Wide application of a novel topoisomerase I inhibitor-based drug conjugation technology.

To establish a novel and widely applicable payload-linker technology for antibody-drug conjugates (ADCs), we have focused our research on applying exatecan mesylate (DX-8951f), a potent topoisomerase I inhibitor, which exhibits extensive antitumor activity as well as significant myelotoxicity, as the payload part. Through this study, we discovered a promising exatecan derivative (DX-8951 derivative, DXd), that has the characteristics of low membrane permeability and shows considerably less myelotoxicity than that shown by exatecan mesylate in an in vitro human colony forming unit-granulocyte macrophage assay. DXd was further used for drug conjugation by using commercially or clinically useful monoclonal antibodies to evaluate the potency of the ADC. The result revealed that the DXd-ADCs targeting CD30, CD33, and CD70 were effective against each of their respective target-expressing tumor cell lines. Moreover, a novel DXd-ADC targeting B7-H3, which is a new target for ADCs, also showed potent antitumor efficacy both in vitro and in vivo. In conclusion, this study showed that this novel topoisomerase I inhibitor-based ADC technology is widely applicable to a diverse number of antibodies and is expected to mitigate myelotoxicity, thereby possibly resulting in better safety profiles than that of existing ADC technologies.

Potent and Selective PTDSS1 Inhibitors Induce Collateral Lethality in Cancers with PTDSS2 Deletion.

SIGNIFICANCE: This study identifies a specific dependency on PTDSS1 for phosphatidylserine synthesis following PTDSS2 deletion and introduces novel PTDSS1 inhibitors as a therapeutic option to induce collateral lethality in cancer with PTDSS2 loss.

Intersubject and Intrasubject Variability of Potential Plasma and Urine Metabolite and Protein Biomarkers in Healthy Human Volunteers.

A limited understanding of intersubject and intrasubject variability hampers effective biomarker translation from in vitro/in vivo studies to clinical trials and clinical decision support. Specifically, variability of biomolecule concentration can play an important role in interpretation, power analysis, and sampling time designation. In the present study, a wide range of 749 plasma metabolites, 62 urine biogenic amines, and 1,263 plasma proteins were analyzed in 10 healthy male volunteers measured repeatedly during 12 hours under tightly controlled conditions. Three variability components in relative concentration data are determined using linear mixed models: between (intersubject), time (intrasubject), and noise (intrasubject). Biomolecules such as 3-carboxy-4-methyl-5-propyl-2-furanpropanoate, platelet-derived growth factor C, and cathepsin D with low noise potentially detect changing conditions within a person. If also the between component is low, biomolecules can easier differentiate conditions between persons, for example cathepsin D, CD27 antigen, and prolylglycine. Variability over time does not necessarily inhibit translatability, but requires choosing sampling times carefully.

Covalent binding and tissue distribution/retention assessment of drugs associated with idiosyncratic drug toxicity.

Bioactivation of a drug to a reactive metabolite and its covalent binding to cellular macromolecules is believed to be involved in clinical adverse events, including idiosyncratic drug toxicities (IDTs). For the interpretation of the covalent binding data in terms of risk assessment, the in vitro and in vivo covalent binding data of a variety of drugs associated with IDTs or not were determined. Most of the "problematic" drugs, including "withdrawn" and "warning" drugs, exhibit higher human liver microsome (HLM) in vitro covalent binding yields than the "safe" drugs. Although some of the problematic drugs that are known to undergo bioactivation other than cytochrome P450-mediated oxidation exhibited only trace levels of HLM covalent binding like safe drugs, a rat in vivo covalent binding study could assess the bioactivation of such drugs. Furthermore, the tissue distribution/retention of the drugs was also examined by rat autoradiography (ARG). The residual radioactivity in the liver observed at 72 or 168 h postdose was found to be well correlated with the rat in vivo covalent binding to liver proteins; thus, the in vivo covalent binding yields of the drugs could be extrapolated from the retention profiles observed by means of ARG. Long-term retention of radioactivity in the bone marrow was observed with some drugs associated with severe agranulocytosis, suggesting a spatial relationship between the toxicity profile and drug distribution/retention. Taken together, the covalent binding and tissue distribution/retention data of the various marketed drugs obtained in the present study should be quite informative for the interpretation of data in terms of risk assessment.

