Exploring the physicochemical properties of the integration of Tristearoyl uridine in Langmuir monolayers: An approach to cell membrane modeling for prodrugs.

Understanding the mechanisms by which drugs interact with cell membranes is crucial for unraveling the underlying biochemical and biophysical processes that occur on the surface of these membranes. Our research focused on studying the interaction between an ester-type derivative of tristearoyl uridine and model cell membranes composed of lipid monolayers at the air-water interface. For that, we selected a specific lipid to simulate nontumorigenic cell membranes, namely 1,2-dihexadecanoyl-sn-glycero-3-phospho-l-serine. We noted significant changes in the surface pressure-area isotherms, with a noticeable shift towards larger areas, which was lower than expected for ideal mixtures, indicating monolayer condensation. Furthermore, the viscoelastic properties of the interfacial film demonstrated an increase in both the elastic and viscous parameters for the mixed film. We also observed structural alterations using vibrational spectroscopy, which revealed an increase in the all-trans to gauche conformers ratio. This confirmed the stiffening effect of the prodrug on the lipid monolayer. In summary, this study indicates that this lipophilic prodrug significantly impacts the lipid monolayer's thermodynamic, rheological, electrical, and molecular characteristics. This information is crucial for understanding how the drug interacts with specific sites on the cellular membrane. It also has implications for drug delivery, as the drug's passage into the cytosol may involve traversing the lipid bilayer.

Differential Bioactivation Profiles of Different GS-441524 Prodrugs in Cell and Mouse Models: ProTide Prodrugs with High Cell Permeability and Susceptibility to Cathepsin A Are More Efficient in Delivering Antiviral Active Metabolites to the Lung.

We assessed factors that determine the tissue-specific bioactivation of ProTide prodrugs by comparing the disposition and activation of remdesivir (RDV), its methylpropyl and isopropyl ester analogues (MeRDV and IsoRDV, respectively), the oral prodrug GS-621763, and the parent nucleotide GS-441524 (Nuc). RDV and MeRDV yielded more active metabolite remdesivir-triphosphate (RDV-TP) than IsoRDV, GS-621763, and Nuc in human lung cell models due to superior cell permeability and higher susceptivity to cathepsin A. Intravenous administration to mice showed that RDV and MeRDV delivered significantly more RDV-TP to the lung than other compounds. Nevertheless, all four ester prodrugs exhibited very low oral bioavailability (<2%), with Nuc being the predominant metabolite in blood. In conclusion, ProTides prodrugs, such as RDV and MeRDV, are more efficient in delivering active metabolites to the lung than Nuc, driven by high cell permeability and susceptivity to cathepsin A. Optimizing ProTides' ester structures is an effective strategy for enhancing prodrug activation in the lung.

Non-canonical substrate recognition by the human WDR26-CTLH E3 ligase regulates prodrug metabolism.

The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5's N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism.

The Catalysis Mechanism of E. coli Nitroreductase A, a Candidate for Gene-Directed Prodrug Therapy: Potentiometric and Substrate Specificity Studies.

E. coli nitroreductase A (NfsA) is a candidate for gene-directed prodrug cancer therapy using bioreductively activated nitroaromatic compounds (ArNO2). In this work, we determined the standard redox potential of FMN of NfsA to be -215 ± 5 mV at pH 7.0. FMN semiquinone was not formed during 5-deazaflavin-sensitized NfsA photoreduction. This determines the two-electron character of the reduction of ArNO2 and quinones (Q). In parallel, we characterized the oxidant specificity of NfsA with an emphasis on its structure. Except for negative outliers nitracrine and SN-36506, the reactivity of ArNO2 increases with their electron affinity (single-electron reduction potential, E17) and is unaffected by their lipophilicity and Van der Waals volume up to 386 Å. The reactivity of quinoidal oxidants is not clearly dependent on E17, but 2-hydroxy-1,4-naphthoquinones were identified as positive outliers and a number of compounds with diverse structures as negative outliers. 2-Hydroxy-1,4-naphthoquinones are characterized by the most positive reaction activation entropy and the negative outlier tetramethyl-1,4-benzoquinone by the most negative. Computer modelling data showed that the formation of H bonds with Arg15, Arg133, and Ser40, plays a major role in the binding of oxidants to reduced NfsA, while the role of the π-π interaction of their aromatic structures is less significant. Typically, the calculated hydride-transfer distances during ArNO2 reduction are smallwer than for Q. This explains the lower reactivity of quinones. Another factor that slows down the reduction is the presence of positively charged aliphatic substituents.

