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Liraglutide is a glucagon-like peptide-1 (GLP-1) analog that has been utilized for the treatment of type 2 diabetes mel- litus. Liraglutide at a higher dose also shows beneficial effects in weight loss, which prompted its widespread use as an anti-obesity drug. The potential of liraglutide to treat Alzheimer’s disease and cognitive impairment has also been suggested. Nevertheless, the pharmacokinetics of liraglutide, including its distribution to the brain, has not been fully characterized. Therefore, this study aimed to develop a simple and sensitive bioanalytical method using liquid chro- matography–tandem mass spectrometry (LC–MS/MS) and determine the pharmacokinetics and brain distribution of liraglutide in rats. Liraglutide in the rat plasma and brain tissue homogenates was extracted by protein precipitation using methanol. A gradient elution profile was used for chromatographic separation with mobile phases comprising 0.3% formic acid in water and 0.3% formic acid in acetonitrile. The mass spectrometry was operated in the positive electrospray ionization with multiple reaction monitoring mode. The lower limit of quantification of the present LC–MS/MS was 1 ng/mL in the plasma and 2 ng/mL in the brain tissue. Following intravenous injection (0.05 mg/ kg, n = 5), plasma concentrations of liraglutide decreased monoexponentially with an average half-life of 3.67 h. The estimated absolute bioavailability of liraglutide after subcutaneous injection was 13.16%. Brain distribution of liraglu- tide was not significant, with the tissue-to-plasma partition coefficient (K ) of liraglutide less than 0.00031. However, the concentrations of liraglutide were significantly different in the different brain regions following IV injection. In the brain, liraglutide concentrations were the highest in the hypothalamus, followed by the cerebellum and cerebrum. The present LC–MS/MS assay and the pharmacokinetic results may be helpful to understand better the effect of lira- glutide in the brain for further preclinical and clinical studies of liraglutide. Keywords Glucagon-like peptide-1, Liraglutide, LC–MS/MS, Pharmacokinetics, Bioavailability, Brain distribution Hyeon Seok Oh and Minkyu Choi contributed equally to this work *Correspondence: Beom Soo Shin email@example.com Full list of author information is available at the end of the article © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Oh et al. Journal of Analytical Science and Technology (2023) 14:19 Page 2 of 11 As the indication of liraglutide is being expanded, the Introduction side effects associated with liraglutide at higher as well Glucagon-like peptide-1 (GLP-1) stimulates insulin as lower doses also need to be considered carefully. The secretion in response to blood glucose, thereby control- most frequently observed adverse effects of liraglutide ling blood glucose levels with a minimal adverse effect of are gastrointestinal symptoms. Other side effects include hypoglycemia (Doyle and Egan 2007; Drucker and Nauck increased levels of serum lipase and amylase, risk of acute 2006). Nevertheless, its extremely short biological half- pancreatitis, gallbladder or biliary disease, liver disease, life of approximately 2 min hampers the clinical utility of and kidney disease (Seo 2021). Thus, it is important to intact GLP-1 for type 2 diabetes mellitus (T2DM) (Tah- determine the proper dosing regimen according to the rani et al. 2010). Therefore, structural analogs of GLP-1 pharmacokinetics as well as the patient’s condition. with extended biological half-life have been developed Nevertheless, the pharmacokinetics of liraglutide, par- and become an emerging therapeutic class of drugs as an ticularly its distribution to the brain, has not been fully alternative to insulin drugs. characterized. So far, most pharmacokinetic studies of Liraglutide (Victoza ) is a GLP-1 analog with an liraglutide, like those of other peptide and protein drugs, extended half-life in which 16-carbon fatty acid is have relied on enzyme-linked immunosorbent assays attached at Lys26, and Arg34 is replaced with Lys while (ELISA) (Jacobsen et al. 