AU647586B2 - Polypeptide fraction affording protection to cells against mechanical damage and assay - Google Patents

Description

CORRECTED

VERSION*

PCr pa, es 1-27, description, replaced by new pages 1-26; pages 28-31, claims, replaced by new pages 27-30; pages 1/3-3/3, drawings, replaced by new pages 1/2-2/2; due to late transmittal by the receiving Office 0 1 I h 2 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 92/11282 C07K 3/02 Al (43) International Publication Date: 9 July 1992 (09.07.92) (21) International Application Number: (22) International Filing Date: 131 Priority data: 54776 4 L 7 17 Decen PCT/US91/09558 DecemL,- 1991 (13.12.91) nber 1990 (17.12.90) US u_-l r\_W \L i.'C (71) Applicant: TEXAS -TEC 1H---UN-I- ftSf-- [US/US]; Broadway and University Avenue, Lubbock, TX 79409

(US).

(72) Inventor: BUTLER, Thomas, C. 4611 10th Street, Lubbock, TX 79416 (US).

(74) Agents: AMZEL, Viviana et al.; Cox Smith Incorporated, 112 East Pecan Street, Suite 2000, San Antonio, TX 78205 (US).

(81) Designated States: AT (European patent), AU, BE (European patent), CA, CH (European patent), DE (European patent), DK, DK (European patent), ES (European patent), FI, FR (European patent), GB (European patent), GR (European patent), IT (European patent), JP, KP, KR, LU (European patent), MC (European patint), NL (European patent), NO, SE (European patent).

Published With international search report.

1' L? (54)Title: POLYPEPTIDE FRACTION AFFORDING PROTECTION TO CELLS AGAINST MECHANICAL DAMAGE AND ASSAY (57) Abstract A cell protective composition comprises a polypeptide fraction capable of inhibiting mechanical damage to cells and heatstable up to about 100 inhibitory of mechanical cell damage at about pH 5.5 to 7.5, inhibitory activity unaffected by ribonuclease and/or deoxyribonuclease enzymes but partially lost to trypsin, molecular weight about 6-100 Kdaltons, denatured by ethanol precipitation, acid labile at pH below about 1.2, and trichloroacetic acid-precipitable and protectively active upon redissolution. The polypeptide fraction may be prepared by separating plasma from blood, heating the plasma to about 90 to 100 OC to form a heat-labile precipitate and a heat-stable supernate, separating the heat-labile precipitate from the heat-stable supernate, and separating a polypeptide fraction of molecular weight about 6 to 100 Kdaltons from the heat-stable supernate. An in vitro method of assessing the degree of protection from mechanical damage afforded cells in a biological sample, comprises placing cells in a medium under conditions effective for growth, adding to the medium particles capable of undergoing a mild mechanical interaction with the cells, adding to the medium a substance to be tested, agitating the medium comprising the cells for a preset period of time under preset conditions, and determining the amount in the medium of a factor associated with the degree of mechanical damage of the cell.

(Referrcd to in P(UT GawLi,; No. 2111992, Section I1 WO 92/11282 PCT/US91/09558 1 POLYPEPTIDE FRACTION AFFORDING PROTECTION TO CELLS AGAINST MECHANICAL DAMAGE AND ASSAY BACKGROUND OF THE INVENTION Field of the Invention This invention relates to a blood cell protective composition comprising a peptide fraction obtained from plasma capable of inhibiting the mechanical damage of blood cells, and more particularly of red blood cells.

This invention also relates to a method of preparing the composition of the invention. The present invention 2 finds its utility in the treatment of various diseases including dysentery, and hemolytic anemia associated with sepsis, heart surgery, atherosclerosis, hemodialysis, and hemolytic-uremic syndrome, all diseases that are associated with mechanical darage to blood cells.

Description of the Background Hemolytic uremic syndrome (HUS) is defined as the following conditions hemolytic anemia, renal failure, and 3 thrombocytopenia. HUS also occurs as a complication of dysenteric infections caused by Shigella dysenteriae 1 and Escherichia coli 0157. Most cases of HUS have been described in patients that received treatment with antimicrobial agents, and most of the affected patients 4 have been young children, elderly, or malnourished subjects. In general, patients with grossly bloody stools have been found to be more likely to develop HUS than patients affected with milder diseases.

WO 92/11282 PCr/US1/09558 2 The pathogenesis of HUS is still not completely understood. Bacterial products of the causative organisms, including lipopolysaccharides and the cytotoxins such as the Shiga toxin and verotoxin, have been proposed as causes of HUS. Leukocytes and leukocytic products appear to play a role in HUS as well. This has been shown in animal models and in patients who often have elevated blood leukocyte counts and increased serum levels of the neutrophil products elastase and alpha-l-antitrypsin and of the cytokine interferon.

