Investigation of the absolute bioavailability, mass balance, metabolism, and excretion of the cholesterol 24-hydroxylase inhibitor soticlestat in healthy volunteers.

AIMS: Our aim was to determine the absolute bioavailability, mass balance, metabolism and excretion of soticlestat (TAK-935). METHODS: An open-label, two-period, single-site, phase 1 study was conducted in six healthy men. In Period 1, a single 300 mg dose of soticlestat was administered orally, followed by a 15-min intravenous infusion of [14 C]soticlestat 50 µg (~1 µCi) 10 min later. In Period 2, a single 300 mg dose (~100 µCi) of [14 C]soticlestat in solution was administered orally. Samples were collected, analysed for radioactivity or unchanged soticlestat, and profiled for metabolites. RESULTS: In Period 1, soticlestat had an absolute bioavailability of 12.6% (90% confidence interval, 7.81-20.23%). In Period 2, there was near-complete recovery of total radioactivity (TRA) following a 300 mg dose of [14 C]soticlestat: urine, 94.8% (standard deviation [SD], 1.35%); faeces, 2.7% (SD, 1.67%). Of TRA, 0.1% (SD, 0.09%) and 0.6% (SD, 0.21%) were recovered as soticlestat and metabolite M-I in urine, respectively. In plasma, soticlestat and M-I reached geometric mean maximum observed concentrations of 1352 ng/mL (geometric percent coefficient of variation [gCV%], 61.3) and 253.2 ng/mL (gCV%, 44.1) after 25 min and declined with mean terminal half-lives (SD) of 5.7 (2.90) and 2.0 (0.15) h, respectively. Soticlestat represented 4.9% of TRA in plasma. Soticlestat was rapidly eliminated primarily via O-glucuronidation to metabolite M3, which was the dominant species in plasma (92.6%) and urine (86%). CONCLUSIONS: This study indicates that soticlestat and its metabolites are rapidly cleared and eliminated, lowering the risk of dose accumulation from repeated dosing and supporting further investigation of soticlestat.

Pharmacokinetics, mass balance, metabolism, and excretion of the liver-targeted acetyl-CoA carboxylase inhibitor PF-05221304 (clesacostat) in humans.

The disposition of the hepatoselective ACC inhibitor PF-05221304 (Clesacostat) was studied after a single 50-mg oral dose of [14C]-PF-05221304 to healthy human subjects.Mass balance was achieved with 89.9% of the administered dose recovered in urine and faeces, over the 11-day study period. The total administered radioactivity excreted in faeces and urine was 81.7 and 8.2%, respectively. Unchanged PF-05221304 accounted for 35.6% of the radioactive dose in faeces, suggesting ∼64% of the administered dose was absorbed.PF-05221304 was principally metabolised via oxidative and reductive pathways involving: (a) N-dealkylation, (b) isopropyl group monohydroxylation to yield enantiomeric metabolites (M2a and M2b), (c) hydroxylation on the 3-azaspiro[5.5]undecan-8-one moiety to metabolites M5 and 519c, and (d) carbonyl group reduction to enantiomeric alcohol metabolites M3, and M4. Secondary metabolites (521a, 521b, and 533), derived from a combination of oxidation and reduction of the primary metabolites accounted for ∼14.8% of the dose. In plasma, unchanged PF-05221304 represented 96.1% circulating radioactivity. Metabolites M1, M2b, and M2a represented 1.94, 1.76, and 0.18% of circulating radioactivity, respectively.Overall, these data suggest that PF-05221304 is well absorbed in humans and eliminated largely via phase I metabolism.

Simplifying the Execution of HepatoPac MetID Experiments: Metabolite Profile and Intrinsic Clearance Comparisons.

