key: cord-0685952-1u73cm0o
authors: Choi, Hae-In; Kim, Taeheon; Lee, Seung-Won; Woo Kim, Jin; Ju Noh, Yoon; Kim, GwanYoung; Jin Park, Hyun-; Chae, Yoon-Jee; Lee, Kyeong-Ryoon; Kim, Soo-Jin; Koo, Tae-Sung
title: Bioanalysis of Niclosamide in Plasma using Liquid Chromatography-Tandem Mass and Application to Pharmacokinetics in Rats and Dogs
date: 2021-07-18
journal: J Chromatogr B Analyt Technol Biomed Life Sci
DOI: 10.1016/j.jchromb.2021.122862
sha: f27efcf666f3aceb43e25cb8853ddb453199df93
doc_id: 685952
cord_uid: 1u73cm0o

Niclosamide, which is an anti-tapeworm drug, was developed in 1958. However, recent studies have demonstrated the antiviral effects of niclosamide against the SARS-CoV-2 virus, which causes COVID-19. In this study, we developed and validated a quantitative analysis method for the determination of niclosamide in rat and dog plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS), and used this method for pharmacokinetic studies. Biological samples were prepared using the protein precipitation method with acetonitrile. Ibuprofen was used as an internal standard. The mobile phase used to quantify niclosamide in rat or dog plasma consisted of 10 mM ammonium formate in distilled water-acetonitrile (30:70, v/v) or 5 mM ammonium acetate-methanol (30:70, v/v). An XDB-phenyl column (5 µm, 2.1 × 50 mm) and a Kinetex® C18 column (5 µm, 2.1 × 500 mm) were used as reverse-phase liquid chromatography columns for rat and dog plasma analyses, respectively. Niclosamide and ibuprofen were detected under multiple reaction monitoring conditions using the electrospray ionization interface running in the negative ionization mode. Niclosamide presented linearity in the concentration ranges of 1-3000 ng/mL (r =0.9967) and 1-1000 ng/mL (r =0.9941) in rat and dog plasma, respectively. The intra- and inter-day precision values were <7.40% and <6.35%, respectively, for rat plasma, and <3.95% and <4.01%, respectively, for dog plasma. The intra- and inter-day accuracy values were <4.59% and <6.63%, respectively, for rat plasma, and <12.1% and <10.9%, respectively, for dog plasma. In addition, the recoveries of niclosamide ranged between 87.8-99.6% and 102-104% for rat and dog plasma, respectively. Niclosamide was stable during storage under various conditions (three freeze-thaw cycles, 6 h at room temperature, long-term, and processed samples). A reliable LC–MS/MS method for niclosamide detection was successfully used to perform pharmacokinetic studies in rats and dogs. Niclosamide presented dose-independent pharmacokinetics in the dose range of 0.3-3 mg/kg after intravenous administration, and drug exposure in rats and dogs after oral administration was very low. Additionally, niclosamide presented high plasma protein binding (>99.8%) and low metabolic stability. These results can be helpful for further developing and understanding the pharmacokinetic characteristics of niclosamide to expand its clinical use.

COVID-19 is a new infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged in December 2019 and quickly became a pandemic [1] . The structure of SARS-CoV-2, which is a single-stranded RNA coronavirus, is similar to that of the SARS-CoV virus, which originated in China in 2002. The spike protein on the membrane surface of these viruses specifically binds to the host cell angiotensin-converting enzyme 2 receptor to rapidly penetrate and proliferate inside cells [2, 3] . Typically, SARS-COV-2 is transmitted via droplets or human contact, and the infected individuals can be asymptomatic or present several respiratory infection symptoms, such as fever, cough, shortness of breath, and sore throat [4] .

Recently, a few vaccines and drugs have been approved for emergency use in COVID-19 patients. Nevertheless, viral mutations occur rapidly; therefore, safe and effective drugs for COVID-19 treatment are still necessary.

Niclosamide, which is an anti-tapeworm drug, was developed in 1958. Niclosamide inhibits glucose absorption, oxidative phosphorylation, and anaerobic metabolism in tapeworms [5] . Recent studies have demonstrated that niclosamide inhibits cancer cells by blocking the WNT signaling pathway [6] . Clinical trials on niclosamide for colorectal and prostate cancers have been conducted, and the results revealed that the drug was toxic only when administered orally in high doses [7] [8] [9] . Furthermore, studies have demonstrated the antiviral effects of niclosamide against MERS-CoV, SARS-CoV, and SARS-CoV-2 at cellular level via autophagy activation through inhibition of the SKP2 signaling pathway [10] [11] [12] . In addition, according to a recent study on 48 drugs previously approved by the US Food and Drug Administration (FDA) for SARS-CoV-2, niclosamide was approximately 40 times more effective than antiviral drugs, such as remdesivir and chloroquine, at cellular level [13] . Moreover, clinical studies on COVID-19 treatments are in progress in the USA, Germany, Australia, and Korea [14] .

