WO1998009633A2

WO1998009633A2 - Pharmaceutical compositions

Info

Publication number: WO1998009633A2
Application number: PCT/EP1997/004796
Authority: WO
Inventors: Paul Leslie Nicklin; Judith Ann Phillips; William Guy Love; Karen Ophelia Hamilton
Original assignee: Ciba Geigy AG; Novartis AG
Current assignee: Novartis AG
Priority date: 1996-09-04
Filing date: 1997-09-03
Publication date: 1998-03-12
Anticipated expiration: 1999-03-04
Also published as: GB9618376D0; WO1998009633A3; AU4457597A; US6465439B1; EP0925066A2

Abstract

A pharmaceutical composition comprising (A) and oligonucleotide having 5 to 50 nucleotide units, which is specifically hybridizable with DNA or RNA derived from a protein kinase C gene, entrapped in (B) sterically stabilised liposomes.

Description

Pharmaceutical Compositions

This invention relates to pharmaceutical compositions, particularly liposomal oligonucleotide compositions, their preparation and their use.

In WO 95/02069 there are described oiigonucleotides specifically hybridizable with DNA or RNA derived from a protein kinase C (PKC) gene, which oiigonucleotides are particularly for use in the diagnosis and treatment of neoplastic, hyperprohferative and inflammatory disorders associated with protein kinase C.

It has now been found that compositions retaining high activity after prolonged circulation in the bloodstream and exhibiting reduced accumulation of oligonucleotide in non-target organs such as the liver and kidney can be prepared by formulation of such oiigonucleotides within sterically stabilised liposomes.

Accordingly, the present invention provides a pharmaceutical composition comprising (A) an oligonucleotide having 5 to 50 nucleotide units specifically hybridizable with DNA or RNA derived from a protein kinase C gene, entrapped in (B) sterically stabilised liposomes.

Hybridisation, in the context of nucleic acid chemistry, means hydrogen bonding, also known as Watson-Cπck base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them Adenine and thymme are examples of complementary bases which form two hydrogen bonds between them. "Specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementary such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding, of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementary to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, or, in the case of in vitro assays, under conditions in which the assays are conducted.

As used in the context of this invention, the term "oligonucleotide" refers to a substance having a plurality of nucleotide units formed from naturally occurring bases and sugars joined by phosphodiester internucleoside (backbone) linkages. The term "oligonucleotide" also includes analogues which function similarly to naturally occurring oiigonucleotides but which have non-naturally occurring monomers (nucleotides) or portions thereof. These oligonucleotide analogues are often preferred over native forms because of properties such as enhanced cellular uptake, enhanced target binding affinity and increased stability in the presence of nucleases.

In preferred embodiments of the invention, the oligonucleotide (A) is specifically hybridizable with the translation initiation codon of the PKC gene, in which case it preferably comprises a CAT sequence, or with the 5' untranslated region or 3' untranslated region of the gene. In other preferred embodiments of the invention, the oligonucleotide (A) is specifically hybridizable with DNA or mRNA encoding a particular PKC isozyme (isoform) or a particular set of PKC isozymes.

The oligonucleotide (A) preferably comprises from 8 to 30 nucleotide units, more preferably 12 to 25 nucleotide units, especially 18 to 22 nucleotide units.

In some preferred oiigonucleotides (A), at least one nucleotide is modified at the 2' position of the sugar moiety. Certain preferred oiigonucleotides (A) are chimeric oiigonucleotides. "Chimeric oiigonucleotides" or "chimeras", in the context of this invention, are oiigonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oiigonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the RNA target) and a region that is a substrate for RNase H cleavage. In one preferred embodiment, a chimeric oligonucleotide comprises at least one region modified to increase target binding affinity and, usually, a region that acts as a substrate for RNAse H. Affinity of an oligonucleotide for its target is routinely determined by measuring the Tm of an oligonucleotide/target pair, which is the temperature at which the oligonucleotide and target dissociate; dissociation is detected spectrophotometrically. The higher the Tm, the greater the affinity of the oligonucleotide for the target. In a more preferred embodiment, the region of the oligonucleotide which is modified to increase target binding affinity comprises at least one nucleotide modified at the 2' position of the sugar, particularly a 2' - alkoxy, 2'- alkoxyalkoxy or 2'-fluoro-modified nucleotide. Such modifications are routinely incorporated into oiigonucleotides and these oiigonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target. In the chimeric oiigonucleotides, the region which is a substrate for RNAse H comprises at least one 2'-deoxynucleotide. RNAse H is a cellular endonuclease that cleaves the RNA strand of RNA.DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus can greatly enhance the efficiency of antisense inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis. In another preferred embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide. Nuclease resistance is routinely measured by incubating oiigonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oiigonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oiigonucleotides. A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. Oiigonucleotides which contain at least one phosphorothioate modification are presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance.