The HepatoPac micropatterned coculture (MPCC) hepatocyte system has been shown to be an effective tool to investigate the qualitative human and preclinical species' metabolite profiles of new drug candidates. However, additional improvements to the overall study conditions and execution, layout, and human-donor count could be made. To that end, we have evaluated several ways to increase the amount of data one can generate per MPCC plate and how to more efficiently execute a MPCC study for the purpose of metabolite generation. Herein, we compare a set of compounds using single- and 10-donor pooled human MPCC hepatocytes. Intrinsic clearance and mean metabolic activities assessed by diverse enzyme markers were comparable between the single- and 10-donor pool. We have confirmed that the generated metabolite profiles were indistinguishable between the single- and 10-donor pool and also that rat MPCC can be performed at 400 µl media volume, which greatly simplifies study execution. Additional tips for successful study execution are also described. SIGNIFICANCE STATEMENT: When using the HepatoPac micropatterned coculture (MPCC) system, sometimes simple experimental condition variables or problematic plate designs can hamper productive study execution. We evaluated conditions to increase the amount of data one can generate per MPCC plate and, perhaps more importantly, execute that study more efficiently with less likelihood of error. We describe some of our key learnings, provide an examination of enzyme activity levels and clearance values, and provide some recommendations to simplify the execution of a HepatoPac experiment.

Natural Product Bis-Intercalator Depsipeptides as a New Class of Payloads for Antibody-Drug Conjugates.

A potent class of DNA-damaging agents, natural product bis-intercalator depsipeptides (NPBIDs), was evaluated as ultrapotent payloads for use in antibody-drug conjugates (ADCs). Detailed investigation of potency (both in cells and via biophysical characterization of DNA binding), chemical tractability, and in vitro and in vivo stability of the compounds in this class eliminated a number of potential candidates, greatly reducing the complexity and resources required for conjugate preparation and evaluation. This effort yielded a potent, stable, and efficacious ADC, PF-06888667, consisting of the bis-intercalator, SW-163D, conjugated via an N-acetyl-lysine-valine-citrulline- p-aminobenzyl alcohol- N, N-dimethylethylenediamine (AcLysValCit-PABC-DMAE) linker to an engineered variant of the anti-Her2 mAb, trastuzumab, catalyzed by transglutaminase.

Synthesis and Antimicrobial Evaluation of Amixicile-Based Inhibitors of the Pyruvate-Ferredoxin Oxidoreductases of Anaerobic Bacteria and Epsilonproteobacteria.

Amixicile is a promising derivative of nitazoxanide (an antiparasitic therapeutic) developed to treat systemic infections caused by anaerobic bacteria, anaerobic parasites, and members of the Epsilonproteobacteria (Campylobacter and Helicobacter). Amixicile selectively inhibits pyruvate-ferredoxin oxidoreductase (PFOR) and related enzymes by inhibiting the function of the vitamin B1 cofactor (thiamine pyrophosphate) by a novel mechanism. Here, we interrogate the amixicile scaffold, guided by docking simulations, direct PFOR inhibition assays, and MIC tests against Clostridium difficile, Campylobacter jejuni, and Helicobacter pylori Docking simulations revealed that the nitro group present in nitazoxanide interacts with the protonated N4'-aminopyrimidine of thiamine pyrophosphate (TPP). The ortho-propylamine on the benzene ring formed an electrostatic interaction with an aspartic acid moiety (B456) of PFOR that correlated with improved PFOR-inhibitory activity and potency by MIC tests. Aryl substitution with electron-withdrawing groups and substitutions of the propylamine with other alkyl amines or nitrogen-containing heterocycles both improved PFOR inhibition and, in many cases, biological activity against C. difficile Docking simulation results correlate well with mechanistic enzymology and nuclear magnetic resonance (NMR) studies that show members of this class of antimicrobials to be specific inhibitors of vitamin B1 function by proton abstraction, which is both novel and likely to limit mutation-based drug resistance.

Determination of Antibody-Drug Conjugate Released Payload Species Using Directed in Vitro Assays and Mass Spectrometric Interrogation.

