A new evaluation system for drug–microbiota interactions

Abstract The drug response phenotype is determined by a combination of genetic and environmental factors. The high clinical conversion failure rate of gene‐targeted drugs might be attributed to the lack of emphasis on environmental factors and the inherent individual variability in drug response (IVDR). Current evidence suggests that environmental variables, rather than the disease itself, are the primary determinants of both gut microbiota composition and drug metabolism. Additionally, individual differences in gut microbiota create a unique metabolic environment that influences the in vivo processes underlying drug absorption, distribution, metabolism, and excretion (ADME). Here, we discuss how gut microbiota, shaped by both genetic and environmental factors, affects the host's ADME microenvironment within a new evaluation system for drug–microbiota interactions. Furthermore, we propose a new top‐down research approach to investigate the intricate nature of drug–microbiota interactions in vivo. This approach utilizes germ‐free animal models, providing foundation for the development of a new evaluation system for drug–microbiota interactions.


INTRODUCTION
Since the introduction of pharmacogenetics by Dr. Archibald Edward Garrod in the early 20th century, the influence of human genetic information on drug response (individual variability in drug response [IVDR]) has been increasingly emphasized.Pharmacogenomics harnesses this information to understand variations in drug sensitivity, metabolism, and adverse drug reactions (ADRs) [1].In recent years, clinical drug research has primarily focused on the development of new drugs targeting disease gene products.Despite the emergence of numerous new drugs, translating innovative ideas into effective and safe therapeutics remains a challenge.Over 50% of Phase III trials have failed to replicate positive results from Phase II, leading to wasted resources and potential harm to participants [2].It is now understood that the drug response phenotype is determined by a complex interplay of genetic and environmental factors.Gut microbiota composition reflects environmental influences such as diet, lifestyle, medication use, and calorie intake [3,4].This study aims to highlight recent advancements in pharmacology regarding how gut microbiota, shaped by both genetic and environmental factors, affects the host's drug absorption, distribution, metabolism, and excretion (ADME) microenvironment within a new framework for evaluating drug-microbiota interactions.By incorporating gut microbiota, we may gain new opportunities for preclinical drug efficacy and safety investigations (Figure 1A).
Pharmacogenomics leverages human genomic information to decode the mechanisms underlying IVDR, particularly focusing on the in vivo processes of drug ADME.Similarly, Pharmacomicrobiomics aims to understand how gut microbiota genomic information contributes to IVDR.Current research has predominantly focused on identifying drug-metabolizing enzymes produced by gut bacteria through isolation [5,6].However, the metabolic function of gut microbiota is influenced by the host microenvironment, as the gut microbiota itself is a key component of this complex environment.This intricate interplay between gut microbiota and the host environment may be the key reason for its involvement in IVDR.This study innovatively investigates how gut microbiota alters the host microenvironment's impact on ADME, focusing on the in vivo function of gut microbiota within the host body.Additionally, the study proposes a new top-down research approach to address the challenges of Pharmacomicrobiomics. Overall, Pharmacomicrobiomics offers not only new insights into the mechanisms of IVDR but also new standards for preclinical drug research.By considering both genetic and environmental factors (as reflected by gut microbiota), this field holds promise.Furthermore, due to the relative ease of manipulating gut microbiota and its high safety profile, Pharmacomicrobiomics has the potential to provide effective solutions for improving drug efficacy and reducing toxicity through the development of a new evaluation system for drug-microbiota interactions.

How does gut microbiota drive individual differences in drug ADME?
Drugs circulating and undergoing metabolism in the bloodstream can reside in the gut for extended periods, where crucial drug-microbiota interactions occur.This interaction is bidirectional: drugs can alter gut microbial composition, growth, and function, while gut microbiota can metabolize and modify drug structure through enzymatic activity.These interactions ultimately influence a drug's bioavailability, biological activity/toxicity, and consequently, the individual's response to the medication.Regardless of the administration route (oral or injected), drugs can directly reach the gut microbiota or indirectly interact with it via the bloodstream.Although studies have identified drug-metabolizing enzymes derived from gut microbiota [5,6] GERM-FREE ANIMAL AND DRUG METABOLISM transplantation (FMT) and probiotics vary significantly [7].ADME processes determine a drug's stability, target site delivery, potential for generating toxic substances, and successful elimination of excess components.Individual variations in the gut microbiota-influenced metabolic environment in vivo can influence drug ADME, regardless of the administration route (Figure 1B).

