The present invention relates to a combination therapy for treating cancers, including solid tumours and lymphomas and leukaemias.

Introduction.

The treatment of many forms of cancer relies on chemotherapy. One of many problems associated with chemotherapy is that it requires the use of drugs which have general toxic effects not only on cancer cells but also on other cells of the human body. It is difficult for the physician to achieve the dose of a chemotherapeutic agent which will be effective in treating tumours but which will not be too toxic to the patient.

There is a continuing need for the development of chemotherapeutic regimes which can achieve this.

Background to the invention.

One class of compounds which have been used in the treatment of certain cancers, particularly breast and ovarian cancers and melanomas are anti-oestrogens. A well known anti-oestrogen compound is tamoxifen (2- [4- (1, 2-diphenyl-l-butenyl)phenoxy-N,N- dimethylethanamine) . This compound is disclosed in GB-A- 1,013,907 and 1,064,629. Other anti-oestrogen compounds, including salts and derivatives of tamoxifen are well known in the art. Examples of such compouxids can be found in EP-A-260 066, EP-A-160 508, EP-A-138 504, EP-A-124 369, EP-A-019 377, EP- A-011 372, EP-A-002 097, GB-A-2, 003, 855 and GB-A-1, 560, 274, the disclosures of which are incorporated herein by reference.

Numerous clinical trials of tamoxifen have been held and this compound has been shown to be effective in the treatment of cancers, in particular tumours which are stimulated by oestrogens. This includes breast and ovarian tumours, and melanomas. Tamoxifen is also known to inhibit protein kinase C.

A separate group of anti-cancer compounds are bryostatins and neristatins. These compounds are macrocyclic lactones found in the marine organisms Bryozoa. Bryozoa are members of the phylum Ectoprocta and are colonial filter feeders. The superficial appearance of Bryozoa has given rise to the common name of false coral.

The structure of bryostatin 1, a bryostatin from Bugula neri tina has been described by Pettit et al, J. Am. Chem. Soc, 104 ,-6846- 48, 1982. Pettit et al report that this compound has anti- cancer activity. Further bryostatins with anti-cancer activity include bryostatin 2, bryostatin 3, bryostatin 4, bryostatin 5, bryostatin 6, bryostatin 7, bryostatin 8, bryostatin 9, bryostatin 9, bryostatin 10, bryostatin 11, bryostatin 12, bryostatin 13 and neristatin 1. The isolation and characterization of these compounds are reported in Pettit et al, J. Org. Chem., 5_2;2854, 1987, EP-A-109 811 (Bryostatins 1- 3), EP-A-178 048 (Bryostatins 4-8), EP-A-264 173 (Bryostatins 9- 13), and EP-A-530 980 (Neristatin 1), the disclosures of which are incorporated herein by reference.

A number of clinical trials of bryostatins have been held, for example as reported by Prendiville et al , British J. Cancer, 68 , 418, 1993.

Bryostatin 1 has been found to be a potent activator or modulator of protein kinase C (PKC) , an enzyme family involved in cell signalling. Other PKC activators or modulators include PMA (phorbol 12-myristate 13-acetate) , diacyl glycerol and compounds disclosed in EP-A-445 271 the disclosure of which is hereby incorporated by reference.

Description of the invention.

The present invention has been derived from observations made during a phase I clinical trial of bryostatin 1 in treating ovarian carcinoma. Two of the patiimts treated with bryostatin 1 had responded (1 partial, 1 minor) . After a period of a few months, both women developed progressive disease. However,

treatment with tamoxifen induced unexpected prolonged responses. The interaction of tamoxifen with bryostatin 1 was investigated at the molecular level. Surprisingly, it was found that the simultaneous treatment of cultured mouse leukaemia cells (P388) has shown a large potentiation of growth inhibition. The P388 leukaemic cells is a model system used by the US National Cancer Institute to assess the activity of potential human anti-cancer compounds.

The present invention thus provides products containing an anti- oestrogen compound and a PKC modulator or activator, preferably a bryostatin or neristatin, as a combined preparation simultaneous, separate or sequential use in cancer therapy.

The present invention also provides use of an anti-oestrogen compound for the manufacture of a medicament for the treatment of cancer in patients being treated with a PKC modulator or activator, preferably a bryostatin or neristatin. The invention also provides the use of a PKC modulator or activator, preferably a bryostatin or neristatin, for the manufacture of a medicament for the treatment of cancer in patients being treated with an anti-oestrogen compound.

The present invention also provides a method of treatment of cancer which comprises administering to a patient in need of treatment an effective amount of an anti-oestrogen compound and a PKC modulator or activator, preferably a bryostatin or neristatin, in a simultaneous, separate or sequential manner.

Detailed description of the invention.

