ABSTRACT
Aim: To quantify the free form of a protein as a target-engagement biomarker in nonhuman primate serum, a Meso Scale Discovery ligand-binding assay was developed and qualified. Results: The initial assay produced an unexpected artifact when used to measure the free target in study samples dosed with drug. By using incurred study samples dosed with high drug levels to test assay performance, we developed an alternative assay that does not suffer from drug interference. Conclusion: Our work demonstrated that an assay designed to measure free target may not necessarily deliver reliable quantitation. In our case, incurred study samples dosed with drug proved to be useful in developing an alternative free assay that does not suffer from drug interference.
Subject(s)
Drug Development , Pharmaceutical Preparations/blood , Animals , Biomarkers/blood , LigandsABSTRACT
Reslizumab and mepolizumab are recently approved monoclonal antibodies for the treatment of severe (uncontrolled) eosinophilic asthma. Both are effective in neutralizing the function of interleukin-5 (IL-5). This study is the first to compare the binding affinity and in vitro potency of both antibodies in head-to-head assays. Two assays assessed binding affinity (using the equilibrium dissociation constant [KD]) of each drug for human IL-5. In the Biacore surface plasmon resonance assay, the association constant (kon) values for human IL-5 for reslizumab and mepolizumab were 3.93 × 106 and 1.83 × 105, respectively. The dissociation constant (koff) values were 4.29 × 10â»4 and 2.14 × 10â»4, respectively. Calculated KD values for human IL-5 for reslizumab and mepolizumab were 109 and 1,170 pM, respectively, representing an approximately 11-fold stronger binding affinity with reslizumab. In the Kinetic Exclusion Assay, the kon values for human IL-5 for reslizumab and mepolizumab were 3.17 × 106 and 1.32 × 105, respectively. The koff values were 1.36 × 10â»5 and 1.48 × 10â»5, respectively. Measured KD values for human IL-5 for reslizumab and mepolizumab were 4.3 and 112 pM, respectively, representing an approximately 26-fold stronger binding affinity for reslizumab. A human-IL-5-dependent cell proliferation assay was developed to assess in vitro potency, based on a human cell line selected for enhanced surface expression of IL-5 receptor-alpha and consistent proliferation response to IL-5. The concentration at which 50% inhibition occurred (IC50) was determined for both antibodies. Reslizumab and mepolizumab inhibited IL-5-dependent cell proliferation, with IC50 values of approximately 91.1 and 286.5 pM, respectively, representing on average 3.1-fold higher potency with reslizumab. In conclusion, comparative assays show that reslizumab has higher affinity binding for and in vitro potency against human IL-5 compared with mepolizumab. However, these results do not take into consideration the different methods of administration of reslizumab and mepolizumab.
ABSTRACT
TL1A is an attractive therapeutic target for the treatment of mucosal inflammation associated with inflammatory bowel disease (IBD) and asthma. Blockade of the TL1A pathway has been shown to reduce inflammatory responses while leaving baseline immunity intact, and to be beneficial in animal models of colitis and asthma. Given the therapeutic potential of blocking this pathway in IBD and asthma, we developed C03V, a human antibody that binds with high affinity to soluble and membrane-bound TL1A. In an assay measuring apoptosis induced by exogenous TL1A, C03V was 43-fold more potent than the next most potent anti-TL1A antibody analyzed. C03V also potently inhibited endogenous TL1A activity in a primary cell-based assay. This potency was linked to the C03V-binding epitope on TL1A, encompassing the residue R32. This residue is critical for the binding of TL1A to its signaling receptor DR3 but not to its decoy receptor DcR3, and explains why C03V inhibited TL1A-DR3 binding to a much greater extent than TL1A-DcR3 binding. This characteristic may be advantageous to preserve some of the homeostatic functions of DcR3, such as TL1A antagonism. In colitis models, C03V significantly ameliorated microscopic, macroscopic and clinical aspects of disease pathology, and in an asthma model it significantly reduced airways inflammation. Notable in both types of disease model was the reduction in fibrosis observed after C03V treatment. C03V has the potential to address unmet medical needs in asthma and IBD.
