CA2376067A1 - Use of paclitaxel and steroid derivatives as aromatase inhibitors for the treatment of cancer - Google Patents
Use of paclitaxel and steroid derivatives as aromatase inhibitors for the treatment of cancer Download PDFInfo
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- CA2376067A1 CA2376067A1 CA002376067A CA2376067A CA2376067A1 CA 2376067 A1 CA2376067 A1 CA 2376067A1 CA 002376067 A CA002376067 A CA 002376067A CA 2376067 A CA2376067 A CA 2376067A CA 2376067 A1 CA2376067 A1 CA 2376067A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
- A61K31/566—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol having an oxo group in position 17, e.g. estrone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
There is provided use of a material selected from (i) microtubule stabilisin g agent; (ii) microtubule disrupter; (iii) a compound of the formula A-B where in A is an oxyhydrocarbyl group and B is a cyclic group; and (iv) a compound of the formula C-D wherein C is a sulphamate group and D is a cyclic group, for the manufacture of a medicament for the inhibition of tumour necrosis factor .alpha. (TNF.alpha.) stimulated aromatase activity.
Description
USE
The present invention relates to inhibition of tumour necrosis factor a stimulated aromatase activity.
Introduction Synthesis of oestrone from androstenedione, by the aromatase enzyme complex, is an important source of oestrogen available to support the growth of hormone-dependent tumours (1). Cytokines, such as interleukin 6 (IL-6) and tumour necrosis factor a (TNFa) and Prostaglandin E2 (PGEZ), can all stimulate aromatase activity (2-4).
Many breast tumours are infiltrated by macrophages and lymphocytes and there is evidence that these cells may be an important source of the factors that can stimulate aromatase activity (5-6).
The role that the immune system has in the development of cancers remains controversial (7). In women receiving long-term immune suppressive therapy, however, the incidence of breast cancer is reduced (8). This suggests that the immune system may have an immunostimulatory role in the development of breast cancer. Support for such a role, possibly acting via cytokine stimulation of oestrogen synthesis, was obtained by comparing the abilities of conditioned medium (CM) collected from white blood cells of an immunosuppressed subject or woman with breast cancer to stimulate aromatase (9). Stimulation of the activity of this enzyme was greatly reduced by CM collected from cells of the immunosuppressed subject.
Furthermore, concentrations of TNFa were barely detectable in CM from cells of the immunosuppressed subject in contrast to the high levels present in CM from cells of a woman with breast cancer. It is likely, therefore, that TNFa has an important role in regulating aromatase activity.
TNFa, like other cytokines, acts by interacting with cell-surface receptors (10).
Using human macrophages, paclitaxel, a compound that stabilises microtubules, was found to rapidly down-regulate TNFa receptors (11). The endogenous oestrogen metabolite, 2-methoxyoestradiol (2-me0E2), was recently shown to have a similar effect to that of paclitaxel on microtubule stability (12, 13).
Aspects of the present invention are defined in the appended claims.
In a first aspect the present invention provides use of a material selected from (i) microtubule stabilising agent; (ii) microtubule disrupter, preferably Paclitaxel; (iii) a compound of the formula A-B wherein A is an oxyhydrocarbyl group and B is a cyclic group, preferably 2-me0E2; and (iv) a compound of the formula C-D
wherein C is an sulphamate group and D is a cyclic group, preferably MeOEMATE; for the manufacture of a medicament for the inhibition of tumour necrosis factor a (TNFa) stimulated aromatase activity.
In the following discussion and throughout the description of the present invention it will be understood by a person skilled in the art that ~ 2-me0E2 is a preferred and is exemplary of a compound of the formula A-B
wherein A is an oxyhydrocarbyl group and B is a cyclic group ~ 2-MeOEMATE is a preferred and is exemplary of a compound of the formula C-D
wherein C is an sulphamate group and D is a cyclic group. Preferably 2-MeOEMATE is exemplary of a compound of the formula C-D-E wherein C is an sulphamate group, D is a cyclic group, and E is an oxyhydrocarbyl group ~ Paclitaxel is a preferred and is exemplary of a microtubule disrupter In the present investigation we have examined the ability of paclitaxel and 2-me0E2 to antagonise TNFa stimulated activity in cultured fibroblasts derived from breast tissues.
The aromatase enzyme, which converts androstenedione to oestrone, regulates the availability of oestrogen to support the growth of hormone-dependent breast tumours.
Biological response modifiers, including cytokines, such as interleukin 6 (IL-6) and tumour necrosis factor a (TNFa) or prostaglandin Ez (PGEZ) can stimulate aromatase activity. These factors may originate from cells of the immune system that infiltrate breast tumours. Paclitaxel, which is used in the treatment of breast cancer, stabilises microtubules and has previously been shown to rapidly down-regulate TNF-receptors on human macrophages. The endogenous oestrogen metabolite, 2-methoxyoestradiol (2-me0E2) also acts to stabilise microtubules. In this study we have examined the ability of paclitaxel, 2-me0E2 and 2-methoxyoestrone-3-O-sulphamate (2-meOEMATE) to antagonise TNFa-stimulated aromatase activity in stromal fibroblasts derived from normal or malignant breast tissues. Paclitaxel inhibited basal and TNFa-stimulated aromatase activities by 88% and 91% respectively. 2-Me0E2 also reduced basal and TNFa-stimulated aromatase activities by 46% and 56% respectively. 2-MeOEMATE also reduced basal and TNFa-stimulated aromatase activities. Paclitaxel, 2-me0E2 and 2-MeOEMATE also inhibited stimulation of aromatase activity by IL-6 plus its soluble receptor and PGE~.
The 16a-hydroxylated derivative of 2-meoE2, 2-methoxyoestriol (2-me0E3), which does not bind to microtubules, was less effective at inhibiting TNFa-stimulated aromatase activity. Increased 2-hydroxylation of oestrogens, and subsequent formation of their 2-methoxy derivatives, may be associated with a reduced risk of breast cancer. It is possible that the pathway of oestrogen metabolism may influence the ability of stromal cells to respond to cytokine stimulation.
The cyclic group may be a single ring or it is a polycyclic ring structure.
Here, the term ''polycyclic" includes fused and non-fused ring structures including combinations thereof.
In one aspect, the cyclic group may contain any one or more of C, H, O, N, P, halogen (including Cl, Br and I), S and P.
At least one of the cyclic groups may be a heterocyclic group (a heterocycle) or a non-heterocyclic group.
At least one of the cyclic groups may be a saturated ring structure or an unsaturated ring stricture (such as an aryl group).
The present invention relates to inhibition of tumour necrosis factor a stimulated aromatase activity.
Introduction Synthesis of oestrone from androstenedione, by the aromatase enzyme complex, is an important source of oestrogen available to support the growth of hormone-dependent tumours (1). Cytokines, such as interleukin 6 (IL-6) and tumour necrosis factor a (TNFa) and Prostaglandin E2 (PGEZ), can all stimulate aromatase activity (2-4).
Many breast tumours are infiltrated by macrophages and lymphocytes and there is evidence that these cells may be an important source of the factors that can stimulate aromatase activity (5-6).
The role that the immune system has in the development of cancers remains controversial (7). In women receiving long-term immune suppressive therapy, however, the incidence of breast cancer is reduced (8). This suggests that the immune system may have an immunostimulatory role in the development of breast cancer. Support for such a role, possibly acting via cytokine stimulation of oestrogen synthesis, was obtained by comparing the abilities of conditioned medium (CM) collected from white blood cells of an immunosuppressed subject or woman with breast cancer to stimulate aromatase (9). Stimulation of the activity of this enzyme was greatly reduced by CM collected from cells of the immunosuppressed subject.
Furthermore, concentrations of TNFa were barely detectable in CM from cells of the immunosuppressed subject in contrast to the high levels present in CM from cells of a woman with breast cancer. It is likely, therefore, that TNFa has an important role in regulating aromatase activity.
TNFa, like other cytokines, acts by interacting with cell-surface receptors (10).
Using human macrophages, paclitaxel, a compound that stabilises microtubules, was found to rapidly down-regulate TNFa receptors (11). The endogenous oestrogen metabolite, 2-methoxyoestradiol (2-me0E2), was recently shown to have a similar effect to that of paclitaxel on microtubule stability (12, 13).
Aspects of the present invention are defined in the appended claims.
In a first aspect the present invention provides use of a material selected from (i) microtubule stabilising agent; (ii) microtubule disrupter, preferably Paclitaxel; (iii) a compound of the formula A-B wherein A is an oxyhydrocarbyl group and B is a cyclic group, preferably 2-me0E2; and (iv) a compound of the formula C-D
wherein C is an sulphamate group and D is a cyclic group, preferably MeOEMATE; for the manufacture of a medicament for the inhibition of tumour necrosis factor a (TNFa) stimulated aromatase activity.