Specific examples of some preferred oiigonucleotides may contain phosphorothioate, phosphotriester, methyl phosphonate, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar ("backbone") linkages, e.g. amide-type linkages. Most preferred are phosphorothioates and those with CH₂-NH-O-CH₂, CH₂- N(CH₃)-O-CH₂, CH₂-O-N(CH₃)-CH₂, CH2-N(CH₃)-N(CH₃)-CH₂ and O-N(CH₃)-CH₂-CH₂ and CH₂- C(O)-NH-CH₂ backbones (where phosphodiester is O-P-O-CH₂). Also preferred are oiigonucleotides having morpholino backbone structures, for example as described in U.S. Patent No. 5, 034, 506. In other preferred embodiments, such as the protein-nucleic acid or peptide-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, as described by P.E. Nielsen, M. Egholm, R.H. Berg, O. Buchardt, Science 1991, 254, 1497. Other preferred oiigonucleotides may contain sugar moieties comprising one of the following at the 2' position: OH; SH; SCH₃; F; OCN; OCH₂OCH₃; O(CH₂CH₂)_mOCH₃ wherein m is 1 , 2 or 3, preferably OCH₂CH₂OCH₃; OCH₂O(CH₂)_n CH₃ ;O(CH₂)_nNH₂ or O(CH₂)_nCH₃ where n is from I to about I0; OCH₂CH₂NRιR₂ wherein R- and R₂ are independently of each other, H or CH₃. Ci to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl; C_M0 lower alkoxy or substituted alkoxy, preferably OCH₃; CI; Br; CN; CF₃; OCF₃; O-, S-, or N-alkyl; O-, S-, or N- alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycioalkaryl; aminoaikylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a cholesteryl group; a conjugate; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Oiigonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. Other preferred embodiments may include at least one modified base form or "universal base" such as inosine. Preferred bases includexanthine, hypoxanthine, adenine, 2-aminoadenine, guanine, 6-thioguanine, uracil, thymine, cytosine, 5-methylcytosine, 5-propynyluracil, 5-fluorouracil and 5-propynylcytosine.

In certain especially preferred embodiments of the invention, all nucleotides of the oligonucleotide (A) are 2'-deoxynucleotides and all backbone linkages are phosphorothioate linkages.

In certain other especially preferred embodiments, the oligonucleotide (A) is a chimeric oligonucleotide having one or more regions with 2'-deoxynucleotides and one or more regions with 2'-modrfied nucleotides, preferably 2'-alkoxynucleotides or 2'- alkoxyalkoxynucleotides, particularly 2'-methoxyethoxynucleotides, the one or more 2'- deoxynucleotide regions preferably having phosphorothioate backbone linkages and the one or more 2'-modified nucleotide regions preferably having phosphodiester or phosphorothioate backbone linkages. These chimeric oiigonucleotides preferably comprise a region of 2'-deoxynucleotides between two regions of 2'-modified nucleotides, the deoxynucleotide region being preferably at least 4 nucleotides long, more preferably at least 6 nucleotides long, especially at least 8 nucleotides long.

The oiigonucleotides used as component (A) of the composition of the invention may be conveniently and routinely made using well-known techniques such as solid phase synthesis. Equipment for such synthesis is available commercially from various sources including Applied Biosystems. The use of such techniques to prepare oiigonucleotides such as the phosphorothioates and alkylated derivatives is well known. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling VA) to synthesize fluorescently labelled, biotinylated or other modified oiigonucleotides such as cholesterol-modified oiigonucleotides.

Specific especially preferred oiigonucleotides, for which the nucleotide sequences and preparation have been published in WO95/02069, include the following:

Oiigonucleotides Targeted to the Human PKC-α isozvme

No Sequence Target Seo. ID No.