Gut microbiota produces druggable metabolites
The gut microbiota exerts a broad and profound influence on the physiological functions of various bodily systems.Individual differences in gut microbiota composition create a unique metabolic environment within the body, impacting drug ADME.Furthermore, the secondary metabolites produced by gut microbiota can serve as druggable targets for regulating bodily functions.Through fermentation, gut microbiota digest complex carbohydrates and proteins, generating vitamins and enteral nutrients.These nutrients modulate host energy metabolism and serve as a carbon source for colonocytes [8].A clinical medication study even demonstrated a clear association between polypharmacy and microbial functions related to short-chain fatty acid (SCFA) metabolism [9].The dynamic interplay between gut microbiota and bile acid biotransformation plays a key role in maintaining glycolipid metabolic homeostasis, while also modulating intestinal immunity, inflammation, and tumorigenesis [10,11].These microbiota-derived metabolites also contribute to the maintenance of liver and kidney function, which are crucial organs for drug metabolism and excretion.

Gut microbiota biotransformation of drugs
The gut microbiota plays a crucial role in various metabolic reactions through enzymes.These reactions include reduction, hydrolysis, functional group transfer, and cleavage.These complex metabolic processes can regulate the duration and intensity of a drug's pharmacological effects, ultimately influencing its clinical benefit [6].Studies have shown that, beyond antibiotics, many nonantibiotic drugs targeting humans can also be affected by gut microbiome metabolism.This includes medications like statins and digoxin [12].Gut microbiota can directly degrade or compete with drugs by producing drug-degrading enzymes [13,14].Indirectly, drugs can influence the activity of gut microbiota's drugmetabolizing enzymes and metabolic pathways, either activating or inactivating them through host drugmetabolizing enzymes [15,16].This concept even applies to reducing drug side effects.For example, higher statin doses increase the risk of liver and muscle problems, but combining them with probiotics can potentially lower the effective dose and minimize these side effects [17].

Drug-induced gut microbiota remodeling
Most drugs undergo metabolism through endogenous enzymes and conjugation reactions.This process can have unintended consequences, leading to changes in gut microbiota composition and colonization patterns.Drugs The clinical significance of gut microbiota in pharmacological research and the regulation of drug-targeting genes.(A) Pharmacomicrobiomics provides new opportunities for preclinical investigations of drug efficacy and safety.① The effect of drugs on disease characterization depends on a combination of genetic and environmental factors.② Pharmacogenomics employs human genomic information (genetic factors) to decode the reasons behind individual variability in drug response (IVDR).③ Pharmacomicrobiomics employs gut microbial genomic information (environmental factors) to decode the reasons behind environmental factors in drug response.④ A better understanding of the role of environmental factors on drug safety and efficacy is expected to provide preclinical research strategies to improve the success of clinical drug development.(B) Involvement of gut microbiota in drug ADME.Up: Gut microbiota is a primary site for the drug ADME.Whether the drug is administered orally or injected, it directly reaches the gut microbiota or via the bloodstream.Down: Gut microbiota produced new drugs or druggable metabolites.It concludes short-chain fatty acids (SCFAs), vitamins, 5hydroxytryptamine (5-TH), γ-aminobutyric acid (GABA), trimethylamine oxide (TMAO), and bile acids, as well as secondary metabolites of related metabolic pathways.Gut microbiota is involved in a variety of metabolic reactions, mainly involving reductive metabolism and hydrolysis.Drug-induced gut microbiota remodeling.Most drugs are metabolized through endogenous enzymes and coupling reactions, resulting in ectopic microbiota colonization and remodeling of the gut microbiota.(C) Interactions between gut microbiota genes and drugtargeted host genes.ARGs: Drugs can influence the expression of antibiotic resistance genes (ARGs) in the gut microbiota.Gut phenotype: The relationship between personalized gut phenotype and drug efficacy based on genetic sequencing of the gut microbiota has been demonstrated.Immunotherapy: Gut microbiota can modulate the safety and efficacy of targeted immunomodulators.Encoding enzyme: CRISPR gene editing of engineered bacteria targets the elimination of ARGs in gut microbiota.
can disrupt the mucosal barrier of the gut, oral cavity, and gastrointestinal tract in two ways.On the one hand, they can trigger inflammation and promote the ectopic colonization of microbiota in unintended locations [18,19].For instance, methotrexate, a common immunosuppressive and anticancer drug, can significantly reduce levels of Bacteroides fragilis while increasing macrophage density in the gut, leading to gastrointestinal harm in clinical settings [20].Conversely, some drugs can have beneficial effects on the gastrointestinal mucosal barrier.Metformin, for example, promotes the production of short-chain fatty acids by gut microbiota, which strengthens intestinal mucosal immunity and contributes to its therapeutic effects in reducing insulin resistance and promoting blood glucose homeostasis [21].These examples highlight how gut microbiota can influence individual differences in drug absorption, distribution, metabolism, and excretion.However, the precise mechanisms by which drugs contribute to these individual variations remain under investigation.

