VESICLES

Abstract

The present invention relates to vesicular formulations for use in the topical administration of a therapeutic, metabolic, cosmetic or structural Agent Of Interest (“AOI”) and methods of administering an AOI.

Claims

1-18. (canceled)

19. A vesicle comprising a phospholipid component, a non-ionic surfactant component and a modified component comprising at least one Agent of Interest (AOI), wherein the modified component is a lipid tethered to the AOI or a surfactant tethered to the AOI, or both; and the AOI is tethered such that, when the AOI is on the external surface of the vesicle, a majority of the AOI is external to the vesicular membrane; and the vesicle is deformable to facilitate topical administration of the AOI through the skin of a patient.

20. The vesicle according to claim 19, wherein the modified component is the surfactant tethered to the AOI.

21. The vesicle according to claim 19, wherein the modified component is the lipid tethered to the AOI.

22. The vesicle according to claim 19, wherein the modified component is both the lipid and the surfactant tethered to the AOI.

23. The vesicle according to claim 19, wherein the vesicle comprises a single AOI.

24. The vesicle according to claim 19, wherein the vesicle comprises a plurality of AOIs.

25. The vesicle according to claim 24, wherein the AOIs are homogeneous.

26. The vesicle according to claim 24, wherein the AOIs are heterogeneous.

27. The vesicle according to claim 19, wherein the AOI is selected from the group consisting of an element, an ion, an inorganic salt, a small molecule, an amino acid, a peptide, a protein, a micronutrient, a macromolecule, a macrocyclic molecule and combinations thereof.

28. The vesicle according to claim 19, wherein the AOI is selected from the group consisting of a skin structural protein, a therapeutic protein, a carbohydrate, a chromophore-containing macromolecule, a vitamin, a metal, a metal salt, a non-metallic element, a non-metallic salt, melanin, a melanin analogue, an anti-inflammatory and combinations thereof.

29. The vesicle according to claim 28, wherein the AOI is selected from the group consisting of a vitamin, a metal, a metal salt and combinations thereof.

30. The vesicle according to claim 28, wherein the AOI is a NSAID selected from the group consisting of diclofenac, naproxen and combinations thereof.

31. The vesicle of claim 19, wherein the phospholipid component is phosphatidyl choline, the non-ionic surfactant component is polysorbate 80, and the AOI is selected from the group consisting of ascorbic acid and tripeptide-1.

32. The vesicle of claim 19, wherein the AOI of the modified component is tethered to the lipid of the modified component via at least one lipid glycerol hydroxyl group of the lipid of the modified component by an ester bond.

33. The vesicle of claim 19, wherein the AOI of the modified component is tethered to the lipid of the modified component by replacement of a lipid phosphatidyl moiety of the lipid of the modified component with the AOI such that the lipid of the modified component has two fatty acid chains together with the tethered AOI.

34. The vesicle of claim 19, wherein the AOI is tethered via an ester bond or an amide bond.

35. The vesicle of claim 19, wherein the AOI is tethered via a polymer chain.

36. The vesicle of claim 35, wherein the polymer chain is a polyethylene glycol polymer.

37. A vesicular formulation comprising a plurality of vesicles according to claim 19 and a pharmaceutically acceptable carrier.

38. A method of delivering an AOI through the skin of a patient, the method comprising topically applying to the skin of the patient the vesicular formulation of claim 37 in an amount sufficient to penetrate the skin to deliver the AOI.

Description

[0100] The invention is described below with reference to the following non-limiting examples and figures, in which:

[0101] FIG. 1 shows the arachidonic substrate concentration plotted against the velocity of reaction for the vesicles tethered to Naproxen or Diclofenac;

[0102] FIG. 2 shows the reciprocal (Lineweaver Burk) plot of FIG. 1;

[0103] FIG. 3 shows the arachidonic substrate concentration plotted against the velocity of reaction for vesicles tethered to Naproxen or Diclofenac after a CMA assy; and

[0104] FIG. 4 shows the reciprocal (Lineweaver Burk) plot of FIG. 3.

EXAMPLE FORMULATIONS

Example Vesicular Formulations

Example Formulation 1

[0105] Formulation 1 comprises sphingomyelin (brain) (47.944 mg/g) as a lipid, Tween 80 (42.05 mg/g) as a surfactant, lactate buffer (pH 4). benzyl alcohol or paraben (5.000 mg/g) as an antimicrobial agent, BHT (0.200 mg/g) and sodium metabisulfite (0.0500 mg/g) as antioxidants, glycerol (30.000 mg/g), EDTA (3.000 mg/g) as a chelating agent, and ethanol (30.000 mg/g).

Example Formulation 2

[0106] Formulation 2 comprises sphingomyelin (brain) (53.750 mg/g) as a lipid, Tween 80 (31.250 mg/g) as a surfactant, lactate (pH 4) buffer, benzyl alcohol or paraben (5.000 mg/g) as an antimicrobial agent, BHT (0.200 mg/g) and sodium metabisulfite (0.500 mg/g) as antioxidants, glycerol (30.000 mg/g), EDTA (3.000 mg/g) as a chelating agent, and ethanol (15.000 mg/g).

Example Formulation 3

[0107] Formulation 3 comprises sphingomyelin (brain) (90.561 mg/g) as a lipid, Tween 80 (79.439 mg/g) as a surfactant, lactate (pH 4) buffer, benzyl alcohol or paraben (5.000 mg/g) as an antimicrobial agent, BHT (0.200 mg/g) and sodium metabisulfite (0.500 mg/g) as antioxidants, glycerol (30.000 mg/g), EDTA (3.000 mg/g) as a chelating agent, and ethanol (30.000 mg/g).

Example Formulation 4

[0108] Formulation 4 comprises phosphatidyl choline (68.700 mg/g) as a lipid, Tween 80 (8.500 mg/g) as a surfactant, phosphate (pH 7.5) buffer, BHT (0.200 mg/g) and sodium metabisulfite (0.500 mg/g) as antioxidants, benzyl alcohol or paraben (5.000 mg/g) as an antimicrobial, glycerol (30.000 mg/g), EDTA (1.000 mg/g) as a chelating agent, and ethanol (36.51 mg/g).

Example Formulation 5

[0109] Formulation 5 comprises phosphatidyl choline (71.460 mg/g) as a lipid, Tween 80 (4.720 mg/g) as a surfactant, phosphate (pH 7.8) buffer. BHA (0.200 mg/g) and sodium metabisulfite (0.500 mg/g) as antioxidants, benzyl alcohol or paraben (5.000 mg/g) as an antimicrobial, glycerol (15.000 mg/g), EDTA (3.000 mg/g) as a chelating agent, and ethanol (35.000 mg/g).