The hemolytic anemia accompanying HUS is not believed to be caused by an immune reaction because patients afflicted with it show negative direct Coomb's tests.

The pathogenesis of the anemia associated with HUS is believed to be microangiopathic because of the presence of fragmented red blood cells and intravascular coagulation. However, direct effects of bacterial toxins on red blood cells have been suggested on the basis of experimental results with phospholipase C of C. perfingens and because of the vacuolations demonstrated in red blood cells.

The protein-losing enteropathy observed in patients with dysentery results in losses of albumin and other proteins into the intestine. A deficiency of the plasma protein fibronectin has been shown in children with HUS and was attributed to the deposition of fibronectin along with fibrinogen and platelet antigen, in the vasculature of the kidneys. A deficiency of plasma factor that stimulates prostacyclin (PGI 2 activity, an inhibitor of platelet aggregation, has been proposed as playing a role in the pathogensis of HUS and may be the basis for the beneficial effect of fresh plasma in the treatment of

HUS.

SUBSTITUTE SHEET WVO 92/11282~ PCT/US91/09558 3 Most patients with HUS show symptoms of malnutrition. This has been associated with hypoproteinemia. A role for hypotonicity of plasma could also occur in patients with shigellosis associated with 10 hyponatremia. However, a degree of hypotonicity necessary to cause the lysis of red cells in vitro probably does not develop in even severe cases of hyponatremia. A decreased life span of red blood cells occurs in anemia associated with various chronic diseases. This may very well correlate with low concentrations of plasma proteins. A role for antibiotic treatment in eliciting HUS is not supported by the results of these in vitro experiments, but it is possible that antibiotic treatment releases for example toxins, such a lipopolyaccharides from dying bacteria, which promote intravascular coagulation and, therefore, increased mechanical trauma to red blood cells.

Up to the presert time, there exist no effective methods of treating these conditions.

Accordingly, there is still a need for an effective method of preventing the mechanical damage of cells in the blood that is applicable to a variety of disease states and/or surgical procedures.

DESCRIPTION OF THE INVENTION This invention relates to a blood cell protective composition comprising a polypeptide fraction capable of inhibiting mechanical damage to cells and having the following characteristics heat-stable up to about 100 0

C;

inhibitory of mechanical cell damage at about pH to SUBSTITUTE SHEET WO 92/11282 PCT/US91/09558 4 inhibitory activity unaffected by ribonuclease and/or deoxyribonuclease but partially lost to trypsin; molecular weight about 6 to 100 Kdaltons; denatured by ethanol precipitation; acid labile at pH below about 1.2; and trichloroacetic acid-precipitable and inhibitory of mechanical cell damage upon redissolution.

This invention also relates to a cell protective solution or suspension that comprises about 1 to 100 mg/ml of the peptide fraction of the invention; and a pharmaceutically-acceptable liquid carrier.

The invention also relates to a method of preparing a peptide fraction capable of inhibiting mechanical damage to cells, comprising separating plasma from blood; heating the plasma to about 90 to 100 0 C for a period of time of, about 15 to 30 min to form a heat-labile precipitate and a heat-stable supernate; separating the heat-labile precipitate from the heat-stable supernate; and separating a peptide fraction of about 6 to 100 Kdaltons molecular weight from the heat-stable supernate.

Still part of the invention is a method of inhibiting the mechanical damage to cells that comprises intravenously administering to a subject in need of such treatment an anti-lytic amount of the cell protective solution of the invention.

This invention also relates to a method of assessing the degree of protection against mechanical damage afforded cells in a biological sample that comprises placing cells in a medium under conditions effective for maintenance of their structural integrity; adding to the medium particles capable of undergoing a mild mechanical interaction with the cells; WO 92/11282 PCT/US91/09558 adding to the medium a substance to be tested; agitating the medium comprising the cells for a preset period of time and preset conditions; and determining the amount in the medium of a factor associated with the degree of mechanical damage of the cell.

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily perceived as the same becomes better understood by reference to the following detailed description when considered in connection with the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 represents the hemolysis of human red blood cells after incubation at 37 0 C for 18 hrs in Tris-buffered saline or plasma under stationary conditions or after shaking in the presence of glass beads.

Figure 2 shows the effect of plasma and two-fold dilutions thereof in Tris-buffered saline to protect red blood cells against mechanical hemolysis.

Figure 3 shows the effect of a supernate of human plasma obtained by heating to 100°C and two-fold dilutions thereof in protecting red blood cells against mechanical hemolysis.

Other objects, advantages and features of the present invention will become apparent to those skilled in the art from the following discussion.

