Identification of novel metabolic pathways of sitagliptin (STG) by LC/MS and LC/MS2 after incubations with rat hepatcoytes

Osama IG Khreit, Helen M Grant, Catherine Henderson, David G Watson, Oliver B Sutcliffe

Research output: Contribution to journalArticle

Abstract

Sitagliptin (STG), a drug for treating Type Ii Diabetes Mellitus (T2DM), has been associated with severe joint pain in some patients. In this paper the metabolic profile of the drug has been investigated in order to determine metabolism and formation of reactive compounds which may contribute to this adverse effect. Metabolism of STG was investigated in vitro by incubation with freshly isolated Sprague-Dawley rat hepatocytes, to characterize Phase I and II metabolites, and the reaction mixture analysed on a zwitter ionic hydrophilic interaction (ZIC®-HILIC) column using LC-MS and LC-MS2 utilising electrospray ionization (ESI) in the positive ion mode. STG was metabolised to yield eleven metabolites, but in total only 3.1% of the parent drug was metabolised over 2 hrs incubation. These metabolites were structurally characterized on the basis of accurate mass analyses and the major metabolic routes for STG determined to be via aromatic oxidation (0.86%) and desaturation of N-C and C-C of the piperazine (0.44%). Novel metabolites of STG detected using these methods included STG N-glucuronide (M6) and a di-ketone metabolite (M4), hydroxylation of both the amine group and aromatic ring followed by formation of glucuronide metabolites (M5, M5’), oxidative desaturation of NH2 and di-hydroxylation of metabolites followed by loss of HF. Also, observed was an N-sulfate metabolite (0.07%) and acetylation followed by glucuronide conjugation was also found in trace amounts (<0.01%). MS2 fragment ions provide additional structural confirmation providing a possible structure for most metabolites such as by fragment ion loss of the glucuronide group (176 Da) from metabolite M5 and loss of the phenolic sulfate (80 Da) of N-Sulfate metabolite (M7). Reduction reaction of piperazine ring probably generates highly electrophilic metabolite of STG, which may be susceptible to produce adverse effects. Furthermore, N-oxidation reaction forming reactive intermediates metabolic to give a hydroxylamine metabolite that may undergo further reactions to yield electrophilic intermediate metabolites.
LanguageEnglish
Article number1000220
Number of pages7
JournalDrug Metabolism and Disposition
Volume7
Issue number4
DOIs
Publication statusPublished - 30 Dec 2016

Fingerprint

Metabolic Networks and Pathways
Glucuronides
Sulfates
Ions
Hydroxylation
Pharmaceutical Preparations
Hydroxylamine
Metabolome
Arthralgia
Acetylation
Sitagliptin Phosphate
Ketones
Hydrophobic and Hydrophilic Interactions
Type 2 Diabetes Mellitus
Amines
Sprague Dawley Rats
Hepatocytes
Parents

Keywords

  • sitagliptin
  • STG
  • joint pain
  • dipeptidyl peptidase 4
  • DPP-4
  • metabolism
  • HILIC
  • mass spectrometers

Cite this

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title = "Identification of novel metabolic pathways of sitagliptin (STG) by LC/MS and LC/MS2 after incubations with rat hepatcoytes",
abstract = "Sitagliptin (STG), a drug for treating Type Ii Diabetes Mellitus (T2DM), has been associated with severe joint pain in some patients. In this paper the metabolic profile of the drug has been investigated in order to determine metabolism and formation of reactive compounds which may contribute to this adverse effect. Metabolism of STG was investigated in vitro by incubation with freshly isolated Sprague-Dawley rat hepatocytes, to characterize Phase I and II metabolites, and the reaction mixture analysed on a zwitter ionic hydrophilic interaction (ZIC{\circledR}-HILIC) column using LC-MS and LC-MS2 utilising electrospray ionization (ESI) in the positive ion mode. STG was metabolised to yield eleven metabolites, but in total only 3.1{\%} of the parent drug was metabolised over 2 hrs incubation. These metabolites were structurally characterized on the basis of accurate mass analyses and the major metabolic routes for STG determined to be via aromatic oxidation (0.86{\%}) and desaturation of N-C and C-C of the piperazine (0.44{\%}). Novel metabolites of STG detected using these methods included STG N-glucuronide (M6) and a di-ketone metabolite (M4), hydroxylation of both the amine group and aromatic ring followed by formation of glucuronide metabolites (M5, M5’), oxidative desaturation of NH2 and di-hydroxylation of metabolites followed by loss of HF. Also, observed was an N-sulfate metabolite (0.07{\%}) and acetylation followed by glucuronide conjugation was also found in trace amounts (<0.01{\%}). MS2 fragment ions provide additional structural confirmation providing a possible structure for most metabolites such as by fragment ion loss of the glucuronide group (176 Da) from metabolite M5 and loss of the phenolic sulfate (80 Da) of N-Sulfate metabolite (M7). Reduction reaction of piperazine ring probably generates highly electrophilic metabolite of STG, which may be susceptible to produce adverse effects. Furthermore, N-oxidation reaction forming reactive intermediates metabolic to give a hydroxylamine metabolite that may undergo further reactions to yield electrophilic intermediate metabolites.",
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year = "2016",
month = "12",
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Identification of novel metabolic pathways of sitagliptin (STG) by LC/MS and LC/MS2 after incubations with rat hepatcoytes. / Khreit, Osama IG; Grant, Helen M; Henderson, Catherine; Watson, David G; Sutcliffe, Oliver B.

