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Metabolism of stability studies and sub cellular fractions
1. ADVANCED PHARMACOLOGY-I
METABOLIC STABILITY STUDIES
USING CELLULAR (Hepatocytes)
AND
SUB-CELLULAR FRACTIONS
PRESENTED BY:-
MANJUNATHA D C
1ST M PHARM
PES COLLEGE OF PHARMACY
BENGALURU
2. INTRODUCTION:-
Metabolic Stability studies in hepatocytes:-
Metabolic stability is defined as the percentage of parent compound lost
over time in the presence of a metabolically active test system. For
metabolic stability assays, the typical test systems are liver micro somes,
liver S9, or hepatocytes (plated or suspended), depending on the goal of
the assay.
3. Drug metabolism and pharmacokinetics(DMPK)have improved success rate
of drug discovery and development in the last two decades1–5.
The contributions and improvement to the success rates by DMPK can be
attributed to at least three reasons .First , DMPK is now routinely
incorporated into early drug discovery, a trend beginning about 15–20years
ago. Second , DMPK scientists have a better understanding of drug
metabolic enzymes , transporters, and differences among species and
individuals. Many research tools and advanced instrumentation are now
available to make relatively reliable predictions from the in vitro to the in
vivo data and from animals to humans.
4. Hepatocytes
The liver plays a central role in drug metabolism and disposition
through its primary metabolic enzymes (both Phase I and Phase II)
and many transporters (including both uptake transporters expressed
on the sinusoidal membrane and efflux transporters expressed on
sinusoidal and canalicular membranes). Therefore , freshly isolated
hepatocytes are regularly used to determine hepatic metabolism and
clearance mediated by liver enzymes37, as well as to assess hepatic
uptake mediated by uptake transporters 38,39.
5. In experiments, the uptake into hepatocytes is measured in
suspension by a centrifugation method ,using oil-layered tubes to
separate hepatocytes from incubation medium. There have also been
reports that cryopreserved hepatocytes can be used for studying
hepatic uptake15. Isolated hepatocytes cannot be directly used for
efflux assay because the efflux transporters lose their function
during the isolation process ; however , primary cultures of
hepatocytes can restore the function of efflux transporters even
though the uptake transporters demonstrate decreased functionality
in primary cultures.
6. Rat hepatocytes
Simultaneous isolation of rat hepatocytes and hepatic macrophage (HM) is
carried out using modified protocol of collagenase digestion (1, 2). In brief,
the rat liver is perfused through portal vein with 100 ml of Ca2+ free Hanks
buffer containing 1ml of EGTA (34mg/ml, Sigma) and 1g of bovine serum
albumin (BSA) at 37˚C for 10 minutes. This is followed by enzymatic
digestion with 60 ml of Hanks buffer containing 0.05% (w/v) collagenase
type IV (Sigma) and CaCl2(58.8mg/ml) for 4 to 7 minutes via recirculation.
The liver is then removed and cut into small pieces.
7. Liver cells are further disassociated by gentle pipetting in the Hanks buffer, and
filtered through a Nylon filter to remove undigested tissues and debris. The
resulting cell suspension is centrifuged at 700 rpm for 1 minute. The
supernatant containing non parenchymal cells is collected for HM purification
(see below). The cell pellet is re-suspended in 10ml of Hanks buffer mixed
with 5 ml Per coll (Sigma #P1644), and centrifuge at 2,500 rpm (150xg) for 5
minutes at 4˚C (3). The cell pellet is washed 2 times with DMEM medium and
centrifuged at 700rpm (50xg) for 1 minute at 4˚C.
8. The non-parenchymal liver cell fraction is separated by centrifugation at 50xg
for 1-2 minutes. After two washes of the fraction by 2500 rpm centrifugation,
it is laid onto the top of four layers of Opti Prep TM gradients (1.085, 1.058,
1.043, 1.034) in Beckman ultracentrifuge tubes (4-6). The tubes are
centrifuged in the SW-41Ti rotor at 21,400 rpm for 35 minutes at 25˚C.
Relatively pure (>83%) HM are collected from the 1.043/1.058 interface and
less pure HM from the 1.058/1.085 interface. The viability of both cell types is
checked by trypan blue exclusion.
9. Isolation from a normal male rat (BW=350-550g) yields 2-3x108 hepatocytes
with the purity and viability exceeding 92%, and 1.8-2.5x108 HM with the
purity > 83% and the viability >95%. HM are further purified by the adherence
method and cultured in PRMI containing 5% FCS and antibiotics at the
density of 3-4x106 cells per 100 mm dish. After adherence method, the HM
purity will exceed 95% with the yield of 2.7x107 cells from a rat.
