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I am currently working on project which involved the prodrug (ester type)
approach to enhance oral bioavailability. However, I faced some problem
with bioanalytical problem with the prodrug. The prodrug hydrolysis very
very quickly at room temperature. It takes us 3-5 minute to finally
process the blood to become plasma. Because of this handling delay, we
loss the capability to accurately detect the amount of prodrug and/or
parent drug in the plasma. We are looking some esterase inhibitor to stop
the reaction once the blood is withdrawal. But so far no luck.
I'm very interested if someone could give me any suggestion about how to
overcome this problem or some experts' names so that I can contact to get
help. I would greatly appreciate any help I could get. Company is also
willing to compensate the cost if it is required.
Jian-ping Tang, Ph.D.
Hoffmann-La Roche
Jian-ping.Tang.at.roche.com
Jian-ping
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[A very popular question - quite a few replies - db]
Date: Wed, 12 May 1999 16:49:41 +1000
From: Laurent Rivory
Reply-To: lrivory.-a-.canc.rpa.cs.nsw.gov.au
Organization: Sydney Cancer Centre - CSAHS
X-Accept-Language: en
MIME-Version: 1.0
To: PharmPK.-a-.pharm.cpb.uokhsc.edu
Subject: Re: PharmPK esterase inhibitor??
Many esters are not stable under physiological condition so the first thing
that needs to be considered is whether conversion is truly enzymatic. If it
isn't (ie if conversion also occurs quickly in PBS pH 7.4) then a "chemical"
strategy is probably best. Try collecting blood into cooled tubes.
Alternatively, hydrolysis may be base catalysed and a low pH buffer may be
suitable.
If hydrolysis is indeed esterase mediated, chances are that it is a serine
hydrolase. In this case, the addition of paraoxon, bis para nitro phenyl
phosphate (BNPP) or PMSF (phenylmethyl sulfonyl fluoride) will likely yield
inhibition of esterase activity and allow you to prepare serum/plasma as
required. A problem is that you will need ~ 100 microM PMSF. To get it int=
o
a small volume (eg 20 microL ) in the collection tube, it will need to be
dissolved in ethanol or DMSO. Rapid mixing will be required during
collection.
Of course, many of these serine hydrolase inhibitors may interfere with the
HPLCassay. You won't know until you try.
A more specific strategy may be used if you have identified the enzyme
responsible. For example, arylesterases may be inhibited by EDTA which is
useful for plasma collection anyway. This may be worth a try.
Good Luck!
Laurent Rivory
Senior Scientist
Sydney Cancer Centre
Sydney, Australia
---
Sender: gmould.at.dial.pipex.com
Date: Wed, 12 May 1999 08:14:44 +0100
From: Graham Mould
MIME-Version: 1.0
To: PharmPK.at.pharm.cpb.uokhsc.edu
CC: Multiple recipients of PharmPK - Sent by
Subject: Re: PharmPK esterase inhibitor??
Re esterase inhibitor
When I used to measure acetylsalicylic acid in blood I added ascorbic
acid beforehand. Try that?
What about freezing straight away?
Graham Mould
Guildford Clinical Pharmacology Unit
Royal Surrey County Hospital
Guildford
Phone: 00 44 (0) 1483 455375
email: gmould.aaa.dial.pipex.com
---
Date: Wed, 12 May 1999 08:05:16 -0400
From: Nabil B. Darwazeh
To: PharmPK.-a-.pharm.cpb.uokhsc.edu
Subject: Re: PharmPK esterase inhibitor??
Mime-Version: 1.0
Can't you process the blood sample at low temperature (ice, refrigarated
centrifuge and HPLC).
Nabil
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Date: Wed, 12 May 1999 15:53:44 +0200
From: "Dr. Bernhard Ladstetter"
Reply-To: Lstetter.-at-.merck.de
Organization: merck.de
MIME-Version: 1.0
To: PharmPK.aaa.pharm.cpb.uokhsc.edu
Subject: Re: PharmPK esterase inhibitor??
