A mechanistic PK/PD model to enable dose selection of the potent anti-tryptase antibody (MTPS9579A) in patients with moderate-to-severe asthma.

Tryptase, a protease implicated in asthma pathology, is secreted from mast cells upon activation during an inflammatory allergic response. MTPS9579A is a novel monoclonal antibody that inhibits tryptase activity by irreversibly dissociating the active tetramer into inactive monomers. This study assessed the relationship between MTPS9579A concentrations in healthy subjects and tryptase levels in serum and nasal mucosal lining fluid from healthy subjects and patients with moderate-to-severe asthma. These data were used to develop a mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model that quantitatively inter-relates MTPS9579A exposure and inhibition of active tryptase in the airway of patients with asthma. From initial estimates of airway tryptase levels and drug partitioning, the PK/PD model predicted almost complete neutralization of active tryptase in the airway of patients with asthma with MTPS9579A doses of 900 mg and greater, administered intravenously (i.v.) once every 4 weeks (q4w). Suppression of active tryptase during an asthma exacerbation event was also evaluated using the model by simulating the administration of MTPS9579A during a 100-fold increase in tryptase secretion in the local tissue. The PK/PD model predicted that 1800 mg MTPS9579A i.v. q4w results in 95.7% suppression of active tryptase at the steady-state trough concentration. Understanding how the exposure-response relationship of MTPS9579A in healthy subjects translates to patients with asthma is critical for future clinical studies assessing tryptase inhibition in the airway of patients with moderate-to-severe asthma.

Dose-dependent inactivation of airway tryptase with a novel dissociating anti-tryptase antibody (MTPS9579A) in healthy participants: A randomized trial.

Tryptase is the most abundant secretory granule protein in human lung mast cells and plays an important role in asthma pathogenesis. MTPS9579A is a novel monoclonal antibody that selectively inhibits tryptase activity by dissociating active tetramers into inactive monomers. The safety, tolerability, pharmacokinetics (PKs), and systemic and airway pharmacodynamics (PDs) of MTPS9579A were assessed in healthy participants. In this phase I single-center, randomized, observer-blinded, and placebo-controlled study, single and multiple ascending doses of MTPS9579A were administered subcutaneously (s.c.) or intravenously (i.v.) in healthy participants. In addition to monitoring safety and tolerability, the concentrations of MTPS9579A, total tryptase, and active tryptase were quantified. This study included 106 healthy participants (82 on active treatment). Overall, MTPS9579A was well-tolerated with no serious or severe adverse events. Serum MTPS9579A showed a dose-proportional increase in maximum serum concentration (Cmax ) values at high doses, and a nonlinear increase in area under the curve (AUC) values at low concentrations consistent with target-mediated clearance were observed. Rapid and dose-dependent reduction in nasosorption active tryptase was observed postdose, confirming activity and the PK/PD relationship of MTPS9579A in the airway. A novel biomarker assay was used to demonstrate for the first time that an investigative antibody therapeutic (MTPS9579A) can inhibit tryptase activity in the upper airway. A favorable safety and tolerability profile supports further assessment of MTPS9579A in asthma. Understanding the exposure-response relationships using the novel PD biomarker will help inform clinical development, such as dose selection or defining patient subgroups.

Analysis of Drug-Drug Interaction Labeling Language and Clinical Recommendations for Newly Approved Drugs Evaluated With Digoxin, Midazolam, and S-Warfarin.

PURPOSE: To best promote drug tolerability and efficacy in the clinic, data from drug-drug interaction (DDI) evaluations and subsequent translation of the results to DDI prevention and/or management strategies must be incorporated into the US Food and Drug Administration (FDA) product labeling in a consistent manner because differences in language might result in varied interpretations. This analysis aimed to assess the consistency in DDI labeling language in New Drug Applications (NDAs). METHODS: NDAs of recently approved drugs (2012-2020) that increase the exposure of digoxin, midazolam, and S-warfarin, index substrates of P-glycoprotein, cytochrome P450 (CYP) 3A, and CYP2C9 activity, respectively, were fully reviewed. Noninhibitors were also evaluated to appreciate the extent of mechanistic extrapolation in case of negative index studies. FINDINGS: After a systematic review of the DDI studies available in NDAs, FDA-approved labeling, and commonly used clinical tertiary resources, differences in DDI results presentation and resulting clinical recommendations were found, even for inhibitors that affect similarly the exposure of the same index substrate. Studies with negative results were often reported in the labels without providing mechanistic interpretation, thus limiting the possible extrapolation of this information to other known substrates. IMPLICATIONS: The variability in language affects how the information was presented to clinicians in tertiary resources. Strategies that aim to improve the translation of mechanistic DDI index studies into consistent labeling recommendations are briefly discussed in this review.

