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WO1993000910A1 - Liponucleosides antiviraux: traitement de l'hepatite b - Google Patents

Liponucleosides antiviraux: traitement de l'hepatite b Download PDF

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Publication number
WO1993000910A1
WO1993000910A1 PCT/US1992/004856 US9204856W WO9300910A1 WO 1993000910 A1 WO1993000910 A1 WO 1993000910A1 US 9204856 W US9204856 W US 9204856W WO 9300910 A1 WO9300910 A1 WO 9300910A1
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Prior art keywords
compound
lipid
compound according
nucleoside
ddc
Prior art date
Application number
PCT/US1992/004856
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English (en)
Inventor
Karl Y. Hostetler
Original Assignee
Vical, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Vical, Inc. filed Critical Vical, Inc.
Priority to EP92914562A priority Critical patent/EP0594677A4/en
Priority to JP5502213A priority patent/JPH07500573A/ja
Priority to AU22268/92A priority patent/AU668873B2/en
Publication of WO1993000910A1 publication Critical patent/WO1993000910A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/548—Phosphates or phosphonates, e.g. bone-seeking
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide

Definitions

  • the present invention relates to the treatment of infections of hepatitis virus using lipid derivatives of antiviral nucleoside analogues. More particularly, the present invention relates to lipid, especially phospholipid, derivatives of antiviral nucleoside analogues which can be integrated into the structure of liposomes, thereby forming a more stable liposomal complex that can deliver greater amounts of antihepatitis drugs to target cells with less toxicity.
  • nucleoside analogues are known to have activity against the hepatitis B virus (HBV) .
  • HBV hepatitis B virus
  • dideoxynucleosides such as dideoxycytidine (ddC) , dideoxyinosine (ddl) , dideoxyadenosine (ddA) , dideoxythymidine (ddT) , dideoxyguanosine (ddG) and dideoxydiaminopurine are active against duck hepatitis B in vitro and in vivo (1,2).
  • acyclovir ACV
  • bromovinyldeoxyuridine BVdU
  • FIAC deoxyfluoro-arabinosyliodocytosine
  • arabinofuranosyladenines (ara-A) and arabinofuranosyl- cytidine ⁇ (ara-C) inhibit the human hepatitis B DNA polymerase, and ara-A has activity when administered to individuals suffering from chronic type B hepatitis (4) . Further, Matthes et al.
  • the antihepatitis B nucleoside analogues described above have very short half lives when administered to humans or animals as the free compound. After 4 to 6 hours, their levels in tissue and plasma are very low or negligible. These nucleoside analogues are also toxic, and their toxicity can be a limiting factor in therapeutic regimens. Clearly, it would be useful to administer the antihepatitis B nucleosides
  • the invention provides, in one embodiment, a compound having antiviral properties, comprising an antihepatitis B nucleoside analogue having a base portion comprising a purine or pyri idine or analogue thereof, and a sugar portion comprising a pentose residue, wherein at least one the portion is a non-naturally occurring nucleoside component; and a lipid moiety linked to the pentose residue; with the proviso that the compound is in the form of a liposo e when the pentose residue is arabinofuranose and the base portion is cytosine or adenine.
  • the non-naturally occurring nucleoside component can be an analogue of a naturally occurring base or pentose by virtue of substitution, deletion, or replacement.
  • the pentose residue is a 2' ,3'-dideoxy, 2' ,3•-didehydro, or halo derivative of ribose, or an acyclic hydroxylated fragment of ribose.
  • the pentose residue is a 2' ,3'-dideoxyribose
  • the nucleoside analogue is 2' ,3'- dideoxycytidine; 2 ', 3 * -dideoxythymidine; 2 ',3'- dideoxyguanosine; 2 * , 3 ' -dideoxyadenosine; 2', 3'- dideoxyinosine; or 2,6-diaminopurine, 2' ,3'-dideoxyriboside.
  • the pentose group is a halo- or an amino derivative of ribose and the nucleoside is 3'- fluoro-5-methyl-deoxycytidine(FddMeCyt) , 3 » -chloro-5-methyl- deoxycyti ine(ClddMeCyt) , 3 ' -amino-5-methyl - deoxycytidine(AddMeCyt) , or 2' ,3 '-dideoxy-3'-fluorothymidine.
  • the nucleoside analogue can alternatively be acyclovir, l-(2'- deoxy-2 ⁇ -fluoro-1- ⁇ -D-arabinofuranosyl)-5-iodocytosine(FIAC) or 1(2'-deoxy-2'-fluoro-1- ⁇ -D-arabinofuranosyl)-5-iodouracil (FIAU) .
  • the nucleoside analogue is 2 • ,3 » -didehydro-2 » ,3'-dideoxythymidine.
  • the compounds described can further comprise a monophosphate, diphosphate, or triphosphate linking group between the 5' position of the pentose residue and the lipid moiety.
  • the lipid moiety of the compound can be a fatty acid, a monoacylglycerol or a diacylglycerol, a phosphatidic acid, a diphosphatidyl glycerol, a bis 1,2-(diacylglycero)-phosphate, a D,L-2,3-diacyloxypropyl-(dimethyl)-6-hydroxyethyl ammonium group, or l-O-stearyl-rac-3-glycerol.
  • the invention provides a method for treating hepatitis B infection in a mammal, comprising administering to the mammal an effective hepatitis B virus-inhibiting dose of any of the compounds disclosed.
  • the invention also provides a method for inhibiting the replication of hepatitis B virus in a cell, comprising contacting the cell with an effective hepatitis B virus- inhibiting dose of any of the compounds disclosed.
  • the hepatitis B virus infection is in a human cell
  • the compound is phosphatidyl-dideoxycytidine (p-ddC) , phosphatidyl-FIAC, FIAC diphosphate diglyceride, phosphatidyl- FIAU, or FIAU diphosphate diglyceride.
  • the infection is in a human cell and the compound comprises l-O-stearyl-rac-3-glycerol attached to an antihepatitis B nucleoside analogue through a phosphate group.
  • the lipid derivatives of nucleoside analogues are incorporated into liposomes.
  • substantially all of the liposomes have a diameter less than about 100 nanometers, with a mean diameter of from about 30 to 80 nanometers.
  • the liposomes are accordingly sized appropriately to pass through the hepatic sinusoids and to be selectively taken up by hepatocytes and targeted to the reservoir of infection.
  • the compound is administered parenterally to a mammal, either intravenously, subcutaneously, intramuscularly, or intraperitoneally. In an alternative embodiment, the compound is administered orally to a mammal.
  • the invention also provides a method for delivering an active mono-, di-, or triphosphate of a nucleotide analogue selected from the group consisting of dideoxycytidine (ddC) , FIAU, FIAC, or FMAU to a cell, comprising delivering to the cell the lipid prodrug of the nucleoside analogue, and permitting the enzymatic cleavage of the prodrug to deliver the active phosphate to the cell.
  • ddC dideoxycytidine
  • FIAU dideoxycytidine
  • FIAC FIAC
  • FMAU FMAU
  • the invention further provides a pharmaceutical composition, comprising an antihepatitis B compound of the invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises a different antiviral agent.
  • Figure 2 is a graph showing the comparative levels of ddC in 1iver
  • Figure 3 is a graph showing the comparative levels of ddC in sciatic nerve
  • Figure 4 is a graph showing the comparative levels of ddC in brain
  • Figure 5 is a graph showing the comparative levels of ddC in lymph node
  • Figure 6 is a graph showing the comparative levels of ddC in spleen.
