Pharmacodynamic Models of Indirect Effects and Irreversible Inactivation with Turnover: Applicability to Mechanism-Based Modeling of Gene Silencing and Targeted Protein Degradation.

Indirect response (IDR) and turnover with inactivation (TI) comprise two arrays of mechanism-based pharmacodynamic (PD) models widely used to describe delayed drug effects. IDR Model-IV (stimulation of response loss) and TI (irreversible loss) have been described with discerning "signature" profiles; classical IDR-IV response-time profiles display slow declines where peak response shifts later with increasing dose, whereas TI profiles feature steep response declines with earlier-shifting nadirs. Herein, we demonstrate mathematical convergence of IDR-IV and TI models upon implementation with identical linear versus nonlinear pharmacologic effect terms. Time of peak response in IDR-IV can in fact shift earlier or later depending on PK or PD parameters (e.g., kel, Smax) and effect type. A generalized dynamic model linking mRNA and protein turnover is proposed. Applicability of IDR-IV and TI, with either linear or nonlinear terms acting on degradation/catabolism/loss of response, is demonstrated through model-fitting PK-PD effects of three proteolysis-targeting chimeras (PROTACs) and two ligand-conjugated small interfering RNAs (siRNA). This work clarifies mathematical properties, convergence, and expected responses of IDR-IV and TI, demonstrates their applicability for targeted gene-silencing and protein-degrading agents, and illustrates how well-designed in vivo studies covering broad dose ranges with richly sampled time-points can influence PK-PD model structure and parameter resolution.

Mechanistic Pharmacokinetics and Pharmacodynamics of GalNAc-siRNA: Translational Model Involving Competitive Receptor-Mediated Disposition and RISC-Dependent Gene Silencing Applied to Givosiran.

Triantennary N-acetyl-D galactosamine (GalNAc)3-conjugated small interfering RNA (siRNA) have majorly advanced the development of RNA-based therapeutics. Chemically stabilized GalNAc-siRNAs exhibit extensive albeit capacity-limited (nonlinear) distribution into hepatocytes with additional complexities in intracellular liver disposition and pharmacology. A mechanism-based pharmacokinetic-pharmacodynamic (PK-PD) model of GalNAc-siRNA was developed to i) quantitate ASGPR-mediated disposition and downstream RNA-induced silencing complex (RISC)-dependent pharmacology following intravenous (IV) and subcutaneous (SC) dosing, ii) assess the kinetics of formed active metabolite, iii) leverage, as an example, published experimental data for givosiran, and iv) demonstrate PK translation across two preclinical species (rat and monkey) with subsequent prediction of human plasma PK. The structural model is based on competition between parent and formed active metabolite for occupancy and uptake via ASGPR into hepatocytes, intracellular sequestration and degradation, and downstream engagement of RNA-induced silencing complex (RISC) governing target mRNA degradation. The model jointly and accurately captured available concentration-time profiles of givosiran and/or AS(N-1)3' givosiran in rat and/or monkey plasma, liver, and/or kidney following givosiran administered both IV and SC. RISC-dependent gene silencing of ALAS1 mRNA was well-characterized. The model estimated an in vivo affinity (KD) value of 27.7 nM for GalNAc-ASGPR and weight-based allometric exponents of -0.27 and -0.24 for SC absorption and intracellular (endolysosomal) degradation rate constants. The model well-predicted reported givosiran plasma PK profiles in humans. PK simulations revealed net-shifts in liver-to-kidney distribution ratios with increasing IV and SC dose. Importantly, decreases in the relative liver uptake efficiency were demonstrated following IV and, to a lesser extent, following SC dosing explained by differential ASGPR occupancy profiles over time.

Pharmacokinetic and Pharmacodynamic Modeling of siRNA Therapeutics - a Minireview.