Preferred PKC modulators or activators are byrostatins or neristatins. Bryostatins and neristatins for use in the invention will generally be the macrocyclic lactone compounds obtainable from the organism Bugula neri tina Such compounds usually have a molecular weight of between about 700 and 1000, e.g. about 800-950. It is also preferred that bryostatins are those which have an activity which provides a 50-90% life extension at a dose of 10-70μg/ (kg/injection dose) in the urine

P388 lymphocytic leukaemia system, as reported by Pettit et al, J. Am. Chem. Soc., ibid.

Cancers which may be treated include solid tumours such as breast, ovarian, lung (including small cell lung carcinoma) , colon and bladder cancers and melanomas, as well as leukaemias and lymphomas.

A preferred anti-oestrogen compound is tamoxifen. Other anti- oestrogen compounds include those with the ability to inhibit PKC to a substantially similar degree (or greater) than tamoxifen.

Anti-oestrogen compounds include tamoxifen derivatives such as a triphenylalkene derivative of the formula:-

wherein R ¹ is alkenyl, cycloalkenyl, cycloalkenylalkyl, halogenoalkenyl, hydroxyalkyl, dihydroxyalkyl, hydroxy- cycloalkyl, dihydroxycycloalkyl, hydroxycycloalkyl-alkyl, epoxyalkyl, alkoxyalkyl or alkylthioalkyl, each of up to 10 carbon atoms, wherein R ² is alkyl of up to 6 carbon atoms, halogen or nitro and wherein ring A may optionally bear a halogen substituent or an alkoxy substituent of up to 4 carbon atoms.

These compounds can exist in the form of a cis- or a trans- isomer, in which the designation cis- and trans- refers to the relative positions of the two unsubstituted phenyl groups about the double bond.

The trans- isomer is usually preferred.

R ¹ may be, for example, allyl, but-2-enyl, 3-methylbut-2-enyl, but-3-enyl, l-methylprop-2-enyl, cyclohex-2-enyl, 2-chloroprop- 2-enyl, 2-hydroxyethyl, 2-hydroxypropyl, 2,3-dihydroxypropyl, 1- methyl-2,3-dihydroxypropyl, 2,3-dihydroxybutyl, 3-methyl-2,3- dihydroxybutyl, 3,4-dihydroxybutyl, 2-hydroxycyclohexyl, 2,3- dihydroxycyclohexyl, 2,3-dihydroxycyclohexyl, 2,3-epoxypropyl, 2,3-epoxybutyl, 3,4-epoxybutyl, 2-methoxy-ethyl or 2- methylthioethyl.

R ² may be, for example, methyl, ethyl, n-propyl, n-butyl, chloro, bromo or nitro.

The optional substituent in ring A may be, for example, a chloro or methoxy substituent.

These compounds may be obtained by reference to EP-A-019 377.

Other anti-oestrogen compounds include triphenylalkene derivatives of the formula:-

wherein either R ¹ is a hydrogen atom or a lower alkyl radical ε^d R ² is a lower alkyl radical, or R ¹ and R ² are joined toget ? with the adjacent nitrogen atom to form a heterocyclic radic. ; n is 2, 3, , 5 or 6; R ³ is a halogen atom or a lower alj;yl radical; R ⁴ is a hydrogen or halogen atom, or a hydroxy or lower alkyl or lower alkoxy radical, or is the buta-1,3-dienyl radical such that together with the benzene ring it forms a naphthyl radical, or R ⁴ has the formula -OCOR ⁵ where R ⁵ is defined below; X is hydrogen or a group -COR ⁵ where R is an alkyl radical of 1

to 10 carbon atoms or an alkoxyalkyl radical of 2 to 10 carbon atoms, or R ⁵ has the formula

wherein m is 0,

1, 2, 3, or 4, wherein R ⁶ is a hydrogen or halogen atom or a cyano, lower alkyl or lower alkoxy radical and wherein p is 1, 2 or 3; or R ⁵ has the formula -NHR ⁷ , wherein R ⁷ is an alkyl radical of 1 to 10 carbon atoms or R ⁷ has the formula

wherein m, p and R ⁶ have the meanings stated above; or a pharmaceutically-acceptable acid-addition salt thereof.

These compounds can exist in the form of a cis or a trans isomer in which the designation cis or trans refers to the relative positions of the acyloxyphenyl radical, and the phenyl radical bearing the group R ⁴ , about the double bond.

A particularly suitable value for R ¹ , R ² , R ³ , R ⁴ or R ⁶ when it is a lower alkyl radical is, for example, an alkyl radical of 1-4 carbon atoms, for example a methyl, ethyl, n-propyl, isopropyl or a butyl radical.