  • the present invention involves lipid derivatives of nucleoside analogues having an inhibiting effect on the replication of hepatitis B virus.
  • these antiviral agents can be stably incorporated into the lipid bilayer of liposomes.
  • These lipid derivatives can be converted into nucleoside analogue triphosphates by constituent cellular metabolic processes, and have antiviral effects in vivo and in vitro.
  • nucleoside analogues of this type particularly lipid derivatives of dideoxycytidine (ddC) and azidothymidine (AZT) , have shown that very large amounts of drug-containing lipid particles are taken up in the liver.
  • ddC dideoxycytidine
  • AZA azidothymidine
  • nucleoside analogues used in preparing the lipid derivatives and liposomes of the present invention will have a purine or pyrimidine base, e.g., adenine, guanine, cytosine or thymine, or an analogue thereof, attached to a pentose, such as ribose, arabinose, or a ribose or arabinose residue and/or derivative.
  • a pentose such as ribose, arabinose, or a ribose or arabinose residue and/or derivative.
  • the attachment is through the nitrogen in the 9-position of the purines or through the nitrogen in the l-position of the pyrimidines. These nitrogens are linked by a ⁇ -N-glycosyl linkage to carbon 1 of the pentose residue.
  • the pentose residue may be a complete pentose, or a derivative such as a deoxypentose or dideoxypentose.
  • the pentose residue can be a fragment of a pentose, such as a hydroxylated 2-propoxymethyl residue or a hydroxylated ethoxymethyl residue.
  • Particular nucleoside residues having these structures include acyclovir and ganciclovir.
  • the pentose may also have an oxygen or sulfur substitution for a carbon atom at, for example, the 3*- position of deoxyribose (BCH-189) .
  • nucleosides are dideoxynucleosides such as 2' ,3'-dideoxycytidine (ddC) , 2' ,3'-dideoxyinosine (ddl) , 2' ,3'-dideoxyadenosine (ddA) , 2',3'-dideoxythymidine (ddT) , 2' ,3 '-dideoxyguanosine (ddG) ; nucleoside analogues such as 9- (2-hydroxyethoxy ethyl)guanine (acyclovir, ACV), E-5-(2- bro ovinyl)-2 '-deoxyuridine (BVdU) , and1-(2•-deoxy-2•-fluoro- 1- ⁇ -arabinosyl)-5-iodocytosine (FIAC); l-(2'-deoxy-2*-fluoro- l- ⁇ -D-arabinofuranosy
  • modified pyrimidine nucleosides comprising phosphorylated forms of 2* ,3 '-dideoxy-3 '-fluorothymidine (FddThd) ; 2• ,3'-didehydro- 2 ' ,3 '-dideoxythymidine (ddeThd) ; 3 '-fluoro-5-methyl- deoxycytidine (FddMeCyt) ; 3'-chloro-5-methyl-deoxycytidine (ClddMeCyt) ; and 3'-amino-5-methyl-deoxycytidine (AddMeCyt) ; as well as isoddA, isoddG, 2-CDG, and BVara-U.
  • nucleosides are 2',3'-dideoxycytidine (ddC) and 9- ⁇ - D-arabinofuranosyl-adenine (ara-A) .
  • ddC 2',3'-dideoxycytidine
  • ara-A 9- ⁇ - D-arabinofuranosyl-adenine
  • Any lipid derivative of the nucleoside analogues disclosed, having an activity against hepatitis B, is within the scope of the invention.
  • the lipid groups are preferably attached to the nucleoside analogues through a phosphate link between the nucleoside analogue and the lipid.
  • the phosphate groups may be either mono-, di-, or triphosphate groups and are generally connected to the 5' carbon of the pentoses in the compounds of the present invention; however, compounds wherein the phosphate groups are attached to the 3' hydroxyl group of the pentose are within the invention if they possess antihepatitis B activity. Where lipids are linked directly to pentose groups, those linkages may also be made either through the 3 1 , or preferably through the 5', pentose carbon.
  • the lipids to which the antiviral nucleosides are bound have hydrophobic acyl groups capable of anchoring ' the liponucleoside in the lipid bilayer.
  • Suitable lipids are, for example, mono- or diacylglycerides, sphingosine and dihydrosphingosine, and long chain fatty acids or alcohols.
  • Other suitable lipids are those having novel structures, for example, ether-linked lipids such as batyl alcohol, l-O- stearyl-r ⁇ c-3-glycerol, or those comprising ammonium groups, for example, D,L-2,3-distearoyloxypropyl (dimethyl)- ⁇ - hydroxyethyl ammonium (6) .
  • Liponucleotides may include more than one lipid moiety attached to the phosphate linker.
  • the aliphatic groups of the lipid moieties preferably have chain lengths of two to twenty-four carbon atoms and can be saturated or unsaturated with up to six double bonds.
  • the aliphatic groups may be attached to the glycerol moiety by acyl ester, ether, thioester, thioether, or vinyl ether bonds.
  • nucleoside analogues comprising phosphatidylnucleosides, nucleoside diphosphate diglycerides, nucleoside acyl phos ⁇ phates, and ceramide phosphonucleosides, are disclosed in copending application Serial No. 07/373,088.
  • Preferred antihepatitis liponucleotides of the invention are phosphatidyl-dideoxyadenosine ⁇ (p-ddA) , phosphatidyl- dideoxycytidines (p-ddC) , phosphatidyl-dideoxyguanosines (p- ddG) , phosphatidyl-dideoxyinosines (p-ddl) , phosphatidyl- dideoxythy idines (p-ddT) , phosphatidyl-9-(2-hydroxymethyl)- guanosines (p-ACV) , phosphatidyl-1-(2'-deoxy-2'-fluoro-1- ⁇ - arabinosyl)-5-iodocytosines (p-FIAC) , phosphatidyl-1-(2'- deoxy-2'-fluoro-l-9-arabinosyl)-5-iodouracils (p-FI
  • the compounds of the invention are formed according to synthetic procedures which couple a phospholipid to a nucleo- side analogue or which couple a phospholipid to a nucleoside analogue monophosphate or diphosphate, wherein the phosphate group of the nucleoside is located on the ribose group of the nucleoside, at either the 3' or preferably the 5' location.
  • the synthesis can be carried out according to general methods applicable to all lipids and all antiviral nucleosides described, as in Examples 1 through 7 of co-pending applica ⁇ tion U.S. Serial No. 07/373,088, but preferably according to the methods of synthesis described in Examples 1-3.
  • Lipids comprising fatty acids, fatty alcohols, gly- cerides, and phospholipids may be purchased from commercial suppliers (Avanti Polar Lipids, Inc., Pelham, Alabama 35124) or may be synthesized according to known methods. Antiviral nucleoside analogues are available from Aldrich, Milwaukee, Wisconsin or from Sigma, St. Louis, Missouri.
  • the lipids are first either freeze-dried by solvent evaporation under vacuum, or in a vacuum oven over P 2 0 5 .
  • the reactions are also carried out under an inert gas, such as, for example, argon, A.
  • Liponucleotides comprising a mono-, di-, or triphosphate link between an antiviral nucleoside analogue and the lipid group may be prepared from phospholipids, phosphorylated nucleoside analogues, or both.
  • Suitable phospholipids comprise phosphoglycerides, sphingolipids, or acyl phosphates.
  • Phosphorylated nucleoside analogues are known.