WO 92/11282 PCT/US91/09558 6 BEST MODE FOR CARRYING OUT THE INVENTION The invention arose from a desire by the inventor to provide a product suitable for the in vivo treatment of a subject, including a human patient, afflicted with a condition associated with mechanical damage of blood cells, particularly red and/or white blood cells.

However, the present technology is also applicable to other types of cells, and more specifically to situations where mechanical damage is to be avoided to those cells.

This invention provides a cell protective composition that comprises a polypeptide fraction capable of inhibiting mechanical damage to cells having the following characteristics heat-stable up to about 100°C; inhibitory activity unaffected by ribonuclease and/or deoxyribonuclease but partially lost to trypsin; inhibitory of mechanical cell damage at about pH to molecular weight about 6 to 100 Kdaltons; denatured by ethanol precipitation; acid labile at pH below about 1.2; and trichloroacetic acid-precipitable and inhibitory of mechanical cell damage upon redissolution.

In a preferred embodiment of the invention, the cell-protective composition is obtained from plasma, and more preferably from human plasma. Typically, either fresh or frozen active blood and/or plasma may be utilized to obtain the cell-protective composition of the invention. In the context of this patent, active blood is defined as human blood collected under procedures of routine blood banking, anticoagulated with citrate solution or heparin, and stored at about 4 0 C as either whole blood or plasma. Outdated blood or plasma of more than 6 weeks after donation, may be used as well.

WO 92/11282 PCT/US91/09558 7 The polypeptide fraction of the invention has been shown to be mostly formed of polypeptides, and to contain up to about 63 to 86% protein, and in some instances to contain greater than about 63% polypeptide and/or protein as determined by the biuret method of measuring total protein.

The hemolytic activity of the polypeptide fraction of the invention is capable of resisting enzymatic degradation by deoxyribonuclease and ribonuclease.

Trypsin treatment of the polypeptide fraction has been shown to cause only partial loss of the inhibitory activity of mechanical damage. The active component of the protective composition is, therefore, not DNA or RNA but one or more of the polypeptide and/or protein components in the fraction.

The treatment of the polypeptide fraction with trichloricidic acid (TCA) was shown to produce a precipitate, which when redissolved and tested for cell protective activity was shown not to have substantially lost it. The addition of high concentration of ethanol to the polypeptide fraction produces a precipitate which upon redissolution may be shown to have substantially lost most of its cell protective activity. Treatment of the polypeptide fraction with a strong acid such as hydrochloric acid to lower the pH to about 1.0 followed by neutralization back to pH 7.0 was shown to form a precipitate, and the remaining supernate was devoid of cell protective activity.

In another preferred embodiment, the cell-protective composition of the invention is substantially devoid of one or more blood component(s) such as albumin, complement, gamma-globulins, haptcglobulins, ceruloplasmin, and alpha-2-macroglobulin. These are all plasma components that may be eliminated from the polypeptide fraction of the invention.

WO 92/11282 PCT/US91/09558 8 In one embodiment of the invention, the cell-protective extract was shown to be free of albumin and gamma globulins by protein electrophoresis.

Complement activity was not present because of the prior heating step. Other proteins, haptoglobins, ceruloplasmin, and alpha-2-macroglobulin, are assumed to be absent from the cell-protective composition or alternatively substantially unnecessary for activity since solutions of these proteins from commercial preparations afforded no cell protection activity.

In addition, low and high molecular weight components may also be eliminated from the composition, including any blood component having a molecular weight of less than about 6 Kdaltons, and more preferably less than about 8 Kdaltons. High molecular weight substances, particularly proteins and/or polypeptides of molecular weight greater than about 300 Kdalton, and more preferably greater than about 100 Kdalton may also be eliminated from the fraction of the invention, by filtration. In a still more preferred embodiment, the polypeptide fraction encompasses polypeptides of about to 80 Kdalton molecular weight.

The polypeptide fraction of the composition of the invention may be prepared by the method described below.

However, the present invention also covers compositions comprising the active component of the composition which are prepared by other methods and have have the ability to inhibit mechanical damage inflicted to cells.

Also provided herein is a cell-protective solution that comprises about 0.01 to 500 mg/ml of the cell-protective composition of the invention; and a pharmaceutically-acceptable liquid carrier.

q I P Q vTi P r P-r iv r-P- WO 92/11282 PCT/US91/09558 9 In a particularly preferred embodiment of the invention, the cell-protective solution comprises about 1 to 200 mg/ml cell-protective composition.

In another particularly preferred E ,bodiment of the invention, the cell-protective solution of the invention comprises about 5 to 100 mg peptide fraction/ml solution, and still more preferably about 10 to 50 mg peptide fractions/ml solution. However, the solution of the invention may be prepared and administered in other concentrations as well.