In the following discussion and throughout the description of the present invention it will be understood by a person skilled in the art that ~ 2-me0E2 is a preferred and is exemplary of a compound of the formula A-B
wherein A is an oxyhydrocarbyl group and B is a cyclic group ~ 2-MeOEMATE is a preferred and is exemplary of a compound of the formula C-D
wherein C is an sulphamate group and D is a cyclic group. Preferably 2-MeOEMATE is exemplary of a compound of the formula C-D-E wherein C is an sulphamate group, D is a cyclic group, and E is an oxyhydrocarbyl group ~ Paclitaxel is a preferred and is exemplary of a microtubule disrupter In the present investigation we have examined the ability of paclitaxel and 2-me0E2 to antagonise TNFa stimulated activity in cultured fibroblasts derived from breast tissues.
The aromatase enzyme, which converts androstenedione to oestrone, regulates the availability of oestrogen to support the growth of hormone-dependent breast tumours.
Biological response modifiers, including cytokines, such as interleukin 6 (IL-6) and tumour necrosis factor a (TNFa) or prostaglandin Ez (PGEZ) can stimulate aromatase activity. These factors may originate from cells of the immune system that infiltrate breast tumours. Paclitaxel, which is used in the treatment of breast cancer, stabilises microtubules and has previously been shown to rapidly down-regulate TNF-receptors on human macrophages. The endogenous oestrogen metabolite, 2-methoxyoestradiol (2-me0E2) also acts to stabilise microtubules. In this study we have examined the ability of paclitaxel, 2-me0E2 and 2-methoxyoestrone-3-O-sulphamate (2-meOEMATE) to antagonise TNFa-stimulated aromatase activity in stromal fibroblasts derived from normal or malignant breast tissues. Paclitaxel inhibited basal and TNFa-stimulated aromatase activities by 88% and 91% respectively. 2-Me0E2 also reduced basal and TNFa-stimulated aromatase activities by 46% and 56% respectively. 2-MeOEMATE also reduced basal and TNFa-stimulated aromatase activities. Paclitaxel, 2-me0E2 and 2-MeOEMATE also inhibited stimulation of aromatase activity by IL-6 plus its soluble receptor and PGE~.
The 16a-hydroxylated derivative of 2-meoE2, 2-methoxyoestriol (2-me0E3), which does not bind to microtubules, was less effective at inhibiting TNFa-stimulated aromatase activity. Increased 2-hydroxylation of oestrogens, and subsequent formation of their 2-methoxy derivatives, may be associated with a reduced risk of breast cancer. It is possible that the pathway of oestrogen metabolism may influence the ability of stromal cells to respond to cytokine stimulation.
The cyclic group may be a single ring or it is a polycyclic ring structure.
Here, the term ''polycyclic" includes fused and non-fused ring structures including combinations thereof.
In one aspect, the cyclic group may contain any one or more of C, H, O, N, P, halogen (including Cl, Br and I), S and P.
At least one of the cyclic groups may be a heterocyclic group (a heterocycle) or a non-heterocyclic group.
At least one of the cyclic groups may be a saturated ring structure or an unsaturated ring stricture (such as an aryl group).
Preferably, at least one of the cyclic groups is an aryl ring.
Preferably, Group A and/or Group C and/or Group E is linked or attached to the aryl ring.
If the cyclic group is polycyclic some or all of the ring components of the compound may be fused together or joined via one or more suitable spacer groups.
The polycyclic compound may comprise a number of fused rings. In this aspect the fused rings may comprise any combination of different size rings, such as 3 six-membered rings (6,6,6), a six-membered ring, a seven-membered ring and a six-membered ring (6,7,6), a six-membered ring and two eight-membered rings (6,8,8) etc.
In one aspect, if the cyclic group is polycyclic, Group A and/or Group C
and/or Group E are attached to the same ring of the polycyclic compound.
Thus, in accordance with one aspect of the present invention, preferably the compound is a polycyclic compound.
Preferably the polycyclic compound will contain, inclusive of all substituents, no more than 50 about carbon atoms, more usually no more than about 30 to 40 carbon atoms.
The polycyclic compound can comprise at least two ring components, or at least three ring components, or at least four ring components.
Preferably, the polycyclic compound comprises four ring components.
Preferred polycyclic compounds have a steroidal ring component - that is to say a cyclopentanophenanthrene skeleton, or bio-isosteres thereof.
As is well known in the art, a classical steroidal ring structure has the generic formula of:
In the above formula, the rings have been labelled in the conventional manner.
Preferably, Group A and/or Group C and/or Group E is linked or attached to the aryl ring.
If the cyclic group is polycyclic some or all of the ring components of the compound may be fused together or joined via one or more suitable spacer groups.
The polycyclic compound may comprise a number of fused rings. In this aspect the fused rings may comprise any combination of different size rings, such as 3 six-membered rings (6,6,6), a six-membered ring, a seven-membered ring and a six-membered ring (6,7,6), a six-membered ring and two eight-membered rings (6,8,8) etc.
In one aspect, if the cyclic group is polycyclic, Group A and/or Group C
and/or Group E are attached to the same ring of the polycyclic compound.
Thus, in accordance with one aspect of the present invention, preferably the compound is a polycyclic compound.
Preferably the polycyclic compound will contain, inclusive of all substituents, no more than 50 about carbon atoms, more usually no more than about 30 to 40 carbon atoms.
The polycyclic compound can comprise at least two ring components, or at least three ring components, or at least four ring components.
Preferably, the polycyclic compound comprises four ring components.
Preferred polycyclic compounds have a steroidal ring component - that is to say a cyclopentanophenanthrene skeleton, or bio-isosteres thereof.
As is well known in the art, a classical steroidal ring structure has the generic formula of:
In the above formula, the rings have been labelled in the conventional manner.
5 An example of a bio-isostere is when any one or more of rings A, B, C and D
is a heterocyclic ring and/or when any one or more of rings A, B, C and D has been substituted and/or when any one or more of rings A, B, C and D has been modified;
but wherein the bio-isostere in the absence of the sulphamate group has steroidal properties.
In this regard, the structure of a preferred polycyclic compound can be presented as:
C. D.
A' B' wherein each ring A', B',, C' and D' independently represents a heterocyclic ring or a non-heterocyclic ring, which rings may be independently substituted or unsubstituted, saturated or unsaturated.
By way of example, any one or more of rings A', B', C' and D' may be independently substituted with suitable groups - such as an alkyl group, an aryl group, a hydroxy group, a halo group, a hydrocarbyl group, an oxyhydrocarbyl group etc.
An example of D' is a five or six membered non-heterocyclic ring having at least one substituent.
is a heterocyclic ring and/or when any one or more of rings A, B, C and D has been substituted and/or when any one or more of rings A, B, C and D has been modified;
but wherein the bio-isostere in the absence of the sulphamate group has steroidal properties.
In this regard, the structure of a preferred polycyclic compound can be presented as:
C. D.
A' B' wherein each ring A', B',, C' and D' independently represents a heterocyclic ring or a non-heterocyclic ring, which rings may be independently substituted or unsubstituted, saturated or unsaturated.
By way of example, any one or more of rings A', B', C' and D' may be independently substituted with suitable groups - such as an alkyl group, an aryl group, a hydroxy group, a halo group, a hydrocarbyl group, an oxyhydrocarbyl group etc.
An example of D' is a five or six membered non-heterocyclic ring having at least one substituent.
In one preferred embodiment, the ring D' is substituted with a ethinyl group.
If any one of rings A', B', C' and D' is a heterocyclic ring, then preferably that heterocyclic ring comprises a combination of C atoms and at least one N atom and/or at least one O atom. Other heterocyclic atoms may be present in the ring.
Examples of suitable, preferred steroidal nuclei rings A'-D' of the compounds of the present invention include rings A-D of dehydroepiandrosterone and oestrogens including oestrone.
Preferred steroidal nuclei rings A'-D' of the compounds of the present invention include rings A-D of:
1 ~ oestrones and substituted oestrones viz:
oestrone 4-OH-oestrone 6a-OH-oestrone 7a-OH-oestrone 16a-OH-oestrone 16 (3-OH-oestrone 17-deoxyoestrone oestrone oestradiols and substituted oestradiols, viz:
4-OH-17(3-oestradiol 6a-OH-17(3-oestradiol 7a-OH-17(3-oestradiol 4-OH-17a-oestradiol 6a-OH-17a-oestradiol 7a-OH-17a-oestradiol 16a-OH-17a-oestradiol 16a-OH-17(3-oestradiol 163-OH-17a-oestradiol 16(3-OH-17(3-oestradiol 17a-oestradiol 17(3-oestradiol 17a-ethinyl-17(3-oestradiol 17(3-ethinyl-17a-oestradiol 17-deoxyoestradiol oestriols and substituted oestriols. viz:
oestriol 4-OH-oestriol 6a-OH-oestriol 7a-OH-oestriol 17-deoxyoestriol dehydroepiandrosterones and substituted dehydroepiandrosterones viz.
dehydroepiandrosterones 6a-OH-dehydroepiandrosterone 7a-OH-dehydroepiandrosterone 16a-OH-dehydroepiandrosterone 163-OH-dehydroepiandrosterone In general terms the ring system A'B'C'D' may contain a variety of non-interfering substituents. In particular, the ring system A'B'C'D' may contain one or more hydroxy, alkyl especially lower (C1-C6) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (C,-C6) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
In an alternative embodiment, the polycyclic compound may not contain or be based on a steroid nucleus. In this regard, the polycyclic compound may contain or be based on a non-steroidal ring system - such as diethylstilboestrol, stilboestrol, coumarins, flavonoids, combrestatin and other ring systems. Other suitable non-steroidal compounds for use in or as the composition of the present invention may be found in US-A-5567831.
