Metabolomic Profiling and Drug Interaction Characterization Reveal Riboflavin As a Breast Cancer Resistance Protein-Specific Endogenous Biomarker That Demonstrates Prediction of Transporter Activity In Vivo.

Advancement of endogenous biomarkers for drug transporters as a tool for assessing drug-drug interactions (DDIs) depends on initial identification of biomarker candidates and relies heavily on biomarker validation and its response to reference inhibitors in vivo. To identify endogenous biomarkers of breast cancer resistance protein (BCRP), we applied metabolomic approaches to profile plasma from Bcrp-/-, multidrug resistance protein (Mdr)1a/1b-/-, and Bcrp/Mdr1a/1b-/- mice. Approximately 130 metabolites were significantly altered in Bcrp and P-glycoprotein (P-gp) knockout mice, indicating numerous metabolite-transporter interactions. We focused on BCRP-specific substrates and identified riboflavin, which was significantly elevated in the plasma of Bcrp single- and Bcrp/P-gp double- but not P-gp single-knockout mice. Dual BCRP/P-gp inhibitor elacridar caused a dose-dependent increase of the area under the plasma concentration-time curve (AUC) of riboflavin in mice (1.51- and 1.93-fold increases by 30 and 150 mg/kg elacridar, respectively). In three cynomolgus monkeys, we observed approximately 1.7-fold increases in the riboflavin concentrations caused by ML753286 (10 mg/kg), which correlated well with the increase of sulfasalazine, a known BCRP probe in monkeys. However, the BCRP inhibitor had no effect on isobutyryl carnitine, arginine, or 2-arachidonoyl glycerol levels. Additionally, clinical studies on healthy volunteers indicated low intrasubject and intermeal variability of plasma riboflavin concentrations. In vitro experiments using membrane vesicles demonstrated riboflavin as a select substrate of monkey and human BCRP over P-gp. Collectively, this proof-of-principle study indicates that riboflavin is a suitable endogenous probe for BCRP activity in mice and monkeys and that future investigation of riboflavin as a blood-based biomarker of human BCRP is warranted. SIGNIFICANCE STATEMENT: Our results identified riboflavin as an endogenous biomarker candidate of BCRP. Its selectivity, sensitivity, and predictivity regarding BCRP inhibition have been explored. The findings of this study highlight riboflavin as an informative BCRP plasma biomarker in animal models. The utility of this biomarker requires further validation by evaluating the effects of BCRP inhibitors of different potencies on riboflavin plasma concentrations in humans. Ultimately, riboflavin may shed light on the risk assessment of BCRP DDIs in early clinical trials.

Bicyclic Ligand-Biased Agonists of S1P1: Exploring Side Chain Modifications to Modulate the PK, PD, and Safety Profiles.

Sphingosine-1-phosphate (S1P) binds to a family of sphingosine-1-phosphate G-protein-coupled receptors (S1P1-5). The interaction of S1P with these S1P receptors has a fundamental role in many physiological processes in the vascular and immune systems. Agonist-induced functional antagonism of S1P1 has been shown to result in lymphopenia. As a result, agonists of this type hold promise as therapeutics for autoimmune disorders. The previously disclosed differentiated S1P1 modulator BMS-986104 (1) exhibited improved preclinical cardiovascular and pulmonary safety profiles as compared to earlier full agonists of S1P1; however, it demonstrated a long pharmacokinetic half-life (T1/2 18 days) in the clinic and limited formation of the desired active phosphate metabolite. Optimization of this series through incorporation of olefins, ethers, thioethers, and glycols into the alkyl side chain afforded an opportunity to reduce the projected human T1/2 and improve the formation of the active phosphate metabolite while maintaining efficacy as well as the improved safety profile. These efforts led to the discovery of 12 and 24, each of which are highly potent, biased agonists of S1P1. These compounds not only exhibited shorter in vivo T1/2 in multiple species but are also projected to have significantly shorter T1/2 values in humans when compared to our first clinical candidate. In models of arthritis, treatment with 12 and 24 demonstrated robust efficacy.

Design, Synthesis, and Structure-Activity Relationships of Novel Tetrahydroisoquinolino Benzodiazepine Dimer Antitumor Agents and Their Application in Antibody-Drug Conjugates.