Subject(s)
Antibodies, Monoclonal/pharmacology , Tumor Necrosis Factor Ligand Superfamily Member 15/antagonists & inhibitors , Animals , Antibodies, Monoclonal/chemistry , Asthma/immunology , Humans , Inflammatory Bowel Diseases/immunologyABSTRACT
A novel series of 3,4-diaza-bicyclo[4.1.0]hept-4-en-2-ones were designed and synthesized as H3R analogs of irdabisant 6. Separation of the isomers, assignment of the stereochemistry by crystallography, and detailed profiling of diastereomers 25 and 26 led to the identification of (1R,6S)-5-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)propoxy]phenyl}-3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one 25 as a potential second generation H3R candidate. Diastereomer 25 had high H3R binding affinity, excellent selectivity, displayed potent H3R functional antagonism and robust wake-promoting activity in vivo, and showed acceptable pharmacokinetic and pharmaceutical profiles for potential further development.
Subject(s)
Drug Inverse Agonism , Histamine Antagonists/chemistry , Histamine Antagonists/pharmacology , Pyridazines/chemistry , Pyridazines/pharmacology , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Receptors, Histamine H3/metabolism , Wakefulness/drug effects , Animals , Dose-Response Relationship, Drug , Drug Design , Histamine Antagonists/pharmacokinetics , Humans , Pyridazines/pharmacokinetics , Pyrrolidines/pharmacokinetics , Rats , Stereoisomerism , Structure-Activity RelationshipABSTRACT
A series of fused cyclopropyl-4,5-dihydropyridazin-3-one (3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one) phenoxypiperidine analogs was designed and synthesized, leading to the identification of (1R,6S)-5-[4-(1-cyclobutyl-piperidin-4-yloxy)-phenyl]-3,4-diaza-bicyclo[4.1.0]hept-4-en-2-one (R,S-4a) as a second-generation pyridazin-3-one H3R antagonist. Compound R,S-4a was a potent H3R functional antagonist in vivo in the rat dipsogenia model, demonstrated potent wake activity in the rat EEG/EMG model, and enhanced short-term memory in the rat social recognition memory model at doses as low as 0.03-0.3 mg/kg po.
Subject(s)
Nootropic Agents/chemistry , Piperidines/chemistry , Pyridazines/chemistry , Receptors, Histamine H3/chemistry , Animals , Cognition Disorders/drug therapy , Disease Models, Animal , Dogs , Drug Evaluation, Preclinical , Drug Inverse Agonism , Half-Life , Haplorhini , Memory, Short-Term/drug effects , Nootropic Agents/pharmacokinetics , Nootropic Agents/pharmacology , Nootropic Agents/therapeutic use , Piperidines/pharmacokinetics , Piperidines/pharmacology , Piperidines/therapeutic use , Pyridazines/pharmacokinetics , Pyridazines/pharmacology , Pyridazines/therapeutic use , Rats , Receptors, Histamine H3/metabolism , Stereoisomerism , Structure-Activity RelationshipABSTRACT
A novel series of 4-pyridazin-3-one and 5-pyridazin-3-one analogues were designed and synthesized as H(3)R antagonists. Structure-activity relationship revealed the 5-pyridazin-3-ones 8a and S-methyl 8b had excellent human and rat H(3)R affinities, and acceptable pharmacokinetic properties. In vivo evaluation of 8a showed potent activity in the rat dipsogenia model and robust wake-promoting activity in the rat EEG/EMG model.
Subject(s)
Histamine Antagonists/chemical synthesis , Histamine Antagonists/pharmacology , Propylamines/pharmacology , Pyridazines/pharmacology , Receptors, Histamine H3/metabolism , Animals , Histamine Antagonists/chemistry , Humans , Male , Molecular Sequence Data , Molecular Structure , Propylamines/chemical synthesis , Propylamines/chemistry , Pyridazines/chemical synthesis , Pyridazines/chemistry , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
A novel class of benzocinnolinones analogs of irdabisant were designed and synthesized as histamine H3R antagonists/inverse agonists. Modifications to the pyridazinone portion of the core and linker led to the identification of molecules with excellent target potency and selectivity with improved rat pharmacokinetic properties and reduced potential hERG liabilities.
