Dehydrocostus lactone alleviates irinotecan-induced intestinal mucositis by blocking TLR4/MD2 complex formation.

BACKGROUND: Irinotecan (CPT-11) is used as chemotherapeutic drug for treatment of colorectal cancer. However, without satisfactory treatments, its gastrointestinal toxicities such as diarrhea and intestinal inflammation severely restrained its clinical application. Roots of Aucklandia lappa Decne. are used as traditional Chinese medicine to relieve gastrointestinal dysfunction and dehydrocostus lactone (DHL) is one of its main active components. Nevertheless, the efficacy and mechanism of DHL against intestinal mucositis remains unclear. PURPOSE: The present study aimed to investigate the protective effects of DHL on CPT-11-induced intestinal mucositis and its underlying mechanisms. METHODS: The protective effect of DHL was investigated in CPT-11-induced mice and lipopolysaccharide (LPS)+CPT-11 induced THP-1 macrophages. Body weight, diarrhea score, survival rate, colon length, and histopathological changes in mice colon and jejunum were analyzed to evaluate the protective effect of DHL in vivo. And DHL on reducing inflammatory response and regulating TLR4/NF-κB/NLRP3 pathway in vivo and in vitro were explored. Moreover, DHL on the interaction between TLR4 and MD2 was investigated. And silencing TLR4 targeted by siRNA was performed to validate the mechanisms of DHL on regulating the inflammation. RESULTS: DHL prevented CPT-11-induced intestinal damage, represented by reducing weight loss, diarrhea score, mortality rate and the shortening of the colon. Histological analysis confirmed that DHL prevented intestinal epithelial injury and improved the intestinal barrier function in CPT-11 induced mice. Besides, DHL significantly downregulated the level of inflammatory cytokines by inhibiting TLR4/NF-κB/NLRP3 signaling pathway in CPT-11-induced mice and LPS+CPT-11-induced THP-1 macrophages. In addition, DHL blocked TLR4/MD2 complex formation. Molecular docking combined with SIP and DARTS assay showed that DHL could bind to TLR4/MD2 and occludes the hydrophobic pocket of MD2. Furthermore, Silencing TLR4 abrogated the effect of DHL on LPS+CPT-11 induced inflammatory response in THP-1 macrophages. Additionally, DHL ameliorate the CPT-11-induced intestinal mucositis without affecting the anti-tumor efficacy of CPT-11 in the tumor xenograft mice. CONCLUSION: This study found that DHL exhibited the anti-inflammatory effects in CPT-11-induced intestinal mucositis by inhibiting the formation of TLR4/MD2 complex and then regulation of NF-κB/NLRP3 signaling pathway. DHL is potentially served as a novel strategy of combined medication with CPT-11.

Insight into the mechanism of Xiao-Chai-Hu-Tang alleviates irinotecan-induced diarrhea based on regulating the gut microbiota and inhibiting Gut ß-GUS.

BACKGROUND: Irinotecan (CPT-11, Camptosar@) is a first-line drug for metastatic colorectal cancer. CPT-11-induced diarrhea, which is closely related to the concentrations of ß-glucuronidase (ß-GUS) and SN-38 in the gut, largely limits its clinical application. PURPOSE: Herein, Xiao-Chai-Hu-Tang (XCHT), a traditional Chinese formula, was applied to mitigate CPT-11-induced toxicity. This study initially explored the mechanism by which XCHT alleviated diarrhea, especially for ß-GUS from the gut microbiota. METHODS: First, we examined the levels of the proinflammatory cytokines and the anti-inflammatory cytokines in the intestine. Furthermore, we researched the community abundances of the gut microbiota in the CPT-11 and XCHT-treated mice based on 16S rRNA high-throughput sequencing technology. Meanwhile, the level of SN-38 and the concentrations of ß-GUS in intestine were examined. We also resolved the 3D structure of ß-GUS from gut microbiota by X-ray crystallography technology. Moreover, we used virtual screening, SPR analysis, and enzyme activity assays to confirm whether the main active ingredients from XCHT could selectively inhibit ß-GUS. RESULTS: In XCHT-treated mice, the levels of the proinflammatory cytokines decreased, the anti-inflammatory cytokines increased, and the community abundances of beneficial Firmicutes and Bacteroidota improved in the gut microbiota. We also found that the concentrations of ß-GUS and the level of SN-38, the major ingredient that induces diarrhea in the gut, significantly decreased after coadministration of XCHT with CPT-11 in the intestine. Additionally, we revealed the structural differences of ß-GUS from different gut microbiota. Finally, we found that EcGUS had good affinity with baicalein and meanwhile could be selectively inhibited by baicalein from XCHT. CONCLUSIONS: Overall, XCHT could relieve the delayed diarrhea induced by CPT-11 through improving the abundance of beneficial gut microbiota and reduced inflammation. Furthermore, based on the three-dimensional structure, baicalein, especially, could be used as a candidate EcGUS inhibitor to alleviate CPT-11-induced diarrhea.