A series of tetrahydroisoquinoline-based benzodiazepine dimers were synthesized and tested for in vitro cytotoxicity against a panel of cancer cell lines. Structure-activity relationship investigation of various spacers guided by molecular modeling studies helped to identify compounds with picomolar activity. Payload 17 was conjugated to anti-mesothelin and anti-fucosylated monosialotetrahexosylganglioside (FucGM1) antibodies using lysosome-cleavable valine-citrulline dipeptide linkers via heterogeneous lysine conjugation and bacterial transglutaminase-mediated site-specific conjugation. In vitro, these antibody drug conjugates (ADCs) exhibited significant cytotoxic and target-mediated selectivity on human cancer cell lines. The pharmacokinetics and efficacy of these ADCs were further evaluated in gastric and lung cancer xenograft models in mice. Consistent pharmacokinetic profiles, high target specificity, and robust antitumor activity were observed in these models after a single dose of the ADC-46 (0.02 µmol/kg).

Determination of Real Time in Vivo Drug Receptor Occupancy for a Covalent Binding Drug as a Clinical Pharmacodynamic Biomarker by Immunocapture-LC-MS/MS.

We report a novel immunocapture (IC)-LC-MS/MS methodology to directly measure real time in vivo receptor occupancy (RO) for a covalent binding drug in blood lysate. A small molecule quencher was added immediately after sample collection to convert the free receptor to a quencher-bound receptor (QB-R) which was measured with the drug-bound receptor (DB-R) simultaneously by LC-MS/MS after immunocapture enrichment, followed by trypsin digestion. Addition of the quencher is necessary to prevent the free receptor from ex vivo binding with the drug. The real time RO was calculated based on the concentrations of DB-R and the free receptor (which is now QB-R) that were obtained from each sample. This strategy has been successfully applied to the measurement of the RO for Bruton's tyrosine kinase (BTK) in the blood lysate of monkeys after dosing with branebrutinib (BMS-986195), a covalent BTK inhibitor being evaluated to treat rheumatoid arthritis. A custom-made quencher, which is more reactive to BTK than branebrutinib, was added in excess amount to bind with all available free BTK to form quencher-bound BTK (QB-BTK) during blood sample collection. To measure a wide range of % BTK RO, including those of <5% or >95%, the required LLOQ at 0.125 nM for QB-BTK and 0.250 nM for drug-bound BTK (DB-BTK) in blood lysate were successfully achieved by using this IC-LC-MS/MS strategy. This proof-of-concept assay demonstrated its suitability with high throughput for real time in vivo BTK RO measurement as a pharmacodynamic (PD) biomarker for clinical drug development.

Critical role of kinase activity of hematopoietic progenitor kinase 1 in anti-tumor immune surveillance.

Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of-function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens.

Identification and Preclinical Pharmacology of ((1 R,3 S)-1-Amino-3-(( S)-6-(2-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (BMS-986166): A Differentiated Sphingosine-1-phosphate Receptor 1 (S1P1) Modulator Advanced into Clinical Trials.

Recently, our research group reported the identification of BMS-986104 (2) as a differentiated S1P1 receptor modulator. In comparison to fingolimod (1), a full agonist of S1P1 currently marketed for the treatment of relapse remitting multiple sclerosis (RRMS), 2 offers several potential advantages having demonstrated improved safety multiples in preclinical evaluations against undesired pulmonary and cardiovascular effects. In clinical trials, 2 was found to exhibit a pharmacokinetic half-life ( T1/2) longer than that of 1, as well as a reduced formation of the phosphate metabolite that is required for activity against S1P1. Herein, we describe our efforts to discover highly potent, partial agonists of S1P1 with a shorter T1/2 and increased in vivo phosphate metabolite formation. These efforts culminated in the discovery of BMS-986166 (14a), which was advanced to human clinical evaluation. The pharmacokinetic/pharmacodynamic (PK/PD) relationship as well as pulmonary and cardiovascular safety assessments are discussed. Furthermore, efficacy of 14a in multiple preclinical models of autoimmune diseases are presented.

Asymmetric Hydroboration Approach to the Scalable Synthesis of ((1R,3S)-1-Amino-3-((R)-6-hexyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (BMS-986104) as a Potent S1P1 Receptor Modulator.

We describe a highly efficient route for the synthesis of 4a (BMS-986104). A key step in the synthesis is the asymmetric hydroboration of trisubstituted alkene 6. Particularly given the known difficulties involved in this type of transformation (6 → 7), the current methodology provides an efficient approach to prepare this class of compounds. In addition, we disclose the efficacy of 4a in a mouse EAE model, which is comparable to 4c (FTY720). Mechanistically, 4a exhibited excellent remyelinating effects on lysophosphatidylcholine (LPC) induced demyelination in a three-dimensional brain cell culture assay.

Identification of Tricyclic Agonists of Sphingosine-1-phosphate Receptor 1 (S1P1) Employing Ligand-Based Drug Design.

