Disposition of Nirmatrelvir, an Orally Bioavailable Inhibitor of SARS-CoV-2 3C-Like Protease, across Animals and Humans.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease inhibitor PF-07321332 (nirmatrelvir), in combination with ritonavir (Paxlovid), was recently granted emergency use authorization by multiple regulatory agencies for the treatment of coronavirus disease 2019 (COVID-19) in adults and pediatric patients. Disposition studies on nirmatrelvir in animals and in human reagents, which were used to support clinical studies, are described herein. Plasma clearance was moderate in rats (27.2 ml/min per kg) and monkeys (17.1 ml/min per kg), resulting in half-lives of 5.1 and 0.8 hours, respectively. The corresponding oral bioavailability was moderate in rats (34%-50%) and low in monkeys (8.5%), primarily due to oxidative metabolism along the gastrointestinal tract in this species. Nirmatrelvir demonstrated moderate plasma protein binding in rats, monkeys, and humans with mean unbound fractions ranging from 0.310 to 0.478. The metabolism of nirmatrelvir was qualitatively similar in liver microsomes and hepatocytes from rats, monkeys, and humans; prominent metabolites arose via cytochrome P450 (CYP450)-mediated oxidations on the P1 pyrrolidinone ring, P2 6,6-dimethyl-3-azabicyclo[3.1.0]hexane, and the tertiary-butyl group at the P3 position. Reaction phenotyping studies in human liver microsomes revealed that CYP3A4 was primarily responsible (fraction metabolized = 0.99) for the oxidative metabolism of nirmatrelvir. Minor clearance mechanisms involving renal and biliary excretion of unchanged nirmatrelvir were also noted in animals and in sandwich-cultured human hepatocytes. Nirmatrelvir was a reversible and time-dependent inhibitor as well as inducer of CYP3A activity in vitro. First-in-human pharmacokinetic studies have demonstrated a considerable boost in the oral systemic exposure of nirmatrelvir upon coadministration with the CYP3A4 inhibitor ritonavir, consistent with the predominant role of CYP3A4 in nirmatrelvir metabolism. SIGNIFICANCE STATEMENT: The manuscript describes the preclinical disposition, metabolism, and drug-drug interaction potential of PF-07321332 (nirmatrelvir), an orally active peptidomimetic-based inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease, which has been granted emergency use authorization by multiple regulatory agencies around the globe for the treatment of coronavirus disease 2019 (COVID-19) in COVID-19-positive adults and pediatric patients who are at high risk for progression to severe COVID-19, including hospitalization or death.

Estimation of Circulating Drug Metabolite Exposure in Human Using In Vitro Data and Physiologically Based Pharmacokinetic Modeling: Example of a High Metabolite/Parent Drug Ratio.

(R)-4-((4-(((4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol (TBPT), a serotonin-4 receptor partial agonist, is metabolized to two metabolites: an N-dealkylation product [(R)-3-(piperidin-4-ylmethoxy)-4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazole (M1)] and a cyclized oxazolidine structure [7-(((4-(((R)-tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)octahydro-3H (M2)]. After administration of TBPT to humans the exposure to M1 was low and the exposure to M2 was high, relative to the parent drug, despite this being the opposite in vitro. In this study, projection of the plasma metabolite/parent (M/P) ratios for M1 and M2 was attempted using in vitro metabolism, binding, and permeability data in static and dynamic physiologically based pharmacokinetic (PBPK) models. In the static model, the fraction of parent clearance yielding the metabolite (which also required taking into account secondary metabolites of M1 and M2), the clearance of the metabolites and parent, and an estimate of the availability of the metabolites from the liver were combined to yield estimated parent/metabolite ratios of 0.32 and 23 for M1 and M2, respectively. PBPK modeling that used in vitro and physicochemical data input yielded estimates of 0.26 and 20, respectively. The actual values were 0.12 for M1/TBPT and 58 for M2/TBPT. Thus, the ratio for M1 was overpredicted, albeit at values less than unity. The ratio for M2/TBPT was underpredicted, and the high ratio of 58 may exceed a limiting ceiling of the approach. Nevertheless, when considered in the context of determining whether a potential circulating metabolite may be quantitatively important prior to administration of a drug for the first time to humans, the approaches succeeded in highlighting the importance of M2 (M/P ratio >> 1) relative to M1, despite M1 being much greater than M2 in vitro.

