ABSTRACT
Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.
Subject(s)
Immunoconjugates , NAV1.7 Voltage-Gated Sodium Channel/metabolism , Peptides/chemistry , Spider Venoms/chemistry , Voltage-Gated Sodium Channel Blockers , Animals , Antibodies/chemistry , Drug Discovery , Humans , Immunoconjugates/chemistry , Immunoconjugates/pharmacokinetics , Male , Mice , Molecular Targeted Therapy , NAV1.7 Voltage-Gated Sodium Channel/immunology , Peptides/pharmacokinetics , Spider Venoms/pharmacokinetics , Voltage-Gated Sodium Channel Blockers/chemistry , Voltage-Gated Sodium Channel Blockers/pharmacokineticsABSTRACT
Inhibitors of the voltage-gated sodium channel NaV1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified NaV1.7-inhibitory peptides GpTx-1 and JzTx-V from tarantula venom screens. Potency and selectivity were modulated through attribute-based positional scans of native residues via chemical synthesis. Herein, we report JzTx-V lead optimization to identify a pharmacodynamically active peptide variant. Molecular docking of peptide ensembles from NMR into a homology model-derived NaV1.7 structure supported prioritization of key residues clustered on a hydrophobic face of the disulfide-rich folded peptide for derivatization. Replacing Trp24 with 5-Br-Trp24 identified lead peptides with activity in electrophysiology assays in engineered and neuronal cells. 5-Br-Trp24 containing peptide AM-6120 was characterized in X-ray crystallography and pharmacokinetic studies and blocked histamine-induced pruritis in mice after subcutaneous administration, demonstrating systemic NaV1.7-dependent pharmacodynamics. Our data suggests a need for high target coverage based on plasma exposure for impacting in vivo end points with selectivity-optimized peptidic NaV1.7 inhibitors.
Subject(s)
Drug Discovery , Histamine/adverse effects , NAV1.7 Voltage-Gated Sodium Channel/metabolism , Peptides/chemistry , Peptides/pharmacology , Pruritus/drug therapy , Spider Venoms/chemistry , Animals , HEK293 Cells , Humans , Mice , Molecular Docking Simulation , NAV1.7 Voltage-Gated Sodium Channel/chemistry , Peptides/pharmacokinetics , Peptides/therapeutic use , Protein Conformation , Protein Folding , Pruritus/chemically induced , Structure-Activity Relationship , Tissue Distribution , Voltage-Gated Sodium Channel Blockers/chemistry , Voltage-Gated Sodium Channel Blockers/pharmacokinetics , Voltage-Gated Sodium Channel Blockers/pharmacology , Voltage-Gated Sodium Channel Blockers/therapeutic useABSTRACT
The voltage-gated sodium channel NaV1.7 is a genetically validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain; however functional NaV1.7 targeting antibodies are not known. In this report, we describe NaV1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the NaV1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of 13 potential cysteine conjugation sites in the antibody scaffold was tuned using a model alkylating agent. Subsequent reactions with the peptide identified cysteine locations with the highest conversion to desired conjugates, which blocked NaV1.7 currents in whole cell electrophysiology. Variations in attachment site, linker, and peptide loading established design parameters for potency optimization. Antibody conjugation led to in vivo half-life extension by 130-fold relative to a nonconjugated GpTx-1 peptide and differential biodistribution to nerve fibers in wild-type but not NaV1.7 knockout mice. This study describes the optimization and application of antibody derivatization technology to functionally inhibit NaV1.7 in engineered and neuronal cells.
