| Cytochrome P450 isoform-specific in vitro methods to predict drug metabolism and interactions | ||
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Preclinical studies consist of animal studies (on the pharmacokinetics and pharmacodynamics of the compound, toxicological studies) and animal and human tissue-derived in vitro studies. Because of the problems in extrapolating the results of animal studies to humans, various in vitro methods have been developed by employing human tissue-derived systems (Wrighton et al. 1993). Also, the authorities have begun to demand increasingly that the issues concerning metabolism and toxicity in test species compared to humans should be actively clarified in early preclinical tests. This is done by utilising liver preparations from humans and trying to find the test species that most closely resemble human metabolism and the production of toxic intermediates (Yuan et al. 1999).
It is important to elucidate the in vitro metabolism and the putative interactions at the time of planning other preclinical and early clinical studies. In the next few chapters, human tissue-derived preclinical in vitro systems will be briefly discussed.
There are several approaches to preclinical metabolism studies. The enzyme sources in these studies are human-derived systems currently under rapid development and evaluation. These systems consist of liver microsomes, hepatocytes and cell lines heterologously expressing drug-metabolising enzymes, liver slices and individually cDNA-expressed enzymes in host cell microsomes. Each of these will be discussed briefly here. Table 3 shows a comparison of different human-derived in vitro methods.
Human liver microsomes are fractionated from subcellular organelles by differential ultracentrifugation. Microsomes are formed from smooth endoplasmic reticulum during tissue homogenisation (Boobis 1995). A microsomal fraction from human liver contains a full complement of P450 enzymes, which makes it a suitable tool for studying inhibitory interactions and CYP-catalysed metabolite formation (Kremers 1999). By employing relevant cofactors and other reaction components, one can readily investigate and distinguish between P450s, flavin-containing monooxygenases (FMO) and glucuronosyl transferases. Microsomes are relatively easy to prepare, and enzymatic activities are stable during prolonged storage (Beaune et al. 1986; Yamazaki et al. 1997), if the original tissue is correctly handled and frozen immediately after excision (Kremers 1999). The validation and harmonisation of assay procedures between different laboratories in this respect, too, would lead to less inter-laboratory variation in the same assays (Boobis et al. 1998; Kremers 1999).
Hepatocytes contain the full compartment of phase I and phase II enzymes, and the whole metabolite pattern can therefore be detected in incubations with hepatocytes. Other effects of an NCE, such as the induction of drug-metabolising enzymes and possible toxic effects, can also be elucidated. (Ferrini et al. 1997; Li et al. 1997; Maurel 1996; Morel et al. 1990).
The utilisation of human primary hepatocytes is restricted, because sufficient liver samples are quite difficult to obtain and hepatocytes are difficult to preserve for later use. Some successful attempts to cryopreserve primary hepatocytes have been described (Guillouzo et al. 1995). A prolonged culture method has also been published, in which hepatocytes are maintained for over 30 days (Kono et al. 1997), preserving some of their drug-metabolising activities and inducibility instead of the standard for up to one week. Hepatocytes can be subjected to several test and washout periods during their lifetime. This method, together with the cryopreservation method, enables more efficient use of a single hepatocyte batch than before.
Table 3. Comparison of in vitro enzyme sources used in preclinical research.
| Enzyme sources | Availability | Advantages | Disadvantages |
|---|---|---|---|
| Microsomes | Relatively good, from transplantations or commercial sources. | Easy to obtain. Also commercially available. Relatively inexpensive technique. | Contains only phase I DMEs and UDP-glucuronosyl transferases. Requires strictly specific substrates and inhibitors or antibodies for individual DMEs. |
| cDNA-expressed individual CYPs | Good, commercially available. | Can be utilised with HTS substrates. The role of individual CYPs in the metabolism of an NCE can be easily studied. | The effect of only one enzyme at a time can be evaluated. |
| Immortalised cell lines | Available at request, not many adequately characterised cell lines exist. | Non-limited source of enzymes. | The expression of most DMEs is poor or absent if characterised at all. |
| Primary hepatocytes | Relatively difficult to obtain, relatively healthy fresh tissue needed. Commercially available. Cryopreservation possible. | Contains the whole complement of DMEs. The induction effect of an NCE can be studied. | Requires specific techniques and well established procedures. The levels of many DMEs decrease rapidly during cultivation. |
| Liver slices | Relatively difficult to obtain, fresh tissue needed. Cryopreservation possible. | Contains the whole complement of DMEs and cell-cell connections. The induction effect of an NCE can be studied. | Requires specific techniques and well established procedures. |
| Data partially adapted from Wrighton et al. (1993) and Skett et al. (1995). | |||
Immortalised cell lines that express one or more drug-metabolising enzymes could be valuable tools in preclinical studies, but for most CYPs this approach has failed (Daujat et al. 1996; Gonzalez & Korzekwa 1995). When whole-cell systems are used, one must know which P450s are really expressed as functional proteins and which are liable to fade out during cultivation. Many of the cell lines used lack the complement of enzymes present in hepatocytes in vivo. On the other hand, when studying direct interactions with the enzyme, whole cell systems could be too complex (Boobis 1995).
