Identification of a novel 10-hydroxyevodiamine prodrug as a potent topoisomerase inhibitor with improved aqueous solubility for treatment of hepatocellular carcinoma.

Natural product evodiamine (Evo) and its synthetic derivatives represent an attractive dual Topo 1/2 inhibitors with broad-spectrum antitumor efficacy. However, the clinical applications of these compounds have been impeded by their poor aqueous solubility. Herein, a series of water-soluble 10-substituted-N(14)-phenylevodiamine derivatives were designed and synthesized. The most potent compound 45 featuring a quaternary ammonium salt fragment achieved robust aqueous solubility and nanomolar potency against a panel of human hepatoma cell lines Huh7, HepG2, SK-Hep-1, SMMC-7721, and SMMC-7721/DOX (doxorubicin-resistant cell). Further studies revealed that 45 could inhibit Topo 1 and Topo 2, induce apoptosis, arrest the cell cycle at the G2/M stage and inhibit the migration and invasion. Compound 45 exhibited potent antitumor activity (TGI = 51.1 %, 10 mg/kg) in the Huh7 xenograft model with acceptable safety profile. In addition, a 21-day long-term dose toxicity study confirmed that the maximum tolerated dose of compound 45 was 20 mg/kg. Overall, this study presented a promising Evo-derived candidate for the treatment of hepatocellular carcinoma.

Fenamates: Forgotten treasure for cancer treatment and prevention: Mechanisms of action, structural modification, and bright future.

Fenamates as classical nonsteroidal anti-inflammatory agents are widely used for relieving pain. Preclinical studies and epidemiological data highlight their chemo-preventive and chemotherapeutic potential for cancer. However, comprehensive reviews of fenamates in cancer are limited. To accelerate the repurposing of fenamates, this review summarizes the results of fenamates alone or in combination with existing chemotherapeutic agents. This paper also explores targets of fenamates in cancer therapy, including COX, AKR family, AR, gap junction, FTO, TEAD, DHODH, TAS2R14, ion channels, and DNA. Besides, this paper discusses other mechanisms, such as regulating Wnt/ß-catenin, TGF-ß, p38 MAPK, and NF-κB pathway, and the regulation of the expressions of Sp, EGR-1, NAG-1, ATF-3, ErbB2, AR, as well as the modulation of the tumor immune microenvironment. Furthermore, this paper outlined the structural modifications of fenamates, highlighting their potential as promising leads for anticancer drugs.

Discovery of novel small molecules targeting the USP21/JAK2/STAT3 axis for the treatment of triple-negative breast cancer.

The deficiency in available targeted agents and frequency of chemoresistance are primary challenges in clinical management of triple-negative breast cancer (TNBC). The aberrant expression of USP21 and JAK2 represents a characterized mechanism of TNBC progression and resistance to paclitaxel (PTX). Despite its clear that high expression of USP21-mediated de-ubiquitination leads to increased levels of JAK2 protein, we lack regulator molecules to dissect the mechanisms that the interaction between USP21 and JAK2 contributes to the phenotype and resistance of TNBC. Here, we report a USP21/JAK2/STAT3 axis-targeting regulator 13c featuring a N-anthraniloyl tryptamine scaffold that showed excellent anti-TNBC potency and promising safety profile. Importantly, the therapeutic potential of using 13c in combination with PTX in PTX-resistant TNBC was demonstrated. This study showcases N-anthraniloyl tryptamine derivatives as a novel anti-TNBC chemotype with a pharmacological mode of action targeting the USP21/JAK2/STAT3 axis and provides a potential therapeutic target for the treatment of TNBC.

Structure-activity relationship study of 1,6-naphthyridinone derivatives as selective type II AXL inhibitors with potent antitumor efficacy.

The role of AXL in various oncogenic processes has made it an attractive target for cancer therapy. Currently, kinase selectivity profiles, especially circumventing MET inhibition, remain a scientific issue of great interest in the discovery of selective type II AXL inhibitors. Starting from a dual MET/AXL-targeted lead structure from our previous work, we optimized a 1,6-naphthyridinone series using molecular modeling-assisted compound design to improve AXL potency and selectivity over MET, resulting in the potent and selective type II AXL-targeted compound 25c. This showed excellent AXL inhibitory activity (IC50 = 1.1 nM) and 343-fold selectivity over the highly homologous kinase MET in biochemical assays. Moreover, compound 25c significantly inhibited AXL-driven cell proliferation, dose-dependently suppressed 4T1 cell migration and invasion, and induced apoptosis. Compound 25c also showed noticeable antitumor efficacy in a BaF3/TEL-AXL xenograft model at well-tolerated doses. Overall, this study presented a potent and selective type II AXL-targeted lead compound for further drug discovery.

