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I have a question regarding design of an experiment to elucidate the
renal clearance (Cumulative amount excreted in urine) of doxorubicin in
an instrumented rat model.
Rats have cannulas placed in jugular vein, common bile duct, and
bladder. Cannulas are exteriorized at the back of the neck. Route of
drug administration is via the tail vein. These freely moving rats are
individually housed in metabolic cages.
Question: how to effectively collect urine? A. just house rats in
metabolic cages, B. collect urine via bladder catheter, or C. use
anaesthetized rats.
Problems with A: if housed in metabolic cages, the protocol "normally"
used is to wash receiver flask (where urine collects, likely evaporates,
and leaves behind a residue - drug may or may not bind to the plastic),
after a sample collection period, with a defined volume of water/normal
saline and these washings are used to determine drug concentrations.
This precludes measurement of cummulative amount excreted, where you
need to know exact volume of urine discharged/collected. Any
alternative approach to calculate cum. amount excreted and renal
clearance?
Problems with B: in freely moving rats with implanted bladder catheter,
protocol normally used particularly with large animal models is collect
urine into volumetric flask by gravity over a period of time. With
small animal models, however, volume of urine discharged is low (100-200
ul/0-4 h) and the cannula is brought via back of the neck and upwards
via the outlet on the top of the cage (to prevent the rat from chewing
the catheter tubing), against gravity and impeding urine flow. Any
thoughts on this?
Problems with C: anesthesia would seem to be a solution and an easier
way of collecting these fluids (urine or bile) via catheters by gravity,
but anesthesia/anesthetics can very well alter the pharmacokinetics of
the agents, so using this strategy is not a desirable option.
Thanks in advance.
----
Rajesh Krishna
University of British Columbia
and BC Cancer Agency
600 West 10 Avenue
Vancouver, BC
V5Z 4E6
http://members.tripod.com/~rkrishna
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You do not need to know the amount of urine to determine the total amount
excreted in urine.
If you wash the cage pan with water you lower the concentration of
Doxorubicin in (urine + water) but the total amount of DOX stays the same.
You measure the concentration, multiply by the volume of (water + urine)
and that gives total amount excreted.
The biggest problem with this technique is as you mentioned, the drying and
sticking to plastic, but you should also check whether or not DOX is stable
in urine at room temperature but that can be tested by spiking a known
amount of DOX into some and letting it sit around.
You can check the sticking to the plastic too.
Susan Shoaf
shoaf.-a-.clinpharm.niaaa.nih.gov - Nat'l Institute on Alcohol Abuse and Alcoholism
I don't speak for NIAAA, Unit of Pharmacokinetic Studies
That's for the PR office
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[A few replies - db]
From: David Claghorn
To: "'PharmPK.aaa.pharm.cpb.uokhsc.edu'"
Subject: RE: PharmPK Re: Renal Clearance
Date: Fri, 12 Jun 1998 10:16:56 -0500
MIME-Version: 1.0
I'm not an expert on Analytical Chemistry, I only took two semesters in
undergrad. But if I remember, you should be able to rinse with DI water
without affecting any of the results, using calculations mentioned by
Dr. Shoaf. I'd stay away from using any saline products.
Just my opinion,
Dave Claghorn
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X-Sender: jbiollaz.aaa.hola.hospvd.ch
Date: Fri, 12 Jun 1998 18:19:40 +0200
To: PharmPK.-at-.pharm.cpb.uokhsc.edu
From: Jerome Biollaz
Subject: Re: PharmPK Renal Clearance
Mime-Version: 1.0
D. could be an answer ...
The dome of the urinary bladder is removed under light ether anesthesia 24
hours before the renal clearance. On the day of the study, the
unanesthetized rats are kept in lucite restraining cages (see attachment).
Urine is collected directly from the urethral orifice into plastic tubes
(the area around the urethral orifice is siliconized).