Fingolimod (1) is the first approved oral therapy for the treatment of relapsing remitting multiple sclerosis. While the phosphorylated metabolite of fingolimod was found to be a nonselective S1P receptor agonist, agonism specifically of S1P1 is responsible for the peripheral blood lymphopenia believed to be key to its efficacy. Identification of modulators that maintain activity on S1P1 while sparing activity on other S1P receptors could offer equivalent efficacy with reduced liabilities. We disclose in this paper a ligand-based drug design approach that led to the discovery of a series of potent tricyclic agonists of S1P1 with selectivity over S1P3 and were efficacious in a pharmacodynamic model of suppression of circulating lymphocytes. Compound 10 had the desired pharmacokinetic (PK) and pharmacodynamic (PD) profile and demonstrated maximal efficacy when administered orally in a rat adjuvant arthritis model.

Discovery of the Selective CYP17A1 Lyase Inhibitor BMS-351 for the Treatment of Prostate Cancer.

Efforts to identify a potent, reversible, nonsteroidal CYP17A1 lyase inhibitor with good selectivity over CYP17A1 hydroxylase and CYPs 11B1 and 21A2 for the treatment of castration-resistant prostate cancer (CRPC) culminated in the discovery of BMS-351 (compound 18), a pyridyl biaryl benzimidazole with an excellent in vivo profile. Biological evaluation of BMS-351 at a dose of 1.5 mg in castrated cynomolgus monkeys revealed a remarkable reduction in testosterone levels with minimal glucocorticoid and mineralcorticoid perturbation. Based on a favorable profile, BMS-351 was selected as a candidate for further preclinical evaluation.

Determination of the molecular weight of poly(ethylene glycol) in biological samples by reversed-phase LC-MS with in-source fragmentation.

PEGylation has been widely used to improve the biopharmaceutical properties of therapeutic proteins and peptides. Previous studies have used multiple analytical techniques to determine the fate of both the therapeutic molecule and unconjugated poly(ethylene glycol) (PEG) after drug administration. A straightforward strategy utilizing liquid chromatography-mass spectrometry (LC-MS) to characterize high-molecular weight PEG in biologic matrices without a need for complex sample preparation is presented. The method is capable of determining whether high-MW PEG is cleaved in vivo to lower-molecular weight PEG species. Reversed-phase chromatographic separation is used to take advantage of the retention principles of polymeric materials whereby elution order correlates with PEG molecular weight. In-source collision-induced dissociation (CID) combined with selected reaction monitoring (SRM) or selected ion monitoring (SIM) mass spectrometry (MS) is then used to monitor characteristic PEG fragment ions in biological samples. MS provides high sensitivity and specificity for PEG and the observed retention times in reversed-phase LC enable estimation of molecular weight. This method was successfully used to characterize PEG molecular weight in mouse serum samples. No change in molecular weight was observed for 48 h after dosing.

Addressing drug effects on cut point determination for an anti-drug antibody assay.

The effect of trough levels of a monoclonal antibody drug (drugB) on screening cut point (CP) determination for an anti-drug antibody (ADA) assay was scrutinized and the conclusions substantiated by data from a phase 3 cancer clinical study. The ADA assay utilized an acid dissociation step and either 0 or 100 µg/ml drugB was added to the samples prior to obtaining the signals used for CP calculations. Serum samples from three different drug-naive populations were tested (healthy individuals, cancer patients enrolled in the drugB clinical trial and cancer patients whose serum samples were available commercially). For the same disease state samples, both the screening CP and confirmation CP were different when calculated during validation or from study sample analysis. It is reasonable to assume that variability was due to the patient heterogeneity, as they could have been at distinct stages of disease progression, and/or taking different medications, amongst other differences. The patients enrolled in the clinical trial were stratified as per protocol and hence represented a more homogeneous population. Drug effects on CP may be population dependent and also assay dependent.

Characterization of protein therapeutics by mass spectrometry: recent developments and future directions.

Mass spectrometry (MS) has become a powerful technology in the discovery and development of protein therapeutics in the biopharmaceutical industry. This review article describes recent developments and future trends in the characterization of protein therapeutics using MS. We discuss top-down MS for the characterization of protein modifications, hydrogen/deuterium exchange MS and ion mobility MS methods for higher order protein structure studies. Quantitative analysis of protein therapeutics (in vivo) by MS as an orthogonal approach to immunoassay for pharmacokinetics studies will also be illustrated.

A tris (2-carboxyethyl) phosphine (TCEP) related cleavage on cysteine-containing proteins.

