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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

van Beusekom, C.D., Schipper, L., Fink-Gremmels, J. Cytochrome P450-mediated hepatic metabolism of new fluorescent substrates in cats and dogs. J. vet. Pharmacol. Therap. 33, 519–527.

This study aimed to investigate the biotransformation of cat liver microsomes in comparison to dogs and humans using a high throughput method with fluorescent substrates and classical inhibitors specific for certain isozymes of the human cytochrome P450 (CYP) enzyme family. The metabolic activities associated with CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A were measured. Cat liver microsomes metabolized all substrates selected for the assessment of cytochrome P450 activity. The activities associated with CYP3A and CYP2B were higher than the activities of the other measured CYPs. Substrate selectivity could be demonstrated by inhibition studies with α-naphthoflavone (CYP1A), tranylcypromine/quercetine (CYP2C), quinidine (CYP2D), diethyldithiocarbamic acid (CYP2E) and ketoconazole (CYP3A) respectively. Other prototypical inhibitors used for characterization of human CYP activities such as furafylline (CYP1A), tranylcypromine (CYP2B) and sulfaphenazole (CYP2C) did not show significant effects in cat and dog liver microsomes. Moreover, IC50-values of cat CYPs differed from dog and human CYPs underlining the interspecies differences. Gender differences were observed in the oxidation of 7-ethoxy-4-trifluoromethylcoumarin (CYP2B) and 3-[2-(N, N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (CYP2D), which were significantly higher in male cats than in females. Conversely, oxidation of the substrates dibenzylfluorescein (CYP2C) and 7-methoxy-4-trifluoromethylcoumarin (CYP2E) showed significant higher activities in females than in male cats. Overall CYP-activities in cat liver microsomes were lower than in those from dogs or humans, except for CYP2B. The presented difference between feline and canine CYP-activities are useful to establish dose corrections for feline patients of intensively metabolized drugs licensed for dogs or humans.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Each year new drugs are developed and licensed for veterinary use. Drugs need to be licensed per animal species, especially drugs for food-producing animals. In companion animals, more substances are available for dogs than for cats, and compounds which are intended for canine or human patients are often also used in feline medicine with only a general adaptation in dosage regimes to (metabolic) body weight (body weight (kg0.75). However, extrapolations solely based on metabolic body weight or body surface area may be insufficient for all substances that are extensively metabolized, as significant interspecies variations in cytochrome P450-activity have been reported (Shimada et al., 1997; Nebbia et al., 2003; Baririan et al., 2006).

Cytochrome P450 enzymes (CYPs) are involved in the metabolism of numerous xenobiotics (including drugs) and endogenous substances. The ultimate goal of biotransformation processes is to convert a substance into metabolites, which are less active than the parent compound or even inactive, less lipid-soluble, more polar and more suitable for elimination by renal and/or biliary excretion (for review see: Anzenbacher & Anzenbacherova, 2001; Fink-Gremmels, 2008; Nebert & Russell, 2002; Zuber et al., 2002).

The CYP superfamily consists of a large number of isozymes, which are classified into gene families on the basis of their amino acid sequence. The most important enzyme families for drug biotransformation are CYP1, CYP2 and CYP3, and to a lesser extent CYP4 (Nebert & Russell, 2002). These families are further divided into subfamilies. The five most important drug metabolizing CYP subfamilies in humans are CYP1A, CYP2C, CYP2D, CYP2E and CYP3A. Each CYP subfamily metabolizes a distinct set of substrates but there is a considerable overlap between the different subfamilies in substrate specificity (Brosen & Rasmussen, 1997).

The expression and activity of these isozymes have been investigated primarily in rodents, as surrogates for humans in drug development (Shimada et al., 1997; Zhao & Ishizaki, 1997; Eagling et al., 1998; Bogaards et al., 2000). For example, similar CYP3A activities were found in male rat liver microsomes and human liver microsomes with respect to the 6-hydroxylation of dexamethasone (Tomlinson et al., 1997). Bogaards et al. confirmed the similarity in CYP3A activity of male rats, mice and humans by measuring the testosterone 6β-hydroxylase activity. They also found similarities in 7-ethoxyresorufin O-dealkylase activity (CYP1A), 7-ethoxy-4-trifluoromethyl-coumarin O-dealkylase activity (CYP2B) and diclofenac 4′-hydroxylase (CYP2C9) between these species (Bogaards et al., 2000).

