ABSTRACT
The 8th GCC Closed Forum for Bioanalysis was held in Baltimore, MD, USA on 5 December 2013, immediately following the 2013 AAPS Workshop (Crystal City V): Quantitative Bioanalytical Methods Validation and Implementation--The 2013 Revised FDA Guidance. This GCC meeting was organized to discuss the contents of the draft revised FDA Guidance on bioanalytical method validation that was published in September 2013 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants, from seven countries, representing 46 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the draft FDA Guidance, and to build unified comments to be provided to the FDA.
Subject(s)
Chemistry Techniques, Analytical/standards , Guidelines as Topic , Validation Studies as Topic , Biomarkers/analysis , Calibration , Ligands , Limit of Detection , Reagent Kits, Diagnostic , Reproducibility of Results , United States , United States Food and Drug AdministrationABSTRACT
The matrix metalloproteinase enzyme MMP-13 plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). An effective MMP-13 inhibitor would provide a disease modifying therapy for the treatment of arthritis, although this goal still continues to elude the pharmaceutical industry due to issues with safety. Our efforts have resulted in the discovery of a series of hydroxamic acid inhibitors of MMP-13 that do not significantly inhibit MMP-2 (gelatinase-1). MMP-2 has been implicated in the musculoskeletal side effects resulting from pan-MMP inhibition due to findings from spontaneously occurring human MMP-2 deletions. Analysis of the SAR of hundreds of previously prepared hydroxamate based MMP inhibitors lead us to 2-naphthylsulfonamide substituted hydroxamates which exhibited modest selectivity for MMP-13 versus MMP-2. This Letter describes the lead optimization of 1 and identification of inhibitors exhibiting >100-fold selectivity for MMP-13 over MMP-2.
Subject(s)
Hydroxamic Acids/pharmacology , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/pharmacology , Sulfonamides/chemistry , Crystallography, X-Ray , Hydroxamic Acids/chemistry , Models, Molecular , Protease Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
The matrix metalloproteinase enzyme MMP-13 plays a key role in the degradation of type II collagen in cartilage and bone in osteoarthritis (OA). An effective MMP-13 inhibitor would therefore be a novel disease modifying therapy for the treatment of arthritis. Our efforts have resulted in the discovery of a series of carboxylic acid inhibitors of MMP-13 that do not significantly inhibit the related MMP-1 (collagenase-1) or tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE). It has previously been suggested (but not proven) that inhibition of the latter two enzymes could lead to side effects. A promising carboxylic acid lead 9 was identified and a convergent synthesis developed. This paper describes the optimization of 9 and the identification of a compound 24f for further development. Compound 24f is a subnanomolar inhibitor of MMP-13 (IC(50) value 0.5 nM and K(i) of 0.19 nM) having no activity against MMP-1 or TACE (IC(50) of >10000 nM). Furthermore, in a rat model of MMP-13-induced cartilage degradation, 24f significantly reduced proteoglycan release following oral dosing at 30 mg/kg (75% inhibition, p < 0.05) and at 10 mg/kg (40% inhibition, p < 0.05).
Subject(s)
Cartilage/drug effects , Matrix Metalloproteinase Inhibitors , Piperidines/pharmacology , Protease Inhibitors/chemical synthesis , Sulfonamides/pharmacology , Animals , Cartilage/metabolism , Cattle , Collagen Type II/metabolism , Crystallography, X-Ray , Inhibitory Concentration 50 , Piperidines/administration & dosage , Piperidines/chemical synthesis , Piperidines/pharmacokinetics , Protease Inhibitors/administration & dosage , Protease Inhibitors/pharmacokinetics , Protease Inhibitors/pharmacology , Proteoglycans/metabolism , Rats , Structure-Activity Relationship , Sulfonamides/administration & dosage , Sulfonamides/chemical synthesis , Sulfonamides/pharmacokineticsABSTRACT
Screening of our compound collection using Staphylococcus aureus Ni-Peptide deformylase (PDF) afforded a very potent PDF inhibitor with an IC(50) in the low nanomolar range but with poor antibacterial activity (MIC). Three-dimensional structural information obtained from Pseudomonas aeruginosa Ni-PDF complexed with the inhibitor suggested the synthesis of a variety of analogues that would maintain high binding affinity while attempting to improve antibacterial activity. Many of the compounds synthesized proved to be excellent PDF-Ni inhibitors and some showed increased antibacterial activity in selected strains.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Bridged Bicyclo Compounds/chemistry , Protease Inhibitors/chemistry , Amidohydrolases/metabolism , Bridged Bicyclo Compounds/pharmacology , Crystallography, X-Ray , Microbial Sensitivity Tests , Protease Inhibitors/pharmacology , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymologyABSTRACT
To improve the pharmacokinetics of a previously reported series of dipeptidyl nitrile cathepsin B inhibitors, the P(2)-P(3) amide group was replaced with an arylamine. Further optimization of this template resulted in highly potent and selective inhibitors with excellent oral availability.
Subject(s)
Cathepsin B/antagonists & inhibitors , Dipeptides/pharmacokinetics , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Administration, Oral , Animals , Biological Availability , Dipeptides/administration & dosage , Dipeptides/chemical synthesis , Dipeptides/pharmacology , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacology , Models, Molecular , Molecular Conformation , Rats , X-Ray DiffractionABSTRACT
Aggrecan is one of the most important structural components of joint cartilage, and members of the metalloprotease (MMP) and ADAM (a disintegrin and metalloproteinase) protease families have been shown to degrade aggrecan in vivo. A robust assay for aggrecan-degrading activity suitable for high-throughput screening (HTS) was set up and measured using AlphaScreen. In this technology, beads brought into proximity through cross-linking and stimulated with laser light generate a signal through luminescent oxygen tunneling, the outcome of which is a time-resolved fluorescent signal. Specific antibodies to the carbohydrate side chains of aggrecan were harnessed to create a scaffold whereby aggrecan could form a cross-link between donor and acceptor AlphaScreen detector beads. Digested aggrecan, which failed to form a cross-link, generated no signal, so that inhibitors of the digestion could be detected as a restoration of signal. The development of this assay and its validation for HTS are described in this report.