How do drug-targeted host genes contribute to the gut microbiota?
Pharmacomicrobiomics has emerged as a natural extension of pharmacogenomics, driven by the growing recognition of gut microbiota as a "second human genome" (Figure 1C).A major concern associated with antibiotic use is the widespread presence of antibiotic resistance genes (ARGs) within gut microbiota.The human gut serves as a reservoir for resistance to β-lactam and plasmid-mediated quinolone antibiotics [22].It has been established that antibiotic use can enrich the abundance of resistant bacteria and ARGs within the gut microbiota.Interestingly, similar effects have been observed with nonantibiotic drugs, including nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, naproxen, and diclofenac, as well as the antiepileptic drug carbamazepine [23,24].Clinical trials are exploring the use of gene-edited bacteria, called clustered regularly interspaced short palindromic repeats (CRISPR) systems, to eliminate antibiotic-resistant bacteria.Current evidence suggests that gut microbiota facilitates the transmission of antibiotic resistance plasmids within the human gut [25].Conversely, novel derivatives obtained by manipulating gut microbiota using techniques like immobilization, enzyme-directed evolution, flow biocatalysis, protein engineering, and combinatorial biosynthesis hold promise in combating the spread of antibiotic resistance [26].For example, FMT and colonization with the gut bacterium Coprobacillus cateniformis have been shown to improve the efficacy of PD-1 immunotherapy by downregulating the PD-L2-RGMb pathway [27].Recent studies have confirmed the association between specific gut microbiota enterotypes and drug efficacy [28].Characterizing an individual's gut microbiota enterotype has the potential to inform personalized and precision medicine approaches.While it has been established that drugtargeted host genes influence gut microbiota, the specific environmental factors involved require further investigation.
Influence of environmental variables on gut microbiota and its implications for developing a novel drug-microbiota interaction evaluation system While correcting environmental factors is crucial, investigating drugs that can mitigate their negative effects on the gut microbiota is equally important.Mounting evidence in recent years has linked gut microbial composition and function to a wide range of diseases.For instance, a 2022 cohort study published in Nature involving 8208 participants found that various environmental factors, both during early life and present times, significantly affect gut microbial composition and function.In fact, the study suggests that environmental factors may have an even greater impact on the gut microbiota than the disease itself [4].These environmental factors encompass both the external natural environment and social environment (Figure 2).