Many reports regarding the pharmacological effects and clinical results of niclosamide, which is an old drug, have been published. However, information on analytical methods used for niclosamide detection in biological samples and pharmacokinetic analysis in animals is limited. Doran and Stevens [15] and Jiang et al. [16] used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for niclosamide detection in water; however, the proposed methods could not be extrapolated to the analysis of biological matrices in general or plasma samples in particular. Chang et al. [17] studied the pharmacokinetics of niclosamide in rats; however, the sensitivity, precision, accuracy, and matrix effect of their method for biological samples has not been validated.

In addition, because their study compared simple pharmacokinetic results with other derivatives, evaluating the pharmacokinetic properties of niclosamide was limited.

In this study we developed and validated a quantitative analysis method for the determination of niclosamide in rat and dog plasma using a LC-MS/MS system in accordance with the European Medicines Agency (EMA) and US FDA guidelines. Furthermore, we used the developed method to perform pharmacokinetic studies to evaluate the in vitro and in vivo mechanisms of niclosamide. 

All biological samples, except the dog-derived samples, were analyzed under the following LC-MS/MS conditions. We used an Agilent 1100 (Agilent Technologies, Santa Clara, CA, USA) HPLC system with a Zorbax Eclipse XDB-Phenyl (5 µm, 2.1 × 50 mm, Agilent, Santa Clara, CA, USA) column connected to a Zorbax Eclipse XDB-C8 (5 µm, 2.1 × 12.5 mm, Agilent, Santa Clara, CA, USA) guard column, which was used as the reverse-phase column and was maintained at 40 °C. The mobile phase consisted of a mixture of 10 mM ammonium formate-acetonitrile 30:70 (v/v), and separation occurred via isocratic elution at a flow rate of 0.3 mL/min with an injection volume of 5 µL. The autosampler temperature was maintained at 10 °C. All analytes were detected using an API 4000 QTrap (AB Sciex, Framingham, MA, USA) triple quadrupole mass spectrometer in the negative ionization mode of the electrospray ionization (ESI) interface. The MS parameters were as follows: ion voltage of −4200 V, temperature of 600 °C, curtain gas pressure of 20 psi, nebulizer gas pressure of 50 psi, turbo gas pressure of 50 psi, entrance potential of −10 V, declustering potentials of −55 and 

To make the standard sample for the calibration curve, 10-fold concentrated working standard solution was prepared via serial dilution of niclosamide with acetonitrile, and the working standard solution for QC samples were independently prepared.

For rat plasma samples, 20 µL of acetonitrile containing IS (1 μg/mL of ibuprofen) and 160 µL of acetonitrile were added to 20 µL of a rat plasma to induce protein precipitation. Thereafter, the mixture was vigorously mixed for 10 min, followed by centrifugation at 13 500 rpm for 10 min. Next, the supernatant was transferred into a vial, and 5 μL of supernatant was injected into the LC-MS/MS system. 

The method validation parameters, namely specificity, linearity, precision and accuracy, matrix effect, recovery, process efficiency, and stability were analyzed according to the guidelines of the EMA and US FDA [18, 19] .

Specificity was evaluated using six lots of untreated blank plasma samples, and the results were compared with those of the respective analyte and IS-spiked samples. The retention times of the analyte and IS were compared to confirm the absence of interference peaks of endogenous substances in plasma. The calibration curves of niclosamide in plasma were obtained by plotting the peak ratios of niclosamide to ibuprofen vs. the nominal concentrations of the calibration standards (1, 2, 5, 10, 30, 100, 300, 1000, 2000, and 3000 ng/mL for rats, or 1, 3, 10, 30, 100, 300, and 1000 ng/mL for dogs). Calibration curves were fitted using least-squares linear regression analysis using a weighted factor (1/x 2 ). Linearity was validated using correlation coefficients (r).

The precision and accuracy of the analytical method were validated using the lower limit of quantification (LLOQ; 1 ng/mL) and QC samples (3, 500, and 2700 ng/mL for rats and 2.5, 50, and 500 ng/mL for dogs).

