Characterization of the global metabolic profile of Canagliflozin in Rat plasma, urine and feces based on HPLCUV-MS Analysis.
Rawa Akasha1*, Abdul Wahab Allaf2, Mohammad Amer Al-Mardini1
1Department of Pharmaceutical, Chemistry and Quality Control, Faculty of Pharmacy, Damascus University, Damascus, Syria.
2Department of Pharmaceutical Chemistry and Quality Control, Faculty of pharmacy, Arab International University, Ghabaghib, Daraa, Syria.
*Corresponding Author E-mail: Rawaokas@gmail.com
ABSTRACT:
Canagliflozin is a newly approved drug for type II diabetes mellitus, in the present work, a strategy based on high performance liquid chromatography combined with mass spectrometry (HPLC-UV/MS) method was proposed to systematically characterize the in vivo metabolites of canagliflozin in Rats. After oral administration of Canagliflozin to rats in a single dose of 3mg/kg, the rat plasma, urine, feces samples were collected and used in order to to determine metabolites. As a result of that and besides the parent drug, a total of 12 metabolites of canagliflozin were detected and identified.it was indicated that the metabolic pathways of canagliflozin in rats included: oxidation (as a main rout), glucuronation, and hydroxylation The result could provide valuable information regarding the metabolism of canagliflozin in rats, which contribute to better understanding of its action mechanism.
KEYWORDS: canagliflozin; metabolites; diabetes; HPLC-UV/MS; sodium glucose co‐transporter 2 inhibitor.
1. INTRODUCTION:
Canagliflozin (Fig.1) was the first SGLT2 inhibitor approved in the United States for the treatment of T2DM. Canagliflozin is a potent inhibitor of SGLT2 activity and has low potency SGLT1 inhibitory activity. )2,3(
Canagliflozin inhibits re-absorption of filtered glucose from the lumen of the proximal tubules of the kidney, thereby leading to lowered renal threshold for glucose and increased urinary glucose excretion (4-7-8)
In human, Absorption of canagliflozin is rapid with peak plasma concentration
(C max) achieved within 1-2 h following oral administration. After multiple dose administration, steady state levels were attained in 4-5 days (4-8-9).
Absolute oral bioavailability of canagliflozin is 65%, and high bound to plasma protein (99%), which is mainly albumin (10).
Following single dose oral administration, the terminal half-life was in the range of 10.6-13.1 h with a large volume of distribution of 119L (8-11).
It is extensively metabolized via O-glucuronidation by the enzymes uridine diphosphate-glucuronosyltransferase1A9 (UGT1A9) and UGT2B4 to O-glucuronide metabolites, M5 and M7, and to a smaller extent by cytochrome P450
(CYP3A4) (M9) )13(.
About 33% of metabolites are eliminated in the urine and approximately 42% are excreted in the feces. (14-13) Up to now, few study on metabolism of canagliflozin in rats has been reported (15-16), therefore, it is crucial and valuable to establish a sensitive and effective analytical method for systematic study on the metabolic profile of canagliflozin in rat plasma, urine and feces after oral administration.
Fig.1: Chemical structure of canagliflozin hemihydrate.
In this study, a strategy for systematic study of the metabolic profile canagliflozin in rat plasma, urine, and feces after oral administration was illustrated based on HPLC-MS and the results of this study may provide meaningful information for further efficacy and safety evaluation of canagliflozin.
2. EXPERIMENTAL:
Canagliflozin standard was purchased from topfond pharmaceutical CO, LTD. APIS, Methanol (HPLC grade) and Acetonitrile (HPLC grade), formic acid (HPLC grade) ammonium acetate were purchased from SIGMA-ALDRICH®. HPLC water was obtained by Siemens Water Technologies LaboStar. All other chemicals and reagents were of analytical grade and commercially available which were used as received.
2.2 Animal experiments:
Male Sprague dawley rats (250 g) were provided by Gemraya Scientific Research Center. Before administration, the rats were acclimated in a controlled animal room for 7 days with standardized humidity (50±10%), temperature (22±2C0). Purified water and standard food were provided freely.
Twenty rats were randomly divided into groups of 5 rats each. canagliflozin was suspended in 0.5% sodium carboxymethyl cellulose and intragastric administrated to rats at a single dose of 3mg/kg. Each group was held in a separate metabolic cage to collect plasma, urine and feces samples from 0 to 24 h after administration of dosing. All samples were frozen at -80OC until analysis.
