1. may cause early death either directly from life-threatening

1. INTRODUCTION

Irinotecan (CPT-11, Camptosar®) is a semi-synthesized
water-soluble prodrug of 7-Ethyl-10-hydroxy-camptothecin (SN-38) derived from
camptothecin, a natural compound isolated from the bark and stem of Camptotheca acuminata.1 Irinotecan is
approved by the FDA as the first-line drug for the treatment of metastatic
colon cancer in combination with 5-FU/leucovorin and is also being actively
tested to treat different types of malignant such as lung, pancreatic, ovarian,
cervical, prostate, and gastrointestinal cancers. 2-6 The mechanism of action is that the active form SN-38
can inhibit topoisomerase I to interrupt DNA synthesis in cancer cells. 7

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

The
efficacy of irinotecan is promising; however, this drug could cause severe
diarrhea. There are two types of
diarrhea induced by irinotecan: early-onset and late-onset diarrhea. The
former, occurring within 24 hours of drug administration, can be effectively
controlled by atropine 8, 9 but the latter, occurring after 24 hours of
administration, may affect patients’ quality of life and may cause early death
either directly from life-threatening sequelae or indirectly from adjustments
in chemotherapy plan. 10, 11 The overall incidence of diarrhea ranges from 60% to
87%, including 20-30% of severe diarrhea (grade III and IV), which appears to
be dose-dependent. 12, 13 The median time to onset ranged from 5 to 11 days
after drug administration and the diarrhea duration last for 2 to 5 days depend
on the dosing schedule. 10, 14

Different
drugs and approaches have been tested or suggested for treatment or prevention
of irinotecan-induced diarrhea including controlling diet and fluids, using
antibiotics, probiotics, adsorbents, opiates (e.g., loperamide), somatostatin
analogue (e.g., octreotide), anti-inflammation agents (e.g., celecoxib) etc. 10, 15 So far, there are just three drugs recommended in
current guidelines: loperamide, deodorized tincture of opium and octreotide to
control the diarrhea symptoms. 16 However, a large portion of cancer
patients (about 10-15% patients) failed to respond to these treatments when
using irinotecan. 17, 18 Therefore, it is necessary to develop safe and
effective drugs to attenuate irinotecan-induced diarrhea.

The mechanisms underlying diarrhea
induced by irinotecan is not entirely understood. The complex etiology of
diarrhea seems to involve changes in the absorption of fluids and electrolytes,
intestinal motor dysfunction, and inflammation of the mucosal membranes lining
the gastrointestinal tract. 19, 20
It is believed that diarrhea is due to the damage of colonic mucosa caused by
the active compound SN-38.14, 20
and decreasing
the exposure of SN-38 in the colon could mitigate irinotecan-induced diarrhea. 21-26 For example, Horikawa et al,
reported that inhibiting biliary secretion of irinotecan and its metabolites
using probenecid could mitigate irinotecan-induced diarrhea in an animal model.27  Another example is that Chester et al reported
that in a clinical trial, irinotecan-induced diarrhea was attenuated by P-gp
inhibitor cyclosporine. 28 However, safety of these chemical inhibitors
is the major challenge. 29

Due
to low side effects, herbal medicines (e.g., PHY906, TJ-14) having been tested
to alleviate irinotecan-induced diarrhea.30-32 The encouraging results inspire us
to evaluate the possibility of XCHT in the attenuation of diarrhea induced by
irinotecan. XCHT
is a well-known Chinese traditional medicine primarily used for hepatic
protection. This herbal mixture contains 7 herbals including Bupleurum falcatum, Panax ginseng, Glycyrrhiza glabra, Zingiber officinale, Scutellaria baicalensis, Zizyphus jujube, and Pinellia
ternate. 33, 34 This famous formula was initially recorded in the
ancient medicinal book named Shanghanlun 2000 years ago.35 In addition, XCHT, (Sho-saiko-to
in Japanese), was introduced into Japan as an oriental classical medicine from
China approximately 1500 years ago, and it is manufactured in Japan as an
ethical drug on a modern industrial scale in which the quality of ingredients
is standardized with Good Manufacturing Practices (GMP) regulation.36 It is estimated that XCHT has been treat more than a
million of patients without any severe side effects. Therefore, XCHT could
highly possible to be used in cancer patient. The purpose of this paper is to determine
the impact of XCHT on the intestinal and systemic exposure of SN-38 and
investigate the mechanism.

2. Materials
and Methods

2.1 Chemicals and
Reagents

Irinotecan,
SN-38, camptothecin (CPT), uridine-5′-diphosphate-?,D-glucuronic acid ester (UDPGA), D-saccharic-1,4-lactone
monohydrate, magnesium chloride, hanks’ balanced salt solution (powder form)
and formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). recombinant
human UGT1A1was purchased from BD Biosciences (Woburn, MA, USA). Solid phase
extraction (C18) columns were purchased from J.T. Baker
(Phillipsburg, NJ, USA). Acetonitrile, methanol and water (LC-MS grade) were
purchased from EMD (Gibbstown, NJ, USA). Water was deionized by a Milli-Q water
purification system of Millipore (Bedford, MA, USA). Intravenous irinotecan
hydrochloride injection was purchased from Teva Pharmaceuticals (Pearl River,
NY, USA). All other materials (typically analytical grade or better) were used as
received.

