ABSTRACT
The 13th GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5th, 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the ICH M10 Bioanalytical Method Validation Draft Guideline and to build unified comments to be provided to the ICH.
Subject(s)
Biomarkers/analysis , Guidelines as Topic , Humans , Reproducibility of Results , Research DesignABSTRACT
Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.
Subject(s)
Aldehyde-Lyases/antagonists & inhibitors , Antirheumatic Agents/chemical synthesis , Imidazoles/chemical synthesis , Isoxazoles/chemical synthesis , Oximes/chemical synthesis , Aldehyde-Lyases/genetics , Animals , Antirheumatic Agents/pharmacokinetics , Antirheumatic Agents/pharmacology , Arthritis, Experimental/drug therapy , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Blood Pressure/drug effects , Cell Movement , Dogs , Heart Rate/drug effects , Imidazoles/pharmacokinetics , Imidazoles/pharmacology , Isoxazoles/pharmacokinetics , Isoxazoles/pharmacology , Lymphocytes/drug effects , Lymphocytes/physiology , Macaca fascicularis , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Oximes/pharmacokinetics , Oximes/pharmacology , Rats , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity RelationshipABSTRACT
During nearly a decade of research dedicated to the study of sphingosine signaling pathways, we identified sphingosine-1-phosphate lyase (S1PL) as a drug target for the treatment of autoimmune disorders. S1PL catalyzes the irreversible decomposition of sphingosine-1-phosphate (S1P) by a retro-aldol fragmentation that yields hexadecanaldehyde and phosphoethanolamine. Genetic models demonstrated that mice expressing reduced S1PL activity had decreased numbers of circulating lymphocytes due to altered lymphocyte trafficking, which prevented disease development in multiple models of autoimmune disease. Mechanistic studies of lymphoid tissue following oral administration of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI) 3 showed a clear relationship between reduced lyase activity, elevated S1P levels, and lower levels of circulating lymphocytes. Our internal medicinal chemistry efforts discovered potent analogues of 3 bearing heterocycles as chemical equivalents of the pendant carbonyl present in the parent structure. Reduction of S1PL activity by oral administration of these analogues recapitulated the phenotype of mice with genetically reduced S1PL expression.
Subject(s)
Aldehyde-Lyases/antagonists & inhibitors , Autoimmune Diseases/drug therapy , Imidazoles/pharmacology , Administration, Oral , Animals , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Imidazoles/administration & dosage , Imidazoles/therapeutic use , Lymphocyte Count , Mice , Structure-Activity RelationshipABSTRACT
Methods for the absolute quantification of a membrane protein are described using isotopically labeled or unlabeled synthetic peptides as standards. Synthetic peptides are designed to mimic peptides that are cleaved from target analyte proteins by proteolytic or chemical digestion, and the peptides selected serve as standards for quantification by LC/MS/MS on a triple quadrupole mass spectrometer. The technique is complementary to relative quantification techniques in widespread use by providing absolute quantitation of selected targets with greater sensitivity, dynamic range, and precision. Proteins that are found to be of interest by global proteome searches can be selected as targets for quantitation by the present method. This method has a much shorter analytical cycle time (minutes versus hours for the global proteome experiments), making it well suited for high-throughput environments. The present approach using synthetic peptides as standards, in conjunction with proteolytic or chemical cleavage of target proteins, allows mass spectrometry to be used as a highly selective detector for providing absolute quantification of proteins for which no standards are available. We demonstrate that quantification is simple and reliable for the integral membrane protein rhodopsin with reasonable recoveries for replicate experiments using low-micromolar solutions of rhodopsin from rod outer segments.