Dear Jian-ping,
we transfer plasma into a tube containing 10 =B5L of the esterase
inhibitor dichlorvos (conc. 1 mg/mL). The process of plasma separation
is done in a cooling centrifuge at 4 C (1100g, 10 min).
I hope this helps.
Regards,
Bernhard J. Ladstetter, Ph.D.
Merck KGaA
Institute of Pharmacokinetics & Metabolism
---
From: "Melethil, Srikumaran K."
To: "'PharmPK.-at-.pharm.cpb.uokhsc.edu'"
Subject: RE: PharmPK esterase inhibitor??
Date: Wed, 12 May 1999 09:21:35 -0500
MIME-Version: 1.0
Dear Jian-ping,
Nice to "see" you in the discussion group.
When we were working with aspirin, we found that using fluoride coated tubes=
to
collect blood prevented hydrolysis of aspirin (see JPB 19, 157-173, 1991).
Let
me know if I can be of further assistance.
Best wishes,
Sri
Sri Melethil, Ph.D.
Professor of Pharmaceutics and Medicine
University of Missouri-KC
School of Pharmacy
Room 203-B, 5005 Rockhilll Road
Kansas City, MO 64110
816-235-1794 (fax: 816-235-5190)
---
Date: Wed, 12 May 1999 07:44:39 -0700
From: caroline Lee
Organization: Agouron Pharmaceuticals Inc.
MIME-Version: 1.0
To: PharmPK.at.pharm.cpb.uokhsc.edu
Subject: Re: PharmPK esterase inhibitor??
We were faced with a similar situation with one of our development compounds=
=2E
One of our groups found that the addition of 0.5 mg of paraoxon inhibited
esterase activity completely for 1 hour on ice. The only drawback with
paraoxon is that it is a potent neurotoxin so care should be taken when usin=
g
this compound (check its MSDS first).
Paraoxon may work for you.
Caroline
Caroline Lee
Agouron Pharmaceuticals, Inc
4245 Sorrento Valley Blvd
San Diego, CA 92121
---
Date: Wed, 12 May 99 10:55:29 -0400
From: "Eddington, Natalie"
Sender: "Eddington, Natalie"
Organization: UMAB - School of Pharmacy
To: pharmpk.-at-.pharm.cpb.uokhsc.edu
Subject: Re: PharmPK esterase inhibitor??
MIME-Version: 1.0
In our pharmacokinetic studies, with the opioid agonist, reminfentanil,
we added acetonitrile immediately after blood sample collection from
rats. Remifentanil is metabolized by non-specific esterases in the
blood. ( Pharm Res, 14,1817,1997.)
Natalie D. Eddington, Ph.D.
Associate Professor
Department of Pharmaceutical Sciences
University of Maryland, School of Pharmacy
100 Penn Street, AHB 540C
Baltimore, MD 21201
(410) 706-6710, (410) 706-6580 (fax)
eddingto.-a-.pharmacy.ab.umd.edu
---
To: PharmPK.at.pharm.cpb.uokhsc.edu
Date: Wed, 12 May 1999 11:51:45 -0500
Subject: Re: PharmPK esterase inhibitor??
X-Juno-Line-Breaks: 0-1,8-10,13,15,17-19,21,23-27,29-34
From: Mickey & Jaimini
What type of ester prodrug are you working with?
I have worked with prodrugs which undergo rapid hydrolysis under
physiological pH although the one I was mainly concerned about did not
undergo enzymatic hydrolysis. However, in an attempt to prove so, I had
worked with the enzyme poison tetraethyl pyrophosphate (TEPP) and the
enzyme denaturant sodium fluoride. Also, we had established the presence
of enzymatic activity in our biological media with the help of a known
substrate - p-nitrophenylacetate.
I have several suggestions for you:
1) Rinse or flush all syringes, needles and tubes etc. that are going to
come into contact with the prodrug, with the enzyme poison TEPP (100
ug/ml). Be careful, TEPP is toxic!.
