Physiologically-Based Pharmacokinetic Modeling of PAXLOVID™ with First-Order Absorption Kinetics.

PURPOSE: PAXLOVID™ is nirmatrelvir tablets co-packaged with ritonavir tablets. Ritonavir is used as a pharmacokinetics (PK) enhancer to reduce metabolism and increase exposure of nirmatrelvir. This is the first disclosure of Paxlovid physiologically-based pharmacokinetic (PBPK) model. METHODS: Nirmatrelvir PBPK model with first-order absorption kinetics was developed using in vitro, preclinical, and clinical data of nirmatrelvir in the presence and absence of ritonavir. Clearance and volume of distribution were derived from nirmatrelvir PK obtained using a spray-dried dispersion (SDD) formulation where it is considered to be dosed as an oral solution, and absorption is near complete. The fraction of nirmatrelvir metabolized by CYP3A was estimated based on in vitro and clinical ritonavir drug-drug interaction (DDI) data. First-order absorption parameters were established for both SDD and tablet formulation using clinical data. Nirmatrelvir PBPK model was verified with both single and multiple dose human PK data, as well as DDI studies. Simcyp® first-order ritonavir compound file was also verified with additional clinical data. RESULTS: The nirmatrelvir PBPK model described the observed PK profiles of nirmatrelvir well with predicted AUC and Cmax values within ± 20% of the observed. The ritonavir model performed well resulting in predicted values within twofold of observed. CONCLUSIONS: Paxlovid PBPK model developed in this study can be applied to predict PK changes in special populations, as well as model the effect of victim and perpetrator DDI. PBPK modeling continues to play a critical role in accelerating drug discovery and development of potential treatments for devastating diseases such as COVID-19. NCT05263895, NCT05129475, NCT05032950 and NCT05064800.

Multifaceted plant responses to circumvent Phe hyperaccumulation by downregulation of flux through the shikimate pathway and by vacuolar Phe sequestration.

Detrimental effects of hyperaccumulation of the aromatic amino acid phenylalanine (Phe) in animals, known as phenylketonuria, are mitigated by excretion of Phe derivatives; however, how plants endure Phe accumulating conditions in the absence of an excretion system is currently unknown. To achieve Phe hyperaccumulation in a plant system, we simultaneously decreased in petunia flowers expression of all three Phe ammonia lyase (PAL) isoforms that catalyze the non-oxidative deamination of Phe to trans-cinnamic acid, the committed step for the major pathway of Phe metabolism. A total decrease in PAL activity by 81-94% led to an 18-fold expansion of the internal Phe pool. Phe accumulation had multifaceted intercompartmental effects on aromatic amino acid metabolism. It resulted in a decrease in the overall flux through the shikimate pathway, and a redirection of carbon flux toward the shikimate-derived aromatic amino acids tyrosine and tryptophan. Accumulation of Phe did not lead to an increase in flux toward phenylacetaldehyde, for which Phe is a direct precursor. Metabolic flux analysis revealed this to be due to the presence of a distinct metabolically inactive pool of Phe, likely localized in the vacuole. We have identified a vacuolar cationic amino acid transporter (PhCAT2) that contributes to sequestering excess of Phe in the vacuole. In vitro assays confirmed PhCAT2 can transport Phe, and decreased PhCAT2 expression in PAL-RNAi transgenic plants resulted in 1.6-fold increase in phenylacetaldehyde emission. These results demonstrate mechanisms by which plants maintain intercompartmental aromatic amino acid homeostasis, and provide critical insight for future phenylpropanoid metabolic engineering strategies.

Dynamic modeling of subcellular phenylpropanoid metabolism in Arabidopsis lignifying cells.

Lignin is a polymer that significantly inhibits saccharification of plant feedstocks. Adjusting the composition or reducing the total lignin content have both been demonstrated to result in an increase in sugar yield from biomass. However, because lignin is essential for plant growth, it cannot be manipulated with impunity. Thus, it is important to understand the control of carbon flux towards lignin biosynthesis such that optimal modifications to it can be made precisely. Phenylalanine (Phe) is the common precursor for all lignin subunits and it is commonly accepted that all biosynthetic steps, spanning multiple subcellular compartments, are known, yet an in vivo model of how flux towards lignin is controlled is lacking. To address this deficiency, we formulated and parameterized a kinetic model based on data from feeding Arabidopsis thaliana basal lignifying stems with ring labeled [13C6]-Phe. Several candidate models were compared by an information theoretic approach to select the one that best matched the experimental observations. Here we present a dynamic model of phenylpropanoid metabolism across several subcellular compartments that describes the allocation of carbon towards lignin biosynthesis in wild-type Arabidopsis stems. Flux control coefficients for the enzymes in the pathway starting from arogenate dehydratase through 4-coumarate: CoA ligase were calculated and show that the plastidial cationic amino-acid transporter has the highest impact on flux.

