 % and 30.5 %, respectively, for 6ßCR, and 33.8 % and 7.5 %, respectively, for 4ßHC. Baseline midostaurin area under the concentration-time curve (AUC) correlated with 4ßHC levels (ρ = -0.72; P = .003), but not with 6ßCR (ρ = 0.0925; P = .6981). CONCLUSIONS: Both 6ßCR and 4ßHC levels showed a good dynamic response range upon strong CYP3A4 induction with rifampicin. Because of lower inter- and intrasubject variability, 4ßHC appeared more reliable and better predictive of CYP3A4 activity compared with 6ßCR. The data from our study further support the clinical utility of these biomarkers.

Liquid chromatography-tandem mass spectrometry assay for therapeutic drug monitoring of the tyrosine kinase inhibitor, midostaurin, in plasma from patients with advanced systemic mastocytosis.

We developed and validated quantitative bioanalytical liquid chromatography-tandem mass spectrometry assay for the protein kinase inhibitor, midostaurin. Plasma samples were pre-treated using a protein precipitation with methanol containing midostaurin-d5 as an internal standard. After centrifugation, 5µL of the supernatant was injected into the chromatographic system. The system consisted of a 3.5µm particle bonded octadecyl silica column, with gradient elution using a mixture of 0.1% (v/v) formic acid in acetonitrile and 10mM ammonium formate in water with 0.1% formic acid. The analyte was quantified using the selected reaction-monitoring mode of a triple quadrupole mass spectrometer equipped with a heated electrospray interface. The assay was validated in a 75-2500ng/mL calibration range. For quality control, within-day and between-day precisions were 1.2-2.8%, and 1.2-6.9%, respectively. The ß-expectation tolerance limit (accuracy) met the limits of acceptance ±15% (±20% for the LLQ). The drug was sufficiently stable under all relevant analytical conditions. The assay has successfully been used to assess drug levels for therapeutic drug monitoring in patients presenting advanced systemic mastocytosis and treated with the promising midostaurin.

Radiotherapy in conjunction with 7-hydroxystaurosporine: a multimodal approach with tumor pO2 as a potential marker of therapeutic response.

Checkpoint inhibitors potentially could be used to enhance cell killing by DNA-targeted therapeutic modalities such as radiotherapy. UCN-01 (7-hydroxystaurosporine) inhibits S and G2 checkpoint arrest in the cells of various malignant cell lines and has been investigated in combination with chemotherapy. However, little is known about its potential use in combination with radiotherapy. We report the effect of 20 Gy radiation given in conjunction with UCN-01 on the pO2 and growth of subcutaneous RIF-1 tumors. Multisite EPR oximetry was used for repeated, non-invasive tumor pO2 measurements. The effect of UCN-01 and/or 20 Gy on tumor pO2 and tumor volume was investigated to determine therapeutic outcomes. Untreated RIF-1 tumors were hypoxic with a tissue pO2 of 5-7 mmHg. Treatment with 20 Gy or UCN-01 significantly reduced tumor growth, and a modest increase in tumor pO2 was observed in tumors treated with 20 Gy. However, irradiation with 20 Gy 12 h after UCN-01 treatment resulted in a significant inhibition of tumor growth and a significant increase in tumor pO2 to 16-28 mmHg from day 1 onward compared to the control, UCN-01 or 20-Gy groups. Treatment with UCN-01 12 h after 20 Gy also led to a similar growth inhibition of the tumors and a similar increase in tumor pO2. The changes in tumor pO2 observed after the treatment correlated inversely with the tumor volume in the groups receiving UCN-01 with 20 Gy. This multimodal approach could be used to enhance the outcome of radiotherapy. Furthermore, tumor pO2 could be a potential marker of therapeutic response.

A highly sensitive method for the detection of PKC412 (CGP41251) and its metabolites by high-performance liquid chromatography.

INTRODUCTION: PKC412 has proven activity in the treatment of acute myelogenous leukemia (AML). The drug is extensively metabolized, and recently it was shown that its metabolites have differing efficacies against malignant cells. Therefore, we established a new isocratic HPLC method, which sensitively detects PKC412 and five of its metabolites. METHOD: Quantification was performed using N-phenyl-1-naphtylamine as an internal standard. We could demonstrate linearity in a range from 10 to 10,000 ng/ml PKC412 with an intra-day variability of less than 7.5% and an inter-day-variability of less than 11%. RESULTS: The assay was used to monitor plasma samples from patients with AML treated within a clinical trial. Here we could demonstrate the ability of the assay to detect five metabolites of PKC412 and describe the application of the assay for drug monitoring in clinical situations. CONCLUSION: The assay described here will enable a discriminative analysis of PKC412 and its metabolites in human plasma samples. First clinical application of the assay suggests different rates of metabolism of the individual metabolites of PKC412 with e.g. accumulation of CGP52421 epimer 2 even after cessation of therapy. Since it is assumed that different metabolites of PKC412 have a very individual mode of actions, determination of PKC412 and its metabolites within clinical studies of the drug will be important.

