LIPOSOME FORMULATION

Abstract

The present invention describes liposome formulations comprising a phospholipid, cholesterol and a fatty acid compound or fatty acid containing compound. Also, various medical uses of the liposome formulation are described.

Claims

1.-53. (canceled)

54. A liposome formulation comprising; i) a phospholipid, ii) cholesterol and iii) a fatty acid compound or a fatty acid containing compound, wherein the fatty acid compound (iii) has the general formula (I):
R.sup.1—[Z—X.sub.i]n-Y  (I) wherein 10 is; a C.sub.6-C.sub.24 alkene with one or more double bonds and/or with one or more triple bonds, and/or a C.sub.6-C.sub.24 alkyne, or a C.sub.6-C.sub.24 alkyl or C.sub.6-C.sub.24 alkyl substituted in one or several positions with one or more compounds selected from the group comprising fluoride, chloride, hydroxy, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylthio, C.sub.2-C.sub.5 acyloxy or C.sub.1-C.sub.4 alkyl, and wherein n is an integer from 1 to 12, and wherein i is an odd number and indicates the position relative to Y, and wherein X.sub.i independent of each other is N, O, S, CH.sub.2 or N—R.sup.3, and wherein Z is CH.sub.2 or CO or X.sub.i, and wherein at least one X.sub.i is N or O or S, or at least one Z is CO, and wherein R.sup.3 is CH.sub.3 or (CH.sub.2).sub.2, wherein Y is CO—COOR.sub.2, CH.sub.2—COOR.sub.2, or CH.sub.2-R.sub.4, and wherein R.sub.4 is carboxylic acid or a derivate thereof, wherein the derivate is a carboxylic ester, a glyceride or a phospholipid wherein R.sub.2, if present, represents hydrogen or C1-C4 alkyl.

55. The liposome formulation according to claim 54, wherein the phospholipid is selected from the group consisting of phosphatidic acid (PA), phosphatidyletanoleamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and a phosphatidylinositol (PIs).

56. The liposome formulation according to claim 54, wherein the molar ratio of phospholipid to cholesterol to fatty acid compound in the liposome is about 1-3 to 1 to 1-2.

57. The liposome formulation according to claim 56, wherein the molar ratio of phospholipid to cholesterol to fatty acid compound in the liposome is about 1.8 to 1 to 1.15.

58. The liposome formulation according to claim 56, wherein the molar ratio of phospholipid to cholesterol to fatty acid compound in the liposome is about 1.8 to 1 to 1.5.

59. The liposome formulation according to claim 54, wherein the size of the liposomes is between 110 and 140 nm.

60. The liposome formulation according to claim 54, wherein Xi is N.

61. The liposome formulation according to claim 54, wherein said compound is Tetradec-12-yn-1-ylglycine.

62. The liposome formulation according to claim 54, wherein said compound is N-tetradecylglycine.

63. The liposome formulation according to claim 54, wherein the compound is 2-(tridec-12-yn-ylthio) acetic acid.

64. A liposome formulation comprising; i) a phospholipid and ii) cholesterol and iii) a fatty acid compound or a fatty acid containing compound for use in the prevention and/or treatment of a disorder or disease, wherein the fatty acid compound (iii) has the general formula (I):
R.sup.1—[Z—X.sub.i]n-Y  (I) wherein R.sup.1 is; a C.sub.6-C.sub.24 alkene with one or more double bonds and/or with one or more triple bonds, and/or a C.sub.6-C.sub.24 alkyne, or a C.sub.6-C.sub.24 alkyl or C.sub.6-C.sub.24 alkyl substituted in one or several positions with one or more compounds selected from the group comprising fluoride, chloride, hydroxy, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylthio, C.sub.2-C.sub.5 acyloxy or C.sub.1-C.sub.4 alkyl, and wherein n is an integer from 1 to 12, and wherein i is an odd number and indicates the position relative to Y, and wherein X.sub.i independent of each other is N, O, S, CH.sub.2 or N—R.sup.3, and wherein Z is CH.sub.2 or CO or X.sub.i, and wherein at least one X.sub.i is N or O or S, or at least one Z is CO, and wherein R.sup.3 is CH.sub.3 or (CH.sub.2).sub.2, wherein Y is CO—COOR.sub.2, CH.sub.2—COOR.sub.2, or CH.sub.2—R4, and wherein R4 is carboxylic acid or a derivate thereof, wherein the derivate is a carboxylic ester, a glyceride or a phospholipid wherein R.sub.2, if present, represents hydrogen or C1-C4 alkyl.

