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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
• • 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
• • 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
• • 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
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV

Definitions

• the present invention relates generally to the treatment of viral infections with nucleoside analogues. More particularly, the present invention relates to the encapsulation of modified antiviral nucleoside analogues in liposomes to enhance the effectiveness of the analogues when administered to mammals.
• nucleoside analogues are designed to inhibit viral functions by preventing the synthesis of new DNA by viral reverse transcriptase during viral replication.
• Nucleosides are the precursors of DNA or RNA.
• a nucleoside consists of a pyrimidine or purine base which is linked to a five-carbon sugar.
• nucleoside triphosphates 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.
• antiviral nucleoside analogues have strategically located differences in chemical structure which inhibit the viral enzyme reverse transcriptase or which prevent further DNA synthesis once the analogue has been attached to the growing DNA chain.
• Azidothymine (AZT) dideoxycytidine
• dideoxyadenosine dideoxyadenosine
• acyclovir dideoxyadenosine
• ribavirin vidarabine
• AIDS Acquired immune deficiency syndrome
• HAV human immune deficiency virus
• AZT Dideoxynucleoside analogues such as AZT are the most potent agents currently known, but in a recent human trial, serious toxicity was noted consisting of anemia (24%) and granulocytopenia (16%) (37, 38).
• CD4 CD4
• helper lymphocytes CD4 helper lymphocytes
• CD4 monocytes and macrophages CD4 monocytes and macrophages.
• the infection of C34 lymphocytes (3-5) results in a cytoiytic infection and contributes to the progressive immunodeficiency of HIV infection (6, 7).
• CD4 monocyte/macrophages have been shown to be infected in vitro (8, 9), and in vivo (10-15). Infection of monocyte/macrophages may also contribute to immunodeficiency and to the pathogenesis of HIV induced encephalopathy.
• these cells may serve as a reservoir for the virus because HIV replication in monocyte/macrophages appears to be more prolonged and less cytolytic than in lymphocytes. (8, 9, 13).
• liposomes which are nearing the clinical arena (22).
• liposomal antimonial drugs are several hundred fold more effective than the free drug in treating leishmaniasis as shown independently by Black and Watson (23) and Alving et al (24).
• Liposome-entrapped amphotericin B appears to be more effective than the free drug in treating immunosuppressed patients with systemic fungal disease (25, 26).
• Other uses for liposome encapsulation include restriction of doxorubicin toxicity (27) and diminution of aminoglycoside toxicity (22).
• nucleoside analogues such as iododeoxyuridine (IUDR), acylovir (ACV) and ribavirin into liposomes for treating diseases other than AIDS (16, 17).
• IUDR iododeoxyuridine
• ACV acylovir
• ribavirin ribavirin
• a new treatment for AIDS in which antiviral nucleoside analogues are trapped within liposomes in a manner which prevents or substantially reduces leakage.
• This reduction in leakage provides reduced toxicity and the use of liposomes ensures that a greater amount of the antiviral nucleoside analogue reaches the macrophages to thereby increase the effectiveness of these drugs against the HIV infection present in such cells.
• the analogue of the neucleoside encapsulated in liposomes further enhances the antiviral effect of the formulation by providing a prephosphorylated antivral nucleoside, bypassing an enzymatically deficient step in macrophages which would otherwise greatly limit the antiviral activity of the drug itself (43).
• the present invention is based on the discovery that leakage of nucleoside analogues from liposomes is greatly reduced if the analogues are converted to phosphate derivatives prior to encapsulation in liposomes. Conversion of the nucleoside analogues to their respective phosphate derivatives is believed not only to prevent leaking of the analogues from the liposomes, but also to increase the effectiveness of the analogues against HIV-infected macrophage cells. As previously mentioned, nucleosides are phosphorylated to the triphosphate form prior to attachment to the growing DNA or RNA chain. Macrophages have been found to have diminished deoxynucleoside kinase activities and a reduced ability to phosphorylate nucleosides.
• free nucleoside analogues i.e., non-phosphorylated analogues
• AZT, ddC or ddA are not particularly effective against HIV present in macrophage cells (43).
• the phosphorylated analogues of the present invention are more effective when presented by the lipsomal delivery system because they by-pass the metabolic block caused by the lack of deoxynucleoside kinase activity in macrophages.
• the present invention basically involves the encapsulation of phosphorylated antiviral nucleoside analogues within liposomes for use in treating viral infections.
• this invention has applications to a wide variety of nucleoside analogues which may be used to treat various viral infections, the following description will be in the context of the treatment of patients suffering from Acquired Immune Deficiency
• AIDS AIDS-related complex
• ARC AIDS-related complex
• HTLV-1 or HTLV-2 HTLV-1 or HTLV-2.
• HIV Human immunodeficiency virus
• HTLV-III/LAV Human immunodeficiency virus
• HIV infects the CD4 (T4) helper lymphocyte resulting in the death of these cells.
