`
`A HANDBOOK
`OF BIOANALYSIS
`AND DRUG
`
`METABOLISM
`Gary Evans
`
`EDITED BY
`
`Ce olXen Iran)
`
`Taylor & Francis Group
`
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`

`

`A HANDBOOK OF
`BIOANALYSIS AND
`DRUG
`METABOLISM
`
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`

`

` Taylor & Franci
`
`Taylor & Francis Group
`http://taylorandfrancis.com
`
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`

`A HANDBOOK OF
`BIOANALYSIS AND
`DRUG
`METABOLISM
`
`EDITED BY
`Gary Evans
`
`Boca Raton London New York
`
`CRC Press is an imprint of the
`Taylor & Francis Group, an informa business
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`CRC Press
`Taylor & Francis Group
`6000 Broken Sound Parkway NW, Suite 300
`Boca Raton, FL 33487-2742
`
`First issued in paperback 2019
`
`© 2004 by Gary Evans
`CRC Press is an imprint of Taylor & Francis Group, an Informa business
`
`No claim to original U.S. Government works
`
`ISBN-13: 978-0-415-27519-4 (hbk)
`ISBN-13: 978-0-367-39442-4 (pbk)
`DOI: 10.1201/9780203642535
`
`Library of Congress Card Number 2005053800
`
`This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made
`to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all
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`

`chapter 15
`
`In vitro techniques
`for investigating
`drug metabolism
`
`Graham Somers, Peter
`Mutch and Amanda
`Woodrooffe
`
`15.1 Introduction
`
`The use of in vitro techniques to study drug metabolism allows comparison of the
`metabolism of a compound across species prior to administration to humans. In
`addition, the use of human in vitro systems provides more relevant information on
`the metabolites likely to be formed in clinical studies. Therefore, interspecies
`comparisons of the metabolite profile of a drug candidate may assist rational
`selection of the most appropriate species for safety assessment studies. Finally,
`potential clinical interactions, i.e. the ability of a drug candidate to affect the
`pharmacokinetics of other coadministered therapies (induction or inhibition of
`drug-metabolising enzymes) can also be investigated using human in vitro systems.
`The liver is a major site for the biotransformation of xenobiotics and so many
`models of drug metabolism have primarily focussed on the liver and the enzymes
`contained within it. Investigation of drug metabolism has involved the use of liver
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`In vitro techniques for investigating drug metabolism 245
`
`preparations which vary in their levels of cellular integrity. Systems range from the
`use of purified enzymes in vitro to whole liver perfusion in situ. For the purpose
`of this chapter, discussions will concentrate around the use of subcellular fractions
`(S9 and microsomes), isolated hepatocytes and liver slices.
`The cytochromes P450 are a large family of enzymes involved in the metabolism
`of a wide range of structurally diverse xenobiotics. Cytochrome P450 enzymes are
`bound to the membranes of the endoplasmic reticulum, and concentrated sources
`used for the investigation of drug metabolism can be prepared from homogenised
`liver by differential centrifugation. The submitochondrial (S9) fraction of liver
`contains both cytosolic and membrane-bound enzymes but as a homogenate rather
`than an ordered cellular environment. The S9 fraction is prepared by sedimenting
`particulate matter, including cell nuclei and mitochondria, from homogenised liver
`g and is a crude tissue preparation; the impure nature of this preparation
`at 9,000
`may lead
`o analytical difficulties. However, a more refined enzyme may be prepared
`from crude liver S9 by further centrifugation. Fragments of the endoplasmic
`reticulum reform into vesicles known as microsomes. The microsomal pellet is
`
`g.
`produced by centrifugation of the S9 fraction at 100,000
`d enzymes
`Microsomes are essentially an enriched source of the membrane-boun
`such as the cytochromes P450 and as such are probably the most commonly used
`system in the pharmaceutical industry to study in vitro cytochrome P450-mediated
`metabolism. Both S9 and microsomes retain enzyme activity following cold storage
`at 80
`
`C. Therefore, S9 and microsome preparations may be made and stored
`ready for use, an advantage for cross comparisons of metabolism between species and
`in the case of humans between different liver donors.