  • the dideoxynucleoside analogue is phosphorylated according to conventional procedures such as the phosphorous oxychloride method of Yoshikawa et al. (7,8) or Toorchen and Topal (9) .
  • the preferred modified analogue is the 5'-monophosphate.
  • Lipids suitable for coupling to nucleosides comprising primarily long chain fatty acids or alcohols, monoglycerides or diglycerides, sphingosines and other lipid species described below, may be phosphorylated by treatment with appropriate agents, for example using phenyl phosphorodichloridate according to the procedure of Brown (10) , by treatment with phosphorus oxychloride, or by other known phosphorylation procedures.
  • a phospholipid such as, for example, a phosphatidic acid
  • a selected nucleoside analogue at either the 3 1 or 5' hydroxyl by means of a coupling agent, such as, for example, 2, 4, 6-tri- isopropylbenzenesulfonyl chloride in the presence of a basic catalyst, for example, anhydrous pyridine, at room tempera ⁇ ture, as in Example ID.
  • a coupling agent such as, for example, 2, 4, 6-tri- isopropylbenzenesulfonyl chloride
  • a basic catalyst for example, anhydrous pyridine
  • Other coupling agents such as dicyclohexyl-carbodii ide can be used.
  • Lipid derivatives can also be synthesized by coupling a phosphatidic acid to an antiviral nucleoside monophosphate through a pyrophosphate bond.
  • the nucleo ⁇ side monophosphate or diphosphate is converted to a derivative having a leaving group, for example, morpholine, attached to the terminal phosphate group, according to the procedure of Agranoff and Suomi (11) .
  • a coupling of the phosphatidic acid and the nucleoside phosphate morpholidate occurs on treatment of a dry mixture of the two reactants with a basic catalyst, such as anhydrous pyridine, at room temperature.
  • the phosphatidic acid can be converted to a derivative having a leaving group, for example, morpholine, as described in Example 2D and 3A.
  • the coupling of the phosphatidic acid morpholidate and the nucleoside phosphate occurs on treatment of the mixture with a basic catalyst.
  • This alternative synthetic method is the subject of a U.S. Patent Application Serial No. 07/706,873, filed May 29, 1991.
  • TLC thin layer chromatography
  • the synthesis of products comprising adenine or cytidine having reactive amino groups may be facilitated by blocking those groups with acetate before the coupling reaction by treatment with acetic anhydride; after the chromatography of the final product, the amino groups are unblocked using dilute ammonium hydroxide.
  • the nucleoside may be any antiviral nucleoside having antihepatitis activity;
  • R._ 2 (as well as R-_ 4 for the bis(diacylglycero) phosphate species) may be any saturated or unsaturated fatty acid having from 2 to 24 carbon atoms. Polyunsaturated, hydroxy, branched chain, and cyclopropane fatty acids are also possible.
  • the stereochemistry of the glycerol moieties can include sn-1 or sn-3 phosphoester bonds or racemic mixtures thereof.
  • acyl ester groups There may be 1 or 2, (as well as 3, or 4 for the bis(diacylglycero) phosphate species) acyl ester groups, or alkyl ether or vinyl ether groups, as required.
  • a variety of other phospholipids may be linked to nucleosides, including, but not limited to phosphatidyl- glycerol, phosphatidylinositol, or any other phospholipid wherein the head group contains an available linking hydroxyl group, in either a natural polyhydroxyl alcohol such as inositol, or one in which it has been substituted by another polyhydroxy alcohol or by a carbohydrate, such as a sugar, again either natural or synthetic.
  • nucleo ⁇ side phosphate will be added by esterification to one or more of the hydroxyls of the alcohol or carbohydrate.
  • Other glycolipids may also serve as the ligand to which the phos ⁇ phate group of the nucleotide is attached by means of esteri- fication to a glycolipid hydroxyl group.
  • Other glycolipids whether or not phospholipids, such as selected cerebrosides or gangliosides, either natural or synthetic, having suitable hydrophobic properties may also be advantageously used. These may also be linked to nucleotides by similar esterification of carbohydrate hydroxyl groups.
  • antiviral nucleosides can be linked to the phosphate groups of the phosphatidylinositol mono-, di- and triphosphates, or to the phosphate-substituted carbohydrate moieties of phospholipids or glycolipids, either natural or synthetic.
  • Phosphatidylserine may be linked to nucleoside analogues directly by esterification of its carboxyl group with the 5'- hydroxyl of the nucleoside ribose group.
  • Synthetic phospholipids which are similar in structure to phosphatidylserine, in the presence of a carboxyl group in the polar headgroup, may be linked in a similar way.
  • Phospholipids having alkyl chains attached by ether or vinyl ether bonds may also be used to prepare nucleotide derivatives.
  • Suitable phospholipids for this purpose comprise naturally occurring acetal phosphatide ⁇ , or plasmalogen ⁇ , co prising a long chain fatty acid group present in an unsaturated vinyl ether linkage.
  • analogues of 1-O-alkyl glycerol or 2-O-alkyl glycerol may be prepared synthetically, and linked to a selected nucleotide.
  • Deriva- tives of l-0-alkyl-glycero-3-phospho-5'-dideoxycytidine are preferred, and may be prepared by condensing ddC monophosphate with various analogues of 1-O-alkyl-glycerol having an alkyl group of 2 to 24 carbon chain length at the 1 position of glycerol.
  • the 1-O-alkyl group may consist of a saturated or unsaturated aliphatic group having a chain length of 2 to 24 carbon atoms.
  • the 1-O-alkyl glycerol residue may be racemic or stereo ⁇ pecific.
  • Thi ⁇ compound may be acylated with fatty acid chlorides or anhydrides resulting in the synthesis of 1- O-alkyl, 2-acyl-glycero-3-phospho-5 '-dideoxycytidine.
  • the1-O-alkyl, 2,3-bi ⁇ (phospho-5'-2' ,3'-deoxycytid- ine)glycerol analogues may be synthesized. These derivatives have the general structure:
  • R 1 i an un ⁇ aturated or ⁇ aturated alkyl chain 1 to 23 carbon atom ⁇ in length in ether or vinyl ether linkage.
  • An ether or vinyl ether link at R 2 is al ⁇ o possible.
  • the group at position 1 of glycerol may also be OH if R 2 is the ether linked alkyl chain.
  • N is any antiviral nucleo ⁇ ide having activity again ⁇ t hepatiti ⁇ B, linked in a 5' phosphodiester link, and A is a chalcogen (O, C or S) .
  • nucleoside analogues are preferred embodiment ⁇ of the present invention, it is possible to utilize non-phosphorus containing lipid derivatives of nucleoside analogues if it is not nece ⁇ sary to provide the infected cell with the nucleoside phosphate in order to achieve an antiviral effect through the proces ⁇ e ⁇ of cellular metabolism.
  • Some examples of compounds of thi ⁇ type would have fatty acid ⁇ e ⁇ terified, or present in alkyl linkage, directly to the 5'-hydroxyl of the nucleoside according to the synthetic method of the invention.
  • a "spacer" molecule having, for example, carboxyl groups at either end and 0 to 10 CH 2 groups in the center, could be esterified to the 5'-hydroxyl of the antivi- ral nucleo ⁇ ide.
  • the other carboxyl of the "spacer” may be esterified to the free hydroxyl of diacylglycerol or any other lipid having an available hydroxyl function.
  • Other linking ("spacer") group ⁇ with ⁇ uitable functional group ⁇ at the ends may also be used to link the diglyceride or other suitable lipid group to the nucleoside, by chemical methods well known to tho ⁇ e skilled in the art.