65. The liposome formulation for use in accordance with claim 64, wherein the disorder or disease is obesity, or wherein the disorder or disease is multi metabolic syndrome termed “metabolic syndrome” which is inter alia characterised by hyperinsulinemia, insulin resistance, obesity, glucose intolerance, Type 2 diabetes mellitus, dyslipidemia and/or hypertension, or wherein the disorder or disease is diabetes, or hyperinsulinemia or restenosis, or the disorder is a proliferative skin disorder, or an inflammatory or autoimmune disorder, or wherein the disorder is a neurodegeneration disorder or a mitochondrial dysfunction or disorders caused by hyperproliferation.

66. The liposome formulation for use according to claim 65, wherein the neurodegenerative disorder is present in an individual with patient dementia.

67. The liposome formulation for use according to claim 65 wherein the neurodegenerative disorder is present in an individual with Alzheimer's disease.

68. The liposome formulation for use according to claim 65, wherein the neurodegenerative disorder is present in an individual with movement disorder.

69. The liposome formulation for use according to claim 65, wherein the neurodegenerative disorder is present in an individual with Parkinson's disease.

70. The liposome formulation for use according to claim 65, wherein the compound is a mitochondrial uncoupling agent for use in a mitochondrial dysfunction.

71. The liposome formulation according to claim 65, wherein the disorder is cancer.

72. The liposome formulation for use according to claim 65, wherein the disease is a liver disease.

73. The liposome formulation for use according to claim 65, wherein the liposome formulation is treated with ultrasound or micro bubbles to increase the uptake and distribution in a tissue.

Description

DESCRIPTION OF THE DIAGRAMS

[0106] Embodiments of the present invention and experimental results will now be described, by way of example only, with reference to the following diagrams wherein:

[0107] FIG. 1 shows viability of NB4 cell line treated with TTA, 2-tr-TTA and PA dissolved in DMSO after 48 hours. NB4 cells at a concentration of 0.1×10.sup.5 cells/mL were incubated with 37.5, 75, 150 and 300 μM of TTA, 2-tr-TTA and PA dissolved in DMSO for 48 hours. Cell viability was assessed with WST-1 assay after 4 hours of incubation, and the absorption was related to the DMSO-control. Cell viability is presented as percentage of DMSO-control (100%). The assay was performed with triplicates for each concentration of the individual SBFA and the result is presented as mean of the triplicates±SD, n=1.

[0108] FIG. 2 shows WST-1 viability assay with MOLM-13 cell line treated with liposomes in PBS from batch 1 for 72 hours. MOLM-13 was treated with (1.8, 3.6, 7.2, 14.5 28.9 and 57.8 μM) TTA in liposomes, (1.4, 2.8, 5.5, 11.2, 22.4 and 44.8 μM) N-TTA in liposomes and (1.5, 3.1, 6.1, 12.3, 24.5 and 49.0 μM) PA in liposomes. The cell line was incubated with liposomes for 72 hours and incubated with WST-1 reagent for 4 hours. Absorbance was measured with a plate reader and absorbance of treated cells is presented as percentage of empty liposomes control (100%). All concentrations were tested in triplicates, and the experiment was repeated three times on 3 independent days (n=3). Data is presented as mean±SD.