Exploring the Therapeutic Effects of Multifunctional N-Salicylic Acid Tryptamine Derivative against Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. Neuroinflammation and oxidative stress play an important role in the whole course of PD, which have been the focus of PD drug development. In our previous research, a series of N-salicylic acid tryptamine derivatives were synthesized, and the biological evaluation showed that the compound LZWL02003 has good anti-neuroinflammatory activity and displayed great therapeutic potency for neurodegenerative disease models. In this work, the neuroprotective efficiency of LZWL02003 against PD in vitro and in vivo has been explored. It was found that LZWL02003 could protect human neuron cells SH-SY5Y from MPP+-induced neuronal damage by inhibiting ROS generation, mitochondrial dysfunction, and cellular apoptosis. Moreover, LZWL02003 could improve cognition, memory, learning, and athletic ability in a rotenone-induced PD rat model. In general, our study has demonstrated that LZWL02003 has good activity against PD in in vitro and in vivo experiments, which can potentially be developed into a therapeutic candidate for PD.

Development of o-aminobenzamide salt derivatives for improving water solubility and anti-undifferentiated gastric cancer.

Background: Gastric cancer is one of the cancers with wide incidence, difficult treatment and high mortality in the world, especially in Asia and Africa. In our previous work, a novel o-aminobenzamide analogue F8 was identified as an early preclinical candidate for treatment of undifferentiated gastric cancer (IC50 of 0.26 µM for HGC-27). However, the poor water solubility of compound F8 prevents its further progress in preclinical studies. Aim: To improve the water solubility and drug-likeness of F8 via salt formation. Method: Different acids and F8 were reacted to obtain different salt forms. Physicochemical property screening, pharmacokinetic property research, and antitumor biological activity evaluation in vitro and in vivo were used to obtain the optimal salt form with the best druggability. Results: our continuous efforts have finally confirmed F8·2HCl as the optimal salt form with maintained in vitro antitumor activity, improved water solubility and pharmacokinetic properties. Importantly, the F8·2HCl displayed superior in vivo antitumor efficacy (TGI of 70.1% in 75 mg/kg) in HGC-27 xenograft model. The further immunohistochemical analysis revealed that F8·2HCl exerts an antitumor effect through the regulation of cell cycle-related protein (CDK2 and p21), apoptosis-related protein Cleaved Caspase-3, proliferation marker Ki67, and cell adhesion molecule E-cadherin. In addition, F8·2HCl showed acceptable safety in the in vivo acute toxicity assay. Conclusion: Salting is an effective means to improve the drug-like properties of compound F8, and F8·2HCl can serve as a promising therapeutic agent against undifferentiated gastric cancer.

N(14)-substituted evodiamine derivatives as dual topoisomerase 1/tubulin-Inhibiting anti-gastrointestinal tumor agents.

Gastrointestinal tumor is an important factor threatening human health. Natural product-based drug discovery is a popular paradigm for expanding the chemical space and identifying new molecular entities that ameliorate human disease. Evodiamine-inspired medicinal chemistry presents therapeutic potential for treating tumors in different tissues via multi-target inhibition. Here, by focusing on the discovery of anti-gastrointestinal tumor drugs, a series of N(14) alkyl-substituted evodiamine derivatives were designed and synthesized. The structure-activity relationship studies culminated in the identification of the N(14)-propyl-substituted evodiamine analog 6b, which showed low nanomolar inhibitory activity against MGC-803 (IC50 = 0.09 µM) and RKO (IC50 = 0.2 µM) cell lines. Moreover, compound 6b was effective in inducing apoptosis, arresting the cell cycle in the G2/M phase, and inhibiting migration and invasion of MGC-803 and RKO cell lines in a dose-dependent manner in vitro. Further antitumor mechanism studies revealed that compound 6b significantly inhibited topoisomerase 1 (inhibition rate of 58.3% at 50 µM) and tubulin polymerization (IC50 = 5.69 µM). Overall, compound 6b represents a promising dual topoisomerase 1/tubulin-targeting lead structure for the treatment of gastrointestinal tumor.