  • the antiviral nucleoside derivatives noted above are incorporated in liposome ⁇ in order to direct these compounds to cells which take up the liposomal composition.
  • liposome preparation procedures such as sonication, or by extrusion.
  • Suitable conventional methods of liposome preparation also include, but are not limited to, those disclosed by Bangham, et al. (13), Olson, et al. (14), Szoka and Papahadjapoulos (15), Mayhew, et al. (16), Kim, et al. (17), Mayer, et al. (18) and Fukunaga, et al. (19).
  • Liposomes suitable for use in the methods of the invention may also be prepared by microfluidization, using, for example, a commer ⁇ cial device (Microfluidizer ® , Newton, MA) .
  • Ligands may also be incorporated to further focus the specificity of the liposome ⁇ .
  • the size of liposomes is related to achieving effective uptake of antihepatitis B nucleoside analogues into the HBV- infected parenchymal cells of the liver, the hepatocyte ⁇ , in the treatment of hepatitis B infection. If one makes liposomes containing a lipid derivative of a nucleotide analogue and having a diameter of less than 100 nanometers, the drug may be targeted in a highly efficient manner to these parenchymal cells.
  • the intrahepatic distribution of systemi- cally admini ⁇ tered liposome ⁇ has been demonstrated by the group of G.
  • liposome-encap ⁇ ulated radioactive inulin as a marker (12) .
  • the uptake of lipo ⁇ omes into parenchymal cells appears to be limited by the morphology of the hepatic sinusoid ⁇ wherein fene ⁇ tration ⁇ of about 100 nm in diameter deny efficient access of particles larger than 100 nm to the underlying hepatocytes.
  • Liposomes of relatively large size are preferentially taken up by the resident macrophages of liver, the Kupffer cells (20, 21) .
  • liposomes containing anti-hepatitis nucleoside analogues should be less than 200 nanometers in diameter, preferably les ⁇ than 100 nanometers in diameter, and in the range of about 30 to 80 nanometers in diameter.
  • liposomes containing the antihepatitis B nucleotide of a diameter les ⁇ than 100 nanometer ⁇ can be expected to target drugs with improved efficiency to the hepatic parenchymal cells to achieve a very large and effective uptake of drug by the liver.
  • Sizing of liposome preparations to a selected diameter is accompli ⁇ hed by extru ⁇ ion through a Nuclepore ® filter of appropriate pore ⁇ ize, as indicated in Example 4, by passage through a ceramic filter as disclosed in U.S. Patent No. 4,737,323 to Martin, or preferably by proces ⁇ ing in a microem- ulsifier apparatus (Microfluidizer ® , BiotechnologyDevelopment Corporation, Newton, MA) , as de ⁇ cribed in Example 6.
  • a microem- ulsifier apparatus Microfluidizer ® , BiotechnologyDevelopment Corporation, Newton, MA
  • Liposome ⁇ can be made from the lipid derivative ⁇ of nucleoside analogues, preferably in combination with any of the conventional synthetic or natural phospholipid liposome materials including phospholipids from natural source ⁇ such as egg, plant or animal ⁇ ource ⁇ ⁇ uch a ⁇ phosphatidylcholines, pho ⁇ phatidylethanola ines, phosphatidylglycerols, sphingo- myelins, phosphatidylserines, or phosphatidylinositols.
  • natural source ⁇ such as egg, plant or animal ⁇ ource ⁇ ⁇ uch a ⁇ phosphatidylcholines, pho ⁇ phatidylethanola ines, phosphatidylglycerols, sphingo- myelins, phosphatidylserines, or phosphatidylinositols.
  • Synthetic phospholipid ⁇ that may also be used, include, but are not limited to, dimyristoylphosphatidylcholine, dioleoyl- pho ⁇ phatidylcholine, dipalmitoylpho ⁇ phatidylcholine and di ⁇ tearoylphosphatidylcholine, andthe corresponding synthetic phosphatidylethanolamines and phosphatidyl-glycerols.
  • DOTAP dimethyl-a moniopropane
  • D,L,-2,3- distearoyloxypropyl(dimethyl)- ⁇ -hydroxyethyl ammonium- (acetate) 1, 2-dioleoyl-3-dimethyl-aminopropyl-j ⁇ - hydroxyethylammonium acetate
  • DORI diester 1, 2-0-dioleyl-3- dimethylaminopropyl- / 9-hydroxyetylammonium acetate
  • the relative amounts of phospholipid and additives used in the liposomes may be varied if desired.
  • the preferred ranges are from about 80 to 95 mole percent phospholipid and 5 to 20 mole percent psychosine or other additive.
  • Cholesterol, cholesterol hemisuccinate, fatty acids or DOTAP may be used in amounts ranging from 0 to 50 mole percent.
  • the amounts of antiviral nucleoside analogue incorporated into the lipid layer of liposomes can be varied with the concentration of their lipids ranging from about 0.01 to about 90 mole percent, preferably up to about 40 or 60 mole percent.
  • the lipid derivatives of the invention can also be prepared for therapeutic use as emulsions or microemulsions, free from emulsifying agents, wherein the formulations described above are present as dispersed phase droplets of very small diameters in an aqueous medium. See, for example, the apparatus for forming such emulsion ⁇ , U.S. Patent No. 4,533,254 to Cook et al., disclosing the formation of such microemulsion ⁇ wherein the dispersed phase droplets range from about 0.010 ⁇ m to about 0.2 ⁇ m in diameter.
  • the liposomes can be administered intravenously, intraperitone- ally, intramuscularly, or subcutaneou ⁇ ly a ⁇ a buffered aqueou ⁇ ⁇ olution; alternatively, they can be administered orally in liquid or solid formulations. Any pharmaceutically acceptable aqueous buffer or other vehicle may be utilized so long as it does not destroy the liposome structure or the activity of the antiviral nucleoside analogue.
  • aqueous buffers examples include 150 Mm NaCl containing 5 Mm sodium phosphate with a pH of about 7.4, or any other physiological buffered salt solution ⁇ .
  • the do ⁇ age for a mammal, including a human, may vary depending upon the extent and severity of the infection and the activity of the administered compound. Dosage levels for antiviral nucleoside analogues are well established.
  • Dosage levels of lipid derivatives of nucleoside analogues should be such that about 0.001 mg/kilogram to 1000 mg/kilogram is administered to the patient on a daily basis and more prefera ⁇ bly from about 0.05 mg/kilogram to about 100 mg/kilogram, measured a ⁇ the nucleoside analogue portion of the liponucleotide.
  • the derivatives described have several unique and novel advantages over the water soluble dideoxynucleoside pho ⁇ phate ⁇ de ⁇ cribed in an earlier copending application, Serial No. 07/099,755. First, they can be formulated more efficiently.
  • Liposomes comprising lipid derivatives of nucleoside analogues have much higher ratios of drug to lipid because they are incorporated into the wall of the liposome instead of being located in the aqueous core compartment.
  • the lipo ⁇ ome ⁇ containing the lipophilic dideoxynucleoside derivatives noted above do not leak during storage, providing improved product stability.
  • these compositions may be lyophilized, stored dry at room temperature, and reconstituted for use, providing improved shelf life. They also permit efficient incorporation of antiviral compounds into liposomal formulations without significant waste of active compound. They also provide therapeutic advantages.
  • Stability of the lipo ⁇ omally incorporated agent causes a larger percentage of the administered antiviral nucleoside to reach the intended target, while the amount being taken up by cells in general is minimal, thereby decreasing the toxic side effects of the nucleosides.
