Multiple-dose pharmacokinetics and opioid effects of a novel analgesic with a deterrent to human opioid abuse (methadone-fluconazole-naltrexone) after oral administration in dogs.

OBJECTIVE: To assess the pharmacokinetics and opioid effects of methadone after administration of multiple doses by means of 2 dosing regimens of methadone-fluconazole-naltrexone. ANIMALS: 12 healthy Beagles. PROCEDURES: Dogs were randomly allocated (6 dogs/group) to receive 1 of 2 oral dosing regimens of methadone-fluconazole-naltrexone. Treatment 1 doses were administered at 0 (methadone-to-fluconazole-to-naltrexone ratio of 1:5:0.25 mg/kg), 14 (1:5:0.25), 24 (0.5:2.5:0.125), and 38 (0.5:2.5:0.125) hours. Treatment 2 doses were administered at 0 (1:5:0.25), 4 (0.5:2.5:0.125), 10 (0.5:2.5:0.125), and 24 (0.5:2.5:0.125) hours. Blood samples, rectal temperatures, and von Frey antinociceptive measurements were obtained at designated times. RESULTS: Compared with baseline, temperatures significantly decreased for treatment 1 group dogs at 2 to ≥ 4 hours and from 16 to ≥ 50 hours (12 hours after last dose) and for treatment 2 group dogs at 2 to ≥ 36 hours (12 hours after last dose), when trough methadone concentrations were ≥ 21.3 ng/mL. Antinociception occurred after the first dose but was not maintained throughout the study. Lesions were noted in some dogs at the application site of the von Frey device. Naltrexone and ß-naltrexol were sporadically detected in plasma, and naltrexone glucuronide was consistently detected. CONCLUSIONS AND CLINICAL RELEVANCE: Opioid effects were noted after oral administration of the first dose, and data suggested that administering a second dose 6 hours later and every 12 hours thereafter was necessary to maintain opioid effects. Antinociception may have been lost because dogs became averse or hyperalgesic to the von Frey device, such that the antinociception model used here may not be robust for repeated measurements in dogs.

Selective Vulnerability to Pyrimidine Starvation in Hematologic Malignancies Revealed by AG-636, a Novel Clinical-Stage Inhibitor of Dihydroorotate Dehydrogenase.

Agents targeting metabolic pathways form the backbone of standard oncology treatments, though a better understanding of differential metabolic dependencies could instruct more rationale-based therapeutic approaches. We performed a chemical biology screen that revealed a strong enrichment in sensitivity to a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic versus solid tumor origin. Differential AG-636 activity translated to the in vivo setting, with complete tumor regression observed in a lymphoma model. Dissection of the relationship between uridine availability and response to AG-636 revealed a divergent ability of lymphoma and solid tumor cell lines to survive and grow in the setting of depleted extracellular uridine and DHODH inhibition. Metabolic characterization paired with unbiased functional genomic and proteomic screens pointed to adaptive mechanisms to cope with nucleotide stress as contributing to response to AG-636. These findings support targeting of DHODH in lymphoma and other hematologic malignancies and suggest combination strategies aimed at interfering with DNA-damage response pathways.

Pharmacokinetics and pharmacodynamics of a novel analgesic with a deterrent to human opioid abuse (methadone-fluconazole-naltrexone) after oral administration in dogs.