In recent years, the CYP activity has been investigated for many animal species that represent veterinary patients and considerable interspecies variations have been found (Shimada et al., 1997; Nebbia et al., 2003; Baririan et al., 2006). Relatively little is known about the biotransformation of dogs (Chauret et al., 1997; Zhao & Ishizaki, 1997; Roussel et al., 1998; Shou et al., 2003; Lu et al., 2005) and in cats this knowledge is even more limited (Maugras & Reichart, 1979; Chauret et al., 1997; Tanaka et al., 2005; Shah et al., 2007). Insight into the species-specific biotransformation by cytochromes is of importance in drug development and in the veterinary clinic, as drugs can be substrates, as well as inducers or inhibitors of CYP isozymes. This can result in a shortening or a prolongation of the duration of action, drug-drug interactions and unexpected side effects (Trepanier, 2006; Fink-Gremmels, 2008).

For cats, the highest cytochrome activities were found in the phenacetin O-deethylase (CYP1A) and the testosterone 6β-hydroxylase (CYP3A). In dogs, these activities were high as well but comparable to the chlorzoxazone 6-hydroxylase activity (CYP2E) (Chauret et al., 1997) and also the aniline p-hydroxylation (CYP2E), benzphetamine N-demethylation and nifedipine oxidation (CYP3A) (Shimada et al., 1997).

In consideration of these obvious differences, it was one of the aims of the present study to provide a summation of the activity of the CYP enzymes of cats and dogs. To this end a rapid screening fluorometric assay developed for human CYP investigation was used. The fluorometric assay is based on a cytochrome catalyzed reaction that converts a substrate into a quantifiable fluorescent product (Crespi et al., 1997).

The second aim of this study was the evaluation of the suitability of this assay, which is fast and does not require extensive analytical skills and technology, in a clinical routine.

In a comparative approach the metabolic activity of the isozymes CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A were measured in cat and dog liver microsomes. The substrates for these isozymes were 3-cyano-7-ethoxycoumarin (CEC), 7-ethoxy-4-trifluoromethylcoumarin (EFC), dibenzylfluorescein (DBF), 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 7-methoxy-4-trifluoromethylcoumarin (7-MFC) and 7-benzyloxy-4-(trifluoromethyl)-coumarin (BFC) respectively. Substrate specificity was estimated using human prototypical inhibitors of these isozymes, with furafylline/α-naphthoflavone as inhibitors for CYP1A, tranylcypromine for CYP2B, tranylcypromine/quercetine/sulfaphenazole for CYP2C, quinidine for CYP2D, diethyldithiocarbamic acid (DETC) for CYP2E and ketoconazole for CYP3A.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Drugs and chemicals

3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methylcoumarin-HCl (AHMC), Dibenzylfluorescein (DBF) and 7-Hydroxy-4-trifluoromethylcoumarin (7-HFC) were purchased from BD Gentest (Woburn, MA, USA). 7-Benzyloxy-4-(trifluoromethyl)-coumarin (BFC), Diethyldithiocarbamic acid (DETC), 7-Ethoxy-4-trifluoromethylcoumarin (EFC), fluorescein, furafylline, glucose-6-phosphate, ketoconazole, 7-methoxy-4-trifluoromethylcoumarin (7-MFC), NADP, sulfaphenazole, tranylcypromine and quinidine were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 3-Cyano-7-ethoxycoumarin (CEC) and 3-Cyano-7-hydroxycoumarin (CHC) were purchased from Ultrafine Chemicals (Manchester, UK).

Quercetine was purchased from Indofine Chemical Company (Hillsborough, NJ, USA). Glucose-6-phosphate dehydrogenase was purchased from Roche Diagnostics GmbH (Mannheim, Germany). and magnesium chloride hexahydrate was from BDH Chemicals Ltd (Poole, England). All other reagents and solvents used were of analytical grade.

Preparations of submitochondrial fractions

Cats

The isolation of submitochondrial fractions (commonly referred to as microsomes) containing predominantly microsomal proteins followed the procedure as described by Rutten et al. with minor modifications (Rutten et al., 1987). In brief, cat liver samples of approximately 10 g were extracted from adult healthy cats (n = 10, five males and five females, aged ± 1 year) directly after euthanasia and were quickly frozen by liquid nitrogen and stored at −70 °C. The cats had served in an authorized study for the development FIV vaccines and had been sacrificed as cell donors. The tissue samples were homogenized with 1.15% KCl, containing 0.1 mm EDTA at 4 °C. The homogenates were centrifuged at 9000 g for 25 min at 4 °C, and the supernatant collected (S9-fraction) was centrifuged at 100 000 g for 1 h and 15 min at 4 °C. The microsomal pellet was resuspended in 1.15% KCl 0.05 m phosphate buffer, pH 7.4, containing 0.1 mm EDTA and 20% glycerol, quickly frozen in liquid nitrogen and stored in Eppendorf-cups at −70 °C until use.

