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. Author manuscript; available in PMC: 2024 May 1.
Published in final edited form as: Br J Pharmacol. 2023 Jan 9;180(10):1362–1378. doi: 10.1111/bph.16020

Opioid-induced microbial dysbiosis disrupts CPT-11 metabolism and increases gastrointestinal toxicity in a murine model

Jingjing Meng 1,*, Yaa F Abu 2,*, Yue Zhang 1, Yuyin Zhou 3, Yun Xie 3, Yan Yan 1, Junyi Tao 1, Sundaram Ramakrishnan 1, Chi Chen 2, Sabita Roy 1,┼
PMCID: PMC10089971  NIHMSID: NIHMS1860761  PMID: 36562107

Abstract

Background and purpose:

Opioids are commonly used for the management of cancer-associated pain and chemotherapy-induced diarrhea. The chemotherapeutic agent CPT-11 causes severe GI toxicity as a result of deconjugation of its inactive metabolite SN-38G by intestinal bacterial β-glucuronidases to the pharmacologically active SN-38. As opioids are known to cause gut microbial dysbiosis, this study evaluated whether CPT-11’s anti-tumor efficacy and GI toxicity are exacerbated by opioid co-administration.

Experiment approach:

8-week-old C57BL/6 male mice were treated with CPT-11 ± opioid co-administration. 16S rRNA sequencing was used for gut microbiome analysis. LC-MS analyses of plasma and intestinal extracts were performed to investigate the pharmacokinetic profile of CPT-11. Histological analysis and qRT-PCR were used to determine the severity of intestinal tissue damage. The in vitro assay using human liver microsome was also performed to confirm the effects of opioids on CPT-11 metabolism.

Key results:

Gut microbiome analysis showed that morphine treatment induced enrichment of β-glucuronidase-producing bacteria in the intestines of CPT-11-treated mice, resulting in SN-38 accumulation and exacerbation of GI toxicity in the small intestine. Oral administration of both antibiotics and a glucuronidase inhibitor protected mice against GI toxicity induced with CPT-11 and morphine co-administration, implicating a microbiome-dependent mechanism. Additionally, morphine and loperamide decreased the plasma concentration of SN-38 and compromised CPT-11’s anti-tumor efficacy, although this seemed to be microbiome-independent.

Conclusion and Implications:

Gut microbiota play a significant role in opioid and chemotherapeutic agent drug-drug interactions. Inhibition of gut microbial glucuronidase may also prevent adverse GI effects of CPT-11 in patients on opioids.

Keywords: Opioids, CPT-11, Chemotherapy, Gut microbiome, drug metabolism

Introduction

Pain is a common symptom associated with cancer. A recent clinical study showed that pain is experienced by 55% of patients during anti-cancer treatment and 66.4% of patients with advanced, metastatic, or terminal staged cancer (Van Den Beuken-Van Everdingen et al., 2016). Among various analgesics, opioids are the most effective medications for severe cancer pain and chemotherapy-induced diarrhea (e.g., loperamide) (Pergolizzi et al., 2015). Accordingly, opioids significantly improve quality of life and patient compliance with active cancer therapy (Andreyev et al., 2014). That being said, recent clinical studies have demonstrated that opioid use is associated with poor survival in advanced cancer patients undergoing chemotherapy (Zheng et al., 2020). Therefore, a comprehensive understanding of how prescription opioids influence anti-tumor efficacy and contribute to off-target effects of chemotherapeutic agents is warranted for potential modification of the current therapeutic approach.

In particular, CPT-11 (Irinotecan) is a chemotherapeutic agent widely utilized in the treatment of different types of solid tumors by inhibiting DNA topoisomerase I (Kciuk et al., 2020). However, CPT-11 causes severe gastrointestinal (GI) toxicity resulting in diarrhea, pain, weight loss, GI bleeding, and infection, precluding treatment in many patients (Wallace et al., 2010). It has been well-demonstrated that this dose-limiting diarrhea is mitigated by intestinal gut microbiota, which play a key role in the absorption and metabolism of CPT-11. While CPT-11 is converted to the pharmacologically active SN-38, it undergoes hepatic glucuronidation by phase II uridine diphosphate glucuronosyltransferase (UGT) 1A1 (UGT1A1) enzymes to create the inactive metabolite SN-38G for excretion in the GI tract (Wallace et al., 2010). However in the GI tract, SN-38G can serve as a substrate for gut microbial β-glucuronidase (GUS) enzymes, which remove the glucuronide moiety to reactivate SN-38, regenerating the toxic metabolite SN-38 in the intestine (Wallace et al., 2010). Considering opioids are commonly used alongside chemotherapy agents for pain management and diarrhea relief, how opioids affect CPT-11 metabolism and GI toxicity is an area of active interest, especially as opioids are well-known to cause intestinal microbial dysbiosis (Meng et al., 2015, 2020; Banerjee et al., 2016) and the quantities and activities of β-glucuronidase-producing bacteria in the intestine are considered to be major contributors to CPT-11-associated GI toxicity. Additionally, morphine-induced gut dysbiosis has further been shown to modulate the efficacy of morphine and compromise its analgesic efficacy (Zhang et al., 2019). However, whether the adverse GI effects of CPT-11 (which have previously been shown to be microbiome-dependent) or the anti-tumor efficacy of CPT-11 might be exacerbated by opioid co-administration remain to be elucidated.

In the present study, we use a murine model to investigate interactions between opioids and the chemotherapeutic agent CPT-11, with the goal of investigating therapeutic strategies for pain and diarrhea management to both preserve the analgesic potential of opioids and the anti-tumor efficacy of CPT-11. Here, we demonstrate that opioids exacerbate CPT-11-induced GI toxicity by modulating gut microbiota in the small intestine. We also show that inhibition of gut microbial glucuronidase may prevent adverse GI effects of CPT-11 in patients on opioids. Our studies further suggest that opioids compromise the anti-tumor efficacy of CPT-11, which may be independent of the gut microbiome.

Methods

Animals:

All animal experiments were approved by the Institutional Animal Care and Use Committee policies at the University of Miami and adhered to all ethical guidelines related to the care of laboratory animals. Animal studies are reported in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020). Eight-week-old male C57BL/6 (WT) mice (25–30g) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) (https://www.jax.org/strain/003752), with age-matched littermate wild type (WT) mice used as controls. Mice were housed five per cage under a controlled temperature (22 ± 2C), humidity (30%–70%), and 12 h light/dark cycle (light at 0700), with food pellets and water ad libitum. Mice were maintained in sterile microisolator cages under pathogen-free conditions. All efforts were made to minimize animal suffering and to reduce the number of animals used. At the conclusion of experiments, mice were killed using CO2 asphyxiation followed by cervical dislocation, as recommended by the Panel of Euthanasia of the American Veterinary Medical Association (AVMA). No anesthesia was used in experiments.