A particularly suitable value for R ¹ and R ² when they are joined together with the adjacent nitrogen atom to form a heterocyclic radical is, for example, a 5- or 6- membered nitrogen-containing heterocyclic radical optionally including an oxygen or sulphur atom as a second hetero-atom, for example a pyrrolidino, piperidino or morpholino radical.

A particularly suitable value for R ³ , R ⁴ or R ⁶ when it is a halogen atom is, for example, a fluorine, chlorine or bromine atom.

A particularly suitable value for R ⁴ or R ⁶ when it is a lower alkoxy radical is, for example, an alkoxy radical of 1-4 carbon atoms, for example a methoxy or ethoxy radical.

A particularly suitable value for R ⁵ when it is an alkyl or alkoxyalkyl radical is, for example, the methyl, ethyl, isopropyl, hexyl, decyl or methoxy-methyl radical.

A particularly suitable salt is, for example, a hydrochloride, sulphate, phosphate, acetate, tartrate or citrate.

These compounds may be obtained by reference to EP-A-002 907 when X is hydrogen and EP-A-101 372 when X is COR ⁵ .

A further class of anti-oestrogen compounds are triphenylalkene compounds of the formula:

where X is I or Br at the 3- or 4- position; R ¹ and R ² are C^ alkyl or R ¹ is hydrogen and R ² is C _1-3 alkyl or together the group R ¹ and R ² form a saturated hetrocycle having from 4 to 6 carbon atoms, or pharmaceutically acceptable acid addition salts thereof. Such salts include >the hydrochloride, sulphate, phosphate, acetate, tartrate or citrate salt. Preferably X is

I at the 4 position and R ¹ and R ² are both methyl or form a pyrrolidino ring. These compounds can exist in the form of a cis- or a trans- isomer. A preferred compound of this formula is E-

1- [4-iodophenyl] -1- [4- (2-pyrrolidino-ethoxy)phenyl] -2-phenyl-l- butene (idoxefene) . These compounds may be obtained by reference to EP-A-260 066.

Another class of anti-oestrogen compounds are steroid derivatives of the formula:-

ST-A-X-R ¹

wherein ST is a 7or-linked steroid nucleus of the general formula: -

wherein there is an optional double bond between carbon atoms 6 and 7, or there are two optional double bonds between carbon atoms 6 and 7 and carbon atoms 8 and 9 of the steroid nucleus; wherein the aromatic ring A may optionally bear one or two halogen or alkyl substituents; wherein R ³ is hydrogen or alkyl, alkanoyl, alkoxycarbonyl, carboxyalkanoyl or aroyl each of up to 10 carbon atoms; wherein R ¹⁶ is hydrogen, alkyl of up to 6 carbon atoms which is preferably in the β-configuration, or hydroxy which is preferably in the cu-configuration; wherein either R ¹⁷ (in the β-configuration) is hydroxy or alkanoyloxy, carboxyalkanoyloxy or aroyloxy each of up to 10 carbon atoms; and R ²⁷ (in the α-configuration) is hydrogen or alkyl, alkenyl or alkynyl each of up to 6 carbon atoms; or R ¹⁷ and R ²⁷ together form oxo (=0) ; wherein R ¹⁸ is alkyl or up to 6 carbon atoms; wherein A is straight- or branched- chain alkylene, alkenylene or alkynylene each of from 3 to 14 carbon atoms, which may have

one or more hydrogen atoms replaced by fluorine atoms, or has the formula

-A^Y-A ¹¹ -

wherein A ¹ and A ¹¹ are each alkylene or alkenylene, optionally fluorinated, having together a total of 2 to 13 carbon atoms and Y- is -0-, -S-, -SO-, -S0 ₂ -, -CO- or -NR- wherein R is hydrogen or alkyl or up to 3 carbon atoms; or A ¹ is alkylene or alkenylene, optionally fluorinated, and A ¹¹ is a direct link or alkylene or alkenylene, .optionally fluorinated, such that A ¹ and A ¹¹ together have a total of 1 to 12 carbon atoms, and Y is -NRC0-, -C0NR-, -COO-, -OCO- or phenylene wherein R has the meaning stated above; wherein R ¹ is hydrogen, or alkyl, alkenyl, cycloalkyl, halogenoalkyl, carboxyalkyl, alkoxycarbonylalkyl, aryl or arylalkyl each of up to 10 carbon atoms, or dialkylaminoalkyl wherein each alkyl is of up to 6 carbon atoms, or R ¹ is joined to R ² as defined below; and wherein X is -CONR ² -, -CSNR ² -, -NR ¹² -CO-,

22 NR

-NR ¹² -CS-, -NR ¹² -C0NR ² -, -NR ¹² -C-NR ² -, -S0 ₂ NR ² - or -CO-; or, when R ¹ is not hydrogen, is -O-, NR ² -,

-(NO)R ² -, -(PO)R ² -, -NR ¹² -COO-, -NR ¹² -S0 ₂ -, -S-,

-SO- or -S0 ₂ -; wherein R ² is hydrogen or alkyl of up to 6 carbon atoms, or R ¹ and R ² together form alkylene or halogenoalkylene such that, with the adjacent nitrogen atom, they form a heterocyclic ring of 5 to 7 ring atoms, one of which atoms may be a second heterocyclic atom selected from oxygen, sulphur and nitrogen; wherein R ¹² is hydrogen or alkyl of up to 6 carbon atoms; and wherein R ²² is hydrogen, cyano or nitro; or a salt thereof when appropriate.