The cell-protective solution of the invention finds its use in in vivo and in vitro methods. In one embodiment, it is provided herein an in vivo method of inhibiting mechanical damage to cells comprising the intravenous administration of the solution of this invention to a patient in need of the treatment.

Accordingly, the protective composition and the cell-protective solution of the invention must be free of toxicity and particles of any blood transmissible diseases such as AIDS, and various other viruses and bacteria. Testing and elimination of blood contaminated samples are conducted as is known in the art.

The pharmaceutically-acceptable liquid carrier may be any such carrier known in the art. By means of example, a saline solution may be utilized, and more preferably an electrolyte-balanced saline solution. However, other pharmaceutically-acceptable liquid carriers known in the art for the intravenous administration of substances may also be utilized as is contemplated within the confines of this invention.

A particularly well suited form of the cell-protective solution of the invention further comprises about 0.01 to 100 mg albumin/ml solution, and more preferably about 5 to 50 mg SUBSTITUTE SHEET WO 92/11282 PCT/US91/09558 albumin/ml solution in addition to the aforementioned composition that is substantially free of albumin.

Other components may also be added to the solution either at the time of preparation or prior to its administration to a patient. Examples of such additional ingredients are known in the art and need not be further described herein. In general, any other ingredient which is salutory and facilitates the acceptance of the intravenous solution by the patient may be added herein as long as it is pharmaceutically-acceptable, non-toxic and free of the blood-transmissible particles described above.

Also provided herein is a method of preparing a polypeptide fraction such as the one described above that is capable of inhibiting mechanical damage to cells, the method comprising separating plasma from blood; heating the plasma to about 90 to 100 0 C for a period of time of, about 15 to 30 min or longer, to form a heat-labile precipitate and a heat-stable supernate; separating the heat-labile precipitate from the heat-stable supernate; and separating a polypeptide fraction of about 6 to 100 Kdalton molecular weight from the heat-stable supernate.

The separation of plasma from mammalian blood, including human blood may be conducted as is known in the art. Briefly, the separation of plasma from blood may be conducted by centrifuging anticoagulated blood at about 1,500 x g for about 15 min. The clear upper plasma layer may be removed, with a pipette, and the lower layers of red blood cells, white blood cells, and platelets discarded.

Once the plasma is separated from the remainder portion of the blood, it may be heated by itself to a temperature of about 85 to 100 0 C, and more preferably to SUBSTITUTE SHEET WO 92/11282 PCT/US91/09558 11 about 90 to 100 0 C for P period of time, of about to 60 min, and more preferably about 15 to 30 min, to separate heat-precipitable components. These precipatible components include albumin, beta globulins, gamma globulins, and complement, among others.

The heat-labile precipitate may be separated from this supernate by implementing technology which is known in the art. By means of example, the separation may be conducted by centrifugation or filtration. Of these, nreferable is centrifugation becat~e no component is generally lost by adhesion to a filter and no extraneous material is added by elution from the filter. However, other means of separating the precipitate from the supernate may also be utilized which are known in the art.

Once the heat-stable supernate has been separated from the precipitate, a polypeptide fraction having about 6 to 100 Kdalton molecular weight may be separated, in successive steps, utilizing membranes having a low cut-off molecular weight of about 6 Kdaltons and a high cut-off molecular weight of about 100 Kdaltons. As shown in the examples accompanying this patent, the fractions of molecular weights lower than about 6 Kdaltons and higher than about 100 Kdaltons do not evidence substantial protective activity of cells exposed to mechaiical damage. In a particularly preferreo embodiment, the low cut-off molecular weight of the membrane is about 8 Kdaltons, and more preferably, the polypeptide fraction separated with the aid of the membranes has a molecular weight cut-off of about 10 to 80 Kdaltons.

The separation of the specified molecular weight polypeptide fraction may be conducted as is known in the art. By means of example, dialysis may be conducted with a membrane sieving molecules of less than about 6 Kdaltons, and more preferably less than about 8 WO 92/11282 PCT/US91/09558 Kdaltons. In a separate step, ultrafi Lation may be conducted with a membrane with a cut-off moleculdr weight of 300 Kdalton, and more preferably about 100 Kdalton.

However, other methods may also be utilized such as gel filtration, ion exchange, and column chromatography, among others.

Once the desired molecular weight range polypeptide fraction is separated from the remaining supernate, it may, optionally, be freeze-dried, particularly, when it is intended for storage. The freeze-dried polypeptide fraction may be stored in a sealed container as is known in the art at a temperature of about -60 to 4 0 C, and particularly below -20°C. The freeze-dried polypeptide fraction has been shown to have a shelf life of at least about 3 months at a temperature of about 4 0 C. The freeze-dried polypeptid fraction may be refrigerated and/or stored at freezing temperatures, as well.

