Differential effects of two phosphodiesterase 4 inhibitors against lipopolysaccharide-induced neuroinflammation in mice.

BACKGROUND: Several phosphodiesterase 4 (PDE4) inhibitors have emerged as potential therapeutics for central nervous system (CNS) diseases. This study investigated the pharmacological effects of two selective PDE4 inhibitors, roflumilast and zatolmilast, against lipopolysaccharide-induced neuroinflammation. RESULTS: In BV-2 cells, the PDE4 inhibitor roflumilast reduced the production of nitric oxide and tumor necrosis factor-α (TNF-α) by inhibiting NF-κB phosphorylation. Moreover, mice administered roflumilast had significantly reduced TNF-α, interleukin-1ß (IL-1ß), and IL-6 levels in plasma and brain tissues. By contrast, zatolmilast, a PDE4D inhibitor, showed no anti-neuroinflammatory effects in vitro or in vivo. Next, in vitro and in vivo pharmacokinetic studies of these compounds in the brain were performed. The apparent permeability coefficients of 3 µM roflumilast and zatolmilast were high (> 23 × 10-6 cm/s) and moderate (3.72-7.18 × 10-6 cm/s), respectively, and increased in a concentration-dependent manner in the MDR1-MDCK monolayer. The efflux ratios were < 1.92, suggesting that these compounds are not P-glycoprotein substrates. Following oral administration, both roflumilast and zatolmilast were slowly absorbed and eliminated, with time-to-peak drug concentrations of 2-2.3 h and terminal half-lives of 7-20 h. Assessment of their brain dispositions revealed the unbound brain-to-plasma partition coefficients of roflumilast and zatolmilast to be 0.17 and 0.18, respectively. CONCLUSIONS: These findings suggest that roflumilast, but not zatolmilast, has the potential for use as a therapeutic agent against neuroinflammatory diseases.

PredPS: Attention-based graph neural network for predicting stability of compounds in human plasma.

Stability of compounds in the human plasma is crucial for maintaining sufficient systemic drug exposure and considered an essential factor in the early stages of drug discovery and development. The rapid degradation of compounds in the plasma can result in poor in vivo efficacy. Currently, there are no open-source software programs for predicting human plasma stability. In this study, we developed an attention-based graph neural network, PredPS to predict the plasma stability of compounds in human plasma using in-house and open-source datasets. The PredPS outperformed the two machine learning and two deep learning algorithms that were used for comparison indicating its stability-predicting efficiency. PredPS achieved an area under the receiver operating characteristic curve of 90.1%, accuracy of 83.5%, sensitivity of 82.3%, and specificity of 84.6% when evaluated using 5-fold cross-validation. In the early stages of drug discovery, PredPS could be a helpful method for predicting the human plasma stability of compounds. Saving time and money can be accomplished by adopting an in silico-based plasma stability prediction model at the high-throughput screening stage. The source code for PredPS is available at https://bitbucket.org/krict-ai/predps and the PredPS web server is available at https://predps.netlify.app.

Pharmacokinetics and Tissue Distribution of 13C-Labeled Succinic Acid in Mice.

Succinic acid is widely used as a food additive, and its effects on sepsis, cancer, ataxia, and obesity were recently reported. Dietary drug exposure studies have been conducted to evaluate the in vivo efficacy of succinic acid, but limited pharmacokinetic information is available. Therefore, this study evaluated the pharmacokinetic profiles and tissue distribution of succinic acid following a single intravenous or oral dose. A surrogate analyte, succinic acid-13C4 (13C4SA), was administrated to distinguish endogenous and exogenous succinic acid. The concentration of 13C4SA was determined by a validated analytical method using mass spectrometry. After a 10 mg/kg intravenous dose, non-compartmental pharmacokinetic analysis in plasma illustrated that the clearance, volume of distribution, and terminal half-life of 13C4SA were 4574.5 mL/h/kg, 520.8 mL/kg, and 0.56 h, respectively. Oral 13C4SA was absorbed and distributed quickly (bioavailability, 1.5%) at a dose of 100 mg/kg. In addition, 13C4SA exposure was the highest in the liver, followed by brown adipose tissue, white adipose tissue, and the kidneys. This is the first report on the pharmacokinetics of succinic acid after a single dose in mice, and these results could provide a foundation for selecting dosing regimens for efficacy studies.

