Federica Sala

A first in human phase I study of the proteasome inhibitor CEP-18770 in patients with advanced solid tumours and multiple myeloma

BACKGROUND The safety, pharmacokinetics (PK) and pharmacodynamics of CEP-18770, a new peptide boronic acid proteasome inhibitor, have been investigated after intravenous administration on days 1, 4, 8 and 11 of every 21d cycle in patients with solid tumours and multiple myeloma (MM). PATIENTS AND METHODS Thirty-eight patients were treated with CEP-18770 at escalating doses from 0.1 to 1.8mg/m(2) where 2 out of 5 patients showed dose limiting toxicities. The maximum tolerated/recommended dose (MTD/RD) of 1.5mg/m(2) was tested in 12 additional patients. Skin rash was dose-limiting and occurred in 53% of patients; other frequent toxicities were asthenia (29%), stomatitis (21%) and pyrexia (16%). No significant peripheral neuropathy was observed. PK in plasma was linear with a half-life of the elimination phase of 62.0±43.5h. Proteasome inhibition in peripheral blood mononuclear cells was dose related in MM patients; it was of 45.4±11.5% at the RD. CONCLUSIONS CEP-18770 showed a favourable safety profile with lack of neurotoxicity and linear plasma PK. The definition of the optimal biological dose and schedule of treatment is actively pursued because of the high incidence of skin toxicity of the twice a week schedule.

Quantification of trabectedin in human plasma: Validation of a high-performance liquid chromatography–mass spectrometry method and its application in a clinical pharmacokinetic study

A rapid, sensitive and specific HPLC-MS/MS method was developed and validated for the quantification of trabectedin in human plasma after using deuterated trabectedin as Internal Standard (IS). After the addition of ammonium sulphate, the analyte was extracted from human plasma with acidified methanol (0.1 M HCl). Chromatographic separation was done on an Accucore XL C₁₈ column (4 μm; 50 mm × 2.1 mm) using a Mobile Phase (MP) consisting of CH₃COONH₄ 10 mM, pH 6.8 (MP A) and CH₃OH (MP B). The mass spectrometer worked with electrospray ionization in positive ion mode and Selected Reaction Monitoring (SRM), using target ions at [M-H₂O+H]⁺ m/z 744.4 for trabectedin and [M-H₂O+H]⁺m/z 747.5 for the IS. The standard curve was linear (R² ≥ 0.9955) over the concentration range 0.025-1.0 ng/ml and had good back-calculated accuracy and precision. The intra- and inter-day precision and accuracy determined on three quality control samples (0.04, 0.08 and 0.80 ng/ml) were <9.9% and between 98.3% and 105.3%, respectively. The extraction recovery was >81% and the lower limit of quantification 0.025 ng/ml. The method was successfully applied to study trabectedin pharmacokinetics in a patient with a liposarcoma who received 1.3 mg/m² as a 24 h continuous i.v. infusion.

Development and validation of a high-performance liquid chromatography-tandem mass spectrometry method for the determination of the novel proteasome inhibitor CEP-18770 in human plasma and its application in a clinical pharmacokinetic study

CEP-18770, [(1R)-1-{[(2S,3R)-3-hydroxy-2-{[(6-phenyl-2-pyridinyl)carbonyl]amino}butanoyl]amino}-3-methylbutyl]boronic acid, is a novel proteasome inhibitor, now under early clinical evaluation as an anticancer agent. To investigate its clinical pharmacokinetics, a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated to measure the drug in human plasma, based on simple protein precipitation with acetonitrile after the addition of irbesartan as internal standard. The method requires a small volume of sample (100 µl) and is rapid and selective, allowing good resolution of peaks in 5 min. It is sensitive, precise and accurate, with overall precision, expressed as coefficient of variation (CV%), always < 10.0%, accuracy in the range 93.8-107.7% and high recovery, close to 100%. The limit of detection is 0.01 ng/ml and the lower limit of quantitation (LLOQ) is 0.20 ng/ml. The assay was validated in the range from the LLOQ up to 50.00 ng/ml. This is the first method developed and validated for analyzing a proteasome inhibitor with a boronic-acid-based structure in human plasma. The method was successfully applied to study the pharmacokinetics of CEP-18770 in cancer patients with solid tumors or multiple myeloma who had received the drug as a short intravenous bolus during the initial Phase I trial.