The polypeptide of the invention may be shipped as a freeze-dried product or as a solution in a pharmaceutically-acceptable liquid carrier as was described above.

Also provided herein is a method of preventing mechanical damage to cells that comprises the intravenous administration to a subject in need of the treatment of an anti-lytic amount of the polypeptide fraction of the invention, optionally in the form of the cell protective solution of the invention. The intravenous administration of the polypeptide fraction of this invention i7 recommended since the polypeptides and/or protein in the fraction would be hydrolyzed in the gastrointestinal tract when administered orally or by other routes, such as by rectal suppository.

The cell protective solution of the invention may be administered to a subject at a concentration of 10 to 500 WO 92/11282 PCT/US91/09558 13 mg polypeptide fraction/ml solution, and more preferably about 50 to 100 mg polypeptide in the fraction/ml solution.

When administered to a subject in need of on-going cell protection from mechanical damage, the cell protective solution may be repeatedly administered at intervals effective to maintain a concentration of the polypeptide fraction in the subject's plasma higher than about 0.5 mg/ml, and more preferably higher than about 1 mg/ml. In most instances, a daily administration of the solution will provide satisfactory results. However, a practitiner would know how to adjust the dose and the interval for the injections for specific patients. The treatment may be continued over a period of time greater than about a week, and even greater than about a month.

In some instances, a human may be treated in accordance with this invention for periods of up to about a year and longer.

The cell protective effect of the polypeptide fraction cf the invention has been shown to be suitable for treatment of a subject that is afflicted with a hemolytic anemia. Specific examples of hemolytic anemias are anemias associated with sepsis, heart surgery such as surgery utilizing a heart-lung bypass machine and surgery associated with the implantation of a prosthetic heart valve, anemias associated with atherosclerosis, particularly those occurring in the elderly, and anemias occurring during the course of, or after, hemodialysis and in patients afflicted from hemolytic-uremic syndrome.

Typically, the patient may be administered about 0.01 to 200 g of the polypeptide fraction per day, and more preferably about 5 to 10 g of the polypeptide fraction per day. However, other amounts may also be administered as adjusted by the practitioner for a desired effect.

WO 92/11282 PCT/US91/09558 14 Another condition afflicting a patient for which the present technology is suitable is that of dysentery, particularly that associated with the Shigella dysenteriae and E. Coli bacterias.

When treating a patient afflicted with dysentery, the regime for the administration of the polypeptide fraction of the invention and/or the cell protective solution described herein is similar to what was described above.

Still part of this invention is an in vitro method of assessing the degree of protection from mechanical damage afforded cells in a biological sample, that comprises placing cells in a medium under conditions effective for maintenance of its structural integrity; adding to the medium particles capable of undergoing a mild mechanical interaction with the cells; adding to the medium a substance to be tested; agitating the medium comprising the cells for a preset period of time and preset conditions; and determining the amount in the medium of a factor associated with the degree of mechanical damage of the cell.

This is a rapid and effective method for assessing the degree of protection of a variety of substances. The method relies on mild damage purposely inflicted to the cells on which the effect is to be tested by particles such as beads, and more preferably glass beads. However, other types of particles and/or materials may also be utilized.

The medium in which the cells are placed may be any medium that will permit the short term growth and survival of the cells. Examples are saline, buffered saline, a variety of cell growth media known in the art such as tissue culture media, and Hank's buffered salt solution, among others.

W| m, WO 92/11282 PCT/US91 /09558 Once the cells are placed in the medium, various similarly prepared samples may be run side-by-side. One will remain without the addition of a substance to be tested and one without the particles whereas a third one, as well as other test samples if there is more than one substance to be tested, will have the particles and the abstance(s) to be tested added to the medium under standardized conditions. Typically, the test may be conducted at room temperature or at a temperature suitable for the maintenance and growth of the cell on which the substance is to be tested. However, all different samples ar: +o be run under similar conditions.

The present in vitro test is suitable for assessing the damage to variety of cell types. Examples are red blood cells, white blood cells, infected macrophages and HIV-infected lymphocytes, among others. However, other cells may also be utilized as long as their integrity or lack thereof can be assessed by the measurement of one or more parameters.

The agitation of the medium comprising the cells and the other ingredients is conducted for a preset period of time and under similar conditions. Typically, the agitation is conducted at about 100 to 200 oscillations/minute, and more preferably about 150 to 180 oscillations/minute at a temperature of, about to 39 0

C.