`The parenchymal cells of the liver (hepatocytes) are a rich source of cytochromes
`P450. Hepatocytes can be isolated from liver tissue by enzymatic dissociation using
`collagenase perfusion. Once isolated, hepatocytes can be used to study drug metab-
`olism and induction or inhibition of drug-metabolising enzymes by xenobiotics.
`The major advantage of hepatocytes over microsomes for the study of drug
`metabolism is that, as a cellular system, hepatocytes contain many enzymes and
`enzyme cofactors not present in the microsomal fraction of the cells. For example,
`several of the enzymes responsible for conjugation of xenobiotics, such as the
`sulphotransferases, are located in the cytoplasm of the cell (see Table 15.1).
`Although some Phase II metabolic pathways can be studied using microsomes
`supplemented with appropriate cofactors (e.g. glucuronidation), they may be more
`easily investigated using hepatocytes.
`Hepatocytes may be used as short-term suspension cultures (four hours) or may
`be placed into culture for investigation of drug-related enzyme induction or enzyme
`inhibition that requires longer periods of exposure to the test compound. One issue with
`hepatocytes that are placed into culture is that they undergo de-differentiation,
`i.e. the loss of specific cell characteristics, and this results in a rapid decrease in their
`content and activity of drug-metabolising enzymes, notably the cytochromes
`P450. As such, primary cultures of hepatocytes are not routinely used for the study
`
` t
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`Table 15.1 Cellular location of the major drug-metabolising enzymes
`
`Cellular
`location
`
`Substrates
`
`Esters
`
`Epoxides
`
`Peptides
`Glutathione conjugates
`
`Azo
`Nitro
`N-oxides
`Arene oxides
`Alkyl halogenides
`Alcohols
`Alkanes
`Alkenes
`Arenes
`Amines
`Thiones
`Thioesters
`Amines
`Alcohols
`Aldehydes
`
`Electrophiles
`Phenols
`Thiols
`Amines
`Carboxylic acid
`Phenols
`Thiols
`Amines
`Phenols
`Amines
`Amines
`Carboxylic acid
`Aromatic hydroxylamine
`
`Reaction
`
`Enzyme
`
`Phase I reactions
`Hydrolysis
`
`Esterases
`
`Eposide hydrolase
`
`Peptidase
`
`Reduction
`
`Mixed function oxidases
`
`Cytosol
`Mitochondria
`Microsomes
`Blood
`Cytosol
`Microsomes
`Microsomes
`Blood
`Lysosomes
`Microsomes
`
`Oxidation
`
`Alcohol dehydrogenases
`Mixed function oxidases
`
`Cytosol
`Microsomes
`
`Monoamine oxidases
`Alcohol dehydrogenases
`Aldehyde dehydrogenases
`
`Mitochondria
`Cytosol
`Cytosol
`
`Phase II reactions
`Glutathione conjugation Glutathione transferases
`Glucuronide conjugation Glucuronyl transferases
`
`Microsomes
`Microsomes
`
`Sulphate conjugation
`
`Sulfotransferases
`
`Cytosol
`
`Methylation
`
`Methyl transferases
`
`Acetyl transferases
`Acetylayion
`Amino acid conjugation Transferases
`
`Cytosol
`Microsomes
`Cytosol
`Cytosol
`Mitochondria
`
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`In vitro techniques for investigating drug metabolism 247
`
`of species differences in drug metabolism but are much more valuable in the study
`of drug-related enzyme induction or enzyme inhibition.
