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.

Simplifying the Execution of HepatoPac MetID Experiments: Metabolite Profile and Intrinsic Clearance Comparisons.

The HepatoPac micropatterned coculture (MPCC) hepatocyte system has been shown to be an effective tool to investigate the qualitative human and preclinical species' metabolite profiles of new drug candidates. However, additional improvements to the overall study conditions and execution, layout, and human-donor count could be made. To that end, we have evaluated several ways to increase the amount of data one can generate per MPCC plate and how to more efficiently execute a MPCC study for the purpose of metabolite generation. Herein, we compare a set of compounds using single- and 10-donor pooled human MPCC hepatocytes. Intrinsic clearance and mean metabolic activities assessed by diverse enzyme markers were comparable between the single- and 10-donor pool. We have confirmed that the generated metabolite profiles were indistinguishable between the single- and 10-donor pool and also that rat MPCC can be performed at 400 µl media volume, which greatly simplifies study execution. Additional tips for successful study execution are also described. SIGNIFICANCE STATEMENT: When using the HepatoPac micropatterned coculture (MPCC) system, sometimes simple experimental condition variables or problematic plate designs can hamper productive study execution. We evaluated conditions to increase the amount of data one can generate per MPCC plate and, perhaps more importantly, execute that study more efficiently with less likelihood of error. We describe some of our key learnings, provide an examination of enzyme activity levels and clearance values, and provide some recommendations to simplify the execution of a HepatoPac experiment.

Application of a Micropatterned Cocultured Hepatocyte System To Predict Preclinical and Human-Specific Drug Metabolism.

Laboratory animal models are the industry standard for preclinical risk assessment of drug candidates. Thus, it is important that these species possess profiles of drug metabolites that are similar to those anticipated in human, since metabolites also could be responsible for biologic activities or unanticipated toxicity. Under most circumstances, preclinical species reflect human in vivo metabolites well; however, there have been several notable exceptions, and understanding and predicting these exceptions with an in vitro system would be very useful. Human micropatterned cocultured (MPCC) hepatocytes have been shown to recapitulate human in vivo qualitative metabolic profiles, but the same demonstration has not been performed yet for laboratory animal species. In this study, we investigated several compounds that are known to produce human-unique metabolites through CYP2C9, UGT1A4, aldehyde oxidase (AO), or N-acetyltransferase that were poorly covered or not detected at all in the selected preclinical species. To perform our investigation we used 24-well MPCC hepatocyte plates having three individual human donors and a single donor each of monkey, dog, and rat to study drug metabolism at four time points per species. Through the use of the multispecies MPCC hepatocyte system, the metabolite profiles of the selected compounds in human donors effectively captured the qualitative in vivo metabolite profile with respect to the human metabolite of interest. Human-unique metabolites that were not detected in vivo in certain preclinical species (normally dog and rat) were also not generated in the corresponding species in vitro, confirming that the MPCC hepatocytes can provide an assessment of preclinical species metabolism. From these results, we conclude that multispecies MPCC hepatocyte plates could be used as an effective in vitro tool for preclinical understanding of species metabolism relative to humans and aid in the choice of appropriate preclinical models.

Chromatographic resolution of atropisomers for toxicity and biotransformation studies in pharmaceutical research.

Atropisomerism can be a complex concept for those who have not encountered it before. This paper discusses the experiments for identification, isolation, thermal stability, toxicity and biotransformation of various species. The identified atropisomers are a series of rotational hindered biaryl, rotational hindered amide, ring flip, and macrocycles atropisomers identified using supercritical fluid chromatography (SFC) and high performance liquid chromatography (HPLC). These technologies offered the advantage of separating various atropoenantiomers, atropdiastereomers and mixed atropisomers with other forms of stereoisomers in both analytical and preparative scales. With ultra-performance convergence chromatography (UPC(2)), the detection of N-oxide atropisomer metabolites can be obtained at very low level thus enabling the observation of conversion in human plasma possible. As the resolution of atropisomers are related to the energy barriers on the rotational axis, a calculated computational protocol was developed to predict the formation. A threshold of 10kcal/mol was established for possible detection of the atropisomers' existence with chromatographic technologies at room temperature or above. The atropisomer with higher energy barrier (>20kcal/mol) were isolated via preparative chromatography and the isolates studied in vitro and in vivo for evaluation of their stability in human plasma. The detailed analytical method development to analyze the biotransformation of the atropisomers in human plasma are also discussed in this paper.

Metabolism and clinical pharmacokinetics of 2-methyl-n-(2'-(pyrrolidinyl-1-ylsulfonyl)-n-[1,1'-biphenyl]-4-yl)propran-1-amine: insights into monoamine oxidase- and CYP-mediated disposition by integration of in vitro ADME tools.

