Methoxyestradiol

Methoxyestradiol Adonia E. Papathanassiu, Shawn J. Green*, Davida K. Grella and B. Kim Lee Sim EntreMed, Inc., 9640 Medical Center Drive, Rockville, MD 20850 USA * corresponding author tel: 301 738 2494, fax: 301 217 9858, e-mail: [email protected] DOI: 10.1006/rwcy.2000.08010.

SUMMARY 2-Methoxyestradiol (2ME2) is a byproduct of estrogen metabolism that was shown to suppress the growth of rapidly dividing endothelial and tumor cells in vitro and in vivo.

or cell cycle arrest. It has no effect on confluent or slowly dividing cells. Systemic administration of 2ME2 results in inhibition of pathophysiological neovascularization, suppression of tumor growth, and reduction of cancer metastasis.

BACKGROUND

CELLULAR SOURCES AND TISSUE EXPRESSION

Discovery

Cellular sources that produce

2ME2 is a byproduct of estrogen metabolism that was widely considered to be devoid of biological properties. A recent observation that fractions of human urine comprising catecholic and nonpolar estrogen metabolites was able to inhibit endothelial cell proliferation led to the discovery of the antimitotic activity of 2ME2. It is presently recognized that 2ME2 inhibits the growth of rapidly dividing endothelial and tumor cells in vitro and in vivo. This renders 2ME2 as the very first endogenous chemotherapeutic agent to be discovered.

Alternative names The systematic name of 2ME2 is: 1,3,5 (10)oestratriene-2,3,17 -triol 2-methyl ether.

Main activities and pathophysiological roles 2ME2 inhibits the growth of actively dividing proliferating cells, including endothelial, smooth muscle cell, and tumor cells via induction of apoptosis

2ME2 is synthesized via hydroxylation and subsequent O-methylation of 17 -estradiol at the 2-position. Estradiol can be hydroxylated at various positions by NADPH-dependent cytochrome P450 present in liver and a variety of other tissues, including placenta, prostate, brain, kidney, heart, liver, testes, ovary, and uterus. The relative amounts of 2- and 4-hydroxyestrogens formed in this process are considered to be estrogen- and tissue-dependent. Following hydroxylation, catechol-O-methyl transferase (COMT) converts 2-hydroxyestradiol to 2ME2. COMT is a ubiquitous enzyme found in a wide variety of organs such as liver, placenta, testis, kidney, spleen, brain, and blood.

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators In vivo generation of 2ME2 is enhanced by compounds known to stimulate 2-hydroxylation of estrogens, such as indole-2-carbinol and sodium phenobarbital, and suppressed by competitive and

836 Adonia E. Papathanassiu, Shawn J. Green, Davida K. Grella and B. Kim Lee Sim noncompetitive inhibitors of COMT. Competitive inhibitors of COMT include endogenously produced catechols, e.g., epinephrine and norepinephrine, and exogenously administered dietary polyphenols such as quercetin and fisetin. Noncompetitive inhibitors of COMT include S-adenosyl-L-homocysteine. In addition, endogenous production 2ME2 is likely influenced by the available levels of S-adenosyl-Lmethionine, an essential cofactor for enzymatic O-methylation of estrogens by COMT. Other endogenous modulators include demethylases and 17 -hydroxysteroid dehydrogenases (17 -HSDs). Demethylases are enzymes that catalyze the conversion of 2ME2 to its precursor 2-hydroxyestradiol, while 17 -HSDs catalyze the interconversions between 2ME2 and 2-methoxyestrone in a celldependent manner. Specifically, type II 17 -HSD, found in the uterus, catalyzes the conversion of estradiol to estrone, whereas type I, found in the breast, catalyzes the conversion of estrone to estradiol.

RECEPTOR UTILIZATION Although an estrogen derivative, 2ME2 has little binding affinity for the classical estrogen receptor (0.5% compared to estradiol) and lacks estrogenic activity in vivo. Studies on the mechanism of the antimitotic action of 2ME2 have shown that 2ME2 inhibits tubulin polymerization by interacting with the colchicine-binding site of tubulin (Ki = 22 mM). However, it is not clear if the interaction between 2ME2 and tubulin is responsible for 2ME2's antiproliferative activity, since concentrations required for inhibition of cell growth are far less than the ones required for competitive inhibition of the binding of colchicine to tubulin.

IN VITRO ACTIVITIES

In vitro findings 2ME2 exerts a variety of in vitro biological activities that include cell cycle arrest and/or apoptosis of rapidly proliferating cells, inhibition of endothelial cell migration, downregulation of endothelial NO production, stimulation of progesterone production in ovarian cells, inhibition of prostaglandin, and inhibition of catecholamine uptake by aortic cells. The effects of 2ME2 on cell proliferation are summarized in Table 1. In vitro findings related to

the mechanism of the cytostatic and/or apoptotic action of 2ME2 are summarized in Table 2.