`Liver slices represent a greater level of structural integrity compared to sub-cellular
`fractions or hepatocytes and may be prepared using instruments such as the Krum-
`dieck tissue slicer. Tissue slices have the advantage that, compared to hepatocytes, they
`retain an intact three-dimensional structure, allowing cellular architecture and hence
`intercellular communication to be maintained. This cell–cell contact between differ-
`ent hepatic cell types is important in maintaining cell differentiation. For studies
`using human liver, tissue slices offer a major advantage over the preparation of
`hepatocytes, in that samples encapsulated by the Glisson’s capsule (required for
`hepatocyte isolation) are not necessary for the preparation of slices and therefore use
`can be made of any fresh human liver samples. Liver slices, like hepatocytes, are
`capable of performing both Phase I and Phase II biotransformations.
`The preparation, uses, advantages and disadvantages associated with the use of
`S9, microsomes, hepatoctyes and tissue slices will be discussed in the next section.
`This chapter has primarily focussed on hepatic in vitro preparations; however, it should
`be noted that not only the liver but a range of other tissues such as kidney, lung and
`intestine contain drug-metabolising enzymes and carry out xenobiotic metabolism
`in vivo. Therefore, in vitro metabolic preparations, especially S9, and microsomes could
`be prepared from any tissue to study extrahepatic drug metabolism.
`
`15.2 Preparation of liver subcellular fractions and hepatocytes
`
`1 5 . 2 . 1 P R E P A R A T I O N O F S U B C E L L U L A R F R A C T I O N S
`
`The most commonly used subcellular fractions in industrial drug metabolism
`studies are the S9 fraction and microsomal preparations. Both preparations are
`produced using differential centrifugation of tissue homogenates. In addition,
`microsomes can be prepared from S9 by calcium aggregation.
`In order to preserve enzyme activity, buffers, tools and centrifuge rotors should
`be maintained cold for the duration of the preparation procedure, either by storage
`on ice or within a refrigerator.
`Excised livers (or portions of liver from larger animals and man) are weighed and
`washed in a suitable ice cold buffer at pH 7.4. The initial buffer is removed and the
`liver is added to the buffer to give a 25 per cent homogenate. The tissue is scissor
`minced and then homogenised using a suitable ‘soft’ homogenisation technique
`such as the ‘pestle and mortar’ Potter Elvhejam homogeniser. More aggressive tissue
`disruption methods tend to reduce the enzyme activity of the final preparation.
`The crude tissue homogenate is then centrifuged at 9,000  g for 20 minutes at
`
`C. This step pellets and therefore removes intact cells, cell debris, nuclei and
`
`4
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`248 Graham Somers, Peter Mutch and Amanda Woodrooffe
`
`mitochondria from the crude cell homogenate. The supernatant represents the sub
`mitochondrial fraction (S9), which may then be quickly frozen in liquid nitrogen
`and stored at 80
`
`C prior to use.
`The microsomal fraction may then be prepared from the S9 fraction by further
` g for
`centrifugation or by calcium precipitation. Centrifugation of the S9 at 100,000
`one hour sediments the microsomal
`vesicles.
`After the supernatant is discarded, micro-
`somes are resuspended in a volume of buffer (pH 7.4) equivalent to the original weight of
`tissue used. The centrifugation step may be repeated to further purify the preparation.
`The preparation generally yields a protein concentration of a few milligrams of
`microsomal protein/millilitre tissue preparation. The purity and activity of the
`microsomal homogenate is dependent upon the preparation technique and will vary
`according to the aggressiveness of the homogenisation technique and subsequent
`temperature of the homogenate and degradation of the enzyme.
`A number of additions to the preparation buffers have been suggested to increase
`the purity or enzyme activity of the final preparation. For example, the addition of
`EDTA in decreasing concentration to the preparation buffers (10 mM in homogenisa-
`tion buffer, 1 mM in wash buffer and 0.1 mM in final buffer) is thought to stabilise
`flavin monooxygenase (FMO) activity (Sadeque et al., 1992). The addition of potas-
`sium chloride (1.15 per cent w/v) to the homogenisation buffer further purifies the
`preparation by the removal of blood and cytoplasmic contaminants (Eriksson et al.,
`1978). The addition of glycerol to the final storage buffer is thought to preserve
`enzyme activity on storage (Guengerich and Martin, 1998). Table 15.2 shows the
`composition of buffers used for the preparation of liver subcellular fractions.