1. In early discovery stages, 2-methyl-N-(2'-(pyrrolidinyl-1-ylsulfonyl)-[1,1'-biphenyl]-4-yl)propan-1-amine (PBPA) demonstrated monoamine oxidase A (MAO-A) and cytochrome P450 (CYP)-mediated clearance. While human liver microsomes predicted low CL(b) PBPA demonstrated a moderate CL(p)/F in humans. The plasma pharmacokinetic (PK) of PBPA was characterized by unexpected high inter-individual variability. Hence, a retrospective analysis was undertaken to understand the disposition processes of PBPA, by applying in vitro mechanistic tools. 2. The in vitro-to-in vivo of rat CL(b) of PBPA was calculated as similar to that of human, suggesting rat to be a better predictor of a MAO-A/CYP substrate, but not dog or monkey; this is consistent with differences in expression of MAO-A in rat, dog, monkey and human. Fraction metabolized (f(m)) of human MAO A (hMAO-A) (50%), CYP3A4 (8%), CYP3A5 (16%) and CYP2D6 (29%) was determined, in vitro. 3. While the fm of CYP3A5 was <50%, Michaelis-Menten kinetics demonstrated that it was a higher capacity pathway compared with MAO-A, 2D6 and 3A4. This was consistent with strong association of dose-normalized plasma C(max) and area under the plasma concentration time curve (AUC(0-tlast)) of PBPA with CYP3A5 genotype, but not with genotype of CYP2D6. 4. This investigation demonstrates the value of integrating in vitro mechanistic tools to gain comprehensive understanding of disposition properties of drug candidates, in a discovery paradigm and prior to the investment in clinical trials.

Design and discovery of a selective small molecule κ opioid antagonist (2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine, PF-4455242).

By use of parallel chemistry coupled with physicochemical property design, a series of selective κ opioid antagonists have been discovered. The parallel chemistry strategy utilized key monomer building blocks to rapidly expand the desired SAR space. The potency and selectivity of the in vitro κ antagonism were confirmed in the tail-flick analgesia model. This model was used to build an exposure-response relationship between the κ K(i) and the free brain drug levels. This strategy identified 2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine, PF-4455242, which entered phase 1 clinical testing and has demonstrated target engagement in healthy volunteers.

Pharmacokinetics, metabolism, and excretion of torcetrapib, a cholesteryl ester transfer protein inhibitor, in humans.

The pharmacokinetics, metabolism, and excretion of torcetrapib, a selective inhibitor of human cholesteryl ester transfer protein, were investigated in healthy human male volunteers after oral administration of [(14)C]torcetrapib (120-mg dose). The total mean recovery of radiolabeled dose after 21 days was 75.7%, and most of the dose (63%) was excreted in the urine. The total circulating radioactivity and unchanged torcetrapib plasma concentrations increased over the first 6 h and then declined slowly with mean terminal elimination half-lives of 373 and 211 h. Metabolism of torcetrapib was extensive in humans. Only 5.2% of the total dose constituted unchanged torcetrapib in the feces, whereas no parent was excreted unchanged in the urine. Similarly, pharmacokinetic analysis of total radioactivity and unchanged torcetrapib revealed that the area under the concentration versus time curve from zero to infinity of torcetrapib accounted for approximately 7.0% of the circulating radioactivity. Torcetrapib was metabolized to numerous metabolites via oxidation. The primary metabolic pathway involved initial oxidative decarbamoylation followed by extensive further oxidation, resulting in the formation of bistrifluoromethylbenzoic acid (M1) and quinaldic acid (M4) metabolites. A mean 40% of the total dose was excreted in the urine as M4 (and its glucuronide and urea conjugates), whereas 7.0% of the total dose was excreted as M1. In vitro studies using human subcellular fractions suggested that the initial metabolism of torcetrapib proceeds via CYP3A-mediated decarbamoylation. Subsequent oxidations lead to the major circulating and excretory metabolites M1 and M4.

Comparison of the bioactivation potential of the antidepressant and hepatotoxin nefazodone with aripiprazole, a structural analog and marketed drug.

