Design, synthesis and biological evaluation of phenol-linked uncialamycin antibody-drug conjugates.

Uncialamycin is one of the structurally simpler and newer members of enediyne family of natural products. It exhibits highly potent activity against several types of bacteria and cancer cells. Described herein is a strategy for the targeted delivery of this cytotoxic agent to tumors using an antibody-drug conjugate (ADC) approach. Central to the design of ADC were the generation of potent and chemically stable uncialamycin analogues and attachment of protease cleavable linkers to newly realized phenolic handles to prepare linker-payloads. Conjugation of the linker-payloads to tumor targeting antibody, in vitro activity and in vivo evaluation are presented.

Fucosyl monosialoganglioside: Quantitative analysis of specific potential biomarkers of lung cancer in biological matrices using immunocapture extraction/tandem mass spectrometry.

RATIONALE: Certain lung cancer patients express elevated Fucosyl Monosialoganglioside (Fuc-GM1) in circulation compared to control groups. Several sensitive methods involving characterization of Fuc-GM1 have been reported. However, a highly specific and sensitive method for quantifying multiple potential Fuc-GM1 biomarkers present in various biological matrices has not been reported to date. METHODS: Individual Fuc-GM1 analogs in a commercially obtained standard mixture were characterized using HPLC/UV/MS and high-resolution mass spectrometry (HRMS). Proprietary antibodies, mAb1 and mAb2, were used to selectively capture and pre-concentrate the soluble and drug-bound forms of Fuc-GM1 molecules present in human serum and whole blood, eliminating the background matrix components. Immunocapture extraction (ICE) followed by HPLC/MS/MS was used to quantify specific Fuc-GM1 analogs in biological matrices. RESULTS: The concentration of individual Fuc-GM1 analogs in the standard mixture was estimated to be 7-34%, using HPLC/UV/MS. Using the standard mixture spiked into the biological matrices (100 µL), the lower limit of quantification (LLOQ) of each analog was 0.2-0.4 ng/mL with a dynamic range of up to 200 ng/mL. The applicability of the ICE-HPLC/MS/MS method was demonstrated by detecting endogenous Fuc-GM1 analogs present in rat blood and in several lung cancer cell lines. CONCLUSIONS: This highly specific and sensitive HPLC/MS/MS method for quantifying individual potential Fuc-GM1 biomarkers in serum and whole blood can play a critical role in patient stratification strategies and during drug treatment. This method can be employed for monitoring both free (soluble) form and antibody drug-bound Fuc-GM1.

Pharmacokinetic characterization of BMS-936561, an anti-CD70 antibody-drug conjugate, in preclinical animal species and prediction of its pharmacokinetics in humans.

CD70 is a tumor necrosis factor (TNF)-like type II integral membrane protein that is transiently expressed on activated T- and B-lymphocytes. Aberrant expression of CD70 was identified in both solid tumors and haematologic malignancies. BMS-936561 (αCD70_MED-A) is an antibody-drug conjugate composed of a fully human anti-CD70 monoclonal antibody (αCD70) conjugated with a duocarmycin derivative, MED-A, through a maleimide-containing citrulline-valine dipeptide linker. MED-A is a carbamate prodrug that is activated by carboxylesterase to its active form, MED-B, to exert its DNA alkylation activity. In vitro serum stability studies suggested the efficiencies of hydrolyzing the carbamate-protecting group in αCD70_MED-A followed a rank order of mouse>rat > >monkey>dog~human. Pharmacokinetics of αCD70_MED-A was evaluated in mice, monkeys, and dogs after single intravenous doses. In mice, αCD70_MED-A was cleared rapidly, with no detectable exposures after 15 min following dosing. In contrast, αCD70_MED-A was much more stable in monkeys and dogs. The clearance of αCD70_MED-A in monkeys was 58 mL/d/kg, ~2-fold faster than that in dogs (31 mL/d/kg). The human PK profiles of the total αCD70 and αCD70_MED-A were predicted using allometrically scaled monkeys PK parameters of αCD70 and the carbamate hydrolysis rate constant estimated in dogs. Comparing the predicted and observed human PK from the phase I study, the dose-normalized concentration-time profiles of αCD70_MED-A and the total αCD70 were largely within the 5(th)-95(th) percentile of the predicted profiles.

The impact of sex and contraceptive therapy on the plasma and intracellular pharmacokinetics of zidovudine.

OBJECTIVES: Zidovudine remains part of combination antiretroviral therapy. Pharmacological studies rely on quantitation of active triphosphates in peripheral blood mononuclear cells. This study evaluated the impact of female sex and contraceptive therapy on zidovudine plasma and intracellular pharmacokinetics and the impact of contraceptive therapy on HIV viral load. METHODS: Serial plasma and intracellular zidovudine pharmacokinetics following oral and intravenous dosing were determined in 18 men and 20 women treated with zidovudine. Women could repeat pharmacokinetics assessment following 2 months oral or injectable contraceptive therapy. Zidovudine plasma and intracellular mono-, di- and triphosphate concentrations were determined by liquid chromatography tandem mass spectrometry. Plasma and cervical viral loads were determined preceding and following 2 months of contraceptive therapy in women. RESULTS: Men exhibited higher area under the concentration versus time curve for intracellular zidovudine and zidovudine-monophosphate following oral and intravenous dosing and higher zidovudine triphosphate following oral dosing. There was no difference between men and women in plasma zidovudine parameters. Furthermore, contraceptive therapy had no effect on zidovudine plasma or intracellular pharmacokinetics or on plasma or cervical HIV-1 RNA levels. CONCLUSIONS: Using an optimized pharmacokinetic design, this study indicated men exhibit significantly higher zidovudine-monophosphate and zidovudine-triphosphate exposure following zidovudine oral administration, having implications for drug toxicity and overall tolerance of zidovudine therapy. The lack of an effect of contraceptive therapy on zidovudine pharmacokinetics is surprising in light of previous pharmacokinetic studies for drugs eliminated primarily through glucuronidation. Contraceptive therapy had no effect on plasma or cervical viral load, results consistent with previous findings.

Novel detection of DNA-alkylated adducts of antibody-drug conjugates with potentially unique preclinical and biomarker applications.

BACKGROUND: MDX-1203 is an antibody-drug conjugate (ADC) currently in clinical trials for the treatment of renal carcinoma. The active ingredient of MDX-1203 is a DNA minor groove-binding cytotoxic drug that forms a covalently linked adduct with an adenine base. Formation of this adenine adduct prevents DNA replication, thus triggering cell death. RESULTS: A method has been developed to successfully isolate, identify and quantitate the adenine adduct using LC-MS/MS. The method is highly useful to validate the mode of action of this class of ADCs. Additionally, we have demonstrated that this method could potentially be utilized to assess the efficacy of the ADC in in vitro studies by measuring the amount of adenine adduct in various cells expressing the antigen. CONCLUSION: Upon validation, this method could serve as an invaluable tool to evaluate compounds in preclinical in vivo models and in utilizing the DNA adduct as a potential biomarker.