Subject(s)
Heterocyclic Compounds, 3-Ring/chemical synthesis , Histamine Agonists/chemical synthesis , Histamine H3 Antagonists/chemical synthesis , Nootropic Agents/chemical synthesis , Pyridazines/chemical synthesis , Pyrrolidines/chemical synthesis , Receptors, Histamine H3/chemistry , Animals , CHO Cells , Cell Membrane/drug effects , Cell Membrane/metabolism , Cricetinae , Dose-Response Relationship, Drug , Drug Inverse Agonism , Heterocyclic Compounds, 3-Ring/pharmacology , Histamine Agonists/pharmacology , Histamine H3 Antagonists/pharmacology , Molecular Structure , Nootropic Agents/pharmacology , Pyridazines/pharmacology , Pyrrolidines/pharmacology , Rats , Receptors, Histamine H3/metabolism , Structure-Activity RelationshipABSTRACT
Structure-activity relationship on a novel ketone class of H(3)R antagonists/inverse agonists is disclosed. Compound 4 showed excellent target potency, selectivity and brain penetration. Evaluation of antagonist 4 in the rat EEG/EMG model demonstrated robust wake activity thereby establishing preclinical proof of concept.
Subject(s)
Histamine Agonists/pharmacology , Ketones/chemistry , Morpholines/chemistry , Receptors, Histamine H3 , Wakefulness/drug effects , Animals , Electroencephalography , Histamine Agonists/chemistry , Humans , Ketones/pharmacology , Male , Molecular Structure , Morpholines/pharmacology , Rats , Structure-Activity RelationshipABSTRACT
A novel class of 1'-cyclobutyl-6-(4-piperidyloxy)spiro[benzopyran-2,4'-piperidine] derivatives with low nanomolar affinity for the human and rat histamine-3 receptors (H(3)Rs) are described. The spirobenzopyran piperidine ether analogs demonstrated excellent H(3)R affinity and selectivity against histamine receptor subtypes (H(1)R, H(2)R, and H(4)R), were stable in liver microsomes, and had selectivity against CYP P450 enzymes. Compounds 10, 13, 15, and 16 demonstrated high H(3)R affinity, in vitro liver microsomal stability, selectivity against CYP isoforms, moreover, these ether analogs exhibited acceptable iv pharmacokinetic (PK) properties but had poor oral exposure in rat.
Subject(s)
Benzopyrans/chemical synthesis , Histamine Antagonists/chemical synthesis , Piperidines/chemical synthesis , Receptors, Histamine H3/metabolism , Spiro Compounds/chemical synthesis , Administration, Oral , Animals , Benzopyrans/pharmacokinetics , Benzopyrans/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Histamine Antagonists/pharmacokinetics , Histamine Antagonists/pharmacology , Humans , Injections, Intravenous , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Piperidines/pharmacokinetics , Piperidines/pharmacology , Rats , Spiro Compounds/pharmacokinetics , Spiro Compounds/pharmacology , Structure-Activity RelationshipABSTRACT
Structure-activity relationships for a series of phenoxypiperidine pyridazin-3-one H(3)R antagonists/inverse agonists are disclosed. The search for compounds with improved hERG and DAT selectivity without the formation of in vivo active metabolites identified 6-[4-(1-cyclobutyl-piperidin-4-yloxy)-phenyl]-4,4-dimethyl-4,5-dihydro-2H-pyridazin-3-one 17b. Compound 17b met discovery flow criteria, demonstrated potent H(3)R functional antagonism in vivo in the rat dipsogenia model and potent wake activity in the rat EEG/EMG model at doses as low as 0.1 mg/kg ip.
