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

Abstract

The invention relates to Inter-alpha inhibitor proteins (IαIp). The invention further relates to processes for purification of IαIp compositions and their use for treatment of human diseases such as sepsis and septic shock, rheumatoid arthritis, cancer and infectious diseases.

Description

60,000 to about 280,000MgCl2 MgCl₂, 1 mM dNTP, 20 U RNase inhibitor, 2.5 mM oligo d(T) 16 primer and 50 U reverse transcriptase. The reverse transcription reaction solution was incubated at 42° C. for 1 h, followed by heating at 95° C. for 5 min. One μl cDNA was amplified with 0.15 μM each of 3′ and 5′ primers, specific for rat bikunin (633 bp) (5′ TGA CGA ATA TGC CAT TTT CC 3′ (SEQ ID NO: 2), 5′CCA 5′ CCA CAG TAC TCC TTG CAC TCC 3′ (SEQ ID NO: 3)) (accession No. S87544), rat glyceraldehydes-3-phosphate-dehydrogenase 7 (G3PDH) (24) (983 bp) (5′ TGA AGG TCG GTG TCA ACG GAT TTG GC 3′ (SEQ ID NO: 4), 5′CAT 5′ CAT GTA GGC CAT GAG GTC CAC CAC 3′ (SEQ ID NO: 5)) in 25 μl of PCR mixture containing 50 mM KCl, 10 mM Tris-HCl, 2 mM MgCl2 MgCl₂, 0.2 mM dNTP and 0.7 U AmpliTaq DNA polymerase. PCR was carried out in a Bio-Rad thermal cycler. Following RT-PCR, 5 μl of the reaction mixture was electrophoresed in 1.2% TBE-agarose gel containing 0.22 μg/ml ethidium bromide. The gel was then developed and band intensities were normalized by G3PDH using the Bio-Rad image system (Hercules, Calif.).

Radioiodination of Protein and Determination of IαIp Half-Life

Purified IαIp was radioiodinated with Na¹²⁵I (Amersham, Arlington Heights, Ill.) using 1,3,4,6-tetrachloro-3a-6a-diphenyl glycoluril (IODO-GEN iodination reagent; Pierce, Rockford, Ill.). The unincorporated ¹²⁵I was removed by applying the reaction mixture to an Excellulose GF-5 desalting column (Pierce). Radioactivity was determined in a gamma counter (Pharmacia-LKB, Piscataway, N.J.). At 12 h after CLP or sham operation, the animals were anesthetized with isoflurane inhalation. A steady state of sedation was maintained with a subsequent intravenous injection of sodium pentobarbital (˜30 mg/kg BW). Polyethylene-50 catheters were placed in the right jugular vein and left femoral artery, and a bolus injection of ¹²⁵I-labeled IαIp (˜500,000 cpm/rat) was administered through the jugular cannula. The remaining radioactivity in the syringe was measured with a gamma counter, and the radioactivity counts were subtracted from the initial preinjection counts to determine the net injected radioactivity. Blood samples were collected immediately after the injection and then every 2 h for a period of 8 h for determining the half-life (t_1/2) of ¹²⁵I-IαIp in the circulation. The radioactivity (cpm) in each sample was measured with a gamma counter. The t_1/2 was calculated according to Wu R, Zhou M, Cui X, et al: Ghrelin clearance is reduced at the late stage of polymicrobial sepsis. Int J Mol Med. 2003; 12:777-782.

Survival Study

CLP was performed as described above. At 1, 5, 10 or 10 and 20 h after CLP, human IαIp concentrate (30 mg/kg BW) or vehicle (normal saline, 1.5 ml/rat, at 1 h after CLP or 10 and 20 h after CLP) was infused intravenously. At 20 h after CLP, the necrotic cecum was excised and the abdominal cavity was washed twice by using 40 ml of warm, sterilized normal saline solution. The abdominal incision then was closed in layers. The procedure of cecal excision in CLP animals was performed to mimic the clinical situation in which septic focus should be removed whenever possible. The animals then were allowed food and water ad libitum and were monitored for 10 days to record survival.

Statistical Analysis

Results are expressed as means ±SE. One-way analysis of variance (ANOVA) and Tukey's test were used to compare different groups of experimental animals. The survival rate was estimated by Kaplan-Meier method and compared by the log-rank test. Differences in values were considered significant if P<0.05. 9

Alterations in Bikunin mRNA Expression after CLP

Bikunin is an active part of IαIp. The liver is the major source of bikunin. Therefore, we chose bikunin mRNA expression in the liver to reflect the production of IαIp. As shown in FIG. 3, mRNA expression of bikunin in the liver did not change at 5 h after CLP, however, a 32% decrease was found at 20 h after CLP as compared with sham operated animals (P<0.05).