ON1 CCC CAA CCA CCT CTT GCT CC 5" 1

Untranslated

ON2 GTT CTC GCT GGT GAG TTT CA 3' 2

Untranslated

ON3 AAA ACG TCA GCC ATG GTC CC Translation 3 init. codon ON4 GGA TTC ACT TCC ACT GCG GG 3' 4

Untranslated

ON5 GAG ACC CTG AAC AGT TGA TC 3' 5

Untranslated

ON6 CCC GGG AAA ACG TCA GCC AT Translation 6 init codon

ON7 CTG CCT CAG CGC CCC TTT GC Internal 7

(C1) domain

ON8 AGT CGG TGC AGT GGC TGG AG Internal 8

(C1) domain

ON9 GCA GAG GCT GGG GAC ATT GA Internal 9

(C1 ) domain

ON10 GGG CTG GGG AGG TGT TTG TT 3' 10

Untranslated

ON11 CAC TGC GGG GAG GGC TGG GG 3' 11

Untranslated

ON12 AGC CGT GGC CTT AAA ATT TT 3' 12

Untranslated

ON13 ATT TTC AGG CCT CCA TAT GG 3' 13

Untranslated

ON14 AAG AGA GAG ACC CTG AAC AG 3' 14

Untranslated ON15 GAT AAT GTT CTT GGT TGT AA 3' 15

Untranslated

ON16 ATG GGG TGC ACA AAC TGG GG Internal 16

(C3) domain

ON 17 GTC AGC CAT GGT CCC CCC CC Translation 17 init. codon

ON18 CGC CGT GGA GTC GTT GCC CG Internal 18

(V1 ) domain

ON19 TCA AAT GGA GGC TGC CCG GC Internal 19

(C3) domain

ON20 TGG AAT CAG ACA CAA GCC GT 3' 20

Untranslated

OLIGONUCLEOTIDES TARGETED TO PKC-β TYPES I AND II

NO SEQUENCE TARGET SEQ. ID NO.

ON 21 CAT CTT GCG CGC GGG GAG CC Translation init. 21

ON22 TGC GCG CGG GGA GCC GGA CC Translation init. 22

ON23 CGA GAG GTG CCG GCC CCG GG Translation init. 23

ON24 CTC TCC TCG CCC TCG CTC GG Translation init. 24

OLIGONUCLEOTIDES TARGETED TO PKC-β TYPE 1

NO. SEQUENCE TARGET SEQ ID NO. ON25 TGG AGT TTG CAT TCA CCT AC 3' Untranslated 25

ON26 AAA GGC CTC TAA GAC AAG CT 3' Untranslated 26

ON27 GCC AGC ATG TGC ACC GTG AA 3' Untranslated 27

ON28 ACA CCC CAG GCT CAA CGA TG 3' Untranslated 28

ON29 CCG AAG CTT ACT CAC AAT TT 3' Untranslated 29

OLIGONUCLEOTIDES TARGETED TO PKC-β TYPE II

NO. SEQUENCE TARGET SEQ ID NO.

ON30 ACT TAG CTC TTG ACT TCG GG 3' Untranslated 30

ON31 ATG CTG CGG AAA ATA AAT TG 3' Untranslated 31

ON32 ATT TTA TTT TGA GCA TGT TC 3' Untranslated 32

ON33 TTT GGG GAT GAG GGT GAG CA 3' Untranslated 33

ON34 CCC ATT CCC ACA GGC CTG AG 3' Untranslated 34

OLIGONUCLEOTIDES TARGETED TO PKC-y

NO. SEQUENCE TARGET SEQ ID NO.

ON35 CGG AGC GCG CCA GGC AGG GA 5' Untranslated 35

ON36 CCT TTT CCC AGA CCA GCC AT Translation init. 36 ON37 GGC CCC AGA AAC GTA GCA GG 5' of start codon 37

ON38 GGA TCC TGC CTT TCT TGG GG 5' Untranslated 38

ON39 CAG CCA TGG CCC CAG AAA CG Translation init. 39

OLIGONUCLEOTIDES TARGETED TO PKC-n

NO. SEQUENCE TARGET SEQ ID NO.

ON40 CGA CAT GCC GGC GCC GCT GC Translation init. 40

ON41 CAG ACG ACA TGC CGG CGC CG Translation init 41

ON42 GCC TGC TTC GCA GCG GGA GA Translation init. 42

ON43 ACA GGT GCA GGA GTC GAG GC Translation init. 43

ON44 GTC CCG TCT CAG GCC AGC CC Translation init. 44

ON45 CCT CAC CGA TGC GGA CCC TC Translation init. 45

ON46 ATT GAA CTT CAT GGT GCC AG Translation init. 46

ON47 TCT CAC TCC CCA TAA GGC TA 3' Untranslated 47

ON48 TTC CTT TGG GTT CTC GTG CC 3^* Untranslated 48 ON49 TTC CAT CCT TCG ACA GAG TT 3' Untranslated 49

ON50 AGG CTG ATG CTG GGA AGG TC 3' Untranslated 50

ON51 GTT CTA AGG CTG ATG CTG GG 3' Untranslated 51

Chimeric 2' methoxy/deoxy P=S oiigonucleotides; underlined = 2' - methoxy; s = P=S linkage; o = P=O linkage:

NO. SEQUENCE SEQ ID NO.

