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Hi all,
I have a question to pose. I am working with a compound that forms a
glucuronide metabolite. It is not confirmed but I am sure it is an
acyl glucuronide. The site of modification was narrowed down by
product ion comparison of parent and metabolite.
In order to develop an in-vitro screening model for these compounds,
the metabolite was synthesized in-vitro (HLM) and at the end of
incubation the supernatant was centrifuged to remove microsomes and
incubated with HSA (1.5 mg/mL) and aliquots were taken at 0, 1, 3 and
23 hour. Two aliquots were taken for all time points, first aliquot
was analyzed neat and second aliquot was protein precipitated with
ACN (1:1) and supernatant was evaporated under nitrogen and
reconstituted to original volume with saline. In summary, the
biosynthesis of metabolite was performed and followed by reactivity
check with HSA.
First aliquot (neat) was analysed by QTOF-MS and UV for parent,
metabolite and HSA. A second aliqout (ppt and dried down) was
analyzed for levels of parent, metabolite by MRM. Results were
similar for both aliquots in terms of parent and metabolite level.
Here are the findings, at the time T0 there is about 45% glucuronide
metabolite formed and about 55% parent left. Upon incubation with
HSA, a time dependent degradation was observed for glucuronide
metabolite from 0, 1, 3 and 23 hour. At 23 hour the metabolite level
was not at detected level. Interestingly, the parent level (peak
area) was increased from 0, 1, 3, and 23 hour. The parent level in
control sample didn't change upon incubation with HSA. The comparison
of multiply charged MaxEnt profile spectrum of HSA at time 0, 1, 3
and 23 hours were compared with HSA standard (MW:66400 Da). There was
no mass shift observed for these samples when compared with HSA
standard. The parent (M+H)+ at 428 m/z. The glucuronide metabilite (M
+H)+ at 604 m/z. Similar results were seen for both aliquots.
A control sample was prepared in parallel and treated identically
except that no cofactor was added during incubation with microsomes.
Upon incubation with HSA the parent level didn't change from T0 to 1,
3, and 23 hour time points.
There was only one metabolite formed and retention time of metabolite
during the assay was consistent (no acyl migration observed). The
time dependent degradation of glucuronidated metabolite does not seem
to produce any isomers but it increases the parent level.
I didn't see any change in mass shift (66400 Da to 67000) for HSA. I
am taking as 66400 Da for HSA MW and added 604 mass units for
glucuronide metabolite. I did see increase in parent level and
decrease in metabolite level from incubation with HSA over incubation
period of 0-23 hours. Does it mean that the metabolite is not binding
to HSA? There is about 12-15 ug/mL metabolite and 1.5 mg/mL HSA
concentration at T0.
Please share your thoughts.
Thanking you,
Anila
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The following message was posted to: PharmPK
Anila,
I'm sure you realise that acyl glucuronides are particularly labile,
even at physiological pH, so it is likely that it is a simple
hydrolysis that you are seeing, thus liberating the aglycone, as a
function of incubation time.
That said, I would not rule out some degree of protein adduction.
Acyl glucuronides can undergo nucleophilic attack on whatever
particular functionality it has an affinity for e.g. SH leading to
thio esters, NH leading to amides etc. You should remember that this
will liberate glucuronic acid and the net effect is that it is the
alglycone solely that forms the protein adduct. This is in addition
to possible case of multiple binding sites on a particular protein.
Therefore you should be looking for an MH+ of, in your case,
604-176Da as the deltaDa from HSA. MAXENT is OK for some protein
spectra deconvulution but why don't you compare TOF spectra using
incubation with a deuterated analogue of the compound? Comparison of
TOF spectra using it and the unlabelled form may highlight this
possible reactivity better. I have seen this work to great effect on
a SELDI-TOF. Just be careful of both the possibly different reaction
rates and site of the Dx when using D-analogues.
Good luck!
Iain
Dr Iain A. Stuart
Preclinical Development
Cyclacel Pharmaceuticals, Inc.
Dundee Technopole
James Lindsay Place
Dundee
DD1 5JJ, UK
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The following message was posted to: PharmPK
Dear Anila,
You do have a nice set of data there. These are
important findings. Based on your in-vitro data you
can relate in-vivo data,if there is any high
accumulation of parent in the in-vivo samples.
You probably already have but just in case if you
haven't, please take a look at the Ketoprofen
literature you will see that this drug behaves
similarly to your data. When considering for daily
dosing this (unstable metabolite and hydrolysis back
to parent) will have to take into consideration.
Best regards,
Howard P
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Hi Anila,
The question you posed in the PharmPK discussion group was most
interesting to me. I have been unsuccessful in producing acyl-
glucuronide with the conditions I am using. I know that acyl
glucuronides are considered unstable and sensitive to pH, perhaps the
reason for their difficulty in synthesizing in-vitro. I used high
liver microsomes (4 mg/mL)but cannot synthesis metabolite yet.
Could you please share your in-vitro assay conditions?
Thanks very much.
Sue
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