Cytochrome P450 activity and inhibition
The presented data of the metabolic CYP activity show that in cats the oxidation of CEC by CYP1A, EFC by CYP2B and BFC by CYP3A represent the highest activities. Similarly, Chauret et al. (1997) found the highest activities for phenacetin-O-deethylase and testosterone 6β-hydroxylase in cats, associated with CYP1A and CYP3A respectively. Shah et al. (2007) found even higher CYP1A activities in cats than in dogs and humans. The oxidation of DBF by CYP2C and AMMC by CYP2D showed the lowest activity. A direct quantitative comparison of the data is not possible, as the substrates differ in the individual assays.
We found gender differences in cytochrome activity, which were not reported by Chauret et al. (1997). Other investigations confirm these gender differences in cytochrome activity although only for the isozymes CYP2D and CYP3A (Shah et al., 2007).
Using the same assay with dog liver microsomes, results showed that CYP1A, CYP2E and CYP3A were the most active isozymes. The results are in line with previous investigations (Chauret et al., 1997; Shimada et al., 1997), although Shimada et al. (1997) found a lower range of phenacetin-O-deethylation activity, representing CYP1A activity.
Comparing data of cat and dog liver microsomes, it could be observed that cat liver microsomes show significant lower CYP1A, CYP2E and CYP3A activities than dog liver microsomes. Conversely, dog liver microsomes had significant lower activities of CYP2B and CYP2D than those of cats. In human liver microsomes the CYP2D activity was below the detection level of the fluorometric assay. This might be attributable to the diverse polymorphism of this isozyme (Heim & Meyer, 1992; Zhou, 2009; Zhou et al., 2009). Both cats and dogs had lower activities of CYP2C than humans, which is in accordance with previous investigations (Chauret et al., 1997). Shah et al. (2007) demonstrated that cats have a negligible tolbutamide hydroxylation activity, suggesting unusual low CYP2C activities. It has to be mentioned that the substrate DBF for CYP2C is also metabolized by CYP3A. The activity which is measured is therefore not solely the activity of CYP2C. This could be a reason for the high CYP2C activity in human liver samples, because of the high content of CYP3A in human liver.
In our investigations CYP2E activity of cat liver microsomes was 12-fold lower than in dogs, although data showed that cats and dogs share the highest homology in amino acid sequence for this isoform compared to other animal species (Tanaka et al., 2005). By contrast, Tanaka et al. (2005) found a three-fold higher CYP2E activity in cats than in dogs in the 6-hydroxylation of chlorzoxazone. In cats two similar CYP2E genes are present, while in many mammalian species only a single gene exists (Tanaka et al., 2005). CYP2E metabolizes for example acetaminophen (Morgan et al., 1983) and volatile anaesthetics, such as halothane, isoflurane and sevoflurane (Gruenke et al., 1988; Kharasch & Thummel, 1993). Besides the knowledge of the deficient glucuronidation in cats (Court & Greenblatt, 1997, 2000) this relative low activity of CYP2E may explain the sensitivity of cats for the side effects of previously described drugs and toxins which are substrates for CYP2E.
To demonstrate substrate specificity, defined inhibitors of individual isozymes are commonly applied. To obtain IC50 values, different concentrations of specific inhibitors of the human CYP isozymes were added to the liver microsomes of both species.
CYP1A associated activity was not inhibited by the human prototypical inhibitor furafylline in cat and dog liver microsomes, while in human liver microsomes the activity decreased to zero in the presence of 100 μM furafylline. The absence of inhibition of phenacetin-O-deethylase (CYP1A) by furafylline was found in cats, but not in dogs, also by Chauret et al. (1997). Our experiments showed that the other well-known inhibitor α-naphthoflavone inhibited CYP1A activity in cats and dogs (data not shown). These findings suggest that either furafylline has a low binding affinity for the isozyme CYP1A, or a second enzyme is involved in the metabolism of the substrate CBC.
CYP2B associated activity in cats was only inhibited by tranylcypromine at high concentrations. In dogs an unexpected rise of the fluorescence of the CYP2B substrate was found after adding tranylcypromine in increasing concentrations. This phenomenon has been described in rats as well, while EFC, the substrate for CYP2B, was not only metabolized by this isozyme but also for 15% by CYP1A2 and 60% by CYP2E1 (Buters et al., 1993). As two phases could be found in Hanes plots in dog and human microsomes, the involvement of at least two different enzymes for EFC deethylation in these species is suggested (Buters et al., 1993). Hence, we hypothesize that in dogs the fluorescent product is further metabolized by another enzyme resulting in a secondary metabolite which increased fluorescence.