Dogs

Beagle dog liver microsomes (n = 8, four males and four females, pooled, aged ≥12 months) were purchased from BD Gentest (Woburn, MA, USA). The microsomes were stored in Eppendorf-cups at −70 °C until use.

Humans

Human liver microsomes (n = 17, ten males and seven females, pooled) were purchased from BD Gentest (Woburn, MA, USA). The microsomes were stored in Eppendorf-cups at −70 °C until use.

The protein concentrations of the microsomal fractions were determined by the method of Lowry using BSA (bovine serum albumin) as a standard (Lowry et al., 1951).

Enzyme assays

The metabolic activity of the isozymes was measured by means of a fluorescence-based method in flat-bottom 96-well plates according to manufacturer’s instructions with minor changes to optimize the conditions for the feline and canine microsomes (http://www.bdbiosciences.com). Briefly, assays were performed by incubating liver microsomes (final protein concentration of 0.25 mg/mL) in a 200 μL volume with 0.5 m potassium phosphate buffer (pH 7.4), NADP (1.3 mm), glucose-6-phosphate (3.3 mm), MgCl2 (3.3 mm), glucose-6-phosphate dehydrogenase (0.4 U/mL) and CEC, EFC, DBF, AMMC, 7-MFC and BFC as substrates for CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A respectively, after a preincubation of 10 min at 37 °C (an overview is given in Table 1). After an incubation time of 60 min at 37 °C for cat and dog liver microsomes, the reactions were stopped by adding 75 μL STOP-solution, BD Gentest (Woburn, MA, USA) (80% acetonitrile/20% 0.5 m Tris base or 2N NaOH). The fluorescence was measured using a Fluostar Optima BMG (B&L Systems, Maarssen, The Netherlands) and the amount of formed product was calculated by means of a standard curve.

Table 1.   Fluorescent detection parameters of the different hepatic CYP isozymes with their specific substrates, products and inhibitors
Fluorescent detection parameters
EnzymeSubstrateProductInhibitorExcitation (bandwidth of filter), nmEmission (bandwidth of filter), nm
  1. BzRes, Resorufin benzyl ether; CEC, 3-Cyano-7-ethoxycoumarin; CHC, 3-Cyano-7-hydroxycoumarin; EFC, 7-Ethoxy-4-trifluoromethylcoumarin; 7-HFC, 7-Hydroxy-4-trifluoromethylcoumarin; DBF, Dibenzylfluorescein; 7-MFC, 7-Methoxy-4-trifluoromethylcoumarin; OMF, 3-O-methylfluorescein; AMMC, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin; MAMC, 7-Methoxy-4-(aminomethyl)coumarin; AHMC, 3-[2-(N,N-diethylamino)ethyl]-7-hydroxy-4-methylcoumarin hydrochloride; HAMC, 7-Hydroxy-4-(aminomethyl)coumarin; DETC, Diethyldithiocarbamic acid; 7-BQ, Benzyloxyquinolone; BFC, 7-Benzyloxy-4-(trifluoromethyl)-coumarin (BD Biosciences, http://www.bdbiosciences.com).

CYP1A1BzResResorufinα-Naphthoflavone409 (20)460 (40)
CYP1A2CBCCHCFurafylline409 (20)460 (40)
CYP2B6EFCHFCTranylcypromine409 (20)530 (25)
CYP2C8DBFFluoresceinQuercetin485 (20)538 (25)
CYP2C97-MFCHFCSulfaphenazole409 (20)530 (25)
DBFFluoresceinSulfaphenazole485 (20)538 (25)
CYP2C19CECCHCTranylcypromine409 (20)460 (40)
DBFFluoresceinTranylcypromine485 (20)538 (25)
OMFFluoresceinTranylcypromine485 (20)538 (25)
CYP2D6AMMCAHMCQuinidine390 (20)460 (40)
MAMCHAMCQuinidine390 (20)460 (40)
CYP2E17-MFCHFCDETC409 (20)530 (25)
CYP3A47-BQQuinolinolKetoconazole409 (20)530 (25)
 BFCHFCKetoconazole409 (20)530 (25)
 BzResResorufinKetoconazole530 (25)590 (35)
 DBFFluoresceinKetoconazole485 (20)538 (25)

To obtain IC50 values, enzyme activity was measured after addition of various concentrations of the inhibitors furafylline/α-naphthoflavone, tranylcypromine, quercetine/tranylcypromine/sulfaphenazole, quinidine, DETC and ketoconazole for CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A respectively. IC50 values were calculated using a nonlinear curve fitting program (graphpad prism version 4.00 for Windows, GraphPad Software, San Diego California USA).

