USE OF CYP1B1 INHIBITORS FOR TREATING CANCER

Abstract: CYP1B1 proteins and their role in metabolising or inactivating anti-cancer drugs is disclosed, together with compositions for treating cancer comprising a substance capable of inhibiting CYP1B1 protein and an anti-cancer drug (e.g. docetaxel, paclitaxel, flutamide, tamoxifen, mitoxantrone, doxorubicin or daunomycin).

Description: USE OF CYPIBI INHIBITORS FOR TREATING CANCER Field of the Invention The present invention relates to CYPIBI proteins and their role m cancer, and more particularly to the use of inhibitors of CYPIBI proteins to ameliorate the inactivation of anti-cancer drugs by CYPIBI present in cancer cells. Background of the Invention The cytochromes P450 are a multi-gene family of constitutive and inducible enzymes which have a central role in the oxidative metabolic activation and detoxification of a wide range Of xenobiotics and several groups of endogenous compounds active in cell regulation and cell signalling, including arachidonic acid, steroid hormones and fatty acids. The major families of P450 involved in xenobiotic metabolism each consist of several individual forms with different regulatory mechanisms and substrate specificities. The majority of P450s are primarily expressed in liver, although individual P450 forms are also expressed in specific extra-hepatic tissues including small intestine, kιdne _ and lung. The human CYP1 gene family, which is one of the major P450 families involved in the metabolism of xenobiotics, is now known to consist of three individual forms classified into two sub-families . The CYP1A subfamily contains two highly homologous and well characterised but distinct members, CYP1A1 and CYP1A2. CYP1A1 is an inducible P450 expressed primarily in extrahepatic tissues while CYP1A2 is a major form of P450 that is constitutively expressed in liver. There is also a second human CYP1 subfamily which contains one member, CYPIBI. This P450 is dioxm-inducible and sequence analysis of CYPIBI shows 40% homology with both CYP1A1 and CYP1A2. Although CYPIBI is assigned to the CYP1 family on the basis of its sequence, it is structurally and functionally distinct from both CYP1A1 and CYP1A2. 097/12246 (University of Aberdeen) discloses that CYPIBI is present m a range of tumour cells and proposes the use of this enzyme as a marker for the diagnosis of cancer. This application further discloses therapies for the treatment of cancer based on the presence of CYPIBI m tumour cells. In one embodiment, the application proposes a selective therapy employing drugs which are activated by CYPIBI present in tumour cells, converting a prodrug into a cytotoxic form capable of killing tumour cells. In other embodiments, 097/12246 proposes the use of CYPIBI present m tumour cells as a marker to guide a therapeutic compound selectively to the cells, e.g. by conjugating a drug to a moiety which is capable of specifically recognising the CYPIBI. 097/12246 further suggests that CYPIBI may be involved in providing an essential function for tumour cells m inactivating endogenous anti-tumour compounds such as 2- methoxyestradiol . In view of this, the application proposes reducing endogenous CYPIBI levels in tumour cells, e.g. by using antisense RNA or suicide inhibitors to inhibit CYPIBI production. WO00/56773 (University of Aberdeen) relates to the fragments of CYPIBI for use m raising antibodies capable of specifically binding CYPIBI, and the use of such antibodies for the diagnosis or treatment of cancers linked to enhanced CYPIBI expression. Summary of the Invention Broadly, the present invention is based on the finding that the presence of CYPIBI in tumour cells contributes to the resistance of tumour cells to anti-cancer drugs. This opens up the possibility of enhancing the effectiveness of cancer therapies employing these anti- cancer drugs by inhibiting CYPIBI or reducing its level m tumour cells thereby ameliorating the effect of the CYPIBI m inactivating or metabolising the drugs. CYPIBI is a member of the cytochrome P450 superfamily of enzymes. There have been some reports in the prior art that a number of the other P450 family members are involved m a biotransformation of anti-cancer drugs used to treat a variety of cancers. Cytochrome P450 mediated metabolism generally leads to iriactivation or reduced activity of the drug, whereas cyclophosphamide, an inactive pro-drug, must firstly undergo a 4-hydroxylatιon reaction by cytochrome P450 enzymes before becoming cytotoxic. However, as set out above, there is not a close relationship between CYPIBI and other P450 family members in terms of their structure or the substrates on which the enzymes act. Accordingly, the finding that CYPIBI causes drug resistance is not predictable based on the results obtained with other P450 enzyjies. Ovarian cancer causes the greatest number of deaths from gynaecological malignant disease in the developed world. The lack of symptoms in the early stages of ovarian cancer means that up to 80% of newly diagnosed patients will have disease that is advanced, where complete surgical resection is not possible and with an overall five year survival of only 30%. Early stage ovarian carcinoma has a good prognosis with 80% five year survival and generally chemotherapy is not recommended, whereas adjuvant chemotherapy with a platinum-based regimen is advocated for patients with advanced disease. The results disclosed herein confirm that CYPIBI expression in ovarian cancer shows a strong correlation with patient survival for treatment with paclitaxel. The recent introduction of the taxane, paclitaxel, for the treatment of ovarian cancer may result in a better progression-free and overall survival. Docetaxel, a semi-synthetic taxane, is currently being evaluated for use in the treatment of ovarian cancer. Some of the results disclosed herein examine the ability of CYPIBI to alter the cytotoxic profile of fourteen anti-cancer drugs commonly used as first line treatment in soliα tumours. In the studies disclosed herein, a Chinese hamster ovary cell line expressing human cytochrome P450 CYPIBI was used to evaluate the cytotoxic profile of several anti- cancer drugs (docetaxel, paclitaxel, cyclophosphamide, doxorubicm, 5-fluorouracil (5FU), cisplatin and carboplatin) commonly used clinically in the treatment of cancer. The MTT (3- [4 , 5-dιmethylthιazol-2yl] -2, 5- diphenyltetrazolium bromide) assay, was used to determine the levels of cytotoxicity. The key finding of this study was that on exposure to docetaxel and paclitaxel, a decrease in sensitivity towards the cyto -pxic effects of the drug was observed m the cell line expressing CYPIBI compared to the parental cell line. This effect was particularly marked for docetaxel (p = 0.03), while the lesser result obtained with paclitaxel may be due to the particular cell line used in the assay or because the metabolic product of paclitaxel retained some cytotoxicity to the cells. Further, this difference in cytotoxicity was reversed by co-mcubation of the cells with both docetaxel and the cytochrome P450 CYP1 inhibitor