Human CYP2C can be subdivided in CYP2C19, CYP2C8 and CYP2C9. These isozymes can be inhibited by tranylcypromine, quercetine and sulfaphenazole respectively (Crespi et al., 1997; Naritomi et al., 2004). We found that in cat, dog and human liver microsomes CYP2C activity was inhibited by tranylcypromine and quercetine. Sulfaphenazole gave no inhibition of the activity of CYP2C in all three species. This can be caused by the usage of the same substrate DBF for these isozymes. When CYP2C9 only represents a small part of the total CYP2C activity, the other two isozymes are able to convert the substrate and as a consequence no numerical decline in CYP2C activity will be found in the presence of a specific inhibitor. Distinction between these CYP2C isoforms was not possible with the used assay. However, by HPLC a very low activity of tolbutamide hydroxylase, indicating CYP2C9 in humans, was found in cats and dogs (Chauret et al., 1997; Shah et al., 2007). CYP2D associated activity could be inhibited by very low concentrations of quinidine in cats. Dog en human CYP2D activity could hardly be detected and inhibition by quinidine did not give an obvious decline in activity. The low CYP2D activity might be attributable to the high rate of polymorphism of this isozyme, which is known for humans (Fukuda et al., 2000; Ingelman-Sundberg, 2004).
CYP2E associated activity could be reduced by DETC in cat, dog and human in comparable manner and no species differences were found in IC50 values.
The oxidation of BFC, associated with CYP3A, was inhibited by addition of ketoconazole in cat liver microsomes. However, in dogs again an unexpected increase in fluorescence was found. The inhibitor ketoconazole was proven to be specific for human and dog CYP3A (Newton et al., 1995; Kuroha et al., 2002; Lu et al., 2005). Ketoconazole as such did not give any fluorescence. The rise in fluorescence suggests that another isozyme metabolizes the chosen substrate BFC as well.
Assessment of the fluorometric assay
The fluorometric assay was selected in consideration of the obvious advantages of simplicity and the short duration of the assay compared to HPLC analysis or other analytical techniques. It is considered to be a high throughput method for investigation of drug biotransformation and substrate conversion. The most important disadvantage is that this assay is not entirely validated for the use of tissue fractions, such as microsomes. The manufacturer’s provision was to use pure isolated isozymes to obtain IC50 values, although Miller et al. reported that the CYPs could be introduced in the assay as single, cDNA-expressed enzymes or as enzyme mixtures, such as liver microsomes (Miller et al., 2000). The first evaluation of the assay with mixed human microsomes, did confirm the principle suitability of the assay, but relatively lower enzyme activities were measured in the microsomal fractions. Both assays are normalized for the protein content of the sample, and the CYP-enzyme proteins in the microsomal fractions explain for a large extent the relatively lower values. Moreover, when enzyme mixtures are used as present in the microsomal fraction, the probe substrate may be converted by one or more enzymes, as most CYP450 enzymes have overlapping substrate specificity (Crespi & Stresser, 2000; Miller et al., 2000; Stresser et al., 2002). This overlapping substrate activity is also reflected in the inconsistent results inferred in the inhibition studies. For example BFC, the substrate for CYP3A, should be highly selective for this isozyme and so this substrate could be used with human liver microsomes as a typical substrate. (Crespi & Stresser, 2000; Miller et al., 2000; Stresser et al., 2002; Donato et al., 2004). However, when ketoconazole was added in increasing concentrations to human liver microsomes, CYP3A activities did only decline to approximately 50% of the normal activity, indicating that BFC is a substrate for more than one CYP-isozyme.
The final objective was the evaluation of the fluorometric assay for its suitability in a clinical environment where individual patients might need to be investigated for their biotransformation activity of certain drugs. The fluorescent assay requires a smaller amount of microsomal protein as compared to common HPLC-based analyses, but the requested amount of 0.25 mg/mL protein is still high and can not be collected from normal thin-needle biopsies.
In conclusion, the presented data provide for the first time a summation of cat CYP450 activities and demonstrate again significant species differences in the activity of individual CYP isozymes between cats and dogs. In clinical practice, the lower CYP activities in cats, combined with the low glucuronidation capacity will result in longer half-lives of many drugs that undergo extensive biotransformation reactions. To avoid undesirable side effects and drug toxicity, longer dosage intervals should be considered for cats when dosage regimes established for dogs or humans are extrapolated to feline patients. The developed assays provide a valuable tool in the preclinical phase of veterinary drug development, as the same protocol can be used for different species, allowing a rapid comparison of results and the identification of species differences.