IE83237B1 - Lipid derivatives of antiviral nucleosides, lipsomal incorporation and method of use - Google Patents

Description

PATENTS ACT, 1992 2187 89 LIPID DERIVATIVES OF ANTIVIRAL NUCLEOSIDES, LIPOSOMAL INCORPORATION AND METHOD OF USE NEXSTAR PHARMACEUTICALS, INC. "‘ 83237 The present invention relates the treatment of viral infections using lipid derivatives of the especially generally to antiviral nucleoside analogues. More particularly, present invention relates to lipid, and phospholipid, derivatives of modified antiviral nucleoside analogues which can be integrated into the structure of liposomes, thereby forming a more stable liposomal complex which can deliver greater amounts of drugs to target cells with less toxicity.

The publications referred to herein are hereby incorporated by reference to the extent permitted by law.

There has been a great deal of interest [in recent years in the use of nucleoside analogues to treat viral infections. A nucleoside consists of a pyrimidine or purine base which is linked to ribose, a five—carbon sugar having a cyclic structure. The antiviral nucleoside analogues closely resemble natural nucleosides and are designed to inhibit viral functions by preventing the synthesis of new DNA or RNA. Nucleosides are enzymatically assembled into DNA or RNA.

During DNA synthesis, free nucleoside triphosphates (nucleosides with three phosphate groups attached) react with the end of a growing DNA chain. The reaction involves the linking of the phosphate group at the 5’ position on the incoming nucleoside triphosphate with the hydroxyl group at the 3’ position of the sugar ring on the end of the forming DNA chain. The other two phosphate groups are freed during the reaction, thereby resulting in the addition of a nucleotide to the DNA chain.

Nucleoside analogues are compounds which mimic the naturally occurring nucleosides sufficiently so that they are able to participate in viral DNA synthesis. However, the antiviral nucleoside analogues have strategically located differences in chemical structure which inhibit viral such as which enzymes reverse transcriptase or prevent further DNA synthesis once the analogue has been attached to the growing DNA chain.

Dideoxynucleosides are antiviral compounds that lack the_ hydroxyl groups normally present at the second and third position of ribose. when a ciideoxynucleoside is incorporated into a growing DNA chain, the absence of the 3-OH group on its ribose group makes it impossible to attach another nucleotide and the chain is terminated.

Dideoxynucleosides are particularly useful in treating retroviral infections where viral replication requires the of viral RNA other deoxynucleosides and nucleosides analogues having only a transcription into DNA by viral reverse transcriptase. nucleoside analogues include fragment of ribose or other pentose connected to the base molecule.

Acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). HIV infects cells bearing the CD4 (T4) surface antigen, such as CD4+ helper lymphocytes, CD4+ monocytes and macrophages and certain other CD4+ cell types. The HIV infection of CD4+ lymphocytes results in cytolysis and cell death which contributes to the immunodeficiency of AIDS; however, CD4+ monocytes and macrophages may not be greatly harmed by the virus. Viral replication in these cells appears to be more prolonged and less cytotoxic than in lymphocytes, and as a result, monocytes and macrophages represent It has important reservoirs of HIV infection. recently been discovered that macrophages may serve as reservoirs of HIV infection even in certain AIDS patients who test negative for the presence of HIV antibodies. No effective cure is available for AIDS, although dideoxynucleosides have been shown to prolong life and to reduce the incidence of certain fatal infections associated with AIDS.

Certain monocyte—derived macrophages, when infected with some strains of HIV, have been found to be resistant to treatment with dideoxycytidine, azidothymidine, and other dideoxynucleosides in Vitro as shown by Richman, et al. (1). levels of dideoxynucleoside The resistance may be due in part to the low which result in a AZT, ddC or ddA. it would be useful to have more effective ways of kinase reduced ability to phosphorylate Clearly, delivering large amounts of effective antiviral compounds to macrophages infected with HIV or other viruses and other cells having viral infections. It would also be useful to have more effective ways of delivering antiviral compounds which not only increase their potency but prolong their efficacy.

Dideoxynucleoside analogues such as AZT are the most potent agents currently known for treating AIDS, but in a evidenced It is recent human trial, serious toxicity was noted, (24%) (16%) (2,3). desirable, therefore, to provide a means for administering by anemia and granulocytopenia AZT and other dideoxynucleosides in a manner such that the toxic side effects of these drugs are reduced. Further, it is desirable to provide selective targeting of the dideoxynucleoside to monocyte/macrophages to enhance the efficiency of the drug against. viral infection in this group of cells. One way to do this is to take advantage of the uptake of liposomes by macrophages.

In 1965, Alex Bangham and coworkers discovered that spontaneously formed dried films of phosphatidylcholine closed bimolecular leaflet vesicles upon hydration these (4)- Eventually, known as structures came to be liposomes.

A number of uses for liposomes have been proposed in medicine. Some of these uses are as carriers to deliver therapeutic agents to target organs. The agents are encapsulated during the process of liposome formation and released cell delivering higher concentrations of therapeutic agents to in vivo when liposomes fuse with the lipids of surface membrane. Liposomes provide a means of target organs. Further, since liposomal delivery focuses therapy at the site of liposome uptake, it reduces toxic side effects.

For example, liposomal antimonial drugs are several hundred—fold more effective than the free drug in treating leishmaniasis as shown independently by Black and Watson (5) and (6). amphotericin B appears to be more effective than the free Alving, et al. Liposome-entrapped drug in treating" immunosuppressed patients with systemic fungal disease (7). other uses for liposome encapsulation include restriction of doxorubicin toxicity (8) and diminution of aminoglycoside toxicity (9). now thought that macrophages are an important reservoir of HIV infection (10, 11). Macrophages are also a primary site of liposome uptake (12, 13). utilize liposomes to enhance the effectiveness of antiviral As previously mentioned, it is Accordingly, it would be desirable to nucleoside analogues in treating AIDS and other viral infections.

The use of liposomes to deliver phosphorylated dideoxynucleoside to AIDS infected cells which have become resistant to therapy has been proposed in order to bypass the low dideoxynucleoside kinase levels.