Antibody-drug conjugates (ADC) are currently an active area of research, focused primarily on oncology therapeutics, but also to a limited extent on other areas such as infectious disease. The success of this type of targeted drug delivery is dependent upon many factors, one of which is the performance of the linker in releasing an active drug moiety under the appropriate conditions. As a tool in the development of linker/payload chemistry, we have developed an in vitro method for the identification of payload species released from ADCs in the presence of lysosomal enzymes. This method utilizes commercially available human liver S9 fraction as the source of these enzymes, and this has certain advantages over lysosomal fractions or purified enzymes. This article describes the characterization and performance of this assay with multiple ADCs composed of known and novel linkers and payloads. Additionally, we report the observation of incomplete degradation of mAb protein chains by lysosomal enzymes in vitro, believed to be the first report of this phenomenon involving an ADC therapeutic.

Application of a Micropatterned Cocultured Hepatocyte System To Predict Preclinical and Human-Specific Drug Metabolism.

Laboratory animal models are the industry standard for preclinical risk assessment of drug candidates. Thus, it is important that these species possess profiles of drug metabolites that are similar to those anticipated in human, since metabolites also could be responsible for biologic activities or unanticipated toxicity. Under most circumstances, preclinical species reflect human in vivo metabolites well; however, there have been several notable exceptions, and understanding and predicting these exceptions with an in vitro system would be very useful. Human micropatterned cocultured (MPCC) hepatocytes have been shown to recapitulate human in vivo qualitative metabolic profiles, but the same demonstration has not been performed yet for laboratory animal species. In this study, we investigated several compounds that are known to produce human-unique metabolites through CYP2C9, UGT1A4, aldehyde oxidase (AO), or N-acetyltransferase that were poorly covered or not detected at all in the selected preclinical species. To perform our investigation we used 24-well MPCC hepatocyte plates having three individual human donors and a single donor each of monkey, dog, and rat to study drug metabolism at four time points per species. Through the use of the multispecies MPCC hepatocyte system, the metabolite profiles of the selected compounds in human donors effectively captured the qualitative in vivo metabolite profile with respect to the human metabolite of interest. Human-unique metabolites that were not detected in vivo in certain preclinical species (normally dog and rat) were also not generated in the corresponding species in vitro, confirming that the MPCC hepatocytes can provide an assessment of preclinical species metabolism. From these results, we conclude that multispecies MPCC hepatocyte plates could be used as an effective in vitro tool for preclinical understanding of species metabolism relative to humans and aid in the choice of appropriate preclinical models.

Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging.

Bioorthogonal reactions, including the strain-promoted azide-alkyne cycloaddition (SPAAC) and inverse electron demand Diels-Alder (iEDDA) reactions, have become increasingly popular for live-cell imaging applications. However, the stability and reactivity of reagents has never been systematically explored in the context of a living cell. Here we report a universal, organelle-targetable system based on HaloTag protein technology for directly comparing bioorthogonal reagent reactivity, specificity, and stability using clickable HaloTag ligands in various subcellular compartments. This system enabled a detailed comparison of the bioorthogonal reactions in live cells and informed the selection of optimal reagents and conditions for live-cell imaging studies. We found that the reaction of sTCO with monosubstituted tetrazines is the fastest reaction in cells; however, both reagents have stability issues. To address this, we introduced a new variant of sTCO, Ag-sTCO, which has much improved stability and can be used directly in cells for rapid bioorthogonal reactions with tetrazines. Utilization of Ag complexes of conformationally strained trans-cyclooctenes should greatly expand their usefulness especially when paired with less reactive, more stable tetrazines.

A tag-free collisionally induced fragmentation approach to detect drug-adducted proteins by mass spectrometry.

