Rapid communications in mass spectrometry : RCM, Jan 30, 2015
The covalent modification of proteins by toxicants, new chemical entities or drug molecules, eith... more The covalent modification of proteins by toxicants, new chemical entities or drug molecules, either by metabolic activation or the presence of inherently reactive functional groups, is commonly implicated in organ toxicity and idiosyncratic reactions. In efforts to better prosecute protein modifications, we investigated a tag-free technique capable of detecting protein-small molecule adducts based solely on the collision-induced dissociation (CID) of the protein-small molecule complex. Detection of proteins using unique CID small molecule (SM) product ions would mitigate common issues associated with tagging technologies (e.g., altered reactivity/affinity of the protein-SM complex). A Waters SYNAPT G2 mass spectrometer (MS) was operated in MS(e) mode with appropriate collision energy conditions during the MS(2) acquisition for fragmentation of protein-small molecule adducts to generate characteristic small molecule product ions. Ibrutinib, an acrylamide-containing small molecule dru...
Carprofen, -6-chloro-a-methylcarbazole-2-acetic acid, is a nonsteroidal anti-inflammatory agent m... more Carprofen, -6-chloro-a-methylcarbazole-2-acetic acid, is a nonsteroidal anti-inflammatory agent marketed in the U.S. as Rimadyl for the treatment of pain and inflammation in dogs. In-vitro experiments with horseradish peroxidase (HRP) and other peroxidase enzymes yielded two major metabolites which were less polar than the parent compound. These metabolites were characterized by LC/MS/MS with electrospray ionization in negative-ion mode. Collision induced dissociation mass spectra suggested that the metabolites were the alcohol and ketone resulting from decarboxylation and the addition of oxygen on the benzylic carbon atom. Neither metabolite has been observed previously in microsomes, hepatocytes, or in-vivo metabolism studies in dogs, rats, or humans. Decarboxylation reactions of HRP with the indole-containing plant hormone auxin (indole-3-acetic acid) and the anti-inflammatory drug indomethacin have been documented in the literature. However, no references were found of carbazole...
Tulathromycin is a novel member of the triamilide class of antibiotics that was developed as a sa... more Tulathromycin is a novel member of the triamilide class of antibiotics that was developed as a safe and effective single-dose treatment of bovine and porcine respiratory disease. An accurate and precise analytical method was developed for the extraction and measurement of tulathromycin in livestock plasma and lung homogenates. Analytes were solid-phase extracted onto a weak cation exchanger after aqueous dilution of samples and addition of heptadeutero-tulathromycin as an internal standard. Following HPLC with a narrow bore C8 column, quantitative detection of tulathromycin was accomplished by monitoring the transition of a doubly charged precursor ion to a singly charged product ion by tandem mass spectrometry using a triple quadrupole mass spectrometer. Procedures were validated for a dynamic range of 0.1 to 25 ng on column. Observed accuracies were between 90 and 110% of nominal and precision (RSD) varying 7% or less. Response and stability experiments showed that deuterated tulathromycin did not parallel the chemical behavior of tulathromycin. Applicability of the method to livestock studies was tested with plasma and lung samples from cattle and swine dosed with tulathromycin at multiple doses. The results demonstrated that the analytical method performed well in a range of sample concentrations spanning over 3 orders of magnitude and provided dose-exposure relationships for cattle and swine.
Multiple crystal structures of CYP3A4 bound with various substrates or inhibitors have been used ... more Multiple crystal structures of CYP3A4 bound with various substrates or inhibitors have been used as templates for docking of new chemical entities to predict sites of metabolism and molecular interactions for drug design. Herein, modeling studies with dirlotapide, a CYP3A4 substrate, indicated that a substantial conformational change of CYP3A4 was necessary to accommodate it within the active site cavity, which is in good agreement with a new published CYP3A4 ritonavir co-crystal structure. Thus, the importance of considering the substrate-induced conformational change in CYP3A4, thermochemical properties of reaction centers, and essential in vitro experimental data support were analyzed for the refinement of computational models.
Analogues related to dirlotapide (1), a gut-selective inhibitor of microsomal triglyceride transf... more Analogues related to dirlotapide (1), a gut-selective inhibitor of microsomal triglyceride transfer protein (MTP) were prepared with the goal of further reducing the potential for unwanted liver MTP inhibition and associated side-effects. Compounds were designed to decrease active metabolite load: reducing MTP activity of likely human metabolites and increasing metabolite clearance to reduce exposure. Introduction of 4'-alkyl and 4'-alkoxy substituents afforded compounds exhibiting improved therapeutic index in rats with respect to liver triglyceride accumulation and enzyme elevation. Likely human metabolites of select compounds were prepared and characterized for their potential to inhibit MTP in vivo. Based on preclinical efficacy and safety data and its potential for producing short-lived, weakly active metabolites, compound 13 (PF-02575799) advanced into phase 1 clinical studies.
