AU618972B2 - Phosphatidyl treatment of viral disease - Google Patents

Description

I _f *2

PCT

OPI DATE 02/05/89 AOJP DATE 15/06/89 APPLN- ID 26019 88

WORI

PCT NUMBER PCT/US88/03332 INTERNATIONAL APPLICATION VULtLl tbU UNUK IHE PAIAlbN 1 CUUPhKAT1IUN TREATY (51) International Patent Classification 4 (11) International Publication Number: WO 89/ 03220 A61K 37/00, C12N 07/00 Al (43) International Publication Date: 20 April 1989 (20.04.89) (21) International Application Number: PCT/US88/03332 (81) Design eStat s: Eu an ptent BE (Europea- atent), H'(Ef6 ean ate DE (Euro- (22) International Filing Date: 29 September 1988 (29.09.88) pean patent), DK, FI, FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), NO, SE (31) Priority Application Number: 106,615 (European patent).

(32) Priority Date: 6 October 1987 (06.10.87) Published (33) Priority Country: US With international search report.

(71) Applicant: HOUSTON BIOTECHNOLOGY INCOR- PORATED [US/US]; 3608 Research Forest Drive, The Woodlands, TX 77381 (US).

(72) Inventor: TILTON, Marti, Jett 3446 Oakwood Terrace, Washington, DC 20010 (US).

(74) Agent: ROWLAND, Bertram, Leydig, Voit Mayer, 350 Cambridge Avenue, Suite 200, Palo Alto, CA 94306 (US).

(54) Title: PHOSPHATIDYL TREATMENT OF VIRAL DISEASE (57) Abstract Phosphatides are shown to have antiviral activity, where tht phosphatides are characterized by having a polyunsaturated fatty acid at the sn2 position. By contacting cells susceptible to or infected with a virus, particularly a retrovirus, the proliferation of the virus is substantially inhibited.

4 WO 89/03220 PCT/US88/03332 -1- PHOSPHATIDYL TREATMENT OF VIRAL DISEASE This application is a continuation In Part of U.S. Application Serial No. 106,615, filed October 6, 1987.

INTRODUCTION

Technical Field The field concerns the treatment of viral *i diseases employing polyunsaturated fatty acid glycerides and derivatives thereof.

Background Viral infections are particularly difficult -to treat. In order for viruses to survive, the viruses have developed many mechanisms to elude the host defenses. The viruses may be highly polymorphic, so that the immune system cannot recognize different strains, leaving the host subject to repetitive infection by the various strains. The virus may be subject to mutation, so that the viral antigens over the course of the infection change, preventing the immune system from responding to the virus due to the varying nature of the antigenic proteins presented to the host. In cases of such viruses, the host has a difficult time to respond to the infection.

In addition, the virus is parasitic in requiring the use of the metabolism of the host cell and its enzymes for proliferation. Since the cellular mechanisms are involved with viral proliferation, it is difficult to be able to treat the host to prevent proliferation, without also affecting the host's cells. Viral drugs are frequently associated with the i I WO 89103220 PCTIUS88/03332 2 rapid rate of replication of the virus as compared to the host cell to inhibit the virus. Inevitably, the host cell also is inhibited from DNA replication, which seriously affects those rapidly multiplying host cells, such as the hematopoietic system.

A further difficulty with protection against viral diseases is that the virus spends a substantial portion of its lifetime within the cell, to some degree protected from the host immune system. While the host has developed the ability to recognize viral proteins on the cell surface and kill virus invaded cells, the ability of the virus to remain in the cell and proliferate prior to cellular death, enhances the viral capability to maintain an infectious state.

It is therefore of substantial interest to be able to develop therapeutic treatments which enhance the host's ability to respond to a viral infection.

Relevant Literature Jett et al., Biochem. Biophys. Res. Commun.

(1983) 114:863-871, incorporated herein by reference, discloses that liposomes containing plant phosphatidyl 4 inositol cholesterol and cholesteryl oleate selectively killed tumor cells from cultured cell lines without harming the normal cells present. Certain synthetic phosphatidyl inositols were found to be equally effective as the plant PI. Jett et al., Cancer Research (1985) 45:4810-4815..