Preferably, Group A or Group C and/or Group E are attached to the same ring of the cyclic compound of the present invention at positions ortho with respect to each other.
Preferably, the polycyclic compound has a steroidal structure and Group A or Group E
is attached to the A ring.
Preferably, the Group A or Group E is attached to the 2 position of the A ring of the steroidal structure.
Preferably, the polycyclic compound has a steroidal structure and Group C is attached to the A ring.
Preferably, the Group C is attached to the 3 position of the A ring of the steroidal structure.
Group A is a oxyhydrocarbyl group.
The term "oxyhydrocarbyl group" as used herein means a group comprising at least C, H and O and may optionally comprise one or more other suitable substituents.
Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the oxyhydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the oxyhydrocarbyl group may contain hetero atoms.
Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur and nitrogen.
If any one of rings A', B', C' and D' is a heterocyclic ring, then preferably that heterocyclic ring comprises a combination of C atoms and at least one N atom and/or at least one O atom. Other heterocyclic atoms may be present in the ring.
Examples of suitable, preferred steroidal nuclei rings A'-D' of the compounds of the present invention include rings A-D of dehydroepiandrosterone and oestrogens including oestrone.
Preferred steroidal nuclei rings A'-D' of the compounds of the present invention include rings A-D of:
1 ~ oestrones and substituted oestrones viz:
oestrone 4-OH-oestrone 6a-OH-oestrone 7a-OH-oestrone 16a-OH-oestrone 16 (3-OH-oestrone 17-deoxyoestrone oestrone oestradiols and substituted oestradiols, viz:
4-OH-17(3-oestradiol 6a-OH-17(3-oestradiol 7a-OH-17(3-oestradiol 4-OH-17a-oestradiol 6a-OH-17a-oestradiol 7a-OH-17a-oestradiol 16a-OH-17a-oestradiol 16a-OH-17(3-oestradiol 163-OH-17a-oestradiol 16(3-OH-17(3-oestradiol 17a-oestradiol 17(3-oestradiol 17a-ethinyl-17(3-oestradiol 17(3-ethinyl-17a-oestradiol 17-deoxyoestradiol oestriols and substituted oestriols. viz:
oestriol 4-OH-oestriol 6a-OH-oestriol 7a-OH-oestriol 17-deoxyoestriol dehydroepiandrosterones and substituted dehydroepiandrosterones viz.
dehydroepiandrosterones 6a-OH-dehydroepiandrosterone 7a-OH-dehydroepiandrosterone 16a-OH-dehydroepiandrosterone 163-OH-dehydroepiandrosterone In general terms the ring system A'B'C'D' may contain a variety of non-interfering substituents. In particular, the ring system A'B'C'D' may contain one or more hydroxy, alkyl especially lower (C1-C6) alkyl, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers, alkoxy especially lower (C,-C6) alkoxy, e.g. methoxy, ethoxy, propoxy etc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.
In an alternative embodiment, the polycyclic compound may not contain or be based on a steroid nucleus. In this regard, the polycyclic compound may contain or be based on a non-steroidal ring system - such as diethylstilboestrol, stilboestrol, coumarins, flavonoids, combrestatin and other ring systems. Other suitable non-steroidal compounds for use in or as the composition of the present invention may be found in US-A-5567831.
Preferably, Group A or Group C and/or Group E are attached to the same ring of the cyclic compound of the present invention at positions ortho with respect to each other.
Preferably, the polycyclic compound has a steroidal structure and Group A or Group E
is attached to the A ring.
Preferably, the Group A or Group E is attached to the 2 position of the A ring of the steroidal structure.
Preferably, the polycyclic compound has a steroidal structure and Group C is attached to the A ring.
Preferably, the Group C is attached to the 3 position of the A ring of the steroidal structure.
Group A is a oxyhydrocarbyl group.
The term "oxyhydrocarbyl group" as used herein means a group comprising at least C, H and O and may optionally comprise one or more other suitable substituents.
Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the oxyhydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the oxyhydrocarbyl group may contain hetero atoms.
Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur and nitrogen.
In one preferred embodiment of the present invention, the oxyhydrocarbyl group is a oxyhydrocarbon group.
Here the term "oxyhydrocarbon" means any one of an alkoxy group, an oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an oxyaryl group. The term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
Preferably the oxyhydrocarbyl group is of the formula C,_60 (such as a Ci_30).
If the compound comprises a steroidal nucleus, preferably the A ring has an oxyhydrocarbyl group at the 2 position.
More preferably the group C~_60 is attached to the 2 position of the A ring of a steroidal nucleus.
Preferably, the oxyhydrocarbyl group is an alkoxy.
The alkyl group of the alkoxy substituent is preferably a lower alkyl group containing from 1 to 5 carbon atoms, that is to say methyl, ethyl, propyl etc.
Preferably, the alkyl group is methyl.
Thus, in a preferred embodiment, if the compound comprises a steroidal nucleus the A
ring has an methoxy substituent at the 2 position.
A preferred compound of the present invention has the formula:
Group A or E
Group wherein rings A, B, C and D are independently optionally substituted.
5 Preferably Group A or Group E is in the 2-position.
Preferably Group C is in the 3-position.
Group C is a "sulphamate group". A "sulphamate group" is a group of the formula O
R~~N~ ~~~0~
O
to R2 wherein each of R~ and R? is independently selected from H or a hydrocarbyl group.
The term "hydrocarbyl group" as used herein means a group comprising at least C
and H and may optionally comprise one or more other suitable substituents.
Examples of such substituents may include halo-, alkoxy-, nitro-, a hydrocarbon group, an N-acyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
Preferably, R, and RZ are independently selected from H or alkyl, cycloalkyl, alkenyl and aryl, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted sulphamate compound may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl, N-acyl, or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R~ and/or R~ is alkyl, the preferred values are those where R, and R2 are each independently selected from lower alkyl groups containing from 1 to 5 carbon atoms, that is to say methyl, ethyl, propyl etc. Preferably R~ and R~ are both methyl. When R, and/or RZ is aryl, typical values are phenyl and tolyl (-PhCH3; o-, m- or p-). Where Ri and RZ represent cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. When joined together R~
and R2 typically represent an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. -0- or -NH-to provide a 5-, 6- or 7- membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
Within the values alkyl, cycloalkyl, alkenyl, acyl and aryl we include substituted groups containing as substituents therein one or more groups which do not interfere with the activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl. A non-limiting example of a hydrocarbyl group is an acyl group.
In some preferred embodiments, at least one of Ri and RZ is H.
Examples of suitable sulphamate compounds for use in the present invention, or examples of suitable compounds that can be converted to suitable sulphamate compounds for use in the present invention, can be found in the art - such as PCT/GB92/01587, PCT/GB97/03352, PCT/GB97/00444, GB 9725749.7, GB
9725750.5, US-A-5567831, US-A-5677292, US-A-5567831, WO-A-96/05216, and WO-A-96/05217.
By way of example, PCT/GB92/01587 teaches novel sulphamate compounds and pharmaceutical compositions containing them for use in the treatment of oestrone dependent tumours, especially breast cancer. These sulphamate compounds are sulphamate esters. Examples of such inhibitors are sulphamate ester derivatives of steroids.
Another compound suitable for use in the present invention has at least the following skeletal structure:
O
Group A o Group C
It is preferred that at least one of R, and RZ is H.
Another compound suitable for use in the present invention has at least the following skeletal structure:
O
Group A c Group C
wherein rings A, B, C and D are independently optionally substituted.
A preferred compound of the present invention has the formula:
H
Group A or E
Vroup C;
wherein rings A, B, C and D are independently optionally substituted.
Preferably Group E is in the 2-position.
Preferably Group C is in the 3-position.
For the present invention, preferably the sulphamate compound is an oxyhydrocarbyl steroidal sulphamate compound, in particular 2-methoxyoestrone-3-O-sulphamate, or a pharmaceutically active salt thereof, including analogues thereof.
2-methoxyoestrone-3-O-sulphamate is an analogue of oestrone-3-O-sulphamate (otherwise known as "EMATE"), which has the following structure:
H O
I
~N~S
11~ O
O
- and can be called 2-methoxy EMATE.
2-methoxy EMATE is the sulphamoylated derivative of a naturally occurring oestrogen metabolite of the present invention, 2-methoxyoestrone. This compound is formed in the liver by the hydroxylation of oestrone by a 2-hydroxylase, with subsequent metabolism to the methoxy derivative by catechol oestrogen methyl transferase.
2-methoxy EMATE has the formula presented as formula below:
H2NS02v In one embodiment, preferably, the sulphamate compound is an oxyhydrocarbyl derivative of oestrone-3-O-sulphamate.
In one embodiment, preferably, the sulphamate compound is a C~_6 (such as a C~_3) alkoxy derivative of oestrone-3-O-sulphamate.
In one embodiment, preferably, the sulphamate compound is a 2-C1_6 (such as a C~_3) alkoxy derivative of oestrone-3-O-sulphamate.