Anti-neuroinflammatory Effects and Brain Pharmacokinetic Properties of Selonsertib, an Apoptosis signal-regulating Kinase 1 Inhibitor, in mice.

Selonsertib is a first-in-class apoptosis signal-regulating kinase 1 (ASK1) inhibitor in clinical trials for treating NASH and diabetic kidney disease due to its anti-inflammatory and anti-fibrotic activities. In the present study, we investigated the anti-neuroinflammatory effects and brain pharmacokinetic properties of selonsertib. It inhibited inflammatory cytokines and NO production by suppressing phosphorylated ASK1 in the LPS-stimulated microglial cell line, BV2 cells. Consistent with the in vitro results, selonsertib attenuated plasma and brain TNF-α levels in the LPS-induced murine neuroinflammation model. In vitro and in vivo pharmacokinetic studies of selonsertib were conducted in support of central nervous system (CNS) drug discovery. In both Caco-2 and MDR-MDCK cells, selonsertib exhibited a high efflux ratio, showing that it is a P-gp substrate. Selonsertib was rapidly and effectively absorbed into the systemic circulation after oral treatment, with a Tmax of 0.5 h and oral bioavailability of 74%. In comparison with high systemic exposure with Cmax of 16.2 µg/ml and AUC of 64 µg·h/mL following oral dosing of 10 mg/kg, the brain disposition of selonsertib was limited, with Cmax of 0.08 µg/g and Kp value of 0.004. This study demonstrates that selonsertib can be a therapeutic agent for neuroinflammatory diseases.

IVIVC of Octreotide in PLGA-Glucose Microsphere Formulation, Sandostatin® LAR.

In vitro-in vivo correlation (IVIVC) analysis reveals a relationship between in vitro release and in vivo pharmacokinetic response of the drug of interest. Sandostatin LAR Depot (SLD) for endocrine tumors and acromegaly is a sustained-release formulation of octreotide, a cyclic oligomer of 8 amino acids, which prolongs therapeutic efficacy and enhances medication compliance of octreotide. Since the efficacy of SLD is dependent on the pharmacokinetic characteristics of octreotide released from a biodegradable matrix polymer, poly(lactide-co-glycolide)-glucose, of SLD, the IVIVC of SLD is critical for predicting an in vivo behavior of the octreotide. In this study, in vitro release of octreotide from SLD was investigated using the release test media each containing 0.02% or 0.5% surfactant and having different pH values of 7.4 and 5.5. In vivo pharmacokinetic profiles of SLD were determined by LC-MS/MS analysis of the systemic blood concentration of octreotide after the SLD injection to rodents. In IVIVC analysis, the Weibull model was adopted as a drug release model for biodegradable microsphere formulation. The IVIVC analyses revealed the in vitro release test condition of SLD with the highest IVIV correlation coefficient. By applying the in vitro release data to the model derived from the IVIVC analysis, pharmacokinetic parameters of SLD could be predicted with the prediction error of ± 10 ~ 15%. IVIVC analysis and pharmacokinetic prediction model of SLD in our study can be an efficient tool for the development of long-acting pharmaceutical dosage forms.

PEGylation improves the therapeutic potential of dimerized translationally controlled tumor protein blocking peptide in ovalbumin-induced mouse model of airway inflammation.