The approval of four small interfering RNA (siRNA) products in the past few years has demonstrated unequivocally the therapeutic potential of this novel modality. Three such products (givosiran, lumasiran and inclisiran) are liver-targeted, using tris N-acetylgalactosamine (GalNAc)3 as the targeting ligand. Upon subcutaneous administration, GalNAc-conjugated siRNAs rapidly distribute into the liver via asialoglycoprotein receptor (ASGPR) mediated uptake in the hepatocytes, resulting in fast elimination from the systemic circulation. Patisiran, on the other hand, has been formulated in a lipid nanoparticle for optimal delivery to the liver. While several publications have described preclinical and clinical pharmacokinetic (PK) and pharmacodynamic (PD) results, including absorption, distribution, metabolism, and elimination (ADME) profiles in selected species as well as limited modeling efforts for siRNA therapeutics, there is no systematic review of the PK and PD models developed for these agents or work summarizing the utility and application(s) of such models in drug development and regulatory review. Here, we provide a mini-review of the current state of modeling efforts for siRNA therapeutics within the early preclinical, translational, and clinical stages of siRNA development. Diverse modeling methods including simple compartmental, mechanistic and systems PK/PD, physiologically-based PK (PBPK), population PK/PD, and dose-response-time models are introduced and reviewed. The utility of such models in development and regulatory review for siRNA therapeutics is also discussed with examples. Finally, the current knowledge gaps in mechanism of action of siRNA and resulting challenges in model development are summarized. The goal of this minireview is to trigger cross-functional discussion amongst all key stakeholders to generate key experimental datasets and align on current assumptions, model structures, and approaches to facilitate development and application of robust PK/PD models for siRNA therapeutics.

Minimal Physiologically-Based Pharmacokinetic (mPBPK) Metamodeling of Target Engagement in Skin Informs Anti-IL17A Drug Development in Psoriasis.

The pharmacologic effect(s) of biotherapeutics directed against soluble targets are driven by the magnitude and duration of free target suppression at the tissue site(s) of action. Interleukin (IL)-17A is an inflammatory cytokine that plays a key role in the pathogenesis of psoriasis. In this work, clinical trial data from two monoclonal antibodies (mAbs) targeting IL-17A for treatment of psoriasis (secukinumab and ixekizumab) were analyzed simultaneously to quantitatively predict their target engagement (TE) profiles in psoriatic skin. First, a model-based meta-analysis (MBMA) for clinical responses was conducted separately for each drug based on dose. Next, a minimal physiologically-based pharmacokinetic (mPBPK) model was built to assess skin site IL-17A target engagement for ixekizumab and secukinumab simultaneously. The mPBPK model captured the observed drug PK, serum total IL-17A, and skin drug concentration-time profiles reasonably well across the different dosage regimens investigated. The developed mPBPK model was then used to predict the average TE (i.e., free IL-17A suppression) in skin achieved over a 12-weeks treatment period for each drug following their respective regimens and subsequently assess the TE-efficacy response relationship. It was predicted that secukinumab achieved 98.6% average TE in the skin at 300 mg q4w SC while ixekizumab achieved 99.9% average TE under 160 mg (loading) followed by 80 mg q2w SC. While direct quantification of free IL-17A levels at the site of action is technically challenging, integrated mPBPK-MBMA approaches offer quantitative predictions of free IL-17A levels at the site of action to facilitate future drug development via IL-17A suppression in psoriasis.

Minimal Physiologically Based Pharmacokinetic-Pharmacodynamic (mPBPK-PD) Model of N-Acetylgalactosamine-Conjugated Small Interfering RNA Disposition and Gene Silencing in Preclinical Species and Humans.

Conjugation of small interfering RNA (siRNA) to tris N-acetylgalactosamine [(GalNAc)3] can enable highly selective, potent, and durable knockdown of targeted proteins in the liver. However, potential knowledge gaps between in vitro experiments, preclinical species, and clinical scenarios remain. A minimal physiologically based pharmacokinetic-pharmacodynamic model for GalNAc-conjugated siRNA (GalNAc-siRNA) was developed using published data for fitusiran (ALN-AT3), an investigational compound targeting liver antithrombin (AT), to delineate putative determinants governing the whole-body-to-cellular pharmacokinetic (PK) and pharmacodynamic (PD) properties of GalNAc-siRNA and facilitate preclinical-to-clinical translation. The model mathematically linked relevant mechanisms: 1) hepatic biodistribution, 2) tris-GalNAc binding to asialoglycoprotein receptors (ASGPRs) on hepatocytes, 3) ASGPR endocytosis and recycling, 4) endosomal transport and escape of siRNA, 5) cytoplasmic RNA-induced silencing complex (RISC) loading, 6) degradation of target mRNA by bound RISC, and 7) knockdown of protein. Physiologic values for 36 out of 48 model parameters were obtained from the literature. Kinetic parameters governing (GalNAc)3-ASGPR binding and internalization were derived from published studies of uptake in hepatocytes. The proposed model well characterized reported pharmacokinetics, RISC dynamics, and knockdown of AT mRNA and protein by ALN-AT3 in mice. The model bridged multiple PK-PD data sets in preclinical species (mice, rat, monkey) and successfully captured reported plasma pharmacokinetics and AT knockdown in a phase I ascending-dose study. Estimates of in vivo potency were similar (∼2-fold) across species. Subcutaneous absorption and serum AT degradation rate constants scaled across species by body weight with allometric exponents of -0.29 and -0.22. The proposed mechanistic modeling framework characterizes the unique PK-PD properties of GalNAc-siRNA. SIGNIFICANCE STATEMENT: Tris N-acetylgalactosamine (GalNAc)3-conjugated small interfering RNA (siRNA) therapeutics enable liver-targeted gene therapy and precision medicine. Using a translational and systems-based minimal physiologically based pharmacokinetic-pharmacodynamic (mPBPK-PD) modeling approach, putative determinants influencing GalNAc-conjugated siRNA (GalNAc-siRNA) functionality in three preclinical species and humans were investigated. The developed model successfully integrated and characterized relevant published in vitro-derived biomeasures, mechanistic PK-PD profiles in animals, and observed clinical PK-PD responses for an investigational GalNAc-siRNA (fitusiran). This modeling effort delineates the disposition and liver-targeted pharmacodynamics of GalNAc-siRNA.