In vitro metabolism/bioactivation of structurally related central nervous system agents nefazodone (hepatotoxin) and aripiprazole (nonhepatotoxin) were undertaken in human liver microsomes in an attempt to understand the differences in toxicological profile. NADPH-supplemented microsomal incubations of nefazodone and glutathione generated conjugates derived from addition of thiol to quinonoid intermediates. Inclusion of cyanide afforded cyano conjugates to iminium ions derived from alpha-carbon oxidation of the piperazine ring in nefazodone and downstream metabolites. Although the arylpiperazine motif in aripiprazole did not succumb to bioactivation, the dihydroquinolinone group was bioactivated via an intermediate monohydroxy metabolite to a reactive species, which was trapped by glutathione. Studies with synthetic dehydroaripiprazole metabolite revealed an analogous glutathione conjugate with molecular weight 2 Da lower. Based on the proposed structure of the glutathione conjugate(s), a bioactivation sequence involving aromatic ortho-or para-hydroxylation on the quinolinone followed by oxidation to a quinone-imine was proposed. P4503A4 inactivation studies in microsomes indicated that, unlike nefazodone, aripiprazole was not a time- and concentration-dependent inactivator of the enzyme. Overall, these studies reinforce the notion that not all drugs that are bioactivated in vitro elicit a toxicological response in vivo. A likely explanation for the markedly improved safety profile of aripiprazole (versus nefazodone) despite the accompanying bioactivation liability is the vastly improved pharmacokinetics (enhanced oral bioavailability, longer elimination half-life) due to reduced P4503A4-mediated metabolism/bioactivation, which result in a lower daily dose (5-20 mg/day) compared with nefazodone (200-400 mg/day). This attribute probably reduces the total body burden to reactive metabolite exposure and may not exceed a threshold needed for toxicity.

The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: evaluation using the MDR1A/1B knockout mouse model.

Thirty-two structurally diverse drugs used for the treatment of various conditions of the central nervous system (CNS), along with two active metabolites, and eight non-CNS drugs were measured in brain, plasma, and cerebrospinal fluid in the P-glycoprotein (P-gp) knockout mouse model after subcutaneous administration, and the data were compared with corresponding data obtained in wild-type mice. Total brain-to-plasma (B/P) ratios for the CNS agents ranged from 0.060 to 24. Of the 34 CNS-active agents, only 7 demonstrated B/P area under the plasma concentration curve ratios between P-gp knockout and wild-type mice that did not differ significantly from unity. Most of the remaining drugs demonstrated 1.1- to 2.6-fold greater B/P ratios in P-gp knockout mice versus wild-type mice. Three, risperidone, its active metabolite 9-hydroxyrisperidone, and metoclopramide, showed marked differences in B/P ratios between knockout and wild-type mice (6.6- to 17-fold). Differences in B/P ratios and cerebrospinal fluid/plasma ratios between wild-type and knockout animals were correlated. Through the use of this model, it appears that most CNS-active agents demonstrate at least some P-gp-mediated transport that can affect brain concentrations. However, the impact for the majority of agents is probably minor. The example of risperidone illustrates that even good P-gp substrates can still be clinically useful CNS-active agents. However, for such agents, unbound plasma concentrations may need to be greater than values projected using receptor affinity data to achieve adequate receptor occupancy for effect.

Sertraline is metabolized by multiple cytochrome P450 enzymes, monoamine oxidases, and glucuronyl transferases in human: an in vitro study.

The oxidative and conjugative metabolism of sertraline was examined in vitro to identify the enzymes involved in the generation of N-desmethyl, deaminated, and N-carbamoyl-glucuronidated metabolites in humans. In human liver microsomes, sertraline was N-demethylated and deaminated by cytochrome P450 (P450) enzymes with overall K(m) values of 98 and 114 microM, respectively, but the intrinsic clearance for N-demethylation was approximately 20-fold greater than for deamination. Using P450 isoform-selective inhibitors and recombinant heterologously expressed enzymes, it was demonstrated that several P450 enzymes catalyzed sertraline N-demethylation, with CYP2B6 contributing the greatest extent, and lesser contributions from CYP2C19, CYP2C9, CYP3A4, and CYP2D6. For deamination, data supported a role for CYP3A4 and CYP2C19. Purified human monoamine oxidases A and B also catalyzed sertraline deamination with comparable K(m) values (230-270 microM). Monoamine oxidase B catalyzed the reaction approximately 3-fold faster than did monoamine oxidase A. Sertraline N-carbamoyl glucuronidation was measured in human liver microsomes in bicarbonate buffer and under a CO2 atmosphere (K(m) = 50 microM) and was catalyzed at the fastest rate by recombinant human UGT2B7. The observation that multiple enzymes appear to be involved in sertraline metabolism suggests that there should be no single agent that could substantially alter the pharmacokinetics of sertraline, nor should there be any single drug-metabolizing enzyme genetic polymorphism (e.g., CYP2D6, CYP2C19, CYP2C9, UGT1A1) that could profoundly impact the pharmacokinetics of sertraline.