Formulations of human tissue kallikrein-1 for parenteral delivery and related methods

a technology of human tissue and kallikrein, which is applied in the direction of drug composition, peptide/protein ingredient, metabolic disorder, etc., can solve the problems of many complications, achieve the effects of improving pharmacokinetics, improving bioavailability, and improving systemic pharmacokinetics

Inactive Publication Date: 2013-11-28
DIAMEDICA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0005]In some embodiments, administering the composition subcutaneously produces improved systemic pharmacokinetics relative to intravenously administration of the composition. In some embodiments, the improved pharmacokinetics comprises increased bioavailability. In some embodiments, the improved pharmacokinetics comprises increased Tmax for a subcutaneous

Problems solved by technology

Untreated, diabetes can

Method used

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  • Formulations of human tissue kallikrein-1 for parenteral delivery and related methods
  • Formulations of human tissue kallikrein-1 for parenteral delivery and related methods
  • Formulations of human tissue kallikrein-1 for parenteral delivery and related methods

Examples

Experimental program
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Effect test

example 1

[0117]A cDNA coding for pre-pro-human KLK1, the 262 amino acid residue sequence depicted in SEQ ID NO:2, was purchased from OriGene™ (Rockville, Md., USA). The KLK1 cDNA (Catalogue No. SC122623) is a human cDNA open reading frame clone, cloned into the multi-cloning site of OriGene's pCMV6-XL5 vector, between a cytomegalovirus (CMV) promoter to control transcription of cDNA coding for pre-pro-human KLK1 and a polyadenylation signal. This KLK1 clone was sequenced and, using translation software, translated to reveal SEQ ID NO:2. This sequence differed at 2 amino acid residues from the human KLK1 sequence in GenBank as Ref No. NP—002248.1 (SEQ ID NO:1). As depicted in SEQ ID NO:2, single nucleotide polymorphisms (SNP's) resulted in an E to Q change at amino acid residue 145 of 262, and an A to V change at amino acid position 188 of 262. All subsequent experiments were performed with KLK1 having the amino acid sequence in SEQ ID NO:2.

[0118]The human KLK1 cDNA in the pCMV6-XL5 was trans...

example 2

[0121]The purified recombinant human KLK1 contained approximately 30% carbohydrate content based on the molecular weight estimated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (see FIG. 1). KLK1 from CHO cells appears as a band having an apparent molecular weight of ˜40 to 49 kDa; such a broad band may result from different glycosylation foams of KLK1 secreted by CHO cells. For KLK1 expressed in 293 cells, two bands appeared on the SDS-PAGE gel at approximately 40 kDa and 45 kDa. The identity of the bands as human KLK1 was confirmed by Western blot analysis using mouse polyclonal antibody raised against a full-length human KLK1 protein (Catalog # H00003816-B01P, KLK1 purified MaxPab mouse polyclonal antibody (B01P), Abnova Corporation, Walnut, Calif., USA) (see FIG. 2). The Western blot confirms the results of the SDS-PAGE gel, in that recombinant human KLK1 from CHO cells appears as a band having an apparent molecular weight of ˜40 to 49 kDa, and KLK1 express...

example 3

[0126]Generation of concentrated formulation of rhKLK1. The following example determined that rhKLK1 could be concentrated to at least 50 mg / mL, and that the resulting high concentration formulation would be stable and not aggregate.

[0127]The rhKLK1 having at least 4 moles sialic acid per mole protein was formulated into formulations having 5 mg / mL, 10 mg / mL, 25 mg / mL and 50 mg / mL Bulk rhKLK1 was concentrated using VIVASPIN® 500 Centrifugal Concentrator 3,000 MWCO PES membrane. The protein concentration of rhKLK1 was determined by absorbance at 280 nm and the samples were close to the target concentration (5.0 mg / mL, 9.9 mg / mL, 26.2 mg / mL and 50.3 mg / mL). Following concentration, the samples were sterile filtered through a 0.2 μm filter. The rhKLK1 samples at the four different concentrations were filled into Schott borosilicate 3 cc, 13 mm serum vials at a fill volume of 0.5 mL, and the vials were sealed with a Daiko 13 mm injection stopper, Flourotec Plus, B2-40 rubber stopper. Th...

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Abstract

Provided are high concentration compositions of tissue kallikrein-1 (KLK1) and methods of parenterally administering such compositions to a subject in need thereof, where absorption into the circulation via, for example, intravenous or subcutaneous administration improves systemic pharmacokinetics, bioavailability, safety, and/or convenience relative to intravenous or other forms of administration. Also provided are recombinant human KLK1 (rhKLK1) polypeptides that can be readily concentrated to high protein concentrations, and substantially pure compositions thereof.

Description

CONTINUING APPLICATION DATA[0001]This application claims the benefit of U.S. Provisional Application Ser. No. 61 / 652,069, filed May 25, 2012, and U.S. Provisional Application Ser. No. 61 / 791,762, filed Mar. 15, 2013, each of which is incorporated by reference herein.BACKGROUND[0002]In healthy individuals, insulin release by the pancreas is strictly coupled to the blood glucose concentration. Elevated blood glucose levels like those occurring after meals are rapidly compensated by a corresponding rise in insulin secretion. Diabetes mellitus, or simply diabetes, is a group of metabolic diseases in which a person has high blood sugar, either because the pancreas does not produce enough insulin, or because cells do not respond to the insulin that is produced. Around 366 million people worldwide suffer from diabetes mellitus. Untreated, diabetes can cause many complications. Acute complications include diabetic ketoacidosis and nonketotic hyperosmolar coma. Serious long-term complication...

Claims

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Application Information

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IPC IPC(8): A61K38/48A61K9/00
CPCA61K38/4853A61K9/0019A61P3/10
Inventor CHARLES, MATTHEW
Owner DIAMEDICA INC
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