• T4 helper lymphocytes depletion of CD4 helper lymphocytes makes the host vulnerable to certain well described opportunistic infections and malignancies. HIV binds to the CD4 receptor of lymphocytes by forming a complex between the 110K viral surface glycoprotein (gp110) and the CD4 antigen (32, 32). Subsequently, the virus is thought to enter the cell by endocytosis as suggested by the finding that productive infection is blocked by NH 4 Cl and amantadine pretreatment of the cells.
• gp110 110K viral surface glycoprotein
• 32, 32 the CD4 antigen
• HIV cells other than the CD4 helper lymphocyte become infected with HIV (28, 29, 30).
• the virus has been shown to replicate in B cells, promyelocytes and monocytes. It also has been shown that mononuclear phagocytes isolated from brain and lung harbored HIV and normal peripheral blood macrophages produced large quantities of virus. Furthermore, human alveolar macrophages and brain macrophages harbor HIV and the viral cytopathic effects on these cells are much less than that observed in HIV- infected helper T4 lymphocytes. It has been proposed that HIV-infected macrophages and monocytes may serve as a reservoir for virus and that this may be a mechanism for viral persistence and dissemination in the infected host (29, 30).
• the present invention utilizes the affinity of macrophages for liposomes as a vehicle for directing liposome encapsulated antiviral nucleoside analogues at macrophages, monocytes and any other infected cells which may take up liposomes. Further, phosphorylation of the nucleoside analogue prior to encapsulation provides advantages in that: 1) the nucleoside analogue is prevented from leaking out of the liposome; and 2) the metabolic block associated with the inability of macrophages to phosphorylate free nucleosides is overcome.
• liposomal encapsulation of the phosphorylated nucleoside is essential to prevent hydrolysis of the phosphate ester by plasma enzymes such as alkaline phosphatase, phosphodiesterases or 5'-nucleotidases.
• the nucleoside analogues can be any of the known analogues used for treating AIDS including 3'-azido-3'-deoxythymidine (azidothymidine or AZT), 2 ', 3 '-dideoxycytidine (dideoxycytidine or ddC), 2'3'-dideoxyadenosine (dideoxyadenosine or ddA), ribavirin or any other suitable dideoxynucleoside analogue.
• AZT is a preferred analogue.
• the analogue is phosphorylated according to conventional procedures such as the phosphorous oxychloride method of Toorchen and Topal (34).
• the preferred modified analogue is the 5'-monophosphate. Since AZT and other dideoxynucleosides have only the 5'-hydroxyl, only the 5'-monophosphate is formed during phosphorylation. Alternatively, the nucleoside 5'-monophosphate thioester is also effective. Diphosphate and triphosphate analogues of antiviral nucleosides are also effective, however, these diphosphate or triphosphate analogues tend to be less stable than the monophosphate and may be hydrolyzed gradually back to the monophosphate derivative in aqueous solution.
• the nucleoside analogue is encapsulated in liposomes.
• the encapsulation can be carried out according to well known liposome encapsulation procedures such as sonication and extrusion. Suitable conventional methods of encapsulation include but are not limited to those disclosed by Bangham et al (18), Olson et al (39), Szoka and Papahadjapoulos (40), Mayhew et al (41), Kim et al (42), Mayer et al (36) and Fukunaga et al (35).
• the liposomes can be made from any of the conventional synthetic or natural phospholipid liposome materials including phospholipius from natural sources such as egg, plant or animal sources such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, phosphatidylserine or phosphatidylinositol.
• natural sources such as egg, plant or animal sources such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, sphingomyelin, phosphatidylserine or phosphatidylinositol.
• Synthetic phospholipids may also be used, such as, but not limited to, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dilauroylphosphatidylethanolamine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylethanolamine and distearoylphosphatidylethanolamine.
• additives such as cholesterol or other sterols, glycolipids, cerebrosides, gangliosides, sphingolipids, glucopsychosine, or psychosine can also be added as is conventionally known.
• the relative amounts of phospholipid and additives used in the liposomes may be varied if desired. The preferred ranges are from about 80-99 mole percent phospholipid and 1 to 20 mole percent psychosine or other additive.
• Cholesterol may be used in amounts ranging from 0 to 50 mole percent.
• the relative amounts of antiviral nucleoside analogue entrapped in liposomes can be varied with the concentration of entrapped analogue in the liposome aqueous compartment ranging from about 0.001 mM to about 300 mM.
• the liposome entrapped phosphorylated nucleoside analogue is administered to patients by any of the known procedures utilized for administering liposomes.
• the liposomes can be administered intravenously, intraperitoneally or intramuscularly as a buffered aqueous solution.
• Any pharmaceutically acceptable aqueous buffer may be utilized so long as it does not destroy the liposome structure or the activity of the encapsulated phosphorylated nucleoside analogue.
• One suitable aqueous buffer is 150 mM NaCl containing 5mM Na-Phosphate with a pH of about 7.4; other physiological buffered salt solutions may also be used.
• the dosage may vary depending upon the extent and severity of the infection. Dosage levels of encapsulated phosphorylated nucleoside analogue should be such that about 0.001 mg/kilogram to 1000 mg/kilogram be administered to the patient on a daily basis.