ON52 AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC 3

ON53 AoAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCoC 3

ON54 AsAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCsC 3

ON55 AsAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCsC 3

Chimeric 2'-propoxy/deoxy P=S oiigonucleotides; underlined = 2'-propoxy; s = P=S linkage, o = P=0 linkage:

NO. SEQUENCE SEQ ID NO:

ON56 AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC 3

ON57 AoAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCoC 3

ON58 AsAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCsC 3 ON59 AsAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCsC

Chimeric 2'-propoxy/deoxy P=S oiigonucleotides targeted to PKC-α 3'-UTR;

Underlined = 2'-propoxy; s = P=S linkage, o = P=0 linkage:

NO. SEQUENCE SEQ. ID NO:

ON60 TsTsCsTsCsGs CsTsGs GsTsGs AsGsTs TsTsC 52

ON61 ToToCoTsCsGs CsTsGs GsTsGs AsGsTo ToToC 52

ON62 TsCsTs CsGsCs TsGsGs TsGsAs GsTsTs TsC 53

ON65 ToCoTo CsGsCs TsGsGs TsGsAs GsToTo ToC 53

Most preferred among the oiigonucleotides hereinbefore described are those having SEQUENCE ID No. 2, 3 or 5.

In compositions of the invention, the oligonucleotide (A) is entrapped in sterically stablised liposomes (B). Examples of sterically stabilised liposomes are those in which part of the lipid is a glycolipid, particularly ganglioside GM, saturated phosphatidylinositol or galactocerebroside sulphate ester, such as those described in WO 88/04924; those in which part of the lipid is derivatised with hydrophilic polymer such as those described in WO 91/05545 or US 5225212; and those comprising a vesicle-forming lipid and a lipid-polymer conjugate having a hydrophobic moiety and a polar head group, such as those described in WO 94/20073. ln a preferred embodiment of the invention, the liposomes (B) comprise at least one underivatised vesicle-forming lipid and at least one vesicle-forming lipid derivatised with hydrophilic polymer which may be, for example, a polymer containing a hydroxy and/or carboxyl group such as a polylactic acid, a polyglycolic acid or, preferably, a polyethyleneglycol. More preferably, the hydrophilic polymer is a polyethyleneglycol having a molecular weight of IOO0 to 5000 daltons, such as I500 to 2500 daltons, especially I800 to 2200 daltons. The hydrophilic polymer is preferably derivatised with a polar head group of a phospholipid, especially a phospholipid having an amino head group, i.e. the derivatised lipid is preferably a phospholipid having an amino group, especially a phosphatidylethanolamine such as dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine or, particularly, distearoyl phosphatidylethanolamine.

Various methods of derivatising an amino-containing lipid with a hydroxyl- and/or carboxyl- containing hydrophilic polymer will be apparent to those skilled in the art. Several such methods are described in WO 91/05545 and US 5 225 212; the phospholipid having an amino group may be derivatised with the hydrophilic polymer by any of these methods. Preferably, the phospholipid having an amino group is derivatised with a hydroxyl- containing hydrophilic polymer such that the polymer is attached to the phospholipid through a carbamate linkage; this may be achieved by reacting a hydroxyl group of the polymer (other hydroxyl groups being capped, if necessary in view of their reactivity, for example by etherification) with diimidazole to give an activated imidazoie - terminated polymer which is then reacted with the amino-containing phospholipid to couple the phospholipid to the hydrophilic polymer through a carbamate group, as described in WO 91/05545 or US 5 225 212. In an especially preferred embodiment of the invention, the derivatised lipid is an amino-containing phospholipid, particularly a phosphatidylethanolamine, coupled through a carbamate group to a polyethyleneglycol capped at one end by an alkoxy group, particularly a methoxy or ethoxy group. Such a derivatised lipid is available commercially.

