IKZF1 gene deletions drive resistance to cytarabine in B-cell precursor acute lymphoblastic leukemia.

IKZF1-deletions occur in 10-15% of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and predict a poor outcome. However, the impact of IKZF1-loss on sensitivity to drugs used in contemporary treatment protocols has remained underexplored. Here we show in experimental models and in patients that loss of IKZF1 promotes resistance to AraC, a key component of both upfront and relapsed treatment protocols. We attribute this resistance, in part, to diminished import and incorporation of cytarabine (AraC) due to reduced expression of the solute carrier hENT1. Moreover, we find elevated mRNA expression of Evi1, a known driver of therapy resistance in myeloid malignancies. Finally, a kinase directed CRISPR/Cas9-screen identified that inhibition of either mediator kinases CDK8/19 or casein kinase 2 can restore response to AraC. We conclude that this high-risk patient group could benefit from alternative antimetabolites, or targeted therapies that resensitize the cells to AraC.

Cortisol as Biomarker for CYP17-Inhibition is Associated with Therapy Outcome of Abiraterone Acetate.

BACKGROUND: Abiraterone acetate is an irreversible 17α-hydroxylase/C17, 20-lyase (CYP17) inhibitor approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC) patients. Inhibition of this enzyme leads to low testosterone and cortisol levels in blood. There is growing evidence that clinical efficacy of abiraterone is related to the rate of suppression of serum testosterone. However, quantification of very low levels of circulating testosterone is challenging. We therefore aimed to investigate whether circulating cortisol levels could be used as a surrogate biomarker for CYP17 inhibition in patients with mCRPC treated with abiraterone acetate. PATIENTS AND METHODS: mCRPC patients treated with abiraterone acetate were included. Abiraterone and cortisol levels were measured with a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). On treatment cortisol and abiraterone concentrations were related to treatment response and progression free survival. RESULTS: In total 117 patients were included with a median cortisol concentration of 1.13 ng/ml (range: 0.03 - 82.2) and median abiraterone trough concentration (Cmin) of 10.2 ng/ml (range: 0.58 - 92.1). In the survival analyses, abiraterone Cmin ≥ 8.4 ng/mL and cortisol < 2.24 ng/mL were associated with a longer prostate-specific antigen (PSA) independent progression-free survival than patients with an abiraterone concentration ≥ 8.4 ng/mL and a cortisol concentration ≥ 2.24 ng/mL (13.8 months vs. 3.7 months). CONCLUSION: Our study shows that cortisol is not an independent predictor of abiraterone response in patients with mCRPC, but it is of added value in combination with abiraterone levels, to predict a response on abiraterone.

Mass balance and metabolite profiling of 14C-guadecitabine in patients with advanced cancer.

Purpose The objective of this mass balance trial was to determine the excretory pathways and metabolic profile of the novel anticancer agent guadecitabine in humans after administration of a 14C-radiolabeled dose of guadecitabine. Experimental design Included patients received at least one cycle of 45 mg/m2 guadecitabine subcutaneously as once-daily doses on Days 1 to 5 of a 28-day cycle, of which the 5th (last) dose in the first cycle was spiked with 14C-radiolabeled guadecitabine. Using different mass spectrometric techniques in combination with off-line liquid scintillation counting, the exposure and excretion of 14C-guadecitabine and metabolites in the systemic circulation, excreta, and intracellular target site were established. Results Five patients were enrolled in the mass balance trial. 14C-guadecitabine radioactivity was rapidly and almost exclusively excreted in urine, with an average amount of radioactivity recovered of 90.2%. After uptake in the systemic circulation, guadecitabine was converted into ß-decitabine (active anomer), and from ß-decitabine into the presumably inactive metabolites M1-M5. All identified metabolites in plasma and urine were ß-decitabine related products, suggesting almost complete conversion via cleavage of the phosphodiester bond between ß-decitabine and deoxyguanosine prior to further elimination. ß-decitabine enters the intracellular activation pathway, leading to detectable ß-decitabine-triphosphate and DNA incorporated ß-decitabine levels in peripheral blood mononuclear cells, providing confirmation that the drug reaches its DNA target site. Conclusion The metabolic and excretory pathways of guadecitabine and its metabolites were successfully characterized after subcutaneous guadecitabine administration in cancer patients. These data support the clinical evaluation of safety and efficacy of the subcutaneous guadecitabine drug product.

Determination of the absolute bioavailability of oral imatinib using a stable isotopically labeled intravenous imatinib-d8 microdose.

