Progress in CYP Enzymes Mechanisms of Induction and Its

Applications

Zhanxu Meng

Tai 'an No. 1 Middle School, Tai 'an, Shandong Province, China

Keywords: Cytochrome P450 (CYP) Enzymes, Induction Mechanisms, Drug Metabolism, In vitro, In vivo.

Abstract: Cytochrome P450 (CYP) enzymes are pivotal in the metabolic processing of drugs, and their modulation via

induction pathways plays an important role in the therapeutic efficacy, safety, and drug interaction profile.

This comprehensive review delineates three focal points concerning CYP enzyme induction. The first domain

scrutinizes the accuracy of in vitro methodologies—predominantly cryopreserved primary human hepatocytes

(PHH) and Human HepaRG cells—in forecasting the in vivo dynamics of CYP enzymes. The second segment

elucidates novel induction paradigms, highlighting the enduring induction of CYP1A enzymes via 3-

methylcholanthrene (MC) in murine models and the atypical induction pathways that involve YAP/TEAD

perturbation and consequent hepatocyte dedifferentiation. The terminal section evaluates the application of

these findings in clinical settings, discussing the kinetic profiling of CYP3A modulation and the transposition

of in vitro CYP repression to actual drug-drug interaction scenarios. The synthesis of these facets contributes

to an enriched understanding of CYP induction mechanisms and their ramifications for drug discovery and

tailored therapeutic approaches. Furthermore, the appraisal accentuates the reliability and pertinence of

cryopreserved PHH and HepaRG cells as in vitro proxies for human CYP enzyme induction studies,

potentially informing regulatory risk evaluation and elucidating drug metabolism and nuclear receptor-

mediated regulatory anomalies in biochemical pathways.

1 INTRODUCTION

Cytochrome P450 (CYP) enzymes play a critical role

in drug metabolism, facilitating the biotransformation

of a wide range of endogenous and exogenous

compounds within the body (Pelkonen et al. 2008).

The regulation of CYP expression is essential for

maintaining physiological balance and ensuring

efficient drug metabolism (Li et al., 2019). Induction

represents a primary mechanism through which CYP

expression levels are modulated, whereby various

xenobiotics and endogenous signalling molecules can

upregulate these enzymes (Li et al., 2019). When the

drug is cleared from the body more quickly, it leads

to lower levels of the drug in the body, which can

reduce its effectiveness. An example of this is when

rifampin is taken alongside sulfonylureas, resulting in

decreased levels of sulfonylureas in the blood and a

diminished ability to lower blood glucose. This

decrease in effectiveness may be caused by the

induction of CYP2C9 enzyme by rifampin. Another

factor contributing to the decreased drug

concentrations and effectiveness could be the

induction of P-glycoprotein by rifampin.

CYPenzymes play a critical role in drug

metabolism, with induction and inhibition as key

modulatory mechanisms. Induction of these enzymes

is characterized by a time-dependent augmentation in

enzyme levels, necessitating a period of adaptation to

stabilize at a new homeostasis (Lin, 2006).

Contrasting the immediate effect of CYP inhibition,

induction involves intricate genetic regulation

leading to an increase in enzyme synthesis.

Activation of CYP enzymes within the families 1 to 3

is a complex process governed by three primary

pathways, responding to exogenous compounds

(Pelkonen et al. 2008). Under basal conditions, these

receptors are sequestered in the cytoplasm. It is bound

to heat shock protein 90 (Hsp90). Ligand binding

instigates a conformational alteration, prompting

dissociation from Hsp90, receptor activation, and

nuclear translocation, thereby kick-starting gene

transcription. Beyond these classical receptor-

mediated mechanisms, CYP enzyme induction also

encompasses pathways such as the direct and indirect

Meng, Z.

Progress in CYP Enzymes Mechanisms of Induction and Its Applications.

DOI: 10.5220/0013846300004914

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of the 2nd International Conference on Renewable Energy and Ecosystem (ICREE 2024), pages 76-86

ISBN: 978-989-758-776-4

glucocorticoid receptor-mediated induction,

highlighting the nuanced regulatory landscape of

CYP enzyme activity (Schuetz et al., 1996 & Pascussi

et al., 2001). Moreover, the modulation of certain

CYP enzymes transcends transcriptional control,

involving post-transcriptional modifications that

stabilize the mRNA and protein, further

complexifying the regulation of CYP enzyme levels

(Chen et al., 2017)

The process of CYP induction holds significant

implications in pharmacology and toxicology,

impacting drug efficacy, safety, and potential

interactions. Understanding the molecular

mechanisms underlying CYP induction is vital for

optimizing drug therapy, predicting drug-drug

interactions (DDIs), and comprehending the effects of

environmental toxins on human health.