A suitable value for the halogen or alkyl substituent in ring A is, for example, fluoro, chloro, bromo, iodo, methyl or ethyl.

A suitable value for R ³ when it is alkyl, alkanoyl, alkoxycarbonyl, carboxyalkanoyl or aroyl is, for example, methyl, ethyl, acetyl, propionyl, butyryl, pivalyl, decanoyl, isopropoxycarbonyl, succinyl or benzoyl. R ³ is preferably hydrogen or alkanoyl or alkoxycarbonyl each of up to 5 carbon atoms.

A suitable value for R ¹⁶ when it is alkyl is, for example, methyl or ethyl. R ¹⁶ is preferably hydrogen.

A suitable value for R ¹⁷ when it is alkanoyloxy, carboxyalkanoyloxy or aroyloxy is, for example, acetoxy, propionyloxy, succinyloxy or benzoyloxy. R ¹⁷ is preferably hydroxy.

A suitable value for R ²⁷ when it is alkyl, alkenyl or alkynyl is, for example, ethyl, vinyl or ethynyl. R ²⁷ is preferably hydrogen.

A suitable value for R ¹⁸ is methyl or ethyl, especially methyl.

The group ST- is preferably oestra-1,3, 5 (10) -triene-3 , 173-diol, 3-hydroxyoestra-l,3, 5 (10) -trien-17-one or 17α-ethynyloestra- 1,3,5(10) -triene-3,17 _/ S-diol, all of which bear the -A-X-R ¹ substituent in the 7o;-position, or a 3-alkanoyl ester thereof.

One preferred value for the group -A- is a straight-chain alkylene group of the formula

-(CH ₂ ) _n -

wherein n is an integer of from 3 to 14, especially from 7 to 11, which may have one of the hydrogen atoms replaced by fluorine, for example to provide the group - (CH ₂ ) ₈ CHFCH ₂ - . The group A may also be a branched-chain alkylene group, for example the group

- (CH ₂ ) ₆ CH(CH ₃ ) - , or a straight-chain alkenylene group, for example of the formula - (CH ₂ ) ₂ CH=CH(CH ₂ ) _m - wherein m is an integer from 0 to 10, especially from 3 to 7.

A second preferred value for the group A is a group of the formula

-A'-Y-A ¹¹ -

wherein A ¹ is straight-chain alkylene or alkenylene each of 2 to 9 carbon atoms, especially alklene of 4 to 6 carbon atoms, -Y- is phenylene (ortho, meta- or, especially, para-) and A ¹¹ is a direct link, ethylene or vinylene, especially ethylene.

A suitable value for R ¹ when it is alkyl, alkenyl or cycloalkyl is, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, t-pentyl, 2,2-dimethylpropyl, 1- methylbutyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1,1- dimethylbutyl, 1, 3-dimethylbutyl, n-heptyl, n-nonyl, n-decyl, n- undecyl, allyl, cyclopentyl or cyclohexyl.

A suitable value for R ¹ when it is aryl or aryalkyl is, for example, phenyl, 2-ethylphenyl, p-fluorophenyl, _-chlorophenyl, m-chlorophenyl, p_-cyanophenyl, p_-methoxyphenyl, benzyl, a- methylbenzyl, p_-chlorobenzyl, p-fluorophenethyl or p_- chlorophenethyl.

A suitable value for R ¹ when it is halogenoalkyl, carboxyalkyl, alkoxycarbonylalkyl or dialkylaminoalkyl is, for example, 2- chloro-2,2-difluoroethyl, 2,2,2-trifluoroethyl, 2,2,3,3,3-penta- fluoropropyl, 3-chloropropyl, 2,2-difluorobutyl, 4,4,4- trifluorobutyl, 1H, lH-heptafluorobutyl , 4,4,5,5,5- pentafluoropentyl, 4,4, 5,5, 6, 6, 6-heptafluorohexyl, 1H, 1H- tridecafluoroheptyl, 5-carboxypentyl, 5-methoxycarbonylpentyl or 3- dimethylaminopropyl.