  • the toxic side effects of the nucleoside ⁇ may be further reduced by targeting the liposomes in which they are contained to actual or potential sites of infection by incorporating ligands specifically binding thereto into the liposomes.
  • the compounds noted above have been constructed in a novel way which may give rise to phosphorylated dideoxynucleosides or other antiviral nucleosides upon further cellular metabolism. This improves their antiviral effect in cells which are known to be resistant to the effects of the free antiviral compounds.
  • use of the present invention may provide a method for delivering biologically active phosphorylated nucleosides to the interior of a cell.
  • the compound ⁇ of the present invention are thus precursors or prodrugs of phosphorylated nucleoside analogues.
  • Lipid derivatives of antiviral agents have a prolonged antiviral effect as compared to the lipid-free agents; therefore they provide therapeutic advantages as medicaments even when not incorporated into liposome ⁇ .
  • Nonliposomal lipid derivatives of antiviral nucleoside analogues may be applied to the skin or ucosa or into the interior of the body, for example orally, intratracheally or otherwise by the pulmonary route, enterally, rectally, nasally, vaginally, lingually, intravenously, intra-arterially, intramuscularly, intraperitoneally, intradermally, or subcutaneou ⁇ ly.
  • the pre ⁇ ent pharmaceutical preparation ⁇ can contain the active agent alone, or can contain further pharmaceutically valuable ⁇ ubstances. They can further comprise a pharmaceutically acceptable carrier, including bioerodible or other time release carriers.
  • composition ⁇ containing lipid derivative ⁇ of antiviral nucleo ⁇ ide ⁇ are produced by conventional di ⁇ olving and lyophilizing proce ⁇ e ⁇ to contain from approximately 0.01% to 100%, preferably from approximately 0.1% to 50%, weight percent, of the active ingredient. They can be prepared a ⁇ ointment ⁇ , salve ⁇ , tablet ⁇ , capsules, powders or sprays, together with effective excipients, vehicles, diluents, fragrances or flavor to make palatable or pleasing to use. Oral administration of liponucleotide analogues may be advantageous in assuring effective liver uptake.
  • Formulations for oral ingestion are in the form of tablet ⁇ , capsules, pills, ampoules of powdered active agent, or oily or aqueous suspensions or solutions.
  • Tablets or other non-liquid oral composition ⁇ may contain acceptable excipient ⁇ , known to the art for the manufacture of pharmaceutical compositions, comprising diluents, such as lactose or calcium carbonate; binding agents such a ⁇ gelatin or starch; and one or more agents ⁇ elected from the group con ⁇ i ⁇ ting of sweetening agents, flavoring agents, coloring or preserving agents to provide a palatable preparation.
  • such oral preparations may be coated by known techniques to further delay disintegration and absorption in the intestinal tract.
  • Aqueous suspension ⁇ may contain the active ingredient in admixture with pharmacologically acceptable excipient ⁇ , compri ⁇ ing suspending agents, such as methyl cellulose; and wetting agents, such as lecithin or long-chain fatty alcohols.
  • the the aqueous suspensions may also contain preservatives, coloring agents, flavoring agent ⁇ and ⁇ weetening agent ⁇ in accordance with indu ⁇ try ⁇ tandards.
  • the preparations may further comprise antioxidants, ⁇ uch as ascorbic acid or tocopherol, and preservatives, such as p- hydroxybenzoic acid esters.
  • Parenteral preparations comprise particularly sterile or sterilized products.
  • Injectable compo ⁇ ition ⁇ may be provided containing the active compound and any of the well known injectable carrier ⁇ .
  • the ⁇ e may contain salts for regulating the osmotic pres ⁇ ure.
  • the parenteral dosage will be appropriately 3/4 to 1/10 of the oral dose, and may be given by intravenous, subcutaneous, intramuscular, or intraperitoneal route.
  • the efficacy of the antihepatitis liponucleotides of the invention was demonstrated in tests carried out both in vitro and in vivo.
  • the in vivo tests were carried out as described in Example 5 and the pharmacokinetic results are shown in Figures 1-6.
  • the efficacy was evaluated by noting the area under the curve (AUC) of dose level v. time.
  • AUC area under the curve
  • the AUC of phosphatidyl-ddC in liver was 42 times greater than that of free ddC, demonstrating clearly the anticipated targeting feature of this novel liponucleotide.
  • the AUC in sciatic nerve and brain, the sites of toxic damage for ddC showed effective tissue levels not sub ⁇ tantially greater than that for the free drug.
  • targeted anti-HBV analogues such as phosphatidyl-ddC may be able to increase efficacy in treating hepatitis B while decreasing neurotoxicity which is the major clinical problem with ddC.
  • Lipid derivatives of antihepatitis nucleoside ⁇ are effective again ⁇ t HBV in cell ⁇ in culture.
  • In vitro tests using 3 different lipid derivatives of FIAC and 3 corresponding lipid derivatives of FIAU were carried out in hepG2.2.15 HBV-infected cells as described in Example 4, Section B and Table 1. The data shows that the FIAC and FIAU liponucleotides have sub ⁇ tantial activity in vitro.
  • the lipid control, dioleoylphosphatidylcholine (DOPC; 40W and 40X) is without activity.
  • DOPC dioleoylphosphatidylcholine
  • In vitro test ⁇ u ⁇ ing a 1,2-dioleoylglycero- 3-phospho-5'-(2' ,3 '-dideoxycytidine) were carried out in hepG2.2.15 HBV-infected cell ⁇ a ⁇ de ⁇ cribed in Example 4, Section C, and Table 2. Again, both ddC and corre ⁇ ponding liponucleotides have substantial activity against the HBV virus in cells in culture, while the lipid control, 1,2- dioleoylphosphatidylcholine (DOPC) i ⁇ without activity.
  • DOPC 1,2- dioleoylphosphatidylcholine
  • Lipid derivative ⁇ of antiviral nucleotides demonstrate an ability to acces ⁇ ⁇ pecialized cell ⁇ and ti ⁇ sues, particularly those of the liver, preferentially, and in this way to target the reservoirs of hepatitis B infection.
  • the data of Example 5, as represented in Figure la-lf, demontrates this targeting advantage in the enhanced level ⁇ of ddC delivered to and maintained in these cells and tissues by lipid derivatives of ddC and the corresponding reduced levels in tissues subject to the toxic effects of ddC.
  • the lipid derivatives of the invention offer an effective therapeutic agent for the treatment of hepatitis B infections. No generally effective therapy now exist ⁇ for thi ⁇ disease.
  • Phosphatidic acids for example, dilauroyl, di yristoyl, and dipalmitoyl phosphatidic acids, were obtained as disodium salt ⁇ from Avanti Polar lipids (Pelham, AL, USA) .
  • Dowex 50 W 50 x 2-200,100-200 mesh
  • 2' ,3'- dideoxycytidine were products from Sigma Chemical Co. (St. Louis, MO, USA) .
  • Morpholine, dicyclohexylcarbodiimide (DCC) and tertiary butyl alcohol (2-methyl-2-propanol, tBuOH) were the highest grade available from Aldrich Chemical Co. (Milwaukee, Wl, USA).
  • DCC dicyclohexylcarbodiimide
  • tBuOH tertiary butyl alcohol
  • DMPA-H prepared as described in B, above, 250 mg, 0.42 mmol
  • cyclohexane 10 ml
  • a round-bottom flask 50 ml
  • the solvent evaporated under reduced pres ⁇ ure at room temperature. This process was repeated four more times and DMPA-H further dried in a vacuum oven at room temperature overnight over P 2 0 5 .