Precision was expressed as the coefficient of variation (CV%), which was obtained as the percentage of the standard deviation (SD) of the peak area ratio divided by the mean of the peak area ratio of niclosamide and IS.

The accuracy was calculated as the relative error (%RE), which was obtained as the percentage of the difference between the measured and nominal concentrations divided by the nominal concentrations of niclosamide. The intra-day precision and accuracy of the analytical method were determined by repeating experiments five times per day, whereas the inter-day precision and accuracy of the analytical method were obtained by repeating experiments for 3 day.

The matrix effect, recovery, and process efficiency were measured for each QC group. A matrix effect experiment was performed to evaluate the enhancement or suppression of analyte ionization owing to the presence of matrix components in the samples. The matrix effect was calculated by dividing the mean of the peak areas of niclosamide spiked in a blank plasma extract (set 2) by the peak area of the analyte added using the mobile phase of the clean analyte solutions (set 1). Recovery was estimated by comparing the mean peak areas of the analyte in the sample extracts (set 3) with those of the samples in set 2. Process efficiency was calculated by comparing the data for sets 1 and 3 [20, 21] .

Sample stability was determined at low and high QC concentrations. Short-term stability was evaluated at room temperature for 6 h, whereas processed sample stability was evaluated by comparison with samples prepared using an autosampler at 10 °C for 24 h. Long-term stability was determined by assaying samples stored at −20 °C for four weeks and samples that underwent three freeze-thaw cycles.

An equilibrium dialysis device (RED ® , Thermo, Waltham, MA, USA) was used to perform the plasma protein binding assay of niclosamide in rats, dogs, and humans. A semi-permeable membrane was used to separate the chamber containing plasma spiked with 2 μg/mL of niclosamide from that containing phosphoric acid buffer (pH 7.4). Incubation was performed in a water bath shaken for 4 h at 100 rpm and 37 °C. Thereafter, 50 μL aliquots from each chamber were collected, pretreated, and analyzed using LC-MS/MS. Plasma protein binding was calculated as follows: 1 − (concentration in buffer/concentration in plasma).

The metabolic stability of niclosamide was examined using rat, dog, and human liver microsomes.

Niclosamide was dissolved in DMSO and diluted to final niclosamide concentrations of 1 µM. Reactions were performed in triplicate in 96-well plates at a final volume of 160 µL in 0.1 M potassium phosphate buffer and 0.5 mg/mL of rat, dog, and human liver microsomes. The plates were incubated at 37 °C before a 1 mM βnicotinamide adenine dinucleotide phosphate solution was added to each well. The reaction was terminated at 0, 10, 30, and 60 min by adding 320 µL of ice-cold acetonitrile containing IS to the wells. The samples were centrifuged at 3000 rpm for 10 min. The supernatant (5 µL) was analyzed using an LC-MS/MS system in the MRM mode. Subsequently, hepatic clearance (CL H ) was estimated using the in vitro intrinsic clearance (CL int, in vitro ) levels in liver, which was calculated using the metabolic stability data and following equations [20] :

CL u,int, vivo = f u mic × CL int, in vitro × (45 mg microsomes/g liver) × (g liver/kg body weight),

and

where T 1/2 , k e , f u mic , f ub , CL u int in vivo , and Q h denote the elimination half-life, elimination rate constant, microsome-and blood-unbound fraction of niclosamide, in vivo intrinsic clearance, and hepatic blood flow,

respectively. To calculate f u,b , the plasma unbound fraction was divided by the blood-to-plasma concentration ratio and then multiplied by (1-0.44), and f u,mic was assumed to be 0.5.

Male Sprague Dawley (SD) rats (7 weeks old) weighing 210-251 g were purchased from Orient Bio Inc. was calculated as follows: k a = 1/MRT po − MRT iv [22, 23] . The area under the concentration of niclosamide in plasma vs. time curve in the 0 to ∞ time interval (AUC inf ) was calculated using the linear trapezoidal rule and standard area extrapolation method. The maximum plasma concentration (C max ) and time C max was reached (T max ) were determined directly from the plasma concentration-time curves.

All data are reported as mean ± SD. The pharmacokinetic parameters were estimated using one-way analysis of variance (ANOVA) and the Prism 7.0 (GraphPad Software, San Diego, CA, USA) software. A result was considered statistically significant when p < 0.05 for both analyses.

