Composition and process for a lipid NANO-particle delivery of oxygen and vital metabolic compounds for hemorrhagic shock

Abstract

Generation of lipid nano-particles containing purified human hemoglobin as an oxygen carrier was accomplished with the co-encapsulation of metabolic agents. Additionally, the nano-particles described are of the long-circulating variety (Pegylation of the exterior surface) which are more tolerable in vivo when compared against their non-pegylated counterparts. The generated nano-particles are lyophilizable, given proper formulation with lyo-protectants, which are storage stable solids ready for reconstitution prior to use.

Description

COMPOSITION AND PROCESS FOR A LIPID NANO-PARTICLE DELIVERY OFOXYGEN AND VITAL METABOLIC COMPOUNDS FOR HEMORRHAGIC SHOCKFIELD OF THE INVENTION

[0001] This invention relates to a composition and process for the generation of, and for the delivery of oxygen and metabolic factors / co-factors to organs in need due to insufficient ability of red blood cells to perform these functions.BACKGROUND OF THE INVENTION

[0002] Physiologically, the prevalent allosteric effector for hemoglobin in humans is 2,3- DPG (2,3-Diphosphoglycerate) while other phosphate-containing molecules have been identified as important effectors in other species. Other phosphate containing allosteric effectors include: IHP (inositol hexa-phosphate), sphingosine monophosphate, ATP (adenine triphosphate), and GTP (guanosine triphosphate), the latter two of which are believed to play important roles in fish and NAD(H) / NADP(H). In human RBC (red blood cells), 2,3-DPG and its variability dominate the allostery for Hb. Although ATP has a presence in RBC, its concentration (1 uM) and the chelation of ATP with Mg2+ions (Mg2+ at 3 uM in RBC reduces the ability of ATP to bind to the Hb) significantly renders ATP to a lesser role as an allosteric effector. GTP is found in RBC but at low concentrations relative to ATP.

[0003] ATP and GTP are key physiological molecules for energy in many cellular functions. In hemorrhagic shock, a substantial loss of ATP from major organs occurs. Intravenous administration of ATP-MgCL has ample president in partial restoration in organs such as the liver and kidney. The short circulatory stability of ATP hinders the effectiveness of direct systemic infusion. Liposomal entrapped ATP-MgCh has been described to deliver the ATP to various organs during ischemia, including the liver during hemorrhagic shock.SUMMARY OF THE INVENTION

[0004] Generation of lipid nano-particles containing purified human hemoglobin as an oxygen carrier was accomplished with the co-encapsulation of metabolic agents. The purpose of the metabolic agents is two-fold: to provide for metabolic energy during metabolism in cells of injured organs, which were the consequence of loss of RBCs, andto act as an allosteric effector for the hemoglobin in its ability to transport oxygen. The allosteric effector provides for a change in the ability of the hemoglobin to bind hemoglobin, with a lower affinity in effected organs (higher p50, release of oxygen) while distinctly higher in the lung capillaries (lower p50, higher oxygen binding). Although available in RBCs, metabolic agents described in this application are not effective as allosteric effectors due to low relative concentrations. Blood substitutes of purified hemoglobin, or polymerized hemoglobin have been reported and contain allosteric effectors such as 2,3-DPG, IHP or pyroxidal-5-phosphate while failing to use the nucleoside metabolites, ATP, UTP, CTP, GTP, NADP(H), FADP etc. In this study we show that the use of nucleoside metabolic agents gives p50 values for hemoglobin, encapsulated in nano-particles, which are ideal for oxygen transfer. These nucleoside metabolic agents are also available for restoration of cellular functions in damaged cells of effected organs.

[0005] Additionally, the nano-particles described herein are of the long-circulating variety (Pegylation of the exterior surface) which are more tolerable in vivo when compared against their non-pegylated counterparts. The generated nano-particles are lyophilizable, given proper formulation with lyo-protectants, which are storage stable solids ready for reconstitution prior to use.

[0006] It is specifically noted that every combination and sub-combination of the features and embodiments described herein is considered to be part of the invention.DETAILED DESCRIPTION OF THE INVENTION

[0007] Example 1: Formation of Lyophilized Lipids

[0008] DPPC DPPGNa, cholesterol, KC1003 -acetate and DSPE-PEG(2000) are removed from freezer (-20 storage) and allowed to warm to RT. The following amounts were weighed (Table 1) into a 500 mL RB flask.