A suitable value for the heterocyclic ring -NR^ ² is, for example, pyrrolidino, piperidino, 4-methylpiperidino, 4- ethylpiperidino, 3-methylpiperidino, 3,3-dimethylpiperidino, 4- chloropiperidino, morpholino or 4- methylpiperazino.

A suitable value for R ² or R ¹² when it is alkyl is, for example, methyl, ethyl or n-butyl.

One appropriate salt is an acid-addition salt of a steroid derivative which possesses an amino function, for example a compound wherein Y is -NR-, X is - -NINRK ² --- oorr Rκ ¹ ~ is dialkylaminoalkyl. A suitable acid-addition salt is, for example, a hydrochloride, hydrobromide, acetate, citrate, oxalate or tartrate.

Another appropriate salt is a base-addition salt of a steroid derivative which possesses a carboxy function, for example a compound wherein R ¹ is carboxyalkyl. A suitable base-addition salt is, for example, a sodium, potassium, ammonium or cyclohexylamine salt.

A preferred steroid derivative has the formula : -

wherein R ¹⁷ is hydroxy and R ²⁷ is hydrogen or ethyl, or R ¹⁷ and R ²⁷ together form oxo;

-A- is -(CH ₂ ) _n -, wherein n is an integer from 3 to 14, especially from 7 to 11 or A is a group;

wherein m is an integer from 2 to 9, especially from 4 to 6, and p is 0 to 2, especially 0 or 2; wherein R ¹ is alkyl, fluoroalkyl or cycloalkyl each of up to 10 carbon atoms; or R ¹ is phenyl, chlorophenyl or benzyl, or is linked to R ² as stated below;

X is -CONR ² , -NR ¹² C0-, -S-, -SO- or -S0 ₂ - wherein R ² is hydrogen or alkyl of up to 3 carbon atoms or together with R ¹ forms alkylene of 5 or 6 carbon atoms, and wherein R ¹² is hydrogen or alkyl of up to 3 carbon atoms.

Most preferably R ¹⁷ is hydroxy, R ²⁷ is hydrogen, A is -(CH ₂ ) ₈ -, X is -SO- and R ¹ is 4,4, 5, 5, 5-pentafluoropentyl (- (CH ₂ ) ₃ C ₂ F ₅ ) . This compound is known as ICI 182780.

These steroid derivatives may be obtained by reference to EP-A- 138 504.

Further anti-oestrogen compounds include phenol derivatives of the formula:-

NU-A-X-R ¹

where NU is a mono- or bis-phenolic nucleus of the general formula: -

wherein one of R ¹³ and R ²³ , and one of R ³³ and R ⁴³ , has the formula R ³ 0-, wherein each R ³ , which may be the same or different, is hydrogen or alkyl, cycloalkyl, alkanoyl, alkoxycarbonyl, carboxyalkanoyl or aroyl each of up to 10 carbon atoms, and

wherein the other of R ¹³ and R ²³ , and the other of R ³³ and R ⁴³ , is hydrogen; or wherein one of R ¹³ and R ²³ , and/or one of R ³³ and R ⁴³ , has the formula R ³ 0-, wherein R ³ is alkanoyl of up to 10 carbon atoms which bears one or two substituents selected from hydroxy, amino, alkylamino, dialklamino, alkoxycarbonylamino, alklthio, alkylsulphinyl, alkylsulphonyl, alkyldithio, carbamoyl, alkylcarbamoyl and dialkylaminoalkoxycarbonyl, wherein each alkyl or alkoxy has up to 6 carbon atoms; or R ³ is (azacycloalkyl) carbonyl or

N-alkoxycarbonyl (azacycloalkyl) carbonyl wherein the azacycloalkyl is of 5 to 7 ring atoms and the alkoxy is of up to 6 carbon atoms; or R ³ is alkanoyl of up to 10 carbon atoms which bears a carboxy substituent and one other substituent selected from those stated above; or R ³ is alkyl of up to 10 carbon atoms which bears an alkanoyloxy substituent or an alkanoyloxy substituent wherein the alkanoyl group itself bears one or two substituents selected from those stated above; or R ³ is sulphobenzoyl, l-alkyl-l,4-dihydro-pyridylcarbonyl or N- (sulphamoylphenyl) carbamoyl; wherein if only one of R ¹³ , R ²³ , R ³³ and R ⁴³ has the formula R ³ 0- another of R ¹³ , R ²³ , R ³³ and R ⁴³ which is in the other aromatic ring B or C from that which contains the R ³ 0- substituent, is hydrogen, halogen, amino, trifluoromethyl, carboxy or carbamoyl, or alkyl, hydroxyalkyl, hydro yfluoroalkyl, alkylamino, dialkylamino, alkanoylamino, alkanoyl, alkoxycarbonyl, alkylcarbamoyl, dialkylcarbamoyl, alkylsulphamoyl or dialkylsulphamoyl each of up to 6 carbon atoms, and wherein the other two of R ¹³ , R ²³ , R ³³ and R ⁴³ are both hydroge ; wherein R ⁴ and R ¹⁴ , which may be the same or different, each is hydrogen or alkyl of up to 5 carbon atoms, or R ⁴ and R ¹⁴ are joined together so that CR ⁴ -CR ¹⁴ is an olefinic double bond; wherein either R ⁵ and R ¹⁵ are both hydrogen and R ⁶ is alkyl of up to 5 carbon atoms; or R ⁵ and R ^β together form a direct link or together form a group