[0109] FIG. 3 shows WST-1 viability assay with AML cell lines treated with liposomes in PBS from batch 2 for 72 hours. MOLM-13 (A) and HL60 (B) was treated with (11.9, 23.9, 47.8, 95.5, 191 and 382 μM) TTA in liposomes and (5.3, 10.6, 21.2, 42.4, 84.8 and 169.6 μM) N-TTA in liposomes. The cell lines were incubated with liposomes for 72 hours and incubated with WST-1 reagent for 4 hours. Absorbance was measured with a plate reader and absorbance of treated cells is presented as percentage of empty liposomes control (set as 100%). All concentrations except HL60 with TTA-liposomes were tested in triplicates, and the experiment was repeated three times on three independent days (n=3). HL60 with TTA-liposomes was tested in triplicates on two independent days (n=2). Data is presented as mean±SD. ****=p<0.0001, **=p<0.01, *=p<0.05.

[0110] FIG. 4 shows .sup.3H-thymidine proliferation assay with MOLM-13 (A and A2) and HL60 (B and B2). Cell lines were treated with (11.9, 23.9, 47.8, 57.3, 76.4, 95.5, 191 and 382 μM) TTA-liposomes and (5.3, 10.6, 21.2, 25.4, 33.9, 42.4, 84.8 and 169.6 μM) N-TTA-liposomes. The cell lines were incubated with liposomes for 48 hours and incubated with .sup.3H-thymidine for 18 hours. .sup.3H-incorporation was measured and related to empty liposome control (set as 100%) All concentrations were tested in triplicates, and the experiment was repeated three times on three independent days (n=3). Data is presented as mean±SD. In A2 and B2 .sup.3H-incorporation is compared to empty liposome control in a two-way ANOVA, ****=p<0.0001, ***=p<0.001, **=p<0.01, *=p<0.05.

[0111] FIG. 5 shows Flow cytometric Annexin/PI apoptosis assay with PA, TTA and N-TTA in liposomes. HL60 (right) and MOLM-13 (left) (both 0.8×10.sup.6 cells/mL) were treated for 48 hours with (A) TTA-liposomes (47, 94, 189, 283 and 378 μM), (B) N-TTA-liposomes (32, 63, 127, 190, 253 μM) and (C) PA-liposomes (48, 97, 193, 290, 386 μM) liposomes from batch 3. SDs are removed for aesthetics reasons and presented in appendix, table x. All concentrations liposomes were tested in triplicates, and the experiment was repeated three times on three independent days.

DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Experimental Section

Example 1

Preparation of N-tetradecylglycine (N-TTA)

[0112] ##STR00001##

Structure of N-tetradecylglycine (Termed N-TTA or TDG in the Present Application).

[0113] ##STR00002##

[0114] Ethyl bromoacetate (7.2 mL, 65 mmol) dissolved in chloroform (50 mL) was added dropwise to a solution of tetradecylamine (26.32 g, 123 mmol) in chloroform (250 mL) over approximately 30 minutes. After the addition was completed the reaction was stirred for an additional hour at ambient temperature.

[0115] The crude reaction mixture was reduced under reduced pressure and the product was purified by column chromatography on silica using a gradient of methanol in dichloromethane.

[0116] Yield: 14.96 g, 49.9 mmol.

[0117] .sup.1H NMR (CDCl.sub.3, 400 MHz): 4.17 (q, 7.1 Hz, 2H), 3.38 (s, 2H), 2.62-2.53 (m, 2H), 1.45 (m, 2H), 1.34-1.18 (m, 25H), 0.85 (t, 6.8 Hz, 3H)

##STR00003##

[0118] Ethyl tetradecylglycinate (19.83 g, 66.2 mmol) was dissolved in methanol (400 mL) and water (80 mL). Lithium hydroxide monohydrate (11.07 g, 264 mmol) was added and the reaction mixture was stirred over night at ambient temperature.

[0119] Formic acid (15 mL) was added dropwise to the reaction mixture and the reaction mixture was reduced under reduced pressure and the product was purified by column chromatography on reversed phase silica using a gradient of acetonitrile in water. Yield: 10.20 g (37.6 mmol).

