Reconstituted HDL Formulation

Abstract

The present invention relates to reconstituted high density lipoprotein (rHDL) formulations comprising an apolipoprotein, a lipid and a lyophilization stabilizer. Said formulations have reduced renal toxicity and good long-term stability, especially in lyophilized form.

Claims

1.-53. (canceled)

54. A reconstituted high density lipoprotein (rHDL) formulation comprising an apolipoprotein, a lipid, and a lyophilization stabilizer, wherein the ratio between the apolipoprotein and the lipid is from about 1:45 to about 1:65 (mol:mol), and the total concentration of all lyophilization stabilizers present in the composition is from about 1.0% to less than 6.0% (w/w of the rHDL formulation).

55. The rHDL formulation according to claim 54, wherein the ratio between the apolipoprotein and the lyophilization stabilizer is selected from the group consisting of (i) from about 1:1 (w:w) to about 1:7 (w:w); (ii) from about 1:1 (w:w) to about 1:3 (w:w); (iii) from about 1:1 (w:w) to about 1:2.4 (w:w); and (iv) from about 1:1 (w:w) to less than 1:2 (w:w).

56. The rHDL formulation according to claim 54, wherein the lyophilization stabilizer is present in a concentration selected from the group consisting of (i) from about 1.0 to 5.9% (w/w); (ii) from about 3.0 to 5.9% (w/w); (iii) from about 4.0 to 5.5% (w/w); (iv) from 4.3 to 5.3% (w/w); and (v) from 4.6 to 4.8% (w/w).

57. The rHDL formulation according to claim 54, wherein the lyophilization stabilizer is selected from a sugar, a sugar alcohol, an amino acid, a mixture of a sugar and a sugar alcohol, a mixture of a sugar and an amino acid, and a mixture of a sugar alcohol and an amino acid.

58. The rHDL formulation according to claim 54, wherein the lyophilization stabilizer comprises a sugar selected from the group consisting of a monosaccharide, disaccharide and trisaccharide.

59. The rHDL formulation according to according to claim 54, wherein the lyophilization stabilizer comprises a disaccharide selected from the group consisting of fructose, trehalose, maltose and lactose.

60. The rHDL formulation according to claim 54, wherein the lyophilization stabilizer comprises a sugar alcohol selected from the group consisting of mannitol, inositol, xylitol, galactitol and sorbitol.

61. The rHDL formulation according to claim 54, wherein the lyophilization stabilizer comprises an amino acid selected from the group consisting of proline, glycine, serine, alanine, lysine, 4-hydroxyproline, L-serine, sodium glutamate, lysine hydrochloride, sarcosine, and y-aminobutyric acid.

62. The rHDL formulation according to claim 54, wherein the lyophilization stabilizer comprises a mixture of a sugar and an amino acid selected from the group consisting of (i) 1% sucrose and 2.2% proline (w/w); (ii) 3% sucrose and 1.5% proline (w/w); and (iii) 4% sucrose and 1.2% proline (w/w).

63. The rHDL formulation according to claim 54, wherein the formulation further comprises a detergent.

64. The rHDL formulation according to claim 63, wherein the detergent comprises sodium cholate.

65. The rHDL formulation according to claim 54, wherein the concentration of the apolipoprotein is from about 5 to about 50 mg/ml.

66. The rHDL formulation according to claim 54, wherein the apolipoprotein comprises apolipoprotein A-I (Apo A-I).

67. The rHDL formulation according to claim 54, wherein the apolipoprotein comprises a fragment of apolipoprotein.

68. The rHDL formulation according to claim 54, wherein the lipid comprises a phosphatidylcholine.

69. The rHDL formulation according to claim 54, wherein the apolipoprotein is recombinant Apo A-I, the lipid is a mixture of sphingomyelin and phosphatidylglycerol, and the lyophilization stabilizer is a mixture of sucrose and mannitol.

70. A rHDL formulation according to claim 54, wherein the formulation is lyophilized.

71. A vial comprising the lyophilized rHDL formulation according to claim 70, wherein the protein content is 1, 2, 4, 6, 8, or 10 g per vial.

72. A method of producing a rHDL formulation comprising an apolipoprotein, a lipid, and a lyophilization stabilizer having a ratio between the apolipoprotein and the lipid of from about 1:45 to about 1:65 (mol:mol), comprising adding the lyophilization stabilizer to a solution comprising the lipid and the apolipoprotein to provide a total concentration of all lyophilization stabilizers present in the rHDL formulation of from about 1.0% to less than 6.0% (w/w) is reached.