Therapeutic drug monitoring of neoadjuvant mFOLFIRINOX in resected pancreatic ductal adenocarcinoma.

BACKGROUND: Despite a potentially curative treatment, the prognosis after upfront surgery and adjuvant chemotherapy for patients with resectable pancreatic ductal adenocarcinoma (PDAC) is poor. Modified FOLFIRINOX (mFOLFIRINOX) is a cornerstone in the systemic treatment of PDAC, including the neoadjuvant setting. Pharmacokinetic-guided (PKG) dosing has demonstrated beneficial effects in other tumors, but scarce data is available in pancreatic cancer. METHODS: Forty-six patients with resected PDAC after mFOLFIRINOX neoadjuvant approach and included in an institutional protocol for anticancer drug monitoring were retrospectively analyzed. 5-Fluorouracil (5-FU) dosage was adjusted throughout neoadjuvant treatment according to pharmacokinetic parameters and Irinotecan (CPT-11) pharmacokinetic variables were retrospectively estimated. RESULTS: By exploratory univariate analyses, a significantly longer progression-free survival was observed for patients with either 5-FU area under the curve (AUC) above 28 mcg·h/mL or CPT-11 AUC values below 10 mcg·h/mL. In the multivariate analyses adjusted by age, gender, performance status and resectability after stratification according to both pharmacokinetic parameters, the risk of progression was significantly reduced in patients with 5-FU AUC ≥28 mcg·h/mL [HR = 0.251, 95% CI 0.096-0.656; p = 0.005] and CPT-11 AUC <10 mcg·h/mL [HR = 0.189, 95% CI 0.073-0.486, p = 0.001]. CONCLUSIONS: Pharmacokinetically-guided dose adjustment of standard chemotherapy treatments might improve survival outcomes in patients with pancreatic ductal adenocarcinoma.

Gegen Qinlian standard decoction alleviated irinotecan-induced diarrhea via PI3K/AKT/NF-κB axis by network pharmacology prediction and experimental validation combination.

BACKGROUND: The frequently occurred chemotherapy-induced diarrhea (CID) caused by irinotecan (CPT-11) administration has been the most representative side-effects of CPT-11, resulting in the chemotherapy suspension or failure. Our previous studies indicated that Gegen Qinlian formula exhibited a significant alleviation effect on CPT-11-induced diarrhea. However, referencing to Japanese Kampo medicine, the TCM standard decoction would supply the gap between ancient preparation application and modern industrial production. METHODS: The LC-MS technology combined with network pharmacology was employed to identify the active ingredients and mechanisms of GQD standard decoction for CPT-11-induced diarrhea. The anti-inflammatory activities associated with intestinal barrier function of GQD standard decoction were studied by SN-38 activated NCM460 cells in vitro and CPT-11-induced diarrhea in vivo. Proteins involved in inflammation, mRNA levels, disease severity scores, and histology involved in intestinal inflammation were analysed. RESULTS: There were 37 active compounds were identified in GQD standard decoction. Network pharmacology analyses indicated that PI3K-AKT signaling pathway were probably the main pathway of GQD standard decoction in CPT-11-induced diarrhea treatment, and PIK3R1, AKT1, NF-κB1 were the core proteins. Moreover, we found that the key proteins and pathway predicted above was verified in vivo and in vitro experiments, and the GQD standard decoction could protect the cellular proliferation in vitro and ameliorate CPT-11-induced diarrhea in mice model. CONCLUSIONS: This study demonstrated the molecular mechanism of 37 active ingredients in GQD standard decoction against CPT-11-induced diarrhea. And the core proteins and pathway were validated by experiment. This data establishes the groundwork for particular molecular mechanism of GQD standard decoction active components, and this research can provide a scientific reference for the TCM therapy of CID.