JB
[I have placed the answer and attached image on-line at
http://www.cpb.uokhsc.edu/pkin/images/cages.html - db]
---
X-Lotus-FromDomain: SGS
From: Ray_French.-a-.sgsgroup.com
To: PharmPK.aaa.pharm.cpb.uokhsc.edu
Date: Fri, 12 Jun 1998 18:47:21 +0100
Subject: Re: PharmPK Re: Renal Clearance
Mime-Version: 1.0
Dox sticks to certain plastics. It also sticks readily to glass. I used to
use glass vessels only, but silanised them all to prevent sticking.
Remember too that Dox is a nasty cytotoxic - use appropriate protection if
you're going to splash it around!
Dox in human urine is pinky-red..can cause some consternation. May occur in
your rats, too, so don't assume that this is blood in the urine if you see
it.
Ray French
_________________
Inveresk Research.
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[A few replies - db]
From: Rajesh Krishna
To: PharmPK
Subject: Renal Clearance - addendum
Date: Tue, 16 Jun 1998 09:53:00 -0700
MIME-Version: 1.0
In my previous mail, last couple of lines got missed somehow.
Apologies.
----------
From: Rajesh Krishna
To: PharmPK
Subject: Re: Renal Clearance
Date: Monday, June 15, 1998 3:27PM
First, I would thank everyone for their valuable input.
>Problems with A: if housed in metabolic cages, the protocol "normally"
>used is to wash receiver flask (where urine collects, likely
evaporates,
>and leaves behind a residue - drug may or may not bind to the plastic),
>after a sample collection period, with a defined volume of water/normal
>saline and these washings are used to determine drug concentrations.
>This precludes measurement of cummulative amount excreted, where you
>need to know exact volume of urine discharged/collected. Any
>alternative approach to calculate cum. amount excreted and renal
>clearance?
You do not need to know the amount of urine to determine the total
amount
excreted in urine. If you wash the cage pan with water you lower the
concentration of Doxorubicin in (urine + water) but the total amount of
DOX stays the same.
You measure the concentration, multiply by the volume of (water + urine)
and that gives total amount excreted.
----
Question: If you dilute the urine with water (or residue) using the
metabolic cage approach (non-cannulated method), you don't really know
the actual volume of discharged urine. You may determine the amount of
drug present in urine, but you don't get a rate parameter, i.e., urine
flow rate (?? volume/time period) and hence cannot obtain a reliable
renal clearance value in ml/min.
Conc of drug in urine (ng/ml) x urinary flow rate (ml/min) = urinary
drug excretion rate (ng/min)
----------------------
Addendum:
----------------------
Plot of urinary drug excretion rate vs concentration of drug in plasma,
slope gives the renal clearance value in ml/min.
Renal clearance (ml/min) = urinary drug excretion rate (ng/min) /
concentration of drug in plasma (jugular vein plasma, ng/ml)
The question still remains: how do you (or can you) determine renal
clearance using the metabolic (non-cannulated method) cage approach.
---
From: "Anaizi, Nasr"
To: "'PharmPK.-a-.pharm.cpb.uokhsc.edu'"
Subject: RE: PharmPK Re: Renal Clearance
Date: Tue, 16 Jun 1998 13:17:26 -0400
MIME-Version: 1.0
Although I have not read all of the inputs on this questions I
would like to briefly address the question below:
"Question: If you dilute the urine with water (or residue)
using the
> metabolic cage approach (non-cannulated method), you don't really know
> the actual volume of discharged urine. You may determine the amount
> of
> drug present in urine, but you don't get a rate parameter, i.e., urine
> flow rate (?? volume/time period) and hence cannot obtain a reliable
> renal clearance value in ml/min.
> Conc of drug in urine (ng/ml) x urinary flow rate (ml/min) = urinary
> drug excretion rate (ng/min)"
To determine the clearance accurately you need only to be able
to:
1- recover the excreted mass over a known period of time
completely and measure it accurately. You will then have an accurate
excretion rate (mass/time).
2- determine the mean (steady state) plasma concentration of the
substance in question over the period of collection. The calculated
clearance will be inaccurate if the concentration is unstable (e.g.,
declining sharply)
There is no need to know the actual (real) urine flow rate, a
virtual one would do just as well. The virtual urine flow rate in this
case would be = wash volume / collection time.