The niclosamide and ibuprofen ions were detected using MRM conditions. The deprotonated precursor ions previously reported [15, 16] . Niclosamide molecules contain two Cl atoms, with a 35 Cl-to-37 Cl ratio of 3:1. The typical Cl isotope ratio of molecules containing two Cl atoms is approximately 9:6:1. When the precursor ion was analyzed using the Q1 scan, three isotope peaks at a ratio of approximately 9:6:1 were observed in the profile of niclosamide [15] . For ibuprofen (the IS), the transition of product ions was observed at m/z 205.0 → 161.1 (Fig. 1) .

The mobile phase and column conditions were changed to optimize the plasma chromatograms. To detect niclosamide in rat plasma using the API 4000 QTrap spectrometer, we used a mixture of 10 mM ammonium formate-acetonitrile (30:70 v/v) as the mobile phase. These solvents are commonly used in the negative ion mode of the ESI interface because weak base conditions favor ionization. Consequently, the sensitivity was higher than that when 0.1% formic acid was used as the mobile phase, allowing niclosamide quantification at lower concentrations. The performance of the commonly used C18 and Phenyl columns in the API 4000 QTrap spectrometer was compared. The sensitivity of the XBD-C18 (Agilent, Santa Clara, CA) column was approximately twice as high that of the XBD-Phenyl column (Agilent, Santa Clara, CA); however, the XBD-Phenyl column was selected owing to its low baseline, adequate quantification range, peak shape, and negligible carryover in rat plasma ( Fig. 2A-C) . Niclosamide in dog plasma samples was detected using a QTrap 6500 LC-MS/MS system with a higher sensitivity than the API 4000 QTrap spectrometer used for rat plasma analysis.

When the same method used for rat plasma analysis was utilized for dog plasma samples, an interference peak was observed in the chromatogram of blank plasma. Therefore, a Kinetex ® C18 (5 μm, 2.1  500 mm) column and 5 mM ammonium acetate-methanol (30:70, v/v) mobile phase were used to improve peak shape and analyte separation in dog plasma samples ( Fig. 2D and E ).

Because the analyte signal was more than five times stronger that of the blank sample, 1 ng/mL was The calibration curve for niclosamide, which was obtained using five rat plasma samples, namely blank plasma, zero, and calibration standard samples, presented a good linearity in the concentration range of 1-3000 ng/mL (r = 0.9967). The equation of the standard curve for niclosamide: y = 0.0391x + 0.0119, was obtained using a weighting factor of 1/x 2 . The calibration standard curve for niclosamide in dog plasma samples presented a good linearity in the range of 1-1000 ng/mL (r = 0.9941), and the equation of the standard curve was y = 0.01600x + 0.001667. Dilution integrity was validated for concentrations up to 10 times higher than the high QC concentration in five replicates; moreover, CV was 4.54%, and RE was −2.48%.

The precision and accuracy values of the method are summarized in Table 1 . For rat plasma, the intra-and inter-day precision values were <7.40% and <6.35%, respectively, whereas the intra-and inter-day accuracy values were <4.59% and <6.63%, respectively, at four concentration levels (lower quality control, middle quality control, and higher quality control concentrations, and LLOQ). For dog plasma, the intra-and inter-day precision values were <5.55% and <5.82%, respectively, and the intra-and inter-day accuracy values were <13.0% and <12.7%, respectively. These results satisfied the acceptance criteria [18, 19] and demonstrated that the method used to analyze rat and dog plasma samples was reproducible and reliable.

The matrix effect, recovery, and process efficiencies of niclosamide and IS in both types of plasma samples were determined at three QC sample concentrations. For rat plasma, the matrix effect ranged between 73.8-87.7% (IS, 81.4%), recovery ranged between 87.8-99.6% (IS, 89.0%), and process efficiency ranged between 72.6-87.2% (IS, 72.4%). If the samples were normalized with IS, the matrix effect, recovery, and process efficiency were improved by 100-108%. For dog plasma, the matrix effect ranged between 94.9-102% (IS, 94.8%), recovery ranged between 102-104% (IS, 79.1%), and process efficiency ranged between 98.1-104%

(IS, 75.0%). These results suggested that protein precipitation was an appropriate pretreatment for efficient niclosamide and ibuprofen extraction from rat and dog plasma.

The stability values of niclosamide in rat and dog plasma are summarized in Table 2 . Niclosamide in rat and dog plasma samples was stable at room temperature for 6 h, for three freeze-thaw cycles, in an autosampler at 10 °C for 24 h, and at −20 °C for one month.