OBJECTIVE: To determine the effects of coadministration of naltrexone, a human opioid abuse deterrent, on the pharmacokinetics and pharmacodynamics of a methadone-fluconazole combination administered orally to dogs. ANIMALS: 12 healthy Beagles. PROCEDURES: Dogs (body weight, 10.7 to 13.9 kg) were randomly allocated to 2 groups in a parallel design study. All dogs received fluconazole (100 mg [7.19 to 9.35 mg/kg], PO). Twelve hours later (time 0), dogs were administered methadone (10 mg [0.72 to 0.93 mg/kg]) plus fluconazole (50 mg [3.62 to 4.22 mg/kg]; methadone-fluconazole) or methadone (10 mg [0.72 to 0.93 mg/kg]) plus fluconazole (50 mg [3.60 to 4.67 mg/kg]) and naltrexone (2.5 mg [0.18 to 0.23 mg/kg]; methadone-fluconazole-naltrexone), PO, in a gelatin capsule. Blood samples were collected for pharmacokinetic analysis, and rectal temperature and sedation were assessed to evaluate opioid effects at predetermined times up to 24 hours after treatment. RESULTS: Most dogs had slight sedation during the 12 hours after drug administration; 1 dog/group had moderate sedation at 1 time point. Mean rectal temperatures decreased significantly from baseline (immediate pretreatment) values from 2 to ≥ 12 hours and 2 to ≥ 8 hours after methadone-fluconazole and methadone-fluconazole-naltrexone treatment, respectively. Geometric mean maximum observed concentration of methadone in plasma was 35.1 and 33.5 ng/mL and geometric mean terminal half-life was 7.92 and 7.09 hours after methadone-fluconazole and methadone-fluconazole-naltrexone treatment, respectively. Naltrexone was sporadically detected in 1 dog. The active naltrexone metabolite, ß-naltrexol, was not detected. The inactive metabolite, naltrexone glucuronide, was detected in all dogs administered methadone-fluconazole-naltrexone. CONCLUSIONS AND CLINICAL RELEVANCE: Opioid effects were detected after oral administration of methadone-fluconazole or methadone-fluconazole-naltrexone. Further studies assessing additional opioid effects, including antinociception, are needed.

The effect of fluconazole on oral methadone in dogs.

OBJECTIVE: To determine the effects of fluconazole on oral methadone pharmacokinetics and central effects mediated by opioid receptors in dogs. STUDY DESIGN: Prospective, incomplete block. ANIMALS: A total of 12 healthy Beagle dogs. METHODS: Dogs were randomly allocated into two groups of six dogs. In total, four treatments (two treatments/group) were administered including: oral methadone (1 mg kg-1); oral fluconazole (5 mg kg-1) every 12 hours starting 24 hours prior to oral methadone (1 mg kg-1); oral fluconazole (2.5 mg kg-1) every 12 hours starting 24 hours prior to oral methadone (1 mg kg-1); and oral fluconazole (5 mg kg-1) every 24 hours starting 12 hours prior to oral methadone (1 mg kg-1). At least 28 days were implemented as a washout period between fluconazole treatments. Rectal temperature (RT), heart rate (HR), respiratory rate (fR), sedation scores and blood samples were obtained for 24 hours after methadone administration. Plasma drug concentrations were measured with liquid chromatography/mass spectrometry. RESULTS: Significantly higher maximum plasma methadone concentration (mean, 25-46 ng mL-1) occurred in all fluconazole-administered treatments than in methadone alone (1.5 ng mL-1). The mean 12 hour methadone plasma concentration in fluconazole treatments was 11-20 ng mL-1. Significantly decreased RT and variable sedation occurred in all fluconazole treatments, but no changes occurred with methadone alone. There were no differences in HR or fR among treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Fluconazole significantly increases the extent and duration of oral methadone exposure in dogs resulting in significant central opioid effects.

Comparison of predicted intrinsic hepatic clearance of 30 pharmaceuticals in canine and feline liver microsomes.

1. Known cytochrome P450 (CYP) substrates in humans are used in veterinary medicine, with limited knowledge of the similarity or variation in CYP metabolism. Comparison of canine and feline CYP metabolism via liver microsomes report that human CYP probes and inhibitors demonstrate differing rates of intrinsic clearance (CLint). 2. The purpose of this study was to utilize a high-throughput liver microsome substrate depletion assay, combined with microsomal and plasma protein binding to compare the predicted hepatic clearance (CLhep) of thirty therapeutic agents used off-label in canines and felines, using both the well-stirred and parallel tube models. 3. In canine liver microsomes, 3/30 substrates did not have quantifiable CLint, while midazolam and amitriptyline CLint was too rapid for accurate determination. A CLhep was calculated for 29/30 substrates in feline microsomes. Overall, canine CLhep was faster compared to the feline, with fold differences ranging from 2-20-fold. 4. A comparison between the well-stirred and parallel tube model indicates that the parallel tube model reports a slighter higher CLhep in both species. 5. The differences in CYP metabolism between canine and feline highlight the need for additional research into CYP expression and specificity.