2.3 Sample preparation:
The feces samples were dried in the dark on filter paper at ambient temperature, weighted and extracted by Ultra sonication using 6 volume of methanol for 15min. then, it was centrifuged at 5000 rpm for 15 min. subsequently, and the supernatant was injected into the LC-MS system for analysis after centrifuge filtration through 0.45mm nylon filter.
The urine samples were extracted with 6 volumes of methanol to precipitate protein, methanolic extracts were vortex-mixed and centrifuged at 5000 rpm for 10 min. Supernatants were evaporated to dryness under a stream of nitrogen, and residues were reconstituted of water-methanol (9:1). Drug-derived materials were solubilized by sonication and vortex-mixing before centrifuge filtration of samples through a 0.45-mm nylon filter.
The rat plasma was mixed with 2 volumes methanol by vortexing for 5 min, the mixture was centrifuged at 5000 rpm for 5 min to separate precipitated proteins, and then the supernatant solution was dried under nitrogen stream. The residue was re-dissolved in 100µl methanol for HPLC-UV/MS analysis.
2.4 Instrument and analytical conditions:
All analyses were performed on a SHIMADZU LC-MS prominence system (Shimadzu, Japan) provided with UV-Vis Detector SPD 20A, MS Detector 2020, binary pump: LC/20AD, degasser, manual injector with an injection loop of 10µl and column heater-cooler CTO-20A, the chromatographic separation was achieved on C18 column (250 x 4.6 mm, 5µm particle size) for feces, urine and plasma analysis. System control and data analysis were carried out using LabSolutions CS (Schimadzu, Japan).
For Rats feces extracts, the mobile phase, consisting of water with 0.1% formic acid (A) and methanol with formic acid 0.1%(B), was delivered using a gradient program as follows: [Time (min)/ Pump B Value (%)] [0.01/15, 15/50, 20/75, 25/100 and 40/100], with a flow rate of 0.5ml/min
For rat’s plasma and urine, the mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B) with a flow rate of 0.5 mL/min. The gradient elution program was as follow:
[Time (min)/ Pump B Value (%)] [0.01/0, 5/5, 10/20, 14/70 and 23/100], the column was then equilibrated for 15 minutes with 0% A.
The column temperature was maintained 250C, and the injection volume for all analyses was 10µl.
The analysis was performed in positive and negative electro-spray ionization mode ESI±, the ion source voltage was 5000 V, the source temperature was 450 °C, the curtain gas flow was 15 psi, and full scan mass range was 100-1500 Da.
The LC-MS method was developed and validated as per ICH guidelines and according to USP 35 guideline recommendations.
3. RESULTS AND DISCUSSIONS:
3.1 Metabolic profile analysis based on LC-MS:
All urine, feces and plasma samples were detected and analyzed under the HPLC-UV/MS conditions mentioned above. The chemical structures of metabolites were characterized according to the retention behaviors, MS data, relevant drug biotransformation knowledge, the blank and medicated biological samples comparison and the fragmentation pattern of the prototype.
The base peak ion chromatograms of feces, plasma and urine and samples were presented in Figs:2,3,4 respectively
As a result of that, besides Canagliflozin a total of 5 metabolites in feces, 3 in plasma 4 in urine were detected.
