WO2014194285A1

WO2014194285A1 - Quassinoid and coumarin compounds for cancer prevention

Info

Publication number: WO2014194285A1
Application number: PCT/US2014/040375
Authority: WO
Inventors: Thejani R. DELGODA; Simone Am BADAL; Eileen BRANTLEY
Original assignee: THE UNIVERSITY OF THE WEST INDIES, a regional institution established by ROYAL CHARTER; Loma Linda University
Priority date: 2013-05-31
Filing date: 2014-05-30
Publication date: 2014-12-04

Abstract

The present invention relates to the discovery that quassinoids and coumarin compounds isolated from the endemic Jamaican Castela macrophylla plant can be used to treat or prevent cancer.

Description

QUASSINOID AND COUMARIN COMPOUNDS FOR CANCER PREVENTION

[0001] This application claims priority to United States Provisional Application No.

61/829,881, filed on May 31, 2013, and United States Provisional Application No.

61/917,798, filed on December 18, 2013 , which are herein incorporated by reference in their entireties.

[0002] All patents, patent applications and publications, and non-patent publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

[0004] Quassinoids, (termed quassin after a man by the name of Quassi who treated fever with the bark of these plants) are bitter principles most occurring in the Simaroubaceae family. Chemically, quassinoids are degraded triterpenes. Coumarin is a fragrant organic chemical compound in the benzopyrone chemical class, and is a colorless crystalline substance in its standard state. It is found naturally in many plants. Quassinoids and coumarins have been associated with anti-inflammatory, antioxidant and anti-proliferative activity.

[0005] Quassinoids have been isolated from Castela turpin (Simaroubaceae) and Castela macrophylla. Although quassinoids have been reported to display many biological activities, isolates specifically from Castela macrophylla have so far been limited in their

pharmacological applications to anti-feedant activities against tobacco budworm,

Plasmodium falciparum and Plamopara viticola. A better understanding of the biological characteristics of quassinoids and coumarin isolates from Castela macrophylla, especially their anti-proliferative activity, can help determine their pharmacological applications. SUMMARY OF THE INVENTION

[0006] In one aspect, the present invention provides a method of preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a quassinoid compound, thereby preventing the cancer.

[0007] In another aspect, the present invention provides a method of preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a coumarin compound, thereby preventing the cancer.

[0008] In one embodiment, the quassinoid is glaucarubolone-15-0-P-D-glucopyranoside. In another embodiment, the quassinoid is holocanthone. In one embodiment, the coumarin is scopoletin. In one embodiment, the compound is extracted from Castela macrophylla.

[0009] In one embodiment, the cancer is selected from colon cancer, liver cancer and breast cancer.

[0010] In one embodiment, the subject is at risk of developing cancer. In another embodiment, the subject is exposed to polyaromatic hydrocarbons (PAHs).

[0011] In one embodiment, the preventing comprises reducing the activity of a CYP450 enzyme. In one embodiment, the activity is reduced by about 20% relative to the activity of the CYP450 enzyme prior to administration of the compound. In another embodiment, the activity is reduced by about 50% relative to the activity of the CYP450 enzyme prior to administration of the compound. In another embodiment, the activity is reduced by about 70% relative to the activity of the CYP450 enzyme prior to administration of the compound.

[0012] In one embodiment, the preventing comprises inhibiting the activity of a CYP450 enzyme by the compound. In one embodiment, the IC50 value of the compound is about 10 μΜ. In one embodiment, the IC50 value of the compound is about 5 μΜ. In another embodiment, the IC50 value of the compound is about 1 μΜ.

[0013] In one embodiment, the preventing comprises reducing the expression of a CYP450 mR A. In one embodiment, the expression is reduced by about 70% relative to the expression of the CYP450 mRNA, prior to administration of the compound. In one embodiment, the expression is reduced by about 50%> relative to the expression of the CYP450 mRNA, prior to administration of the compound. In another embodiment, the expression is reduced by about 30% relative to the expression of the CYP450 mRNA, prior to administration of the compound.

[0014] In one embodiment, the CYP450 consists of CYPlAl, CYP1A2, CYP1B1, CYP2C19, CYP2D6 or CYP3A4.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figures 1A-B. Figures 1A. Structures of the investigated isolates from the plant Castela macrophylla. Figure IB. Anticancer activity of the isolates in cancer cell lines in comparison to Tamoxifen and 5-fluorouracil following 24 h of exposure. Anticancer activity was determined by the MTS assay as outlined in Materials and Methods.

[0016] Figures 2A-D. Cytotoxicity of Gg in comparison to tamoxifen following 24, 48 and 72 h of treatment (Figures 2A-C) and Gg-induced apoptosis (Figure 2D) in MCF-7 breast cancer cells. In Figures 2A-C, cells were analysed for cell survival using the Alamar Blue™ assay following treatment as outlined in Materials and Methods. Statistical significance as indicated by * P < 0.05, **P < 0.01 or***P < O.Olversus vehicle control. In Figure 2D, cells were exposed to media containing Gg or .025% DMSO for 24 h before being analysed for apoptosis using the AnnexinV-7AAD assay as described in Materials and Methods. Data represent the mean percentage ± SEM of three independent experiments performed in triplicate. Statistical significance as indicated by * P < 0.05 versus vehicle control.

[0017] Figures 3 A-C. Figure 3A. Characterisation of the inhibition of (-)-Glaucarubolone glucoside on CYP enzyme activities. Human recombinant CYP IB 1 -catalysed 7- ethoxyresorufin activity (0.37μΜ), CYPs 1 Al catalysed 7-ethoxy-3-cyanocoumarin deethylase activity (0.5μΜ), were determined in the presence of varying concentrations of (- )-Glaucarubolone ranging between 0 and 20μΜ, as described in Materials and Methods for IC₅₀ determinations. Control enzyme activity (mean ± SEM) for CYPs 1B1 and 1A1 were 0.34 ± 0.08, 0.86 ± 0.01 μΜ/min/pmolof C YP respectively. Eadie-Hofstee plots of CYPs 1A1 (Figure 3B) and 1B1 (Figure 3C) by (-)-Glaucaruboloneglucoside. Ethoxy-3- cyanocoumarin deethylaseactivityof recombinant CYPlAl and 7-ethoxyresorufin activity of recombinant CYPs 1B1 were determined in the presence and absence six different concentrations of (-)-Glaucaruboloneglucoside (stated as I in the key). Data are expressed as mean percentage ± SEM of control enzyme activity for three independent experiments. [0018] Figure 4. Effect of Gg on Benzo-a-pyrene-induced CYP1A in non-malignant MCF-IOA breast epithelial cells. MCF-7 cells were exposed to benzo-a-pyrene alone or in combination with Gg at indicated concentrations for 24 h. Cells were harvested, R A extracted and quantitative real-time PCR analysis performed in accordance with Materials and methods to evaluate CYPlAl and CYP1A2 mRNA expression. Data represent the mean ± SEM of three independent experiments. Statistical significance as indicated by ** P< O.Olor ***P < 0.001 versus treatment with B[a]P only.

[0019] Figure 5. Structures of the investigated isolates from the plant Castela macrophylla.

[0020] Figure 6. Effect of Gg on 1,6-BPQ-mediated increases in ROS in non-malignant MCF-IOA breast epithelial cells. MCF-IOA cells were exposed to 1,6-BPQ alone or in combination with Ggfor 2 hours before being analysed for ROS production using flow cytometry as described in Materials and Methods. Data represent the mean ± SEM of three independent experiments. Statistical significance as indicated by * P< 0.05 or ** P < 0.01 when comparing indicated data points.

[0021] Figures 7A-C. Microscopic evaluation of breast cancer cells in the presence of Gg or tamoxifen. Figure 7A represents breast MCF-7 cancer cells in the absence of Gg or tamoxifen, Figure 7B represents breast MCF-7 cancer cells in the presence of Gg and Figure 7C represents breast MCF-7 cancer cells in the presence of tamoxifen. Figures illustrate greater potency of Gg in cell reduction viability when compared to tamoxifen.

[0022] Figure 8. Cytotoxicity of 4-hydroxytamoxifen in MCF-7 cells following 72 h of treatment. Cells were analysed for survival using the using the Alamar Blueassay following treatment as outlined in Materials and Methods. Data represent the mean percentage ± SEM of two independent experiments.

[0023] Figure 9. Graphical Abstract: A schematic representation to summarize points of intervention of Gg of B[a]P activation. B[a]P molecule is depicted at the top of the figure, while only a terminal benzo ring is shown for the rest of the schematic. Major enzymes involved are indicated. EH: epoxide hydrolase; CYP: cytochrome P450. In the figure, only (+)B[a]P-7,8-diol-9,10-epoxide-2, the most reactive metabolite formation is depicted, following the more sterochemically preferred pathway. Information from (Bolt and Ross 2008; Shimada and Fujii-Kuriyama 2004), were gathered. DETAILED DESCRIPTION OF THE INVENTION

Abbrevations and Definitions

[0024] The abbreviation "Gg" designates the compound glaucarubolone-15-0-P-D- glucopyranoside. The chemical structure of Gg is available in the art.

[0025] The singular forms "a", "an" and "the" include plural reference unless the context clearly dictates otherwise.

[0026] As used herein the term "about" is used herein to mean approximately, roughly, around, or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

Description

Background

Cancer

[0027] Cancer is a broad group of various diseases, all involving unregulated cell growth. In cancer, cells divide and grow uncontrollably, forming malignant tumors, and invade nearby parts of the body. The cancer may also spread to more distant parts of the body through the lymphatic system or bloodstream.

[0028] Breast cancer is one of the major cancers affecting women, with an estimated 232,340 new cases of invasive breast cancer expected to be diagnosed in women in 2013. Although mortality rates have declined over the past decade, this disease still accounts for nearly 40,000 deaths annually. Current treatments for breast cancer include surgery, chemotherapy, radiation therapy, targeted therapy, hormone therapy, and bone-directed therapy. For additional information, see http://www.cancer.org/index.

[0029] Colorectal cancer is one of the most common cancers diagnosed in both men and women in the United States. Estimates for the number of colorectal cancer cases in the United States for 2013 are 102,480 new cases of colon cancer and 40,340 new cases of rectal cancer. For additional information, see http://www.cancer.org/index. [0030] Liver cancer is more common in men than in women. The average age when liver cancer is found is 62. Liver cancer is much more common in countries in sub-Saharan Africa and Southeast Asia than in the United States. In many of these countries it is the most common type of cancer. Estimates for primary liver and bile duct cancers in the United States for 2013 are about 30,640 new cases of primary liver cancer and bile duct cancer. For additional information, see http://www.cancer.org/index.

[0031] Cytochrome P450 Family

[0032] Cytochrome P450 (CYP) is a heme containing enzyme superfamily that catalyzes the oxidative biotransformation of lipophilic substrates to hydrophilic metabolites facilitating their removal from cells. See e.g., Simone Badal, Mario Shields and Rupika Delgoda (2012). Cytochrome P450 Enzyme Inhibitors from Nature, Enzyme Inhibition and Bioapplications, Prof. Rakesh Sharma (Ed.), ISBN: 978-953-51-0585-5, InTech, available from:

http ://www.intechopen. com/books/ enzyme-inhibition-and-bioapplications/ cytochrome-p450- enzyme-inhibitors-from-nature, which is incorporated by reference herein in its entirety.

[0033] Eighteen mammalian CYP enzyme structures are known and 15 of these are of human origin, including 1A2, 2A6, 2A13, 2B4 (rabbit), 2B6, 2C5 (rabbit), 2C8, 2C9, 2D6, 2E1, 2R1, 3A4, 7A1, 8A1, 19A1, 24A1 (rat), 46A1, 51A1 (see Badal et al, 2012). CYPs sharing >40% sequence identity are categorized within the same family, while those with >55% sequence identity are placed within the same subfamily.

[0034] CYP2 is the largest CYP450 family in mammals, with 13 subfamilies and 16 genes in humans. CYPs2C8, 2C9, 2C18 and 2C 19 jointly metabolize more than 50 drugs whilst CYP2D6 metabolizes more than 70 drugs. CYP3A is the most abundantly expressed CYP450 gene in the human liver and gastrointestinal tract, and is known to metabolize more than 120 commonly prescribed pharmaceutical agents. For additional information, see Badal et al, 2012.

[0035] Chemoprevention is the ability of compounds to protect healthy tissues via the prevention, inhibition or reversal of carcinogenesis. The members of the CYP1 family (i.e. CYPslAl, 1A2 and 1B1) are capable of metabolizing procarcinogens, such as polyaromatic hydrocarbons (PAH), into their reactive and cancer-causing carcinogenic forms. Inhibiting the formation of the carcinogen can thus prevent the initiation of the tumour. Therefore, inhibitors of CYP1 enzymes are thought to possess chemopreventive or chemoprotective properties. The prevention of DNA-PAH adduct formation, among other potential properties, can confirm the use of a compound as a chemopreventor.

[0036] The CYP1 family has been linked to the activation of pro-carcinogens, which is facilitated by the regulation of the aryl hydrocarbon receptor. As such, research has shown that inhibiting CYP1 enzymes plays a key role in protecting healthy cells from the harmful effects of activated carcinogens (Badal et al., 2012).

[0037] Quassinoids and Coumarin Compounds

[0038] Quassinoids, (termed quassin after a man by the name of Quassi who treated fever with the bark of these plants) are bitter principles most occurring in the Simaroubaceae family. Chemically, quassinoids are degraded triterpenes. Coumarin is a fragrant organic chemical compound in the benzopyrone chemical class, and is a colorless crystalline substance in its standard state. It is found naturally in many plants. Quassinoids and coumarins have been associated with anti-inflammatory, antioxidant and anti-proliferative activity.

[0039] Quassinoids have been isolated from Castela turpin (Simaroubaceae) and Castela macrophylla. Although quassinoids have been reported to display many biological activities, isolates specifically from Castela macrophylla have so far been limited in their

Plasmodium falciparum and Plamopara viticola.

Methods of Prevention

[0040] The present invention provides methods of preventing cancer.

[0041] In one aspect, the present invention provides a method of preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a quassinoid compound, thereby preventing the cancer.

[0042] In another aspect, the present invention provides a method of preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a coumarin compound, thereby preventing the cancer. [0043] In one aspect, the present invention provides a method of preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a crude extract comprising a quassinoid compound, thereby preventing the cancer.

[0044] In another aspect, the present invention provides a method of preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a crude extract comprising a coumarin compound, thereby preventing the cancer.

[0045] In one embodiment, the quassinoid is glaucarubolone-15-0-P-D-glucopyranoside. In another embodiment, the quassinoid is holocanthone. In another embodiment, the quassinoid is glaucarubolone.

[0046] In one embodiment, the coumarin is scopoletin.

[0047] In one embodiment, the quassinoid consists of a quassinoid comprising a glycoside on ring D. In another embodiment, the quassinoid consists of a quassinoid comprising a glucoside on ring D. In another embodiment, the quassinoid consists of a quassinoid comprising a hydroxyl group on ring D. In another embodiment, the quassinoid consists of a quassinoid comprising a hydrogen on ring D. In another embodiment, the quassinoid consists of a quassinoid comprising an acetyl moiety on ring D.

[0048] In one embodiment, the glycoside is linked to the C12 position on ring D. In another embodiment, the glucoside is linked to the C12 position on ring D. In another embodiment, the hydroxyl group is linked to the C12 position on ring D. In one embodiment, the hydrogen is linked to the C12 position on ring D. In another embodiment, the acetyl moiety is linked to the C12 position on ring D.

[0049] In one embodiment, the compound is extracted from Castela macrophylla. In one embodiment, the coumarin or the quassinoid is extracted from Castela macrophylla. In another embodiment, the coumarin or quassinoid is commercially available.

[0050] In one embodiment, the compound induces apoptosis in a cancer cell.

[0051] In one embodiment, the cancer is selected from colon cancer, liver cancer and breast cancer. In another embodiment, the cancer is selected from kidney cancer, brain cancer, liver cancer, colorectal cancers, progressive lung adenocarcinoma, as well as lymphomas, leukemias, adenocarcinomas, gliomas and sarcomas. The cancer can be, for example, B cell lymphoma, colon cancer, lung cancer, renal cancer, bladder cancer, T cell lymphoma, myeloma, leukemia, chronic myeloid leukemia, acute myeloid leukemia, chronic

lymphocytic leukemia, acute lymphocytic leukemia, hematopoietic neoplasias, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, uterine cancer, renal cell carcinoma, hepatoma, adenocarcinoma, pancreatic cancer, liver cancer, prostate cancer, head and neck carcinoma, thyroid carcinoma, soft tissue sarcoma, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancer, angiosarcoma, hemangiosarcoma, bone sarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, testicular cancer, uterine cancer, cervical cancer, gastrointestinal cancer, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Waldenstroom's macroglobulinemia, papillary adenocarcinomas,

cystadenocarcinoma, bronchogenic carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, lung carcinoma, epithelial carcinoma, cervical cancer, testicular tumor, glioma, astrocytoma, meduUoblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, leukemia, melanoma, neuroblastoma, small cell lung carcinoma, bladder carcinoma, lymphoma, multiple myeloma, or medullary carcinoma.

[0052] In one embodiment, the subject is at risk of developing cancer. In another embodiment, the subject is not at risk of developing cancer.