PURPOSE: The aim of this study was to ascertain whether the absolute bioavailability of oral imatinib (Glivec®) during steady state plasma pharmacokinetics in cancer patients could be determined through a concomitant intravenous administration of a single 100 µg microdose of deuterium labeled imatinib (imatinib-d8). Secondly, the usefulness of liquid chromatography-tandem mass spectrometry (LC-MS/MS) was investigated for simultaneous analysis of orally and intravenously administered imatinib. METHODS: Included patients were on a stable daily dose of 400 mg oral imatinib prior to study participation. On day 1, patients received a 100 µg intravenous imatinib-d8 microdose 2.5 h after intake of the oral dose. Plasma samples were collected for 48 h. Imatinib and imatinib-d8 concentrations were simultaneously quantified using a validated LC-MS/MS assay. The absolute bioavailability was calculated by comparing the dose-normalized exposure with unlabeled and stable isotopically labeled imatinib in plasma. RESULTS: A total of six patients were enrolled. All patients had a history of gastrointestinal stromal tumors (GIST). The median absolute bioavailability of oral imatinib at steady state was 76% (range 44-106%). Imatinib and imatinib-d8 plasma concentrations were quantified in all collected plasma samples, with no samples below the limit of quantification for imatinib-d8. CONCLUSION: The absolute bioavailability of imatinib was successfully estimated at steady state plasma pharmacokinetics using the stable isotopically labeled microdose trial design. This study exhibits the use of a stable isotopically labeled intravenous microdose to determine the absolute bioavailability of an oral anticancer agent in patients with LC-MS/MS as the analytical tool.

Pharmacokinetics and excretion of (14)C-omacetaxine in patients with advanced solid tumors.

Background Omacetaxine mepesuccinate is indicated in adults with chronic myeloid leukemia resistant and/or intolerant to ≥ 2 tyrosine kinase inhibitor treatments. This phase I study assessed the disposition, elimination, and safety of (14)C-omacetaxine in patients with solid tumors. Methods The study comprised a 7-days pharmacokinetic assessment followed by a treatment period of ≤ six 28-days cycles. A single subcutaneous dose of 1.25 mg/m(2) (14)C-omacetaxine was administered to six patients. Blood, urine, and feces were collected through 168 h or until radioactivity excreted within 24 h was <1 % of the dose. Total radioactivity (TRA) was measured in all matrices and concentrations of omacetaxine, 4'-desmethylhomoharringtonine (4'-DMHHT), and cephalotaxine were measured in plasma and urine. For each treatment cycle, patients received 1.25 mg/m(2) omacetaxine twice daily for 7 days. Results Mean TRA recovered was approximately 81 % of the dose, with approximately half of the radioactivity recovered in feces and half in urine. Approximately 20 % of the dose was excreted unchanged in urine; cephalotaxine (0.4 % of dose) and 4' DMHHT (9 %) were also present. Plasma concentrations of TRA were higher than the sum of omacetaxine and known metabolites, suggesting the presence of other (14)C-omacetaxine-derived compounds. Fatigue and anemia were common, consistent with the known toxicity profile of omacetaxine. Conclusion Renal and hepatic processes contribute to the elimination of (14)C-omacetaxine-derived radioactivity in cancer patients. In addition to omacetaxine and its known metabolites, other (14)C-omacetaxine-derived materials appear to be present in plasma and urine. Omacetaxine was adequately tolerated, with no new safety signals.

Metabolite profiling of 14C-omacetaxine mepesuccinate in plasma and excreta of cancer patients.

Omacetaxine mepesuccinate (hereafter referred to as omacetaxine) is a protein translation inhibitor approved by the US Food and Drug Administration for adult patients with chronic myeloid leukemia with resistance and/or intolerance to two or more tyrosine kinase inhibitors. The objective was to investigate the metabolite profile of omacetaxine in plasma, urine and faeces samples collected up to 72 h after a single 1.25-mg/m2 subcutaneous dose of 14C-omacetaxine in cancer patients. High-performance liquid chromatography mass spectrometry (MS) (high resolution) in combination with off-line radioactivity detection was used for metabolite identification. In total, six metabolites of omacetaxine were detected. The reactions represented were mepesuccinate ester hydrolysis, methyl ester hydrolysis, pyrocatechol conversion from the 1,3-dioxole ring. Unchanged omacetaxine was the most prominent omacetaxine-related compound in plasma. In urine, unchanged omacetaxine was also dominant, together with 4'-DMHHT. In feces very little unchanged omacetaxine was found and the pyrocatechol metabolite of omacetaxine, M534 and 4'-desmethyl homoharringtonine (4'-DMHHT) was the most abundant metabolites. Omacetaxine was extensively metabolized, with subsequent renal and hepatic elimination of the metabolites. The low levels of the metabolites found in plasma indicate that the metabolites are unlikely to contribute materially to the efficacy and/or toxicity of omacetaxine.

The pharmacokinetics and pharmacodynamics of ibogaine in opioid use disorder patients.

