Transporter-Enzyme Interplay in the Pharmacokinetics of PF-06835919, A First-in-class Ketohexokinase Inhibitor for Metabolic Disorders and Non-alcoholic Fatty Liver Disease.

Excess dietary fructose consumption promotes metabolic dysfunction thereby increasing the risk of obesity, type 2 diabetes, non-alcoholic steatohepatitis (NASH), and related comorbidities. PF-06835919, a first-in-class ketohexokinase (KHK) inhibitor, showed reversal of such metabolic disorders in preclinical models and clinical studies, and is under clinical development for the potential treatment of NASH. In this study, we evaluated the transport and metabolic pathways of PF-06835919 disposition and assessed pharmacokinetics in preclinical models. PF-06835919 showed active uptake in cultured primary human hepatocytes, and substrate activity to organic anion transporter (OAT)2 and organic anion transporting-polypeptide (OATP)1B1 in transfected cells. "SLC-phenotyping" studies in human hepatocytes suggested contribution of passive uptake, OAT2- and OATP1B-mediated transport to the overall uptake to be about 15%, 60% and 25%, respectively. PF-06835919 showed low intrinsic metabolic clearance in vitro, and was found to be metabolized via both oxidative pathways (58%) and acyl glucuronidation (42%) by CYP3A, CYP2C8, CYP2C9 and UGT2B7. Following intravenous dosing, PF-06835919 showed low clearance (0.4-1.3 mL/min/kg) and volume of distribution (0.17-0.38 L/kg) in rat, dog and monkey. Human oral pharmacokinetics are predicted within 20% error when considering transporter-enzyme interplay in a PBPK model. Finally, unbound liver-to-plasma ratio (Kpuu) measured in vitro using rat, NHP and human hepatocytes was found to be approximately 4, 25 and 10, respectively. Similarly, liver Kpuu in rat and monkey following intravenous dosing of PF-06835919 was found to be 2.5 and 15, respectively, and notably higher than the muscle and brain Kpuu, consistent with the active uptake mechanisms observed in vitro. Significance Statement This work characterizes the transport/metabolic pathways in the hepatic disposition of PF-06835919, a first-in-class KHK inhibitor for the treatment of metabolic disorders and NASH. Phenotyping studies using transfected systems, human hepatocytes and liver microsomes signifies the role of OAT2 and OATP1B1 in the hepatic uptake and multiple enzymes in the metabolism of PF-06835919. Data presented suggest hepatic transporter-enzyme interplay in determining its systemic concentrations and potential enrichment in liver, a target site for KHK inhibition.

LC/MS Methods for Studying Lysosomal ADC Catabolism.

A critical component of antibody-drug conjugate (ADC) development is identification or verification of the active released entity upon cellular uptake and exposure to lysosomal enzymes. Coupled with LC/MS, commercial human lysosomal preparations can be used as an in vitro tool to explore the release characteristics of new ADCs, and gain information on potential metabolic or chemical liabilities of new payload structures. A general method for approaching this is described for cathepsin B-cleavable as well as non-cleavable ADCs, and opportunities for tailoring the method to specific cases are indicated.

Development of Fluorophore-Labeled Thailanstatin Antibody-Drug Conjugates for Cellular Trafficking Studies.

As the antibody-drug conjugate (ADC) field grows increasingly important for cancer treatment, it is vital for researchers to establish a firm understanding of how ADCs function at the molecular level. To gain insight into ADC uptake, trafficking, and catabolism-processes that are critical to ADC efficacy and toxicity-imaging studies have been performed with fluorophore-labeled conjugates. However, such labels may alter the properties and behavior of the ADC under investigation. As an alternative approach, we present here the development of a "clickable" ADC bearing an azide-functionalized linker-payload (LP) poised for "click" reaction with alkyne fluorophores; the azide group represents a significantly smaller structural perturbation to the LP than most fluorophores. Notably, the clickable ADC shows excellent potency in target-expressing cells, whereas the fluorophore-labeled product ADC suffers from a significant loss of activity, underscoring the impact of the label itself on the payload. Live-cell confocal microscopy reveals robust uptake of the clickable ADC, which reacts selectively in situ with a derivatized fluorescent label. Time-course trafficking studies show greater and more rapid net internalization of the ADCs than the parent antibody. More generally, the application of chemical biology tools to the study of ADCs should improve our understanding of how ADCs are processed in biological systems.

Optimization of Tubulysin Antibody-Drug Conjugates: A Case Study in Addressing ADC Metabolism.