In Vivo Functional Effects of CYP2C9 M1L, a Novel and Common Variant in the Yup'ik Alaska Native Population.

Alaska Native people are under-represented in genetic research but have unique gene variation that may critically impact their response to pharmacotherapy. Full resequencing of CYP2C9 in a cross-section of this population identified CYP2C9 Met1Leu (M1L), a novel, relatively common single nucleotide polymorphism hypothesized to confer CYP2C9 poor metabolizer phenotype by disrupting the start codon. M1L is present at a minor allele frequency of 6.3% in Yup'ik Alaska Native people and thus can contribute to the risk of an adverse drug response from narrow-therapeutic-index CYP2C9 substrates such as (S)-warfarin. This study's objective was to characterize the catalytic efficiency of the Leu1 variant enzyme in vivo by evaluating the pharmacokinetic behavior of naproxen, a probe substrate for CYP2C9 activity, in genotyped Yup'ik participants. We first confirmed the selectivity of (S)-naproxen O-demethylation by CYP2C9 using activity-phenotyped human liver microsomes and selective cytochrome P450 inhibitors and then developed and validated a novel liquid chromatography mass spectrometry method for simultaneous quantification of (S)-naproxen, (S)-O-desmethylnaproxen, and naproxen acyl glucuronide in human urine. The average ratio of (S)-O-desmethylnaproxen to unchanged (S)-naproxen in urine was 18.0 ± 8.0 (n = 11) for the homozygous CYP2C9 Met1 reference group and 10.3 ± 6.6 (n = 11) for the Leu1 variant carrier group (P = 0.011). The effect of M1L variation on CYP2C9 function and its potential to alter the pharmacokinetics of drugs metabolized by the enzyme has clinical implications and should be included in a variant screening panel when pharmacogenetic testing in the Alaska Native population is warranted. SIGNIFICANCE STATEMENT: The novel CYP2C9 Met1Leu variant in Alaska Native people was recently identified. This study validated (S)-naproxen as a CYP2C9 probe substrate to characterize the in vivo functional activity of the CYP2C9 Leu1 variant. The results of this pharmacogenetic-pharmacokinetic study suggest that the CYP2C9 Leu1 variant exhibits loss of enzyme activity. This finding may be important to consider when administering narrow-therapeutic-index medications metabolized by CYP2C9 and also compels further investigation to characterize novel genetic variation in understudied populations.

Methods and sensors for functional genomic studies of cell-cycle transitions in single cells.

Much of our understanding of the regulatory mechanisms governing the cell cycle in mammals has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our current understanding of cell proliferation, these approaches mask the genetic signatures of rare subpopulations such as quiescent (G0) and very slowly dividing (SD) cells. Results described in this study and those of others using single-cell analysis reveal that even in clonally derived immortalized cancer cells, ∼1-5% of cells can exhibit G0 and SD phenotypes. Therefore to enable the study of these rare cell phenotypes we established an integrated molecular, computational, and imaging approach to track, isolate, and genetically perturb single cells as they proliferate. A genetically encoded cell-cycle reporter (K67p-FUCCI) was used to track single cells as they traversed the cell cycle. A set of R-scripts were written to quantify K67p-FUCCI over time. To enable the further study G0 and SD phenotypes, we retrofitted a live cell imaging system with a micromanipulator to enable single-cell targeting for functional validation studies. Single-cell analysis revealed HT1080 and MCF7 cells had a doubling time of ∼24 and ∼48 h, respectively, with high duration variability in G1 and G2 phases. Direct single-cell microinjection of mRNA encoding (GFP) achieves detectable GFP fluorescence within ∼5 h in both cell types. These findings coupled with the possibility of targeting several hundreds of single cells improves throughput and sensitivity over conventional methods to study rare cell subpopulations.