Novel Pure αVß3 Integrin Antagonists That Do Not Induce Receptor Extension, Prime the Receptor, or Enhance Angiogenesis at Low Concentrations.

The integrin αVß3 receptor has been implicated in several important diseases, but no antagonists are approved for human therapy. One possible limitation of current small-molecule antagonists is their ability to induce a major conformational change in the receptor that induces it to adopt a high-affinity ligand-binding state. In response, we used structural inferences from a pure peptide antagonist to design the small-molecule pure antagonists TDI-4161 and TDI-3761. Both compounds inhibit αVß3-mediated cell adhesion to αVß3 ligands, but do not induce the conformational change as judged by antibody binding, electron microscopy, X-ray crystallography, and receptor priming studies. Both compounds demonstrated the favorable property of inhibiting bone resorption in vitro, supporting potential value in treating osteoporosis. Neither, however, had the unfavorable property of the αVß3 antagonist cilengitide of paradoxically enhancing aortic sprout angiogenesis at concentrations below its IC50, which correlates with cilengitide's enhancement of tumor growth in vivo.

Discovery and Optimization of Potent and CNS Penetrant M5-Preferring Positive Allosteric Modulators Derived from a Novel, Chiral N-(Indanyl)piperidine Amide Scaffold.

The pharmacology of the M5 muscarinic acetylcholine receptor (mAChR) is the least understood of the five mAChR subtypes due to a historic lack of selective small molecule tools. To address this shortcoming, we have continued the optimization effort around the prototypical M5 positive allosteric modulator (PAM) ML380 and have discovered and optimized a new series of M5 PAMs based on a chiral N-(indanyl)piperidine amide core with robust SAR, human and rat M5 PAM EC50 values <100 nM and rat brain/plasma Kp values of ∼0.40. Interestingly, unlike M1 and M4 PAMs with unprecedented mAChR subtype selectivity, this series of M5 PAMs displayed varying degrees of PAM activity at the other two natively Gq-coupled mAChRs, M1 and M3, yet were inactive at M2 and M4.

A Two-Step Immunocapture LC/MS/MS Assay for Plasma Stability and Payload Migration Assessment of Cysteine-Maleimide-Based Antibody Drug Conjugates.

Plasma stability assessment under physiological temperature is an essential step for developing and optimizing antibody drug conjugate (ADC) molecules, especially those with cleavable linkers. The assessment of plasma stability often requires monitoring multiple analytes using a combination of bioanalytical assays for free payloads, conjugated payloads (or conjugated antibodies), total antibodies, and payloads that have migrated from antibodies to plasma constituent proteins. Bioanalytical assays are needed in early drug discovery to quickly screen diverse ADC candidates of different antibody constructs, linker variants, and antibody anchor sites. To improve the sensitivity and selectivity of LC/MS/MS-based assays for the assessment, immunocapture has been widely used for extracting ADCs and unconjugated antibodies from plasma samples. In this study, a novel two-step immunocapture LC/MS/MS assay was described to allow the quantification of conjugated payloads, total antibodies, and migrated payloads forming adducts with albumin in the plasma samples for stability assessment. A target antigen immobilized on magnetic beads was used to exhaustively extract the ADC and antibody-associated species. The remaining supernatant was then extracted further with anti-albumin beads for recovering the albumin-associated adducts for quantification. The method was optimized for higher efficiency and cost-effectiveness using microwave enhanced papain-based enzymatic cleavage for measuring conjugated payloads of ADCs and lysyl endopeptidase cleavage in the total antibody assay. A maleimide linker-based ADC with a proprietary payload, TAK-001, was used to demonstrate the streamlined workflow of the ADC stability assessment. The method could provide valuable evaluation of the stability of the ADC as well as the quantitative assessment of the albumin adducts formed from the linker-payload migration in mouse and human plasma. Furthermore, the method should be readily adaptable for other ADCs using thiol-maleimide conjugation chemistry.