SUMMARY OF THE INVENTION Prophylactic or therapeutic amounts of phosphatides or phosphatidyl derivatives are administered to a host for viral treatment. The phosphatides are characterized by being triesters, having carboxylates at the snl and sn2 positions, where the carboxylate at the sn2 position is at least about carbon atoms and has at least 2 sites of aliphatic 3 unsaturation, where the sn3 position has a phosphate, which phosphate may be substituted or unsubstituted on oxygen. The compounds are effective against viral diseases when utilized either as pure compounds or as naturally occurring mixtures.

According to a first embodiment of this invention, there is provided a method for inhibiting proliferation of a virus in a host cell, said method comprising: contacting said host cell with a viral proliferation inhibiting amount of a diacyl phosphatide, wherein the acyl group at the sn2 position is polyunsaturated.

According to a second embodiment of this invention, there is provided a method for inhibiting proliferation of a virus in a host cell, said method comprising: contacting said host cell with a viral proliferation inhibiting 15 amount of a composition comprising at least one diacyl phosphatide of the S formula: 0 -0-P-OT RCO CH OV 2 2 c 202CR 2 wherein: T is a group which does not interfere with phospholipase A 2 activity; V is a hydrogen or physiologically acceptable counterion;

R

1

CO

2 is a polyunsaturated fatty acid group of at least carbon atoms; and

SR

2 CO2 is an aliphatic fatty acid group of at least 2 carbon atoms.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS Mammalian hosts are treated with a prophylactic or therapeutic dose of a phosphatide to prevent the proliferation of virus in the host. The phosphatide is characterized by having at least 17 carbon atoms and usually fewer than 60 carbon atoms, more usually fewer than about carbon atoms, where the snl and sn2 positions are substituted with carboxylic acids, the sn2 position having a carboxylic acid having at least 2 aliphatic sites of unsaturation, usually olefinic sites.

3A For the most part, the phosphatides will be phospholipase A 2 substrates and have the following formula: 0

CH

2

-O-P-OT

'1 l R CO 2 CH OV C 02 2 wherein: T is hydrogen, physiologically acceptable counterion, e.g. salt, such as sodium, potassium, calcium, etc., inositol, any of its phosphate derivatives, mono-, di- or tri-, including cyclic phosphate, choline, i serine, ethanolamine, glycerol, or other group which does not interfere with phospholipase A 2 activity; S 10 V is hydrogen or a physiologically acceptable counterion; i :-i

I

1/536Z I- 7 'WO 89/03220 PCT/US88/03332 4 RIC0 2 is a polyunsaturated aliphatic fatty acid group of at least about 10 carbon atoms, preferably at least about 12 carbon atoms, usually not more than about 36 carbon atoms, more usually not more than about 28 carbon atoms, having at least 2 sites of aliphatic unsaturation and may have 6 or more, usually not more than 5, more usually not more than 4, generally from 2 to 4 sites of aliphatic unsaturation;

R

2

CO

2 is a fatty acid group of at least 2 carbon atoms and not more than about 36 carbon atoms, generally ranging from about 2, usually 12 to 24 carbon atoms, and may be saturated or unsaturated, having from 0 to 5, usually 0 to 4, more usually 0 to 3 sites, generally 0 to 1 of aliphatic unsaturation.

The aliphatic unsaturation may be at any site in the fatty acid and may be ethylenic or acetylenic, conjugated or unconjugated, preferably conjugated, naturally occurring or synthetic. There may be from 0 to 2, usually 0 to 1 substituents, particularly oxy, e.g. hydroxyl. The chains may be branched or straight chain, usually straight. For the unsaturated acids, unsaturated acids include linoleic, a- or y-linolenic, arachidonic, geranic, 9,11-octadecatrienoic acid, dehydrogeranic acid, elaeostearic acid, 6,9,12octadecatrienoic, stearidonic, clupanadonic, etc. For the saturated or mono-unsaturated fatty acids, the acids include acetic, propionic, butyric, lauric, myristic, palmitic, palmitoleic, oleic, stearic, etc.