In one embodiment, preferably, the sulphamate compound is 2-methoxyoestrone-3-O-sulphamate.
The invention will be now be further described by way of example only with reference to the figures which show:
Fig 1 Inhibition of basal and TNFa-stimulated aromatase activity by paclitaxel (Pax) in breast tissue fibroblasts. Paclitaxel was added to cells which were cultured for 24h in 2% stripped foetal calf serum. TNFa was added, in the presence of dexamethasone (100nmol/1) and cells cultured for a further 48h in the same medium. Controls and cells with paclitaxel, but not TNFa, were also cultured in the presence of dexamethasone for a 48h period. Aromatase activity was measured in intact monolayers after washing cells with phosphate buffered saline. (means ~ SD, n=3; a, p<0.001 versus controls; b, p<0.001 versus TNFa-stimulated aromatase activity).
Fig 2 Inhibition of TNFa (20ng/inl), IL-6 plus IL-6sR (SOng + 100ng/ml) or PGEZ
(10~M) 5 stimulated aromatase activity in fibroblasts by paclitaxel (Pax, 10~M) or 2-methoxyoestradiol (2-me0E2, 10~,M). (means ~ SD, n=3). The experimental protocol used was as described in the legend to Fig 1. Aromatase activity in cells treated for 48h with TNFa, IL-6 plus IL-6sR or PGEZ was significantly higher (p<0.01 - p<0.001 ) than in control cells. All inhibitions were significant (p<0.001 ) 10 compared with aromatase activity in TNFa-stimulated cells in the absence of paclitaxel or 2-methoxyoestradiol.
Fig 3 Dose response for inhibition of TNFa-stimulated aromatase activity (expressed as 15 of TNFa-stimulated activity) by paclitaxel (Pax), 2-methoxyoestradiol (2-me0E2) or 2-methoxyoestriol (2-me0E3) in fibroblasts. (means ~ SD, n=3). The experimental protocol used was as described in the legend to Fig 1. All compounds significantly inhibited TNFa stimulation of aromatase activity (p<0.001 ) with the exception of 2-methoxyoestriol at S~M (NS).
Fig 4 Ability of compounds to block stimulation of aromatase activity by TNFa, IL-6+IL-6sR or PGE2.
Fig 5 Dose response for inhibition of TNFa-stimulated aromatase activity.
EXAMPLE
Materials and Methods Samples of breast adipose or tumour tissue were obtained from women undergoing reduction mammoplasty or lumpectomy after obtaining their informed consent.
Fibroblasts were cultured as previously described (2). Briefly, they were cultured in Eagles' modified minimal essential medium containing Hepes buffer (20mmol/1), 10% foetal calf serum (FCS) and supplements. Cells were routinely passaged 2-3 times after which replicate 25cm2 flasks were seeded with fibroblasts and grown to confluency. 2-MeOEMATE was prepared in as described in Appendix I. (For experiments, cells were cultured in phenol red-free medium containing 2%
stripped FCS for 24h in the presence of paclitaxel, 2-me0E2 or 2-MeOEMATE before the addition of TNFa, IL-6 plus IL-6 soluble receptor (IL-6sR) or PGE2 and cultured for a further 48h in this medium. TNFa, IL-6 and IL-6sR (R&D Systems Ltd, Abingdon, Oxford, UK) or PGE~ (Sigma, Poole, Dorset, UK) were used in the presence of dexamethasone (100nmol/l, Sigma). Paclitaxel, 2-me0E2 and other chemicals were also obtained from Sigma.
At the end of the treatment period aromatase activity was measured in intact monolayers using [1(3-3H] androstenedione (15-30Ci/mmol, NEN-Du Pont, Stevenage, Herts, UK) over a 3-20h period (2,3). The number of cells was measured by counting cell nuclei using a Coulter counter. Experiments were carried out in triplicate and results shown are representative of 2-3 investigations.
Statistics Student's t test was used to assess the significance of differences in mean values of treated and control cells.
Results The ability of paclitaxel to inhibit TNFa stimulated aromatase activity was initially examined using fibroblasts derived from reduction mammoplasty tissue (Fig 1).
In these cells TNFa, in the presence of dexamethasone, stimulated aromatase activity by 375%. Both paclitaxel and 2-me0E2 inhibited basal aromatase activity by 88%
and 46% respectively. In addition, TNFa stimulated aromatase was also significantly reduced by these compounds by 91% and 56% respectively. This ability appears to be specific to agents that stabilise microtubules. Colchicine, which inhibits microtubule polymerisation, or Cytocholasin B, which binds to microfilaments, were without effect (data not shown).
As other factors, such as IL-6 and PGE~, also act via interaction with cell surface receptors, the ability of paclitaxel, 2-me0E2 and 2-MeOEMATE to antagonise aromatase stimulation by these factors and TNFa was also examined (Fig 2 and Fig 4). TNFa, IL-6 plus IL-6sR or PGEZ all significantly enhanced aromatase activity in tumour-derived fibroblasts. Paclitaxel, 2-me0E2 and 2-MeOEMATE inhibited basal aromatase activity and TNFa stimulated activity. In addition, however, they were also found to antagonise stimulation of aromatase activity by IL-6 plus IL-6sR
or PGE2.
The relative potencies of paclitaxel, 2-me0E2 and 2-MeOEMATE to antagonise TNFa stimulated aromatase activity were compared in a dose-response study (Fig and Fig 5). In addition, the ability of the 16a-hydroxy derivative of 2-me0E2, me0E3, which does not appear to bind to microtubules (14), to inhibit TNFa stimulated aromatase activ ity was also examined.
While paclitaxel, 2-me0E2 and 2-MeOEMATE inhibited TNFa stimulated aromatase, it was evident that paclitaxel is a more potent antagonist. At 0.1 ~M
paclitaxel inhibited stimulation by 90% whereas 2-me0E2 at this concentration only reduced the stimulation by S1%. 2-Me0E3, while showing some inhibitory effect at the highest concentration tested did not significantly reduce TNFa stimulation of aromatase activity at S~M.
Discussion Results from this investigation have revealed agents that alter microtubule stability, paclitaxel, 2-me0E2 and 2-MeOEMATE, not only inhibit basal aromatase activity but greatly reduce TNFa stimulated activity. Paclitaxel is used in the treatment of breast cancer but, as far as we are aware, this is the first report demonstrating its ability to inhibit basal and cytokine stimulated aromatase activity. This property is restricted microtubule stabilising agents as colchicine or cytocholasin B, which have different effects on the microskeleton, were unable to inhibit TNFa stimulated aromatase activity.
TNFa, IL-6 plus IL6sR and PGE2 are the three main factors identified so far that can regulate aromatase activity in fibroblasts derived from subcutaneous adipose or breast tissues. Microtubules may be required for the synthesis of cytokine receptors or for their translocation to the plasma membrane (15). It is likely, therefore, that the effect that paclitaxel, 2-me0E2 and 2-MeOEMATE have on the ability of TNFa, IL-6 or PGEZ to stimulate aromatase activity may also result from an effect on the synthesis/translocation of the receptors involved in their signalling. The ability of paclitaxel, 2-me0E2 and 2-MeOEMATE to reduce basal (i.e. unstimulated) aromatase activity may result from blocking the autocrine/ paracrine action of cytokines and PGE~, which are known to be produced by these fibroblasts, on aromatase activity (6, 16).
Paclitaxel is used for the treatment of breast cancer but its toxicity precludes its long term use. The finding that 2-me0E2, an endogenous oestrogen metabolite, and 2 MeOEMATE may have similar properties to paclitaxel suggests they may have considerable therapeutic potential (17). Oral administration of 2-me0E2 to mice inoculated with B16 melanoma, Meth A sarcoma or MDA-MB-435 breast cancer .
cells significantly reduced tumour growth (18, 19).
The results from this investigation also suggest a possible mechanism by which the immune system could develop an immunostimulatory role. In the presence of adequate production and/or administration of 2-me0E2 or 2-MeOEMATE, cytokine receptors in breast tissues would be down-regulated and thus cytokine stimulation of aromatase activity inhibited. Reduced production and/or administration of 2-me0E2 or 2-MeOEMATE would enable cytokines to stimulate oestrogen synthesis in breast tissues. Bradlow and his colleagues have obtained convincing evidence that a reduction in the formation of 2-hydroxyoestrogens and an increase in synthesis of 16a-hydroxy metabolites is associated with an increased risk of breast cancer (20, 21 ). The observation in the present study that the 16a-hydroxy derivative of me0E2 had only a limited ability to suppress TNFa stimulated aromatase activity would appear to support Bradlow's findings.
Stromal fibroblasts cultured from adipose tissue have the ability to differentiate into adipocytes. TNFa, while stimulating aromatase activity in fibroblasts, inhibits their differentiation into adipocytes. High concentrations of oestradiol (10-100~,M) can inhibit TNFa stimulated aromatase activity in adipose stromal cells and it has been postulated that a feed-back loop may exist to prevent excessive oestrogen synthesis in these cells (22). Peroxisome prolierator activated receptor y (PPARy) ligands, such as thiozolidinedione, can also stimulate adipocyte differentiation and also inhibit TNFa stimulated aromatase activity. As high concentrations of oestradiol were required to inhibit TNFa stimulated aromatase activity in human adipose stromal cells it is tempting to speculate that oestradiol may act after conversion to 2-me0E2.
Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.
References 1. Reed, M.J., Owen, A.M., Lai, L.C., Coldham, N.G., Ghilchik, M.W., Shaikh, N.A., and James, V.H.T. (1989) Int. J. Cancer 44, 233-237.
5 2. Reed, M.J., Coldham, N.G., Patel, S., Ghilchik, M.W., and James, V.H.T.
(1992) J. Endocrinol. 132, RS-R8.
3. Macdiarmid, F., Wang, D., Duncan, L.J., Purohit, A., Ghilchik, M.W., and Reed, M.J. (1994) Molec. Cell. Endocrinol. 106, 17-21.
4. Zhao, Y., Agarwal, V., Mendelson, C.R., and Simpson, E.R. (1996) 10 Endocrinology 137, 5739-5742.
5. Kelly, P.M., Davison, R.S., Bliss, E., and McGee, J.O. (1988) Br. J. Cancer 57, 174-177.
6. Purohit, A., Ghilchik, M.W., Duncan, L.J., Wang, D.Y., Singly A., Walker, M.M., and Reed, M.J. (1995) J. Clin. Endocrinol. Metab. 80, 3052-3058.
15 7. Reed, M.J., and Purohit, A. (1997) Endocrine Rev. 18, 701-715.
8. Stewart, T., Tsai, S.-C.J., Grayson, H., Henderson, R., and Opelz, G.
(1995) Lancet 346, 796-798.
9. Singh, A., Purohit, A., Duncan, L.J., Mokbel, K., Ghilchik, M.W., and Reed, M.J. (1997) J. Steroid Biochem. Molec. Biol. 61, 185-192.
20 10. Tartaglia, L.A., and Goeddel, D.V. (1992) Immamol. Today 13, 151-153.
Here the term "oxyhydrocarbon" means any one of an alkoxy group, an oxyalkenyl group, an oxyalkynyl group, which groups may be linear, branched or cyclic, or an oxyaryl group. The term oxyhydrocarbon also includes those groups but wherein they have been optionally substituted. If the oxyhydrocarbon is a branched structure having substituent(s) thereon, then the substitution may be on either the hydrocarbon backbone or on the branch; alternatively the substitutions may be on the hydrocarbon backbone and on the branch.
Preferably the oxyhydrocarbyl group is of the formula C,_60 (such as a Ci_30).
If the compound comprises a steroidal nucleus, preferably the A ring has an oxyhydrocarbyl group at the 2 position.
More preferably the group C~_60 is attached to the 2 position of the A ring of a steroidal nucleus.
Preferably, the oxyhydrocarbyl group is an alkoxy.
The alkyl group of the alkoxy substituent is preferably a lower alkyl group containing from 1 to 5 carbon atoms, that is to say methyl, ethyl, propyl etc.
Preferably, the alkyl group is methyl.
Thus, in a preferred embodiment, if the compound comprises a steroidal nucleus the A
ring has an methoxy substituent at the 2 position.
A preferred compound of the present invention has the formula:
Group A or E
Group wherein rings A, B, C and D are independently optionally substituted.
5 Preferably Group A or Group E is in the 2-position.
Preferably Group C is in the 3-position.
Group C is a "sulphamate group". A "sulphamate group" is a group of the formula O
R~~N~ ~~~0~
O
to R2 wherein each of R~ and R? is independently selected from H or a hydrocarbyl group.
The term "hydrocarbyl group" as used herein means a group comprising at least C
and H and may optionally comprise one or more other suitable substituents.
Examples of such substituents may include halo-, alkoxy-, nitro-, a hydrocarbon group, an N-acyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen.
Preferably, R, and RZ are independently selected from H or alkyl, cycloalkyl, alkenyl and aryl, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
When substituted, the N-substituted sulphamate compound may contain one or two N-alkyl, N-alkenyl, N-cycloalkyl, N-acyl, or N-aryl substituents, preferably containing or each containing a maximum of 10 carbon atoms. When R~ and/or R~ is alkyl, the preferred values are those where R, and R2 are each independently selected from lower alkyl groups containing from 1 to 5 carbon atoms, that is to say methyl, ethyl, propyl etc. Preferably R~ and R~ are both methyl. When R, and/or RZ is aryl, typical values are phenyl and tolyl (-PhCH3; o-, m- or p-). Where Ri and RZ represent cycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc. When joined together R~
and R2 typically represent an alkylene group providing a chain of 4 to 6 carbon atoms, optionally interrupted by one or more hetero atoms or groups, e.g. -0- or -NH-to provide a 5-, 6- or 7- membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.
Within the values alkyl, cycloalkyl, alkenyl, acyl and aryl we include substituted groups containing as substituents therein one or more groups which do not interfere with the activity of the compound in question. Exemplary non-interfering substituents include hydroxy, amino, halo, alkoxy, alkyl and aryl. A non-limiting example of a hydrocarbyl group is an acyl group.
In some preferred embodiments, at least one of Ri and RZ is H.
Examples of suitable sulphamate compounds for use in the present invention, or examples of suitable compounds that can be converted to suitable sulphamate compounds for use in the present invention, can be found in the art - such as PCT/GB92/01587, PCT/GB97/03352, PCT/GB97/00444, GB 9725749.7, GB
9725750.5, US-A-5567831, US-A-5677292, US-A-5567831, WO-A-96/05216, and WO-A-96/05217.
By way of example, PCT/GB92/01587 teaches novel sulphamate compounds and pharmaceutical compositions containing them for use in the treatment of oestrone dependent tumours, especially breast cancer. These sulphamate compounds are sulphamate esters. Examples of such inhibitors are sulphamate ester derivatives of steroids.
Another compound suitable for use in the present invention has at least the following skeletal structure:
O
Group A o Group C
It is preferred that at least one of R, and RZ is H.
Another compound suitable for use in the present invention has at least the following skeletal structure:
O
Group A c Group C
wherein rings A, B, C and D are independently optionally substituted.
A preferred compound of the present invention has the formula:
H
Group A or E
Vroup C;
wherein rings A, B, C and D are independently optionally substituted.
Preferably Group E is in the 2-position.
Preferably Group C is in the 3-position.
For the present invention, preferably the sulphamate compound is an oxyhydrocarbyl steroidal sulphamate compound, in particular 2-methoxyoestrone-3-O-sulphamate, or a pharmaceutically active salt thereof, including analogues thereof.
2-methoxyoestrone-3-O-sulphamate is an analogue of oestrone-3-O-sulphamate (otherwise known as "EMATE"), which has the following structure:
H O
I
~N~S
11~ O
O
- and can be called 2-methoxy EMATE.
2-methoxy EMATE is the sulphamoylated derivative of a naturally occurring oestrogen metabolite of the present invention, 2-methoxyoestrone. This compound is formed in the liver by the hydroxylation of oestrone by a 2-hydroxylase, with subsequent metabolism to the methoxy derivative by catechol oestrogen methyl transferase.
2-methoxy EMATE has the formula presented as formula below:
H2NS02v In one embodiment, preferably, the sulphamate compound is an oxyhydrocarbyl derivative of oestrone-3-O-sulphamate.
In one embodiment, preferably, the sulphamate compound is a C~_6 (such as a C~_3) alkoxy derivative of oestrone-3-O-sulphamate.
In one embodiment, preferably, the sulphamate compound is a 2-C1_6 (such as a C~_3) alkoxy derivative of oestrone-3-O-sulphamate.
In one embodiment, preferably, the sulphamate compound is 2-methoxyoestrone-3-O-sulphamate.
The invention will be now be further described by way of example only with reference to the figures which show:
Fig 1 Inhibition of basal and TNFa-stimulated aromatase activity by paclitaxel (Pax) in breast tissue fibroblasts. Paclitaxel was added to cells which were cultured for 24h in 2% stripped foetal calf serum. TNFa was added, in the presence of dexamethasone (100nmol/1) and cells cultured for a further 48h in the same medium. Controls and cells with paclitaxel, but not TNFa, were also cultured in the presence of dexamethasone for a 48h period. Aromatase activity was measured in intact monolayers after washing cells with phosphate buffered saline. (means ~ SD, n=3; a, p<0.001 versus controls; b, p<0.001 versus TNFa-stimulated aromatase activity).
Fig 2 Inhibition of TNFa (20ng/inl), IL-6 plus IL-6sR (SOng + 100ng/ml) or PGEZ
(10~M) 5 stimulated aromatase activity in fibroblasts by paclitaxel (Pax, 10~M) or 2-methoxyoestradiol (2-me0E2, 10~,M). (means ~ SD, n=3). The experimental protocol used was as described in the legend to Fig 1. Aromatase activity in cells treated for 48h with TNFa, IL-6 plus IL-6sR or PGEZ was significantly higher (p<0.01 - p<0.001 ) than in control cells. All inhibitions were significant (p<0.001 ) 10 compared with aromatase activity in TNFa-stimulated cells in the absence of paclitaxel or 2-methoxyoestradiol.
