WO2001044494A2 - P450 enzyme substrate - Google Patents

Abstract

1-Pyrenacetic acid was identified as a new substrate of CYP2C9, a cytochrome P450 enzyme.

Description

ENZYME SUBSTRATE

This invention relates to the use of a compound as a substrate for cytochrome P450 enzymes. The majority of metabolism based drug interactions are a result of inhibition of cytochrome P450 enzymes. Drug interactions involving individual P450 enzymes can be predicted using in vitro methods. Typical in vitro P450 enzyme assays involve incubation of an appropriate substrate with a source of enzyme. Traditionally, time consuming chromatographic methods have been used for metabolite detection in these incubations. More recently the availability of fluorimetric plate readers has facilitated the higher throughput of enzyme assays in general. Adapting P450 assays to fluorescent plate reader technology requires the identification of substrates with appropriate fluorescent products for individual enzymes. Among the xenobiotic-metabolising cytochromes P450, CYP2C9 is one of those commonly responsible for the metabolism of drugs. 3-Cyano-7-ethoxycoumarin has been described for high throughput CYP2C9 inhibition screening (Crespi et al, Anal. Biochem., 1997, 248, 188-190). However, the rate of 3-cyano-7- ethoxycoumarin metabolism by CYP2C9 is low, therefore a more appropriate CYP2C9 substrate is required to enable higher throughput inhibition screening.

International Patent Application WO 00/22159 discloses 7-alkoxycoumarins as CYP2C9 substrates.

1 -Pyrenacetic acid has now been identified as an improved substrate for CYP2C9 and is of use for configuring high throughput inhibition screening assays.

According to the invention there is provided an assay for identifying inhibitors of the enzyme CYP2C9 which comprises contacting the enzyme and a compound of formula (I):

(I) with a test compound and measuring inhibition of hydroxylation of the compound of formula (I) by the enzyme.

Generally the rate of hydroxylation of the compound of formula (I) in the absence of test compound will be known, as will the extent of hydroxylation at given time points. The assay may identify inhibition of hydroxylation continuously or at specified time points. Hydroxylation of the compound of formula (I) following incubation with CYP2C9 gives a readily quantifiable fluorescent product of formula (II):

(II) which can be scanned with suitable excitation and emission wavelengths, for example an excitation wavelength of 410nm and an emission wavelength of 460nm.

In the compound of formula (II) the hydroxyl group may be present on any one of the available aromatic carbon atoms.

The assay may be carried out either in solution or utilising a solid support in which case the enzyme may be attached to a solid support. When the assay is carried out in solution suitable solvents include methanol and acetonitrile.

The assay is preferably performed in a solution buffered to a pH of 7.4 or 7.5, e.g. using a potassium phosphate or Tris HCl buffer. The assay may also be performed in potassium phosphate buffer containing 10 mM MgCl2. The assay is preferably performed at a temperature of 37°C.

The test compound may be pre-incubated with enzyme prior to the addition of the substrate, or alternatively the substrate may be added simultaneously with the test compound. Final concentrations of enzyme and substrate are calculated so as to achieve a suitable rate of processing for carrying out the assay. If desired, the reaction may be stopped, for example by addition of acid or solvent.

As will be apparent to those skilled in the art cofactors for the CYP2C9 enzyme will be present in the assay system, cofactors for CYP2C9 are NADP, glucose-6-phosphate and glucose- 6-dehydrogenase. NADH or NADPH may be used instead of NADP. The assay may conveniently be initiated by addition of the cofactor solution, preferably prewarmed to 37°C, to the test compound / enzyme / substrate mixture.

The fluorescent product of formula (II) may be analysed using any conventional system of fluorescence detection, for example a multi-well plate/fluorescent plate reader.

The compound of formula (I), CAS registry no. [64709-55-3], is commercially available. Since the inhibition of cytochrome P450 enzymes is often the mechanism for drug/drug interactions, the assay according to the invention is particularly useful for identifying compounds which may give rise to adverse drug/drug interactions. The assay can therefore be used in combination with the chemical modification of test compounds to increase a test compounds potential for use as a pharmaceutical.

