Implementation of Systematic Bioanalysis of Antibody-Drug Conjugates for Preclinical Pharmacokinetic Study of Ado-Trastuzumab Emtansine (T-DM1) in Rats.

Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies covalently bound to cytotoxic drugs by a linker. They are designed to selectively bind target antigens and present a promising cancer treatment without the debilitating side effects of conventional chemotherapies. Ado-trastuzumab emtansine (T-DM1) is an ADC that received US FDA approval for the treatment of HER2-positive breast cancer. The purpose of this study was to optimize methods for the quantification of T-DM1 in rats. We optimized four analytical methods: (1) an enzyme-linked immunosorbent assay (ELISA) to quantify the total trastuzumab levels in all drug-to-antibody ratios (DARs), including DAR 0; (2) an ELISA to quantify the conjugated trastuzumab levels in all DARs except DAR 0; (3) an LC-MS/MS analysis to quantify the levels of released DM1; and (4) a bridging ELISA to quantify the level of anti-drug antibodies (ADAs) of T-DM1. We analyzed serum and plasma samples from rats injected intravenously with T-DM1 (20 mg/kg, single dose) using these optimized methods. Based on these applied analytical methods, we evaluated the quantification, pharmacokinetics, and immunogenicity of T-DM1. This study establishes the systematic bioanalysis of ADCs with validated assays, including drug stability in matrix and ADA assay, for future investigation on the efficacy and safety of ADC development.

Identification and Characterization of mRNA Biomarkers for Sodium Cyanide Exposure.

Biomarkers in exposure assessment are defined as the quantifiable targets that indicate the exposure to hazardous chemicals and their resulting health effect. In this study, we aimed to identify, validate, and characterize the mRNA biomarker that can detect the exposure of sodium cyanide. To identify reliable biomarkers for sodium cyanide exposure, critical criteria were defined for candidate selection: (1) the expression level of mRNA significantly changes in response to sodium thiocyanate treatment in transcriptomics results (fold change > 2.0 or <0.50, adjusted p-value < 0.05); and (2) the mRNA level is significantly modulated by sodium cyanide exposure in both normal human lung cells and rat lung tissue. We identified the following mRNA biomarker candidates: ADCY5, ANGPTL4, CCNG2, CD9, COL1A2, DACT3, GGCX, GRB14, H1F0, HSPA1A, MAF, MAT2A, PPP1R10, and PPP4C. The expression levels of these candidates were commonly downregulated by sodium cyanide exposure both in vitro and in vivo. We functionally characterized the biomarkers and established the impact of sodium cyanide on transcriptomic profiles using in silico approaches. Our results suggest that the biomarkers may contribute to the regulation and degradation of the extracellular matrix, leading to a negative effect on surrounding lung cells.

Preclinical immunogenicity testing using anti-drug antibody analysis of GX-G3, Fc-fused recombinant human granulocyte colony-stimulating factor, in rat and monkey models.

Human granulocyte colony-stimulating factor (G-CSF, this study used Fc-fused recombinant G-CSF; GX-G3) is an important glycoprotein that stimulates the proliferation of granulocytes and white blood cells. Thus, G-CSF treatment has been considered as a crucial regimen to accelerate recovery from chemotherapy-induced neutropenia in cancer patients suffering from non-myeloid malignancy or acute myeloid leukemia. Despite the therapeutic advantages of G-CSF treatment, an assessment of its immunogenicity must be performed to determine whether the production of anti-G-CSF antibodies causes immune-related disorders. We optimized and validated analytical tools by adopting validation parameters for immunogenicity assessment. Using these validated tools, we analyzed serum samples from rats and monkeys injected subcutaneously with GX-G3 (1, 3 or 10 mg/kg once a week for 4 weeks followed by a 4-week recovery period) to determine immunogenicity response and toxicokinetic parameters with serum concentration of GX-G3. Several rats and monkeys were determined to be positive for anti-GX-G3 antibodies. Moreover, the immunogenicity response of GX-G3 was lower in monkeys than in rats, which was relevant to show less inhibition of toxicokinetic profiles in monkeys, at least 1 mg/kg administrated group, compared to rats. These results suggested the establishment and validation for analyzing anti-GX-G3 antibodies and measurement of serum levels of GX-G3 and anti-GX-G3 antibodies, which was related with toxicokinetic profiles. Taken together, this study provides immunogenicity assessment which is closely implicated with toxicokinetic study of GX-G3 in 4-week repeated administrated toxicological studies.