This review provides a detailed examination of

CYP induction, highlighting its implications for drug

metabolism and toxicity testing. Anchored by three

primary perspectives, the analysis begins with an

exploration of in vitro methodologies and their role in

predicting in vivo CYP induction, a topic extensively

addressed by Bernasconi et al. in 2019 (Bernasconi et

al., 2019). The translation of in vitro findings to in

vivo contexts is critical, particularly in understanding

how induced CYP activity may affect metabolic

processes. Recent advances have refined these in

vitro techniques, capitalizing on human-based

systems to simulate and study enzyme interactions.

CYP P450 enzymes, key players in xenobiotic

metabolism, are underscored for their expansive

presence and metabolic diversity. They are

instrumental in detoxifying xenobiotics by

metabolizing harmful substances into benign

products or, conversely, converting non-toxic

compounds into harmful metabolites. Beyond

detoxification, these enzymes are essential for

synthesizing various endogenous compounds.

Therefore, the xenobiotic-induced alteration of CYP

enzyme activity has far-reaching effects on metabolic

stability and can precipitate adverse biological

consequences. This review underscores two pivotal

studies utilizing in vitro approaches to forecast drug

interactions and refine clinical outcomes. The first

study by Bernasconi et al. investigates the effects of

tipranavir/ritonavir on enzymatic and transporter

functions (Bernasconi et al., 2019). The second, by

Dumond et al., presents cocktail phenotyping, a novel

strategy for evaluating drug interaction potentials.

Both studies furnish essential insights into the

complex interplay of drug interactions, contributing

to the enhancement of therapeutic protocols (Dumond

et al., 2010). Consequently, the review will critically

analyze and synthesize the findings, emphasizing

their significant contributions to the field of in vitro

prediction methods.

Furthermore, the review delves deeper into novel

mechanisms of CYP enzyme induction, specifically

highlighting the extended induction of CYP1A

enzymes by MC in murine models, a phenomenon

elucidated by Jiang et al. in 2009 (Jiang et al., 2009).

The research has shed light on the persistent

transcriptional activation of promoters associated

with these enzymes. Considering the potent

carcinogenicity of MC, a polycyclic aromatic

hydrocarbon (PAH) prevalent in various

environmental matrices, it's crucial to understand

how its metabolism by CYP enzymes results in

intermediates capable of DNA binding and potential

carcinogenesis.

The induction of CYP1A1 & A2 enzymes by MC

and the eventual decline post-exposure cessation

present an enigmatic aspect of CYP regulation. This

review aims to dissect the mechanisms behind the

lasting transcriptional activation of these CYP

enzymes, which have significant implications for

environmental health. Moreover, the review

addresses a comparative analysis of YAP/TEAD

inhibitors within bidimensional and tridimensionality

primary human hepatocyte cultures conducted by

Oliva-Vilarnau et al. in 2023 (Oliva-Vilarnau et al.,

2023). These inhibitors, initially developed as cancer

therapeutics, have been observed to stimulate CYP

enzymes in 2D hepatocyte cultures. Intriguingly, such

induction is absent in 3D spheroid cultures,

underscoring the importance of considering

alternative induction pathways and adopting

organotypic culture systems in drug development to

predict CYP enzyme modulation.

Clinical trials form the apex of this review,

particularly the meticulous quantification of CYP3A

modulation dynamics through continuous midazolam

(MDZ) infusion and the correlation of in vitro P450

downregulation with in vivo DDIs, especially

regarding 13-cis-Retinoic Acid (13cisRA) (Li et al.,

2019 & Stevison et al., 2019). The time-dependent

modulation of CYP enzymes is critical in the design

of DDI studies, which in turn influences the safe and

efficacious application of drugs metabolized by

CYP3A.

This compendium of studies coalesces into a

comprehensive understanding of CYP enzyme

induction, from experimental exploration to clinical

relevance, imparting vital insights that have the

potential to refine drug development and pave the

way for tailored therapeutic approaches in

personalized medicine.

Progress in CYP Enzymes Mechanisms of Induction and Its Applications

2 USING IN VITRO

METHODS FOR

PREDICTING IN VIVO

BEHAVIOUR

In vitro methodologies represent a cornerstone in the

preclinical assessment of CYP enzyme expression

upon exposure to pharmaceuticals. These

methodologies utilize various hepatocyte-based

models as proxies to the in vivo environment, offering

a simplified yet controlled setting to circumvent the

complexities and ethical considerations of animal

testing.

According to the Food and Drug Administration

(FDA) in U.S., a spectrum of in vitro hepatic models

has been established. This suite includes fresh and

cryopreserved primary hepatocytes, hepatocytes with

stable or transient transfections, hepatic cell lines, and

assays utilizing reporter genes. These platforms

enable pharmaceutical entities to gauge the induction

potential of new compounds, aligning their evaluation

processes with the regulatory framework set forth by

the FDA. A compound is flagged for further clinical

drug-drug interaction studies and in vivo scrutiny if it

elicits a CYP enzyme induction surpassing 40%

relative to a positive control, as per FDA guidelines.