Dimerized translationally controlled tumor protein (dTCTP) initiates a variety of allergic responses in mouse models and that dTCTP-binding peptide 2 (dTBP2) attenuates the allergic inflammation by targeting dTCTP. However, the usefulness of peptide-based drugs is often limited due to their short half-lives, rapid degradation, and high levels of clearance after systemic administration. In this study, we chemically conjugated dTBP2 with 10 kDa polyethylene glycol (PEG) to improve its therapeutic potential. N-terminal mono-PEGylated dTBP2 (PEG-dTBP2) was characterized by in vitro bioactivity assay, pharmacokinetics study, and in vivo efficacy. When compared to the unmodified dTBP2, PEG-dTBP2 reduced proinflammatory cytokine IL-8 secretion in human bronchial cells by 10 to 15% and increased plasma half-life by approximately 2.5-fold in mice. This study specifically demonstrated that PEG-dTBP2 shows higher inhibitory action against ovalbumin (OVA)-induced airway inflammation in mice compared to dTBP2. Importantly, PEG-dTBP2, when administered once at 1 mg/kg, significantly reduced the migration of inflammatory cells and the levels of cytokines in the bronchoalveolar lavage fluids as well as OVA-specific IgE levels in serum. In addition, the degree of goblet cell hyperplasia and mucus secretion were significantly attenuated in the PEG-dTBP2 group compared with the control group. These results suggest that PEG-dTBP2 can be considered a potential candidate drug for regulating allergic inflammation.

Identification of New Non-BBB Permeable Tryptophan Hydroxylase Inhibitors for Treating Obesity and Fatty Liver Disease.

Serotonin (5-hydroxytryptophan) is a hormone that regulates emotions in the central nervous system. However, serotonin in the peripheral system is associated with obesity and fatty liver disease. Because serotonin cannot cross the blood-brain barrier (BBB), we focused on identifying new tryptophan hydroxylase type I (TPH1) inhibitors that act only in peripheral tissues for treating obesity and fatty liver disease without affecting the central nervous system. Structural optimization inspired by para-chlorophenylalanine (pCPA) resulted in the identification of a series of oxyphenylalanine and heterocyclic phenylalanine derivatives as TPH1 inhibitors. Among these compounds, compound 18i with an IC50 value of 37 nM was the most active in vitro. Additionally, compound 18i showed good liver microsomal stability and did not significantly inhibit CYP and Herg. Furthermore, this TPH1 inhibitor was able to actively interact with the peripheral system without penetrating the BBB. Compound 18i and its prodrug reduced body weight gain in mammals and decreased in vivo fat accumulation.

Synthesis and biological evaluation of tyrosine derivatives as peripheral 5HT2A receptor antagonists for nonalcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD), attributed to excessive fat accumulation in the liver, is reportedly prevalent worldwide. NAFLD is one of the leading causes of chronic liver disease, including non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatic cellular carcinoma (HCC). The peripheral roles of serotonin (5-hydroxytryptamine, 5HT) were found to regulate hepatic lipid metabolism. Among serotonin receptor subtypes, 5HT2A receptor is known to regulate hepatic lipid metabolism. Hepatic lipid accumulation and hepatic triglyceride (TG) were reduced in liver-specific 5HT2A receptor knockout (5HT2A receptor LKO) mice upon high-fat diet (HFD) feeding. In the present study, we explored a series of new peripherally acting 5HT2A receptor antagonists. Compound 14a displayed good in vitro activity, with an IC50 value of 0.17 nM. Compound 14a exhibited good microsomal stability, no significant CYP and hERG inhibition, and 5HT receptor subtype selectivity. The brain-to-plasma ratio of 14a was below the lower limit of quantification, indicating limited blood-brain barrier (BBB) penetration. HFD-fed 14a treated mice showed decreased liver steatosis and lobular inflammation. These results demonstrate the potential of newly synthesized peripheral 5HT2A receptor antagonists for treating NAFLD.

Role of the efflux transporters Abcb1 and Abcg2 in the brain distribution of olaparib in mice.