Bioassays used The following bioassays were used for the in vitro experiments.  Proliferation assay. Subconfluent cultures of endothelial or tumor cells are cultured with growth factor(s) in the presence or absence of an angiogenic inhibitor. The assay is terminated 72± 96 hours later. Cell growth is determined by cell counting or DNA synthesis via thymidine or uridine incorporation.  Wound migration assay. Confluent endothelial cell monolayers are wounded with a razor blade. Cells from the wound area migrate in the presence of growth factors. After a short incubation in the presence or absence of an angiogenic inhibitor, the cells are fixed and stained and the number of migrating cells are counted under a light microscope. The following bioassays were used for the in vivo experiments:  Chorioallantoic membrane (CAM). Disks containing various concentrations of an angiogenic inhibitor are placed on the chorioallantoic membrane of 6-day-old chicken embryos. After 48 hours of incubation, CAMs are checked for the presence of avascular zones in the periphery of the disk using a stereomicroscope.  Mouse cornea micropocket assay. A pellet containing sucrose octasulfate, hydron, and 80±100 ng bFGF is placed into the cornea micropocket of a mouse and the cornea angiogenesis is evaluated by slit-lamp microscopy.  Primary tumor model. Mice or rats are implanted with a specific number of a certain tumor cell type. Systemic administration of an angiogenic inhibitor is initiated after the tumors have grown to a palpable size. Tumor size is assessed with a caliper and the tumor volume is determined.  Metastatic tumor model. Mice are implanted with tumor cells that exhibit a metastatic potential. The tumors are allowed to grow to a certain size before they are surgically removed. After tumor resection, systemic treatment with an angiogenic inhibitor is initiated. At the end of the experiment, the number of pulmonary metastases and/or the lung weight of treated mice are compared to those of mice that received a control treatment or did not undergo tumor resection.

Methoxyestradiol 837 Table 1

Antimitotic activity of 2ME2

Inhibitory concentration

Cell type

< 0.5 mM

Endothelial cell type: Human umbilical vein (HUVEC) Bovine brain capillary (BCE) (Fotsis et al., 1994) Bovine pulmonary artery (PAEC) (Yue et al., 1997) Human tumor cell type: Colon (HCT-116) (Cushman et al., 1995), neuroblastoma (SF-539) (Cushman et al., 1995), melanoma (UACC-62) (Cushman et al., 1995), ovarian (OVCAR-3) (Cushman et al., 1995), breast (MDA-MB-435, MCF-7) (Zhu and Conney, 1998) lymphoblast (Jurkat) (Attalla et al., 1996) Murine tumor cell type: Melanoma (B16BL6, B16F10)

0.5±1 mM

Tumor cell type: Human lung (HOP-2) (Mukhopadhyay and Roth, 1997), human renal (SN 12-C) (Cushman et al., 1995), murine endothelial (EOMA)

1±5 mM

Nontumor cell type: Human skin fibroblast (HFK2) (Fotsis et al., 1994), hamster lung (V79) (Aizu-Yokota et al., 1995), porcine ovarian granulosa (Spicer and Hammond, 1989), rabbit smooth muscle aorta (Nishigaki et al., 1995) Human tumor cell type: Lung (H460, A549) (Mukhopadhyay and Roth, 1997), neuroblastoma (SH-SY5Y) (Cushman et al., 1995), prostate (DU-145) (Cushman et al., 1995), breast (MDA-MB-231) (Zhu and Conney, 1998), lymphoblast (TK6, WTK1) (Seegers et al., 1997), cervical (HeLa) (Seegers et al., 1989) Murine tumor cell type: Endothelial (H5V) (Josefsson and Tarkowski, 1997)

Table 2 Potential molecular targets of 2ME2 in various cell types Molecular target

Cell type

Reference

Induction of p53 and/or p21WAF1/CIP1

Endothelial; tumor

Mukhopadhyay and Roth, 1997

Induction of SAPK/JNK, FAS

Endothelial (BPAEC)

Yue et al., 1997

Induction of cAMP

Breast tumor (MCF-7)

Lotering et al., 1992

Activation of p34

Breast tumor (MCF-7)

Lotering et al., 1996

Inhibition of tubulin polymerization

Chinese hamster (V79)

Aizu-Yokota et al., 1995

Inhibition of calmodulin-dependent phosphodiesterase

Breast tumor (MCF-7)

Lotering et al., 1992

cdc2

838 Adonia E. Papathanassiu, Shawn J. Green, Davida K. Grella and B. Kim Lee Sim

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

model, presumably by suppressing production of endothelial NO, which is a well-known proinflammatory mediator.