CPT-11-induced GI toxicity was established and evaluated in an in vivo experimental system in mice as previously described (Chen et al., 2013). This model of CPT-11-induced GI toxicity parallels intestinal toxicity observed in humans due to the accumulation of SN-38 in intestinal tissue through enterohepatic circulation (Chen et al., 2013). Mice were randomized into six groups and received intraperitoneal injection of morphine (15 mg/kg), clonidine (30 μg/kg), or saline b.i.d (Day 1–7 b.i.d.; Day 8 once/day prior to sacrificing animals). On the fourth day, mice concomitantly received 75 mg/kg CPT-11 or vehicle once per day for five consecutive days (Day 4–8). For β-Glucuronidase inhibitor studies, 1-((6,8-Dimethyl-2-oxo-1,2-dihydroquinolin-3-yl)methyl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea (Calbiochem®) was administered by oral gavage b.i.d. at 10 μg per mouse 1 day prior to CPT-11 treatment (Fig. 5A). This dose regimen has been shown to effectively inhibit bacterial β-glucuronidase and protect mice against enteropathy induced by CPT-11 (Saitta et al., 2014). For loperamide treatment, mice were gavaged with 5 mg/kg loperamide or water once per day one day after the start of CPT-11 injection (Fig 6A). This model is clinically relevant in the treatment of CPT-11-induced diarrhea. According to established protocol, the pan-antibiotics cocktail (Zhang et al., 2019) was prepared in drinking water. The antibiotics treatment was given 7 days before morphine treatment and continued throughout the following eight days of morphine and CPT-11 treatment (Fig. 4A). For CPT-11 and morphine co-administration anti-tumor efficacy studies, a total of 5 × 105 MC38 colon adenocarcinoma cells (RRID: CVCL_B288) were injected subcutaneously (s.c.) into the left flank of mice seven days before CPT-11 treatment. Experimenters were blinded to all experimental conditions for histological scoring, intestinal transit time calculations, and determination of CPT-11, SN-38, and SN-38G concentrations in the plasma and luminal contents.

Fig. 5. β-Glucuronidase inhibitor Inh-1 alleviates GI toxicity induced by morphine and CPT-11.

Fig. 5

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MC: morphine +CPT-11 (n=5), MC+GI: morphine+ CPT-11+ Inh-1 (n=5). (A) Schematic of treatment regimen (B) Measurements of mice body weight in MC (n =5) and MC + GI (n=5) groups. (C) Percentage of weight change after CPT-11 treatment in MC (n =5) and MC + GI (n=5) groups. (D) Representative (n=5/treatment group) H&E-stained section of mouse ileum. (E) Histological score of ileal sections in MC (n =5) and MC + GI (n=5) groups. (F) SN-38 concentration in the small intestine of MC (n =5) and MC + GI (n=5) groups. (G) SN-38G concentration in the small intestine of MC (n =5) and MC + GI (n=5) groups (H) The ratio of SN-38 to SN-38G in the small intestine of MC (n =5) and MC + GI (n=5) groups. Data were analyzed by student t test with data represented as the mean ± SD. P < 0.05 used for statistical significance.

Fig. 6. Loperamide reduces SN-38 concentration in plasma and increases SN-38 conversion in small intestine.

Fig. 6

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(A) CPT-11 and loperamide treatment regimen. (B) Representative H&E-stained section of mouse ileum with n=8/treatment group. (C-E) Histological score, weight, and % weight change among groups were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test, with data represented as the mean ± SD. (C) Histological score of ileal sections (n=8/treatment group). (D) Body weight of each group (n=8/treatment group) (E) Percentage of weight change after CPT-11 treatment (n=8/treatment group). (F-K) Plasma or ileal concentrations of CPT-11 and metabolites in CPT-11 (n=8) or Lop + CPT-11 (n=8) treated animals were analyzed by student t test. Animals were sacrificed three hours following CPT-11 (75 mg/kg) injection. Data were analyzed by student t test, with data represented as the mean ± SD. (F) Plasma concentration of CPT-11 in CPT-11 (n=8) or Lop + CPT-11 (n=8) treated animals three hours following CPT-11 injection. (G) Plasma concentration of SN-38 in CPT-11 (n=8) or Lop + CPT-11 (n=8) treated animals three hours following CPT-11 injection. (H) The ratio of plasma SN-38 to CPT-11 in CPT-11 (n=8) or Lop + CPT-11 (n=8) treated animals three hours following CPT-11 injection. (I) SN-38 concentration in the small intestine of CPT-11 (n=8) or Lop + CPT-11 (n=8) treated animals three hours following CPT-11 injection (J) SN-38G concentration in the small intestine of CPT-11 (n=8) or Lop + CPT-11 (n=8) treated animals three hours following CPT-11 injection (K) The ratio of SN-38 to SN-38G in the small intestine of CPT-11 (n=8) or Lop + CPT-11 (n=8) treated animals three hours following CPT-11 injection. P < 0.05 used for statistical significance. Lop, loperamide.

Fig. 4. Depletion of gut microbiota using antibiotics reverses morphine’s effects on CPT-11 metabolism.

Fig. 4

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WC: water+ CPT-11, AC: antibiotics+CPT-11, WMC: water+ morphine +CPT-11, AMC: antibiotics + morphine+ CPT-11. (A) Schematic of treatment regimen (B-D) Weight, % weight change, and histological score among WC (n=8), AC (n=8), WMC (n=8), and AMC (n=8) groups were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test, with data represented as the mean ± SD. (B) Measurements of mice body weight in WC (n=8), AC (n=8), WMC (n=8), and AMC (n=8) groups. (C) The percentage of weight change after CPT-11 treatment in WC (n=8), AC (n=8), WMC (n=8), and AMC (n=8) groups. (D) Representative (n=8/treatment group) H&E-stained section of mouse ileum. (E) Histological score of ileal sections in WC (n=8), AC (n=8), WMC (n=8), and AMC (n=8) groups (F-H) Ileal concentration of SN-38, SN-38G and SN-38/SN-38G in animals sacrificed three hours following CPT-11 (75 mg/kg) injection. Ileal concentrations in WC (n=8), AC (n=8), WMC (n=8), and AMC (n=8)-treated groups were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test, with data represented as the mean ± SD. (F) SN-38 concentration in the small intestine of WC (n=8), AC (n=8), WMC (n=8), and AMC (n=8) groups. (G) SN-38G concentration in the small intestine of WC (n=8), AC (n=8), WMC (n=8), and AMC (n=8) groups (H) The ratio of SN-38 to SN-38G in the small intestine of WC (n=8), AC (n=8), WMC (n=8), and AMC (n=8) groups. P < 0.05 used for statistical significance.

