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. 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 1:20 to about 1:120 (mol:mol), the lyophilization stabilizer comprises sucrose in an amount selected from (i) from 1.0% w/w to 1.3% w/w of the rHDL formulation and (ii) from 4.0% w/w to 4.8% w/w of the rHDL formulation, and the total concentration of all lyophilization stabilizers present in the rHDL formulation is from about 1.0% w/w to less than 6.0% w/w of the rHDL formulation.

2. The rHDL formulation of claim 1, wherein the lyophilization stabilizer comprises sucrose in an amount selected from (i) from 1.0% w/w to 1.3% w/w of the rHDL formulation and (ii) from 4.0% w/w to 4.8% of the rHDL formulation, and further 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.

3. The rHDL formulation of claim 1, wherein the lyophilization stabilizer comprises sucrose in an amount selected from (i) from 1.0% w/w to 1.3% w/w of the rHDL formulation and (ii) from 4.0% w/w to 4.8% of the rHDL formulation, and further comprises proline.

4. The rHDL formulation of claim 1, wherein the sucrose is the only lyophilization stabilizer in the rHDL formulation and is present in an amount from 4.0% w/w to 4.8% w/w of the rHDL formulation.

5. The rHDL formulation according to claim 1, wherein the formulation further comprises a detergent.

6. The rHDL formulation according to claim 5, wherein the detergent comprises sodium cholate.

7. The rHDL formulation according to claim 1, wherein the concentration of the apolipoprotein is from about 5 to about 50 mg/ml.

8. The rHDL formulation according to claim 1, wherein the apolipoprotein comprises apolipoprotein A-I (Apo A-I).

9. The rHDL formulation according to claim 1, wherein the apolipoprotein comprises recombinant apolipoprotein A-I (Apo A-I).

10. The rHDL formulation of claim 1, wherein the apolipoprotein comprises Apo A-I purified from plasma.

11. The rHDL formulation according to claim 1, wherein the apolipoprotein comprises a fragment of Apo A-I.

12. The rHDL formulation according to 1, wherein the lipid comprises a phosphatidylcholine.

13. The rHDL formulation of claim 1, wherein the formulation has a pH in the range of 6 to 8.

14. A lyophilized preparation of the rHDL formulation of claim 1.

15. A vial comprising the lyophilized rHDL formulation of claim 14 in an amount to provide from 1 to 10 g apolipoprotein per vial.

16. A kit comprising a vial comprising the lyophilized rHDL formulation of claim 14 and instructions for use.

17. A method of treating a disease, disorder or condition responsive to therapeutic administration of rHDL in a human in need thereof, comprising administering the rHDL formulation of claim 1 to the human.

18. The method of claim 17, wherein the disease, disorder or condition is selected from one or more of atherosclerosis, cardiovascular disease, diabetes, and hypercholesterolaemia.

19. A method of preparing the rHDL formulation of claim 1, comprising combining the apolipoprotein and lipid at a ratio of from 1:20 to 1:120 (mol:mol) with the lyophilization stabilizer in an amount to provide a total concentration of all lyophilization stabilizers present in the rHDL formulation of from about 1.0% (w/w) to less than 6.0% (w/w) of the rHDL formulation.

20. The method of claim 19, further comprising lyophilizing the rHDL formulation to obtain a lyophilized rHDL formulation.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1: Molecular size distribution of formulations containing 5 to 10% w/w sucrose.

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

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

(4) FIG. 2C: Molecular size distribution of formulations containing sucrose and proline and 7.5% sucrose.

(5) FIGS. 3A and 3B: LCAT activity for 4 to 10% w/w sucrose formulations.

(6) FIG. 3C: LCAT activity for 1, 2, 3, 4 and 7.5% w/w sucrose formulations.

(7) FIG. 3D: LCAT activity for formulations containing sucrose and proline.

(8) FIG. 4A to 4B: Impact of sucrose concentration on cholesterol efflux.

(9) FIG. 4C: Impact of formulations containing sucrose and proline on cholesterol efflux.

(10) FIGS. 5A to 5H: Turbidity of formulations with different sucrose concentrations and formulations containing sucrose and proline.

(11) FIG. 6: Picture of lyo cakes with different sucrose concentration.

(12) FIG. 7: Picture of lyo cakes with different sucrose concentrations and sucrose and proline.

EXAMPLES

Example 1: Preparation of the Samples

(13) 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% sucrose 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.

(14) In some formulations proline was added to the desired concentration. Proline maintains an isotonic formulation.

Example 2: Molecular Size Distribution

(15) 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 μg protein were applied, and elution profiles were recorded at 280 nm.

(16) 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.

(17) 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).

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

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

Example 3: LCAT Activation

(20) 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 A1 (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.

(21) 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 expressed as % of the Reference sample.

(22) 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

(23) Reverse cholesterol transport (RCT) 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.

(24) 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.

(25) 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

(26) 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.

(27) 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).

(28) 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.

(29) Relative increases in the turbidity of a solution upon 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

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

(31) 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

(32) 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 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.

(33) 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)

(34) 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- 104 116 109 95 103 99 activation 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)