The subject compound may be a single compos3tion or a mixture of compounds, as to the lipids at the 1 and 2 positions (phosphate being the 3 position), or as to the substituent bound to phosphate. Conveniently, naturally occurring compositions may be employed such as plant compositions, such as soybean, rape seed, safflower, corn, sunflower, etc., as the inositols, glycerols, cholines, etc. The naturally occurring compositions may be extracted, purified, i i MOo 89/03220 PCT/US88/03332 fractionated, or subjected to other treatment.

The compounds of the present invention vary widely. Specific compounds of interest include 1palmitoyl-2-linoleoyl-phosphatidylcholine; 1-palmitoyl- 2-linoleoyl-phosphatidylinositol; l-palmitoyl-2linolenoyl-phosphatidylinositol; l-palmitoyl-2linoleoyl-phosphatidylethanolamine; l-acetyl-2arachidonyl-phosphatidylcholine; l-butyryl-2epicosatetraenoyl-phosphatidylinositol-4-phosphate; 1lauryl-2-linoleoyl-phosphatidylethanolamine; and the like. Preferred compounds are of the natural Lenantiomer, however, the unnatural D-enantiomer, or racemic mixtures, may find use.

While particular polar groups are preferred, for example inositol or choline, the polar head group identity is much less important than the requirement for unsaturation at the sn2 position. Thus unsaturation at the sn2 position is essential for activity of the compound. Variations in the polar head groups can be tolerated as long as the sn2 position remains unsaturated.

The compounds of interest can be used as crude components or pure compounds. Crude components include naturally occurring phosphatide mixtures which may be obtained from any source, including plant glycerides, fish glycerides or combinations thereof. Of particular interest is the naturally occurring phosphatide mixture from plants, preferably soybeans.

Pure compounds can also be utilized. By pure compound is intended the purified phosphatide of interest, from a naturally occurring phosphatide mixture, or a pure compound prepared synthetically. An analysis of pure l-palmitoyl-2-linoleoylphosphatidylinositol from soybean, which has been demonstrated to be particularly effective against virus proliferation, is given in Table 1.

The subject compositions, whether crude or 4"-IC WO 89/03220 PCT/US88/03332 6 pure, can be utilized singularly or in combination with other pure compounds or crude mixtures of compounds.

For example, 1-palmitoyl-2-linoleoyl-phosphatidylinositol inhibits viral diseases when utilized either as a pure compound or as the naturally occurring mixture from soybeans.

The subject compositions may be used by themselves or in conjunction with other fatty acids, particularly when prepared as vesicles or liposomes.

Where prepared as liposomes, the subject compositions will be at least about 5 mole percent, more usually at least about 25 mole percent and preferably in the range of about 25 to 90 mole percent, more preferably in the range of about 40 to 75 mole percent. Desirably, the subject compositions should be free of phosphatides which have a saturated or mono-olefinic carboxylic acid at the sn2 position. The liposomes may be made in accordance with conventional ways, by combining the various lipids, evaporating solvent, suspending the dried lipid in an aqueous medium, and subjecting the medium to rapid agitation, for example, by ultrasonic sound. Other lipids which may be used include cholesterol, polyunsaturated fatty acid esters, e.g.

methyl esters, etc. The resulting vesicles may then be isolated and used. See, for example, Kim and Martin, Biochem. et Biophys, Acta (1981) 646:1-9 and U.S.

Patent Nos. 4,311,712; 4,310,506; 4,302,459; 4,261,975; 4,241,046; 4,235,871; 4,229,360; 4,224,179; 4,053,385; 4,016,290 and 3,957,971.

The amount of the nonphosphatidyl lipid component of the liposome can vary widely. Although the amount is not critical, some nonphosphatidyl lipid content is beneficial as it prevents excessive toxicity to uninfected cells and may serve to stabilize the liposome. Nonphosphatidyl lipids include steroid compounds commonly used for liposome preparation including cholesterol, polyunsaturated fatty acid I i I W;O 89/03220 PCT/US88/03332 7 esters and the like. Best results are observed with drug to cholesterol ratios of about 1:0.1 to about 1:1.5, preferably with ratios of about 1:0.3 to about 1:1.