Decreased Tissue Omega-6/Omega-3 Fatty Acid Ratio Prevents Chemotherapy-Induced Gastrointestinal Toxicity Associated with Alterations of Gut Microbiome.

Gastrointestinal toxicity (GIT) is a debilitating side effect of Irinotecan (CPT-11) and limits its clinical utility. Gut dysbiosis has been shown to mediate this side effect of CPT-11 by increasing gut bacterial ß-glucuronidase (GUSB) activity and impairing the intestinal mucosal barrier (IMB). We have recently shown the opposing effects of omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFA) on the gut microbiome. We hypothesized that elevated levels of tissue n-3 PUFA with a decreased n-6/n-3 PUFA ratio would reduce CPT-11-induced GIT and associated changes in the gut microbiome. Using a unique transgenic mouse (FAT-1) model combined with dietary supplementation experiments, we demonstrate that an elevated tissue n-3 PUFA status with a decreased n-6/n-3 PUFA ratio significantly reduces CPT-11-induced weight loss, bloody diarrhea, gut pathological changes, and mortality. Gut microbiome analysis by 16S rRNA gene sequencing and QIIME2 revealed that improvements in GIT were associated with the reduction in the CPT-11-induced increase in both GUSB-producing bacteria (e.g., Enterobacteriaceae) and GUSB enzyme activity, decrease in IMB-maintaining bacteria (e.g., Bifidobacterium), IMB dysfunction and systemic endotoxemia. These results uncover a host-microbiome interaction approach to the management of drug-induced gut toxicity. The prevention of CPT-11-induced gut microbiome changes by decreasing the tissue n-6/n-3 PUFA ratio could be a novel strategy to prevent chemotherapy-induced GIT.

Substrate-dependent Inhibition of Hypericin on Human Carboxylesterase 2: Implications for Herb-drug Combination.

BACKGROUND: Hypericin is the main active ingredient of St. John's wort, a Chinese herb commonly used for treating depression. Previous studies shown that hypericin can strongly inhibit human cytochrome P450 (CYP) enzyme activities; however, its potential interactions that inhibit human carboxylesterases 2 (hCE2) are unclear. PURPOSE: This study aimed to investigate the inhibitory effect of hypericin on hCE2. METHODS: The inhibition mechanism of hypericin on hCE2 was studied by using N-(2-butyl-1,3-dioxo-2,3-dihydro- 1H-phenalen-6-yl)-2-chloroacetamide (NCEN). The type of inhibition of hypericin on hCE2 and the corresponding inhibition constant (Ki) value were determined. The inhibition of hypericin on hCE2 in living cells was discussed. The risk of herb-drug interactions (HDI) of hypericin in vivo was predicted by estimating the area under the drug concentration-time curve (AUC) in the presence or absence of hypericin. To understand the inhibition mechanism of hypericin on the activity of hCE2 in-depth, molecular docking was performed. RESULTS: The half-maximal inhibitory concentration (IC50) values of hypericin against the hydrolysis of NCEN and irinotecan (CPT-11) were calculated to be 26.59 µM and 112.8 µM, respectively. Hypericin inhibited the hydrolysis of NCEN and CPT-11. Their Ki values were estimated as 10.53 µM and 81.77 µM, respectively. Moreover, hypericin distinctly suppressed hCE2 activity in living cells. In addition, the AUC of hCE2 metabolic drugs with metabolic sites similar to NCEN was estimated to increase by up to 5 % in the presence of hypericin. More importantly, the exposure of CPT-11 in the intestinal epithelium was predicted to increase by 2 % - 69 % following the oral coadministration of hypericin. Further, molecular simulations indicated that hypericin could strongly interact with ASP98, PHE307, and ARG355 to form four hydrogen bonds within hCE2. CONCLUSION: These findings regarding the combination of hypericin-containing herbs and drugs metabolized by hCE2 are of considerable clinical significance.

CPT11 with P-glycoprotein/CYP 3A4 dual-function inhibitor by self-nanoemulsifying nanoemulsion combined with gastroretentive technology to enhance the oral bioavailability and therapeutic efficacy against pancreatic adenocarcinomas.