Example Formulation 6

[0110] Formulation 6 comprises phosphatidyl choline (71.460 mg/g) as a lipid, Tween 80 (4.720 mg/g) as a surfactant, phosphate (pH 7.8) buffer, BHA (0.200 mg/g) and sodium metabisulfite (0.500 mg/g) as antioxidants, glycerol (50.000 mg/g), EDTA (3.000 mg/g) as a chelating agent, and ethanol (15.000 mg/g).

Example Formulation 7

[0111] Formulation 8 comprises phosphatidyl choline (71.4600 mg/g) as a lipid, Tween 80 (4.720 mg/g) as a surfactant, phosphate (pH 7.5) buffer, BHA (0.200 mg/g) and sodium metabisulfite (0.500 mg/g) as antioxidants, glycerol (50.000 mg/g), EDTA (3.000 mg/g) as a chelating agent, and ethanol (35.000 mg/g).

Example Formulation 8

[0112] Formulation 8 comprises phosphatidyl choline (64.516 mg/g) as a lipid, Tween 80 (35.484 mg/g) as a surfactant, phosphate (pH 6.7) buffer, BHA (0.200 mg/g) as antioxidant, benzyl alcohol or paraben (4.200 mg/g) as an antimicrobial, glycerol (30.000 mg/g), EDTA (3.000 mg/g) as a chelating agent, and ethanol (30.000 mg/g).

Example Formulation 9

[0113] Phosphatidylcholine (64.516 mg/g) as a lipid, Tween 80 (35.484 mg/g) as a surfactant, phosphate (pH 6.7) buffer, BHA (0.200 mg/g) as an antioxidant, benzyl alcohol (5.250 mg/g) or paraben (4.200 mg/g) as a solvent, glycerol (30.000 mg/g), EDTA (3.000 mg/g) as a chelating agent, and ethanol (30.000 mg/g).

Example Formulation 10

[0114] Phosphatidyl choline (71.460 mg/g) as a lipid, Tween 80 (4.720 mg/g) as a surfactant, phosphate (pH 6.7) buffer, BHA (0.200 mg/g) as antioxidant, benzyl alcohol or paraben (10.000 mg/g) as a solvent, glycerol (50.000 mg/g), EDTA (3.000 mg/g) as a chelating agent, and ethanol (30.000 mg/g).

Example Vesicular Formulations with an AOI Attached

Example Formulation 11

[0115] Formulation 9 comprises phosphatidyl choline (68.700 mg/g) as a lipid, Tween 80 (8.500 mg/g) as a surfactant, collagenyl phosphatidylcholine (1 mg/g) as a AOI, phosphate (pH 7.5) buffer, BHT (0.200 mg/g) and sodium metabisulfite (0.500 mg/g) as antioxidants, benzyl alcohol or paraben (5.000 mg/g) as an antimicrobial, glycerol (30.000 mg/g), EDTA (1.000 mg/g) as a chelating agent, and ethanol (36.51 mg/g).

Example Formulation 12

[0116] Formulation 10 comprises phosphatidyl choline (68.700 mg/g) as a lipid, Tween 80 (8.500 mg/g) as a surfactant, collagenyl phosphatidylcholine (0.5 mg/g) as a AOI, phosphate (pH 7.5) buffer, BHT (0.200 mg/g) and sodium metabisulfite (0.500 mg/g) as antioxidants, benzyl alcohol or paraben (5.000 mg/g) as an antimicrobial, glycerol (30.000 mg/g), EDTA (1.000 mg/g) as a chelating agent, and ethanol (36.51 mg/g).

Example Formulation 13

[0117] Formulation 11 comprises phosphatidyl choline (68.700 mg/g) as a lipid, Tween 80 (8.500 mg/g) as a surfactant, collagenyl Tween (0.5 mg/g), phosphate (pH 7.5) buffer, BHT (0.200 mg/g) and sodium metabisulfite (0.500 mg/g) as antioxidants, benzyl alcohol or paraben (5.000 mg/g) as an antimicrobial, glycerol (30.000 mg/g), EDTA (1.000 mg/g) as a chelating agent, and ethanol (36.51 mg/g).

Example 14 and Manufacture and Testing Thereof

[0118] Formulation 14 comprises phosphatidyl choline (68.700 mg/g) as a lipid, Tween 80 (6.800 mg/g) as a surfactant, ascorbyl palmitate (0.530 mg/g) as an AOI, citrate phosphate (pH 5.4) buffer, BHT (0.200 mg/g) and sodium metabisulfite (0.500 mg/g) as antioxidants, EDTA (1.000 mg/g) as a chelating agent, and ethanol (48.87 mg/g).

SUMMARY

[0119] A Transfersome preparation has been successfully manufactured to contain covalently bonded ascorbic acid at 20% polysorbate 80 molar substitution. Test results showed that the size distribution, deformability characteristics and charge of the transfersomes were unaffected by the inclusion of the ascorbic acid, that the L-ascorbyl palmitate ester was accessible on the external surface of the transfersome to a carboxylesterase enzyme, that the ascorbyl palmitate transfersomes were active in an Fe.sup.3+ reducing assay and that they retained their reducing activity after deforming to pass through pores that were smaller than their average size.

Manufacture

[0120] Transfersomes were prepared using soybean phosphatidylcholine (Lipoid SPC S-100) and polysorbate 80, containing L-ascorbyl palmitate (Sigma 7618). A control batch of transfersomes was also made. Butylhydroxytoluene, EDTA and sodium metabisulphite were added to the transfersomes to minimize oxidation of L-ascorbyl palmitate.

Preparation of L-Ascorbyl Palmitate Transfersomes

[0121] A 50 g batch of L-ascorbyl palmitate transfersomes was prepared with soybean phosphatidylcholine: polysorbate 80: L-ascorbyl palmitate molar ratios of 13.3:0.8:0.2

[0122] Using gentle heat and stirring, soybean phosphatidylcholine (3.44 g), polysorbate 80 (0.34 g), butylhydroxytoluene (0.01 g) and L-ascorbyl palmitate (0.0265 g) were dissolved in ethanol to give a total weight of 6.26 g.

[0123] 25 mM citrate phosphate buffer pH5.4, with 0.1% EDTA and 0.05% sodium metabisulphite, (43.74 g) was stirred vigorously at 35° C. while the soybean phosphatidylcholine preparation was added from a syringe fitted with a wide gauge needle. The mixture was stirred for approximately 15 minutes.

[0124] The transfersomes were prepared by extrusion through a 0.2 μm filter, followed by a 0.1 μm filter and a further 0.1 μm filter using a Sartorius 47 mm filter system at 35° C. with nitrogen at 4 bar pressure. Each filter had a glass fibre pre-filter on top. Transfersomes were stored in the dark at +5° C.