If desired, the liposomes may be modified to direct the liposomes to particular types of cells using site directing molecules. Thus, specific binding members, receptors such as antibodies or ligands, for particular receptors may be employed, where the target cell is associated with a particular surface protein. For example, with the AIDS virus, the AIDS virus is primarily directed to cells having the CD4 surface protein. By having anti-CD4 bound to the surface of the liposome, the liposome may be directed primarily to T-helper cells.

The particular ligand or antibody may be conjugated to the liposome in accordance with conventional ways, either by conjugating the site directing molecule to a lipid for incorporation into the lipid bilayer or by providing for a linking group on a lipid present in the bilayer for linking to a functionality of the site directing compound.

The subject compositions may be formulated in a wide variety of ways, employing physiologically acceptable media, such as deionized water, phosphate buffered saline, balanced salt solution, Ringers solution or other appropriate solution. The amount of the active phosphatide may vary widely in the medium, generally ranging from about 100 mg/ml to 1 mg/ml.

Depending upon the manner of administration, the Sfrequency, the nature of the target, and the like, usually from about 0.5 mg to 800 mg of the phosphatide will be used per kg of host, inversely related to the weight of the host. Other compositions may be present, such as stabilizers, buffers to provide a physiological pH, e.g. 6-8.5, or the like.

The subject compositions may be used for '7w 0 89/03220 PCT/US88/03332 treatment of a variety of viral diseases, such as treatment of lenti viruses, e.g. HIV, papilloma, influenza, herpes, or the like.

The subject compositions may be used by themselves or in conjunction with other drugs for the treatment of viral diseases. Where the subject compositions have been prepared as liposomes, other drugs may be included in the lumen of the liposome, so as to be delivered at the same time to the cell and may be subject to endocytosis. Drugs of interest include cytotoxic drugs, drugs inhibiting DNA replication, etc. Various drugs which may find use include methotrexate, y-interferon, azidothymidine, azidocytidine, vinca alkaloids, adriamycin, dideoxyadenosine or derivatives thereof, 5-fluorouracil, ribovirin, acyclovir, and cytokines. These drugs will be used at concentrations in accordance with normal administration or in smaller amounts where the combined effect of the phosphatides and ancillary drug provide the therapeutic result with lesser amounts.

The subject compounds are found to be effective in inhibiting the proliferation of viruses intracellularly, while having little or no effect on normal cells. Thus, the subject compounds may be administered systemically by injection into the vascular system, parenterally, by inhalation, orally, or the like. The particular manner of administration may vary with the site of the virus, whether the administration is prophylactic or therapeutic, the dosage level, or the like.

The subject compositions may also be used in vitro to inhibit viral infection in cell cultures, compare activities of viruses, elucidate the mechanism of viral replication and the use of cellular processes, and the like.

The following examples are offered by way of illustration and not by way of limitation.

WO 89/03220 PCT/US8/03332 9

EXPERIMENTAL

In order to establish the effect of plant phosphatidyl esters as inhibitors of viral growth, the following study was carried out. Samples of the druls to be tested were formulated into liposomes and used in tests on the same day as they were prepared. The efficacy of each sample was determined by its ability to inhibit the replication of the HIV virus in infected H9 cells. Both the viability of the cells and the production of p24 antigen (a measure of viral concentration) were assayed.

The following are the procedures and materials employed.

Liposome preparation Using sterile technique, the phosphatidyl ester solution (1 ml, 9.1 mg, 12 pM) in chloroform (Aldrich, HPLC grade) was transferred to a serum bottle, and to it was added 1 ml of a 6 uM solution of cholesterol (recrystallized, CalbioChem), in chloroform. Prior to use, the phosphatidyl ester was stored in a manner that would prevent oxidation. The solvent was removed under reduced pressure at 40 0 C, and the bottle was stored in a vacuum desiccator overnight. The next day, the film adhering to the walls of the bottle was resuspended in 2 ml RPMI 1640 medium (GIBCO, without serum) by intermittent vortexing at room temperature. The typical cloudy suspension formed. The preparation was sealed under nitrogen and used for testing.