Translational PK/PD and model-informed development of JNJ-67842125, a Fab reversal agent for JNJ-64179375, a long-acting thrombin inhibitor.

BACKGROUND AND PURPOSE: Antigen-binding fragment (Fab ) reversal agents were developed to reverse, in bleeding emergency, the long-acting anticoagulant effect of JNJ-64179375 (JNJ-9375), a monoclonal antibody that binds exosite-1 on thrombin. EXPERIMENTAL APPROACH: The pharmacokinetic and pharmacodynamic (PK/PD) activities of three reversal agents of varying in vitro binding affinities to JNJ-9375 were characterised in cynomolgus monkeys. The time course of JNJ-9375 anticoagulant activity and reversal effects of each agent were evaluated. A mechanism-based PK/PD model, which integrated free serum concentrations of reversal agent, total and free serum concentrations of JNJ-9375, and thrombin time, was developed to quantitatively relate JNJ-9375 neutralisation to reversal of induced thrombin time prolongation. Model-based allometric scale-up of the lead reversal agent and the PK/PD relationship of JNJ-9375 in healthy volunteers were utilised to predict clinical dosing regimens. KEY RESULTS: Lowering of free JNJ-9375 by the reversal agents corresponded with reversal of thrombin time prolongation. Total JNJ-9375 displayed typical mAb clearance at 2.75 ml·day-1 ·kg-1 , whereas reversal agents cleared faster between 1400 and 2400 ml·day-1 ·kg-1 . The model-estimated in vivo KD values for JNJ-9375 reversal agents were 9 nM (ICHB-256), 0.4 nM (ICHB-281) and 13.7 pM (ICHB-164), in rank-ordered agreement of their KD values determined in vitro. The three reversal agents exhibited different neutralisation characteristics in vivo, governed primarily by their binding kinetics to JNJ-9375. The model predicted a priori free JNJ-9375 kinetics after dosing ICHB-164 (JNJ-67842125) and JNJ-9375 under a different regimen. CONCLUSION AND IMPLICATIONS: The results enabled selection of JNJ-67842125 as the reversal agent for JNJ-9375.

Circadian rhythms: influence on physiology, pharmacology, and therapeutic interventions.

Circadian rhythms are ubiquitous phenomena that recur daily in a self-sustaining, entrainable, and oscillatory manner, and orchestrate a wide range of molecular, physiological, and behavioral processes. Circadian clocks are comprised of a hierarchical network of central and peripheral clocks that generate, sustain, and synchronize the circadian rhythms. The functioning of the peripheral clock is regulated by signals from autonomic innervation (from the central clock), endocrine networks, feeding, and other external cues. The critical role played by circadian rhythms in maintaining both systemic and tissue-level homeostasis is well established, and disruption of the rhythm has direct consequence for human health, disorders, and diseases. Circadian oscillations in both pharmacokinetics and pharmacodynamic processes are known to affect efficacy and toxicity of several therapeutic agents. A variety of modeling approaches ranging from empirical to more complex systems modeling approaches have been applied to characterize circadian biology and its influence on drug actions, optimize time of dosing, and identify opportunities for pharmacological modulation of the clock mechanisms and their downstream effects. In this review, we summarize current understanding of circadian rhythms and its influence on physiology, pharmacology, and therapeutic interventions, and discuss the role of chronopharmacometrics in gaining new insights into circadian rhythms and its applications in chronopharmacology.

Model-Based Cellular Kinetic Analysis of Chimeric Antigen Receptor-T Cells in Humans.