[0120] .sup.1H NMR (MeOH-d.sub.4, 400 MHz): 3.49 (s, 2H), 3.03-2.90 (m, 2H), 1.73-1.63 (m, 2H), 1.43-1.23 (m, 22H), 0.90 (t, 6.8 Hz, 3H)

Example 2—Preparation of Tetradec-12-yn-1-ylglycine hydrochloride (tr-N-TTA)

[0121] ##STR00004##

Structure of tetradec-12-yn-1-ylglycine (termed tr-N-TTA or tr-TDG in the Present Application)

tert-Butyl tetradec-12-yn-1-ylglycinate (AKB:DP-5:61-EH-1)

[0122] ##STR00005##

[0123] A mixture of bromo/iodotetradec-2-yne (45 g, 146 mmol) and glycine t-butyl ester hydrochloride (26.9 g, 161 mmol) in ACN, 600 ml, was added DIPEA (63.6 ml, 365 mmol) and the reaction mixture was refluxed for 4 hours. After cooling to room temperature, the mixture was concentrated under reduced pressure. Flash chromatography on silica gel eluting with heptane/EtOAc (95:5)-(70:30)-(65:35) afforded 13 g (28%) of the title compound as a yellow oil and 19 g of the starting material as bromotetradec-2-yne. .sup.1H NMR (400 MHz, CDCl.sub.3) δ 3.26 (s, 2H), 2.55 (t, J=7.2, 2H), 2.16-1.92 (m, 2H), 1.75 (t, J=2.5, 3H), 1.54-1.38 (m, 14H), 1.24 (s, 13H).

tert-Butyl tetradec-12-yn-1-ylglycinate (AKB:DP-5:61-EH-2)

[0124] ##STR00006##

[0125] A mixture of bromotetradec-2-yne (13.6 g, 49.9 mmol) and glycine t-butyl ester hydrochloride (9.2 g, 54.9 mmol) in CAN, 200 ml, was added K.sub.2CO.sub.3 (17.3 g, 125 mmol) and NaI (7.5 g, 50 mmol) and refluxed overnight. The reaction mixture was cooled to room temperature, filtered and concentrated under reduced pressure. Flash chromatography on silica gel eluting with heptane/EtOAc (95:5)-(70:30)-(65:35) afforded 5.2 g (32%) of the title compound as a yellow oil and 13.8 g of the starting material.

Tetradec-12-yn-1-ylglycine hydrochloride (EH:DP-4:82)

[0126] ##STR00007##

[0127] A mixture of tert-butyl tetradec-12-yn-1-ylglycinate (25.8 g, 79.7 mmol) in dioxane, 300 ml, was added 6 M HCl (80 ml) and stirred at room temperature overnight before it was stirred at 55° C. for 6 hours. The reaction mixture was cooled to room temperature and stirred overnight. Precipitated product was filtered off and washed with EtOAc, 200 ml, and dried under reduced pressure to afford 22 g (91%) as a colorless powder.

[0128] .sup.1H NMR (400 MHz, DMSO-d6) δ 9.27 (bs, 1H), 3.80 (s, 2H), 2.96-2.78 (m, 2H), 2.57-2.43 (m, 2H), 2.19-1.99 (m, 2H), 1.71 (t, J=2.5, 3H), 1.63 (s, 2H), 1.49-1.14 (m, 14H).

[0129] .sup.13C NMR (101 MHz, DMSO-d6) δ 167.92, 79.28, 75.58, 46.70, 46.69, 28.89, 28.85, 28.72, 28.49 (2C), 28.44, 28.23, 25.89, 25.10, 18.01, 3.07.