Development of a liquid chromatography-tandem mass spectrometry method for the determination of Grayanotoxins in rat blood and its application to toxicokinetic study.

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of Grayanotoxin I (GTX I) and Grayanotoxin III (GTX III) in rat whole blood. Grayanotoxins (GTXs) and clindamycin as internal standard (IS) were extracted from rat blood via solid-phase extraction using PEP solid-phase extraction cartridges. Chromatographic separation of the analytes was achieved on a Kinetex C18 (100 × 2.1 mm, 2.6 µm) reversed-phase column using a gradient elution with the mobile phase of 1% acetic acid in water and methanol at a flow rate of 0.2 mL/min. Electrospray ionization mass spectrometry was operated in the positive ion mode with multiple reaction monitoring. The calibration curves obtained were linear over the concentration range of 1-100 ng/mL with a lower limit of quantification of 1 ng/mL for GTXs. The relative standard deviation of intra-day and inter-day precision was below 6.8% and accuracy ranged from 94.8 to 106.6%. The analytes were stable in the stability studies. The validated method was successfully applied to the quantification and toxicokinetic study of GTXs in rats for the first time after oral administration of 11.52 mg/kg mad honey and 0.35 mg/kg GTX III, respectively.

Fenretinide metabolism in humans and mice: utilizing pharmacological modulation of its metabolic pathway to increase systemic exposure.

BACKGROUND AND PURPOSE: High plasma levels of fenretinide [N-(4-hydroxyphenyl)retinamide (4-HPR)] were associated with improved outcome in a phase II clinical trial. Low bioavailability of 4-HPR has been limiting its therapeutic applications. This study characterized metabolism of 4-HPR in humans and mice, and to explore the effects of ketoconazole, an inhibitor of CYP3A4, as a modulator to increase 4-HPR plasma levels in mice and to increase the low bioavailability of 4-HPR. EXPERIMENTAL APPROACH: 4-HPR metabolites were identified by mass spectrometric analysis and levels of 4-HPR and its metabolites [N-(4-methoxyphenyl)retinamide (4-MPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR)] were quantified by high-performance liquid chromatography (HPLC). Kinetic analysis of enzyme activities and the effects of enzyme inhibitors were performed in pooled human and pooled mouse liver microsomes, and in human cytochrome P450 (CYP) 3A4 isoenzyme microsomes. In vivo metabolism of 4-HPR was inhibited in mice. KEY RESULTS: Six 4-HPR metabolites were identified in the plasma of patients and mice. 4-HPR was oxidized to 4-oxo-4-HPR, at least in part via human CYP3A4. The CYP3A4 inhibitor ketoconazole significantly reduced 4-oxo-4-HPR formation in both human and mouse liver microsomes. In two strains of mice, co-administration of ketoconazole with 4-HPR in vivo significantly increased 4-HPR plasma concentrations by > twofold over 4-HPR alone and also increased 4-oxo-4-HPR levels. CONCLUSIONS AND IMPLICATIONS: Mice may serve as an in vivo model of human 4-HPR pharmacokinetics. In vivo data suggest that the co-administration of ketoconazole at normal clinical doses with 4-HPR may increase systemic exposure to 4-HPR in humans.

Phase I trial of MS-275, a histone deacetylase inhibitor, administered weekly in refractory solid tumors and lymphoid malignancies.