Mitochondria-Targeted Prodrug Nanoassemblies for Efficient Ferroptosis-Based Therapy via Devastating Ferroptosis Defense Systems.

Ferroptosis is a form of regulated cell death accompanied by lipid reactive oxygen species (ROS) accumulation in an iron-dependent manner. However, the efficiency of tumorous ferroptosis was seriously restricted by intracellular ferroptosis defense systems, the glutathione peroxidase 4 (GPX4) system, and the ubiquinol (CoQH2) system. Inspired by the crucial role of mitochondria in the ferroptosis process, we reported a prodrug nanoassembly capable of unleashing potent mitochondrial lipid peroxidation and ferroptotic cell death. Dihydroorotate dehydrogenase (DHODH) inhibitor (QA) was combined with triphenylphosphonium moiety through a disulfide-containing linker to engineer well-defined nanoassemblies (QSSP) within a single-molecular framework. After being trapped in cancer cells, the acidic condition provoked the structural disassembly of QSSP to liberate free prodrug molecules. The mitochondrial membrane-potential-driven accumulation of the lipophilic cation prodrug was delivered explicitly into the mitochondria. Afterward, the thiol-disulfide exchange would occur accompanied by downregulation of reduced glutathione levels, thus resulting in mitochondria-localized GPX4 inactivation for ferroptosis. Simultaneously, the released QA from the hydrolysis reaction of the adjacent ester bond could further devastate mitochondrial defense and evoke robust ferroptosis via the DHODH-CoQH2 system. This subcellular targeted nanoassembly provides a reference for designing ferroptosis-based strategy for efficient cancer therapy through interfering antiferroptosis systems.

Clostridium Bacteria: Harnessing Tumour Necrosis for Targeted Gene Delivery.

Necrosis is a common feature of solid tumours that offers a unique opportunity for targeted cancer therapy as it is absent from normal healthy tissues. Tumour necrosis provides an ideal environment for germination of the anaerobic bacterium Clostridium from endospores, resulting in tumour-specific colonisation. Two main species, Clostridium novyi-NT and Clostridium sporogenes, are at the forefront of this therapy, showing promise in preclinical models. However, anti-tumour activity is modest when used as a single agent, encouraging development of Clostridium as a tumour-selective gene delivery system. Various methods, such as allele-coupled exchange and CRISPR-cas9 technology, can facilitate the genetic modification of Clostridium, allowing chromosomal integration of transgenes to ensure long-term stability of expression. Strains of Clostridium can be engineered to express prodrug-activating enzymes, resulting in the generation of active drug selectively in the tumour microenvironment (a concept termed Clostridium-directed enzyme prodrug therapy). More recently, Clostridium strains have been investigated in the context of cancer immunotherapy, either in combination with immune checkpoint inhibitors or with engineered strains expressing immunomodulatory molecules such as IL-2 and TNF-α. Localised expression of these molecules using tumour-targeting Clostridium strains has the potential to improve delivery and reduce systemic toxicity. In summary, Clostridium species represent a promising platform for cancer therapy, with potential for localised gene delivery and immunomodulation selectively within the tumour microenvironment. The ongoing clinical progress being made with C. novyi-NT, in addition to developments in genetic modification techniques and non-invasive imaging capabilities, are expected to further progress Clostridium as an option for cancer treatment.

Development of tenofovir monobenzyl ester phosphonoamidate prodrugs with improved anti-hepatitis B virus activity and intrahepatic tenofovir enrichment.

Various tenofovir (TFV) prodrugs have been developed by introducing masking groups to the hydroxyls of the monophosphonate group to enhance intestinal absorption efficiency and therapeutic effects. However, the reported TFV prodrugs have drawbacks such as low bioavailability, systemic toxicity caused by their breakdown in non-targeted tissues, and potential low intracellular conversion efficiency. In the present study, we developed a class of TFV monobenzyl ester phosphonoamidate prodrugs without substitutions on the benzene ring. Compared with previous TFV prodrugs, compounds 3a and 3b developed in the present study showed higher anti-hepatitis B virus activity, stronger stability and higher levels of intrahepatic enrichment of the metabolic product (TFV), indicating the potential of these compounds as novel prodrugs with high efficiency and low systemic toxicity for the treatment of hepatitis B.