Therapeutic efficacies of orally administrated hydrophobic chemodrugs are decreased by poor water solubilities and reduced oral bioavailabilities by P-glycoprotein (P-gp) and CYP450. In this study, CPT11 alone or combined with dual-function inhibitors (baicalein (BA) silymarin (SM), glycyrrhizic acid (GA), and glycyrrhetinic acid (GLA)) of P-gp and CYP450 loaded in a lecithin-based self-nanoemulsifying nanoemulsion preconcentrate (LBSNENP) to improve the solubility and inhibit the elimination by P-gp and CYP450. Results revealed that the LBSNENP composed of Capryol 90, lecithin/Tween 80/Cremophor EL, and propylene glycol at a weight ratio of 18:58:24 (designated PC90C10P0) was optimally selected. Encapsulating CPT11 with PEO-7000K in PC90C10P10/30 further enhanced the resultant hydrogel to be gastro-retainable and to release CPT11 in a sustained manner. Pharmacokinetic study of CPT11-loaded PC90C10P0 administered orally revealed an absolute bioavailability (FAB, vs. intravenous CPT11) of 7.8 ± 1.01% and a relative bioavailability (FRB1, vs. oral solution of CPT11) of 70.7 ± 8.6% with a longer half-life (T1/2) and mean residence time (MRT). Among the dual-function inhibitors, SM was shown to be the most influential in increasing the oral bioavailability of CPT11. SM also increased the plasma concentration of the SN-38 active metabolite, which formed from the enhanced plasma concentration of CPT11. It is concluded that treatment with CPT11 loaded in PC90C10P0 with or without solubilization with SM could expose tumors to higher plasma concentrations of both CPT11 and SN-38 leading to enhancement of tumor growth inhibition with no signs of adverse effects.

Total Flavonoids of Glycyrrhiza uralensis Alleviates Irinotecan-Induced Colitis via Modification of Gut Microbiota and Fecal Metabolism.

Irinotecan (CPT-11)-induced gastrointestinal toxicity strongly limits its anticancer efficacy. Glycyrrhiza uralensis Fisch., especially flavonoids, has strong anti-inflammatory and immunomodulatory activities. Herein, we investigate the protective effect of the total flavonoids of G. uralensis (TFGU) on CPT-11-induced colitis mice from the perspective of gut microbiota and fecal metabolism. The body weight and colon length of mice were measured. Our results showed that oral administration of TFGU significantly attenuated the loss of body weight and the shortening of colon length induced by CPT-11. The elevated disease activity index and histological score of colon as well as the up-regulated mRNA and protein levels of TNF-α, IL-1ß, and IL-6 in the colonic tissue of CPT-11-treated mice were significantly decreased by TFGU. Meanwhile, TFGU restored the perturbed gut microbial structure and function in CPT-11-treated mice to near normal level. TFGU also effectively reversed the CPT-11-induced fecal metabolic disorders in mice, mainly call backing the hypoxanthine and uric acid in purine metabolism. Spearman's correlation analysis further revealed that Lactobacillus abundance negatively correlated with fecal uric acid concentration, suggesting the pivotal role of gut microbiota in CPT-11-induced colitis. Since uric acid is a ligand of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, TFGU was further validated to inhibit the activation of NLRP3 inflammasome by CPT-11. Our findings suggest TFGU can correct the overall gut microbial dysbiosis and fecal metabolic disorders in the CPT-11-induced colitis mice, underscoring the potential of using dietary G. uralensis as a chemotherapeutic adjuvant.