Molecules demonstrating significant induction

propensities might be withdrawn from the

development pipeline to pre-empt adverse drug

interactions (Ghosh et al., 2023).

An initial high-throughput screen can detect

enhanced activation of nuclear receptors leading to

upregulated CYP enzyme synthesis. One approach

involves coalescing hepatoma cells with a CYP3A4

promoter region and a luciferase reporter or using a

human pregnane X receptor (PXR) coupled with the

CYP3A4-luciferase construct. Given CYP3A4's

susceptibility to PXR-mediated induction, regulatory

protocols advocate for its in vitro examination during

early drug development. A negative outcome for

CYP3A4 can generally rule out the induction potential

for CYP2C, as PXR also governs this enzyme. Hepatic

and immortalized cell lines that maintain hepatocyte

characteristics and deliver reproducible findings are

integral to the pharmaceutical industry. Among these,

the HepaRG and Fa2N-4 cell lines are frequently used,

with mRNA measurement serving as an accepted

endpoint for induction in immortalized cells.

Hepatoma lines, like HepG2, HepaRG, and BC2, see

extensive application within industry settings (Ghosh

et al., 2023 & Ingelman-Sundberg, 2004).

Primary hepatocytes are endorsed by industry and

regulatory bodies due to their preservation of in vivo-

like CYP metabolism post-isolation. For prolonged

phenotypic stability, 3D cultures are preferred over 2D

monolayers. With primary human hepatocyte cultures,

CYP mRNA, protein levels, and microsomal activity

can be accurately quantified. Furthermore, to address

inter-individual metabolic variance, hepatocyte

cultures sourced from multiple donors are utilized

(Ghosh et al., 2023).

Despite their merits, the application of primary or

cryopreserved hepatocytes is constrained by

challenges such as limited availability, potential loss

of enzymatic function due to cryopreservation, single-

receptor pathway analysis limitations, and variability

across different batches. As a result, immortalized and

hepatic cell lines have surfaced as practical

substitutes. These alternatives furnish manifold

benefits: they facilitate the examination of multiple

receptor-mediated mechanisms, offer an inexhaustible

supply via cell propagation, and ensure uniform

inducer responses. Current regulatory guidance also

transitions the focus from enzyme activity to mRNA

expression as the definitive endpoint for in vitro

assays. This shift underscores mRNA levels as a more

dependable metric for gauging CYP induction (Ghosh

et al., 2023).

2.1 Vitro Methods: Human

Cytochrome P450 Enzyme

Induction

The 2019 research by Bernasconi et al. pursued the

validation of two in vitro methodologies designed to

assess chemical compounds' propensity to activate

CYP enzymes—particularly CYP1A2, CYP2B6, and

CYP3A4 (Bernasconi et al., 2019). These

investigations employed two cellular models:

cryopreserved PHH and HepaRG human cells.

The choice of cryopreserved PHH was predicated

on their diverse array of native drug-processing

enzymes and necessary cofactors, establishing them

as a versatile tool for exploring toxicokinetic and

toxicodynamic phenomena. PHH stands as a robust in

vitro proxy for the human liver's metabolic processes.

In parallel, HepaRG cells are recognized for their

hepatic-like functionality, mirroring the metabolic

processes of actual human hepatocytes. This includes

the synthesis of key liver enzymes, the operation of

nuclear receptors, and the facilitation of xenobiotic

transporters. The criterion for validating these

models' metabolic competence was the induction of

CYP enzymes—a vital marker for evaluating the

cellular expression machinery's integrity and

functionality.

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The methodology detailed by the authors

leveraged differentiated HepaRG cells, preserved

through cryopreservation, to monitor the stimulation

of specific CYP enzymes. This process involved the

application of chemical agents to the cells and the

subsequent measurement of CYP1A2, CYP2B6, and

CYP3A enzyme induction via metabolite

quantification using advanced liquid

chromatography/mass spectrometry techniques.

Results indicated that CYP enzyme induction is

not merely a sensitive marker for protein synthesis

but is also a critical parameter for determining hepatic

metabolic capacity. Prior validations have confirmed

the reliability of both PHH and HepaRG cells in

gauging.the functional induction of the specified CYP

enzymes. The robustness of these results is further

corroborated by ring trial data demonstrating

consistent reproducibility across different

laboratories. These findings underscore the methods'

translatability and applicability across research

facilities equipped with cell culture and analytical

chemistry capabilities. Moreover, these in vitro

techniques have been corroborated for their

predictive accuracy regarding in vivo CYP enzyme

induction by various chemicals, evidenced by their

correct identification of reference inducers and the

successful prediction of in vivo human CYP

induction for a majority of the chemicals tested.