Escherichia coli cytosine deaminase: Structural and biotechnological aspects.

Suicide gene therapy involves introducing viral or bacterial genes into tumor cells, which enables the conversion of a nontoxic prodrug into a toxic-lethal drug. The application of the bacterial cytosine deaminase (bCD)/5-fluorocytosine (5-FC) approach has been beneficial and progressive within the current field of cancer therapy because of the enhanced bystander effect. The basis of this method is the preferential deamination of 5-FC to 5-fluorouracil by cancer cells expressing cytosine deaminase (CD), which strongly inhibits DNA synthesis and RNA function, effectively targeting tumor cells. However, the poor binding affinity of toward 5-FC compared to the natural substrate cytosine and/or inappropriate thermostability limits the clinical applications of this gene therapy approach. Nowadays, many genetic engineering studies have been carried out to solve and improve the activity of this enzyme. In the current review, we intend to discuss the biotechnological aspects of Escherichia coli CD, including its structure, functions, molecular cloning, and protein engineering. We will also explore its relevance in cancer clinical trials. By examining these aspects, we hope to provide a thorough understanding of E. coli CD and its potential applications in cancer therapy.

Synthesis and evaluation of indomethacin prodrugs with a diester structure that are metabolically activated by human carboxylesterases.

1. Carboxylesterase (CES) has been studied extensively, mostly with substrates in the monoester structures. We investigated the relationship between indomethacin diester prodrugs and metabolic activation by microsomes and recombinant human CES.2. Eight indomethacin diester prodrugs were synthesised in two steps. They were used as substrates and hydrolysis rates were calculated.3. As a result, the major hydrolysis enzyme was CES. The hydrolysis rate of recombinant CES2A1 was comparable to that of recombinant CES1A1.4. In this study, by changing the structure of the prodrug to a diester structure, it was found that CES2 activity was equivalent to CES1 activity.5. It should be noted that the use of diester prodrugs in prodrug discovery, where organ-specific hydrolysis reactions are expected, may not yield the expected results.

Bacterial amidohydrolases and modified 5-fluorocytidine compounds: Novel enzyme-prodrug pairs.

Gene-directed enzyme prodrug therapy is an emerging strategy for cancer treatment based on the delivery of a gene that encodes an enzyme that is able to convert a prodrug into a potent cytotoxin exclusively in target cancer cells. However, it is limited by the lack of suitable enzyme variants and a scarce choice of chemical bonds that could be activated. Therefore, this study is aimed to determine the capability of bacterial amidohydrolases YqfB and D8_RL to activate novel prodrugs and the effect such system has on the viability of eukaryotic cancer cells. We have established cancer cell lines that stably express the bacterial amidohydrolase genes and selected several N4-acylated cytidine derivatives as potential prodrugs. A significant decrease in the viability of HCT116 human colon cancer cell lines expressing either the YqfB or the D8_RL was observed after exposure to the novel prodrugs. The data we acquired suggests that bacterial YqfB and D8_RL amidohydrolases, together with the modified cytidine-based prodrugs, may serve as a promising enzyme-prodrug system for gene-directed enzyme prodrug therapy.

Diester Prodrugs of a Phosphonate Butyrophilin Ligand Display Improved Cell Potency, Plasma Stability, and Payload Internalization.

Activation of Vγ9Vδ2 T cells with butyrophilin 3A1 (BTN3A1) agonists such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) has the potential to boost the immune response. Because HMBPP is highly charged and metabolically unstable, prodrugs may be needed to overcome these liabilities, but the prodrugs themselves may be limited by slow payload release or low plasma stability. To identify effective prodrug forms of a phosphonate agonist of BTN3A1, we have prepared a set of diesters bearing one aryl and one acyloxymethyl group. The compounds were evaluated for their ability to stimulate Vγ9Vδ2 T cell proliferation, increase production of interferon γ, resist plasma metabolism, and internalize into leukemia cells. These bioassays have revealed that varied aryl and acyloxymethyl groups can decouple plasma and cellular metabolism and have a significant impact on bioactivity (>200-fold range) and stability (>10 fold range), including some with subnanomolar potency. Our findings increase the understanding of the structure-activity relationships of mixed aryl/acyloxymethyl phosphonate prodrugs.