Mechanisms of Gegen Qinlian Pill to ameliorate irinotecan-induced diarrhea investigated by the combination of serum pharmacochemistry and network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine suggests the use of natural extracts and compounds is a promising strategy to prevent irinotecan (CPT-11)-induced gut toxicity and resulting diarrhea. Previous work from our lab indicated the protective effect of Gegen Qinlian decoction; given this, we further speculated that Gegen Qinlian Pill (GQP) would exhibit similar therapeutic effects. The effective material basis as well as potential mechanisms underlying the effect of GQP for the treatment of CPT-11-induced diarrhea have not been fully elucidated. AIM OF THE STUDY: The application of natural extracts or compounds derived from Chinese medicine is deemed to a promising strategy to prevent irinotecan (CPT-11)-induced gut toxicity. The aim of this study was to investigated the beneficial effects of GQP on CPT-11-induced gut toxicity and further explored its anti-diarrheal mechanism. METHODS: First, the beneficial effect of GQP in alleviating diarrhea in mice following CPT-11 administration was investigated. We also obtained the effective ingredients in GQP from murine serum samples using HPLC-Q-TOF-MS analysis. Based on these active components, we next established an interaction network linking "compound-target-pathway". Finally, a predicted mechanism of action was obtained using in vivo GQP validation based on Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. RESULTS: A total of 19, GQP-derived chemical compounds were identified in murine serum samples. An interaction network linking "compound-target-pathway" was then established to illuminate the interaction between the components present in serum and their targets that mitigated diarrhea. These results indicated GQP exerted a curative effect on diarrhea and diarrhea-related diseases through different targets, which cumulatively regulated inflammation, oxidative stress, and proliferation processes. CONCLUSION: Taken together, this study provides a feasible strategy to elucidate the effective constituents in traditional Chinese medicine formulations. More specifically, this work detailed the basic pharmacological effects and underlying mechanism behind GQP's effects in the treatment of CPT-11-induced gut toxicity.

Identification of the bioactive components of Banxia Xiexin Decoction that protect against CPT-11-induced intestinal toxicity via UPLC-based spectrum-effect relationship analyses.

ETHNOPHARMACOLOGICAL RELEVANCE: Irinotecan (CPT-11) is a valuable chemotherapeutic compound, but its use is associated with severe diarrhea in some patients. The CPT-11 prodrug is converted into the active 7-ethyl-10-hydroxycamptothecin (SN-38) metabolite, which can then be retained for extended periods in the intestine, leading to the onset of diarrhea and related symptoms. Banxia Xiexin Decoction (BXD) is commonly employed for the treatment of gastroenteritis in traditional Chinese medicine (TCM), and in clinical settings, it is used to prevent diarrhea in patients undergoing CPT-11 treatment. To date, however, there have been no studies specifically examining which components of BXD can alleviate the gastrointestinal symptoms associated with CPT-11 administration. AIM: This study aimed to identify the main herbal components of BXD associated with protection against CPT-11-induced intestinal toxicity in a murine model system. MATERIALS AND METHODS: SN-38 levels were measured by UPLC-ESI-MS/MS in samples collected from mice subjected to CPT-11-induced diarrhea that had been administered BXD or different components thereof. Pearson correlation and Grey relational analyses were then used to explore spectrum-effect relationships between reductions in intestinal SN-38 levels and specific chemical fingerprints in samples from mice administered particular combinations of BXD component herbs. RESULTS: We found that different herbal combinations were associated with significant differences in intestinal SN-38 reductions in treated mice. Our spectrum-effect analysis revealed that BXD components including chrysin 6-C-arabinoside-8-C-glucoside, coptisine, hydroxyl oroxylin A 7-O-glucuronide (hydroxyl wogonoside), baicalin, an isomer of 5,6,7-trihydroxyl-flavanone-7-O-glucuronide, berberine, palmatine, and chrysin-7-O-glucuronide were all directly linked with reductions in intestinal SN-38 levels. We therefore speculate that these compounds are the primary bioactive components of BXD, suggesting that they offer protection against CPT-11-induced diarrhea. CONCLUSION: By utilizing UPLC to analyze SN-38 levels in mice treated with a variety of herbal combinations, we were able to effectively explore BXD spectrum-effect relationships and to thereby establish the components of this medicinal preparation that were bioactive and capable of preventing CPT-11-induced diarrhea in mice. This and similar spectrum-effect studies represent a robust means of exploring the mechanistic basis for the pharmacological activity of TCM preparations.

Identify old drugs as selective bacterial ß-GUS inhibitors by structural-based virtual screening and bio-evaluations.

