Patient Centric Microsampling to Support Paxlovid Clinical Development: Bridging and Implementation.

Nirmatrelvir is a potent and selective severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) main protease inhibitor. Nirmatrelvir co-packaged with ritonavir (as PAXLOVID) received US Food and Drug Administration (FDA) Emergency Use Authorization (EUA) on December 22, 2021, as an oral treatment for coronavirus disease 2019 (COVID-19) and subsequent new drug application approval on May 25, 2023. Pharmacokinetic (PK) capillary blood sampling at-home using Tasso-M20 micro-volumetric sampling device was implemented in the program, including three phase II/III outpatient and several clinical pharmacology studies supporting the EUA. The at-home sampling complemented venous blood sampling procedures to enrich the PK dataset, to decrease the need for patients' site visit for PK sampling, and to allow different sampling approaches for flexibility and convenience. To demonstrate concordance/equivalence, bridging between venous plasma and Tasso dried blood results was conducted by comparing concentrations and derived PK parameters from both sampling approaches. In addition, a two-compartment population PK model was utilized to bridge the plasma and Tasso data by estimating the PK parameters using blood-to-plasma ratio as a slope parameter. Operational challenges were successfully managed to implement at-home PK sampling in global phase II/III trials. Sample quality was generally very good with less than 3% samples deemed as "not usable" from over 800 samples collected in all the studies. Experience gained from sites and patients will guide future broader implementations.

Using Mitra sampling to support first-in-human pharmacokinetic evaluations for PF-07059013.

Aim: A sensitive and selective method for the determination of PF-07059013 in dried blood collected by Mitra™ tips was developed and qualified from 50 to 50,000 ng/ml. Materials & methods: PF-07059013 is isolated from 10 µl of human dried blood by extraction with methanol and analyzed by HPLC-MS/MS. Results & conclusions: In addition to routine validation elements, impact of hematocrit and Mitra tip's lot-to-lot variation on assay accuracy were evaluated. The qualified method was used in one clinical study with excellent performance. Correlation coefficient between blood concentrations obtained from liquid-incurred blood samples and dried-incurred blood samples is 0.95. Clinical Trial Registration: NCT04323124 (ClinicalTrials.gov).

Effects of nirmatrelvir/ritonavir on midazolam and dabigatran pharmacokinetics in healthy participants.

AIMS: To evaluate pharmacokinetics (PK) and safety after coadministration of nirmatrelvir/ritonavir or ritonavir alone with midazolam (a cytochrome P450 3A4 substrate) and dabigatran (a P-glycoprotein substrate). METHODS: PK was studied in 2 phase 1, open-label, fixed-sequence studies in healthy adults. Single oral doses of midazolam 2 mg (n = 12) or dabigatran 75 mg (n = 24) were administered alone and after steady state (i.e. ≥2 days) of nirmatrelvir/ritonavir 300 mg/100 mg and ritonavir 100 mg. Midazolam and dabigatran plasma concentrations and adverse events were analysed for each treatment. RESULTS: After administration of midazolam with nirmatrelvir/ritonavir (test) or alone (reference), midazolam geometric mean area under the concentration-time curve extrapolated to infinity (AUCinf ) and maximum plasma concentration (Cmax ) increased 14.3-fold and 3.7-fold, respectively. Midazolam coadministered with ritonavir (test) or alone (reference) resulted in 16.5-fold and 3.9-fold increases in midazolam geometric mean AUCinf and Cmax , respectively. After administration of dabigatran with nirmatrelvir/ritonavir (test) or alone (reference), dabigatran geometric mean AUCinf and Cmax increased 1.9-fold and 2.3-fold, respectively. Dabigatran coadministered with ritonavir (test) or alone (reference) resulted in a 1.7-fold increase in dabigatran geometric mean AUCinf and Cmax . Midazolam or dabigatran exposures were generally comparable when coadministered with nirmatrelvir/ritonavir or ritonavir alone, with a slightly higher dabigatran Cmax with nirmatrelvir/ritonavir vs. ritonavir alone. Nirmatrelvir/ritonavir was generally safe when administered with or without midazolam or dabigatran. No serious or severe adverse events were reported. CONCLUSION: Coadministration of midazolam or dabigatran with nirmatrelvir/ritonavir increased systemic exposure of midazolam or dabigatran. Midazolam exposures were comparable when coadministered with nirmatrelvir/ritonavir or ritonavir alone, suggesting no incremental effect of nirmatrelvir. Dabigatran Cmax was slightly higher when coadministered with nirmatrelvir/ritonavir compared with of ritonavir alone, suggesting a minor incremental effect of nirmatrelvir.