Heterologous Expression and Functional Characterization of Novel CYP2C9 Variants Identified in the Alaska Native People.

CYP2C9 is a major form of human liver cytochrome P450 that is responsible for the oxidative metabolism of several widely used low-therapeutic index drugs, including (S)-warfarin and phenytoin. In a cohort of Alaska Native people, ultrarare or novel CYP2C9 protein variants, M1L (rs114071557), N218I (rs780801862), and P279T (rs182132442, CYP2C9*29), are expressed with higher frequencies than the well characterized CYP2C9*2 and CYP2C9*3 alleles. We report here on their relative expression in lentivirus-infected HepG2 cells and the functional characterization of purified reconstituted enzyme variants expressed in Escherichia coli toward (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen. In the infected HepG2 cells, robust mRNA and protein expression were obtained for wild-type, N218I, and P279T variants, but as expected, the M1L variant protein was not translated in this liver-derived cell line. His-tagged wild-type protein and the N218I and P279T variants, but not M1L, expressed well in E. coli and were highly purified after affinity chromatography. Upon reconstitution with cytochrome P450 oxidoreductase and cytochrome b5, the N218I and P279T protein variants metabolized (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen to the expected monohydroxylated or O-demethylated metabolites. Steady-state kinetic analyses revealed that the relative catalytic efficiency ratios of (S)-warfarin metabolism by the P279T and N218I variants were 87% and 24%, respectively, of wild-type CYP2C9 protein. A similar rank ordering was observed for metabolism of phenytoin, flurbiprofen, and (S)-naproxen. We conclude that carriers of the variant N218I and, especially, the M1L alleles would be at risk of exacerbated therapeutic effects from drugs that rely on CYP2C9 for their metabolic clearance. SIGNIFICANCE STATEMENT: Novel gene variants of CYP2C9-M1L, and N218I, along with P279T (CYP2C9*29)-are expressed in Alaska Native people at relatively high frequencies. In vitro characterization of their functional effects revealed that each variant confers reduced catalytic efficiency toward several substrates, including the low-therapeutic index drugs (S)-warfarin and phenytoin. These data provide the first functional information for new, common CYP2C9 variants in this understudied population. The data may help guide dose adjustments in allele carriers, thus mitigating potential healthcare disparities.

VKORC1 and Novel CYP2C9 Variation Predict Warfarin Response in Alaska Native and American Indian People.

Alaska Native and American Indian (AN/AI) people have unique pharmacogene variation that may affect warfarin disposition and therapeutic response. We performed targeted genotyping for cytochrome P450 (CYP)2C9, vitamin K epoxide oxidase reductase complex subunit 1 (VKORC1), CYP4F2, CYP4F11, and gamma-glutamyl carboxylase (GGCX) variants in AN/AI people receiving warfarin. The primary outcome was stable warfarin dose, defined as one dose, and associated international normalized ratio within the target range, at least 6 months after starting therapy, with two matching doses at least 2 weeks apart. Genotype-phenotype relationships were assessed by multivariate regression analysis, adjusted for self-reported heritage, age, gender, and concurrent statin use. VKORC1 genotype explained 34% of dose variability, with VKORC1 -1639G>A and 1173C>T associated with a 1.7 mg/day (P = 1.4e-05) dose reduction. Additionally, CYP2C9 N218I was suggestively significant (P = 0.077), with heterozygotes requiring 1.1 mg/day less than reference individuals. Self-reported heritage was significantly associated with dose, largely driven by differences in the diagnostic VKORC1 allele frequencies among AN/AI people.

P450 Pharmacogenetics in Indigenous North American Populations.

Indigenous North American populations, including American Indian and Alaska Native peoples in the United States, the First Nations, Métis and Inuit peoples in Canada and Amerindians in Mexico, are historically under-represented in biomedical research, including genomic research on drug disposition and response. Without adequate representation in pharmacogenetic studies establishing genotype-phenotype relationships, Indigenous populations may not benefit fully from new innovations in precision medicine testing to tailor and improve the safety and efficacy of drug treatment, resulting in health care disparities. The purpose of this review is to summarize and evaluate what is currently known about cytochrome P450 genetic variation in Indigenous populations in North America and to highlight the importance of including these groups in future pharmacogenetic studies for implementation of personalized drug therapy.