Development and validation of a high-performance liquid chromatography-tandem mass spectrometry method for the determination of the novel inhibitor of angiogenesis E-3810 in human plasma and its application in a clinical pharmacokinetic study.

E-3810, 6-[[7-[(1-aminocyclopropyl)methoxy]-6-methoxy-4-quinolyl]oxy]-N-methyl-naphthalene-1-carboxamide, is a novel, potent, dual inhibitor of vascular endothelial growth factor and fibroblast growth factor receptors with antiangiogenic properties, now under early clinical evaluation as an anticancer agent. To investigate its clinical pharmacokinetics, a high-performance liquid chromatography-tandem mass spectrometry method was developed and validated to measure the drug in human plasma on the basis of simple protein precipitation with methanol after addition of deuterated E-3810 as internal standard. The method requires a small volume of sample (100 µl) and is rapid and selective, allowing good resolution of peaks in 5 min. It is sensitive, precise, and accurate, with overall precision, expressed as CV%, always ≤7.1%, accuracy in the range 92.7%-104.4%, and high recovery, close to 100%. The limit of detection is 0.01 ng/ml, and the lower limit of quantitation is 2.0 ng/ml. The assay was validated in the range from the lower limit of quantitation up to 500.0 ng/ml. This is the first method developed and validated for analyzing E-3810 in human plasma. The method has been successfully applied to study E-3810 pharmacokinetics in cancer patients with solid tumors who are receiving daily oral doses of the drug during the phase I trial.

Pharmacokinetic profile of imatinib mesylate and N-desmethyl-imatinib (CGP 74588) in children with newly diagnosed Ph+ acute leukemias.

Dear Editor, Because of the rarity of childhood Philadelphia chromosome-positive (Ph+) leukemias, data on imatinib pharmacokinetics in children are scant. Imatinib pharmacokinetics were reported in a limited number of leukemic children receiving drug in doses of 260–570 mg/m per day [1]. Despite a wide inter-patient variability, the plasma drug levels were similar to those reported in adult patients treated with standard doses of 400–600 mg/day [2–5]. No data were provided on the pharmacokinetics of the main circulating metabolite of imatinib, N-desmethyl-imatinib (CGP 74588), that had already been determined in adults [2, 6]. Ph+ childhood leukemia is less sensitive to imatinib than adult CML and this is likely to be due to major biological diVerences between these leukemias. However, the diVerent sensitivity may at least partly be related to diVerent bioavailability, metabolism and pharmacokinetic features of imatinib in children and adults that might require adjustment of doses and schedules. In our study, we determined the pharmacokinetics of imatinib and CGP 74588 in three children (two females and one male, aged 11, 15 and 6 years, respectively), with newly diagnosed Ph+ ALL treated according to the induction phase of the EsPhALL (European intergroup study on post-induction treatment of Ph+ ALL) protocol and in one female child (aged 6 years) with CML in lymphoblastic phase. Imatinib was administered at the dosage of 300 mg/m per day in all patients. They were treated in diVerent Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) centers: Monza, Turin and Florence. The Institutionals Ethical Board approved the study, conducted in accordance with the Helsinky declaration of Principle. Patient four merits more detail: male, 6 years old, Ph+ ALL. Three days after starting the induction phase Ib according to the EsPhALL protocol (cyclophosphamide, cytarabine, imatinib 300 mg/m per day £ 28 days administered orally, dissolved in apple juice), the patient developed a neurologic disorder, with asthenia, drowsiness, slowed speech and aphasia, with negative neuroimaging. Chemotherapy and imatinib were suspended, until full spontaneous clinical recovery; 25 days later, imatinib was restarted, initially at half of full dose (a new pharmacokinetic study was carried out when therapy restarted), then 70% and, Wnally, at full dose. This time the drug was taken orally, by swallowing the whole capsule. The patient underwent stem cell transplant and died due to treatment-related toxicity. For the pharmacokinetic study, blood samples were collected at pretreatment (before the morning dose) and 0.5, 1, 2, 4, 8 and 24 h after the Wrst dose and after at least one week of daily doses, presumably when the plasma imatinib concentration achieved steady-state. E. Marangon (&) · F. Sala · M. D’Incalci · M. Zucchetti Laboratory of Cancer Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy e-mail: marangon@marionegri.it