Subject(s)
Immunoconjugates/pharmacology , NAV1.7 Voltage-Gated Sodium Channel/metabolism , Peptides/pharmacology , Voltage-Gated Sodium Channel Blockers/pharmacology , Animals , HEK293 Cells , Humans , Immunoconjugates/chemistry , Immunoconjugates/pharmacokinetics , Male , Mice , Models, Molecular , Peptides/chemistry , Peptides/pharmacokinetics , Tissue Distribution , Voltage-Gated Sodium Channel Blockers/chemistry , Voltage-Gated Sodium Channel Blockers/pharmacokineticsABSTRACT
Several reports have recently emerged regarding the identification of heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. The optimization of a series of internal NaV1.7 leads that address a number of metabolic liabilities including bioactivation, PXR activation, as well as CYP3A4 induction and inhibition led to the identification of potent and selective inhibitors that demonstrated favorable pharmacokinetic profiles and were devoid of the aforementioned liabilities. The key to achieving this within a series prone to transporter-mediated clearance was the identification of a small range of optimal cLogD values and the discovery of subtle PXR SAR that was not lipophilicity dependent. This enabled the identification of compound 20, which was advanced into a target engagement pharmacodynamic model where it exhibited robust reversal of histamine-induced scratching bouts in mice.
Subject(s)
Isoquinolines/chemistry , NAV1.7 Voltage-Gated Sodium Channel/metabolism , Sulfonamides/chemistry , Voltage-Gated Sodium Channel Blockers/chemistry , Animals , Cell Line , Cytochrome P-450 CYP3A/biosynthesis , Cytochrome P-450 CYP3A Inhibitors/chemistry , Cytochrome P-450 CYP3A Inhibitors/pharmacokinetics , Cytochrome P-450 CYP3A Inhibitors/pharmacology , Dogs , Enzyme Induction , Histamine , Humans , Isoquinolines/administration & dosage , Isoquinolines/pharmacokinetics , Male , Mice, Inbred C57BL , Pregnane X Receptor , Pruritus/chemically induced , Pruritus/prevention & control , Rats , Receptors, Steroid/agonists , Structure-Activity Relationship , Sulfonamides/administration & dosage , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacology , Voltage-Gated Sodium Channel Blockers/pharmacokinetics , Voltage-Gated Sodium Channel Blockers/pharmacologyABSTRACT
Because of its strong genetic validation, NaV1.7 has attracted significant interest as a target for the treatment of pain. We have previously reported on a number of structurally distinct bicyclic heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. Herein, we report the discovery and optimization of a series of atropisomeric quinolinone sulfonamide inhibitors [ Bicyclic sulfonamide compounds as sodium channel inhibitors and their preparation . WO 2014201206, 2014 ] of NaV1.7, which demonstrate nanomolar inhibition of NaV1.7 and exhibit high levels of selectivity over other sodium channel isoforms. After optimization of metabolic and pharmacokinetic properties, including PXR activation, CYP2C9 inhibition, and CYP3A4 TDI, several compounds were advanced into in vivo target engagement and efficacy models. When tested in mice, compound 39 (AM-0466) demonstrated robust pharmacodynamic activity in a NaV1.7-dependent model of histamine-induced pruritus (itch) and additionally in a capsaicin-induced nociception model of pain without any confounding effect in open-field activity.
Subject(s)
NAV1.7 Voltage-Gated Sodium Channel/metabolism , Quinolones/chemistry , Sulfonamides/chemistry , Voltage-Gated Sodium Channel Blockers/chemistry , Analgesics/chemistry , Analgesics/pharmacokinetics , Analgesics/pharmacology , Animals , Capsaicin , Cell Line , Dogs , Histamine , Mice, Inbred C57BL , Molecular Docking Simulation , Pain/chemically induced , Pain/prevention & control , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/metabolism , Pruritus/chemically induced , Pruritus/prevention & control , Quinolones/administration & dosage , Quinolones/chemical synthesis , Quinolones/pharmacokinetics , Quinolones/pharmacology , Rats , Structure-Activity Relationship , Sulfonamides/administration & dosage , Sulfonamides/chemical synthesis , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacology , Voltage-Gated Sodium Channel Blockers/pharmacokinetics , Voltage-Gated Sodium Channel Blockers/pharmacologyABSTRACT
[This corrects the article DOI: 10.1021/acsmedchemlett.6b00243.].