Plasma inhibitory activity (PIA): a pharmacodynamic assay reveals insights into the basis for cytotoxic response to FLT3 inhibitors.

We have developed a useful surrogate assay for monitoring the efficacy of FLT3 inhibition in patients treated with oral FLT3 inhibitors. The plasma inhibitory activity (PIA) for FLT3 correlates with clinical activity in patients treated with CEP-701 and PKC412. Using the PIA assay, along with in vitro phosphorylation and cytotoxicity assays in leukemia cells, we compared PKC412 and its metabolite, CGP52421, with CEP-701. While both drugs could effectively inhibit FLT3 in vitro, CEP-701 was more cytotoxic to primary samples at comparable levels of FLT3 inhibition. PKC412 appears to be more selective than CEP-701 and therefore less effective at inducing cytotoxicity in primary acute myeloid leukemia (AML) samples in vitro. However, the PKC412 metabolite CGP52421 is less selective than its parent compound, PKC412, and is more cytotoxic against primary blast samples at comparable levels of FLT3 inhibition. The plasma inhibitory activity assay represents a useful correlative tool in the development of small-molecule inhibitors. Our application of this assay has revealed that the metabolite CGP52421 may contribute a significant portion of the antileukemia activity observed in patients receiving oral PKC412. Additionally, our results suggest that nonselectivity may constitute an important component of the cytotoxic effect of FLT3 inhibitors in FLT3-mutant AML.

Patients with acute myeloid leukemia and an activating mutation in FLT3 respond to a small-molecule FLT3 tyrosine kinase inhibitor, PKC412.

Leukemic cells from 30% of patients with acute myeloid leukemia (AML) have an activating mutation in the FLT3 (fms-like tyrosine kinase) gene, which represents a target for drug therapy. We treated 20 patients, each with mutant FLT3 relapsed/refractory AML or high-grade myelodysplastic syndrome and not believed to be candidates for chemotherapy, with an FLT3 tyrosine kinase inhibitor, PKC412 (N-benzoylstaurosporine), at a dose of 75 mg 3 times daily by mouth. The drug was generally well tolerated, although 2 patients developed fatal pulmonary events of unclear etiology. The peripheral blast count decreased by 50% in 14 patients (70%). Seven patients (35%) experienced a greater than 2-log reduction in peripheral blast count for at least 4 weeks (median response duration, 13 weeks; range, 9-47 weeks); PKC412 reduced bone marrow blast counts by 50% in 6 patients (2 of these to < 5%). FLT3 autophosphorylation was inhibited in most of the Corresponding patients, indicating in vivo target inhibition at the dose schedule used in this study. PKC412 is an oral tyrosine kinase inhibitor with clinical activity in patients with AML whose blasts have an activating mutation of FLT3, suggesting potential use in combination with active agents, such as chemotherapy.

A phase I trial of daily oral 4'- N -benzoyl-staurosporine in combination with protracted continuous infusion 5-fluorouracil in patients with advanced solid malignancies.

PURPOSE: 4'- N -Benzoyl-staurosporine (PKC412) is an orally available staurosporine derivative that inhibits protein kinase C. The objectives of this phase I trial were to determine the maximum tolerated dose (MTD), the dose limiting toxicities (DLTs), and the pharmacokinetics of PKC412 when co-administered with 5-Fluorouracil (5-FU). EXPERIMENTAL DESIGN: PKC412 was given daily with a 21-day continuous i.v. infusion of 5-FU 200 mg/m2/day, repeated every 4 weeks. The PKC412 dose was escalated by a modified continual reassessment method. The steady-state plasma pharmacokinetics of 5-FU, PKC412, and two of its circulating metabolites were determined during the first cycle of therapy. RESULTS: A total of 33 patients were treated with 70 cycles (median: 2, range: 1-4) of PKC412 at doses ranging from 25 to 225 mg/day. No significant toxicities were encountered with doses up to 150 mg/day. Among nine patients treated with 225 mg/day of PKC412, one experienced grade 3 fatigue and nausea, another developed grade 3 hyperglycemia, and three had grade 2 emesis and stomatitis, leading to early treatment discontinuation. Minor responses consisting of a 40-45% tumor reduction were observed in two patients, one with gall bladder carcinoma and one with breast cancer. Mean values of steady-state pharmacokinetic variables for both PKC412 and 5-FU were comparable to single agent studies. CONCLUSIONS: The recommended phase II dose of PKC412 is 150 mg/day when combined with a continuous infusion of 200 mg/m2/day 5-FU. The dose limiting toxicity was grade 2 emesis and stomatitis and the regimen showed indications of activity. There was no evidence of a pharmacokinetic interaction between the two drugs.