Preparation of Control Transfersomes

[0125] A 50 g batch of control transfersomes was prepared with a soybean phosphatidylcholine: polysorbate 80 molar ratio of 13.3:1

[0126] Using gentle heat and stirring, soybean phosphatidylcholine (3.44 g), polysorbate 80 (0.425 g) and butylhydroxytoluene (0.01 g) were dissolved in ethanol to give a total weight of 6.26 g.

[0127] 25 mM citrate phosphate buffer pH5.4, with 0.1% EDTA and 0.05% sodium metabisulphite, (43.74 g) was stirred vigorously at 35° C. while the soybean phosphatidylcholine preparation was added from a syringe fitted with a wide gauge needle. The mixture was stirred for approximately 15 minutes.

[0128] The control transfersomes were extruded as described for L-ascorbyl palmitate transfersome batch PD-14-0035. Transfersomes were stored in the dark at +5° C.

Analytical Methods

Particle Size Measurement

[0129] The average particle size and the particle size distribution for the transfersome preparations were determined by dynamic light scattering using a photon correlation spectrometer. When coherent light is passed through a suspension of particles, light is scattered in all directions. By measurement and correlation of the scattered light intensity of a particle suspension, it is possible to determine the size and size distribution of the particles in the suspension.

[0130] The mean particle size and particle size distribution for each sample were determined using an ALV-5000/E photon correlation spectrometer. Samples were diluted in de-ionised water to give a detectable signal within the range of 50-500 kHz, and then analysed over six measurements, each of 30 seconds duration. The temperature was controlled at 25° C. The data was subjected to a regularised fit cumulative second order analysis to give the mean particle size (reported as r or the mean radius) as well as the particle sizing distribution for the sample (reported as w or width). The mean radius was multiplied by 2 to give the mean diameter (nm).

[0131] The polydispersity index (PDI) for each sample was calculated according to the following equation:

[00001]$\mathrm{PDI}={\left(\frac{w}{r}\right)}^2$

where: w=width and r=average radius.

Continuous Membrane Adaptability Assay

[0132] The continuous membrane adaptability (CMA) assay used applied pressure to provide activation energy to transfersomes to enable them to deform and pass through a filter pore that is smaller than the average size of the transfersomes.

[0133] An Anodisc 13 membrane filter (pore size 20 nm) was mounted on a filtration support in the base of a filtering device and the upper stainless steel barrel was attached. 3 ml transfersome sample pre-equilibrated at 25° C. was placed in the barrel and heat transmitting tube connected to a thermocirculator (25° C.) was wrapped around it. The barrel was connected to a pressure tube connected to a Nitrogen cylinder. Using a series of valves, the system was primed with set-point of 9.5 bar pressure to give 7.5 bar starting pressure. The filtration device was placed over a collection vessel sited on a precision weighing balance that was connected to an Excel computer program. A Bronkhurst pressure controller was used to control and monitor the pressure and when the system valves were opened and timing started, the increasing mass of transfersome filtrate collected on the balance was recorded against the decreasing pressure and increasing time.

[0134] The time, pressure, mass data was evaluated in a MathCAD program to determine a P* value. P* is a measure of the activation pressure required for pore penetration and therefore a measure of transfersome membrane stiffness and deformability. The average particle size of the transfersomes was measured by photon correlation spectroscopy before and after the CMA filtration.

Ascorbic Acid Assay

[0135] Ascorbyl palmitate and ascorbic acid concentrations were measured using an Ascorbic Acid Assay Kit (Abcam ab65656). In this assay, Fe.sup.3+ is reduced to Fe.sup.2+ in the presence of antioxidants such as ascorbic acid. The Fe.sup.2+ is chelated with a colorimetric probe to produce a product with absorbance at 593 nm.

[0136] To determine the total ascorbyl palmitate concentration, transfersomes were solubilised by dilution 1:7:2 v/v with ethanol and 5% Triton X-100. An ascorbyl palmitate standard curve was prepared by initial dilution in ethanol to a concentration range 0.0125 to 0.25 mM, then further diluted 7:1:2 v/v in water and 5% Triton X-100 to a final concentration range of 0.01 to 0.175 mM. A 0 mM ascorbyl palmitate blank was included. Standards and samples were loaded onto a microtitre plate and mixed 1:1 v/v with a reaction mixture containing kit buffer, Fe′ and colorimetric probe. After 1 minute incubation at room temperature, the plate was read at 593 nm. The 0 mM ascorbyl palmitate blank was subtracted from all standards and samples and the absorbance for control ‘empty’ transfersomes was subtracted from that of the ascorbyl palmitate transfersomes. The final absorbance was compared against the ascorbyl palmitate standard curve to obtain the total ascorbyl palmitate (ascorbic acid) concentration (mM).

[0137] To determine the external ascorbic acid concentration, an ascorbic acid standard curve was prepared by diluting ascorbic acid in water to a concentration range of 0.025 to 0.2 mM. Standards and transfersome samples were loaded onto a microtitre plate and mixed 1:1 v/v with reaction mixture containing kit buffer, Fe.sup.3+ and colorimetric probe. After 1 minute incubation at room temperature, the plate was read at 593 nm. The plate blank was subtracted from all standards and samples and the absorbance for control ‘empty’ transfersomes was subtracted from that of the ascorbyl palmitate transfersomes. The final absorbance was compared against the ascorbic acid standard curve to obtain ascorbic acid concentration. The concentration was compared with the total ascorbyl palmitate (ascorbic acid) concentration to calculate the % ascorbic acid tethered on the external surface of the ascorbyl palmitate transfersomes.

Carboxylesterase Digest and Rp-HPLC

[0138] Release of ascorbic acid from transfersomes containing ascorbyl palmitate was performed by enzymatic digestion of the ester using Carboxylesterase 1 isoform B (Sigma E0287). 960 units of enzyme were added per ml of transfersomes, before incubation at +37° C. Samples were taken at 2 and 4 hours and the released ascorbic acid extracted by adding 1 volume of acetonitrile/methanol/formic acid (80 v/20 v/0.2 v) followed by sonication for 5 minutes and centrifugation to pellet insoluble components. Supernatant samples were then filtered through a 0.2 μm membrane before diluting 1 in 10 with ultra-high purity water.

[0139] Samples were assayed by a reversed phase high pressure liquid chromatography (RP-HPLC) method using a Luna C18(2) 100 A 5 μm 4.6×250 mm column and Waters 2695 separation module at +25° C. and a gradient method as per the table below where eluent A was 20 mM potassium phosphate pH3.0 and eluent B was acetonitrile. Detection was performed at a wavelength of 260 nm using a Waters 2487 detector.