The derivatised lipid is generally present in a minor molar amount relative to the total lipid content of the liposomes, preferably in an amount of I to 20 mole % of the total lipid content, although a lower amount, for example 0.1 mole %, may be appropriate when the derivatised lipid has a high molecular weight. The major part of the lipid content of the liposomes generally comprises one or more underivatised vesicle-forming lipids such as are used in conventional liposomes. Such lipids include, for example, lipids having two hydrocarbon chains, usually in acyl groups, and a polar head group, including phospholipids, for example phosphatidylcholines such as dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, l-palmitoyl-2-oleoyl phosphatidylcholine, phosphatidylethanolamines such as those mentioned hereinbefore, and phosphatidic acids such as dimyristoyl phosphatidic acid and dipalmitoyl phosphatidic acid. Other conventionally used lipids include sterols, particularly cholesterol, and glycolipids such as those mentioned hereinbefore. Preferably, the underivatised lipid comprises a mixture of a phospholipid, especially a phosphatidylcholine, and a sterol, especially cholesterol.

In the abovementioned preferred embodiment, the sterically stabilised liposomes (B) preferably comprise 4-I0 mol % of the derivatised lipid, 40-80 mol % of the underivatised phospholipid and 20-50 mol % of the sterol. In especially preferred liposomes (B), the molar ratio of derivatised lipid: underivatised phospholipid: sterol is I:I0:5.

In another preferred embodiment of the invention, the liposomes (B) comprise (i) a glycolipid together with (ii) a vesicle-forming phospholipid or sphingolipid or mixture thereof and, optionally, (iii) a sterol and/or an acylglycerol lipid. The glycolipid is preferably a negatively charged glycolipid, especially ganglioside G i (monosialoganglioside) or hydrogenated phosphatidylinositol. The vesicle-forming phospholipid may be one or more of the phospholipids hereinbefore mentioned, preferably a phosphatidylcholine, a phosphatidylethanolamine or a mixture thereof. Especially preferred phospholipids are distearoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine. The sphingolipid is preferably sphingomyelin and is preferably used together with a phospholipid. The sterol may be, for example, ergosterol or, preferably, cholesterol. The acylglycerol lipid may be an ester of glycerol containing two fatty acid acyl groups each having at least 12 carbon atoms, for example lauroyl, myristoyl, palmitoyl or oleoyl groups, and one acyl group of formula R¹CO-, where R¹ is a residue, containing up to 10 carbon atoms, of a monocarboxylic acid of formula R¹COOH after removal of the -COOH group or, preferably, of formula -COR²COOH where R² is a residue, containing up to 10 carbon atoms, preferably 1 to 4 carbon atoms, of a dicarboxylic acid of formula HOOC-R²-COOH, especially succinic acid, after removal of both -COOH groups. An especially preferred acylglycerol is 1 , 2- dipalmitoyl-sn-3-succinyl glycerol.

In this second preferred embodiment of the invention, the liposomes preferably comprise (i) a negatively charged glycolipid together with (ii) a vesicle-forming phospholipid and/or sphingolipid and (iii) a sterol or acylglycerol lipid, especially (i) ganglioside G i or hydrogenated phosphatidylinositol together with (ii) distearoyl phosphatidylcholine or dioleoyl phosphatidylethanolamine or a mixture thereof with sphingomyelin and (iii) cholesterol or 1 ,2-dipalmitoyl-sn-3-succinylglycerol.

The liposomes may comprise from 2 to 20 mol% of the glycolipid (i) and 80 to 98 mol% of (ii) the phospholipid, sphingolipid or mixture thereof. In preferred embodiments, where the liposomes also comprise a sterol or acylglycerol, they may comprise 2 to 20 mol %, preferably 4 to I0 mol %, of the glycolipid, 40 to 80 mol %, preferably 60 to 80 mol %, of the phospholipid, sphingolipid or mixture thereof and I0 to 50 mol %, preferably 20 to 40 mol%, of the sterol or 5 to 40 mol %, preferably I0 to 30 mol %, of the acylglycerol.

Specific especially preferred liposomes (B) are those described hereinafter in the Examples.

The oligonucleotide-containing liposomes of the invention can be prepared using known methods for the preparation of drug-containing liposomes. For example, in one method, the lipid composition is dissolved in an organic solvent, such as an alcohol, ether, halohydrocarbon or mixture thereof, the solvent is removed from the resulting solution, for example by rotary evaporation or freeze drying, and the resulting lipid film is hydrated by dispersing in an aqueous medium, such as phosphate-buffered saline or an aqueous solution of a sugar, e.g. lactose, which medium also contains the oligonucleotide (A), to give an aqueous suspension of liposomes in the form of multilamellar vesicles (MLV's). The aqueous liposome suspension may be treated to reduce the liposome size, for example to give small unilamellar vesicles (SUV's), using known methods, for example by sonication or by extrusion through one or more membranes, e.g. polycarbonate membranes, having a selected pore size. Liposomes according to the invention preferably have on average a particle size below 500nm, more preferably 50 to 200 nm, especially 80 to I20 nm. It is generally desirable to have as high a weight ratio of oligonucleotide to lipid as possible consistent with liposome stability. The maximum for this weight ratio may vary depending on the nature and composition of the lipid component, but in general this maximum is likely to be about 1 :20. Ratios between 1 :40 and 1 :400 can be used with good results.