Histological Evaluation and TUNEL Assay:

Hematoxylin and eosin (H&E) staining was performed by The Division of Comparative Pathology. Each section was blindly examined and scored using a histological scoring system, previously described (Kaczmarek et al., 2012): To assess the damage induced by intestinal inflammation, the villi were scored (ranging from 0 to 12 points) based on epithelial morphology, villus shape, retracted stroma, infiltration, and crypt shape. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay was performed using Click-iT® Plus TUNEL assay kit (Invitrogen™) following the manufacture’s protocol.

Real-time PCR:

Total RNA from intestine and liver was extracted using TRIzol (Invitrogen). cDNA was synthesized using the M-MLV Reverse Transcription kit (Promega) following manufacturer’s protocol. Primers for IL-6, IL-1β, and rpL32A were purchased from Invitrogen. Quantitative real-time polymerase chain reaction was performed using LightCycler® 480 SYBR Green I Master (Roche). rpL32A was used to normalize values. The results were analyzed by the relative quantity (ΔΔCt) method. Primer sequences: IL-6: 5’-CCGGAGAGGAGACTTCACAG-3’, 5’-TCCACGATTTCCCAGAGAAC-3’; IL-1β: 5’-GGCAGGCAGTATCACTCATT-3’,5’-AAGGTGCTCATGTCCTCATC-3’; rpL32A:5’- GCTGGAGGTGCTGCTGATGT-3’, 5’- ACTCTGATGGCCAGCTGTGC-3’.

Pharmacokinetic studies:

On Day 8, after the described co-administered experimental treatments, mice were injected with CPT-11 (timepoint 0). Thereafter, each mouse was restrained by scruffing, and the facial vein was punctured with a Blood Lancet with a penetration depth of 3 mm (Assistent®, Germany) to collect blood 10, 20, 30, and 60 min after CPT-11 injection in time course studies. After centrifugation of blood, plasma was isolated and frozen at −20 °C for CPT-11, SN-38, and SN-38G analysis in plasma. Determination of CPT-11, SN-38 and SN-38G in the intestine (ileal or large intestinal contents) or plasma (in loperamide-treated animals) was conducted three hours post CPT-11 and co-administered treatments, after animals were sacrificed. After centrifugation of intestinal contents, supernatants were isolated and frozen at −20 °C until LC-MS analysis.

Sample preparation and LC-MS analysis:

Intestinal contents were suspended in ten volumes (v/w) of 50% acetonitrile, and extracted by vortexing and sonication for 10 min. The suspension was centrifuged at 18,000 × g for 10 minutes to separate supernatant and precipitate. Serum samples were mixed with 9 volumes of 66% aqueous acetonitrile (ACN), and then centrifuged at 18,000 × g for 10 min to obtain the supernatants. All sample extracts were transferred into sample vials for LC-MS analysis. A 5 μL aliquot of sample extract was injected into an AcquityTM UPLC system (Waters, Milford, MA). For CPT-11, SN-38, and SN-38G determination, LC separation was achieved in a BEH C18 column by mobile phases of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in ACN (B). Gradient of mobile phases ranged from 99.5% A to 5% A and back to 99.5% A over the 10 min run. Flow rate was 0.5 mL/min. LC eluent was introduced into an SYNAPT QTOF mass spectrometer (Waters) for accurate mass measurement and ion counting. Capillary voltage and cone voltage for electrospray ionization (ESI) was maintained at 3 kV and 30 V for positive-mode detection. Source temperature and desolvation temperature were set at 120°C and 350°C, respectively. Nitrogen was used as both cone gas (50 L/h) and desolvation gas (600 L/h), and argon as collision gas. For accurate mass measurement, the mass spectrometer was calibrated with sodium formate solution (range m/z 50–1000) and monitored by real-time intermittent injection of the lock mass leucine enkephalin ([M+H]+ = 556.2771 m/z). Mass chromatograms and mass spectral data were acquired and processed by MassLynxTM software (Waters) in centroided format. Additional structural information was obtained by tandem MS (MS/MS) fragmentation with collision energies ranging from 15 to 40 eV. The quantities of CPT-11, SN-38, and SN-38G were determined by calculating the ratio between the peak area of individual compound and the peak area of internal standard and then fitting with a standard curve using the QuanLynxTM software (Waters). CPT-11, SN-38, and SN-38G concentrations were normalized to the weight of intestinal contents in each mouse.

Imaging for intestinal βG activity:

Fluorescein di-β-D-glucuronide (FDGlcU) was reconstituted in water. On the day of imaging, mice were gavaged with 50 μL of FDGlcU (7.3 μmol/kg). After four hours, intestines were obtained and measured by the IVIS® imaging system (Chen et al., 2017).

Intestinal Transit Time:

8-week-old C57BL/6 (WT) mice were fasted for 6 hours and subsequently, gavaged with 70-kDa fluorescein isothiocyanate–dextran (750 mg/kg) or PBS. Intestinal concentrations of 70-kDa FITC dextran was determined 0, 15, 40 min after gavage. Small intestine was cut evenly into 3 segments (corresponding to duodenum, jejunum, ileum) and liberated luminal contents were homogenized for 45 sec; Tissue and coarse particles were removed by centrifugation (300× g, 3 min), as described previously (Woting and Blaut, 2018). Fluorescence was measured spectrophotometrically (Infinite M200 PRO, Tecan, Crailsheim, Germany) in 96-well plates (excitation: 485 nm, emission: 528 nm). FITC dextran concentrations were calculated with standard concentrations prepared in PBS ranging from 0 to 1250 μg/mL 70-kDa FITC dextran. The fluorescence signal of luminal 70-kDa FITC dextran in each segment was related to the sum of the fluorescence signals in all segments of the gastrointestinal tract.

16S rRNA gene sequencing:

DNA was extracted from small intestinal contents using DNeasy PowerSoil Pro Kit (Qiagen, Maryland, USA). Sequencing was performed by the University of Minnesota Genomics Center. The hypervariable regions V4 region of 16S rRNA gene was PCR amplified using the forward primer 515F (GTGCCAFCMGCCGCGGTAA), reverse primer 806R (GGACTACHVGGGTWTCTAAT). The amplicons were sequenced with the Illumina MiSeq v.3 platform, generating 300-bp paired-end reads.

