Background
Cholelithiasis is a highly prevalent disease, particularly in developed countries, with incidence rates of 10–15% in the adult population, indicating that 20–25 million Americans have gallstones [
1]. Laparoscopic cholecystectomy is considered the cornerstone treatment for symptomatic cholelithiasis. However, clinicians are occasionally confronted with inoperable patients because of the operative risks or refusal of patients. To better cope with such situations, several topical gallstone-dissolving agents have been developed, which are administrated via the percutaneous transhepatic route.
Of all the gallstone-dissolving compounds, methyl tert-butyl ether (MTBE) is a representative solvent and the only such drug currently used. MTBE is an alkyl ether similar to diethyl ether, an anesthetic agent. Unlike the lower boiling point of diethyl ether (35 °C), MTBE has a relatively higher boiling point (55 °C); thus, it is liquid at body temperature [
2,
3]. Acceptable dissolubility of MTBE was validated by a variety of studies, including a large survey of 803 patients across 21 European hospitals that demonstrated a dissolution rate of 96.6% (724/804) with a low rate of toxic side effects and complications [
4]. However, the widespread use of MTBE is limited by its side effects, such as nausea, upper abdominal pain, duodenitis, mild-to-moderate anesthesia, and hemolysis [
5‐
14]. A substantial proportion of these side effects could be attributed to its relatively low boiling point and the resultant higher evaporation; the evaporation rate of MTBE is 8.0 (i.e., 8.0 times higher than that of standard
n-butyl acetate), and thus is classified as “fast evaporating”.
In this study, we first developed a gallstone-dissolving compound with an aromatic moiety, named 2-methoxy-6-methylpyridine (MMP). To date, various compounds have been used to dissolve gallstones, including MTBE, heparin, bile acid,
d-limonene, mono-octanoin, ethylenediamine tetraacetic acid (EDTA), and sodium hexametaphosphate, all of which are composed of aliphatic chains [
15,
16]. MTBE has an asymmetric chemical structure that contains an ether functional group. With an oxygen atom in between, one side of MTBE is the bulky tert-butyl group and the other side is a simple methyl group. In MMP, the bulky aliphatic tert-butyl group of MTBE is replaced with pyridine, an aromatic group. The compounds with an aromatic ring tend to have a relatively higher boiling point and lower vapor pressure [
17]. MMP, thus, has a relatively higher boiling point (156 °C) and evaporates less, raising the possibility of having lesser toxicities while maintaining similar dissolubility as MTBE. In this study, we intended to determine whether MMP could be an alternative to MTBE by comparing their gallstone dissolubility and toxicities.
Methods
Chemicals and reagents
MTBE was obtained from Sigma-Aldrich (St. Louis, MO). MMP was produced in the Korea Research Institute of Chemical Technology (KRICT, Daejeon, Republic of Korea).
Nuclear magnetic resonance spectroscopy
Nuclear magnetic resonance (NMR) was used to measure the chelation effect of the gallstone-dissolving compounds, MTBE and MMP. 1-h NMR spectra were recorded on Bruker INOVA 400 MHz NMR spectrometers (BRUKER, Fȁllanden, Switzerland) at 25 °C. Chemical shifts are reported in parts per million (ppm). Data for 1-h-NMR are reported as follows: chemical shift (δ ppm) (integration, multiplicity, and coupling constant [Hz]). Multiplicities are reported as follows: s = singlet, d = doublet, t = triplet, q = quartet, sep = septet, dd = doublet of doublets, and m = multiplet. The residual solvent peak was used as an internal reference.
Thermogravimetric analysis
The thermogravimetric analysis (TGA), differential thermal analysis (DTA), and derivative of TGA (DTG) curves were recorded for each solvent using Thermo plus EVO II TG 8120 series thermal analyzer (RIGAKU, Tokyo, Japan). All three thermo-curves, TG, DTG, and DTA, were recorded simultaneously within temperatures of 25–100 °C in an air atmosphere. The thermo-curves were recorded for heating rates of 5 °C/min. In all the thermo-curves, the sample mass initially weighed between 4 and 5 mg. The kinetic parameters were calculated using the data obtained from the recorded thermo-curves by employing non-mechanistic Kissinger evaluation method.