Propagation of HIV-infected Cell Line The cell line which was utilized can be described as a cloned T-cell population H9, derived from the HT cell line, with the same clone persistently infected with HIV isolate HTLV-IIIB2 (H9/HIV). Cells WO 89/03220 PCT/US88/03332 were cultured in RPMI 1640 medium (GIBCO) supplemented with 10% decomplemented fetal bovine serum and 10 mM Lglutamine (complete medium). The H9/HIV cells were morphologically indistinguishable from uninfected cells when examined by light microscopy. Virus production by the H9/HIV cells was monitored by electron microscopy, and a standard reverse transcriptase assay was performed at weekly intervals. Periodic "feeding" of H9/HIV cells with uninfected H9 cells was performed when viral production dropped.

p24 Antigen Assay Each assay was done in microtiter travs with a fixed concentration of H9/HIV cells in each well to which were added dilutions of the test samples. After four days incubation, the p24 antigen levels were measured using a Dupont ELISA kit. Each test sample was run in quadruplicate. The resulting four optical density readings for each sample were converted into ng/ml of p24 antigen produced by comparison with the standard curve generated from known concentrations provided by Dupont. The values in ng/ml were then averaged and a standard deviation determined. Drugfree controls were run with each assay.

Proliferation Assay The proliferation of both infected and noninfected H9 cells in the presence of the samples was determined. The assays were done in quadruplicate in microtiter trays with a fixed concentration of H9 or H9/HIV cells in each well to which were added dilutions of each test sample. Cell proliferation was determined by incorporation of 3 H]-thymidine and was reported as counts per minute (cpm SD). Drug-free controls were run with each assay.

WO 89/03220 PCT/US88/03332 11

RESULTS

The results, in general, indicated that the polar head group of the phosphatidyl derivative was not as important as the requirement for unsaturation at the sn2 position and that crude, or naturally occurring mixtures, as well as pure phosphatidyl compounds were effective against viral proliferation. Pure soybean phosphatidylinositol was demonstrated to be a potent inhibitor of viral proliferation (Table The crude phosphatidylinositol mixture was also shown to be effective against viral diseases, although it was not as effective as pure phosphatidylinositol. Table 3 gives the results of crude phosphatidylinositol and components of the crude mixture on HIV proliferation.

Additionally, phosphatidylcholine was also demonstrated to be an inhibitor of viral proliferation (Table 4).

Table 5 indicates that while some cholesterol in the liposome preparation is beneficial, the amount of cholesterol in the preparation is not critical. The results show that best results are seen at drug to cholesterol ratios of about 1:0.3 to about 1:1.

WO 89/03220 PCr/US88/03332 12 Table 1 FATTY ACID DISTRIBUTION Acid Residue commosition Palmitoyl 16:0 31.6 Palmitoleoyl 16:1 Stearoyl 18:0 10.9 Oleoyl 18:1 5.7 Linoleoyl 18:2 45.6 Linolenoyl 18:3 5.7 Table 2 PURIE SOYBEAN PHOSPHATIDYLINOSITOL 2:1 WITH CHOLESTEROL Code Drug Conc. (UM) Amount of p 24 Produced ng /ml Normalized to 1 00 -p 24 ng/ml H IV- infected H9 Cells Non-infected H9 Cells Proliferation (cpm SD) 191-21 192-37 Control 100 Control 50 6 Control 50 12 6 81.0 19.1 15.5 26.5 115.5 5 7.0 2 24.5 4 69.5 6 93.0 3 96.0 11 10.0 3 29.0 4 51.0 3 84.0 5 100.0 4.3 6.1 1.7 21.2 3.5 60.2 5.2 80.5 2.6 100.0 11.5 10.4 3.1 30.2 4.2 53.1 3.1 87.5 5.2 100 23.6 19.1 32.7 188,125 101,698 129,246 123,782 79,144 1,294 15,913 78,494 102,241 76,074 592 25,548 59,541 74,317 3,100 9,662 4,155 14,366 2,254 144 1,373 2,014 2,356 3,311 228 892 459 971 188,690± 88,041± 112,828± 114,374± 253,171± 216,344± 225,557± 234,470± 262,268± 13,541 i, 3, 513w 9,402 17,755 17,106 3,313 8,906 7,349 7,054 19 2-4 2 243,935 10,649 165,348 20,190 189,313 12,466 218,347 14,922 246, 637 17,653 Each set of dilutions represents one during each experiment.