[0009] Table 1 Lipids for solid mixture

[0010] The lipids were then dissolved in t-BuOH (50 mL ). The hazy solution of lipids in t-BuOH was transferred to a lyophilization jar and frozen in freezer at -80°C

[0011] A lyophilizer chiller was set at < -95°C and vacuum <150 millitorr. The jar with frozen lipids in t-BuOH was attached to lyophilizer and opened to full vacuum. The temperature and pressure in the lyophilizer were checked until steady state achieved 4-6 days. Lyo-cake stored at -80°C until use in the hydration step.

[0012] Example 2: Hydration of Lyo-Cake with Hb

[0013] Purified Hemoglobin thawed ahead of time and kept at 5°C prior to use. (5 mM Hb in 10 mM phosphate buffered water at pH 6.5-6.75. The following additives to the Hb were weighed out: 1) ATP (adenosine triphosphate disodium salt) at 6.0 mg / mL of Hemoglobin and dissolved in WFI (at 500 mg / mL) 2) MgC12 (anhydrous) at 0.36 mg / mL of Hemoglobin and dissolved in WFI (at 100 mg / mL)-exothermic dissolution 3) NaCl at 2.5 mg / mL of Hemoglobin 4) Sucrose at 10 mg / mL Hemoglobin and 5) HPCD (hydroxypropyl cyclodextrin) at 25 mg / mL Hemoglobin.

[0014] The Hemoglobin was filtered first through a 0.22 micron sterile filter followed by a 0.1 micron sterile filter in the biohood. To the filtered Hemoglobin add (in order) was added 1) ATP with swirling, 2) MgC12 with swirling 3) NaCl as a solid, swirl to dissolve 4) Sucrose as a solid, swirl to dissolve 5) HPCD. The mixture was sonicated in a batch sonicator for 20 minutes at ambient temperature. Filter the Hb mixture through a 0.1 micron sterile filter and then a 0.03 sterile membrane filter.

[0015] 200 mL of the filtered Hb mixture was added to the lyophilized lipids (lyophilized lipids produced as a powder, clumps broken before addition of Hb) at ambient temperature and at a rate that prevents formation of clumps. The mixture was stirred 100 rpm for a minimum 2 hrs at room temperature.

[0016] Example 3: Extrusion of Hydrated Lipid Vesicles

[0017] The Hb / hydrated lipid vesicles from the hydration step was added to a 800 mL extruder at room temperature and passed through double stacked 0.8 micron membranes stacked membranes a2 times. The resulting mixture was then passed through, 2 times each, in sequence double stacked 0.6 mi con, 0.4 micron, and finally 0.2 micron membranes. The resulting particle suspension was saturated with CO and chilled at 5°C.

[0018] Example 4: Cross Flow for EM Purification

[0019] PBS, sterile filtered at pH 7.0 was used to remove unencapsulated materials using a 750 K NMWC fiber. Crude particles from extruder was pre-cooled to 4°C in refrigerator, diluted with the PBS buffer and diafiltered with 7X volume turnover for full removal. The resulting material was concentrated to 100 mL using ultrafiltration. Concentrated retentate removed from reservoir and saturated with CO prior to storage at 4°C.

[0020] Example 5: PEG-Post Insertion - KC240808-PP

[0021] To a 250 mL bottle was weighed 4.4 g DSPE-PEG(2000) and dissolved into 110 mL of purified water. The solution was filtered through a 0.1 micron sterile filter.

[0022] 175 mL of the purified particle suspension of Hb (KC240808) and 100 mL of the40 mg / mL solution of DSPE-PEG(2000) were added to a IL three neck flask fitted with a gas inlet, gas outlet (attached to a ballon) and a thermometer. With magnetic stirring the flask was filled with CO at ambient temperature. CAUTION: CO is highly poisonous and should only be used in a hood designated for use of CO and has an audible CO alarm in proximity to the hood

[0023] The CO was released into the fume hood and the fill-release steps repeated 3X. After the flask was purged, the ballon is partially inflated with CO. The mixture was heated in a water bath to 60°C under the CO atmosphere for 1 hr. Reaction was allowed to cool to ice temperature (ice bath) under CO, once chilled the CO was removed and the resulting mixture purified by cross-low filtration to remove any residual DSPE- PEG(2000) using PBS.

[0024] Table 2: KC24808-PP Properties

[0025] Example 6: Lyophilization of Cleam EM

[0026] 2.74 g of HPCD (EMPROVE EXPERT Cyclodextrin HPB) and 1 .82 g of BSA(Bovine Serum Albumin, low-endotoxin) were weighed out in a 250 mL bottle. This mixture was dissolved into 75 mL purified water for injection and then filtered through a 0.1 micron filter.