  • the solvent was evaporated under reduced pressure to yield a yellow gum which was redissolved in a small volume of methanol in chloroform (1:9 by volume) and applied to a column of silica gel (45 g, Kieselgel 60, EM Science) .
  • the column was topped with a small amount of sand (500 mg) to prevent the sample from floating during elution.
  • the column was eluted with 8% methanol in chloroform (1.5L). After a forerun (rejected) , then dimyristoylphosphatidyl-5'- (2*3 '-dideoxy)cytidine (DMPA-ddC) was obtained.
  • DMPA-ddC dimyristoylphosphatidyl-5'- (2*3 '-dideoxy)cytidine
  • DMPA-ddC dimyristoylphosphatidyl 5 * (2'3 '-dideoxy)cytidine
  • the Rf values were: 0.11 (chloroform:methanol:water:ammonia 80:20:1:1) ; 0.38 (chloroform:methanol:ammonia:water 70:30:3:2) ; 0.15 (chloroform:methanol: water 65:25:4) ; UV absorption maximum 273 nm (e 5,800).
  • EXAMPLE la 1 , 2-dilauroylglycerophospho-5 ' - (2 ' , 3 * - dideoxycytidine) , DLP-ddC;
  • EXAMPLE lc 1,2- dipalmitoylglyceropho ⁇ pho-5 ' - ( 2 • , 3 ' -dideoxycytidine) , DPM-ddC ;
  • EXAMPLE Id 1 , 2- ⁇ tearoylglyceropho ⁇ pho-5 ' - (2 ' , 3 ' - dideoxycytidine) , DSP-ddC;
  • EXAMPLE le 1, 2-dilauroylglyceropho ⁇ pho-5 '- (2 • -deoxy-2 ' - fluoro-l-i3-arabinosyl)-5-iodocytosine, DLP-FIAC; EXAMPLE If: l,2-dimyri ⁇ toylglycerophospho-5 * - (2 ' -deoxy-2 * -f luoro-1-S- arabinosyl) -5-iodocyto ⁇ ine, DMP-FIAC; EXAMPLE lg: 1,2- dipalmitoylglycerophospho-5 ' - (2 ' -deoxy-2 ' -f luoro-1- ⁇ - arabino ⁇ yl)-5-iodocyto ⁇ ine, DPM-FIAC; EXAMPLE lh: 1,2- stearoylglycerophospho-5 ' - (2 ' -deoxy-2 ' -f luoro-1-
  • EXAMPLE li 1,2-dilauroylglycerophospho-5'-(2'-deoxy-2*- fluoro-l-9-arabinosyl)-5-iodouracil, DLP-FIAU;
  • EXAMPLE 1j l,2-dimyristoylglycerophospho-5'-(2 '-deoxy-2 '-fluoro-1- ⁇ - arabino ⁇ yl)-5-iodouracil, DMP-FIAU;
  • EXAMPLE Ik 1,2- dipalmitoylglyceropho ⁇ pho-5 ' - (2 ' -deoxy-2 '-fluoro-1- ⁇ - arabino ⁇ yl)-5-iodouracil, DPM-FIAU; and
  • EXAMPLE 11 1,2- stearoylglycerophospho-5'-(2'-deoxy-2'-fluoro-1- ⁇ -arabinosyl)-
  • DSP-FIAU 5-iodouracil
  • DCC dicyclohexylcarbodiimide
  • Dipalmitoylphosphatidic acid (950 mg, 1.47 mmol) was prepared from its disodium salt, essentially as described in Example 1, Part B. Free phosphatidic acid was dissolved in 30 ml chloroform, and the obtained solution was transferred to a two-neck round bottom flask, which contained 30 ml tert- butanol, morpholine (0.53 ml, 6 mmol), and distilled water (0.1 ml, 6 mmol). This mixture was gently refluxed and a solution of dicyclohexylcarbodiimide (1.20 g, 5.9 mmol) in 30 ml tert-butanol was added stepwise from a dropping funnel within 2 hours.
  • the ⁇ olvent was evaporated under vacuum and the residue was added to 50 ml water. This aqueou ⁇ ⁇ u ⁇ pen ⁇ ion wa ⁇ extracted five-ti e ⁇ with 75-ml portion ⁇ of chloroform.
  • FIAU (800 g, 2.16 mmol) was dis ⁇ olved in trimethyl phosphate (2 ml) at 45°C with vigorous stirring.
  • the reaction mixture was cooled to 0°C under argon and added phosphoru ⁇ oxychloride (2 ml, 20 mmol) via ⁇ yringe.
  • the reaction mixture was first stirred at 0°C for one hour, and then kept at -20°C for 12 hours.
  • the reaction was monitored by TLC (acetic acid:n-butanol:water, 1:4:1 v/v).
  • FIAU-MP precipitated as a white crystal.
  • the supernatant wa ⁇ di ⁇ carded and the precipitate wa ⁇ washed with anhydrous ether (5x10 ml) .
  • HPLC retention time of FIAU-MP was 15.3 min using a
  • anhydrous 1,2-dipalmitoyl- sn-glycero-3-phosphoromorpholidate 400 mg, 0.55 mmol
  • FIAU-MP 200 mg, 0.48 mmol
  • anhydrous pyridine 15 ml
  • the solution was evaporated to dryness in vacuum 5-times from anhydrous pyridine, and then 7 ml of anhydrous pyridine were added. This solution was stirred at room temperature overnight under argon. The progress of the reaction was monitored by TLC (chloroform:methanol:ammonium hydroxide:water, 70:38:8:2, v/v).
  • reaction mixture was then evaporated from toluene (4x10 ml) .
  • This residue was dissolved in 15 ml of chloroform:methanol:water (2:3:1, v/v), and acidified to pH 3 with 0.1N hydrochloric acid.
  • Two layers formed, and the aqueou ⁇ layer wa ⁇ washed with chloroform (2x10 ml) .
  • the combined organic layers were evaporated to dryness, and the residue was dissolved in chloroform:methanol:water (2:3:1, v/v) and applied to a DEAE Sephadex (acetate form) column (2.8 x 30 cm).
  • HPLC retention time of FIAU-DP-DPG diammonium salt was 12.65 min. using a 250x4.6 mm, 5 micron Brownlee silica column elutedwithhexane:2-propanol:ammonium hydroxide: water (43:57:3:7, v/v) as the developing system.
