Download Results Introduction Method Conclusions Acyl migration

Assessing the Reactivity of Acyl Glucuronide Metabolites
Hokkanen J, Komu M & Tolonen A.
Admescope Ltd, Oulu, Finland www.admescope.com
Introduction
Results
The goal of this work was to set up an assay to assess the reactivity of acyl glucuronide
metabolites in liver microsomes. With the aid of UPLC/TOF-MS the isomeric AGs
formed via acyl migration can be separated. Furthermore the disappearance by
hydrolysis can be differentiated from acyl migration.
Degradation of Zomepirac 1-O-β-AG
Peak area, % of 0 min
Glucuronide conjugation of the drugs is most commonly considered as a detoxification
route, but in the presence of carboxylic acid moiety in a parent drug or a metabolite,
reactive acyl glucuronide conjugates (AGs) can be formed. These conjugates can
mediate idiosyncratic toxicity by binding to cellular enzymes, proteins and DNA. The
glucuronide moiety of acyl glucuronides migrates to form several isomeric products,
including an aldehyde form. The disappearance rate of acyl glucuronide metabolites is
shown to correlate with the toxicity risk. 1,2,3
100
1-O-b-AG
1-O-b-AG relative
other AGs
50
0
0
1
2
3
Fig 2. Peak areas of 1-O-acyl glucuronides relative
to peak area at 0 min.
Disappearance of 1-O--AG
is illustrated with black,
formation of isomeric acyl
glucuronides by acyl migration with red and relative
disappearance of 1-O--AG
with blue.
4
Incubation time (h)
Acyl migration
HOOC
HO
HO
O
O
OH
R
HOOC
HO
HO
O
OH
R
O
O
Table 1: Half-lives for 1-O--acyl glucuronides of reference compounds
HOOC
HO
O
O
HOOC
HO
O
OH
OH
O
HOOC
HO
N
O
Protein
R
O
O
OH
O
OH
OH
R
O
OH
R
Fig 1. An isomerization process in which the acyl group migrates from one position to another
one, revealing reactive aldehyde functionality, is called acyl migration. Acyl group is
highlighted in red and aldehyde formation/ protein binding in blue.
Category1-2
Compound
Observed
T1/2 (h)
Relative
T1/2 (h)a
T1/2 (h)
in buffer (pH 7.4)1-2
Montelukast
4.3
NA, > 40
37.5
Safe
Repaglinide
4.8
13.7
11.5
Safe
Valproic acid
4.4
29.8
79
Safe
Gemfibrozil
6.2
NA, > 40
44 - 71.4
Safe
Telmisartan
3.7
32.3
26 - 45.6
Safe
Diclofenac
0.2
0.3
0.5 - 0.7
Warning
Indomethacin
1.2
2.2
1.4 - 1.7
Warning
S-Naproxen
2.0
4.4
1.8 - 2.2
Warning
Tolmetin
0.5
0.4
0.3 - 0.4
Warning
Furosemide
2.6
3.1
3.2 - 5.3
Warning
Isoxepac
0.6
0.6
0.3
Withdrawn
Zomepirac
0.5
0.6
0.4 - 0.5
Withdrawn
a) Hydrolysis
is neglected, NA = by hydrolysis only, no acyl migration observed
Method
Conclusions
The study compounds were incubated at 100 µM concentration with pooled human
liver microsomes (1.0 mg/ml) in the presence of cofactor UDPGA (1 mM) for 40 min for
the formation of AG conjugate. These pre-incubations were diluted with a phosphate
buffer containing UDP to suppress the formation of AG in the secondary incubation.
The secondary incubations were sampled up to 6h and the samples were analysed
using UPLC/TOF-MS to monitor the chemical stability of the AG formed in the
microsomal incubation. The observed data was used to calculate the half-lives of the
acyl glucuronide metabolites. In addition, half-lives based on acyl migration only were
calculated to eliminate the effect of ester hydrolysis (chemical or esterase based) from
the disappearance rate, by using the relation of the initial 1-O-β-AG to the total
abundance of all isomeric AGs.
The stability of 1-O--AGs formed in the human liver microsomal incubations were
studied and compared to the literature values. The safe compounds possess longer
half-lives and thus can be differentiated from compounds with known toxicity risks.
The relative half-lives for 1-O--AGs were calculated relative to combined peak area of
all AGs to neglect the effect of disappearance caused by hydrolysis, which occured due
to the microsome-originated esterase enzymes present in the secondary incubations.
With these values the difference between safe and toxic compounds was even more
pronounced, than with observed half-lives of initial 1-O--AGs only.
The method is a valuable tool for assessing the reactivity of acyl glucuronide
metabolites without a need for synthetized glucuronide metabolites.
The relative degradation rate of 1-O-β-AG was determined by fitting the data to equation:
𝐴1−𝑂−𝛽−𝐴𝐺
(
) = 𝐴0 ∗ 𝑒 −𝐾𝑡
𝐴𝐴𝐺,𝑡𝑜𝑡𝑎𝑙
where A1-O-β-AG is the abundance of 1-O-β-acyl glucuronide at each time point and AAG,total is the abundance of
all acyl glucuronides (1-O-β-AG and acyl migration products) at each time point. A0 is the abundance of 1-Oβ-AG in the beginning of secondary incubation, K is the degradation rate constant and t is the incubation
time in secondary incubation. Fitting was done using GraphPad Prism 5.04 software (GraphPad Software Inc).
References:
1. Sawamura R. et al (2010) Drug Metab Disposit, 38; 1857- 1864.
2. Ebner T. et al (1999) Drug Metab. Dispos. 27; 1143-1149.
3. Chen Z. et al (2007) J Pharm Toxicol Meth. 55; 91-95
The half-life for 1-O-β-acyl glucuronide was obtained from the degradation rate constant:
𝑡1/2 = 𝑙𝑛2/𝐾
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