-CH ₂ -, -CH(CH ₃ )-, -CH ₂ CH ₂ -, -(CH ₂ ) ₃ -, -CH=CH ² -, -S-, -0-, -0-CR ₂ -, -0-C0-, -NR-CH ₂ - or -N=CH- wherein R, the two values of which may be the same or different; in -OCR ₂ -, is hydrogen or alkyl of up to 3 carbon atoms and R ¹⁵ is hydrogen; or R ¹⁵ and R ⁶ together form -CH ₂ - and R ⁵ is hydrogen; and wherein the aromatic rings B and C each may optionally bear one or more halogen or alkyl substituents; wherein A is straight- or branched-chain alkylene, alkenylene or alkynylene each of from 4 to 12 carbon atoms; or A has the formula:-

-A^Y^ ¹¹ - or -A ¹ -Y ² -A ²¹ - or -A ¹ -Y ³ -A ²¹ - wherein A ¹ and A ¹¹ are each alkylene or alkenylene having together a total of 3 to 11 carbon atoms and Y ¹ is -0-, -S-, -S0- , -S0 ₂ - or -CO-; or A ¹ is alkylene or alkenylene and A ²¹ is a direct link or alkylene, alkenylene or cycloalkylene, such that A ¹ and A ²¹ together have a total of 2 to 10 carbon atoms, and Y ² is -NRCO-, -C0NR-, -COO- or -OCO-, wherein R has the meaning stated above, or Y ³ is phenylene, naphthylene or heterocyclene which may optionally bear one or more halogen or alkyl substituents, or heterocyclene which bears one or more alkoxy or oxo substituents, or A has the formula:-

-A ¹ -Y ¹ -A ²¹ -Y ³ -A ³¹ - or -A ¹ -Y ³ -A ²¹ -Y ¹ -A ¹¹ - wherein A ¹ and A ¹¹ are each alkylene or alkenylene, and A ²¹ and A ³¹ are each a direct link or alkylene or alkenylene, such that A ¹ , A ²¹ and A ³¹ together, or A ¹ , A ²¹ and A ¹¹ together, have a total of 1 to 9 carbon atoms, and Y ¹ and Y ³ have the meanings stated above; wherein R ¹ is hydrogen, or alkyl, alkenyl, cycloalkyl, halogenoalkyl, aryl or arylalkyl each cf up to 10 carbon atoms, or R ¹ is joined to R ² as defined below; and wherein X is -CONR ² -, -CSNR ² -, ~NR ¹² CO-,

NR -NR ¹² CS-, -NR ¹² CONR ² -, -NR ¹² -C-NR ² -,

-S0 ₂ NR ² - or -CO-, or, when R ¹ is -not hydrogen, is-NR ¹² COO-, -S-, -SO- or -S0 ₂ -, wherein R ² is hydrogen or alkyl of up to 6 carbon atoms, or R ¹ and R ² together form alkylene such that, with the adjacent nitrogen atom, they form a heterocyclic

ring of 5 to 7 ring atoms, one of which may be a second heterocyclic atom selected from oxygen, sulphur and nitrogen; wherein R ¹² is hydrogen or alkyl of up to 6 carbon atoms; and wherein R ²² is hydrogen, cyano or n.itro; or a salt thereof when appropriate.

Preferably, the anti-oestrogen compound is tamoxifen or a physiologically acceptable salt thereof. The bryostatin is preferably a bryostatin obtainable from the marine organism Bugula neri tina . In addition to bryostatins 1-13 and the neristatins, acyl derivatives of bryostatins may be used. This is because bryostatins and neristatins have free hydroxyl groups and replaceable acyl groups such that acyl ester of the compounds may be prepared. The preparation of such compounds may be made by reference to EP-A-264 173.

