Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1996-05-17
1998-07-07
Weber, Jon P.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
530395, 530415, C07K 1481, A61K 3817
Patent
active
057770816
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a process for producing an inter-alpha-trypsin inhibitor (ITI) concentrate from a human plasma fraction and the concentrate obtained by said process which is suitable for therapeutic use.
BACKGROUND OF THE INVENTION
ITI is a serine-protease inhibitor found in plasma. Its name derives from its activity as a trypsin inhibitor and from its electrophoretic mobility located between that of .alpha..sub.1 and .alpha..sub.2 globulins (thus "inter-alpha").
ITI belongs to the "Kunitz-type inhibitors" family (1-4) and consists of 3 polypeptidic chains, two heavy H1 and H2 and one light, called bikunin, the whole being linked by a glycosaminoglycan (GAG) chain. The light chain is responsible for the antiprotease activity. ITI is a glycoprotein with a molecular mass of 220,000 Da.
ITI is synthesized in the liver and circulates in plasma at a concentration of about 0.5 g/l.
ITI is very sensitive to the action of numerous proteases which degrade it into derivatives of lower molecular weight. Among them UTI (urinary trypsin inhibitor derived from bikunin) is finally found in urine which has allowed its isolation and the study of its properties (5,6); this fragment is responsible for many ITI activities and, in particular, its antiprotease activity.
The physiological role of ITI is not yet totally understood. It shows antiprotease activities, in particular as trypsin and chymotrypsin inhibitor, as inhibitor of elastase and cathepsin G which are liberated by the activation of neutrophil polynuclear cells, as plasmin and kallikrein inhibitor. It could also have a growth factor activity for endothelial cells (7).
Moreover a series of studies on UTI indicate that this part of the molecule can play a role in inflammatory-type disorders such as pancreatitis, polyarthritis, septic choc and secondary symptoms associated with treatment of certain cancers (8-10).
ITI could be considered as a pro-inhibitor able to generate active fragments, for example by the action of proteases liberated during inflammatory reactions, said fragments being able to diffuse into the intercellular spaces and to participate to tissue protection against proteases.
It could thus be advantageous to get a preparation of native ITI, for therapeutic use, which could have a longer half-life and an in situ activity closer to the natural activity than that of UTI (11).
Several papers describe attempts to purify native ITI from plasma, either with ammonium sulfate or PEG followed by anion exchange chromatography, or by affinity chromatography with a zinc chelator or on Blue-Sepharose (12-14). Those various processes require the addition of protease inhibitors to avoid rapid degradation of ITI; these inhibitors are usually incompatible with a therapeutic use; moreover the yields are generally very low.
SUMMARY OF THE INVENTION
The Applicant has thus tried to develop a new process for purifying ITI, from human plasma, applicable on an industrial scale and resulting in a molecule with similar biological and structural properties as the native molecule.
The Applicant has taken advantage of a process for producing a high purity concentrate of blood coagulation Factor IX that he has developed (15, 16, and EP 0 317 376) and that provides a fraction which has not been used industrially until now and which is particularly rich in ITI. It is one aim of the present invention to develop a process for purifying ITI from this fraction, without interfering with the yield of recovery of other blood components.
The Applicant has thus revealed an affinity of ITI for sulfate glycosaminoglycans (like heparin) and has taken advantage of this unexpected property to develop a separation process by affinity chromatography on an immobilized sulfate glycosaminoglycan column, for example on a Sepharose.RTM. gel, with a mild elution in the presence of 0.2M NaCl.
The Applicant has shown that this process was particularly efficient on a large scale (i.e. with plasma volumes possibly greater than 2,000 liters).
The Applicant has inc
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Salier et al. (1981) J. Immunol. Meth., 47(2), "Inter-.alpha.-Trypsin-Inhibitor (ITI): Use of Immunoabsorbents for Preparation of Anti-ITI Antiserum, ITI-Free Human Serum and Purified ITI", pp. 239-248.
Salier et al. (1983) Anal. Biochem., 133(2), "Inter-.alpha.-Trypsin-Inhibitor (ITI): Use of New Antisera for Qualitative Studies and Discrete Quantitation of ITI and its Derivatives", pp. 336-343.
Jochum et al. (1983) Hoppe Seyler's Z. Physiol. Chem. 364(12), "Inter-.alpha.-Trypsin-Inhibitor of Human Serum: An Inhibitor of Polymorphonuclelar Granulocyte Elastase", pp. 1709-1715.
Alberts et al. (1983) "Molecular Biology of the Cell", Garland Publishing, New York, pp. 702-705.
Michalski et al. (1994) Vox Sang. 67(4), "Preparation and Properties of a Therapeutic Inter-.alpha.-Trypinsin-Inhibitor Concentrate from Human Plasma", pp. 329-336.
Michalski Catherine
Mizon Jacques
Association Pour L'Essor de la Transfusion Sanguine Dans la Regi
Weber Jon P.
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