α-naphthoflavone (ANF) . This study is the first to indicate, that the presence of CYPIB-- in cells decreases their sensitivity to the cytotoxic effects of a specific anti-cancer drug. Further studies with kidney tumours showed that addition of the CYPIBI inhibitor α- naphthoflavone inhibited metabolism of EROD by CYPIBI 11 the tumour cells. Several cytochrome P450 enzymes are involved in the metabolism of a range of anti-cancer drugs, such as cyclophosphamide, paclitaxel and docetaxel [9-14] Cytochrome P450 meαiated metabolism usually results in reduced activity or mactivation of the anti-cancer drugs but m some cases bioactivation to a more cytotoxic metabolite occurs. One example of detoxification of anti-cancer drugs can be shown by the taxanes. The major pathway of metabolism of paclitaxel, an anti-cancer drug used in the treatment of breast, ovarian and non-small cell lung cancer, is catalysis by CYP2C8 and involves the hydroxylation of position 6 on the taxane ring [15]. The metabolite 6-hydroxytaxol is 30 fold less cytotoxic than the parent compound paclitaxel [16], and 6-hydroxytaxol is further metabolised by CYP3A4 [13] . Docetaxel, a semi-synthetic taxane, currently undergoing phase II and phase III trials for use m first-line therapy of ovarian cancer is metabolised by CYP3A to apparently less cytotoxic metabolites [12,13]. Accordingly, in a first aspect, the present invention provides the use of a substance capable of inhibiting CYPIBI protein and an anti-cancer drug for the preparation of a medicament for the treatment of cancer. Preferably, the substance capable of inhibiting CYPIBI protein inhibits the activity of CYPIBI enzyme m metabolising the anti-cancer drug or a pro-drug form thereof, or a metabolic product of the anti-cancer drug or pro-drug, thereby inactivating, detoxifying or otherwise modifying it so that it loses some or all of its anti-cancer activity (e.g. cytotoxicity to cancer cells), or in the case of a pro-drug, the capacity to be converted to active drug. Examples of CYPIBI inhibitors are well known in the art and include flavones and flavonoids such as α-naphthoflavone (ANF) , accacatm, diosmetm, hesperetm and homoer odictyol , and 2- ethynylpyrene . See, for example, Doostdar et al, Toxicology, 144:31-38, 2000 and Shimada et al, Chem. Res. Toxicol., 11:1048-1056, 1998. Preferred inhibitors are selective, that is they inhibit CYPIBI while having a reduced inhibitory effect, or more preferably substantially no effect, on the function of endogenous enzymes present in normal cells. " In addition to using known inhibitors, the skilled person can readily screen libraries of compounds to look for further inhibitors for use m accordance with the present invention. The anti-cancer compound may be a drug or a pro-drug and is preferably a substrate of CYPIBI. Examples of suitable anti-cancer drugs for use in conjunction with the CYPIBI inhibitors include docetaxel, paclitaxel, flutamide, tamoxifen, mitoxantrone, doxo-ααbicm or daunomycm, and more especially taxanes such as docetaxel and paclitaxel. The results disclosed herein show that all of these drugs inhibit the action of CYPIBI on its substrate EROD, and further experiments with paclitaxel and docetaxel confirm this result in cell assays. The CYPIBI inhibitor and the anti-cancer agents may be formulated together in a composition or separately for simultaneous or sequential administration. Preferably, the types of cancer treatable using the present invention are those characterised by the presence of CYPIBI m tumour cells and more especially, the presence of CYPIBI at an elevated level. Examples of such types of cancer include breast cancer, kidney cancer, colorectal cancer, prostate cancer, liver cancer or ovarian cancer. Accordingly, m a further aspect, the present invention provides a composition comprising a substance capable of inhibiting CYPIBI protein and an anti-cancer drug, m combination with a physiologically acceptable carrier. In a further aspect, the present invention provides a kit comprising: (a) in a first container, a substance capable of inhibiting CYPIBI protein; and (b) in a second container, an anti-cancer drug; wherein the substances are formulated with a physiologically acceptable carrier and are for simultaneous or sequential administration. The kit may also include instructions for administering the components of the kit. In a further aspect, tne present invention provides a method for treating cancer, the method cojnprising administering to a patient m need a combination of a substance capable of inhibiting CYPIBI protein and an anti-cancer drug. In a further aspect, the present invention provides a method of screening for CYPIBI inhibitors for use m combination with anti-cancer drugs, the method comprising: (a) contacting a candidate suostance with CYPIBI protein under conditions where the candidate substance and CYPIBI can interact; (b) measuring the activity of the CYPIBI protein and comparing the value obtained to standards; and (c) selecting candidate compounds which have the effect of inhibiting CYPIBI. In one embodiment, the method involves contacting CYPIBI with a candidate compound and a substrate of CYPIBI (e.g. EROD) and the effect of the candidate compound can be determined by monitcring the effect of the candidate compound in inhibiting the breakdown of the substrate by CYPIBI. Optionally, the method may comprise the additional step of testing the candidate compounds, e.g. in combination with CYPIBI and one or more anti-cancer drugs, to determine the effect the candidate inhibitor has in reducing the effect of CYPIBI in inactivating or metabolising the anti-cancer drug. Examples of assays include the in vitro interaction assays described below and cell based assays measuring the effect of the inhibitors in reducing the loss of cytotoxicity of the anti-cancer drugs caused by CYPIBI action. In carrying out these methods, it may be ^ convenient to screen a plurality of candidate compounds, e.g. as present in a library, at the same time, e.g. by contacting a mixture of different candidate compounds with the interacting peptides, and then in the event of a positive result resolving which member of the mixture is active. These techniques are used in high throughput screening (HTS) to increase the numbers of compounds, e.g. resulting from a combinatorial chemistry program or present in a library derived from a natural source material . The precise format of the assays of the invention may be varied by those of skill in the art using routine skill and knowledge. For example, interaction between substances may be studied in vitro by labelling one witn a detectable label and bringing it into contact with the other which has been immobilised on a solid support. Tie amount of candidate substance or compound which may be added to an assay of the invention will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.01 to 100 nM concentrations of putative inhibitor compound may be used, for example from 0.1 to 10 nM. In a further aspect, the present invention provides a method of determining the diagnosis, prognosis or responsiveness to treatment of a patient having ovarian cancer, the method comprising determining a presence or amount of CYPIBI protein in a sample from a patient comprising ovarian cancer cells and correlating the presence or amount to control values. Examples of assays for use m this aspect of the invention are disclosed on 097/12246 and antibodies suitable for use m immunoassays are disclosed m O00/56773. Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures. Brief Description of the Figures Figure 1. Survival curves of cells treated with A, docetaxel; B, paclitaxel; C, cyclophosphamide; D, doxorubicm; E, 5-FU; F, carboplatm and G, cisplatm. Parental V79MZ cells and those transfected with CYPIBI (V79MZhlBl) were incubated with increasing concentrations of the appropriate drug for 24h. Cell viability was then determined by the MTT assay and the percentage of surviving cells relative to the respective controls (cells treated with solvent only), was calculated for each drug concentration. There was significantly different cytotoxicity for docetaxel (A) m CYPIBI expressing and non-expressing cell lines and a lesser effect was observed with paclitaxel, perhaps because the action of CYPIBI resulted m a product which retained some cytotoxicity. There was no cytotoxicity observed m cells exposed to cyclophosphamide (C) . Each 96 well plate allowed eight concentrations of the appropriate drug per plate, with eight replicates (i.e. eight separate wells) per concentration, (i.e. 8 measurements for each concentration of drug per plate, There were three triplicate plates per experiment resulting in a total of 24 measurements of absorbance per concentration of drug) . The standard deviation was less than 5% of the mean absorbance for all drugs used, and at each concentration of drug. Figure 2 Survival curve of cells treated with docetaxel in the presence or absence of the cytochrome P450 inhibitor ANF. Parental V79MZ cells and those transfected with CYPIBI (V79MZhlBl) were mcubated with increasing concentrations of docetaxel for 24 hr. ANF was added at the following concentration 1, 10, or 100 μM to the transfected V79MZhlBl cells. Cell viability was then determined by the MTT assay and the percentages of surviving cells, relative to the respective solvent controls, were calculated. ANF at a concentration of 100 μM totally abolished the differential cytotoxicity observed in the V79MZhlBl transfected cells. ANF, itself, exhibited no cytotoxic effects on the parental (V79MZ) and CYPIBI (V79MZhlBl) expressing cell line, at all of the concentrations used. Figure 3. Survival of patients treated with docetaxel as part of their anti-cancer drug regimen and classified according to CYPIBI status of their primary tumours. The presence of strong/moderate CYPIBI lmmunoreactivity results m a poorer overall survival compared with weak or absent CYPIBI lmmunoreactivity . Detailed Description Pharmaceutical Compositions The compounds described herein or their derivatives can be formulated pharmaceutical compositions, and administered to patients in a variety of forms, in particular to treat cancer, and more especially breast cancer, colorectal cancer, prostate cancer, liver cancer or ovarian cancer. Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant or an inert diluent. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. Such compositions and preparations generally contain at least 0.1wt% of the compound. Parental administration includes administration by the following routes: intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraocular, transepithelial, mtrapeπtoneal and topical (including dermal, ocular, rectal, nasal, inhalation and aerosol), and rectal systemic routes. For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution whicn is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, solutions of the compounds or a derivative thereof, e.g. in physiological saline, a dispersion prepared with glycerol, liquid polyethylene glycol or oils. In addition to one or more of the compounds, optionally m combination with other active ingredient, the compositions can comprise one or more of a pharmaceutically acceptable excipient, carrier, buffer, stabiliser, lsotonicizmg agent," preservative or anti- oxidant or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. orally or parentally. Liquid pharmaceutical compositions are typically formulated to have a pH between about 3.0 and 9.0, more preferably between about 4.5 and 8.5 and still more preferably between about 5.0 and 8.0. The pH of a composition can be maintained by the use of a buffer such as acetate, citrate, phosphate, succinate, Tris or histid e, typically employed in the range from about 1 mM to 50 mM. The pH of compositions can otherwise be adjusted by using pnysiologically acceptable acids or bases. Preservatives are generally included in pharmaceutical compositions to retard microbial growth, extending the shelf life of the compositions and allowing multiple use packaging. Examples of preservatives include phenol, meta-cresol, oenzyl alcohol, para-hydroxybenzoic acid and its esters, methyl paraben, propyl paraben, benzalconium chloride and oenzetnomum chloride. Preservatives are typically employed in the range of about 0.1 to 1.0 % (w/v) . Preferably, t e pharmaceutical compositions are given to an individual m a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prop h ylaxis may be considered therapy) , this being sufficient to show benefit to the individual. Typically, th s will be to cause a therapeutically useful activity providing oenefit to the individual. The actual amount of the compounds administered, and rate and time- course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pnarmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980. By way of example, the compositions may be administered to patients in dosages of between about 0.01 and lOOmg of active compound per kg of body weight, and more preferably between about 0.5 and lOmg/kg of body weight . Materials and Methods Tissue Samples of ovarian cancer (n=172) submitted to the Department of Pathology, University of Aberdeen for diagnosis, over a five year period (1993-1998), were used in this study, with ethical approval from the Joint Grampian Health Board and University of Aberdeen Research Ethics Committee. Of the 172 cases of ovarian cancer, 167 cases were of the primary ovarian tumour, 43 of these cases had samples of both primary and metastatic disease, while 5 cases had samples of metastatic deposits with no corresponding ovarian tumour for investigation, (i.e. a total of 48 cases of metastatic disease and 167 cases of primary ovarian tumour). In 49 cases contralateral normal ovary was also submitted for histopathological examination and available for study. All the tissue samples had been fixed in 10% neutral buffered formalin for 24 hours and then routinely -processed to paraffin wax. The diagnosis of ovarian cancer was performed with hematoxylm and eosin stained sections using standard histopathological criteria by a consultant histopathologist with special interest