Once the preset period of time for the test has been completed, agitation may be stopped and the amount of a factor that is associated with the degree of mechanical damage to the cells being tested in the medium may be determined. By means of example, if the cells are red blood cells containing hemoglobin, the test may entail the determination of the presence of hemoglobin in the medium as well as the amount present. Thus, if the cells have not been damaged the amount of hemoglobin detected WO 9/128 PCT/US91/09558 16 in the medium will be minimal whereas where great damage has occurred, the cells will have lost a good proportion of its content of hemoglobin.

The hemoglobin will thus be detected in the medium as shown in the examples. The determination of hemoglobin in the medium may be conducted by measuring the optical density of the various samples containing the cells at about 412 nm. Similarly, for other types of cells, a parameter can be found which correlates with the degree of damage inflicted to them. Leukocyte esterase may be neasured for the measurement of damage to white blood cells and cytokines such as interleukin-1l may be assayed for infected macrophages.

The relationship of the results shown in the examples provided vith this patent to HUS is that the plasma of patients afflicted with dysentery becomes deficient in components that may protect red blood cells against hemolysis.

Having now generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein for purposes of illustration only and are not intended to limiting of the invention or any embodiment thereof, unless so specified.

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WO 92/11282 PPrUS9] /09558 17

EXAMPLES

Example 1: Hemolytic Assay Blood was obtained from normal human volunteers by venipuncture in heparin sodium (Lyphomed Inc., Rosemont, IL) in a concentration of 50 units/ml. The blood was centrifuged at 1,500 g for 10 min and the plasma was removed. The red blood cell (RBC) layer was resuspended in 3 volumes of sterile normal saline, mixed, centrifuged again at 1,500xg for 10 min, and the supernate discarded. A total of four saline washes were carried out. The RBC were stored at 4°C and used within two days of collection.

One-tenth ml of RBC was added to test tubes containing 1 ml tris-buffered 0.135 M saline at pH (TBS), plasma, or other tested solution. After mixing, the tubes were incubated at 37 0 C for 18 hrs. The tubes were inverted twice to resuspend the red cells and were then centrifuged at 700xg for 10 min. The O.D.s of the supernates were read at 412 nm in a spectrophotometer.

Maximal release of hemoglobin was determined by using deionized water in place of TBS and the results expressed as the proportion of maximal hemoglobin released (Carr,C.

Jr., and Morrison,D.C., "Lipopolysaccharide interaction with rabbit erythrocyte membranes", Infect.Immun.

43:600-606(1984)) Five autoclaved 3mm glass beads were added to each tube for the detection of the effect mechanical hemolysis. The tubes were placed in a shaking water bath at a 450 angle and agitated for 18 hrs. at 170 oscillations per min. The tubes were then centrifuged at 700xg for 10 min and the supernates read as described above to determine the release of hemoglobin. The per cent protection of tested solutions against mechanical cE)Pe)~L ~Plrrr~l WO 92/11282 PCT/US91/09558 18 hemolysis was expressed as the O.D. of red blood cells shaken in TBS minus the O.D. of cells shaken in the test solution, divided by the O.D. of cells shaken in TBS, times 100.

Example 2: Preparation of protein solutions Human plasma and dilutions of plasma in TBS were made from blood obtained from normal volunteers in heparin to a final concentration of 50 units/ml. Autologous plasma was used with RBC in hemolytic assays. Plasma was heated in a water bath at 56 0 C for 20 min to inactivate complement. Plasma was dialyzed against normal saline in a dialysis tube with MW cut-off 6,000-8,000 (Spectra/por, Los Angeles, CA). Purified human plasma proteins were obtained to test their effect in the protection against mechanical hemolysis after dissolving in TBS the following components.

Human albumin, essentially free of fatty acid and globulin (Sigma, St. Louis, MO).

Immune globulin,USP (Travenol Laboratories, Glendale, CA).

Haptoglobin (Sigma,St. Louis, MO).

Ceruloplasmin Type VI (Sigma, St. Louis, MO).

Alpha-2-macroglobulin (Sigma, St. Louis, MO).

Outdated fresh frozen plasma was obtained from the blood bank at Texas Tech University Health Sciences Center to prepare a heat-stable extract of plasma. The plasma was placed in a boiling water bath for 30 min.