2016; Agerso et al. 2002). Despite maintaining the pharmacological activities comparable the high sensitivity, ligand binding assays (LBA), includ- to native GLP-1 (Drucker and Nauck 2006; Chen et al. ing ELISA methods in general, have disadvantages in 2016). The structural modification protects liraglutide their application in pharmacokinetic studies (Dong et al. from metabolic degradation by dipeptidyl peptidase-4 2018; Meng et al. 2017; Pinho et al. 2019). Due to the fail- (DPP-4), which is responsible for the rapid elimina- ure to discern if the epitope is present in the parent or tion of GLP-1 (Jacobsen et al. 2016). Moreover, liraglu- metabolite, as well as inherent cross-reactivity, ELISA tide binds to serum albumin, which further reduces the may overestimate the concentrations of the parent drug. metabolism possibly by preventing the enzyme binding, Moreover, ELISA is susceptible to a significant matrix and decreases renal elimination (Jacobsen et al. 2016; effect, which makes it difficult to be applied to another Deacon 2009). Thus, liraglutide has a half-life of 10–14 h biological matrix for tissue distribution studies. Potential after subcutaneous injection in humans and is adminis- penetration of liraglutide across the blood–brain bar- tered once daily for the treatment of T2DM (Drucker and rier was indicated by the determination of the liraglutide Nauck 2006; Jacobsen et al. 2016). content in the brain via ELISA after intraperitoneal injec- In addition, liraglutide has extra benefits in weight tion in mice (Hunter and Holscher 2012). Nevertheless, loss, which motivates its use in treating obesity (Nuffer ELISA assay only allowed relative comparison of brain and Trujillo 2015). The mechanism by which liraglutide levels of liraglutide after saline injection and liraglutide exerts weight loss is attributed to its combinatorial effects injection instead of measuring its actual concentrations on the brain and the gut. While stimulating insulin and in the brain (Hunter and Holscher 2012). Direct assess- suppressing glucagon secretion, liraglutide inhibits gas- ment of brain distribution of liraglutide by quantifying its tric emptying as well as increases satiety and reduces exact concentration in the brain has never been pursued. appetite by binding to the GLP-1 receptors in the brain Moreover, a variety of research is ongoing toward (Nuffer and Trujillo 2015; Shah and Vella 2014; Turton the development of its analogs or other GLP-1 recep- et al. 1996). Thus, high-dose liraglutide (Saxenda ) was tor agonists. The development of longer-acting and dif - approved for the treatment of obesity by the US Food ferent types of dosage forms for liraglutide and other and Drug Administration, which became the most widely GLP-1 receptor analogs are of particular interest (Chen prescribed anti-obesity drug in 2020 (Neeland et al. et al. 2016; Meng et al. 2017). For example, dissolv- 2021). ing microneedle-assisted drug delivery system (Rabiei Furthermore, liraglutide has shown promising effects et al. 2021), longer-acting liraglutide precursor peptide in treating Alzheimer’s disease and cognitive impair- (Ahmadi et al. 2022), and controlled release thermogel ments, which are also likely to be mediated by GLP-1 formulation of liraglutide (Chen et al. 2016) have been receptors in the brain. After peripheral injection, lira- reported. The development of formulations with reduced glutide reduced plaque formation, protected memory frequency of injection and better patient compliance and synaptic plasticity, and decreased inflammation in would further protract the therapeutic utility of liraglu- the mouse model of Alzheimer’s disease (McClean et al. tide. For the research on new formulations and analogs 2011). Improvement in spatial working memory and res- of liraglutide, a robust bioanalytical method, as well as toration of episodic memory was also observed (Vargas- rigorous pharmacokinetic evaluation, is highly required. Soria et al. 2021). Oh et al. Journal of Analytical Science and Technology (2023) 14:19 Page 3 of 11 Recently, liquid chromatography–tandem mass spec- 50 µL of the IS working solution (50 ng/mL) in a 1.5 