m gynaecological pathology. The tumours were graded according to criteria described by FIGO. The median age of patients in this study was 63 years with an age range from 30-89 years. Information on therapeutic treatment and clinical outcome was available for 170 patients. The following anti- cancer drugs (cisplatm, carboplatic, cyclophosphamide, paclitaxel, and docetaxel) were used to treat the patients. Most patients received either cisplatm or carboplatm. The other three drugs were usually given in combination with a platinum based drug. Following diagnosis, the disease status of patients was assessed at regular intervals by two gynaecological oncologists with the median overall survival of patients being 17 months. Localisation of CYPIBI in ovarian cancer Immunohistochemical detection of CYPIBI with a monoclonal antibody to CYPIBI was performed using a tyramme signal amplification method as described previously [3]. Sites of immunoreactivity were demonstrated colorimetrically with diaminobenzidine and hydrogen peroxide (Liquid DAB plus, Dako Ltd High ycombe, Bucks, UK) . Positive control tissue was sections of breast cancer, which had been previously shown to contain CYPIBI by immunohistochemistry [3], the negative control used Tris buffered saline (TBS) in place of the primary monoclonal antibody. To establish the presence or absence of CYPIBI and its distribution, intensity and cellular localisation, the sections were examined using bright field light microscopy by two independent observers. CYPIBI immunoreactivity in the tumours was assessed as strong (3), moderate (2), -weak fl), or negative (0). Tumours exhibiting CYPIBI immunoreactivity in more than 5% of the cell were considered as positive. Cell lines and cell culture A Chinese hamster ovary fibroblast cell line (V79MZ) and a clone expressing human CYPIBI (V79MZhlBl) [21], were grown at 37°C, 5%C0 2 and at 90% saturated humidity, in DMEM (Dulbecco's modified eagles medium) high glucose type supplemented with 1 mM sodium pyruvate, 10% fetal calf serum, 100 U penicillin/ml and 100 μg streptomycin/ml. Neither cell line expresses endogenous P450s, although cytochrome P450 reductase is present in both cell lines [22,23,24 . The parental and CYPIBI expressing cell lines double in cell number every 10-12h [23] and were sub-cultured at 1:50 ratio (i.e. 1 ml of cells to 50 ml of fresh media) every 4-5 days. The cells were not allowed to reach confluency at any time, to ensure optimal cell physiological conditions and maximal cytochrome P450 activity [23]. Cells were routinely used at 3 rd -5 th passage for all experiments [21]. Immunoblotting of CYPIBI protein in V79 total cellular homogenate Both cell lines (V79MZ and V79MZhlBl) were grown to 60- 80% confluence and a total cellular homogenate from each prepared. Cellular protein was determined according to the method of Bradford [25]. Samples of cellular homogenates were then resolved by SDS-PAGE on a 10% polyacrylamide gel using a Hoefer SE 600 (Amersham Pharmacia Biotech) vertical gel electrophoresis system. This was followed by transfer to nitrocellulose membrane (Hybond-ECL, Amersham Pharmacia Biotech) . Sites of immunoreactivity were detected with a monoclonal antibody to human CYPIBI [3] . This antibody was raised against a 15 amino acid peptide corresponding to amino acid residues 437-451 of the human CYPIBI protein [3] . CYPIBI immunoreactivity was visualised by an enhanced chemiluminescence detection system (-Amersham Pharmacia Biotech) . Microsomes prepared from human lymphoblastoid cells which have been transfected to stably express human CYPIBI (Gentest) were used as the positive control [26] . Cell viability assay Paclitaxel, cyclophosphamide, doxorubicic 5-FU, cisplatin, carboplatin and ANF were purchased from Sigma. Docetaxel was obtained from Rhone-Poulenc Rorer. Optimal growth conditions were established. The same conditions were used for both cell lines; V79MZ and V79MZhlBl cells at 60-80% confluence were harvested and seeded at 0.5-1 x 10 3 cells per well in 96 well culture plates. Cells were grown for 48 hours, media was then removed and each drug added at increasing concentrations in the appropriate solvent (0.1% ethanol for docetaxel, paclitaxel, cyclophosphamide and carboplatin, 0.1% DMSO for cisplatin, sterile water for 5-FU and doxorubicin) . Cells treated with solvent alone acted as a negative control. Enzyme inhibition studies were undertaken by co- administration of docetaxel and the known P450 CYP1 inhibitor ANF (Sigma) [27] at different concentration in the media. Stock solutions of ANF (1 mM, 10 mM and 100 mM) were dissolved in DMSO and added to the media to give final concentrations of 1 μM, 10 μM and 100 μM ANF (in each case the final concentration of DMSO in media was 0.1%). Following 24h exposure to each drug (with or without inhibitor) , the media were removed and replaced with fresh media without drug. In this study, cells were then grown for three doubling times (36h) with the media changed at 24hr. Cell viability was then assessed using the MTT assay, which is comparable to using a clonogenic assay [23,28,29]. Media were removed from the wells and replaced with 200 μl of fresh media, followed by addition of 50 μl of MTT solution (50 mg/ml of MTT (Sigma) in sterile PBS) , the cells were incubated at 37°C in a humidified atmosphere with 5%C0 2 /95% air for 4h. The MTT containing media was then removed and 200μl of DMSO plus 25 μl of glycine buffer (0.1 M glycine/ 0.1 M NaCl pH10.5) added to the cells in each well. This procedure overcomes any effect that cell density or culture medium may have on the absorption spectrum [28] . The absorbance of the formazan produced by the viable cells was measured once for each well at 540nm on a Labsystems EMS microplate reader (Life Sciences International). To calculate the cell viability cells treated with solvent alone were assigned a value of 100% absorbance indicating zero cytotoxicity, i.e. 100% viability. The cytotoxic profile of each drug was evaluated in triplicate 96 well plates. Each plate included several controls (media only, cells only and cells treated with solvent alone) . The 96 well plate format allowed eight concentrations of the appropriate drug per plate, with eight replicates (i.e. eight separate wells) per drug concentration, -i.e. 8 measurements of absorbance for each concentration of drug per plate. There were three triplicate plates per experiment resulting in a total of 24 measurements of absorbance per concentration of drug. Plate reader variability was found to be negligible. P4501B1 has a role in metabolism of anticancer agents Recent immunohistochemical analysis of P4501B1 protein in a variety of solid tumours identified tumour-specifIC enzyme expression (Murray et al, 1998). This provides a mechanism of resistance to currently used anticancer agents, as well as, an approach for targeted prodrug therapy. 7-ethoxyresoruf deethylase activity (EROD) was used to define interactions with cytochrome 1B1 (CYPIBI) . 