Thanks
Nasr Anaizi
U of Rochester Med Cntr
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Mime-Version: 1.0
Date: Tue, 16 Jun 1998 16:57:08 -0400
From: GOPAL.KRISHNA.-a-.spcorp.com (GOPAL KRISHNA)
To: PharmPK.-a-.pharm.cpb.uokhsc.edu
Subject: Re: PharmPK Re: Renal Clearance
I would like to add to what Susan already alluded to in her reply. To
determine cumulative amount excreted in the urine all we need to know
is the amount (not the volume of urine) excreted unchanged over a
given time interval. Therefore, wash/rinse should not change anything
as long as we know the final volume so that we can calculate the total
amount present in that volume. The amount for each time interval can
then be added and further divided by the corresponding time. This will
provide dx/dt (unit, mass/time). The ln(dx/dt) when plotted at the
midpoint of the urine collection interval can provide an estimate of K
and t1/2. Only thing we need to be careful about is that the urinary
collection time interval should not exceed half-life of the drug.
As far as clearance is concerned, it can be determined by dividing
excretion rate (dx/dt) with the plasma concentration of the drug at
the midpoint of the urinary collection interval.
Unless I am missing something, hope this helps.
Gopal
---
From: "CHARLES"
Organization: School of Pharmacy
To: PharmPK.-at-.pharm.cpb.uokhsc.edu
Date: Wed, 17 Jun 1998 09:45:45 +1000
MIME-Version: 1.0
Subject: Re: PharmPK Re: Renal Clearance
X-Confirm-Reading-To: "CHARLES"
X-pmrqc: 1
Priority: normal
I agree... Urine flow rate always should be considered since it can
signficantly influence renal clearance (and, therefore, the systemic
clearance if fraction unchanged is appreciable) particularly for
drugs which are extensively reabsorbed in the tubules -- Forced
diuresis in clinical toxicity increases renal clearance because of
the markedly diminished concentration gradient between plasma and
(normally) higher drug concentrations in the tubular fluids after
filtration (Correlations between urine flow rate and renal clearance
have been reported for methylxanthines and antihistamines).
Adding rinsings to the collected samples could mask or confound any
such effects.
Cheers,
BC
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[A few replies - db]
Date: Wed, 17 Jun 1998 18:38:53 -0400
From: jianhua liu
MIME-Version: 1.0
To: PharmPK.at.pharm.cpb.uokhsc.edu
CC: Multiple recipients of PharmPK - Sent by
Subject: Re: PharmPK (No subject)
How about accumulative amount of urine excretion vs accumulative AUC Plot?
---
From: Rajesh Krishna
To: PharmPK
Subject: Renal Clearance
Date: Wed, 17 Jun 1998 14:42:00 -0700
MIME-Version: 1.0
Hi all:
Thanks everybody for all the responses.
Regarding calculation of renal clearance, the following method is
frequently used:
Using metabolic cages, allow animal to urinate on the receiver flask.
After a time period, wash receiver flask with defined volume of water.
Multiply concentration of drug present in washings with volume of water
added to get total amount of drug in urine/time period to get mg/min.
Divide this with midpoint Cp to get CLr. The generated CLr value is
only an average estimate.
As this method suggests, renal clearance is not dependent on urinary
output or urinary flow rate (as urine volume discharged is not required
for calculations). Perhaps the method is okay for drugs that do not
alter urine flow rate. As B Charles suggests (below), there is a
relationship between urine flow rate and renal clearance for many drugs.
I am curious to know if such relationships are possible in the following
scenario:
For the pharmacological reversal of multidrug resistance, an anticancer
drug (DOX) is co-administered with a P-glycoprotein (PGP) blocker
(Cyclosporin A, PSC 833, for example). We know that PGP is present in
normal non-tumor tissues, such as the kidney and the liver. Therefore,
co-administration of PGP blocker with DOX can interefere with DOX renal
excretion processes via blockade of kidney PGP. We don't really know
the effect of the PGP blocker on DOX urinary output. In such
circumstances, is the calculation of CLr using the above method reliable
(acceptable with a reasonable degree of confidence)?
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