Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Reexamination Certificate
2002-01-02
2004-07-13
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
C544S111000
Reexamination Certificate
active
06762034
ABSTRACT:
This invention relates to compounds, processes for preparing them and their use as enzyme substrates.
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, CYP3A4 is one of those commonly responsible for the metabolism of drugs.
Resorufin benzyl ether has been described for high throughput CYP3A4 inhibition screening (Crespi et al,
Anal. Biochem
., 1997, 248, 188-190). However, the rate of Resorufin benzyl ether metabolism by CYP3A4 is low, therefore a more appropriate CYP3A4 substrate is required to enable higher throughput inhibition screening.
A compound has now been identified which is an improved substrate for CYP3A4 and which is of use for configuring high throughput inhibition screening assays.
According to the present invention there is provided an assay for testing for inhibitors of the enzyme CYP3A4 which comprises contacting the enzyme and a compound of formula (I):
with a test compound and measuring inhibition of O-dealkylation of the compound of formula (I) by the enzyme.
Generally the rate of O-dealkylation of the compound of formula (I) in the absence of test compound will be known, as will the extent of O-dealkylation at given time points. The assay may test for inhibition of O-dealkylation continuously or at specified time points.
O-Dealkylation of the compound of formula (I) following incubation with CYP3A4 gives a readily quantifiable fluorescent product of formula (II):
which can be scanned with suitable excitation and emission wavelengths, for example an excitation wavelength of 530 nm and an emission wavelength of 590 nm.
The assay may be carried out either in solution or utilising a solid support. When the assay is carried out in solution suitable solvents include methanol, acetonitrile and DMSO.
The test compound may be pre-incubated with enzyme prior to the addition of the substrate, or alternatively the substrate may be added simultaneously. 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. 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) is novel and as such also forms part of the invention.
The compound of formula (I) may be prepared by conventional methods, for example as shown in Scheme 1:
Thus according to a further aspect of the invention there is provided a process for the production of a compound of formula (I) which comprises reaction of a compound of formula (III):
wherein L is a leaving group, e.g. Br, with 1-phenylpiperazine. The reaction is preferably performed in the presence of dimethylformamide.
The starting materials resorufin, &agr;,&agr;′-dibromo-m-xylene and 1-phenyl piperazine are 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 CYP3A4 enzyme inhibitory activity of a compound, comprising the steps of identifying the compound as an inhibitor of CYP3A4 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 CYP3A4 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.
REFERENCES:
patent: 4954630 (1990-09-01), Klein et al.
patent: 6143492 (2000-11-01), Makings et al.
Crespi, C. L., et al. “Microtiter Plate Assays for Inhibition of Human Drug-Metabolizing Cytocromes P450”Analytical Biochemistry, U.S. Academic Press, vol. 248, 1997, pp. 188-190 (XP002115753).
Kenworthy, K.E., Bloomer, J.C., Clarke, S.E., Houston, J.B., “CYP3A4 Drug Interactions: Correlation of 10 in vitro probe substrates”, British Journal of Clinical Pharmacology, vol. 48, Nov. 1, 1999, pp. 716-727 (XP000921042).
Ono, S., Hatanaka, H. et al., “Specificity for substate and inhibitor probes for cytochrome P450s: evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes”, XENOBIOTICA, vol. 26, No. 7, 1996, pp. 681-693 (XP000921045).
Bloomer Jacqueline Carol
Elliott Richard Leonard
Leach Colin Andrew
Furman Theodore R.
Saucier Sandra E.
SmithKline Beecham p.l.c.
Stein-Fernandez Nora
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