10. Mouse hepatocyte
Simultaneous isolation of mouse hepatocytes and HM is similar to the
procedures used for the rat, with following adjustment. Mouse liver is
perfused through superior vena cava with EMEM at 5ml/minute for 10
minutes, and then with digestion buffer (DMEM containing 0.044% (w/v)
collagenase) for 6-10 minutes. The liver is removed and agitated in a rotary
shaker for 10-15 minutes to further digest and dissociate the cells in DMEM
medium containing 10μg/ml D nase I. The cell suspension is centrifuged at
700rpm (50xg) for 1min at 4˚C.
11. The resulting cell pellet is used for hepatocyte purification as described above. The
supernatant containing non parenchymal cells is centrifuged at 2500rpm for 5
minutes, and the pellet is wash twice with DMEM medium. The final cell suspension
is laid on the top of four Opti PrepTM gradients (1.085, 1.058, 1.043, 1.034) and
centrifuged in the SW-41Ti rotor at 20,000 rpm for only15 minutes at 25˚C. The
viability of both cell types is checked by trypan blue exclusion. Isolation from a
normal male mouse (BW=25-30g) yields 1.5-4.5x107 hepatocytes with the purity
and viability exceeding 90%, and 0.5-1.5x107 HM with the purity > 80% and the
viability >95%. The adherence method will increase HM purity to 94% with the
yield of 3.9x106 cells.
12. Sub-cellular fractions
Along with the expressed enzyme systems , sub-cellular fractions prepared from
drug metabolizing tissues (liver and gut), including cytosol , S9 and microsomal
fractions , are used in drug metabolism studies to address various questions . The
cytosol, which is isolated as a supernatant from the S9 fraction, contains a group
of soluble drug-metabolizing enzymes responsible for specific routes of drug
metabolism. It is the simplest system of the three.The S9 fraction contains both
cytosol and microsomes , and represents an early complete collection of all drug
metabolizing enzymes.
13. However , the presence of so many enzymes can sometimes dilute the
activities of the enzyme of interest . The microsomal fraction contains
membrane-bound CYPs and primary conjugation enzymes such as UGTs.
These enzymes are responsible for the metabolism of over90% of marketed
drugs, thus this is the most frequently utilized enzyme system. The cytosol is
often used to conduct mechanistic studies for identifying the soluble enzymes
involved in particular metabolic pathways, such as SULT, N-acetyl
transferase (NAT), certain glutathione transferases (GST),AO, and xanthine
oxidase (XO).
14. For example, region selective sulfation of the phyto estrogens daidzein and
geniste in was investigated using human liver cytosol and purified,
recombinant human SULT isoforms11. Additionally, cytosol assays are used
to complement microsomal studies for assessing drug metabolism
pathways12.
15. Sub cellular fractions in rats
. Weigh out 0.5 g of Rat Liver . Add 5 ml of 0.25 M Sucrose in 10mM Tris-
HCl (pH 7.5). Chop up the liver with a clean single edge razor and transfer
to homogenation tube. Homogenize liver while keeping it cool in the ice
bucket. After the homogenization, pour though cheesecloth into the
centrifuge tube
First take three 100ul samples of the broken cells from the centrifuge tube.
Place these 100 ul samples into each of three separate small microfuge tubes
(label them well for your group). Bring them up to the front of the class for
freezer storage and later analysis. Decant the solution into a centrifuge tube
and try to keep most of the glass beads out of the new tube. The rest of the
sample (4.7 ml) will be centrifuged at 3000 rpm (1000 X g) for 5 minutes to
pellet cell fragments, unlysed cells and nuclei.
16. After centrifugation, pour the supernate into a new centrifuge tube (for
further centrifugation at 10,000 rpm (9,500 X g) for 10 minutes. While this
centrifugation is going, resuspend the pellet in 1 ml of the Sucorse/Tris
buffer (pH 7.5) and put an about 300 ul into each of three separate small
microfuge tubes. Bring them up to the front of the class for freezer storage
and later analysis. This is your nuclei sample.
After centrifugation, pour the supernate into a new centrifuge tube (for
further centrifugation at 12,000 rpm (14,000 X g) for 25 minutes. While this
centrifugation is going, resuspend the pellet in 1 ml of Tris buffer (pH 7.5)
and put an about 300 ul into each of three separate small microfuge tubes.
Bring them up to the front of the class for freezer storage and later analysis.
This is your mitochondria sample.
17. Take the supernate (cytosol components) and pipet into three large tubes
and bring them up to the front of the class for freezer storage and later
analysis. Resuspend the pellet (containing plasma membrane) in 1 ml of
Tris buffer (pH 7.5) and put an about 300 ul into each of three separate
small microfuge tubes. Bring them up to the front of the class for freezer
storage and later analysis.