  • the compound had an Rf of 0.23 on silica 60A F254 TLC plate eluted with chloroform: ethanol:ammonium hydroxide:water
  • EXAMPLE 2a dideoxycytidine diphosphate dilauroylglycerol, ddC-DP-DLG
  • EXAMPLE 2b dideoxycytidine diphosphate dimyristoylglycerol, ddC-DP-DMG
  • EXAMPLE 2c dideoxycytidine diphosphate dipal itoylglycerol, ddC-DP-DPG
  • EXAMPLE 2d dideoxycytidine diphosphate distearoylglycerol, ddC-DP-DSG;
  • EXAMPLE 2e 1-(2'-deoxy-2 '-fluoro-1- ⁇ -arabinosyl)-5- iodocytosine diphosphate dilauroylglycerol, FIAC-DP-DLG;
  • EXAMPLE 2f 1-(2'-deoxy-2•-fluoro-1- ⁇ -arabino ⁇ yl)-5- iodocyto ⁇ ine dipho ⁇ phate dimyristoylglycerol, FIAC-DP-DMG;
  • EXAMPLE 2g 1-(2 '-deoxy-2 '-fluoro-l- / 9-arabino ⁇ yl)-5- iodocyto ⁇ ine diphosphate dipalmitoylglycerol, FIAC-DP-DLG;
  • EXAMPLE 2h 1-(2•-deoxy-2'-fluoro-1-jS-arabino ⁇ yl)-5- iodocyto ⁇ ine diphosphate distearoylgly
  • EXAMPLE 2i 1-(2'-deoxy-2'-fluoro-1- ⁇ -arabinosyl)-5-iodouracil diphosphate dilauroylglycerol, FIAU-DP-DLG;
  • EXAMPLE 2j l-(2'- deoxy-2*-fluoro-l-3-arabinosyl)-5-iodouracil diphosphate dimyristoylglycerol, FIAU-DP-DMG;
  • EXAMPLE 21 l-(2'-deoxy- 2 ' -fluoro-l-3-arabino ⁇ yl) -5-iodouracil dipho ⁇ phate distearoylglycerol, FIAU-DP-DLG are prepared according to the above method except for using an appropriate specie ⁇ of pho ⁇ phatidic acid in the coupling ⁇ tep.
  • a human hepatoblastoma cell line tran ⁇ fected with a pla ⁇ mid carrying HBV DNA, HEPG2.2.15, carrying 4 copie ⁇ of the HBV genome as chromosomally integrated sequences and chroni ⁇ cally producing HBV was incubated with varying concentrations of test compounds as indicated in Tables 1 and 2 below for 10 days at 37°C. Samples of culture medium were periodically collected and stored for later extracellular HBV DNA analysis. On the 10th day, test cells were lysed for analysis of intracellular HBV genomic forms. HBV DNA was analyzed in a quantitative manner for (i) overall levels of both intracel ⁇ lular and extracellular DNA) ; and (ii) the relative rate of HBV replication (intracellular DNA only) .
  • HEPG2.2.15 cells were seeded in 6-well culture plates and grown to confluence over a 10 day period in medium with 5% FBS.
  • Test compounds comprising antiviral nucleotides and corresponding antiviral liponucleotides were added daily for a continuous 10 day period in medium with 1% dialyzed FBS.
  • This reduced serum level does not affect HBV replication in confluent cultures of 2.2.15 cell ⁇ and help ⁇ to eliminate uncontrolled variations of endogenous low molecular weight compounds, such as nucleo ⁇ ide ⁇ , present in FBS
  • Culture medium changed daily during the treatment period, wa ⁇ collected and stored for analysis of extracellular (virion) HBV DNA from days 0, 3, 6, and 10 of the treatment period.
  • the culture medium was changed daily during the treatment period to (1) prevent the buildup of potentially toxic metabolites derived from the test compounds; and (2) provide an analysi ⁇ of HBV virion produc- tion during di ⁇ crete 24-hour intervals which enables a quantitative compari ⁇ on of any effect on virion production.
  • Compounds were tested in duplicate cultures at 3 concentra- tions, covering a 100-fold range.
  • Treated cells were lysed following the 10th day of treatment for the analysis of intracellular HBV genomic forms. Untreated cells were maintained as negative controls.
  • HBV DNA analysis The analysi ⁇ of HBV DNA i ⁇ performed u ⁇ ing blot hybridization technique ⁇ (Southern and slot blot) and ( 3 2P)-labelled HBV specific probes.
  • HBV DNA levels were measured by comparison to known amount ⁇ of HBV DNA ⁇ tandard ⁇ applied to every nitrocellulose filter (gel or slot blot) .
  • An AMBIS Beta Scanner which measures the radioactive decay of the hybridized probe directly from the nitrocellulose membranes, wa ⁇ u ⁇ ed for the quantitative analysis. Standard curves, generated by multiple analyses, were u ⁇ ed to correlate CPM ea ⁇ urement ⁇ with relative level ⁇ of target HBV DNA.
  • the level ⁇ of HBV DNA in each of three classes of intracellular viral genomic forms was individually quantitated; integrated HBV DNA (Integ) , episomal monomeric genomes (Mono) , and HBV DNA replication intermediates (Rl) .
  • the levels of the episomal monomeric HBV genes and Rl were used as an indicator of the relative level of HBV replication. Inhibition of HBV DNA replication is indicated by the loss of Rl without changes in the level of integrated DNA.
  • the levels of HBV virion DNA released into the medium were analyzed by a slot blot hybridization procedure. HBV DNA levels were then compared to levels at day 0 to determine the efficacy of drug treatment.
  • Integrated HBV DNA was used to normalize the relative amounts of DNA in each lane because the levels of this class of HBV DNA were expected to remain constant on a per cell basis, and were thus used as a verifying parameter to insure that equal amounts of cellular DNA were compared between separate samples.
  • the manner in which the hybridization analyses were performed for these experiments results in an equivalence of approximately 1.0 pg/ml of extracellular HBV DNA to 3-5 genomic copies per cell and 1.0 pg/ml of extracellular HBV DNA to 3 x 10 5 viral particles/ml.
  • Typical values for extracellular HBV DNA in untreated cells generally range from 50 to 150 pg/ml; intracellular HBV DNA replication intermediates in untreated cells generally range from 50 to 10 pg/ ⁇ g cell DNA (average approximately 760 pg/. g) •
  • HBV DNA Cells carrying HBV DNA and chronically producing HBV DNA, as described above, were treated with test compounds as indicated in Table 1 and HBV DNA determined in accordance with the protocol.
  • ara-A adenosine-9- ⁇ -D-arabinofuranoside
  • FMAU 1 -(2'-deoxy-2'-fluoro-1 - ⁇ -D-arabinofuranosyl) -5 -methyiuracil
  • FIAU 1-(2'-deoxy-2'-fluoro-1- ⁇ -D-arabinofuranosyl)-5-iodouracil
  • FIAC 1 -(2'-deoxy -2' -f iuoro-1 - ⁇ -arabinosyl)-5-iodocytosine
  • DMP-FIAU dimyristoylphosphatidyl-FIAU
  • DPP-FIAU dipalmitoyl phosphatidyl-FlAU
  • FIAU-DP-DPG FIAU diphosphate dipalmitoyl glycerol
  • DMP-FIAC dir ⁇ yristoylphosphatidyl- FIAC
  • DPP-FIAC dipalmitoylphosphatidyl-FIAC
  • FIAC-DP-DPG FIAC diphosphate dipalmitoylglycerol
  • DOPC Control: dioleoylphosphatidylcholine liposomes without added drug.
  • tAnalysis of intracellular HBV DNA was 24 hours following the 10th day of treatment. Sum arv of Table 1 Result ⁇
  • HBV DNA Cells carrying HBV DNA and chronically producing HBV DNA, as described above, were treated with test compounds as indicated in Table 2 and HBV DNA determined in accordance with the protocol.
  • DOPC dioleoylphosphatidylcholine (a lipid control without drug)
  • DOP-2', 3'ddC dioleoylphosphatidyl-ddC
  • the positive treatment control 2' ,3 ' ddC(2' ,3'- dideoxycytosine) produced a significant depression of HBV DNA replication.
  • the test compound dioleoylphosphatidyl-ddC (DOP-ddC) is a potent inhibitor of HBV DNA replication.