Data and Statistical Analysis:

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). Statistical analyses were performed with GraphPad 9.3.1 software (GraphPad Software Inc., San Diego, CA, USA). Data are presented as means ± SD, with data points from individual animals (n). Statistical analysis was undertaken using individual replicate values, when sample sizes were at least n = 5. One-way analysis of variance was used for statistical evaluation of differences between groups (n>2) with Bonferroni’s post hoc. Analysis of variance for repeated measures with Tukey’s post hoc test was used for temporal comparisons in any given group. Post-hoc tests were only run if F achieved the necessary level of statistical significance (p <0.05). Student’s t-test was used wherever appropriate (two experimental groups). Fold change of IL-6 or IL-1β was expressed by normalizing to that in saline-treated mice. All P values were two-tailed. A P value <0.05 was considered statistically significant.

For bioinformatic analysis, sequence quality control and feature table construction was performed with the DADA2 package in R (Callahan et al., 2016). QIIME2 pipeline was used for diversity analyses (Bolyen et al., 2019) and Greengenes reference database (13_8) were used for taxonomic assignment (McDonald et al., 2012). LDA Effect Size (LEfSe) was used to detect differentially enriched taxa across groups (Segata et al., 2011). The threshold on the logarithmic LDA score for discriminative features was set to 2. For statistical analysis of microbiome data, Kruskal-Wallis test was used to detect if α diversity differed across treatments. Permutational multivariate analysis of variance (PERMANOVA) was used to detect if β diversity differed across treatments. Benjamini-Hochberg method was used for controlling false discovery rate (q value). For other experiments, Student t test or ANOVA followed by Bonferroni correction was used (GraphPad Prism). P < 0.05 was considered to be statistically significant.

Materials:

Morphine sulfate was obtained from National Institutes of Health [NIH]/National Institute on Drug Abuse [NIDA] (Bethesda, MD). Loperamide and 70 kDa FITC-Dextran were purchased from Sigma Aldrich (St. Louis, MO, USA). Click-iT® Plus TUNEL assay kit (Invitrogen™) and FDGlcU were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The β-Glucuronidase inhibitor 1-((6,8-Dimethyl-2-oxo-1,2-dihydroquinolin-3-yl)methyl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea (Calbiochem®), referred to as Inh-1 (Wallace et al., 2010; Saitta et al., 2014), was purchased from MilliporeSigma (Burlington, MA, USA). Irinotecan hydrochloride (CPT-11) was purchased from TCI America (Portland, OR, USA) and Thermo Fisher Scientific (Waltham, MA, USA). Clonidine hydrochloride was purchased from VWR (Radnor, Pennsylvania, USA).

Nomenclature of targets and ligands:

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22 (Alexander et al., 2021b, 2021a).

Results

Morphine treatment exacerbates CPT-11-induced GI toxicity.

To determine the effects of morphine and CPT-11 co-administration on GI toxicity, small intestinal (ileal) and large intestinal samples were collected for histological analysis in saline or morphine-treated mice co-administered CPT-11 (Fig. 1A). Histological examination indicated that co-administration of CPT-11 and morphine induced severe epithelial damage and infiltration of immune cells in the ileum (Fig. 1B). Quantitative analysis of histological scores showed that morphine potentiated CPT-11-induced tissue damage in the ileum (Fig. 1C). In contrast, less epithelial disruption was observed in the large intestine of CPT-11-treated mice, and morphine treatment showed no effects on CPT-11-induced tissue damage (Sup. Fig. 1). Small intestinal epithelial damage was further studied by evaluation of TUNEL-stained sections (Fig. 1 B and D). Notably, morphine and CPT-11 co-administration induced a 2-fold increase in apoptotic cell number compared to CPT-11 alone (Fig. 1D). PCR analysis also showed that CPT-11-induced-upregulation of pro-inflammatory cytokines IL-6 and IL-1β was significantly exacerbated by morphine treatment (Fig. 1 E and F); morphine and CPT-11 co-administration induced a 3.5-fold increase in IL-6 and a 2.4-fold increase in IL-1β compared to CPT-11 alone. Collectively, these results indicate that morphine treatment exacerbates CPT-11-induced GI toxicity.

Fig. 1. Morphine treatment enhances CPT-11-induced GI toxicity.

Fig. 1

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(A) Schematic of morphine and CPT-11 treatment regimen. Animals received a.m. doses of respective treatments before being sacrificed on Day 8. (B) Representative H&E- and TUNEL- stained section of mouse ileum from 8 mice/treatment group (C) Histological score of small intestinal (ileal) sections in saline (n=8), morphine (n=8), CPT-11 (n=8), and morphine +CPT-11 (n=8) treated mice. (D) Number of TUNEL positive cells in ileal sections from saline (n=6), morphine (n=6), CPT-11 (n=16), and morphine + CPT-11 (n=16) treated mice. (E) IL-6 mRNA expression in ileum from saline (n=8), morphine (n=8), CPT-11 (n=7), and morphine + CPT-11 (n=7) treated mice. (F) IL-1β mRNA expression in ileum from saline (n=8), morphine (n=8), CPT-11 (n=7), and morphine + CPT-11 (n=7) treated mice. (G) Representative H&E-stained section of mouse ileum from 5 mice/treatment group (H) Histological score of ileal sections from saline (n=5), morphine (n=5), clonidine (n=5), CPT-11 (n=5), morphine + CPT-11 (n=5), and clonidine + CPT-11 (n=5) treated mice. All data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test, with data represented as the mean ± SD. P < 0.05 used for statistical significance.

Morphine exacerbation of CPT-11-induced GI toxicity is distinct from that induced by prolonged GI transit

It is well known that morphine prolongs GI transit, resulting in constipation. As such, it is plausible that morphine aggravation of CPT-11-induced GI toxicity may be due to its effects on gut transit time and parallel the effects of constipation. To address this, morphine, saline, or clonidine-treated mice were gavaged with the impermeable 70 kDa FITC-Dextran to monitor gut transit time (Fig. 1A). As expected, both clonidine, an adrenergic 2-receptor agonist, and morphine treatment significantly prolonged gut transit time compared to saline-treated mice, with no significant difference in transit time between morphine and clonidine-treated mice (Sup. Fig. 2).

To further probe whether increased gut transit time may influence the degree of CPT-11-induced GI toxicity, ileal samples from morphine, clonidine, or saline-treated mice co-administered CPT-11 were collected for histological analysis. Consistent with our previous findings, histological examination indicated that the combination of CPT-11 and morphine induced severe epithelial damage and infiltration of immune cells in the small intestines, compared to saline and clonidine + CPT-11 treated groups (Fig. 1G). Additionally, quantitative analysis of histological scores showed that morphine significantly potentiated CPT-11-induced tissue damage in the small intestines, whereas damage induced by mice co-administered clonidine or saline were not statistically different from each other (Fig. 1H). Collectively, these results indicate that morphine treatment exacerbates CPT-11-induced GI toxicity, and that this effect is distinct from that observed with prolonged GI transit.