Irinotecan (CPT-11) is a cytotoxic drug that has wide applicability and usage in cancer treatment. Despite its success, patients suffer dose-dependent diarrhea, limiting the drug's efficacy. No effective therapy is available for this unmet medical need. The bacterial ß-glucuronidase (ß-GUS) plays pivotal role in CPT-11-induced diarrhea (CID) via activating the non-toxic SN-38G to toxic SN-38 inside intestine. By using structural-based virtual screening, three old drugs (N-Desmethylclozapine, Aspartame, and Gemifloxacin) were firstly identified as selective bacterial ß-GUS inhibitors. The IC50 values of the compounds in the enzyme-based and cell-based assays range from 0.0389 to 3.6040 and 0.0105 to 5.3730 µM, respectively. The compounds also showed good selectivity against mammalian ß-GUS and no significant cytotoxicity in bacteria. Molecular docking and molecular dynamics simulations were performed to further investigate the binding modes of compounds with bacterial ß-GUS. Binding free energy decomposition revealed that the compounds formed strong interactions with E413 in catalytic trail from primary monomer and F365' on the bacterial loop from the other monomer of bacterial ß-GUS, explaining the selectivity against mammalian ß-GUS. The old drugs identified here may be used as bacterial ß-GUS inhibitors for CID or other bacterial ß-GUS-related disorders.

Protective effect of curcumin against irinotecan­induced intestinal mucosal injury via attenuation of NF­κB activation, oxidative stress and endoplasmic reticulum stress.

Irinotecan (CPT­11) is a DNA topoisomerase I inhibitor which is widely used in clinical chemotherapy, particularly for colorectal cancer treatment. However, late­onset diarrhea is one of the severe side­effects of this drug and this restricts its clinical application. The present study aimed to investigate the protective effects of curcumin treatment on CPT­11­induced intestinal mucosal injury both in vitro and in vivo and to elucidate the related mechanisms involved in these effects. For this purpose, mice were intraperitoneally injected with CPT­11 (75 mg/kg) for 4 days to establish a model of late­onset diarrhea. Curcumin (100 mg/kg) was intragastrically administered 8 days before the injection of CPT­11. Injury to small intestinal tissues was examined by H&E staining. The protein expression of prolyl 4­hydroxylase subunit beta (P4HB) and peroxiredoxin 4 (PRDX4) was detected by immunohistochemistry, as well as western blot analysis. IEC­6 cell viability was detected by MTT assay. Flow cytometry was performed to examine the cell apoptotic rate, mitochondrial membrane potential and reactive oxygen species (ROS) generation. Immunofluorescence was used to observe the localization of nuclear factor (NF)­κB. The levels of cleaved caspase­3, glucose­regulated protein, 78 kDa (GRP78), P4HB, PRDX4 and CHOP were detected by western blot analysis. The results revealed that in vivo, curcumin effectively attenuated the symptoms of diarrhea and abnormal intestinal mucosa structure induced by CPT­11 in nude mice. Treatment with curcumin also increased the expression of P4HB and PRDX4 in the tissue of the small intestine. In vitro, curcumin, exhibited little cytotoxicity when used at concentrations <2.5 µg/ml for 24 h in IEC­6 cells. At this concentration, curcumin also improved cell morphology, inhibited apoptosis, maintained mitochondrial membrane potential and reduced the elevated levels of ROS induced by CPT­11 (20 µg/ml). Furthermore, curcumin abolished NF­κB signal transduction and protected the cells from CPT­11­induced apoptosis by upregulating the expression of molecular chaperones, such as GRP78, P4HB and PRDX4, and suppressing the levels of the apoptosis­related proteins, CHOP and cleaved caspase­3. On the whole, our data indicate that curcumin exerted protective effects against CPT­11­induced intestinal mucosa injury. The protective effects of curcumin are mediated by inhibiting the activation of NF­κB, and suppressing oxidative stress and endoplasmic reticulum stress.

Preventative effects of selenium-enriched Bifidobacterium longum on irinotecan-induced small intestinal mucositis in mice.

Intestinal mucositis is a frequent side effect in cancer patients who are treated with chemotherapy. There are no effective treatment strategies to date. To find a novel way to alleviate mucositis, the effects of selenium-enriched Bifidobacterium longum (Se-B. longum) in preventing irinotecan (CPT-11)-induced intestinal mucositis in a mouse model were investigated. We tested the ability of Se-B. longum (Se 0.6 mg/kg, 5×108 cfu/mice) to reduce small intestinal mucositis induced by CPT-11 (75 mg/kg, daily) injected intraperitoneally for four consecutive days in mice. Se-B. longum significantly decreased mortality induced by CPT-11 from 71.4% to 16.7%. CPT-11 induced body weight loss, which was alleviated by preventative and simultaneous administration of Se-B. longum. Se-B. longum significantly decreased the severity of diarrhoea from 11 to 4% compared to the CPT-11 group. Inflammation, including intestinal shortening and upregulation of tumour necrosis factor-α and interleukin-1ß induced by CPT- 11, were prevented by Se-B. longum. Se-B. longum is effective in preventing small intestinal mucositis induced by CPT-11 and therefore has potential to be used clinically by cancer patients.

