WO2004012723A1

WO2004012723A1 - Tamoxifen containing not more than 0.006% e-isomer

Info

Publication number: WO2004012723A1
Application number: PCT/GB2003/003204
Authority: WO
Inventors: Jonathan Murphy; Anthony Atkinson; Philip Burke; Jonathan Dickens; Judith Mcardell
Original assignee: Kymed Gb Ltd.
Priority date: 2002-08-01
Filing date: 2003-07-25
Publication date: 2004-02-12
Also published as: AU2003281838A1; GB0217873D0

Abstract

Tamoxifen containing not more than 0.006% by weight of its geometric E-isomer, and pharmaceutically-acceptable acid addition salts thereof, has use in the manufacture and formulation of a medicament having a significantly reduced risk of causing endometrial hyperplasia for the treatment or prevention of hormone dependent tumours.

Description

TAMOXIFEN CONTAINING NOT MORE THAN 0 . 006% E-ISOMER

This invention relates to tamoxifen. More particularly, it relates to the use of tamoxifen containing not greater than 0.006% by weight of its geometric E- isomeric form.

Tamoxifen is a triphenylethylene derivative having the structural formula I

wherein one of Ri and R₂ is phenyl and the other is ethyl. This compound exists as two geometrical isomers: the Z (trans) isomer wherein, in formula I above, R-i is phenyl and R₂ is ethyl and the E (cis) isomer wherein, in formula I above, R^ is ethyl and R₂ is phenyl.

Acting as a potent oestrogen antagonist, tamoxifen is the established first line human therapy for the treatment of all stages of oestrogen positive breast cancer. Tamoxifen's efficacy is extensively supported by a clinical 'use' database spanning in excess of thirty years and has recently been reinforced by several clinical articles reporting the dramatic reduction in the mortality rate for breast cancer as a direct result of tamoxifen treatment. For example, a recent study funded by the National Cancer Institute involving 13,000 women in the USA and Canada found that tamoxifen decreased the risk of breast cancer by as much as half among women at high risk of developing the disease. More than 180,000 cases of breast cancer are diagnosed each year in the USA and Canada and about 44,000 women are expected to die from the disease. Tamoxifen therapy has been one of the greatest success stories in the pharmacological management of breast cancer and it remains the treatment of choice for most clinicians.

While the action of tamoxifen is complex, pharmacological data demonstrates that the Z-isomer is a potent anti-oestrogen in humans as well as various other species, whilst at the same time apparently having very weak and atypical oestrogenic activity. In contrast, the E-isomer acts as a potent oestrogen. Z-isomer tamoxifen entirely free of E-isomer is not currently available for therapy.

Throughout the world, pharmacopoeial monographs define the pharmaceutical quality of Tamoxifen. The British Pharmacopoeia demands that for Tamoxifen Citrate BP, the E (cis) isomer and related substances are restricted by the pharmacopoeial monograph to an upper level of 0.3 per cent. Similar monographs limiting the levels of the E-isomer exist elsewhere.

Tamoxifen is known to affect the endometrium. Endometrial hyperplasia determined by tamoxifen can be expected in about 50% of women receiving the drug and may develop as quickly as 2 to 6 months from the start of the tamoxifen treatment. Clinicians acknowledge that hyperplasia is a significant precursor to trte development of endometrial cancer. Howeveffnα direct link>^ has been established between the E-isomer being responsible for tamoxifen induced endometrial hyperplasia.

In the USA, tamoxifen is also approved for the prevention (prophylaxis) of breast cancer in women at a high risk of developing the disease. However, elsewhere approval is still limited to treatment due to the ongoing safety concerns of tamoxifen, which includes its effects on the endometrium.

The present invention is based on our discovery that the risk of endometrial hyperplasia is directly linked to the levels of the E-isomer in the tamoxifen preparation administered and thereby provides a means to significantly reduce the risk of endometrial hyperplasia in a woman patient if the tamoxifen administered to the patient contains not more than 0.006% by weight of the E-isomer. Furthermore the weak and atypical oestrogen activity observed and recorded previously for the Z-isomer is, as a result of this invention, identified as being due to the presence of low amounts (variable 0.05 to 0.3%) by weight) of the E-isomer in the previous material used.