Chimeric antigen receptor (CAR)-T cell therapy has achieved considerable success in treating B-cell hematologic malignancies. However, the challenges of extending CAR-T therapy to other tumor types, particularly solid tumors, remain appreciable. There are substantial variabilities in CAR-T cellular kinetics across CAR-designs, CAR-T products, dosing regimens, patient responses, disease types, tumor burdens, and lymphodepletion conditions. As a "living drug," CAR-T cellular kinetics typically exhibit four distinct phases: distribution, expansion, contraction, and persistence. The cellular kinetics of CAR-T may correlate with patient responses, but which factors determine CAR-T cellular kinetics remain poorly defined. Herein, we developed a cellular kinetic model to retrospectively characterize CAR-T kinetics in 217 patients from 7 trials and compared CAR-T kinetics across response status, patient populations, and tumor types. Based on our analysis results, CAR-T cells exhibited a significantly higher cell proliferation rate and capacity but a lower contraction rate in patients who responded to treatment. CAR-T cells proliferate to a higher degree in hematologic malignancies than in solid tumors. Within the assessed dose ranges (107 -109 cells), CAR-T doses were weakly correlated with CAR-T cellular kinetics and patient response status. In conclusion, the developed CAR-T cellular kinetic model adequately characterized the multiphasic CAR-T cellular kinetics and supported systematic evaluations of the potential influencing factors, which can have significant implications for the development of more effective CAR-T therapies.

Seeking Nonspecific Binding: Assessing the Reliability of Tissue Dilutions for Calculating Fraction Unbound.

It has become commonplace (270+ article citations to date) to measure the fraction unbound (FrUn) of drugs in tissue homogenates and diluted plasma and then use a Correction Factor Equation (CFE) to extrapolate to the undiluted state. The CFE is based on assumptions of nonspecific binding with experimental use of very low drug concentrations. There are several possible determinants of apparent nonspecific binding as measured by methods such as equilibrium dialysis: true macromolecule binding and lipid partitioning along with receptor, enzyme, and transporter interactions. Theoretical calculations based on nonlinear protein binding indicate that the CFE will be most reliable to obtain FrUn when added drug concentration is small, binding constants are weak, protein concentrations are relatively high, and tissue dilution is minimal. When lipid partitioning is the sole factor determining apparent tissue binding, the CFE should be perfectly accurate. Use of very low drug concentrations, however, makes it more likely that specific binding to receptors and other targets may occur, and thus FrUn may reflect some binding to such components. Inclusion of trapped blood can clearly cause minor to marked discrepancies from purely tissue binding alone, which can be corrected. Furthermore, assessment of the occurrence of ionization/pH shifts, drug instability, and tissue metabolism may be necessary. Caution is needed in the use and interpretation of results from tissue dilution studies and other assessments of nonspecific binding, particularly for very strongly bound drugs with very small FrUn values and in tissues with metabolic enzymes, receptors, and trapped blood. SIGNIFICANCE STATEMENT: The use of tissue, plasma, and cell preparations to help obtain fraction unbound and tissue-to-plasma partition coefficients in pharmacokinetics has grown commonplace, especially for brain. This report examines theoretical, physiological, and experimental issues that need consideration before trusting such measurements and calculations.

Transitioning from Basic toward Systems Pharmacodynamic Models: Lessons from Corticosteroids.

Technology in bioanalysis, -omics, and computation have evolved over the past half century to allow for comprehensive assessments of the molecular to whole body pharmacology of diverse corticosteroids. Such studies have advanced pharmacokinetic and pharmacodynamic (PK/PD) concepts and models that often generalize across various classes of drugs. These models encompass the "pillars" of pharmacology, namely PK and target drug exposure, the mass-law interactions of drugs with receptors/targets, and the consequent turnover and homeostatic control of genes, biomarkers, physiologic responses, and disease symptoms. Pharmacokinetic methodology utilizes noncompartmental, compartmental, reversible, physiologic [full physiologically based pharmacokinetic (PBPK) and minimal PBPK], and target-mediated drug disposition models using a growing array of pharmacometric considerations and software. Basic PK/PD models have emerged (simple direct, biophase, slow receptor binding, indirect response, irreversible, turnover with inactivation, and transduction models) that place emphasis on parsimony, are mechanistic in nature, and serve as highly useful "top-down" methods of quantitating the actions of diverse drugs. These are often components of more complex quantitative systems pharmacology (QSP) models that explain the array of responses to various drugs, including corticosteroids. Progressively deeper mechanistic appreciation of PBPK, drug-target interactions, and systems physiology from the molecular (genomic, proteomic, metabolomic) to cellular to whole body levels provides the foundation for enhanced PK/PD to comprehensive QSP models. Our research based on cell, animal, clinical, and theoretical studies with corticosteroids have provided ideas and quantitative methods that have broadly advanced the fields of PK/PD and QSP modeling and illustrates the transition toward a global, systems understanding of actions of diverse drugs. SIGNIFICANCE STATEMENT: Over the past half century, pharmacokinetics (PK) and pharmacokinetics/pharmacodynamics (PK/PD) have evolved to provide an array of mechanism-based models that help quantitate the disposition and actions of most drugs. We describe how many basic PK and PK/PD model components were identified and often applied to the diverse properties of corticosteroids (CS). The CS have complications in disposition and a wide array of simple receptor-to complex gene-mediated actions in multiple organs. Continued assessments of such complexities have offered opportunities to develop models ranging from simple PK to enhanced PK/PD to quantitative systems pharmacology (QSP) that help explain therapeutic and adverse CS effects. Concurrent development of state-of-the-art PK, PK/PD, and QSP models are described alongside experimental studies that revealed diverse CS actions.