OBJECTIVE: Ibogaine is a hallucinogenic drug that may be used to treat opioid use disorder (OUD). The relationships between pharmacokinetics (PKs) of ibogaine and its metabolites and their clinical effects on side effects and opioid withdrawal severity are unknown. We aimed to study these relationships in patients with OUD undergoing detoxification supported by ibogaine. METHODS: The study was performed in 14 subjects with OUD. They received a single dose of 10mg/kg ibogaine hydrochloride. Plasma PKs of ibogaine, noribogaine, and noribogaine glucuronide were obtained during 24 h. Cytochrome P450 isoenzyme 2D6 (CYP2D6) genotyping was performed. The PKs were analyzed by means of nonlinear mixed effects modeling and related with corrected QT interval (QTc) prolongation, cerebellar ataxia, and opioid withdrawal severity. RESULTS: The PK of ibogaine were highly variable and significantly correlated to CYP2D6 genotype (p < 0.001). The basic clearance of ibogaine (at a CYP2D6 activity score (AS) of 0) was 0.82 L/h. This increased with 30.7 L/h for every point of AS. The relation between ibogaine plasma concentrations and QTc was best described by a sigmoid Emax model. Spearman correlations were significant (p < 0.03) for ibogaine but not noribogaine with QTc (p = 0.109) and cerebellar effects (p = 0.668); neither correlated with the severity of opioid withdrawal symptoms. CONCLUSIONS: The clearance of ibogaine is strongly related to CYPD2D6 genotype. Ibogaine cardiac side effects (QTc time) and cerebellar effects are most likely more driven by ibogaine rather than noribogaine. Future studies should aim at exploring lower doses and/or applying individualized dosing based on CYP2D6 genotype.

Development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry method for the quantification of the antimalarial drug pyronaridine in human whole blood.

Malaria remains a major health concern, aggravated by emerging resistance of the parasite to existing treatments. The World Health Organization recently endorsed the use of artesunate-pyronaridine to treat uncomplicated malaria. However, there is a lack of clinical pharmacokinetic (PK) data of pyronaridine, particularly in special populations such as children and pregnant women. Existing methods for the quantification of pyronaridine in biological matrices to support PK studies exhibit several drawbacks. These include limited sensitivity, a large sample volume required, and extensive analysis time. To overcome these limitations, an ultra-performance reversed-phase liquid chromatography tandem-mass spectrometry method to determine pyronaridine was developed and validated according to international guidelines. The method enabled fast and accurate quantification of pyronaridine in whole blood across a clinically relevant concentration range of 0.500-500 ng/mL (r2 ≥ 0.9963), with a required sample volume of 50 µL. Pyronaridine was extracted from whole blood using liquid-liquid extraction, effectively eliminating the matrix effect and preventing ion enhancement or suppression. The method achieved a satisfactory reproducible sample preparation recovery of 77%, accuracy (as bias) and precision were within ±8.2% and ≤5.3%, respectively. Stability experiments demonstrated that pyronaridine was stable for up to 315 days when stored at -70°C. Adjustments to the chromatographic system substantially reduced carry-over and improved sensitivity compared to prior methods. The method was successfully applied to quantify pyronaridine in whole blood samples from a selection of pregnant malaria patients participating in the PYRAPREG clinical trial (PACTR202011812241529) in the Democratic Republic of the Congo, demonstrating its suitability to support future PK studies. Furthermore, the enhanced sensitivity allows for the determination of pyronaridine up to 42 days post-treatment initiation, enabling assessment of the terminal elimination half-life.

Drug Transporters ABCB1 (P-gp) and OATP, but not Drug-Metabolizing Enzyme CYP3A4, Affect the Pharmacokinetics of the Psychoactive Alkaloid Ibogaine and its Metabolites.