Natural environment contributions to the drug-microbiota interactions
Natural climate and geographic environments shape unique microbial ecosystems, facilitating the direct transfer of microbes to humans.Furthermore, climatic extremes can influence the diversity of gut microbiota composition.Interestingly, specific gut microbes can undergo adaptive changes that benefit the host's response to these extreme environments.For example, factors like low temperature, high heat, and high altitude can all influence the composition of gut microbiota, leading to changes in the activity of drug-metabolizing enzymes harbored by these gut microbes [29][30][31].Additionally, hypoxia can impact the direct and indirect effects of gut microbiota on its own or host drug metabolism.These effects are mediated by various factors, including gut microbiota-derived drug-metabolizing enzymes, metabolites, and immune modulation [32].

Environmental contamination contributions to the drug-microbiota interactions
External environmental contaminants, including ARGs, PM2.5 particulates, and microplastics, may represent another significant factor influencing drug metabolism within the gut microbiota.It is important to note that ARGs and pollution do not directly inactivate antibiotic efficacy, but rather contribute to the development and spread of antibioticresistant bacteria, thereby reducing the effectiveness of these drugs [33][34][35].The metabolic function of gut microbiota is a critical factor influencing both drug effectiveness and the potential for side effects.For instance, studies have shown that ephedra polysaccharides can alleviate PM2.5-induced airway inflammation resembling asthma in mice by modulating gut microbiota composition and SCFA production [36].Microplastics can disrupt gut microbiota metabolism, potentially contributing to host insulin resistance, liver damage, and blood cell abnormalities [37][38][39].Interestingly, gut microbiota also plays a role in metabolizing and influencing the efficacy of drugs used to mitigate radiation damage.Examples include yellow wine polyphenolic compounds, baicalein, quercetin, D-galactose, and the botanical Lycium barbarum [40][41][42][43].

Social environmental factors contribute to the drug-microbiota interactions
The gut microbiota plays a crucial role in cognitive and behavioral processes influenced by psychological stress.This interplay primarily involves the hypothalamic-pituitary-adrenal axis (HPA axis), neuroimmune regulation, and the immune system.Compelling evidence suggests that gut commensal segmented filamentous bacteria can exacerbate cardiovascular disorders associated with psychological stress by influencing the HPA axis [44,45].Research also indicates that chronic stress can alter the expression and activity of drug-metabolizing enzyme genes within gut microbiota [46].Gut microbiotaderived metabolites, including probiotics, prebiotics, and neurotransmitters such as 5-hydroxytryptophan (5-HT), indole, glutamate, gamma-aminobutyric acid (GABA), and SCFAs, can alleviate chronic stressinduced cognitive impairment and emotional responses [47,48].Interestingly, the efficacy of clinical antidepressants like (R)-ketamine, Lanicemine, Venlafaxine, Fluoxetine, and Agomelatine (AGO) has been closely linked to the composition of gut microbiota [49,50].More interestingly, researchers have found that drugs that regulate gut microbiota, such as antibiotics (Rifaximin, Isoalantolactone, and Minocycline) and proton pump inhibitors (Esomeprazole, etc.), can also influence psychological stress relief [51][52][53].Traditional Chinese medicine formulas, herbal medicines, and their individual components (monomers or compounds) may also alleviate stress-related disorders by impacting gut microbiota [54,55].