Modeling Corticosteroid Pharmacokinetics and Pharmacodynamics, Part II: Sex Differences in Methylprednisolone Pharmacokinetics and Corticosterone Suppression.

Methylprednisolone (MPL), a corticosteroid of intermediate potency, remains an important immunomodulatory agent for autoimmune diseases. Although sex differences in corticosteroid pharmacokinetics/pharmacodynamics (PK/PD) have been documented in humans, comprehensive preclinical assessments of such differences have not been conducted. Limited in vitro evidence indicates possible sex differences in corticosteroid PK and PD. Therefore, it is hypothesized that comparative PK/PD assessments of MPL disposition and selected PD actions in both sexes will provide insights into factors controlling sex differences in steroid responses. This report focused on the plasma and tissue pharmacokinetics of MPL and its adrenal suppressive effects. Because time-dependent (estrous) regulation of sex hormones in females can influence drug responses, female rats were studied in the proestrus (high estradiol/progesterone) and estrus (low estradiol/progesterone) phases of the reproductive cycle. Cohorts of male and female rats were given a 50 mg/kg bolus dose of MPL intramuscularly. Plasma and liver concentrations of MPL as well as plasma corticosterone concentrations were assayed using high-performance liquid chromatography. An enhanced minimal physiologically-based PK/PD model was developed to characterize MPL kinetics and corticosterone dynamics. The clearance of MPL was ∼3-fold higher in males compared with females, regardless of estrous phase, likely attributable to sex-specific hepatic metabolism in males. Strong inhibitory effects on adrenal suppression were observed in all animals. These temporal steroid profiles in plasma and tissues will be used to drive receptor/gene-mediated PD effects of MPL in both sexes, as described in a companion article (Part III). SIGNIFICANCE STATEMENT: Sex is a relevant factor influencing the pharmacokinetics (PK) and pharmacodynamics (PD) of drugs. Few preclinical PK/PD studies, however, include sex as a variable. Sex differences in the PK and adrenal suppressive effects of the synthetic corticosteroid, methylprednisolone, were assessed in male and female rats as a function of the 4-day rodent reproductive cycle. Drug exposure was 3-fold higher in females, regardless of estrous stage, compared with males. An extended minimal physiologically-based PK/PD model utilizing in vitro and in vivo measurements was developed and applied. These studies provide a framework to account for sex-dependent variability in drug and endogenous agonist (corticosterone) exposures, serving as a prelude to more intricate assessments of sex-related variability in receptor/gene-mediated PD corticosteroid actions.

Modeling Corticosteroid Pharmacokinetics and Pharmacodynamics, Part I: Determination and Prediction of Dexamethasone and Methylprednisolone Tissue Binding in the Rat.