Owing to plasma protein binding through equilibrium dialysis, niclosamide in rat, dog, and human plasma samples presented high plasma protein bindings of 99.86 ± 0.006%, 99.83 ± 0.015%, and 99.84 ± 0.042%, respectively; these values were not significantly different.

The mean residual concentration-time profiles of niclosamide in rat, dog, and human hepatic microsomes are presented in Fig. 3 . Owing to its stability in rat, dog, and human liver microsomes, a 1 μM niclosamide solution was incubated for 60 min, and 39.7%, 7.49%, and 2.95%, respectively, of the initial niclosamide amount was recovered. The half-lives of niclosamide in rat, dog, and human hepatic microsomes were 44.9, 16.0 and 11.8 min, respectively. These results indicated that niclosamide was metabolically unstable with a halflife of 45 min, and the half-life was shorter in larger mammals. The estimated k e , CL int, in vitro , and CL H values in rat liver microsomes were 0.015 min −1 , 6661 mL/(h·kg), and 12.1 mL/(h·kg), respectively. For dog liver microsomes, k e , CL int, in vitro , and CL H were 0.043 min −1 , 25841 mL/(h·kg), and 45.9 mL/(h·kg), respectively; and for human liver microsomes, k e , CL int, in vitro , and CL H were 0.059 min −1 , 17980 mL/(h·kg), and 31.9 mL/(h·kg), respectively. The estimated in vitro CL H values were compared with the CL values of the in vivo study, and the clearance scaling factors in rats and dogs were determined to be approximately 86.0 and 54.4, respectively.

Using the scaling factors for rat and dog liver microsomes, it was estimated that CL for human liver microsomes ranged between 1735-2743 mL/(h·kg). In addition, the significant difference between in vitro CL H and in vivo CL values (12.1 vs. 1041 mL/(h·kg) for rat liver microsomes and 45.9 vs. 2496 mL/(h·kg) for dog liver microsomes) indicated that the major elimination route of niclosamide was not phase 1 metabolism mediated by CYP enzymes. Additionally, it was confirmed that the half-life of niclosamide was approximately 10 times shorter when the microsomes were treated with uridine diphosphate glucuronic acid, indicating that phase 2 metabolism studies should be performed (data not included).

The mean plasma concentration-time profiles of niclosamide after IV (0.3, 1, and 3 mg/kg), PO (1 mg/kg), and IM (1 mg/kg) administration to rats are presented in Fig. 4A . The mean plasma concentration-time profiles of niclosamide after IV (2 mg/kg) and PO (100 mg/kg) administration to dogs are illustrated in Fig. 4B . The pharmacokinetic parameters of niclosamide in rats and dogs are summarized in Table 3 .

After IV administration of 0.3, 1, and 3 mg/kg niclosamide to rats, C max was 1035 ± 166, 3088 ± 481, and 11920 ± 1144 ng/mL, respectively, at the first sampling time (0.083 h), and AUC last was 301 ± 49.2, 903 ± 150, and 3375 ± 254 ng·h/mL, respectively. This indicated that upon increasing the niclosamide nominal dose by 3.3 and 10 times, C max and AUC last increased in a dose-dependent manner, namely 3.0 and 11.5 times for C max and 3.0 and 11.2 times for AUC last . Furthermore, the CL values at the niclosamide doses of 0.3, 1, and 3 mg/kg were 1012 ± 164, 1130 ± 188, and 892 ± 66.9 mL/(h·kg), respectively, which were moderate compared with the hepatic blood flow rate of rats (3300 mL/(h·kg)) [23] , and did not change significantly with the niclosamide dose. The V ss value was low <400 mL/kg in the niclosamide dose range of 0.3-3 mg/kg. This suggested that niclosamide was confined mainly to the plasma pool with limited tissue distribution because of its high plasma protein-binding properties. The one-way ANOVA results indicated that the T 1/2 , MRT, CL, and V ss values were not significantly different (p > 0.05) with increasing niclosamide dose, and the pharmacokinetic properties were dose-independent in the niclosamide dose range of 0.3-3 mg/kg.