Optimization of the choline transporter (CHT) inhibitor ML352: Development of VU6001221, an improved in vivo tool compound.

This Letter describes the further lead optimization of the CHT inhibitor probe, ML352 (VU0476201), and the development of VU6001221, an improved in vivo tool. A multi-dimensional optimization effort encountered steep SAR, and ultimately, subtle tuning of the electronics of the central phenyl core provided VU6001221, a CHT inhibitor with comparable potency for choline uptake inhibition as ML352, yet improved PK and CNS penetration. Moreover, VU6001221 enabled evaluation, for the first time, of a CHT inhibitor in a standard preclinical rodent cognition model, novel object recognition (NOR). We observed VU6001221 to elicit a dose-responsive increase in NOR, raising the possibility of agonism of synaptic α7 nicotinic ACh receptors by elevated extracellular choline, that if confirmed would represent a novel molecular strategy to enhance cognition.

Subcutaneous meloxicam suspension pharmacokinetics in mice and dose considerations for postoperative analgesia.

Meloxicam is a cyclooxygenase (COX) inhibitor with a higher selectivity for cyclooxygenase-2 (COX-2) than for cyclooxygenase-1 (COX-1). In the laboratory setting, this nonsteroidal anti-inflammatory drug (NSAID) is commonly selected for analgesia in mice and administered every 24 h. This study characterizes the plasma concentration achieved from a dose of 1.6 mg/kg of meloxicam administered once every 24 h subcutaneously for 72 h in male and female C57BL/6 mice. These values were compared, over time, to reference COX-2 inhibition constants for meloxicam. No significant differences in trough plasma concentrations were noted between genders. The plasma concentrations were below the COX-2 IC50 after 12 h. To maintain a plasma concentration at or above the COX-2 whole blood IC50, the study results suggest an administration frequency of every 12 h when using a dose of 1.6 mg/kg in C57BL/6 mice.

Design of 4-Oxo-1-aryl-1,4-dihydroquinoline-3-carboxamides as Selective Negative Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 2.

Both orthosteric and allosteric antagonists of the group II metabotropic glutamate receptors (mGlus) have been used to establish a link between mGlu2/3 inhibition and a variety of CNS diseases and disorders. Though these tools typically have good selectivity for mGlu2/3 versus the remaining six members of the mGlu family, compounds that are selective for only one of the individual group II mGlus have proved elusive. Herein we report on the discovery of a potent and highly selective mGlu2 negative allosteric modulator 58 (VU6001192) from a series of 4-oxo-1-aryl-1,4-dihydroquinoline-3-carboxamides. The concept for the design of this series centered on morphing a quinoline series recently disclosed in the patent literature into a chemotype previously used for the preparation of muscarinic acetylcholine receptor subtype 1 positive allosteric modulators. Compound 58 exhibits a favorable profile and will be a useful tool for understanding the biological implications of selective inhibition of mGlu2 in the CNS.

Development of Novel, CNS Penetrant Positive Allosteric Modulators for the Metabotropic Glutamate Receptor Subtype 1 (mGlu1), Based on an N-(3-Chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfuran-2-carboxamide Scaffold, That Potentiate Wild Type and Mutant mGlu1 Receptors Found in Schizophrenics.