[0053] In another embodiment, the subject is exposed to polyaromatic hydrocarbons (PAHs). Such PAHs are produced by cigarette smoke, environmental pollutants, and other sources. In one embodiment, the PAH is benzo[a]pyrene (BaP).

[0054] In one embodiment, the BaP is metabolized into a BaP-quinone (BPQ). In one embodiment, the BPQ is 1,6- BPQ.

[0055] In one embodiment, the preventing comprises reducing the rate of a CYP450 enzyme, relative to the rate of a CYP450 enzyme prior to administration of the compound. In one embodiment, the rate is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, relative to the rate of a CYP450 enzyme, prior to administration of the compound. In another embodiment, the rate is reduced by about 5%-10%, or about 10%-15%, or about 15%-20%, or about 20%-25%, or about 25%-30%, or about 30%-35%, or about 35%-40%, or about 40%-45%, or about 45%- 50%, or about 50%-55%, or about 55%-60%, or about 60%-65%, or about 65%-70%, or about 70%-75%, or about 75%-80%, or any range in between, relative to the rate of a CYP450 enzyme, prior to administration of the compound.

[0056] In one embodiment, the preventing comprises reducing the rate of a CYP450 enzyme relative to the rate of a CYP450 enzyme, prior to administration of the compound. In one embodiment, the rate is reduced by about 0.25-fold, about 0.5-fold, about 1-fold, about 2- fold, about 3-fold, about 4-fold, or about 5-fold, or by about 0.25-fold to 0.5-fold, about 0.5 to 1-fold, about 1 to 2-fold, about 2 to 3 fold, about 3 to 4-fold, or about 4 to 5-fold, or any range in between, relative to the rate of a CYP450 enzyme, prior to administration of the compound.

[0057] In one embodiment, the preventing comprises reducing the activity of a CYP450 enzyme, relative to the activity of a CYP450 enzyme prior to administration of the compound. In one embodiment, the activity is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, relative to the activity of a CYP450 enzyme, prior to administration of the compound. In another embodiment, the activity is reduced by about 5%-10%, or about 10%-15%, or about 15%- 20%, or about 20%-25%, or about 25%-30%, or about 30%-35%, or about 35%-40%, or about 40%-45%, or about 45%-50%, or about 50%-55%, or about 55%-60%, or about 60%- 65%), or about 65%-70%, or about 70%-75%, or about 75%-80%, or any range in between, relative to the activity of a CYP450 enzyme, prior to administration of the compound.

[0058] In one embodiment, the preventing comprises reducing the activity of a CYP450 enzyme relative to the activity of a CYP450 enzyme, prior to administration of the compound. In one embodiment, the activity is reduced by about 0.25-fold, about 0.5-fold, about 1-fold, about 2-fold, about 3-fold, about 4-fold, or about 5-fold, or by about 0.25-fold to 0.5-fold, about 0.5 to 1-fold, about 1 to 2-fold, about 2 to 3 fold, about 3 to 4-fold, or about 4 to 5 -fold, or any range in between, relative to the activity of a CYP450 enzyme, prior to administration of the compound. [0059] In one embodiment, the preventing comprises inhibiting the activity of a CYP450 enzyme by the compound. In one embodiment, the IC50 value of the compound is about 0.25 μΜ, 0.5 μΜ, 1 μΜ, 2 μΜ, 3 μΜ, 4 μΜ, 5 μΜ, 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, 20 μΜ, 25 μΜ, 30 μΜ, 35 μΜ, 40 μΜ, 45 μΜ, 50 μΜ, 55 μΜ, 60 μΜ, or more. In another embodiment, the IC50 value of the compound is about 0.25 μΜ-0.5 μΜ, 0.5 μΜ -1 μΜ, 1 μΜ - 2 μΜ, 2 μΜ -3 μΜ, 3 μΜ -4 μΜ, 4 μΜ -5 μΜ, 5 μΜ -6 μΜ, 6 μΜ-7 μΜ, 7 μΜ-8 μΜ, 8 μΜ-9 μΜ, 9 μΜ -10 μΜ, 10 μΜ -11 μΜ, 11 μΜ -12 μΜ, 12 μΜ -13 μΜ, 13 μΜ -14 μΜ, 14 μΜ -15 μΜ, 15 μΜ -20 μΜ, 20 μΜ -25 μΜ, 25 μΜ -30 μΜ, 30 μΜ -35 μΜ, 35 μΜ -40 μΜ, 40 μΜ -45 μΜ, 45 μΜ -50 μΜ, 50 μΜ -55 μΜ, 55 μΜ -60 μΜ, or any range in between.

[0060] In one embodiment, the IC₅₀ value of the compound is less than or equal to 1 μΜ. In one embodiment, the IC₅₀ value of the compound is less than or equal to 5 μΜ. In one embodiment, the IC₅₀ value of the compound is less than or equal to 10 μΜ. In another embodiment, the IC₅₀ value of the compound is between about 1 μΜ and 5 μΜ. In another embodiment, the IC₅₀ value of the compound is between about 1 μΜ and 10 μΜ. In another embodiment, the IC₅₀ value of the compound is greater than or equal to 10 μΜ.

[0061] In one embodiment, the preventing comprises reducing the expression of a CYP450 mRNA relative to the expression of the CYP450 mRNA, prior to administration of the compound. In one embodiment, the expression is reduced by about 5%, 10%, 15%, 20%>, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, relative to the expression of the CYP450 mRNA, prior to administration of the compound. In another embodiment, the expression is reduced by about 5%>-10%>, or about 10%>-15%>, or about 15%-20%, or about 20%-25%, or about 25%-30%, or about 30%-35%, or about 35%- 40%, or about 40%-45%, or about 45%-50%, or about 50%-55%, or about 55%-60%, or about 60%-65%, or about 65%-70%, or about 70%-75%, or about 75%-80%, or any range in between, relative to the expression of the CYP450 mRNA, prior to administration of the compound.

[0062] In one embodiment, the preventing comprises reducing the expression of a CYP450 protein relative to the expression of the CYP450 protein prior to administration of the compound. In one embodiment, the expression is reduced by about 5%>, 10%>, 15%>, 20%>, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, relative to the expression of the CYP450 protein prior to administration of the compound. In another embodiment, the expression is reduced by about 5%-10%, or about 10%-15%, or about 15%-20%, or about 20%-25%, or about 25%-30%, or about 30%-35%, or about 35%- 40%, or about 40%-45%, or about 45%-50%, or about 50%-55%, or about 55%-60%, or about 60%-65%, or about 65%-70%, or about 70%-75%, or about 75%-80%, or any range in between, relative to the expression of the CYP450 protein prior to administration of the compound.

[0063] In one embodiment, the CYP450 consists of CYP1A1, CYP1A2, CYP1B1, CYP2C19, CYP2D6 or CYP3A4. In another embodiment, the CYP450 consists of a member of the CYP1 family of enzymes.

[0064] In one embodiment, the expression is reduced in the presence of PAH. In another embodiment, the expression is not reduced in the absence of PAH. In one embodiment, the expression is reduced in the presence of BaP. In another embodiment, the expression is not reduced in the absence of BaP.

[0065] In one embodiment, intracellular reactive oxygen species (ROS) levels are increased in the presence of a BPQ relative to intracellular reactive oxygen species (ROS) levels prior to the presence of the BPQ. In one embodiment, the reactive oxygen species (ROS) levels are increased by about 0.25-fold, about 0.5-fold, about 1-fold, about 2-fold, about 3-fold, about 4-fold, or about 5-fold, or by about 0.25-fold to 0.5-fold, about 0.5 to 1-fold, about 1 to 2-fold, about 2 to 3 fold, about 3 to 4-fold, or about 4 to 5 -fold, or any range in between, relative to the intracellular reactive oxygen species (ROS) levels prior to presence of the BPQ.

[0066] In one embodiment, the preventing comprises reducing intracellular reactive oxygen species (ROS) levels relative to intracellular reactive oxygen species (ROS) levels prior to administration of the compound. In one embodiment, the levels are reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%), or 95%), relative to intracellular reactive oxygen species (ROS) levels prior to administration of the compound. In another embodiment, the levels are reduced by about 5%-10%, or about 10%-15%, or about 15%-20%, or about 20%-25%, or about 25%-30%, or about 30%-35%, or about 35%-40%, or about 40%-45%, or about 45%-50%, or about 50%- 55%, or about 55%-60%, or about 60%-65%, or about 65%-70%, or about 70%-75%, or about 75%-80%, or any range in between, relative to intracellular reactive oxygen species (ROS) levels prior to administration of the compound. In another embodiment, the levels are reduced by about 0.25-fold, about 0.5-fold, about 1-fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 4-fold, or about 5-fold, or by about 0.25-fold to 0.5-fold, about 0.5 to 1-fold, about 1 to 2-fold, about 2 to 3 fold, about 3 to 4-fold, or about 4 to 5-fold, or any range in between, relative to intracellular reactive oxygen species (ROS) levels prior to administration of the compound.

[0067] In another embodiment, the preventing comprises reducing the ability of a BPQ to increase intracellular reactive oxygen species (ROS) levels relative to the ability of a BPQ to increase intracellular reactive oxygen species (ROS) levels prior to administration of the compound. In one embodiment, the ability is reduced by about 5%, 10%, 15%, 20%>, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, relative to the ability of a BPQ to increase intracellular reactive oxygen species (ROS) levels prior to administration of the compound. In another embodiment, the ability is reduced by about 5%- 10%, or about 10%- 15%, or about 15%-20%, or about 20%-25%, or about 25%-30%, or about 30%-35%, or about 35%-40%, or about 40%-45%, or about 45%-50%, or about 50%- 55%, or about 55%-60%, or about 60%-65%, or about 65%-70%, or about 70%-75%, or about 75%-80%, or any range in between, relative to the ability of a BPQ to increase intracellular reactive oxygen species (ROS) levels prior to administration of the compound. In another embodiment, the ability is reduced by about 0.25-fold, about 0.5-fold, about 1- fold, about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 4-fold, or about 5-fold, or by about 0.25 -fold to 0.5 -fold, about 0.5 to 1-fold, about 1 to 2-fold, about 2 to 3 fold, about 3 to 4-fold, or about 4 to 5 -fold, or any range in between relative to the ability of a BPQ to increase intracellular reactive oxygen species (ROS) levels prior to administration of the compound.

Methods of Treatment

[0068] The present invention provides methods of treating cancer.

[0069] In one aspect, the present invention provides a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a quassinoid compound, thereby treating the cancer. [0070] In another aspect, the present invention provides a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a coumarin compound, thereby treating the cancer.

[0071] In one aspect, the present invention provides a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a crude extract comprising a quassinoid compound, thereby treating the cancer.

[0072] In another aspect, the present invention provides a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a crude extract comprising a coumarin compound, thereby treating the cancer.

[0073] In one embodiment, the quassinoid is glaucarubolone-15-0-P-D-glucopyranoside. In another embodiment, the quassinoid is holocanthone. In another embodiment, the quassinoid is glaucarubolone.

[0074] In one embodiment, the quassinoid consists of a quassinoid comprising a glycoside on ring D. In another embodiment, the quassinoid consists of a quassinoid comprising a glucoside on ring D. In another embodiment, the quassinoid consists of a quassinoid comprising a hydroxyl group on ring D. In another embodiment, the quassinoid consists of a quassinoid comprising a hydrogen on ring D. In another embodiment, the quassinoid consists of a quassinoid comprising an acetyl moiety on ring D.

[0075] In one embodiment, the glycoside is linked to the C12 position on ring D. In another embodiment, the glucoside is linked to the C12 position on ring D. In another embodiment, the hydroxyl group is linked to the C12 position on ring D. In one embodiment, the hydrogen is linked to the C12 position on ring D. In another embodiment, the acetyl moiety is linked to the C12 position on ring D.

[0076] In one embodiment, the coumarin is scopoletin.

[0077] In one embodiment, the compound induces apoptosis in a cancer cell.

[0078] In one embodiment, the coumarin or the quassinoid is extracted from Castela macrophylla. In another embodiment, the coumarin or quassinoid is commercially available. [0079] In one embodiment, the cancer is selected from colon cancer, liver cancer and breast cancer. In another embodiment, the cancer is selected from kidney cancer, brain cancer, liver cancer, colorectal cancers, progressive lung adenocarcinoma, as well as lymphomas, leukemias, adenocarcinomas, gliomas and sarcomas. The cancer can be, for example, B cell lymphoma, colon cancer, lung cancer, renal cancer, bladder cancer, T cell lymphoma, myeloma, leukemia, chronic myeloid leukemia, acute myeloid leukemia, chronic

[0080] In one embodiment, the treating comprises reducing the viability of a cancer cell of the subject, relative to the viability of the cancer cell of the subject prior to administration of the compound. In one embodiment, the viability is reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, relative to the viability of the cancer cell of the subject prior to administration of the compound. In another embodiment, the viability is reduced by about 5%>-10%>, or about 10%-15%, or about 15%-20%, or about 20%-25%, or about 25%-30%, or about 30%-35%, or about 35%-40%, or about 40%-45%, or about 45%-50%, or about 50%-55%, or about 55%- 60%, or about 60%-65%, or about 65%-70%, or about 70%-75%, or about 75%-80%, or any range in between, relative to the viability of the cancer cell of the subject prior to

administration of the compound.

[0081] In one embodiment, the treating comprises reducing the viability of a cancer cell of the subject, relative to the viability of the cancer cell of the subject prior to administration of the compound. In one embodiment, the viability is reduced by about 0.25-fold, 0.5-fold, 1- fold, 2-fold, 3-fold, 4-fold, or about 5-fold, or by about 0.25-fold to 0.5-fold, about 0.5 to 1- fold, about 1 to 2-fold, about 2 to 3 fold, about 3 to 4-fold, or about 4 to 5 -fold, or any range in between, relative to the viability of the cancer cell of the subject prior to administration of the compound.

[0082] In one embodiment, the treating comprises increasing the apoptosis of a cancer cell of the subject, relative to the apoptosis of the cancer cell of the subject prior to administration of the compound. In one embodiment, the apoptosis is increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, relative to the apoptosis of the cancer cell of the subject prior to administration of the compound. In another embodiment, the apoptosis is increased by about 5%>-10%>, or about 10%-15%, or about 15%-20%, or about 20%-25%, or about 25%-30%, or about 30%-35%, or about 35%-40%, or about 40%-45%, or about 45%-50%, or about 50%-55%, or about 55%- 60%, or about 60%-65%, or about 65%-70%, or about 70%-75%, or about 75%-80%, or any range in between, relative to the apoptosis of the cancer cell of the subject prior to

administration of the compound.

[0083] In one embodiment, the treating comprises increasing the apoptosis of a cancer cell of the subject, relative to the apoptosis of the cancer cell of the subject prior to administration of the compound. In one embodiment, the apoptosis is increased by about 0.25-fold, about 0.5-fold, about 1-fold, about 2-fold, or about 3-fold, or about 4-fold, or about 5-fold, or by about 0.25-fold to about 0.5-fold, about 0.5 to 1-fold, about 1 to 2-fold, about 2 to 3 fold, about 3 to 4-fold, or about 4 to 5 -fold, or any range in between, relative to the apoptosis of the cancer cell of the subject prior to administration of the compound.

[0084] In one embodiment, the IC50 value of the compound is about 0.25 μΜ, 0.5 μΜ, 1 μΜ, 2 μΜ, 3 μΜ, 4 μΜ, 5 μΜ, 6 μΜ, 7 μΜ, 8 μΜ, 9 μΜ, 10 μΜ, 11 μΜ, 12 μΜ, 13 μΜ, 14 μΜ, 15 μΜ, 16 μΜ, 17 μΜ, 18 μΜ, 19 μΜ, 20 μΜ, 25 μΜ, 30 μΜ, 35 μΜ, 40 μΜ, 45 μΜ, 50 μΜ, 55 μΜ, 60 μΜ, or more. In another embodiment, the IC50 value of the compound is about 0.25 μΜ-0.5 μΜ, 0.5 μΜ -1 μΜ, 1 μΜ - 2 μΜ, 2 μΜ -3 μΜ, 3 μΜ -4 μΜ, 4 μΜ -5 μΜ, 5 μΜ -6 μΜ, 6 μΜ-7 uM, 7 μΜ-8 uM, 8 μΜ-9 μΜ, 9 μΜ -10 μΜ, 10 μΜ -11 μΜ, 11 μΜ -12 μΜ, 12 μΜ -13 μΜ, 13 μΜ -14 μΜ, 14 μΜ -15 μΜ, 15 μΜ -20 μΜ, 20 μΜ -25 μΜ, 25 μΜ -30 μΜ, 30 μΜ -35 μΜ, 35 μΜ -40 μΜ, 40 μΜ -45 μΜ, 45 μΜ -50 μΜ, 50 μΜ -55 μΜ, 55 μΜ -60 μΜ, or any range in between.

[0085] In one embodiment, the IC50 value of the compound is less than or equal to 1 μΜ. In one embodiment, the IC50 value of the compound is less than or equal to 5 μΜ. In one embodiment, the IC50 value of the compound is less than or equal to 10 μΜ. In another embodiment, the IC50 value of the compound is between about 1 μΜ and 5 μΜ. In another embodiment, the IC50 value of the compound is between about 1 μΜ and 10 μΜ. In another embodiment, the IC50 value of the compound is greater than or equal to 10 μΜ.