Determination of gallstone components
We used the following assay kits to determine the components of gallstones; Cholesterol Enzymatic Assay Kit (Xpressbio, Fredrick, MD) for cholesterol, Triglyceride Quantification Kit (Cell Biolabs, San Diego, CA) for triglycerides, Bilirubin Assay Kit (Cell Biolabs) for bilirubin, and Phosphate Colorimetric Assay Kit (Biovision, Mountain View, CA) for bilirubin. The gallstones were homogenized with the substances described in each assay kit and the supernatant was collected for assay. After incubation for a determined period at 37 °C in the assay buffer, the absorbance was measured using a microplate reader (model 680; Bio-Rad, Hercules, CA).
Measurement of in vitro gallstone dissolubility
We collected gallstones following cholecystectomy in patients with gallstones. The study was approved by the Ethics Committee of Daejeon St Mary’s hospital, the Catholic University of Korea (IRB code: DC09FZZZ0045). We first determined the cholesterol contents in the collected gallstones using Cholesterol Enzymatic Assay Kit (Xpressbio, Fredrick, MD). Based on the cholesterol content, the gallstones were categorized into cholesterol (cholesterol content > 70%), mixed (70–30%), and pigmented gallstones (< 30%). The gallstones were air-dried, weighed, and preserved in saline. Subsequently, they were matched for size, weight, and shape for preparation for the dissolubility measurements. The three types of gallstones, with a mean weight of 43 ± 4 mg, were placed in separate glass containers, to which 10 ml aliquots of MTBE or MMP were added; the aliquots were aspirated and replaced every hour. The glass containers were gently stirred at 50 rpm on the reactor (VS-8480SF; Vision Co., Daejeon, Republic of Korea) at 37 °C for 24 h. Gallstone dissolubility according to each solvent was determined by measuring the dry weights of the gallstones at three different intervals (4, 8, and 24 h). Specifically, in vitro gallstone dissolubility (%) of each solvent was defined as (the average weight of untreated gallstones − the average weight of gallstones after solvent treatment)/the average weights of untreated gallstones.
Cell viability assay
Cell viability of human gallbladder epithelial cells (hGBECs) was evaluated with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (water soluble tetrazolium salt; ST-1) assay using EZ-Cytox Cell Proliferation Assay kit (Itsbio, Seoul, Republic of Korea) according to the manufacturer’s instructions. Additionally, we performed another series of cell viability assay for the following cells using Cyto X cell viability assay kit (LPS solution Co., Daejeon, Republic of Korea); Vero (African green monkey kidney, ATCC#81-CCL), L929 (mouse fibroblast, ATCC #2148-CRL), NIH 3T3 (mouse embryonic fibroblast, ATCC #1658-CRL), and CHO-K1 (Chinese hamster ovary, ATCC #61-CCL) cells.
In vivo animal experiments
In vivo dissolubility study using a hamster model of gallstones
All animal studies were carried out in compliance with the guidelines of the Institute for Laboratory Animal Research in Korea. This animal study was approved by the Institutional Animal Care and Use Committee of the Clinical Research Institute at Daejeon St. Mary’s Hospital at the Catholic University of Korea (IRB No. CMCDJ-AP-2016-004). For in vivo validation of each solvent, we used 10-week-old female Syrian golden hamsters (
Mesocricetus auratus, Harlan Sprague–Dawley Indianapolis, IN). Hamsters in each group were fed a different diet as follows for 4 months: the hamsters in the control group (N = 10) were fed a normal diet, while those in group CG (the group with cholesterol gallstones, N = 50) were fed a general rodent diet with 0.5% cholesterol, and those in group PG (the group with pigmented gallstones, N = 50) were fed a diet rich in carbohydrates. The diet compositions of each group are shown in Additional file
1: Table S1. The body weights of the hamsters were measured weekly. After 4 months of diet, the hamsters with gallstones were identified using abdominal ultrasonography and selected for further experiments. We directly infused the control material (DMSO) and each solvent into the gallbladder of the hamsters with cholesterol (n = 40) and pigmented gallstones (n = 40), respectively, after laparotomy under general anesthesia. After laparotomy under general anesthesia, each solvent was infused into the gallbladder using a 30-gauge syringe; after complete aspiration of the bile, the gallbladder was cautiously filled with a similar volume (0.1 ml) of dimethyl sulfoxide (DMSO), MTBE, and MMP, respectively. After 24 h, the hamsters were euthanized, and the gallstones and several pieces of tissues (liver, kidney, and gallbladder) were collected for further evaluations. Gallstone dissolubility of each solvent was determined by comparing the weights of solvent-treated gallstones and control (DMSO)-treated gallstones. Specifically, in vivo gallstone dissolubility (%) of each solvent was defined as (the average weight of DMSO-treated gallstones − the average weight of gallstones after solvent treatment)/the average weights of DMSO-treated gallstones.