Subject(s)
Histamine Antagonists/chemistry , Histamine Antagonists/pharmacology , Piperidines/chemistry , Pyridazines/chemistry , Receptors, Histamine H3 , Wakefulness/drug effects , Animals , Disease Models, Animal , Models, Molecular , Molecular Structure , Piperidines/pharmacology , Pyridazines/pharmacology , RatsABSTRACT
A novel class of 4-alkoxy-[1'-cyclobutyl-spiro(3,4-dihydrobenzopyran-2,4'-piperidine)] analogues were designed and synthesized as H(3)R antagonists. Structure-activity relationship identified sulfone 27 with excellent H(3)R affinities in both humans and rats, and acceptable pharmacokinetic properties. Further, compound 28 achieved single digit nanomolar H(3)R affinities in both species with minimum hERG activity.
Subject(s)
Histamine Antagonists/chemistry , Piperidines/chemistry , Receptors, Histamine H3/chemistry , Spiro Compounds/chemical synthesis , Animals , Chemistry, Pharmaceutical/methods , Drug Design , Drug Interactions , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Humans , Inhibitory Concentration 50 , Kinetics , Liver/metabolism , Mice , Models, Chemical , Rats , Spiro Compounds/pharmacology , Structure-Activity RelationshipABSTRACT
Optimization of the R(2) and R(6) positions of (5-{4-[3-(R)-2-methylpyrrolin-1-yl-propoxy]phenyl}-2H-pyridazin-3-one) 2a with constrained phenoxypiperidines led to the identification of 5-[4-(cyclobutyl-piperidin-4-yloxy)-phenyl]-6-methyl-2H-pyridazin-3-one 8b as a potent, selective histamine H(3) receptor antagonist with favorable pharmacokinetic properties. Compound 8b had an excellent safety genotoxocity profile for a CNS-active compound in the Ames and micronucleus tests, also displayed potent H(3)R antagonist activity in the brain in the rat dipsogenia model and robust wake activity in the rat EEG/EMG model.
Subject(s)
Piperidines/pharmacology , Pyridazines/pharmacology , Receptors, Histamine H3/chemistry , Animals , Humans , Molecular Structure , Piperidines/chemical synthesis , Piperidines/chemistry , Pyridazines/chemical synthesis , Pyridazines/chemistry , Rats , Receptors, Histamine H3/metabolism , Structure-Activity RelationshipABSTRACT
H(3)R structure-activity relationships for a new class of 4,5-dihydropyridazin-3-one H(3)R antagonists/inverse agonists are disclosed. Modification of the 4,5-dihydropyridazinone moiety to block in vivo metabolism identified 4,4-dimethyl-6-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)-propoxy]-phenyl}-4,5-dihydro-2H-pyridazin-3-one 22 as a lead candidate demonstrating potent in vivo functional H(3)R antagonism in the rat dipsogenia model and robust wake promoting activity in the rat EEG/EMG model.
Subject(s)
Histamine Agonists/chemical synthesis , Pyridazines/chemistry , Receptors, Histamine H3/chemistry , Animals , Area Under Curve , Dose-Response Relationship, Drug , Drug Design , Electroencephalography/methods , Electromyography/methods , Histamine Agonists/pharmacology , Kinetics , Models, Chemical , Pyridazines/chemical synthesis , Pyridazines/pharmacology , Rats , Stereoisomerism , Structure-Activity Relationship , Time FactorsABSTRACT
Previous studies have shown that (5-{4-[3-(R)-2-methylpyrrolin-1-yl-propoxy]phenyl}-2H-pyridazin-3-one) 2 had high affinity for both the human (hH(3)R K(i) = 2.8 nM) and rat H(3)Rs (rH(3)R K(i) = 8.5 nM) but displayed low oral bioavailability in the rat. Optimization of the 5-pyridazin-3-one R(2) and R(6) positions to improve the pharmacokinetic properties over 2 led to the identification of 5-{4-[3-(R)-2-methylpyrrolidin-1-yl)propoxy]phenyl}-2-pyridin-2-yl-2H-pyridazin-3-one 29. Compound 29 displayed high affinity for both human and rat H(3)Rs (hH(3)R K(i) = 1.7 nM, rH(3)R K(i) = 3.7 nM) with a greater than 1000-fold selectivity over the other histamine receptor subtypes and favorable pharmacokinetic properties across species (F = 78% rat, 92% dog, 96% monkey). It showed low binding to human plasma proteins, weakly inhibited cytochrome P450 isoforms, and displayed an excellent safety profile for a CNS-active compound. 29 displayed potent H(3)R antagonist activity in the brain in a rat dipsogenia model and demonstrated enhancement of cognitive function in a rat social recognition model at low doses. However, the development of compound 29 was discontinued because of genotoxicity.