mL trometry (LC–MS/MS) assay has become a promising Protein LoBind Tubes (Eppendorf, Hamburg, Ger- alternative to LBAs for the bioanalysis of peptides and many). To prepare calibration samples in the brain tissue, protein drugs. LC–MS/MS has fundamental advantages, 50 µL of the rat brain tissue homogenate was spiked with including high sensitivity, specificity, reproducibility, and 50 µL of the liraglutide standard working solution and high throughput, and serves as the most favorable bio- 50 µL of the IS working solution (50 ng/mL). Following analytical technique for pharmacokinetic studies. Recent the addition of 150 µL of methanol as a precipitation sol- advances in LC–MS/MS have been overcoming its limi- vent, the mixture was mixed on a vortex mixer for 1 min tations of poor ionization, significant endogenous inter - and centrifuged at 15,000 × g for 10 min. An aliquot of ference, and low sensitivity in the bioanalysis of peptides 10 µL of the supernatant was injected onto the LC–MS/ and proteins (Dong et al. 2018; Meng et al. 2017; Pinho MS. et al. 2019; Ewles and Goodwin 2011). There are several Quality control (QC) samples were prepared to provide LC–MS/MS methods available for the determination of four concentration levels, i.e., high, medium, low, and liraglutide in the plasma (Dong et al. 2018; Meng et al. the lower limit of quantification (LLOQ), of liraglutide 2017; Sauter et al. 2019; Zhai et al. 2020). However, no based on the calibration standard ranges for each biologi- LC–MS/MS methods have been reported to determine cal matrix by using the same procedure as the calibration liraglutide concentrations in the tissues. standards. QC concentration levels were 800, 80, 4, and Therefore, the purpose of this study was to develop a 1 ng/mL for the plasma matrix, and 160, 40, 8, and 2 ng/ simple and sensitive LC–MS/MS analysis for the quanti- mL for the brain tissue homogenates. fication of liraglutide in the plasma as well as in the brain tissue. The LC–MS/MS analysis was fully validated in two Sample preparation different biological matrices and allowed to evaluate the An aliquot of 50 µL of biological samples, i.e., rat plasma pharmacokinetics and brain distribution of liraglutide in or brain tissue homogenates, was mixed with 50 µL of IS rats. The developed bioanalysis and its pharmacokinetic working solution (50 ng/mL) and 200 µL of methanol. information may be helpful in better understanding its After vortexing for 1 min, the mixture was centrifuged at action for further preclinical and clinical studies. 15,000 × g for 10 min. An aliquot of 10 µL of the superna- tant was injected onto the LC–MS/MS for analysis. Materials and methods LC–MS/MS Materials The LC–MS/MS analysis of liraglutide was performed on Liraglutide (99.2%) was purchased from Chengdu Sheng- an LC–MS/MS system consisting of Agilent 1260 HPLC nuo Biopharm Co., Ltd (Chengdu, China). Semaglu- (Agilent Technologies, Santa Clara, CA) and Agilent 6490 tide (98%), which was used as the internal standard (IS), mass spectrometer (Agilent Technologies). Chromato- was purchased from Zhejiang Peptites Biotech Co., Ltd graphic separation was performed on a bioZen 2.6 µm (Hangzhou, China). Formic acid was a product of Sigma- Peptide XB-C18 (100 × 2.1 mm, Phenomenex, Torrence, Aldrich Co. (St. Louis, MO). Other reagents, including CA) by using gradient elution. The mobile phase con - methanol, acetonitrile, and distilled water, were all high- sisted of 0.3% formic acid in distilled water (MP-A) and performance liquid chromatography (HPLC)-grade and 0.3% formic acid in acetonitrile (MP-B). The gradient elu - purchased from J.T. Baker, Inc. (Phillipsburg, NJ). tion profile and flow rate are shown in Table 1. The total Stock solutions, calibration standards, and quality control samples Table 1 Gradient elution condition for liraglutide LC–MS/MS The stock solution of liraglutide (1 mg/mL) was prepared by dissolving 5 mg of liraglutide in 5 mL of methanol. Time (min) Mobile phase A Mobile phase B Flow rate The stock solution of liraglutide was serially diluted with (mL/min) 0.3% Formic acid in 0.3% Formic acid methanol, yielding standard working solutions of 1000, distilled water (%) in acetonitrile (%) 500, 100, 50, 10, 5, 2, and 1 ng/mL for plasma and 200, 0.00 65 35 0.3 100, 50, 20, 10, 5, and 2 ng/mL for brain tissue homogen- 5.00 20 80 0.3 ates. Similarly, IS working solution at 50 ng/mL was pre- 5.01 0 100 0.3 pared by diluting the IS stock solution (1 mg/mL) with 9.00 0 100 0.3 methanol. 