Retroviral Replicating Vectors Mediated Prodrug Activator Gene Therapy in a Gastric Cancer Model.

Retroviral replicating vectors (RRVs) selectively replicate and can specifically introduce prodrug-activating genes into tumor cells, whereby subsequent prodrug administration induces the death of the infected tumor cells. We assessed the ability of two distinct RRVs generated from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which infect cells via type-III sodium-dependent phosphate transporters, PiT-2 and PiT-1, respectively, to infect human gastric cancer (GC) cells. A quantitative RT-PCR showed that all tested GC cell lines had higher expression levels of PiT-2 than PiT-1. Accordingly, AMLV, encoding a green fluorescent protein gene, infected and replicated more efficiently than GALV in most GC cell lines, whereas both RRVs had a low infection rate in human fibroblasts. RRV encoding a cytosine deaminase prodrug activator gene, which converts the prodrug 5-flucytosine (5-FC) to the active drug 5-fluorouracil, showed that AMLV promoted superior 5-FC-induced cytotoxicity compared with GALV, which correlated with the viral receptor expression level and viral spread. In MKN-74 subcutaneous xenograft models, AMLV had significant antitumor effects compared with GALV. Furthermore, in the MKN-74 recurrent tumor model in which 5-FC was discontinued, the resumption of 5-FC administration reduced the tumor volume. Thus, RRV-mediated prodrug activator gene therapy might be beneficial for treating human GC.

An Activatable Prodrug Nanosystem for Ultrasound-Driven Multimodal Tumor Therapy and Metastasis Inhibition.

Ultrasound, featuring deep tissue penetration and noninvasiveness, offers a new opportunity to activate functional materials in a tumor-selective manner. However, very few direct ultrasound-responsive redox systems are applicable under therapeutic ultrasound (1 MHz). Herein, the investigations on nanoprodrug of DHE@PEG-SS-DSPE are reported, which exhibit glutathione-activated release of dihydroethidium (DHE) in tumor cells. DHE is stable with good biosafety and is transformed into cytotoxic ethidium to induce DNA damage under medical ultrasound irradiation, accompanied by the generation of reactive oxygen species. Further, DHE@PEG-SS-DSPE could effectively induce ferroptosis through glutathione depletion, lipid peroxide accumulation, and downregulation of glutathione peroxidase 4. In vivo studies confirmed that DHE@PEG-SS-DSPE nanoparticles effectively inhibit both the growth of solid tumors and the expression of metastasis-related proteins in mice, thus effectively inhibiting lung metastasis. This DHE-based prodrug nanosystem could lay a foundation for the design of ultrasound-driven therapeutic agents.

Plasma Protein Binding Determination for Unstable Ester Prodrugs: Remdesivir and Tenofovir Alafenamide.

Remdesivir (RDV) and tenofovir alafenamide (TAF) are prodrugs designed to be converted to their respective active metabolites. Plasma protein binding (PPB) determination of these prodrugs is important for patients with possible alteration of free fraction of the drugs due to plasma protein changes in renal impairment, hepatic impairment, or pregnancy. However, the prodrugs' instability in human plasma presents a challenge for accurate PPB determination. In this research work, two approaches were used in the method development and qualification for PPB assessment of RDV and TAF. For RDV, dichlorvos was used to inhibit esterase activity to stabilize the prodrug in plasma during equilibrium dialysis (ED). The impact of dichlorvos on protein binding was evaluated and determined to be insignificant by comparing the unbound fraction (fu) determined by the ED method with dichlorvos present and the fu determined by an ultrafiltration method without dichlorvos. In contrast to RDV, TAF degradation in plasma is ∼3-fold slower, and TAF stability cannot be improved by dichlorvos. Fit-for-purpose acceptance criteria for the TAF PPB method were chosen, and an ED method was developed based on these criteria. These two methods were then qualified and applied for PPB determinations in clinical studies.

