A simple <i>in chemico</i> method for testing skin sensitizing potential of chemicals using small endogenous molecules

Nepal, Mahesh Raj,Shakya, Rajina,Kang, Mi Jeong,Jeong, Tae Cheon Elsevier 2018 Toxicology letters Vol.289 No.-

<P><B>Abstract</B></P> <P>Among many of the validated methods for testing skin sensitization, direct peptide reactivity assay (DPRA) employs no cells or animals. Although no immune cells are involved in this assay, it reliably predicts the skin sensitization potential of a chemical <I>in chemico</I>. Herein, a new method was developed using endogenous small-molecular-weight compounds, cysteamine and glutathione, rather than synthetic peptides, to differentiate skin sensitizers from non-sensitizers with an accuracy as high as DPRA. The percent depletion of cysteamine and glutathione by test chemicals was measured by an HPLC equipped with a PDA detector. To detect small-size molecules, such as cysteamine and glutathione, a derivatization by 4-(4-dimethylaminophenylazo) benzenesulfonyl chloride (DABS-Cl) was employed prior to the HPLC analysis. Following test method optimization, a cut-off criterion of 7.14% depletion was applied to differentiate skin sensitizers from non-sensitizers in combination of the ratio of 1:25 for cysteamine:test chemical with 1:50 for glutathione:test chemical for the best predictivity among various single or combination conditions. Although overlapping HPLC peaks could not be fully resolved for some test chemicals, high levels of sensitivity (100.0%), specificity (81.8%), and accuracy (93.3%) were obtained for 30 chemicals tested, which were comparable or better than those achieved with DPRA.</P> <P><B>Highlights</B></P> <P> <UL> <LI> An <I>in vitro</I> method to differentiate skin sensitizers and non-sensitizers is proposed by using cysteamine and glutathione. </LI> <LI> The study follows a simple covalent binding of a test chemical with low-molecular weight endogenous compounds. </LI> <LI> The proposed method presents sufficiently high sensitivity, specificity, and accuracy when compared with the existing methods. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Alternative Methods for Testing Botulinum Toxin: Current Status and Future Perspectives

( Mahesh Raj Nepal ),( Tae Cheon Jeong ) 한국응용약물학회 2020 Biomolecules & Therapeutics(구 응용약물학회지) Vol.28 No.4

Botulinum toxins are neurotoxic modular proteins composed of a heavy chain and a light chain connected by a disulfide bond and are produced by Clostridium botulinum. Although lethally toxic, botulinum toxin in low doses is clinically effective in numerous medical conditions, including muscle spasticity, strabismus, hyperactive urinary bladder, excessive sweating, and migraine. Globally, several companies are now producing products containing botulinum toxin for medical and cosmetic purposes, including the reduction of facial wrinkles. To test the efficacy and toxicity of botulinum toxin, animal tests have been solely and widely used, resulting in the inevitable sacrifice of hundreds of animals. Hence, alternative methods are urgently required to replace animals in botulinum toxin testing. Here, the various alternative methods developed to test the toxicity and efficacy of botulinum toxins have been briefly reviewed and future perspectives have been detailed.

Role of Intestinal Microbiota in Metabolism of Voglibose In Vitro and In Vivo

Mahesh Raj Nepal,강미정,김건호,차동호,김주현,정태천 대한당뇨병학회 2020 Diabetes and Metabolism Journal Vol.44 No.6

Background Voglibose, an α-glucosidase inhibitor, inhibits breakdown of complex carbohydrates into simple sugar units in intestine. Studies showed that voglibose metabolism in the liver might be negligible due to its poor intestinal absorption. Numerous microorganisms live in intestine and have several roles in metabolism and detoxification of various xenobiotics. Due to the limited information, the possible metabolism of voglibose by intestinal microbiota was investigated in vitro and in vivo. Methods For the in vitro study, different concentrations of voglibose were incubated with intestinal contents, prepared from both vehicle- and antibiotics-treated mice, to determine the decreased amount of voglibose over time by using liquid chromatography-mass spectrometry. Similarly, in vivo pharmacodynamic effect of voglibose was determined following the administration of voglibose and starch in vehicle- and antibiotic-pretreated non-diabetic and diabetic mice, by measuring the modulatory effects of voglibose on blood glucose levels. Results The in vitro results indicated that the remaining voglibose could be significantly decreased when incubated with the intestinal contents from normal mice compared to those from antibiotic-treated mice, which had less enzyme activities. The in vivo results showed that the antibiotic pretreatment resulted in reduced metabolism of voglibose. This significantly lowered blood glucose levels in antibiotic-pretreated mice compared to the control animals. Conclusion The present results indicate that voglibose would be metabolized by the intestinal microbiota, and that this metabolism might be pharmacodynamically critical in lowering blood glucose levels in mice.