Compounds

[0086] The present disclosure provides methods for the treatment and/or prevention of cancer, which comprise administration of one or more compounds. The compounds of the invention include compounds isolated from the endemic Jamaican Castela macrophylla plant, including, but not limited to, quassinoids and coumarins. The compounds of the invention include compounds that are commercially available, including, but not limited to, quassinoids and coumarins.

[0087] The compounds of the invention include inhibitors of members of the cytochrome P450 (CYP450) family. The compounds of the invention include activators of the

cytochrome P450 (CYP450) family. The compounds include, but are not limited to, inhibitors of CYP1A1, CYP1A2, CYP1B1, CYP2C19, CYP2D6 or CYP3A4.

[0088] Any suitable inhibitor or activator of a member of the CYP450 family may be used. Such compounds may be, for example, small molecule compounds, peptide agents, peptidomimetic agents, antibodies (including, but not limited to monoclonal, poycloncal, humanized, and fully human antibodies, as well as antibody fragments), inhibitory RNA molecules (such as siRNA) and the like. One of skill in the art will understand that these and other types of agents may be used to inhibit or activate a member of the cytochrome P450 (CYP450) family. [0089] Examples of small molecules include, but are not limited to, quassinoids and coumarins. Examples of quassinoids include, but are not limited to, glaucarubolone glucoside, glaucarubolone-15-0-P-D-glucopyranoside and holocanthone.

[0090] Other examples of quassinoids include quassinoids comprising a glycoside on ring D, quassinoids comprising a glucoside on ring D, quassinoids comprising a hydroxyl group on ring D, quassinoids comprising a hydrogen on ring D, and quassinoids comprising an acetyl moiety on ring D. The glycoside, glucoside, hydroxyl group, hydrogen and acetyl moiety are linked to the C12 position on ring D.

[0091] Examples of coumarins include, but are not limited to, scopoletin.

Pharmaceutical Compositions and Administration for Therapy and Prevention

[0092] Compounds of the invention can be administered to the subject once (e.g., as a single injection or deposition). Alternatively, compounds of the invention can be

administered once or twice daily to a subject in need thereof for a period of from about two to about twenty-eight days, or from about seven to about ten days. Compounds of the invention can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. Furthermore, compounds of the invention can be co-administrated with another therapeutic. Where a dosage regimen comprises multiple administrations, the effective amount of the compound(s) administered to the subject can comprise the total amount of the compound(s) administered over the entire dosage regimen.

[0093] Compounds can be administered to a subject by any means suitable for delivering the compounds to cells of the subject. For example, compounds can be administered by methods suitable to transfect cells. Transfection methods for eukaryotic cells are well known in the art, and include direct injection of a nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate precipitation, and transfection mediated by viral vectors.

[0094] The compositions of this invention can be formulated and administered to reduce the symptoms associated with cancer by any means that produces contact of the active ingredient with the agent's site of action in the body of a subject, such as a human or animal (e.g., a dog, cat, or horse). They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

[0095] The compounds of the invention may be administered to a subject in an amount effective to treat or prevent cancer. One of skill in the art can readily determine what will be an effective amount of the compounds of the invention to be administered to a subject, taking into account whether the compound is being used prophylactically or therapeutically, and taking into account other factors such as the age, weight and sex of the subject, any other drugs that the subject may be taking, any allergies or contraindications that the subject may have, and the like. For example, an effective amount can be determined by the skilled artisan using known procedures, including analysis of titration curves established in vitro or in vivo. Also, one of skill in the art can determine the effective dose from performing pilot experiments in suitable animal model species and scaling the doses up or down depending on the subjects weight etc. Effective amounts can also be determined by performing clinical trials in individuals of the same species as the subject, for example starting at a low dose and gradually increasing the dose and monitoring the effects on cancer. Appropriate dosing regimens can also be determined by one of skill in the art without undue experimentation, in order to determine, for example, whether to administer the agent in one single dose or in multiple doses, and in the case of multiple doses, to determine an effective interval between doses.

[0096] A therapeutically effective dose of a compound that treats or prevents cancer can depend upon a number of factors known to those of ordinary skill in the art. The dose(s) of the compounds can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the compound to have upon the target of interest. These amounts can be readily determined by a skilled artisan. These amounts include, for example, mg or microgram ^g) amounts per kilogram (kg) of subject weight, such as about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg, or about 11 mg/kg, or about 12 mg/kg, or about 13 mg/kg, or about 14 mg/kg, or about 15 mg/kg, or about 16 mg/kg, or about 17 mg/kg, or about 18 mg/kg, or about 19 mg/kg, or about 20 mg/kg, or about 21 mg/kg, or about 22 mg/kg, or about 23 mg/kg, or about 24 mg/kg, or about 25 mg/kg, or about 26 mg/kg, or about 27 mg/kg, or about 28 mg/kg, or about 29 mg/kg, or about 30 mg/kg, or about 31 mg/kg, or about 32 mg/kg, or about 33 mg/kg, or about 34 mg/kg, or about 35 mg/kg, or about 36 mg/kg, or about 37 mg/kg, or about 38 mg/kg, or about 39 mg/kg, or about 40 mg/kg, or about 41 mg/kg, or about 42 mg/kg, or about 53 mg/kg, or about 44 mg/kg, or about 45 mg/kg, or about 46 mg/kg, or about 47 mg/kg, or about 48 mg/kg, or about 49 mg/kg, or about 50 mg/kg, or about 51 mg/kg, or about 52 mg/kg, or about 53 mg/kg, or about 54 mg/kg, or about 55 mg/kg, or between about 1 mg/kg to 2 mg/kg, 2 mg/kg to 3 mg/kg, 3 mg/kg to 4 mg/kg, 4 mg/kg to 5 mg/kg, 5 mg/kg to 6 mg/kg, 6 mg/kg to 7 mg/kg, 7 mg/kg to 8 mg/kg, 8 mg/kg to 9 mg/kg, or 9 mg/kg to 10 mg/kg, or between about 10 mg/kg to 15 mg/kg, or between about 15 mg/kg to 20 mg/kg, or between about 20 mg/kg to 25 mg/kg, or between about 25 mg/kg to 30 mg/kg, or between about 30 mg/kg to 35 mg/kg, or between about 35 mg/kg to 40 mg/kg, or between about 40 mg/kg to 45 mg/kg, or between about 45 mg/kg to 50 mg/kg, or between about 50 mg/kg to 55 mg/kg, or between about 55 mg/kg to 60 mg/kg, or any range in between. These amounts also include a unit dose of a compound, for example, at least about 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1 g, 1.5 g, 2 g, 2.5 g, 3 g, 3.5 g, 4 g, 4.5 g, 5 g, 5.5 g, 6 g, or more. Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.

[0097] Pharmaceutical compositions for use in accordance with the invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration.

Techniques and formulations generally can be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa (20^th Ed., 2000), the entire disclosure of which is herein incorporated by reference. For systemic administration, an injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen- free. These pharmaceutical formulations include formulations for human and veterinary use.

[0098] According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.

[0099] A pharmaceutical composition containing a compound of the invention can be administered in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed herein. The compositions can be administered alone or in combination with at least one other agent, such as a stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

[00100] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [00101] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by incorporating an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00102] Sterile injectable solutions can be prepared by incorporating the compound (e.g., a small molecule, peptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00103] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

[00104] Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[00105] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In some embodiments, the compound can be applied via transdermal delivery systems, which slowly releases the active compound for percutaneous absorption. Permeation enhancers can be used to facilitate transdermal penetration of the active factors in the conditioned media.

Transdermal patches are described in for example, U.S. Pat. No. 5,407,713; U.S. Pat. No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No.

5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No. 4,921,475.

[00106] Administration of the compound is not restricted to a single route, but may encompass administration by multiple routes. For instance, exemplary administrations by multiple routes include, among others, a combination of intradermal and intramuscular administration, or intradermal and subcutaneous administration. Multiple administrations may be sequential or concurrent. Other modes of application by multiple routes will be apparent to the skilled artisan. [00107] The compounds of the invention may be formulated into compositions for administration to subjects for the treatment and/or prevention of cancer. Such compositions may comprise the compounds of the invention in admixture with one or more

pharmaceutically acceptable diluents and/or carriers and optionally one or more other pharmaceutically acceptable additives. The pharmaceutically-acceptable diluents and/or carriers and any other additives must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the subject to whom the composition will be administered. One of skill in the art can readily formulate the compounds of the invention into compositions suitable for administration to subjects, such as human subjects, for example using the teaching a standard text such as Remington's

Pharmaceutical Sciences, 18th ed, (Mack Publishing Company: Easton, Pa., 1990), pp. 1635- 36), and by taking into account the selected route of delivery.

[00108] Examples of diluents and/or carriers and/or other additives that may be used include, but are not limited to, water, glycols, oils, alcohols, aqueous solvents, organic solvents, DMSO, saline solutions, physiological buffer solutions, peptide carriers, starches, sugars, preservatives, antioxidants, coloring agents, pH buffering agents, granulating agents, lubricants, binders, disintegrating agents, emulsifiers, binders, excipients, extenders, glidants, solubilizers, stabilizers, surface active agents, suspending agents, tonicity agents, viscosity- altering agents, carboxymethyl cellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methyl cellulose, powders, saline, sodium alginate. The combination of diluents and/or carriers and/or other additives used can be varied taking into account the nature of the active agents used (for example the solubility and stability of the active agents), the route of delivery (e.g. oral, parenteral, etc.), whether the agents are to be delivered over an extended period (such as from a controlled-release capsule), whether the agents are to be coadministered with other agents, and various other factors. One of skill in the art will readily be able to formulate the compounds for the desired use without undue experimentation.

[00109] The compounds of the invention may be administered to a subject by any suitable method that allows the agent to exert its effect on the subject in vivo. For example, the compositions may be administered to the subject by known procedures including, but not limitated to, by oral administration, sublingual or buccal administration, parenteral administration, transdermal administration, via inhalation, via nasal delivery, vaginally, rectally, and intramuscularly. The compounds of the invention may be administered parenterally, or by epifascial, intracapsular, intracutaneous, subcutaneous, intradermal, intrathecal, intramuscular, intraperitoneal, intrasternal, intravascular, intravenous, parenchymatous, or sublingual delivery. Delivery may be by injection, infusion, catheter delivery, or some other means, such as by tablet or spray. In one embodiment, the compounds of the invention are administered to the subject by way of delivery directly to the muscle tissue of interest, such as by way of a catheter inserted into, or in the proximity of the subject's muscle of interest, or by using delivery vehicles capable of targeting the drug to the muscle.

[00110] For oral administration, a formulation of the compounds of the invention may be presented as capsules, tablets, powders, granules, or as a suspension or solution. The formulation may contain conventional additives, such as lactose, mannitol, cornstarch or potato starch, binders, crystalline cellulose, cellulose derivatives, acacia, cornstarch, gelatins, disintegrators, potato starch, sodium carboxymethylcellulose, dibasic calcium phosphate, anhydrous or sodium starch glycolate, lubricants, and/or or magnesium stearate.

[00111] For parenteral administration (i. e. , administration by through a route other than the alimentary canal), the compounds of the invention may be combined with a sterile aqueous solution that is isotonic with the blood of the subject. Such a formulation may be prepared by dissolving the active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering the solution sterile. The formulation may be presented in unit or multi-dose containers, such as sealed ampoules or vials. The formulation may be delivered by injection, infusion, or other means known in the art.

[00112] For transdermal administration, the compounds of the invention may be combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone and the like, which increase the permeability of the skin to the compounds of the invention and permit the compounds to penetrate through the skin and into the bloodstream. The compounds of the invention also may be further combined with a polymeric substance, such as ethylcellulose, hydroxypropyl cellulose, ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to provide the composition in gel form, which are dissolved in a solvent, such as methylene chloride, evaporated to the desired viscosity and then applied to backing material to provide a patch. [00113] In some embodiments, the compounds of the invention are provided in unit dose form such as a tablet, capsule or single-dose injection or infusion vial.

Combination Therapy

[00114] According to the methods of the invention, a compound of the invention can be administered to a subject either as a single agent, or in combination with one or more other agents. In one embodiment, a compound of the invention is administered to a subject as a single agent. In one embodiment, a compound of the invention is administered to a subject alone. In one embodiment, a compound of the invention is administered to a subject in combination with one or more other agents.

[00115] In certain embodiments, a compound of the invention may be used in combination with other agents that are used for the treatment or prevention of cancer. In certain embodiments, a compound of the invention may be used in combination with other agents that are not used for the treatment or prevention of cancer. In one embodiment, the cancer is breast cancer. In another embodiment, the cancer is colon cancer. In yet another

embodiment, the cancer is liver cancer.

[00116] In one embodiment, a compound of the invention may be delivered to a subject as part of the same pharmaceutical composition or formulation containing one or more additional active agents. In another embodiment, a compound of the invention may be delivered to a subject in a composition or formulation containing only that active agent, while one or more other agents are administered to the subject in one or more separate

compositions or formulations. In one embodiment, the other agents are not used for the treatment or prevention of cancer. In another embodiment, the other agents are used for the treatment or prevention of cancer. In one embodiment, the cancer is breast cancer. In another embodiment, the cancer is colon cancer. In yet another embodiment, the cancer is liver cancer.

[00117] A compound of the invention and the other agents that are used for the treatment or prevention of cancer may be administered to the subject at the same time, or at different times. A compound of the invention and the other agents that are not used for the treatment or prevention of cancer may be administered to the subject at the same time, or at different times. For example, a compound of the invention and the other agents may be administered within minutes, hours, days, weeks, or months of each other, for example as part of the overall treatment regimen of a subject. In some embodiments, a compound of the invention may be administered prior to the administration of other agents. In other embodiments, a compound of the invention may be administered subsequent to the administration of other agents.

[00118] A compound of the invention may also be used in combination with known therapies for cancer, such as, but not limited to, chemotherapy, radiation therapy, hormone therapy, and targeted therapy. Such targeted therapy includes, but is not limited to, the use of monoclonal antibodies and small molecules. Example therapies include, but are not limited to, Gleevec® (imatinib mesylate), Iressa® (gefitinib), Sutent® (sunitinib, Velcade®

(bortezomib), Campath® (alemtuzumab), Erbitux® (cetuximab), Rituxan® (rituximab), Herceptin® (trastuzumab), Avastin® (bevacizumab), Thalomid® (thalidomide) and

Revlimid® (lenalidomide). Other example therapies include aromatase inhibitors, such as anastrozole (Arimidex®), exemestane (Aromasin®), and letrozole (Femara®), as well as progestins, such as megestrol acetate (Megace®), anti-estrogens, such as fulvestrant

(Faslodex®), tamoxifen, and toremifene (Fareston®), as well as compounds such as 5- fluorouracil (5-FU), and Doxorubicin (Adriamycin®).

[00119] Compounds of the invention, as described above, including, but not limited to, compounds isolated from the endemic Jamaican Castela macrophylla plant, such as quassinoids and coumarins, may be used in combination with each other for the treatment or prevention of cancer. In one embodiment, the cancer is breast cancer. In another

embodiment, the cancer is colon cancer. In yet another embodiment, the cancer is liver cancer.

[00120] In some embodiments, the administration of a compound of the invention in combination with one or more other agents has an additive effect, in comparison with administration of the compound of the invention alone, or administration of the one or more other agents alone. In other embodiments, the administration of a compound of the invention in combination with one or more other agents has a synergistic effect, in comparison with administration of the compound of the invention alone, or administration of the one or more other agents alone. In some embodiments, the administration of a compound of the invention in combination with one or more other agents can help reduce side effects, in comparison with administration of the compound of the invention alone, or administration of the one or more other agents alone. [00121] In some embodiments, the compound of the invention is used as an adjuvant therapy. In other embodiments, the compound of the invention is used in combination with an adjuvant therapy.

Subjects

[00122] According to the methods of the invention, the subject or patient can be any animal that has or is diagnosed with cancer. According to the methods of the invention, the subject or patient can be any animal at risk of developing cancer. In one embodiment, the cancer is breast cancer. In another embodiment, the cancer is colon cancer. In ey another

embodiment, the cancer is liver cancer.

[00123] According to the methods of the invention, the subject or patient can be any animal that is predisposed to or is at risk of developing cancer. In preferred embodiments, the subject is a human subject. In some embodiments, the subject is a rodent, such as a mouse. In some embodiments, the subject is a cow, pig, sheep, goat, cat, horse, dog, and/or any other species of animal used as livestock or kept as pets.

[00124] In some embodiments, the subject has not been diagnosed with cancer. In some embodiments, the subject is already suspected to have cancer. In other embodiments, the subject is being treated for cancer, before being treated according to the methods of the invention. In other embodiments, the subject is not being treated for cancer, before being treated according to the methods of the invention.