Attempts have also been made to incorporate nucleoside acylovir (ACV) analogues, such as iododeoxyuridine (IUDR), and ribavirin into liposomes for treating diseases other than AIDS. However, these attempts have not been entirely satisfactory because these relatively small water soluble nucleoside analogues tend to leak out of the liposome (14, 15), effectiveness. other to leak out of difficulties rapidly resulting in decreased »targeting disadvantages include the tendency liposomes in the presence of in liposome formulation and stability, low degree of liposomal loading, and hydrolysis of liposomal dideoxynucleoside phosphates when exposed to acid hydrolases after cellular uptake of the liposomes.

Attempts have also been made to combine nucleoside analogues, such as arabinofuranosylcytosine (ara-C) and arabinofuranosyladenine (ara-A), with phospholipids in order to enhance their catabolic stability as chemotherapeutic agents in the treatment of various types of cancer (16). The resulting agents showed a decreased toxicity and increased stability over the unincorporated nucleoside analogues. However, the (16) resulting agents exhibited poor cellular uptake and poor drug absorption (17).

EP-A-0122151 (36) discloses the preparation of primary or secondary alcohol derivatives of phospholipids by means of an enzymatic transfer technique that exchanges the alcohol structural moiety of a phosphdlipid species for a primary or secondary alcohol through the enzymatic activity of phospholipase DH. serum,« The phospholipid may be selected from, amongst others, a particular defined range of 1,20- diacyl and l,3—O-diacyl glycerol monophosphate phospholipids and the primary alcohol may be selected from, amongst others, the nucleosides cytidine, uridine, arabinosylcytosine, adenosine, guanosine, cyclocytidine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, deoxyuridine and inosine. However, EP—A—Ol22l5l contains no teaching of any pharmacological advantages of such compounds and only very few of the possible individual compounds arising from the theoretically possible selections are taught or suggested.

J. Med. Chem. 25 (1982) pages 1322-1329 (Ryu et al) (37) discloses the preparation of certain particular phospholipid conjugates of ara—C and certain other nucleosides. The phospholipid—nucleoside linkage can be 3—diphosphate or monophosphate, depending on the particular conjugate being described, and in certain ones of the conjugates the nucleosides cytidine, arabinosylcytosine, arabinosyladenine and 7—deazaadenosine are present. A pharmacological activity against mouse myeloma is reported for certain conjugates. No suggestion is made of any antiviral or antiretroviral activity.

EP-A-0262876 (38) discloses the preparation of certain particular 1,2-O-diacyl and 1,2-O- dialkyl glycerol monophosphate phospholipid derivatives of nucleosides and reports that such derivatives have strong antitumor activity and "superior" solubility in aqueous medium. No suggestion is made of any antiviral or antiretroviral activity. The nucleosides 5-fluoro- uridine, 5—fluorocytidine, bredinin, tubercidin, arabinosyl-cytosine, arabinosyl—5— fluorocytosine, arabinosylfluorocytosine, arabinosyl—adenine, arabinosyl—thymine, 5- fluoro-2’-deoxyiibo-uridine and Neplanocin-A are mentioned by way of example although only 5-fluorouridine, Neplanocin-A and arabinosylfluorocytosine are present in any of the specific compounds taught.

GB—A-2168350 (39) discloses the preparation of certain particular l-O-a1l glycerol diphosphate phospholipid derivatives of certain nucleosides in which the sugar is ribose, 2’—deoxyribose, arabinose or 2’,2’—dihydroxyribose and the base is adenine, cytosine, S-fluorouracil, 5-azacytosine, 6-niercaptopurine or 7-deazaadenine, and reports generally that such derivatives have anticancer (antitumor) and antiviral activity although only an antitumor activity of two compounds in lymphoid leukemic mice is supported by specific data.

In order to use nucleoside analogues incorporated into liposomes for treating viral infections more effectively, it is desirable to increase the stability of the association between the liposome and the nucleoside analogue.

In order to further enhance the effectiveness of these antiviral liposomes, it would be desirable to target the liposomes to infected cells or sites of infection. liposomal delivery may be obtained by Greater specificity in incorporating monoclonal antibodies or other ligands into the liposomes. Such ligands will target the liposomes to sites of liposome uptake capable of binding the ligands.

Two different approaches for incorporating antibodies into liposomes to create immunoliposomes have been described: that of Huang and coworkers (18) involving the synthesis of et al. (19) palmitoyl antibody, and that of Leserman, involving the linkage of thiolated antibody to liposome- incorporated phosphatidylethanolamine (PB).

The methods apply dideoxynucleosides used in the treatment of AIDS and other disclosed here not only to retroviral diseases, but also to the use of antiviral nucleosides in the treatment of diseases caused by other viruses, such as herpes simplex virus (HSV), human herpes virus 6, cytomegalovirus (CMV), hepatitis B virus, Epstein- Thus, the term "nucleoside analogues" is used herein to refer to Barr virus (EBV), and varicella zoster virus (VZV). compounds that can inhibit viral replication at various steps, including inhibition of viral reverse transcriptase or which can be incorporated into viral DNA or RNA, where they exhibit a chain—terminating function.

Summary of the Invention The invention enables compounds and compositions to be prov- ided for use in treating viral infections, including HIV (AIDS), herpes simplex virus (HSV), human herpes virus 6, cytomegalovirus (CMV), hepatitis B virus, Epstein-Barr virus (EBV), and varicella zoster virus (VZV). A composition may contain, in addition to a pharmaceutically acceptable carrier, a lipophilic antiviral compound prepared by chemically linking an antiviral nucleoside analogue ix) at least one lipid species. The antiviral nucleoside analogue is linked to the lipid through a provides a method for incorporating monophosphate, diphosphate or triphosphate group. invention, further, such lipid derivatives of antiviral agents into liposomes for improved delivery of the antiviral agent. A liposome comprises a relatively spherical bilayer which is comprised wholly or in part of the above-described lipid derivatives The pharmacologically inactive lipids. of antiviral agents. liposome may also contain Further, the liposome may contain a ligand, such as a monoclonal antibody to a viral binding site (such as CD4), or other binding protein.

Such a ligand provides additional specificity in the delivery site of the antiviral agent. The invention provides a method for incorporating such ligands into antiviral liposomes.