[0130] MS (pos) 290 [M-HCl+Na].sup.+

Example 3—Preparation of 2-tr-TTA

2-(Tridec-2-yn-1-yloxy)tetrahydro-2H-pyran (AKB:TM-1:57)

[0131] ##STR00008##

[0132] A mixture of 2-(prop-2-yn-1-yloxy)tetrahydro-2H-pyran (67.5 ml, 480 mmol) in dry THF (200 ml) was cooled to 0° C. under N.sub.2-atmosphere before BuLi 1.6 M in hexanes (300 ml, 480 mmol) was added drop wise. 1-Bromodecane (100 ml, 483 mmol) was added followed by DMSO (1000 ml). The cooling bath was removed and the slurry was stirred for 220 minutes. The reaction mixture was cooled to 0° C. before water (250 ml) was added drop wise. Diethyl ether (600 ml) was added and the phases was separated. The organic phase was washed with a (1:1) mixture of water/brine (400 ml×4), dried (Na.sub.2SO.sub.4), filtered and concentrated under reduced pressure. Dry-flash chromatography on silica gel eluting with heptane-heptane:EtOAc (100:1) afforded 88.18 g (65%) of the title compound. .sup.1H NMR (200 MHz, CDCl.sub.3) δ 4.80-4.77 (m, 1H), 4.40-4.02 (m, 2H), 3.95-3.70 (m, 1H), 3.55-3.44 (m, 1H), 2.31-2.06 (m, 2H), 1.99-1.05 (m, 22H), 0.85 (t, J=6.2, 3H).

Tridec-2-yn-1-ol (AKB:TM-1:59)

[0133] ##STR00009##

[0134] A mixture of 2-(Tridec-2-yn-1-yloxy)tetrahydro-2H-pyran (AKB:TM-1:57) (85.21 g, 303.8 mmol) and PPTS (9.6 g, 38.2 mmol) in EtOH (770 ml) was stirred at 50° C. for 18 hrs and concentrated under reduced pressure. The residue was diluted with CH.sub.2Cl.sub.2 (500 ml) and washed with water (200 ml). The water phase was extracted with CH.sub.2Cl.sub.2 (500 ml). The combined organic phase was dried (Na.sub.2SO.sub.4), filtered and concentrated under reduced pressure. TLC showed remaining starting material. A mixture of the residue and PPTS (7.03 g, 28 mmol) in EtOH (600 ml) was stirred for 17 hrs at 50° C. and concentrated under reduced pressure. The residue was diluted with CH.sub.2Cl.sub.2 (500 ml) and washed with water (200 ml). The water phase was extracted with CH.sub.2Cl.sub.2 (500 ml). The combined organic phase was dried (Na.sub.2SO.sub.4), filtered and concentrated under reduced pressure. Dry-flash chromatography on silica gel eluting with heptane:EtOAc (100:1)-(95:5)-(80:20) afforded 46.06 g (77%) of the title compound as a colorless waxy solid. .sup.1H NMR (200 MHz, CDCl.sub.3) δ 4.27-4.21 (m, 2H), 2.23-2.15 (m, 2H), 1.65-1.25 (m, 17H), 0.90-0.82 (m, 3H).

Tridec-12-yn-1-ol (AKB:TM-1:63)

[0135] ##STR00010##

[0136] Sodium hydride 60% dispersion in mineral oil (38.82 g, 970.5 mmol) in 1,3-diaminopropane (500 ml) was stirred at 70° C. for 1 hr. The mixture was cooled to room temperature before a solution of tridec-2-yn-1-ol (AKB:TM-1:59) (23.95 g, 122 mmol) in 1,3-diaminopropane (250 ml). The reaction mixture was stirred at 55° C. under N.sub.2-atmosphere for 20 hrs. The mixture was cooled in an ice-bath and water 1000 ml was added. The mixture was extracted with diethyl ether (500 ml×4), washed with 1 M HCl (500 ml), water (500 ml) and brine (300 ml), dried Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. Dry-flash chromatography on silica gel eluting with heptane-heptane:EtOAc (95:5)-(80:20) afforded 19.76 g (83%) of the title compound. .sup.1H NMR (200 MHz, CDCl.sub.3) δ 3.62 (dd, J=11.7, 6.4, 2H), 2.16 (td, J=6.9, 2.6, 2H), 1.91 (t, J=2.6, 1H), 1.70-1.05 (m, 18H).