Targeted Metabolomics of the Phenylpropanoid Pathway in Arabidopsis thaliana using Reversed Phase Liquid Chromatography Coupled with Tandem Mass Spectrometry.

INTRODUCTION: The phenylpropanoid pathway is a source of a diverse group of compounds derived from phenylalanine, many of which are involved in lignin biosynthesis and serve as precursors for the production of valuable compounds, such as coumarins, flavonoids, and lignans. Consequently, recent efforts have been invested in mechanistically understanding monolignol biosynthesis, making the quantification of these metabolites vital. OBJECTIVE: To develop an improved and comprehensive analytical method for (i) extensively profiling, and (ii) accurately quantifiying intermediates of the monolignol biosynthetic network, using Arabidopsis thaliana as a model system. METHOD: A liquid chromatography-tandem mass spectrometry with electrospray ionization was developed to quantify phenylpropanoid metabolites in Arabidopsis wildtype and cinnamoyl CoA reductase1 (CCR1) deficient lines (ccr1). RESULTS: Vortexing at high temperatures (65°C) enhanced release of phenylpropanoids, specifically the more hydrophobic compounds. A pH of 5.3 and ammonium acetate buffer concentration of 2.5 mM resulted in an optimal analyte response across standards. Ion suppression was estimated using standard spike recovery studies for accurate quantitation. The optimized method was used to profile Arabidopsis wildtype and ccr1 stems. An increase in hydroxycinnamic acid derivatives and a decrease in the hydroxycinnamyl aldehydes and alcohols in ccr1 lines, supports a shift of flux from lignin synthesis to other secondary metabolites and phenylpropanoid derivatives. CONCLUSIONS: Compared to existing targeted profiling techniques, our method is capable of quantifying a wider range of intermediates (15 out of 22 in WT Arabidopsis stems) at low in vivo concentrations (~50 pmol/g-FW for certain compounds), while requiring minimal sample preparation. Copyright © 2017 John Wiley & Sons, Ltd.

PBPK Modeling of PAXLOVIDTM: Incorporating Rotamer Conversion Kinetics to Advanced Dissolution and Absorption Model.

PAXLOVID™ is a combination medicine of nirmatrelvir tablets co-packaged with ritonavir tablets. Nirmatrelvir is a peptidomimetic inhibitor of SARS-CoV2 main protease (Mpro), developed for the treatment of COVID-19. Ritonavir is co-administered as a pharmacokinetics (PK) enhancer to inhibit CYP3A mediated metabolism increasing exposures of nirmatrelvir. In the solid form, nirmatrelvir exists in a stable single conformational state (ANTI form). However, nirmatrelvir exhibits atropisomerism in solution whereby upon dissolution the ANTI rotational isomer reversibly converts to another conformation state (SYN form). Nirmatrelvir rotamer conversion follows pseudo first order kinetics with a conversion half-life of approximately 15 min in aqueous solutions, which is on a similar time scale of diffusion mediated dissolution from the solid form. In vitro dissolution studies further indicated that rotamer conversion is one of the processes controlling nirmatrelvir dissolution. It was hypothesized that rotamer conversion kinetics would affect oral absorption of nirmatrelvir in vivo. Consequently, a physiologically based pharmacokinetic (PBPK) model for Paxlovid was developed in Simcyp™ using the advanced dissolution, absorption, and metabolism model (ADAM) by incorporating rotamer conversion kinetics to achieve a more mechanistic description of nirmatrelvir oral absorption. The results demonstrate that the established absorption model with rotamer kinetics adequately described observed clinical data from various nirmatrelvir doses, dosage forms, and dosing regimens. The predicted vs. observed AUCinf and Cmax ratios were within 2-fold. The model has been internally used to inform clinical studies and dose recommendations for pediatrics.

Self-Assembly Properties of an Amphiphilic Phosphate Ester Prodrug Designed for the Treatment of COVID-19.

PF-07304814 is a water-soluble phosphate ester prodrug of a small molecule inhibitor for the SARS CoV-2 3CL protease designed for the treatment of COVID-19. The amphiphilicity and self-assembly behavior of the prodrug was investigated computationally and experimentally via multiple orthogonal techniques to better design formulations for intravenous infusion. The self-assembly of PF-07304814 into micellar structures enabled an increase in the solubility of lipophilic impurities by up to 1900x in clinically relevant formulations. The observed solubilization could help extend the drug product shelf-life and in use stability through inhibition of precipitation, without the need for solubilizing excipients. The work presented in this manuscript provides a roadmap for the characterization of prodrug self-assembly and highlights the potential for prodrug modifications to enhance solubility of both active ingredients and impurities and to extend drug product shelf-life.