TABLE-US-00005 Time Flow (minutes) (ml/min) % Eluent A % Eluent B 0 1 95 5 10 1 87 13 11 1 35 65 15 1 35 65 16 1 95 5 25 1 95 5

[0140] In addition, a standard curve of ascorbic acid in the range of 0.4 to 100 μg/ml was analysed using the same RP-HPLC method. The ascorbic acid peak was integrated in the resulting chromatograms for the samples and standards. The peak areas of the standards were analysed with linear regression to produce an equation for the standard curve. The peak areas for the samples were then used to determine the ascorbic acid concentration from the equation for the standard curve taking into account the dilution from the extraction method. The concentration was compared with the total ascorbic acid concentration to calculate the % ascorbic acid released from the external surface of the ascorbyl palmitate transfersomes.

Paper Electrophoresis

[0141] The charge characteristics of transfersome preparations were investigated using paper electrophoresis where a paper strip was suspended between two buffer filled reservoirs, the test sample was applied to the strip and an electrical current applied across the strip. Charged particles migrated across the strip, with the direction and distance travelled being determined by the net charge of the particles at the buffer pH.

[0142] Volumes (100 μl) of each test sample were applied to the centre of individual 2×20 cm Whatman filter strips (pre-wetted in running buffer; 2.3 mg/ml sodium chloride, 1.5 mg/ml calcium chloride, 1.3 mg/ml glycyl glycine, 25 mg/ml mannitol, 10 mg/ml sucrose and 0.5 mg/ml methionine at pH5.75) and run at 130V for 2 hours. Each strip was stained for PEG (a component of polysorbate 80) with 5% w/v barium chloride and 0.05M iodine and then dried. The extent of travel of the transfersomes away from the centre point for each sample was measured for both the anode and cathode sides of the strip to determine the vesicle net charge.

Results and Discussion

[0143] Results are summarised in Table 4. Samples of the ascorbyl palmitate transfersomes were 0.2 μm sterile filtered and retested post filtration in order to recheck the integrity of the samples for information.

TABLE-US-00006 TABLE 4 Control and Ascorbyl Palmitate Transfersomes Analysis Ascorbyl Ascorbyl Palmitate Palmitate Ascorbyl Trans- Trans- Control Palmitate fersomes fersomes Trans- Trans- Post 0.2 um Post CMA Test fersomes fersomes Filtration Assay Photon Correlation Spectrometry: Average Particle 138.64 141.32 140.90 74.26 Diameter (nm) Polydispersity 0.050 0.063 0.062 0.127 Index CMA Assay: Filtration 20.3 13.3 N/A N/A Recovery % Deformability P* 1.622 1.698 N/A N/A Ascorbic Acid Assay: Total Ascorbyl N/A 0.61 mM/ 0.67 mM/ 0.79 mM/ Palmitate 253 μg/ml 278 μg/ml 327 μg/ml Concentration Total Ascorbic N/A 0.61 mM/ 0.67 mM/ 0.79 mM/ Acid (AA) 107 μg/ml 118 μg/ml 139 μg/ml Concentration External AA N/A 0.13 mM/ 0.12 mM/ 0.14 mM/ Concentration 23 μg/ml 21 μg/ml 25 μg/ml % External AA N/A 21% 18% 18% Carboxylesterase Digest/HPLC: Released External N/A 16 μg/ N/A 13 μg/ AA Concentration ml (15%) ml (9%) (2 hours 37° C.) Released External N/A 38 μg/ N/A 26 μg/ AA Concentration ml (36%) ml (19%) (4 hours 37° C.) Paper Net positive Net positive N/A N/A Electrophoresis charge charge

Particle Size Measurement

[0144] The average particle diameter and polydispersity index were similar for the control transfersomes and for those containing ascorbyl palmitate. This indicated that 20% substitution of polysorbate 80 with the ester in the transfersomes had not affected the size characteristics. There was no significant change in size post 0.2 μm sterile filtration.

Continuous Membrane Adaptability Assay

[0145] The deformability P* value was virtually the same for the transfersomes containing the ascorbyl palmitate and the control transfersomes, indicating that 20% substitution of polysorbate 80 with the ester had not significantly affected the deformability properties of the transfersomes. The filtration % recovery was slightly higher for the control transfersomes which could indicate that the inclusion of ascorbyl palmitate had a very slight stiffening effect on the vesicle membrane.

[0146] The average particle diameter post-CMA filtration decreased by almost 50% compared with pre-filtration for both the control transfersomes and for the transfersomes containing the ascorbyl palmitate ester. The polydispersity index was slightly higher, indicating a broader size distribution. These characteristics are as expected for transfersome vesicles.

Ascorbic Acid Assay

[0147] The total ascorbyl palmitate concentration in ascorbyl palmitate transfersomes was determined as 0.61 mM. This equates to 253 μg/ml ascorbyl palmitate or 107 μg/ml ascorbic acid. The concentration was approximately 50% of that at the start of the manufacturing process, indicating that losses had occurred, probably through a combination of filtration and ascorbic acid oxidisation. However, results showed that active ascorbic acid capable of reducing Fe.sup.3+ was present in the final transfersome preparation.

[0148] The concentration of ascorbic acid that reacted on the external surface of the ascorbyl palmitate transfersomes was determined as 0.13 mM. This equates to 23 μg/ml or 21% of the total ascorbic acid concentration being externally tethered.

[0149] There was no significant change in total or external ascorbic acid concentration post 0.2 μm sterile filtration.

[0150] The total ascorbyl palmitate concentration of transfersomes that had been subjected to the continuous membrane adaptability (CMA) assay was slightly higher than pre-CMA. The external ascorbic acid concentration was virtually the same pre/post-CMA. This showed that transfersomes that had deformed to pass through a pore size that was smaller than their average diameter did not lose any of their reducing activity.

Carboxylesterase Digest and Rp-HPLC

[0151] Incubation of transfersomes containing ascorbyl palmitate ester with carboxylesterase 1 enzyme resulted in the release of 15% (16 μg/ml) of the total ascorbic acid after 2 hours incubation at 37° C. and 36% (38 μg/ml) after 4 hours at 37° C. Ascorbic acid that was tethered to the external surface of the transfersome was therefore accessible to the enzyme. The concentrations obtained for external ascorbic acid were similar to those obtained in the ascorbic acid assay.

[0152] Transfersomes that had been subjected to the CMA deformability filtration assay were also incubated with carboxylesterase 1 enzyme resulting in the release of 9% (13 μg/ml) of the total ascorbic acid after 2 hours incubation at 37° C. and 19% (26 μg/ml) after 4 hours at 37° C. It is unclear why the percentage release was lower post CMA, but possibly the change in vesicle size reduced the accessibility of the ascorbyl palmitate to the enzyme.

Paper Electrophoresis

[0153] Control transfersomes and transfersomes containing ascorbyl palmitate both migrated towards the cathode of the electrophoresis apparatus, demonstrating a net positive charge. The presence of ascorbyl palmitate did not therefore alter the charge characteristics of the transfersomes.