The plasma and tissue binding properties of two corticosteroids, dexamethasone (DEX) and methylprednisolone (MPL), were assessed in the rat in anticipation of developing physiologically based pharmacokinetic and pharmacokinetic/pharmacodynamic models. The tissue-to-plasma partition coefficients (K P) of DEX and MPL were measured in liver, muscle, and lung in vivo after steady-state infusion and bolus injection in rats. Since K P is often governed by reversible binding to macromolecules in blood and tissue, an attempt was made to assess K P values of DEX and MPL by in vitro binding studies using rat tissue homogenates and to compare these estimates to those obtained from in vivo kinetics after dosing. The K P values of both steroids were also calculated in rat tissues using mechanistic tissue composition-based equations. The plasma binding of DEX and MPL was linear with moderate binding (60.5% and 82.5%) in male and female rats. In vivo estimates of steroid uptake appeared linear across the tested concentrations and K P was highest in liver and lowest in muscle for both steroids. Assessment of hepatic binding of MPL in vitro was severely affected by drug loss at 37°C in male liver homogenates, whereas DEX was stable in both male and female liver homogenates. With the exception of MPL in liver, in vitro-derived K P estimates reasonably agreed with in vivo values. The mechanistic equations modestly underpredicted K P for both drugs. Tissue metabolism, saturable tissue binding, and active uptake are possible factors that can complicate assessments of in vivo tissue binding of steroids when using tissue homogenates. SIGNIFICANCE STATEMENT: Assuming the free hormone hypothesis, the ratio of the unbound drug fraction in plasma and in tissues defines the tissue-to-plasma partition coefficient (K P), an important parameter in physiologically based pharmacokinetic modeling that determines total drug concentrations within tissues and the steady-state volume of distribution. This study assessed the plasma and tissue binding properties of the synthetic corticosteroids, dexamethasone and methylprednisolone, in rats using ultrafiltration and tissue homogenate techniques. In vitro-in vivo and in silico-in vivo extrapolation of K P was assessed for both drugs in liver, muscle, and lung. Although the extrapolation was fairly successful across the tissues, in vitro homogenate studies severely underpredicted the K P of methylprednisolone in liver, partly attributable to the extensive hepatic metabolism.

Modeling Corticosteroid Pharmacokinetics and Pharmacodynamics, Part III: Estrous Cycle and Estrogen Receptor-Dependent Antagonism of Glucocorticoid-Induced Leucine Zipper (GILZ) Enhancement by Corticosteroids.

Our previous report examined the pharmacokinetics (PK) of methylprednisolone (MPL) and adrenal suppression after a 50 mg/kg IM bolus in male and female rats, and we described in detail the development of a minimal physiologically based pharmacokinetic/pharmacodynamic (mPBPK/PD) model. In continuation of such assessments, we investigated sex differences in genomic MPL responses (PD). Message expression of the glucocorticoid-induced leucine zipper (GILZ) was chosen as a multitissue biomarker of glucocorticoid receptor (GR)-mediated drug response. Potential time-dependent interplay between sex hormone and glucocorticoid signaling in vivo was assessed by comparing the enhancement of GILZ by MPL in the uterus [high estrogen receptor (ER) density] and in liver (lower ER density) from male and female rats dosed within the proestrus (high estradiol/progesterone) and estrus (low estradiol/progesterone) phases of the rodent estrous cycle. An expanded-systems PD model of MPL considering circadian rhythms, multireceptor (ER and GR) control, and estrous variations delineated the determinants controlling receptor/gene-mediated steroid responses. Hepatic GILZ response was ∼3-fold greater in females, regardless of estrous stage, compared with males, driven predominantly by increased MPL exposure in females and a negligible influence of estrogen interaction. In contrast, GILZ response in the uterus during proestrus in females was 60% of that observed in estrus-phased females, despite no PK or receptor differences, providing in vivo support to the hypothesis of estrogen-mediated antagonism of glucocorticoid signaling. The developed model offers a mechanistic platform to assess the determinants of sex and tissue specificity in corticosteroid actions and, in turn, reveals a unique PD drug-hormone interaction occurring in vivo. SIGNIFICANCE STATEMENT: Mechanisms relating to sex-based pharmacodynamic variability in genomic responses to corticosteroids have been unclear. Using combined experimental and systems pharmacology modeling approaches, sex differences in both pharmacokinetic and pharmacodynamic mechanisms controlling the enhancement of a sensitive corticosteroid-regulated biomarker, the glucocorticoid-induced leucine zipper (GILZ), were clarified in vivo. The multiscale minimal physiologically based pharmacokinetics/pharmacodynamic model successfully captured the experimental observations and quantitatively discerned the roles of the rodent estrous cycle (hormonal variation) and tissue specificity in mediating the antagonistic coregulation of GILZ gene synthesis. These findings collectively support the hypothesis that estrogens antagonize pharmacodynamic signaling of genomic corticosteroid actions in vivo in a time- and estrogen receptor-dependent manner.

ATLAS mPBPK: A MATLAB-Based Tool for Modeling and Simulation of Minimal Physiologically-Based Pharmacokinetic Models.

Minimal physiologically-based pharmacokinetic (mPBPK) models are frequently used to model plasma pharmacokinetic (PK) data and utilize and yield physiologically relevant parameters. Compared with classical compartment and whole-body physiologically-based pharmacokinetic modeling approaches, mPBPK models maintain a structure of intermediate physiological complexity that can be adequately informed by plasma PK data. In this tutorial, we present a MATLAB-based tool for the modeling and simulation of mPBPK models (ATLAS mPBPK) of small and large molecules. This tool enables the users to perform the following: (i) PK data visualization, (ii) simulation, (iii) parameter optimization, and (iv) local sensitivity analysis of mPBPK models in a simple and efficient manner. In addition to the theoretical background and implementation of the different tool functionalities, this tutorial includes simulation and sensitivity analysis showcases of small and large molecules with and without target-mediated drug disposition.