73. A method of preventing or treating a disease, disorder or condition in a human, comprising administering the rHDL formulation according to claim 54 to the human to thereby prevent or treat the disease, disorder or condition.

Description

BRIEF DESCRIPTION OF DRAWINGS:

[0055] FIG. 1: Molecular size distribution of formulations containing 5 to 10% w/w sucrose.

[0056] FIG. 2A: Direct comparison of molecular size distribution of formulations containing 4 and 7.5% w/w sucrose.

[0057] FIG. 2B: Molecular size distribution of formulations containing 1, 2, 3, 4 and 7.5% (w/w) sucrose.

[0058] FIG. 2C: Molecular size distribution of formulations containing sucrose and proline and 7.5% sucrose.

[0059] FIGS. 3A and 3B: LCAT activity for 4 to 10% w/w sucrose formulations.

[0060] FIG. 3C: LCAT activity for 1, 2, 3, 4 and 7.5% w/w sucrose formulations.

[0061] FIG. 3D: LCAT activity for formulations containing sucrose and proline.

[0062] FIG. 4A to 4B: Impact of sucrose concentration on cholesterol efflux.

[0063] FIG. 4C: Impact of formulations containing sucrose and proline on cholesterol efflux.

[0064] FIGS. 5A to 5H: Turbidity of formulations with different sucrose concentrations and formulations containing sucrose and proline.

[0065] FIG. 6: Picture of lyo cakes with different sucrose concentration.

[0066] FIG. 7: Picture of lyo cakes with different sucrose concentrations and sucrose and proline.

EXAMPLES

Example 1

Preparation of the Samples

[0067] To make the samples for the following experiments, sodium cholate (New Zealand Pharmaceuticals) was dissolved in buffer (10 mM NaCl, 1 mM EDTA, 10 mM TRIS, pH 8.0) and stirred until clear. Soybean phosphatidylcholine (Phospholipid GmbH) was added to an appropriate volume of the cholate and stirred for 16 h at room temperature. The Apo A-I solution was diluted to a protein concentration of 9.0 mg/mL (determined by OD280) with 10 mM NaCl and mixed with an appropriate volume of the lipid solution to obtain protein to lipid ratio in the range of 1:45 to 1:65. The mixture was stirred at 2-8° C. for 30 min to 16 h. The HDL mimetics were prepared by cholate dialysis using 1% as a diafiltration buffer. The eluate was concentrated to a protein concentration of 33 to 38 g protein /L. Sucrose was added to obtain the desired concentration (1%, 2%, 3%, 4%, 5%, 6.5%, 7%, 10% w/w). The pH of the solution was adjusted, with 0.2 M NaOH to pH 7.50 ±0.1 after which WFI (water for injection) was added to obtain a protein concentration of 30 mg/mL. The final formulations were then sterile filtered through a 0.2+0.1 μm filter and filled into 100 mL glass vials at 1.7 g protein per vial and lyophilized.

[0068] In some formulations proline was added to the desired concentration. Proline maintains an isotonic formulation.

Example 2

Molecular Size Distribution

[0069] Particle formation was determined using HPLC-SEC and assessed by the molecular size distribution of the various formulations. Size exclusion chromatography (HPLC-SEC) was performed on a Superose 6 HR 10/30 column (GE Healthcare) with 140 mmol/1 NaCl, 10 mmol/1 Na-phosphate, 0.02% NaN3, pH7.4, with a flow rate of 0.5 ml/min. Samples of about 90 .sub.ug protein were applied, and elution profiles were recorded at 280 nm.

[0070] Little difference was observed for formulations containing 5-10% w/w sucrose in the final formulation (FIG. 1), indicating that formulations containing % 5% w/w sucrose did not affect particle stability after reconstitution. FIG. 1 shows a complete chromatogram of (1) internal control, 2: 5% w/w sucrose, 3: 6.5% w/w sucrose, 4: 7.5% w/w sucrose and 5: 10% w/w sucrose.

[0071] In addition a direct comparison between a 7.5% w/w sucrose formulation and 4% w/w sucrose formulation demonstrated that these formulations exhibit a similar molecular size distribution (FIG. 2A).