Accordingly, the present invention provides the use of tamoxifen containing not more than 0.006% by weight of its E-isomer, and pharmaceutically-acceptable acid addition salts thereof, in the manufacture and formulation of a medicament having a significantly reduced risk of causing endometrial hyperplasia, for the treatment, and prevention, of hormone dependent tumours, particularly of the breast.

The tamoxifen containing not more than 0.006%, preferably not more than 0.005%), by weight of the E-isomer is preferably prepared and used in the form of a pharmaceutically-acceptable acid addition salt. Examples of such salts include the hydrochloride, sulphate, phosphate, acetate, tartrate, oxalate and citrate salts of the tamoxifen. Preferably, the acid addition salt is the citrate salt.

Substantially pure Z-isomer tamoxifen, i.e. tamoxifen containing not greater than 0.006%, preferably not greater than 0.005%, by weight of E- isomer, and the pharmaceutically-acceptable acid addition salts thereof, may be made by processes such as stereoselective syntheses (which may require the use of expensive catalysts) as described in J. Chem. Soc, Perkin Traτfsr^ri#' 1997, 1101 and J. Org. Chem. 1990, 55, 6184 or ones that involve the chromatographic separation of an E/Z mixture of isomers as described in J. Chem. Res., 1985 (S) 116, (M) 1342, 1986 (S) 58, (M) 771.

GB-A-2,327,673 describes a process of preparing the Z-isomer of tamoxifen in a substantially pure form. The method disclosed in this document involves the preparation of a mixture of Z- and E- isomers of a tamoxifen precursor, dissolving the precursor mixture in a solvent, such as hexanol, and then allowing recrystallization of the precursor from the solvent, whereby the recrystallized precursor contains a greater proportion of the desired Z-isomer than the mixture prior to dissolution in the solvent, repeating (if desired) the recrystallization procedure in the same or in a different solvent (for instance, methanol) to purify the Z-isomer of the tamoxifen precursor further and then subjecting the Z-isomer ~of the precursor to a reaction to produce substantially pure tamoxifen Z-isomer. The method described in GB-A-2, 327,673 may suitably be used to prepare tamoxifen containing not more than 0.006%, and preferably not more than 0.005%, by weight of the E- isomer of tamoxifen as used in the present invention.

The tamoxifen containing not more than 0.006%, preferably not more than 0.005%), by weight of the E-isomer and the pharmaceutically-acceptable acid addition salts thereof, is useful in the treatment of hormone dependent tumours, particularly hormone dependent breast cancers, but also prostate cancer. Typically, the tamoxifen containing not more than 0.006%>, preferably not more than 0.005%, by weight of the E-isomer and the pharmaceutically- acceptable acid addition salts thereof will be used at dosages currently used in conventional tamoxifen therapy, for example from 10-40 mg/day, preferably from 20 to 40 mg/day.

EXAMPLES

Example 1

Preparation of the substantially pure Z-isomer of tamoxifen

Reaction Schematic 2-Chloroethoxybenzene (1)

Equipment

10 L Rb flange Flask Phenol (Lancaster)

Overhead Mechanical Stirrer 1 ,2-Dichloroethane (Fisher)

Heating Mantle Tetra-n-buytl Ammonium

Reflux Condenser Hydrogen Sulphate (Lancaster)

Sodium Hydroxide

Tetra-π-butylammonium hydrogen sulphate (31.8g, 0.09mol) was added to a stirred solution of phenol (503.3g, 5,35mol) in 1,2-dichloroethane (3.2L) in a 10L-flanged flask. 3M NaOH(aq) (2.64L, 7.8mmol) was added to the stirred reaction mixture over 15min, and the reaction mixture was heated under reflux. After 1h at reflux, 6M NaOH(aq) (640mL, 3.8mol) was added over 10min, and heating was continued for a further 1h. The reaction mixture was cooled to room temperature. The organic phase was washed with water (1.5L), brine (1.5L), dried, filtered and evaporated to a yellow oil (273g).