Novel SN38 derivative-based liposome as anticancer prodrug: an in vitro and in vivo study.

BACKGROUND: Many novel drug delivery systems have been extensively studied to exploit the full therapeutic potential of SN38, which is one of the most potent antitumor analogs of camptothecins (CPTs), whose clinical application is seriously hindered by poor water solubility, low plasmatic stability, and severe toxicity, but results are always unsatisfactory. METHODS: In this study, combining the advantages of prodrug and nanotechnology, a lipophilic prodrug of SN38, SN38-PA, was developed by conjugating palmitic acid to SN38 via ester bond at C10 position, and then the lipophilic prodrug was encapsulated into a long-circulating liposomal carrier by film dispersion method. RESULTS: The SN38-PA liposomes were characterized as follows: an average particle size of 80.13 nm, an average zeta potential of -33.53 mv, and the entrapment efficiency of 99%. Compared with CPT-11, SN38-PA liposome was more stable in close lactone form, more efficient in conversion rate to SN38, and more potent in cytotoxicity against tumor cells. Pharmacokinetic study showed that SN38-PA liposome had significantly enhanced plasma half-life (t1/2) value of SN38 and increased area under the curve (AUC) of SN38, which was 7.5-fold higher than that of CPT-11. Biodistribution study showed that SN38-PA liposome had more active metabolite SN38 in each tissue. Finally, the pharmacodynamic study showed that SN38-PA liposome had higher antitumor effect with the antitumor inhibition rate of 1.61 times than that of CPT-11. CONCLUSION: These encouraging data merit further investigation on this novel SN38-PA liposome.

Traditional Chinese medicine Gegen Qinlian decoction ameliorates irinotecan chemotherapy-induced gut toxicity in mice.

BACKGROUND: Gegen Qinlian decoction (GQT), is a classic traditional Chinese medicine formula chronicled in Shang Han Lun, and is widely used to treat diarrhea and inflammation symptoms in various gastrointestinal disorders. Although it has been found to inhibit delayed-onset mice diarrhea resulted from irinotecan (CPT-11) administration in preliminary experiments, the underlying mechanisms and chemical components remain elusive. METHODS: The effective fraction of GQT by macroporous resin elution was obtained and screened using a diarrhea mouse model induced by CPT-11 and quantified by UPLC analysis. The protective effect of GQT extract towards alleviating diarrhea in mice following CPT-11 administration was further investigated. The levels of inflammatory cytokines and intestinal tight junction related proteins in colonic tissues were determined. The inhibitory effect of GQT extract against hCE2 was evaluated by a fluorescence-based method. Lastly, the synergistic effect of GQT extract combined with CPT-11 against tumor growth in a colorectal tumor mouse model, induced by HT-29 colon cancer cells xenograft subcutaneously, was investigated. RESULTS: The obtained GQT extract, which profoundly ameliorated the gut toxicity induced by CPT-11, contained puerarin, liquiritin, berberine, and baicalin of 27.2 mg/g, 4.6 mg/g, 491.4 mg/g, and 304.2 mg/g, respectively. After 5 days of administration of GQT extract to mice with diarrhea induced by CPT-11, aberrantly elevated levels of pro-inflammatory cytokines, including IL-1ß, COX-2, ICAM-1, and TNF-α, were significantly decreased. Meanwhile, GQT extract also exhibited a remarkable anti-oxidative stress effect, involving activating the Keap1/Nrf2 pathway, and up-regulating the intestinal barrier function by enhancing the expression of tight junction proteins ZO-1, HO-1, and occludin. Additionally, a potent inhibitory effect of GQT extract against hCE2 was observedin vitro, with its IC50 value of 0.187 mg/ml, suggesting alleviating activity on hCE2-mediated severe diarrhea in patients suffered from CPT-11. Moreover, GQT extract was shown to improve inhibition of the colonic tumor growth synergistically with CPT-11. CONCLUSION: The present study indicates that GQT extract can ameliorate CPT-11 induced gut toxicity in mice and improve CPT-11 efficacy in colorectal cancer treatment.