Morphine treatment disrupts CPT-11 metabolism

CPT-11, as a prodrug, is hydrolyzed by carboxylesterase (CES), generating the active product, SN-38. SN-38 is then inactivated via UGT1A1-catalyzed glucuronidation to SN-38G, which is excreted into the intestinal lumen in bile. In the intestinal lumen, β-glucuronidase produced by gut bacteria can convert SN-38G to SN-38, with accumulation of SN-38 in intestines associated with GI toxicity (Fig. 2A). To determine if morphine enhances the toxicity of SN-38 in intestinal epithelial cells, intestinal organoids were cultured and treated with CPT-11 in the presence or absence of morphine (Sup. Fig. 3). The images of intestinal organoids showed that CPT-11 inhibited the growth of crypt stem cells. Interestingly, morphine did not show any direct effects on CPT-11-treated organoids (Sup. Fig. 3).

Fig. 2. Morphine treatment induces SN-38 accumulation in the small intestine.

Fig. 2

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(A) Metabolic pathway of CPT-11 (B-D) Ileal concentration of CPT-11 and metabolites in morphine and saline treated animals sacrificed three hours following CPT-11 (75 mg/kg) injection. Ileal concentrations in CPT-11 (n=9) or morphine + CPT-11 (n=7) groups were analyzed by student t test, with data represented as the mean ± SD. (B) Ileal concentration of SN-38 in CPT-11 (n=9) or morphine + CPT-11 (n=7) treatment groups. (C) Ileal concentration of SN-38G in CPT-11 (n=9) or morphine + CPT-11 (n=7) treatment groups. (D) The ratio of ileal SN-38G to SN-38 in CPT-11 (n=9) or morphine + CPT-11 (n=7) treatment groups. P < 0.05 for statistical significance.

To determine whether morphine influences the metabolism of CPT-11 to exacerbate GI toxicity, we examined the pharmacokinetic profile of CPT-11 in morphine or saline treated mice co-administered CPT-11. The intestinal concentration of CPT-11 metabolites SN-38 and SN-38G was analyzed three hours post CPT-11 (75 mg/kg) co-administration (Fig. 2B–D). Interestingly, a 3-fold elevation in SN-38 concentration was detected in the small intestine of morphine-treated mice (Fig. 2B), while the concentration of SN-38G was not significantly different in saline- or morphine-treated mice (Fig. 2C). The lower ratio of SN-38G to SN-38 in the small intestine of morphine-treated mice suggested that morphine treatment resulted in more conversion of SN-38G to SN-38 in the small intestine (Fig. 2D). In contrast, morphine didn’t show any effect on SN-38 concentration in the large intestine (Sup. Fig. 4). Together, these results suggest that reactivation of SN-38G by β-glucuronidase in the intestine is enhanced by morphine treatment. To determine whether morphine influences the pharmacokinetics of CPT-11 by modulating enzymes involved in CPT-11 metabolism, the expression levels of CES and UGT1A1 in liver and small intestines were determined by PCR analysis (Sup. Fig. 5). The results showed that CPT-11 downregulated the expression of liver CES1d and intestinal UGT1A1. However, morphine did not show any effect on expression of CES and UGT in the liver or small intestine.

CPT-11 and morphine co-administration results in altered gut microbiota in the small intestine.

To determine if morphine-induced SN-38 accumulation in the intestine is mediated by gut microbiota, small intestinal contents were collected for gut microbiome analysis based on Illumina sequencing of microbial 16S-rRNA genes. Analysis of weighted Unifrac distance showed that the microbiota of saline-, morphine-, CPT-11-, and morphine + CPT-11-treated mice clustered distinctly from each other (q<0.05 for all pairwise comparison) (Fig. 3A and Sup. Fig. 6). Additionally, morphine treatment significantly altered the microbiome in the presence (q<0.05) and absence of (q<0.05) CPT-11 co-administration. CPT-11 treatment alone also resulted in significant alterations to the microbiome (q<0.05) compared to saline-treated mice. We further show that morphine-induced alterations in the gut microbiota were significantly different from clonidine-induced alterations (Sup. Fig 7–9), garnering more support for other constipation-independent mechanisms. Analysis of weighted Unifrac distance showed that the microbiota from the saline-, morphine-, and clonidine-treated mice all clustered distinctly from each other (q<0.05) (Sup Fig 3A). In addition, the alpha diversity (measured by Faith phylogenic diversity) from clonidine-treated mice was not significantly different from saline, whereas the alpha diversity of morphine and saline-treated mice were significantly different from each other (p<0.05) (Sup. Fig. 7B). LEfSe analysis between morphine and clonidine treated groups additionally showed a long list of differentially enriched bacteria taxa both with and without CPT-11 co-administration (Sup. Fig 8–9). Together, these analyses suggest that CPT-11 and morphine co-administration result in altered gut microbiota in the small intestine, with morphine-induced gut microbiota alterations distinct from those induced in a clonidine model of constipation.

Fig. 3. Morphine treatment modulates gut microbiota in the small intestine.

Fig. 3

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(A) Principal coordinate analysis (PCoA) of samples using the weighted UniFrac distances show distinct clustering (q<0.05 for all pairwise comparison) in CPT-11 (n=7), morphine (n=6), morphine + CPT-11 (n=7), and saline (n=6) treatment groups. (B) LEfSe (Linear Discriminant Analysis Effect Size) analysis of CPT-11 (n=7) and morphine + CPT-11 (n=7) treated mice show that morphine induced enrichment of Clostridium and Ruminococcus in CPT-11-treated mice. (C) Representative images from small intestine of CPT-11 (n=6) or morphine+ CPT-11 (n=6) treated mice gavaged with FDGlcU (D) Fluorescence intensity of CPT-11 (n=6), or morphine+ CPT-11 (n=6) treated mice gavaged with FDGlcU. Student t test was used to evaluate significant differences in fluorescence intensity between CPT-11 and morphine + CPT-11 treatment groups, with data represented as mean ± SD. All other data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test. P < 0.05 used for statistical significance.

Additionally, LEfSe analysis of bacterial composition at the genus level indicated that morphine treatment induced the enrichment of Clostridium and Ruminococcus in CPT-11-treated animals (Fig. 3B and Sup. Fig. 10). Among the diversity of gut microbes encoding glucuronidase enzymes, Clostridium and Ruminococcus are among some of the resources of β-glucuronidase in the intestine (Pollet et al., 2017; Creekmore et al., 2019). To confirm that β-glucuronidase is up-regulated by morphine in CPT-11-treated mice, mice were gavaged with a fluorescent probe, fluorescein di-β-D-glucuronide (FDGlcU). In this assay, hydrolysis of FDGlcU by β-glucuronidase generates a fluorescent signal. Accordingly, small intestines were imaged with an IVIS® spectrum optical imaging system to detect the intensity of the fluorescence. Compared with CPT-11 treated mice, fluorescence intensity showed a significant 2.3- fold increase in mice co-administered CPT-11 and morphine (Fig. 3D), indicating increased activity of β-glucuronidase. This data further provides evidence that alongside altering the small intestinal microbiota, co-administration of morphine and CPT-11 increases β-glucuronidase activity in the small intestine.