Inception and development of a LC-MS/MS assay for the multiplexed quantitation of nine human drug transporter biomarkers.

Background: It has become common practice to assess solute carrier transporter (SLC)-mediated drug-drug interactions (DDIs) by quantitating various individual endogenous compounds as biomarkers in human plasma and urine. The goal of this work was to develop biomarker multiplex assays that could be utilized during first in human studies to support the simultaneous assessment of clinical DDI risk across various SLCs. Methodology: Hydrophilic interaction chromatography-MS/MS methods were developed, and validations were performed. Results: The multiplex assays were applied to a first in human study. Placebo/reference subject biomarker data were consistent with single assay in-house and published data. Conclusion: This work demonstrates the utility of these multiplex methods to support the concurrent evaluation of clinical DDI risk across various SLCs.

Development of a sensitive LC-MS/MS assay to support human microdose study for an oral agonist of the GLP-1 receptor.

Aim: The purpose of this work was to determine the feasibility of supporting a clinical microdose study for PF-06882961 (danuglipron), an oral small molecule agonist of the GLP-1 receptor, by LC-MS/MS. Methodology: Statistical instrument parameter optimization using response surface methodology was employed to develop a LC-MS/MS method for the analyte, PF-06882961. Results: An LC-MS/MS method was developed and validated to support a proof of concept microdose pharmacokinetics preclinical study in monkeys, administered PF-06882961 (0.005 mg total, average dose = 0.0007 mg/kg) via intravenous bolus injection. Conclusion: The present study demonstrated the feasibility of analyzing human microdose plasma samples for PF-06882961 by LC-MS/MS, instead of accelerator mass spectrometry, thereby reducing cost and eliminating synthesis and exposure to 14C labeled material.

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A Novel Milli-fluidic Liver Tissue Chip with Continuous Recirculation for Predictive Pharmacokinetics Applications.

A crucial step in lead selection during drug development is accurate estimation and optimization of hepatic clearance using in vitro methods. However, current methods are limited by factors such as lack of physiological relevance, short culture/incubation times that are not consistent with drug exposure patterns in patients, use of drug absorbing materials, and evaporation during long-term incubation. To address these technological needs, we developed a novel milli-fluidic human liver tissue chip (LTC) that was designed with continuous media recirculation and optimized for hepatic cultures using human primary hepatocytes. Here, we characterized the LTC using a series of physiologically relevant metrics and test compounds to demonstrate that we could accurately predict the PK of both low- and high-clearance compounds. The non-biological characterization indicated that the cyclic olefin copolymer (COC)-based LTC exhibited negligible evaporation and minimal non-specific binding of drugs of varying ionic states and lipophilicity. Biologically, the LTC exhibited functional and polarized hepatic culture with sustained metabolic CYP activity for at least 15 days. This long-term culture was then used for drug clearance studies for low- and high-clearance compounds for at least 12 days, and clearance was estimated for a range of compounds with high in vitro-in vivo correlation (IVIVC). We also demonstrated that LTC can be induced by rifampicin, and the culture age had insignificant effect on depletion kinetic and predicted clearance value. Thus, we used advances in bioengineering to develop a novel purpose-built platform with high reproducibility and minimal variability to address unmet needs for PK applications.

Metabolism and Excretion of Nirmatrelvir in Humans Using Quantitative Fluorine Nuclear Magnetic Resonance Spectroscopy: A Novel Approach for Accelerating Drug Development.