In: Drug Metabolism and Disposition, Vol. 7, No. 4, 1000220, 30.12.2016.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Identification of novel metabolic pathways of sitagliptin (STG) by LC/MS and LC/MS2 after incubations with rat hepatcoytes

AU - Khreit, Osama IG

AU - Grant, Helen M

AU - Henderson, Catherine

AU - Watson, David G

AU - Sutcliffe, Oliver B

PY - 2016/12/30

Y1 - 2016/12/30

N2 - Sitagliptin (STG), a drug for treating Type Ii Diabetes Mellitus (T2DM), has been associated with severe joint pain in some patients. In this paper the metabolic profile of the drug has been investigated in order to determine metabolism and formation of reactive compounds which may contribute to this adverse effect. Metabolism of STG was investigated in vitro by incubation with freshly isolated Sprague-Dawley rat hepatocytes, to characterize Phase I and II metabolites, and the reaction mixture analysed on a zwitter ionic hydrophilic interaction (ZIC®-HILIC) column using LC-MS and LC-MS2 utilising electrospray ionization (ESI) in the positive ion mode. STG was metabolised to yield eleven metabolites, but in total only 3.1% of the parent drug was metabolised over 2 hrs incubation. These metabolites were structurally characterized on the basis of accurate mass analyses and the major metabolic routes for STG determined to be via aromatic oxidation (0.86%) and desaturation of N-C and C-C of the piperazine (0.44%). Novel metabolites of STG detected using these methods included STG N-glucuronide (M6) and a di-ketone metabolite (M4), hydroxylation of both the amine group and aromatic ring followed by formation of glucuronide metabolites (M5, M5’), oxidative desaturation of NH2 and di-hydroxylation of metabolites followed by loss of HF. Also, observed was an N-sulfate metabolite (0.07%) and acetylation followed by glucuronide conjugation was also found in trace amounts (<0.01%). MS2 fragment ions provide additional structural confirmation providing a possible structure for most metabolites such as by fragment ion loss of the glucuronide group (176 Da) from metabolite M5 and loss of the phenolic sulfate (80 Da) of N-Sulfate metabolite (M7). Reduction reaction of piperazine ring probably generates highly electrophilic metabolite of STG, which may be susceptible to produce adverse effects. Furthermore, N-oxidation reaction forming reactive intermediates metabolic to give a hydroxylamine metabolite that may undergo further reactions to yield electrophilic intermediate metabolites.

AB - Sitagliptin (STG), a drug for treating Type Ii Diabetes Mellitus (T2DM), has been associated with severe joint pain in some patients. In this paper the metabolic profile of the drug has been investigated in order to determine metabolism and formation of reactive compounds which may contribute to this adverse effect. Metabolism of STG was investigated in vitro by incubation with freshly isolated Sprague-Dawley rat hepatocytes, to characterize Phase I and II metabolites, and the reaction mixture analysed on a zwitter ionic hydrophilic interaction (ZIC®-HILIC) column using LC-MS and LC-MS2 utilising electrospray ionization (ESI) in the positive ion mode. STG was metabolised to yield eleven metabolites, but in total only 3.1% of the parent drug was metabolised over 2 hrs incubation. These metabolites were structurally characterized on the basis of accurate mass analyses and the major metabolic routes for STG determined to be via aromatic oxidation (0.86%) and desaturation of N-C and C-C of the piperazine (0.44%). Novel metabolites of STG detected using these methods included STG N-glucuronide (M6) and a di-ketone metabolite (M4), hydroxylation of both the amine group and aromatic ring followed by formation of glucuronide metabolites (M5, M5’), oxidative desaturation of NH2 and di-hydroxylation of metabolites followed by loss of HF. Also, observed was an N-sulfate metabolite (0.07%) and acetylation followed by glucuronide conjugation was also found in trace amounts (<0.01%). MS2 fragment ions provide additional structural confirmation providing a possible structure for most metabolites such as by fragment ion loss of the glucuronide group (176 Da) from metabolite M5 and loss of the phenolic sulfate (80 Da) of N-Sulfate metabolite (M7). Reduction reaction of piperazine ring probably generates highly electrophilic metabolite of STG, which may be susceptible to produce adverse effects. Furthermore, N-oxidation reaction forming reactive intermediates metabolic to give a hydroxylamine metabolite that may undergo further reactions to yield electrophilic intermediate metabolites.

KW - sitagliptin

KW - STG

KW - joint pain

KW - dipeptidyl peptidase 4

KW - DPP-4

KW - metabolism

KW - HILIC

KW - mass spectrometers

U2 - 10.4172/2157-7609.1000220

DO - 10.4172/2157-7609.1000220

M3 - Article

VL - 7

JO - Drug Metabolism and Disposition

T2 - Drug Metabolism and Disposition

JF - Drug Metabolism and Disposition

SN - 0090-9556

IS - 4

M1 - 1000220

ER -