Natural product evodiamine-inspired medicinal chemistry: Anticancer activity, structural optimization and structure-activity relationship.

It is a well-known phenomenon that natural products can serve as powerful drug leads to generate new molecular entities with novel therapeutic utility. Evodiamine (Evo), a major alkaloid component in traditional Chinese medicine Evodiae Fructus, is considered a desirable lead scaffold as its multifunctional pharmacological properties. Although natural Evo has suboptimal biological activity, poor pharmacokinetics, low water solubility, and chemical instability, medicinal chemists have succeeded in producing synthetic analogs that overshadow the deficiency of Evo in terms of further clinical application. Recently, several reviews on the synthesis, structural modification, mechanism pharmacological actions, structure-activity relationship (SAR) of Evo have been published, while few reviews that incorporates intensive structural basis and extensive SAR are reported. The purpose of this article is to review the structural basis, anti-cancer activities, and mechanisms of Evo and its derivatives. Emphasis will be placed on the optimizing strategies to improve the anticancer activities, such as structural modifications, pharmacophore combination and drug delivery systems. The current review would benefit further structural modifications of Evo to discover novel anticancer drugs.

Comprehensive analysis of prognostic value, relationship to cell cycle, immune infiltration and m6A modification of ZSCAN20 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common tumor across the globe with a high mortality rate. ZSCAN20 is a ZNF transcription factor, a key determinant of gene expression. Nonetheless, the mechanism of ZSCAN20 as a potential clinical biomarker and therapeutic target for HCC is not understood. Here, TIMER, TCGA, ICGC databases and immunohistochemical (IHC) and Western Blot found ZSCAN20 mRNA and protein levels were upregulated. Additionally, Kaplan-Meier Plotter, GEPIA and TCGA databases showed high ZSCAN20 expression was related to the short survival time of HCC patients. Multivariate Cox analysis exposed that ZSCAN20 can act as an independent prognostic factor. We observed methylation level of ZSCAN20 was associated with the clinicopathological characteristics and prognosis of HCC patients through UALCAN. Furthermore, enrichment examination exposed functional association between ZSCAN20 and cell cycle, immune infiltration. Functional experiments showed that interference with ZSCAN20 significantly reduced the invasion, migration and proliferation abilities of HCC cells. An immune infiltration analysis showed that ZSCAN20 was associated with immune cells, particularly T cells. The expression of ZSCAN20 was correlated with poor prognosis in the Regulatory T-cell. And Real-Time RT-PCR analysis found interference with ZSCAN20 significantly reduced the expression of some chemokines. Finally, the TCGA and ICGC data analysis suggested that the ZSCAN20 expression was greatly related to m6A modifier related genes. In conclusion, ZSCAN20 can serve as a prognostic biomarker for HCC and provide clues about cell cycle, immune infiltration, and m6A modification.

Pure laparoscopic radical nephroureterectomy for complicated renal pelvis carcinoma combined with horseshoe kidney: A case report and literature review.

We reported a case of pure laparoscopic radical nephroureterectomy for complicated renal pelvis carcinoma combined with horseshoe kidney (HSK). The aim was to present a case report and review of the literature about renal pelvis carcinoma combined with HSK. The case report includes a history of patient data. The pure laparoscopic radical nephroureterectomy was provided with the informed consent of the patient. A 53-year-old patient was diagnosed with a right renal pelvis mass with HSK. We performed laparoscopic radical nephroureterectomy with partial cystectomy and horseshoe renal isthmus amputation. Histopathological features, computed tomography urography (CTU), and angiography (CTA) confirmed the diagnosis of renal pelvis carcinoma combined with HSK. The tumor was removed, and the patient had an uneventful recovery. Renal pelvis carcinoma combined with HSK is a rare case. Due to severe anatomical abnormalities, this disease is a major challenge for urologists. We share our successful case for readers to learn from.

Comparison of a histone deacetylase inhibitor plus exemestane with exemestane alone in hormone receptor-positive advanced breast cancer that progressed on prior endocrine therapy: A meta-analysis.