Hyaluronic acid-based prodrug nanomedicines for enhanced tumor targeting and therapy: A review.

Hyaluronic acid (HA) represents a natural polysaccharide which has attracted significant attention owing to its improved tumor targeting capacity, enzyme degradation capacity, and excellent biocompatibility. Its receptors, such as CD44, are overexpressed in diverse cancer cells and are closely related with tumor progress and metastasis. Accordingly, numerous researchers have designed various kinds of HA-based drug delivery platforms for CD44-mediated tumor targeting. Specifically, the HA-based nanoprodrugs possess distinct advantages such as good bioavailability, long circulation time, and controlled drug release and retention ability and have been extensively studied during the past years. In this review, the potential strategies and applications of HA-modified nanoprodrugs for drug molecule delivery in anti-tumor therapy are summarized.

The current status in computational exploration of Pt(IV) prodrug activation by reduction.

Octahedral PtIV complexes are considered highly promising candidates for overcoming some shortcomings of clinically approved PtII drugs. PtIV compounds, owing to their inertia, appear to be capable of resisting premature aquation and undesired binding to essential plasma proteins and have shown remarkable potential for both oral administration and for reducing side effects. Additionally, their pharmacological properties can be finely tuned by choosing appropriate axial ligands. The reduction inside the cell by biological reducing agents to the correponding active cytotoxic PtII species, accompanied by the loss of the axial ligands, is considered an essential step of their mechanism and has been extensively studied. However, a detailed understanding of the mechanism by which PtIV prodrugs are activated, which should be highly beneficial for their proper design, is lacking, and many contradictory results continue to be collected. In the hope of contributing to the advancement of knowledge in this field, this perspective focuses on the insights gained from computational studies carried out with the aim of finding answers to the many still open questions concerning the reduction of PtIV complexes in biological environments.

Targeted bacteria-mediated therapy of mouse colorectal cancer using baicalin, a natural glucuronide compound, and E. coli overexpressing ß-glucuronidase.

The side effects of common chemotherapeutic drugs that damage healthy tissues account for one of the most important problems in cancer research that needs careful addressing. Bacterial-Directed Enzyme Prodrug Therapy (BDEPT) is a promising strategy that uses bacteria to direct a converting enzyme to the tumor site and activate a systemically injected prodrug selectively within the tumor; so that the side effects of the therapy would significantly decrease. In this study, we evaluated the efficacy of baicalin, a natural compound, as a glucuronide prodrug in association with an engineered strain of Escherichia coli DH5α harboring the pRSETB-lux/ßG plasmid in a mouse model of colorectal cancer. E. coli DH5α-lux/ßG was designed to emit luminescence, and overexpress the ß-glucuronidase. Unlike the non-engineered bacteria, E. coli DH5α-lux/ßG showed the ability to activate baicalin, and the cytotoxic effects of baicalin on the C26 cell line were increased in the presence of E. coli DH5α-lux/ßG. Analyzing the tissue homogenates of mice bearing C26 tumors inoculated with E. coli DH5α-lux/ßG indicated the specific accumulation and multiplication of bacteria in the tumor tissues. While both baicalin and E. coli DH5α-lux/ßG could inhibit tumor growth as monotherapy, an enhanced inhibition was observed when animals were subjected to combination therapy. Moreover, no significant side effects were observed after histological investigation. The results of this study indicate that baicalin has the capability of being used as a suitable prodrug in the BDEPT, however further research is required before it can be applied in the clinic.

DprE2 is a molecular target of the anti-tubercular nitroimidazole compounds pretomanid and delamanid.

Mycobacterium tuberculosis is one of the global leading causes of death due to a single infectious agent. Pretomanid and delamanid are new antitubercular agents that have progressed through the drug discovery pipeline. These compounds are bicyclic nitroimidazoles that act as pro-drugs, requiring activation by a mycobacterial enzyme; however, the precise mechanisms of action of the active metabolite(s) are unclear. Here, we identify a molecular target of activated pretomanid and delamanid: the DprE2 subunit of decaprenylphosphoribose-2'-epimerase, an enzyme required for the synthesis of cell wall arabinogalactan. We also provide evidence for an NAD-adduct as the active metabolite of pretomanid. Our results highlight DprE2 as a potential antimycobacterial target and provide a foundation for future exploration into the active metabolites and clinical development of pretomanid and delamanid.

