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Preparation and composition of inter-alpha inhibitor proteins from human plasma for therapeutic use

A technology of alpha inhibitors and compositions, applied in the fields of inhibiting metastasis and treating sepsis

Active Publication Date: 2008-04-09
POROTHERA BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has become difficult to treat sepsis quickly and reliably with conventional drugs

Method used

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  • Preparation and composition of inter-alpha inhibitor proteins from human plasma for therapeutic use
  • Preparation and composition of inter-alpha inhibitor proteins from human plasma for therapeutic use
  • Preparation and composition of inter-alpha inhibitor proteins from human plasma for therapeutic use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Characterization of H4 as part of the IαIp complex

[0124] SELDI-TOF mass spectrometry analysis indicated that heavy chain 4 (H4) was also present in a 125 kDa band (PαI) and a 250 kDa band (IαI) (data not shown). The presence of H4 is more pronounced in the 250 kDa band. This indicates that another complex protein other than IαI (H1+H2+LC) and PαI (H3+LC) may be present in some compositions of the invention. To date, no complex form of H4 has been described. Free H4 is less than 125 or 250 kDa. Due to the mass spectrometry results, we thought that H4 might be complexed with bikunin (light chain) or others.

Embodiment 2

[0126] Animal Models of Sepsis

[0127] Male Sprague-Dawley rats (275-325 g) were housed in a temperature-controlled room on a 12-h light / dark cycle and fed a standard Purina rat solid diet. Before the induction of sepsis, rats were fasted overnight but had access to water ad libitum. Rats were anesthetized with isoflurane inhalation, the ventral neck, abdomen and groin were shaved and washed with 10% povidone-iodine. A 2-cm midline laparotomy was performed. The cecum is exposed, ligated at the end of the ileocecal valve to avoid ileus, punctured twice with an 18-gauge needle, and gently squeezed to allow a small amount of fecal matter to flow out of the hole and return to the abdominal cavity; the abdominal incision is then sutured layer by layer . Sham-operated animals (ie, control animals) were subjected to the same procedure except that the cecum was neither ligated nor punctured. Animals were revived immediately after surgery with subcutaneous 3ml / 100gBW saline. Anim...

Embodiment 3

[0150] Purification of IαIp

[0151] After applying dialysis or ultrafiltration / diafiltration, the eluate was extracted with DEAE Sephadex A50 solid phase, and the pH6.0 containing 0.28M sodium chloride 0.005M sodium citrate / 0.0055M sodium phosphate buffer was used The column elutes weakly bound components (described in Hoffer et al., Journal of Chromatography B 669 (1995) 187-196). In the previous step, the column was washed with 0.005M sodium citrate / 0.0055M sodium phosphate buffer containing 0.20M sodium chloride at pH 6.0.

[0152] After dialysis or ultrafiltration / diafiltration (UF / DF) against 0.005M sodium phosphate buffer at pH 7.0, the eluate was applied to a hydroxyapatite column. IαIp does not bind to the column and is collected as the effluent fraction. Contaminant proteins, mainly FII, FVII, and FX, can be eluted using a gradient of increasing concentrations of sodium phosphate buffer. IαI / PαI contains more than 90% of the target protein, mainly IαI and PαI.

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Abstract

The present invention relates to an inner-alpha inhibitor protein (I alpha Ip). The invention also relates to a method for purifying the I alpha Ip composition and the usage for treating the human diseases such as pyaemia, septicemic shock, atrophic arthritis, cancer and infectious disease.

Description

[0001] related application [0002] This application contains relevant information disclosed in Provisional Patent Application Serial No. 60 / __, filed on November 8, 2003, entitled "Preparation and Compositions of Endo-alpha Inhibitor Proteins from Human Plasma for Therapeutic Use" subject matter, the disclosure of which is hereby incorporated by reference in its entirety. [0003] governmental support [0004] This invention was supported in part by National Institutes of Health grants R01 GM053008, R01 GM057468 and R43 GM065667. Background of the invention [0005] The intra-alpha inhibitor protein (IαIp) family is a group of plasma-associated serine protease inhibitors. Members of this family are composed of heavy and light chain polypeptide subunits covalently linked by glycosaminoglycans. The light chain, also known as bikunin, is responsible for the molecule's serine protease inhibitor activity. The name "bikunin" reflects the presence of two Kunitz-type protease inh...

Claims

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Application Information

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IPC IPC(8): A61K35/14A61K35/16A23J1/00C07K17/00
CPCG01N2800/52
Inventor Y·－P·林D·乔斯克D·C·希克森
Owner POROTHERA BIOLOGY
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