ABSTRACT
Human genetic evidence has identified the voltage-gated sodium channel NaV1.7 as an attractive target for the treatment of pain. We initially identified naphthalene sulfonamide 3 as a potent and selective inhibitor of NaV1.7. Optimization to reduce biliary clearance by balancing hydrophilicity and hydrophobicity (Log D) while maintaining NaV1.7 potency led to the identification of quinazoline 16 (AM-2099). Compound 16 demonstrated a favorable pharmacokinetic profile in rat and dog and demonstrated dose-dependent reduction of histamine-induced scratching bouts in a mouse behavioral model following oral dosing.
ABSTRACT
Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Pyridones/chemical synthesis , Pyridones/pharmacology , Triazoles/chemical synthesis , Triazoles/pharmacology , Animals , Antineoplastic Agents/pharmacokinetics , Crystallography, X-Ray , Drug Design , Drug Discovery , Humans , Mice , Models, Molecular , Pyridones/pharmacokinetics , Structure-Activity Relationship , Triazoles/pharmacokinetics , Xenograft Model Antitumor AssaysABSTRACT
Efforts to improve upon the physical properties and metabolic stability of Aurora kinase inhibitor 14a revealed that potency against multidrug-resistant cell lines was compromised by increased polarity. Despite its high in vitro metabolic intrinsic clearance, 23r (AMG 900) showed acceptable pharmacokinetic properties and robust pharmacodynamic activity. Projecting from in vitro data to in vivo target coverage was not practical due to disjunctions between enzyme and cell data, complex and apparently contradictory indicators of binding kinetics, and unmeasurable free fraction in plasma. In contrast, it was straightforward to relate pharmacokinetics to pharmacodynamics and efficacy by following the time above a threshold concentration. On the basis of its oral route of administration, a selectivity profile that favors Aurora-driven pharmacology and its activity against multidrug-resistant cell lines, 23r was identified as a potential best-in-class Aurora kinase inhibitor. In phase 1 dose expansion studies with G-CSF support, 23r has shown promising single agent activity.
Subject(s)
Aurora Kinases/antagonists & inhibitors , Drug Discovery , Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm/drug effects , Neoplasms/drug therapy , Phthalazines/pharmacology , Protein Kinase Inhibitors/pharmacology , Animals , Cell Proliferation/drug effects , Female , Humans , Mice , Mice, Nude , Molecular Structure , Neoplasms/enzymology , Neoplasms/pathology , Rats , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, we report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM), selective inhibitors of c-Met with desirable pharmacokinetic properties that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.
Subject(s)
Drug Discovery , Prostatic Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Quinolines/pharmacology , Triazoles/pharmacology , Animals , Cell Proliferation/drug effects , Drug Design , Hepatocyte Growth Factor/metabolism , Humans , Male , Mice , Microsomes, Liver/drug effects , Models, Molecular , Molecular Structure , Phosphorylation/drug effects , Prostatic Neoplasms/pathology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Quinolines/chemistry , Quinolines/pharmacokinetics , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Tissue Distribution , Triazoles/chemistry , Triazoles/pharmacokinetics , Xenograft Model Antitumor AssaysABSTRACT
1. This study was designed to evaluate how the absence of P-glycoprotein (Pgp, Mdr1a), breast cancer-resistance protein (Bcrp, Abcg2) or both affects drug distribution into sciatic nerves, brain and cerebrospinal fluid (CSF) in rats. 2. Pgp substrate (loperamide), BCRP substrates (dantrolene and proprietary compound X) and dual substrates (imatinib and proprietary compound Y) were well distributed into sciatic nerves with comparable nerve to plasma concentration ratios between wild-type and knockout (KO) rats. 3. Brain exposure increased substantially in Mdr1a(-/-) rats for loperamide and in Mdr1a(-/-)/Abcg2(-/-) rats for imatinib and compound Y, but minimally to modestly in Abcg2(-/-) rats for dantrolene and compound X. The deletion of Mdr1a or Abcg2 alone had little effect on brain distribution of compound Y. 4. While CSF to unbound brain concentration ratio remained ≥3 in the KO animals for dantrolene, compounds X and Y, it was reduced to 1 in the Mdr1a(-/-)/Abcg2(-/-) rats for imatinib. 5. The data indicate that Pgp and Bcrp do not play significant roles in drug distribution into peripheral nerve tissues in rats, while working in concert to regulate brain penetration. Our results further support that CSF concentration may not be a good surrogate for unbound brain concentration of efflux substrates.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP-Binding Cassette Transporters/metabolism , Brain/metabolism , Dantrolene/pharmacokinetics , Loperamide/pharmacokinetics , Sciatic Nerve/metabolism , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Animals , Dantrolene/pharmacology , Loperamide/pharmacology , Male , Rats , Rats, Sprague-Dawley , Rats, TransgenicABSTRACT