The invention is defined in the appended claims.

In one preferred embodiment, the liponucleotide compound is a phosphatidyldideoxynucleoside or a dideoxynucleoside diphosphate diglyceride. In another, the lipid species may comprise at least one acyl ester, ether, or vinyl ether derivative of glycerol.

In another embodiment, the nucleoside analogue is a purine or pyrimidine linked through a B-N-glycosyl bond to a pentose residue that lacks at least one of the 2’ or 3’ carbons, but retains the 5’ carbon, and the phosphate group is bound to the 5’ carbon (i.e., what would have been the pentose moiety). In another ' carbon in a complete embodiment of the invention, the lipid species is an N—acy1 sphingosine.

In some preferred embodiments, the acyl or alkyl groups of the lipid species, of whatever linkage, as for ether or vinyl ether, comprise 2 to 24 at least one of the acyl example ester, carbon atoms. In one variation, or alkyl groups is saturated. In another, at least one of the acyl or alkyl groups has up to six double bonds.

In still another embodiment, the lipid moiety is a glyceride and the glyceride has two acyl groups that are the same or different.

In addition to the compound, liposomes formed at least in part from the liponucleotide compounds may further comprise phospholipids selected from the group consisting of phosphatidylcholine, phosphatidyl— ethanolamine, phosphatidyl glycerol, phosphatidylserine, phosphatidylinositol and sphingomyelin.

In one embodiment of the invention, the percentage of antiviral agent is 0.01 to 100 percent by weight of the liposome.

In another embodiment, the liposome further comprises a ligand bound to a lipid substrate. The ligand may be an antibody, such as a monoclonal antibody to a viral antigen.

The viral antigen could be gp4l or gpllo of HIV, or could be any other suitable viral antigen. In one embodiment, the ligand is CD4 receptor protein, or CD4 protein itself. the protein or other substance that binds CD4.

Alternatively, ligand is an antibody to CD4 or a In one embodiment, the nucleoside which is a thereof, and the 2’,3’-didehydro, or an acyclic hydroxylated fragment analogue is a nitrogenous base purine, pyrimidine, or a derivative pentose residue is a 2’,3’-dideoxy, azido or halo derivative of ribose, of ribose. The pentose residue may thus be a 2',3’- dideoxyribose, and the nucleoside analogue may be 2',3’- dideoxycytidine, 2’,3’-dideoxythymidine, 2',3’- dideoxyguanosine, 2’,3’-dideoxyadenosine, 2',3’- dideoxyinosine, or 2,6 diaminopurine, 2',3’- dideoxyriboside.

In another embodiment, the pentose residue is a 2',3’- the 2',3’- didehydrothymidine, 2’,3’-didehydrocytidine carbocyclic, or didehydroribose and nucleoside is ’,3'—didehydroguanosine.

In still another embodiment, the pentose residue is an azide derivative of ribose, and the nucleoside is 3’—azido- ’-deoxythymidine, 3’—azido—3’-deoxyguanosine, or ,6-diaminopurineazido-2’,3’dideoxyriboside.

In still another embodiment of the invention, the pentose residue is a halo derivative of ribose and the nucleoside is ’ — f l u o r o — 2 ’ ’-fluoro—3’—deoxythymidine, ' —di.de<3xy 2,6- diaminopurine-3’-fluoro-2’,3’—dideoxyriboside. The ’,3’-dideoxy-2’-fluoro—ara-adenosine, or invention also includes halo derivatives of the purine or pyrimidine rings, such as, for example, -chloro—deoxyadenosine. Alternatively, the pentose residue is an acyclic hydroxylated fragment of ribose, and the nucleoside is 9—(4,-hydroxy-1’,2’-butadienyl) adenine, 3-(4,—hydroxy—1’,2’-butadienyl) 9-(2- p h o s p h o n y 1 m e t h o x y e t h y l ) a d e n i n e o r cytosine, phosphonomethoxydiaminopurine.

In accordance with another embodiment of the invention, 1—(2'— deoxy—2’-fluoro5—D—arabinofuranosyl)iodocytosine (FIAC) or iodouracil (FIAU). the nucleoside analogue is acyclovir, gancyclovir, (2’—deoxy-2’-fluoro—1-fl-D—arabinofuranosyl) In all of the foregoing compounds, a monophosphate, diphosphate, or triphosphate linking group may be provided between the 5’ position of the pentose residue and the lipid species. In still further embodiments of the invention, the lipid species is a monoacylglycerol or a_ diacylglycerol. Other examples of lipid species include D,L—l,2~diacyloxyethyl—(dimethyl)—beta— hydroxyethyl ammonium groups.

In accordance with another embodiment of the present invention, the lipid species comprises from 1 to 4 fatty each the moiety comprising from 2 to 24 fatty acid acid moieties, carbon atoms. Advantageously, at least one moiety of the lipid species is unsaturated, and has from 1 to 6 double bonds.

A particular example of these compounds is 1,2- diacylglycerophospho—5'—(2',3'—dideoXy)thymidine.

Specific examples of these compounds are provided having the formula: (L)m—(w)n-A-Q-z wherein Z is the base portion of the nucleoside analogue, Q is the pentose residue, A is O or S, W is phosphate, n = 1 to 3, and L is the lipid moiety wherein m = 1 to 5, and wherein each L is linked directly to a W.

Also included are compounds having the formula: % L-W-L wherein Z is the substituted or unsubstituted purine or pyrimidine group of the nucleoside analogue, Q is the pentose residue, W is phosphate, A is O or S, and L is the lipid moiety.

In one embodiment of the invention, with reference to each IJ is independently selected the foregoing formulas, from the group consisting of CH=CH-(CH2)12-CH3 H2C‘R1 H2C'R1 H on H —R2, , and H —R2 £ RC(O)NH- H H2 - (CH3)2"N(CH2)2' HZC" wherein R, R1 and R2 independently have from O to 6 and have the structure CH3’ (CH2) a- (CH=CH-CH2)b- (CH2 ) C-Y wherein the sum of a and c is from 1 to 23, sites of unsaturation, and b is 0 to , and wherein Y is C(O)O—, C, C=C—0-, C(O)S-, C-S-, or c=c—s-.