Fig 3 Dose response for inhibition of TNFa-stimulated aromatase activity (expressed as 15 of TNFa-stimulated activity) by paclitaxel (Pax), 2-methoxyoestradiol (2-me0E2) or 2-methoxyoestriol (2-me0E3) in fibroblasts. (means ~ SD, n=3). The experimental protocol used was as described in the legend to Fig 1. All compounds significantly inhibited TNFa stimulation of aromatase activity (p<0.001 ) with the exception of 2-methoxyoestriol at S~M (NS).
Fig 4 Ability of compounds to block stimulation of aromatase activity by TNFa, IL-6+IL-6sR or PGE2.
Fig 5 Dose response for inhibition of TNFa-stimulated aromatase activity.
EXAMPLE
Materials and Methods Samples of breast adipose or tumour tissue were obtained from women undergoing reduction mammoplasty or lumpectomy after obtaining their informed consent.
Fibroblasts were cultured as previously described (2). Briefly, they were cultured in Eagles' modified minimal essential medium containing Hepes buffer (20mmol/1), 10% foetal calf serum (FCS) and supplements. Cells were routinely passaged 2-3 times after which replicate 25cm2 flasks were seeded with fibroblasts and grown to confluency. 2-MeOEMATE was prepared in as described in Appendix I. (For experiments, cells were cultured in phenol red-free medium containing 2%
stripped FCS for 24h in the presence of paclitaxel, 2-me0E2 or 2-MeOEMATE before the addition of TNFa, IL-6 plus IL-6 soluble receptor (IL-6sR) or PGE2 and cultured for a further 48h in this medium. TNFa, IL-6 and IL-6sR (R&D Systems Ltd, Abingdon, Oxford, UK) or PGE~ (Sigma, Poole, Dorset, UK) were used in the presence of dexamethasone (100nmol/l, Sigma). Paclitaxel, 2-me0E2 and other chemicals were also obtained from Sigma.
At the end of the treatment period aromatase activity was measured in intact monolayers using [1(3-3H] androstenedione (15-30Ci/mmol, NEN-Du Pont, Stevenage, Herts, UK) over a 3-20h period (2,3). The number of cells was measured by counting cell nuclei using a Coulter counter. Experiments were carried out in triplicate and results shown are representative of 2-3 investigations.
Statistics Student's t test was used to assess the significance of differences in mean values of treated and control cells.
Results The ability of paclitaxel to inhibit TNFa stimulated aromatase activity was initially examined using fibroblasts derived from reduction mammoplasty tissue (Fig 1).
In these cells TNFa, in the presence of dexamethasone, stimulated aromatase activity by 375%. Both paclitaxel and 2-me0E2 inhibited basal aromatase activity by 88%
and 46% respectively. In addition, TNFa stimulated aromatase was also significantly reduced by these compounds by 91% and 56% respectively. This ability appears to be specific to agents that stabilise microtubules. Colchicine, which inhibits microtubule polymerisation, or Cytocholasin B, which binds to microfilaments, were without effect (data not shown).
As other factors, such as IL-6 and PGE~, also act via interaction with cell surface receptors, the ability of paclitaxel, 2-me0E2 and 2-MeOEMATE to antagonise aromatase stimulation by these factors and TNFa was also examined (Fig 2 and Fig 4). TNFa, IL-6 plus IL-6sR or PGEZ all significantly enhanced aromatase activity in tumour-derived fibroblasts. Paclitaxel, 2-me0E2 and 2-MeOEMATE inhibited basal aromatase activity and TNFa stimulated activity. In addition, however, they were also found to antagonise stimulation of aromatase activity by IL-6 plus IL-6sR
or PGE2.
The relative potencies of paclitaxel, 2-me0E2 and 2-MeOEMATE to antagonise TNFa stimulated aromatase activity were compared in a dose-response study (Fig and Fig 5). In addition, the ability of the 16a-hydroxy derivative of 2-me0E2, me0E3, which does not appear to bind to microtubules (14), to inhibit TNFa stimulated aromatase activ ity was also examined.
While paclitaxel, 2-me0E2 and 2-MeOEMATE inhibited TNFa stimulated aromatase, it was evident that paclitaxel is a more potent antagonist. At 0.1 ~M
paclitaxel inhibited stimulation by 90% whereas 2-me0E2 at this concentration only reduced the stimulation by S1%. 2-Me0E3, while showing some inhibitory effect at the highest concentration tested did not significantly reduce TNFa stimulation of aromatase activity at S~M.
Discussion Results from this investigation have revealed agents that alter microtubule stability, paclitaxel, 2-me0E2 and 2-MeOEMATE, not only inhibit basal aromatase activity but greatly reduce TNFa stimulated activity. Paclitaxel is used in the treatment of breast cancer but, as far as we are aware, this is the first report demonstrating its ability to inhibit basal and cytokine stimulated aromatase activity. This property is restricted microtubule stabilising agents as colchicine or cytocholasin B, which have different effects on the microskeleton, were unable to inhibit TNFa stimulated aromatase activity.
TNFa, IL-6 plus IL6sR and PGE2 are the three main factors identified so far that can regulate aromatase activity in fibroblasts derived from subcutaneous adipose or breast tissues. Microtubules may be required for the synthesis of cytokine receptors or for their translocation to the plasma membrane (15). It is likely, therefore, that the effect that paclitaxel, 2-me0E2 and 2-MeOEMATE have on the ability of TNFa, IL-6 or PGEZ to stimulate aromatase activity may also result from an effect on the synthesis/translocation of the receptors involved in their signalling. The ability of paclitaxel, 2-me0E2 and 2-MeOEMATE to reduce basal (i.e. unstimulated) aromatase activity may result from blocking the autocrine/ paracrine action of cytokines and PGE~, which are known to be produced by these fibroblasts, on aromatase activity (6, 16).
Paclitaxel is used for the treatment of breast cancer but its toxicity precludes its long term use. The finding that 2-me0E2, an endogenous oestrogen metabolite, and 2 MeOEMATE may have similar properties to paclitaxel suggests they may have considerable therapeutic potential (17). Oral administration of 2-me0E2 to mice inoculated with B16 melanoma, Meth A sarcoma or MDA-MB-435 breast cancer .
cells significantly reduced tumour growth (18, 19).
The results from this investigation also suggest a possible mechanism by which the immune system could develop an immunostimulatory role. In the presence of adequate production and/or administration of 2-me0E2 or 2-MeOEMATE, cytokine receptors in breast tissues would be down-regulated and thus cytokine stimulation of aromatase activity inhibited. Reduced production and/or administration of 2-me0E2 or 2-MeOEMATE would enable cytokines to stimulate oestrogen synthesis in breast tissues. Bradlow and his colleagues have obtained convincing evidence that a reduction in the formation of 2-hydroxyoestrogens and an increase in synthesis of 16a-hydroxy metabolites is associated with an increased risk of breast cancer (20, 21 ). The observation in the present study that the 16a-hydroxy derivative of me0E2 had only a limited ability to suppress TNFa stimulated aromatase activity would appear to support Bradlow's findings.
Stromal fibroblasts cultured from adipose tissue have the ability to differentiate into adipocytes. TNFa, while stimulating aromatase activity in fibroblasts, inhibits their differentiation into adipocytes. High concentrations of oestradiol (10-100~,M) can inhibit TNFa stimulated aromatase activity in adipose stromal cells and it has been postulated that a feed-back loop may exist to prevent excessive oestrogen synthesis in these cells (22). Peroxisome prolierator activated receptor y (PPARy) ligands, such as thiozolidinedione, can also stimulate adipocyte differentiation and also inhibit TNFa stimulated aromatase activity. As high concentrations of oestradiol were required to inhibit TNFa stimulated aromatase activity in human adipose stromal cells it is tempting to speculate that oestradiol may act after conversion to 2-me0E2.
Various modifications and variations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.
References 1. Reed, M.J., Owen, A.M., Lai, L.C., Coldham, N.G., Ghilchik, M.W., Shaikh, N.A., and James, V.H.T. (1989) Int. J. Cancer 44, 233-237.
5 2. Reed, M.J., Coldham, N.G., Patel, S., Ghilchik, M.W., and James, V.H.T.
(1992) J. Endocrinol. 132, RS-R8.
3. Macdiarmid, F., Wang, D., Duncan, L.J., Purohit, A., Ghilchik, M.W., and Reed, M.J. (1994) Molec. Cell. Endocrinol. 106, 17-21.
4. Zhao, Y., Agarwal, V., Mendelson, C.R., and Simpson, E.R. (1996) 10 Endocrinology 137, 5739-5742.
5. Kelly, P.M., Davison, R.S., Bliss, E., and McGee, J.O. (1988) Br. J. Cancer 57, 174-177.
6. Purohit, A., Ghilchik, M.W., Duncan, L.J., Wang, D.Y., Singly A., Walker, M.M., and Reed, M.J. (1995) J. Clin. Endocrinol. Metab. 80, 3052-3058.
15 7. Reed, M.J., and Purohit, A. (1997) Endocrine Rev. 18, 701-715.
8. Stewart, T., Tsai, S.-C.J., Grayson, H., Henderson, R., and Opelz, G.
(1995) Lancet 346, 796-798.