Ultimately, the decision to employ PHH or HepaRG

cells hinges on the specific evaluative needs of the

chemical assessment in question.

However, there have been discrepancies in

prediction for certain chemicals in PHH.

Carbamazepine, sulfinpyrazone, and rifampicin in

PHH did not align with in vivo human CYP induction

as expected (Table 2). This could be attributed to the

absence of sufficient human data or variability in the

hepatocyte batches used in the studies.

Figure 1: a comparative analysis of the predictive

capabilities of HepaRG cells and PHHin evaluating the

induction of CYP1A2, CYP2B6, and CYP3A4 (Bernasconi

et al., 2019).

The current validation study supports the

reliability and relevance of cryopreserved PHH and

cryopreserved HepaRG cells as in vitro tools for

assessing human CYP enzyme induction. These in

vitro methodologies are proving valuable in

regulatory risk assessment, offering insights into

metabolic processes, thyroid disruption, and nuclear-

receptor-mediated changes in biochemical pathways.

Utilizing these approaches helps researchers

understand the potential risks of xenobiotic exposure,

guiding the development of more accurate and

effective risk assessment frameworks.

2.2 CYP450 & P-Glycoprotein

Interactions’ Prediction

The Dumond et al. conducted a comprehensive study

to evaluate the potential for DDIs in the combination

therapy of tipranavir/ritonavir (TPV/r), used to treat

HIV-1 infections. Tipranavir, a protease inhibitor

with high efficacy against drug-resistant strains, is

known to induce certain cytochrome P450 enzymes,

particularly CYP3A (Dumond et al., 2010). To offset

this, it is coadministered with ritonavir (RTV), a

CYP3A inhibitor that helps sustain therapeutic levels

of tipranavir. This combination, however,

complicates the accurate prediction of drug

interactions because of its varying effects on

metabolic enzymes and transporters, a challenge that

this study aimed to address.

The researchers utilized a modified cocktail

phenotyping approach to evaluate potential drug

interactions resulting from the coadministration of

TPV and RTV. The study employed the caffeine,

warfarin, omeprazole, dextromethorphan,

intravenous MDZ, and vitamin K, to gauge the

activity of key hepatic and intestinal proteins and

explore the impact of TPV/RTV on them.

In vitro analyses had indicated that the TPV/RTV

combination inhibits several CYP enzymes, such as

CYP3A4, CYP1A2, CYP2C19, and CYP2D6. Yet, in

clinical settings with HIV-1-infected patients on

TPV/RTV and other CYP3A4 substrates, a decrease

in exposure to coadministered protease inhibitors was

observed, contrary to expectations. The study aimed

to resolve this inconsistency by investigating how

TPV/RTV influences various CYP enzymes and by

exploring genotype-phenotype correlations and the

genetic factors that contribute to drug interactions.

The recent investigation has furnished a

multifaceted analysis of the drug-drug interaction

potential attributed to tipranavir/ritonavir

(TPV/RTV) via a comprehensive utilization of probe

substrates and extensive genotyping. This

Progress in CYP Enzymes Mechanisms of Induction and Its Applications

encompasses the genotypic characterization of

critical CYP isoforms and P-glycoprotein (P-gp),

endeavoring to articulate the interaction landscape at

various pharmacokinetic junctures—baseline,

subsequent to acute exposure, and upon achievement

of a steady state. Study participants were

administered a cocktail of probe substrates and

digoxin, both via oral and intravenous routes, across

three distinct stages to delineate the

pharmacodynamic profile: baseline, after the triad of

initial TPV/r dosages, and at the steady-state

concentration.

Empirical results divulged that an inaugural dose

of TPV/r exerts negligible modulation on CYP1A2

and CYP2C9 activities. Contrastingly, it mediates a

mild inhibitory effect on CYP2C19 and P-gp, while

imposing a pronounced inhibition on the activity of

CYP2D6 and CYP3A enzymes. The investigative

outcomes underscore a spectrum of induction and

inhibition effects, thereby enriching the

comprehension of drug interaction mechanisms

inherent to TPV/RTV. Such insights are

indispensable for the refinement of clinical

deployment strategies for TPV/RTV.

The research emphasizes the efficaciousness of a

phenotyping methodology in the prognostication of

complex drug interactions and advocates for the

application of biomarker probes in clinical

pharmacokinetics. However, the translation of these

findings to alternative therapeutic agents that display

similar mixed inhibitory and inductive propensities

should be approached with circumspection. The

solicitation of further investigative efforts is

necessary to unravel the intricate web of interactions

that TPV/RTV may engage with a diverse array of

pharmacological entities.