13-Bromotridec-1-yne (AKB:TM-1:65)

[0137] ##STR00011##

[0138] A solution of tridec-12-yn-1-ol (35.27 g, 180 mmol) in dry CH.sub.2Cl.sub.2 (700 ml) was cooled to 0° C. before addition of triphenylphosphine (51.86 g, 197.7 mmol) followed by tetrabromomethane (65.62 g, 197.9 mmol). The reaction mixture was stirred at 0° C. under N.sub.2-atmosphere for 2 hrs. Silica gel was added and the mixture was concentrated under reduced pressure. Dry-flash chromatography on silica gel eluting with heptane afforded 45.55 g (98%) of the title compound as a colorless liquid which solidified upon storage in the freezer. .sup.1H NMR (200 MHz, CDCl.sub.3) δ 3.38 (t, J=6.8, 2H), 2.16 (td, J=6.9, 2.6, 2H), 1.91 (t, J=2.6, 1H), 1.81 (dd, J=14.7, 6.8, 2H), 1.62-1.11 (m, 16H).

14-Bromotetradec-2-yne (AKB:TM-1:67)

[0139] ##STR00012##

[0140] A solution of 13-bromotridec-1-yne (AKB:TM-1:65) (44.68 g, 172.4 mmol) in dry THF (500 ml) was cooled to −10° C. under N.sub.2-atmosphere before BuLi 1.6 M in hexanes (118.5 ml, 189.6 mmol) was added drop wise. The reaction mixture was stirred for 10 minutes before TMEDA (56.5 ml, 376.3 mmol) was added drop wise followed by drop wise addition of methyl iodide (57 ml, 915.6 mmol). A white solid precipitated and extra THF was added in order to stir the reaction mixture. The cooling bath was removed and the reaction mixture was stirred for 18 hrs. Water (500 ml) was added and the phases were separated. The water phase was extracted with diethyl ether (500 ml×2), washed with 1 M HCl (aq) (300 ml), dried (Na.sub.2SO.sub.4), filtered and concentrated under reduced pressure to afford the crude title compound as a mixture of the bromo- and iodo-compound.

2-(Tetradec-12-yn-1-ylthio)acetic acid (AKB:TM-1:69/GH:DP-3:42)

[0141] ##STR00013##

[0142] Potassium hydroxide (25.05 g, 446 mmol) was dissolved in MeOH (270 ml) before a solution of 2-mercaptoacetic acid (14 ml, 201.4 mmol) in MeOH (270 ml) was added drop wise. The reaction mixture was stirred for 10 minutes before 14-bromotetradec-2-yne/14-iodotetradec-2-yne (AKB:TM-1:67) (49.74 g) was added drop wise. The 14-bromotetradec-2-yne/14-iodotetradec-2-yne flask was washed out with MeOH (100 ml). The reaction mixture was stirred at 50° C. for 16 hrs, cooled to 0° C. and 1 M and 6 M HCl (aq) was added to pH 1-2 and water 250 ml was added. The mixture was extracted with diethyl ether (1000 ml×2), dried (MgSO.sub.4), filtered and concentrated under reduced pressure. Recrystallization from heptane/EtOAc afforded 22.9 g of the title compound as a light yellow solid. The mother liquor was dissolved in diethyl ether and precipitated with heptane to afford another 10.8 g of the title compound. Total yield 33.7 g (69% from 13-bromotridec-1-yne). .sup.1H NMR (400 MHz, CDCl.sub.3) δ 11.58 (s, 1H), 3.18 (s, 2H), 2.65-2.51 (m, 2H), 2.06-2.02 (m, 2H), 1.71 (t, J=2.6, 3H), 1.61-1.48 (m, 2H), 1.42-1.36 (m, 2H), 1.25 (d, J=36.2, 14H). MS (neg): 283 [M-H].sup.−

Example 4—Preparation of 1-tr-TTA

[0143] 1-tr-TTA was obtained in a similar process as described in example 3, but the third last step can be omitted.