1 ,2-Dichloroethane (3L) was- addled -to- the

and the reaάifόn mixture was heated under reflux for 24h. (Tic indicated reaction still incomplete). Tetra-n-butylammmonium hydrogen sulphate (18g, O.Oδmol) was added and the reaction mixture stirred at reflux for 48h. The reaction mixture was cooled to room temperature and the organic phase was separated, washed with 2M NaOH (2x500mL), water (500ml_) and brine (500mL), dried (MgSO ), filtered and evaporated under reduced pressure to afford an oil (ca. 500g).

The combined crude product was distilled under reduced pressure to yield the product (1) as a colourless oil, bp 87°/3 mm - 98°/ 10 mm (535g, 64%). 1 -[4-(2-Chloroethoxy)phenyl]-2-phenylbutan-1 -one (2)

Equipment

3 L Rb Flask 2-Phenyl butyric acid (Lancaster)

Magnetic Stirrer Trifluoroacetic anhydride (Aldrich)

Iso-hexane (Fisher)

A mixture of 2-chloroethoxybenzene (1) (534g, 3.4mol), 2-phenylbutyric acid (497g, 3.0mol) and trifluoroacetic anhydride (674g, 3.2mol) was stirred in a 3L r.b. flask at room temperature for 84h. The brown oil was poured into water (3.8L), and triturated until crystals appeared. The solid was collected by filtration, washed with water, and then dried under vacuum. The crude material was recrystallized from iso-hexane (2.5L) collected by filtration, washed with iso-hexane and air dried, to yield the product (2) (792g, 77%), mp 65°C.

1 -[4-(2-Chloroethoxy)phenyl]-1 ,2-phenylbutan-1 -one (3)

Equipment.....

10 L Rb flange Flask Phenylmagnesium Chloride

Overhead Mechanical Stirrer (2.0M in THF) (Aldrich)

Heating Mantle Diethyl Ether (Fisher)

Reflux Condenser Hydrochloric Acid (2M)

Pressure Equalising Addition Funnel

Inlet/ Outlet for Argon

In a 10L flange, under argon atmosphere, a solution of (2) (780g, 2.6mol) in dry ether (3.0L) was added over 70min to phenylmagnesium chloride (2M solution in THF) (2.4L, 4.8mol) at room temperature. After completion of the addition, the reaction mixture was refluxed for 48h. The resulting solution was poured onto dil. HCI (10L) and extracted with ether (3x 2.5L). The combined organic phase was dried (MgSO ), filtered and evaporated (rotary evaporator) to yield the product (3) (1.34Kg, >100%) as an oil.

1 -[4-(2-Chloroethoxy)phenyl]-1 ,2-phenylbut-1 -ene (4)

Equipment

10 L Rb flange Flask Hydrochloric Acid 36% (w/v)

Overhead Mechanical Stirrer Ethanol (Fisher)

Hydrochloric acid (36% w/v, 100mL, 1.2mol) was added to a stirred solution of the crude alcohol (3) (1.34Kg, 3.5mol) in ethanol (3.0L) at room temperature. The reaction mixture was stirred at room temperature, in darkness, for 18h. The reaction mixture set into a solid mass and the mechanical stirred seized, ethanol (2L) was added to slurry the solid which was then filtered, washed with ethanol, and air dried overnight. The crude material (still containing residual ethanol) was split into two portions. Each portion (ca. 1 Kg) was dissolved in hot methanol (ca. 7L) in a 20L flask, and left overnight to crystallise. The product was collected by filtration and washed with methanol to give .the product as_^a white crystalline solid (594g). Nmr analysis of 4his_. product indicated the presence of a small amount of the E-isomer. HPLC confirmed this and indicated a ratio of 97:3 Z:E. A second recrystallization from methanol afforded the product (4) as a white crystalline solid (41 Og), the unwanted E-isomer was not detected by nmr or HPLC.