  • Extracellular HBV DNA was undetectable by day 9 (>1000-fold depression versus the day 0 values) and approximately a 100- fold depression of intracellular HBV DNA replication intermediates was observed after 9 days of treatment at the highest concentrations used.
  • EXAMPLE 4 Pharmacokinetics of Phosphatidyl-[.a]ddC in Mice
  • Dioleoylphosphatidyl-ddC was synthesized by coupling [ 3 H]ddC (5 MC/mol) to dioleoylphosphatidic acid as described in Example 1.
  • Unila ellar lipid vesicles were prepared using dioleoylphosphatidylcholine/cholesterol/phosphatidyl-[ 3 H]ddC in a molar ratio of 67/30/3 by the method of Mayer, L.D., Hope, M.J., and Cullis, P.R. (16) , using a 200 nanometer NucleporeTM filter. Smaller vesicles may be prepared by the same method using a 100 nanometer NucleporeTM filter, or by microfluidization as described by Mayhew, E., et al. (16).
  • mice were treated intraperitoneally (i.p.) with 3 mg/kg of [ 3 H]ddC or an equimolar dose of phosphatidyl-[ 3 H]ddC and the animals were sacrificed at various time ⁇ and blood and ti ⁇ ue ⁇ were obtained for analysis.
  • DdC was cleared rapidly from plasma in the first hour while phosphatidyl-ddC reached much higher level ⁇ (50 versus 16 nmol/ml) and persisted much longer in the circulation (Figure 2a) .
  • the area under the curve (AUC) for phosphatidyl-ddC was sub ⁇ tantially larger than that of ddC.
  • the peak liver level for ddC was 18 nmol/gm at 15 min.
  • phosphatidyl-ddC reached a peak level at 1 hr of about 80 nmol/gm.
  • the AUC in liver for phosphatidyl-ddC was 42 times greater than that of ddC ( Figure 2b) .
  • Spleen levels were also much higher with phosphatidyl-ddC (AUC 85 times greater than ddC, Figure 2f) .
  • AUC 85 times greater than ddC, Figure 2f phosphatidyl-ddC
  • the levels were not greatly increased with phosphatidyl- ddC as compared to ddC; AUC values in sciatic nerve were 1.7 times greater than with ddC ( Figure 2c) , and the differences were not significant at most time points.
  • Brain levels of phosphatidyl-ddC were increa ⁇ ed ⁇ lightly (2.7X; Figure 2d); however, it should be noted that the brain was not perfused to remove blood, which may be responsible for the apparent increase.
  • a patient suffering from active hepatitis B infection is treated by parenteral administration of liposomally incorporated DMP-FIAU at a dose of 1 mg/kilo/day of the active FIAU moiety until a clinical re ⁇ pon ⁇ e indicating the inhibition of production of hepatitis B virus is observed.
  • Liposome ⁇ incorporating DMP-FIAU are prepared u ⁇ ing a microemul ⁇ ification apparatus (Microfluidizer ® , Newton,
  • the dose of FIAU can be adjusted from 1/lOOth of the indicated dose to 10 times the indicated dose, as determined by clinical judgment.
  • An effective respon ⁇ e to hepatiti ⁇ therapy with DMP-FIAU lipid prodrug i ⁇ measured by absence of detectable virus particles in the serum of the patient and clinical improvement in the patient.
  • a quantity of 6.42 micromoles of dioleoylphosphatidyl ⁇ choline, 3.85 micromoles of cholesterol, and 1.28 micromoles of dioleoylphosphatidyl-dideoxycytidine (DOP-ddC) were mixed in a ⁇ terile 2.0 ml glass vial and the solvent was removed in vacuo in a rotary evaporator.
  • dioleoyl- phosphatidyldideoxycytidine was replaced by DMP-FIAU or FIAC- DP-DPG; control liposome ⁇ were prepared by omitting the antiviral liponucleotide.
  • the dried film wa ⁇ put under high vacuum overnight at room temperature to remove traces of ⁇ olvent.
  • the lipid film was hydrated at 30°C with 0.3 ml of sterile 10 Mm sodium acetate buffer (pH 5.0) containing isotonic dextro ⁇ e and the ampule was sealed.
  • the mixture was vortexed intermittently for 10 minutes followed by sonication using a Heat Systems Ultrasonics sonicator with a cup horn generator (431B) at output control setting #9 for 90 to 120 minutes at which time the sample is clarified, and diluted with buffer for use.
  • the lipid film was hydrated by the addition of 1.0 ml of sterile phosphate buffered saline, and the mixture was shaken gently at 20°C for 20 minutes, followed by ten 30-second cycle ⁇ of vortexing to form multilamellar lipo ⁇ omes.
  • the suspension of liposomes was subjected to 5 cycles of extrusion through two stacked Nuclepore ® polycarbonate filters having pore diameters of 200nm, to form a homogenously sized liposomal population.
  • a liposomal population having an approximate mean diameter of 0.2 ⁇ m to 0.1 ⁇ m or less is prepared by processing the hydrated lipid suspension in a Microfluidizer ® apparatus.
  • the size distribution of the particles can be regulated by varying the time of microemulsification from about 2 up to about 10 minutes as described by Mayhew, E. et al. (16) .
  • nucleoside analogue ⁇ and lipid derivatives thereof can be substituted in the Examples to obtain similar results. It should be further emphasized that the present syntheses are broadly applicable to formation of compounds from essentially all antihepatitis B nucleosides for use in the invention.

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Abstract

L'invention concerne des composés permettant de traiter les infections de l'hépatite B. Les composés consistent en des analogues de nucléosides présentant une activité antihépatite B qui sont liés, ordinairement par un 5' phosphate de résidu de pentose, à un des lipides du groupe sélectionné. La nature lipophile de ces composés procure un avantage quant à l'utilisation de l'analogue de nucléoside seul, rendant possible leur incorporation à la structure lamellaire des liposomes, soit seuls soit en combinaison avec des molécules lipidiques similaires. Sous la forme de liposomes de taille appropriée, ces agents antihépatite B sont de préférence absorbés par les cellules du foie abritant le virus cible.
PCT/US1992/004856 1991-07-12 1992-06-03 Liponucleosides antiviraux: traitement de l'hepatite b WO1993000910A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP92914562A EP0594677A4 (en) 1991-07-12 1992-06-03 Antiviral liponucleosides: treatment of hepatitis b
JP5502213A JPH07500573A (ja) 1991-07-12 1992-06-03 抗ウィルス性リポヌクレオシド：ｂ型肝炎の治療
AU22268/92A AU668873B2 (en) 1991-07-12 1992-06-03 Antiviral liponucleosides: treatment of hepatitis B

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US73027391A 1991-07-12 1991-07-12
US730,273 1991-07-12

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WO1993000910A1 true WO1993000910A1 (fr) 1993-01-21

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JP (1) JPH07500573A (fr)
AU (1) AU668873B2 (fr)
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US7456155B2 (en) 2002-06-28 2008-11-25 Idenix Pharmaceuticals, Inc. 2′-C-methyl-3′-O-L-valine ester ribofuranosyl cytidine for treatment of flaviviridae infections
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US5484911A (en) * 1993-04-01 1996-01-16 Health Research, Inc. Nucleoside 5'-diphosphate conjugates of ether lipids
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AU668873B2 (en) 1996-05-23
EP0594677A4 (en) 1997-09-17
CA2112803A1 (fr) 1993-01-21
AU2226892A (en) 1993-02-11
EP0594677A1 (fr) 1994-05-04
JPH07500573A (ja) 1995-01-19

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