Addressing evaluation gaps in drug-microbiota interactions: exploring novel methodologies
The ability of human gut microbiota to metabolize small molecule medications contributes to variations in clinical drug efficacy.Pharmacomicrobiomics offers a promising avenue for preclinical studies to optimize drug effectiveness.However, current research faces limitations due to the complex interplay between gut microbiota and drugs in vivo.Traditionally, researchers have focused on isolating single bacterial strains from the gut to study drug metabolism mechanisms in vitro.While in vitro culture allows in-depth analysis, it fails to capture the in vivo function of the microbiota-its interaction with host genes and its role in shaping the metabolic and immune environment within the body.To gain a complete picture, it is crucial to identify key gut microbiota-driven pharmacodynamic ingredients within the unique in vivo environment of host cells and tissues, rather than relying solely on in vitro cultures.Next-generation sequencing technologies have empowered researchers to understand the role of gut microbiota in drug metabolism in vivo.However, these techniques often lack the ability to establish causal relationships between specific microbes and drug metabolism.Sequencing data may reveal enzymes with similar functions across different bacterial species, making it difficult to pinpoint the functionally relevant bacteria.Furthermore, research has broadened its focus beyond the digestive and respiratory systems, recognizing the potential role of microbiota in other tissues in disease onset and progression [56,57].This raises the question of whether drug-targeting microbiota resides not only in the gut but also at the site of drug delivery.Precise germ-free animal models offer a potential solution to the challenges mentioned above (Figure 3A).These models, with their in vivo gut microbiota function, allow researchers to investigate the interaction between drugs and gut microbiota.Colonizing germ-free animals with single, functionally characterized microbes can provide direct evidence for gut microbiota's involvement in drug metabolism in vivo, helping to elucidate the causal relationship between specific microbes and clinical drug efficacy and safety.Germ-free treatment of genetically engineered animals can provide even deeper insights into the mechanisms of gut microbiota-mediated drug metabolism.Additionally, addressing the challenge of "in vivo" gut microbiota function is crucial for understanding how gut microbiota influences environmental factors, leading to variations in drug effectiveness and IVDR.Furthermore, identifying microbes at the single-cell level can provide technical support for isolating functionally relevant, yet difficult-to-cultivate, microbiota strains, given the low sequence conservation and functional redundancy within microbial genes.Ultimately, advancing Pharmacomicrobiomics research requires the development of in-depth coculture models that integrate tissues, cells, and microbes, reflecting the "in vivo" function of gut microbiota.

Leveraging in vivo gut microbiota function for the design of next-generation drug ingredients involved in drug-microbiota interaction
Genetically engineered germ-free disease models offer a top-down approach to understanding the "in vivo" function of drug metabolism by gut microbiota (Figure 3B).These models can be used to screen for the active ingredients of bacterially modified drugs that enter the bloodstream [58].This approach sheds light on how gut microbiota communities, functioning as a metabolic GERM-FREE ANIMAL AND DRUG METABOLISM | 7 of 14 "factory," modify pharmaceutical compounds through the action of microbial enzymes [59].However, it is important to recognize that the metabolic effects of gut microbiota on drugs are also influenced by the host's unique immune and metabolic microenvironment.While drugs, whether oral or intravenous, are primarily metabolized in the liver before entering the intestine for absorption, the impact of gut microbiota on drug efficacy after this initial metabolism often goes overlooked.These modifications can occur through the action of microbial enzymes within the complex gut microbiota community.
To effectively analyze the pharmacodynamic mechanisms of Traditional Chinese Medicine (TCM) formulas, TCM compounds, and other natural products with complex components, researchers may benefit from developing efficient techniques for genetic engineering and establishing aseptic/humanized microbiota animal models.This approach is particularly valuable for studying the interactions between gut microbiota, host, and drugs.Similar to the approach used in serum pharmacology for TCM and other complex natural compounds [60], researchers can utilize drugcontaining serum from GF disease model animals.This strategy allows not only for screening small molecule pharmacodynamic substances modified by gut microbiota enzymes but also for more refined in vitro studies investigating the interplay between host cell-gut microbiota-drug.