The mother liquors from the first recrystallization were evaporated to dryness and recrystallized from methanol to give the product (4) as an off-white crystalline solid 180g. HPLC analysis indicated a Z:E ratio of 87:13. (Z)-1 -[4-(2-Dimethylaminoethoxy)phenyl]-1 ,2-diphenylbut-1 -ene (5)

Equipment

20 L Rb flange Flask Dimethylamine Hydrochloride

Overhead Mechanical Stirrer (Aldrich)

Heating Mantle Potassium Carbonate

Reflux Condenser Ethanol (Fisher)

The chloro-compound (4) (480g, 1.12mol), dimethylamine hydrochloride (4.58Kg), ethanol (7.8L) and anhydrous potassium carbonate (3.9Kg) were placed in a 20L-flanged flask fitted with a stirrer and reflux condenser. The mixture was heated under reflux. Reaction mixture was filtered, washed with ethanol. The filtrate was evaporated to a solid, which contained some inorganic salts. The residue was suspended in chloroform, filtered and evaporated to give the crude product as a yellow solid (520g). This was reciγstallized from methanol to give a white crystalline (213g, 51%) m.p. 92°C. The mother liquor were concentrated and recrystallized from methanol to obtain a second crop, (128g, 31 %), m.p. 92°C.

Tamoxifen Citrate

Equipment

100ml Rb flange Flask Citric Acid (BDH)

Magnetic Stirrer Acetone (Fisher)

Citric acid (2.0g, 10.4mmol) was dissolved in acetone (10ml). Tamoxifen (5) (7.0g, 18.8mmol) was added to the stirred solution. After 5 min a precipitate had formed which was filtered, washed with cold acetone and dried to give tamoxifen citrate (8.gg, 81%). Example 2

In vivo mouse studies comparing the pharmacological effects of substantially pure Z-tamoxifen with a clinically acceptable tamoxifen containing substantial levels of the E-isomer

Experiments were carried out to compare the effects of tamoxifen Z- and E-isomers on the skeleton and uterus in vivo as assessed in mice. Oestrogen-like effects on bone formation and resorption, respectively, were assessed.

The formulations of tamoxifen (as citrate salt) used in these experiments were as follows:

Formulation Composition E-isomer content

(% w/w)

A Substantially pure Z-isomer tamoxifen 0.006

B As formulation A but with added E-isomer 0.303

N.B. The E-isomer content of Formulation B was selected to be typical of that of commercially available tamoxifen at the upper pharmacopoeial limits than can be currently used clinically.

Analysis was performed as shown below:

Standard Preparation

A suitable quantity of tamoxifen citrate was dissolved in 35% (v/v) methanol/water to give 0.6 mg/mL solution.

Test Preparation

Test samples were prepared at 1.0 mg/mL as above. For formulated tablets 1.517 mis of 35% (v/v) methanol/water per mg of stated tamoxifen content were added. The tablet was disrupted in a sonic water bath for 10 min and insoluble residue was removed by centrifugation. This yielded a solution of 1 mg/mL tamoxifen citrate. Chromatographic System

Hplc solvent D was 50 mM sodium phosphate pH3. Solvent B was acetonitrile.

Procedure

For each sample, inject 2, 20 and 100 μL were injected in triplicate. Using peak area recorded at 254 nm from the 2 μL injection the Z-isomer concentration was calculated by reference to the USP standard. Using 20 or 100 μL injections (depending on a suitable response) the E-isomer concentration was calculated by reference to the USP standard.