18. Sub cellular fractions in mouse
Materials and Reagents
Mice
PBS
Phosphatase inhibitor (e.g. PhosStop)
HEPES
EDTA
Distilled water
Glucose
Protease inhibitors (e.g. Complete)
Solution A
2.5 M sucrose stock
Sucrose solution (0.8 M and 1.6 M)
19. Equipment
A pair of scissor
A brain scooper
Falcon tubes (15 ml)
Centrifuge
Ultracentrifuge
Ultracentrifuge rotor
Ultracentrifuge tubes for T70.1 Ti rotor (Polycarbonate, 16 x 76 mm)
Ultracentrifuge tubes for SW41 Ti rotor (14 x 89 mm) (Seton Open-Top Polyclear centrifuge
tubes,
Thomas® Teflon Pestle
1 and 10 ml syringes
Needles (18 and 27 G)
Gradient Master 107 ip
Refractometer
20. Procedure
Mouse is sacrificed by cervical dislocation; Restrain the mouse on a hard, flat surface. Hold
a strong stick or metal rod firmly against the base of the skull, and the tail firmly with the
other hand. Pull the mouse body away from the head in one single quick motion. Verify the
dislocation by feeling for a separation between cervical vertebras.
Cut the head off the mouse, and open the skin of the head with a pair of scissor.
Open the skull of the mouse using a pair of scissor, and gently lift out the exposed brain
using a brain scooper and transfer to ice-cold PBS.
Brains are quickly rinsed in ice-cold PBS.
Brains are put in 10 ml solution A containing proteinase inhibitors and continue to point 9
when all brains have been isolated.
Repeat steps 1-7 for the remaining mice.
The brains are homogenized using a Thomas® Teflon Pestle
21. Transfer the homogenized brain tissue to a 15 ml Falcon tube.
Centrifuge at 1,000 x g for 10 min at 4 °C.
Transfer the supernatant (there are typically several layers of supernatant – take them all)
to a new 15 ml Falcon tube.
Centrifuge at 3,000 x g for 10 min at 4 °C.
Transfer the supernatant to an ultracentrifugation tube.
Centrifuge at 13,000 rpm for 10 min using an ultracentrifuge with a T70.1 Ti rotor.
Transfer the supernatant to a new ultracentrifugation tube and centrifuge at 50,000 rpm
for 45 min at 4 °C (using the same rotor
Discard the supernatant.
Dissolve pellet in 600 μl solution A containing proteinase inhibitors. Use a 1 ml syringe
with an 18 G needle first, thereafter a 27 G needle to dissolve pellet.
22. Carefully layer 500 μl of the homogenate (= dissolved pellet) on top of a continuous 0.8 to
1.6 M sucrose gradient and centrifuge at 25,000 rpm for 18 h/overnight, using a SW41 Ti
swing bucket rotor.
The gradient is made with a Gradient Master 107 ip.
Insert a tube in the marker block and mark a line on the tube at the half-full mark for short,
4 mm cap.
Add 5.5 ml 0.8 M sucrose solution to the tube.
Add 1.6 M sucrose beneath the 0.8 M sucrose solution using a cannula (a cannula is
received together with the Gradient Master) and a 10 ml syringe until the bottom of the 0.8
M sucrose solution reaches the recently marked line.
Adjust the level of the Gradient Master before use, to ensure that the plate is in level.
Prepare the 10-57% linear gradient, by choosing “SW41” in the ”gradient menu” list of
BioComp Gradient Master. This program will mix the gradient at 50 degrees for 10 min,
followed by 1 min at 80 degrees.
23. Next day the gradient is fractionated into 24 samples (500 μl/sample), using a 1 ml pipette.
Alternatively, one can use a Piston Gradient Fractionator to collect the fractions.
Store the fractions at -20 °C until Western blot analysis.
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3. Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29-83.
Epub1976/01/01. PubMed PMID: 177845.
4. Li Y, Lua I, Asahina K. MACS isolation and culture of mouse liver mesothelial cells.
Bioprotocol. 2013;http://www.bio-protocol.org/wenzhang.aspx?id=815.
5. Seglen, P. O. (1976) Preparation of isolated rat liver cells. Methods Cell. Biol. 13, 29–83.
6. Gustafsen, C., Glerup, S., Pallesen, L. T., Olsen, D., Andersen, O. M., Nykjaer, A., Madsen, P.
and Petersen, C. M. (2013). Sortilin and SorLA display distinct roles in processing and
trafficking of amyloid precursor protein. J Neurosci 33(1): 64-71.