9.01 65 35 0.4 To prepare calibration samples of liraglutide in the 13.50 65 35 0.4 plasma, 50 µL of the rat blank plasma was spiked with 13.51 65 35 0.3 50 µL of the liraglutide standard working solution, and Oh et al. Journal of Analytical Science and Technology (2023) 14:19 Page 4 of 11 run time was 15 min, and the column oven temperature and conducted in accordance with the standard operat- was set at 50 °C. ing procedures (SOPs). The electrospray ionization (ESI) source was operated in the positive mode. The mass spectrometer was con - Pharmacokinetic studies ducted in the multiple reaction monitoring (MRM) mode Male Sprague–Dawley rats (7 weeks, 194–238 g; DBL, with a dwell time of 200 ms per MRM channel. For MRM Eumseong, Korea) were fasted overnight before drug analysis, the MS parameters were set as follows: gas tem- administration. Liraglutide dissolved in normal saline perature, 350 °C; gas flow, 17 L/min; nebulizer gas pres - was administered by intravenous (IV) injection via penile sure, 40 psi. The transition m/z ions for MRM analysis vein at 0.05 mg/kg (n = 5) or subcutaneous (SC) injec- were selected as follows: 938.5 → 1063.7 for liraglutide tion at 1 mg/kg (n = 5). The injection volume was 0.5 mL/ and 1029.4 → 1302.9 for semaglutide. The fragment volt - kg and 1 mL/kg for IV and SC injection, respectively. age was 380 V for both liraglutide and IS. The collision Approximately 0.3 mL of the venous blood samples was energy was set at 24 eV for liraglutide and 38 eV for IS. collected at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, and 36 h MassHunter Quantitative Analysis (Agilent Technolo- after drug administration directly from the jugular vein. gies) was used to process the mass spectrometric data. Plasma samples were obtained by centrifugation of the blood samples at 15,000 × g for 10 min. The obtained plasma samples were immediately frozen and stored at Assay validation − 70 °C until analysis. The assay was validated following the guidance published by US FDA (US FDA 2018) to demonstrate the specific - Brain distribution studies ity, sensitivity, linearity, accuracy, precision, extraction For brain distribution studies, liraglutide (1 mL/kg) was recovery, and stability. Specificity was evaluated by com - administered via IV injection at 5 mg/kg or SC injection paring the blank biological matrix, i.e., rat plasma and at 500 mg/kg. Brain tissues were excised at 1 h after IV brain tissue homogenates, spiked with the liraglutide injection or 12 h after SC injection of liraglutide (n = 6, and IS to the blank biological matrix. The linearity of the each). From the excised brain, parts of the brain, i.e., cer- method was evaluated over the concentration ranges of ebrum, cerebellum, and hypothalamus, were separated, 1–1000 ng/mL in the rat blank plasma and 2–200 ng/mL accurately weighed, and homogenized (Tissue Tearor, in the brain tissue homogenates. The calibration curve Biospec Products Inc., Bartlesville, OK) with appropriate for each biological matrix was constructed from the peak volumes of isotonic saline. The tissue homogenates were area ratios of liraglutide to IS versus theoretical concen- stored at − 70 °C until analysis. tration via the weighted regression method (1/x). Accuracy and precision were determined by assay- Data analysis ing the three replicates of matrix-matched QC samples The plasma concentration vs. time data were analyzed on the same day (intra-day) and three consecutive days by a non-compartmental method to obtain the pharma- (inter-day). Accuracy was calculated as the percentage cokinetic parameters of liraglutide. The bioavailability of the mean back-calculated concentration compared was calculated as the ratio of dose-normalized area under to the nominal concentration. Precision was defined as the plasma concentration–time