High-resolution sequencing for single-cell recognition provides technology for new evaluation system for drug-microbial interaction research
Gut-liver humanization models that incorporate human gut microbiota and drug-metabolizing enzymes can offer a more clinically relevant platform for studying the interactions between gut microbiota, drugs, and humans (Figure 3C).High-throughput sequencing plays a crucial role in characterizing gut microbiota by analyzing various aspects like the metagenome, transcriptome, metabolome, and isotope nucleic acid probe labeling coupled sequencing.However, most current sequencing methods decode all the genetic information within a sample, making it difficult to pinpoint the specific function of a single microbe in a complex gut microbiota community due to functional redundancy and low gene conservation among gut microbes.Microbe-seq offers a powerful alternative-a high-throughput technique that generates individual microbial genomes from complex communities.This allows researchers to investigate these communities at single-cell resolution.Using Microbeseq, scientists have successfully obtained over 20,000 single-amplified genomes (SAGs) from a single human donor [61,62].This unprecedented resolution allows for "in vivo" functional analysis of drug transformation at the individual cell level.Microbe-seq holds promise for identifying gut microbiota that influence drug efficacy and toxicological effects in vivo.By eliminating the need for large-scale microbial purification and culturing, this technique paves the way for function-oriented, precise bacterial culture methods.
Precise function-oriented microbes culture enables a new evaluation system for drug-microbial interaction Despite the remarkable progress achieved in gut microbiota research over the past few years, a significant portion of intestinal microbes remain unculturable.This presents a challenge for fully understanding the in vivo mechanisms of microbial drug metabolism and for large-scale production of beneficial, microbially modified drug molecules.The human gut microbiome boasts a vast array of taxa, each carrying unique genes and gene families.However, a notable characteristic is the presence of phylogenetically diverse taxa that share similar genes and perform comparable functions [63].This highlights the potential of "microbiota function-oriented host-microbiota crosstalk" as a new area of investigation.Microbe-seq technology offers a powerful tool for single-bacterium identification and sorting.This, combined with a strategy called culturomics, provides significant technical support for elucidating the role of gut microbiota in vivo function in clinical drug efficacy and safety (Figure 3D).Culturomics incorporates various growth conditions with rapid microbial identification techniques [64].Furthermore, genetic manipulation techniques like CRISPR-Cas9 enable editing of bacterial and fungal strains within the complex gut microbiome.This allows researchers to investigate the specific role of individual genes within this intricate system [63,65].These advancements highlight the growing sophistication of methods for mining functional bacteria.Functional genomics is another promising approach.Recent studies have demonstrated its potential for characterizing physiologically active biomolecules.For example, this approach can be applied to identify bacterial polysaccharide consumption loci targeting pectins, with expression levels corresponding to strain-specific liberation [66].Understanding the activity and substrate specificity of bacterial drug-modifying enzymes is crucial, as these enzymes significantly impact the activity of drug components.Existing research has successfully characterized the activity and substrate specificity of bacterial enzymes from culturable bacteria, particularly in the context of inflammatory diseases [67].Finally, the construction of germ-free animals with specific functional phenotypes offers a promising avenue for translating the "in vitro" function of microbially modified small molecule drugs to their "in vivo" function.

