Biomarker Assay Validation by Mass Spectrometry.

Decades of discussion and publication have gone into the guidance from the scientific community and the regulatory agencies on the use and validation of pharmacokinetic and toxicokinetic assays by chromatographic and ligand binding assays for the measurement of drugs and metabolites. These assay validations are well described in the FDA Guidance on Bioanalytical Methods Validation (BMV, 2018). While the BMV included biomarker assay validation, the focus was on understanding the challenges posed in validating biomarker assays and the importance of having reliable biomarker assays when used for regulatory submissions, rather than definition of the appropriate experiments to be performed. Different from PK bioanalysis, analysis of biomarkers can be challenging due to the presence of target analyte(s) in the control matrices used for calibrator and quality control sample preparation, and greater difficulty in procuring appropriate reference standards representative of the endogenous molecule. Several papers have been published offering recommendations for biomarker assay validation. The situational nature of biomarker applications necessitates fit-for-purpose (FFP) assay validation. A unifying theme for FFP analysis is that method validation requirements be consistent with the proposed context of use (COU) for any given biomarker. This communication provides specific recommendations for biomarker assay validation (BAV) by LC-MS, for both small and large molecule biomarkers. The consensus recommendations include creation of a validation plan that contains definition of the COU of the assay, use of the PK assay validation elements that support the COU, and definition of assay validation elements adapted to fit biomarker assays and the acceptance criteria for both.

Method for Continuous Monitoring of Electrospray Ion Formation.

A method for continuously monitoring the performance of electrospray ionization without the addition of hardware or chemistry to the system is demonstrated. In the method, which we refer to as SprayDx, cluster ions with solvent vapor natively formed by electrospray are followed throughout the collection of liquid chromatography-selected reaction monitoring data. The cluster ion extracted ion chromatograms report on the consistency of the ion formation and detection system. The data collected by the SprayDx method resemble the data collected for postcolumn infusion of analyte. The response of the cluster ions monitored reports on changes in the physical parameters of the ion source such as voltage and gas flow. SprayDx is also observed to report on ion suppression in a fashion very similar to a postcolumn infusion of analyte. We anticipate the method finding utility as a continuous readout on the performance of electrospray and other atmospheric pressure ionization processes. Graphical Abstract ᅟ.

Applications of low-flow LC-SRM for the analysis of large molecules in pharmaceutical R&D.

Although ligand-binding assays are frequently employed to measure large molecules, the use of LC-SRM assays is increasingly popular due to the inherent selectivity advantage and the ability to operate without exquisitely selective antibodies. Until recently LC-SRM assays have been unable to compete with ligand-binding assays in terms of sensitivity. However, the use of low-flow chromatography prior to mass spectrometry has played a crucial role in increasing the sensitivity of LC-SRM platforms and enabling measurements of large molecules that had previously been unmeasurable. In this article, we highlight some technical advances, describe strategies for employing low-flow chromatography, and review recent literature that describes implementation of low-flow LC-SRM to support large-molecule analysis in pharmaceutical R&D.

Riluzole prodrugs for melanoma and ALS: design, synthesis, and in vitro metabolic profiling.

Riluzole (1) is an approved therapeutic for the treatment of ALS and has also demonstrated anti-melanoma activity in metabotropic glutamate GRM1 positive cell lines, a mouse xenograft assay and human clinical trials. Highly variable drug exposure following oral administration among patients, likely due to variable first pass effects from heterogeneous CYP1A2 expression, hinders its clinical use. In an effort to mitigate effects of this clearance pathway and uniformly administer riluzole at efficacious exposure levels, several classes of prodrugs of riluzole were designed, synthesized, and evaluated in multiple in vitro stability assays to predict in vivo drug levels. The optimal prodrug would possess the following profile: stability while transiting the digestive system, stability towards first pass metabolism, and metabolic lability in the plasma releasing riluzole. (S)-O-Benzyl serine derivative 9 was identified as the most promising therapeutically acceptable prodrug.

Kinesin spindle protein (KSP) inhibitors. 9. Discovery of (2S)-4-(2,5-difluorophenyl)-n-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide (MK-0731) for the treatment of taxane-refractory cancer.

Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.

Kinesin spindle protein (KSP) inhibitors. Part 6: Design and synthesis of 3,5-diaryl-4,5-dihydropyrazole amides as potent inhibitors of the mitotic kinesin KSP.

3,5-diaryl-4,5-dihydropyrazoles were discovered to be potent KSP inhibitors with excellent in vivo potency. These enzyme inhibitors possess desirable physical properties that can be readily modified by incorporation of a weakly basic amine. Careful adjustment of amine basicity was essential for preserving cellular potency in a multidrug resistant cell line while maintaining good aqueous solubility.

Kinesin spindle protein (KSP) inhibitors. Part 7: Design and synthesis of 3,3-disubstituted dihydropyrazolobenzoxazines as potent inhibitors of the mitotic kinesin KSP.