Effects of Baicalin on Oral Pharmacokinetics of Caffeine in Rats

( Keumhan Noh ),( Mahesh Raj Nepal ),( Ki Sun Jeong ),( Sun A Kim ),( Yeon Ji Um ),( Chae Shin Seo ),( Mi Jeong Kang ),( Pil Hoon Park ),( Wonku Kang ),( Hye Gwang Jeong ),( Tae Cheon Jeong ) 한국응용약물학회 2015 Biomolecules & Therapeutics(구 응용약물학회지) Vol.23 No.2

Scutellaria baicalensis is one of the most widely used herbal medicines in East Asia. Because baicalein and baicalin are major components of this herb, it is important to understand the effects of these compounds on drug metabolizing enzymes, such as cytochrome P450 (CYP), for evaluating herb-drug interaction. The effects of baicalin and baicalein on activities of ethoxyresoru- fin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), benzyloxyresorufin O-debenzylase (BROD), p-nitrophenol hydroxylase and erythromycin N-demethylase were assessed in rat liver microsomes in the present study. In addition, the pharmacokinetics of caffeine and its three metabolites (i.e., paraxanthine, theobromine and theophylline) in baicalin-treated rats were compared with untreated control. As results, EROD, MROD and BROD activities were inhibited by both baicalin and baicalein. However, there were no significant differences in the pharmacokinetic parameters of oral caffeine and its three metabolites between control and baicalin-treated rats. When the plasma concentration of baicalin was determined, the maximum concentration of baicalin was below the estimated IC50 values observed in vitro. In conclusion, baicalin had no effects on the pharmacokinetics of caffeine and its metabolites in vivo, following single oral administration in rats.

Impact of gut microbiota on drug metabolism: an update for safe and effective use of drugs

노금한,강유라,Mahesh Raj Nepal,Rajina Shakya,강미정,강원구,이상규,정혜광,정태천 대한약학회 2017 Archives of Pharmacal Research Vol.40 No.12

The intestinal mucosa and liver have long beenconsidered as the main sites of drug metabolism, and thecontribution of gut microbiota to drug metabolism has beenunder-estimated. However, it is now generally acceptedthat the gut microbiota plays an important role in drugmetabolism prior to drug absorption or during enterohepaticcirculation via various microbial enzymatic reactionsin the intestine. Moreover, some drugs are metabolized bygut microbiota to specific metabolite(s) that cannot beformed in the liver. More importantly, the metabolism ofdrugs by gut microbiota prior to absorption can alter thesystemic bioavailability of certain drugs. Therefore,understanding drug metabolism by gut microbiota is criticalfor explaining changes in the pharmacokinetics ofdrugs, which may cause significant alterations in drug-inducedpharmacodynamics and toxicities. In this review, wedescribe recent progress with regard to the role of metabolismby gut microbiota in some drug-induced alterationsof either pharmacological or toxicological effects toemphasize the clinical importance of gut microbiota forsafe and effective use of drugs.

Development of alternative tests to identify skin sensitizers and non-sensitizers in chemico

Tae Cheon Jeong(정태천),Mahesh Raj Nepal,Geon Ho Kim,Dong Ho Cha,Mi Jeong Kang 환경독성보건학회 2019 한국독성학회 심포지움 및 학술발표회 Vol.2019 No.10

Inflammation-triggered local drug release ameliorates colitis by inhibiting dendritic cell migration and Th1/Th17 differentiation

Regmi, Shobha,Pathak, Shiva,Nepal, Mahesh Raj,Shrestha, Prakash,Park, Junhyeung,Kim, Jong Oh,Yong, Chul Soon,Choi, Dong-Yong,Chang, Jae-Hoon,Jeong, Tae Cheon,Orive, Gorka,Yook, Simmyung,Jeong, Jee-Heo Elsevier 2019 Journal of controlled release Vol.316 No.-

<P><B>Abstract</B></P> <P>Enteric-coated formulations using Eudragit® polymers have been extensively used for delivering drugs to the lower gastrointestinal tract. However, these drug-delivery systems cannot accurately deliver the therapeutic cargoes to colon because of early degradation of the polymers at alkaline pH of the small intestine. Here, we describe a precise method of delivering drugs to inflammation sites in colon using an oral drug delivery system. Tacrolimus (FK506)-loaded microspheres were prepared using a thioketal-based polymer that releases drug in response to reactive oxygen species (ROS), which are abundantly produced at the sites of inflammation in acute colitis. Orally-administered FK506-loaded thioketal microspheres (FK506-TKM) led to a substantial accumulation of FK506 in inflamed colon and effectively alleviated dextran-sulfate sodium (DSS)-induced murine colitis. At the molecular level, FK506-TKM significantly inhibited infiltration of CD4<SUP>+</SUP> and CD8<SUP>+</SUP> T lymphocytes in colon and differentiation of CD4<SUP>+</SUP> T cells into Th1 and Th17 cells in colon-draining mesenteric lymph nodes <I>via</I> restricting dendritic cell migration from colon. Our findings indicate orally-administered thioketal-based drug delivery system as a promising means of treating acute inflammatory bowel diseases.</P> <P><B>Highlights</B></P> <P> <UL> <LI> FK506-loaded ROS-responsive microspheres were prepared and characterized. </LI> <LI> FK506 was accumulated at inflammed colon after oral administration. </LI> <LI> Local delivery of FK506 inhibited colonic dendritic cell migration. </LI> <LI> Effector T cells were attenuated at the colon-draining mesenteric lymph nodes. </LI> <LI> Thioketal polymer-based local drug delivery alleviate inflammation. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Identification of a N7-guanine adduct of 1-bromopropane in calf thymus DNA by mass spectrometry