Bioanalytical method validation and sample analysis for nirmatrelvir in dried blood collected using the Tasso-M20 device.

Aim: A sensitive and selective method for the determination of nirmatrelvir in dried human blood collected by Tasso-M20 was developed and validated from 20.0 to 20,000 ng/ml. Materials & methods: Nirmatrelvir and its stable-labeled internal standard were isolated from approximately 20 µl of blood dried on one volumetric absorptive pad inside the Tasso-M20 device by extraction with methanol, followed by dilution of the supernatant. The extracts were analyzed by high-performance liquid chromatography coupled with tandem mass spectrometric detection. Results & conclusion: The method was fully validated. Hematocrit levels do not impact assay accuracy. Stabilities to cover sample drying and storage at a variety of conditions were conducted. The validated method was used in multiple clinical studies with excellent performance.

2021 White Paper on Recent Issues in Bioanalysis: Mass Spec of Proteins, Extracellular Vesicles, CRISPR, Chiral Assays, Oligos; Nanomedicines Bioanalysis; ICH M10 Section 7.1; Non-Liquid & Rare Matrices; Regulatory Inputs (Part 1A - Recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC & Part 1B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 1A) covers the recommendations on Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC. Part 1B covers the Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine. Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) and Part 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparabil ity & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 10 and 11 (2022), respectively.

Metabolism and Disposition of a Novel B-Cell Lymphoma-2 Inhibitor Venetoclax in Humans and Characterization of Its Unusual Metabolites.

Venetoclax (ABT-199), a B-cell lymphoma-2 (Bcl-2) protein inhibitor, is currently in clinical development for the treatment of hematologic malignancies. We characterized the absorption, metabolism, and excretion of venetoclax in humans. After a single oral dose of [14C]venetoclax to healthy volunteers, the recovery of total radioactive dose was 100%, with feces being the major route of elimination of the administered dose, whereas urinary excretion was minimal (<0.1%). The extent of absorption was estimated to be at least 65%. Venetoclax was primarily cleared by hepatic metabolism (∼66% of the administered dose). ∼33% of the administered dose was recovered as the parent drug and its nitro reduction metabolite M30 [2-((1H-pyrrolo[2,3-b]pyridin-5-yl)oxy)-N-((3-amino-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-4-(4-((4'-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1'-biphenyl]-2-yl)methyl)piperazin-1-yl)benzamide] (13%) in feces. Biotransformation of venetoclax in humans primarily involves enzymatic oxidation on the dimethyl cyclohexenyl moiety, followed by sulfation and/or nitro reduction. Nitro reduction metabolites were likely formed by gut bacteria. Unchanged venetoclax was the major drug-related material in circulation, representing 72.8% of total plasma radioactivity. M27 (oxidation at the 6 position of cyclohexenyl ring followed by cyclization at the α-carbon of piperazine ring; 4-[(10aR,11aS)-7-(4-chlorophenyl)-9,9-dimethyl-1,3,4,6,8,10,10a,11a-octahydropyrazino[2,1-b][1,3]benzoxazin-2-yl]-N-[3-nitro-4-(tetrahydropyran-4-ylmethylamino)phenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) was identified as a major metabolite, representing 12% of total drug-related material. M27 was primarily formed by cytochrome P450 isoform 3A4 (CYP3A4). Steady-state plasma concentrations of M27 in human and preclinical species used for safety testing suggested that M27 is a disproportionate human metabolite. M27 is not expected to have clinically relevant on- or off-target pharmacologic activities.

Toxicokinetic evaluation of atrasentan in mice utilizing serial microsampling: validation and sample analysis in GLP study.

BACKGROUND: A semi-automated 96-well protein precipitation followed by HPLC-MS/MS method for the determination of atrasentan (2R-[4-methoxyphenyl]-4S-[1,3-benzodioxol-5-yl]-1-[N,N-di-(N-butyl)-aminocarbonyl-methyl]-pyrrolidine-3R-carboxylic acid) in mouse whole blood was developed, validated and utilized in GLP toxicokinetic evaluations. Six 40-µl whole blood samples were collected from a single mouse over the course of a 12 h blood collection window. To avoid sample volume losses, whole blood was selected as the matrix in place of the more typically used plasma. A 10-µl assay volume was used to ensure sufficient volumes are available for dilutions, repeats and incurred sample reanalysis. The samples (10-µl aliquot) were fortified with stable-labeled internal standard (d18-atrasentan) and lysed thoroughly prior to protein precipitation. The chromatographic separation was performed on a Zorbax(®) SB-C18 (50 x 2.1 mm; 5 µm) HPLC column with a mobile phase consisting of 25 mM ammonium acetate and 0.25% (v/v) acetic acid in 50/50 (v/v) acetonitrile/water. The MS measurement was conducted under positive ion mode using multiple-reaction monitoring of m/z 511→354 for analyte and 529→354 for stable-labeled internal standard. The peak area ratio (analyte:stable-labeled internal standard) was used to quantitate atrasentan. RESULTS: A dynamic range of 5-1400 ng/ml was established after validation. The challenges associated with a small-volume whole-blood assay involved anticoagulant overloading with commercial blood collection tubes, managing phospholipids to ensure a robust assay and automation. In-depth discussions are provided in this article. The validated method was then used for GLP toxicokinetic evaluations. To demonstrate the method reproducibility, approximately 10% of the incurred samples from the study were repeated in singlet. Excellent assay reproducibility was demonstrated where 100% of samples met incurred sample reanalysis acceptance criteria. CONCLUSION: Good quality exposure data were obtained from every serial sampled mouse in the study.