Evidence for Shikonin acting as an active inhibitor of human carboxylesterases 2: Implications for herb-drug combination.

Shikonin, a natural naphthoquinone compound derived from the herb Lithospermum erythrorhizon, is widely used for its various pharmacological activities. However, its potential interactions with other medications by inhibiting human carboxylesterases 2 (hCE2) remain unknown. In this study, the inhibitory effects of shikonin on the activity of hCE2 in human liver microsomes are investigated by using fluorescein diacetate (FD), N-(2-butyl-1,3-dioxo-2,3-dihydro-1H-phenalen-6-yl)-2-chloroacetamide (NCEN), and CPT-11 as substrates of hCE2. The results demonstrate that shikonin significantly inhibits the activity of hCE2 when FD and NCEN are used as substrates, whereas the half inhibition concentration value of shikonin increased by 5-30 times when CPT-11 was used as the substrate. The inhibition types of shikonin against hCE2 activity reflected by 3 substrates were all best fit to noncompetitive manners. In addition, shikonin was found to distinctly suppress endogenous hCE2 activity, characterized with attenuated fluorescence. Furthermore, for drugs metabolized by hCE2 with the similar binding sites with FD or NCEN, the estimated magnitudes of area under the curve variation were approximately 9-357% in the presence of shikonin. Also, the area under the curve of CPT-11 could be increased by 1-14% following administration of shikonin. These findings have clear clinical implications for the combination of shikonin and hCE2-metabolizing prodrugs.

Assessment of the chemotherapeutic potential of a new camptothecin derivative, ZBH-1205.

CPT-11 (irinotecan) is a derivative of camptothecin which is a natural product derived from the Chinese tree Camptotheca acuminta and widely used in antitumor therapy. Here, the in vitro anti-tumor activity and associated mechanisms of a novel derivative of camptothecin, ZBH-1205, were investigated in a panel of 9 human tumor cell lines, as well as in HEK 293 and SK-OV-3/DPP, a multi-drug resistant (MDR) cell line, and compared to CPT-11 and 7-ethyl-10-hydroxy-camptothecin (SN38). Comparisons between the different compounds were made on the basis of IC50 values as determined by the MTT assay, and flow cytometry was used to evaluate cell cycle progression, apoptosis, and the levels of pro- and active caspase-3 among different treatment groups. Interaction between the molecules and topoisomerase-1 (Topo-1)-DNA complexes was detected by a DNA relaxation assay. Our results demonstrated that IC50 values for ZBH-1205 ranged from 0.0009 µmol/L to 2.5671 µmol/L, which were consistently lower than IC50 values of CPT-11 or SN38 in the panel of cell lines, including SK-OV-3/DPP. Furthermore, ZBH-1205 was more effective than CPT-11 or SN38 at stabilizing Topo-1-DNA complexes and inducing tumor cell apoptosis. Therefore, ZBH-1205 is a promising chemotherapeutic agent to be further assessed in large-scale clinical trials.

Thymoquinone induces caspase-independent, autophagic cell death in CPT-11-resistant lovo colon cancer via mitochondrial dysfunction and activation of JNK and p38.

Chemotherapy causes unwanted side effects and chemoresistance, limiting its effectiveness. Therefore, phytochemicals are now used as alternative treatments. Thymoquinone (TQ) is used to treat different cancers, including colon cancer. The irinotecan-resistant (CPT-11-R) LoVo colon cancer cell line was previously constructed by stepwise CPT-11 challenges to untreated parental LoVo cells. TQ dose-dependently increased the total cell death index and activated apoptosis at 2 µM, which then diminished at increasing doses. The possibility of autophagic cell death was then investigated. TQ caused mitochondrial outer membrane permeability (MOMP) and activated autophagic cell death. JNK and p38 inhibitors (SP600125 and SB203580, respectively) reversed TQ autophagic cell death. TQ was also found to activate apoptosis before autophagy, and the direction of cell death was switched toward autophagic cell death at initiation of autophagosome formation. Therefore, TQ resulted in caspase-independent, autophagic cell death via MOMP and activation of JNK and p38 in CPT-11-R LoVo colon cancer cells.