curve (AUC ) after SC inf the coefficient of variance for each concentration. The injection and IV injection, i.e., Bioavailability = (AUC / sc extraction recovery was calculated by comparing the Dose )/(AUC /Dose ). The tissue-to-plasma partition sc iv iv peak responses of liraglutide and IS in the biological coefficient (K ) was calculated by the ratio of liraglutide matrices spiked before and after the extraction. The sta - concentration in the brain tissue homogenates to that in bility was examined under four different conditions as the plasma. follows: short-term, 4 h at room temperature; long-term, 7 days at − 70 °C; autosampler, 24 h in the autosampler; Statistical analysis freeze/thaw, 3 freeze/thaw cycles. All stability data were Statistical analyses were performed using one-way expressed as the percentage of the mean response of the ANOVA. Comparisons among different groups were fol - QC samples for stability tests vs. those of the freshly pre- lowed using Tukey post hoc analysis. Differences with pared samples. p < 0.05 were denoted as statistically significant. Application to in vivo studies Results and discussion The animal study protocol was approved by the Ethics An LC–MS/MS assay was developed and validated for Committee for the Treatment of Laboratory Animals at quantifying liraglutide concentrations in rat plasma as Sungkyunkwan University (SKKUIACUC2021-09-01-2) well as brain tissue. The application of the LC–MS/MS Oh et al. Journal of Analytical Science and Technology (2023) 14:19 Page 5 of 11 method was demonstrated through in vivo pharmacoki- 1029.4 → 1302.9 for IS were monitored. The selected netic studies to characterize the pharmacokinetics and MRM transition of liraglutide is consistent with literature brain distribution of liraglutide after IV and SC injections reports (Meng et al. 2017; Sauter et al. 2019). in rats. Chromatographic conditions were optimized to achieve the best chromatographic resolution of liraglu- tide with minimum matrix interference. BioZen 2.6 µm Sample preparation Peptide XB-C18 (100 × 2.1 mm, Phenomenex) column Liraglutide, like other peptides, is susceptible to adsorp- with a mobile phase consisting of 0.3% formic acid in tion to plastic or glass wares, which provides one of the distilled water and 0.3% formic acid in acetonitrile was major challenges for the development of bioanalyti- finally selected, which led to a symmetric and sharp peak cal method. Since adsorption is mostly unpredictable shape and less endogenous interference. However, sig- or more prominent at the lower concentrations, pep- nificant carryover was observed with the isocratic elu - tide adsorption results in unreliable and unreproducible tion, probably due to the adsorption of liraglutide to the results in the analysis (Verbeke et al. 2020). The linear - parts of the LC–MS/MS system. The gradient elution ity of the working standard solutions of liraglutide in the profile was therefore employed and optimized to prevent biological matrix is often poor due to the strong peptide inconsistent carryover (Table 1). The gradient profile in adsorption (Dong et al. 2018). Thus, we utilized Protein which the gradual increase of the organic phase for 9 min LoBind Tubes (Eppendorf ) during the sample prepara- followed by a quick return to the initial condition led to tion and optimized the subsequent analytical procedures reproducible peak response. Representative MRM chro- to minimize the peptide adsorption. By using the low matograms of liraglutide and IS in the biological matrices adsorption tubes, the impact of nonspecific adsorption of are shown in Fig. 2. The retention times of liraglutide and liraglutide could be minimized. IS in the rat plasma were 6.0 min and 5.5 min, respec- To extract liraglutide from the plasma samples, simple tively, in both plasma and brain tissue. protein precipitation with methanol was used. Compared to solid-phase extraction which has been applied in pre- vious studies (Meng et al. 2017; Shah et al. 2017), protein Method validation precipitation is advantageous because it is rapid, simple, Specificity, linearity, and sensitivity less labor-intensive, and economically