Of the systems presented here, precision-cut liver slices resemble most closely the in vivo situation, which contains not only the enzymes of the whole liver but also the connections between individual cells. The technique of maintaining liver slices is demanding. The thickness of a slice has to be minimal within the limits of the optimal number of cell layers and oxygen and nutrient transportation (Vickers et al. 1995). Recently, methods for cryopreservation of tissue slices have been developed (for example Fisher et al 1993; Ekins et al. 1996; de Kanter et al. 1998; Glöckner et al. 1998; de Graaf et al. 2000). These methods allow more flexible use of slices, because viability and CYP activities seem to be preserved quite well (de Graaf et al. 2000; Renwick et al. 2000).
Drug-metabolising enzymes are available commercially as heterologously expressed enzyme systems. In these preparations, an individual enzyme is produced in the ER of an eucaryote host cell. The expression of human liver CYPs in different artificial systems has become easier due to the rapid development of recombinant DNA techniques (Gonzalez et al. 1991). The systems employed for the production of cDNA-expressed CYPs include bacteria (Fisher et al. 1992; Gillam et al. 1993), yeast (Guengerich et al. 1991a; Peyronneau et al. 1992), mammalian cell lines (Guengerich 1995b) and baculovirus systems (Asseffa et al. 1989). cDNA-expressed enzymes are a valuable tool in the search for the enzymes participating in the metabolism of an NCE. Because the enzymes are studied in isolation from other hepatic enzymes and because they lack the whole complement of hepatic enzymes, the in vivo predictive value of the data obtained from heterologously expressed enzyme systems has been debated (Rodriguez 1999).
If the inhibition of the metabolism of an unknown compound is studied in a single enzyme system, one should take into account that in vivo there are different amounts of individual enzymes in the human liver. The cofactor supply may also affect the relative contribution of certain CYPs. As it was pointed out above, the contribution of one enzyme to the specific metabolic route may not be so significant as it seems on the basis of cDNA-expressed enzymes (Rodriguez 1999).
As an affinity-screening tool, cDNA-expressed enzymes are valuable. It is also the most useful system allowing a high-throughput screening (HTS) technology for P450 studies today, because of the difficulties and high costs in the detection of multiple substrates and metabolites produced in the HTS applications of other techniques, such as human liver microsomes (White 2000). The detection of these multiple metabolites requires novel, highly sensitive mass spectrometry tools, whereas cDNA-expressed systems can utilise the conventional measurement of fluorescence metabolite production for multiple enzymes (see, for example, www.gentest.com).
The metabolic stability of an NCE determines, to a great extent, its future as a drug candidate (see, for example, Kuhnz & Gieschen 1998). If an NCE is rapidly metabolised in human liver preparations, its bioavailability in vivo is most probably too low for it to be a drug candidate. This naturally depends on the administration route of the drug. By determining the time and concentration dependence of metabolite formation from an NCE on the disappearance of the NCE in vitro in an appropriate system, its metabolic fate and half-life in vivo can be predicted. Similar studies performed in human and test species give valuable information for the selection of test species for pharmacokinetic and toxicological in vivo studies.
Today, there are effective methods for metabolite identification and subsequent construction of metabolic routes. Metabolite identification can be developed from incubations with human liver preparations, homogenates or microsomes (Kremers 1999). For example, mass-spectrometric (MS) methods employing HPLC as a separative tool have evolved into extremely sensitive and accurate techniques. By these methods, it is possible to determine with high accuracy the exact molecular masses and metabolite structures. As analytical methods, these techniques are at their best in skilful and highly experienced hands.
Sample preparation for MS studies is a critical step, since the available chemical information of an NCE is often limited. The incubation conditions and the reaction-terminating reagent have to be chosen so as not to alter the parent compound or the metabolites chemically and to keep the recovery of the substrate and the metabolites close to 100%. Otherwise, it is impossible to predict the pathways for biotransformation.
After characterising the metabolic stability and metabolic routes of an NCE, the in vivo prediction requires clarification of the drug-metabolising enzymes that participate in the in vitro biotransformation of the NCE. The methods usually employed for this purpose will be presented below.
After determining the initial velocity conditions and enzyme kinetic parameters, the CYPs involved in the metabolism of an NCE can be characterised by chemical and antibody inhibitors selective or specific for respective CYPs. It is important to select the substrate concentration correctly: near or preferably below the determined Km values, if the analytical method for metabolite detection allows it. In case the in vivo concentrations are known, it is recommendable to use substrate concentrations close to the therapeutic level, if at all feasible.
Numerous compounds have been characterised for their inhibitory potency against different CYPs. Many of them are selective for only the desired enzyme at relatively low concentrations, as for example, furafylline for CYP1A2 (Sesardic et al. 1990; Clarke et al. 1994; Bourrie et al. 1996; Racha et al. 1998); CYP2C9 is selectively inhibited by sulfaphenazole (Bourrie et al. 1996); quinidine is potent inhibitor for CYP2D6 though metabolised via CYP3A4 (Bourrie et al. 1996); pyridine seems to be quite selective for CYP2E1 (Hargreaves et al. 1994; own unpublished results), and there are many selective inhibitors for CYP3A4, of which ketoconazole is the most widely used, although it also affects other CYPs (Schmider et al. 1995; Bourrie et al. 1996). For CYP2A6 and CYP2C19, the search for selective chemical inhibitors is still under way (Draper et al. 1997; III).