`An alternative method for the preparation of microsomes is to use calcium
`precipitation. This method is based on the calcium-dependent aggregation of the
`endoplasmic reticulum. Calcium chloride is added to the post-mitochondrial frac-
`tion to give a final concentration of 8 mM. The mixture is left to stand for five
`minutes with occasional mixing. Centrifugation at 27,000  g for 15 minutes will
`yield the microsomal pellet which may be resuspended as previously described.
`The protein concentration of both S9 and microsomes should be determined
`prior to use in order to normalise incubation conditions between preparations (i.e.
`ensure the same amount of protein is added to each incubation). Protein measurement
`kits are commercially available and include the Lowry method (Lowry et al., 1951)
`and the bicinchoninic acid method (Smith et al., 1985).
`
`Table 15.2 Buffers used for the preparation of S9 and microsomes
`0.1 M phosphate buffer pH7:4 þ 1:15% KCI
`0.1 M phosphate buffer pH 7:4 þ 1:15% KCI
`0.1 M phosphate buffer pH 7:4 þ 20% glycerol
`
`Homogenisation buffer
`
`Wash buffer
`
`Storage buffer
`
`(10 mM EDTA optional)
`
`(1 mM EDTA optional)
`
`(0.1 mM EDTA optional)
`
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`In vitro techniques for investigating drug metabolism 249
`
`The levels of active cytochrome P450 may be determined by the difference in
`spectrum (Omura and Sato, 1964). Determination of active cytochrome P450 gives
`an extra level of confidence in the activity of the preparation as well as greater con-
`sistency in incubation conditions if the incubations are normalised for cytochrome
`P450 concentration rather than microsomal protein concentration. In general,
`different spectra with S9 are not recommended as non-specific absorption and light
`scattering can occur due to the turbidity of this preparation.
`
`Summary of the preparation of S9 and microsomal protein (by
`ultracentrifugation)
`
`
`
`C.
`1 Carry out all steps at 4
`2 Weigh the fresh or thawed liver sample.
`3 Add four times the volume of homogenisation buffer to liver weight.
`4 Scissor mince tissue then homogenise using a Potter Elvhejam or similar
`mechanical tissue homogeniser.
` g for 20 minutes.
`5 Centrifuge the homogenate at 9,000
`6 Combine the supernatants (S9 fraction)
`and snap freeze in liquid nitrogen. Store
`S9 fraction at 80
`
`C.
`7 For the preparation of the microsomal fraction, centrifuge the S9 fraction at
`100,000 g for 1 hour.
`8 Discard the supernatant and resuspend the pellet in buffer. Centrifuge the
`
`g for 1 hour.
`homogenate at 100,000
`9 Discard the supernatant
`and resuspend the pellet in storage buffer (volume
`equivalent to the original tissue weight).