Olaparib is a first-in-class poly (ADP-ribose) polymerase oral inhibitor used to treat various tumors. In this study, we clarified the roles of ABCB1/Abcb1 and ABCG2/Abcg2 transporters in restricting olaparib distribution to the brain. Olaparib was efficiently transported by human ABCG2, human ABCB1, and mouse Abcg2 in vitro. In the in vivo disposition study of olaparib using single or combination knockout mice, the systemic exposure of olaparib did not differ significantly between the strains over an 8-h period. However, the brain-to-plasma unbound concentration ratio of olaparib increased 5.6- and 8.1-fold in Abcb1a/1b and Abcb1a/1b;Abcg2 knockout mice, respectively, compared with wild-type mice. The Abcg2 single knockout mice exhibited a similar brain-to-plasma unbound concentration ratio to wild-type mice. Moreover, the brain distribution of olaparib could be modulated by the ABCB1/ABCG2 dual inhibitor elacridar to reach a similar degree of inhibition to Abcb1a/1b-/-. These findings suggest that olaparib is actively transported by both human and mouse ABCB1/Abcb1 and ABCG2/Abcg2; while Abcb1a/1b is a major determinant of olaparib brain penetration in mice, Abcg2 is likely to be a minor contributor. Concomitant treatment with temozolomide slightly increased the brain distribution of olaparib in mouse, but the clinical impact of the interaction was expected to be limited.

PredMS: a random forest model for predicting metabolic stability of drug candidates in human liver microsomes.

MOTIVATION: Poor metabolic stability leads to drug development failure. Therefore, it is essential to evaluate the metabolic stability of small compounds for successful drug discovery and development. However, evaluating metabolic stability in vitro and in vivo is expensive, time-consuming and laborious. In addition, only a few free software programs are available for metabolic stability data and prediction. Therefore, in this study, we aimed to develop a prediction model that predicts the metabolic stability of small compounds. RESULTS: We developed a computational model, PredMS, which predicts the metabolic stability of small compounds as stable or unstable in human liver microsomes. PredMS is based on a random forest model using an in-house database of metabolic stability data of 1917 compounds. To validate the prediction performance of PredMS, we generated external test data of 61 compounds. PredMS achieved an accuracy of 0.74, Matthew's correlation coefficient of 0.48, sensitivity of 0.70, specificity of 0.86, positive predictive value of 0.94 and negative predictive value of 0.46 on the external test dataset. PredMS will be a useful tool to predict the metabolic stability of small compounds in the early stages of drug discovery and development. AVAILABILITY AND IMPLEMENTATION: The source code for PredMS is available at https://bitbucket.org/krictai/predms, and the PredMS web server is available at https://predms.netlify.app. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The KDM4B-CCAR1-MED1 axis is a critical regulator of osteoclast differentiation and bone homeostasis.

Bone undergoes a constant and continuous remodeling process that is tightly regulated by the coordinated and sequential actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Recent studies have shown that histone demethylases are implicated in osteoblastogenesis; however, little is known about the role of histone demethylases in osteoclast formation. Here, we identified KDM4B as an epigenetic regulator of osteoclast differentiation. Knockdown of KDM4B significantly blocked the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. Mice with myeloid-specific conditional knockout of KDM4B showed an osteopetrotic phenotype due to osteoclast deficiency. Biochemical analysis revealed that KDM4B physically and functionally associates with CCAR1 and MED1 in a complex. Using genome-wide chromatin immunoprecipitation (ChIP)-sequencing, we revealed that the KDM4B-CCAR1-MED1 complex is localized to the promoters of several osteoclast-related genes upon receptor activator of NF-κB ligand stimulation. We demonstrated that the KDM4B-CCAR1-MED1 signaling axis induces changes in chromatin structure (euchromatinization) near the promoters of osteoclast-related genes through H3K9 demethylation, leading to NF-κB p65 recruitment via a direct interaction between KDM4B and p65. Finally, small molecule inhibition of KDM4B activity impeded bone loss in an ovariectomized mouse model. Taken together, our findings establish KDM4B as a critical regulator of osteoclastogenesis, providing a potential therapeutic target for osteoporosis.

Peripheral Selective Oxadiazolylphenyl Alanine Derivatives as Tryptophan Hydroxylase 1 Inhibitors for Obesity and Fatty Liver Disease.