EXAMPLES

[00125] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

[00126] Example 1: Glaucarubolone glucopyranoside, a natural quassinoid exhibits potent impact on breast cancer cell viability and inhibit pro-carcinogen bioactivating CYP enzymes in vitro

[00127] Quassinoids and coumarins have been associated with anti-inflammatory, antioxidant and anti-proliferative activity in the past. Emerging evidence indicates that they may also act as chemopreventive agents. In this study, the potential for new quassinoids and a coumarin isolated from the endemic Jamaican Castela macrophylla plant to exhibit antiproliferative activity was investigated using three cancerous and one normal cell lines and to inhibit cytochrome P450s (CYPs) known to convert polyaromatic hydrocarbons (PAHs) into carcinogenic metabolites. The quassinoid, glaucarubolone- 15-0-P-D-glucopyranoside (Gg) displayed dose and time dependant reduction on the viability of breast cancer MCF-7 cells with greater potency than tamoxifen and induced cell death significant at 1 μΜ with 24 hour exposure. In addition, Gg inhibited the activities of CYPslAl and 1B1 enzymes with Kis 7.1 and 5.6 μΜ, both according to non-competitive kinetics and comparison with structural analogs identifies the importance of the glycoside side chain. Gg also attenuated at nano molar values the ability of PAH benzo[a]pyrene to induce CYPIA gene expression in MCF-7 cells. Taken together, these data indicate that improved chemotherapeutic and

chemopreventive regimes can be achieved with the inclusion of the plant isolate Gg.

[00128] Introduction

[00129] Castela turpin (Simaroubaceae) has been reported to accumulate quassinoids (1, 2) and of the 15 species found in the tropical Americas (3) only Castela macrophylla is endemic to Jamaica (4). Although quassinoids have been reported to display many biological activities, isolates specifically from Castela macrophylla are limited in their pharmacological applications to anti-feedant activities against tobacco budworm, Plasmodium falciparum and Plamopara viticola (5). [00130] The biological activities of three quassinoids and a coumarin isolated from Castela macrophylla were evaluated for their potential as anti-cancer and potential chemopreventive properties. The cytotoxicity of four isolated compounds was evaluated against three cancerous cell lines and one normal cell line. The capacity of quassinoids to inhibit CYP enzyme activity, particularly in light of the available generalized active site model for the quassinoid bound to CYP1A1 (13), was also evaluated. Inhibitors of CYP1 enzymes are associated with potential chemopreventive properties (14, 15, 16).

[00131] Materials and Methods

[00132] Chemicals & Reagents

[00133] Benzo-a-pyrene (BaP) and all chemicals for the MTS and CYP inhibition assays were purchased from Sigma-Aldrich (St.Louis, MO). Dimethylsulfoxide (DMSO) was purchased from American Type Culture Collection (ATCC, Manassas, VA, US), 7-Amino- actinomycin D (7AAD) was purchased from eBioscience (San Diego, CA) and Annexin V- FITC was purchased from Pharmingen (San Diego, CA). All CYP substrates and metabolites were purchased from Gentest Corporation (Worburn, MA, USA). All cell lines along with their respective media and supplements were purchased from ATCC (Manassas, VA, USA). Escherichia coli membranes expressing human CYP1A1, CYP1A2, CYP1B1, CYP2D6, CYP3A4 and CYP2C19 co-expressed with CYP reductase were purchased from Cypex Ltd. (Dundee, U.K).

[00134] Plant isolates

[00135] Castela macrophylla (Simaroubaceae) was collected in Hellshire Hills, St.

Catherine, Jamaica, in June 2003 and a voucher specimen, (#UWI-Mona 34, 982) was deposited in the Herbarium at the University of the West Indies, Mona, Jamaica. The leaves, twigs and thorns were air-dried, chopped and exhaustively extracted with hexane, acetone and methanol. The resulting residues were subjected to column chromatography on silica gel. Elution with various concentrations (ranging between 5-100% acetone -hexane) and recrystallization yielded pure compounds (-)-glaucarubolone, glaucarubolone-15-0-P-D- glucopyranoside (Gg) , (-)-holocanthone and scopoletin as described elsewhere (2).

[00136] Cell Culture and cytotoxicity assays [00137] Cells (CCD 18 Co normal colon, HepG2 hepatoma and MCF-7 breast carcinoma) were maintained in ATCC-formulated Eagle's Minimum Essential Medium and HT29 colon carcinoma cells were maintained in McCoy's 5a Medium Modified supplemented with 10% foetal bovine serum (Atlas; Fort Collins CO), lOmM HEPES solution, lOOmM L-glutamine penicillin streptomycin solution, 3 g/L glucose, and 1.5 g/L of sodium bicarbonate. For the apoptosis studies and certain cytotoxicity assays, MCF-7 cells were maintained in RPMI medium supplemented with 10% FBS and penicillin and streptomycin antibiotics. Cells were maintained at 37°C with 5% C0₂ in Corning 75cm² culture flasks. Cells were exposed to a given isolate or known anticancer agent for 24 h. Following the appropriate treatments, cell proliferation was evaluated using an MTS assay according to the manufacturer's instructions (30). All assays were performed at least three times and their O.D. measured at 590 nm (31). Cell viability was measured as a percentage of the controls containing respective solvents. In some experiments, MCF-7 plated in 96 well plates were exposed to tamoxifen or Gg at varying concentrations and exposure times (24-72 h) before being analyzed for cytotoxicity using the Alamar Blue™ Assay as described in detail elsewhere (32).

[00138] AnnexinV-7AAD apoptosis assay

[00139] The ability of Gg to induce apoptosis was investigated in MCF-7 cells using a protocol similar to what has been previously described in detail elsewhere (32, 33). Briefly, cells were exposed to varying concentrations of Gg for 24 h. Following treatment, cells were washed with cold PBS, harvested and counted and aliquots placed in 96 well plates. Cells were then resuspended in assay buffer and stained with Annexin-V(which stains for cell in early apoptosis and distinguishes them from those that are viable) and 7-amino-actinomycin D (7AAD; which stains for necrotic or late apoptotic cells). Cells were analyzed using flow cytometry soon after addition of 7AAD to preserve sufficient cell integrity.

[00140] CYP inhibition assays

[00141] The test compounds were evaluated for their ability to inhibit the catalytic activity of human CYP1 enzymes by means of high throughput fluorometric inhibition assays conducted in 96 well microtitre plates as described elsewhere (34). 7 ethoxyresorufin (ERes) was used as a substrate for detecting activities of CYP IB 1 and 7-ethoxy-3-cyanocoumarin (CEC) was used as a substrate for both CYPs 1 Al and 1 A2. Further, the substrates,3-[2- (N,N-Diethyl-N-methylamino)ethyl]-7-methoxy-4methylcoumarin (AMMC), 7-Benzyloxy- 4-trifluoromethylcoumarin (BFC), and CEC were used as substrates for CYPs 2D6, 3A4 and 2C19 respectively. The reactions were monitored fluorometrically at 37°C, using a Varian Cary Eclipse Fluorescence spectrophotometer. All inhibitors were dissolved in a solvent of 20% acetonitrile in water and less than 0.3% of acetonitrile was used in the final assay.

[00142] RNA Extraction & Quantitative RT-PCR Analysis

[00143] Total RNA was isolated from MCF-7 breast cancer cells treated with media containing 0.02 % DMSO, 2 μΜ BaP or BaP in combination with 10, 50 or 100 nM Gg for 24 h using the Aurum™ Total RNA Mini kit (Bio-Rad, Hercules, CA). The quantity of the RNA extracts was determined by measuring the absorbance at 260 nm and 280 nm using a Beckman DU 800 Spectrophotometer (Beckman, Brea, CA). RNA quality was further analyzed, and RNA quality indicator (RQI) values were determined using the Experion Automated Electrophoresis system (Bio-Rad, Hercules, CA). cDNA was prepared from 1 μg of total RNA from MCF-7 breast cancer cells to measure CYP1A1 or CYP1A2 expression. RNA was extracted using the iScript^® cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's instruction. The cDNA was used as a template for real time quantitative PCR analysis using the CFX-96 PCR instrument (Bio-Rad, Hercules, CA). The primers for the reference genes GAPDH and GUSB as well as CYP1A1 and CYP1A2 genes were obtained from SA Biosciences (Frederick, MD). The PCR reactions were set up in accordance with the manufacturer's recommendations. Relative fold-changes in gene expression were calculated using the ^AACT method.

[00144] Immunoblotting

[00145] MCF-7 cells were exposed to media containing 0.02 % DMSO, 2 μΜ Benzo-a- pyrene or Benzo-a-pyrene in combination with 10, 50 or 100 nM Gg for 24 h before cells were harvested and pellets lysed. Protein content was evaluated using the BCA assay as previously described (32). Proteins (25 μg) were resolved by SDS-PAGE and transferred to PVDF membranes probed with specific antibodies against CYPl Al and CYPl A2 (1 :500 and 1 :250 respectively, Santa Cruz Biotechnology) followed by anti-rabbit or anti-mouse alkaline phosphatase-conjugated IgG according to the Western Breeze protocol. Blots were developed using the enhanced chemiluminescence method with actin serving as the loading control.

[00146] Data & Statistical analysis [00147] IC₅₀ and K values were determined by fitting the data in Sigma Plot (version 10.0) and enzyme kinetics module, using non linear regression analysis. The apparent values were determined on the basis of visual inspection of Eadie Hofstee and various statistics to evaluate goodness of fit, such as the size of the sum of squares of residuals, Akaike information criterion, and standard error (Enzyme kinetics module, version 1.3). The data listed represent the average values from three different determinations. Statistical significance between three or more groups was determined using one-way AN OVA with the Tukey- Kramer multiple comparison test. Statistical analysis was performed using GraphPad Prism 4.0 Graph Pad Software, Inc. San Diego, California, USA, www.graphpad.com.

[00148] Results

[00149] In vitro cvtoxicity of Castela macrophylla plant isolates

[00150] Three quassinoids and a coumarin illustrated in Figure 1, isolated from Castela macrophylla plant were investigated for their bioactivity value in this study. The impact of these compounds on cell viability was assessed using a panel of cell lines including colon normal (CCDI8C0), colon cancer (HT29), liver cancer (HepG2) and breast cancer (MCF-7) cells using the MTS assay (Figure 2). IC₅₀ values (that concentration needed to inhibit 50% cell viability) were calculated for all test isolates and known drug entities, tamoxifen and 5- fluorouracil (which served as positive controls, Table 1).

Table 1. IC₅₀ values (μΜ) obtained from the interaction of isolates from the Castela macrophylla with various cancer and normal cell lines along with positive controls.

Compound Cell Lines

CCD 18 Co HT29 HepG MCF-7

(-)- NI NI NI NI

Glaucarubolone

(-)-Holocanthone <5 9.91 ± 0.52 >40 1 1.031 ± 1.13

(-)- 40.14 ± 3.41 NI >50 8.65± 1.1 1

Glaucarubolone

glucoside

Scopoletin NI 19.28 ± 0.348 NI NI

Tamoxifen NA NA NA 17.28 ± 0.06

Fluorouracil 55.51 ± 3.71 23.50 ± 1.12 ND ND

Doxorubicin NA NA 18.61± 0.58 NA

Key: NI: No inhibition (<10% inhibition at 60μΜ), NA: Not applicable, ND: Not determined. [00151] Scopoletin potently and selectively reduced the viability of HT29 colon cancer cells. Its IC₅₀ value of 19.28 ± 0.34 μΜ compared well with that of 5-fluorouracil (23.50 ±

1.12μΜ), yet this isolate did not affect the viability of normal colon cells (<10% inhibition at 60μΜ) in contrast to 5-fluorouracil which displayed cytotoxicity with an IC₅₀ of 55.51 ± 3.71μΜ (Table 1).

[00152] Gg and holocanthone impacted the viability of MCF-7 cells, at potency greater than that observed with the breast cancer agent tamoxifen (IC₅₀s, 8.65 ± 1.11 and 11.03 ± 1.13 compared to 17.28 ± 0.06μΜ respectively). Microscopic evaluation of breast cancer cells in the presence of Gg or tamoxifen at 5 (B&C), 15 (D&E), 40 (F&G) and 60μΜ (H&I) respectively illustrates comparable impact on MCF7 cells by Gg and tamoxifen at 60μΜ although impact by Gg starts at a lower concentration (at 40μΜ).

[00153] Holocanthone inhibited colon cancer cells more potently than the known colon cancer therapeutic 5-fluorouracil (IC₅₀S 9.91 ± 0.52 compared to 23.50 ± 1.12μΜ

respectively), although it also largely affected normal colon cell viability (IC₅₀ <5μΜ).

[00154] None of the three quassinoids demonstrated noticeable impact on liver cancer cells while glaucarubolone had no impact on all 4 cell lines evaluated.

[00155] Gg-mediated impact and apoptosis in MCF-7 cells

[00156] To determine whether the cytotoxicity of Gg increased in a time-dependent fashion, MCF-7 cells were exposed to Gg in comparison to Tamoxifen for 24, 48 or 72 h with varying concentrations and evaluated cell viability using the Alamar Blue assay. It was found that Gg induced a time- and dose-dependent decrease in cell viability which was greater than that observed with tamoxifen (Figure 2).

[00157] Whether Gg was able to induce apoptosis in MCF-7 breast cancer cells was evaluated, since the cells showed substantial sensitivity to this quassinoid. Using the

Annexin V-7AAD assay, a dose-dependent increase in early apoptosis was detected in MCF- 7 cells following 24 h treatment with Gg (Figure 2) which was significant at 1 μΜ (26.5 ± 9.5%). Interestingly, this concentration is more than 8-fold lower than the IC₅₀ value.

Apoptosis was also apparent after 48 h of Gg treatment.

[00158] Inhibition of cytochrome P450 (CYP) enzyme activity imparted by Gg [00159] Given the involvement of CYP1 enzymes in procarcinogen activation, the inhibitory impact of Gg on the activities of these enzymes was evaluated using fluorogenic probe substrates (Figures 3 A, C and Table 2). Gg moderately inhibited the activities of CYPslAl and IB 1 (IC_50, 6.93 ± 0.31 μΜ and 9.17 ± 0.91 μΜ respectively). Further kinetic

characterisations (Figure 3C) using Eadie-Hofstee plots yielded non-competitive inhibition of CYPslAl and IBl with IQs of 7.1 ± 1.44 and 5.6 ± 0.66μΜ respectively. Characterization of the interaction of Gg with other CYP enzymes, summarized in Table 2 reveals that while the inhibitions against CYPs 2C19 (IC₅₀, 19.40 ± 1.1 ΙμΜ) and 2D6 (IC_50, 18.02 ± 0.51μΜ) were weak (>10μΜ) for Gg, it appears to show moderate potency against the activity of CYP3A4 (IC₅₀, 1.31 ± 0.49μΜ).

Table 2. IC₅₀ values (μΜ) obtained from the interaction of isolates from the plant Castela macrophylla with CYP enzymes

Compound CYP Isoforms

1A1 1A2 IB l 2C19 2D6 3A4

(-)-Glaucarubolone *ND *ND *ND *ND *ND *ND (-)-Holocanthone *ND *ND *ND *ND *ND *ND

(-)-Glaucarubolone 6.93 ± *ND 9.17 ± 19.40 ± 18.02 ± 1.31 ± glucoside 0.31 0.91 1.1 1 0.51 0.49

Key *ND: Not determined due to quenching of metabolite fluorescence or instrinsic fluorescence

[00160] The intrinsic fluorescence of glaucarubolone glucoside (> 20% of control) at the wavelength used for detecting CYP1A2 activity prevented accurate IC₅₀ calculation while control experiments on holocanthone and glaucarubolone indicated that they severely quenched the fluorescence of the formed metabolite for all substrates used in CYP1 activity determination.

[00161] To verify the accuracy of experimental techniques employed to detect CYP inhibition, assays with known inhibitors were carried out, with furafylline (against CYP1A2), ketoconazole (against CYPslAl, 1B1 and 3A4), (-)-N-3-Benzyl-phenobarbital (NBPB, against CYP2C19) and quinidine (against CYP2D6) and the obtained IC₅₀ values (0.8 ± 0.2, 0.04 ± 0.01, 6.3 ± 1.7, 0.06 ± 0.01, 0.3 ± 0.01, 0.03 ± 0.0 ΙμΜ respectively) compared well with published values (0.99 <10, <10, 0.06, 0.25 and 0.04μΜ respectively (17, 18, 19, 20). Michaelis constant, Km, was determined for each marker substrate under the specified experimental conditions, in order to determine suitable substrate concentrations for assessing inhibitory potential of test compounds.

[00162] Effect of Gg on BenzoTalpyrene-induced CYP1A mRNA and protein expression

[00163] Substances that directly inhibit the activity of cytochrome P450s often also inhibit the ability of polyaromatic hydrocarbons (PAHs) to induce mRNA and protein expression of these enzymes. One such PAH that is known to convert into carcinogenic metabolites is Benzo[a]pyrene (BaP). As such, MCF-7 breast cancer cells were exposed to BaP in the presence or absence of varying concentrations of Gg for 24 h and then analyzed CYPlAl and CYP1A2 mRNA and protein expression for. Figure 4 shows that BaP alone induced a robust 70-fold increase in expression relative to untreated controls. Gg induced a dose-dependent inhibition of BaP-induced CYPlAl mRNA expression and sustained inhibition of CYP1A2 mRNA expression. In contrast, Gg caused a slight but insignificant decrease in Gg-induced CYPlAl protein expression and did not decrease Gg-induced CYP1A2 protein expression in MCF-7 cells (Figure 4).