`10 Snap freeze aliquots (1 or 2 ml) in liquid nitrogen and store at 80
`
`
`C
`
`1 5 . 2 . 2 P R E P A R A T I O N O F H E P A T O C Y T E S
`
`There are three methods routinely used for the preparation of hepatocytes. The
`methods involve removal of calcium from the tissue followed by treatment of the
`tissue with a solution containing collagenase. Calcium removal initiates the separ-
`ation of cell–cell adhesion via calcium-dependent desmosomes. The collagenase
`treatment digests the architecture of the liver and allows the hepatocytes to be
`released. The first method is based on that of Berry and Friend (1969) and involves
`the perfusion of the liver in situ. The second technique was developed in 1976 by
`Fry and co-workers and involves digestion of liver slices, thus avoiding the need for
`perfusion. The benefit of this method is that an intact Glisson’s capsule around the
`liver is not required, so use can be made of tissue that would otherwise be wasted. The
`third method is an adaptation of the in situ collagenase perfusion technique (Strom
`et al., 1982; Oldham et al., 1985). Small, end-of-lobe liver fragments surrounded
`
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`250 Graham Somers, Peter Mutch and Amanda Woodrooffe
`
`by an intact Glisson’s capsule are perfused by cannulating the exposed vessels on
`the cut surface of the sample. This third method is the method routinely used in our
`laboratories because it can be used to isolate hepatocytes from several different
`species, including human, with minimal changes to the basic technique. An in situ
`perfusion technique would not be suitable to use with larger species such as the dog.
`The method described below is used for the isolation of hepatocytes from end-of-
`lobe liver samples (wedge biopsies). Table 15.3 details the composition of buffers and
`
`Table 15.3 Buffers and solutions for the isolation of hepatocytes
`10  EBSS
`
`Isolation buffer:
`Perfusion buffer
`
`(without calcium and magnesuim)
`7.5% sodium bicarbonate solution
`purified water
`
`100 ml
`30 ml
`870 ml
`pH adjusted to 7.4
`
`Isolation buffer:
`Chelating buffer
`
`Perfusion buffer
`25 mM EGTA (in 0.1 M NaOH)
`
`490 ml
`10 ml pH adjusted to 7.4
`
`Isolation buffer:
`Collagenase buffer
`
`Dispersal buffer
`
`Culture medium
`(for suspensions)
`
`Culture medium
`(for monolayers)
`
`Perfusion buffer
`1 M CaCl2
`Trypsin inhibitor
`Collagenase H
`
`Sodium chloride
`Potassium chloride
`HEPES
`Purified water
`BSA
`
`William’s medium E
`200 mM L-glutamine
`
`William’s medium E
`200 mM L-glutamine
`
`Penicillin (10,000 U/ml)/Streptomycin
`(10,000 mg/ml)
`Insulin (250 U/ml)
`d-aminolevulinic acid (1 mM)
`Transferrin (5 mg/ml)
`Hydrocortisone (3.6 mg/ml)
`Zinc sulphate (5 mM)
`
`Chee’s essential medium
`200 mM L-glutamine
`Penicillin (10,000 U/ml)/
`Streptomycin (10,000 mg/ml)
`
`Culture medium
`(for monolayers)
`
`150 ml
`300 ml
`~10 mg
`12 units (rat) 24 units
`(dog, human, pig)
`
`4.15 g
`0.26 g
`1.19 g
`500 ml pH adjusted to 7.4
`5.0 g
`
`500 ml
`10 ml
`
`500 ml
`5 ml
`
`5 ml
`0.5 ml
`0.5 ml
`0.5 ml
`0.5 ml
`0.5 ml
`
`500 ml
`5 ml
`5 ml
`
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`In vitro techniques for investigating drug metabolism 251
`
`solutions required for the isolation of hepatocytes. Figures 15.1 and 15.2 show the
`apparatus used for the isolation procedure and the perfusion of whole lobes of rat liver.
`
`All buffers and solutions should be maintained at 37
`C with the exception of the
`
`dispersal buffer which should be kept at 4
`C. All buffers used during the perfusion
`should be maintained at pH 7.4 by continuous gassing with carbogen (95 per cent
`oxygen, 5 per cent carbon dioxide).