The precipitate was dispersed with a stirring rod and the WO 92/11282 PCT/US91/09558 19 tubes were centrifuged at 2,500xg for 1 hr. The supernate was decanted and filtered through a 0.2 micron pore size ultramembrane filter using a tangential flow system with a peristaltic pump (Minitan System, Millipore Corp., Bedford, MA). The active principle in the heat-stable supernate was concentrated by filtering through an ultramembrane with a cut-off molecular weight (MW) of 100,000, dialyzing the filtrate with a membrane with MW cut-off of 6,000-8,000 for 2 days with frequent changes of deionized water at 4 0 C and lyophilizing the dialysand. The total protein concentration was measured by the biuret method (Doumas, Bayse, Carter, Peters, and Schaffer, "A candidate reference method for determination of total protein in serum 1. Development and validation", Clin. Chem.

27:1642-1650 (1981)), on an Ektachem 700XR analyzer (Kodak, Rochester, NY) and agarose gel electrophoresis (Laurell, "Electrophoresis, specific protein assays, or both in measurement of plasma proteins", Clin.

Chem. 19:99-102 (1973)), was performed with a Beckman Paragon Electrophoresis System (Beckman Instruments, Berea, CA).

Example 3: Measurement of viscosity Viscosity was measured at ambient temperature using an Ostwald viscometer (Lowe,G.D.O., and Barbenel,J.C., "Plasma and blood viscosity", in Clinical Blood Rheology, Vol. Lowe ed., CRC Press, Boca Raton, pp.18-20 (1988)). Results were expressed as relative viscosity, which was the time required by a liquid to pass two separate marks divided by the time required to travel the same distance by deionized water. The time for each solution was recorded as the mean of two consecutive times that agreed within 3 sec of each C~lbc? Plr l .IP-- WO 92111282 PCT/US91/09558 other. Viscosities of solutions of dextran with an average MW 72,000 (Sigma, St. Louis, MO) in saline were measured.

Example 4: Promotion of hemolysis by mechanical trauma and protection against hemolysis by plasma The addition of glass beads to RBC in TBS with shaking for 18 hrs at 37 0 C resulted in a sharp increase in hemoglobin release to about 30% (see, Figure 1).

Shaking human plasma instead of TBS in the presence of beads protected the red cells against hemolysis.

Serial dilutions of plasma in TBS showed a dose-dependent protection that was detectable down to 6% plasma (see, Figure 2).

Solutions of plasma in saline were prepared and compared with solutions of dextran in saline for viscosity and protection against mechanical hemolysis.

Although dextran solutions provided some protection against hemolysis, the amounts of protection afforded the cells were much less than that of plasma in concentrations with comparable viscosities. These data are shown in Table 1 below.

WO 92/11282 PCT/US91/09558 21 Table 1: Relative Viscosities and Protection Against Mechanical Hemolysis of Solutions Containing Plasma, Dextran, and Heat-Stable Extract of Fresh Frozen Plasma (FFP).

Substance Relative Protection Tested Viscosity v. Mechanical Hemolysis*+ Plasma, whole 1.75 56 Plasma 50% in .15M NaC1 1.30 Plasma 25% in .15M NaCI 1.11 Dextran 3% in .15M NaCl 1.98 33 Dextran 1.5% in .15M Nal 1.41 12 Dextran 0.75% in .15M NaCI 1.18 1 Heat-stable extract of FFP mg/ml in .15M NaC1 1.11 53 Tris-buffered .135M NaCl 1.00 0 Optical density (OD) at 412 nm of tris-buffer saline (TBS) minus OD of tested solution, divided by OD of TBS, times 100.

Measurements Relative to TLis-Buffered 0.135M NaC1 Example 5: Protection against mechanical hemolysis by plasma proteins Plasma was heated to 56°C for 20 min to determine whether complement or other heat-labile proteins in plasma are responsible for protection against mechanical hemolysis. No reduction in protective activity of plasma S after heating occurred as may be determined from Table 2 below.

WO 92/11282 PCT/US91/09558 22 Table 2: Protection in Mechanical Hemolysis Provided by Human Plasma, Human Plasma after Heating Dialysis, and Selected Plasma Proteins Protection v.

Substance Tested Mechanical Hemolysis* (mean SEM) Human plasma 75 Human Plasma (56 0 C for 20 min) 74 2 Human plasma (dialysis against saline) 59 4 Human serum albumin 4g/100ml 59 2 2g/100ml 53 4 Ig/100ml 47 12 0.5g/100ml 32 1 Human immune serum globulin 4g/100ml 10 2g/100ml 15 Ig/100ml 15 13 Haptoglobin (2mg/ml) 6 6 Ceruloplasmin Type VI (2mg/ml) 6 6 Alpha-2-macroglobulin (2mg/ml) Less than zero Optical density (OD) at 412 nm of Tris-buffered saline (TBS) minus OD of tested solution, divided by OD of TBS times 100.

The dialysis of plasma against normal saline using a membrane with a MW cut-off of 6,000-8,000 daltons did not cause loss of the majority of protective activity.