[00164] Discussion

[00165] Two quassinoids, glaucarubolone glycoside (Gg) and holocanthone, and a coumarin (scopoletin) have been shown to display potent anti-proliferative properties comparable to or more potent than, known chemotherapeutic drugs. Further, the coumarin has the added benefit of displaying no toxicity to normal colon cells in contrast to the drug 5-fluorouracil. Many side effects of chemotherapy can be attributed to the destruction of normal cells (21) and the apparent absence of such on the examined normal colon cells highlights scopoletin as a candidate suitable for further experimentation.

[00166] Gluacarubolone did not impact cell viability of any of the cancer cells lines, although its structural isomers, Gg and holocanthone did. It appears then that the conjugation (glycosylation or acetylation) is critical for bioactivity, and can be due to the enhanced cell membrane permeability that the conjugation allows. [00167] Exhibiting some degree of specificity amongst the cancer cells, Gg was twice more potent towards breast cancer cells than tamoxifen but approximately three times weaker towards liver cancer cells than doxorubicin and showed no activity against colon cancer cells. As expected, increasing the duration of exposure of MCF-7 cells to Gg resulted in a decrease in cell viability. Cytotoxicity imparted on the breast cancer cells appear to be induced by apoptosis, a mechanism also observed by tamoxifen (22). Gg (Ι μΜ, 24h) induced significant early apoptosis in MCF-7 breast cancer cells at a concentration more than 8-fold lower than the IC₅₀ for this isolate. The clinically available anti-breast cancer agent Tamoxifen (ΙμΜ, 24h) has previously been shown to induce apoptosis in MCF-7 cells (23) though it is considerably less potent than Gg in these cells. Gg appeared to exhibit selective cytotoxicity towards the MCF-7 breast cancer cell line, which indicates its potential use in the prevention or treatment of breast cancer. Gg also showed weak inhibition of CYP2D6, and since this enzyme plays a key role in the metabolism of Tamoxifen (24), combination therapy with the pharmaceutical will have little risk of metabolism based drug interactions. Although most warnings for drug interactions for Tamoxifen users are against CYP2D6 inhibitors, one does have to bear in mind that CYP3 A4 is also involved in its metabolism. The propensity for Gg to cause moderate inhibition of this enzyme indicates that care be exercised if it is used in combination with Tamoxifen.

[00168] Holocanthone affected both colon and breast cancer cells more potently than their respective chemotherapeutic drugs, 5-Fluorouracil and Tamoxifen. However its activity against liver cancer cells was weaker than the known chemotherapeutic drug Doxorubicin. 5- fluorouracil is usually used in combination with other drugs such as Leucovorin (25) when treating colon cancer and its treatment is not confined to colon cancer but encompasses breast, pancreas and cervical cancer among others. Similarly, holocanthone 's broad cytotoxicity against several cell lines may make it useful as a broad spectrum anticancer drug candidate. Its ability to inhibit colon cancer cells to a greater extent than 5-fluorouracil (10 compared to 23μΜ), warrants further investigations. Though derivatives of holocanthone far more selective for malignant compared to non-malignant cells can serve as more suitable anticancer agents.

[00169] The quilinolones examined in this study showed no activity towards any of the cells examined, in contrast to the known quilinolone, 5-fluorouracil. The latter's possession of a fluorine moiety may be central to its bioactivity. Research efforts are aimed at modifying the structure of 5-Fluorouracil to counteract the emergence of chemoresistance to this drug. Successful modifications can ultimately result in derivatives that can help increase patient survival which is currently as low as 10% in certain cancer types.

[00170] Inhibitors of CYPl enzymes have been accepted as chemo-protectors and several natural compounds such as flavonoids including resveratrol (14, 26, 27) and organosulfur compounds have been categorised as such. Previous work (13, 28) described an active site model for quassinoid bound to CYP1A1, identifying a key residue required for effective interactions with the enzyme. The generalised model identified a hydrogen bond donor in position 1 which can aid binding with the backbone carbonyl of Asp313. The oxygens on rings C (2) and A (3) can bind with the sidechains of Thrl 11 and Serl24 respectively, and hydrophobic groups, possibly on ring positions 4 and 5 can facilitate interactions with Phel23, Ile386 and Leu496. Without being bound by theory, glaucarubolone can interact competitively with CYP1A1. Glaucarubolone has a glucoside moiety at position 1 replacing the hydroxyl group found in quassin, and holoconthone has an acetyl group preventing them from acting as a hydrogen bond donors. Without being bound by theory, both these compounds are unlikely to interact with the active site of CYPl Al . Indeed, this is confirmed by the non-competitive binding kinetics displayed by Gg in Figure 2.

[00171] Non competitive inhibitors generally follow the relation IC50=Ki , and in the studies herein, the obtained IC50 value for Gg on CYPslAl and 1B1 yielded similar Ki values further confirming the graphical representation and computer assisted generations using Eadie Hoftsee plots.

[00172] Inhibitors of CYPl enzymes frequently inhibit the mRNA and protein expression of CYPl enzymes in cells that are induced by PAHs. The data herein indicate that Gg inhibited BaP-induced CYP1A1 and CYP1A2 mRNA expression in MCF-7 cells. Gg alone did not appreciably affect the endogenous mRNA expression of any of the CYPl enzymes within the breast cancer cells. Gg also demonstrated low cytoxicity with normal cells which is an added benefit for lead compounds search as chemopreventors. It was found that Gg was unable to significantly inhibit BaP-induced CYPl A protein expression. Though substances exist that inhibit the CYP enzyme activity yet fail to inhibit CYP protein expression induced by a pro- carcinogen (35), without being bound by theory, the nanomolar concentrations of Gg were insufficient to inhibit BaP-induced protein expression. In addition, it is likely that longer durations of co-exposure are required to inhibit BaP-induced protein expression levels. [00173] BaP, like many other PAHs, binds with high affinity to the cytosolic aryl hydrocarbon receptor (AHR) which then complexes with the related AHR nuclear translocator (ARNT) protein in the nucleus, commencing the transcriptional activation of CYP1A1 and CYP1A2. AHR undergoes post-translational modification for optimal transactivation potential, and whether Gg inhibits the kinases that phosporylates the AHR, like some have postulated polyphenols to do (36), is yet to be determined. What is clear though is that BaP induction of CYP1A gene expression is reduced in the presence of Gg. Such reductions in CYP1A coupled with direct inhibition of the enzymes' activities in the presence of Gg can be expected to decrease the levels of carcinogenic metabolites formed through CYP1A activity.

[00174] Three isolates examined in this study demonstrated potent anticancer activity greater than or equal to clinically available chemotherapeutic agents. Scopoletin, a coumarin isolate from Castela macrophylla showed high potency against colon cancer cells with minimal impact on normal cells, making it a candidate worth further investigations. The quassinoids, glucarabulone glucoside and holocanthone also from the same plant showed growth inhibitory potency against breast cancer cells. Gg displayed dose and time dependant impact on MCF-7 cells, caused apoptosis in these cells at low micromolar values and further impacted the carcinogen activating enzymes, CYPslAl and 1B1 with non competitive kinetics. BaP induction of CYP1A1 mRNA was lowered in the presence of Gg and thus chemopreventive and chemotherapeutic potential are implied by the bioactivity displayed by these compounds, and validate the on-going search for natural products from endemic tropical biodiversity.

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[00176] Example 2: Quassinoid natural product, glaucarubulone glycoside limits damaging effects of benzo[a]pyrene in-vitro: implications for chemoprevention

[00177] Quassinoids and coumarins have been associated with anti-inflammatory, antioxidant and anti-proliferative activity. Emerging evidence indicates that quassinoids and coumarins may also act as chemopreventive agents. In this study, the potential for new quassinoids and a coumarin isolated from the endemic Jamaican Castela macrophylla plant to inhibit cytochrome P450s (CYPs), in particular CYPslAl and 1B1 enzymes, known to convert polyaromatic hydrocarbons (PAHs) into carcinogenic metabolites, was investigated. The quassinoid glaucarubulone- 15-0-P-D-glucopyranoside (Gg) inhibited the activity of CYPslAl and 1B1 enzymes (IC₅₀, 6.93 ± 0.31μΜ and 9.17 ± 0.91μΜ respectively);

according to non-competitive kinetics. Gg also attenuated (by a near 70 fold) the ability of PAH benzo-a-pyrene to induce CYPIA gene expression in MCF-7 cells at a nanomolar level. Gg suppressed the ability of benzo-a-pyrene metabolite benzo-a-pyrene- 1,6-quinone to increase reactive oxygen species in non-malignant MCF-IOA breast epithelial cells. Finally, Gg reduced viability of MCF-7 cells with greater potency than tamoxifen (IC₅₀S, 8.65 ± 1.11 compared to 17.28 ± 0.06μΜ) and induced MCF-7 breast cancer cell death in a time and dose dependant manner. Taken together, these data indicate that improved chemotherapeutic and chemopreventive regimes can be achieved with the inclusion of the plant isolate Gg.

[00178] Introduction

[00179] Exposure to poly cyclic aromatic hydrocarbons (PAHs) is hypothesized as being a risk factor in developing breast cancer following evidence in rodents (1). Benzo(a)pyrene (BaP) is a widespread PAH environmental contaminant formed as by-products of combustion and found in chargrilled meats for example. BaP is activated via oxidative metabolism catalysed by the cytochrome P450 (CYP) enzyme family and microsomal epoxide hydrolase to form the carcinogenic metabolites. The ultimate carcinogen, 7,8-dihydroxy-9,10-epoxy- 7,8,9, 10-tetrahydrobenzo(a)pyrene (BPDE) which is formed following CYPslAl and 1B1 catalysis has been found to form stable adducts with DNA and detected in human breast tumours. BaP induces CYP1 enzymes, thereby enhancing its own metabolism. Additionally, BaP-quinones (BPQs), important metabolites of BaP have been linked with the production of reactive oxygen species and shown to activate epidermal growth factor receptors in mammary epithelial cells thus acting as a tumor promoter (2). Compounds capable of inhibiting the generation of BaP metabolites or their damaging effects can thereby carry chemopreventive worth. Natural compounds found in the tropical plant species Castela macrophylla were investigated for such a worth.

[00180] Castela turpin (Simaroubaceae) has been reported to accumulate quassinoids (3, 4) and of the 15 species found in the tropical Americas (5) only Castela macrophylla is endemic to Jamaica (6). Although quassinoids have been reported to display many biological activities (7), isolates specifically from Castela macrophylla are limited in their

Plasmodium falciparum and Plasmoparavitico la (8).

[00181] The capacity of three quassinoids and a coumarin isolated from Castela

macrophylla to inhibit the activity of CYP enzymes, particularly in light of the available generalized active site model for the quassinoid bound to CYP1A1 (9), and potential chemopreventive properties (10-12) associated with such inhibitors, was evaluated. The most potent compound's impact on BaP effect on CYP1 induction, and the generation of ROS by BPQs, were also further evaluated. The cytotoxicity of these quassinoid compounds against three cancerous cell lines including breast cancer cells and one normal cell line, were also evaluated, and the chemopreventing and anticancer potential of one of these quassinoid natural products were highlighted.

[00182] Materials and Methods

[00183] Chemicals & Reagents

[00184] Benzo-a-pyrene(BaP), benzo-a-pyrene-l,6-quinone (1,6-BPQ) and all chemicals for the MTS and CYP inhibition assays were purchased from Sigma-Aldrich (St. Louis, MO). Dimethylsulfoxide (DMSO) was purchased from American Type Culture Collection (ATCC, Manassas, VA, US), 7-Amino-actinomycin D (7AAD) was purchased from eBioscience (San Diego, CA) and Annexin V-FITC was purchased from Pharmingen (San Diego, CA). All CYP substrates and metabolites were purchased from Gentest Corporation (Worburn, MA, USA). All cell lines along with their respective media and supplements were purchased from ATCC (Manassas, VA, USA). ATCC certifies that the cells were authenticated prior to shipment using short tandem repeat profiling to verify that they are of the correct lineage and uncontaminated with other cell types. In addition, the cells were routinely screened to ensure they were free from mycoplasma contamination and other types of microorganisms.

Escherichia coli membranes expressing human CYP1A1, CYP1A2, CYP1B1, CYP2D6, CYP3A4 and CYP2C19 co-expressed with CYP reductase were purchased from Cypex Ltd. (Dundee, U.K).

[00185] Plant isolates

[00186] Castela macrophylla (Simaroubaceae) was collected in Hellshire Hills, St.

Catherine, Jamaica, in June 2003 and a voucher specimen, (#UWI-Mona 34, 982) was deposited in the Herbarium at the University of the West Indies, Mona, Jamaica. The leaves, twigs and thorns were air-dried, chopped and exhaustively extracted with hexane, acetone and methanol. The resulting residues were subjected to column chromatography on silica gel. Elution with various concentrations (ranging between 5-100% acetone -hexane) and recrystallization yielded pure compounds (-)-glaucarubolone, glaucarbulone-15-0-P-D- glucopyranoside (Gg), (-)-holocanthone and scopoletin as described elsewhere (4).

[00187] Cell Culture and cytotoxicity assays

[00188] Cells (CCD 18 Co normal colon, HepG2 hepatoma and MCF-7 breast carcinoma) were maintained in ATCC-formulated Eagle's Minimum Essential Medium and HT29 colon carcinoma cells were maintained in McCoy's 5a Medium Modified supplemented with 10% foetal bovine serum (Atlas; Fort Collins CO), lOmM HEPES solution, lOOmM L-glutamine penicillin streptomycin solution, 3 g/L glucose, and 1.5 g/L of sodium bicarbonate. For the apoptosis studies and certain cytotoxicity assays, MCF-7 cells were maintained in RPMI medium supplemented with 10% FBS and penicillin and streptomycin antibiotics. Cells were maintained at 37°C with 5% C0₂ in Corning 75cm² culture flasks. Cells were exposed to a given isolate or known anticancer agent for 24 h. Following the appropriate treatments, cell proliferation was evaluated using an MTS assay according to the manufacturer's instructions (13). All assays were performed at least three times and their O.D. measured at 590 nm (14). Cell viability was measured as a percentage of the controls containing respective solvents. In some experiments, MCF-7 plated in 96 well plates were exposed to tamoxifen or Gg at varying concentrations and exposure times (24-72 h) before being analyzed for cytotoxicity using the Alamar Blue™ Assay as described in detail elsewhere (14, 15).

[00189] AnnexinV-7AAD apoptosis assay

[00190] The ability of Gg to induce apoptosis in MCF-7 cells was investigated similarly to a method previously described in detail elsewhere (15, 16). Briefly, cells were exposed to varying concentrations of Gg for 24 h. Following treatment, cells were washed with cold PBS, harvested and counted and aliquots placed in 96 well plates. Cells were then re- suspended in assay buffer and stained with Annexin-V(which stains for cell in early apoptosis and distinguishes them from those that are viable) and 7-amino-actinomycin D (7AAD;

which stains for necrotic or late apoptotic cells). Cells were analyzed using flow cytometry soon after addition of 7AAD to preserve sufficient cell integrity.

[00191] CYP inhibition assays

[00192] The test compounds were evaluated for their ability to inhibit the catalytic activity of human CYP1 enzymes by means of high throughput fluorometric inhibition assays conducted in 96 well microtitre plates as described elsewhere (17). 7 ethoxyresorufin (ERes) was used as a substrate for detecting activities of CYP IB 1 and 7-ethoxy-3-cyanocoumarin (CEC) was used as a substrate for both CYPs 1 Al and 1 A2. Further, the substrates 3-[2- (N,N-Diethyl-N-methylamino)ethyl]-7-methoxy-4methylcoumarin (AMMC), 7-Benzyloxy- 4-trifluoromethylcoumarin (BFC), and CEC were used as substrates for CYPs 2D6, 3A4 and 2C19 respectively. The reactions were monitored fluorometrically at 37°C, using a Varian Cary Eclipse Fluorescence spectrophotometer. All inhibitors were dissolved in a solvent of 20% acetonitrile in water and less than 0.3% of acetonitrile was used in the final assay.

[00193] RNA Extraction & Quantitative RT-PCR Analysis

[00194] Total RNA was isolated from MCF-7 breast cancer cells treated with media containing 0.02 % DMSO, 2 μΜ BaP or BaP in combination with 10, 50 or 100 nMGg for 24 h using the Aurum™ Total RNA Mini kit (Bio-Rad, Hercules, CA). The quantity of the RNA extracts was determined by measuring the absorbance at260 nm and 280 nm using a

Beckman DU 800 Spectrophotometer (Beckman, Brea, CA). RNA quality was further analyzed, and RNA quality indicator (RQI) values were determined using the Experion Automated Electrophoresis system (Bio-Rad, Hercules, CA). cDNA was prepared from 1 μg of total RNA from MCF-7 breast cancer cells to measure CYP1A1 or CYP1A2 expression. RNA was extracted using the iScript®cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's instruction. The cDNA was used as a template for real time quantitative PCRanalysis using the CFX-96 PCR instrument (Bio-Rad, Hercules, CA). The primers for the reference genes GAPDH and GUSB as well as CYP1A1 and CYP1A2 genes were obtained from SA Biosciences (Frederick, MD). The PCR reactions were set up in accordance with the manufacturer's recommendations. Relative fold-changes in gene expression were calculated using the ^AACT method.