3 NOVEL MECHANISMS OF

INDUCTION

3.1 Induction of CYP Enzymes in Mice

In the realm of toxicological research, the elucidation

of mechanisms underlying the induction of CYP

enzymes by xenobiotic substances remains a domain

of significant scientific inquiry. Jiang et al. embarked

on an incisive exploration of the molecular

mechanisms driving the persistent induction of

CYP1A1 and CYP1A2 enzymes after 3-

methylcholanthrene (MC) exposure, a potent

carcinogenic constituent among polycyclic aromatic

hydrocarbons (PAHs) (Jiang et al., 2009). The

investigators postulated a theory suggesting that MC

catalyzes a long-standing transcriptional activation of

the CYP1A1 and CYP1A2 gene promoters, thus

inciting extended enzyme activity (Chen et al., 2017;

Jiang et al., 2009 & Gibson et al., 2002).

To substantiate their hypothesis, the researchers

designed a study utilizing both adult male wildtype

(WT) mice and genetically modified counterparts,

engineered to harbor human CYP1A1 or murine

CYP1A2 promoter sequences. The investigative

protocol entailed the administration of MC to these

models, with subsequent assessments focused on

gauging promoter-specific transcriptional activity.

The study treated mice with MC or a vehicle

control (corn oil) daily for four consecutive days. The

researchers utilized bioluminescent imaging to assess

luciferase reporter gene expression, serving as a

proxy for promoter activity, at intervals of 1, 8, 15,

and 22 days after cessation of MC treatment.

The results indicated that MC treatment

substantially increased luciferase expression driven

by both CYP1A1 and CYP1A2 promoters in the

transgenic mice. This elevated luciferase activity

persisted for up to 22 days, with a more significant

effect observed in the CYP1A1-luc mice. The MC-

induced increases in CYP1A1 and CYP1A2 activity,

as demonstrated by luciferase expression, supported

the initial hypothesis regarding sustained

transcriptional activation (Chen et al., 2017; Jiang et

al., 2009 & Gibson et al., 2002).

Endogenous CYP1A1 and CYP1A2 expression

was persistently induced in WT, CYP1A1-luc, and

CYP1A2-luc mice, corroborating the sustained

impact of MC exposure. Analysis of the findings

indicated a 15-fold increase in CYP1A1-luc

expression (Figure 2B). In contrast, the induction of

CYP1A2-luc by MC was less pronounced (Figure 2C,

D).

These data underscore the differential and

prolonged effects of MC exposure on the induction of

CYP1A1 & CYP1A2 enzymes. The utilization of

transgenic mouse models expressing luciferase

reporters driven by the CYP1A1 and CYP1A2

promoters represents a powerful tool for elucidating

the molecular mechanisms underlying persistent

enzyme induction, particularly in the context of PAH-

induced carcinogenesis.

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Figure 2: Bioluminescent imaging of CYP1A1-luc & CYP1A2-luc mice of MC treatment (Dumond et al., 2010).

3.2 Novel CYP Induction Mechanism

by YAP/TEAD Inhibitors in

Human Hepatocytes

DDIs which is mediated by the induction of

CYPenzymes represent a significant challenge in

drug development, necessitating comprehensive

assessment during preclinical studies. A recent study

focuses on the Hippo signalling pathway, which is

known to govern cell fate, proliferation, and

apoptosis, emphasizing its significance in the

regulation of cellular processes (Zhang et al., 2024).

At the molecular fulcrum of the Hippo signalling

cascade lies the intricate intracellular trafficking of

Yes-associated protein (YAP) and transcriptional

coactivator with PDZ-binding motif (TAZ), which

transits between the cytoplasm and nucleus.

Ordinarily, in the quiescent state of the Hippo

pathway, YAP/TAZ are permitted nuclear ingress,

instigating the transcription of genes that promulgate

cell proliferation and survival. This nuclear

localization embodies the core of a cellular growth-

promoting regime. In stark contrast, the activation of

the Hippo pathway signals for a shift—YAP/TAZ are

sequestered within the cytoplasm, an event that

curtails their role as transcriptional regulators and, by

extension, serves as a biochemical clamp on cellular

proliferation. The dichotomy of YAP/TAZ

localization thus constitutes a critical regulatory axis

in cell growth control, embodying a cellular

barometer that modulates gene expression in

accordance with the proliferative or inhibitory cues of

the Hippo pathway (Chen et al., 2017; Jiang et al.,

2009 & Gibson et al., 2002).

The YAP/TEAD (TEA domain family member)

signalling pathway has gained attention as a potential

therapeutic target in oncology due to its role in cell

growth regulation. Various inhibitors of YAP/TEAD

are progressing through clinical development, each

with distinct chemical structures and mechanisms of

action. The pathway is also influenced by cell-cell

contacts and biomechanical factors. YAP/TEAD

activity is further modulated by cell geometry, which

contributes to the complexity of the system.