Acids which may be used in the acylation of bryostatins and neristatins include saturated or unsaturated, straight or branched chain aliphatic carboxylic acids, containing from 2 to 20, preferably 2 to 6 carbon atoms. Examples include acetic, propionic and butyric acids. Other acids include saturated or unsaturated alicyclic acids, containing from 4 to 12 carbon atoms; saturated or unsaturated alicyclic aliphatic carboxylic acids, containing from 6 to 14 carbon atoms; aromatic carboxylic acids, eg benzoic acid, toluic acid, isobutylbenzoic acid or methylbutylbenzoic acid, aromaticaliphatic carboxylic acids, e.g. phenylacetic acid, phenylpropionic acid or phenylvaleric acid.

The acyl derivative will be selected such that the bryostatin or neristatin will have substantially the same or greater activity than the parent compound, for example as measured in the P388 model system mentioned above. Desirably the bryostatin is bryostatin 1.

Compounds for use in the invention may be administered in the form of salts. Salts which may be conveniently used in therapy include physiologically acceptable base salts, eg derived from an appropriate base, such as alkali metal (e.g. sodium) ,

alkaline earth metal (e.g. magnesium) salts, ammonium and NR ₄

(wherein R is C _1-4 alkyl) salts. Salts further include, where for example the compound has and amine group, physiologically acceptable acid addition salts, including the hydrochloride and acetate salts.

The compounds may be administered to mammals including humans by any route appropriate to the condition to be treated, suitable routes including oral, rectal, nasal, topical (including buccal and sublingual) , vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) . It will be appreciated that the preferred route may vary with, for example, the condition of the recipient.

The amount of each compound required will depend upon a number of factors including the severity of the condition to be treated and the identity of the recipient and will ultimately be at the discretion of the attendant physician. The doses preferably will be selected to provide the observed synergistic effect between the PKC modulator or activator and the anti-oestrogen compound.

In general, however, for each of the above- and below-mentioned utilities and indications, a suitable, effective dose of PKC modulators or activators will be in the range 0.001 to 250 mg/m ² body surface area of recipient per day, preferably in the range 0.01 to 100 mg/m ² body surface area per day. Anti-oestrogen compounds such as tamoxifen and derivatives thereof are most preferably administered in the range 5 to 50 mg, e.g. 20 to 40 mg per day. Bryostatins are desirably administered in the range from 0.1 to 100, preferably from 0.1 or 1.0 to 50, more preferably from 20 to 30 μg/m ² /dose. Doses may be administered from once a week to one or more (e.g. 2 or 3) times a day. Unless otherwise indicated all weights of active ingredient are calculated as the parent compound; for salts and esters thereof the figures would be increased proportionately.

The desired dose may if desired be presented as two, three, four or more sub-doses administered at appropriate intervals

throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing 0.001 to 100 mg of active ingredient per unit dosage form.

While it is possible for the compounds to be administered alone it is preferable to present them as pharmaceutical formulations. The formulations of the present invention comprise at least one active ingredient, as above defined, together with one or more acceptable carriers .thereof and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipients thereof.

The formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual) , vaginal or parenteral

(including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. Formulations suitable for oral use may also include buffering agents designed-to neutralise stomach acidity. Such buffers may be chosen from a variety of organic or inorganic agents such as weak acids or bases admixed with their conjugated salts.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bac eriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Injection solutions and suspensions may be prepared extemporaneously from sterile powders, granules and tablets of the kind previously described.

Bryostatins will preferably be administered intravenously or as a bolus injection. Intravenous administration includes administration in the form of a slow infusion, for example given over from 1 to 72, e.g from 1 to 24 or 1 to 12 hours.

The anti-oestrogen compound is preferably administered orally (e.g. in tablet form) . Suitable doses are as mentioned above, e.g about from 5 to 50, e.g from 20 to 40 mg per day.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient (s) .

The anti-oestrogen compound and bryostatin may be administered simultaneously, separately or sequentially. For simultaneous administration the compounds may be formulated in a single preparation. Alternatively the- compounds may be formulated separately and mixed immediately prior to administration. Simultaneous administration may also occur by introducing the compounds into the patient by different routes at the same time, eg orally and by injection.

If the compounds are administered sequentially, this may be done by administering one compound immediately after the other, or there may be a delay of several minutes, hours, days or months between the doses. For example, doses of the anti-oestrogen compound and bryostatin may be given on alternate days. A single dose of each may be administered, or, more usually, several doses of each over the course of a few days, e.g. 4, 6, 7, 10, 14, 21 or 35 days.

Products of the invention containing the anti-oestrogen compound and the PKC modulator or activator may be packaged together in the form of a kit or presentation pack. Such a kit or pack may be particularly useful where separate or sequential administration of the two components are contemplated. The kit or package may comprise the components in unit dosage form, and/or instructions for use and/or calendar markings indicating the timing of doses to be taken.

The present invention is of particular use in the treatment of cancers such as ovarian and breast cancers and melanomas.

Compounds for use in the invention may be obtained by reference to the published literature, including the patent documents and journal references mentioned herein.