In vivo acute toxicity test in mice
Seven-week-old female Institute of Cancer Research (ICR) mice were purchased from Orient Bio, Inc. (Seongnam, Republic of Korea). The mice were allowed to acclimatize to their new environment for 7 days and were maintained in an environment with temperature 23 ± 3 °C, humidity 50 ± 10%, 12-h light–dark cycle with 150–300 lx, and ventilation at 10–20 times/h. For the acute toxicity study, the mice were divided into 3 groups (n = 7, respectively). After starvation for 24 h, the control group was given 5% DMSO, while the treatment groups received MTBE and MMP, respectively, at 2000 mg/kg prepared in a corn oil through oral gavage. The general behavior (skin, respiration, tremors, lethargy, or sleep), body weight, and mortality were monitored for 14 days.
Measurement of Zebrafish locomotor activity according to each solvent
Zebrafish were maintained under standard conditions as previously reported [
18‐
20]. To monitor the locomotor activity, larval zebrafish at 5 days post-fertilization were placed in individual wells of a 96-well plate with 100 μl embryonic medium. Then, 100 μl of MTBE and MMP were added to a final concentration of 1 mM, respectively. After the treatment, changes were monitored at 28 °C for 60 min using DanioVision and EthoVision 10 XT locomotion tracking software (Noldus, Netherlands).
Statistical analysis
All data were analyzed with SPSS 11.0 software (SPSS Inc., Chicago, IL), and are presented as mean ± standard deviation (SD). Statistical comparisons between the groups were determined using the Kruskal–Wallis test. P < 0.05 was considered statistically significant. All authors had access to the study and reviewed and approved the final manuscript.
Additional materials and methods
Additional and more detailed information regarding the experimental procedures are fully described in Additional file
2: Materials and methods.
Discussion
This is the first validation report of MMP, the gallstone-dissolving compound containing an aromatic moiety. To determine the dissolubility and toxicities of MMP, both in vitro and in vivo experiments were performed using human gallstones and an animal model of gallstones, respectively. Throughout the experiments, MMP demonstrated dissolubility similar to or better than that of MTBE towards both cholesterol and pigmented gallstones, while maintaining lesser toxicities. We believe that the chelate effect of MMP resulted in its improved dissolubility, while its higher boiling point (156 °C) resulted in fewer toxicities. We, thus, believe that MMP could be an attractive alternative to MTBE.
Until now, numerous investigators have developed, or applied, a variety of compounds in dissolving gallstones. The compounds include MTBE, heparin, bile acid, d-limonene, mono-octanoin, EDTA, and sodium hexametaphosphate, all of which are composed of aliphatic chains [
15]. Of these, MTBE is the only compound currently available. Although clinical studies have not reported critical toxicities of MTBE to date [
4,
24,
25], several studies call into question the safety of MTBE. After administration, MTBE could be absorbed into the duodenum, leading to somnolence, nausea or vomiting [
5‐
9,
11‐
14], it can also be systemically absorbed, resulting in hemolysis and reversible kidney injury [
26]. It was reported that inhalation of MTBE led to a statistically significant increase in kidney tumors and liver tumors in rats [
27]. Furthermore, MTBE has resulted in the development of cancers in many organs and tissues, which were similar to those caused by exposure to equal doses of carcinogens such as benzene, vinyl chloride, and 1,3-butadiene [
28]. Oral exposure to MTBE has been shown to cause dose-dependent, statistically significant increases in carcinomas such as lymphoma, leukemia, and Leydig cell carcinoma of the testes in rats [
29]. These dismal outcomes of MTBE motivated us to attempt to develop a novel gallstone-dissolving compound.