Oliva-Vilarnau et al. conducted a pivotal study to

evaluate the potential for CYP induction by assessing

multiple YAP/TEAD inhibitors with varying

selectivity profiles for TEAD isoforms (Oliva-

Vilarnau et al., 2023). This study explored both

traditional 2D cultures & 3D spheroids of PHH. The

results indicated that YAP/TEAD inhibition caused

extensive CYP enzyme induction in 2D monolayers

but significantly reduced induction in 3D spheroids,

suggesting a critical relationship between cell

geometry and CYP regulation.

Further in-depth analysis through RNA

sequencing revealed that YAP/TEAD signalling was

more pronounced in 2D cultures compared to 3D,

likely due to alterations in mechanoenzyme. The

hyperactivation of YAP/TEAD in these cultures

contributed to increased activity of other interacting

transcription factors. This hyperactivation led to

hepatocyte dedifferentiation, with a corresponding

increase in hepatic function, including CYP enzyme

induction. This induction was, therefore, an indirect

consequence of YAP/TEAD inhibition.

These findings underscore the relevance of the

Hippo pathway in drug development and its broader

implications in pharmacokinetics. It highlights the

necessity for organotypic 3D cultures in preclinical

studies to better simulate clinical conditions and

accurately assess pharmacokinetic profiles.

Consequently, the results advocate for advanced

testing models to refine the drug development process

and enhance safety evaluations.

Progress in CYP Enzymes Mechanisms of Induction and Its Applications

Figure 3: Comparative Gene Expression Response to YAP/TEAD Inhibition in 2D vs. 3D Primary Human Hepatocyte

Cultures (Oliva-Vilarnau et al., 2023)

A-B: The figure presents volcano plots illustrating

the gene expression impact of Compound 3 (CMPD-

3) on PHHcultured in two dimensions (2D) (A) and

three dimensions (3D) (B). Differentially expressed

genes (DEGs) are highlighted.

C: A Venn diagram details the intersection of

DEGs between 2D and 3D PHH cultures under

CMPD-3 influence, underscoring the distinctive gene

expression profiles elicited by YAP/TEAD inhibition

in differing culture formats.

D-E: Volcano plots convey the gene expression

ramifications of Compound 4 (CMPD-4) on PHH in

2D and 3D contexts. As with CMPD-3, DEGs

connected to ADME are denoted in yellow, and those

linked to Hippo signalling are in green.

F: Another Venn diagram showcases the

commonalities and discrepancies of DEGs across 2D

and 3D PHH cultures when subjected to CMPD-4,

highlighting the dependency of cellular responses to

YAP/TEAD inhibition on the cultural environment.

4 CLINICAL TRIALS

In the domain of drug metabolism research, in vitro

assays are a cornerstone; however, their predictive

validity for in vivo CYP induction is inherently

limited. Hence, in vivo assays emerge as the gold

standard, offering a more faithful reflection of CYP

induction within a living system. The direct

measurement of enzyme quantity and activity in vivo

presents practical challenges, especially in humans.

Consequently, an indirect methodology prevails,

primarily involving comparative analysis of a drug's

AUC before and after the introduction of a novel drug

entity or potential inductive agent.

Preclinical evaluation often recruits animal

models—ranging from mice and rats to monkeys and

dogs—to ascertain CYP induction as a precursor to

human testing. It is crucial to recognize the distinct

discrepancies in enzyme systems and receptor

affinities across species, which often result in

divergent metabolic responses. For example,

omeprazole's induction effect on CYP1A2 is

exclusive to humans and does not extend to murine or

rat models. To mitigate such species-specific

limitations, research has pivoted towards the

development of humanized mice through genetic

engineering or the engraftment of human hepatocytes

into immunocompromised mice, circumventing the

confounding influence of murine hepatic enzymes.

Animal models, notwithstanding the interspecies

variation in CYP induction, serve to generate initial

pharmacokinetic profiles. Nevertheless, human

subjects remain the epitome for assessing CYP

induction. In human studies, the characterization of

enzyme induction is conducted using selected CYP

probe substrates, adhering to rigorous criteria such as

enzyme specificity, minimal cross-enzyme inhibition,

and optimal pharmacokinetic characteristics, like

minimal rapid metabolism or shorter half-lives.

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An alternative in vivo measurement strategy

involves evaluating pharmacological parameters like

the EC50 and Emax, mindful of the interindividual

variances that arise due to CYP polymorphisms,

which can influence the response to induction probes.

Per FDA directives, data from in vitro assays and

preliminary clinical assessments should inform the

decision to advance to comprehensive human in vivo

or clinical evaluations. A drug is earmarked for in

vivo investigation only if it elicits an induction

exceeding 40%. Furthermore, the FDA's regulatory

framework allows for the exclusion of certain

enzymes from in vivo scrutiny if in vitro results are

conclusively negative (Ghosh et al., 2023).