[0027] To a 20 mL sterile de-pyrogenated vial was added 5.3 mL of EM and 5.3 mL of the HPCD / BSA solution. The vials were argon purged prior to lyophilization. The lyophilization cycle (Table 3) was carried out giving Red solid lyophilized cakes which were stored at 5 °C.

[0028] Table 3: Lyophilization Cycle

[0029] Example 7: ATP as Allosteric Effector and Metabolism Additive

[0030] 5 mL of purified hemoglobin at 5 mM in 10 mM phosphate buffer at pH 6.75 is filtered through a 0.1 micron filter. To the filtered Hb is added as appropriate (see Table) HPCD, Glucose, NaCl, MgC12 and ATP. The mixture is sonicated for 1 minute in a bath sonicator and then filtered through a 0.05 micron syringe filter.

[0031] Into a 20 mL reaction vessel is weighed 240 mg of solid lipid mixture (DPPC, DPPGNa, Cholesterol, KC1003 and DSPE-PEG(2000) in 54.3:8:30:7.5:0.3 molar ratio). The Hb mixture from above is added to the lipids and mixed on an orbital shaker for 2 hours.

[0032] The crude vesicles were then extruded under nitrogen at ambient temperature using first stacked 0.8 / 0.6 micron membranes (2 passes) and then 2 passes through 0.4 / 0.2 / 0.2 stacked membranes.

[0033] The extruded mixture was purified by use of Sepharose 4B-CL using PBS to elute the particles.

[0034] For measurement of oxygen loading and offloading (p50 values), samples were gas exchanged under visible light using pure oxygen.

[0035] Table 4: NaCl, Glucose and ATP Components of Selected Specimens from Example 7

[0036] Table 5: Characteristics of EM with ATP as allosteric effector and metabolism additive

[0037] Table 6: ATP with MgC12 as allosteric effector and metabolism additive

[0038] Table 7: Characteristics of EM with ATP and MgC12

[0039] Example 8: NADP(H) as Allosteric Effector and Metabolism Additive

[0040] 5 mL of purified hemoglobin at 5 mM in 10 mM phosphate buffer at pH 6.75 is filtered through a 0.1 micron filter. To the filtered Hb is added as appropriate (see Table) HPCD, Glucose, NaCl, NADP(H) and MgC12. The mixture is sonicated for 1 minute in a bath sonicator and then filtered through a 0.05 micron syringe filter.

[0041] Into a 20 mL reaction vessel is weighed 240 mg of solid lipid mixture (DPPC, DPPGNa, Cholesterol, KC1003 and DSPE-PEG(2000) in 54.3:8:30:7.5:0.3 molar ratio). The Hb mixture from above is added to the lipids and mixed on an orbital shaker for 2 hours.

[0042] The crude vesicles were then extruded under nitrogen at ambient temperature using first stacked 0.8 / 0.6 micron membranes (2 passes) and then 2 passes through 0.4 / 0.2 / 0.2 stacked membranes.

[0043] The extruded mixture was purified by use of Sepharose 4B-CL using PBS to elute the particles.

[0044] Table 8: EM with NADP(H)

[0045] Table 9: Selected characteristics of NADP(H) containing EM

[0046] Table 10: Additional Metabolic Compounds as Effectors-CompositionGTP = Guanosine Triphosphate UTP = Uridine TriphosphateCTP = Cytidine Triphosphate

[0047] Table 11: Additional Metabolic Compounds as Allosteric Effectors

[0048] Table 12: Composition of Comparative Example with 2,3-DPG

[0049] Table 13: Comparative example with 2,3-DPG

[0050] Notwithstanding the specific embodiments, features, elements, combinations and sub-combinations disclosed herein, it is expressly considered and here disclosed that every single element, every single feature, and every combination and sub-combination thereof disclosed herein may be combined with every other element, feature, combination and sub-combination disclosed herein.

[0051] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as outlined in the present disclosure and defined according to the broadest reasonable reading of the claims that follow, read in light of the present specification.

Claims

Claims1. A composition for the delivery of oxygen and metabolic factors / co-factors to organs in need due to insufficient ability of RBCs to perform these functions, the composition comprising a nanoparticle encapsulating an oxygen carrier and a metabolic agent.

2. The composition of claim 1, further comprising a lipid.

3. The composition of claim 1, wherein Hemoglobin is the oxygen carrier.

4. The composition of claim 1, wherein the metabolic factor is a nucleotide triphosphate.

5. The composition of claim 4, wherein the nucleotide triphosphate serves as an allosteric effector of hemoglobin in oxygen delivery.