[00195] Reactive oxygen species (ROS) analysis

[00196] Intracellular ROS levels were measured in MCF-IOA breast epithelial cells exposed to 1 μΜ Gg, 2 μΜ 1,6-BPQ or their combination for 2 has described previously (2). In brief, cells were exposed to 10 μΜ H₂DCF-DA dye for 15 min. The media was then removed and replaced with the treatments mentioned above. Then the cells were rinsed with PBS, pelleted, re-suspended in PBS and analyzed by a FACScan flow cytometer. In all cases 5,000 events were collected.

[00197] Data & Statistical analysis

[00198] IC₅₀ and K; values were determined by fitting the data in Sigma Plot (version 10.0) and enzyme kinetics module, using non-linear regression analysis. The apparent K; values were determined on the basis of visual inspection of EadieHofstee and various statistics to evaluate goodness of fit, such as the size of the sum of squares of residuals, Akaike information criterion, and standard error (Enzyme kinetics module, version 1.3). The data listed represent the average values from three different determinations. Statistical significance between three or more groups was determined using one-way ANOVA with the Tukey-Kramer multiple comparison test. Statistical analysis was performed using GraphPad Prism 4.0 Graph Pad Software, Inc. San Diego, California, USA, www.graphpad.com.

[00199] Results

[00200] Three quassinoids and a coumarin illustrated in Figure 5, isolated from Castela macrophylla plant were investigated for their chemopreventive and anti-cancer value in this study. [00201] Inhibition of cytochrome P450 (CYP) enzyme activity imparted by Gg

[00202] Given the involvement of CYPl enzymes in procarcinogen activation, the ability of Gg to inhibit the activity of these enzymes was determined using fluorogenic probe substrates (Figure 3 A; Table 3). The intrinsic fluorescence of glaucaruboloneglucoside (> 20% of control) at the wavelength used for detecting CYP1A2 activity prevented accurate IC₅₀ calculation while control experiments on holocanthone and glaucarubolone indicated that they severely quenched the fluorescence of the formed metabolite for all substrates used in CYPl activity determination. Gg moderately inhibited the activities of CYPslAl and IBl (IC₅₀, 6.93 ± 0.3 ΙμΜ and 9.17 ± 0.9 ΙμΜ respectively).

Table 3. IC₅₀ values (μΜ) obtained from the interaction of isolates from the plant

Castelamacrophylla with CYP enzymes

1A! !A2 2 1§ 2 § 4

^S 5 9Λ1 ± iS.02 *

gj∞ftsife 0.31 ϋ-$ί Li t 0.51 Ci.49

¾S «!«¾s *MD «HD * *ND *HP

Key: *ND: Note determined due to quenching of metabolite fluorescence or intrinsic fluorescence

[00203] Further kinetic characterisations (Figure 3B) using Eadie-Hofstee plots yielded non-competitive inhibition of CYPslAl and IBl with IQs of 7.1 ± 1.44 and 5.6 ± 0.66μΜ respectively. Characterisation of the interaction with other CYP enzymes, summarized in Table 3 reveals that while Gg weakly inhibited the activity of CYPs 2C19 (IC₅₀, 19.40 ± 1.1 ΙμΜ) and 2D6 (IC₅₀, 18.02 ± 0.51μΜ; >10μΜ), it moderately inhibited the activity of CYP3A4 (IC₅₀, 1.31 ± 0.49μΜ). [00204] To verify the accuracy of experimental techniques employed to detect CYP inhibition, assays were performed with known inhibitors furafylline (against CYP1 A2), ketoconazole (against CYPslAl, 1B1 and 3A4), (-)-N-3-Benzyl-phenobarbital (NBPB, against CYP2C19) and quinidine (against CYP2D6). The IC₅₀ values (0.8 ± 0.2, 0.04 ± 0.01, 6.3 ± 1.7, 0.06 ± 0.01, 0.3 ± 0.01, 0.03 ± 0.0 ΙμΜ respectively) were consistent with published values (0.99 <10, <10, 0.06, 0.25 and 0.04μΜ respectively (18-21). The Michaelis constant, K_M, was determined for each marker substrate under the specified experimental conditions, to determine suitable substrate concentrations for assessing inhibitory potential of test compounds.

[00205] Effect of Gg on Benzo-a-pyrene-induced CYP1A mRNA expression

[00206] BaP represents a PAH known to convert into carcinogenic metabolites and also known to induce CYP I thereby further facilitating such conversions into activated

carcinogens. As such, MCF-7 breast cancer cells were exposed to BaP in the presence or absence of varying concentrations of Gg for 24 h and CYP1A1 and CYP1A2 mRNA expression determined. BaP induced a robust nearly 70-fold increase in expression relative to untreated controls (Figure 4). On the other hand, Gg induced a dose-dependent inhibition of BaP-induced CYP1A1 mRNA expression and sustained inhibition of CYP1A2 mRNA expression.

[00207] Effect of Gg on 1,6-BPQ-mediated increases in reactive oxygen species in MCF- 10A cells

[00208] Increases in intracellular reactive oxygen species (ROS) frequently occurs just before DNA becomes damaged within susceptible cells. Increases in ROS production within non-malignant cells can promote the onset of mutagenesis due to increases in DNA lesions. A previous report indicated that the BaP metabolite 1,6-BPQ increases ROS production in MCF-IOA cells (2). Thus, ROS levels were evaluated in MCF-IOA cells exposed to media containing 0.15 DMSO, Gg alone, 1,6-BPQ or a combination of 1,6-BPQ and Gg. Figure 6 indicates 1,6-BPQ brought about the expected 2-fold increase in ROS levels. However, Gg effectively counteracted 1,6-BPQ-mediated increases in ROS production in these cells. This further substantiates the chemopreventive potential for Gg and supports previous assertions that this plant isolate also exhibits antioxidant actions.

[00209] In vitro cytoxicity of Castela macrophylla plant isolates [00210] The impact of these compounds on cell viability was assessed using a panel of cell lines including colon normal (CCDI8C0), colon cancer (HT29), liver cancer (HepG2) and breast cancer (MCF-7) cells using the MTS assay. IC₅₀ values (that concentration needed to inhibit 50% cell viability) were calculated for all test isolates and known drug entities, tamoxifen and 5-fluorouracil (which served as positive controls, Table 1). Scopoletin potently and selectively reduced the viability of HT29 colon cancer cells. Its ICsovalue of 19.28 ± 0.34 M compared well with that of 5-fluorouracil (23.50 ± 1.12μΜ), yet this isolate did not affect the viability of normal colon cells (<10% inhibition at 60μΜ) in contrast to 5- fluorouracilwhich displayed cytotoxicity with an IC₅₀ of 55.51 ± 3.71μΜ (Table 1).

[00211] Gg and holocanthone impacted the viability of MCF-7 cells, at potency greater than that observed with the breast cancer agent tamoxifen (IC₅₀S, 8.65 ± 1.11 and 11.03 ± 1.13 compared to 17.28 ± 0.06μΜ respectively). Figure 7 illustrates microscopic evaluation of breast cancer cells in the presence of Gg or tamoxifen at 40μΜ respectively which illustrates effect impact of Gg at that concentration. Holocanthone reduced the viability of colon cancer cells more potently than the known colon cancer therapeutic 5-fluorouracil (IC₅₀S 9.91 ± 0.52 compared to 23.50 ± 1.12μΜ respectively), although it also largely affected normal colon cell viability (IC₅₀ <5μΜ). None of the three quassinoids demonstrated noticeable impact on liver cancer cells while glaucarubolone had no impact on either of the four cell lines evaluated.

[00212] Gg-mediated cytotoxicity and ability to induce apoptosis in MCF-7 cells

[00213] To determine whether the cytotoxicity of Gg increased in a time-dependent fashion, MCF-7 cells were exposed to Gg and compared its effect to tamoxifen for 24, 48 or 72 h with varying concentrations and evaluated cell viability using the Alamar Blue™ assay. Gg induced a time- and dose-dependent decrease in cell viability which was greater than that observed with tamoxifen (Figure 2). Next the ability of Gg to induce apoptosis in MCF-7 breast cancer cells was examined since they showed substantial sensitivity to this quassinoid. Using the Annexin V-7AAD assay, a dose-dependent increase in early apoptosis was detected in MCF-7 cells treated with Gg (Figures 2B-D), which was significant at 1 μΜ (26.5 ± 9.5%). Interestingly, this concentration is more than 8-fold lower than the IC₅₀ value. Apoptosis was also apparent after 48 h of Gg treatment.

[00214] Discussion [00215] Inhibitors of CYPl enzymes are recognized to play a role as chemoprotectors (22) and several natural compounds such as flavonoids including resveratrol (10, 23, 24) and organosulfur compounds have been categorised as such. Gg displayed moderate potency in inhibiting the activities of both CYPslAl and 1B1, critically important in the activation of BaP. Previous studies (9, 25) described an active site model for quassinoid bound to

CYP1A1, identifying a key residue required for effective interactions with the enzyme. The generalised model identified a hydrogen bond donor in position 1 which can aid binding with the backbone carbonyl of Asp313. The oxygens on rings C (2) and A (3) can bind with the side chains of Thrl 11 and Serl24 respectively, and hydrophobic groups, possibly on ring positions in A can facilitate interactions with Phel23, Ile386 and Leu496. Gg has a glucoside moiety on ring D replacing the hydroxyl group found in quassin, preventing it from acting as a hydrogen bond donor. This can explain the lack of affinity to the active site as implicated by the non-competitive binding kinetics in Figure 6B.

[00216] Non-competitive inhibitors generally follow the relation IC₅o=Ki (26). In the studies herein, the obtained IC₅₀ value for Gg on CYPslAl and 1B1 yielded similar K; values further confirming the graphical representation and computer assisted generations using EadieHoftsee plots. BaP like many other PAHs binds with high affinity to the cytosolic aryl hydrocarbon receptor (AhR) which then complexes with the related AhR nuclear translocator (ARNT) protein in the nucleus, commencing the transcriptional activation of CYP1A1 and CYP1A2. AhR undergoes post-translational modification for optimal transactivation potential, and whether Gg inhibits the kinases that phosphorylates the AhR, like some have postulated polyphenols to do (27), is yet to be determined. What is clear though is that BaP induction of CYPIA gene expression is reduced in the presence of Gg. Yet alone, it did not appreciably affect the endogenous mRNA expression of any of the CYPl enzymes within the breast cancer cells. Such reductions in CYPIA coupled with direct inhibition of the enzymes' activities in the presence of Gg can be expected to decrease the levels of carcinogenic metabolites formed through CYPIA activity.

[00217] Finally, the ability of Gg to inhibit and counteract the increase in ROS levels by BaP metabolite 1,6-BPQ, in non-tumorigenic MCF-IOA breast epithelial cells supports the chemopreventive as well as the anti-oxidant actions of Gg.

[00218] Additionally, Gg also demonstrated low cytoxicity towards normal cells (Table 1), which is an added benefit for the search of lead compounds as chemopreventive agents. The two quassinoidsglaucarubolone glycoside (Gg) and holocanthone, and a coumarin

(scopoletin) have been shown to display potent cytotoxic properties comparable to or more potent than, known chemotherapeutic drugs. The coumarin showed no toxicity towards normal colon cells in contrast to the drug 5-fluorouracil, making it an attractive candidate for further development. Many side effects of chemotherapy can be attributed to the destruction of normal cells (28) and the absence of such on the examined normal colon cells highlights scopoletin as a candidate suitable for further experimentation.

[00219] Gluacarubolone did not impact cell viability of any of the cancer cells lines, although its structural isomers, Gg and holocanthone did. The conjugation (glycosylation or acetylation) can be critical for bioactivity, and can be due to the enhanced cell membrane permeability that the conjugation allows.

[00220] Exhibiting some degree of specificity amongst the cancer cells, Gg was twice more potent towards breast cancer cells than tamoxifen but approximately three times weaker towards liver cancer cells than doxorubicin. In addition, Gg showed no impact on colon cancer cells. As expected, increasing the duration of exposure of MCF-7 cells to Gg resulted in a decrease in cell viability. Gg-mediated cytotoxicity in breast cancer cells can involve apoptosis. Gg (ΙμΜ, 24h) induced significant early apoptosis in MCF-7 breast cancer cells at a concentration more than 8-fold lower than the IC₅₀ for this isolate. The clinically available anti-breast cancer agent tamoxifen (ΙμΜ, 24h) has previously been shown to induce apoptosis in MCF-7 cells (29) though it is considerably less potent than Gg in these cells. Gg can exhibit selective cytotoxicity towards the MCF-7 breast cancer cell line, which indicates its potential use in the prevention or treatment of breast cancer. Gg also showed weak inhibition of CYP2D6, and since this enzyme plays a key role in the metabolism of tamoxifen, combination therapy with the pharmaceutical should have little risk of metabolism based drug-drug interactions (30). Although most warnings for drug-drug interactions for tamoxifen users are against CYP2D6 inhibitors, CYP3A4 is also involved in its metabolism and represents a predominant enzyme in the metabolism of a myriad of agents. The propensity for Gg to cause moderate inhibition of CYP3A4 indicates that care be exercised if it is used in combination with tamoxifen and/or other drugs predominantly reliant on this enzyme for clearance.

[00221] Holocanthone affected both colon and breast cancer cells more potently than their respective chemotherapeutic drugs, 5-fluorouraciland tamoxifen. However its activity against liver cancer cells was weaker than the known chemotherapeutic drug doxorubicin. 5- fluorouracilis usually used in combination with other drugs such as leucovorin (31) when treating a variety of malignancies which include those of the colon, breast, pancreas and cervix. Similarly, holocanthone's broad cytotoxicity against several cell lines can make it useful as a broad-spectrum anticancer drug candidate. Its ability to reduce the viability of colon cancer cells to a greater extent than 5-fluorouracil (10 compared to 23 μΜ) warrants further investigation. Holocanthone derivatives show greater selectivity towards malignant compared to non-malignant cells, which supports their development as suitable anticancer agents.

[00222] Conclusion

[00223] Three isolates examined herein demonstrated potent anticancer activity greater than or equal to clinically available chemotherapeutic agents. Scopoletin, a coumarin isolate from Castela macrophylla showed high potency against colon cancer cells with minimal impact on normal cells, making it a candidate worth further investigations. The quassinoids, glucarabulone glucoside and holocanthone also from the same plant showed growth inhibitory potency against breast cancer cells, and the former showed moderate inhibition of CYP1A1 and CYPlBl enzymes with non-competitive binding kinetics. Glucarabulone glucoside's ability to thwart 1,6-BPQ-mediated increases in ROS production in MCF-IOA cells indicates its antioxidant actions can at least partially contribute to its potential to serve as a chemopreventive agent, in addition to its ability to inhibit the induction of CYP1 mRNA by BaP. Chemotherapeutic and chemopreventive potential are implied by the bioactivity displayed by these compounds, and validate the on-going search for natural products from endemic tropical biodiversity.

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17. Crespi C, Miller V, Penman B. Microtitre plate assays for inhibition of human, drug metabolising cytochromes P450. Analytical Biochemistry 1997;248: 188-90.

18. Powrie RH. High-throughput inhibition screening of five major human cytochrome P450 enzymes using an in-vitor substrate cocktail. CXR biosciences .Available at

<http://wwwcxrbiosciencescom/cmsimages/media/pdfs/Microsoft%20PowerPoint%20- %20P450%20inhibition%20posterpdf>2007;Accessed on April 20,2010.

19. Stresser DM, Broudy MI, Ho T, et a/.Highly selective inhibition of human CYP3A in vitro by azmulin and evidence that inhibition is irreversible. Drug MetabDispos 2004;32: 105- 12.

20. Cali J. Screen for CYP450 inhibitors using P450-GLOTM luminescent cytochrome P450 assays, cell notes issue available at wwwpromegacom 2003.

21. Cai X, Wang R, Edom R, et al. Validation of (-)-N~3-benzyl-phenobarbital as a selective inhibitor of CYP2C19 in human liver microsomes. Drug Metabolism and

Disposition 2004;32:584-6.

22. Badal S, Delgoda R. Role of the modulation of CYP1A1 expression and activity in chemoprevention. Acepted for publication in Applied Toxicology 2013.

23. Leung H, Wang Y, Chan H, Leung L. Developing a high throughput system for the screening of cytochrome P450 1 Al - Inhibitory polyphenols. Toxicol in vitro 2007;21 :996- 1002.

24. Ren W, Qiao Z, Wang H, Zhu L, Zhang L. Flavonoids: Promising anticancer agents. Medicinal Research Reviews 2003;23:519-34.

25. Badal S, Shields M, Niazi U, Jacobs H, Sutcliffe M, Delgoda R. Screening Natural Products for CYP1 Inhibitors Proceedings of the 17 th International symposium on Microsomes and Drug Oxidation. Medimond Publishers, Saratoga Springs, New York, USA 2008:20-4.

26. Cheng Y, Prusoff W. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (150) of an enzymatic reaction. BiochemPharmacol 1973;22:3099-108.

27. Mukai R, Fukuda I, Nishiumi S, et al. Cacao polyphenol extract suppresses transformation of an aryl hydrocarbon receptor in C57BL/6 mice. J Agric Food Chem

2008;56: 10399-405.

28. Wang W, Chen M, Chuang C, Jeang K, Huang L. Molecular biology of human immunodeficiency virus type 1. J Microbiollmmunol Infect 2000;33: 131-40.

