Brooke M. Rock

Background: B-cell maturation antigen is a pivotal therapeutic target for multiple myeloma (MM). Membrane-bound BCMA can be cleaved by γ-secretase and shed as soluble BCMA (sBCMA). sBCMA can act as a neutralizing sink to compete with drug, as well as serve as a diagnostic/prognostic biomarker for MM. Antibody-capture based methods, such as enzyme-linked immunosorbent assay (ELISA) and immunoaffinity-liquid chromatography-multiple reaction monitoring (IA-LC-MRM), have been reported and well adopted to measure sBCMA in clinical samples. However, both methods are biased by capturing antibodies. Methods: We have used various LC-MS workflows to characterize and quantify endogenous sBCMA in MM patient samples, including bottom-up peptide mapping, intact analysis, IA-based, and reagent-free (RF)-LC-MRM quantitation. Results: We have confirmed that sBCMA contains a variable N-terminus and a C-terminus that extends to the transmembrane domain, ending at amino acid 61. Leveraging an in-house synthesized G-1-61 sBCMA recombinant standard, we developed a RF-LC-MRM method for unbiased sBCMA quantitation in MM patient samples. By comparing the results from RF-LC-MRM with ELISA and IA-LC-MRM, we demonstrated that RF-LC-MRM measures a more complete pool of endogenous sBCMA compared to the antibody-based methods. Conclusions: This work fills the knowledge gap of the exact sequence of endogenous sBCMA for the first time, which differs from the current commercially available standard. Additionally, this work highlights the necessity of identifying the actual sequence of an endogenous soluble target such as sBCMA, both for bioanalytical purposes and to underpin pharmacodynamic measurements.

Background: Current heart failure therapies unload the failing heart without targeting the underlying problem of reduced cardiac contractility. Traditional inotropes (ie, calcitropes) stimulate contractility via energetically costly augmentation of calcium cycling and worsen patient survival. A new class of agents—myotropes—activates the sarcomere directly, independent of calcium. We hypothesize that a novel myotrope TA1 increases contractility without the deleterious myocardial energetic impact of a calcitrope dobutamine. Methods: We determined the effect of TA1 in bovine cardiac myofibrils and human cardiac microtissues, ex vivo in mouse cardiac fibers and in vivo in anesthetized normal rats. Effects of increasing concentrations of TA1 or dobutamine on contractile function, phosphocreatine and ATP concentrations, and ATP production were assessed by ³¹ P nuclear magnetic resonance spectroscopy on isolated perfused rat hearts. Results: TA1 increased the rate of myosin ATPase activity in isolated bovine myofibrils and calcium sensitivity in intact mouse papillary fibers. Contractility increased dose dependently in human cardiac microtissues and in vivo in rats as assessed by echocardiography. In isolated rat hearts, TA1 and dobutamine similarly increased the rate-pressure product. Dobutamine increased both developed pressure and heart rate accompanied by decreased phosphocreatine-to-ATP ratio and decreased free energy of ATP hydrolysis (ΔG _~ATP ) and elevated left ventricular end diastolic pressure. In contrast, the TA1 increased developed pressure without any effect on heart rate, left ventricular end diastolic pressure, phosphocreatine/ATP ratio, or ΔG _~ATP . Conclusions: Novel myotrope TA1 increased myocardial contractility by sensitizing the sarcomere to calcium without impairing diastolic function or depleting the cardiac energy reserve. Since energetic depletion negatively correlates with long-term survival, myotropes may represent a superior alternative to traditional inotropes in heart failure management.

Enzymes are the catalysts of biological systems and are extremely efficient. A typical enzyme accelerates the rate of a reaction by factors of at least a million compared to the rate of the same reaction in the absence of the enzyme. In contrast to traditional catalytic enzymes, the family of cytochrome P450 (CYPs) enzymes are catalytically promiscuous and thus they possess remarkable versatility in substrates. The great diversity of reactions catalyzed by CYP enzymes appear to be based on two unique properties of these heme proteins, the ability of their iron to exist under multiple oxidation states with different reactivities and a flexible active site that can accommodate a wide variety of substrates. Herein, is a discussion of two distinct type of kinetics observed with CYP enzymes. The first example is of CYP complex kinetic profiles when multiple CYP enzymes form the sample product. The second is sequential metabolism, in other words, the formation of multiple products from one CYP enzyme. Given the degree of CYP enzyme promiscuity, it is hardly surprising that there is also a high degree of complex kinetic profiles generated during the catalytic cycle.

Oligonucleotide therapeutics use short interfering RNA (siRNA) or antisense oligonucleotide (ASO) molecules to exploit endogenous systems—neutralizing target RNA to prevent subsequent protein translation. While the potential clinical application is vast, delivery efficiency and extrahepatic targeting is challenging. Bioanalytical assays are important in building understanding of these complex relationships. The literature currently lacks description of robust and sensitive methods to measure siRNA and ASOs in complex biological matrices. Described herein is a non-enzymatic hybridization-based immunoassay that enables quantification of individual siRNA strands (antisense or sense) in serum, urine, bile, and liver and kidney homogenates. Assay utility is also demonstrated in ASOs. The assay improves upon previous works by abolishing enzymatic steps and further incorporating Locked Nucleic Acid (LNA) nucleotide modifications to increase analyte hybridization affinity and improve sensitivity, specificity, and robustness. We report an assay with an ultrasensitive dynamic range of 0.3 to 16,700 pM for siRNA in serum. The assay was submitted to full qualification for accuracy and precision in both serum and tissue matrices and assay performance was assessed with single and mixed analytes. The reliable LNA-hybridization-based approach removes the need for matrix sample extraction, enrichment or amplification steps which may be impeded by more advanced chemical modifications.