Currently, endocrine therapy is the standard treatment for hormone receptor-positive advanced breast cancer (ABC). Despite the high sensitivity of anti-estrogen therapy, many breast cancer patients still experience disease progression, relapse, and reduced overall survival (OS) because of endocrine resistance. Several underlying mechanisms of this phenomenon include a change in hormone receptor expression, mutations in ESR1 and modification of important signaling pathways, but thus far none of these can be defined as the complete explanation. Additionally, it has been shown that in some breast cancers, expression of the estrogen receptor (ER) can be repressed by epigenetic modifications such as DNA methylation and histone deacetylation, and this could be a mechanism for endocrine resistance. Interestingly, although the efficacy of the combination of histone deacetylase (HADC) inhibitors and exemestane in hormone receptor-positive ABC that progressed on prior endocrine therapy has been investigated in several studies, whether pharmacologic blocking of HDAC activity acts as a therapeutic strategy remains highly controversial. Herein, we conducted a meta-analysis to evaluate the efficacy and safety of an HDAC inhibitor plus exemestane vs. exemestane alone in this setting. Our meta-analysis demonstrated that the combination group exhibited significantly prolonged progression-free survival (PFS) [hazard ratio (HR)=0.776, 95% confidence interval (CI)=0.675-0.892, P=0.000] and an improved objective response rate (ORR) (RR=1.612, 95% CI=1.085-2.396, P=0.018) compared to those treated with exemestane alone. Additionally, in terms of OS, the combination group failed to achieve a significant clinical OS benefit (HR=0.811, 95% CI=0.596-1.104, P=0.183). Although grade 3/4 toxicities were more common in the combination group, those toxicities were mostly asymptomatic and manageable. In conclusion, the addition of an HDAC inhibitor to exemestane significantly improves PFS over exemestane alone in hormone receptor-positive ABC patients who progressed on previous endocrine therapy. Identification of novel biomarkers to select patients who will benefit from this combination strategy is a high priority.

N-2-(Phenylamino) Benzamide Derivatives as Dual Inhibitors of COX-2 and Topo I Deter Gastrointestinal Cancers via Targeting Inflammation and Tumor Progression.

Given the close association between inflammation and cancer, combining anti-inflammation therapy is prominent to improve the anticancer effect. Based on I-1, a series of agents targeting COX-2 and Topo I were designed by combining fenamates and phenols. The optimal compound 1H-30 displayed an enhanced inhibitory effect on COX-2 compared to tolfenamic acid and I-1 and showed better inhibition of Topo I than I-1. Importantly, 1H-30 showed potential anticancer effects and suppressed the activation of the NF-κB pathway in cancer cells. 1H-30 inhibited the nuclear translocation of NF-κB and suppressed the production of NO, COX-2, and IL-1ß in RAW264.7. In vivo, 1H-30 showed acceptable pharmacokinetic parameters, decreased the tumor growth without affecting the body weight, down-regulated COX-2 and MMP-9, and induced apoptosis in the CT26.WT tumor-bearing mice. Accordingly, 1H-30 as a potential Topo I/COX-2 inhibitor which possessed anti-inflammatory and anticancer effects, with inhibition of the NF-κB pathway, is promising for gastrointestinal cancer therapy.

E3 ubiquitin ligase RBX1 drives the metastasis of triple negative breast cancer through a FBXO45-TWIST1-dependent degradation mechanism.

Triple-negative breast cancer (TNBC) patients are at high risk of recurrence and metastasis in the early stages, although receiving standard treatment. However, the underlying mechanism of TNBC remains unclear. Here, the critical effect of E3 ubiquitin ligase RBX1 in the metastasis of TNBC was reported for the first time. We discovered that RBX1 expression was evidently raised in the tissues of TNBC. Our clinical research displayed that high RBX1 expression was markedly related to poor distant invasion and survival. Functional analysis exhibited that RBX1 facilitated metastasis of TNBC cells through increasing EMT. Furthermore, we demonstrated that RBX1 knockdown increased the levels of the Twist family bHLH transcription factor 1 (TWIST1), is a significant regulator in the EMT process in some cancers. It can be observed an evident positive correlation between the TWIST1 and RBX1 levels, further confirming that EMT induced by RBX1 in TNBC cells is determined by TWIST1. Mechanistically, RBX1 modulates the expression of TWIST1 via modulating FBXO45, directly binding to FBXO45, and facilitating its degradation and ubiquitination. Briefly, our findings confirm that RBX1 is probably a new biomarker of TNBC carcinogenesis, thus suggesting that targeting the RBX1/FBXO45/TWIST1 axis may be an underlying strategy for TNBC treatment.