favorable. Initially, Examination of the blank matrix and blank matrix spiked acetonitrile was tested as a precipitation solvent from the with liraglutide at LLOQ and upper limit of quantifica - rat biological samples for liraglutide. However, the use tion (ULOQ) indicated no interfering endogenous or of acetonitrile resulted in poor sensitivity and recovery, exogenous peaks at the retention times corresponding to likely due to the coprecipitation of liraglutide with plasma liraglutide and IS (Fig. 2). The calibration curve of lira - and tissue proteins (Dong et al. 2018). Thus, methanol glutide was linear over the calibration standard concen- was selected as a precipitation solvent, resulting in over tration range from 1 to 1000 ng/mL in the plasma and 92.17% recovery for liraglutide and over 92.61% for IS 2–200 ng/mL in the brain tissue homogenates with a cor- from the rat plasma and brain homogenates. relation coefficient > 0.999. The LLOQ of liraglutide was 1 ng/mL in the plasma and 2 ng/mL in the brain tissue homogenates, which were defined as the lowest concen - LC–MS/MS conditions tration in the calibration range. The sensitivity of lira - As the molecular weight of liraglutide is large glutide in the rat plasma in this study is compatible with (m.w. = 3751.2), multiply charged precursor ions are previous studies with LLOQ of 1 ng/mL (Meng et al. often selected for MRM analysis (Dong et al. 2018; Meng 2017; Zhai et al. 2020) or 0.5 ng/mL (Dong et al. 2018). et al. 2017; Sauter et al. 2019; Zhai et al. 2020). In the The present method provided sufficient sensitivity to present study, the most abundant precursor ion in the 4+ evaluate the pharmacokinetics and tissue distribution of positive Q1 mass scan spectrum was [M + 4H] at m/z 4+ liraglutide in rats. 938.5 for liraglutide and [M + 4H] at m/z 1029.4 for IS (semaglutide). Figure 1 shows the product ion mass spec- tra of protonated liraglutide and IS. The most prominent Accuracy and precision fragment ion was m/z 1063.7 of the protonated liraglu- Table 2 shows the accuracy and precision determined tide, and m/z 1302.9 for protonated IS. The m/z 1063.7 by using matrix-matched QC samples at four differ - 4+ fragment of [M + 4H] at m/z 938.5 for liraglutide has ent concentration levels for rat plasma and brain tissue been suggested as the doubly charged C-terminal frag- homogenates. The intra- and inter-day accuracies were ment after cleavage of the peptide bond between posi- 92.09–106.36% in the plasma and 91.91–105.19% in the tions 15 and 16 (Sauter et al. 2019). Therefore, the MRM brain tissue. The intra- and inter-day precisions were transitions of m/z 938.5 → 1063.7 for liraglutide and m/z within 10.99% and 12.52% in the plasma and brain tissue, Oh et al. Journal of Analytical Science and Technology (2023) 14:19 Page 6 of 11 4+ 4+ Fig. 1 Product ion spectra of protonated A liraglutide ([M + 4H] , m/z = 938.5) and B semaglutide ([M + 4H] , m/z = 1029.4) respectively. The obtained accuracy and precision of the post-extraction. Table 3 summarizes the calculated current assay satisfied the FDA guidance on bioanalytical extraction recovery of liraglutide and IS in the rat methods validation (US FDA 2018). plasma and brain tissue. The extraction recovery in the plasma was 92.17–104.59% for liraglutide and 92.61% for IS. The extraction recovery in the brain tis- Extraction recovery sue was 94.09–99.76% for liraglutide and 95.76% for IS. The extraction recovery was calculated as the ratio These extraction recoveries indicated that the extrac- of the average peak area obtained from three rep- tion of liraglutide in the rat plasma was efficient and licates of standard solutions at each QC concen- reproducible. tration level spiked in pre-extraction to those in Oh et al. Journal of Analytical Science and Technology (2023) 14:19 Page 7 of 11 Fig. 2 Representative chromatograms of liraglutide (left) and IS (right) obtained from A blank plasma, B blank brain tissue, C LLOQ concentration of liraglutide and IS in the plasma, D LLOQ concentration of liraglutide and IS in the brain tissue, E ULOQ concentration of liraglutide and IS in the plasma, and F ULOQ concentration