Depletion of gut microbiota using antibiotics reverses morphine’s effects on CPT-11 metabolism.

To confirm the role of gut microbiota in the metabolism of CPT-11, wild type mice were administered antibiotics to deplete the gut microbiota and were subsequently treated with morphine and CPT-11. Antibiotics treatment was maintained throughout the duration of the experiment (Fig. 4A). Results from these studies show that gut bacteria depletion markedly reduces weight loss induced by CPT-11 and CPT-11 + morphine (Fig. 4B and C). While morphine + CPT-11-treated animals showed an 8.0% reduction in weight, pretreatment with antibiotics only resulted in 1.48% weight loss in these animals.

Histological analysis of intestinal sections showed that antibiotics prevent CPT-11-induced small intestinal tissue damage (Fig. 4D and E), which is consistent with lower SN-38 concentrations observed in the small intestines of antibiotics-treated mice. Interestingly, SN-38 formation was significantly blocked by gut bacteria depletion (Fig. 4 F to H). In the absence of antibiotics, morphine + CPT-11-treated animals showed a 2.4- fold increase in SN-38 concentration in the small intestine compared to CPT-11 alone treated animals. However, pre-treatment with antibiotics in morphine +CPT-11-treated animals resulted in a 90% reduction in SN-38 concentration compared to CPT-11 treatment alone. This suggests that the gut microbiome plays a significant role in CPT-11 metabolism.

Pharmacological inhibition of bacterial β-glucuronidase activity in mice attenuates GI toxicity induced by CPT-11 and morphine co-administration.

To further confirm the role of bacterial β-glucuronidase in GI toxicity induced by CPT-11 and morphine co-administration, mice were gavaged with the β-glucuronidase inhibitor Inh-1 prior to CPT-11 treatment (Fig. 5A). While morphine + CPT-11-treatment resulted in 10.3% weight loss, co-administration of Inh-1 significantly decreased weight loss induced by morphine+ CPT-11 to only 5.9% (Fig. 5B and C). Inh-1 treatment also rescued the intestinal epithelial damage observed with morphine and CPT-11 (Fig. 5D and E). Quantification of SN-38 in the small intestine further confirmed that inhibition of β-glucuronidase significantly blocks conversion of SN-38G to SN-38 (Fig. 5 F to H). SN-38 concentration in the small intestine was reduced by 86.3% with Inh-1 co-administration, while the concentration of SN-38G was not significantly different from morphine + CPT-11 treated animals.

Loperamide exerts similar effects on metabolism of CPT-11 as morphine

To determine whether other μ-receptor binding opioids exert similar effects on CPT-11 metabolism, mice were administered oral loperamide in the context of CPT-11 treatment (Fig. 6A). Loperamide, which is used to treat chemotherapy-related diarrhea, is an opioid that acts on the μ-opioid receptors in the myenteric plexus. Pharmacokinetic analysis of CPT-11 indicated that oral loperamide had similar effects on CPT-11 metabolism as morphine. Oral loperamide inhibited the conversion of CPT-11 to SN-38 (Fig. 6F–H), resulting in a 46% decrease in plasma SN-38 concentration (Fig. 6G), which was consistent with our in vitro study using human liver microsome (Sup. Fig. 11).

Interestingly, the effects of loperamide were not sufficient to induce SN-38 or SN-38G accumulation in the small intestine (Fig. 6I–K). Consistent with the lowered SN-38 plasma concentration relative to morphine, loperamide did not significantly exacerbate small intestinal tissue damage (Fig. 6 B and C) and weight loss (Fig. 6D and E). Gut microbiome analysis further showed that loperamide treatment modulates the small intestinal bacterial composition (Sup. Fig. 12A and B, 13), though effects were not as significant as those from morphine treatment. Loperamide induced enrichment of select β-glucuronidase-producing bacteria such as Clostridium but showed no effect on Ruminococcus (Sup. Fig. 12C, 13).

Morphine treatment compromises the anti-tumor efficacy of CPT-11

To determine the effects of morphine on the anti-tumor efficacy of CPT-11, MC38 cells (murine colon adenocarcinoma cells) were subcutaneously injected into wild-type C57BL/6 mice. The anti-cancer efficacy of CPT-11 was compared in morphine or saline-treated mice that were co-administered CPT-11 (Fig. 7A). Both morphine and CPT-11 induced weight loss, with morphine treatment exacerbating CPT-11-induced weight loss (Fig. 7B/C). CPT-11 alone significantly reduced tumor sizes by 41.6% compared with saline controls (Fig. 7D). However, morphine co-administration attenuated the anti-tumor efficacy of CPT-11, with no significant differences in tumor weight observed between morphine + CPT-11 and saline-treated animals (Fig. 7D and Sup. Fig.14).

Fig. 7. Morphine treatment compromises anti-tumor efficacy of CPT-11.

Fig. 7

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(A) Schematic of morphine and CPT-11 treatment regimen. (B-D) Weight, % weight change, and tumor weight among saline (n=8) morphine (n=8), CPT-11 (n=8), and morphine + CPT-11 (n=8) groups were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons test, with data represented as the mean ± SD. (B) Measurements of mice body weight in each group (n=8/treatment group) for study duration. (C) Percentage of weight change following CPT-11 treatment (n=8/treatment group). (D) Measurements of tumor weight in each group (n=8/treatment group). (E-H) Plasma concentration of CPT-11 and metabolites immediately following CPT-11 (75 mg/kg) injection and morphine or saline co-administration (time 0). Plasma concentrations assessed at 10, 20, 30, and 60 minutes in CPT-11 (n=5) or morphine + CPT-11 (n=5) groups were analyzed by student t test, with data represented as the mean ± SD. (E) Plasma concentration of CPT-11 following CPT-11 injection (n=5/treatment group) (F) Plasma concentration of SN-38 following CPT-11 injection (n=5/treatment group). (G) Plasma concentration of SN-38G following CPT-11 injection (n=5/treatment group). (H) The ratio of SN-38 to CPT-11 in plasma following CPT-11 injection (n=5/treatment group). P < 0.05 for statistical significance.