Example Formulation 15 and Manufacture and Testing Thereof

[0154] Formulation 15 comprises either phosphatidyl choline (68.700 mg/g) as a lipid, Tween 80 (7.66 mg/g) as a surfactant, palmitoyl tripeptide 1 (0.370 mg/g) as an AOI, phosphate (pH 7.7) buffer and ethanol (48.10 mg/g), or phosphatidyl choline (68.700 mg/g) as a lipid, Tween 80 (7.66 mg/g) as a surfactant, palmitoyl tetrapeptide 7 (0.450 mg/g) as an AOI, phosphate (pH 7.7) buffer and ethanol (48.40 mg/g).

SUMMARY

[0155] Transfersome preparations have successfully been manufactured to contain covalently bonded peptides; tetrapeptide-7 and tripeptide-1; at 10% polysorbate 80 molar substitution. Test results showed that the size distribution, deformability characteristics and charge of the transfersomes were unaffected by the inclusion of the peptides.

Manufacture

[0156] Transfersomes were prepared using soybean phosphatidylcholine (Lipoid SPC S-100) and polysorbate 80 containing either palmitoyl tetrapeptide-7 (PAL-GQPR) or palmitoyl tripeptide-1 (PAL-GHK) (Sinoway Industrial Co. Ltd). A control batch of transfersomes was also made.

Preparation of Palmitoyl Peptide Transfersomes

[0157] A 50 g batch of palmitoyl tetrapeptide-7 transfersomes and a 50 g batch of palmitoyl tripeptide-1 transfersomes were prepared with soybean phosphatidylcholine: polysorbate 80: palmitoyl peptide molar ratios of 13.3:0.9:0.1

[0158] Using gentle heat and stirring, soybean phosphatidylcholine (3.44 g), polysorbate 80 (0.383 g) and EITHER palmitoyl tetrapeptide-7 (0.0224 g) palmitoyl tripeptide-1 (0.0186 g) were dissolved in ethanol to give a total weight of 6.26 g.

[0159] Phosphate buffer, pH7.7 (43.74 g) was stirred vigorously at 35° C. while the soybean phosphatidylcholine preparation was added from a syringe fitted with a wide gauge needle. The mixture was stirred for approximately 15 minutes.

[0160] The transfersomes were prepared by extrusion through a 0.2 μm filter, followed by a 0.1 μm filter and a further 0.1 μm filter using a Sartorius 47 mm filter system at 35° C. with nitrogen at 4 bar pressure. Each filter had a glass fibre pre-filter on top. Transfersomes were stored in the dark at +5° C.

Preparation of Control Transfersomes

[0161] A 50 g batch of control transfersomes was prepared with a soybean phosphatidylcholine: polysorbate 80 molar ratio of 13.3:1.

[0162] Using gentle heat and stirring, soybean phosphatidylcholine (3.44 g) and polysorbate 80 (0.425 g) were dissolved in ethanol to give a total weight of 6.26 g.

[0163] Phosphate buffer, pH7.7 (43.74 g) was stirred vigorously at 35° C. while the soybean phosphatidylcholine preparation was added from a syringe fitted with a wide gauge needle. The mixture was stirred for approximately 15 minutes.

[0164] The control transfersomes were extruded as described for palmitoyl peptide transfersomes batches. Transfersomes were stored in the dark at +5° C.

Analytical Methods

Particle Size Measurement

[0165] The average particle size and the particle size distribution for transfersome preparations were determined by dynamic light scattering using a photon correlation spectrometer. When coherent light is passed through a suspension of particles, light is scattered in all directions. By measurement and correlation of the scattered light intensity of a particle suspension, it is possible to determine the size and size distribution of the particles in the suspension.

[0166] The mean particle size and particle size distribution for each sample were determined using an ALV-5000/E photon correlation spectrometer. Samples were diluted in de-ionised water to give a detectable signal within the range of 50-500 kHz, and then analysed over six measurements, each of 30 seconds duration. The temperature was controlled at 25° C. The data was subjected to a regularised fit cumulative second order analysis to give the mean particle size (reported as r or the mean radius) as well as the particle sizing distribution for the sample (reported as w or width). The mean radius was multiplied by 2 to give the mean diameter (nm).

[0167] The polydispersity index (PDI) for each sample was calculated according to the following equation:

[00002]$\mathrm{PDI}={\left(\frac{w}{r}\right)}^2$

where: w=width and r=average radius.

Continuous Membrane Adaptability Assay

[0168] The continuous membrane adaptability (CMA) assay used applied pressure to provide activation energy to transfersomes to enable them to deform and pass through a filter pore that is smaller than the average size of the transfersomes.

[0169] An Anodisc 13 membrane filter (pore size 20 nm) was mounted on a filtration support in the base of a filtering device and the upper stainless steel barrel was attached. 3 ml of transfersome sample pre-equilibrated at 25° C. was placed in the barrel and heat transmitting tube connected to a thermocirculator (25° C.) was wrapped around it. The barrel was connected to a pressure tube connected to a Nitrogen cylinder. Using a series of valves, the system was primed with set-point of 9.5 bar pressure to give 7.5 bar starting pressure. The filtration device was placed over a collection vessel sited on a precision weighing balance that was connected to an Excel computer program. A Bronkhurst pressure controller was used to control and monitor the pressure and when the system valves were opened and timing started, the increasing mass of transfersome filtrate collected on the balance was recorded against the decreasing pressure and increasing time.

[0170] The time, pressure, mass data was evaluated in a MathCAD program to determine a P* value. P* is a measure of the activation pressure required for pore penetration and therefore a measure of transfersome membrane stiffness. The average particle size of the transfersomes was measured by photon correlation spectroscopy before and after the CMA filtration.

Peptide Concentration (CBQCA) Assay

[0171] The concentration of the peptide portion of palmitoyl tripeptide-1 with the amino acid sequence glycine-histidine-lysine was measured by derivitisation of the primary amine group of the lysine amino acid with the reagent 3-(4-carboxybenzoyl)quinolone-2-carboxaldehyde (CBQCA) to yield a fluorescent product.

[0172] Samples of palmitoyl tripeptide-1 transfersomes and control transfersomes were diluted in a range of 1 in 400 to 1 in 3200 in 0.1 mM sodium borate buffer pH 9.3. Since it was not possible to solubilise palmitoyl tripeptide 1 in aqueous conditions suited to this assay; the determination of concentration of tripeptide 1 in transfersomes was made against a bovine serum albumin (BSA) standard curve. BSA of known concentration was prepared to yield a range of 6.7 μg/ml to 0.33 mg/ml. Derivitisation of the primary amines of the standards and samples was performed in a micro-plate format at room temperature with CBQCA reagent in the presence of potassium cyanide for 1 hour. Measurement was performed by reading with a BMG Fluostar Optima fluorometer with excitation wavelength 485 nm and fluorescence emission wavelength 520 nm.