of liraglutide and IS in the brain tissue Table 2 Intra- and inter-day accuracy and precision of liraglutide analysis in the rat plasma and brain tissue Matrix Concentration (ng/mL) Intra-day (n = 3) Inter-day (n = 3) Accuracy (%) Precision (%) Accuracy (%) Precision (%) Plasma 800 102.31 5.46 100.75 6.09 80 94.46 5.21 95.03 7.08 4 92.09 4.17 95.97 7.61 1 92.96 10.99 106.36 3.21 Brain 160 93.79 2.44 93.63 0.90 40 91.91 5.98 92.60 3.14 8 92.31 4.41 97.08 8.83 2 100.26 12.52 105.19 6.88 Stability was 96.93–111.61% in the plasma and 86.60–109.38% The stability evaluation of liraglutide determined under in the brain tissue, with no significant deviations in all four different storage conditions, i.e., short-term, long- the tested conditions. These data demonstrated that term, autosampler, and freeze/thaw cycles, is sum- liraglutide was stable for application in the routine marized in Table 4. The average stability of liraglutide analysis. Oh et al. Journal of Analytical Science and Technology (2023) 14:19 Page 8 of 11 Table 3 Extraction recovery (%) of liraglutide and the internal average half-life of 3.67 ± 0.40 h. After SC injection, standard (IS) in the rat plasma and brain tissue liraglutide plasma concentration gradually increased and reached its peak concentration at 2.00–6.00 h, then Matrix Analyte Concentration Extraction (ng/mL) recovery (%) decreased with an average half-life of 3.55 ± 0.51 h. The obtained half-life of liraglutide is well comparable with Plasma Liraglutide (n = 3) 800 96.58 the literature-reported half-life of liraglutide in rats, 80 92.17 i.e., 3.95–4.01 h (Dong et al. 2018; Zhai et al. 2020). In 4 104.59 humans, the slow absorption with T of about 12 h max IS (n = 9) 50 92.61 and extended half-life (approximately 13 h) of liraglutide Brain Liraglutide (n = 3) 160 98.33 were more recognizable (Jacobsen et al. 2016), which is 40 99.76 compatible in beagle dogs (Sauter et al. 2019). Finally, the 8 94.06 absolute bioavailability of liraglutide after SC injection IS (n = 9) 50 95.76 was 13.16% in rats. Pharmacokinetics of liraglutide in rats Brain distribution of liraglutide in rats The developed LC–MS/MS assay was applied to in vivo It has been suggested that the benefits of liraglutide for pharmacokinetic studies of liraglutide to assess the phar- weight loss and Alzheimer’s disease are associated with macokinetics and bioavailability of liraglutide in rats. Fig- GLP-1 receptors in the brain (McClean et al. 2011; Farr ure 3 shows the obtained plasma concentration vs. time et al. 2016; Adams et al. 2018; Lee and Kim 2020). How- profiles following IV and SC injections of liraglutide in ever, there is limited information regarding the distri- rats. The non-compartmental pharmacokinetic param - bution of liraglutide to the brain. Although it has been eters of liraglutide are summarized in Table 5. reported that liraglutide crosses the blood–brain barrier, Following IV injection, plasma concentrations of lira- but to a lesser extent than lixisenatide (Hunter and Hols- glutide showed a mono-exponential decline with an cher 2012), the brain distribution of liraglutide has never Table 4 Stability (%) of liraglutide in the rat plasma and brain tissue under four different conditions (mean ± SD, n = 3) Matrix Concentration Short-term stability Long-term stability Autosampler stability Freeze/thaw stability (ng/mL) (%) (%) (%) (%) Plasma 800 103.93 ± 2.51 104.84 ± 3.03 100.00 ± 3.39 100.98 ± 0.67 80 109.73 ± 4.77 106.74 ± 1.75 109.32 ± 2.14 103.62 ± 2.39 4 107.91 ± 8.80 111.61 ± 2.09 96.93 ± 2.54 102.14 ± 9.85 Brain 160 100.63 ± 1.45 103.64 ± 4.21 104.64 ± 3.02 103.07 ± 6.51 40 96.44 ± 1.96 95.28 ± 6.60 92.24 ± 8.69 86.60 ± 2.99 8 109.38 ± 9.94 90.42 ± 9.70 87.46 ± 11.79 92.85 ± 12.34 Short-term, 4 h at room temperature; long-term, 7 days at − 70 °C; autosampler, 24 h in the autosampler; freeze/thaw, 3 freeze/thaw cycles Fig. 3 Plasma concentration–time profiles of liraglutide following intravenous (IV, 0.05 mg/kg) and subcutaneous (SC, 1 mg/kg) injection in rats A on a linear plot and B on a semi-log plot (mean ± SD, n = 5) Oh et al. Journal of Analytical Science and Technology (2023) 14:19 Page 9 of 