Coculture, models mimicking gut microbiota function: A novel platform for drug-microbiota interaction evaluation
Three-dimensional in vitro cell culture systems known as "organoids" offer a powerful tool for studying drug effects.These models closely resemble the source tissues or organs in vivo by replicating their complex spatial morphology and differentiated cell types [68].Microbiota-loaded organoids provide an excellent platform for coculturing host cells and microbes, allowing researchers to investigate drug pharmacology within the context of "microbiota-host" interactions (Figure 3E).The brain, bone, and blood have long been considered sterile environments.However, with the advent of highresolution sequencing techniques, growing evidence suggests that microbiota, including bacteria, fungi, and viruses, colonize various organs and play a crucial physiological role within the host's local tissue microenvironment [57,69].Particularly, noteworthy is the presence of microbiota in tumor tissues, cells, and blood, where they appear to influence host immunity and cancer cell metastasis [69,70].An intriguing question remains unanswered: will drugs delivered to specific host tissues or cells undergo secondary metabolism and modification by local microbiota, potentially impacting their efficacy?While current research has not yet focused on the role and mechanisms of local microbiota in influencing the effectiveness and toxicity of drugs targeted for localized delivery, these are undoubtedly issues that will capture the attention of researchers in the near future.This study addresses two key issues regarding clinical drugs and gut microbiota.First, individual variability in the human body arises from a combination of genetic and environmental factors.A critical question remains: how do these variations in the human microenvironment, including gut microbiota, affect the safety and efficacy of medications?Second, pharmacogenomics sheds light on how individual genetic variations influence drug absorption, distribution, metabolism, and excretion.However, research on the impact of environmental factors, particularly gut microbiota, on drug ADME remains limited.Given the gut microbiota's crucial role in gene-environment interactions affecting drug ADME, it presents a valuable and understudied avenue for preclinical drug safety and efficacy investigations.Our proposed new evaluation system for drugmicrobiota interaction shows significant promise, as evidenced by the findings above.Future research can explore several exciting avenues.First, by comparing conventional and germ-free animals, disease models with humanized gut microbiota, and single-strain animal drug metabolism models, researchers can develop preclinical evaluation technology services.This approach can also optimize drug safety and efficacy testing, identify potential microbial targets for existing drugs, and aid in discovering new drug mechanisms and uses.Second, research could focus on evaluating the effectiveness of clinical interventions targeting gut microbiota in vivo, such as vaccines, antibodies, and cell-based therapies.Finally, the development of a comprehensive technical service platform integrating animal models, microbiome technology, and differential drug response analysis would be a valuable resource for studying drugmicrobiota interactions.In conclusion, the strong operability and high safety of gut microbial regulation in germfree animal models make them a promising new tool for evaluating drug-microbiota interactions.
AUTHOR CONTRIBUTIONS Tian-Hao Liu, Chen-Yang Zhang, Hang Zhang, Xiu-Wu Bian, Wei Hong, Yu-Zheng Xue participated in study design.Tian-Hao Liu, Xue Li, Ya-Hong Zhou, Jing Jin, Shi-Qiang Liang conducted study operation.Chen-Yang Zhang, Tian-Hao Liu, Yu-Zheng Xue, Feng-Lai Yuan helped to draft and revise the manuscript.All authors have read the final manuscript and approved it for publication.

DATA AVAILABILITY STATEMENT
No new data was generated in this study.Supplementary materials (graphical abstract, slides, videos, Chinese translated version and update materials) may be found in the online DOI or iMeta Science (http://www.imeta.science/).

ETHICS STATEMENT
No animals or humans were involved in this study.

F I G U R E 2
Environmental factors influencing gut microbiota.(A) Natural conditions: natural environment contributions to the gut microbiota.The climatic extremes including cold and heat exposure or stress and high-altitude hypoxia environment can alter the specific bacteria in gut and have an impact on the host response to extreme environments.(B) Environmental pollution: Environmental contamination including PM2.5 pollution, microplastic pollution, and antibiotic resistance genes (ARGs) contributes to the gut microbiota and human health.(C) Psychological factor and stress: There is an important relationship between gut microbiota and cognitive and behavioral processes caused by psychological stress, which mainly include hypothalamic-pituitary-adrenal axis (HPA-axis), neuroimmune regulation, immune system, and endocannabinoid (eCB).Moreover, psychiatric factors are one of the most important influences on drug metabolism.(D) Lifestyle: The intervention effect of lifestyle including western diets, modern lifestyles, sedentary behaviors, smoking habits, and alcohol dependence on the gut microbiota is evident.
Drug preclinical research strategies based on precise germ-free animal models.(A) Precise germ-free animal models.Single functional microbe colonization, germ-free treatment of genetically engineered disease animals, and liver-gut humanization model system provide support for studying the in vivo function of gut microbiota in drug metabolism.(B) Gut microbiota in vivo function.The drugcontaining serum from GF disease model animal can not only screen small molecule pharmacodynamic substances modified by gut microbiota enzyme but also achieve more refined research of "host cell-gut microbiota-drug" by the intervention on host cells in vitro.(C) High-resolution sequencing for single-cell recognition.The single-cell resolution sequencing of the microbiota (Microbe-seq) can realize the high-precision cognition on the cell individual level of the in vivo function of drug transformation.(D) Function-oriented precise bacterial culture.The construction of GF animals with specific functional phenotypes will realize the transformation of small molecule drugs modified by microbial enzymes from in vitro function to in vivo function.(E) Coculture based on the in vivo function of gut microbiota.The microbiota-loaded organoids are excellent media for host "cells and microbes" coculture.