Methods of identifying modulators of the estrogen receptor

Details Methods of identifying modulators of the estrogen receptor Methods of identifying modulators of the estrogen receptor

1999-06-17

2001-03-20

Marschel, Ardin H. (Department: 1631)

Chemistry: analytical and immunological testing

Including titration or ph determination

C436S172000, C436S063000, C436S111000, C564S324000

Reexamination Certificate

active

06204067

ABSTRACT:

FIELD OF THE INVENTION
The present invention pertains to ways of identifying compounds that can modulate the estrogen receptor without having the undesirable side-effects of pharmaceuticals such as tamoxifen.
BACKGROUND OF THE INVENTION
Tamoxifen is the most commonly used treatment for breast cancer [Jaiyesimi et al.,
Journal of Clinical Oncology
13:513-529 (1995)]. In addition, it is currently being considered for widespread use in healthy women for breast cancer prevention [Jordan,
Proceedings of the Society for Experimental Biology
&
Medicine
208:144-149 (1995); Fisher et al.,
J.Natl.Canc.Inst
. 90:1371-1388 (1998)]. Yet, despite its widespread use, its mechanisms of action remain obscure. Tamoxifen is a known estrogen receptor modulator that acts as an antagonist or partial-agonist. But it has also been reported to have many pleiotropic effects both in vivo and in vitro that cannot be explained by an interaction with the estrogen receptor [Kellen,
Tamoxifen: Beyond the Antiestrogen
, Birkhäuser, Boston (1996)]. For example, tamoxifen has been shown to enhance drug sensitivity of multidrug resistant cells [Chatterjee and Harris,
British Journal of Cancer
62:712-717 (1990); Berman et al.,
Blood
77:818-825 (1991); Berman et al.,
Leukemia
9:1631-1637 (1995); Weinlander et al.,
Journal of Cancer Research
&
Clinical Oncology
123:452-455 (1997); Altan et al.,
J.Exp.Med
. 187: 1583-1598 (1998)], inhibit bone resorption and osteoporosis both in vivo and in vitro [Love et al.,
N.Engl.J.Med
. 326:852-856 (1992)] and inhibit a number of channels, including the volume activated chloride channel [Zhang et al.,
Journal of Clinical Investigation
94:1690-1697 (1994); Ehring et al.,
J.Gen.Physiol
. 104:1129-1161 (1994)] and calcium channels[Greenberg et al.,
Cancer Res
. 47: 70-74 (1987); Song et al.,
Journal of Pharmacology
&
Experimental Therapeutics
277: 1444-1453 (1996); Williams et al.,
J.Biol.Chem
. 271:12488-12495 (1996); Turner et al.,
Endocrinology
122:1146-1150 (1988)]. These effects have been attributed to inibition of P-glycoprotein [Callaghan and Higgins
Br.J.Cancer
71:294-299 (1995)], calmodulin [Williams et al.,
J.Biol.Chem
. 271:12488-12495 (1996)], and direct channel interaction [Zhang et al.,
Journal of Clinical Investigation
94:1690-1697 (1994)] respectively. Thus, the administration of tamoxifen as an estrogen receptor modulator results in a number of undesirable side-effects which are totally independent of the modulation of the estrogen receptor.
As mentioned above, tamoxifen has been shown to enhance drug sensitivity of multidrug resistant cells. Different drug-resistant cells overexpress a variety of membrane proteins including a subunit of a vacuolar H
+
-ATPase [Ma, L et al.,
Biochem Biophys. Res. Commun
., 182:675-681 (1992)], a protein with homology to CFTR [Cole et al.,
Science
, 258:1650-1654 (1992)] and the P-glycoprotein, a 170-180 kD plasma membrane glycoprotein [Gottesman et al.,
Annu. Rev. Biochem
., 62:385-427 (1993)]. The most generally accepted hypothesis for MDR suggests the P-glycoprotein uses ATP to power a molecular pump that removes chemotherapeutic molecules from the cell [Dano et al.,
Biochem Biophys
., 323:466-483 (1973) and reviewed in Gottesman et al.,
Annu. Rev. Biochen
., 62:385-427 (1993)]. This model proposes that chemotherapeutic agents diffuse down a concentration gradient into the cell and that the pump either transports the drugs out of the cytosol or serves as a flippase to expel them from the bilayer [Higgins et al.,
TIBS
