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Metabolite identification study plays an important role in determining the sites of metabolic liability of new chemical entities (NCEs) in drug discovery for lead optimization. Here we compare the two predictive software, MetaSite and StarDrop, available for this purpose. They work very differently but are used to predict the site of oxidation by major human cytochrome P450 (CYP) isoforms. Neither software can predict non-CYP catalyzed metabolism nor the rates of metabolism. For the purpose of c

Mass spectrometry is a highly selective and high throughput analytical technique that is ideally suited for the identification and purity determination of large numbers of compounds prepared using combinatorial chemistry or for the dereplication of natural products. Compounds may be characterized based on molecular weight, elemental composition and structural features based on fragmentation patterns. When coupled to a separation technique such as high-performance liquid chromatography (HPLC) or

The novel prenyl transferase-mediated, site-specific, antibody-drug conjugate LCB14-0110 is comprised of a proprietary beta-glucuronide linker and a payload (Monomethyl auristatin F, MMAF, an inhibitor for tubulin polymerization) attached to human epidermal growth factor receptor 2 (HER2)-targeting trastuzumab. A LC-MS/MS method was developed to quantify the antibody-conjugated drug (acDrug) for in vitro linker stability and preclinical pharmacokinetic studies. The method consisted of affinity c

Two alkenyl catechols were rapidly identified as probable cytotoxic constituents from the petroleum ether extract of Semecarpus anacardium using HPLC–electrospray/MS analysis linked to bioassay. These compounds were then isolated from the same extract using semi-prep HPLC and were proven to be 1,2-dihydroxy-3-pentadeca-7′,10′-dienylbenzene and 1,2-dihydroxy-3-pentadec-8′-enylbenzene, respectively, by comparison with physico-chemical data in the literature. Both isolates showed cytotoxic activiti

A simple liquid chromatography-quadrupole-time-of-flight-mass spectrometric assay (LC-TOF-MS/MS) has been developed for the evaluation of metabolism and pharmacokinetic (PK) characteristics of monomethyl auristatin F (MMAF) in rat, which is being used as a payload for antibody-drug conjugates. LC-TOF-MS/MS method was qualified for the quantification of MMAF in rat plasma. The calibration curves were acceptable over the concentration range from 3.02 to 2200 ng/mL using quadratic regression. MMAF

A pulsed ultrafiltration-mass spectrometric screening method has been developed to evaluate the metabolic stability of drugs. Pooled human liver microsomes containing cytochrome P450 enzymes were trapped by an ultrafiltration membrane in a stirred flow-through chamber, and eight beta-blocker drugs including acebutolol, alprenolol, atenolol, metoprolol, oxprenolol, pindolol, propranolol, and timolol were flow-injected through the chamber along with the cofactor NADPH. The ultrafiltrate was collec

Tozadenant is one of the selective adenosine A2a receptor antagonists with a potential to be a new Parkinson's disease (PD) therapeutic drug. In this study, a liquid chromatography-mass spectrometry based bioanalytical method was qualified and applied for the quantitative analysis of tozadenant in rat plasma. A good calibration curve was observed in the range from 1.01 to 2200 ng/mL for tozadenant using a quadratic regression. In vitro and preclinical in vivo pharmacokinetic (PK) properties of t

Antibody–drug conjugate (ADC) linkers play an important role in determining the safety and efficacy of ADC. The Ortho Hydroxy-Protected Aryl Sulfate (OHPAS) linker is a newly developed linker in the form of a di-aryl sulfate structure consisting of phenolic payload and self-immolative group (SIG). In this study, using two bioanalytical approaches (namely “bottom-up” and “middle-up” approaches) via the liquid chromatography-quadrupole time-of-flight mass spectrometric (LC-qTOF-MS) method, in vitr

A single hybrid affinity-captured-LC-TOF-MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine-citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65-613.00 ng/mL with an equation y = ax² + bx + c for the antibody-co

A liquid chromatographic-electrospray ionization-time-of-flight/mass spectrometric (LC-ESI-TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro-elution solid-phase extraction (SPE) for sample preparation and LC-ESI-TOF/MS in the positive ion mode for analysis. Phenanthroline (10 mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amou

MMAE is a potent antimitotic drug used as payload of an antibody-drug conjugate which shows potent activity in preclinical and clinical studies against a range of lymphomas, leukemia and solid tumors. Liquid chromatography-high resolution mass spectrometric method was developed for the quantification of MMAE and its preclinical pharmacokinetics. The method consisted of protein precipitation using acetonitrile (ACN) for sample preparation and liquid chromatography - quadrupole - time-of-flight -

Parkinson's disease is one of the most common neurodegenerative diseases. Adenosine regulates the response to other neurotransmitters in the brain regions related to motor function. In the several subtypes of adenosine receptors, especially, adenosine 2A receptors (A_2ARs) are involved in neurodegenerative conditions. ZM241385 is one of the selective non-xanthine A2AR antagonists with high affinity in the nanomolar range. This study describes the in vitro and in vivo pharmacokinetic pr