experiment; each data point is generated in quadruplicate 00 wc i i :-i Table 3 COMPONENTS OF CRUDE PI Code Identity Control Drug Cone. (pM) Amount of p24 Produced ng/ml Proliferation (cpm SD) HIV-infected Non-infected H9 Cells H9 Cells 115.5 5 192-36 Crude soybean PI 12.5 50.5 70.0 89.5 7.0 24.5 69.5 93.0 79,144 1,272 52,362 88,231 90,655 1,294 15,913 78,494 102,241 192-37 Pure soybean PI 192-39 Soybean Phosphatidic acid 192-41 1 PE: 1 PI: 1 CH 2,254 617 976 2,162 2,078 144 1,373 2,014 2,356 1,378 3,735 3,452 2,841 333 620 2,522 442 194,490 212,467 240,298 245,979 216,344 225,557 234,470 262,268 190,927 209,715 257,829 258,533 135,818 156,417 212,454 241,950 10,991 13,315 15,314 20,207 3,513, 9,402 17,755' 17,106 7,180 15,529 9,690 7,220 9,136 11,273 16,012' 12,606 253,171 13,541 29.0 89.0 100.0 109.0 11.0 29.0 80.0 100.0 1 15 16 7 4 3 6 14 24,956 81,373 82,335 77,736 487 5,028 53,183 67,399 00 00 Is)I

I

Table 3 Continued Code Identity Drug Cone. (pM) Amount of p 24 Produced ng/ml HIV-infected H9 Cells Proliferation (cpm SD) Non-infected H9 Cells 192-38 Cholesterol: PI 2:1 87.0 7 111.0 13 115.5 15 105.5 3 65,195 4,700 81,014 1,117 85,341 3,178 83,119 2,039 239,205 19,694 248,959 13,820 254,910 13,832 292,269 18,697 Unless otherwise indicated, samples are 2 PL: 1 CH PI phosphatidylinositol PE l-palmitoyl-2-linoleoyl phosphatidylethanolamine CH cholesterol PL phospholipid Each set of dilutions represents one experiment; each data point is generated in quadruplicate during each experiment

I

Table 4 PHOSPHATIDYLCHOLINE ACTIVITY Code Identity Control Drug Conc. (pM) Amount of p 24 Produced Proliferation (cpm SD) HIV-infected H9 Cells 79,144 2,254 Non-infected H9 Cells 253,171 13,541 115.5 5 192-40 Dipalmitoyl PC 115.5 117.0 118.0 129.0 76,066 78,185 74,064 74,261 3,461 1,341 913 3,362 261,731 248,777 250,747 251,794 14,253 7,233 8,032 10,033 Control 96.0 11 76,074 3,311 243,935 192-46 192-47 l-Palmitoyl-2- Linoleoyl PC Soybean PC 15.0 34.0 51.0 78.0 14.5 43.0 54.0 64.0 81.0 12.8 21.5 25.5 1,290 18,054 51,151 60,497 377 2,287 1,324 3,646 121,628 209,071 219,385 225,583 238,055 235,182 222,164 158,960 1,269 149 26,730 1,366 66,095 2,773 188,125 3,100 32,316 742 118,313 6,956 169,602 11,025 10,649 3,181 10,679 14,129 5,661 3,400 10,506 12,535 7,001 3,313 718- 9,678 4,386 Control l-Palmitoyl-2- Linoleoyl PC 188,690 192-24 100 50 40,940 104,337 152,019