The discovery and development of novel pharmaceutical therapies is rapidly transitioning from a small molecule-dominated focus to a more balanced portfolio consisting of small molecules, monoclonal antibodies, engineered proteins (modified endogenous proteins, bispecific antibodies, and fusion proteins), oligonucleotides, and gene-based therapies. This commentary, and the special issue as a whole, aims to highlight these emerging modalities and the efforts underway to better understand their unique pharmacokinetic and absorption, disposition, metabolism, and excretion (ADME) properties. The articles highlighted herein can be broadly grouped into those focusing on the ADME properties of novel therapeutics, those exploring targeted-delivery strategies, and finally, those discussing oligonucleotide therapies. It is also evident that whereas the field in general continues to progress toward new and more complex molecules, a significant amount of effort is still being placed on antibody-drug conjugates. As therapeutic molecules become increasingly complex, a parallel demand for advancements in experimental and analytical tools will become increasingly evident, both to increase the speed and efficiency of identifying safe and efficacious molecules and simultaneously decreasing our dependence on in vivo studies in preclinical species. The research and commentary included in this special issue will provide researchers, clinicians, and the patients we serve more options in the ongoing fight against grievous illnesses and unmet medical needs. SIGNIFICANCE STATEMENT: Recent trends in drug discovery and development suggest a shift away from a small molecule-dominated approach to a more balanced portfolio that includes small molecules, monoclonal antibodies, engineered proteins, and gene therapies. The research presented in this special issue of Drug Metabolism and Disposition will serve to highlight advancements in the understanding of the mechanisms that govern the pharmacokinetic and drug metabolism properties of the novel therapeutic modalities.

Despite the exquisite potential of siRNA as a therapeutic, the mechanism(s) responsible for the robust indirect exposure-response relationships have not been fully elucidated. To understand the siRNA properties linked to potent activity, requires the disposition of siRNA to be characterized. A technical challenge in the characterization is the detection and quantitation of siRNA from biological samples. Described herein, a Locked Nucleic Acid (LNA) Hybridization-Ligation ECL ELISA was designed for ultra-sensitive quantification of both sense and antisense strands of siRNA independent of structural modifica-tions. This assay was applied to measure siRNA in serum and tissue homogenate in preclinical species. We observed rapid clearance of siRNA from the systemic circulation which contrasted the prolonged accumulation within the tissue. The assay was also able to distinguish and quantify free siRNA from RNA-induced silencing complex (RISC) and Argonaute 2 (Ago2) associated with therapeutic siRNA. We utilized an orthogonal method, LC-MS, to investigate 3′ exonuclease activity toward the antisense strand metabolism. Taken together, we have demonstrated that the LNA Hybridization-Ligation ECL ELISA is arobust analytical method with direct application to measuring the exposure of siRNA therapeutics seamlessly across biological matrices.

Aim: PCSK9 and Lp(a) have been identified as potential biomarkers for cardiovascular disease. The ability to measure protein turnover rates will provide insights into the dynamic properties of these proteins and lead to better understanding of their biological roles. We aimed to implement the stable isotope-labeled tracers ([2H3]-leucine) and develop a novel LC-selected reaction monitoring (SRM) mass spectrometry (MS) method to study the kinetics of PCSK9 and Lp(a). Results: A sensitive method using immunoaffinity enrichment coupled with LC-SRM MS was developed to measure the production and degradation rates of PCSK9 and Lp(a) in naive nonhuman primate serum. Comparable results were obtained from two different routes of tracer administration. Conclusion: Immunoaffinity enrichment coupled with LC-SRM MS demonstrated success in in vivo kinetic measurements of proteins with relatively slow turnover rate (Lp[a]) or low abundance (PCSK9) in serum.

Ritonavir is a human immunodeficiency virus (HIV) protease inhibitor and an inhibitor of cytochrome P450 3A4, the major human hepatic drug-metabolizing enzyme. Given the potent inhibition of CYP3A4 by ritonavir, subtherapeutic doses of ritonavir are used to increase plasma concentrations of other HIV drugs oxidized by CYP3A4, thereby extending their clinical efficacy. However, the mechanism of inhibition of CYP3A4 by ritonavir remains unclear. To date, data suggests multiple types of inhibition by ritonavir, including mechanism-based inactivation by metabolic-intermediate complex formation, competitive inhibition, irreversible type II coordination to the heme iron, and more recently heme destruction. The results presented here demonstrate that inhibition of CYP3A4 by ritonavir occurs by CYP3A4-mediated activation and subsequent formation of a covalent bond to the apoprotein. Incubations of [³H]ritonavir with reconstituted CYP3A4 and human liver microsomes resulted in a covalent binding stoichiometry equal to 0.93 ± 0.04 moles of ritonavir bound per mole of inactivated CYP3A4. The metabolism of [³H]ritonavir by CYP3A4 leads to the formation of a covalent adduct specifically to CYP3A4, confirmed by radiometric liquid chromatography–trace and whole-protein mass spectrometry. Tryptic digestion of the CYP3A4-[³H]ritonavir incubations exhibited an adducted peptide (255-RMKESRLEDTQKHR-268) associated with a radiochromatic peak and a mass consistent with ritonavir plus 16 Da, in agreement with the whole-protein mass spectrometry. Additionally, nucleophilic trapping agents and scavengers of free oxygen species did not prevent inactivation of CYP3A4 by ritonavir. In conclusion, ritonavir exhibited potent time-dependent inactivation of CYP3A, with the mechanism of inactivation occurring though a covalent bond to Lys257 of the CYP3A4 apoprotein.