[0173] The fluorescent reading of a blank sample of 0.1 mM sodium borate buffer pH 9.3 was subtracted from all the data. The resulting fluorescent measurement from the BSA standards was analysed with linear regression to produce an equation for the standard curve. The amount of peptide in the palmitoyl tripeptide-1 transfersomes relative to the BSA curve was then determined after subtraction of the fluorescence of the control transfersomes at the equivalent dilution.

Paper Electrophoresis

[0174] The charge characteristics of transfersome preparations were investigated using paper electrophoresis where a paper strip was suspended between two buffer filled reservoirs, the test sample was applied to the strip and an electrical current applied across the strip. Charged particles migrated across the strip, with the direction and distance travelled being determined by the net charge of the particles at the buffer pH.

[0175] Volumes (100 μl) of each test sample were applied to the centre of individual 2×20 cm Whatman filter strips (pre-wetted in running buffer; 2.3 mg/ml sodium chloride, 1.5 mg/ml calcium chloride, 1.3 mg/ml glycyl glycine, 25 mg/ml mannitol, 10 mg/ml sucrose and 0.5 mg/ml methionine at pH5.75) and run at 130V for 2 hours. Each strip was stained for PEG (a component of polysorbate 80) with 5% w/v barium chloride and 0.05M iodine and then dried. The extent of travel of the transfersomes away from the centre point for each sample was measured for both the anode and cathode sides of the strip to determine the vesicle net charge.

Results and Discussion

[0176] Results are summarised in Table 5.

TABLE-US-00007 TABLE 5 Palmitoyl Peptide Transfersome Analysis Batch Palmitoyl Palmitoyl Control Tetrapeptide 7 Tripeptide 1 Test Transfersomes Transfersomes Transfersomes Photon Correlation Spectrometry: Average Particle 142.70 142.84 141.12 Diameter (nm) Polydispersity 0.071 0.053 0.062 Index CMA Assay: Filtration % 14.7 14.3 14.0 Recovery Deformability 1.725 1.595 1.759 P* Average Particle 74.3 72.28 74.68 Diameter Post CMA (nm) Polydispersity 0.11 0.12 0.099 Index Post CMA Theoretical 0 mM/ 0.65 mM/ 0.65 mM/ Peptide 0 μg/ml 296 μg/ml 221 μg/ml Concentration Peptide N/A Non-detectable Peptide detected Concentration due to lack of (424 μg/ml) (CBQCA primary amines assay) in peptide Paper Net positive Net positive Net positive Electrophoresis charge charge charge

Particle Size Measurement

[0177] The average particle diameter and polydispersity index were similar for the control transfersomes and for those containing the palmitoyl peptides. This indicated that 10% substitution of polysorbate 80 with a palmitoyl peptide in the transfersomes had not affected the size characteristics.

Continuous Membrane Adaptability Assay

[0178] The deformability P* value was similar for the control transfersomes and for those containing the palmitoyl peptides. The value for the palmitoyl tetrapeptide 7 transfersomes was slightly lower, indicating that 10% substitution of polysorbate 80 with the ester might have had a slight softening effect on the membrane making the vesicles more deformable. However, this was not evidenced in the filtration % recovery which was similar for the palmitoyl peptide transfersomes compared to the control, so the lower P* is possibly not significant.

[0179] The average particle diameter post-CMA filtration decreased by almost 50% compared with pre-filtration for both the control transfersomes and for the transfersomes containing the palmitoyl peptide. The polydispersity index was slightly higher, indicating a broader size distribution. These characteristics are as expected for transfersome vesicles.

Peptide Concentration (CBQCA) Assay

[0180] Palmitoyl tetrapeptide 7 transfersomes did not produce a result in the CBQCA assay due to a lack of lysine residues in the sequence to react with the reagent. However, palmitoyl tripeptide 1 was detectable since it contains a lysine. Since it was not possible to solubilise palmitoyl tripeptide 1 in aqueous conditions suited to the assay; the determination of concentration of tripeptide 1 in transfersomes had to be made against a bovine serum albumin (BSA) standard. BSA is a 66 kDa protein with 58 lysine residues; ˜1 per 1138 Da of peptide. The peptide contains 1 lysine in 340 Da. The peptide was detected and an attempt was made to quantify the amount by correcting for the difference in concentration of lysines between BSA and peptide, however the total peptide still appeared to be overestimated; 424 μg/ml compared to theoretical 221 μg/ml.

Paper Electrophoresis

[0181] Control transfersomes and transfersomes containing a palmitoyl peptide all migrated towards the cathode of the electrophoresis apparatus, demonstrating a net positive charge. The presence of palmitoyl tetrapeptide 7 or palmitoyl tripeptide 1 did not therefore alter the charge characteristics of the transfersomes.

Example Formulation 16 and Manufacture and Testing Thereof

[0182] Formulation 16 comprises either phosphatidyl choline (68.700 mg/g) as a lipid, Tween 80 (6.55 mg/g) as a surfactant, naproxen-polysorbate (2.195 mg/g) as an AOI, phosphate (pH 7.7) buffer and ethanol (47.56 mg/g), or phosphatidyl choline (68.700 mg/g) as a lipid, Tween 80 (5.80 mg/g) as a surfactant, diclofenac-polysorbate (2.96 mg/g) as an AOI, phosphate (pH 7.7) buffer and ethanol (47.54 mg/g).

SUMMARY

[0183] Transfersome preparations have successfully been manufactured to contain covalently bonded, non-steroidal anti-inflammatory drugs (NSAIDs); Naproxen and Diclofenac; at 20% polysorbate 80 molar substitution. Test results showed that the size distribution, deformability characteristics and charge of the transfersomes were unaffected by the inclusion of the NSAIDs, that the NSAID esters were accessible on the external surface of the transfersome to a carboxylesterase enzyme and that the NSAID transfersomes had a greater inhibitory effect in a COX-1 enzyme inhibition assay than control transfersomes alone. NSAID transfersomes retained their inhibitory activity after deforming to pass through pores that were smaller than their average size.

Manufacture

[0184] Transfersomes were prepared using soybean phosphatidylcholine (Lipoid SPC S-100) and polysorbate 80, containing either Naproxen-polysorbate 80 ester (Key Organics DK-0035-3) or Diclofenac-polysorbate 80 ester (Key Organics DK-0036-3). A control batch of transfersomes was also made.

Preparation of NSAID Transfersomes

[0185] A 20 g batch of Naproxen-polysorbate transfersomes and a 20 g batch of Diclofenac-polysorbate transfersomes were prepared with soybean phosphatidylcholine: polysorbate 80: NSAID-polysorbate 80 molar ratios of 13.3:0.8:0.2 (accounting for purity of the NSAID-polysorbate 80 esters).

