Pharmaceutical Formulations of PEGylated Liposomes and Blood Coagulation Factors

Abstract

The present invention provides a pharmaceutical composition for subcutaneous administration comprising a blood factor and a colloidal particle comprising about 0.5 to 20 mole percent of an amphipathic lipid derivatized with a biocompatible hydrophilic polymer, wherein the blood factor is not encapsulated in said colloidal particle.

Claims

1. A method for treating a patient suffering from a blood clotting disease or trauma, comprising: administering subcutaneously a pharmaceutical composition comprising blood factor FVIII and a colloidal particle comprising about 0.5 to 20 mole percent of an amphipathic lipid derivatized with a biocompatible hydrophilic polymer, wherein the blood factor is not encapsulated in said colloidal particle.

2. The method of claim 1 wherein the colloidal particles are substantially neutral and the polymer carries substantially no net charge.

3. The method of claim 1 wherein the colloidal particle has a mean particle diameter of between about 0.03 to about 0.4 microns (m).

4. The method of claim 3 wherein the colloidal particle has a mean particle diameter of approximately 0.1 microns (m).

5. The method of claim 1 wherein said amphipathic lipid is a phospholipid from natural or synthetic sources.

6. The method of claim 5 wherein said amphipathic lipid is phosphatidylethanolamine (PE).

7. The method of claim 1 wherein said amphipathic lipid is a carbamate-linked uncharged lipopolymer.

8. The method of claim 7 wherein said amphipathic lipid is aminopropanediol distearoyl (DS).

9. The method of claim 1 wherein said colloidal particles further comprise a second amphipathic lipid obtained from either natural or synthetic sources.

10. The method of claim 9 wherein said second amphipathic lipid is phosphatidylcholine.

11. The method of claim 10 wherein the colloidal particle comprises palmitoyl-oleoyl phosphatidyl choline (POPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine (DSPE) in a ratio (POPC:DSPE) of from 85 to 99:15 to 1.

12. The method of claim 11 wherein the ratio of POPC:DPSE is from 90 to 99:10 to 1.

13. The method of claim 12 wherein the ratio of POPC:DPSE is 97:3.

14. The method of claim 9 wherein cholesterol is supplemented to the composition.

15. The method of claim 1 wherein said biocompatible hydrophilic polymer is selected from the group consisting of polyalkylethers, polylactic acids and polyglycolic acids.

16. The method of claim 15 wherein said biocompatible hydrophilic polymer is polyethylene glycol.

17. The method of claim 16 wherein the polyethylene glycol has a molecular weight of between about 500 to about 5000 Daltons.

18. The method of claim 17 wherein the polyethylene glycol has a molecular weight of approximately 2000 Daltons.

19. The method of claim 16 wherein the derivatized amphipathic lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly-(ethyleneglycol)].

20. The method of claim 16 wherein the derivatized amphipathic lipid is 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] (DSPE-PEG 2000).

21. (canceled)

22. The method of claim 1 wherein the composition additionally comprises another therapeutically active compound.

23.-27. (canceled)

Description

EXAMPLE 1

Synthesis of Liposomes

[0138] Mixed lipids were prepared from palmitoyl-oleoyl phosphatidyl choline (POPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] derivatized with PEG-2000 (PEG with molecular weight 2000 Daltons) (DSPE-PEG 2000), as follows: [0139] Molecular weight of POPC: 760.08 g/mol [0140] Molecular weight of DSPE-2kPEG: 2789.5 g/mol

[0141] The final preparation had a concentration of 100mM phospholipids. A 15% w/v mixture of lipids was made with a 97:3 molar ratio of POPC:DSPE-2kPEG. The following were weighed and mixed: [0142] 2.04 g POPC [0143] 0.232 g DSPE-2kPEG [0144] 14.9 mL tert-butanol (melted in a 35 C. water bath), all placed in a 100 mL Schott bottle.

[0145] The mixture was maintained at 35 C. in a water bath and stirred intermittently until all solids had dissolved/dispersed. The final material was a clear colourless mixture. The mixture was frozen at 80 C. overnight.

[0146] The operation was maintained in a fume hood to allow containment during the post-use clean-up of dried/condensed solvent. The Christ Alpha 1-2 LD freeze-drier and vacuum pump were warmed up for 20 minutes, and the frozen lipid/solvent mixture was removed from 80 C. storage and dried overnight.

[0147] The dried lipids were recovered from the drier the following morning. They appeared as a dry crystalline cake. A 100 mM lipid solution was required for further processing. The quantities of lipid present calculate through as around 82 moles of DSPE-2kPEG and 2.69 mmoles of POPC; so around 2.77 mmoles of lipids. Thus 27.7 mL of diluent was required. 27.7 mL of 50 mM sodium citrate buffer was added to the dried lipids, and the resulting mixture was stirred and heated to around 35 C. After around 120 minutes, a white emulsion with no obvious large solids resulted. This was subjected to extrusion as below.