Example 5—Preparation of Liposomes

[0144] Liposomes (lipid vesicles) were prepared with a technique called lipid extrusion. The basic principle of this method is to press a lipid suspension through a polycarbonate filter with defined pore size at a temperature above the lipids transition temperature. Before the extrusion process, a thin lipid film (also called lipid cakes) is produced. When the lipid film is rehydrated, the stacks of crystalline bilayers within the lipid film swell. During agitation, the lipids sheets self-assembly into large multilamellar vesicles (MLV). With decreasing pore size, the extrusion pressure increases. At higher pressure, the vesicles are broken down and the phospholipid bilayer is reorganized, resulting in unilammelar vesicles.

[0145] Hydrogenated egg phosphatidylcholine (HEPC), cholesterol (CHO) and fatty acid compound (PA, N-TTA and TTA) were weighed out separately and dissolved in chloroform. N-TTA is not soluble in chloroform alone and was therefore dissolved in a 1:1 mixture of methanol and chloroform. The dissolved lipids were mixed in a molar ratio of 1.81 HEPC:1CHO and 0.5-2 PA/N-TTA/TTA in a 100 or 250 mL Duran round bottom flasks. Next, a thin lipid film was made by slowly evaporating the solvents by using Laborota 4000 rotary evaporator at light vacuum, 200 mbar, and 60 rpm for 1.5-2.5 hours depending on the volume of solvents. To ensure a solvent-free lipid film, full vacuum (0 mbar) was applied the last 30 minutes.

[0146] The lipid film was then rehydrated in 70° C. PBS 70° C. by alternating between a Vortex Genie and 70° C. water bath. The lipid suspension was protected with plastic film until large unilammelar vesicles (LUVs) were prepared with a mini extruder set from Avanti® Polar lipids. The mini extruder was placed on a DRI-BLOCK® heating block, ensuring approximately 70° C. through the extruding process, as this temperature is above the lipids transition temperature. In the extruding process, the hydrated lipid film was passed through Whatman® Nucleopore® Track-Etched polycarbonate membranes with decreasing pore size. Firstly, the suspension was passed 11 times through a 400 nm pore size membrane. Secondly, the suspension was pressed 11 times through a 200 nm pore size membrane, and lastly the suspension was passed 22 times through a 100 nm pore size membrane. The membranes and Avanti® filter supports were regularly replaced with new, intact membranes during this process. This resulted in liposomes between 110-140 nm. Finally, liposomes were stored in sterile Eppendorf tubes protected from light at 4° C.

[0147] The liposomes were stored for maximum 6 weeks, and the liposome solution was always mixed before use in experiments and analysis. Empty liposomes were prepared similarly as liposomes with PA/N-TTA/TTA, except these FAs were not added. Round-bottom flasks and PBS was autoclaved before use. Clean gloves were always used in preparation of lipids and in handling of the equipment in order to avoid contamination with lipids from the human skin and environment. When handling organic solvents, glass pipettes were always used.

[0148] The quantity of PA, TTA and N-TTA in the liposomes was estimated with GLC-FID.

[0149] After preparation of B SFAs, the B SFAs were investigated for their cytotoxic potential on NB4, MOLM-13 and HL60. The cytotoxic effect was investigated by three different methods, i) WST-1 viability assay, ii) .sup.3H-thymidine proliferation assay and iii) Apoptosis assay performed with flow cytometry.

Example 6—Assessment of Cell Viability by WST-1 Assay

[0150] As a preliminary test, NB4 was exposed to TTA, 2-tr-TTA and PA dissolved in DMSO in concentrations between 37.5 to 300 μM for 48 hours. WST-1 assay with TTA, PA and 2-tr-TTA dissolved in DMSO was only performed once on NB4, and was not tested further in this project due to the lack of significant antiproliferative effect (data not shown). It was decided to not try higher concentrations of TTA PA and 2-tr-TTA dissolved in DMSO because of DMSO's cellular toxicity. In this project, one aim was to compare TTA with N-TTA and 2-tr-N-TTA. Because N-TTA and 2-trN-TTA were seemingly insoluble in DMSO, it was considered unreasonable to continue with DMSO as a solvent. Compared to DMSO-control there was no significant decrease in cell viability after treatment with B SFAs dissolved in DMSO (p>0.05). As presented in FIG. 1, 2-tr-TTA seemed to have a stimulating effect, especially at a concentration of 75 μM. More interestingly, PA seemed to decrease metabolic activity more than TTA.