The impact of inhibitor depletion on the determination of shifted IC50 (IC50 determined after 30 minutes of preincubation with inhibitor) is examined. In addition, IC50-shift data are analyzed using a mechanistic model that incorporates the processes of inhibitor depletion, as well as reversible and time-dependent inhibition. Anomalies such as a smaller-than-expected shift in IC50 and even increases in IC50 with preincubation were explained by the depletion of inhibitor during the preincubation. The IC50-shift assay remains a viable approach to characterizing a wide range of reversible and time-dependent inhibitors. However, as with more traditional time-dependent inactivation methods, it is recommended that IC50-shift experimental data be interpreted with some knowledge of the magnitude of inhibitor depletion. For the most realistic classification of time-dependent inhibitors using IC50-shift methods, shifted IC50 should be calculated using observed inhibitor concentrations at the end of the incubation rather than nominal inhibitor concentrations. Finally, a mechanistic model that includes key processes, such as competitive inhibition, enzyme inactivation, and inhibitor depletion, can be used to describe accurately the observed IC50 and shifted IC50 curves. For compounds showing an IC50 fold shift >1.5 based on the observed inhibitor concentrations, reanalyzing the IC50-shift data using the mechanistic model appeared to allow for reasonable estimation of Ki, KI, and kinact directly from the IC50 shift experiments.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 CYP3A , Cytochrome P-450 CYP3A Inhibitors , Humans , Inhibitory Concentration 50 , Models, Biological , Nicardipine/pharmacology , Saquinavir/pharmacology , Time FactorsABSTRACT
Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of a structurally diverse series of carbon-linked quinoline triazolopyridinones, which demonstrates nanomolar inhibition of c-Met kinase activity. This novel series of inhibitors exhibits favorable pharmacokinetics as well as potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.
Subject(s)
Antineoplastic Agents/chemical synthesis , Protein Kinase Inhibitors/chemical synthesis , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Pyridones/chemical synthesis , Quinolines/chemical synthesis , Triazoles/chemical synthesis , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Drug Discovery , Hepatocyte Growth Factor/metabolism , Humans , Male , Mice , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Models, Molecular , Phosphorylation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-met/metabolism , Pyridones/pharmacology , Quinolines/pharmacology , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Triazoles/pharmacologyABSTRACT
We previously reported that the accuracy of clearance (CL) prediction could be differentiated by permeability. CL was drastically under-predicted by in vitro metabolic intrinsic clearance (CL(int)) for compounds with low permeability (<5 × 10(-6) cm/s). We determined apparent uptake CL(int) by measuring initial disappearance from medium using attached rat hepatocytes and metabolic CL(int) by measuring parent depletion in suspended rat hepatocytes (cells and medium). Uptake and metabolic CL(int) were comparable for highly permeable metabolic marker compounds. In contrast, uptake CL(int) was 3- to 40-fold higher than metabolic CL(int) for rosuvastatin, bosentan, and 15 proprietary compounds, which had low permeability, suggesting that uptake could be a rate-determining step in hepatic elimination for these poorly permeable compounds. The prediction of hepatic CL was improved significantly when using uptake CL(int) for the compounds with low permeability. The average fold error was 2.2 and 6, as opposed to >11 and >47 by metabolic CL(int), with and without applying a scaling factor of 4, respectively. Uptake CL(int) from attached hepatocytes can be used as an alternative approach to predict hepatic clearance and to understand the significance of hepatic uptake in elimination in an early drug discovery setting.