[0186] Using gentle heat and stirring, soybean phosphatidylcholine (1.374 g) with EITHER polysorbate 80 (0.131 g) and Naproxen-polysorbate (0.0439 g) OR polysorbate 80 (0.116 g) and Diclofenac-polysorbate (0.0592 g) were dissolved in ethanol to give a total weight of 2.50 g.

[0187] Phosphate buffer, pH7.7 (17.50 g) was stirred vigorously at 35° C. while the soybean phosphatidylcholine preparation was added from a syringe fitted with a wide gauge needle. The mixture was stirred for approximately 15 minutes.

[0188] The transfersomes were prepared by extrusion through a 0.2 μm filter, followed by a 0.1 μm filter and a further 0.1 μm filter using a Sartorius 47 mm filter system at 35° C. with nitrogen at 4 bar pressure. Each filter had a glass fibre pre-filter on top. Transfersomes were stored in the dark at +5° C.

Preparation of Control Transfersomes

[0189] A 50 g batch of control transfersomes was prepared with a soybean phosphatidylcholine: polysorbate 80 molar ratio of 13.3:1

[0190] Using gentle heat and stirring, soybean phosphatidylcholine (3.44 g) and polysorbate 80 (0.425 g) were dissolved in ethanol to give a total weight of 6.26 g.

[0191] Phosphate buffer, pH7.7 (43.74 g) was stirred vigorously at 35° C. while the soybean phosphatidylcholine preparation was added from a syringe fitted with a wide gauge needle. The mixture was stirred for approximately 15 minutes.

[0192] The control transfersomes were extruded as described for NSAID transfersomes batches. Transfersomes were stored in the dark at +5° C.

Analytical Methods

Particle Size Measurement

[0193] The average particle size and the particle size distribution for the transfersome preparations were determined by dynamic light scattering using a photon correlation spectrometer.

[0194] When coherent light is passed through a suspension of particles, light is scattered in all directions. By measurement and correlation of the scattered light intensity of a particle suspension, it is possible to determine the size and size distribution of the particles in the suspension.

[0195] The mean particle size and particle size distribution for each sample were determined using an ALV-5000/E photon correlation spectrometer. Samples were diluted in de-ionised water to give a detectable signal within the range of 50-500 kHz, and then analysed over six measurements, each of 30 seconds duration. The temperature was controlled at 25° C. The data was subjected to a regularised fit cumulative second order analysis to give the mean particle size (reported as r or the mean radius) as well as the particle sizing distribution for the sample (reported as w or width). The mean radius was multiplied by 2 to give the mean diameter (nm).

[0196] The polydispersity index (PDI) for each sample was calculated according to the following equation:

[00003]$\mathrm{PDI}={\left(\frac{w}{r}\right)}^2$

where: w=width and r=average radius.

Continuous Membrane Adaptability Assay

[0197] The continuous membrane adaptability (CMA) assay used applied pressure to provide activation energy to transfersomes to enable them to deform and pass through a filter pore that is smaller than the average size of the transfersomes.

[0198] An Anodisc 13 membrane filter (pore size 20 nm) was mounted on a filtration support in the base of a filtering device and the upper stainless steel barrel was attached. 3 ml transfersome sample pre-equilibrated at 25° C. was placed in the barrel and heat transmitting tube connected to a thermocirculator (25° C.) was wrapped around it. The barrel was connected to a pressure tube connected to a nitrogen cylinder. Using a series of valves, the system was primed with set-point of 9.5 bar pressure to give 7.5bar starting pressure. The filtration device was placed over a collection vessel sited on a precision weighing balance that was connected to an Excel computer program. A Bronkhurst pressure controller was used to control and monitor the pressure and when the system valves were opened and timing started, the increasing mass of transfersome filtrate collected on the balance was recorded against the decreasing pressure and increasing time.

[0199] The time, pressure, mass data was evaluated in a MathCAD program to determine a P* value. P* is a measure of the activation pressure required for pore penetration and therefore a measure of transfersome membrane stiffness and deformability. The average particle size of the transfersomes was measured by photon correlation spectroscopy before and after the CMA filtration.

Carboxylesterase Digest and Rp-HPLC

[0200] Release of the tethered non-steroidal anti-inflammatory drugs (NSAIDs); Diclofenac or Naproxen; from the external surface of transfersomes containing polysorbate 80 esters of either of the two compounds was performed by enzymatic digestion of the ester using carboxylesterase 1 isoform B (Sigma E0287). 960 units of enzyme were added per ml of transfersomes, before incubation at +37° C. Samples were taken at 4 hours and the released NSAID extracted by adding 1 volume of acetonitrile/methanol/formic acid (80 v/20 v/0.2 v) followed by sonication for 5 minutes and centrifugation to pellet insoluble components. Supernatant samples were then filtered through a 0.2 μm membrane before diluting 1 in 10 with ultra-high purity water.

[0201] Samples were assayed by a reversed phase high pressure liquid chromatography (RP-HPLC) method using a Kinetex C18 5 μm 100 A 4.6×150 mm column and Waters 2695 separation module at +25° C. and a gradient method as per the table below where eluent A was 0.1% trifluoroacetic acid in ultra-high purity water and eluent B was 0.1% trifluoroacetic acid in acetonitrile. Detection for both of the NSAIDs was performed at a wavelength of 254 nm using a Waters 2487 detector.

TABLE-US-00008 Time Flow % Eluent % Eluent (minutes) (ml/min) A B 0 1.2 95 5 15 1.2 5 95 20 1.2 5 95 21 1.2 95 5 25 1.2 95 5

[0202] In addition, a standard curve of each of the NSAIDs in the range of 0.4 to 91 μg/ml was analysed using the same RP-HPLC method. The NSAID peaks were integrated in the resulting chromatograms for the samples and standards. The peak areas of the standards were analysed with linear regression to produce equations for the standard curves. The peak areas for the samples were then used to determine the released NSAID concentration from the equation for the respective standard curve taking into account the dilution from the extraction method. The concentration was compared with the theoretical total NSAID concentration to calculate the % NSAID released from the external surface of the NSAID transfersomes.

Cyclooxygenase-1 Inhibition Assay

[0203] The cyclooxygenase 1 (COX-1) inhibition assay measures the ability of drugs such as NSAIDs to inhibit the activity of the COX-1 enzyme. COX-1 catalyses the conversion of arachidonic acid to prostaglandin H.sub.2. During the reaction the enzyme consumes oxygen. The velocity of oxygen consumption (nmol/ml/min) is a measure of the rate of reaction and is reduced in the presence of inhibitors.