I

Table 4 Continued Code Identity Control Drug Conc. (pM) Amount of p 2 4 Produced ng/ml HIV-infected H9 Cells 72,626 3,089 Proliferation (cpm SD) Non-infected H9 Cells 215-1 l-Palmitoyl-2linoleoyl PC 56.0 1 12.0 4 25.0 2 41.0 3 43.0 5 855 24,800 57,696 62,653 265 307 1,833 3,547 367,072 27,502 166,025 12,544 263,458 31,364 310,665 40,392 271,755 17,738 All samples, 2 PC: 1 cholesterol PC phosphatidylcholine Each set of dilutions represents one experiment; each data point is generated in quadruplicate during each experiment Table EFFECT OF CHOLESTEROL ON PHOSPHATIDYLINOSITOL ACTIVITY Code Identity Control PI: no CH Drug Conc. (vM) Amount of p24 Produced ng/ml Proliferation (cpm SD) HIV-infected Non-infected H19 Cells H9 Cells 192-45 192-42 2 PI: 1 CH 96.0 11 30.0 2 53.0 2 67.0 5 85.0 8 10.0 3 29.0 4 51.0 3 84.0 5 6.0 3 26.0 6 54.0 8 69.0 13 76,074 3,311 23,090 1,273 54,215 1,367 69,304 3,518 6 2, 174 959 592 25,548 59,541 74,317 1,068 10,394 46,755 66,962 243,935 10,649 228 892 459 971 139,123 152,742 211,469 183,759 165,348 189,313 218,347 246,637 189,166 22,552 182,475 243,933 13,007 8,962 9,869 14,312 20,190 12,466 14,922 17,653 7,271 141893 81819 15,258 19 2-4 3 2 PI: 1.5 CH 297 1,230 2,684 1,625 00i 00 w0 0~ w m q i Table 5 Continued Code Identity 2 PI: 2 CH Drug Cone. (pM) Amount of p24 Produced ng/ml 192-44 Proliferation (cpm SD) 7.5 30.0 60.0 80.0 HIV-infected H9 Cells 423 45 7,340 992 47,491 4,117 64,082 4,027 Non-infected H9 Cells 182,931 16,349 174,769 33,954 163,623 6,189 266,983 20,679 PI purified soybean phosphatidylinositol CH cholesterol Each set of dilutions represents one experiment; each data point is generated in quadruplicate during each experiment WO 89/03220 PCT/US88/03332 It is evident from the above results that by employing polyunsaturated carboxylic acid containing phosphatides, where the sn2 fatty acid is polyunsaturated, viral infection can be substantially inhibited.

The above results demonstrate that in the presence of the subject phosphatides, the retrovirus HIV is specifically inhibited. The work demonstrates that both crude and pure components can be utilized to inhibit virus proliferation. Further, the phosphatide composition must be unsaturated at the sn2 position for activity. The subject invention provides an important adjunct in the treatment, both prophylactic and therapeutic, of viral infections, particularly retroviral infections.

All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (21)

1. A method for inhibiting proliferation of a virus in a host cell, said method comprising: contacting said host cell with a viral proliferation inhibiting amount of a diacyl phosphatide, wherein the acyl group at the sn2 position is polyunsaturated.

2. A method according to Claim 1, wherein said virus is a retrovirus.

3. A method according to Claim 1, wherein said phosphatide is a plant phosphatide composition.

4. A method according to Claim 3, wherein said phosphatide is l-palmitoyl-2-linoleoyl phosphatidylinositol.

5. A method according to Claim 1, wherein said phosphatide is 1-palmitoyl-2-linoleoyl phosphatidylcholine.

6. A method for inhibiting proliferation of a virus in a host cell, said method comprising: contacting said host cell with a viral proliferation inhibiting amount of a composition comprising at least one diacyl phosphatide of the formula: 0 CH 2 -O-P-OT R CO2CH OV CH202CR2 wherein: T is a group which does not interfere with _XII WO 89/03220 Pcr/us88/03332 22 phospholipase A 2 activity; V is a hydrogen or physiologically acceptable counterion; RICO 2 is a polyunsaturated fatty acid group of at least abet- 10 carbon atoms; and R 2 C0 2 is an aliphatic fatty acid group of at least 2 carbon atoms.

7. A method according to Claim 6, wherein said composition further comprises a nonphosphatidyl lipid component wherein said phosphatide to nonphosphatidyl lipid component is provided at ratios of ab-et 1:0.1 to about 1:1.5.

8. A method according to Claim 7, wherein said nonphosphatidyl lipid component is selected from cholesterol and polyunsaturated fatty acid esters.