Statistical analysis

Data were analysed using a one-way analysis of variance (anova) followed by the Bonferroni post test (graphpad prism version 4.00 for Windows, GraphPad Software, San Diego California USA) with P < 0.05 denoting a significant difference.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Enzyme assays

In a primary validation assay CYP450 enzyme activities measurable in human liver microsomes were compared with published data collected with human liver microsomes (Table 2). The activity measured in the human microsomes is generally lower than in the published data. However, the relative enzyme activities were comparable. The results of the investigated CYP activities of pooled cat, dog and human liver microsomes are summarized in Table 3. The CYP activities of male and female cats and dogs are shown in Table 4 & 5 respectively.

Table 2.   Comparison of measured CYP activities in HLM (human liver microsomes) in pm/(mg·protein * min) ± SD and the reference values presented in the literature for HLM
IsozymeSubstrateVmaxHLMVmax as reported by the manufacturer
  1. The SD of the reference values were all within 10% of the mean. Human CYP2D activity was below the limit of quantification.

CYP1ACEC200.5 ± 3.8542 (Stresser et al., 2002)
CYP2BEFC59.0 ± 6.045 (Donato et al., 2004)
CYP2CDBF95.9 ± 1.4289 (Stresser et al., 2002)
CYP2DAMMC< LOQ  3.12 (Stresser et al., 2002)
CYP2E7-MFC326.2 ± 9.91744 (Stresser et al., 2002)
CYP3ABFC92.2 ± 4.8205 (Stresser et al., 2002)
   40 (Donato et al., 2004)
Table 3.   Activity of the different hepatic CYP isozymes in cats (n = 10), dogs (n = 8) and human (n = 17, as in Table 2) in pm/(mg·protein * min) ± SD
IsozymeActivity pooled catsActivity pooled dogsActivity pooled humans
  1. Values are the mean of triplicate analyses. Statistical significance was determined by a one-way anova with a Bonferroni post test. P < 0.05 indicates a significant difference.

  2. *significantly different from cats

  3. significantly different from dogs

  4. significantly different from humans.

CYP1A101.2 (±5.1)†,‡204.4 (±6.3)*200.5 (±3.8)*
CYP2B131.3 (±3.1)†,‡53.5 (±3.0)*59.0 (±6.0)*
CYP2C31.2 (±0.8)†,‡33.6 (±0.7)*,‡95.9 (±1.4)*,†
CYP2D12.6 (±1.7)4.9 (±0.3)*< LOQ
CYP2E40.5 (±4.7)†,‡473.4 (±16.5)*,‡326.2 (±9.9)*,†
CYP3A65.0 (±3.7)†,‡90.9 (±4.3)*92.2 (±4.8)*
Table 4.   Cytochrome P450 activities of male (n = 5) and female (n = 5) cats in pm/(mg·protein * min) ± SD
IsozymeActivity male catsActivity female cats
  1. Statistical significance was determined by a t-test, with P < 0.05 as significant different.

  2. *Significantly different from male cats.

CYP1A102.0 (±4.7)99.7 (±6.0)
CYP2B158.5 (±3.6)142.7 (±4.9)*
CYP2C27.9 (±1.0)38.2 (±1.4)*
CYP2D15.0 (±1.7)11.8 (±1.4)*
CYP2E35.3 (±3.2)46.2 (±3.3)*
CYP3A64.9 (±8.8)70.2 (±4.5)
Table 5.   Cytochrome P450 activities of male (n = 4) and female (n = 4) dogs (Beagle) in pmol/(mg protein * min) ± SD
IsozymeActivity male dogsActivity female dogs
  1. Statistical significance was determined by a t-test, with P < 0.05 as significant different.

  2. *Significantly different from male dogs.

CYP1A175.4 (±7.2)220.6 (±11.0)*
CYP2B74.2 (±0.8)33.4 (±1.0)*
CYP2C34.9 (±0.5)35.6 (±0.5)
CYP2D2.8 (±0.2)5.3 (±0.5)*
CYP2E413.2 (±12.3)624.3 (±36.4)*
CYP3A94.5 (±0.2)87.8 (±9.4)

Cats showed significant lower activities towards the oxidation of the substrates CEC (CYP1A), 7-MFC (CYP2E) and BFC (CYP3A) compared to dogs and humans. The oxidation activities of EFC (CYP2B) and AMMC (CYP2D) were significant lower in dog liver microsomes than in cat, although the activity associated with CYP2D is extremely low in all animal species. The oxidation of DBF (CYP2C) did not differ significantly between cats and dogs, but a 3-fold higher activity was seen in human microsomes. The most striking difference was found in the oxidation of 7-MFC (CYP2E), as cats seem to have a 12-fold lower activity compared to dogs and an 8-fold lower activity compared to humans.