In one embodiment of the foregoing compounds, the pentose residue comprises ribose, dideoxyribose, didehydroribose, or an azido or halosubstituted ribose, attached at the 9 position of the purine or at the 1 position of the pyrimidine.

A method for synthesizing the lipid derivatives of antiviral nucleosides comprises the step of reacting an antiviral nucleoside, having a ribose hydroxyl group, with a phospholipid in the presence of a coupling reagent whereby the nucleoside is joined to the phospholipid by a phosphate bond at the position of the ribose hydroxyl group. In one preferred embodiment, the phospholipid is aa diacyl phosphate. In another, the phospholipid is a phosphatidic acid or a ceramide. A further method of synthesizing the lipid derivatives of antiviral nucleosides comprises the steps of reacting an antiviral nucleoside monophosphate with a reagent HL, wherein I4 represents a leaving group, to form a nucleoside P04-L, reacting’ the nucleoside P04-L with a phosphatidic acid to bind the acid to the nucleoside through a pyrophosphate bond. In one variation of the method, the nucleoside monophosphate is AZT 5’-monophosphate.

Still a further method of synthesizing a glyceride derivative of a nucleoside analogue comprises the step of joining a monoglyceride or diglyceride and an antiviral in the In one embodiment, the nucleoside monophosphate with a coupling agent presence of a basic catalyst. glyceride is 1-O-stearoylglycerol and the nucleoside is RZT monophosphate.

A method for preparing a suspension of the liposomes for use in treating viral and retroviral infections in a mammal comprises providing the lipophilic antiviral agent comprising at least one lipid species attached to a nucleoside analogue through a monophosphate, at the 5’ diphosphate or triphosphate linking group position of the pentose residue of the nucleoside, combining the lipophilic antiviral agent and a pharmacologically acceptable aqueous solvent to form a mixture, and forming liposomes from the lipophilic antiviral agent. The liposomes may be formed, for example, by sonication, extrusion or microfluidization. In one preferred embodiment, the combining step further comprises including in the combination a pharmacologically inactive This inactive lipid can be, lipophilic lipid. for example, a phosphatidylethanolamine, a sphingolipid, a sterol or a The method also may include treating with including in the glycerophosphatide. the immunoliposomes, or liposomes thio-antibodies to produce combination an lipophilic lipid which is, in part, comprised of a ligand.

Thus, the liposome may include a ligand bound to a lipid substrate.

The invention enables a method for treating retroviral and viral infections in a mammal, such as a human, by administering a sufficient quantity of the antiviral nucleoside analogues described herein to deliver a therapeutic dose of the antiviral agent to the mammal. the method retroviral and viral infections in a mammal, In a preferred embodiment, is used. to treat wherein the with The present retrovirus has become resistant to therapy conventional forms of an antiviral agent. invention also enables a method for treatment of patients having strains of HIV that have developed resistance to AZT or reduced sensitivity to AZT, comprising the step of administering a compound of the present invention to such patient in an effective, retrovirus-inhibiting dosage. Also enabled by the present invention is a method for treating a viral infection in a mammal, comprising the step of administering an effective amount of a compound as described herein to a mammal. The infection may be a herpes simplex infection, and the compound may be phosphatidylacyclovir. Alternatively, the virus may be HIV retrovirus, and the compound may be 5’-palmitoylAzT.

The method includes use where the retrovirus is a strain of HIV that has developed resistance to a nucleoside analogue.

Also enabled by the present invention is a method for prolonging the antiviral effect of a nucleoside analogue in a mammal, comprising administering the nucleoside analogue to the mammal in the form of the nucleoside-lipid derivatives disclosed herein. Also enabled is a method for avoiding or overcoming resistance of the retrovirus to nucleoside analogues through administering the analogue in the form of the lipid derivative compounds disclosed herein.

The present invention enables use of compounds medicament for treatment of a human viral infection. The compositions of the invention may comprise a compound of the invention and. a pharmaceutically' acceptable carrier.

Compositions of the invention may comprise a compound of the invention and at least one other antiviral compound.

Liposomal delivery of antiretroviral and antiviral drugs results in higher dosing of macrophage and monocyte cells which take up liposomes readily. The unique advantages of the present invention are that the lipid derivatives of the antiviral nucleosides are incorporated predominantly into the phospholipid layer of the liposome rather than in the aqueous compartment. This allows larger quantities of antiviral analogue to be incorporated in liposomes than is the case when water soluble phosphate esters of the nucleosides are used. Complete incorporation of the antiviral derivative" into will be obtained, thus improving both the drug to lipid ratio and liposomes the efficiency of formulation. Further, there will be no leakage of the antiviral lipid analogues from the liposome during storage. Finally, liposomal therapy" using ‘these compounds allows larger amounts of antiviral compound to be delivered to the infected macrophage and monocyte cells.

Therapy with liposomal compounds containing site specific still compounds to be delivered with increased specificity. ligands allows greater amounts of antiviral Another novel advantage of this invention is that each class of lipid derivatives of antiviral nucleosides disclosed below is believed to give rise directly to antiviral phosphorylated or non—phosphorylated nucleosides upon cellular metabolism.

A further advantage of this invention is that. the novel lipid derivatives are incorporated into the cell, protecting the cell for prolonged periods of time, up to or exceeding 48 hours after the drug is removed.

These and other advantages and features of the present will fully following description and appended claims. invention become more apparent from the Brief Description of the Drawings Figures 1-5 are graphs plotting p24 production by HIV-infected cells as a function of the amount of the compound of the present invention administered in vitro.

Detailed Description of the Invention The present invention involves lipid derivatives of nucleoside analogues which can be incorporated into the lipid bilayer of liposomes. These derivatives are converted into nucleoside analogues by constituent cellular metabolic processes, and have antiviral effects in vivo and in vitro.

Suitable lipid derivatives of nucleoside analogues comprise phosphatidyl nucleosides, nucleoside diphosphate diacylglycerols, and ceramide phosphonucleosides. The lipid derivatives of these compounds provide one or two hydrophobic acyl groups to anchor the nucleoside in the lipid bilayer of the liposome.