[0072] FIGS. 2B and 2C show the results for sucrose concentrations 1, 2, 3, and 4% (w/w) and formulations comprising sucrose and proline.

[0073] All tested formulations are stable. The sucrose content of 4 to 7.5% w/w was optimum and did not affect the particle to stability after reconstitution.

Example 3

LCAT Activation

[0074] A measure of the effectiveness of the rHDL particles in various formulations was determined by measuring the LCAT activity. HDL particles are capable of sequestering cholesterol from plaques formed along artery walls or cells by interaction with the ATP-binding cassette transporter Al (ABCA1). Lecithin-cholesterol acyltransferase (LCAT), a plasma enzyme converts the free cholesterol into cholesteryl ester (a more hydrophobic form of cholesterol), which is then sequestered into the core of the HDL particle before being transported to the liver to be metabolized. If the sucrose content in the final formulation affected the efficacy of the rHDL particle, LCAT activity would decrease.

[0075] The lecithin-cholesterol acyltransferase (LCAT) activity esterification was assayed as described by Stokke and Norum (Scand J Clin Lab Invest. 1971; 27(1):21-7). 150 μl pooled human plasma (CSL Behring) was incubated with 10 μl rHDL sample and 150 μl PBS in the presence of 20 μl [4-14C]cholesterol (7.5 μCi/ml) for 1.5 h at 4° C. To initiate the esterification of cholesterol, half of the reaction mixture was placed at 37° C. for 30 min while the other half was further incubated at 4° C. for 30 min (to determine background noise). For both samples the cholesterol and cholesteryl ester is extraction by liquid liquid extraction with n-hexane. The cholesteryl ester was separated from unesterified cholesterol using a solid phase extraction column (SampliQ Amino, Agilent) and measured by scintillation counting. The count rate of the sample stored at 4° C. is subtracted from the count rate of the sample stored at 37° C. The same procedure is also performed with a reference sample. The LCAT activity is is expressed as % of the Reference sample.

[0076] FIGS. 3A and 3B show LCAT activity for 4-10% w/w sucrose formulations. FIG. 3C shows LCAT activity for 1-4% w/w sucrose formulations. Very little difference is seen in LCAT activity when the sucrose ranges from 5-10% w/w in the final formulation (FIG. 3A), however a slight decreasing trend is evident when the sucrose is further reduced to 4% w/w (FIG. 3B). FIG. 3D shows LCAT activity for formulations comprising sucrose and proline. No apparent trend in LCAT activity is observed for formulations containing sucrose and proline. Thus the efficacy of the HDL particle in sucrose/proline formulations is maintained.

Example 4

Cholesterol Efflux

[0077] Reverse cholesterol transport (ROT) is a pathway by which accumulated cholesterol is transported from the vessel wall to the liver for excretion. Cells efflux free cholesterol to lipid-poor Apo A-I via the ABCA1 pathway. The cholesterol efflux assay measures the capacity of HDL to accept cholesterol released from cells. It is anticipated that if sucrose content affected particle formation and/or integrity, differences would affect cholesterol efflux.

[0078] Cholesterol efflux from murine macrophage cell lines J774 and RAW 264.7 is highly responsive to cAMP stimulation, which leads to the up-regulation of ABCA1 (Bortnick et. al.,. J Biol Chem. 2000; 275(37):28634-40). RAW264.7 cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured in DMEM (Dulbecco's modified Eagle's medium, Gibco) supplemented with 10% (v/v) foetal calf serum (FCS, Gibco), 2 mM glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin in a humidified CO.sub.2 incubator at 37°. For efflux experiments, cells were seeded into 24-well plates at a density of 0.35×10.sup.6 cells per well. The following day, cells were labeled with [1,2-.sup.3H]cholesterol (1 μCi/mL, GE) in DMEM supplemented with 5% (v/v) FCS. After a labelling period of 36 h, cells were washed with phosphate buffered saline (PBS) and then incubated in DMEM containing 0.2% fatty-acid-free bovine serum albumin (BSA) in the absence or presence of 0.3 mM 8-bromoadenosine 3′,5′-cyclic monophosphate sodium salt-cAMP (8Br-cAMP) for 16 h to up-regulate ABCA1. Following two washes with PBS, cells were incubated with different cholesterol acceptors in DMEM/ 0.2% fatty-acid-free BSA medium. After 5-6 h of incubation, plates were centrifuged at 500 g for 10 minutes to remove any floating cells and cellular debris. Radioactivity in cell supernatants was measured by liquid scintillation counting. Total cell-associated [.sup.3H]cholesterol was determined after extraction of cells in control wells for at least 30 minutes with 0.1 M Triton X-100. Cholesterol efflux was expressed as the percentage of the radioactivity released from cells into the medium relative to the total radioactivity in cells and medium. The difference in efflux between control and 8Br-cAMP-stimulated cells was taken as a measure of ABCA1-dependent efflux.