Discovery of carbamate-based N-salicyloyl tryptamine derivatives as novel pleiotropic agents for the treatment of Alzheimer's disease.

In this work, based on the potential anti-AD molecule previously studied by our group, we continue to introduce different substituents at different positions to improve both drug-like properties and on target activities. 33 N-salicyloyl tryptamine-carbamate hybrids were designed, synthesized and evaluated as cholinesterase inhibitors. H327 was the most potent BChE inhibitor (eqBChE IC50 = 0.057 ± 0.005 µM), and showed threefold improved inhibitory potency than the positive drug rivastigmine (eqBChE IC50 = 0.19 ± 0.001 µM). In addition, H327 as a pseudo-irreversible BChE inhibitor was endowed with neuroprotective, antioxidative and anti-neuroinflammatory properties. Cytotoxicity and acute toxicity tests confirmed the safety of compound H327. The pharmacokinetics study showed that compound H327 had a longer T1/2 time and higher bioavailability than the lead compound 1 g. Compound H327 was able to cross the blood-brain barrier (BBB) in vivo. Moreover, the behavioral tests showed that compound H327 could significantly improve scopolamine-induced cognitive impairment in vivo. Overall, these results demonstrated that compound H327 is a promising multi-target agent for the treatment of AD.

Discovery of dual inhibitors of topoisomerase I and Cyclooxygenase-2 for colon cancer therapy.

Novel tolfenamic acid derivatives based on the structure of I-1 were designed and synthesized to improve its poor target inhibition and solubility. Among them, W10 was identified as a potent dual-target inhibitor of Topo I (IC50 = 0.90 ± 0.17 µM) and COX-2 (IC50 = 2.31 ± 0.07 µM) with improved water solubility (32.33 µg/mL). Moreover, W10 also exhibited fairly potent anti-proliferative and pro-apoptosis activity via the mitochondrial pathway, as well as suppressed aberrant NF-κB/IκB activation in colon cancer cells in vitro. Additionally, W10 possessed favorable pharmacokinetic properties and excellent antitumor effects in vivo. In general, our study has demonstrated the potency of a novel Topo I/COX-2 dual inhibitor, which can potentially be developed into a chemotherapeutic candidate for colon cancer.

Design, synthesis and biological evaluation of 3-arylisoquinoline derivatives as topoisomerase I and II dual inhibitors for the therapy of liver cancer.

To explore novel topoisomerase inhibitors with high activity and druggability, 3-aryl isoquinoline alkaloids based on the corydamine modification and preliminary SARs of isoquinoline alkaloids in our previous works were re-designed. Currently, the design strategy is mainly revolved around the rigidity and flexibility of the molecular side chain and the molecular size. Consequently, not only the activity and druggability of the compound could be further improved, also the mechanism behind could been discovered. In vitro pharmacological studies, the outstanding nature with the excellent activity and the researchable depth of azepane-substituted compound 7 has been found through the vitro cytotoxicity test (IC50 = 1.93 µM in HuH7 cells and 2.10 µM in LM9 cells) and topoisomerase test. It was found that compound 7 had dual inhibitory effects on topoisomerase I and II, and its inhibitory activity on topoisomerase II is stronger than the positive drug etoposide. From the perspective of molecular docking, it had been verified that compound 7 could insert between DNA base pairs, which was consistent with the results of the DNA unwinding experiment. And the comet experiment confirmed 7 caused DNA damage. Meanwhile, compound 7 could inhibit cell proliferation, invasion and migration, and induce apoptosis by inhibiting PI3K/Akt/mTOR signaling pathway. Therefore, this study may lay a foundation for the discovery of 3-arylisoquinoline compounds with anti-liver cancer potential.

Design, synthesis and biological evaluation of novel o-aminobenzamide derivatives as potential anti-gastric cancer agents in vitro and in vivo.