The invention includes a method of modulating the expression of protein kinase C in cells which comprises contacting the cells with a composition of the invention as hereinbefore defined. The invention also includes a method of treating a condition associated with expression of protein kinase C which comprises administering a composition of the invention as hereinbefore defined to a mammal, particularly a human, or cells thereof, in need of such treatment.

The composition of the invention may be administered by pulmonary delivery or, preferably, parenterally, for example intravenously, subcutaneously, intraperitoneally or intramuscularly. Conditions which may be treated with the composition include hyperproliferative disorders such as psoriasis and mammalian cancer, particularly human cancer such as lung cancer, breast cancer, colorectal cancer and skin cancer.

For these indications, the appropriate dosage will, of course, vary depending upon the method of administration and on the severity and responsiveness of the condition to be treated. Individual doses and the administration regime can best be determined by individual judgement of a particular case of illness. However, in general, satisfactory results in animals are indicated to be obtained at daily dosages from about 0.6mg/kg to about 6mg/kg.

In larger mammals, for example humans, an indicated daily dose in the range of about 4.2 mg to about 420 mg conveniently administered, for example in divided doses of up to 3 times per day.

The invention is illustrated by the following Examples. Example 1

A derivatised lipid, prepared by coupling distearoyl phosphatidylethanolamine to a methoxy- capped polyethylene glycol of molecular weight 2000 through a carbamate group (DSPE- MPEG 2000 available from Genzyme), distearoyl phosphatidylcholine (available from Sigma Chemical) and cholesterol are dissolved, at a molar ratio of I:I0:5, in chloroform. The solvent is removed by rotary evaporation to leave a lipid film. This film (250mg) is hydrated with Hanks' balanced salt solution (2ml) buffered to pH 7.4 with 25mM 4-(2- hydroxyethyl)piperazine-1 -ethane sulphonic acid (HEPES) and containing oligonucleotide ON2 as hereinbefore defined (5mg) in tritiated form. The resulting MLV's are subjected to ten liquid nitrogen-water freeze-thaw cycles and then sonicated (wavelength 6μm) for 2 minutes to give small unilamellar vesicles (SUVs) having an average diameter of 80 to 100nm. The resulting sterically stabilised liposomes are purified to remove unentrapped oligonucleotide by size exclusion chromatography using a Sephadex G-I50 column and a 25m M sodium borate elution buffer.

The liposomes are subjected to pharmacokinetic testing as follows: male Wistar rats (240- 270g) are fed ad libitum with a standard laboratory diet (Rat and Mouse diet, Bantin and Kingman, Hull, UK) and kept under controlled conditions (12 hour light cycle; 20°C). The animals are lightly sedated by intramuscular injection of 20ml fentanyl (Hyponorm, Janssen Pharmaceuticals Ltd, Oxford UK) to immobilise them. A suspension of the tritiated oligonucleotide - containing liposomes in phosphate buffered saline, containing 1.0μCi of the tritiated oligonucleotide, is administered to the rats by tail vein injection. The rats are then maintained in metabolism cages for up to 48 hours with free access to food and water. Animals are sacrificed at intervals of 6, 12, 24 and 48 hours by overdosing with sodium pentabarbitone. The organs of interest are collected, weighed and their [³H] content determined by combustion in a tissue oxidiser followed by liquid scintillation counting (LS 6500; Beckman Instruments, UK). The [³H] content of each sample is adjusted for total tissue weight and expressed as a percentage of the dose administered to each animal.