Thus according to further aspects of the invention there are provided a method for reducing the CYP2C9 enzyme inhibitory activity of a compound, comprising the steps of identifying the compound as an inhibitor of CYP2C9 in the assay described above; and thereafter producing a chemically modified version of the test compound in which the functionality suspected to be responsible for CYP2C9 inhibition is eliminated or changed; and novel compounds produced according to this method. The chemical modification of test compounds according to this method can be performed using techniques well known to those skilled in the art.

The novel compounds produced according to this aspect of the invention may find application as pharmaceuticals. A compound produced according to this method will be readily identifiable as novel by performing routine literature and database searches. The pharmaceutical activity of such compounds can be readily ascertained using conventional biological screening methods known to those skilled in the art.

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth. The invention is illustrated by the following example and figure.

Figure 1 shows the sulphaphenazole inhibition of 1-pyrenacetic acid metabolism by CYP2C9.

Example 1 Assay methodology

Materials:

6 mM 1-Pyrenacetic acid (i.e. 1.61 mg/ml methanol, Aldrich Chemical Company) - store at approx. -20°C in the dark

2 % (w/v) NaHC03 - store at approx. 4°C 50 mM potassium phosphate buffer, pH 7.4

Freshly prepared cofactor solution:- approx. the following per mL of 2 % (w/v) NaHCC>3

1.7 mg NADP, monosodium salt

7.8 mg glucose-6-phosphate, monosodium salt

6 Units glucose-6-phosphate dehydrogenase, Type VII from Bakers Yeast Method:

1) Pre-warm the plate reader oven to 37°C and pre-warm the lamp for at least 10 minutes. 2) Mix 2.5 μL 6 mM 1-pyrenacetic acid, 7.5 μL (75 μg) CYP2C9 microsomal protein and 210 μL buffer per incubate (giving 60 μM 1-pyrenacetic acid and 300 μg/mL protein final concentration).

3) To each well of a 96-well plate add 220 μL of incubation mix and 5 μL of test compound solution (or 5 μL of appropriate solvent for control wells - methanol or acetonitrile may be used). For the sulphaphenazole experiment 0.001, 0.033, 0.1, 0.33, 1.0. 3.3 and 10 μM solutions of sulphaphenazole in were used.

4) Pre-incubate the multi-well plate in the plate reader at 37°C for 5 minutes. Pre-warm the cofactor solution at 37°C for 5 minutes. 5) Add 25 μL cofactor solution to each well and scan with an excitation wavelength of 410 nm and an emission wavelength of 460 nm with a gain of 80. Scan for 10 cycles at 1 minute intervals. Results

Confirmation of 1-pyrenacetic acid as a CYP2C9 substrate was achieved using sulphaphenazole, a diagnostic CYP2C9 inhibitor (Baldwin et al, Xenobiotica, 1995, 25, 261-270). With sulphaphenazole, 1-pyrenacetic acid metabolism was inhibited with an IC50 of 0.44 μM (Figure 1), an inhibition value typical of other, well characterised, CYP2C9 substrates.

Claims

1. An assay for identifying inhibitors of the enzyme CYP2C9 which comprises contacting the enzyme and a compound of formula (I):

(I) with a test compound and measuring inhibition of hydroxylation of the compound of formula (I) by the enzyme.

2. The assay according to claim 1 wherein inhibition of hydroxylation of the compound of formula (I) by the enzyme is measured by quantifying the compound of formula (II):

(II)

3. The assay according to claim 2 wherein the compound of formula (II) is quantified by fluorescence detection.

4. The assay according to claim 3 wherein the compound of formula (II) is quantified by scanning at excitation wavelength of 410 nm and an emission wavelength of 460 nm.

5. A method for reducing the CYP2C9 enzyme inhibitory activity of a compound, comprising the steps of identifying the compound as an inhibitor of CYP2C9 in an assay according to any one of claims 1 to 4; and thereafter producing a chemically modified version of the test compound in which the functionality suspected to be responsible for CYP2C9 inhibition is eliminated or changed.

6. A novel compound produced according to the method of claim 5.