Typically human absorption, distribution, metabolism, and excretion (ADME) studies are executed using radiolabeled (e.g., carbon-14) material, the synthesis of which is a time-consuming activity. In this study, we were able to assess the metabolism and excretion of unlabeled nirmatrelvir (PF-07321332) within the first-in-human study via a novel application of quantitative fluorine (19 F) nuclear magnetic resonance (NMR) spectroscopy in place of a standard radiolabel ADME study. Six healthy participants received a single 300-mg oral dose of nirmatrelvir (in combination with ritonavir), and excreta were collected up to 10 days. Virtually all drug-related material was recovered within 5 days, and mass balance was achieved with 84.9 ± 8.9% (range = 70.7-95.5%) of the administered dose recovered in urine and feces. The excretion of fluorine-containing material in urine and feces was 47.0% and 33.7%, respectively. Unchanged nirmatrelvir represented 82.5% of the normalized drug-related material with a carboxylic acid metabolite M5, derived from hydrolysis of the P2 amide bond, present at 12.1% of dose. Nirmatrelvir was the only drug-related entity observed in plasma. Approximately 4.2% of the dose was excreted as metabolite M8 (measured by liquid chromatography-mass spectrometry), which was 19 F NMR silent due to hydrolysis of the trifluoroacetamide moiety. Hydrolysis of nirmatrelvir to M5 and M8 was shown to occur in cultures of human gut microflora. This successful demonstration of quantitative 19 F NMR spectroscopy to establish the mass-balance, excretion, and metabolic profile of nirmatrelvir offers an advantageous means to execute human ADME studies for fluorine-containing compounds early in drug development.

Innovative Randomized Phase I Study and Dosing Regimen Selection to Accelerate and Inform Pivotal COVID-19 Trial of Nirmatrelvir.

Coronavirus disease 2019 (COVID-19) is a continued leading cause of hospitalization and death. Safe, efficacious COVID-19 antivirals are needed urgently. Nirmatrelvir (PF-07321332), the first orally bioavailable, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) Mpro inhibitor against the coronaviridae family, has demonstrated potent preclinical antiviral activity and benign safety profile. We report safety, tolerability, and pharmacokinetic data of nirmatrelvir with and without ritonavir as a pharmacokinetic enhancer, from an accelerated randomized, double-blind, placebo-controlled, phase I study. Two interleaving single-ascending dose (SAD) cohorts were evaluated in a three-period crossover. Multiple-ascending dose (MAD) with nirmatrelvir/ritonavir twice daily (b.i.d.) dosing was evaluated over 10 days in five parallel cohorts. Safety was assessed, including in a supratherapeutic exposure cohort. Dose and dosing regimen for clinical efficacy evaluation in phase II/III clinical trials were supported by integrating modeling and simulations of SAD/MAD data with nonclinical data and a quantitative systems pharmacology model (QSP). In SAD, MAD, and supratherapeutic exposure cohorts, nirmatrelvir/ritonavir was safe and well-tolerated. Nirmatrelvir exposure and half-life were considerably increased by ritonavir, enabling selection of nirmatrelvir/ritonavir dose and regimen for phase II/III trials (300/100 mg b.i.d.), to achieve concentrations continuously above those required for 90% inhibition of viral replication in vitro. The QSP model suggested that a 5-day regimen would significantly decrease viral load in SARS-CoV-2-infected patients which may prevent development of severe disease, hospitalization, and death. In conclusion, an innovative and seamless trial design expedited establishment of phase I safety and pharmacokinetics of nirmatrelvir/ritonavir, enabling high confidence in phase II/III dose selection and accelerated pivotal trials' initiation (NCT04756531).

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Discovery of a Novel Inhibitor of Coronavirus 3CL Protease for the Potential Treatment of COVID-19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, in vitro , and in vivo antiviral activity data that supports the clinical evaluation of this compound as a potential COVID-19 treatment.

Application of hydrophilic interaction chromatography retention coefficients for predicting peptide elution with TFA and methanesulfonic acid ion-pairing reagents.

Hydrophilic retention coefficients for 17 peptides were calculated based on retention coefficients previously published for TSKgel silica-60 and were compared with the experimental elution profile on a Waters Atlantis HILIC silica column using TFA and methanesulfonic acid (MSA) as ion-pairing reagents. Relative peptide retention could be accurately determined with both counter-ions. Peptide retention and chromatographic behavior were influenced by the percent acid modifier used with increases in both retention and peak symmetry observed at increasing modifier concentrations. The enhancement of net peptide polarity through MSA pairing shifted retention out by nearly five-fold for the earliest eluting peptide, compared with TFA. Despite improvements in retention and efficiency (N(eff)) for MSA over TFA, a consistent reduction in calculated selectivity (alpha) was observed. This result is believed to be attributed to the stronger polar contribution of MSA masking and diminishing the underlying influence of the amino acid residues of each associated peptide. Finally, post-column infusion of propionic acid and acetic acid was evaluated for their potential to recover signal intensity for TFA and MSA counter-ions for LC-ESI-MS applications. Acetic acid generally yielded more substantial signal improvements over propionic acid on the TFA system while minimal benefits and some further reductions were noted with MSA.