2) Keep the syringes and the tubes into which you will collect the blood
into, extremely cold.
3) If need be, you can add a known amount of TEPP into your test tubes
into which you will collect blood.
4) Centrifuge under cold temperature.
Though, I had not experimented, you may want to try another
cholinesterase inhibitor - physostigmine.
Bundgaard and co-workers have tested their prodrugs in the presence of
this enzyme inhibitor.
Following are a couple of references which may be of help:
1) Hansen, J.; Mork, N.; Bundgaard, H. Int. J. Pharm. 1992, 81, 253-261.
2) Patel, J.; Prankerd, R. J.; Sloan, K. B. J. Pharm. Sci. 1994, 83, 10,
1477-1480.
Jaimini Patel, Ph.D.
2404 Rick Whinery Dr
Austin, TX 78728
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Mime-Version: 1.0
Date: Wed, 12 May 1999 09:49:44 -0800
To: PharmPK.at.pharm.cpb.uokhsc.edu
From: Mohsen Hedaya
Subject: Re: PharmPK esterase inhibitor??
Dear Jian-ping,
We have done several PK studies with cocaine which is an ester rapidly
hydrolyzed by the esterases in the blood. We have used saturated sodium
fluoride solution to inhibit the blood esterases. For more details please
see the published method:
Pan, W.J. and Hedaya M.A. "Sensitive and Specific High-Performance Liquid
Chromatographic Assay with Ultraviolet Detection for The Determination of
Cocaine and Its Metabolites in Rat Plasma" J. Chromatography B, 703:129-138
(1997)
I hope this can be useful
Mohsen
Mohsen A. Hedaya, Pharm.D. Ph.D.
Department of Pharmaceutical Sciences
College of Pharmacy
Washington State University
Pullman, WA 99164-6510
Phone: (509) 335 5622
Fax: (509) 335 0162
e.mail: hedaya.-at-.mail.wsu.edu
---
From: DGarg8838.aaa.aol.com
Date: Wed, 12 May 1999 19:55:56 EDT
Subject: Re: PharmPK esterase inhibitor??
To: PharmPK.aaa.pharm.cpb.uokhsc.edu
MIME-Version: 1.0
Sodium Floride may work to prevent in vitro hydrolysis. There are vacutainer
tubes containing sodium flouride and these can be tried.
Dyal Garg
561-737-3954
---
Date: Wed, 12 May 1999 17:05:29 -0700
From: "Julie Zhang O'Brien"
To:
Subject: Re: PharmPK esterase inhibitor??
Mime-Version: 1.0
It seems like you are having the problem we've encountered before. I think
you'd better make sure that your prodrug hydrolysis is not caused by water
only or temperature or your extraction process. After these factors are
excluded, then you'll start working on finding the esterase inhibitors.
There're different types of esterase inhibitors you could try to see which
one works the best for you (I don't know what you've tried already). We
used paraoxon, phosphoric acid diethyl 4-nitrophenyl ester, and it works
perfect for our prodrug. The thing is once you find an appropriate
inhibitor, you have to do experiment to see which concentration of the
inhibitor can give you the best result because according to my experience
you need to change the inhibitor's concentration for different prodrugs.
Here's two articles might be helpful for you:
1) "Paraoxonase has a major role in the hydrolysis of prulifloxacin
(NM441), a prodrug of a new antibacterial agent" Katsuhiko Tougou, Akio
Nakamura, Shuji Watanabe, Yoshio Okuyama, and Akira Morino, Drug Metab
Dispos 1998 April, 26 (4) 355-359
2) "Determination of bambuterol, a prodrug of terbutaline, in plasma and
urine by gas chromatography/mass spectrometry" Claes Lindberg, Sven Jonsson
and Jan Paulson, Anders Tunek. Biomedical and Environmental Mass
Spectrometry 1990, vol 19, 218-224.
Good luck.
Julie
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