Subject(s)
Capillary Permeability/physiology , Hepatocytes/physiology , Metabolic Clearance Rate/physiology , Xenobiotics/pharmacokinetics , Animals , Bosentan , Chromatography, Liquid , Cyclosporine/pharmacology , Dogs , Fluorobenzenes/pharmacokinetics , Madin Darby Canine Kidney Cells , Male , Pyrimidines/pharmacokinetics , Quinidine/pharmacology , Rats , Rats, Sprague-Dawley , Rifampin/pharmacology , Rosuvastatin Calcium , Sulfonamides/pharmacokinetics , Tandem Mass Spectrometry , Verapamil/pharmacokinetics , Xenobiotics/metabolismABSTRACT
AMG 900 is an orally available small molecule that is highly potent and selective as a pan-aurora kinase inhibitor. AMG 900 is currently undergoing phase 1 clinical evaluation in patients with advanced solid tumors. The metabolism of AMG 900 was investigated in both male and female rats. We conducted studies in bile-duct catheterized (BDC) rats where bile, urine and plasma were analyzed to obtain metabolism profiles for each gender. These studies identified gender differences in the metabolism profiles in bile. Bile contained the majority of the drug related material and contained little unchanged AMG 900 which indicated that metabolism was the prominent process in drug elimination. Although bile contained the same metabolites for both genders, the amount of specific metabolites differed. Male rats metabolized AMG 900 primarily through hydroxylation with subsequent sulfate conjugation on the pyrimidinyl-pyridine side-chain whereas female rats favored a different oxidation site on the thiophene ring's methyl group, which is then metabolized to a carboxylic acid with subsequent conjugation to an acyl glucuronide. CYP phenotyping identified the prominent isoforms as being gender specific or biased in the oxidative metabolism of AMG 900. The metabolism in male rats favored both CYP2C11 and CYP2A2 whereas females favored the CYP2C12. The prominent sulfate conjugate identified in the male rat bile could also be due to male biased metabolism since it has been reported that sulfate conjugation is more prevalent in male rats. All the prominent rat metabolism routes for AMG 900 either have male or female bias. These differences in the rat AMG 900 metabolism profiles in bile can be explained by gender specific P450CYP isoforms.
Subject(s)
Phthalazines/administration & dosage , Phthalazines/pharmacokinetics , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacokinetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Administration, Oral , Animals , Aryl Hydrocarbon Hydroxylases/metabolism , Aurora Kinases , Bile/metabolism , Biotransformation , Cytochrome P450 Family 2 , Female , Glucuronides/metabolism , Hydroxylation , Male , Molecular Structure , Phthalazines/blood , Phthalazines/chemistry , Phthalazines/urine , Protein Kinase Inhibitors/blood , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/urine , Rats , Rats, Sprague-Dawley , Sex Factors , Steroid 16-alpha-Hydroxylase/metabolism , Steroid Hydroxylases/metabolism , Sulfates/metabolismABSTRACT
AMG 900 is a small molecule being developed as an orally administered, highly potent, and selective pan-aurora kinase inhibitor. The aim of the investigations was to characterize in vitro and in vivo pharmacokinetic (PK) properties of AMG 900 in preclinical species. AMG 900 was rapidly metabolized in liver microsomes and highly bound to plasma proteins in the species tested. It was a weak Pgp substrate with good passive permeability. AMG 900 exhibited a low-to-moderate clearance and a small volume of distribution. Its terminal elimination half-life ranged from 0.6 to 2.4 h. AMG 900 was well-absorbed in fasted animals with an oral bioavailability of 31% to 107%. Food intake had an effect on rate (rats) or extent (dogs) of AMG 900 oral absorption. The clearance and volume of distribution at steady state in humans were predicted to be 27.3 mL/h/kg and 93.9 mL/kg, respectively. AMG 900 exhibited acceptable PK properties in preclinical species and was predicted to have low clearance in humans. AMG 900 is currently in Phase I clinical testing as a treatment for solid tumours. Preliminary human PK results appear to be consistent with the predictions.