Tryptophan hydroxylase 1 (TPH1) has been recently suggested as a promising therapeutic target for treating obesity and fatty liver disease. A new series of 1,2,4-oxadiazolylphenyl alanine derivatives were identified as TPH1 inhibitors. Among them, compound 23a was the most active in vitro, with an IC50 (half-maximal inhibitory concentration) value of 42 nM, showed good liver microsomal stability, and showed no significant inhibition of CYP and hERG. Compound 23a inhibited TPH1 in the peripheral tissue with limited BBB penetration. In high-fat diet-fed mice, 23a reduced body weight gain, body fat, and hepatic lipid accumulation. Also, 23a improved glucose intolerance and energy expenditure. Taken together, compound 23a shows promise as a therapeutic agent for the treatment of obesity and fatty liver diseases.

LightBBB: computational prediction model of blood-brain-barrier penetration based on LightGBM.

MOTIVATION: Identification of blood-brain barrier (BBB) permeability of a compound is a major challenge in neurotherapeutic drug discovery. Conventional approaches for BBB permeability measurement are expensive, time-consuming and labor-intensive. BBB permeability is associated with diverse chemical properties of compounds. However, BBB permeability prediction models have been developed using small datasets and limited features, which are usually not practical due to their low coverage of chemical diversity of compounds. Aim of this study is to develop a BBB permeability prediction model using a large dataset for practical applications. This model can be used for facilitated compound screening in the early stage of brain drug discovery. RESULTS: A dataset of 7162 compounds with BBB permeability (5453 BBB+ and 1709 BBB-) was compiled from the literature, where BBB+ and BBB- denote BBB-permeable and non-permeable compounds, respectively. We trained a machine learning model based on Light Gradient Boosting Machine (LightGBM) algorithm and achieved an overall accuracy of 89%, an area under the curve (AUC) of 0.93, specificity of 0.77 and sensitivity of 0.93, when 10-fold cross-validation was performed. The model was further evaluated using 74 central nerve system compounds (39 BBB+ and 35 BBB-) obtained from the literature and showed an accuracy of 90%, sensitivity of 0.85 and specificity of 0.94. Our model outperforms over existing BBB permeability prediction models. AVAILABILITYAND IMPLEMENTATION: The prediction server is available at http://ssbio.cau.ac.kr/software/bbb.

Design, Synthesis, and Biological Evaluation of New Peripheral 5HT2A Antagonists for Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is increasingly prevalent worldwide, causing serious liver complications, including nonalcoholic steatohepatitis. Recent findings suggest that peripheral serotonin (5-hydroxytryptamine, 5HT) regulates energy homeostasis, including hepatic lipid metabolism. More specifically, liver-specific 5HT2A knockout mice exhibit alleviated hepatic lipid accumulation and hepatic steatosis. Here, structural modifications of pimavanserin (CNS drug), a 5HT2A antagonist approved for Parkinson's disease, led us to synthesize new peripherally acting 5HT2A antagonists. Among the synthesized compounds, compound 14a showed good in vitro activity, good liver microsomal stability, 5HT subtype selectivity, and no significant inhibition of CYP and hERG. The in vitro and in vivo blood-brain barrier permeability study proved that 14a acts peripherally. Compound 14a decreased the liver weight and hepatic lipid accumulation in high-fat-diet-induced obesity mice. Our study suggests new therapeutic possibilities for peripheral 5HT2A antagonists in NAFLD.

A Novel Way of Preventing Postoperative Pancreatic Fistula by Directly Injecting Profibrogenic Materials into the Pancreatic Parenchyma.