Alterations in t_1/2 of ¹²⁵I-IαIp after CLP

Half-life of IαIp was estimated by measuring the changes of blood levels of radioactive labeled IαIp injected at 12 h after the onset of sepsis. As indicated in FIG. 4, the t_1/2 of 125I-IαIp was significantly increased from 5.6±0.3 h to 11.8±2.7 h (P<0.05) after CLP.

Effects of IαIp on Survival Rate

The survival rate after CLP and cecal excision with single time vehicle administration (at 1 h after CLP) was 75% at day 2 and decreased to 50% at days 5-10 FIG. 5). Administration of human IαIp at 1 h after CLP, however, improved the survival rate to 92% throughout the 10-day observation period (P<0.05; FIG. 5). Although administration of human IαIp at 5 or 10 h after CLP improved the survival rate to 64% and 73% respectively, these improvements were not statistically significant (FIG. 6). The survival rate after CLP and cecal excision with two times vehicle administration (at 10 and 20 h after CLP) was 56% at day 2 and decreased to 44% at days 5-10 (FIG. 7), which was not significantly different as compared with one time vehicle administration (FIG. 5). Administration of human IαIp at 10 and 20 h after CLP, however, improved the survival rate to 81% throughout the 10-day observation period, which was significantly different as compared with vehicle group (P<0.05; FIG. 7).

Sepsis is a clinical syndrome characterized by systemic inflammation, coagulopathy, respiratory failure, myocardial dysfunction, renal insufficiency, and neurocognitive defects. It is generally assumed that this syndrome results from an excessive triggering of endogenous inflammatory mediators by the invading microorganisms. These mediators include substances released by activated monocytes, macrophages, endothelial cells and neutrophils such as cytokines, reactive oxygen species and proteases. In severe inflammatory response, various blood and tissue cells, including polymorphonuclear granulocytes, release lysosomal proteinases extracellularly and into the circulation. Such proteases as well as normally intracellular oxidizing agents produced during phagocytosis, can trigger tissue and organ damage and enhance the nonspecific proteolysis of plasma clotting and complement factors. The release of neutrophil proteinases, especially human leukocyte elastase, has been implicated in the progress of complications in subjects with sepsis. Their plasma levels are in close correlation with the severity of infection-induced inflammation and highly predictive of forthcoming organ failure.

During septic shock in humans, in addition to elevated activities of proteases, decreased plasma levels of IαIp have been reported. Subjects with severely decreased concentrations of IαIp have a higher mortality rate. Our results indicate that the gene expression of bikunin in the liver is significantly lower in the CLP animals than in the sham-operated animals. The expression of mRNA related to proteins in the inter-alpha-inhibitor family has been examined in various tissues in primates, pigs, and rodents. These studies indicate the genes which are the source of all members of the IαIp family are primarily transcribed in the liver. Our results also show the gene expression of bikunin in other organs (i.e., intestine and kidneys) was not significantly altered at 5 or 20 h after CLP as compared to the shams (data not shown).

The significant downregulation observed only in the liver at 20 h after CLP suggests that this organ might be an important source of IαIp and furthermore, in late stages of sepsis, bikunin gene expression in the liver is significantly decreased. Therapeutically, bikunin has been reported to have beneficial effects in humans as a prophylactic treatment to prevent pancreatitis after gastrectomy or to attenuate organ injury after cardiac surgery. Studies that examined the effects of bikunin in an acute canine model of lethal E. coli bacteremia showed similar results with an improvement in hemodynamic variables and normalization of cardiac output and mean arterial pressure. Because the plasma half-life of bikunin is very short (approximately 10 min), it appears important to prolong the half-life of bikunin to maintain the sustained beneficial effects of this agent. Our results show that the half-life of IαIp in sham-operated animals is 5.6 h. When we injected radioactive labeled IαIp at 12 h after the onset of sepsis, we found the half-life of IαIp was prolonged to 11.8 h. These results indicate that IαIp clearance was significantly decreased during sepsis. However, even with decreased clearance the plasma levels of IαIp remain significantly lower in septic subjects. Our previous study has shown that administration of low purity IαIp early after the onset of sepsis (i.e., 1 h post-CLP) maintained cardiac output and systemic oxygen delivery and increased systemic oxygen consumption and systemic oxygen extraction ratio. Moreover, IαIp downregulated TNF-a production and attenuated hepatocellular injury and lactic acidosis at 20 h after CLP. In addition, IαIp administration at 1 h after the onset of sepsis improved survival in septic animals.