• Labeled AZT-5'-monophosphate [ 3 H]AZT-MP) was prepared according to the phosphorous oxychloride method of Toorchen and Topal (34). Twenty nanomoles cf AZT (260 microcuries) was dried under nitrogen. Twenty microliters of trimethylphosphate and 4 microliters of triethylamine were added and the mixture cooled to -10°C. Four microliters of phosphorus oxychloride was added and the mixture was allowed to react at -10 °C for 30 minutes. The reaction was stopped by addition of an equal volume of 0.5 M aqueous triethylamine. AZT was converted to AZT-MP in a yield of greater than 90 percent as judged by HPLC on Altex C18 Ultrosphere-ODS column.
• liposomal AZT monophosphate The effect of liposomal AZT monophosphate on HIV replication in MT-2 and U937 cells and human macrophages in culture was tested as follows:
• a thin film of 27 mg cholesterol and 110 mg egg phosphatidylcholine was prepared by rotary evaporation in vacuo and 1 ml of RPMI medium containing 60 nmol of AZT-MP and 6 uCi [ 3 H]AZT-MP was added and the mixture was shaken at 20°C. for 20 min followed by 10 cycles of vortexing to produce MLV containing [ 3 H]AZT-MP.
• a Lipex Extruder Lipex Biomembranes, Inc.
• small unilamellar vesicles of 100 nanometer diameter were prepared by the method of Mayer et al (36). This procedure is based on extrusion of large multilamellar vesicles through two stacked polycarbonate filters (Nucleopore, Pleasanton, CA).
• the resulting liposome preparation was applied to a 1 ⁇ 15 cm column of Sepharose 4B and eluted with RPMI buffer.
• EV 100 liposomes containing [ 3 H]AZT-MP were added in various concentration to MT-2 and U937 cells growing in ELISA plates; to other wells were added AZT-MP and AZT. After 3 days the MT-2 cells (6 ⁇ 10 5 cells/well) were examined for cytopathic effects (CPE) and the results of CPE grading are shown in Table 3. TABLE 3
• Liposomes containing [ 3 H]AZT-monophosphate were prepared in RPMI medium by the method of Mayer et al (36) and the liposomal AZT-MP encapsulated was determined by the 3 H content of the pooled liposomal peak. Liposomes containing [ 3 H] -AZT-MP were added to MT-2 cells infected with HIV in a final volume of 0.200 ml of RPMI medium containing 10% fetal calf serum.
• Liposome A consisted of 67 mole % egg phosphatidylcholine and 33 mole % cholesterol and Liposome B was made up of 6.6 mole % psychosine and 60 mole % egg phosphatidylcholine and 33.3 mole % cholesterol. Grading of CPD was done as described by Haertle et al (44). In this system 1+ corresponds to 1 to 3 syncytia (giant cells) per well.
• 2+ corresponds to 3-10 syncytia per well and up to 20% cell death; 3+ corresponds to 10-30 syncytia per well and 20-70% drop in cell viability; 4+ corresponds to more than 30 syncytia per well and at least a 70% fall in viable cell count (44).
• Liposomes containing [ 3 H]AZT-MP were prepared as in Table 1 and added to U937 cells infected with HIV in 0.200 ml of RPMI containing 10% fetal calf serum.
• Liposome A consisted of 67 mole % egg phosphatidylcholine and 33 mole % cholesterol and liposome B contained 6.6 mole % psychosine and 60 mole % egg phosphatidylcholine and 33 mole % cholesterol. Results are mean of 2 replicates.
• Liposome of egg phosphatidylcholine containing [ 3 H]AZT-MP in the indicated concentration were prepared as in Tab ⁇ e 1 and added to human macrophages. After 3 days in culture the supernatants were assayed for gp24.
• Liposome A consisted of 67 mole % egg phosphatidylcholine and 33 mole % cholesterol and liposome B contained 6.6 mole % psychosine and 60 mole % egg phosphatidylcholine and 33 mole % cholesterol. Additional examples of practice are as follows:
• Liposomes of egg phosphatidylcholine/cholesterol (2/1) (100 nanometer diameter) containing [ 3 H]AZT-MP were prepared in RPMI medium by the extrusion method of Mayer et al (36), and the amount of [ 3 H]AZT-MP encapsulated was determined by 3 H counts in the voiding peak obtained by gel permeating chromatography using Sepharose 4B. Varying amounts of AZT-MP were added and the effect determined on cells infected with HIV by measuring production of HIV antigen gp24 using the DuPont Elisa assay kit. Two liposome types were prepared -- liposome A and liposome B.
• Liposome A consisted of 67 mole % egg phosphatidylcholine and 33 mole % cholesterol and liposome B contained 6.6 mole % psychosine and 60 mole % egg phosphatidylcholine and 33 mole % cholesterol. The results of the tests are shown in Table 6.
• the CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus.
• T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV.
• the T4 gene enclodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 47:333.

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• 1988
  • 1988-09-19 EP EP19880908811 patent/EP0380558A4/en not_active Ceased
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