9. Singh, A., Purohit, A., Duncan, L.J., Mokbel, K., Ghilchik, M.W., and Reed, M.J. (1997) J. Steroid Biochem. Molec. Biol. 61, 185-192.
20 10. Tartaglia, L.A., and Goeddel, D.V. (1992) Immamol. Today 13, 151-153.
11. Ding, A.H., Porteu, F., Sanchez, E., and Nathan, C.F. (1990) Science 248, 3 73 .
12. Attalla, H., Makela, T.P., Adlercreutz, H., and Andersson, L.C. (1996) Biochem.
Biophysc. Res. Comm. 228, 467-473.
Biophysc. Res. Comm. 228, 467-473.
13. Attalla, H., Westberg, J.A., Andersson, L.C., Adlercreutz, H., and Makela, T.P.
(1998) Biochem. Biophys. Res. Comm. 247, 616-619.
(1998) Biochem. Biophys. Res. Comm. 247, 616-619.
14. Yue, T.-L., Wang, X., Londen, C.S., Gupta, S., Pillarisetti, K., Gu, J.-L., Hart, T.K., Lykso, P.G., and Feuerstein, G.Z. (1997) Molec. Pharmacol. ~1, 951-962.
1~. Ding, A.H., Porteu, F., Sanchez, E., and Nathan, C.F. (1990) J. Exp. Med.
171, 715-727.
16. Schrey, M.P., and Patel, K.V. (1995) Br. J. Cancer 72, 1412-1419.
17. Zhu, B.T., and Convey, A.H. (1998) Cancer Res. 58, 2269-2277.
18. Fotsis, T., Zhang, Y., Pepper, M.S., Adlercreutz, H., Montesano, R., Nawroth, P.P., and Schweigerer, L. (1994) Nature 368, 273-239.
19. Klauber, N., Parangi, S., Flynn, E., Hamel, E., and D'Amato, R.J. (1997) Cancer Res. 57, 81-86.
20. Bradlow, H.L. Sepkovic, D.W., Telang, N.T., and Osborne, M.P. (1995) Ann.
N. Y. Acad. Sci. 728, 180-200.
21. Bradlow, H.L., Telang, N.T., Sepkovic, D.W., and Osborne, M.P. (1996) J.
Endocrinol. 150, S259-5265.
22. Simpson, E., Rubin, G., Clyne, C., Robertson, K., O'Donnell, L., Davis, S., and Jones, M. (1999) Endocr. Rel.-Cancer 6, 131-137.
APPENDIX I
Synthesis of 2-methoxyoestrone-3-O-sulphamate (2-methoxy EMATE) 2-methoxy EMATE was synthesised by treating a solution of 2 methoxyoestrone in anhydrous dimethylformamide with sodium hydride at 0°C. After evolution of hydrogen had ceased sulphamoyl chloride (2 equiv.) was added and the reaction mixture was allowed to warm to room temperature overnight. The compound was purified by silica gel flash chromatography, was a single pure spot by TLC and exhibited satisfactory spectroscopic and microanalytical data.
In this regard, 2-Methoxy oestrone (75 mg, 0.250 mmol) gave a crude product (103 mg) which was fractionated on silica (50 g) with chloroform/acetone (8:1 ) and upon evaporation the second fraction gave a pale white residue (83 mg, 81 %) which was recrystallized in ethylacetate/hexane ( 1:2) to give 1 as white crystals (69 mg) .m.p =
177-180°C, Rts= 0.29 and 0.54 for chloroform/ acetone 8:1 and 4:1 respectively and 0.46 and 0.31 for ethylacetate/hexane 2:1 and 1:l respectively. vmax (KBr) 3400, 3300 (-NHZ), 1610 (C=O), and 1380 (-SON-) cm 1. 8H (CDCl3) 0.922 (3H, s, C-18-CH3), 1.24- 2.87 (15H, m), 3.88 (3H, s, C-2-OCH3), 5.0 (2H, br s, exchanged with D20,- SOZNH ), 6.93 (1H, s, C-1- H) and 7.06 (1H, s, C-4-H). MS: m/z (+ve ion FAB in m- NBA, rel. intensity) 379.1 [100, (M)+], 300.0 [25, (M-SO~NHZ)+]. MS:
mlz (-ve ion FAB in m-NBA, rel. intensity) 378.0 [100, (M-H)-]. Acc. MS: mlz (FAB+) = 380.1515 C,9HZ6NO;S requires 380.1532 Found C, 60.0; H, 6.7; N, 3.67;
C,9HZ;NO;S requires C, 60.14; H, 6.64; N, 3.69%.
1~. Ding, A.H., Porteu, F., Sanchez, E., and Nathan, C.F. (1990) J. Exp. Med.
171, 715-727.
16. Schrey, M.P., and Patel, K.V. (1995) Br. J. Cancer 72, 1412-1419.
17. Zhu, B.T., and Convey, A.H. (1998) Cancer Res. 58, 2269-2277.
18. Fotsis, T., Zhang, Y., Pepper, M.S., Adlercreutz, H., Montesano, R., Nawroth, P.P., and Schweigerer, L. (1994) Nature 368, 273-239.
19. Klauber, N., Parangi, S., Flynn, E., Hamel, E., and D'Amato, R.J. (1997) Cancer Res. 57, 81-86.
20. Bradlow, H.L. Sepkovic, D.W., Telang, N.T., and Osborne, M.P. (1995) Ann.
N. Y. Acad. Sci. 728, 180-200.
21. Bradlow, H.L., Telang, N.T., Sepkovic, D.W., and Osborne, M.P. (1996) J.
Endocrinol. 150, S259-5265.
22. Simpson, E., Rubin, G., Clyne, C., Robertson, K., O'Donnell, L., Davis, S., and Jones, M. (1999) Endocr. Rel.-Cancer 6, 131-137.
APPENDIX I
Synthesis of 2-methoxyoestrone-3-O-sulphamate (2-methoxy EMATE) 2-methoxy EMATE was synthesised by treating a solution of 2 methoxyoestrone in anhydrous dimethylformamide with sodium hydride at 0°C. After evolution of hydrogen had ceased sulphamoyl chloride (2 equiv.) was added and the reaction mixture was allowed to warm to room temperature overnight. The compound was purified by silica gel flash chromatography, was a single pure spot by TLC and exhibited satisfactory spectroscopic and microanalytical data.
In this regard, 2-Methoxy oestrone (75 mg, 0.250 mmol) gave a crude product (103 mg) which was fractionated on silica (50 g) with chloroform/acetone (8:1 ) and upon evaporation the second fraction gave a pale white residue (83 mg, 81 %) which was recrystallized in ethylacetate/hexane ( 1:2) to give 1 as white crystals (69 mg) .m.p =
177-180°C, Rts= 0.29 and 0.54 for chloroform/ acetone 8:1 and 4:1 respectively and 0.46 and 0.31 for ethylacetate/hexane 2:1 and 1:l respectively. vmax (KBr) 3400, 3300 (-NHZ), 1610 (C=O), and 1380 (-SON-) cm 1. 8H (CDCl3) 0.922 (3H, s, C-18-CH3), 1.24- 2.87 (15H, m), 3.88 (3H, s, C-2-OCH3), 5.0 (2H, br s, exchanged with D20,- SOZNH ), 6.93 (1H, s, C-1- H) and 7.06 (1H, s, C-4-H). MS: m/z (+ve ion FAB in m- NBA, rel. intensity) 379.1 [100, (M)+], 300.0 [25, (M-SO~NHZ)+]. MS:
mlz (-ve ion FAB in m-NBA, rel. intensity) 378.0 [100, (M-H)-]. Acc. MS: mlz (FAB+) = 380.1515 C,9HZ6NO;S requires 380.1532 Found C, 60.0; H, 6.7; N, 3.67;
C,9HZ;NO;S requires C, 60.14; H, 6.64; N, 3.69%.
Claims (20)
1. Use of a material selected from (i) microtubule stabilising agent;
(ii) microtubule disrupter;
(iii) a compound of the formula A-B wherein A is an oxyhydrocarbyl group and B
is a cyclic group; and (iv) a compound of the formula C-D wherein C is an sulphamate group and D is a cyclic group for the manufacture of a medicament for the inhibition of tumour necrosis factor .alpha.
(TNF.alpha.) stimulated aromatase activity.
(ii) microtubule disrupter;
(iii) a compound of the formula A-B wherein A is an oxyhydrocarbyl group and B
is a cyclic group; and (iv) a compound of the formula C-D wherein C is an sulphamate group and D is a cyclic group for the manufacture of a medicament for the inhibition of tumour necrosis factor .alpha.
(TNF.alpha.) stimulated aromatase activity.
2. Use according to claim 1 wherein (iii) is a compound of the formula C-D-E
wherein C is an sulphamate group, D is a cyclic group, and E is an oxyhydrocarbyl group
wherein C is an sulphamate group, D is a cyclic group, and E is an oxyhydrocarbyl group
3. Use according to claim 1 or 2 wherein the cyclic group has a polycyclic ring structure.
4. Use according to claim 2 or 3 wherein Group A and/or Group C and/or Group E
is linked or attached to the ring.
is linked or attached to the ring.