`Exposed vessels on the cut surface of the liver tissue (or vessels entering the
`individual lobes for rat liver) are cannulated using 20 gauge (rat) or 16 gauge cannulae
`(human, dog, pig). The tissue is perfused with chelating buffer for approximately
`5 minutes at a flow rate of approximately 6 ml/min/cannula (rat) or 12 ml/min/cannula
`(human, dog, pig). The chelating buffer is then washed out by perfusion buffer for
`approximately 5 minutes. The tissue is then perfused with collagenase buffer, which
`should be recirculated after 3–4 minutes. The collagenase perfusion should continue
`until the cells beneath the Glisson’s capsule become spongy to the touch. This
`should take approximately 20–25 minutes for rat liver and anything up to 1 hour for
`the isolation of human hepatocytes. The time required for human hepatocyte isolation
`varies depending upon the age, status and fibrotic nature of the liver sample used.
`
`Chelating buffer
`
`Perfusion buffer
`
`Collagenase buffer
`
`Peristaltic pump
`
`Thermocirculator
`
`Bubble trap
`
`Heated water jacket
`
`Figure 15.1 Apparatus used for the isolation of hepatocytes.
`
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`252 Graham Somers, Peter Mutch and Amanda Woodrooffe
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`Figure 15.2 Isolation of hepatocytes: perfusion of rat liver lobes.
`
`Once the extracellular matrix has dissociated sufficiently the perfusion is stopped,
`avoiding possible rupture of the Glisson’s capsule.
`The tissue is placed into a shallow dish containing dispersal buffer and using
`forceps, the Glisson’s capsule is carefully peeled away releasing isolated cells into the
`dispersal buffer. The forceps can be used to gently ‘comb’ the cells to help release
`them from the tissue.
`The cell suspension is filtered through nylon bolting cloth (64 mm pore size)
`pre-wetted with dispersal buffer containing DNAse I (5 mg/100 ml) to remove
`large clumps of tissue and cells. DNAse removes DNA that has been released from
`damaged cells which causes cells to clump together. All subsequent manipulations
`
`of the cell suspension are carried out at 4
`C. The low temperature minimises the
`activity of any cytotoxic enzymes that may have been released from damaged cells
`and to preserve drug-metabolising enzyme activity. Viable cells are sedimented by
`centrifugation (50  g for 5 min) at 4
`
`C. The cells are washed twice more as
`previously described but without DNAse I in the final wash.
`The final cell pellet is resuspended in a suitable incubation buffer such as
`William’s medium E (plus appropriate supplements) and the viability of the
`preparation assessed using an appropriate cell viability test, of which the simplest
`is the trypan blue exclusion test. Trypan blue is a large molecular weight dye which
`
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`In vitro techniques for investigating drug metabolism 253
`
`is excluded from viable cells with an intact cell membrane. However, non-viable
`cells with a damaged membrane will take up the dye resulting in a blue-stained
`nucleus. After treatment with trypan blue, the total number of cells isolated in
`combination with the number of viable and non-viable cells can easily be deter-
`mined using light microscopy and a haemocytometer. Generally, for small animal
`species, a viability of >90 per cent is required before use.
`The remaining cell suspension is now ready to be diluted appropriately for use.
`
`Summary of hepatocyte isolation (two-step collagenase digestion)
`
`1 Perfuse tissue with chelating buffer.
`2 Perfuse tissue with perfusion buffer.
`3 Perfuse tissue with collagenase buffer until extracellular matrix has dissociated.
`4 Place tissue into dispersal buffer.
`5 Break open Glisson’s capsule and tease out cells into the buffer.
`g, 5 minutes).
`6 Filter cells, with DNAse I and centrifuge (50
`g, 5 minutes).
`7 Filter cells, with DNAse I and centrifuge (50
`
`g, 5 minutes).
`8 Filter cells, without DNAse I and centrifuge (50
`9 Re-suspend cell pellet in medium.
`10 Determine cell viability and total cell number using trypan blue exclusion.
`11 Dilute remaining cells.
`12 Use in suspension or plate cells out as monolayers.
