Use of hydrophobic organic acids to increase hydrophobicity of proteins and protein conjugates

• US 10,046,058 B2
• Filed: 11/30/2015
• Issued: 08/14/2018
• Est. Priority Date: 12/02/2014
• Status: Active Grant

• Current Assignee: Rezolute, Inc.
• Original Assignee: Rezolute, Inc.
• Inventors: Rosendahl, Mary S., Mantripragada, Sankaram B., Gomez, Eliana B.
• Primary Examiner(s): Orwig, Kevin S
• Application Number:
  US14/954,591
• Publication Number:
  US 20160151510A1
• Time in Patent Office: 988 Days
• Field of Search: None
• CPC Class Codes: A61K 31/194 having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
• CPC Class Codes: A61K 38/08 Peptides having 5 to 11 amino acids A61K38/043 - A61K38/046 take precedence
• CPC Class Codes: A61K 38/16 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof enzyme inhibitors A61K38/005
• CPC Class Codes: A61K 38/26 Glucagons
• CPC Class Codes: A61K 38/27 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 38/29 Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
• CPC Class Codes: A61K 47/12 Carboxylic acids; Salts or anhydrides thereof
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 9/0019 Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner non-active ingredients are additionally classified in A61K47/00
• CPC Class Codes: A61K 9/1647 Polyesters, e.g. poly(lactide-co-glycolide)
• CPC Class Codes: A61K 9/1682 Processes
• CPC Class Codes: A61K 9/5031 obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/02 of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
• CPC Class Codes: A61P 5/06 of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: A61P 5/18 of the parathyroid hormones

Abstract

Examples may include a microsphere. The microsphere may include a biodegradable polymer. Furthermore, the microsphere may include a protein mixture selected from the group consisting of a protein-polyethylene glycol conjugate, the protein-polyethylene glycol conjugate and the hydrophobic anion of the organic acid, a protein and the hydrophobic anion of the organic acid, and combinations thereof. Examples may also include a method of making the microspheres. The method may further include reacting a polyethylene glycol with a protein to form a protein-PEG conjugate. In addition, the method may include protonating an amino group on the protein-PEG conjugate with a hydrophobic organic acid to form the protein-PEG conjugate salt. Furthermore, the method may include mixing the protein-PEG conjugate salt in an organic solvent with a biodegradable polymer. The method may also include emulsifying the mixture. Additionally, the method may include hardening the emulsified mixture into the controlled-release microspheres.

Claims

1. A microsphere comprising:

a biodegradable polymer selected from the group consisting of a polylactide;

a polyglycolide;

a poly(d,l-lactide-co-glycolide);

a polycaprolactone;

a polyorthoester;

a copolymer of a polyester and a polyether; and

a copolymer of polylactide and polyethylene glycol; and

a protein mixture selected from the group consisting of a protein-polyethylene glycol conjugate and a hydrophobic anion of an organic acid, a protein and the hydrophobic anion of the organic acid, and combinations thereof, wherein the organic acid comprises pamoic acid, docusate hydrogen, furoic acid, or mixtures thereof;

and wherein the microsphere is formed by a process comprising;

adding the organic acid to the protein-polyethylene glycol conjugate or the protein in a water-immiscible solvent in an organic phase to form a water-immiscible solution, wherein the addition occurs without the presence of water.

2. The microsphere of claim 1, wherein the protein of the protein-polyethylene glycol conjugate or the protein comprises human growth hormone.

3. The microsphere of claim 1, wherein the protein of the protein-polyethylene glycol conjugate or the protein comprises glucagon-like peptide-1.

4. The microsphere of claim 3, further comprising an insulin-polyethylene glycol conjugate.

5. The microsphere of claim 1, wherein the protein of the protein-polyethylene glycol conjugate or the protein is selected from the group consisting of insulin, parathyroid hormone, a fragment of parathyroid hormone, enfuvirtide, and octreotide.

6. The microsphere of claim 1, wherein:

the protein mixture comprises the protein-polyethylene glycol conjugate, and

the polyethylene glycol of the protein-polyethylene glycol conjugate comprises methoxy polyethylene glycol.

7. The microsphere of claim 1, wherein:

the protein mixture comprises the hydrophobic anion of the organic acid, and

the organic acid comprises pamoic acid, and the hydrophobic anion comprises a pamoate anion.

8. The microsphere of claim 1, wherein:

the protein mixture comprises the protein-polyethylene glycol conjugate and the hydrophobic anion of the organic acid, and

a molar ratio of the hydrophobic anion of the organic acid to the protein-polyethylene glycol conjugate ranges from 1;

1 to 11;

1.

9. The microsphere of claim 1, wherein:

the protein mixture comprises the protein-polyethylene glycol conjugate, and

the protein-polyethylene glycol conjugate comprises a monoPEGylated conjugate.

10. The microsphere of claim 1, wherein the biodegradable polymer comprises poly(d, l-lactide-co-glycolide).

11. The microsphere of claim 1, wherein the biodegradable polymer has a molecular weight from 7,000 Da to 17,000 Da.

14. The microsphere of claim 1, wherein:

the protein mixture comprises the protein-polyethylene glycol conjugate and the hydrophobic anion of the organic acid, and

a molar ratio of the hydrophobic anion of the organic acid to the protein-polyethylene glycol conjugate ranges from 3;

1 to 8;

1.

15. The microsphere of claim 1, wherein the hydrophobic anion has a water-octanol partition coefficient log P greater than 1.

16. The microsphere of claim 1, wherein the process further comprises protonating at least one amino group on the protein or the protein-polyethylene glycol conjugate.

12. A method of making controlled-release microspheres containing a protein-PEG conjugate salt;

the method comprising;

providing an aqueous protein solution comprising a protein and a pH buffer;

reacting a polyethylene glycol with the protein to form a protein-PEG conjugate;

protonating an amino group on the protein-PEG conjugate with a hydrophobic organic acid in a water-immiscible solvent in an organic phase without the presence of water to form a water-immiscible solution, wherein the protonation forms the protein-PEG conjugate salt having a hydrophobic anion;

mixing the protein-PEG conjugate salt in an organic solvent with a biodegradable polymer to form a mixture, wherein;

the hydrophobic anion of the protein-PEG conjugate salt increases the solubility of the protein-PEG conjugate salt in the organic solvent,

the biodegradable polymer is selected from the group consisting of a polylactide;

a polyglycolide;

a poly(d,l-lactide-co-glycolide);

a polycaprolactone;

a polyorthoester;

a copolymer of a polyester and a polyether; and

a copolymer of polylactide and polyethylene glycol, and the organic acid comprises pamoic acid, docusate hydrogen, furoic acid, or mixtures thereof;

emulsifying the mixture of the protein-PEG conjugate salt and the biodegradable polymer in an aqueous solution to form an emulsified mixture; and

hardening the emulsified mixture of the protein-PEG conjugate salt and the biodegradable polymer into the controlled-release microspheres.

13. The method of claim 12, wherein the hydrophobic organic acid comprises pamoic acid.