Pharmacokinetics and metabolism of the staurosporine analogue CGP 41 251 in mice.

Studies with CGP 41 251 (I), an N-benzoylstaurosporine derivative and PKC-alpha inhibitor, revealed that oral administration of 400 microg/day of the compound to wild type mice on four successive days reversed multi drug resistance (Killion et al. Oncology Research 7: 453-459, 1995). In our study, the same regimen of administration was followed with the primary objective to establish the pharmacokinetics and metabolism of the compound and to substantiate at which plasma concentrations of CGP 41 251 multi drug resistance (MDR) reversal can be expected. Concentrations of CGP 41 251 and metabolites in plasma were determined by a validated high performance liquid chromatography (HPLC) method with fluorescence detection. Structural characterization of the metabolites was performed with HPLC and mass spectrometric detection. In our experiment extensive metabolism of CGP 41 251 was found. The presence of five hydroxylated metabolites of CGP 41 251 (I) was confirmed and two metabolites were structurally elucidated as CGP 50 750 (III) and CGP 52 421 (V). Maximal concentrations of 73 ng/ml, 1.9 ng/ml and 126 ng/ml for CGP 41 251 (I), III and V were found, respectively. The mass spectra of the other three metabolites indicate that these are oxidized nitrogens or hydroxylated compounds. As yet, the oxidation or hydroxylation sites have not been established. This study has revealed new information about CGP 41 251 pharmacokinetics and metabolism. Target levels between 10-100 ng/ml may be important to achieve in further clinical trials with CGP 41 251 as MDR modulator.

High-performance liquid chromatographic analysis of staurosporine in vivo. Its translocation and pharmacokinetics in rats.

The protein kinase inhibitor staurosporine (Stsp) has been used extensively to study physiological functions, biochemical mechanisms, and cancer therapy. Using an HPLC assay for Stsp developed in our laboratory, we find that only 0.7% of Stsp remains in circulating blood of rats 5 min after injection. In vitro, Stsp is adsorbed to red blood cells (RBC) weakly and reversibly. In vivo, all but 1.2-2.5% of Stsp injected is adsorbed by the heart and lungs in one passage through them, indicating that the endothelium acts as a major Stsp sink. Following initial adsorption, pharmacokinetic studies demonstrated that Stsp had a half-life of 51.6 min in plasma and 75.3 min in RBC. Thus, plasma Stsp was in the cancer therapy range of 1-10 ng/ml for 2.7 h following a bolus injection. This data indicates that a bolus injection of Stsp must be followed by a continuous infusion of low Stsp concentration for several days to produce the G1 arrest in cells necessary to stop cell proliferation.

High-performance liquid chromatographic analysis of the new antitumour drug N-benzoylstaurosporine (CGP 41 251) and four potential metabolites in micro-volumes of plasma.

A high-performance liquid chromatographic (HPLC) assay is described for the determination of the new antitumour drug N-benzoylstaurosporine (CGP 41 251; I) and four of its potential metabolites in micro-volumes (100 microliters) of plasma. After addition of an internal standard, the compounds were isolated from plasma by liquid-liquid extraction with diisopropyl ether. Chromatography was carried out using a 5 microns LiChrospher C-18 end-capped column (125 x 4.0 mm i.d.) and binary gradient elution with acetonitrile and a triethylamine-containing phosphate buffer (pH 3.6) as solvents. Fluorimetric detection was performed with excitation and emission wavelengths set at 286 and 386 nm, respectively. The absolute recovery was more than 98% for all of the investigated compounds. The limit of detection (LOD) for I and three metabolites was 0.1 ng ml-1 and the lower limit of quantitation (LLQ) was 0.2 ng ml-1 in 100 microliters of plasma. The LOD and LLQ for the fourth metabolite was 0.25 and 0.5 ng ml-1, respectively. The between-day and within-day precisions were always < 15% for all the analytes. A limited pharmacokinetic study in mice treated and with I demonstrated that the method is appropriate for this purpose.