5. Use according to claim 3 or 4 wherein the polycyclic ring structure comprises three six-membered rings.
6. Use according to claim 3, 4 or 5 wherein Group A and/or Group C and/or Group E
are attached to the same ring of the polycyclic ring structure.
are attached to the same ring of the polycyclic ring structure.
7. Use according to any one of claims 3 to 6 wherein the polycyclic ring structure is a steroidal ring structure
8. Use according to any one of claims 2 to 7 wherein Group A or Group C and/or Group E are attached to the same ring of the cyclic compound of the present invention at positions ortho with respect to each other.
9. Use according to claim 7 or 8 wherein Group A or Group E is attached to the position of the A ring of the steroidal structure.
10. Use according to any one of claims 7 to 9 wherein Group C is attached to the 3 position of the A ring of the steroidal structure.
11. Use according to any one of claims 1 to 10 wherein Group A is of the formula C1-6O (such as a C1-3O).
12. Use according to any one of claims 1 to 11 wherein Group A is an alkoxy.
13. Use according to any one of claims 7 to 12 wherein Group A is a methoxy substituent at the 2 position of the steroidal ring structure.
14. Use according to any one of claims 1 to 13 wherein Group C is a group of the formula wherein each of R1 and R2 is independently selected from H or a hydrocarbyl group.
15. Use according to claim 14 wherein R1 and R3 are independently selected from H
or alkyl, cycloalkyl, alkenyl and aryl, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
or alkyl, cycloalkyl, alkenyl and aryl, or together represent alkylene, wherein the or each alkyl or cycloalkyl or alkenyl or optionally contain one or more hetero atoms or groups.
16. Use according to claim 14 or 15 wherein at least one of R1 and R2 is H.
17. Use according to any one of claims 1 to 16 wherein (iv) is a C1-6 (such as a C1-3) alkoxy derivative of oestrone-3-O-sulphamate, preferably a 2-C1-6 alkoxy derivative of oestrone-3-O-sulphamate.
18. Use according to any one of claims 1 to 17 wherein (iv) is 2-methoxyoestrone-3-O-sulphamate.
19. Use according to any one of claims 1 to 18 wherein (ii) is paclitaxel.
20. Use according to any one of claims 1 to 19 wherein (iii) is 2-methoxyoestradiol.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9913536.0 | 1999-06-10 | ||
| GBGB9913536.0A GB9913536D0 (en) | 1999-06-10 | 1999-06-10 | Use |
| PCT/GB2000/002186 WO2000076487A2 (en) | 1999-06-10 | 2000-06-05 | Use of paclitaxel and steriod derivatives as aromatase inhibitors for the treatment of cancer |
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| CA2376067A1 true CA2376067A1 (en) | 2000-12-21 |
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| Application Number | Title | Priority Date | Filing Date |
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| CA002376067A Abandoned CA2376067A1 (en) | 1999-06-10 | 2000-06-05 | Use of paclitaxel and steroid derivatives as aromatase inhibitors for the treatment of cancer |
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| Country | Link |
|---|---|
| US (1) | US20020055462A1 (en) |
| EP (1) | EP1207888A2 (en) |
| JP (1) | JP2004513064A (en) |
| AU (1) | AU5234600A (en) |
| CA (1) | CA2376067A1 (en) |
| GB (1) | GB9913536D0 (en) |
| WO (1) | WO2000076487A2 (en) |
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| US20040013613A1 (en) * | 2001-05-18 | 2004-01-22 | Jain Rajeev A | Rapidly disintegrating solid oral dosage form |
| EP2266542A3 (en) * | 1998-10-01 | 2013-07-31 | Elan Pharma International Limited | Controlled release nanoparticulate compositions |
| US8236352B2 (en) * | 1998-10-01 | 2012-08-07 | Alkermes Pharma Ireland Limited | Glipizide compositions |
| US8293277B2 (en) * | 1998-10-01 | 2012-10-23 | Alkermes Pharma Ireland Limited | Controlled-release nanoparticulate compositions |
| US7521068B2 (en) * | 1998-11-12 | 2009-04-21 | Elan Pharma International Ltd. | Dry powder aerosols of nanoparticulate drugs |
| US7078395B1 (en) | 1999-06-16 | 2006-07-18 | Sterix Limited | Methods for treating or preventing cancer by preventing, inhibiting or arresting cell cycling |
| AU783910B2 (en) | 1999-04-30 | 2005-12-22 | Sterix Limited | Use of estrone derivatives as antitumour agents |
| US20090104273A1 (en) * | 1999-06-22 | 2009-04-23 | Elan Pharma International Ltd. | Novel nifedipine compositions |
| US20040115134A1 (en) * | 1999-06-22 | 2004-06-17 | Elan Pharma International Ltd. | Novel nifedipine compositions |
| US20020106348A1 (en) * | 2000-07-12 | 2002-08-08 | Peng Huang | Cancer therapeutics involving the administration of 2-methoxyestradiol and an agent that increases intracellular superoxide anion |
| US7198795B2 (en) * | 2000-09-21 | 2007-04-03 | Elan Pharma International Ltd. | In vitro methods for evaluating the in vivo effectiveness of dosage forms of microparticulate of nanoparticulate active agent compositions |
| US8026229B2 (en) | 2001-08-13 | 2011-09-27 | Sterix Limited | Antitumor-active 2-alkoxyestradiol sulfamates |
| US20040101566A1 (en) * | 2002-02-04 | 2004-05-27 | Elan Pharma International Limited | Novel benzoyl peroxide compositions |
| EP1471887B1 (en) * | 2002-02-04 | 2010-04-21 | Elan Pharma International Ltd. | Nanoparticulate compositions having lysozyme as a surface stabilizer |
| US20080220075A1 (en) * | 2002-03-20 | 2008-09-11 | Elan Pharma International Ltd. | Nanoparticulate compositions of angiogenesis inhibitors |
| DE60309300T3 (en) * | 2002-03-20 | 2011-02-24 | Elan Pharma International Ltd. | NANOPARTICLE COMPOSITIONS OF ANGIOGENIC INHIBITORS |
| DE10307103A1 (en) * | 2003-02-19 | 2004-09-09 | Schering Ag | Anti-tumor effective 2-substituted D-homostra-1,3,5 (10) -trien-3-yl sulfamate |
| DE10307104A1 (en) | 2003-02-19 | 2004-09-23 | Schering Ag | Anti-tumor effective 2-substituted Estra-1,3,5 (10) -trien-3-yl sulfamate |
| EP2987730B1 (en) | 2014-08-16 | 2018-11-21 | Hübner GmbH & Co. KG | Interface module for passenger bridge |
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| GB9625334D0 (en) * | 1996-12-05 | 1997-01-22 | Imperial College | Compound |
| GB9118478D0 (en) * | 1991-08-29 | 1991-10-16 | Imperial College | Steroid sulphatase inhibitors |
| GB9118465D0 (en) * | 1991-08-29 | 1991-10-16 | Imperial College | Steroid sulphatase inhibitors |
| WO1994026254A1 (en) * | 1993-05-17 | 1994-11-24 | The Liposome Company, Inc. | Incorporation of taxol into liposomes and gels |
| US5643900A (en) * | 1993-07-02 | 1997-07-01 | Fotsis; Theodore | Method for treatment of pathological conditions associated with angiogenesis and preparation therefor |
| US5571933A (en) * | 1994-11-17 | 1996-11-05 | Duquesne University Of The Holy Ghost | Derivatives of estra 1,3,5(10)triene-17-one, 3-amino compounds and their use |
| GB2331988B (en) * | 1997-12-04 | 2003-04-16 | Imperial College | Polycyclic sulphamate inhibitors or oestrone sulphatase |
| GB2331987B (en) * | 1997-12-04 | 2002-11-27 | Imperial College | Polycyclic sulphamate inhibitors of oestrone sulphatase |
| JP2002517449A (en) * | 1998-06-10 | 2002-06-18 | ステリックス リミテッド | Pharmaceutical composition having tumor necrosis factor A and 2-methoxyestrone-3-O-sulfamate for inhibition of estrone sulfatase |
| AU783910B2 (en) * | 1999-04-30 | 2005-12-22 | Sterix Limited | Use of estrone derivatives as antitumour agents |
-
1999
- 1999-06-10 GB GBGB9913536.0A patent/GB9913536D0/en not_active Ceased
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2000
- 2000-06-05 CA CA002376067A patent/CA2376067A1/en not_active Abandoned
- 2000-06-05 WO PCT/GB2000/002186 patent/WO2000076487A2/en active Search and Examination
- 2000-06-05 JP JP2001502821A patent/JP2004513064A/en not_active Withdrawn
- 2000-06-05 AU AU52346/00A patent/AU5234600A/en not_active Abandoned
- 2000-06-05 EP EP00937052A patent/EP1207888A2/en not_active Withdrawn
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2001
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| WO2000076487A2 (en) | 2000-12-21 |
| AU5234600A (en) | 2001-01-02 |
| EP1207888A2 (en) | 2002-05-29 |
| JP2004513064A (en) | 2004-04-30 |
| WO2000076487A3 (en) | 2001-08-09 |
| GB9913536D0 (en) | 1999-08-11 |
| US20020055462A1 (en) | 2002-05-09 |
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