Observations from two structurally related series of KSP inhibitors led to the proposal and discovery of dihydropyrazolobenzoxazines that possess ideal properties for cancer drug development. The synthesis and characterization of this class of inhibitors along with relevant pharmacokinetic and in vivo data are presented. The synthesis is highlighted by a key [3+2] cycloaddition to form the pyrazolobenzoxazine core followed by diastereospecific installation of a quaternary center.

Kinesin spindle protein (KSP) inhibitors. Part 8: Design and synthesis of 1,4-diaryl-4,5-dihydropyrazoles as potent inhibitors of the mitotic kinesin KSP.

Inspired by previous efforts in the pyrazolobenzoxazine class of KSP inhibitors, the design and synthesis of 1,4-diaryl-4,5-dihydropyrazole inhibitors of KSP are described. Crystallographic evidence of binding mode and in vivo potency data is also highlighted.

The use of Qtrap technology in drug metabolism.

Advances in mass spectrometry continue to bring new and exciting capabilities to the study of drug metabolism. This review covers the hybrid linear ion trap - triple quadrupole mass spectrometer, the QTrap. While still a recent addition to the arsenal of mass spectrometry techniques available to the metabolism scientist, reports in the literature highlight the advantages of the system for metabolite identification. The system combines the selective scans of the triple quadrupole with the high speed, high sensitivity of the ion trap allowing metabolites to be found and characterized in a single scan. Additionally, the system has MS(3) and time delayed fragmentation scans that aid in structure elucidation. Since the fragmentation occurs in the collision cell of the triple quadrupole, the traditionally rich fragmentation of the collision cell fragmentation is preserved. In addition to helping to make traditional processes more efficient, work has been done that shows the potential of the instrument to change traditional DMPK approaches. Researchers have reported methods that allow for both qualitative analysis of circulating metabolites and quantification of parent drug within the same analysis. The approaches reported show how the instrument can be used to collect more information from every sample and potentially streamline typical drug metabolism assays.

Determination of potato carboxypeptidase inhibitor in African Green Monkey plasma using 96-well SPE and LC-MS/MS.

Potato carboxypeptidase inhibitor (CPI), a peptide with multiple isoforms (MW>4000 Da) was determined from African Green Monkey plasma using a PE Sciex API-3000 LC-MS/MS in the positive ionization mode with the turbo ionspray interface (450 degrees C). Samples were prepared using an Oasis MCX 96-well solid phase extraction plate and chromatographed on an Allure C18 HPLC Column (50 mm x 1.0 mm, 5 microm) using gradient elution. Upon analysis of the extracts using LC-MS/MS, the concentration of CPI was calculated using a single MS/MS transition (m/z 830.5-->221.0) that was reflective of the mass concentration (microg/mL) of main the CPI isoforms present in plasma from monkeys after they were given an intravenous dose of CPI. The assay was linear for CPI over concentrations of 0.05-10 microg/mL when extracting 200-microL aliquots of African Green Monkey plasma. The assay was applied to the determination of CPI in African Green Monkey plasma samples in two separate analytical runs (correlation of standard curves, r1=0.9991 and r2=0.9953). Quality control (QC) samples were run at 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 microg/mL for each assay. Average ranges (n=12) for accuracy and precision for all concentrations of QCs during the two runs were 92.0-102.0% of expected potency and 10.4-21.8% (coefficient of variations), respectively.

Negative ion tandem mass spectrometry for the detection of glutathione conjugates.

Characterization of S-linked conjugates of the endogenous tripeptide glutathione (gamma-glutamyl-cysteinylglycine, GSH) represents a valuable indirect approach for the identification of chemically reactive, electrophilic intermediates formed during the metabolism of both foreign compounds and endogenous substances. In most cases, GSH adducts generated in vitro or excreted in the bile of animals are detected by the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS), employing survey scans based on characteristic fragmentations of this class of conjugates. However, a limitation of current LC-MS/MS approaches, which typically employ electrospray ionization with analysis of positive ions, is that no single survey scan exhibits broad utility in the detection of unknown GSH adducts, since different structural classes of conjugate (aromatic, benzylic, aliphatic, thioester, etc.) behave differently upon collision-induced dissociation (CID) of the respective [M + H]+ parent ions. In the present study, we evaluated MS/MS in the negative ion mode as an alternative approach and report herein that the spectra obtained by CID of the [M - H]- ions of a number of representative GSH adducts, as well as GSH itself, are dominated by fragments originating from the glutathionyl moiety of the tripeptide. In particular, the anion at m/z 272, corresponding nominally to deprotonated gamma-glutamyl-dehydroalanyl-glycine, was abundant in the negative ion spectra of free GSH and all GSH conjugates examined, suggesting that scanning for precursors of this ion may provide a generally applicable technique for the detection of adducts of unknown structure. The utility of this novel detection strategy was demonstrated in a series of in vitro and in vivo experiments where compounds known to undergo metabolic activation were examined for their propensity to form conjugates with GSH. In all cases, scanning for precursors of m/z 272 in the negative ion mode revealed the presence of the expected adducts and in some instances revealed additional conjugates that had not been reported previously. Positive ion MS/MS, on the other hand, was more useful than the corresponding negative ion scans in providing information on the molecular structure of GSH conjugates.