2.2 Biosynthesis of
SN-38 glucuronides 

SN-38
glucuronide was biosynthesized using recombinant human UGT1A1 according to the
procedure reported by us previously.37 Briefly, SN-38 was incubated with UGT1A1 for 24 hours
in HBSS buffer, which was then extracted with methylene chloride twice to
remove the parent compound (i.e., SN-38). The aqueous layer was then purified
through solid phase extraction. The final SN-38G was purified using HPLC and
the purity was determined by UPLC (> 98%, based on chromatography peak in
UV, 210 and 254 nm).

2.3 Animals

Male Wistar rats (280 to 320 g) were purchased from
Harlan Laboratory (Indianapolis, IN). Animals were kept in an environmentally
controlled room (temperature: 25 ± 2°C, humidity: 50 ± 5%, 12 h dark-light
cycle) for at least 1 week before the experiments. The rats were fasted
overnight before the day of the experiment. The experimental procedures were approved by the
University of Houston’s Institutional Animal Care and Uses Committee (IACUC).

2.4 Intestinal
perfusion experiment

To determine the
biliary and intestinal secretion, we performed intestinal perfusion studies. The
experiment followed our previously procedures with minor modification. 38, 39
Briefly, irinotecan (5mg/kg/150 min in dextran) was infused through jugular
vein cannulation using a perfusion pump (model PHD 2000; Harvard
Apparatus Inc., Holliston, MA). A segment of jejunum was perfused with a HBSS buffer
(control group) or XCHT in HBSS (200 mg/ml) using another infusion pump at a
flow rate of 0.191 ml/min. After a 30-min washing period, which was
usually sufficient to achieve the steady-state, four samples were collected
from the outlet cannula periodically (every 30 min). Bile (~ 0.4 ml) samples were
collected from bile-duct cannulation at each time points. All the samples were stored at – 80°C until analysis. 

2.5 Pharmacokinetic
study

To determine the
impact of XCHT on the blood concentrations of irinotecan/SN-38/SN-38G, we
conducted PK studies. Irinotecan was administered at a dose of 5 mg/kg via
intravenous injection through the tail vein. XCHT was administrated through
oral gavage at 500 mg/kg. Animals were housed in metabolic cages. Blood samples
(~50 µL) were collected into heparinized tubes at 0, 15, 30, 60, 120, 240, 360,
480, and 1440 min by snipping the tails.
Feces were collected at 24 hours. All the samples were stored at – 80°C until analysis. 

2.6 Sample
Preparation

The bile samples (5 µL) were diluted 100 times by 50%
methanol and then 50 µL 100 nM CPT in 50% methanol-water was added to each
sample as internal standard. Perfusate samples (50 µL) were diluted with 50%
methanol for 2-fold and 50 µL of testosterone in 94% acetonitrile-6% acetic
acid was added as internal standard. Standard curve samples were prepared in
the same manner. The samples were vortexed, centrifuged for 15 min at 15,500
rpm for LC-MS/MS injection. The injection volume is 10 µL.   

The
plasma samples (20 µL) were spiked with 20 µL of internal standard (100 nM CPT
in 50% methanol) and vortexed for 1 min after extracted with
methanol-acetonitrile (1:1 v/v) solution. All solutions were vortexed and
centrifuged at 15,500 rpm for 15 min. The supernatant for each sample was transferred
to a new microcentrifuge tube and the solvent was removed under N2
flow. The residue was reconstituted in 80 µL of 50% methanol and centrifuged at
15,500 rpm for 15 min for LC-MS/MS injection. The injection volume is 10
µL.  

To
prepare fecal samples, the lyophilized feces (1.0 gram) was homogenized with 10
mL of potassium phosphate buffer (pH 7.4). After centrifuge (15,000 rpm, 4 ?C,
15 min), the supernatant (20 µL) was spiked with 20 µL of internal standard
(100 nM CPT in 50% methanol) and extracted with acetonitrile (720 µL). The
solvent was removed after centrifuge and the residue was re-constituted into 80
µL of 50% methanol for injection. The injection volume is 10 µL.

2.7 UPLC-MS/MS Analysis of irinotecan, SN-38
and SN-38G

The
UPLC system and condition were: Waters AcquityTM UPLC with a diode
array detector (DAD, Waters, Milford, MA, USA); Acquity UPLC BEH C18
Column (2.1 mm × 50 mm, 1.7 µm,); 0.1% of formic acid in water was used as mobile
phase A (MPA) and 100% acetonitrile was used as mobile phase B (MPB); gradient:
10% B ? 25% B (0 – 0.5 min), 25% B ? 40% B (0.5 – 1 min), 40% B (1 – 2.5 min),
40% B ? 10% B (2.5 – 4.5 min); flow rate, 0.4 ml/min; Column temperature was 60°C.

Mass
spectrometer was API 5500 Qtrap triple quadruple mass spectrometer coupled with
a TurboIonSprayTM (Applied Biosystem- MDS SCIEX, Framingham, MA,
USA). The system was operated in positive electrospray ionization (ESI) and
multiple reactions monitoring (MRM) scan mode. Data were acquired and processed
using Analyst®1.5.2 software (AB SCIEX). The instrument parameters were:
ion-spray voltage, 5.5 kV; source temperature, 500?C; curtain gas,
20 psi; gas 1, 20 psi; gas 2; 20 psi, collision gas, medium.

2.8 Statistical
Analysis

Student’s t-test was
used to analyze the data of irinotecan, SN-38, and SN-38G. The prior level of
significance was set at p