Human IαIp proteins were isolated as a by-product of industrial scale plasma fractionation. The isolation method highly, simultaneously enriches the major plasma form of bikunin-containing proteins (IαI and PαI). Thus, in this preparation, a physiologic composition of plasma IαIp is obtained. The results of the mortality study performed indicate that administration of IαIp at 1 h after CLP improved the survival rate from 50% to 92% at 10 days after CLP and cecal excision. Although administration of human IαIp at 5 or 10 h after CLP improved the survival rate to 64% and 73% respectively, these improvements were not statistically significant.

Administration of human IαIp at 10 and 20 h after CLP, however, significantly improved the survival rate from 44% to 81%. Thus, IαIp appear to be a useful adjunct for improving survival during the progression of polymicrobial sepsis. In summary, bikunin gene expression in the liver decreased during sepsis and the half-life of IαIp increased from 5.6±0.3 h to 11.8±2.7 h, suggesting downregulation of bikunin in sepsis despite a decrease clearance. Administration of IαIp at 1 h after CLP improved the survival rate from 50% to 92%, whereas there was no significant improvement when IαIp was administrated at 5 or 10 h after CLP. However, double injection of IαIp at 10 and 20 h after CLP increased the survival rate from 44% to 81%. Delayed but repeated administration of human IαIp improve survival after CLP.

Claims

1. A process for producing a blood plasma-derived IαIp composition comprising a mixture of inter-alpha inhibitor protein (IαI) and pre-alpha protein (PαI), wherein the IαI and the PαI are present in said mixture in a physiological proportion, the process comprising:

isolating from blood plasma a plasma fraction containing IαI and PαI, wherein the IαI and PαI are present in a physiological proportion; and

purifying the plasma fraction to obtain an IαIp composition with a purity of IαIp ranging from about 85% to about 100% pure, wherein the purifying comprises hydroxylapatite chromatography.

2. The process of claim 1, wherein the isolating comprises solid phase extraction or chromatographing blood plasma.

3. The process of claim 1, wherein the plasma fraction comprises a side fraction obtained from the purification of clotting factor IX or from the purification of a prothrombin complex concentrate.

4. The process of claim 1, wherein the plasma fraction is isolated as a cryosupernatant resulting from cryoprecipitation of blood plasma.

5. The process of claim 1, wherein the plasma fraction is cryo-poor plasma.

6. The process of claim 1, wherein the plasma fraction is human, primate, bovine, porcine, feline, or canine.

7. The process of claim 1, further comprising obtaining blood, obtaining blood plasma, obtaining a side fraction obtained from the purification of clotting factor IX, obtaining a side fraction from the purification of a prothrombin complex concentrate, obtaining a cryosupernatant resulting from cryoprecipitation of blood plasma or obtaining cryo-poor plasma.

8. The process of claim 1, wherein the purifying further comprises affinity chromatography.

9. The process of claim 1, wherein the IαI and PαI present in the plasma fraction have an apparent molecular weight of between about 60,000 to about 280,000 kDa.

10. The process of claim 1, further comprising:

purifying the plasma fraction; virus inactivating the plasma fraction and/or the purified IαIp; the addition of stabilizers; pasteurization of the purified IαIp;

or anion-exchange chromatography of the purified IαIp.

11. The process of claim 10, wherein the further purifying the plasma fraction is by passing through heparin affinity column and collecting the flow through (unbound) fraction; the virus inactivating is by a solvent/detergent treatment or thermal inactivation; and the anion-exchange chromatography of the purified IαIp is diethylaminoethyl (DEAE) Sepharose.

12. The process of claim 11, wherein the thermal inactivation comprises pasteurization at a temperature of between about 55 to about 65° C. or dry heat at 70 to 120° C. omega-amino acids, sugar, or combinations thereof.

13. A composition of IαIp comprising a mixture of inter-alpha inhibitor protein (IαI) and pre-alpha protein (PαI), wherein the IαI and the PαI are present in said mixture in a physiological proportion and: have a high trypsin inhibitory specific activity between about 1000 to about 2000 IU/mg; have a half life of greater than one hour; comprise a light chain of inter-alpha inhibitor protein associated with at least one of three heavy chains H1, H2 and H3; or comprise a light chain of inter-alpha inhibitor protein associated with at least one of three heavy chains H1, H2, H3 and H4.

