Microsoft word - solvo microdialysis services_final version.doc

BIOTECHNOLOGY
IN VIVO MICRODIALYSIS PREDISCREENTM SERVICES
Microdialysis techniques have been developed to monitor changes
in the chemistry of the extracellular space in living tissue. These
techniques can be used for: a) to measure drug and metabolite
concentrations in the interstitial space in brain and peripheral
tissues including blood; b) continuous monitoring of
neurotransmitter release to various stimuli; c) to measure
concentrations of many analytes associated with tissue damage in
CNS and in various organs. The microdialysis techniques require the
introduction of an ultra thin, semi-permeable tube, a so-called probe
in the tissue (Fig 1, Fig 2). The probe is connected to a precision
pump, which provides a steady flow of a tissue-compatible fluid
through the probe at a very low flow rate (1–5 µl/min). Open circles on Fig. 1 depict the various endogenous compounds in the extracellular fluid or in blood; the closed circles depict exogenous
compounds (drugs to be tested or calibrators for determination of
the in vivo recovery) which can be delivered by the perfusion fluid.
Squares represent extracellular macromolecules that may bind
analytes. Smaller molecules in the tissue - including the non-protein
bound fraction of drug content in the extracellular fluid - will
passively diffuse across the surface of the membrane and thus enter
the flow of the perfusate, which is sampled at regular intervals and
analyzed either on-line or off-line. Over the last decade in vivo
microdialysis (MD) has been increasingly applied to monitor drug
distribution at peripheral tissue sites and the penetration of various
agents across the blood brain barrier (Zhou and Gallo, The AAPS
Journal 2005, Helmy et al., Current Medicinal Chemistry 2007).
Applications of MD are being explored to address specific safety
issues and MD data are likely to become an important part of new
drug submissions to drug regulatory agencies (Chaurasia et al., The In vivo microdialysis in anesthetized rats. Blood Brain Barrier (BBB) package
SOLVO’s new microdialysis service is an integral in vivo part of our BBB package. The package consists of an
assortment of crossvalidated in vivo and in vitro tools for studying the drug-transporter and/or drug-drug interactions in
this important barrier. The BBB package focuses on two key efflux transporters, MDR1 and BCRP. Membrane based
ATPase and vesicular transport assays represent a HTS assessment that provide transporter specific data to confirm
in vivo detected interactions. Living cell based BBB models, like dye efflux transport assays adapted for an
immortalized human brain capillary endothelial cell line (hCMEC) or primary rat brain capillary endothelial cells (RBEC)
are also validated with specific interactors and dedicated to explore efflux transporter related inter-species differences.
Reference substrates used in MDR1 and BCRP data validation
The reporter substrate for BCRP is dehydroepiandrosterone sulfate (DHEAS). DHEA is a natural steroid
prohormone, while DHEAS – its sulfated form – acts as a reservoir for the prodrug. It has been described as a BCRP
substrate by several papers1-3. BCRP transports both hydrophobic and hydrophilic compounds among which has a
preference for sulfate conjugates. The reference inhibitor for BCRP-mediated DHEAS transport is Chlorothiazide
(CHT). CHT, a commonly used diuretic4, was shown by our lab to specifically interact with the BCRP transporter5.
MDR1 is the most commonly studied efflux transporter in the BBB. The reporter substrate in SOLVO’s microdialysis
package, Quinidine (QND), has first become known for its interaction with digoxin absorption in the intestine6, but it was
also revealed that QND is indeed a transported substrate of MDR17. QND is an antiarrythmic drug producing many
adverse effects when coadministered with other Pgp interactors. The reference inhibitor LY335979 (Zosuquidar) is a
molecule specifically designed to inhibit MDR18. It has an affinity for MDR1 in the nanomolar range, being a very potent
inhibitor of the efflux transporter. Verapamil is a high permeability molecule used as a reference substrate and inhibitor
in SOLVO MDR1 assays, including ATPase assay and Calcein assay.