Development and validation of two liquid chromatography-tandem mass spectrometry methods for the determination of silibinin and silibinin hemisuccinate in human plasma.

To investigate the pharmacokinetics of silibinin and silibinin hemisuccinate in human plasma, two high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) methods were developed and validated. The methods require a small volume of sample (100μL), and the recovery of the analytes was complete with a good reproducibility (CV% 1.7-9.5), after a simple protein precipitation. Naringenin was used as internal standard. The chromatographic methods provided a good separation of diastereoisomers A and B of both silibinin and silibinin hemisuccinate onto a Chromolith Performance RP18e 100mm×3mm column, with a resolution of peaks from plasma matrix in less than 6min. The methods precision values expressed as CV% were always ≤6.2% and the accuracy was always well within the acceptable 15% range. Quantification was performed on a triple-quadrupole tandem mass spectrometer by Selected Reaction Monitoring (SRM) mode, in a negative ion mode, via electrospray ionization (ESI). The lower limit of quantitation was set at 5.0ng/mL (silibinin) and 25.0ng/mL (silibinin hemisuccinate), and the linearity was validated up to 1000.0 and 12,500.0ng/mL, for silibinin and silibinin hemisuccinate, respectively, with correlation coefficients (R(2)) of 0.991 or better. The methods were suitable for pharmacokinetic studies and were successfully applied to human plasma samples from subjects treated intravenously with Legalon(®) SIL at the dose of 20mg/kg, expressed as silibinin.

Abstract B64: Gene expression profile of a liposarcoma mixoid cell line selected in vitro for resistance to Trabectedin

Introduction: Trabectedin is a marine alkaloid originally isolated from Ecteinascidia turbinata approved in Europe for the second line of therapy in soft tissue sarcomas (STS). Here we characterize and compare the gene expression profile of 402‐91/ET Res liposarcoma cells obtained after continuous exposure to trabectedin in vitro with that of their parental cell line 402‐91. Methods: Drug sensitivity was measured by colony assay and Flow Cytometric analysis was used to analyze cell cycle perturbations. Microarray analysis between sensitive and 402‐91/ET Res cells was performed using commercially available arrays onto which 44K oligos were pre‐spotted. Gene expression analysis was performed with “R” package software. Pathway analysis was performed using Metacore software. qRT‐PCR was used for data validation. Statistical analysis was performed using Graphpad software . Results: 402‐91/ET Res cells were selected in vitro for their stable resistance to trabectedin by step‐wise increase in the drug concentration (25 nM) lasting 14 months. Resistance to Trabectedin was stable over 22 months without compound exposure. Cells were cross resistant to melphalan, but more sensitive to taxanes, vinblastine, temozolomide and UV rays. Resistance was not associated to the expression of MDR proteins as well as different uptake/efflux. Cell cycle analysis revealed that trabectedin is able to induce a G2‐M block in the sensitive cell line but not in the resistant one. The expression levels of the FUS‐CHOP gene were comparable between the 402‐91 and 402‐91/ET Res cells but with a different trans‐activation ability. Microarray analysis identified 511 probes set able to discriminate between sensitive and resistant cell lines. Pathway analysis revealed that most of the genes altered in the 402‐91/ET Res cells were associated with liposarcoma phenotype and functionally involved in the CREB pathway as well as control of apoptosis. Conclusion: Data suggests that in the 402‐91/ET Res cells FUS‐CHOP trans‐activation activity has been impaired. Studies are in progress to address whether these mechanisms are implicated in sensitivity of liposarcoma cell lines to trabectedin. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B64.