The psychedelic alkaloid ibogaine is increasingly used as an oral treatment for substance use disorders, despite being unlicensed in most countries and having reported adverse events. Using wild-type and genetically modified mice, we investigated the impact of mouse (m)Abcb1a/1b and Abcg2 drug efflux transporters, human and mouse OATP drug uptake transporters, and the CYP3A drug-metabolizing complex on the pharmacokinetics of ibogaine and its main metabolites. Following oral ibogaine administration (10 mg/kg) to mice, we observed a rapid and extensive conversion of ibogaine to noribogaine (active metabolite) and noribogaine glucuronide. Mouse Abcb1a/1b, in combination with mAbcg2, modestly restricted the systemic exposure (plasma AUC) and peak plasma concentration (Cmax) of ibogaine. Accordingly, we found a ∼2-fold decrease in the relative recovery of ibogaine in the small intestine with fecal content in the absence of both transporters compared to the wild-type situation. Ibogaine presented good intrinsic brain penetration even in wild-type mice (brain-to-plasma ratio of 3.4). However, this was further increased by 1.5-fold in Abcb1a/1b;Abcg2 -/- mice, but not in Abcg2 -/- mice, revealing a stronger effect of mAbcb1a/1b in restricting ibogaine brain penetration. The studied human OATP transporters showed no major impact on ibogaine plasma and tissue disposition, but the mOatp1a/1b proteins modestly affected the plasma exposure of ibogaine metabolites and the tissue disposition of noribogaine glucuronide. No considerable role of mouse Cyp3a knockout or transgenic human CYP3A4 overexpression was observed in the pharmacokinetics of ibogaine and its metabolites. In summary, ABCB1, in combination with ABCG2, limits the oral availability of ibogaine, possibly by mediating its hepatobiliary and/or direct intestinal excretion. Moreover, ABCB1 restricts ibogaine brain penetration. Variation in ABCB1/ABCG2 activity due to genetic variation and/or pharmacologic inhibition might therefore affect ibogaine exposure in patients, but only to a limited extent. The insignificant impact of human CYP3A4 and OATP1B1/1B3 transporters may be clinically advantageous for ibogaine and noribogaine use, as it decreases the risks of undesirable drug interactions or interindividual variation related to CYP3A4 and/or OATP activity.

Development, validation, and clinical application of a high-performance liquid chromatography-tandem mass spectrometry assay for the quantification of total intracellular ß-decitabine nucleotides and genomic DNA incorporated ß-decitabine and 5-methyl-2'-deoxycytidine.

DNA hypermethylation is an epigenetic event that is commonly found in malignant cells and is used as a therapeutic target for ß-decitabine (ß-DEC) containing hypomethylating agents (eg Dacogen® and guadecitabine). ß-DEC requires cellular uptake and intracellular metabolic activation to ß-DEC triphosphate before it can get incorporated into the DNA. Once incorporated in the DNA, ß-DEC can exert its hypomethylating effect by trapping DNA methyltransferases (DNMTs), resulting in reduced 5-methyl-2'-deoxycytidine (5mdC) DNA content. ß-DEC DNA incorporation and its effect on DNA methylation, however, have not yet been investigated in patients treated with ß-DEC containing therapies. For this reason, we developed and validated a sensitive and selective LC-MS/MS method to determine total intracellular ß-DEC nucleotide (ß-DEC-XP) concentrations, as well as to quantify ß-DEC and 5mdC DNA incorporation relative to 2'-deoxycytidine (2dC) DNA content. The assay was successfully validated according to FDA and EMA guidelines in a linear range from 0.5 to 100 ng/mL (ß-DEC), 50 to 10,000 ng/mL (2dC), and 5 to 1,000 ng/mL (5mdC) in peripheral blood mononuclear cell (PBMC) lysate. An additional calibrator at a concentration of 0.1 ng/mL was added for ß-DEC to serve as a limit of detection (LOD). Clinical applicability of the method was demonstrated in patients treated with guadecitabine. Our data support the use of the validated LC-MS/MS method to further explore the intracellular pharmacokinetics in patients treated with ß-DEC containing hypomethylating agents.

Development and validation of LC-MS/MS methods for the quantification of the novel anticancer agent guadecitabine and its active metabolite ß­decitabine in human plasma, whole blood and urine.

Guadecitabine (SGI-110), a dinucleotide of ߭decitabine and deoxyguanosine, is currently being evaluated in phase II/III clinical trials for the treatment of hematological malignancies and solid tumors. This article describes the development and validation of bioanalytical assays to quantify guadecitabine and its active metabolite ߭decitabine in human plasma, whole blood and urine using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Since ߭decitabine is rapidly metabolized further by cytidine deaminase, plasma and whole blood samples were kept on ice-water after collection and stabilized with tetrahydrouridine (THU) directly upon sample collection. Sample preparation consisted of protein precipitation for plasma and whole blood and dilution for urine samples and was further optimized for each matrix and analyte separately. Final extracts were injected onto a C6-phenyl column for guadecitabine analysis, or a Nova-Pak Silica column for ߭decitabine analysis. Gradient elution was applied for both analytes using the same eluents for each assay and detection was performed on triple quadrupole mass spectrometers operating in the positive ion mode (Sciex QTRAP 5500 and QTRAP 6500). The assay for guadecitabine was linear over a range of 1.0-200 ng/mL (plasma, whole blood) and 10-2000 ng/mL (urine). For ߭decitabine the assay was linear over a range of 0.5-100 ng/mL (plasma, whole blood) and 5-1000 ng/mL (urine). The presented methods were successfully validated according to the latest FDA and EMA guidelines for bioanalytical method validation and applied in a guadecitabine clinical mass balance trial in patients with advanced cancer.