The observed masses, elemental compositions and characteristic fragment ions of the proposed metabolites were all listed in Table 1
Table.1 Identified metabolites of canagliflozin based on LC-UV/MS analysis and previous study
|
Metabolite |
M/Z |
Diagnostic product Ions |
formula |
Metabolite description |
Matrix |
|
M1 |
654 |
624,521,443,371,326 |
C24H25FO6S |
Oxidation Glucuronidation |
U,F |
|
M5 |
638 |
585,529,482,456,398,374,330,274,224,182,141 |
C₃₀H₃₃FO₁₁S |
O-glucuronidation |
P |
|
M6 |
476 |
467, 459,454,325, |
C24H23FO6S |
Carboxylation |
F,U |
|
M7 |
638 |
600,585,529,456,398,374,330 |
C₃₀H₃₃FO₁₁S |
O-glucuronidation |
P |
|
M8 |
478 |
469,458,441,428,407 |
C24H25FO6S |
Hydroxylation |
F,U |
|
M9 |
478 |
460,446,434,423,408 |
C24H25FO6S |
Hydroxylation |
P,U,F |
|
M12 |
492 |
487,474,454,430,410,403 |
C24H25FO7S |
Oxidation |
F |
|
CANA |
479 |
445,432,409,377,367,347, 265, 231,143 |
C24H25FO5S |
Parent |
F,P,U |
|
|
|
|
|
|
|
|
M1 |
|
|
M6 |
|
|
M8 |
|
|
M9 |
|
|
M12 |
|
Fig.2.: Product ion mass spectra of feces metabolites
|
M5 |
|
|
M7 |
|
|
M9 |
|
Fig.3.Product ion mass spectra of Plasma metabolites
|
M1 |
|
|
M6 |
|
|
M8 |
|
|
M9 |
|
Fig.4.Product ion mass spectra of urine metabolites
|
|
|
* |
|
|
|
|
|
|
*(M9,M12 were too low to quantify)
Fig.5: Liquid chromatography-UV of Canagliflozin and its metabolites in Rat feces*, Plasma, urine
3.2 Mass spectral fragmentation of Canagliflozin:
In this study both positive and negative ESI (Electrospray ionization) modes were used to obtain higher responses of canagliflozin and other relative compounds in the MS spectra, and authentic canagliflozin was characterized by signal of deprotonated molecule([M-H]-) at m/z 479 (Fig.5,6) which gave production at m/z 445 (C24H26O5NFS) from loss of NH3, which further undergoes sequential loss of H2O to form ions at m/z 409 (C24H22O3NFS), and 377. Product ions at m/z 367 (C22H20O2FS), and 347 (C22H18OFS) were postulated to derive from cleavage of the 6-(hydroxymethyl) tetrahydro-2H-pyran-3,4,5-triol moiety based on chemical formula from accurate mass measurements.
The product ion at m/z 265 undergoes sequential losses of H2O to form ions at 231 (C14H15O3). Alternatively, the product ion at m/z 265 undergoes cleavage of the 6-(hydroxymethyl) tetrahydro-2H-pyran-3,4,5-triol moiety to form ions at m/z 173 (C12H11O) and 143 (C10H11O). (Fig.6,7)
|
|
|
|
Fig.6 Product ion mass spectra of canagliflozin and Extracted ion chromatogram (EICs) in rat plasma
|
|
|
|
Fig.7 Product ions mass spectra of canagliflozin in rat feces.
3.3 Identification of metabolites:
A total of 12 metabolites of canagliflozin were detected and tentatively identified. Among them Canagliflozin and its metabolites were initially identified by HPLC-UV/MS in rat feces, plasma and urine (Figure 2,3,4). In addition, It has been detected two O-glucuronide metabolites (M5, M7), two kinds of hydroxylation metabolites (M8 and M9), one carboxylation metabolite(M6), one oxidation and glucuronide metabolite (M1).and finally one oxidation metabolite (M12)
We founded that the major metabolites were in rats feces (65%, Fig.5)
The concentrations of M5, M7 and M9 were high in rat plasma after 4 hours, and the M5 level declined markedly after 8 hours. None of these metabolites was detectable after 24 hours.
M6 carboxy metabolite was the main metabolite in rats (42%, Fig.5).
4. CONCLUSION:
The investigation of the metabolic profile of canagliflozin in vivo based on HPLC-UV/MS has been carried out. After intragastric gavages to male rats, 12 metabolites including 5 in feces, 3 in plasma, 4 in urine.
Results showed that metabolic pathways of canagliflozin in rats included:
O- glucuronide, glucuronation oxidation and hydroxylation fecal excretion was the primary elimination route of canagliflozin and its metabolites in animals (M1,M6,M8,M9,M12).urinary excretion was a minor elimination pathway for canagliflozin and metabolites in rats.The components detected in rat urine were M1, M6, M8, and M9.
Oxidation is the major metabolic pathway which yielding metabolites differing from human glucuronide metabolites in vivo (M1, M6, M8, M12).
5. ACKNOWLEDGMENT:
The authors are thankful to the colleagues at the central laboratory in the faculty of science for the cooperation to perform this work.
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Received on 15.07.2019 Modified on 29.08.2019
Accepted on 26.09.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(1): 399-403.
DOI: 10.5958/0974-360X.2020.00078.5