In vivo studies

Animals, which were housed in groups of eight received a standard diet (Mouse Standard Diet; B&K Ltd., Humberside, UK) and water ad libidum, and were kept with a cycle of 12 h light and 12 h darkness. The duration of each experiment was 28 days, after which animals were sacrificed by cervical dislocation, body and uterine weight were recorded, and long bones removed for further analysis. To provide a double fluorochrome label, tetracycline hydrochloride (25 mg/kg; Sigma) and calcein (30 mg/kg; Sigma) were injected ^" intraperitoneally at four and one days before sacrifice respectively, unless otherwise stated. Long bones were cleared of soft tissue, and subsequently fixed in 70%> ethanol. Bone mineral density (BMD) was measured by DXA, and expressed as g/cm². BMD was obtained of the whole femur and distal femoral metaphysis, using a peripheral DXA scanner with dedicated small animal software (Lunar PIXImus). Uteri were surgically removed by standard methods and weighed. All experimental procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Experiment

Ten week-old female CBA-I mice were divided into nine groups (eight animals per group), and given vehicle, 17β-oestradiol (E) or either of the two formulations (A or B) of tamoxifen by daily sc injection for 28 days. A double fluorochrome label was also administered 1 and 4 days prior to termination of the study. Uteri were weighed after isolation. A tamoxifen dose of 0.4 mg/kg/day in the mouse is comparable to a typical human clinical dose for treating breast tumours. 17β-oestradiol is included, as it has known estrogenic effects.

Groups

1. Vehicle

2. 17β-oestradiol 0.1 mg/kg/day

3. 17β-oestradiol 1 mg/kg/day

4. A-tamoxifen 0.1 mg/kg/day

5. A-tamoxifen 1 mg/kg/day

6. A-tamoxifen 10 mg/kg/day

7. B-tamoxifen 0.1 mg/kg/day

8. ^"B-tamoxifen 1 mg/kg/day

9. B-tamoxifen 10 mg/kg/day

Results

Female mice, oestradiol 1 mg/kg/day led to a significant increase in uterine weight compared to animals receiving vehicle, while the low E-isomer formulation (A) of tamoxifen was associated with a small dose-dependent reduction in uterine weight, which reached significance at 10mg/kg (Table 1). In contrast to the low E-isomer formulation, the typical E-isomer formulation (B) was associated with an increase in uterine weight. Oestradiol was associated with a dose-dependent increase in BMD of the whole femur and distal femoral metaphysis (Table 2), the high E-isomer formulation (B) of tamoxifen also led to a dose-dependent increase in BMD, which appeared to peak at 1 mg/kg, although the extent of this increase was significantly less than that observed following oestrogen treatment. Though the low E-isomer content tamoxifen also increased BMD at both sites compared to vehicle, the magnitude of this increase appeared to decline at higher doses, at which the response was significantly less than that after treatment with the typical E- isomer tamoxifen formulation. The effect of low E-isomer tamoxifen on uterine weight was different from typical E-isomer tamoxifen and showed a reduction in uterine weight which became significant at high levels of tamoxifen administration. Table 1

Group Body weight gain (%) Uterine weight (mg)

Vehicle 20 +/- 7 116+/- 18

17β-oestradiol0.1 mg/kg/day 34 +/- 3 115+/- 8

17β-oestradio! 1 mg/kg/day 33 +/- 6 148+/- 5*

B-tamoxifen 0.1 mg/kg/day 28 +/- 6 111 +/- 8

B-tamoxifen 1 mg/kg/day 11 +/- 3 127+/- 18

B-tamoxifen 10 mg/kg/day 16+/- 6 106+/- 8

A-tamoxifen 0.1 mg/kg/day 26 +/- 4 97 +/- 6

A-tamoxifen 1 mg/kg/day 24 +/- 3 94 +/- 6

A-tamoxifen 10 mg/kg/day 24 +/- 4 87 +/- 7 *

Results show mean +/- SEM for uterine and body weight for female mice. * p < 0.05 versus vehicle, by one-way analysis of variance