Normal physiological roles

IN THERAPY

It has been suggested that endogenous formation of 2ME2 protects target organs against estrogen-induced cancers. This hypothesis is based on the observation that estrogen-induced tumors can be indirectly correlated with reduced levels of 2ME2. For example, epidemiological studies have shown that development of breast cancer can be linked to sustained emotional stress and the release of endogenous catecholamines; catecholamines are known to inhibit COMT and formation of 2ME2 from 2-hydroxyestradiol. Additional epidemiological studies have shown that Caucasian females with inherited low activity of COMT are at risk for estrogen-associated breast cancer.

Interactions with cytokine network 2ME2 was shown to downregulate nitric oxide (NO) production by endothelial cells (H5V) in vitro. In contrast, 2ME2 had no effect on IL-6 production by the same cells after stimulation with TNF or IFN .

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects The exact levels of endogenous circulating 2ME2 are not known. Studies regarding the plasma concentration of total methoxyestrones suggest that, under physiological conditions, circulating levels of 2ME2 are in the picomolar range; these levels may reach the nanomolar range during late pregnancy.

Preclinical ± How does it affect disease models in animals? The in vivo biological properties of 2ME2 are highlighted in Table 3.

Effects of therapy: cytokine, antibody to cytokine inhibitors, etc. Studies with animals bearing type II collagen-induced arthritis indicate that 2ME2 treatment does not affect antitype II collagen antibody generation (IgG1, IgG2a, IgG3, and IgM) or IL-6 production (Josefsson and Tarkowski, 1997). In addition, 2ME2 therapy does not alter the number of leukocytes in the bone marrow, peripheral blood, and thymus, although it diminishes the number of Ig+ splenocytes. Furthermore, 2ME2 does not affect T cell-mediated DTH response to oxazolone.

Pharmacokinetics Intravenous bolus administration of 3 mg/kg 2ME2 in male and female CDF1 mice led to initial plasma concentrations of > 1.2 mM 2ME2 with rapid elimination to nondetectable levels within 1.5 hours (Squillace et al., 1998). Oral administration of 100 mg/kg 2ME2 resulted in even lower plasma concentrations. Low levels of 2ME2 were attributed to low bioavailability ( 1.5%) due to high first-pass metabolism rather than poor oral absorption, since a high fraction of orally given 2ME2 was absorbed by both male ( 85%) and female ( 75%) mice. In vivo metabolism of 2ME2 Table 3 Induction of permanent dormancy by repeated cycles of endostatin protein treatmenta

Role in experiments of nature and disease states

Primary tumor model

Number of treatment cycles

Lewis lung carcinoma (LLC)

6

2ME2 suppresses tumor growth directly, by reducing tumor cell proliferation, and indirectly, by reducing tumor neovascularization via inhibition of endothelial cell migration, proliferation, and tube formation. In addition, 2ME2 inhibits the development of type II collagen-induced arthritis in the respective animal

T241 fibrosarcoma

4

B16F10 melanoma

2

a

The study was described by Boehm et al. (1997). Recombinant murine endostatin protein treatment was dosed at 20 mg/kg per day.

Methoxyestradiol 839 included conversion to 2-methoxyestrone and conjugation via glucuronidation and/or sulfation. In vitro studies suggested that formation of 2-methoxyestrone by human liver microsomes was NAD-dependent and required the presence of 17 -hydroxysteroid dehydrogenase (Kuffel et al., 1998). In addition, hydroxylation of 2ME2 by NADPH-dependent cytochrome P450 was only observed in murine liver microsomal metabolism of 2ME2, while cytochrome P450-dependent catalyzed O-demethylation of 2ME2 was only observed in human liver microsomal metabolism.

Toxicity Administration of therapeutic doses of 2ME2 for prolonged periods of time did not induce hair loss, gastrointestinal disturbance, or reduction of circulating leukocytes of disease animals (Josefsson and Tarkowski, 1997). 2ME2 lacks estrogenic activity, measured by uterine growth of ovariectomized or prepubertal rats continuously exposed to high concentrations of the drug (Martucci and Fishman, 1979). The diestrous stage of ovariectomized mice was also not altered after treatment with 2ME2, whereas estradiol resulted in rapid induction of estrus phase. Moreover, chronic administration of 2ME2 failed to induce renal carcinogenesis in Syrian hamsters (Liehr et al., 1986). The only side-effect of 2ME2 reported thus far is a minor weight loss when the drug is given at doses of 150 mg/kg per day (Klauber et al., 1997).

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LICENSED PRODUCTS 2ME2 can be purchased by several vendors including Sigma Chemicals Co. (Saint Louis, MI) and Steraloids Inc. (Wilton, NH).