Today, there are several commercial sources for CYP-specific inhibitory antibodies. The products are usually well characterised and not very expensive. Inhibitory antibodies are usually targeted towards a sequence in or near the substrate-binding site. The antigen can be selected in such way that the resulting antibody only inhibits the target CYP. Inhibitory antibodies raised specifically against a certain CYP form are a good tool in distinguishing between CYPs established as equally possible NCE-metabolising CYPs by other methods. Antibodies can also be used if there are no sufficiently specific chemical inhibitors available for a certain enzyme (for example CYP2B6, Stresser & Kupfer 1999).
Similarly to CYP-specific inhibitory antibodies, cDNA-expressed enzymes are convenient tools when a specific activity or a selective chemical inhibitor cannot be used in metabolic studies. With the expressed enzymes, the relative roles of individual CYPs cannot be quantitatively estimated, mainly due to the interindividual variation in the levels of individual active CYPs in the liver (Guengerich 1995b). cDNA-expressed CYPs can also be used in the HTS methodology when the goal is to screen large numbers of compounds (White 2000).
Recombinant enzymes can be used to ascertain the role of a certain CYP in the metabolism of an NCE. Still, the biotransformation of an NCE by a single CYP does not necessarily mean its participation in the reaction in vivo (IV). When taking into account the proportion of a certain CYP in the human liver, the activity obtained for the whole liver can be extrapolated to the prediction of the participation of this enzyme (Rodrigues 1999).
For correlation analysis, a well-characterised bank of human liver samples is needed. The number of individual livers should be at least ten (preferably more), to demonstrate the interindividual variation in the battery of activities. In correlation analysis, the measured CYP-specific activities are correlated against the rate of the metabolic pathway of an NCE in every individual liver sample. Another approach is to correlate the levels of an individual CYP determined by Western blot analysis against the NCE activity (Beaune et al. 1986; Guengerich & Shimada 1991; Guengerich 1995b). When correlating the levels of each CYP protein to the activity of an NCE, one should take into account that any inactive protein could also contribute to the estimation of protein levels in each liver. This could lead to an erroneous correlation, because the inactive CYP proteins do not participate in the metabolism of the NCE (Guengerich 1995b). If there is a sufficient number of individual samples, statistical significance can also be considered. Still, a correlation plot would give all the information needed for the evaluation of the participating CYPs. The higher the correlation between the activities, the larger the likelihood that the respective CYP enzyme is responsible for the metabolism of the NCE. For correlation analysis, the initial velocity conditions and the substrate concentrations near Km, or preferably the concentrations found in vivo should be used to avoid non-specific metabolism of substrates (Guengerich 1995b).
Particularly with CYP3A4, the correlation analysis is somewhat complicated because of the peculiar nature of the CYP3A4 catalytic mechanism (Harlow & Halpert 1998; Guengerich 1999).
From the therapeutic point of view, it is also important to know which drug-metabolising enzymes the substance under development has affinity to. For example, a compound inhibiting CYP3A4 could affect the in vivo concentrations of numerous drugs and other xenobiotics metabolised via this CYP, since over 50% of the drugs on the market are biotransformed by CYP3A4 (Bertz and Granneman 1997; Pelkonen & Breimer 1994). In recent years, severe adverse reactions due to interactions with CYP3A4 have led to the withdrawal of some drugs from the market. For example, terfenadine coadministrated with azole antimycotics caused potentially lethal ventricular arrhythmias (Monahan et al. 1990). Another example is mibefradil, the plasma concentrations of which were highly elevated by CYP3A4 inhibitors, leading to serious side effects (Kleinbloesem et al. 1995; Siepmann et al. 1995). Such economically consequential decisions could have been avoided if the affinity of the new compounds to CYPs had been recognised and correctly interpreted at the early phases of the drug development process (Siepmann & Kirch 2000).
The effect of an NCE on CYP-specific activities is studied by co-incubating series of dilutions of the study substance with a reaction mixture and a specific substrate. The effect of an NCE is described as the concentration of the studied compound causing 50% inhibition of the CYP-specific activities (Boobis 1995). If there is affinity towards some CYP-enzyme, the apparent Ki can be determined. By comparing the effects of an NCE on the CYP specific-activities to the respective effects of diagnostic inhibitors, a tentative prediction of the in vivo situation can be made.
Spectral interaction studies have been used from the early years of P450 research to recognise P450 ligands. Compounds that have affinity to a CYP-active site coordinate towards the heme iron of the protein, causing a change in the spin equilibrium of heme iron. The binding can produce several types of spectral changes that can be monitored by spectrophotometric scanning according to the procedure presented by, for example, Jefcoate (1978).