Remediation of undesirable secondary interactions encountered in hydrophilic interaction chromatography during development of a quantitative LC-MS/MS assay for a dipeptidyl peptidase IV (DPP-IV) inhibitor in monkey serum.

PF-00734200 (3,3-Difluoropyrrolidin-1-yl)-((2S,4S)-4-(4-(pyrimidin-2-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone) is an inhibitor of dipeptidyl peptidase IV (DPP-IV) for the treatment of diabetic complications and other disorders. A sensitive and selective LC-MS/MS assay capable of quantifying PF-00734200 in monkey serum was required to support regulated safety studies. Due to the polar nature of this compound and for ease of sample processing, hydrophilic interaction chromatography (HILIC) was identified as an ideal assay technique. During the initial phase of method development significant peak tailing was observed. The effects of polar organic modifier percentage, buffer concentration, column particle size, and flow rate were assessed to determine the final optimal conditions. PF-00734200 demonstrated a strong dependence on buffer concentration with respect to height equivalent to a theoretical plate (HETP), capacity factor (k'), and tailing factor (T). Improvements in chromatography were observed with increasing buffer concentration due to reduction of electrostatic secondary interactions with ionized silanols. A plot of logk' versus percentage organic modifier at an elevated buffer concentration, produced a linear fit with a correlation coefficient of 0.996, indicating that the primary chromatographic retention mechanism was partitioning. A LC-MS/MS assay was successfully developed and validated for GLP bioanalysis of PF-00734200 in monkey serum utilizing the optimized HILIC conditions. Additionally, carryover was effectively minimized through fortification of ethylene glycol to the sample extract.

Rapid determination of the applicability of hydrophilic interaction chromatography utilizing ACD labs log D suite: a bioanalytical application.

Hydrophilic interaction chromatography (HILIC) is an effective technique for retaining and separating polar compounds. This approach offers several advantages for bioanalytical liquid chromatography/mass spectrometry, considering that a majority of active pharmaceutical ingredients are polar amines. HILIC employs high concentrations of relatively polar organic mobile phase components (i.e. acetonitrile), providing enhanced desolvation and electrospray ionization efficiency, as well as allowing direct injection of many protein precipitation, liquid/liquid, and solid phase extracts. A set of 30 probe compounds was evaluated to demonstrate a relationship between a compound's HILIC capacity factor (k'), and pH dependent distribution coefficient (D), using three sets of generic isocratic conditions. Plots of logk' versus logD(pH3.0) produced correlation coefficients of 0.751, 0.696, and 0.689 at acetonitrile mobile phase concentrations of 85%, 90%, and 95% (v/v), respectively. For bioanalytical applications a k'>2 is typically targeted to ensure adequate retention of a given analyte relative to extracted matrix components. Using k'> or =2 as a measure of HILIC applicability, the linear relationships for each of the three acetonitrile levels predicted whether or not HILIC was able to meet this criterion for at least 90% of the compounds tested. Overall, the relationship between k' and logD can serve as a valuable tool for identifying the applicability of HILIC and a starting point for the chromatographic conditions, prior to the initiation of any laboratory activities. Additionally, this relationship can assist with the selection of appropriate chemical analog internal standards.

Evaluation of the relationship between a pharmaceutical compound's distribution coefficient, log D and adsorption loss to polypropylene in urine and CSF.

BACKGROUND: The distribution coefficient, D, is a physicochemical property used to determine the partitioning of compounds between aqueous and hydrophobic media at a given pH. RESULTS: A clear relationship was observed between the calculated pH-dependent distribution coefficient of six representative pharmaceutical probe compounds and their propensity to partition between a relatively hydrophobic polypropylene surface and the aqueous matrices, human urine or human cerebrospinal fluid (CSF). Compound log D cut-off values of 1.5 and 3.8 for urine and CSF, respectively, were determined using a threshold of less than 20% adsorption to the polypropylene surface. CONCLUSION: The ability to forecast the adsorption of a given compound to a polypropylene container with urine and CSF offers an effective means for screening potential issues and identifying when additional testing and corrective measures may need to be applied.