[0079] FIGS. 4A and 4B show that as sucrose concentration decreases from 7.5% w/w to 4% w/w the cholesterol efflux increased. No apparent difference in cholesterol efflux was observed between the proline containing formulations and the 7.5% sucrose formulation (FIG. 4C).

Example 5

Turbidity

[0080] The term turbidity is used to describe the cloudiness or haze in a solution. Strictly, turbidity arises from the multiple scattering events of visible light by elements present in the solution. Since turbidity arises from the net scattered light, it depends on the sample path length, protein concentration and size of the protein/aggregates/particles. Given that all reduced sucrose formulations contained the same protein concentration upon reconstitution and were measured with the same path length, differences in turbidity can be attributed to differences in the size and/or number of protein/aggregates/particles resulting from the various sucrose formulations.

[0081] Turbidity was determined with a LED nephelometer (Hach 2100AN Turbiditimeter, Loveland, Colo.) using formacin as a standard. Results are given as relative light scattering (NTU).

[0082] Formulations containing 4-10% w/w sucrose produced similar turbid solutions upon reconstitution (FIGS. 5A & 5B). Sucrose concentrations of less than 4% showed increased turbidity (FIGS. 5E and 5G). Based on turbidity, sucrose concentrations of 4% (w/w) and above are optimum.

[0083] Relative increases in the turbidity of a solution upon is storage, is often cited as an indication of aggregation in protein biopharmaceuticals. FIGS. 5C, 5D, 5F and 5H show that little to no increase in turbidity are seen upon storage in liquid form, thereby indicating stability of the particles.

Example 6

Lyo Cake Appearance

[0084] Sucrose formulations with 4% w/w and 7.5% w/w sucrose produced the most stable lyo cakes (FIG. 6).

[0085] Sucrose formulations with 1 to 4% w/w, and formulations containing sucrose and proline, also produced stable lyo cakes (FIG. 7).

Example 7

Stability of rHDL Formulations

[0086] The stability of lyophilized rHDL formulations (prepared as per Example 1) was examined before and after storage (protected from light) at 40 C for 12 weeks. Parameters s tested included pH, turbidity, LCAT activation, HPLC-SEC (aggregate content, % lipoprotein in single peak and its relative retention time) and cholesterol efflux (C-efflux) (Tables 1 & 2). The results indicate that the formulations remain stable over the storage period.

TABLE-US-00001 TABLE 1 t = 0 t = 12 weeks 1385.E009.09- 1% 2% 3% 4% 7.5% 1% 2% 3% 4% 7.5% 13/40° C. sucrose sucrose sucrose sucrose sucrose sucrose sucrose sucrose sucrose sucrose Turbidity 12.4 10.4 8.63 6.87 6.06 13.7 11.1 7.24 5.41 5.16 LCAT- 97 102 104 110 111 94 101 100 98 105 activation HPLC - SEC- <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 Aggregates HPLC-SEC - 98.8 99.2 99.5 99.6 99.6 99.3 99.7 99.6 99.6 99.6 Lipoprotein peak C-efflux (total 110 95 103 119 102 109 85 114 116 84 efflux)

TABLE-US-00002 TABLE 2 t = 0 t = 12 weeks 1385.E009.14- 1% sucrose/ 3% sucrose/ 4% sucrose/ 1% sucrose/ 3% sucrose/ 4% sucrose/ 16/40° C. 2.2% proline 1.5% proline 1.2% proline 2.2% proline 1.5% proline 1.2% proline Turbidity 8.20 8.48 7.48 8.74 6.14 6.12 LCAT-activation 104 116 109 95 103 99 HPLC - SEC <0.2 <0.2 <0.2 <0.2 <0.2 <0.2 Aggregates HPLC-SEC 98.6 99.4 99.5 99.2 99.5 99.7 Lipoprotein peak C-efflux (total 114 80 97 120 108 99 efflux)