The present invention also enables lipid derivatives capable of providing additional acyl groups, and hence greater anchoring strength for nucleoside analogues. The increase in anchoring strength makes it possible to utilize nucleoside analogues of greater polarity in liposome formulations. Accordingly we disclose additional nucleoside structures of this type for use in liposomal therapies.

Nomenclature: The lipid derivatives of the present invention are made up of complex structures which can only be rigorously defined by cumbersome terminology. For purposes of clarity, the descriptions of lipid and nucleosides components and their combinations will be in terms of commonly used trivial names, familiar to those in the art. t h e vie l l 3’-azido-3’—deoxythymidine, will be frequently referred to as AZT. Similarly the derivative of AZT comprising a 1,2 diacylglycerol-3—phosphate will be frequently phosphatidylAZT or pAZT. Parallel of dideoxythymidine or dideoxycytidine will For example, known drug, moiety, referred to as derivatives correspondingly be referred to as phosphatidylddT or pddT and phosphatidylddc and pddC. Derivatives of halogenated nucleosides will‘ be for phosphatidyl-3’BrddT. referred to as, example, The nucleoside analogues for use in the invention can be any nucleoside that does not occur naturally in the species to be treated for viral infection. It may comprise a naturally occurring purine or pyrimidine base attached to an analogue of a naturally occurring ribose group. It may likewise comprise an analogue of a purine or pyrimidine base attached to a ribose or deoxyribose group which is present in naturally occurring nucleosides. Alternatively, both the base and the ribose moieties of the nucleoside analogues may be analogues of those found in nature. A nucleoside analogue may also comprise either a normal base or a base analogue attached to a non-ribose sugar moiety.

Analogues of both the purine or pyrimidine base and the ribose group can differ from a corresponding naturally occurring moiety by having new substituent groups attached thereto, by having naturally occurring substituent groups deleted therefrom, or by having atoms normally present replaced by others. Examples of analogues formed by substitution are 2,6-diaminopurine and 3’-azido- ’deoxyribose; by deletion, 6—oxypurine or didehydroribose; by replacement, 8—azaguanine.

Nucleoside analogues may also comprise a purine or pyrimidine base attached to the pentose moiety in a non- such as, for example through naturally occurring linkage, the nitrogen at the 3 position rather than the 1 position of the pyrimidines.

In general, the nucleoside analogues used in preparing the liposomes of the present invention will have e.g., cytosine or thymine, or an analogue thereof, attached to a a purine or pyrimidine base, adenine, guanine, pentose, such as ribose or a ribose residue and/or derivative. The attachment is through the nitrogen in the position of the purines and through the nitrogen in the 1 position of the pyrimidines. These nitrogens are linked by a B—N~glycosyl linkage to carbon 1 of the pentose residue.

The pentose residue may be a complete pentose, or a derivative such as a deoxy- or dideoxypentose. In addition, 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).

The phosphate groups 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 antiviral activity.

It is important to recognize that in compounds having pentose residues that are not complete pentoses, the phosphate groups are connected to the carbon that would have been the 5’ carbon if the pentose were complete. In the 2' and/or 3' missing: nevertheless, they are considered to be nucleoside these pentose fragments, carbons may be derivatives within the meaning of present invention, and the carbon atom to which the phosphate groups are connected will generally be referred to herein as the 5’ carbon for purposes of consistency of usage.

The antiviral activity of the conjugates described herein may reside in any component of the lipid- nucleoside complex, that is, in a nucleoside base analogue, in a ribose analogue, or in the substitution of another pentose for ribose. It may also reside in the complex as a whole, wherein, for example, a weakly antiviral analogue or one possessing imperceptible or latent viral activity becomes more potent following its incorporation into a lipid derivative of a nucleotide.

Nucleosides known to have such activity are members of the class comprising 3’-azido-2’,3’-dideoxypyrimidine nucleosides, for example, AZT, AZT-P—AZT, AZT-P-ddA, AZT-P- ddI, AzddClU, AzddMeC, AzddMeC N4-OH, AzddMeC N4Me, AZT-P- CyE—ddA, AzddEtU(CS-85), AzddU(CS—87), AzddC(CS-91), AzddFC, AzddBrU, and AzddIU; the class comprising 3'- halopyrimidine dideoxynucleosides, for example, 3-FddClU, 3-FddU, 3-FddT, 3~FddBrU, 3-FddEtU7 the 2’,3'—didehydro-2’,3’-dideoxynucleosides (D4 D4T, D4C, D4MeC, and D4A7 the 2’,3'-unsubstituted dideoxypyrimidine -F-ddC, ddC and ddT; comprising 2’,3'—unsubstituted dideoxypurines nucleosides, for example, ddA, ddG, ddI, and ddMeA(N6 methyl); comprising substituted dideoxypurine nucleosides, for example, 3- N3ddDAPR, 3-N3ddG, 3-FddDAPR, 3-FddG, 3-FddaraA, and 3- FddA, wherein Me is methyl, Et is ethyl and CyEt is cyanoethyl. other suitable nucleotide analogues may be antiviral like (DHPG), analogues, as Preferred dideoxy and class comprising nucleosides), for example, class comprising nucleosides, for example, the class ddDAPR(diaminopurine), and the class sugar- agents acyclovir or ganciclovir Or other described below. used in the treatment of AIDS, derivatives are those ’—azido-3’—deoxythymidine (azidothymidine or (ddT); 2',3'-dideoxycytidine 2’,3’—dideoxyadenosine (ddA); 2’,3’- (ddG). AZT, ddT, and ddc are most preferred analogues at present. The didehydropyrimidines, carbocyclic 2’,3'- The 3’-azido including AZT)7 (ddc); dideoxyguanosine ’,3’-dideoxythymidine and as well as carbovir, a are also preferred.

(AZG) '—fluoro didehydroguanosine, derivatives of deoxyguanosine and the pyrimidine, deoxyuridine, and the derivatives of deoxythymidine and deoxyguanosine are preferred as well.

Among the 2’,6'-diaminopurines, the 2',3'-deoxyriboside and its 3'—fluoro and 3'-azido derivatives are preferred. Also preferred is 2-chloro-deoxyadenosine.