Quantitative Determination of ABT-925 in Human Plasma by On-Line SPE and LC-MS/MS: Validation and Sample Analysis in Phase II Studies.

A fully automated 96-well On-Line Solid Phase Extraction (SPE) followed by High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometric (MS/MS) method for the determination of ABT-925 (2-{3-[4-(2-tert-Butyl-6-trifluoromethyl-pyrimidin-4-yl)-piperazin-1-yl)-propyl-sulfanyl}-3H-pyrimidin-4-one fumarate) in human plasma was developed, validated and utilized in Phase II clinical studies. 50 µL of plasma sample was fortified with internal standard (IS, d8-ABT-925) and extracted on-line with Cohesive Turbo Flow Cyclone P HTLC column. The chromatographic separation was performed on Aquasil C18 (3 µm 50 × 3 mm) HPLC column with a mobile phase consisting of 50/50/0.1 (v/v/v) ACN/H2O/formic acid. The mass spectrometric measurement was conducted under positive ion mode using multiple reaction monitoring (MRM) of m/z 457.4 → 329.4 for analyte and m/z 465.5 → 337.5 for IS.The peak area ratio (analyte/IS) was used to quantitate ABT-925. A dynamic range of 0.0102 µg/mL to 5.24 µg/mL was established after the validation. The validated method was then used for two Phase II studies. To demonstrate the method reproducibility, approximately 10% of the incurred samples from one study were repeated in singlet. The repeated values were compared to the initial values. All repeated values agreed within ±15% of the mean values.

ABT-335, the choline salt of fenofibric acid, does not have a clinically significant pharmacokinetic interaction with rosuvastatin in humans.

ABT-335 is the choline salt of fenofibric acid under clinical development as a combination therapy with rosuvastatin for the management of dyslipidemia. ABT-335 and rosuvastatin have different mechanisms of actions and exert complementary pharmacodynamic effects on lipids. The current study assessed the pharmacokinetic interaction between the 2 drugs following a multiple-dose, open-label, 3-period, randomized, crossover design. Eighteen healthy men and women received 40 mg rosuvastatin alone, 135 mg ABT-335 alone, and the 2 drugs in combination once daily for 10 days. Blood samples were collected prior to dosing on multiple days and up to 120 hours after day 10 dosing for the measurements of fenofibric acid and rosuvastatin plasma concentrations. Coadministering 40 mg rosuvastatin had no significant effect on the steady-state Cmax, Cmin, or AUC24 of fenofibric acid (P > .05). Coadministering ABT-335 had no significant effect on the steady-state Cmin or AUC24 of rosuvastatin (P > .05) but increased Cmax by 20% (90% confidence interval: 12%-28%). All 3 regimens were generally well tolerated with no clinically significant changes in clinical laboratory values, vital signs, or electrocardiograms during the study. Results from this study demonstrate no clinically significant pharmacokinetic interaction between ABT-335 at the full clinical dose and rosuvastatin at the highest approved dose.

Comparing similar spectra: from similarity index to spectral contrast angle.

We investigated a spectral-contrast-angle (theta) method to determine whether mass spectra of structural isomers are the same or significantly different. This method represents collisionally activated dissociation (CAD) spectra as vectors in space. Mass spectra of different isomers are represented as different vectors, having characteristic lengths and direction. The derived spectral contrast angle, which is a measure of the angle between two vectors corresponding to two closely related spectra, is a measure of whether the mass spectra are the same or significantly different. We compare this method with the similarity index (SI) method and show that the spectral contrast angle method is superior and can differentiate between very similar spectra in cases where the SI cannot. Both methods can be implemented simply in situations where the analyst is called on to decide, on the basis of mass or product-ion spectra, whether reference and unknown compounds are the same or to evaluate the reproducibility of spectra comprised of many peaks.