RATIONALE: The covalent modification of proteins by toxicants, new chemical entities or drug molecules, either by metabolic activation or the presence of inherently reactive functional groups, is commonly implicated in organ toxicity and idiosyncratic reactions. In efforts to better prosecute protein modifications, we investigated a tag-free technique capable of detecting protein-small molecule adducts based solely on the collision-induced dissociation (CID) of the protein-small molecule complex. Detection of proteins using unique CID small molecule (SM) product ions would mitigate common issues associated with tagging technologies (e.g., altered reactivity/affinity of the protein-SM complex). METHODS: A Waters SYNAPT G2 mass spectrometer (MS) was operated in MS(e) mode with appropriate collision energy conditions during the MS(2) acquisition for fragmentation of protein-small molecule adducts to generate characteristic small molecule product ions. RESULTS: Ibrutinib, an acrylamide-containing small molecule drug, was shown to form adducts with rat serum albumin in ex vivo experiments and these adducts were detected by relying solely on the CID product ions generated from ibrutinib. Additionally, ibrutinib produced three CID product ions, one of which was a selective protein-ibrutinib fragment ion not produced by the compound alone. CONCLUSIONS: Herein we describe a tag-free mass spectral detection technique for protein-small molecule conjugates that relies on the unique product ion fragmentation profile of the small molecule. This technique allows the detection of macromolecular ions containing the adducted small molecule from complex protein matrices through mass range selection for the unique product ions in the CID spectra.

γ-Secretase modulator (GSM) photoaffinity probes reveal distinct allosteric binding sites on presenilin.

γ-Secretase is an intramembrane aspartyl protease that cleaves the amyloid precursor protein to produce neurotoxic ß-amyloid peptides (i.e. Aß42) that have been implicated in the pathogenesis of Alzheimer disease. Small molecule γ-secretase modulators (GSMs) have emerged as potential disease-modifying treatments for Alzheimer disease because they reduce the formation of Aß42 while not blocking the processing of γ-secretase substrates. We developed clickable GSM photoaffinity probes with the goal of identifying the target of various classes of GSMs and to better understand their mechanism of action. Here, we demonstrate that the photoaffinity probe E2012-BPyne specifically labels the N-terminal fragment of presenilin-1 (PS1-NTF) in cell membranes as well as in live cells and primary neuronal cultures. The labeling is competed in the presence of the parent imidazole GSM E2012, but not with acid GSM-1, allosteric GSI BMS-708163, or substrate docking site peptide inhibitor pep11, providing evidence that these compounds have distinct binding sites. Surprisingly, we found that the cross-linking of E2012-BPyne to PS1-NTF is significantly enhanced in the presence of the active site-directed GSI L-685,458 (L458). In contrast, L458 does not affect the labeling of the acid GSM photoprobe GSM-5. We also observed that E2012-BPyne specifically labels PS1-NTF (active γ-secretase) but not full-length PS1 (inactive γ-secretase) in ANP.24 cells. Taken together, our results support the hypothesis that multiple binding sites within the γ-secretase complex exist, each of which may contribute to different modes of modulatory action. Furthermore, the enhancement of PS1-NTF labeling by E2012-BPyne in the presence of L458 suggests a degree of cooperativity between the active site of γ-secretase and the modulatory binding site of certain GSMs.

Generation of major human excretory and circulating drug metabolites using a hepatocyte relay method.

The prediction of human drug metabolites using in vitro experiments containing human-derived reagents is an important approach in modern drug research; however, this can be challenging for drugs that are slowly metabolized. In this report, we describe the use of a recently developed human hepatocyte relay method for the purpose of predicting human drug metabolite profiles. Five compounds for which in vivo human metabolism data were available were selected for the investigation of this method, and the results were compared with data gathered in hepatocyte suspensions as well as previous data from a micropatterned hepatocyte coculture method. The hepatocyte relay method demonstrated an improved performance (generation of 75% of human in vivo metabolites) for those drugs for which previous methods showed a relatively low rate of success (50% of human in vivo metabolites). Metabolites included those arising from both oxidative and conjugative reactions and metabolites that required sequential reactions. Two 4-hour relays were shown to adequately generate metabolites, and no further benefit was derived from more relays. Overall, it can be concluded that the hepatocyte relay assay method can be successfully used in the generation of relevant human metabolites, even for challenging drugs.

An isotopically tagged azobenzene-based cleavable linker for quantitative proteomics.

Putting a number on it: Cleavable linkers are widely utilized in proteomics applications. In particular, the azobenzene-based linker cleaves under mild conditions that are mass-spectrometry-compatible. Here, we adapt this linker for quantitative proteomic applications by incorporating an isotopic label. These light- and heavy-tagged linkers enable the identification and quantitation of labeled peptides from multiple proteomes.