Nonpeptide alpha(v)beta3 antagonists: identification of potent, chain-shortened 7-oxo RGD mimetics.

Potent, novel 7-oxo alpha(v)beta3 antagonists have been prepared. These antagonists offer decreased plasma protein binding and excellent pharmacokinetic profiles.

Nonpeptide alphavbeta3 antagonists. Part 11: discovery and preclinical evaluation of potent alphavbeta3 antagonists for the prevention and treatment of osteoporosis.

3-(S)-Pyrimidin-5-yl-9-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-nonanoic acid (5e) and 3-(S)-(methylpyrimidin-5-yl)-9-(5,6,7,8-tetrahydro-[1,8]naphthyridin-2-yl)-nonanoic acid (5f) were identified as potent and selective antagonists of the alpha(v)beta(3) receptor. These compounds have excellent in vitro profiles (IC(50) = 0.07 and 0.08 nM, respectively), significant unbound fractions in human plasma (6 and 4%), and good pharmacokinetics in rat, dog, and rhesus monkey. On the basis of the efficacy shown in an in vivo model of bone turnover following once-daily oral administration, these two compounds were selected for clinical development for the treatment of osteoporosis.

Nonpeptide alpha V beta 3 antagonists. Part 9: Improved pharmacokinetic profile through the use of an aliphatic, des-amide backbone.

A series of alphaVbeta3 receptor antagonists lacking the amide bond of previously-reported 'chain-shortened' compounds is described. Replacement of the lone amide bond with two methylene groups in this series yields more lipophilic compounds that have longer half-lives, lower clearance, and greater oral bioavailability when administered to dogs.

Nonpeptide alpha V beta 3 antagonists. Part 10: In vitro and in vivo evaluation of a potent 7-methyl substituted tetrahydro-[1,8]naphthyridine derivative.

Subtle modifications were incorporated into the structure of clinical candidate 1. These changes were designed to maintain potency and selectivity while inducing changes in physical properties leading to improved pharmacokinetics in three species. This approach led to the identification of 4 as a potent, selective alphaVbeta3 receptor antagonist that was selected for clinical development based on an improved PK profile and efficacy demonstrated in an in vivo model of bone turnover.

Non-peptide alpha(v)beta(3) antagonists: identification of potent, chain-shortened RGD mimetics that incorporate a central pyrrolidinone constraint.

Antagonists of the integrin receptor alpha(v)beta(3) are expected to have utility in the treatment of osteoporosis through inhibition of bone resorption. A series of potent, chain-shortened, pyrrolidinone-containing alpha(v)beta(3) receptor antagonists is described. Two sets of diasteromeric pairs of high-affinity antagonists demonstrated marked differences in log P values, which translated into differing dog pharmacokinetic properties. One member of this set was demonstrated to be effective in reducing bone resorption in rats.

Nonpeptide alphavbeta3 antagonists. 8. In vitro and in vivo evaluation of a potent alphavbeta3 antagonist for the prevention and treatment of osteoporosis.

3(S)-(6-methoxypyridin-3-yl)-3-[2-oxo-3-[3-(5,6,7,8-tetrahydro-[1,8]-naphthyridin-2-yl)propyl]imidazolidin-1-yl]propionic acid 6 was identified as a potent and selective antagonist of the alpha(v)beta(3) receptor. This compound has an excellent in vitro profile (IC(50) = 0.08 nM), a significant unbound fraction in human plasma (12%), and good pharmacokinetics in rat, dog, and rhesus monkey. On the basis of the efficacy shown in three in vivo models of bone turnover, the compound was selected for clinical development. To support the ongoing metabolism and safety studies, a novel strategy was employed in which a series of oxidized derivatives of 6 were prepared by exposure of 6 (or the methyl ester) to chemical oxidizing agents. These products proved useful in the identification of active metabolites generated by either in vitro or in vivo metabolism.

Collection of selected reaction monitoring and full scan data on a time scale suitable for target compound quantitative analysis by liquid chromatography/tandem mass spectrometry.

A hybrid linear ion trap/triple quadrupole mass spectrometer was used to demonstrate the value of collecting full scan qualitative data during quantitative analysis of target compounds. We present examples of the additional information that can be obtained from plasma samples analyzed primarily for target compound concentrations. This information includes detection of circulating metabolites, dosing vehicle, interfering matrix components, and potential interfering drug conjugates. Additionally, the quantitative results from selected reaction monitoring (SRM) analysis and from combined full scan and SRM analysis (SRM/EMS) were compared. The quantitative data in both scan modes are acceptable in terms of sensitivity, accuracy and precision. One can conclude from this work that the hybrid linear ion trap/triple quadrupole mass analyzer can provide in a single analysis both useful qualitative data, and accurate and precise quantitative data from the samples routinely prepared and analyzed for target drug concentrations.