This paper aims to validate if intrapancreatic injection of penicillin G can enhance hardness and suture holding capacity (SHC) of the pancreas through prompting the fibrosis process. Soft pancreatic texture is constantly mentioned as one of the most contributory predictors of postoperative pancreatic fistula (POPF). Soft pancreas has poor SHC and higher incidence of parenchymal tearing, frequently leading to POPF. From a library of 114 antibiotic compounds, we identified that penicillin G substantially enhanced pancreatic hardness and SHC in experimental mice. Specifically, we injected penicillin G directly into the pancreas. On determined dates, we measured the pancreatic hardness and SHC, respectively, and performed molecular and histological examinations for estimation of the degree of fibrosis. The intrapancreatic injection of penicillin G activated human pancreatic stellate cells (HPSCs) to produce various fibrotic materials such as transforming growth factor-ß1 (TGF-ß1) and metalloproteinases-2. The pancreatic hardness and SHC were increased to the maximum at the second day after injection and then it gradually subsided demonstrating its reversibility. Pretreatment of mice with SB431542, an inhibitor of the TGF-ß1 receptor, before injecting penicillin G intrapancreatically, significantly abrogated the increase of both pancreatic hardness and SHC caused by penicillin G. This suggested that penicillin G promotes pancreatic fibrosis through the TGF-ß1 signaling pathway. Intrapancreatic injection of penicillin G promotes pancreatic hardness and SHC by enhancing pancreatic fibrosis. We thus think that penicillin G could be utilized to prevent and minimize POPF, after validating its actual effectiveness and safety by further studies.

Neurochemical and behavioral analysis by acute exposure to bisphenol A in zebrafish larvae model.

Bisphenol A (BPA) is a chemical monomer widely used in the production of hard plastics for food containers and personal items. Through improper industrial control and disposal, BPA has become a pervasive environmental contaminant, and toxicological studies have shown potent xenobiotic endocrine disruptor activity. Prenatal exposure in particular can lead to infertility and nervous system disorders characterized by behavioral aggression, depression, and cognitive impairment, thus necessitating careful hazard assessment. In this study, we evaluated BPA accumulation rate, blood-brain barrier (BBB) permeability, lethality, cardiotoxicity, behavioral effects, and impacts on multiple neurochemical pathways in zebrafish larvae. The bioconcentration factor (BCF) ranged from 1.95 to 10.0, resulting in a high rate of accumulation in the larval body. Also, high BBB permeability allowed BPA to accumulate at similar rates in both zebrafish and adult mouse (blood to brain concentration ratios of 3.2-6.7 and 1.8 to 5.5, respectively). In addition, BPA-exposed zebrafish larvae exhibited developmental deformities, reduced heart rate, and impaired behavioral patterns, including decreased total distance traveled, slower movement velocity, and altered color-preference. These impairments were associated with inhibition of the phenylalanine to dopamine synthesis pathway and an imbalance between excitatory and inhibitory neurotransmitter systems. Our results suggest that behavioral alteration in BPA-exposed zebrafish result from high accumulation and ensuing dysregulation of serotonergic, kynurenergic, dopaminergic, cholinergic, and GABAergic neurotransmitter systems. In conclusion, similarities in toxic responses to mammalian models highlight the utility of the zebrafish larva as a convenient model for screening environmental toxins.

Optimization of cyclic sulfamide derivatives as 11ß-hydroxysteroid dehydrogenase 1 inhibitors for the potential treatment of ischemic brain injury.

The 11ß-hydroxysteroiddehydrogenase type 1(11ß-HSD1), acortisolregenerating enzyme that amplifies tissue glucocorticoidlevels, plays an important role in diabetes, obesity, and glaucoma and is recognized as a potential therapeutic target for various disease conditions. Moreover, a recent study demonstrated that selective 11ß-HSD1 inhibitor can attenuate ischemic brain injury. This prompted us to optimize cyclic sulfamide derivative for aiming to treat ischemic brain injury. Among the synthesized compounds, 6e has an excellent in vitro activivity with an IC50 value of 1 nM toward human and mouse 11ß-HSD1 and showed good 11ß-HSD1 inhibition in ex vivo study using brain tissue isolated from mice. Furthermore, in the transient middle cerebral artery occlusion model in mice, 6e treatment significantly attenuated infarct volume and neurological deficit following cerebral ischemia/reperfusion injury. Additionally, binding modes of 6e for human and mouse 11ß-HSD1 were suggested.