11 Table 5 Pharmacokinetic parameters of liraglutide following brain concentration of liraglutide was only detectable intravenous (IV, 0.05 mg/kg) and subcutaneous (SC, 1 mg/kg) after high doses of liraglutide either by IV (5 mg/kg) or injection in rats (mean ± SD, n = 5) SC (500 mg/kg) injections. Table 6 and Fig. 4 show the liraglutide concentration in the different brain regions at IV (n = 5) SC (n = 5) 1 h after IV (5 mg/kg) or 12 h after SC (500 mg/kg) injec- Dose (mg/kg) 0.05 1.00 tions in rats. The tissue concentrations are presented in t (h) 3.67 ± 0.40 3.55 ± 0.51 1/2 ng/g tissue, which was calculated as the measured con- C or C (ng/mL) 822.08 ± 227.19 669.64 ± 186.57 0 max centration in the tissue homogenate by LC–MS/MS (ng/ T (h)* – 3.00 (2.00–6.00) max mL) divided by the density of tissue homogenate (g tis- AUC (ng·h/mL) 3429.30 ± 875.27 9166.62 ± 2465.16 all sue/mL) (Song et al. 2022). The corresponding plasma AUC (ng·h/mL) 3505.33 ± 815.04 9223.30 ± 2417.73 inf concentration was 102,801.74 ± 6627.17 ng/mL and CL or CL/F (mL/min/kg) 0.25 ± 0.06 1.92 ± 0.52 181,525.94 ± 8838.02 ng/mL after IV and SC injections, V (mL/kg) 73.12 ± 20.68 – ss respectively. Bioavailability – 13.16% Following either IV or SC injection, the brain tissue- *Data were presented as the median (minimum–maximum) to-plasma partition coefficient (K ) of liraglutide was estimated as less than 0.00031, indicating the insig- nificant distribution of liraglutide to the brain. On the been directly evaluated by measuring its exact concentra- other hand, the concentrations of liraglutide were dif- tion in the brain. ferent in the different regions of the brain. In the brain, Therefore, the potential utility of this LC–MS/MS assay liraglutide concentrations were the highest in the hypo- to evaluate the brain distribution of liraglutide has been thalamus, followed by the cerebellum and cerebrum. demonstrated by in vivo tissue distribution studies. As Significant distribution of liraglutide to the hypothala - the literature suggested (Hunter and Holscher 2012), mus compared to other brain regions is consistent Table 6 Distribution of liraglutide in the different regions of the brain following intravenous (IV, 5 mg/kg) and subcutaneous (SC, 500 mg/kg) injections in rats Brain region IV (5 mg/kg, n = 6) SC (500 mg/kg, n = 6) Concentration (ng/g) K Concentration (ng/g) K p p Cerebrum 16.00 ± 4.51 0.00016 ± 0.00005 22.14 ± 7.34 0.00012 ± 0.00004 † † Cerebellum 26.30 ± 6.84 0.00026 ± 0.00007 26.26 ± 6.38 0.00014 ± 0.00003 Hypothalamus 31.62 ± 5.74* 0.00031 ± 0.00004* 32.31 ± 9.28 0.00018 ± 0.00005 p < 0.05 versus cerebrum; *p < 0.05 versus cerebrum and cerebellum Fig. 4 Brain distribution of liraglutide in rats represented by A brain concentration and B tissue-to-plasma partition coefficient (K ) following intravenous (IV ) injection and C brain concentration, and D K following subcutaneous (SC) injection in rats (mean ± SD, n = 6). p < 0.05 versus cerebrum; *p < 0.05 versus cerebrum and cerebellum Oh et al. Journal of Analytical Science and Technology (2023) 14:19 Page 10 of 11 Author details with the previous studies using labeled liraglutide School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi 16419, in mice (Secher et al. 2014; Gabery et al. 2020). Fol- Korea. College of Pharmacy, Wonkwang University, Iksan, Jeonbuk 54538, 3 4 lowing peripheral injection of labeled liraglutide, i.e., Korea. D&D Pharmatech, Seongnam, Gyeonggi 13486, Korea. College VT750 VT750 of Pharmacy, Daegu Catholic University, Gyeongsan, Gyeongbuk 38430, Korea. liraglutide , the signal intensity of liraglutide had distributed primarily to circumventricular organs Received: 13 February 2023 Accepted: 6 March 2023 and parts of the hypothalamus (Secher et al. 2014; Gabery et al. 2020). References Adams JM, Pei H, Sandoval DA, Seeley RJ, Chang RB, Liberles SD, et al. 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Journal of Analytical Science & Technology – Springer Journals
Published: Mar 20, 2023
Keywords: Glucagon-like peptide-1; Liraglutide; LC–MS/MS; Pharmacokinetics; Bioavailability; Brain distribution
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