7-Ethoxyresorufm is a known substrate for P4501B1. EROD activity was used to screen the binding affinity of anticancer agents and test compounds with CYPIBI. For this purpose, inhibition constants (Ki) have been calculated using m vi tro kinetics from CYP1B1- mediated EROD activity. EROD activity and assay has been described previously by various authors (Lubet et al . , 1985, Arch. Biochem. Biophys . 238: pp 43^48; Gentest Corporation, 6 Henshaw St., oburn, MA01801 USA, data sheet given with CYPIBI, catalogue number P220) . 7- Ethoxyresorufm is enzymatically deethylated to a product called resorufm. The appearance of resorufin was monitored by a fluorescence detector set at 550nm and 582nm (excitation and emission wavelengths). Various concentrations of 7-ethoxyresorufin (0.05, 0.1 and 0.5μM) as well as various concentrations of anti-cancer agents and test (control) compounds (depending on the efficiency of the inhibition) were added to the incubation system. Incubations contained: phosphate buffer at pH7.4, 7- ethoxyresorufm, CYPIBI Supersomes from Gentest Co (catalogue number P220) and nicotine adenine d ucleotide phosphate (NADP) as cofactor. Biotransformation studies Flutamide and paclitaxel (taxol) were tested as potential substrates of the CYPIBI. The results show that both drugs are biotransformed by CYPIBI, as metabolites were produced by CYPIBI incubation but not control incubations . Kidney tumour assays The effect of a CYPIBI inhibitor was confirmed in an assay using kidney tumour samples. A buffer and NADP regenerating system was allowed ' to equilibrate at 37°C and EROD (5 μM final concentration) was then added. This was followed by the addition of either P450 supersomes (control) or the appropriate tumour sample (1 mg) . Supersomes were incubated for 15 minutes and tumour samples for 40 minutes. The CYPIBI inhibitor α- naphthoflavone was added to some samples just prior to the addition of EROD and P450 sample. Statistics Statistical analysis was performed using both Statistics for Windows 95 and SPSS version 7.5 for Windows 95. Results Localisation of CYPIBI in ovarian cancer CYPIBI immunoreactivity was identified in the majority (153/167; 92%) of primary ovarian cancer sections and was specifically localised to the cytoplasm of tumour cells. There was no detectable CYPIBI expression in any of the normal ovarian tissue samples. In a high percentage of the ovarian cancers there was either strong (85/167; 50.9%) or moderate (39/167; 23.4%) immunoreactivity for CYPIBI (Table 4) . In addition, the presence of CYPIBI was observed in the majority (45/48; 95%) of metastatic deposits with a high proportion showing moderate (22/48;45.8%) to strong ( 18/48 ; 37.5% ) immunoreactivity (Table 5) . A similar level of CYPIBI expression was exhibited for the different histological subtypes in both primary and metastatic tumour (Tables 4 and 5) . In the cases where both primary ovarian tumour and metastatic deposits were available, a significant correlation for CYPIBI expression (p=0.05 Spearman correlation test) was observed) . The presence of moderate to strong CYPIBI in the tumours of the subset of 19 patients who had received docetaxel as part of their anti-cancer drug regime had an adverse effect on overall survival (Fig. 3) . The presence of CYPIBI had no influence on the survival of patients who had received other anti-cancer drug regimes. In parallel with the in vi tro studies of CYPIBI on the cytotoxic profile of the anti-cancer drugs, a comprehensive investigation was conducted into the presence of CYPIBI m primary ovarian tumour and metastatic deposits and the influence it has on the overall survival of patients on different therapeutic regimens. A monoclonal antibody to CYPIBI was used to demonstrate the localisation of CYPIBI to ovarian tumour cells and lack of expression in normal ovarian tissue. The over-expression of CYPIBI was observed in all histological subtypes of epithelial ovarian cancer and at high frequency (>85%). This finding is m agreement with a previous study, which utilised a polyclonal antibody to CYPIBI to demonstrate the over-expression of CYPIBI m a small number (7/7) of ovarian serous adenocarc omas (7). A high frequency of CYPIBI over-expression was also observed in the majority of metastatic deposits in this current study, and was highly correlated with paired primary tumour. These findings also support the concept of CYPIBI being a molecular target for the development of new treatment approaches to ovarian cancer. During the time period in which patients on this study were diagnosed (1993-1998), considerable changes were observed in the chemotherapeutic regimen used for the treatment of ovarian cancer. Prior to 1994 the main treatment of choice was a cisplatin/cyclophosphamide based-regimen (19). Although a platinum agent is still regarded as essential for primary treatment, the less toxic agent carboplatin is now the platinum agent of choice. Following the introduction of paclitaxel to oncological practice, the principal first line therapy for ovarian cancer is currently carboplatin plus paclitaxel (19). Docetaxel, is currently under assessment including the "SCOTROC" trial (Scottish randomised trial on ovarian cancer comparing carboplatin plus paclitaxel versus carboplatin plus docetaxel), prior to possible licensing for treatment in ovarian cancer. These changes observed in primary treatment have resulted in a skewed grouping of patients on the various chemotherapeutic regimens in this study. However, the results of this investigation indicate that in the subset of patients treated with docetaxel either as a single agent or in combination with a platinum agent, the presence of CYPIBI in the tumour resulted in a poorer overall survival, supporting the in vi tro data. In summary this study provides evidence to support the concept that the presence of CYPIBI in tumour cells may have an important role in drug resistance, especially to docetaxel . Cytotoxic effects of treatment with anti-cancer drugs A protein band of approximately 52kDa was identified in the cellular homogenate from the V79MZhlBl cell line, corresponding to the expected molecular size observed with lymphoblastoid cells which also express human CYPIBI [3]. No immunoreactive band was observed in the parental V79MZ cell line. This confirms the presence of CYPIBI immunoreactivity in the CYPIBI transfected cells and an absence of CYPIBI the parental non-transfected cells. The influence of CYPIBI on cytotoxicity was evaluated for seven of the fourteen anti-cancer agents investigated. The range of concentrations for each drug used in this study was based on previous experiments with other P450s [23] . A signif cantly greater Cat least four-fold) decreased sensitivity to docetaxel was observed m cells expressing CYPIBI compared with non-CYPlBl expressing cells (Fig. IA. and Table 1) . The cytochrome P450 CYP1 inhibitor ANF was used at serial concentrations (1, 10 and 100 μM) to determine if the differential cytotoxicity demonstrated in the cells expressing CYPIBI was due to metabolism by CYPIBI (Fig. 2) . Neither cell line (parental or CYPIBI expressing) showed cytotoxicity on exposure to any of the