9. A method according to Claim 8, wherein said nonphosphatidyl lipid component is cholesterol.

A method according to Claim 9, wherein T is hydrogen, a physiologically acceptable counterion, inositol or phosphorylated derivatives thereof, choline, serine, ethanolamine, or glycerol.

11. A method according to Claim 9, wherein RICO 2 is of from 12 to 28 carbon atoms having from 2 to 4 sites of unsaturation.

12. A method according to Claim 11, wherein R 1 C0 2 is linoleoyl.

13. A method according to Claim 10, wherein R 2 CO 2 is of from 12 to 24 carbon atoms.

14. A method according to Claim 10, wherein T 23 is inositol or phosphorylated derivatives thereof.

A method according to Claim 10, wherein R CO 2 is linoleic acid, R2CO2 is palmitic acid, and T is selected from choline and inositol.

16. A method according to Claim 15, wherein T is inositol.

17. A method according to Claim 15, wherein T is choline.

18. A method according to any one of Claims 10 to 17, wherein said composition comprises vesicles, wherein said diacyl phosphatides are incorporated into the lipid layer of said vesicle. 10

19. A method according to Claim 18, wherein each of said vesicles comprise a cytotoxic drug.

20. A method according to Claim 18 or Claim 19, wherein each of said vesicles comprise a binding member specific for a surface membrane protein, which member is bound to the surface of each of said vesicles. 15

21. A method for inhibiting proliferation of a virus in a host S* cell, said method comprising: contacting said host cell with a composition containing an amount effective as a virus proliferation inhibitor for said host cell of a diacyl phosphatide as defined in Claim 1, which composition is substantially as hereinbefore described with reference to any one of the liposome preparations of Tables 2 to DATED this FOURTEENTH day of OCTOBER 1991 Houston Biotechnology Incorporated Patent Attorneys for the Applicant SPRUSON FERGUSON LMM/536Z INTERNATIONAL SEARCH REPORT International Application No.pCT/US 8 8 0 3 3 3 2 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, indicate all) 6 According to Inlernational Patent Classifcation (IPC) or to both National Classification and IPC IPC(4): A61K 37/00C12N 07/00 U.S. CL.: 435/240.2 435/235 I i II. FIELDS SEARCHED Minimum Documentation Searched 7 Classification System Classification Symbols U.S. 435/240.2,435/235 424/450 436/829 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included in the Fields Searched a Computer Searched CAS 1967-1988, Biosis 1969-1988, APS 1975-

1988. Phosphatidylinositol, phosphatidylcholine, phosphatidy- ethanolamine, microemulsion,liposome.vesiclevirus or viral. III. DOCUMENTS CONSIDERED TO BE RELEVANT 9 Category Citation of Document, 11 with indication, where appropriate, of the relevant passages 12 Relevant to Claim No. 13 Y CELL, 32, 1983 Boston, USA, 1-38 "Inhibition of VSV Binding and Infectivity by Phosphatidylserine: Is Phosphatidylserine a VSV-binding site", pages 639-646. Y JOURNAL OF IMMUNOLOGY, 136, 1986, 1-38 Baltimore, MD USA, "Targeting of Drug Loaded Immunoliposomes of Herpes Simplex Virus Infected Corneal Cells: An Erfective means of Inhibiting virus Replication In Vitro", pages 681-685. Y BIOCHEMICAL and BIOPHYSICAL RESEARCH 1-38 COMMUNICATIONS, 114, 1983. Duluth, MN, USA, "Selective Cytotoxicity of Tumor Cells Induced by Liposomes Containing Plant Phosphatidylserine", pages 863-871. SSpecial categories of cited documents: o later document published after the international filing datl document defining the general state of the art which is not or priority date and not in conflict ,ith the application but considered to be of particular relevance cited to understand the principle or theory underlying the conidered to e particular relevance invention earlier document but published on or after the international document of particular relevance: the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which is cited to establish the publication date of another document of particular relevance; the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the international filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 08 December 1988 0 5 JAN 1989 International Searching Authority Sign re ofAut oized O ffer ISA/US ER Form PCT/ISA/210 (cand shee) (Rev.11-87)