To determine whether morphine treatment compromises the anti-tumor efficacy of CPT-11 by influencing the metabolism of CPT-11, we examined the plasma pharmacokinetic profile of CPT-11 in mice co-administered saline or morphine. The serum concentration of CPT-11, SN-38, and SN-38G at 10-, 20-, 30-, and 60-minutes following CPT-11 injection was determined (Fig. 7E–H). Higher serum concentrations of CPT-11 and lower serum concentrations of SN-38 were observed in morphine-treated mice, indicating that morphine inhibits the conversion of CPT-11 to SN-38 (Fig. 7E–H). In vitro assays using human liver microsome also confirmed that morphine treatment inhibits the hydrolysis of CPT-11, resulting in less SN-38 formation (Sup. Fig. 11). Interestingly, as statistically significant differences in SN-38 plasma levels were observed as soon as 10 minutes post drug administration (Fig. 7F), this may suggest fast absorption of CPT-11 which limits the potential direct effect of microbiota metabolism.

Collectively, these results indicate that morphine treatment leads to reduced conversion of CPT-11 to its active form SN-38, which compromises the anti-tumor efficacy of CPT-11. Additionally, our data suggests that morphine-induced disruption of CPT-11’s anti-cancer activity may be microbiome-independent, though this needs to be directly examined.

Discussion

Here, we demonstrate that morphine treatment exacerbates CPT-11-induced gut toxicity by modulating intestinal microbiota. To our knowledge, this is the first study which investigates the interactions between prescription opioids and the chemotherapeutic agent CPT-11. This study has clear clinical implications: prescription opioids are the most commonly used drugs in the management of cancer pain and chemotherapy-induced diarrhea (Bhatt et al., 2020). Thus, understanding how CPT-11 and co-administered opioids influence GI toxicity and anti-tumor efficacy is of great importance. The gut microbiome, in particular, has been shown to play a crucial role in the development of GI toxicity in CPT-11-treated patients (Bernareggi, 2001; Cai et al., 2010; Wallace et al., 2010). Parvez et al reported that β-glucuronidase protein levels were correlated with SN-38 reactivation rate (Parvez et al., 2021). Our study also confirms the crucial role of microbial glucuronidase in CPT-11-induced GI toxicity: morphine treatment increased β-glucuronidase in the small intestine of CPT-11-treated mice, resulting in SN-38 accumulation, which exacerbated tissue damage. Conversely, morphine’s effects were blocked by pan-antibiotic treatment, further validating the role of gut microbiota in reactivation of toxic metabolites of CPT-11. Taken together, these results suggest that opioid treatment exacerbates CPT-11-induced GI toxicity via opioid-induced microbial dysbiosis. While we observed SN-38 accumulation in both the small and large intestine, which is consistent with the literature, morphine potentiation of CPT-11-induced GI toxicity was only observed in the small intestine (Fig 2, Sup. Fig 1, 4). Multiple reports have detailed the profound effects of opioids in inducing microbial dysbiosis in the small intestine in particular (Meng et al., 2013; Banerjee et al., 2016; Wang et al., 2018; Zhang et al., 2019), and CPT-11-induced intestinal mucositis in the ileum (Ikuno et al., 1995; Itagaki et al., 2005; Arifa et al., 2016), which may explain site-specific effects.

As opioids prolong GI transit time, and bacterial densities in the intestine are highly dependent on peristalsis, we alternatively considered the hypothesis that prolongation of intestinal transit time could alter the bacteria in the small intestine, which may be sufficient to exacerbate CPT-11-induced GI toxicity. Using clonidine to induce constipation, we show that while morphine and CPT-11 co-administration exacerbates GI toxicity, the effects of clonidine on CPT-11-induced GI toxicity were not significantly different from that in CPT-11 alone. However, further experiments are needed to directly rule out the potential confounding effect of opioids on GI transit with robust models of constipation that solely act on the GI tract. Still, our data provides evidence that co-administration of opioids and CPT-11 has distinct effects from clonidine, suggesting that opioid exacerbation of GI toxicity extends pasts its effects on GI transit time. For example, in addition to inducing SN-38 accumulation in the intestine, other mechanisms may also contribute to exacerbated GI toxicity in CPT-11-treated mice by morphine. Cario et al have reported that CPT-11-induced GI dysfunction and pain are mediated by TLR4-dependent mechanisms (Cario, 2016). TLR4 deletion in CPT-11-treated mice resulted in improvements in weight loss, intestinal tissue damage, intestinal permeability, glial activation in the lumbar spinal cord, and pain sensation. In our previous studies, we have also shown that morphine treatment can induce gut bacterial translocation and intestinal inflammation by up-regulating TLR4 expression levels in intestinal epithelial cells (Meng et al., 2013). We further have shown that morphine-induced gut dysbiosis further induced inflammation in the spinal cord, leading to compromised analgesic efficacy (Zhang et al., 2019). Therefore, it is plausible that additive effects of morphine and CPT-11 may lead to severe inflammation in both the intestine and spinal cord, resulting in exacerbated GI toxicity and abnormal pain sensation, which paradoxically requires more opioids for the management of pain and diarrhea.

We further show that glucuronidase inhibition by Inh-1 effectively protected morphine+CPT-11-treated mice against intestinal tissue damage, consistent with previous studies with CPT-11 and Inh-1 treatment (Wallace et al., 2010; Saitta et al., 2014). Although intestinal bacterial depletion by broad spectrum antibiotics can remove gut microbial glucuronidase, it also leads to disrupted intestinal homeostasis with subsequent negative consequences such as opportunistic infection or other autoimmune disorders (Willing et al., 2011). Thus, a growing number of studies propose pharmacological inhibition of gut bacterial β-glucuronidase by other mechanisms. Scientists have developed various compounds to inhibit glucuronidase and showed that these inhibitors alleviate CPT-11-induced GI toxicity without impairing its anti-tumor efficacy (Wallace et al., 2010; Saitta et al., 2014; Bhatt et al., 2020). Importantly, the results of our study further provide in vivo evidence to confirm that oral glucuronidase inhibitors may be a promising strategy to prevent CPT-11-induced GI toxicity, especially for patients on prescription opioids. While this study did not delineate how opioids may induce small intestinal dysbiosis to increase gut bacterial β-glucuronidase, future studies utilizing shotgun metagenomic sequencing will need to uncover the potential role of opioids in β-glucuronidase expression. Of note, morphine can be glucoronidated to morphine-3-glucuronide and morphine-6-glucuronide, which may serve as a substrate for gut microbial glucuronidases, increasing the abundance of these enzymes in the intestinal lumen, though this hypothesis remains to be tested.