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31. Saltz L, Cox J, Blanke C, et a/.Irinotecan plus fluorouracil and leucovorin for metastatic colorectal cancer. N Engl J Med 2000;343:905-14.

[00225] Example 3: Glaucarubulone glycoside attenuates procarcinogen-mediated CYP1 gene induction and suppresses cancer cell growth

[00226] Quassinoids and coumarins have in the past been associated with anti-inflammatory, anti-oxidant and anti-proliferative activity. Emerging evidence indicates that these

compounds may also act as chemopreventive agents. In this study, the potential for new quassinoids and a coumarin isolated from the endemic Jamaican Castela macrophylla plant to inhibit cytochrome P450s (CYPs), in particular CYP1A1 and CYP1B1 enzymes, known to convert polyaromatic hydrocarbons (PAHs) into carcinogenic metabolites, was investigated. The quassinoid glaucarubulone- 15-0-P-D-glucopyranoside (Gg) inhibited the activity of human CYP1A1 and CYP1B1 enzymes (IC50, 6.93 ± 0.31μΜ and 9.17 ± 0.91μΜ respectively) according to non-competitive kinetics. Gg also attenuated (nearly 70-fold) the ability of PAH, benzo-a-pyrene(B[a]P) to induce CYP1A gene expression in MCF-7 breast cancer cells as determined by real-time RT-PCR. In addition, Gg suppressed the ability of B[a]P metabolite benzo-a-pyrene-l,6-quinone to increase reactive oxygen species levels in non-malignant MCF-IOA breast epithelial cells. Gg reduced viability of MCF-7 cancer cells (IC50 = 0.121=1= 0.08μΜ) to a greater extent than the reference anticancer agent tamoxifen (IC50> 10 μΜ) as assessed by the Alamar Blue™ assay, without any impact on non- malignant cells. Finally, using the AnnexinV-7AAD assay, it was determined that Gg induced MCF-7 breast cancer cell death in a dose-dependant manner. Taken together, these data indicate that the plant isolate Gg possesses chemopreventive and chemotherapeutic actions, which warrants its continued development.

[00227] Introduction

[00228] Humans and other living organisms are constantly exposed to an array of pollutants and potentially genotoxic environmental chemicals. Poly cyclic aromatic hydrocarbons (PAHs) are ubiquitous chemicals produced by incomplete pyrolysis of organic materials and are commonly released via combustion of fossil fuels. They are found in considerable quantities in vehicle exhaust, cigarette smoke, particulate matter in urban air and charcoal broiled food. Lipophilic PAHs easily diffuse into cells and bind with high affinity to the cytosolic aryl hydrocarbon receptor (AHR) followed by a second related protein, AHR nuclear translocator (ARNT) (Moorthy 2008; Sissung et al. 2006). This PAH activated heterodimer is capable of inducing the transcription of many genes in the Ah locus, including the cytochrome P450 (CYP) 1 enzyme family comprised of three members:CYPlAl, CYPlA2 and CYP IB 1.

[00229] Benzo[a]pyrene, B[a]P, is a well studied, potent pro-carcinogen present in high concentrations in PAH polluted environments. Of the three major pathways of B[a]P bio- activation, the dihydrodiolepoxide (DDE) pathway involving CYPs and epoxide hydrolase (EH), contributes substantially to the formation of highly mutagenic and reactive B[a]P metabolites that form DNA adducts (Baird et al. 2005; Shimada and Fujii-Kuriyama 2004). In this pathway, CYPl enzymes (particularly CYPlAl and CYP IB 1) carry out epoxidation at the 7 and 8 positions followed by hydrolysis to form B[a]P-trans-7,8-dihydrodiol. An additional CYP-catalyzed epoxidation results in the formation of the ultimate carcinogen, 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BDPE) (Shimada 2006; Shimada and Fujii-Kuriyama 2004). The route of exposure along with tissue specific characteristics will influence the bioactivation process of B[a]P, and CYP1A1 has been identified as crucial in the formation of the tetrahydro-BDPE in human lung cells (Uppstad et al. 2010).

[00230] Several dihydrodiol dehydrogenase members of the aldo-ketoreductase (AK ) superfamily promote another form of bioconversion of B[a]P which yields reactive and redox active B[a]P-7,8-dione. Following the first round of activation by CYP and EH, AKR produces quinones that can undergo redox cycling via peroxidases and CYPs. This can result in continuous release of superoxide anion radicals, hydrogen peroxide and subsequent oxidative DNA damage. Reactive oxygen species (ROS) are released during quinone formation, which have been associated with cell damage and apoptosis (Bolt and Ross 2008; Bolton et al. 2000). Indeed benzoquinones formed by B[a]P metabolism have been shown to increase cell proliferation, generate ROS and transactivate the epidermal growth factor receptor in MCF-IOA breast epithelial cells, providing a plausible mechanism for B[a]P- mediated tumour promotion (Burdick et al. 2003).

[00231] Oxidative stress can include activation of pro-inflammatory mediators that are known to disrupt expression and activity of drug metabolizing enzymes in various tissues (Aitken et al. 2006). Inflammation down-regulates the expression and activities of many CYP enzymes, including CYP1A1 involved in B[a]P metabolism. The reduction of CYP1A1 expression has been linked to the activation of NF-κΒ, which is induced by pro-inflammatory cytokines (Tian et al. 2002). In contrast, CYP IB 1 activity is enhanced during inflammation to further exacerbate damage to lung cells (Smerdova et al. 2013). Thus, in the activation of B[a]P into its DDE or quinone forms, both which can lead to the formation of DNA adducts, the activity of CYP1A1 and/or CYP1B1 can play a critical role in various tissues, under varying conditions. In the hope of identifying novel leads that can successfully intervene in the metabolic activation of B[a]P, inhibitors capable of targeting both these enzymes were identified, and B[a]P's ability to induce CYP1 transcription, a crucial step that enables the procarcinogen to derive sufficient CYP1 enzymes for catalysis into its deleterious forms, was simultaneously targeted.

[00232] Since quassinoids and coumarins have previously demonstrated anti-inflammatory properties (Hall et al. 1983; Liang et al. 2011; Menghini et al. 2010), we searched the Castela turpin (Simaroubaceae) family reported to accumulate quassinoids (Grieco et al. 1993; Jacobs et al. 2007) for novel compounds. Of the 15 species found in the tropical

Americas(Mabberley \991),Castela macrophylla is endemic to Jamaica (Adams 1972).

Isolates specifically from this plant were limited in their pharmacological applications to anti- feedant activities against tobacco budworm, Plasmodium falciparum and Plasmopara viticola (Hoffman et al. 1992), and no further biological activities have been reported.

[00233] Previously, a generalised model outlining the key features for probable binding of quassionoid to the CYP1A1 active site was proposed (Shields et al. 2009). In the hope of identifying novel leads that can successfully intervene in the metabolic activation of B[a]P, the capacity of a novel quassinoid, two related quassinoids and a coumarin isolated from Castela macrophylla, to inhibit the activity of CYP1 enzymes, was evaluated. The compounds so investigated in this study are illustrated in Figure 5. B[a]P's ability to induce CYP1 transcription, a crucial step in the bioconversion of pro-carcinogen B[a]P into deleterious metabolites, was also targeted. The ability of quassinoid Glaucarubulone glycoside (Gg) to counteractB[a]P-mediated CYP1A m NA induction in MCF-7 breast cancer cells and the propensity of l,6-B[a]P quinone(B[a]P metabolite) to increase ROS generation in non-tumorigenic MCF-IOA cells were also evaluated. Finally, the ability of Gg to elicit anticancer actions, particularly in MCF-7 breast cancer cells, was investigated. This work sheds new insight into the potential for Gg and related plant isolates to serve as agents that can impact molecular targets of toxicity and thus be useful in the treatment and prevention of cancer.

[00234] Materials and Methods

[00235] Chemicals & Reagents

[00236] Benzo-a-pyrene(B[a]P), benzo-a-pyrene-l,6-quinone (l,6-BPQ)and all chemicals for the MTS and CYP inhibition assays were purchased from Sigma- Aldrich (St.Louis, MO).Dimethylsulfoxide (DMSO) was purchased from American Type Culture Collection (ATCC, Manassas, VA, US), 7-Amino-actinomycin D (7AAD) was purchased from eBioscience (San Diego, CA) and Annexin V-FITC was purchased from Pharmingen (San Diego, CA). All CYP substrates and metabolites were purchased from Gentest Corporation (Worburn, MA, USA). All cell lines along with their respective media and supplements were purchased from ATCC (Manassas, VA, USA). ATCC certifies that the cells were

authenticated prior to shipment using short tandem repeat profiling to verify that they are of the correct lineage and uncontaminated with other cell types. In addition, the cells were routinely screened to ensure they were free from mycoplasma contamination and other types of microorganisms. Escherichia coli membranes expressing human CYP1A1, CYP1A2, CYP1B1, CYP2D6, CYP3A4 and CYP2C19 co-expressed with CYP reductase were purchased from Cypex Ltd. (Dundee, U.K).

[00237] Plant isolates

[00238] Castela macrophylla (Simaroubaceae) was collected in Hellshire Hills, St.

Catherine, Jamaica, in June 2003 and a voucher specimen, (#UWI-Mona 34, 982) was deposited in the Herbarium at the University of the West Indies, Mona, Jamaica. The leaves, twigs and thorns were air-dried, chopped and exhaustively extracted with hexane, acetone and methanol. The resulting residues were subjected to column chromatography on silica gel. Elution with various concentrations (ranging between 5-100% acetone -hexane) and recrystallization yielded pure compounds (-)-glaucarubolone, glaucarbulone-15-0-P-D- glucopyranoside (Gg), (-)-holocanthone and scopoletin as described elsewhere (Jacobs et al. 2007).

[00239] CYP inhibition assays

[00240] The test compounds were evaluated for their ability to inhibit the catalytic activity of human CYP1 enzymes by means of high throughput fluorometric inhibition assays conducted in 96 well microtitre plates as described elsewhere (Crespi et al. 1997). 7 ethoxyresorufin (ERes) was used as a substrate for detecting activities of CYP IB 1 and 7- ethoxy-3-cyanocoumarin (CEC) was used as a substrate for both CYPs 1 Al and 1 A2.

Further, the substrates 3-[2-(N,N-Diethyl-N-methylamino)ethyl]-7-methoxy- 4methylcoumarin (AMMC), 7-Benzyloxy-4-trifluoromethylcoumarin (BFC), and CEC were used as substrates for CYPs 2D6, 3A4 and 2C19 respectively. The reactions were monitored fluorometrically at 37°C, using a Varian Cary Eclipse Fluorescence spectrophotometer. All inhibitors were dissolved in a solvent of 20% acetonitrile in water and less than 0.3% of acetonitrile was used in the final assay.

[00241] Cell Culture

[00242] Cells (CCD 18 Co normal colon, HepG2 hepatoma and MCF-7 breast carcinoma) were maintained in ATCC-formulated Eagle's Minimum Essential Medium and HT29 colon carcinoma cells were maintained in McCoy's 5a Medium Modified supplemented with 10% foetal bovine serum (Atlas; Fort Collins CO), lOmM HEPES solution, lOOmM L-glutamine penicillin streptomycin solution, 3 g/L glucose, and 1.5 g/L of sodium bicarbonate. For the apoptosis studies and certain cytotoxicity assays, MCF-7 cells were maintained in RPMI medium supplemented with 10% FBS and penicillin and streptomycin antibiotics. Cells were maintained at 37°C with 5% C0₂ in Corning 75cm² culture flasks.

[00243] RNA Extraction & Quantitative RT-PCR Analysis

[00244] Total RNA was isolated from MCF-7 breast cancer cells treated with media containing 0.02 % DMSO or 2 μΜ B[a]P alone or in combination with 10, 50 or 100 nMGg for 24 h using the Aurum™ Total RNA Mini kit (Bio-Rad, Hercules, CA). The quantity of theRNA extracts was determined by measuring the absorbance at 260 nm and 280 nm using a Beckman DU 800 Spectrophotometer(Beckman, Brea, CA). RNA quality was further analysed, and RNA quality indicator (RQI) values were determined using the Experion Automated Electrophoresis system (Bio-Rad, Hercules, CA). cDNA was prepared from 1 μg of total RNA from MCF-7 breast cancer cells to measure CYP1A1 or CYP1A2 expression. RNA was extracted using the iScript®cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's instruction. The cDNA was used as a template for real time quantitative PCR analysis using a CFX-96 PCR instrument (Bio-Rad, Hercules, CA). The primers for the reference genes GAPDH and GUSB as well as the CYP1A1 and CYP1A2 genes were obtained from SA Biosciences (Frederick, MD). The PCR reactions were set up in accordance with the manufacturer's recommendations. Relative fold-changes in gene expression were calculated using the ^AACT method.

[00245] Reactive oxygen species (ROS) analysis

[00246] Intracellular ROS levels were measured in MCF-IOA breast epithelial cells exposed to 0.025% DMSO (control) or2 μΜ 1,6-BPQ alone or combined with 1 μΜ Ggfor 2h as described previously (Burdick et al. 2003). In brief, cells were exposed to 10 μΜ H₂DCF- DA dye for 15 min. The media was then removed and replaced with the treatments mentioned above. Then the cells were rinsed with PBS, pelleted, resuspended in PBS and analysed by a FACScan flow cytometer. In all cases 5,000 events were collected.

[00247] Cytotoxicity assays [00248] Following the appropriate treatments, cell proliferation was evaluated using an MTS assay according to the manufacturer's instructions (Palmari et al. 1996). All assays were performed at least three times and their O.D. measured at 590 nm (Heusch and Maneckjee 1999). Cell viability was measured as a percentage of the controls containing respective solvents. In some experiments, MCF-7 plated in 96 well plates were exposed to media containing 0.025% DMSO, tamoxifen or Gg at varying concentrations and time points(24-72 h) before cytotoxicity analysis using the Alamar Blue™ assay as described in detail elsewhere (Heusch and Maneckjee 1999; McLean et al. 2008).

[00249] AnnexinV-7AAD apoptosis assay

[00250] The ability of Gg to induce apoptosis in MCF-7 cells was investigated similarly to a method previously described in detail elsewhere (Lee et al. 2009; McLean et al. 2008).

Briefly, cells were exposed to varying concentrations of Gg for 24 h. Following treatment, cells were washed with cold PBS, harvested and counted and aliquots placed in 96 well plates. Cells were then re-suspended in assay buffer and stained with Annexin-V(which stains for cells in early apoptosis and distinguishes them from those that are viable) and 7- amino-actinomycin D (7AAD; which stains for necrotic or late apoptotic cells). Cells were analysed using flow cytometry soon after 7AAD addition.

[00251] Data & Statistical analysis

[00252] IC₅₀ and K; values were determined by fitting the data in Sigma Plot (version 10.0) and enzyme kinetics module, using non- linear regression analysis. The apparent K; values were determined on the basis of visual inspection of Eadie-Hofstee and various statistics to evaluate goodness of fit, such as residual sum of squares, Akaike information criterion, and standard error (Enzyme kinetics module, version 1.3). The data listed represent the average values from three different determinations. Statistical significance between three or more groups was determined using one-way AN OVA with the Tukey-Kramer multiple comparison test. Statistical analysis was performed using GraphPad Prism 4.0 Graph Pad Software, Inc. San Diego, California, USA, www.graphpad.com.

[00253] Results

[00254] Gg inhibits cytochrome P450 (CYP) enzyme activity [00255] Given the involvement of CYP1 enzymes, in particular CYPslAl and 1B1 in procarcinogen activation, the ability of the test compounds to inhibit the activity of these enzymes was determined using fluorogenic probe substrates (Figure 3(A); Table 3). The intrinsic fluorescence of glaucarubolone glucoside, Gg, (> 20% of control) at the wavelength used for detecting CYP1A2 activity prevented accurate IC₅₀ calculation while control experiments on holocanthone and glaucarubolone indicated that they severely quenched the fluorescence of the formed metabolite for all substrates used in CYP1 activity determination. Gg inhibited the activities of CYPslAl and 1B1 (IC₅₀ values of 6.93 ± 0.31μΜ and 9.17 ± 0.9 ΙμΜ respectively). Further kinetic characterisations (Figure 3B) using Eadie-Hofstee plots yielded non-competitive inhibition of CYPslAl and 1B1 with Kjs of 7.1 ± 1.44 and 5.6 ± 0.66μΜ respectively. These compounds were further profiled against other drug metabolising CYP enzymes to gain an understanding required by the FDA for new therapeutic entities, as impact on certain drug metabolizing enzymes has implications in drug-drug interactions. Such results summarized in Table 3 reveal that while Gg weakly inhibited the activity of CYPs 2C 19 (IC₅₀ = 19.40 ± 1.11 μΜ) and 2D6 (IC₅₀ = 18.02 ± 0.5 ΙμΜ) this isolate more strongly inhibited CYP3A4 activity (IC₅₀ = 1.31 ± 0.49μΜ).