Indirect pharmacodynamic models for responses with circadian removal.

Rhythmicity in baseline responses over a 24-h period for an indirect pharmacological effect R(t) can arise from either a periodic time-dependent input rate [Formula: see text] or a periodic time-dependent loss constant [Formula: see text]. If either [Formula: see text] or [Formula: see text] follows some nonstationary biological rhythm (e.g., circadian), then the response R(t) also displays a periodic behavior. Indirect response models assuming time-dependent input rates [Formula: see text] have been utilized to capture drug effects on various physiological responses such as hormone suppression, immune cell trafficking, and gene expression in tissues. This paradigm was extended to consider responses with circadian-controlled loss [Formula: see text] mechanisms. Theoretical equations describing this model are presented and simulations were performed to examine expected response behaviors. The model was able to capture the chronobiology and pharmacodynamics of applicable drug responses, including the uricosuric effects of lesinurad in humans, suppression of the beta amyloid (Aß) peptide by a gamma-secretase inhibitor in mouse brain, and the modulation of extracellular dopamine by a dopamine transporter inhibitor in rat brain. This type of model has a mechanistic basis and shows utility for capturing drug responses displaying nonstationary baselines controlled by removal mechanism(s).

Modeling Corticosteroid Pharmacogenomics and Proteomics in Rat Liver.

Corticosteroids (CS) regulate the expression of numerous genes at the mRNA and protein levels. The time course of CS pharmacogenomics and proteomics were examined in livers obtained from adrenalectomized rats given a 50-mg/kg bolus dose of methylprednisolone. Microarrays and mass spectrometry-based proteomics were employed to quantify hepatic transcript and protein dynamics. One-hundred, sixty-three differentially expressed mRNA and their corresponding proteins (163 genes) were clustered into two dominant groups. The temporal profiles of most proteins were delayed compared with their mRNA, attributable to synthesis delays and slower degradation kinetics. On the basis of our fifth-generation model of CS, mathematical models were developed to simultaneously describe the emergent time patterns for an array of steroid-responsive mRNA and proteins. The majority of genes showed time-dependent increases in mRNA and protein expression before returning to baseline. A model assuming direct, steroid-mediated stimulation of mRNA synthesis was applied. Some mRNAs and their proteins displayed down-regulation following CS. A model assuming receptor-mediated inhibition of mRNA synthesis was used. More complex patterns were observed for other genes (e.g., biphasic behaviors and opposite directionality in mRNA and protein). Models assuming either stimulation or inhibition of mRNA synthesis coupled with dual secondarily induced regulatory mechanisms affecting mRNA or protein turnover were derived. These findings indicate that CS-regulated gene expression manifested at the mRNA and protein levels are controlled via mechanisms affecting key turnover processes. Our quantitative models of CS pharmacogenomics were expanded from mRNA to proteins and provide extended hypotheses for understanding the direct, secondary, and downstream mechanisms of CS actions.

Receptor/gene/protein-mediated signaling connects methylprednisolone exposure to metabolic and immune-related pharmacodynamic actions in liver.

A multiscale pharmacodynamic model was developed to characterize the receptor-mediated, transcriptomic, and proteomic determinants of corticosteroid (CS) effects on clinically relevant hepatic processes following a single dose of methylprednisolone (MPL) given to adrenalectomized (ADX) rats. The enhancement of tyrosine aminotransferase (TAT) mRNA, protein, and enzyme activity were simultaneously described. Mechanisms related to the effects of MPL on glucose homeostasis, including the regulation of CCAAT-enhancer binding protein-beta (C/EBPß) and phosphoenolpyruvate carboxykinase (PEPCK) as well as insulin dynamics were evaluated. The MPL-induced suppression of circulating lymphocytes was modeled by coupling its effect on cell trafficking with pharmacogenomic effects on cell apoptosis via the hepatic (STAT3-regulated) acute phase response. Transcriptomic and proteomic time-course profiles measured in steroid-treated rat liver were utilized to model the dynamics of mechanistically relevant gene products, which were linked to associated systemic end-points. While time-courses of TAT mRNA, protein, and activity were well described by transcription-mediated changes, additional post-transcriptional processes were included to explain the lack of correlation between PEPCK mRNA and protein. The immune response model quantitatively discerned the relative roles of cell trafficking versus gene-mediated lymphocyte apoptosis by MPL. This systems pharmacodynamic model provides insights into the contributions of selected molecular events occurring in liver and explores mechanistic hypotheses for the multi-factorial control of clinically relevant pharmacodynamic outcomes.