14. The composition of claim 13, wherein trypsin inhibitory specific activity is between about 1400 to about 2000 IU/mg.

15. The composition of claim 13, wherein the IαIp composition has a half life of at least 5 hours.

16. The composition of claim 13, wherein the IαIp composition has a half life of at least 10 hours.

17. A composition of IαIp comprising a mixture of inter-alpha inhibitor protein (IαI) and pre-alpha protein (PαI), wherein the IαI and the PαI are present in said mixture in a physiological proportion, said composition having been prepared by the process according to claim 1.

18. The composition of claim 17, further comprising an additional therapeutic agent.

19. The composition of claim 18, wherein the additional therapeutic agent is an anti-inflammatory agent, an anti-coagulant or an immunomodulator.

20. A pharmaceutical composition comprising a therapeutically effective amount of the composition of claim 17, and a pharmaceutically acceptable carrier.

21. A method of treating an inflammation related disorder comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 17, wherein the inflammation related disorder is selected from an acute inflammatory disease, sepsis, septic shock, rheumatoid arthritis, meningitis, Crohn's Disease, chronic obstructed pulmonary disease and rhinitis.

22. The method of claim 21, wherein the IαIp is administered as a tablet, capsule, or injectables.

23. A method of treating an acute inflammatory disease, sepsis, septic shock, rheumatoid arthritis, meningitis, Crohn's Disease, chronic obstructed pulmonary disease and rhinitis in a subject, comprising:

(a) determining the pre-treatment level of one or more of the following levels in a subject:

(i) the level of IαI;

(ii) the level of PαI;

(iii) the level of IαIp;

(iv) the level of H3;

(v) the level of H4;

(vi) the level of H1;

(vii) the level of H2; and

(viii) the level of LC; and

(b) administering a therapeutically effective amount of the composition of claim 17 to the subject.

24. A method of monitoring the progress of a subject being treated with an IαIp therapy, comprising:

(a) determining the pre-treatment level of one or more of the following levels, in a subject:

(i) the level of IαI;

(ii) the level of PαI;

(iii) the level of IαIp;

(iv) the level of H3;

(v) the level of H4;

(vi) the level of H1;

(vii) the level of H2; and

(viii) the level of LC;

(b) administering a therapeutically effective amount of the composition of claim 17 to the subject; and

(c) determining the level of one or more of the levels in the subject after an initial period of treatment with the composition,

wherein an increase of the level in the subject following treatment with the composition indicates that the subject is likely to have a favorable clinical response to treatment with IαIp.

25. A kit comprising a composition according to claim 17 and instructions for therapeutic use.

26. A method of treating sepsis or septic shock in a human in need thereof comprising administering to the human more than one dose of a pharmaceutical composition comprising 1 to 50 milligrams (mg) inter-alpha inhibitor proteins (IαIps) per kilogram (kg) body weight of the human, wherein the IαIps comprise 5% to 95% by weight of the composition and comprise a mixture of 60% to 80% inter-alpha inhibitor (IαI) and 40% to 20% pre-alpha inhibitor (PαI), the composition is suitable for administration to the human, and each dose of the composition is administered every 4 to 120 hours from about 1 to about 6 times per day.

27. The method of claim 26, wherein said IαI and PαI comprise a light chain (L) associated with at least one heavy chain selected from H1, H2, H3, and H4.

28. The method of claim 27, wherein the composition has a high trypsin inhibitory specific activity of 1000 to 2000 IU/mg.

29. The method of claim 28, wherein the trypsin inhibitory specific activity of said composition is 1400 to 2000 IU/mg.

30. The method of claim 26, wherein the composition has a half-life of greater than one hour.

31. The method of claim 30, wherein the composition has a half-life of at least 5 hours.

32. The method of claim 31, wherein the composition has a half-life of at least 10 hours.

33. The method of claim 26, wherein the IαI and PαI present in said composition have an apparent molecular weight of 60,000 to 280,000 Da.

34. The method of 26 further comprising, prior to said administering, determining the pre-treatment level of one or more of the following IαIps in the human: IαI, PαI, H3, H4, H1, H2, and LC.

35. The method of claim 34 further comprising determining the level of one or more of IαI, PαI, H3, H4, H1, H2, and LC in the human after an initial period of treatment with the composition.

36. The method of claim 26, wherein the composition further comprises a stabilizer.

37. The method of claim 26, wherein the composition is administered as a tablet, capsule, or injectable.

38. The method of claim 26, wherein the composition is administered by intravenous infusion.

39. The method of claim 26, wherein the composition is administered at least twice.

40. The method of claim 26, wherein the composition is administered until symptoms of the sepsis or septic shock improve.