[00256] To verify the accuracy of experimental techniques employed to detect CYP inhibition, assays were performed with known inhibitors furafylline (against CYP1A2), ketoconazole (against CYPslAl, 1B1 and 3A4), (-)-N-3-Benzyl-phenobarbital (NBPB, against CYP2C19) and quinidine (against CYP2D6). The determined IC₅₀ values (0.8 ± 0.2, 0.04 ± 0.01, 6.3 ± 1.7, 0.06 ± 0.01, 0.3 ± 0.01, 0.03 ± 0.0 ΙμΜ respectively) were consistent with published values (0.99,<10, <10, 0.06, 0.25 and 0.04μΜ respectively (Cai et al. 2004; Cali 2003; Powrie 2007; Stresser et al. 2004). The Michaelis constant, K_M, was determined for each marker substrate under the specified experimental conditions, to determine suitable substrate concentrations for assessing inhibitory potential of test compounds.

[00257] Gg attenuates Benzo-a-pyrene-induced CYP1A mRNA expression

[00258] B[a]P represents a PAH known to induce CYPlvia the AhR locus thereby facilitating its own catalysis into its activated and carcinogenic forms (Shiizaki et al. 2013). MCF-7 breast cancer cells exposed to B[a]P in the presence or absence of varying

concentrations of Gg for 24h were used and CYPlAl and CYP1A2 mRNA expression levels were evaluated. B[a]P induced a robust (nearly 70-fold) increase in expression relative to untreated controls (Figure 4). On the other hand, Gg inhibited B[a]P-induced CYPlAl mRNA expression in a dose-dependent fashion and caused sustained inhibition of B[a]P- induced CYP1A2 mRNA expression.

[00259] Gg counteracts 1 ,6-BPQ-mediated increases in reactive oxygen species in MCF- 10A cells

[00260] Increases in intracellular reactive oxygen species (ROS) frequently occur just before DNA becomes damaged within susceptible cells. Increases in ROS production within non- malignant MCF-IOA cells can promote the onset of mutagenesis due to increases in DNA lesions. The B[a]P metabolite l,6-B[a]P quinone (1,6-BPQ), formed via the AKR pathway following initial CYP activation is known to increase ROS production in MCF-IOA cells (Burdick et al. 2003). Thus, ROS levels were evaluated in MCF-IOA cells exposed to media containing 0.01% DMSO, 1 μΜ 1,6-BPQ or a combination of 1,6-BPQ and Gg. Figure 6 indicates 1,6-BPQ brought about the expected 2-fold increase in ROS levels. However, Gg effectively counteracted 1,6-BPQ-mediated increases in ROS production within these cells.

[00261] Isolates derived from Castela macrophylla inhibit cell viability

[00262] The data presented above indicate Gg possesses chemopreventive properties. Many chemopreventive agents demonstrate chemotherapeutic actions. Therefore, normal colorectal (CCDI8C0), colon cancer (HT29), liver cancer (HepG2) and breast cancer (MCF-7) cells were treated with Gg, and their viability was later determined using the MTS assay. IC₅₀ values (that concentration needed to inhibit cell viability by 50%) were calculated for all test isolates and established anticancer agentstamoxifen,doxorubicin and 5-fluorouracil (which served as positive controls, Table 1).

[00263] Gg was rather inactive in CCD18Conormal colon cells though slightly more toxic than 5-fluorouracil. In addition, Gg was also inactive in non-tumorigenic MCF-IOA breast epithelial cells (IC50 > 10 μΜ). Gg displayed cytotoxicity in MCF-7 cancer cells with approximately 2-fold greater potency than tamoxifen (IC₅o=8.65 ± 1.1 ΙμΜ vs. to 17.28 ± 0.06μΜ) following 24 h treatment as determined by the MTS assay. Scopoletin potently and selectively reduced the viability of HT29 colon cancer cells. Its ICsovalue of 19.28 ± 0.34 μΜ was comparable to 5-fluorouracil (23.50 ± 1.12μΜ), yet this isolate did not affect the viability of CCDI8C0 normal colon cells (<10% inhibition at 60μΜ). In contrast, 5- fluorouracil displayed more cytotoxicity in thesecells with an IC₅₀ of 55.51 ± 3.71μΜ (Table 1). Holocanthone reduced the viability of MCF-7 cells, with similar potency asGg (IC₅₀ = 11.03 ± 1.13 μΜ vs.8.65 ± 1.11 μΜ) and more potently than tamoxifen. Halocanthone inhibited HT29 cells to a comparable degree as 5-fluorouracil (IC₅₀ = 9.91 ± 0.52μΜ vs. 11.03 ± 1.13μΜ), but was also highly cytotoxic to the CCD18Cocells (Κ₅₀<5μΜ). None of the three quassinoids demonstrated noticeable impact on the HepGliver cancer cells while glaucarubolone had no impact on either of the four cell lines evaluated.

[00264] Gg induces dose-dependent cytotoxicity and apoptosis in MCF-7 cells

[00265] To determine whether Gg reduced cell viability in a dose- and time-dependent fashion, MCF-7 cells were exposed to Gg or tamoxifen for 24, 48 or 72 h at varying concentrations. Then cell viability was evaluated using the Alamar Blue™ assay. Gg induced a time- and dose-dependent decrease in cell viability which was greater than that observed with tamoxifen (Figures 2A-C). This disparity was especially evident following 72 h of exposure (ICso=121nM vs. >10μΜ). Although the active metabolite of Tamoxifen, 4- hydroxytamoxifen (40HTam) displayed more potent activity than the parent drug tamoxifen, 40HTam was still considerably less potent than Gg (Figure 8) with an IC₅₀ = 5.25 μΜ. Next, the ability of Gg to induce apoptosis in MCF-7 breast cancer cells was examined, since they showed substantial sensitivity to this quassinoid. The cells were treated for 24 h with media containing 0.025% DMSO or varying concentrations of Gg. Using the Annexin V- 7AAD assay, a dose-dependent increase in early apoptosis was detected (Figure 2D), which was significant at 1 μΜ (26.5 ± 9.5%). Interestingly, this concentration is approximately 8- fold lower than the IC₅₀ value determined after 24 h of treatment. Apoptosis was also apparent after 48 h of Gg treatment.

[00266] Discussion

[00267] Since CYP1 enzymes contribute to DDE and dione production to form DNA adducts and hence illicit the carcinogenicity of the PAH B[a]P, much attention has been focused on identifying CYP1 inhibitors as potential chemoprotectors (Badal and Delgoda 2014), alongside other CYP inhibitors with therapeutic value (Francis and Delgoda 2014). Indeed, several classes of natural compounds such as flavonoids including resveratrol (Leung et al. 2007; Ren et al. 2003; Skupinska et al. 2009) and coumarins (Cai et al. 1997) have been categorised as such. In this study, the quassinoid Gg was identified as imparting influence at several key stages of B[a]P activation, as proposed in the scheme illustrated in the graphical abstract. Gg, moderately inhibited the activities of CYPs 1A1 and 1B1; the former is critical for B[a]P activation under normal conditions (Shimada and Fujii-Kuriyama 2004) and the latter under inflammatory conditions (Smerdova et al. 2013). Additionally, Gg repressed B[a]P- mediated CYPIA mRNA induction in MCF-7 cells and 1 ,6-BPQ-mediated ROS productionin MCF-IOA cells.

[00268] In previous studies (Badal et al. 2008; Shields et al. 2009), key residues required for effective interactions for quassinoid bound to the CYP1A1 active site were identified. The generalised model identified a hydrogen bond donor in position 1 which can aid binding with the backbone carbonyl of Asp313. The oxygens on rings C (2) and A (3) can bind with the sidechains of Thrl 11 and Serl24 respectively. Hydrophobic groups, possibly on ring positions in A can facilitate interactions with Phel23, Ile386 and Leu496. Gg has a glucoside moiety on ring D replacing the hydroxyl group found in quassin, preventing it from acting as a hydrogen bond donor. This can explain the lack of affinity to the active site as implicated by the non-competitive binding kinetics in Figure 3B. Non-competitive inhibitors generally follow the relation IC₅o⁼Ki (Cheng and Prusoff 1973). In the studies herein, the obtained IC₅₀ value for Gg on CYPslAl and 1B1 yielded similar Ki values further confirming the graphical representation and computer assisted generations using Eadie-Hoftsee plots.

[00269] B[a]P like many other PAHs binds with high affinity to the cytosolic aryl hydrocarbon receptor (AhR) which then complexes with the related AhR nuclear translocator (ARNT) protein in the nucleus, commencing the transcriptional activation of CYP1A1 and CYP1A2. AhR undergoes post-translational modification for optimal transactivation potential, and, without being bound by theory, Gg can inhibit the kinases that phosphorylates the AhR, like some have postulated polyphenols to do (Mukai et al. 2008). B[a]P induction of CYPIA gene expression is reduced in the presence of Gg. Yet alone, it did not appreciably affect the endogenous mRNA expression of any of the CYP1 enzymes within the breast cancer cells. Such reductions in CYPIA coupled with direct inhibition of the enzymes' activities in the presence of Gg can be expected to decrease the levels of carcinogenic metabolites formed through CYPIA activity.

[00270] The ability of Gg to counteract the increase in ROS levels by B[a]P metabolite 1,6- BPQ, in non-tumorigenic MCF-IOA breast epithelial cells indicates Gg possesses

chemopreventive and anti-oxidant actions. The graphical abstract summarises the ability of Ggto interfere with B[a]PCYP-mediated activationand ROS production at three critical stages which likely diminishes DNA damage. [00271] Additionally, Gg also demonstrated low cytoxicity towards normal cells (Table 1), which is an added benefit for the search of lead compounds as chemopreventive agents. Gg, holocanthone, and scopoletin displayed cancer selective cytotoxic properties comparable to or more potent than, known chemotherapeutic drugs. The coumarin(scopoletin) showed no toxicity towards normal colon cells in contrast to the drug 5-fluorouracil, making it an attractive candidate for further development. Many side effects of chemotherapy can be attributed to the destruction of normal cells(Wang et al. 2000) and the apparent absence of such on the examined normal colon cells highlights scopoletin as a candidate suitable for further experimentation.

[00272] Gluacarubolone did not impact cell viability of any of the cancer cells lines, although it's structural isomers, Gg and holocanthone did. The conjugation (glycosylation or acetylation) can be critical for bioactivity, and can be due to the enhanced cell membrane permeability that the conjugation allows.

[00273] Exhibiting some degree of specificity amongst the cancer cells, Gg was twice more potent towards breast cancer cells than tamoxifen following 24 h of treatment though this isolate was less potent at inhibiting liver cancer cell growth than doxorubicin. After 72 h of exposure,Gg exhibited 250- and 100-fold greater cytotoxicity in MCF-7 cells thantamoxifen and its active metabolite 40HTam respectively (Figure 8). Such cytotoxicity in breast cancer cells appears to involve apoptosis. Gg (ΙμΜ, 24h) induced significant early apoptosis in MCF-7 breast cancer cells at a concentration more than 8-fold lower than the IC₅₀ for this isolate. The clinically available anti-breast cancer agent tamoxifen (ΙμΜ, 24h) has previously been shown to induce apoptosis in MCF-7 cells (Karami-Tehrani and Salami 2003) though it is considerably less potent than Gg in these cells. Interestingly, Gg displayed selective cytotoxicity towards the MCF-7 breast cancer cell line, which indicates its potential use in the prevention or treatment of breast cancer.

[00274] Gg also showed weak inhibition of CYP2D6, and since this enzyme plays a key role in the metabolism of tamoxifen, combination therapy with the pharmaceutical should have little risk of metabolism based drug-drug interactions (Bonanni et al. 2006). Although most warnings for drug-drug interactions for tamoxifen users are against CYP2D6 inhibitors, CYP3 A4 is also involved in its metabolism and represents a predominant enzyme in the metabolism of a myriad of agents. Since Gg moderately inhibits CYP3A4, adjustments may be needed when this isolate is combined with tamoxifen and/or other drugs predominantly reliant on this enzyme for bioactivation.

[00275] Holocanthone affected both colon and breast cancer cells more potently than their respective chemotherapeutic drugs, 5-fluorouraciland tamoxifen. However its activity against liver cancer cells was weaker than the known chemotherapeutic drug doxorubicin. 5- fluorouracilis usually used in combination with other drugs such as leucovorin (Saltz et al. 2000) when treating a variety of malignancies which include those of the colon, breast, pancreas and cervix. Similarly, holocanthone 's broad cytotoxicity against several cell lines may make it useful as a broad-spectrum anticancer drug candidate. Its ability to reduce the viability of colon cancer cells to a greater extent than 5-fluorouracil(10 compared to 23 μΜ) warrants further investigation. Holocanthone derivatives show greater selectivity towards malignant compared to non-malignant cells, which supports their development as suitable anticancer agents.

[00276] Three isolates examined in this study demonstrated potent anticancer activity greater than or equal to clinically available chemotherapeutic agents. Scopoletin, a coumarin isolate from Castela macrophylla showed high potency against colon cancer cells with minimal impact on normal cells, making it a candidate worth further investigations. The quassinoids, glucarabuloneglucoside and holocanthone showed growth inhibitory potency against breast cancer cells, and the former showed moderate inhibition of CYP1A1 and CYP1B1 enzymes with non-competitive binding kinetics. Gg thwarted 1,6-BPQ-mediated increases in ROS production in MCF-IOA cells. This indicates its antioxidant actions in addition to its ability to inhibit B[a]P-mediated CYPl induction contribute to its potential to serve as a chemopreventive agent. Considering both its ability to directly inhibit the activities of CYPslAl and 1B1 and selectively impact viability of malignant cells by inducing apoptosis, Gg displays potential as a chemopreventive and chemotherapeutic lead. It further validates the on-going search for leads from natural products from endemic tropical biodiversity.

[00277] References

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Claims

CLAIMS In the claims:

1. A method of preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a quassinoid compound, thereby preventing the cancer.

2. A method of preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a coumarin compound, thereby preventing the cancer.

3. A method of preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a crude extract comprising a quassinoid compound, thereby preventing the cancer.

4. A method of preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a crude extract comprising a coumarin compound, thereby preventing the cancer.

5. The method of claim 1 or 3, wherein the quassinoid is glaucarubolone-15-Ο-β- D-glucopyranoside.

6. The method of claim 1 or 3, wherein the quassinoid is holocanthone.

7. The method of claim 1 or 3, wherein the quassinoid consists of a quassinoid comprising a glycoside on ring D.

8. The method of claim 1 or 3, wherein the quassinoid consists of a quassinoid comprising an acetyl moiety on ring D.

9. The method of claim 2 or 4, wherein the coumarin is scopoletin.

10. The method of claim 1, 2, 5, 6, 7, 8 or 9, wherein the compound is extracted from Castela macrophylla.

11. The method of any one of claims 1 to 10, wherein the compound induces apoptosis in a cancer cell.

12. The method of any one of claims 1 to 11, wherein the cancer is selected from colon cancer, liver cancer and breast cancer.

13. The method of any one of claims 1 to 12, wherein the subject is at risk of developing cancer.

14. The method of any one of claims 1 to 13, wherein the subject is exposed to polyaromatic hydrocarbons (PAHs).

15. The method of any one of claims 1 to 14, wherein the preventing comprises reducing the activity of a CYP450 enzyme.

16. The method of claim 15, wherein the activity is reduced by about 20% relative to the activity of the CYP450 enzyme prior to administration of the compound.

17. The method of claim 15, wherein the activity is reduced by about 50% relative to the activity of the CYP450 enzyme prior to administration of the compound.

18. The method of claim 1, 3, 5, 6, 7, 8, 10, 11, 12, 13 or 14, wherein the preventing comprises inhibiting the activity of a CYP450 enzyme by the compound.

19. The method of claim 18, wherein the IC₅₀ value of the compound is about 10 uM.

20. The method of claim 18, wherein the IC₅₀ value of the compound is about 5 uM.

21. The method of claim 18, wherein the IC₅₀ value of the compound is about 1 uM.

22. The method of any one of claims 1 to 14, wherein the preventing comprises reducing the expression of a CYP450 mRNA.

23. The method of claim 22, wherein the expression is reduced by about 70%> relative to the expression of the CYP450 mRNA, prior to administration of the compound.

24. The method of claim 22, wherein the expression is reduced by about 50% relative to the expression of the CYP450 mRNA, prior to administration of the compound.

25. The method of claim 22, wherein the expression is reduced by about 30% relative to the expression of the CYP450 mRNA, prior to administration of the compound.

26. The method of claim 22, 23, 24 or 25, wherein the expression is reduced in the presence of PAH.

27. The method of claim 22, 23, 24 or 25, wherein the expression is reduced in the presence of BaP.

28. The method of any one of claims 15 to 27, wherein the CYP450 consists of a member of the CYP1 family of enzymes.

29. The method of any one of claims 15 to 27, wherein the CYP450 consists of CYP1A1, CYP1A2, or CYP1B1.

30. The method of any one of claims 1 to 14, wherein the preventing comprises reducing intracellular reactive oxygen species (ROS) levels relative to intracellular reactive oxygen species (ROS) levels prior to administration of the compound.

31. The method of claim 30, wherein the intracellular reactive oxygen species (ROS) levels are reduced by about 40%>.

32. The method of any one of claims 1 to 14, wherein the preventing comprises reducing the ability of a BPQ to increase intracellular reactive oxygen species (ROS) levels relative to the ability of a BPQ to increase intracellular reactive oxygen species (ROS) levels prior to administration of the compound.

33. The method of claim 32, wherein the ability is reduced by about 1.5-fold.

34. A method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a coumarin compound, thereby treating the cancer.

35. The method of claim 34, wherein the coumarin is scopoletin.