PURPOSE: MS-275 is a histone deacetylase inhibitor that has shown potent and unique anticancer activity in preclinical models. The aims of this phase I trial were to determine the dose-limiting toxicities and maximum tolerated dose of oral MS-275 in humans administered with food on a once weekly schedule and to study the pharmacokinetics of oral MS-275. EXPERIMENTAL DESIGN: Patients with refractory solid tumors and lymphoid malignancies were treated with oral MS-275 on a once weekly schedule for 4 weeks of a 6-week cycle. Samples for pharmacokinetic and pharmacodynamic analyses were collected during cycle 1. Protein acetylation in subpopulations of peripheral blood mononuclear cells was measured using a multivariable flow cytometry assay. RESULTS: A total of 22 patients were enrolled, and 19 were considered evaluable for toxicity. The maximum tolerated dose was 6 mg/m(2). No National Cancer Institute Common Toxicity Criteria grade 4 toxicities were observed. Dose-limiting grade 3 toxicities were reversible and consisted of hypophosphatemia, hyponatremia, and hypoalbuminemia. Non-dose-limiting grade 3 myelosuppression was also observed. The mean terminal half-life of MS-275 was 33.9 +/- 26.2 and the T(max) ranged from 0.5 to 24 h. Although there was considerable interpatient variability in pharmacokinetics, the area under the plasma concentration versus time curve increased linearly with dose. CONCLUSIONS: MS-275 is well tolerated at a dose of 6 mg/m(2) administered weekly with food for 4 weeks every 6 weeks. Drug exposure increases linearly with dose, and protein acetylation increased in all the subpopulations of peripheral blood mononuclear cells following MS-275 administration.

Factors affecting the pharmacokinetic profile of MS-275, a novel histone deacetylase inhibitor, in patients with cancer.

AIMS: To evaluate elimination pathways of the histone deacetylase inhibitor MS-275 in vitro and screen for relationships between demographic factors that may affect its pharmacokinetics in vivo. PATIENTS AND METHODS: Substrate specificity of MS-275 for the liver-specific organic anion transporting polypeptides (OATPs) was assessed using Xenopus laevis oocytes, and in vitro metabolism was evaluated using human liver microsomes. In vivo pharmacokinetic data were obtained from 64 adult patients (36 male/28 female; median age, 57 years) receiving MS-275 orally (dose range, 2 to 12 mg/m2). RESULTS: Accumulation of [G-3H]MS-275 by oocytes expressing OATP1B1 or OATP1B3 was not significantly different from water-injected controls (p = 0.82). Furthermore, no metabolites could be detected after incubation of MS-275 in human liver microsomes, suggesting that hepatic metabolism is a minor pathway of elimination. The mean (+/- SD) apparent oral clearance of MS-275 was 38.5 +/- 18.7 L/h, with a coefficient of variation (%CV) of 48.7%. When clearance was adjusted for body-surface area (BSA), the inter-individual variability was similar (%CV = 50.1%). In addition, in a linear-regression analysis, except for adjusted ideal body weight (p = 0.02, |r| = 0.29), none of the studied measures (BSA, lean-body mass, ideal body weight, body-mass index, height, weight, age, and sex) was a significant covariate (p > 0.13; |r| < 0.11) for oral clearance. CONCLUSIONS: The current analysis has eliminated a number of candidate covariates from further consideration as important determinants of MS-275 absorption and disposition. Furthermore, MS-275 can be added to the list of cancer drugs where BSA-based dosing is not more accurate than fixed dosing.

Determination of the heat shock protein 90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin in plasma by liquid chromatography-electrospray mass spectrometry.

A rapid method was developed for the quantitative determination of the novel heat shock protein 90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; NSC707545), in human plasma. Calibration curves were constructed, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a one-step extraction with ethyl acetate of 0.5-ml samples. The analysis was performed in the range of 1-100 ng/ml on a column (75 mm x 2.1 mm internal diameter with 3.5 microm C18 particle size), using 55% methanol in water containing formic acid as the mobile phase. The column effluent was monitored by mass spectrometry with positive electrospray ionization. The values for precision and accuracy were always <8% and <10% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of 17-DMAG in a cancer patient.

Phase I and pharmacokinetic study of MS-275, a histone deacetylase inhibitor, in patients with advanced and refractory solid tumors or lymphoma.