Among the acyclic sugar derivatives, 9-(4,-hydroxy- ’,2’-butadienyl)adenine (adenallene) and its cytosine Preferred acyclic derivatives 9—(2- and phosphonomethoxyethyl equivalent are preferred. having a purine or diaminopurine base are phosphonylmethoxyethyl)adenine deoxydiaminopurine (PMEDADP).

Stereoisomers of these nucleosides, such as 2’-fluoro- ara—ddA, may be advantageous because of their resistance to acid—cata1yzed hydrolysis of the glycosidic bond, which prolongs their antiviral activity. In such cases, they are preferred.

For treating herpes, cytomegalovirus and hepatitis B utilize the 1-(2'-deoxy—2'-fluorofi—D— arabinofuranosyl)-5—iodocytosine (FIAC) fluoro-1—fi-D—arabinofuranosyl)-5—iodouracil (FIAU). infections, one may lipid derivatives of acyclovir, ganciclovir, or 1(2’-deoxy-2'- The lipids are attached to the nucleoside analogues through phosphate linkages. Lipid derivatives comprising a phosphate link between a nucleoside analogue and lipid may be prepared from phospholipids, phosphorylated nucleoside analogs, or both. Suitable phospholipids comprise phosphoglycerides or sphingolipids.

Lipid derivatives of nucleoside analogue in which lipids are linked either through mono-, di—, or triphosphate groups may be prepared from phosphorylated nucleoside analogues. Phosphoryl ated nucleoside analogues are known. The dideoxynucleoside analogue is phosphorylated according to conventional procedures such as the phosphorous oxychloride method of Toorchen and Topal (20). The monophosphate. preferred modified analogue is the 5’- Since AZT, ddc and other dideoxynucleosides have only the 5'—hydroxyl, only the 5’-monophosphate is formed during phosphorylation; however, in other analogues in which the 3’hydroxyl is present, a 3’-monophosphate can be formed. The diphosphate and triphosphate analogues of antiviral nucleosides may also be used.

The aliphatic groups of the lipid moieties preferably have chain lengths of two to twenty-four carbon atoms and have zero to six double bonds. The aliphatic groups may be attached to the glycerol moiety by acyl, ether or vinyl ether bonds.

Synthetic Methods: The lipid—nucleotide compounds described herein can be synthesized according to general methods applicable to all lipids and all antiviral nucleosides described below, as demonstrated specifically in Examples 1 through 6.

Starting materials comprising fatty acids, alcohols, glycerides and phospholipids may be purchased from commercial Pelham, Alabama ) or may be synthesized according to known methods. suppliers (Avanti Polar Lipids, Inc., Antiviral nucleoside analogues are available from Aldrich, Milwaukee, Wisconsin or from Sigma, St. Louis, Missouri.

It is important that all traces of water he removed from the reactants in order for the coupling reactions to proceed. Therefore, the lipids are first either freeze- dried by solvent evaporation under vacuum, or in a vacuum oven over P205. The reactions are also carried out under an inert gas, such as, for example, argon.

The compounds described herein can be formed according to synthetic procedures which couple a phospholipid to a nucleoside analogue or which couple a phospholipid to a nucleoside analogue monophosphate or diphosphate, wherein the phosphate group is located on the ribose group of the nucleoside, at either the 3’ or preferably the 5’ location. coupling to nucleosides, Lipids suitable for TO B comprising primarily monoglycerides or diglycerides, ceramides 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 (32) .

Example 6, or by other known phosphorylation procedures. by treatment with phosphorus oxychloride as in In the first type of synthesis, a phospholipid, such as, for example, a phosphatidic acid, is coupled to a either the 3’ or 5’ agent, selected nucleoside analogue at hydroxyl by means of a coupling such as, for example, 2, 4, 6—triisopropylbenzenesulfonyl chloride in the presence of a basic catalyst, for example, anhydrous pyridine, at room temperature. Other coupling agents, such as dicyclohexylcarbodiimide can be used.

Lipid derivatives may also be synthesized by coupling a phosphatidic acid to an antiviral nucleoside monophosphate through a pyrophosphate bond. In this procedure, the nucleoside 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 (21) and as illustrated in Example 4, for preparing" a derivative of AZT and Example 6, for a derivative of ddA.

A coupling of the phosphatidic acid and the nucleoside phosphate morpholidate occurs on treatment of a dry mixture of the two anhydrous pyridine, at room temperature.

The followed chromatography and appropriate solvents. reactants with a basic catalyst, such as reactions are using thin layer (TLC) When the reaction, as determined by TLC is complete, the product is extracted with an organic solvent and purified by chromatography on a support suitable for lipid separation, for example, silicic acid. comprising adenine or The synthesis of products cytidine having reactive amino groups may be facilitated by N) U? blocking those groups with acetate before the coupling after the chromatography of the final product, the amino groups are reaction by treatment with acetic anhydride; unblocked using ammonium hydroxide (Example 3).

Lipid Derivatives: The compounds have a lipid portion sufficient to be able to incorporate the material in a stable way into a liposomal bilayer or other macromolecular array.

Some preferred lipid derivatives of nucleoside analogues fall into four general classes: . Antiviral Dhosphatidvlnucleosidesz The structure of these antiviral lipid compounds is shown below: where N is a "chain terminating" dideoxynucleoside such as AZT, ddC, ddA, ddI, acyclovir or ganciclovir, A is a chalcogen (O or S), and or another antiviral nucleoside such as which may be the same or different, from 0 to 6 having the structure R1 and R2, are C1 to C24 aliphatic groups, having sites of unsaturation, and CH3-(CH2)a-(CH=CH-CH2)b—(CH2)C-Y wherein the sum of a and c is from 1 to 23; and b is O to 6; and wherein Y is C(O)O‘, C-0‘, C=C—O‘, C(0)S—, C—S-, C=C—S—, forming acyl ester, ether or vinyl ether bonds, B‘ CD respectively, between the aliphatic groups and the glycerol moiety. These aliphatic groups in acyl ester linkage therefore comprise naturally occurring saturated fatty acids, such as lauric, myristic, palmitic, stearic, arachidic and lignoceric, and the naturally occurring unsaturated fatty acids palmitoleic, oleic, linoleic, linolenic and arachidonic. Preferred embodiments comprise 1-ether, 2-acyl ester embodiments, the aliphatic groups can be branched chains of the same carbon a monoester or diester, or a phosphatidyl derivative. In other number, and comprise primary or secondary alkanol or alkoxy groups, cyclopropane groups, and internal ether linkages.