The above test procedure is repeated, using a solution of the tritiated oligonucleotide ON2, instead of liposomes containing tritiated ON2, in phosphate buffered saline. The test results (average for 3 animals) are as follows:

Percentage dose of [3H] ON2 in tissues after dosing in solution

Tissue 6 hour 12 hour 24 hour 48 hour urine 0.99 1.77 3.24 6.53 liver 40.54 35.00 45.47 35.16 kidney 9.24 7.48 10.39 13.04 spleen 1.66 1.05 1.48 1.48 heart 0.03 0.02 0.05 0.03 muscle 3.99 1.80 4.09 3.07 skin 2.05 1.06 2.53 1.62 bone 17.00 11.59 14.70 16.15 fat 0.44 0.08 0.13 0.46

Percentage dose of [3H] ON2 in tissues after dosing in Sterically Stabilised Liposomes

Tissue 6 hour 12 hour 24 hour 48 hour urine 0.03 0.12 0.47 1.67 liver 9.88 10.16 9.26 7.83 kidney 1.40 0.91 1.28 1.05 spleen 18.02 22.48 23.41 20.51 heart 0.44 0.43 0.36 0.31 muscle 8.09 13.07 31.47 27.81 skin 2.95 1.91 2.82 2.03 bone 6.52 6.06 9.89 6.61 fat 1.93 1.98 1.77 0.84

Claims

1 . A pharmaceutical composition comprising (A) an oligonucleotide having 5 to 50 nucleotide units, which is specifically hybridizable with DNA or RNA derived from a protein kinase C gene, entrapped in (B) sterically stabilised liposomes.

2. A composition according to claim 1 , in which the oligonucleotide (A) is specifically hybridizable with a translation initiation site, 5' untranslated region or 3' untranslated region of the protein kinase C gene.

3. A composition according to claim 1 or 2, in which the oligonucleotide (A) has 8 to 30 nucleotide units.

4. A composition according to claim 3, in which the oligonucleotide (A) has 12 to 25 nucleotide units.

5. A composition according to claim 4, in which the oligonucleotide (A) has 18 to 22 nucleotide units.

6. A composition according to any of claims 1 to 5 in which at least one nucleotide of the oligonucleotide (A) is modified at the 2' position of the sugar moiety.

7. A composition according to any of claims 1 to 5 in which the oligonucleotide (A) is a chimeric oligonucleotide which contains a first region having at least one nucleotide modified to enhance target affinity and a second region which is a substrate for RNAse H.

8. A composition according to claim 7, in which a nucleotide modified to enhance target affinity is modified at the 2' position of the sugar moiety.

9. A composition according to claim 6 or 8, in which the modified nucleotide has an alkoxy, alkoxyalkoxy or fluoro substituent at the 2' position.

10. A composition according to claim 7, 8 or 9, in which the oligonucleotide (A) is a chimeric oligonucleotide and the region which is a substrate for RNAse H comprises at least one 2'-deoxynucleotide.

1 1. A composition according to any of the preceding claims, in which the oligonucleotide (A) has at least one phosphorothioate linkage.

12. A composition according to claim 1 , in which, in the oligonucleotide (A), all nucleotides are 2'-deoxynucleotides and all backbone linkages are phosphorothioate linkages.

13. A composition according to any of claims 1 to 11 , in which the oligonucleotide (A) is a chimeric oligonucleotide having one or more regions with 2'-deoxynucleotides and one or more regions with 2'-alkoxynucleotides or 2'-alkoxyalkoxynucleotides.

14. A composition according to claim 13, in which the 2'-alkoxyalkoxynucleotides are 2'- methoxyethoxynucleotides.

15. A composition according to claim 13 or 14, in which the one or more regions with 2'- deoxynucleotides have phosphorothioate backbone linkages and the one or more regions with 2'-alkoxynucleotides or 2'-alkoxyalkoxynucleotides have phosphodiester or phosphorothioate backbone linkages.

16. A composition according to any of claims 13 to 15, in which the oligonucleotide (A) comprises a region of 2'-deoxynucleotides between two regions of 2'-alkoxynucleotides or 2'-alkoxyalkoxynucleotides.

17. A composition according to claim 16, in which the deoxynucleotide region has at least 4 nucleotides.

18. A composition according to claim 17, in which the deoxynucleotide region has at least 6 nucleotides.

9. A composition according to claim 18, in which the deoxynucleotide region has at least 8 nucleotides.

20. A composition according to any of the preceding claims, in which the oligonucleotide (A) is targeted to PKC-α.

21. A composition according to any of claims 1 to 19, in which the oligonucleotide (A) comprises a nucleotide sequence selected from SEQ. ID Nos. 1 to 53.

22. A composition according to any of claims 1 to 20, in which the oligonucleotide (A) comprises a nucleotide sequence selected from SEQ. ID Nos. 2, 3 and 5.