[0148] A Sartorius 47 mm stainless steel pressure filtration housing was assembled and wrapped with a water jacket (wrapped tubing fed via a thermocirculator) maintained at 35 C. The housing was fitted with a polycarbonate track-etched membrane (details below), covered by a glass-fibre prefilter (Whatman GF/D). The emulsion was poured into the housing and extruded under 4 bar nitrogen gas, with the filtrate collected into 50mL tubes. The duration of each extrusion was timed and noted.

[0149] The filtration sequence was: 0.8 m, 0.4 m, 0.2 m, 0.2 m, 0.1 m and 0.1 m (i.e. single passes through the larger filters and two passes through the smaller 0.2 and 0.1 m filters), with the filtrate warmed back to 35 C. between passes. The liposomes were extruded, with tabulated data is below:

TABLE-US-00001 TABLE 1 Pore size (m) Duration Recovery (g) 0.8 <4 sec 28.19 0.4 <4 sec 26.91 0.2 50 sec 23.76 0.2 22 sec 21.77 0.1 12 minutes 20.18 0.1 4 minutes 19.47

[0150] The resulting extruded lipids were stored at +5 C. 15 mL of Extruded Liposomes were removed from the chilled stock and dispensed into a sterile 50 mL tube within a MicroBiological Safety Cabinet. The size of the extruded liposomes was analysed using an ALV5000 photon correlation spectrometer. The average radius was determined to be 75.400.86 nm and the average peak width 22.213.86 nm, giving an average diameter of 150.80 nm and polydispersity index of 0.087.

EXAMPLE 2

Pharmacokinetics/Pharmacodynamics of Recombinant Human FVIII Reconstituted with PEGylated Liposomes in Haemophilia A Dogs Following Subcutaneous Administration

[0151] A dog with haemophilia A (identified as dog number 1) received subcutaneous doses of PEGylated liposomes associated with Factor VIII (PEGLip FVIII SQ), as follows:

[0152] The objectives of this study were to determine the PK and PD in a haemophilia A dog of full-length rFVIII reconstituted in PEGylated liposomes administered subcutaneously (SQ).

[0153] Full-Length rFVIII

[0154] Lyophilised, full-length rFVIII (Helixate NexGen, Lot 270LR8WB) was used as the test article.

[0155] PEGylated Liposome Formulation

[0156] PEGylated Liposomes in citrate buffer were produced in accordance with Example 1 above according to the method of Baru et al. (2005). The Liposome formulation had the following composition; 50 mM sodium citrate pH 7.0 containing 100 mM phospholipids; comprising a 97:3 molar ratio mixture of palmitoyl-oleoylphosphatidylcholine (POPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] (DSPE-PEG 2000).

[0157] The experimental test subject dog was from the haemophilia A colony housed at the University of Alabama Medical School. All dogs have congenital severe haemophilia A. The test subject weighed 16.4 kg and was naive to human proteins.

[0158] Prior to dosing, the dog was tested to verify normal health status, including complete blood chemistry, serum chemistry profile fibrinogen, fibrinogen derived peptides (FDPs), thrombin time and urinary analysis

[0159] The design of this study was a single SQ dose feasibility trial in a single individual.

[0160] Full-length, recombinant human FVIII (Helixate NexGen, 2,000 IU) was reconstituted with 13.3 ml of PEGylated liposomal diluent. The reconstituted rFVlll was mixed gently at ambient temperature for 5-10 min to allow the protein to adsorb to the liposomes before use. Once reconstituted, the suspension had a FVIII activity of 150 IU/ml.

[0161] The test individual was dosed SQ at 100 IU/kg. Calculation of the volume of drug to be administered was carried out according to the following equation:

Dose volume (ml)=(ab)/c [0162] Where: a is the target dose (100 IU/kg) [0163] b is the weight of the dog (kg) [0164] c is the rFVIII activity (150 IU/ml)

[0165] Following dosing, the test animal was observed for clinical signs. Unexpected toxicities were screened for by performing CBC and serum chemistry tests at 48 hr and 5 days post-dose. Fibrinogen, FDPs and the thrombin time (TT) were evaluated to test for increased thrombosis risk.

[0166] Blood samples (5 ml) were taken from the dog dosed SQ at the following times points after administration: [0167] Pre-drug administration and at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 hours post-dose.

[0168] Whole blood (non-citrated; 1 ml) was used for the whole blood clotting assay and the activated clotting time assay. The remaining 4m1 blood samples were transferred into tubes containing 0.109M tri-sodium citrate anticoagulant (9:1 v/v) on ice.