The Importance of Tracking "Missing" Metabolites: How and Why?

Technologies currently employed to find and identify drug metabolites in complex biological matrices generally yield results that offer a comprehensive picture of the drug metabolite profile. However, drug metabolites can be missed or are captured only late in the drug development process. This could be due to a variety of factors, such as metabolism that results in partial loss of the molecule, covalent bonding to macromolecules, the drug being metabolized in specific human tissues, or poor ionization in a mass spectrometer. These scenarios often draw a great deal of attention from chemistry, safety assessment, and pharmacology. This review will summarize scenarios of missing metabolites, why they are missing, and associated uncovering strategies from deeper investigations. Uncovering previously missed metabolites can have ramifications in drug development with toxicological and pharmacological consequences, and knowledge of these can help in the design of new drugs.

BMS-708,163 targets presenilin and lacks notch-sparing activity.

The "Notch-sparing" γ-secretase inhibitor (GSI) BMS-708,163 (Avagacestat) is currently in phase II clinical trials for Alzheimer's disease. Unlike previously failed GSIs, BMS-708,163 is considered to be a promising drug candidate because of its reported Notch-sparing activity for the inhibition of Aß production over Notch cleavage. We now report that BMS-708,163 binds directly to the presenilin-1 N-terminal fragment and that binding can be challenged by other pan-GSIs, but not by γ-secretase modulators. Furthermore, BMS-708,163 blocks the binding of four different active site-directed GSI photoaffinity probes. We therefore report that this compound acts as a nonselective γ-secretase inhibitor.

Amixicile, a novel inhibitor of pyruvate: ferredoxin oxidoreductase, shows efficacy against Clostridium difficile in a mouse infection model.

Clostridium difficile infection (CDI) is a serious diarrheal disease that often develops following prior antibiotic usage. One of the major problems with current therapies (oral vancomycin and metronidazole) is the high rate of recurrence. Nitazoxanide (NTZ), an inhibitor of pyruvate:ferredoxin oxidoreductase (PFOR) in anaerobic bacteria, parasites, Helicobacter pylori, and Campylobacter jejuni, also shows clinical efficacy against CDI. From a library of ∼250 analogues of NTZ, we identified leads with increased potency for PFOR. MIC screens indicated in vitro activity in the 0.05- to 2-µg/ml range against C. difficile. To improve solubility, we replaced the 2-acetoxy group with propylamine, producing amixicile, a soluble (10 mg/ml), nontoxic (cell-based assay) lead that produced no adverse effects in mice by oral or intraperitoneal (i.p.) routes at 200 mg/kg of body weight/day. In initial efficacy testing in mice treated (20 mg/kg/day, 5 days each) 1 day after receiving a lethal inoculum of C. difficile, amixicile showed slightly less protection than did vancomycin by day 5. However, in an optimized CDI model, amixicile showed equivalence to vancomycin and fidaxomicin at day 5 and there was significantly greater survival produced by amixicile than by the other drugs on day 12. All three drugs were comparable by measures of weight loss/gain and severity of disease. Recurrence of CDI was common for mice treated with vancomycin or fidaxomicin but not for mice receiving amixicile or NTZ. These results suggest that gut repopulation with beneficial (non-PFOR) bacteria, considered essential for protection against CDI, rebounds much sooner with amixicile therapy than with vancomycin or fidaxomicin. If the mouse model is indeed predictive of human CDI disease, then amixicile, a novel PFOR inhibitor, appears to be a very promising new candidate for treatment of CDI.

Development of clickable active site-directed photoaffinity probes for γ-secretase.