4.1 Time Course Quantification of

CYP3A Modulation with Micro

Dosed MDZ

The enzymatic activity of CYP3A is important in the

biotransformation and clearance of various

pharmaceuticals, predominantly in hepatic and

enteric regions. Perpetrator drugs that modulate the

activity of CYP3A enzymes can profoundly influence

the pharmacokinetic profile of CYP3A substrates,

potentially culminating in clinically relevant DDIs.

Traditional studies on DDIs tend to prioritize static

exposure levels as endpoints, thus overlooking the

dynamic nature of these enzymatic interactions over

time. A comprehensive understanding of the temporal

modulation of CYP3A could shed light on critical

periods of altered enzymatic activity, thereby

enhancing the strategic planning and oversight of

DDIs in clinical settings.

To bridge this knowledge gap, Stevison, F et al.

incorporated the use of surrogate probe substrates,

such as MDZ, in their investigation of drug

interactions (Stevison et al., 2019). Given that MDZ

is extensively metabolized by CYP3A, its

pharmacokinetic profile serves as a reliable reflection

of CYP3A activity. The linearity of MDZ

pharmacokinetics across a broad dosage spectrum

facilitates the assessment of CYP3A activity without

eliciting notable pharmacodynamic responses.

This clinical investigation aimed to delineate the

temporal patterns and degree of in vivo modulation of

hepatic CYP3A activity by various perpetrator drugs.

The researchers implemented a continuous micro

dosing regimen involving intravenous MDZ, which

allowed for precise quantification of metabolic DDIs

in a healthy cohort. The study also endeavored to

characterize different modulatory mechanisms.

The study's protocol included 24 healthy

participants who received an initial bolus of

intravenous MDZ. Subjects were stratified into four

cohorts, each receiving a distinct CYP3A perpetrator

drug: voriconazole, rifampicin, or efavirenz, with two

placebo-controlled individuals per group. Following

the MDZ infusion, perpetrator drugs were introduced

after a 2-hour interval. Regular blood sampling

facilitated the measurement of MDZ and its primary

metabolite, 1'-hydroxyMDZ. The study's foremost

aim was to quantify the temporal modulation of

CYP3A activity by comparing MDZ clearance

among the treatment and placebo cohorts.

The findings demonstrated unique temporal

signatures and intensities of CYP3A modulation by

each perpetrator drug. Notably, efavirenz, recognized

as a CYP3A enhancer, displayed a swift onset of

modulation, attaining peak impact within 2 to 3 hours.

Conversely, rifampicin, a CYP3A inductor,

manifested a protracted onset, with maximal impact

noted after 28 to 30 hours, and subsequently a rapid

return to baseline within 1 to 2 hours. Voriconazole,

in both oral and intravenous forms, displayed a

sustained inhibitory effect on CYP3A, maintaining

suppression over the duration of the sampling

interval, which extended to 8 hours post-

administration. The study charted the differential

peak clearance alterations induced by efavirenz,

rifampicin, and both administrations of voriconazole,

as depicted in Figure 4.

Figure 4: The percentage change in MDZ clearance. over

time in comparison to the placebo group, following the

administration of perpetrator drugs (Stevison et al., 2019).

The perpetrator drugs are categorized into

different arms. The vertical dashed line indicates the

time when the perpetrator drug was administered.

Progress in CYP Enzymes Mechanisms of Induction and Its Applications

4.2 Translating Cytochrome P450

Downregulation from In Vitro to In

Vivo

Surrogate probe substrates such as MDZ are

instrumental in drug interaction research, providing a

scientifically robust method to evaluate CYP enzyme

activity. MDZ, primarily metabolized by the CYP3A

isozyme, serves as an accurate barometer for CYP3A

functionality due to its linear pharmacokinetics across

extensive dosing ranges, thus enabling the assessment

of CYP3A without eliciting marked pharmacological

outcomes (Ingelman, 2004).

A 2019 investigation by Li et al. delved into the

impact of all-trans-retinoic acid (atRA), the

metabolite of vitamin A on CYP2D6 expression in

diverse experimental systems (Li et al., 2019). Prior

studies identified that atRA diminishes CYP2D6

expression in cellular models and murine systems by

activating the transcriptional corepressor small

heterodimer partner (SHP). Yet, whether this

suppressive action translates to human physiology

remained ambiguous. Notably, atRA does not

interfere with the enzymatic function of CYP2D6,

offering a unique vantage point to study DDIs that

arise exclusively from transcriptional downregulation

(Chen et al., 2017; Jiang et al., 2009 & Gibson et al.,

2002).