, 17:18-21 (1992)]. More recently it has been shown that changes of organelle pH associated with multidrug resistance (MDR) in tumor cells can lead to multi-drug resistance [see U.S. Pat. No. 5,851,789, Issued on Dec. 22, 1998, and U.S. application Ser. No. 09/080,739, filed May 18, 1998, the disclosures of which are each hereby incorporated by reference herein in their entireties] and that the effect of tamoxifen on MDR is related to the de-acidification of the cellular organelles.
Therefore, there is a need to identify modulators of the estrogen receptor that do not result in the same side-effects as tamoxifen. Thus, there is a need to determine the effects of tamoxifen that are independent of its role in modulation of the estrogen receptor. Furthermore, there is a need to identify the role tamoxifen plays in causing these effects. Finally, there is a need to identify assays for screening potential estrogen receptor modulators that can modulate the estrogen receptor as tamoxifen does, but do not cause the other undesirable effects.
The citation of any reference herein should not be construed as an admission that such reference is available as “Prior Art” to the instant application.
SUMMARY OF THE INVENTION
The present invention provides methods of selecting compounds/agents that have the positive pharmaceutical effects of tamoxifen, without the undesirable side-effects. These compounds can be known analogs of estrogen and/or effectors (e.g., agonists or antagonists) of the estrogen receptor, or alternatively, the compounds can be tested for estrogen receptor binding activity prior to/or after performing the methods disclosed herein.
Therefore the present invention provides a method of selecting a modulating agent of the estrogen receptor that does not dissipate a transmembrane pH gradient to the extent that tamoxifen does. One such embodiment comprises contacting an agent with a vesicle that is surrounded by a membrane under conditions in which tamoxifen dissipates a transmembrane pH gradient and then determining the level (e.g., magnitude) of the transmembrane pH gradient. In this case, the transmembrane pH gradient forms because the pH inside of the membrane differs from that outside of the membrane.
In a preferred embodiment the agent has previously been shown to be able to modulate the estrogen receptor. A modulating agent that does not dissipate the transmembrane pH gradient to the extent that tamoxifen does is identified and/or selected. In a preferred embodiment the modulating agent that is selected does not measurably (and/or significantly) dissipate the transmembrane pH gradient.
An alternative embodiment further provides the step of determining the level (e.g., magnitude) of surface charge on the membrane. A modulating agent is then selected when it neither dissipates a transmembrane pH gradient to the extent that tamoxifen does, nor alters the surface charge on the membrane to the extent that tamoxifen does. Preferably, the modulating agent neither measurably (and/or significantly) dissipates a transmembrane pH gradient nor measurably (and/or significantly) alters the surface charge on the membrane.
The determination of the level of the transmembrane pH gradient can be performed using a number of different methods including directly with an electrode or a calibratable pH indicator or pH sensitive probe. In one particular embodiment, the level of the transmembrane pH gradient is performed with a fluorescent probe. In an embodiment exemplified below the fluorescent probe is acridine orange.
In one embodiment, the vesicle is a mammalian cell. In a particular embodiment of this type the mammalian cell is a human cell. In another embodiment, the vesicle is a organelle. In one such embodiment the organelle is a mammalian organelle. In a particular embodiment of this type the organelle is a secretory compartment. In a particular embodiment exemplified below, the mammalian organelle is a recycling endosome. In one such method, determing the level of the transmembrane pH gradient is performed with fluorescent-labeled transferrin.
In another embodiment the vesicle is a yeast vacuole. In still another embodiment the vesicle is an inverted bacterial membrane vesicle. In yet another embodiment the vesicle is a liposome. In one such method, determining the level of the transmembrane pH gradient is performed using

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