This class of compounds may be prepared, for example, from the reaction of a diacylphosphatidic acid and an antiviral nucleoside analogue in pyridine as described for the preparation of 1,2 dimyristoylglycerophospho-5'-(3'- azido—3'-deoxy)thymidine in Example 1. H Upon liposomal uptake, the compounds are believed to undergo metabolism by the phospholipases present in the cell. For case of a example, in the specific diacylphosphatidyl derivative of a nucleoside, phospholipase C would act to give a diacylglycerol and the nucleoside monophosphate as shown below: I 9 " H-—c—-o-L-3.

O O c """‘O-c-—R, ".___¢._.g_.. ¢.——fl‘ o H——¢l-—-o—-|f-- A --—N H‘—C—c- O9 H 09 H H"‘C—-O—¢__" + _°G__L_ A ____N TO K} Alternatively, the same phosphatidylnucleoside may be hydrolyzed by phospholipase A and lysophospholipase followed by phosphodiesterase to give glycerol and nucleoside monophosphate by the sequence shown below: N o N . O H——c—-a——g——R, H-—c-o-Q-—R, o I] mosruoumse A u——c-—o-—c——R‘ H-C-W 9 O I u——c—-o-r-—-A --N H cga H u " N-c-—on """'"‘ f Puo;Puo)w5 __ j __ __ ,____ 'fl?€1'UH u c on fi— dp r -A N _ i ,,__C_,, oe u—c—o-—r-—A ——u H H TO CD . Antiviral nucleoside diphosphate diqlvcerides: The chemical structure of this class of compounds is shown below: H2C—R1 H —-—R2 where N, A and R1 and R2 are as described above.

Nucleoside diphosphate diglycerides are known. The antiviral nucleoside diphosphate diglycerides may be acid and the antiviral by the method of modified by Prottey and prepared from phosphatidic nucleotide monophosphomorpholidates (21) as This type of synthesis is presented in Agranoff and Suomi Hawthorne (22).

Example 4 for the synthesis of AZT 5’-diphosphate dipalmitoyl glycerol. this class of compounds will take part in several types of reactions Upon liposomal delivery to cells, since it is an analogue of CDP—dig1yceride, an important naturally—occurring intermediate in the biosynthesis of phosphatidylglycerol, cardiolipin and phosphatidylinositol as shown below:

Claims (12)

1. Use in the preparation of a composition for the treatment of a viral or retroviral infection in a mammal of a liponucleotide compound comprising a conjugate of an antiviral nucleoside analogue which does not occur naturally in 5 the mammal to be treated and which is characterized by the ability to exert antiviral activity on DNA or RNA viruses, and a lipid moiety linked to the 5' position of the nucleoside analogue ‘ ' wherein the conjugate has the formula (L)m-(W)n-A-Q?_Z 1 0 wherein I Z is the base portion of a nucleoside analogue; Q is a pentose residue or an acyclic fiagment thereof or a carbocyclic analogue; _ A is O or S; 1 5 ’ W is phosphate; n is l to 3; g and L is a lipid moiety, wherein in = l to 5; wherein L is linked directly to a W, p and wherein L is selected from - H3-C ‘ R] CH=CH'(CH2)l2‘CH3 HZC " R] I _ I I HC - R2 ; HCOH ; and HC — R2 I I ' I ' H2-C - RC(O)NH-CH_ (CH3);-N-(CH2); I ® .H2c _ 2 0 ' wherein ‘R, R, and R2 independentlyhave from 0 to 6 sites of unsaturation, and have the structure I CH3”(CHa)a'(CH=CH’CH2)b'(CH2)c‘Y wherein the sum of a and c is from 1 to 23; and b is 0 to 6; and wherein Y is —C(O)O-, 67 -C-O-, —c=c—o-, —c(o)s—, , or —c=c—s-.

2. Use according to claim 1, wherein the non-naturally occurring nucleoside component is an analogue with modification of a naturally occurring base or pentose by virtue of substitution, deletion or replacement.

3.- Use according to claims 1 or 2, wherein the pentose residue is a 2',3'-deoxy, 2',3'- dideoxy—2',3'-didehydro, azido or halo derivative of ribose, or an acyclic hydroxylated fragment of ribose.

4. Use according to claims 1 to 3, wherein the group -A-Q-Z is selected from the group consisting of 2',3'-dideoxycytidine; 2',3'-dideoxythymidine; 2',3'-dideoxyguanosine; 2',3'-dideoxyadenosine; 2',3'—dideoxyinosine; 2,6-diaminopurine-2',3'-dideoxyriboside; 2‘,3'-dideoxy-2',3'-didehydrothymidine; 2‘,3'—dideoxy-2',3'—didehydrocytidine carbocyclic; 2',3‘—dideoxy-2',3'-didehydroguanosine; 3‘-azido-3'—deoxythymidine; 3'—azido-3'-deoxyguanosine; 2,6—diaminopurine-3'-azido-2',3'-dideoxyriboside 3'-fluoro-3'-deoxythymidine; 3'—fluoro-2',3'-dideoxyguanosine; 2',3'—dideoxy-2'—fluoro-ara-adenosine; 2,6-diaminopurine-3'—fluoro—2',3'-dideoxyriboside 9-(4'-hydroxy-1',2'-butadienyl)adenine; 3-(4'-hydroxy—l',2‘-butadienyl)cytosine; 9-(2-phosphonylmethoxyethyl)adenine; 3-phosphonomethoxyethyl-2,6—diaminopurine; acyclovir or ganciclovir.