[0204] The COX inhibition assay was set up using a Hansatech Oxygraph system that comprised a calibrated Clark oxygen electrode connected to Oxygraph Plus software. A reaction mixture containing 0.1 mM potassium phosphate pH7.2, 2.0 mM phenol, 1 μM hematin was stirred in the reaction chamber at 37° C. until a stable oxygen baseline was attained. 340 units of COX-1 enzyme (Cayman Chemicals CAY60100) was added and allowed to equilibrate for 1 minute before the addition of arachidonic acid substrate. A series of control reactions were performed using arachidonic acid at final concentrations 8, 16, 32 and 6401 For each reaction, the maximum reaction rate was measured on the Oxygraph oxygen curve.

[0205] To determine the inhibitory effect of transfersome samples; control, Naproxen or Diclofenac transfersomes were pre-mixed with arachidonic acid for 10 minutes at room temperature prior to the addition of the arachidonic acid mixture to the reaction. The concentrations were chosen so that the final arachidonic acid concentrations in the reaction were 8, 16, 32 and 64 μM.

[0206] The arachidonic acid concentration was plotted against the reaction velocity (nmol Oxygen/ml/min) for the control, control transfersomes and NSAID transfersomes reactions and a value was calculated for % inhibition by transfersomes by comparing the reaction velocity at the four substrate concentrations and averaging the decrease in rate. Lineweaver-Burk reciprocal plots (1/arachidonic acid concentration against 1/reaction velocity) were also plotted.

[0207] The COX-1 inhibition assay was also performed on samples of transfersomes that had been processed in the continuous membrane adaptability (CMA) assay that used applied pressure to provide activation energy to enable the vesicles to deform and pass through pores that were smaller than their average diameter.

Paper Electrophoresis

[0208] The charge characteristics of transfersome preparations were investigated using paper electrophoresis where a paper strip was suspended between two buffer filled reservoirs, the test sample was applied to the strip and an electrical current applied across the strip. Charged particles migrated across the strip, with the direction and distance travelled being determined by the net charge of the particles at the buffer pH.

[0209] Volumes (100 μl) of each test sample were applied to the centre of individual 2×20 cm Whatman filter strips (pre-wetted in running buffer; 2.3 mg/ml sodium chloride, 1.5 mg/ml calcium chloride, 1.3 mg/ml glycyl glycine, 25 mg/ml mannitol, 10 mg/ml sucrose and 0.5 mg/ml methionine at pH5.75) and run at 130V for 2 hours. Each strip was stained for PEG (a component of polysorbate 80) with 5% w/v barium chloride and 0.05M iodine and then dried. The extent of travel of the transfersomes away from the centre point for each sample was measured for both the anode and cathode sides of the strip to determine the vesicle net charge.

Results and Discussion

[0210] Results are summarised in Table 6.

TABLE-US-00009 TABLE 6 Control and NSAID Transfersomes Analysis Control Naproxen Diclofenac Transfersomes Transfersomes Transfersomes Photon Correlation Spectrometry: Average Particle 137.98 135.90 135.34 Diameter (nm) Polydispersity Index 0.067 0.058 0.045 CMA Assay: Filtration % Recovery 18.0 42.3 41.8 Deformability P* 1.632 1.321 1.339 Average Particle 72.72 77.22 73.02 Diameter Post CMA (nm) Polydispersity Index 0.13 0.078 0.093 Post CMA Carboxylesterase Digest/HPLC: Theoretical NSAID 0 mM/ 1.30 mM/ 1.30 mM/ Concentration 0 μg/ml 299 μg/ml 385 μg/ml Released External N/A 10 μg/ml 21 μg/ml NSAID Concentration (3.3%) (5.5%) COX-1 Inhibition Assay: Inhibition 53% 75% 62% Inhibition Post-CMA 33% 59% 40% Paper Net positive Net positive Net positive Electrophoresis charge charge charge

Particle Size Measurement

[0211] The average particle diameter and polydispersity index were similar for the control transfersomes and for those containing an NSAID-polysorbate 80 ester. This indicated that 20% substitution of polysorbate 80 with either Naproxen-polysorbate 80 or Diclofenac-polysorbate in the transfersomes had not affected the size characteristics.

Continuous Membrane Adaptability Assay

[0212] The deformability P* value was slightly lower for the transfersomes containing an NSAID-polysorbate ester than for the control transfersomes, indicating that 20% substitution of polysorbate 80 with either Naproxen-polysorbate 80 or Diclofenac-polysorbate had a slight softening effect on the membrane, making the vesicles more deformable. This was also evidenced in the filtration % recovery which increased for the Naproxen or Diclofenac transfersomes compared to the control.

[0213] The average particle diameter post CMA filtration decreased by almost 50% compared with pre-filtration for both the control transfersomes and for the transfersomes containing an NSAID-polysorbate ester. The polydispersity index was slightly higher, indicating a broader size distribution. These characteristics are as expected for transfersome vesicles.

Carboxylesterase Digest and Rp-HPLC

[0214] Incubation of transfersomes containing an NSAID-polysorbate ester with carboxylesterase 1 enzyme resulted in the release of between 3 and 6% of the total NSAID concentration, indicating that a small proportion of the Naproxen or Diclofenac that was tethered to the external surface of the transfersome was accessible to the enzyme.

Cyclooxygenase-1 Inhibition Assay

[0215] Transfersomes containing an NSAID-polysorbate ester inhibited the velocity of reaction of cyclooxygenase 1 (COX-1) enzyme by a greater percentage than control transfersomes. Control transfersomes were expected to inhibit the COX-1 enzyme and tethering known COX-1 inhibitors, Naproxen or Diclofenac, to the external surface of the transfersome has further enhanced that inhibitory effect.

[0216] FIGS. 1 and 2 show the arachidonic substrate concentration plotted against the velocity of reaction and the reciprocal (Lineweaver Burk) plots respectively.

[0217] Transfersomes that had deformed to pass through a pore that was smaller than their average size in the continuous membrane adaptability (CMA) assay retained the ability to inhibit the COX-1 enzyme.

[0218] FIGS. 3 and 4 show the arachidonic substrate concentration plotted against the velocity of reaction and the reciprocal (Lineweaver Burk) plots respectively for the samples post-CMA.

[0219] The % inhibition of the COX-1 enzyme was slightly lower post-CMA assay for all 3 transfersome preparations. This was hypothesised to be due to a decrease in the concentration of transfersomes and associated NSAIDs caused by filtration, rather than to a loss in activity of the NSAID. An indication of comparative transfersome concentration was gained from photon correlation spectrometry. The intensity of the frequency signal for the post-CMA samples had decreased in comparison to pre-CMA samples, but was found to be similar for control and NSAID transfersomes, despite the varying filtration recoveries post-CMA.

Paper Electrophoresis

[0220] Control transfersomes and transfersomes containing an NSAID-polysorbate ester all migrated towards the cathode of the electrophoresis apparatus, demonstrating a net positive charge. The presence of Naproxen or Diclofenac did not therefore alter the charge characteristics of the transfersomes.