6. The composition of claim 5, wherein the nucleotide triphosphate facilitates the release of oxygen in organs.

7. The composition of claim 5, wherein the nucleoside triphosphate is Adenosine triphosphate.

8. The composition of claim 5, wherein the nucleoside triphosphate is Guanosine triphosphate.

9. The composition of claim 1, wherein the metabolic factor is a nucleotide diphosphate.

10. The composition of claim 9, wherein the nucleotide diphosphate is Adenosine diphosphate.

11. The composition of claim 9, wherein the nucleotide diphosphate is Guanosine diphosphate.

12. The composition of claim 9, wherein the nucleotide diphosphate is NAD(H).

13. The composition of claim 9, wherein the nucleotide diphosphate is NADP(H).

14. The composition of claim 9, wherein the nucleotide diphosphate also serves as an allosteric effector of hemoglobin in oxygen delivery.

15. The composition of claim 1 in the form of a solid.

16. The composition of claim 1 in the form of an injectable suspension reconstituted from a lyophilized solid.

17. The composition of claim 1 further comprises a lyo-protectant.

18. The composition of claim 16 further comprising an encapsulated lyo-protectant.

19. The composition of claim 17 which is a saccharide, disaccharide, or polysaccharide.

20. The composition of claim 18 which is a saccharide, disaccharide, polysaccharide.

21. The composition of claim 17 wherein the lyo-protectant also serves as a metabolic agent for the sequestering of cholesterol.

22. The composition of claim 18 wherein the lyo-protectant also serves as a metabolic agent for the sequestering of cholesterol.

23. The composition of claim 21, wherein the lyo-protectant is HPCD.

24. The composition of claim 22, wherein the lyo-protectant is HPCD.

25. The composition of claim 17 wherein the lyo-protectant is Albumin.

26. The composition of claim 17, further comprising Albumin, wherein the composition is a saccharide, disaccharide, polysaccharide.

27. The composition of claim 26 wherein Albumin and HPCD are co-lyo-protectants.

28. The composition of claim 1, further comprising a volume expander (oncotic agent).

29. The composition of claim 28, wherein Albumin is the volume expander (oncotic agent).

30. The composition of claim 15 further comprising an agent which facilitates storage stability.

31. The composition of claim 30, wherein the reducing agent is a sugar.

32. The composition of claim 31, wherein sucrose is the reducing agent.

33. The composition of claim 31, wherein Glucose or Dextrose is the reducing agent.

34. The composition of claim 1, wherein Magnesium 2+ is a co-factor.

35. The composition of claim 34, wherein MgCE, MgSC , Mg(NOa)2, and / orMg(carboxylate)2 is a source of Mg21.

36. The composition of claim 1 wherein the metabolic factor / co-factor is Magnesium nucleotide triphosphate.

37. The composition of claim 34, wherein the metabolic factor / co-factor is Mg-ATP.

38. The composition of claim 34, wherein the metabolic factor / co-factor is Mg-GTP.

39. The composition of claim 2 further comprising a component which facilitates the delivery of oxygen in organs as a pH-shuttle for the allosteric effector.

40. The composition of claim 39, which incorporates a pH-sensitive amino acid, dipeptide or polypeptide.

41. The composition of claim 40, wherein the component is KC1003.

42. The composition of claim 1, further comprising Cholesterol.

43. The composition of claim 1, further comprising a Pegylated lipid.

44. The composition of claim 1, further comprising a negatively charged lipid.

45. The composition of claim 1, further comprising a diacyl phosphatidylcholine.

46. The composition of claim 43 wherein the pegylated lipid is DSPE-PEG(2000).

47. The composition of claim 44, wherein the negatively charged lipid is DPPG.

48. The composition of claim 45, wherein the phosphatidylcholine is DPPC.

49. The composition of claim 3 further comprising KC1003, Cholesterol, a pegylated lipid, a negatively charged lipid and a diacyl phosphatidylcholine.

50. The composition of claim 49 further comprising DSPE-PEG(2000), DPPG and DPPC.

51. The composition of claim 1 further comprising KC1003, cholesterol, DSPE- PEG(2000), DPPG, DPPC, HPCD, sucrose, hemoglobin, ATP, Mg2+and albumin.