`
`
`
`1 5 . 2 . 3 P R E P A R A T I O N O F L I V E R S L I C E S
`
`There are a number of different techniques for producing liver slices, and a similar
`choice of incubation systems. A number of commercial tissue slicers are available,
`such as the Brendel-Vitron and Krumdieck machines, which allow thin slices of
`liver (thickness ca. 250 mm) to be reproducibly cut from cores of tissue. These cores
`may be prepared freehand or by using a mechanised borer. The cores are then placed
`in the machine, and slices cut using a microtome, which allows the thickness of the
`prepared slices to be controlled. The core, and the slices prepared from it, is kept
`submerged in ice-cold physiological buffer. Following this, prepared slices are
`harvested and used in the incubation system, the choice of which may depend on
`the aim of the experiment. If slices need to be prepared aseptically this may be
`possible as some mechanised slicers are autoclavable.
`
`Summary of liver slice preparation
`
`1 Obtain fresh liver.
`2 Cut cylindrical cores from the tissue.
`
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`254 Graham Somers, Peter Mutch and Amanda Woodrooffe
`
`3 Place cores into the tissue slicer and prepare slices in ice-cold physiological
`buffer.
`4 Transfer slices into incubation system of choice.
`
`15.3 Use of subcellular fractions, hepatocytes and liver slices to study
`drug metabolism
`
`1 5 . 3 . 1 S U B C E L L U L A R F R A C T I O N S
`
`There are a number of uses for subcellular fractions within the pharmaceutical
`industry. However, the major uses within drug metabolism are to screen potential
`drug candidates for metabolic stability and as ‘metabolite factories’ to produce
`potential metabolites for identification with more ease than by extraction and
`analysis of drug-related material from biological fluids following in vivo drug
`administration.
`Qualitative experiments investigating the metabolic profile of potential drug
`candidates (Adams et al., 1981; Acheampong et al., 1996) have been used to confirm
`the presence of metabolites in animals and man and therefore validate long-term
`and expensive toxicology testing.
`Incubations are prepared by addition of the compound, enzyme preparation,
`buffer and appropriate enzyme cofactors. The addition of organic solvents to enzyme
`incubations should be kept to a minimum as organic solvents may affect enzyme
`activity (Chauret et al., 1998). Choice of the appropriate enzyme cofactor is also an
`important consideration. For example, the preparation of microsomes produces an
`endoplasmic reticulum-rich fraction containing membrane-bound cytochromes
`P450. All soluble enzyme cofactors are lost during the process. Therefore, oxida-
`tions by cytochromes P450 will not proceed without the addition of the reduced
`form of the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) or an
`NADP(H)-regenerating system to the microsomal incubations.
`Following incubation, and simple ‘cleanup’ techniques such as the addition of
`organic solvents to precipitate proteins, the metabolites obtained may be analysed
`using high-performance liquid chromatography (HPLC), in conjunction with mass
`spectrometry (LC–MS) or nuclear magnetic resonance (LC–NMR). In this way,
`detailed information of the metabolic profile of a compound may be determined.
`Species-specific metabolism or the production of pharmacologically active or toxic
`metabolites may be quickly screened and investigated.
`Quantitative metabolism may be used in the initial stages of research to screen
`and then rank potential drug candidates according to their metabolic stability. The
`advent of combinatorial chemistry has enabled chemists to rapidly produce large
`numbers of novel synthetic compounds. Metabolic stability (or instability) is
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`In vitro techniques for investigating drug metabolism 255
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`a crucial factor in the development of any new medicine, so high-throughput
`methodologies employing subcellular fractions have now been developed to enable
`the metabolic stability of large numbers of compounds to be investigated in the
`minimum amount of time (Eddershaw and Dickins, 1999). Compounds with
`inappropriate metabolic stability can therefore be removed from research and
`development at an early stage.