5. Bansaghy et al manuscript in preparation 7. Muller et al FEBS Letters 1994;343:168 8. Dantzig et al Cancer Res 1996;56 :4171 SOLVO Biotechnológiai ZRt. 2040 Budaörs, Gyár u. 2. Tel: 23-503-940, [email protected] www.solvo.com BIOTECHNOLOGY
TYPES OF MICRODIALYSIS STUDIES PERFORMED AT SOLVO
1. Simultaneous MD-sampling of jugular vein and
I. Determination of brain penetration of test molecules in rats:
frontal cortex of rats after dosing with QND (i.v.) We have the capability to perform double microdialysis
experiments using anesthetized rats to simultaneously assess
unbound levels of test molecules in blood and in brain. We implant a femoral vein catheter for dosing; peripheral and brain microdialysis probes for sampling blood and brain. After the implantation of the femoral vein catheter and a jugular vein probe, the rat is placed in a stereotaxic frame (Fig 2 in section-1) for implanting a brain microdialysis probe in the specified brain region and for running the microdialysis (MD) experiment. Vascular and brain MD sampling are performed 90 min before and 210 min after dosing the animal intravenously (i.v.). This type of double MD experiment is shown in Fig 1 and Fig 2 of this section using two doses of QND and DHEAS. In these experiments CMA/20 probes were used for vascular, and CMA/12 probes for brain sampling with a perfusion rate of 1.0 µl/min. (Double MD experiments with i.p. administration of test molecules are also being routinely performed in our Microdialysis Laboratory.)
Prior to in vivo MD experiments on new compounds, in vitro experiments are performed to check the adsorption of the compound to the tubing and to determine the recovery of the probes. 2. Simultaneous MD-sampling of jugular vein and
II. Determination of test molecule interactions with transporters:
frontal cortex of rats after dosing with DH II/1: This type of in vivo MD studies are performed if in vitro
transporter interaction or other studies suggest (see section-2) that a
test molecule might be an inhibitor of a specific efflux transporter. At
least 3 groups will be used (n=5 per group): Group-1: animals receive a vehicle injection at -20 min and a reference substrate injection at 0 time; Group-2: animals receive an injection of a test molecule at -20 min and a reference substrate at 0 time; Group-3: a reference inhibitor of the transporter at -20 min and a reference substrate injection at 0 time. Two doses of test molecule/reference inhibitor/reference substrate will be used upon request. Results of experiments of this type using LY335979 and chlorotiazide as inhibitors are shown on Fig 3. Validation of the MDR1 – verapamil interaction in vivo is in progress. II/2: This type of in vivo MD studies
are performed if in vitro transporter interaction or other studies suggest (see section-2) that a test molecule might be a substrate of
12.5 mg/kg
a specific efflux transporter. At least 4 groups will be used (n=5 per group): Group-1: vehicle injection at -20 min and a test molecule injection at 0 time; Group-2: an injection with a reference inhibitor of the transporter at -20 min and a test molecule at 0 time; Group-3: vehicle injection at -20 min and a reference substrate injection at 0 Inhibitors of MDR1 and BCRP alter the ratio time; Group-4: injections with a reference inhibitor of the transporter at -20 min and with a reference substrate at 0 time. Two doses of test molecule/reference inhibitor will be used upon request. III. Determination of brain penetration of test molecules and
simultaneous monitoring of neurotransmitter release in
specified brain regions: Upon request, brain probes will be placed
in anesthetized rats into brain regions that are rich in specific neurotransmitters. Aliquots of brain dialysate samples and blood dialysate samples will be analyzed for test molecules; further aliquots from the same brain samples will be analyzed for specific neurotransmitters to explore possible drug interactions and/or adverse reactions. Water soluble drugs/test molecules can be applied -50-2 -1 0 1 2 3 -540 -1 0 1 2 3 4 -2 -1 0 1 2 3 4 -1 0 1 2 3 4 In vivo MD studies currently available: Double MD studies described above in anesthetized rats with i.v.
and/or i.p. drug administration; Brain MD studies in rats with i.p. drug administration using CMA/120 system
for freely moving animals.
Additional MD studies available from Q4 in 2008: Double MD studies in rats using a movement-
responsive animal system; Double MD studies in anesthetized mice with i.p. drug administration; Brain MD
studies in mice with i.p. drug administration using a mouse MD system from Linton Instrumentation.
SOLVO Biotechnológiai ZRt. 2040 Budaörs, Gyár u. 2. Tel: 23-503-940, [email protected] www.solvo.com

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