Rapid communications in mass spectrometry : RCM, Jan 30, 2015
The covalent modification of proteins by toxicants, new chemical entities or drug molecules, eith... more The covalent modification of proteins by toxicants, new chemical entities or drug molecules, either by metabolic activation or the presence of inherently reactive functional groups, is commonly implicated in organ toxicity and idiosyncratic reactions. In efforts to better prosecute protein modifications, we investigated a tag-free technique capable of detecting protein-small molecule adducts based solely on the collision-induced dissociation (CID) of the protein-small molecule complex. Detection of proteins using unique CID small molecule (SM) product ions would mitigate common issues associated with tagging technologies (e.g., altered reactivity/affinity of the protein-SM complex). A Waters SYNAPT G2 mass spectrometer (MS) was operated in MS(e) mode with appropriate collision energy conditions during the MS(2) acquisition for fragmentation of protein-small molecule adducts to generate characteristic small molecule product ions. Ibrutinib, an acrylamide-containing small molecule dru...
Carprofen, -6-chloro-a-methylcarbazole-2-acetic acid, is a nonsteroidal anti-inflammatory agent m... more Carprofen, -6-chloro-a-methylcarbazole-2-acetic acid, is a nonsteroidal anti-inflammatory agent marketed in the U.S. as Rimadyl for the treatment of pain and inflammation in dogs. In-vitro experiments with horseradish peroxidase (HRP) and other peroxidase enzymes yielded two major metabolites which were less polar than the parent compound. These metabolites were characterized by LC/MS/MS with electrospray ionization in negative-ion mode. Collision induced dissociation mass spectra suggested that the metabolites were the alcohol and ketone resulting from decarboxylation and the addition of oxygen on the benzylic carbon atom. Neither metabolite has been observed previously in microsomes, hepatocytes, or in-vivo metabolism studies in dogs, rats, or humans. Decarboxylation reactions of HRP with the indole-containing plant hormone auxin (indole-3-acetic acid) and the anti-inflammatory drug indomethacin have been documented in the literature. However, no references were found of carbazole...
Tulathromycin is a novel member of the triamilide class of antibiotics that was developed as a sa... more Tulathromycin is a novel member of the triamilide class of antibiotics that was developed as a safe and effective single-dose treatment of bovine and porcine respiratory disease. An accurate and precise analytical method was developed for the extraction and measurement of tulathromycin in livestock plasma and lung homogenates. Analytes were solid-phase extracted onto a weak cation exchanger after aqueous dilution of samples and addition of heptadeutero-tulathromycin as an internal standard. Following HPLC with a narrow bore C8 column, quantitative detection of tulathromycin was accomplished by monitoring the transition of a doubly charged precursor ion to a singly charged product ion by tandem mass spectrometry using a triple quadrupole mass spectrometer. Procedures were validated for a dynamic range of 0.1 to 25 ng on column. Observed accuracies were between 90 and 110% of nominal and precision (RSD) varying 7% or less. Response and stability experiments showed that deuterated tulathromycin did not parallel the chemical behavior of tulathromycin. Applicability of the method to livestock studies was tested with plasma and lung samples from cattle and swine dosed with tulathromycin at multiple doses. The results demonstrated that the analytical method performed well in a range of sample concentrations spanning over 3 orders of magnitude and provided dose-exposure relationships for cattle and swine.
Multiple crystal structures of CYP3A4 bound with various substrates or inhibitors have been used ... more Multiple crystal structures of CYP3A4 bound with various substrates or inhibitors have been used as templates for docking of new chemical entities to predict sites of metabolism and molecular interactions for drug design. Herein, modeling studies with dirlotapide, a CYP3A4 substrate, indicated that a substantial conformational change of CYP3A4 was necessary to accommodate it within the active site cavity, which is in good agreement with a new published CYP3A4 ritonavir co-crystal structure. Thus, the importance of considering the substrate-induced conformational change in CYP3A4, thermochemical properties of reaction centers, and essential in vitro experimental data support were analyzed for the refinement of computational models.
Analogues related to dirlotapide (1), a gut-selective inhibitor of microsomal triglyceride transf... more Analogues related to dirlotapide (1), a gut-selective inhibitor of microsomal triglyceride transfer protein (MTP) were prepared with the goal of further reducing the potential for unwanted liver MTP inhibition and associated side-effects. Compounds were designed to decrease active metabolite load: reducing MTP activity of likely human metabolites and increasing metabolite clearance to reduce exposure. Introduction of 4'-alkyl and 4'-alkoxy substituents afforded compounds exhibiting improved therapeutic index in rats with respect to liver triglyceride accumulation and enzyme elevation. Likely human metabolites of select compounds were prepared and characterized for their potential to inhibit MTP in vivo. Based on preclinical efficacy and safety data and its potential for producing short-lived, weakly active metabolites, compound 13 (PF-02575799) advanced into phase 1 clinical studies.
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