23. A composition according to any of the preceding claims, in which the liposomes (B) comprise at least one underivatised vesicle-forming lipid and at least one vesicle-forming lipid which is derivatised with a hydrophilic polymer.

24. A composition according to claim 23, in which the hydrophilic polymer is a polyethyleneglycol.

25. A composition according to claim 23 or 24, in which the derivatised lipid is a phospholipid having an amino group.

26. A composition according to claim 25, in which the hydrophilic polymer is attached to the phospholipid through a carbamate linkage.

27. A composition according to claim 25 or 26, in which the amino-containing phospholipid is a phosphatidylethanolamine.

28. A composition according to claim 27, in which the amino-containing phospholipid is distearoyl phosphatidylethanolamine .

29. A composition according to any of claims 23 to 28, in which the derivatised lipid comprises 1-20 mole % of the total lipid content of the liposomes.

30. A composition according to any of claims 23 to 29, in which the underivatised lipid is a lipid having two hydrocarbon chains and a polar head group and/or a sterol.

31. A composition according to claim 30, in which the lipid having two hydrocarbon chains and a polar head group is a phosphatidylcholine.

32. A composition according to claim 31 , in which the phosphatidylcholine is distearoyl phosphatidylcholine.

33. A composition according to any of claims 30 to 32, in which the sterol is cholesterol.

34. A composition according to any of claims 23 to 33, in which the liposomes comprise 4- 10 mol % derivatised lipid, 40-80 mol % underivatised lipid and 20-50 mol % sterol.

35. A composition according to claim 34, in which the molar ratio of derivatised lipid: underivatised lipid: sterol is I:I0:5.

36. A composition according to any of claims 1 to 22, in which the liposomes (B) comprise (i) a glycolipid together with (ii) a vesicle-forming phospholipid or sphingolipid or mixture thereof and, optionally, (iii) a sterol and/or an acylglycerol lipid.

37. A composition according to claim 36, in which the glycolipid is a negatively charged glycolipid.

38. A composition according to claim 37, in which the liposomes comprise (i) a negatively charged glycolipid together with (ii) a vesicle-forming phospholipid and/or sphingolipid and (iii) a sterol or acylglycerol lipid.

39. A composition according to claim 37 or 38, in which the glycolipid is ganglioside GM_T or hydrogenated phosphatidylinositol.

40. A composition according to any of claims 36 to 39, in which the vesicle-forming phospholipid is a phosphatidylcholine or a phosphatidylethanolamine.

41. A composition according to claim 40, in which the phospholipid is distearoyl phosphatidylcholine or dioleoyl phosphatdiylethanolamine.

42. A composition according to any of claims 36 to 41 , in which the sphingolipid is sphingomyelin.

43. A composition according to any of claims 36 to 42, in which the acylglycerol lipid has two fatty acid acyl groups each having at least 12 carbon atoms and one acyl group of formula R¹CO-, where R¹ is a residue, containing up to I0 carbon atoms, of a monocarboxylic acid of formula R¹COOH after removal of the -COOH group, or of formula OC-R²-COOH where R² is a residue, containing up to 10 carbon atoms, of a dicarboxylic acid of formula HOOC-R²-COOH after removal of both -COOH groups.

44. A composition according to claim 43, in which the acylglycerol is 1 , 2-dipalmitoyl-sn-3- succinyl glycerol.

45. A composition according to any of claims 36 to 44, in which the liposomes comprise 2 to 20 mol % of the glycolipid, 40 to 80 mol % of the phospholipid, sphingolipid or mixture thereof and 10 to 50 mol % of the sterol or 10 to 30 mol % of the acylglycerol.

46. A composition according to claim 45, in which the liposomes comprise 4 to 10 mol % of the glycolipid, 60 to 80 mol % of the phospholipid, sphingolipid or mixture thereof and 20 to 40 mol % of the sterol or 10 to 30 mol % of the acylglycerol.

47. A composition according to any of the preceding claims, in which the liposomes have an average particle size of 50 to 200 nm.

48. A composition according to claim 47, in which the liposomes have an average particle size of 80 to I20 nm.

49. A composition according to claim 1 , substantially as described in any of the Examples.

50. Use of a composition according to any of the preceding claims in the preparation of a medicament for the treatment of a condition associated with expression of protein kinase C.

51. A method of modulating the expression of protein kinase C in cells which comprises contacting the cells with a composition according to any of claims 1 to 49.

52. A method of treating a condition associated with expression of protein kinase C which comprises administering a composition according to any of claims 1 to 49 to a mammal, or cells thereof, in need of such treatment.