Introduced in the late 1980s as a reducing reagent, Tris (2-carboxyethyl) phosphine (TCEP) has now become one of the most widely used protein reductants. To date, only a few studies on its side reactions have been published. We report the observation of a side reaction that cleaves protein backbones under mild conditions by fracturing the cysteine residues, thus generating heterogeneous peptides containing different moieties from the fractured cysteine. The peptide products were analyzed by high performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). Peptides with a primary amine and a carboxylic acid as termini were observed, and others were found to contain amidated or formamidated carboxy termini, or formylated or glyoxylic amino termini. Formamidation of the carboxy terminus and the formation of glyoxylic amino terminus were unexpected reactions since both involve breaking of carbon-carbon bonds in cysteine.

Disposition of [1'-(14)C]stavudine after oral administration to humans.

The disposition of stavudine, a potent and orally active nucleoside reverse transcriptase inhibitor, was investigated in six healthy human subjects. Before dosing humans with [1'-(14)C]stavudine, a tissue distribution study was performed in Long-Evans rats. Results from this study showed no accumulation of radioactivity in any of the tissues studied, indicating that the position of the (14)C-label on the molecule was appropriate for the human study. After a single 80-mg (100 microCi) oral dose of [1'-(14)C]stavudine, approximately 95% of the radioactive dose was excreted in urine with an elimination half-life of 2.35 h. Fecal excretion was limited, accounting for only 3% of the dose. Unchanged stavudine was the major drug-related component in plasma (61% of area under the plasma concentration-time curve from time zero extrapolated to infinite time of the total plasma radioactivity) and urine (67% of dose). The remaining radioactivity was associated with minor metabolites, including mono- and bis-oxidized stavudine, glucuronide conjugates of stavudine and its oxidized metabolite, and an N-acetylcysteine (NAC) conjugate of the ribose (M4) after glycosidic cleavage. Formation of metabolite M4 was shown in human liver microsomes incubated with 2',3'-didehydrodideoxyribose, the sugar base of stavudine, in the presence of NAC. In addition, after similar microsomal incubations fortified with GSH, two GSH conjugates, 3'-GS-deoxyribose and 1'-keto-2',3'-dideoxy-3'-GS-ribose, were observed. This suggests that 2',3'-didehydrodideoxyribose underwent cytochrome P450-mediated oxidation leading to an epoxide intermediate, 2',3'-ribose epoxide, followed by GSH addition. In conclusion, absorption and elimination of stavudine were rapid and complete after oral dosing, with urinary excretion of unchanged drug as the predominant route of elimination in humans.

Normalization strategies for metabonomic analysis of urine samples.

Unlike plasma and most biological fluids which have solute concentrations that are tightly controlled, urine volume can vary widely based upon water consumption and other physiological factors. As a result, the concentrations of endogenous metabolites in urine vary widely and normalizing for these effects is necessary. Normalization approaches that utilized urine volume, osmolality, creatinine concentration, and components that are common to all samples ("total useful MS signal") were compared in order to determine which strategies could be successfully used to differentiate between dose groups based upon the complete endogenous metabolite profile. Variability observed in LC/MS results obtained from targeted and non-targeted metabonomic analyses was highly dependent on the strategy used for normalization. We therefore recommend the use of two different normalization techniques in order to facilitate detection of statistically significant changes in the endogenous metabolite profile when working with urine samples.

Utility of the hybrid LTQ-FTMS for drug metabolism applications.

Fourier Transform Ion Cyclotron Mass Spectrometry (FTMS) provides the highest mass accuracy and mass resolving power of the currently available mass spectrometers. One of the main drawbacks in its use for absorption, distribution, metabolism and excretion (ADME) applications has been its incompatibility with standard HPLC columns and flow rates. Hybrid instruments, such as the LTQ-FT, provide the much needed bridge between the excellent performance and capabilities of the FT mass spectrometers and the well-established, tested and validated features of quadrupoles and ion traps. The hybrid instruments are compatible with standard HPLC flow rates, have high-throughput and automation compatibility, and also provide data dependant MSn. The ability to maintain the fidelity of an externally calibrated accurate mass measurement across an HPLC peak, where the analyte concentrations are rapidly changing, is a significant advance for this technology, as is the ability to perform data dependent MS/MS experiments on the chromatographic time scale. The MSn and accurate mass capabilities are routinely utilized to rapidly confirm the identification of expected metabolites or to elucidate the structures of unusual or unexpected metabolites. The combination of traditional high-flow chromatography and robust, externally calibrated accurate mass determination for both parent and product ions makes the LTQ-FTMS a very powerful analytical tool for the characterization of metabolites, identification of metabolic soft-spots and for metabonomics studies.