The following examples illustrate the invention.

Example 1. Phase 1 clinical trial.

In Manchester, United Kingdom, several responses were seen in a phase I clinical trial of bryostatin 1. The trial was conducted as reported by Prendiville et al, British Journal of Cancer, ■S8>418, 1993. Two of the responses were seen in women with advanced, heavily pre-treated ovarian carcinoma (1 x partial response, 1 x minor response) . Progressive disease following the bryostatin treatment occurred at 4 and 6 months, respectively. The woman who progressed following the minor response was treated with Paclitaxel which afforded a further partial response of 4 months. Both women subsequently developed progressive disease.

The women were subsequently treated with Tamoxifen (20 mg/day) . In both women the disease stabilized for a further 10 and 12 months respectively.

Example 2. Combinations of bryostatin and tamoxifen in vitro.

Bryostatin 1 and tamoxifen were used in combination to treat a culture of mouse P388 leukaemic cells. The results are shown in Table 1.

Table 1: I C _∞ concentrations of Bryostatin 1 and Tamoxifen, singly and in combination.

Compound ICso

Bryostatin 36 ng/ml

Tamoxifen 4.1 A/g/ml

Bryostatin in presence of 1 μgl \ 0.1 ng/ml tamoxifen

Tamoxifen in presence of 1 //g/ml 0.2 μg/ml bryostatin

It was found that the IC ₅₀ (concentration required to inhibit cell growth by 50%) is reduced dramatically when the compounds

are used together. The results are especially surprising when it is appreciated that bryostatin 1 at 1 ng/ml and tamoxifen at 1 μg/ml are non-growth inhibitory.

Example 3.

The growth inhibition studies on combinations of bryostatin 1 and Tamoxifen were repeated. An enhancement of growth inhibition over two orders of magnitude was observed. As a control, staurosporine (a known PKC inhibitor) was used as a substitute for Tamoxifen. This showed only an enhancement of 1.7 fold compared to 200-fold for Tamoxifen. In contrast the PKC activator PMA (phorbol 12-myristate 13-acetate) can potentiate the growth inhibitory effects of Tamoxifen to a level similar to that observed for bryostatin 1 (25-fold compared to 34-fold) , but

Tamoxifen only slightly potentiates the growth inhibitory effects of PMA (3.9-fold) compared to the 200-fold observed for bryostatin - Tamoxifen combinations. The results are summarized in Table 2:

TABLE 2: Effect of drug combinations on the growth of cells in vitro. The final column represents the IC ₅₀ of drug 2 (alone) divided by the IC ₅₀ of drug 2 when given in combination with non- inhibitory concentration of drug 1. The larger this value the greater the toxicity of the combination.

Cell Line Drug 1 Drug 2 Enhancement of Growth Inhibition

<-' P388 Bryostatin 1 (1 ng/ml) Tamoxifen 34

P388 Tamoxifen (1//g/ml) Bryostatin 1 200

P388 Staurosporine (1 ng/ml) Tamoxifen 1.2

5 P388 Staurosporine (1 ng/ml) Bryostatin 1 1.7

P388 PMA (lOng/ml) Tamoxifen 25

P388 Tamoxifen (1//g/ml) PMA 3.9

0 Example 4 . ] Effect of combined druαs on PKC activitv .

The protein kinase C (PKC) activity of P388 cells cultured in vitro was measured as described by Prendiville et al , British J. Cancer, 22.; 573, 1994, at 24 hours following treatment with 5 tamoxifen and bryostatin singly and in combination at different concentrations. The results are shown in Table 3.

Bryostatin 1 Tamoxifen % Growth Relative PKC activity (ng/ml) ( g/ml) Inhibition (t = 24hrs)

- - 0 1.0

0.1 - 0 28 ± 6

1 - 0 62 ± 8

10 - 30 0.6 ± 0.8

30 - 50 0.5 ± 0.5

- 0.2 0 52 ± 7

- 1 15 90 ± 11

- 4.1 50 1.2 ± 1

0.1 1 20 157 ± 27

1 0.2 25 8 ± 1

10 4 >90 0

30 1 >90 0

Table 3: Effect of Bryostatin 1 and Tamoxifen, alone and in combination, on the PKC activity of P388 cells. All PKC activities are expressed relative to control P388 cells. The results are expressed as mean + S.D. of triplicate samples. Growth inhibition values were calculated from the isobologram curves.

These data show that combinations of bryostatin 1 and Tamoxifen which cause high levels of growth inhibition results in a loss of PKC activity 24 hours after treatment. However, treatment with non-inhibitory concentrations of bryostatin 1 and Tamoxifen, given alone or in combination, results in increased PKC activity.

PKC activation is an important component of cell replication and thus its inhibition is of significance in the treatment of tumour cells.