Superior gallstone dissolubility and safety of tert-amyl ethyl ether over methyl-tertiary butyl ether.

BACKGROUND: The use of methyl-tertiary butyl ether (MTBE) to dissolve gallstones has been limited due to concerns over its toxicity and the widespread recognition of the safety of laparoscopic cholecystectomy. The adverse effects of MTBE are largely attributed to its low boiling point, resulting in a tendency to evaporate. Therefore, if there is a material with a higher boiling point and similar or higher dissolubility than MTBE, it is expected to be an attractive alternative to MTBE. AIM: To determine whether tert-amyl ethyl ether (TAEE), an MTBE analogue with a relatively higher boiling point (102 °C), could be used as an alternative to MTBE in terms of gallstone dissolubility and toxicity. METHODS: The in vitro dissolubility of MTBE and TAEE was determined by measuring the dry weights of human gallstones at predetermined time intervals after placing them in glass containers with either of the two solvents. The in vivo dissolubility was determined by comparing the weights of solvent-treated gallstones and control (dimethyl sulfoxide)-treated gallstones, after the direct infusion of each solvent into the gallbladder in both hamster models with cholesterol and pigmented gallstones. RESULTS: The in vitro results demonstrated a 24 h TAEE-dissolubility of 76.7%, 56.5% and 38.75% for cholesterol, mixed, and pigmented gallstones, respectively, which represented a 1.2-, 1.4-, and 1.3-fold increase in dissolubility compared to that of MTBE. In the in vitro experiment, the 24 h-dissolubility of TAEE was 71.7% and 63.0% for cholesterol and pigmented gallstones, respectively, which represented a 1.4- and 1.9-fold increase in dissolubility compared to that of MTBE. In addition, the results of the cell viability assay and western blot analysis indicated that TAEE had a lower toxicity towards gallbladder epithelial cells than MTBE. CONCLUSION: We demonstrated that TAEE has higher gallstone dissolubility properties and safety than those of MTBE. As such, TAEE could present an attractive alternative to MTBE if our findings regarding its efficacy and safety can be consistently reproduced in further subclinical and clinical studies.

Efficacy and safety of a novel topical agent for gallstone dissolution: 2-methoxy-6-methylpyridine.

BACKGROUND: Although methyl-tertiary butyl ether (MTBE) is the only clinical topical agent for gallstone dissolution, its use is limited by its side effects mostly arising from a relatively low boiling point (55 °C). In this study, we developed the gallstone-dissolving compound containing an aromatic moiety, named 2-methoxy-6-methylpyridine (MMP) with higher boiling point (156 °C), and compared its effectiveness and toxicities with MTBE. METHODS: The dissolubility of MTBE and MMP in vitro was determined by placing human gallstones in glass containers with either solvent and, then, measuring their dry weights. Their dissolubility in vivo was determined by comparing the weights of solvent-treated gallstones and control (dimethyl sulfoxide)-treated gallstones, after directly injecting each solvent into the gallbladder in hamster models with cholesterol and pigmented gallstones. RESULTS: In the in vitro dissolution test, MMP demonstrated statistically higher dissolubility than did MTBE for cholesterol and pigmented gallstones (88.2% vs. 65.7%, 50.8% vs. 29.0%, respectively; P < 0.05). In the in vivo experiments, MMP exhibited 59.0% and 54.3% dissolubility for cholesterol and pigmented gallstones, respectively, which were significantly higher than those of MTBE (50.0% and 32.0%, respectively; P < 0.05). The immunohistochemical stains of gallbladder specimens obtained from the MMP-treated hamsters demonstrated that MMP did not significantly increase the expression of cleaved caspase 9 or significantly decrease the expression of proliferation cell nuclear antigen. CONCLUSIONS: This study demonstrated that MMP has better potential than does MTBE in dissolving gallstones, especially pigmented gallstones, while resulting in lesser toxicities.