Drug Dissolution in Oral Drug Absorption: Workshop Report.

The in-person workshop "Drug Dissolution in Oral Drug Absorption" was held on May 23-24, 2023, in Baltimore, MD, USA. The workshop was organized into lectures and breakout sessions. Three common topics that were re-visited by various lecturers were amorphous solid dispersions (ASDs), dissolution/permeation interplay, and in vitro methods to predict in vivo biopharmaceutics performance and risk. Topics that repeatedly surfaced across breakout sessions were the following: (1) meaning and assessment of "dissolved drug," particularly of poorly water soluble drug in colloidal environments (e.g., fed conditions, ASDs); (2) potential limitations of a test that employs sink conditions for a poorly water soluble drug; (3) non-compendial methods (e.g., two-stage or multi-stage method, dissolution/permeation methods); (4) non-compendial conditions (e.g., apex vessels, non-sink conditions); and (5) potential benefit of having both a quality control method for batch release and a biopredictive/biorelevant method for biowaiver or bridging scenarios. An identified obstacle to non-compendial methods is the uncertainty of global regulatory acceptance of such methods.

A 13C isotope labeling method for the measurement of lignin metabolic flux in Arabidopsis stems.

BACKGROUND: Metabolic fluxes represent the functional phenotypes of biochemical pathways and are essential to reveal the distribution of precursors among metabolic networks. Although analysis of metabolic fluxes, facilitated by stable isotope labeling and mass spectrometry detection, has been applied in the studies of plant metabolism, we lack experimental measurements for carbon flux towards lignin, one of the most abundant polymers in nature. RESULTS: We developed a feeding strategy of excised Arabidopsis stems with 13C labeled phenylalanine (Phe) for the analysis of lignin biosynthetic flux. We optimized the feeding methods and found the stems continued to grow and lignify. Consistent with lignification profiles along the stems, higher levels of phenylpropanoids and activities of lignin biosynthetic enzymes were detected in the base of the stem. In the feeding experiments, 13C labeled Phe was quickly accumulated and used for the synthesis of phenylpropanoid intermediates and lignin. The intermediates displayed two different patterns of labeling kinetics during the feeding period. Analysis of lignin showed rapid incorporation of label into all three subunits in the polymers. CONCLUSIONS: Our feeding results demonstrate the effectiveness of the stem feeding system and suggest a potential application for the investigations of other aspects in plant metabolism. The supply of exogenous Phe leading to a higher lignin deposition rate indicates the availability of Phe is a determining factor for lignification rates.

Rethinking how volatiles are released from plant cells.

For plant volatile organic compounds (VOCs) to be emitted, they must cross membrane(s), the aqueous cell wall, and sometimes the cuticle, before moving into the gas phase. It is presumed that VOC movement through each barrier occurs via passive diffusion. However, VOCs, which are primarily nonpolar compounds, will preferentially partition into membranes, making diffusion into aqueous compartments slow. Using Fick's first law, we calculated that to achieve observed VOC emission rates by diffusion alone would necessitate toxic VOC levels in membranes. Here, we propose that biological mechanisms, such as those involved in trafficking other hydrophobic compounds, must contribute to VOC emission. Such parallel biological pathways would lower barrier resistances and, thus, steady-state emission rates could be maintained with significantly reduced intramembrane VOC concentrations.

Identification of a plastidial phenylalanine exporter that influences flux distribution through the phenylalanine biosynthetic network.

In addition to proteins, L-phenylalanine is a versatile precursor for thousands of plant metabolites. Production of phenylalanine-derived compounds is a complex multi-compartmental process using phenylalanine synthesized predominantly in plastids as precursor. The transporter(s) exporting phenylalanine from plastids, however, remains unknown. Here, a gene encoding a Petunia hybrida plastidial cationic amino-acid transporter (PhpCAT) functioning in plastidial phenylalanine export is identified based on homology to an Escherichia coli phenylalanine transporter and co-expression with phenylalanine metabolic genes. Radiolabel transport assays show that PhpCAT exports all three aromatic amino acids. PhpCAT downregulation and overexpression result in decreased and increased levels, respectively, of phenylalanine-derived volatiles, as well as phenylalanine, tyrosine and their biosynthetic intermediates. Metabolic flux analysis reveals that flux through the plastidial phenylalanine biosynthetic pathway is reduced in PhpCAT RNAi lines, suggesting that the rate of phenylalanine export from plastids contributes to regulating flux through the aromatic amino-acid network.