Gender differences in CYP-activity were observed as the oxidation of EFC (CYP2B) and AMMC (CYP2D) showed significant higher activities in male cats than in female cats. Conversely, oxidation of the substrates DBF (CYP2C) and 7-MFC (CYP2E) showed significant higher activities in female than in male cats. For dogs these gender differences were found with the oxidation of CEC (CYP1A), AMMC (CYP2D) and 7-MFC (CYP2E), where female cytochrome activities were significant higher than male activities. Only the oxidation of EFC (CYP2B) was higher in male dogs than in females.

A general comparison of individual CYP activity revealed that cat liver microsomes metabolized all substrates recommended for the assessment of human CYP activities. The activities associated with CYP1A, CYP2B and CYP3A were the most pronounced enzyme activities. In dogs, all selected substrates were metabolized as well and activities associated with CYP1A, CYP2E and CYP3A were found to represent the most active isozymes.

Inhibition studies

The effect of the prototypical inhibitors on the CYP mediated metabolism in cat and dog liver microsomes is shown in Fig. 1. The initial activities, i.e. without addition of an inhibitor, are set to 100%.

image

Figure 1.  Inhibition profiles of cat (•), dog (bsl00066) and human (□) liver microsomes. Initial activity (i.e. in the absence of inhibitor) was converted to 100%. Inhibition of (a) CYP1A activity by furafylline; (b) CYP2B activity by tranylcypromine; (c) CYP2C19 activity by tranylcypromine; (d) CYP2C8 activity by quercetine; (e) CYP2C9 by sulfaphenazole; (f) CYP2D activity by quinidine; (g) CYP2E activity by DETC; (h) CYP3A activity by ketoconazole.

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In cats, inhibition of the activity associated with CYP1A could be realized by α-naphthoflavone but not by furafylline. Inhibition of CYP1A activity by furafylline was only seen at concentrations above 10 μM but these concentrations exceeded the maximum solubility of the stock solution in acetonitrile, which was used for all inhibitors. CYP2B activity could be inhibited by addition of high concentrations of tranylcypromine. CYP2C was inhibited by quercetine and high concentrations of tranylcypromine. Sulfaphenazole had no effect on the CYP2C activity in the cat liver microsomes, with DBF as substrate. CYP2D activity declined rapidly after the addition of quinide. CYP2E inhibition could be observed in the presence of DETC and CYP3A was inhibited by ketoconazole.

In dogs, the activity associated with CYP1A was inhibited by high concentrations of furafylline. After addition of the inhibitor tranylcypromine to the dog liver microsomes, the fluorescence indicating for CYP2B activity unexpectedly increased. CYP2C could only marginally be inhibited by tranylcypromine and quercetine. Sulfaphenazole gave no inhibition at all. CYP2D and CYP2E were inhibited by quinidine and DETC respectively. CYP3A was not inhibited by ketoconazole as the fluorescence increased unexpectedly after adding ketoconazole.

Comparing cats and dogs, inhibition of the activities associated with CYP1A by furafylline, CYP2C by quercetine/sulfaphenazole and CYP2E by DETC did not differ. While CYP2B and CYP3A in cat liver microsomes were inhibited by tranylcypromine and ketoconazole respectively, these activities surprisingly increased in dog liver microsomes under the same circumstances. CYP2D activity in cats was more sensitive to quinidine than in dogs.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The presented investigations had two objectives: First, to present a comparison of the hepatic CYP isozyme activities in cats and dogs. Secondly, to assess the suitability of the rapid fluorometric assays to measure CYP activities in liver samples extracted from veterinary patients.

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.

CYP2D activity was hardly detectible in the liver microsomes of all animal species and these findings are in accordance with previous investigations (Chauret et al., 1997; Shimada et al., 1997). It was found that in dogs, bufuralol-1-hydroxylase activity, associated with CYP2D, was comparable with human CYP2D activity, albeit being lower than in other animals (Sharer et al., 1995; Roussel et al., 1998; Bogaards et al., 2000).

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.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The authors thank J.L. de Nijs-Tjon for her technical assistance. Initial results of this study were presented at the 10th EAVPT Conference, held in Turin, Italy, September 2006, proceeding pages 117-118.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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