In our investigations, we further tested the effects of loperamide, which is the first-line choice for the control of chemotherapy-induced diarrhea, on gut microbiota and CPT-11 metabolism (Regnard et al., 2011). Our results show that although four-day treatment with oral loperamide also modulated the gut microbiota in CPT-11-treated mice, these effects were less significant than treatment with morphine injections; oral loperamide did not induce SN-38 accumulation or exacerbate toxicity in the intestine, as seen with morphine. These results suggest that it may be safe to use low-dose and short-term loperamide to control CPT-11-induced diarrhea.

Thus, judicious use of opioids for pain management may be warranted in CPT-11-treated patients given the increased risk of severe GI adverse effects. While studies presented here show the profound role of opioids and CPT-11 on the gut microbiome, it still must be acknowledged that differences in the microbiome between humans and rodents may limit direct translation of these studies. However, major trends reflected in this data such as morphine-induced microbial dysbiosis (Jalodia et al., 2022) and gut microbiota-mediated accumulation of SN-38 in intestines following CPT-11 administration (Sparreboom et al., 1998) have been shown in humans, though differences in bacterial taxa with morphine or CPT-11 administration may change depending on human or rodent models utilized. Additionally, further investigations are warranted to determine whether opioid treatment interferes with the metabolism of other anti-cancer agents inactivated by UDP-glycosyltransferases (e.g., Regorafenib, Belinostat, Binimetinib, and Encorafenib) through similar mechanisms presented in this study (Allain et al., 2020; Ervin et al., 2020). Of interest, in parallel with CPT-11, it has been shown that reactivation of the inactive regorafenib-glucuronide to regorafenib by gut microbial β-glucuronidase enzymes in the gastrointestinal tract contributes to the chemotherapeutic agent regorafenib’s GI toxicity, which is inhibited with β-glucuronidase inhibition (Ervin et al., 2020). How opioid treatment affects the pharmacokinetics of other anticancer glucuronidated drugs that serve as a substrate for specific gut microbial glucuronidase enzymes will need to be elucidated.

In our studies, we also uncovered anti-cancer effects of CPT-11 in mice co-administered morphine that appear to be microbiome independent. We observed that both morphine and loperamide decreased SN-38 concentration in plasma and inhibited conversion of CPT-11 to SN-38 in human liver microsome. Our in vivo studies further show that morphine treatment compromises anti-tumor efficacy of CPT-11, which is consistent with a lower SN-38 concentration in plasma. However, statistically significant differences in SN-38 plasma levels were already observed 10 minutes after drug administration suggesting fast absorption of the compound, which limits the potential direct effect of microbiota metabolism, though this needs to be directly examined. Interestingly, CPT-11 didn’t affect the analgesic efficacy of morphine (Sup. Fig. 15). These results are consistent with previous reports showing that loperamide is an inhibitor of carboxylesterase (CES) (Quinney et al., 2005). This is an important observation since CES is responsible for activating various medications especially chemotherapeutic agents. For example, in addition to CPT-11, the chemotherapeutic agent capecitabine requires activation by CES to generate active 5-fluorouracil (Hatfield et al., 2013). Therefore, a comprehensive understanding of how opioids compromise the anti-tumor efficacy of different anticancer agents is necessary to devise alternate analgesic strategies for patients on chemotherapy. Additionally, pharmacokinetic data shown here also emphasize the temporal nature of the microbiota on CPT-11 drug metabolism. While we did not show a time course for intestinal kinetics of CPT-11 to reduce animals used for experimentation, future studies utilizing multiple timepoints to assess concentrations of intestinal CPT-11 and its metabolites will need to be conducted. Additionally, while our data shows that morphine significantly alters CPT-11 metabolism, CPT-11 did not appear to alter morphine metabolism (Sup. Fig. 16), though future studies should further be performed to better elucidate morphine and CPT-11 drug-drug interactions at different time points as well.

In summary, the present study demonstrates that morphine and loperamide treatment inhibits activation of CPT-11, resulting in compromised anti-tumor efficacy of CPT-11. Additionally, morphine treatment also exacerbates CPT-11-induced GI toxicity by modulating intestinal bacterial glucuronidase activity, suggesting judicious use of opioid therapy in cancer patients treated with CPT-11. Development of an efficient gut bacterial β-glucuronidase inhibitor may be a promising therapeutic intervention for prevention of severe GI toxicity induced by CPT-11 and other glucuronidated chemotherapeutics, especially for cancer patients who are using opioids for pain management.

Supplementary Material

supinfo1
supinfo2

Bullet point summary.

What is already known

  • Opioids are commonly used for the management of cancer-associated pain and chemotherapy (e.g., CPT-11)-induced diarrhea.

  • Whether opioids exacerbate CPT-11-induced-GI-toxicity via gut microbiota modulation or limit CPT-11’s anti-tumor efficacy is unknown.

What this study adds

  • Opioids exacerbate CPT-11-induced GI toxicity by modulating gut microbiota in the small intestine.

  • Opioids compromise the anti-tumor efficacy of CPT-11, which may be independent of the gut microbiome.

Clinical significance

  • Gut microbiota play a significant role in opioid and chemotherapeutic agent drug-drug interactions.

  • Inhibition of gut microbial glucuronidase may prevent CPT-11-induced GI toxicity in patients on opioids.

List of Hyperlinks for Crosschecking.

Acknowledgements:

National Institutes of Health, Grant/Award Numbers: F31DA053795, R01 DA050542, R01 DA047089, R01 DA043252 and R01 DA044582. Sylvester Comprehensive Cancer Center at the University of Miami, Tumor Biology Research Grant.

List of abbreviations:

CPT-11

Camptothecin-11

SN-38

7-ethyl-10-hydroxycamptothecin

SN-38G

SN-38 glucuronide

CES

carboxylesterase

UGT

uridine diphosphate (UDP) glucuronosyltransferase

UGT1A1

UDP-glucuronosyltransferase 1A1

Inh-1

1-((6,8-Dimethyl-2-oxo-1,2-dihydroquinolin-3-yl)methyl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea

FDGlcU

Fluorescein di-β-D-glucuronide

Footnotes

Conflict of interest statement: The authors declare no conflict of interest.

Ethics and Integrity Statements

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Natural Products Research, Design and Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

Data availability statement:

The data that support the findings of this study are openly available in NCBI BioProject at http://ncbi.nlm.nih.gov/bioproject, reference number PRJNA781203.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supinfo1
NIHMS1860761-supplement-supinfo1.pdf (163.9KB, pdf)
supinfo2
NIHMS1860761-supplement-supinfo2.pdf (3MB, pdf)

Data Availability Statement

The data that support the findings of this study are openly available in NCBI BioProject at http://ncbi.nlm.nih.gov/bioproject, reference number PRJNA781203.

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