On-line capillary isoelectric focusing-mass spectrometry for quantitative analysis of peptides and proteins.

On-line capillary isoelectric focusing-mass spectrometry (cIEF-MS) was applied to determine concentrations of peptides and proteins using angiotensin II and human tetrasialo-transferrin as the model samples. The concentration of the carrier ampholyte was optimized for both resolution and ion intensity. cIEF-MS employing 1% Pharmalyte 3-10 and a sheath liquid containing water/methanol/acetic acid (50/49/1) resolved angiotensin I and II (5 microM each, DeltapI=0.2) at an Rs value of 2.29. The determined concentration of angiotensin II (0.1-5 microM) well correlated (R=0.999) with that obtained by the conventional RP-HPLC method. The limit of detection was 0.22 microM, which was about 10 times lower than that by UV detection (2 microM). The repeatability and accuracy were <15 and <11%, respectively. cIEF-MS was also applied to determine human tetrasialo-transferrin concentration. The good linearity (R2=0.998) was also observed between the transferrin concentration (0.5-1.2 g/L) and peak area ratio (IS; beta-lactoglobulin B) with acceptable accuracy (<1.9%) and repeatability ( approximately 10% at 1g/L).

Glutathione S-transferase pi trapping method for generation and characterization of drug-protein adducts in human liver microsomes using liquid chromatography-tandem mass spectrometry.

Covalent binding of reactive metabolites (RMs) to proteins is thought to play an important role in the processes leading to adverse drug reactions. Therefore, there is great interest in methodologies that enable the characterization of covalent binding of drugs to proteins. To facilitate the study of drug-protein adducts, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for characterizing RM-modified proteins formed through drug bioactivation in human liver microsomes (HLMs), which are commonly used for the in vitro drug bioactivation studies. The technique was illustrated by the trapping of RMs of acetaminophen (APAP) and raloxifene with human glutathione S-transferase pi (hGSTP) as a model target protein. After hGSTP-supplemented HLM incubations, the modified/unmodified hGSTP fractions were collected by high-performance liquid chromatography. hGSTP fractions were digested with trypsin, and then analyzed by linear ion trap-orbitrap mass spectrometry followed by a SEQUEST database search. Characteristic MS/MS fragment ions of RM-modified peptides were identified by searching for possible adducted-mass shifts. The method successfully revealed that RMs of both drugs adducted to Cys-47 of hGSTP and the mass shifts corresponded to modification by the N-acetyl-p-benzoquinone imine form of APAP and diquinone methide form of raloxifene, respectively. The developed method would be a possible tool for widespread use for the generation and characterization of drug-protein adducts in HLMs and has the potential to assess the risk of covalent binding of drugs to proteins.

Identification of cytochrome P450 3A4 modification site with reactive metabolite using linear ion trap-Fourier transform mass spectrometry.

Covalent binding of reactive metabolites to cytochrome P450s (P450s) often causes their mechanism-based inactivation (MBI), resulting in drug-drug interactions or toxicity. The detection and identification of the P450 sites to which reactive metabolites bind would elucidate MBI mechanisms. We describe a proteomic approach using nano-LC/linear ion trap-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to characterize the binding of a reactive metabolite of raloxifene, which is a known P450 3A4 inhibitor, to the P450 3A4 isozyme. LTQ-FT analyses revealed that the metabolic reaction of raloxifene in a reconstituted P450 3A4 system formed a reactive metabolite adduct to P450 3A4 apoprotein, accompanied by a mass shift of 471 Da relative to intact P450 3A4 apoprotein. The reaction mixtures were digested with trypsin, and then the tryptic digests were analyzed by nano-LC-MS/MS. This technique revealed that VWGFYDGQQPVLAITDPDMIK (position 71-91) was a tryptic peptide modified by the reactive metabolite derived from raloxifene. The site of adduction with the reactive metabolite was further postulated to be the nucleophilic OH group of Tyr-75 of P450 3A4. A proteomic approach using LTQ-FT can yield direct information on the P450 3A4 modification site without radiolabeled compounds. In addition, this information can elucidate mechanisms involved in the covalent binding of reactive metabolites and the inactivation of P450 3A4.