Reduction of in-source collision-induced dissociation and thermolysis of sulopenem prodrugs for quantitative liquid chromatography/electrospray ionization mass spectrometric analysis by promoting sodium adduct formation.

Six chromatographically resolved sulopenem prodrugs were monitored for their potential to undergo both in-source collision-induced dissociation (CID) and thermolysis. Initial Q1 scans for each prodrug revealed the formation of intense [Prodrug2 + H]+, [Prodrug2 + Na]+, [Prodrug + Na]+, and [Sulopenem + Na]+ ions. Non-adduct-associated sulopenem ([Sulopenem + H]+) along with several additional lower mass ions were also observed. Product ion scans of [Prodrug3 + Na]+ showed the retention of the sodium adduct in the collision cell continuing down to opening of the beta-lactam ring. In-source CID and temperature experiments were conducted under chromatographic conditions while monitoring several of the latter ion transitions (i.e., adducts, dimers and degradants/fragments) for a given prodrug. The resulting ion profiles indicated the regions of greatest stability for temperature and declustering potential (DP) that provided the highest signal intensity for each prodrug and minimized in-source degradation. The heightened stability of adduct ions, relative to their appropriate counterpart (i.e., dimer to dimer adduct and prodrug to prodrug adduct ions), was observed under elevated temperature and DP conditions. The addition of 100 microM sodium to the mobile phase further enhanced the formation of these more stable adduct ions, yielding an optimal [Prodrug + Na]+ ion signal at temperatures from 400 to 600 degrees C. A clinical liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay for sulopenem prodrug PF-04064900 in buffered whole blood was successfully validated using sodium-fortified mobile phase and the [PF-04064900 + Na]+ ion for quantitation. A conservative five-fold increase in sensitivity from previously validated preclinical assays using the [PF-04064900 + H]+ precursor ion was achieved.

Initial rate analysis of zoniporide hydrolysis degradants using high-performance liquid chromatography coupled with mass spectrometric detection.

Zoniporide, N-(5-Cyclopropyl-1-quinolin-5-yl-1H-pyrazole-4-carbonyl)-guanidine methanesulfonic acid, is a sodium-hydrogen exchanger type 1 (NHE-1) inhibitor. This compound forms two major hydrolysis degradants (Degradants I and II) and therefore was formulated as an IV concentrate lyophile. The purpose of this study was to perform initial rate analysis on formation of Degradants I and II, to determine if a liquid Zoniporide formulation is feasible. Solutions of Zoniporide were placed on stability at ambient temperature (30 degrees C), refrigerated temperature (5 degrees C), and frozen temperature (-20 degrees C). Initial formation rates were determined for Degradants I and II by using high-performance liquid chromatography/mass spectrometry (HPLC/MS). The initial formation rates were used to predict the time required for a degradant level of 0.1% relative to Zoniporide to be reached. The predicted times for Degradant I to reach 0.1% were 9 days, 330 days, and 30,962 days at temperatures of 30 degrees C, 5 degrees C, and -20 degrees C, respectively, indicating that refrigerated or frozen storage would be required for a liquid Zoniporide formulation to be feasible and reach the target shelf life. The initial formation rates of Degradant II were approximately 1-order of magnitude lower. In Addition, samples of the Zoniporide solutions were assayed for concentrations of Degradant I and Zoniporide after 2.5 years of storage at 5 degrees C and -20 degrees C to determine the validity of the initial formation rate predictions. The mean experimentally determined Degradant I concentration at 5 degrees C was within 13% of the predicted concentration, and the mean experimentally determined Degradant I concentration at -20 degrees C was within 37% of the predicted concentration. This agreement between the experimentally determined and predicted Degradant I concentrations indicates that the initial rate method is sufficiently accurate and can be used to rapidly evaluate various formulation conditions with respect to stability.