As part of our efforts to develop new classes of tubulin inhibitor payloads for antibody-drug conjugate (ADC) programs, we developed a tubulysin ADC that demonstrated excellent in vitro activity but suffered from rapid metabolism of a critical acetate ester. A two-pronged strategy was employed to address this metabolism. First, the hydrolytically labile ester was replaced by a carbamate functional group resulting in a more stable ADC that retained potency in cellular assays. Second, site-specific conjugation was employed in order to design ADCs with reduced metabolic liabilities. Using the later approach, we were able to identify a conjugate at the 334C position of the heavy chain that resulted in an ADC with considerably reduced metabolism and improved efficacy. The examples discussed herein provide one of the clearest demonstrations to-date that site of conjugation can play a critical role in addressing metabolic and PK liabilities of an ADC. Moreover, a clear correlation was identified between the hydrophobicity of an ADC and its susceptibility to metabolic enzymes. Importantly, this study demonstrates that traditional medicinal chemistry strategies can be effectively applied to ADC programs.

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.

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.

Deconstruction of activity-dependent covalent modification of heme in human neutrophil myeloperoxidase by multistage mass spectrometry (MS(4)).

Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS(4)) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.

Dirlotapide as a model substrate to refine structure-based drug design strategies on CYP3A4-catalyzed metabolism.

Multiple crystal structures of CYP3A4 bound with various substrates or inhibitors have been used as templates for docking of new chemical entities to predict sites of metabolism and molecular interactions for drug design. Herein, modeling studies with dirlotapide, a CYP3A4 substrate, indicated that a substantial conformational change of CYP3A4 was necessary to accommodate it within the active site cavity, which is in good agreement with a new published CYP3A4 ritonavir co-crystal structure. Thus, the importance of considering the substrate-induced conformational change in CYP3A4, thermochemical properties of reaction centers, and essential in vitro experimental data support were analyzed for the refinement of computational models.

Discovery of microsomal triglyceride transfer protein (MTP) inhibitors with potential for decreased active metabolite load compared to dirlotapide.

Analogues related to dirlotapide (1), a gut-selective inhibitor of microsomal triglyceride transfer protein (MTP) were prepared with the goal of further reducing the potential for unwanted liver MTP inhibition and associated side-effects. Compounds were designed to decrease active metabolite load: reducing MTP activity of likely human metabolites and increasing metabolite clearance to reduce exposure. Introduction of 4'-alkyl and 4'-alkoxy substituents afforded compounds exhibiting improved therapeutic index in rats with respect to liver triglyceride accumulation and enzyme elevation. Likely human metabolites of select compounds were prepared and characterized for their potential to inhibit MTP in vivo. Based on preclinical efficacy and safety data and its potential for producing short-lived, weakly active metabolites, compound 13 (PF-02575799) advanced into phase 1 clinical studies.

The use of 18O-exchange and base-catalyzed N-dealkylation with liquid chromatography/tandem mass spectrometry to identify carbinolamide metabolites.

Oxidation of N-alkyl-substituted amides is a common transformation observed in metabolism studies of drugs and other chemicals. Metabolism at the alpha carbon atom can produce stable carbinolamide compounds, which may be abundant enough to require complete confidence in structural assignments. In a drug discovery setting, rapid structural elucidation of test compounds is critical to inform the compound selection process. Traditional approaches to the analysis of carbinolamides have relied upon the time-consuming synthesis of authentic standards or purification of large enough quantities for characterization by nuclear magnetic resonance (NMR). We describe a simple technique used in conjunction with liquid chromatography/tandem mass spectrometry (LC/MS/MS) which demonstrates the chemical identity of a carbinolamide by its distinctive ability to reversibly exchange [(18)O]water through an imine intermediate. A key advantage of the technique is that the chromatographic retention times of metabolites are preserved, allowing direct comparisons of mass chromatograms from non-treated and [(18)O]water-treated samples. Metabolites susceptible to the treatment are clearly indicated by the addition of 2 mass units to their original mass. An additional test which can be used in conjunction with (18)O-exchange is base-catalyzed N-dealkylation of N-(alpha-hydroxy)alkyl compounds. The use of the technique is described for carbinolamide metabolites of dirlotapide, loperamide, and a proprietary compound.

A rapid and specific derivatization procedure to identify acyl-glucuronides by mass spectrometry.

A simple procedure is described to identify acyl-glucuronides by coupled liquid chromatography/mass spectrometry after derivatization to a hydroxamic acid with hydroxylamine. The reaction specificity obviates the need for isolation of the acyl-glucuronide from an extract. Glucuronides derived from carbamic acids, and alkyl- and aromatic amines, are inert to the derivatization reaction conditions, making the hydroxamic acid derivative a fingerprint for acyl-glucuronides.