A novel AKR1C3 specific prodrug AST-3424 and its combination therapy in hepatocellular carcinoma.

OBJECTIVE: AST-3424 is a novel specific aldo-keto reductase 1C3 (AKR1C3) prodrug that releases a DNA alkylating reagent upon reduction by AKR1C3. This study aimed to evaluate the efficacy and safety of AST-3424 in patient-derived tumor xenograft (PDTX) model and orthotopic model against hepatocellular carcinoma (HCC). MATERIALS AND METHOD: PDTX models derived from three HCC patients and orthotopic mice models using HepG2 cells were developed. The mice were treated with AST-3424 alone or combined with other drugs (oxaliplatin, apatinib, sorafenib and elemene in PDTX models, oxaliplatin and 5- fluorouracil in orthotopic models). The tumor volume and weight, as well as the mice weight were assessed. The liver tumor and transplanted tumor were removed for histological, immunohistochemical and Western blot detection in orthotopic model experiments. RESULTS: AST-3424 could inhibit tumor growth in HCC PDTX models and orthotopic models, with no difference in safety compared with other marketed drugs, and the drug combination did not increase toxicity. The inhibitory effect of combination treatment was more obvious than which used alone. The reduction of AKR1C3 expression was negatively correlated with AST-3424 dose. CONCLUSION: AST-3424 had a promising effect against HCC in PDTX model and orthotopic model with good safety. It could promote the sensitivity of other drugs without increasing toxicity. Clinical trials are warranted to further certify its antitumor effect and safety.

The Potentially Significant Role of CYP3A-Mediated Oxidative Metabolism of Dabigatran Etexilate and Its Intermediate Metabolites in Drug-Drug Interaction Assessments Using Microdose Dabigatran Etexilate.

Dabigatran etexilate (DABE), a double ester prodrug of dabigatran, is a probe substrate of intestinal P-glycoprotein (P-gp) commonly used in clinical drug-drug interaction (DDI) studies. When compared with its therapeutic dose at 150 mg, microdose DABE (375 µg) showed approximately 2-fold higher in DDI magnitudes with CYP3A/P-gp inhibitors. In this study, we conducted several in vitro metabolism studies to demonstrate that DABE, at a theoretical gut concentration after microdosing, significantly underwent NADPH-dependent oxidation (~40%-50%) in parallel to carboxylesterase-mediated hydrolysis in human intestinal microsomes. Furthermore, NADPH-dependent metabolism of its intermediate monoester, BIBR0951, was also observed in both human intestinal and liver microsomes, accounting for 100% and 50% of total metabolism, respectively. Metabolite profiling using high resolution mass spectrometry confirmed the presence of several novel oxidative metabolites of DABE and of BIBR0951 in the NADPH-fortified incubations. CYP3A was identified as the major enzyme catalyzing the oxidation of both compounds. The metabolism of DABE and BIBR0951 was well described by Michaelis-Menten kinetics, with Km ranging 1-3 µM, significantly below the expected concentrations following the therapeutic dose of DABE. Overall, the present results suggested that CYP3A played a significant role in the presystemic metabolism of DABE and BIBR0951 following microdose DABE administration, thus attributing partly to the apparent overestimation in the DDI magnitude observed with the CYP3A/P-gp inhibitors. Therefore, DABE at the microdose, unlike the therapeutic dose, would likely be a less predictive tool and should be considered as a clinical dual substrate for P-gp and CYP3A when assessing potential P-gp-mediated impacts by dual CYP3A/P-gp inhibitors. SIGNIFICANT STATEMENT: This is the first study demonstrating a potentially significant role of cytochrome P450-mediated metabolism of the prodrug DABE following a microdose but not a therapeutic dose. This additional pathway, coupled with its susceptibility to P-glycoprotein (P-gp), may make DABE a clinical dual substrate for both P-gp and CYP3A at a microdose. The study also highlights the need for better characterization of the pharmacokinetics and metabolism of a clinical drug-drug interaction probe substrate over the intended study dose range for proper result interpretations.