[0169] The activated Partial Thromboplastin Time (aPTT), Activated Clot Time (ACT) and Thromboelastogram (TEG) assays were conducted on the citrated whole blood.

[0170] Plasma was prepared by centrifugation of the remaining citrated blood and the resulting plasma samples were stored in aliquots of approximately 100 l at 80 C.

[0171] Assays

[0172] (i) Non-Citrated Whole Blood: Whole Blood Clotting Assay

[0173] Blood samples were divided between 2 vacutubes, (20.5 ml) and observed carefully with periodic and judicious levelling of the tube until a clot was determined by interruption of flow in the fully horizontal position. The quality of the clot was observed by holding the tube in the fully inverted position. The whole blood clotting time was recorded as the mean of the total time from sample extraction until visual observation of blood clot for both samples and the quality of the clot in the inverted position was be noted.

[0174] (ii) Citrated Whole Blood: Thromboelastogram (TEG) Assay

[0175] TEG was performed with re-calcified citrated whole blood using a Hemostasis Analyzer Model 5000 (Haemoscope Corporation) thromboelastograph according to the manufacturers' recommendations. Briefly, 1 ml of citrated whole blood was placed in a commercially available (TegeHemostasis System Kaolin, Haemonetics) vial containing kaolin. Mixing was ensured by gentle inversion of the kaolin-containing vials 5 times. Pins and cups were placed in the TEG analyzer in accordance with the standard procedure recommended by the manufacturer. Each standard TEG cup was placed in the 37 C. pre-warmed instrument holder and was filled with 20 l of calcium chloride (0.2 M). Then, 340 l of kaolin-activated citrated whole blood was added for a total volume of 360 l.

[0176] (iii) Activated Clotting Time (ACT) and Activated Partial Thromboplastin Time (aPTT)

[0177] The ACT and aPTT tests were carried out using a Haemachron Jr coagulation analyzer (International Technidyne Corps.) according to the manufacturer's instructions.

[0178] (iv) Plasma: FVIII Activity Assay (Chromogenic)

[0179] FVIII plasma activity was determined using the Coatest Assay (Dia Pharma, West Chester, Ohio). Plasma samples were diluted 1:20 to 1:80 with assay diluent and assayed according to the manufacturers instructions. Standard curves were established using normal hemostasis reference plasma (american diagnostica inc, Stamford, Conn.) and the purified PEG-FVIII protein.

[0180] (v) Plasma: FVIII ELISA

[0181] The concentration of FVIII antigen in plasma samples will be determined by ELISA using the Visulize FVIII antigen kit from Affinity Biologicals (Ancaster, Ontario, Canada) according to the manufacturer's instructions.

[0182] (vi) Plasma: Immunogenicity

[0183] Bethesda assays were conducted on 1:4, 1:10 and 1:20 dilutions of test plasma into FVIII deficient human plasma. Equal volumes of the diluted test plasma and normal human reference plasma were incubated at 37 C. for 2 hours and the Bethesda titre determined using the aPTT assay and a normal human plasma standard curve as described above.

TABLE-US-00002 TABLE 2 Dog Number 1 Dog weight (kg) 16.4 Dose (IU/kg) 100 rFVIII batch number Lot 270LR8WB Volume of PEGLip diluent used (ml) 13.3 ml Volume administered (ml) 10.93

[0184] Results of the study are shown in Table 3.

TABLE-US-00003 TABLE 3 Time WBCT FVIII [FVIII] Date Time post- WBCT 1 WBCT 2 average ACT-LR aPTT-cit TEG activity) (ELISA) Bethesda (dd/mm/yy) (hh:mm) dose (h) (min) (min) (min) (sec) (sec) (r:min) (IU/ml) (% normal) assay (U) 26/11/2013 0 22 34 28 367 189.1 ND ND 03/04/2014 0 28 28 28 347 300 60 0 ND ND 08/04/2014 12:00 pm 0.50 20 22 21 332 300 1 12:30 pm 1.00 10 10.5 10.25 270 158.1 30.2 0.6 2.1 1:30 pm 2.00 8.5 10 9.25 193 129.8 21.9 1.4 4 3:25 pm 4.00 10 12 11 211 126.2 15.6 1.9 4.3 7:35 pm 8.00 7 8 7.5 200 99.4 21.9 1.4 5.2 10:45 pm 11.25 9.5 10.5 10 207 94.7 20.9 2.1 5.4 09/04/2014 11:15 am 23.75 12 12 12 213 178.4 18.1 3.5 5:05 pm 29.50 12 15 13.5 273 140.8 22.6 0.4 3.1 10/04/2014 12:00 pm 48.00 18 18 18 326 156.1 60 3.1 11/04/2014 09:40 70.16 26 24 25 305 387.7 60 2.4 ND ND = Not Detectable