5. Use according to any one of the preceding claims, wherein the nucleotide compound is present in a liposome or is capable of forming a liposome by itself. 20 6 8

6. An antiviral or antiretroviral liponucleotide of formula (L)m°(W)1i'A'Q‘Z wherein is the base portion of a nucleoside analogue; is a pentose residue or an acyclic fragment thereof or a carbocyclic analogue; is O or S; is phosphate; is 1 to 3; is a lipid moiety; is 1 ar=g>oN wherein the lipid moiety is selected from HfC-R4 CH=CH{CHQmCH, PQC-R, I I I HC-R2 ; HCOH ;mm ~HC-R2 I I I H,c- Rc«»NH431 (CHQfN{CHgf I 69 HZC ‘ wherein R, R, and R2 independently have from 0 to 6 sites of unsaturation, and have the structure CH:-(CHz);.-(CH=CH-CH2)»-(CHz)c-Y wherein the sum of a and c is from 1 to 23; and b is O to 6; and wherein Y is -C(O)O'-, -C- O-, —C=C-O-, —C(O)S—, -C-S-, or -C=C-S-, with the proviso that the compound is in the form of a liposome, when the pentose residue is arabinofuranose and the base portion is cytosine or adenine, with the proviso that when the lipid is a 1,2-diradylglycerol, and the linkage is 3- monophosphate, the nucleoside analogue is not 5-fluoro-uridine, 5-fluorocytidine, bredinin, tubercidin, arabinosyl-cytosine, arabinosylfluorocytosine, arabinosyl fluorocytosine, arabinosyl-adenine, arabinosyl-thymine, 5-fluoro-2'-deoxyribo-uridine, and neplanocin A; with the proviso that when the lipid moiety is 1,2-diradyl or 1,3-diradylglycerol, and the linkage is monophosphate, the nucleoside is not cytidine, uridine, arabinosylcytosine, adenosine, guanosine, cyclocytidine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, deoxyuridine, or inosine; with the proviso that when the lipid moiety is 1,2-di-O-acyl glycerol, and the linkage is 3- diphosphate, the nucleoside is not cytidine, arabinosylcytosine, arabinosyladenine, 7- deazaadenosine; provided that when the lipid moiety is 1,2-di-O-alkylglycerol, and the linliage is 3—diphosphate, the nucleoside is not arabinosyl'cytosine; provided that when the lipid moiety is 1,2-di-O-acylglycerol, and the linkage is monophosphate, the nucleoside is not arabinosylcytosine; — and with the proviso that when the lipid moiety is 1-O-alky1O-acylglycerol, the sugar is ribose, 2'-deoxyribose, arabinose or 2',2'-dihydroxyribose, and the linkage is diphosphate, the nucleoside does not contain a base which is adenine, cytosine, 5-fluorouracil, 5- azacytosine, 6-mercaptopurine or 7-deazaadenine.

7. A compound according to claim 6, wherein the group A-Q-Z is selected from the nucleosides comprising 2',3’-dideoxycytidine; 2',3‘-dideoxythymidine; 2',3’-dideoxyguanosine; 2',3 '-dideoxyadenosine; 2',3‘-dideoxyinosine; 2,6—diaminopurine-2',3'—dideoxyriboside; 2',3'-dideoxy-2',3'-didehydrothymidine; 2',3'-dideoxy—2',3'-didehydrocytidine carbocyclic; 2',3'—dideoxy-2',3'-didehydroguanosine; 3‘-azido-3'-deoxythymidine; . 3 '-azido—3'-deoxyguanosine; 2,6-diarninopurine-3'~azido-2',3'-dideoxyriboside 3'-fluoro-3'-deoxythymidine; 3'-fluoro-2',3'-dideoxyguanosine; 2',3'-dideoxy—2'-fluoro—ara-adenosine; 2,6-diaminopurine—3 '-fluoro-2',3'-dideoxyriboside 9-(4'-hydroxy-1',2'—butadienyl)adenine; 3-(4'-hydroxy-1',2'-butadienyl)cytosine; 9-(2-phosphonylmethoxyethyl)adenine; '70 3-phosphonomethoxyethyl-L6-diaminopurine; acyclovir or . ganciclovir.

8. Compounds of claim 6 selected from the group consisting of phosphatidyl(3'-azido-3'-deoxy)thymidine (pAZT); phosphatidyl(2',3'-dideoxy)cytidine (pddC); phosphatidyl(2',3'-dideoxy)thymidine (pddT); (3’—azido-3'-deoxy)thymidine diphosphate diglyceride (AZTdpdg); phosphatidylacyclovir (pACV); » 1-O~stearylglycero-racphospho-5'—(3'azido,3'-deoxy)thymidine.

9. A liposome formed at least in pan from a liponucleotide compound comprising a conjugate of an antiviral nucleoside analogue which does not occur naturally in the mammal to be treated and which is characterized by the ability to exert antiviral activity on DNA or RNA viruses, and a lipid moiety linked to the 5' position of the nucleoside analogue wherein the conjugate has the formula (L)m'(W)n'A'Q'Z wherein Z is the base portion of a nucleoside analogue; Q is a pentose residue or an acyclic fragment thereof or a carbocyclic' analogue; A . is O or S; W is phosphate; n is 1 to 3; and L is a lipid moiety, wherein m = l to 5; wherein L is linked directly to a W, and wherein L is selected from H2-C - R, CH=CH-(CH2),2-CH3 HZC - R, HIC - R, ' ; Hcou ; and H|C - R2 H2-cl: — RC(O)NH-‘CH 6 (CH3)2-1l\I-(CH,_)2- . H2|C- wherein R, R, and R2 independently have from 0 to 6 sites of unsaturation, and have the structure CH3-(CH2),-(CH=CH-CH2),,—(CI-I2)c-Y wherein the sum of a and c is from 1 to 23; and b is 0 to 6; and wherein Y is -C(O)O-, -C- O-, -C=C-O-, -C(O)S-, -C-S-, or -C=C-S-.

10. A liposome formed at least in part from a liponucleotide according to the formula of claim 6.

11. A pharmaceutical composition comprising a liponucleotide compound according to claim 6 and a pharmaceutically acceptable carrier.

12. A pharamceutical composition comprising a liponucleotide compound according to claim 6 and at least one other antiviral compound together with pharrnaceutically acceptable carriers. F. R. KELLY & co, AGENTS FOR THE APPLICANTS