concentrations of ANF. In contrast to the decreased sensitivity observed with docetaxel, no significant difference m cytotoxicity was observed between CYPIBI expressing and non-expressing cells after exposure to paclitaxel (Fig. IB. and Table 1). No cytotoxicity was observed in either cell line after exposure to cyclophosphamide (Fig. 1C and Table 1) . In addition, no significant difference m cytotoxicity was observed between V79MZ and V79MZhlBl cells after exposure to doxorubicm, 5-FU, carboplatin or cisplatm (Fig. 1D-1G; Table 1) . P4501B1 has a role in metabolism of anti-cancer agents The metabolism of anti-cancer drugs by CYPIBI was studied using an inhibition of EROD assay. Control incubations (with similar amounts of drug vehicle) have shown that inhibition of EROD activity was due to the anti-cancer and test compounds mentioned in the attached results. Ki values were determined by the Dixon's replots: see Table 2. The low Ki values show that CYPIBI has a strong binding affinity for many anti-cancer agents and could biotransform these drugs. Many anti-cancer agents showed strong binding affinity (Ki values) on cytochrome 1B1. Moreover, the two anticancer agents tested in biotransformation studies, paclitaxel and flutamide, are biotransformed by CYPIBI. Therefore, CYPIBI, which is over-expressed in tumour cells, could have important consequences in the development of new drugs of new therapies and in the prediction of therapy outcome. Inhibition of CYPIBI As initial screening for the inhibition of CYPIBI activity, resorufm production was measured with 1 μM ethoxyresorufm in the presence of 0 μM -as control) or 100 μM anticancer agent. In these conditions, vmblastme, vmcristme, 5-fluorouracil, etoposide, and cyclophosphamide did not inhibit CYPIBI activity. In contrast, flutamide, paclitaxel, mitoxantrone, docetaxel, doxorubicm, daunomycm, and tamoxifen inhibited CYPIBI activity by decreasing the production of resorufm by 53 to 99%. Further inhibition studies performed with three concentrations of ethoxyresorufm and six concentrations of drugs identified flutamide, mitoxantrone, docetaxel, and paclitaxel as competitive inhibitors with K. values of 1.0, 11.6, 28.0, and 7.85 μM respectively (Table 2). Noncompetitive or mixed inhibition was observed for daunomycm, doxorubicm, and tamoxifen, and K. values were 2.1, 2.6, and 5.0 μM, respectively (Table 2). Similarly, known CYP inhibitors and putative CYPIBI substrates were also initially screened at 100 μM and agents with an apoarent interaction were further characterised as described above. Erythromycm and cyclosporme did not inhibit CYPIBI activity (10%; Table 2). In contrast, testosterone and estradiol were competitive inhibitors of EROD (K-=1.9 and 411.8 μM respectively) . Potent non-competitive inhibition by ketoconazole (K. = 0.3 μM) and α-naphthoflavone (K, - 2.8 μM) was observed. Flutamide Metabolism Flutamide was a potent competitive inhibitor of CYPIBI, suggesting that it is a putative substrate. In vitro incubations of flutamide were performed with human liver microsomes or various cDNA-expressed human CYPs. Two flutamide metabolites were produced in the presence of a NADP-regenerat g system. One metabolite was observed after incubation with CYPIBI, CYP1A1, or CYP1A2. This metabolite has been identified as 2-hydroxyflutamide, following metabolic studies of CYPIBI and CYP1A1 activities. As previously reported (Shet et al, 1997), the production of the other metabolite is via CYP3A4 activity. The 2-hydroxylatιon of flutamide was produced by microsomes containing human CYPIBI, CYP1A1, and CYP1A2. Production of 2-hydroxyflutamide by CYPIBI was described by Michaelis-Menten kinetics, and K m and V may values were calculated by Eadie-Hofstee plots. Flutamide was a competitive inhibitor of CYPIBI, CYP1A1, and CYP1A2 activities, with K values ranging from 1.0 to 10.3 μM. For CYPIBI and CYP1A2, similar K m and K, values were obtained. In contrast, K m and K : values for CYP1A1 are 5 and 10 times higher than CYPIBI, respectively. V ma values for 2-hydroxylatιon of flutamide are different for all three enzymes, likely reflecting differences in cytochrome c reductase activity in the microsome preparation used. In this study, the cytochrome c reductase activity was 310, 1600, and 2330 mnol/mm x mg of proteins for CYPIBI, CYP1A1, and CYP1A2, respectively. Kidney tumour cell assay These results of this assay confirms that an inhibitor of CYPIBI can inhibit the protein when it is present m kidney tumour samples, providing " confirmation that the inhibitors can be employed with anti-cancer agents. Three separate tumour samples were used and the CYPIBI activity in metabolising EROD was measured in the presence and absence of the inhibitor α-naphthoflavone . Sample 1: CYPIBI activity 252 fmol/mm/mg protein plus lOnM ANF 145 fmol/mm/mg protein Sample 2: CYPIBI activity 993 fmol/m /mg protein plus lOnM ANF 470 fmol/min/mg protein Sample 3: CYPIBI activity 880 fmol/min/mg protein plus lOnM ANF 570 fmol/mm/mg protein Benzpyrene assay This assay can be used to measure CYCP1B1 activity in tumour samples, and in particular in frozen sections of tumour. In the assay, fresh, frozen sections of tumour were incubated with benzpyrene (10-100μM and NADPH for 1 hour at 37°C and then mounted m alkaline glycerol jelly. The presence of 1B1 was visualised by fluorescence microscopy using excitation at 400-450nm and detecting emission at 520nm through a dicπroic mirror having a 510nm frequency cut off. Yellow-green fluorescence is observed m tumour cells. Discussion In this study a Chinese hamster cell line which stably expresses human CYPIBI [21] was used as a bioassay to assess the effect of CYPIBI on the cytotoxicity of a range of anti-cancer drugs. Although several of the drugs used in this study are clinically relevant in the treatment of ovarian cancer, i.e. cisplatm, carboplatin and 5-FU [30,31], they have no known interactions with cytochrome P450 enzymes. However, these drugs were used to provide controls to assess the validity of the cytotoxicity assay. The pro-drug cyclophosphamide provided an appropriate negative control (i.e. non- cytotoxic to either parental or CYPIBI expressing cells) as it requires activation by other cytochrome P450 enzymes (CYP2B6 and CYP3A4) before becoming cytotoxic, but we have shown previously that it does not interact with CYPIBI [32] . Doxorubicm is known to be metabolised to more cytotoxic compounds by the action, of other cytochrome P450 enzymes (CYP3A) [20], whereas the taxanes paclitaxel and docetaxel are both metabolised to pharmacologically less active metabolites by cytochrome P450 enzymes; (CYP2C8 and CYP3A4 for paclitaxel and CYP3A4 for docetaxel) [12,13,14,15,33]. Previous studies have demonstrated that V79MZ cells when stably transfected with other cytochrome P450 enzymes have exhibited appropriate cytotoxicity when exposed to individual anti-cancer drugs establishing this cell line as an appropriate model for investigating anti-cancer drug cytotoxicity [23] . It is widely known that both primary cultures and human tumour cell lines