[0151] Liposomes containing TTA, N-TTA and PA was prepared as described above, and the potential anti-proliferative effect the investigated with WST-1 assay as described above. FIG. 2 show viability after treatment with liposomes from batch 1. FIG. 3 displays viability after treatment with liposomes from batch 2. In both experiments, cell lines were incubated with liposomes for 72 hours, and incubated with WST-1 reagent for 4 hours. Absorption, obtained by plate reader, was related to absorption from cells treated with empty liposomes and presented as percent viability of empty liposomes control. Liposomes from batch 1 showed no significant inhibitory effect on metabolic activity on MOLM-13 (p>0.05).

[0152] Batch 2 of liposomes was prepared with a higher concentration of BSFAs than batch one due to lack of inhibitory effect on metabolic activity in batch one measured with WST-1. Results from WST-1 assay is presented in FIG. 3. Compared to empty liposomes (set as 100%), liposomes from batch 2, especially liposomes with TTA, show a considerable reduction of viability. For liposomes with TTA, viability seemed to drop with higher concentrations than 47.8 μM. N-TTA in liposomes seemed to a lesser extent than TTA-liposomes to decrease viability in MOLM-13 and HL60. N-TTA-liposomes did not give a significant decrease in viability (p>0.05). For both MOLM-13 and HL60, 382 μM (p<0.0001), 191 μM (p<0.01) and 95.5 μM (p<0.05, only in MOLM-13) significantly decreased viability compared to empty liposomes. Simultaneously, cell lines were exposed to the same concentration with PBS as the cells in the experiment with liposomes were. 10% PBS had no significant inhibitory effect on the cells.

[0153] The anti-proliferative effect of liposomes with N-TTA and TTA was studied with .sup.3H-thymidine incorporation assay on HL60 and MOLM-13, and the anti-leukaemic effect was compared to WST-1 assay with liposomes from the same batch. The cells were treated with TTA-liposomes in concentrations from 11.9-382.0 μM, and N-TTA-liposomes in concentrations from 5.3-169.6 μM. The cell lines were incubated with the liposomes for 48 hours, and further incubated with .sup.3H-thymidine solution for 18 hours. The .sup.3H-thymidine incorporation was compared to .sup.3H-thymidine incorporation in empty liposomes (set as 100%). FIG. 4 shows mean proliferation compared to empty liposome control±SD. A dose-dependent anti-proliferative effect was seen in both cell lines after treatment with both TTA-liposomes and N-TTA-liposomes. As demonstrated with the large SD in FIG. 4B, results varied greatly for HL60. The results from MOLM-13 were considerably less wide-ranging. Compared to control with empty liposomes, there was observed a significant decrease in cell proliferation in HL60 and MOLM-13 treated with 382, 191 and 95.5 μM of TTA-liposomes (p<0.0001). In addition, proliferation was significantly decreased in MOLM-13 with 76.4 μM (p<0.0001) and 57.3 μM (p<0.05) of TTA-liposomes. Whereas, N-TTA-liposomes significantly decreased cell proliferation after treatment with 169.6 μM (p<0.0001) in both cell lines and 84.8 μM (p<0.05, HL60 and p<0.001, MOLM-13).

[0154] In order to quantify apoptotic, dead, and viable cells after treatment with TTA-, N-TTA- and PA-liposomes, annexin/PI apoptosis assay was performed on MOLM-13 and HL60 with flow cytometry after 48 hours of incubation with liposomes from batch 3.

[0155] HL60 and MOLM-13 was treated with (47, 94, 189, 283 and 378 μM) TTA-liposomes, (48, 97, 193, 290, 386 μM) PA-liposomes and (32, 63, 127, 190, 253 μM) N-TTA-liposomes.

[0156] The results are shown in FIG. 5. The results show high degree of apoptosis among the cells after treatment with liposomes with TTA, especially in concentrations higher than 100 μM.