Mechanistic Multi-Tissue Modeling of Glucocorticoid-Induced Leucine Zipper Regulation: Integrating Circadian Gene Expression with Receptor-Mediated Corticosteroid Pharmacodynamics.

The glucocorticoid-induced leucine zipper (GILZ) is an important mediator of anti-inflammatory corticosteroid action. The pharmacokinetic/pharmacodynamic/pharmacogenomic effects of acute and chronic methylprednisolone (MPL) dosing on the tissue-specific dynamics of GILZ expression were examined in rats. A mechanism-based model was developed to investigate and integrate the role of MPL and circadian rhythms on the transcriptional enhancement of GILZ in multiple tissues. Animals received a single 50-mg/kg intramuscular bolus or a 7-day 0.3-mg/kg/h subcutaneous infusion of MPL and were euthanized at several time points. An additional group of rats were euthanized at several times and served as 24-hour light/dark (circadian) controls. Plasma MPL and corticosterone concentrations were measured by high-performance liquid chromatography. The expression of GILZ and glucocorticoid receptor (GR) mRNA was quantified in tissues using quantitative real-time reverse-transcription polymerase chain reaction. The pharmacokinetics of MPL were described using a two-compartment model. Mild-to-robust circadian oscillations in GR and GILZ mRNA expression were characterized in muscle, lung, and adipose tissues and modeled using Fourier harmonic functions. Acute MPL dosing caused significant down-regulation (40%-80%) in GR mRNA and enhancement of GILZ mRNA expression (500%-1080%) in the tissues examined. While GILZ returned to its rhythmic baseline following acute dosing, a new steady-state was observed upon enhancement by chronic dosing. The model captured the complex dynamics in all tissues for both dosing regimens. The model quantitatively integrates physiologic mechanisms, such as circadian processes and GR tolerance phenomena, which control the tissue-specific regulation of GILZ by corticosteroids. These studies characterize GILZ as a pharmacodynamic marker of corticosteroid actions in several tissues.

Functional proteomic analysis of corticosteroid pharmacodynamics in rat liver: Relationship to hepatic stress, signaling, energy regulation, and drug metabolism.

Corticosteroids (CS) are anti-inflammatory agents that cause extensive pharmacogenomic and proteomic changes in multiple tissues. An understanding of the proteome-wide effects of CS in liver and its relationships to altered hepatic and systemic physiology remains incomplete. Here, we report the application of a functional pharmacoproteomic approach to gain integrated insight into the complex nature of CS responses in liver in vivo. An in-depth functional analysis was performed using rich pharmacodynamic (temporal-based) proteomic data measured over 66h in rat liver following a single dose of methylprednisolone (MPL). Data mining identified 451 differentially regulated proteins. These proteins were analyzed on the basis of temporal regulation, cellular localization, and literature-mined functional information. Of the 451 proteins, 378 were clustered into six functional groups based on major clinically-relevant effects of CS in liver. MPL-responsive proteins were highly localized in the mitochondria (20%) and cytosol (24%). Interestingly, several proteins were related to hepatic stress and signaling processes, which appear to be involved in secondary signaling cascades and in protecting the liver from CS-induced oxidative damage. Consistent with known adverse metabolic effects of CS, several rate-controlling enzymes involved in amino acid metabolism, gluconeogenesis, and fatty-acid metabolism were altered by MPL. In addition, proteins involved in the metabolism of endogenous compounds, xenobiotics, and therapeutic drugs including cytochrome P450 and Phase-II enzymes were differentially regulated. Proteins related to the inflammatory acute-phase response were up-regulated in response to MPL. Functionally-similar proteins showed large diversity in their temporal profiles, indicating complex mechanisms of regulation by CS. SIGNIFICANCE: Clinical use of corticosteroid (CS) therapy is frequent and chronic. However, current knowledge on the proteome-level effects of CS in liver and other tissues is sparse. While transcriptomic regulation following methylprednisolone (MPL) dosing has been temporally examined in rat liver, proteomic assessments are needed to better characterize the tissue-specific functional aspects of MPL actions. This study describes a functional pharmacoproteomic analysis of dynamic changes in MPL-regulated proteins in liver and provides biological insight into how steroid-induced perturbations on a molecular level may relate to both adverse and therapeutic responses presented clinically.