Subject(s)
Phthalazines/pharmacology , Phthalazines/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacokinetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Small Molecule Libraries/pharmacology , Small Molecule Libraries/pharmacokinetics , Administration, Oral , Animals , Aurora Kinases , Biological Availability , Blood Proteins/metabolism , Cell Line , Fasting , Humans , Injections, Intravenous , Male , Phthalazines/blood , Phthalazines/chemistry , Protein Binding/drug effects , Protein Serine-Threonine Kinases/metabolism , Protein Transport/drug effects , Species SpecificityABSTRACT
Compound 1, (7-methoxy-N-((6-(3-methylisothiazol-5-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine) is a potent, selective inhibitor of c-Met (mesenchymal-epithelial transition factor), a receptor tyrosine kinase that is often deregulated in cancer. Compound 1 displayed desirable pharmacokinetic properties in multiple preclinical species. Glutathione trapping studies in liver microsomes resulted in the NADPH-dependent formation of a glutathione conjugate. Compound 1 also exhibited very high in vitro NADPH-dependent covalent binding to microsomal proteins. Species differences in covalent binding were observed, with the highest binding in rats, mice, and monkeys (1100-1300 pmol/mg/h), followed by dogs (400 pmol/mg/h) and humans (144 pmol/mg/h). This covalent binding to protein was abolished by coincubation with glutathione. Together, these in vitro data suggest that covalent binding and glutathione conjugation proceed via bioactivation to a chemically reactive intermediate. The cytochrome (CYP) P450 enzymes responsible for this bioactivation were identified as cytochrome P450 3A4, 1A2, and 2D6 in human and cytochrome P450 2A2, 3A1, and 3A2 in rats. The glutathione metabolite was detected in the bile of rats and mice, thus demonstrating bioactivation occurring in vivo. Efforts to elucidate the structure of the glutathione adduct led to the isolation and characterization of the metabolite by NMR and mass spectrometry. The analytical data confirmed conclusively that the glutathione conjugation was on the 4-C position of the isothiazole ring. Such P450-mediated bioactivation of an isothiazole or thiazole group has not been previously reported. We propose a mechanism of bioactivation via sulfur oxidation followed by glutathione attack at the 4-position with subsequent loss of water resulting in the formation of the glutathione conjugate. Efforts to reduce bioactivation without compromising potency and pharmacokinetics were undertaken in order to minimize the potential risk of toxicity. Because of the exemplary pharmacokinetic/pharmacodynamic (PK/PD) properties of the isothiazole group, initial attempts were focused on introducing alternative metabolic soft spots into the molecule. These efforts resulted in the discovery of 7-(2-methoxyethoxy)-N-((6-(3-methyl-5-isothiazolyl)[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine (compound 2), with the major metabolic transformation occurring on the naphthyridine ring alkoxy substituent. However, a glutathione conjugate of compound 2 was produced in vitro and in vivo in a manner similar to that observed for compound 1. Furthermore, the covalent binding was high across species (360, 300, 529, 208, and 98 pmol/mg/h in rats, mice, dogs, monkeys, and humans, respectively), but coincubation with glutathione reduced the extent of covalent binding. The second viable alternative in reducing bioactivation involved replacing the isothiazole ring with bioisosteric heterocycles. Replacement of the isothiazole ring with an isoxazole or a pyrazole reduced the bioactivation while retaining the desirable PK/PD characteristics of compounds 1 and 2.