Basically, MMP is an MTBE analog with an aromatic moiety. MTBE is an asymmetric chemical compound with an ether functional group; one side of MTBE has a bulky tert-butyl group and the other side has a simple methyl group. MMP has a structure wherein the bulky aliphatic tert-butyl group of MTBE is replaced with an aromatic functional group while keeping the methyl group intact on the other side. The compounds with an aromatic ring have a relatively higher boiling point and lower vaporized pressure. MMP thus boils at a relatively higher temperature (156 °C) and, thus, evaporates less at room temperature, resulting in lesser toxicities while preserving the dissolubility of MTBE.
Our study suggests that the improved dissolubility of MMP is partly because it acts as a chelating ligand. Chelate is a complex compound consisting of a central metal atom and a large molecule (a ligand) that is attached to the metal atom in a cyclic manner. The chelate effect, thus, refers to the enhanced affinity of chelate ligands for a metal ion compared to the affinity of the non-chelating ligands. The chelate effect promotes gallstone dissolubility, as demonstrated by the enhanced dissolubility of calcium stones by EDTA, the representative chelating agent [
21]. MTBE does not have a chelating property because it has only one heteroatom with a lone pair of electrons. In contrast, MMP has two nearby heteroatoms with lone pair electrons, thus enabling to chelate the calcium component in gallstones. Additionally, the aromatic ring in MMP, named pyridine, has the advantage of being miscible with water and virtually all organic solvents, thereby facilitating gallstone dissolution. In the NMR test, we found that when the equivalent of Co(ClO
4)
2·6H
2O was increased to 1.0–2.0 equivalents relative to the solvents, MMP exhibited considerably higher chemical shift than did MTBE, suggesting the enhanced affinity by chelate effect between MMP and Co
2+. Our in vitro and in vivo studies demonstrated that MMP showed higher dissolubility than MTBE in both models of cholesterol and, more prominently, pigmented gallstones. Therefore, we believe that MMP potentiates the dissolubility of calcium-rich pigmented gallstones by chelating the calcium component, which needs to be validated in further studies.
MTBE or MMP are basically cholesterol solvents; therefore, they exhibit higher dissolubility for cholesterol gallstones than pigmented gallstones. In our in vitro study, whereas MTBE showed 65.7%, 54.2%, and 29.0% 24-h-dissolubility, MMP showed 88.2%, 61.0%, and 50.8% 24-h-dissolubility for cholesterol, mixed, and pigmented gallstones, respectively. The higher dissolubility for cholesterol gallstones is clinically preferable because most gallstones predominantly consist of cholesterol (> 85%) in the developed countries [
30,
31].
Currently, topical gallstone-dissolving agents are restrictively available for patients who refuse surgery or are at high risk of surgery, possibly due to the concerns for their serious toxicities as well as the wide recognition of laparoscopic cholecystectomy as a safe operation. In addition, it remains controversial whether the complete removal of gallbladder can be justified in patients with gallstones with a functioning gallbladder, because although it is minimal, surgery inevitably accompanies a certain degree of operative risks and occasionally leads to postcholecystectomy syndrome [
32]. Finally, although still controversial, numerous epidemiological investigations and meta-analyses indicate that cholecystectomy can be a risk factor for gastrointestinal cancers [
33‐
37]. The reason is frequently attributed to the deleterious effects of bile acid exposure; it acts like a carcinogen, in terms of enhancing DNA damage, stimulating mutation, inducing apoptosis in the short term, and ultimately promoting apoptosis resistance in the long term [
33]. Therefore, we believe that there are still indications for gallstone-dissolving solvents in lieu of laparoscopic cholecystectomy.
Several obstacles are placed in front of the popular use of gallstone dissolution therapy. The first concern is high incidence of stone recurrence. In a study of enrolling 803 patients with gallstones who had received contact litholysis using MTBE, stone recurrence rate was about 40% in solitary stones and about 70% in multiple stones over 5 years [
4]. Another challenge is to obtain the minimally invasive way of introducing the solvent into gallbladder. The last, but not least concern is safety because it is almost inevitable to have the proportion of solvent to be absorbed into duodenum or systemic circulation.
This study has several limitations. In the in vivo experiments using hamsters, although there was random assignment of each group, blind assessment of outcomes by investigators was not achieved. In addition, during experiments using hamsters, we could not compare the weights of gallstones before and after treatment because it was nearly impossible to measure the weights of gallstones before the treatment. Instead, in vivo gallstone dissolubility of each solvent was determined by comparing the weights of solvent-treated gallstones and control (DMSO)-treated gallstones.
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