The atRA isomer 13cisRA, is particularly suited

for scrutinizing the translational aspects of CYP

downregulation. Following administration, 13cisRA

is isomerized to atRA and subsequently metabolized

into 4-oxo-13cisRA in humans. Consequently, any

DDIs observed post-13cisRA administration could be

ascribed to 13cisRA itself or a collective effect of

these metabolites.

The primary objective of Li et al.'s research was to

elucidate the impact of three compounds, namely

13cisRA, atRA, and 4-oxo-13cisRA, on the

expression of CYP2D6 within hepatic cells in humans

(Li et al., 2019). Additionally, the study aimed to

determine whether the findings from in vitro

experiments could serve as predictors of potential

DDIs in clinical settings. The research findings

suggested a theoretical reduction of approximately

50% in CYP2D6 activity following the

administration of 13cisRA, as observed in in vitro

assays. However, analysis of clinical data,

specifically the area under the plasma concentration-

time curve for dextromethorphan, a substrate of

CYP2D6, revealed only a minor increase in the drug's

metabolic clearance after 13cisRA therapy. Similarly,

in murine models, the administration of 4-oxo-

13cisRA resulted in increased mRNA expression of

several Cyp2d isoforms; however, this effect did not

strongly correlate with in vivo modulation of

CYP2D6 activity.

Moreover, a modest in vitro induction of CYP3A4

in PHH was associated with a correspondingly minor

induction in vivo, thereby presenting a disparity

between in vitro observations of CYP downregulation

and the manifestation of clinical DDIs. These

observations accentuate the need for an enriched

comprehension of the mechanisms governing CYP

downregulation to refine the prediction and

management of DDIs in a clinical context.

5 CONCLUSION

The scientific quest to understand the induction of

CYP enzymes has garnered considerable focus within

the realms of drug development and toxicology

(Carroccio, 1994). The goal is to foster a deeper, more

precise scientific understanding. Although there has

been considerable progress in decoding the

mechanisms that underlie CYP induction, the

intricacies of these molecular processes demand

further elucidation. This need is heightened by the

ongoing introduction of novel pharmaceuticals and

environmental agents, each necessitating

comprehensive evaluations to assess their potential

for CYP induction and the resultant risk of

undesirable side effects.

The field has seen substantial advancements with

the introduction of innovative models for the study of

CYP induction. These models operate both within

controlled laboratory environments (in vitro) and

living organisms (in vivo), significantly bolstering

our capacity to examine and anticipate the behaviour

of CYP induction. They have markedly enriched our

knowledge of the signalling pathways and the diverse

array of factors that govern CYP induction, including

genetic variability and environmental influencers.

Nevertheless, the predictive accuracy for CYP

induction in relation to newly synthesized compounds

is still encumbered by the multifaceted nature of these

biological pathways.

Currently, the drug development industry is

equipped with well-established experimental

protocols for investigating the induction of CYP

enzymes. The data derived from these laboratory

analyses provide a foundational guide for subsequent

biological investigations and are integral to the

creation of predictive models that mirror the

pharmacokinetic processes observed in actual

physiological conditions. Despite these advanced in

silico tools, empirical studies in human populations

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are indispensable for a conclusive portrayal of the

effects of CYP induction and inhibition, a

requirement that is particularly salient for the

attainment of regulatory approval. It is fortuitous that

ongoing innovations in research methodologies have

sharpened the ability to detect potential drug

interactions that are mediated by CYP enzymes early

in the drug development cycle, thereby mitigating

unforeseen adverse effects in clinical settings. Early

recognition of such interactions is crucial, steering the

course of drug development away from entities that

exhibit strong inhibitory or inductive effects on CYP

enzymes. Nonetheless, we must acknowledge the

presence of yet unidentified agents, possibly present

in our diet, herbal treatments, and environmental

exposures.

The profound enhancement of our understanding

of CYP-mediated interactions has refined the drug

development paradigm and the implementation of

computational tools and databases to support

medication prescribing practices has greatly

facilitated their clinical deployment. These

advancements are particularly vital considering the

vast repository of data on DDIs, which presents a

formidable challenge for clinicians to master single-

handedly. It is pertinent to note that while the design

of drugs has traditionally cantered on improving

metabolic stability to diminish CYP-related

interactions, it is also essential to consider the

potential for interactions mediated by biological

transport mechanisms.

Although the capability to predict CYP inhibition

and induction is generally reliable, exceptional cases

continue to emerge that defy expectations. For

instance, the synergistic interaction between non-

activating compounds and the PXR, resulting in

receptor activation, exemplifies the complexity of

predicting drug interactions. These synergies may

manifest among pharmaceutical agents or in

scenarios of exposure to complex environmental

mixtures, relevant in toxicology. Hence, despite the

substantial body of knowledge regarding CYP

inhibition and induction accrued over the years,

ongoing research is imperative. The landscape of

drug interaction remains dynamic, with the ever-

present prospect of unearthing new insights.

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