rapidly lose the ability to constitutively express cytochrome P450 enzymes in culture. Indeed, we have shown that the MCF-7 human breast carcinoma and PE04 ovarian adenocarcmoma derived cell lines do not express CYPIBI (unpublished observations) even though we have shown over-expression of CYPIBI m both breast and ovarian tumours [1]. The lack of constitutive expression of cytochrome P450s in tumour cell lines was overcome by the use of a stably transfected cell line expressing CYPIBI. CYPIBI activity in these cells was previously shown to be approximately 10 pmol/mm/mg of protein by the EROD assay [21], and is likely to be comparable with that observed in human tumours . These results show that docetaxel is metabolised by expressed human CYPIBI [32] . Docetaxel is a semi- synthetic taxane derived from the European yew, and is currently under investigation, for use as first line treatment of ovarian cancer [12-13,34]. The key finding of the current study was the significant differential cytotoxicity observed on exposure to docetaxel, the cytotoxicity observed in CYPIBI expressing cells was four-fold less than that observed in non-CYPlBl expressing cells. In addition co-treatment of these cells with the known CYP1 P450 inhibitor ANF [27] resulted in the complete reversal of differential cytotoxicity observed m these cells, i.e. the effect is attributed to metabolism of docetaxel by CYPIBI. The resistance to the cytotoxic effects of docetaxel m those cells expressing CYPIBI may have important clinical implications . Inhibition of CYPIBI in tumours may offer a specific mechanism for overcoming the resistance to docetaxel and other drugs. Development of a specific inhibitor to CYPIBI is clinically important as ANF also inhibits CYP1A1 and CYP1A2 [27] . Since CYPIBI is over-expressed m tumour but not m normal tissue, increasing the tumour sensitivity to anti-cancer drugs by CYPIBI inhibition would not be expected to have an effect on normal tissues . In summary, this study provides evidence for the concept that the presence of CYPIBI in tumour cells may have an important role in drug resistance. Table 1. a IC50 values for N79MZ and N79MZhlBl cell lines treated with several anticancer drugs Drug N79MZ (control vector) N79MZhBl p * -value Docetaxel 22nM lOOnM 0.03 Paclitaxel 35nM 60nM ΝS Cyclophosphamide ΝC ΝC ΝD Doxorubicin 80nM 90nM 0.79 5-Fluorouracil 70μM 80μM ΝS Carboplatin 80μM lOOμM ΝS Cisplatin 4.4μM 6μM ΝS Statistical comparison of dose response curves (IC50s) using Mann- Whitney U test. a IC50= Drug concentration at which 50% of the cells are viable. The data represent the means of 24 determinations per drug concentration. ΝC = No cytotoxicity (no cytotoxicity observed with cyclophosphamide), ΝD = Not determined, NS = not statistically significant for paclitaxel, 5FU, cisplatin or carboplatin. The statistical software package we used did not provide p values in many cases when the result was non significant. Table 2. Cytochrome IBl : Ki values Ki values calculated from Dixon re-plots of EROD activity mediated by cDNA expressed cytochrome IBl Anticancer Agents Inhibition Type Ki Values Flutamide competitive mean 0.99μM sd 0.07μM Tamoxifen non-competitive mean 5.02μM sd 0.08μM Mitoxantrone competitive mean 1 1.63μM sd 0.03μM Paclitaxel competitive mean 7.85μM sd 0.91 μM Docetaxel competitive mean 28.03μM sd 9.78μM Doxorubicin mixed mean 2.58μM sd 0.18μM Daunomycin mixed mean 2.12μM sd O.l l μM Test Compounds Inhibition Type Ki Values α-naphthoflavone non-competitive mean 2.80nM sd 0.53nM Testosterone competitive mean 411.83μM sd 40.38μM Estradiol competitive mean 1.85μM sd O.OόμM Ketoconazole non-competitive mean 0.27μM sd O.Ol μM Table 3. Clinicopathological characteristics of patients with ovarian carcinoma Age of Patients Median (Range) 63 years (30-89 years) Stage 1 44/172(25%) 2 15/172(9%) - 103/172(60%) 4 10/172(6%) Histology Serous cystadenocarcinoma 102/172(59%) Endometrioid carcinoma 35/172(21%) Mucinous cystadenocarcinoma 24/172(14%) Clear cell adenocarcinoma 7/172(4%) Malignant mixed Mullerian tumour 4/172(2%) Table 4. The number of cases showing CYPIBI expression in the different histological types of primary ovarian carcinoma Table 5. The number of cases of CYPIBI expression in the different histological types of metastatic epithelial ovarian carcinoma References The references cited herein are all expressly incorporated by reference. [1] Murray et al. Tumour-specifIC expression of cytochrome P450 CYPIBI. Cancer Res 1997;57:3026-31. [2] Murray et al. The regulation, function and tissue specific expression of cytochrome P450 CYPIBI. Annu Rev Pharmacol Toxicol 2001 (m press). [3] McFadyen et al. Immunohistochemical localisation of cytochrome P450 CYPIBI m breast cancer with monoclonal antibodies specific for CYPIBI. J Histochem Cytochem 1999;47:1457-64. [4] McKay et al. Expression of cytochrome P450 CYPIBI in breast cancer. FEBS Letters 1995;374:270-72. [5] Hayes et al. 17b-Estradιol hydroxylation catalysed by human cytochrome P450 IBl. Proc Natl Acad Sci USA 1996;93:9776-81. [6] Liehr et al. 4-Hydroxylatιon of estradiol by human uterine myometrium and myoma microsomes: implications for the mechanism of uterine tumourigenesis. Proc Natl Acad Sci USA 1995;92:9220-24. [7] Spmk et al. The effects of 2,3,7,8- tetrachlorodibenzo-p-dioxm on estrogen metabolism m MCF-7 breast cancer cells: evidence for induction of a novel 17b-estradιol 4-hydroxylase . J Steroid Biochem Molec Biol. 1994;51:251-58. [8] Liehr et al. 4-Hydroxylatιon of estrogens as a marker of human mammary tumours. Proc Natl Acad Sci USA 1996;93:3294-96. [9] Le Blanc et al . Interaction of anticancer drugs with hepatic onooxygenase enzymes. Drug Metab Rev 1989;20:395-439. [10] Kivisto et al. Tne role of human cytochrome P450 enzymes in the metabolism of anti-cancer agents: implications for drug interactions. Br J Clin Pharmacol 1995;40:523-30. [11] Iyer et al. Pharmacogenetics and cancer chemotherapy. Eur J Cancer 1998;34:1493-99. [12] Marre et al. Hepatic biotransformation of docetaxel (Taxotere) m vitro: involvement of the CYP3A subfamily in humans. Cancer Res 1996;56:1296-302. [13] Crommentuyn et al. In-vitro metabolism of anticancer drugs, methods and applications: paclitaxel, docetaxel, tamoxifen and lfosfamide. Cancer Treat Rev 1998;24:345-66. [14] Sonmchsen et al . Clinical pharmacokmetics of paclitaxel. Clin Pharmacokmet 1994;27:256-69. [15] Rahman et al . Selective biotransformation of taxol to 6a-hydroxytaxol by human cytochrome P450 CYP2C8. Cancer Res 1994;54:5543-46. [16] Harris et al. Isolation structural determination, and biological activity of 6a-hydroxytaxol, the principle human metabolite of taxol. J Med Chem 1994;37:706-9. [17] Chang et al. Differential activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes. Cancer Res 1993;53:5629-37. [18] Bohnenstengel et al. Characterization of the cytochrome P450 involved in side-chain oxidation of cyclophosphamide in humans. Eur J Clin Pharmacol 1996;51:297-301. [19] Goeptar et al. Cytotoxicity of mitomycm C and adriamycm in freshly isolated rat hepatocytes: the role of cytochrome P450. Cancer Res 1994;54:2411-18. [20] Lewis et al. Role of cytochrome P450 from the human CYP3A gene family in the potentiation of morphilmo doxorubicm by human liver microsomes. Cancer Res 1992;52:4379-84. [21] Luch et al. Stable expression of human cytochrome P450 IBl m V79 Chinese hamster cells and metabolically catalyzed DNA adduct formation of dibenzo a, 1] pyrene . Chem Res Toxicol 1998;11: 686-95. [22] Engst et al. Benzylic hydroxylation of 1- methylpyrene and 1-ethylpyrene by human and rat cytochromes P450 individually expressed in V79 Chinese hamster cells. Carcmogenesis 1999;20:1777-85. [23] Philip et al. Use of V79 cells with stably transfected cytochrome P450 cDNAs in studying the metabolism and effects of cytotoxic drugs. Cancer Chemother Pharmacol 1999;43:59-67. [24] Doehmer. V79 Chinese hamster cells genetically engineered for cytochrome P450 and their use in mutagenicity and metabolism studies. Toxicology 1993;82:105-118. [25] Bradford. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54. [26] Crespi et al. Development of a human lymphoblastoid cell line constitutively expressing human CYPIBI cDNA: substrate specificity with model substrates and promutagens. Mutagenesis 1997;12:83-89. [27] Shimada et al. Selectivity of polycyclic inhibitors for human cytochrome P450s 1A1, 1A2 , and IBl. Chem Res Toxicol 1998;11:1048-56. [28] Plumb et al. Effects of the pH dependence of 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium based assay. Cancer Res 1989;49:4435-40. [29] Kuilman et al. Cytochrome P450 mediated metabolism and cytotoxicity of aflotoxin B x in bovine hepatocytes. Toxicol in vitro 2000;14:321-27. [30] Adams et al . Chemotherapy for ovarian cancer-a consensus statement on standard practice. Br J Cancer 1998;78: 1404-06. [31] Morgan et al . Phase II trial of combination intraperitoneal cisplatin and 5-fluorouracil in previously treated patients with advanced ovarian cancer: long-term follow-up. Gynecol Oncol 2000;77:433-38. [32] Rochat et al. Human CYPIBI and anti-cancer agent metabolism: mechanism for tumour specific drug interaction? J Pharmacol Exp Ther 2001;296: 537-541. [33] Shou et al . Role of human cytochrome P450 3A4 and 3A5 in the metabolism of taxotere and its derivatives: enzyme specificity, teπnαividual distribution and metabolic contribution in human liver. Pharmacogenetics 1998;8:391-401. [34] Crown et al. The taxanes: -an update. The Lancet 2000;355:1176-78.

Claims: Claims : 1. Use of a substance capable of inhibiting CYPIBI protein and an anti-cancer drug for the preparation of a medicament for the treatment of cancer. 2. The use of claim 1, wherein the substance capable of inhibiting CYPIBI protein inhibits an activity of CYPIBI enzyme m metabolising or inactivating the anti-cancer drug or a pro-drug form thereof, or a metabolic product of the anti-cancer drug or pro-drug. 3. The use of claim 2, wherein CYPIBI causes a loss of cytotoxicity of the anti-cancer -drug, or in the case of a pro-drug, the capacity to be converted to active drug. 4. The use of any one of the preceding claims, wherein the substance capable of inhibiting CYPIBI protein is a flavonoid, a flavone or ethenyl pyrene. 5. The use of claim 4, wherein the substance is α- naphthoflavone . 6. The use of any one of the preceding claims, wherein the anti-cancer drug is a docetaxel, paclitaxel, flutamide, tamoxifen, mitoxantrone, doxorubicm or daunomycm. 7. The use of any one of the preceding claims, wherein the medicament is used for the treatment of breast cancer, kidney cancer, colorectal cancer, prostate cancer, liver cancer or ovarian cancer. 8. The use of any one of the preceding claims, wherein the substance capable of inhibiting CYPIBI protein and the anti-cancer agent are formulated together m a composition . 9. The use of any one of claims 1 to 7, wherein the substance capable of inhibiting CYPIBI protein and the anti-cancer agent are formulated for sequential administration . 10. A composition comprising a substance capable of inhibiting CYPIBI protein and an anti-cancer drug, in combination with a physiologically acceptable carrier. 11. The composition of claim 10, wherein the substance capable of inhibiting CYPIBI protein inhibits an activity of CYPIBI enzyme in metabolising or inactivating the anti-cancer drug or a pro-drug form thereof, or a metabolic product of the anti-cancer drug or pro-drug. 12. The composition of claim 11, wherein CYPIBI causes a loss of cytotoxicity of the anti-cancer drug, or in the case of a pro-drug, the capacity to be converted to active drug. 13. The composition of any one of claims 10 to 12, wherein the substance capable of inhibiting is a flavonoid, a flavone or ethenyl pyrene . 14. The composition of claim 13, wherein the substance is α-naphthoflavone . 15. The composition of any one of claims 10 to 14, wherein the anti-cancer drug is docetaxel, paclitaxel, flutamide, tamoxifen, mitoxantrone, doxorubicm or daunomycm. 16. A kit comprising: (a) in a first container, a substance capable of inhibiting CYPIBI protein; and, (b) m a second container, an anti-cancer drug, wherein the substances are formulated with a physiologically acceptable carrier and are for simultaneous or sequential administration. 17. A method for treating cancer, the method comprising administering to a patient in need a combination of a substance capable of inhibiting CYPIBI protein and an anti-cancer drug. 18. A method of screening for GYPlBl inhibitors for use in combination with anti-cancer drugs, the method comprising: (a) contacting a candidate substance with CYPIBI protein under conditions where the candidate substance and CYPIBI can interact; (b) measuring the activity of the CYPIBI protein and comparing the value obtained to standards; and (c) selecting candidate compounds which have the effect of inhibiting CYPIBI. 19. The method of claim 18, further comprising the step of testing the candidate compounds m combination with CYPIBI and one or more anti-cancer drugs to determine whether the effect the candidate inhibitor has in reducing the effect of CYPIBI in inactivating or metabolising the anti-cancer drug. 20. The method of claim 19, wherein the testing step employs an in vitro interaction assays or a cell based assays measuring the effect of the inhibitors in reducing the loss of cytotoxicity of the anti-cancer drugs caused by CYPIBI action. 21. A method of determining the diagnosis, prognosis or responsiveness to treatment of a patient having ovarian cancer, the method comprising determining a presence or amount of CYPIBI protein m a sample from a patient comprising ovarian cancer cells and correlating the presence or amount to control values.

Inventors: MCFADYEN MORAG, MELVIN WILLIAM THOMAS, MURRAY GRAEME IAN, MCFADYEN, MORAG, MELVIN, WILLIAM, THOMAS, MURRAY, GRAEME, IAN
Applicants: MCFADYEN MORAG, MELVIN WILLIAM THOMAS, MURRAY GRAEME IAN, UNIV ABERDEEN, MCFADYEN, MORAG, MELVIN, WILLIAM, THOMAS, MURRAY, GRAEME, IAN, THE UNIVERSITY COURT OF THE UNIVERSITY OF ABERDEEN
Application year: 2001
Main classification: A61K31
IPC codes: A61K31, A61K45, A61P35, A61P43, G01N33