The therapeutic potential of selective mGlu1 activation is vastly unexplored relative to the other group I mGlu receptor, mGlu5; therefore, our lab has focused considerable effort toward developing mGlu1 positive allosteric modulators (PAMs) suitable as in vivo proof of concept tool compounds. Optimization of a series of mGlu1 PAMs based on an N-(3-chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfuran-2-carboxamide scaffold provided 17e, a potent (mGlu1 EC50 = 31.8 nM) and highly CNS penetrant (brain to plasma ratio (Kp) of 1.02) mGlu1 PAM tool compound, that potentiated not only wild-type human mGlu1 but also mutant mGlu1 receptors derived from deleterious GRM1 mutations found in schizophrenic patients. Moreover, both electrophysiological and in vivo studies indicate the mGlu1 ago-PAMs/PAMs do not possess the same epileptiform adverse effect liability as mGlu5 ago-PAMs/PAMs and maintain temporal activity suggesting a broader therapeutic window.

A Screen of Approved Drugs Identifies the Androgen Receptor Antagonist Flutamide and Its Pharmacologically Active Metabolite 2-Hydroxy-Flutamide as Heterotropic Activators of Cytochrome P450 3A In Vitro and In Vivo.

Once thought to be an artifact of microsomal systems, atypical kinetics with cytochrome P450 (CYP) enzymes have been extensively investigated in vitro and found to be substrate and species dependent. Building upon increasing reports of heterotropic CYP activation and inhibition in clinical settings, we screened a compound library of clinically approved drugs and various probe compounds to identify the frequency of heterotropism observed with different drug classes and the associated CYP enzymes thereof (1A2, 2C9, 2D6, and 3A4/5). Results of this screen revealed that the prescribed androgen receptor antagonist flutamide activated the intrinsic midazolam hydroxylase activity of CYP3A in human hepatic microsomes (66%), rat and human hepatocytes (36 and 160%, respectively), and in vivo in male Sprague-Dawley rats (>2-fold, combined area under the curve of primary rat in vivo midazolam metabolites). In addition, a screen of the pharmacologically active metabolite 2-hydroxy-flutamide revealed that this principle metabolite increased CYP3A metabolism of midazolam in human microsomes (30%) and hepatocytes (110%). Importantly, both flutamide and 2-hydroxy-flutamide demonstrated a pronounced increase in the CYP3A-mediated metabolism of commonly paired medications, nifedipine (antihypertensive) and amiodarone (antiarrhythmic), in multispecies hepatocytes (100% over baseline). These data serve to highlight the importance of an appropriate substrate and in vitro system selection in the pharmacokinetic modeling of atypical enzyme kinetics. In addition, the results of our investigation have illuminated a previously undiscovered class of heterotropic CYP3A activators and have demonstrated the importance of selecting commonly paired therapeutics in the in vitro and in vivo modeling of projected clinical outcomes.

Progress towards small molecule menin-mixed lineage leukemia (MLL) interaction inhibitors with in vivo utility.

A series of substituted hydroxymethyl piperidine small molecule inhibitors of the protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) are described. Initial members of the series showed good inhibitory disruption of the menin-MLL1 interaction but demonstrated poor physicochemical and DMPK properties. Utilizing a structure-guided and iterative optimization approach key substituents were optimized leading to inhibitors with cell-based activity, improved in vitro DMPK parameters, and improved half-lives in rodent PK studies leading to MLPCN probe ML399. Ancillary off-target activity remains a parameter for further optimization.

VFV as a New Effective CYP51 Structure-Derived Drug Candidate for Chagas Disease and Visceral Leishmaniasis.

Sterol 14α-demethylases (CYP51) are the enzymes essential for sterol biosynthesis. They serve as clinical targets for antifungal azoles and are considered as targets for treatment of human Trypanosomatidae infections. Recently, we have shown that VNI, a potent and selective inhibitor of trypanosomal CYP51 that we identified and structurally characterized in complex with the enzyme, can cure the acute and chronic forms of Chagas disease. The purpose of this work was to apply the CYP51 structure/function for further development of the VNI scaffold. As anticipated, VFV (R)-N-(1-(3,4'-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide, the derivative designed to fill the deepest portion of the CYP51 substrate-binding cavity, reveals a broader antiprotozoan spectrum of action. It has stronger antiparasitic activity in cellular experiments, cures the experimental Chagas disease with 100% efficacy, and suppresses visceral leishmaniasis by 89% (vs 60% for VNI). Oral bioavailability, low off-target activity, favorable pharmacokinetics and tissue distribution characterize VFV as a promising new drug candidate.