PURPOSE: The objective of this study was to define the maximum-tolerated dose (MTD), the recommended phase II dose, the dose-limiting toxicity, and determine the pharmacokinetic (PK) and pharmacodynamic profiles of MS-275. PATIENTS AND METHODS: Patients with advanced solid tumors or lymphoma were treated with MS-275 orally initially on a once daily x 28 every 6 weeks (daily) and later on once every-14-days (q14-day) schedules. The starting dose was 2 mg/m2 and the dose was escalated in three- to six-patient cohorts based on toxicity assessments. RESULTS: With the daily schedule, the MTD was exceeded at the first dose level. Preliminary PK analysis suggested the half-life of MS-275 in humans was 39 to 80 hours, substantially longer than predicted by preclinical studies. With the q14-day schedule, 28 patients were treated. The MTD was 10 mg/m2 and dose-limiting toxicities were nausea, vomiting, anorexia, and fatigue. Exposure to MS-275 was dose dependent, suggesting linear PK. Increased histone H3 acetylation in peripheral-blood mononuclear-cells was apparent at all dose levels by immunofluorescence analysis. Ten of 29 patients remained on treatment for > or = 3 months. CONCLUSION: The MS-275 oral formulation on the daily schedule was intolerable at a dose and schedule explored. The q14-day schedule is reasonably well tolerated. Histone deacetylase inhibition was observed in peripheral-blood mononuclear-cells. Based on PK data from the q14-day schedule, a more frequent dosing schedule, weekly x 4, repeated every 6 weeks is presently being evaluated.

Determination of CC-5013, an analogue of thalidomide, in human plasma by liquid chromatography-mass spectrometry.

A high-performance liquid chromatographic assay with MS detection has been developed for the quantitative determination of the anti-angiogenic agent CC-5013 in human plasma. Sample pretreatment involved liquid-liquid extraction with acetonitrile/1-chlorobutane (4:1, v/v) solution containing the internal standard, umbelliferone. Separation of the compounds of interest was achieved on a column packed with Waters C18 Nova-Pak material (4 microm particle size; 300 mm x 3.9 mm internal diameter) using acetonitrile, de-ionized water, and glacial acetic acid in ratios of 20:80:0.1 (v/v/v) (pH 3.5) delivered at an isocratic flow rate of 1.00 ml/min. Simultaneous MS detection was performed at m/z 260.3 (CC-5013) and m/z 163.1 (umbelliferone). The calibration curve was fit to a linear response-concentration data over a range of 5-1000 ng/ml using a weighting factor of 1/x. Values for accuracy and precision, obtained from four quality controls analyzed on three different days in replicates of five, ranged from 98 to 106% and from 5.5 to 15.5%, respectively. The method was successfully applied to study the pharmacokinetics of CC-5013 in a cancer patient receiving the drug as single daily dose.

Determination of the cyclic depsipeptide FK228, a histone deacetylase inhibitor, by liquid chromatography-mass spectrometry.

An analytical method was developed for the quantitative determination of the novel histone deacetylase inhibitor, depsipeptide FK228 (formerly FR901228; NSC 630176), in human plasma. Calibration curves were constructed in the range of 0.5-100 ng/ml, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a liquid-liquid extraction with ethyl acetate using 500 microl aliquots of plasma. The analyte was separated on a column (50 mm x 4.6 mm i.d.) packed with 3.5 microm C8 material, and eluted with methanol-10 mM ammonium formate (55:45; v/v; pH 8). The column effluent was monitored by mass spectrometry with electrospray ionization. The values for precision and accuracy were always < or =7.88% and <3.33% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of FK228 in a cancer patient.

Determination of MS-275, a novel histone deacetylase inhibitor, in human plasma by liquid chromatography-electrospray mass spectrometry.

A rapid method was developed for the quantitative determination of the novel histone deacetylase inhibitor, MS-275, in human plasma. Calibration curves were constructed in the range of 1-100 ng/ml, and were analyzed using a weight factor proportional to the nominal concentration. Sample pretreatment involved a one-step protein precipitation with acetonitrile of 0.1 ml samples. The analysis was performed on a column (75 mm x 4.6 mm i.d.) packed with 3.5 microm Phenyl-SB material, using methanol-10mM ammonium formate (55:45 (v/v)) as the mobile phase. The column effluent was monitored by mass spectrometry with positive electrospray ionization. The values for precision and accuracy were always < or =5.58 and <11.4% relative error, respectively. The method was successfully applied to examine the pharmacokinetics of MS-275 in a cancer patient.

Isolation and structures of guaianolides from Carpesium macrocephalum.

Two new guaianolides, 4alpha,10alpha-dihydroxy-1beta(H),5beta(H)-guai-11(13)-en-8alpha,12-olide (2) and 4beta,10beta-dihydroxy-5alpha(H)-1,11(13)-guaidien-8alpha,12-olide (3), from Carpesium macrocephalum were isolated, and their structures were elucidated on the basis of spectroscopic studies.