Subject(s)
Naphthyridines/metabolism , Pyridazines/metabolism , Thiazoles/metabolism , Animals , Chromatography, High Pressure Liquid , Cytochrome P-450 Enzyme System/metabolism , Dogs , Drug Evaluation, Preclinical , Glutathione/chemistry , Humans , Magnetic Resonance Spectroscopy , Mice , Microsomes, Liver/metabolism , Molecular Conformation , Naphthyridines/chemistry , Naphthyridines/pharmacokinetics , Naphthyridines/toxicity , Protein Binding , Pyridazines/chemistry , Pyridazines/pharmacokinetics , Pyridazines/toxicity , Rats , Risk Factors , Spectrometry, Mass, Electrospray Ionization , Thiazoles/chemistry , Thiazoles/toxicityABSTRACT
The discovery of aurora kinases as essential regulators of cell division has led to intense interest in identifying small molecule aurora kinase inhibitors for the potential treatment of cancer. A high-throughput screening effort identified pyridinyl-pyrimidine 6a as a moderately potent dual inhibitor of aurora kinases -A and -B. Optimization of this hit resulted in an anthranilamide lead (6j) that possessed improved enzyme and cellular activity and exhibited a high level of kinase selectivity. However, this anthranilamide and subsequent analogues suffered from a lack of oral bioavailability. Converting the internally hydrogen-bonded six-membered pseudo-ring of the anthranilamide to a phthalazine (8a-b) led to a dramatic improvement in oral bioavailability (38-61%F) while maintaining the potency and selectivity characteristics of the anthranilamide series. In a COLO 205 tumor pharmacodynamic assay measuring phosphorylation of the aurora-B substrate histone H3 at serine 10 (p-histone H3), oral administration of 8b at 50 mg/kg demonstrated significant reduction in tumor p-histone H3 for at least 6 h.
Subject(s)
Antineoplastic Agents/chemical synthesis , Phthalazines/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridines/chemical synthesis , Pyrimidines/chemical synthesis , Administration, Oral , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Aurora Kinase B , Aurora Kinases , Biological Availability , Blood Proteins/metabolism , Cell Line, Tumor , Drug Screening Assays, Antitumor , Female , Histones/metabolism , Humans , In Vitro Techniques , Male , Mice , Mice, Nude , Microsomes, Liver/metabolism , Models, Molecular , Neoplasm Transplantation , Phthalazines/pharmacokinetics , Phthalazines/pharmacology , Protein Binding , Pyridines/pharmacokinetics , Pyridines/pharmacology , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Transplantation, HeterologousABSTRACT
In vitro intrinsic metabolic clearance (CL(int)) is used routinely for compound selection in drug discovery; however, in vitro CL(int) often underpredicts in vivo clearance (CL). Forty-one proprietary compounds and 16 marketed drugs were selected to determine whether permeability and efflux status could influence the predictability of CL from in vitro CL(int) obtained from liver microsomal and hepatocyte incubations. For many of the proprietary compounds examined, rat CL was significantly underpredicted using the well stirred model incorporating both fraction of unbound drug in blood and fraction of unbound drug in the microsomal or hepatocyte incubation. Further analysis revealed that the accuracy of the prediction was differentiated by permeability and P-glycoprotein- (P-gp) and mouse breast cancer resistance protein (mBcrp)-mediated efflux. For proprietary compounds with passive permeability greater than 5 x 10(-6) cm/s and efflux ratios less than 5 in both P-gp- and mBcrp-expressing cells, CL(int) provided reasonable prediction. The average -fold error (AFE) was 1.8 for rat liver microsomes (RLMs) and 2.3 for rat hepatocytes. In contrast, CL was dramatically underpredicted for compounds with passive permeability less than 5 x 10(-6) cm/s; AFEs of 54.4 and 29.2 were observed for RLM and rat hepatocytes, respectively. In vivo CL was also underpredicted for compounds that were good efflux substrates (permeability >5 x 10(-6) cm/s). The AFEs were 7.4 and 8.1 for RLM and rat hepatocytes, respectively. A similar relationship between permeability, efflux status, and human CL prediction reported in the literature was observed for 16 marketed drugs. These data show that permeability and efflux status are determinants for the predictability of CL from in vitro metabolic CL(int).