We have developed clickable active site-directed photoaffinity probes for γ-secretase which incorporate a photoreactive benzophenone group and an alkyne handle for subsequent click chemistry mediated conjugation with azide-linked reporter tags for visualization (e.g., TAMRA-azide) or enrichment (e.g., biotin-azide) of labeled proteins. Specifically, we synthesized clickable analogs of L646 (2) and L505 (3) and validated specific labeling to presenilin-1N-terminal fragment (PS1-NTF), the active site aspartyl protease component within the γ-secretase complex. Additionally, we were able to identify signal peptide peptidase (SPP) by Western blot analysis. Furthermore, we analyzed the photo-labeled proteins in an unbiased fashion by click chemistry with TAMRA-azide followed by in-gel fluorescence detection. This approach expands the utility of γ-secretase inhibitor (GSI) photoaffinity probes in that labeled proteins can be tagged with any number of azide-linked reporters groups using a single clickable photoaffinity probe for target pull down and/or fluorescent imaging applications.

Nitazoxanide inhibits biofilm formation by Staphylococcus epidermidis by blocking accumulation on surfaces.

Coagulase-negative species of Staphylococcus are often associated with opportunistic hospital-acquired infections that arise from the colonization of indwelling catheters. Here we show that the antiparasitic drug nitazoxanide (NTZ) and its active metabolite, tizoxanide (TIZ), are inhibitory to the growth of Staphylococcus epidermidis and other staphylococci, including methicillin-resistant Staphylococcus aureus strains, under aerobic and microaerobic conditions (MICs, 8 to 16 microg/ml). At sub-MIC levels, NTZ and TIZ also inhibited biofilm production under static conditions by strains of S. epidermidis and Staphylococcus haemolyticus with a 50% inhibitory concentration of approximately 2.5 microg/ml (8 microM). The 5-nitro group was required for biological activity, and a hydrophilic derivative of NTZ (AMIX) also inhibited biofilm formation. NTZ did not disperse the existing biofilm but did block further accumulation. Sub-MICs of NTZ had no effect on primary attachment to surfaces at either 4 or 37 degrees C. The inhibitory action of NTZ and TIZ, but not vancomycin, on biofilm production could be reversed by the addition of zinc salts (2.5 to 40 microM) but not other metals, suggesting that NTZ might target the zinc-dependent accumulation-associated protein (Aap) that mediates accumulation on surfaces. However, neither NTZ nor TIZ formed chelation complexes with zinc salts, based on spectrophotometric and nuclear magnetic resonance analyses, and addition of excess zinc to NTZ-grown bacteria (apo-Aap) did not restore the accumulation phenotype. Our studies suggest that sub-MIC levels of NTZ may affect the assembly or function of cell structures associated with the biofilm phenotype.

Biological activity of modified and exchanged 2-amino-5-nitrothiazole amide analogues of nitazoxanide.

Head group analogues of the antibacterial and antiparasitic drug nitazoxanide (NTZ) are presented. A library of 39 analogues was synthesized and assayed for their ability to suppress growth of Helicobacter pylori, Campylobacter jejuni, Clostridium difficile and inhibit NTZ target pyruvate:ferredoxin oxidoreductase (PFOR). Two head groups assayed recapitulated NTZ activity and possessed improved activity over their 2-amino-5-nitrothiazole counterparts, demonstrating that head group modification is a viable route for the synthesis of NTZ-related antibacterial analogues.

Antibiofilm activity of a diverse oroidin library generated through reductive acylation.

A diverse 20-compound library of analogues based on the marine alkaloid oroidin were synthesized via a reductive acylation strategy. The final target was then assayed for inhibition and dispersion activity against common proteobacteria known to form biofilms. This methodology represents a significant improvement over the generality of known methods to acylate substrates containing 2-aminoimidazoles and has the potential to have broad application to the synthesis of more advanced oroidin family members and their corresponding analogues.

Evaluation of dihydrooroidin as an antifouling additive in marine paint.

Methods used to deter biofouling of underwater structures and marine vessels present a serious environmental issue and are both problematic and costly for government and commercial marine vessels worldwide. Current antifouling methods include compounds that are toxic to aquatic wildlife and marine ecosystems. Dihydrooroidin (DHO) was shown to completely inhibit Halomonas pacifica biofilms at 100 µM in a static biofilm inhibition assay giving precedence for the inhibition of other marine-biofilm-forming organisms. Herein we present DHO as an effective paint-based, non-cytotoxic, antifouling agent against marine biofouling processes in a marine mesocosm.