Subject(s)
Nootropic Agents/chemical synthesis , Propylamines/chemical synthesis , Pyridazines/chemical synthesis , Receptors, Histamine H3/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Dogs , Drug Inverse Agonism , Histamine Agonists/chemical synthesis , Histamine Agonists/pharmacokinetics , Histamine Agonists/pharmacology , Histamine H3 Antagonists/chemical synthesis , Histamine H3 Antagonists/pharmacokinetics , Histamine H3 Antagonists/pharmacology , Humans , Macaca fascicularis , Male , Memory, Short-Term/drug effects , Mutagenicity Tests , Nootropic Agents/pharmacokinetics , Nootropic Agents/pharmacology , Propylamines/pharmacokinetics , Propylamines/pharmacology , Pyridazines/pharmacokinetics , Pyridazines/pharmacology , Radioligand Assay , Rats , Rats, Long-Evans , Rats, Sprague-Dawley , Recognition, Psychology/drug effects , Stereoisomerism , Structure-Activity Relationship , Thirst/drug effects , Tissue DistributionABSTRACT
6-{4-[3-(R)-2-Methylpyrrolidin-1-yl)propoxy]-phenyl}-2H-pyridazin-3-one 6 (Irdabisant; CEP-26401) was recently reported as a potent H(3)R antagonist with excellent drug-like properties and in vivo activity that advanced into clinical evaluation. A series of pyridone analogs of 6 was synthesized and evaluated as H(3)R antagonists. Structure-activity relationships revealed that the 5-pyridone regiomer was optimal for H(3)R affinity. N-Methyl 9b showed excellent H(3)R affinity, acceptable pharmacokinetics and pharmaceutical properties. In vivo evaluation of 9b showed potent activity in the rat dipsogenia model and robust wake-promoting activity in the rat EEG model.
Subject(s)
Histamine H3 Antagonists/chemical synthesis , Histamine H3 Antagonists/pharmacology , Pyridazines/chemical synthesis , Pyridazines/pharmacology , Pyrrolidines/chemical synthesis , Pyrrolidines/pharmacology , Animals , Disease Models, Animal , Histamine H3 Antagonists/chemistry , Humans , Molecular Structure , Protein Binding/drug effects , Pyridazines/chemistry , Pyrrolidines/chemistry , Rats , Structure-Activity RelationshipABSTRACT
Pyridazinone 1 was recently reported as a potent H(3)R antagonist with good drug-like properties and in vivo activity. A series of constrained amine analogs of 1 was synthesized to identify compounds with improved pharmacokinetic profiles. From these efforts, a new class of (S)-2-pyrrolidin-1-ylmethyl-1-pyrrolidinyl amides was identified.
Subject(s)
Amines/chemistry , Histamine H3 Antagonists/pharmacology , Pyridazines/pharmacology , Animals , Dose-Response Relationship, Drug , Histamine H3 Antagonists/chemical synthesis , Histamine H3 Antagonists/chemistry , Humans , Molecular Structure , Pyridazines/chemical synthesis , Pyridazines/chemistry , Rats , Receptors, Histamine H3/metabolism , Stereoisomerism , Structure-Activity RelationshipABSTRACT
H(3)R structure-activity relationships on a novel class of pyridazin-3-one H(3)R antagonists/inverse agonists are disclosed. Modifications of the pyridazinone core, central phenyl ring and linker led to the identification of molecules with excellent target potency, selectivity and pharmacokinetic properties. Compounds 13 and 21 displayed potent functional H(3)R antagonism in vivo in the rat dipsogenia model and demonstrated robust wake activity in the rat EEG/EMG model.