Although gastric cancer has become a major public health problem, oral agents applied in clinics for gastric cancer therapy are scarce. Therefore, to explore new oral chemical entities with high efficiency and low toxicity, 41 o-aminobenzamide derivatives based on the scaffolds of MS-275 and SAHA were designed, synthesized, and evaluated for their anti-gastric cancer abilities in vitro and in vivo. Structure-activity relationships were discussed, leading to the identification of compounds F8 (IC50 = 0.28 µM against HGC-27 cell) and T9 (IC50 = 1.84 µM against HGC-27 cell) with improved cytotoxicity, anti-gastric cancer proliferation potency, induction of cell apoptosis and cell cycle arrest ability, inhibition of cell migration and invasion. What is worth mentioning is that compound F8 was more efficient and less toxic than the positive drug capecitabine in vivo on the HGC-27-xenograft model. Meanwhile, compound F8 exhibited suitable pharmacokinetic properties and less acute toxicity (LD50 > 1000 mg/kg). Besides, western blotting analysis, IHC analysis, differentially expressed proteins analysis and ABPP experiment indicated that compound F8 could modulate molecular pathways involved in apoptosis and cell cycle progression. Consequently, compound F8 is a strong candidate for the development of human gastric cancer therapy.

Novel piperazine based benzamide derivatives as potential anti-glioblastoma agents inhibiting cell proliferation and cell cycle progression.

Highly efficacious and tolerable agents for the treatment of glioblastoma (GBM), the most common and aggressive primary brain tumor, are urgently needed. Herein, we reveal the design, synthesis and biological evaluation of several piperazine based benzamide derivatives, which are based on the non-classical isostere principle and combination principle for GBM therapy. After structure-activity relationship (SAR) study, compound L19 was demonstrated as the most promising compound with IC50 values of 0.15 µM, 0.29 µM, 1.25 µM against GBM C6, U87-MG, U251 cells, respectively. Moreover, compound L19 could inhibit the proliferation, migration and invasion, as well as induce apoptosis and cell cycle arrest of GBM cell lines in vitro. From mechanism perspective, compound L19 could regulate the cell cycle-related proteins and influence the p16INK4a-CDK4/6-pRb pathway by western blotting experiment. What is worth mentioning is that compound L19 could penetrate the blood-brain barrier (BBB) with an exceptional brain-to-plasma ratio of 1.07 in vivo. Besides, the superior anti-glioblastoma potency in vivo of compound L19 was identified on U87-MG-xenograft model without any apparent host toxicity. Overall, the potential of compound L19 warrants further pre-clinical investigation for GBM therapy.

Influence of two-period cross-over design on the bioequivalence study of gefitinib tablets in beagle dogs.

Generally, two-period cross-over design is used in bioequivalence (BE) study. High intra-subject variability of gefitinib was reported in a clinical BE study, and significant changes in gefitinib exposures were observed among different periods in our previous BE study in dogs. Therefore, commercial gefitinib tablets from the same batch were used in the present study and assigned to two groups: the testing drug (GF1) group and the reference drug (GF2) group. A single-oral-dose, two-period cross-over study with a 7-day washout (approximately 24 half-lives) under fasting condition was conducted in 12 dogs to explore the factors. The results showed that the mean values of AUC(0-t), AUC(0-∞), Cmax and Tmax of these two GF1 and GF2 groups were similar. However, the GF1 and GF2 did not meet the acceptance criteria of bioequivalence with 90% confidence intervals, since the values obtained were 76.22%-117.43% for AUC(0-t) and 87.55%-131.59% for Cmax. ANOVA revealed a significant difference between the two periods (P < 0.05). Interestingly, the mean AUC(0-t) of gefitinib in the period 2 was 2.3-fold greater than that in the period 1, while Cmax in the period 2 was 1.7-fold higher than that in the period 1. However, the volume of distribution was significantly decreased, becoming 0.4-fold lower in the period 2. No statistically significant difference in the half-life and Tmax was observed between the two periods (P < 0.05). The pharmacokinetic alteration might come from the different physiological absorption and/or metabolism between periods. Since a 7-day washout interval was applied, DDI risk from P450s and/or P-gp would not play a significant role in the non-bioequivalence. As regard the variability, the intra-subject variation crossing the periods was much larger than the inter-subject variation within each period. The absorption and/or metabolism function of the gut bacteria might play an important role in the increasing exposure of gefitinib in the second period, especially with the comparison with the analysis from the high-fat-diet treatments in humans. Therefore, further studies might be needed to evaluate whether the assessment of bioequivalence could be facilitated by a much longer washout interval allowing the recovery of gut bacteria.