Pharmacokinetic studies for different administration routes were performed by administering single IV, PO, and IM doses of 1 mg/kg niclosamide to SD rats. The k a value was calculated to be 0.34 h −1 using the following equation [22] : k a = 1/MRT po − MRT iv ; k a was lower than k e (0.70 h −1 ), which was estimated through linear regression analysis, suggesting that niclosamide presented flip-flop kinetics. The oral bioavailability of niclosamide in rats was 5.51 ± 1.02% and plasma exposure was very low, indicating that absorption was limited considering that the CL value was moderate compared with the hepatic blood flow rate of rats. The IM absorption rate of niclosamide in rats was high with a T max of 5 min, and bioavailability was higher than that achieved via PO administration.

For dogs, after IV administration of 2 mg/kg of niclosamide, a C max of 2543 ± 386 ng/mL was achieved 5 min after administration; furthermore, the mean CL value was 2496 ± 360 mL/(h·kg). Considering that the liver blood flow of dogs was 1860 mL/(h·kg) [23] , the mean CL was high. The mean V ss value was low (661 ± 61.7 mL/kg), indicating that niclosamide was not well distributed in tissues. For a single PO dose of 100 mg/kg of niclosamide, C max and T max were 109 ± 14.0 ng/mL and 0.833 ± 0.290 h, respectively, and absorption rate was moderate. Moreover, T 1/2 was moderate (~1.03 ± 0.89 and 1.66 ± 0.17 h after IV and PO administration, respectively). Flip-flop kinetics were also observed for dogs, indicating that the absorption rate was lower than the elimination rate. The oral bioavailability of niclosamide (0.54%) was very low and was ascribed to the high CL value.

In conclusion, a reliable LC-MS/MS method for the detection of niclosamide in rat and dog plasma was proposed. The method was in line with the US FDA and EMA guidelines and was successfully used for pharmacokinetic studies. Niclosamide presented linear pharmacokinetics in an IV dose range of 0.3-3 mg/kg in rats; moreover, drug exposure in rats and dogs following PO administration was very low. In addition, niclosamide presented a low metabolic stability in rat, dog, and human liver microsomes. These results can be helpful for further developing and understanding the pharmacokinetic characteristics of niclosamide to expand its clinical use.

Conceptualization 

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

THK, GYK, and HJP are an employee of Daewoong Pharmaceuticals, and SJ Kim is an employee of Daewoong Therapeutics Inc., and all other authors declare that there is no conflict of interest.

 Several Clinical studies of niclosamide on COVID-19 are active.

 Information on analytical methods in biological samples is limited.

 A reliable LC-MS/MS method was developed and validated in rat and dog plasma.

 The developed LC-MS/MS method is simple, rapid, sensitive, and accurate.

 This method was successfully applied to pharmacokinetic study in rats and dogs.

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Biological samples were prepared using the protein precipitation method with acetonitrile. Ibuprofen was used as an internal standard. The mobile phase used to quantify niclosamide in rat or dog plasma consisted of 10 mM ammonium formate in distilled water-acetonitrile (30:70, v/v) or 5 mM ammonium acetate-methanol (30:70, v/v). An XDB-phenyl column (5 µm, 2.1 × 50 mm) and a Kinetex ® C18 column (5 µm, 2.1 × 500 mm) were used as reverse-phase liquid chromatography columns for rat and dog plasma analyses, respectively. Niclosamide and ibuprofen were detected under multiple reaction monitoring conditions using the electrospray ionization interface running in the negative ionization mode. Niclosamide presented linearity in the concentration ranges of 1-3000 ng/mL (r =0.9967) and 1-1000 ng/mL (r =0.9941) in rat and dog plasma, respectively. The intra-and inter-day precision values were <7.40% and <6.35%, respectively, for rat plasma, and <3.95% and <4.01%, respectively, for dog plasma. The intra-and inter-day accuracy values were <4.59% and <6.63%, respectively, for rat plasma, and <12.1% and <10.9%, respectively, for dog plasma. In addition, the recoveries of niclosamide ranged between 87.8-99.6% and 102-104% for rat and dog plasma, respectively. Niclosamide was stable during storage under various conditions (three freeze-thaw cycles, 6 h at room temperature, long-term, and processed samples). A reliable LC-MS/MS method for niclosamide detection was successfully used to perform pharmacokinetic studies in rats and dogs. Niclosamide presented doseindependent pharmacokinetics in the dose range of 0.3-3 mg/kg after intravenous administration, and drug exposure in rats and dogs after oral administration was very low. Additionally, niclosamide presented high plasma protein binding (>99.8%) and low metabolic stability

Investigation and data curation: GYK, HJP and YJC; Writing-original draft preparation: HIC; Writing-review and editing: YJC; Visualization and supervision: SJK and TSK

This work was supported by Chungnam National University.