Pritam Thapa,김은경,Mahesh Raj Nepal,정기선,강미정,노금한,이상규,정혜광,이준호,정태천,이응석 대한독성 유전단백체 학회 2016 Molecular & cellular toxicology Vol.12 No.1

As a replacement for chlorofluorocarbons that cause ozone depletion, 1-bromopropane has been widely used in work place. In the present study, the formation of N7-guanine adduct in DNA by 1-bromopropane was evaluated in vitro to elucidate the possible mechanism of its toxic action. N7-Propyl guanine was chemically synthesized and structurally characterized by NMR, UV, HPLC, and liquid chromatographyelectrospray ionization mass spectrometry (LC- ESI MS) for using as a reference standard. An incubation of 2ʹ-deoxyguanosine with 1-bromopropane produced N7-propyl adduct, which was identified by UV, HPLC and ESI-MS. In addition, N7-guanine adduct was also identified from the incorporation of calf thymus DNA with 1-bromopropane at the physiological condition by LC-ESI MS. Furthermore, the production of adduct was proportional to the amounts of 1-bromopropane used. These results indicated that the molecular mechanism underlying toxic effects of 1-bromopropane would be associated with the adduct formation on DNA at least in part.

Role of Intestinal Microbiota in Baicalin-Induced Drug Interaction and Its Pharmacokinetics

( Keumhan Noh ),( Youra Kang ),( Mahesh Raj Nepal ),( Ki Sun Jeong ),( Do Gyeong Oh ),( Mi Jeong Kang ),( Sangkyu Lee ),( Wonku Kang ),( Hye Gwang Jeong ),( Tae Cheon Jeong ) 영남대학교 약품개발연구소 2016 영남대학교 약품개발연구소 연구업적집 Vol.26 No.-

Since many glycoside compounds in natural products are hydrolyzed by intestinal microbiota when administered orally, it is of interest to know whether their pharmacological effects are derived from the glycoside itself or from the aglycone form in vivo. An interesting example is baicalin versus baicalein, the aglycone of baicalin, which is contained in some herbs from Labiatae including scutellaria baicalensis Georgi and scutellaria Linne. The herbs have been extensively used for treatment of inflammatory diseases in Asia. Although there have been numerous reports regarding the pharmacological effects of baicalin and baicalein in vivo and in vitro, some reports indicated that the glycoside form would hardly be absorbed in the intestine and that it should be hydrolyzed to baicalei.n in advance for absorption. Therefore, the role of metabolism by intestinal rnicrobiota should also be considered in the metabolism of baicalin. In addition, baicalin contains a glucuronide moiety in its structure, by which baicalin and baicalein show complex pharmacokinetic behaviors, due to the interconversion between them by phase II enzymes in the body. Recently, concerns about drug interaction with baicalin and/ or baicalein have been raised, because of the co-administration of Scutellnria species with certain drugs. Herein, we reviewed the role of intestinal rnicrobiota in pharmacokinetic characteristics of baicalin and baicalein, with regards to their pharmacological and toxicological effects.

Single-dose intraperitoneal delivery of FK506-encapsulated polymeric microspheres for the alleviation of murine colitis

Shiva Pathak,Shobha Regmi,Mahesh Raj Nepal,Prakash Shrestha,Jeong Uk Choi,Simmyung Yook,Jee-Heon Jeong 한국공업화학회 2020 Journal of Industrial and Engineering Chemistry Vol.91 No.-

Based on the severity of the disease progression, the treatment of ulcerative colitis involvesadministration of single or multiple therapeutic agents. In refractory cases of ulcerative colitis, FK506(FK) is the drug of choice in clinics. To maintain a long-term stable blood concentration of FK and toreduce its dosing frequency, we propose single-dose injection of FK-loaded biodegradable microspheres(FK-Ms). FK-Ms were intraperitoneally-injected into mice fed with dextran-sulfate sodium (DSS). Pharmacokinetics study revealed presence of FK in blood for over 20 days. The single intraperitonealinjection of the drug-loaded microspheres effectively alleviated DSS-induced murine colitis. Mechanistically, the single injection of FK-Ms inhibited the infiltration of T cells into colon andattenuated differentiation of T cells into interferon-g secreting Th1 and interleukin-17A secreting Th17cells in colon-draining mesenteric lymph nodes. Therefore, administration of prolonged-release typemicrospheres can be considered as an alternative for the effective treatment of inflammatory boweldiseases.