Table 2

Group Femoral BMD Metaphyseal BMD

Vehicle 5.0+/- 10 99 +/- 3

17β-oestradiol0.1 mg/kg/day 63 +/- 0.8 * 129+/- 3*

17β-oestradiol 1 mg/kg/day 68.5+/- 1.7* 145+/- 5*

B-tamoxifen 0.1 mg/kg/day 60.2+/- 1.2* 114+/- 4*

B-tamoxifen 1 mg/kg/day 63.4+/- 1.9 *# 124+/- 3*

B-tamoxifen 10 mg/kg/day 61.8+/- 1.4 *# 122+/- 4 *#

A-tamoxifen 0.1 mg/kg/day 60.1 +/-0.8* 117+/- 3*

A-tamoxifen 1 mg/kg/day 59.6+/- 1.4* 116+/- 2*

A-tamoxifen 10 mg/kg/day 57.6+/- 1.2 110+/- 2*

Results show mean +/- SEM bone mineral density (BMD; mg/cm2) for total femur, and distal femoral metaphysis, for female mice . *p < 0.05 versus vehicle, # p < 0.05 versus equivalent dose of A-tamoxifen, by one-way analysis of variance

Discussion

Tamoxifen Formulation

In female mice, the low E-isomer formulation of tamoxifen (Formulation A) led to a dose-dependent reduction in uterine weight, suggestive of significant antagonist activity particularly in comparison with the typical E-isomer tamoxifen formulation (Formulation B) which increased uterine weight. Overall, these findings are consistent with previous observations, which suggest that tamoxifen formulations act as a partial oestrogen agonist.

Though the typical E-isomer tamoxifen significantly increased bone mass in female mice, the extent of this response was somewhat larger than that previously observed with SERMs, i.e. approximately 50% of that following oestrogen. Since oestrogen's tendency to markedly increase bone mass in female mice largely reflects stimulation of new bone formation rather than suppression of resorption, it is tempting to speculate that the typical E-isomer tamoxifen also shares the former action, albeit with significantly less pόteficy.

While both tamoxifen formulations produced similar increments in BMD in female mice at lower doses, the response to the low E-isomer tamoxifen appeared to diminish at higher doses, at which significant differences between effects of the two formulations emerged. Although the basis for this observation is unclear, it is possible that at higher doses low E-isomer tamoxifen exerts additional antagonistic effects (due to the massively increased dose of the Z-isomer), which counteract its effects at other targets. Summary

In female mice (which have a normal oestrogen level) both tamoxifen formulations produce a similar increase in BMD at low doses. However, this increase appeared to diminish as the dose of the low E-isomer content tamoxifen was increased above 1 mg/kg i.e. 2.5x the typical human clinical dose. In female mice, the typical E-isomer content tamoxifen caused an increase in uterine weight at typical therapeutic doses, whereas the low E- isomer tamoxifen did not. Low E-isomer tamoxifen therefore was protective of bone and uterus at normal therapeutic dose levels.

Although the results given and discussed above are derived from mouse studies, it is believed, due to the body of knowledge existing on the action of tamoxifen, that results comparable to those derived from the mouse studies can be achieved in the human body.

Claims

1. The use of tamoxifen containing not more than 0.006% by weight geometric E-isomer of tamoxifen, and pharmaceutically-acceptable acid addition salts thereof, in the manufacture and formulation of a medicament having a significantly reduced risk of causing endometrial hyperplasia, for the treatment, or prevention, of hormone dependent tumours.

2. The use according to claim 1, wherein the tamoxifen contains not more than 0.005%) by weight of the geometric E-isomer of tamoxifen.

3. The use according to either claim 1 or claim 2 for the treatment, or prevention, of hormone dependent breast cancer.

4. The use according to either claim 1 or claim 2 for the treatment, or prevention, of hormone dependent prostate cancer.

5. A rnethod-of treating or preventing, a hormone dependent tumour in a patient which comprises administering to the patient an effective amount of a medicament having a significantly reduced risk of causing endometrial hyperplasia comprising tamoxifen containing not more than 0.006% by weight of its E-isomer, and pharmaceutically- acceptable acid addition salts thereof.