`
`1 5 . 3 . 2 H E P A T O C Y T E S
`
`Hepatocytes provide the pharmaceutical industry with a whole cell system to study
`the metabolism of drug candidates. Hepatocytes can be cultured in suspension in
`the presence of a test compound for up to 4 hours. The culture medium routinely
`used in our laboratories is William’s medium E containing 4 mM L-glutamine. The
`medium does not contain phenol red as this indicator dye is metabolised by several
`Phase II drug-metabolising enzymes and therefore could compete for metabolism
`by these enzymes and prevent Phase II metabolism of the test compound (Driscoll
`et al., 1982).
`
`1 5 . 3 . 3 L I V E R S L I C E S
`
`As a whole cell preparation, liver slices offer an alternative to hepatocytes and
`perfused liver in the study of metabolism.
`Liver slices have a variety of uses in the study of drug metabolism. The ease and
`speed of their preparation facilitates the retention of metabolic activity and they are
`therefore useful as predictive tools when studying the biotransformations of drugs
`in vitro. They can be used as ‘metabolite factories’ to produce sufficient quantities of
`metabolite for characterisation and identification. Because the amount of liver tissue
`per slice will never be identical, however, there can be greater individual variability
`between slices compared with hepatocyte incubations when used in quantitative
`studies, for instance in drug interaction studies.
`The choice of incubation system may depend on the aim of the experiment.
`A dynamic organ culture system may be used, where the slice is supported on a wire
`mesh in a vial-containing medium and placed on rollers in an oven maintained
`
`C so that the slice moves in and out of the liquid phase (Smith et al., 1986).
`at 37
`Alternatively, slices may be placed in a conical flask, or in 12- or 24-well plates, and
`incubated with agitation in a temperature-controlled incubator. Whichever system
`is used, a pre-incubation of the slice in fresh culture medium (generally up to
`2 hours duration) is carried out to allow sloughing of cells from the cut surfaces of the
`slice and ensure homeostasis. For short-term incubations, such as simple assessment
`of xenobiotic biotransformation in a manner similar to the use of hepatocyte
`suspensions, the slices may be incubated in a tissue culture medium such as
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`256 Graham Somers, Peter Mutch and Amanda Woodrooffe
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`Williams’ medium E (supplemented with glutamine) containing the test com-
`pound. Generally for a short-term culture, sufficient oxygen may be introduced
`to the system by pre-gassing the medium with carbogen. Alternatively, vials with a
`hole drilled in the lid to facilitate oxygen transfer may be placed in the incubator.
`For longer-term incubations, such as studying the biotransformation of slowly
`metabolised xenobiotics or the potential
`for induction of drug-metabolising
`enzymes (Lake et al., 1997), the medium is usually supplemented with a number
`of additives such as foetal calf serum, hormones and antibiotics. In addition,
`for longer-term cultures, the medium may need to be changed regularly and
`adequate oxygenation maintained by placing the culture plates in a tissue culture
`incubator.
`At the end of incubation both the medium and the slice are usually analysed, as
`metabolites may be retained within the slice.
`
`15.4 In vitro–in vivo correlations
`
`All the systems discussed so far can be used to measure the metabolic clearance of a
`compound in vitro. The data obtained from any of these systems can then be scaled
`using various models to provide an estimate of the clearance of that particular drug
`in vivo.
`Subcellular fractions, especially microsomes (Houston, 1994; Houston and
`Carlile, 1997; Iwatsubo et al., 1997) have been used to estimate rates of metabolic
`clearance of a particular drug in vitro in an attempt to estimate rates of metabolic
`clearance of a drug in vivo. This may be achieved either by quantifying rates of
`metabolite production, or by calculation of the rate of disappearance of substrate
`from the incubation by metabolism. Disappearance plots have the advantage that
`knowledge of the metabolic profile of a compound is not required before investiga-
`tion commences, thus enabling the study of drug candidates whose metabolic
`profile is unknown. However, the disadvantage of this approach is that substrate
`may be removed from the incubation matrix by methods other than metabolism
`such as non-specific irreversible binding to cellular proteins.
`The first step in quantitative prediction of the metabolic clearance of a drug
`candidate in vivo is achieved by determination of