Synthesis and structure-activity relationships of a series of 4-methoxy-3-(piperidin-4-yl)oxy benzamides as novel inhibitors of the presynaptic choline transporter.

The synthesis and SAR of 4-methoxy-3-(piperidin-4-yl) benzamides identified after a high-throughput screen of the MLPCN library is reported. SAR was explored around the 3-piperidine substituent as well as the amide functionality of the reported compounds. Starting from the initial lead compounds, 1-7, iterative medicinal chemistry efforts led to the identification of ML352 (10m). ML352 represents a potent and selective inhibitor of CHT based on a drug-like scaffold.

Characterization of bovine phenol sulfotransferases: evidence of a major role for SULT1B1 in the liver.

1. Cattle are an important component of the human food chain. Drugs used either legally or illegally in cattle may therefore enter the food chain and it is thus important to understand pathways of drug metabolism in this species, including sulfation catalyzed by the sulfotransferases (SULTs). 2. In this study, we have analyzed the sulfation of 4-nitrophenol and other compounds in male and female bovine liver and characterized recombinant bovine SULT isoforms 1A1 and 1B1 expressed in Escherichia coli. 3. We found that, in contrast to most other mammalian species, the major phenol sulfotransferase SULT1A1 is not expressed in bovine liver. Rather SULT1B1 seems to be a major form in both male and female bovine liver. 4. We also identified kinetic differences between bovine and human SULT1A1 and, using the human SULT1A1 crystal structure, identified two amino acid positions in the active site of bovine SULT1A1 (Ile89Val and Phe247Val) that may be responsible for these differences.

Discovery of a highly selective PLD2 inhibitor (ML395): a new probe with improved physiochemical properties and broad-spectrum antiviral activity against influenza strains.

Further chemical optimization of the halopemide-derived family of dual phospholipase D1/2 (PLD1/2) inhibitors afforded ML395 (VU0468809), a potent, >80-fold PLD2 selective allosteric inhibitor (cellular PLD1, IC50 >30,000 nM; cellular PLD2, IC50 =360 nM). Moreover, ML395 possesses an attractive in vitro DMPK profile, improved physiochemical properties, ancillary pharmacology (Eurofins Panel) cleaner than any other reported PLD inhibitor, and has been found to possess interesting activity as an antiviral agent in cellular assays against a range of influenza strains (H1, H3, H5 and H7).

Substituted indoles as selective protease activated receptor 4 (PAR-4) antagonists: Discovery and SAR of ML354.

Herein we report the discovery and SAR of an indole-based protease activated receptor-4 (PAR-4) antagonist scaffold derived from a similarity search of the Vanderbilt HTS collection, leading to MLPCN probe ML354 (VU0099704). Using a novel PAC-1 fluorescent αIIbß3 activation assay this probe molecule antagonist was found to have an IC50 of 140nM for PAR-4 with 71-fold selectivity versus PAR-1 (PAR-1IC50=10µM).

Discovery and Characterization of ML398, a Potent and Selective Antagonist of the D4 Receptor with in Vivo Activity.

Herein, we report the structure-activity relationship of a chiral morpholine-based scaffold, which led to the identification of a potent and selective dopamine 4 (D4) receptor antagonist. The 4-chlorobenzyl moiety was identified, and the compound was designated an MLPCN probe molecule, ML398. ML398 is potent against the D4 receptor with IC50 = 130 nM and K i = 36 nM and shows no activity against the other dopamine receptors tested (>20 µM against D1, D2S, D2L, D3, and D5). Further in vivo studies showed that ML398 reversed cocaine-induced hyperlocomotion at 10 mg/kg.