52. The composition of claim 51 in the form of a lyophilized solid.

53. The composition of claim 51 in the form of a reconstituted suspension of a lyophilized solid.

54. A method for using the composition of claim 1 for treatment of hemorrhagic shock.

55. A method for using the composition of claim 1 for treatment of ischemia.

56. A method for using the composition of claim 51 for treatment of hemorrhagic shock.

57. A method for using the composition of claim 51 for treatment of ischemia.

58. A method for using the composition of claim 49 for treatment of59. A method for the manufacture of the composition of claim 1 comprising the steps of hydration of a lipid mixture with an aqueous solution forming, vesicles, size reduction of vesicles formed during the hydration step, removal of non-encapsulated materials in the aqueous solution.

60. The composition of any of claims 1-14 in the form of an aqueous solution.

61. The composition of claim 60 further comprising a lyo-protectant.

62. The composition of claim 61 which is a saccharide, a disaccharide, or a polysaccharide.

63. The composition of claim 61, wherein the lyo-protectant also serves as a metabolic agent for the sequestering of cholesterol.

64. The composition of claim 63, wherein the lyo-protectant is HPCD.

65. The composition of claim 60 further comprising an agent which facilitates storage stability.

66. The composition of claim 65, wherein the agent is a reducing sugar.

67. The composition of claim 66, wherein the reducing sugar is Sucrose.

68. The composition of claim 66, wherein the reducing sugar is Glucose or Dextrose.

69. The composition of claim 60, wherein Magnesium 2+ is a co-factor.

70. The composition of claim 69 comprising MgCh, MgSCE, Mg(NCh)2, Mg(carboxylate)2 as a source of Mg2+.

71. The composition of claim 60, wherein the metabolic factor / co-f actor is Magnesium nucleotide triphosphate.

72. The composition of claim 60, wherein the metabolic factor / co-f actor is Mg-ATP or Mg-GTP73. The composition of claim 60, further comprising a component which facilitates the delivery of oxygen in organs as a pH-shuttle for the allosteric effector.

74. The composition of claim wherein the pH shuttle incorporates a pH-sensitive amino acid, dipeptide or polypeptide.

75. The composition of claim 73, wherein the component is KC1003.

76. The composition of claim 60 further comprising Cholesterol.

77. The composition of claim 60, further comprising a Pegylated lipid.

78. The composition of claim 60, further comprising a negatively charged lipid.

79. The composition of claim 60, further comprising a diacyl phosphatidylcholine.

80. The composition of claim 77, wherein the pegylated lipid is DSPE-PEG(2000).

81. The composition of claim 78 wherein the negatively charged lipid is DPPG.

82. The composition of claim 79 wherein the diacyl phosphatidylcholine is DPPC.

83. The composition of claim 60, comprisingKC1003, Cholesterol, a pegylated lipid, a negatively charged lipid and a diacyl phosphatidylcholine.

84. The composition of claim 83 comprising KC1003, cholesterol, DSPE-PEG(2000), DPPG and DPPC.

85. The composition of claim 60 comprising KC1003, cholesterol, DSPE-PEG(2000), DPPG, and DPPC in a lipid mixture and HPCD, sucrose, hemoglobin, ATP, and Mg2+in an aqueous phase.

86. The composition of claim 85, wherein components of the aqueous phase are entrapped in vesicles comprised of the lipid mixture.

87. The method of claim 59, further comprising extrusion of the vesicles for size reduction.

88. The method of claim 87, wherein the extrusion of vesicles forms essentially uni- lamillar particles of < 250 nm (mean size distribution).

89. The composition of claim 85 further comprising extruded vesicles.

90. The method of claim 59, further comprising removal of non-encapsulated components from the aqueous phase by TFF.

91. The method of claim 59 further comprising removal of non-encapsulated components from the aqueous phase by size exclusion chromatography.

92. A component of claim 63 which serves as a volume expander (oncotic agent).

93. The method of claim 59, further comprising thermal addition of Lipid-PEG conjugate to nano-particles and removal of excess lipid-PEG.

94. The method of claim 93 comprising extrusion of the vesicles for size reduction.

95. The method of claim 93, comprising extrusion of vesicles to forms essentially uni- lamillar particles of < 250 nm (mean size distribution).

96. The method of claim 93, comprising removal of non-encapsulated components from an aqueous phase by TFF.

97. The method of claim 93, comprising removal of non-encapsulated components from an aqueous phase by size exclusion chromatography.

98. The method of claim 93, further comprising thermal addition of Lipid-PEG to nanoparticles.

99. Lyophilization of a mixture containing lipid nano-particles of KC1003, Cholesterol, DPPC, DPPG, DSPE-PEG(2000) with encapsulated hemoglobin, ATP, HPCD, sucrose and Mg2+with thermally inserted DSPE-PEG(2000) and an aqueous phase containing HPCD and Albumin.

Images