Human serum albumin provided partial protection, but when compared to plasma, the dose-response relationship indicated that serum albumin had a considerably weaker protective activity than plasma (see, Table A.above).

1 ^1 WO 92/11282 PCT/US91/09558 23 Immune serum globulin in concentrations that occur in plasma showed very weak protective activity, and the plasma proteins haptoglobin, ceruloplasmin, and alpha-2-macroglobulin gave no protection.

C

Example Heat-stable extract of plasma Fresh frozen plasma was placed in a boiling water bath for 30 min and a clear liquid supernate was obtained after centrifugation at 2,000xg for min. The total protein concentrations of two batches were 290 and 266 mg/100 ml and the sodium concentration of one batch 146 meq/L. The protective activity against mechanical hemolysis afforded by the heat-stable supernate was 70% and serial dilutions in TBS down to 1:16 showed significant activity (see, Figure 3).

Filtrates of the heat-stable supernate were prepared using ultramembrane filtration. The filtrates that passed through membranes with MW cut-offs of 300,000 and 100,000 showed as much protective activity as the original supernate. The pro:ective activity was concentrated by dialyzing the 100,000 MW filtrate of the heat-stable supernate against deionized water and lyophilizing the dialysis. A solution of 20 mg lyophilized concentrate per ml in TBS gave 60% protection against mechanical hemolysis, compared to 49% protection given by 20 mg albumin per ml. The lyophilized concentrate was determined to be 86% protein by content. Protein electrophoresis showed a heavy band in the alpha-2-globulin position without detectable albumin or other globulins.

7 Example Protein Content of Polypeptide Fraction The portion of the extract obtained in Example ifwas shown to be about 63-86% by weight protein using human serum protein .s a standard. The protein content was measured by the biuret method using a solution of copper sulfate and measuring OD at 540 mm (Sigma Diagnostics, St. Louis, MO).

l c WO 92/11282 PCT/US91/09558 24 Example I: Characterization of Polypeptide Activity The hemolytic activity of the extract obtained in Example 5 resisted enzymatic degradation by deoxyribonuclease and ribonuclease under standard conditions. Trypsin treatment caused a partial loss of protective activity.

Incubations with these enzymes (Sigma, St. Louis, MO) were carried out at about 37°C for 1 to 2 hours with appropriate controls and, when appropriate, with the addition of trypsin inhibitor.

This indicates that the active principle is neither DNA nor RNA and that the protective activity is dependent on protein(s) in the extract.

Example Trichloroacetic Treatment of Polypeptide Fraction Treatment of 4.5 ml of the plasma extract of J with 0.5 ml 50% trichloracetic acid (TCA) produced a precipitate, which when redissolved in 4.5 ml saline still contained protective activity. Thus, the protein in the protective principle is TCA-precipitable.

Examplej 0: Alcohol Denaturation of 30 Polypeptide Fraction Treatment of 2 ml of the plasma extract of Example with 8 ml of 100% ethanol produced a precipitate, which when redissolved substantially lacked protective ectivity.

This indicates that the active principle is denatured by alcohol.

Example Acid Liability of Polypeptide Fraction A solution of 100 mg extract in 4 ml of 0.15 M NaC1 was treated with hydrochloric acid to reduce the pH to and then neutralized to pH 7.0. This treatment WO 92/11282 PCT/US91/09558 resulted in the formation of a precipitate and absence of protective activity in the remaining solution.

This indicates that the protective principle is acid-labile.

1L Example 14: Effect of Viscosity of Sthe Plasma Solution The mechanism of protection against mechanical hemolysis provided by plasma was examined by testing whether the normal viscosity of plasma might provide a cushioning effect against collision of red blood cells with particles. There was a poor correlation between viscosity and protective activities of dilutions of plasma and dextran in isotonic saline, indicating that viscosity does not appear to be the principal mechanism of protection. Furthermore, a solution containing the heat-stable extract of plasma of the invention was shown to have a low relative viscosity but provided significant protection from hemolysis. In addition to protecting against mechanical hemolysis, plasma all- protected red cells against hypotonic lysis. Thus, the components of plasma that protect against hemolysis may give a surface coating to red cells that serves to repair tears and breaks in the membrane caused by stretching during exposure to hypotonic solutions or caused by collisions with injurious particles. The better protection afforded by plasma than by dextran might be attributed to negatively charged proteins that attach reversibly to red cell membranes and bind sodium ions to give an osmotically active layer that protects red cells from accumulating excess water.

The invention now being fully described, it will apparc t to one of ordinary skill in the art that many changes and modifications can be made thereto without WO 92/11282 PCr/US91/09558 26 departing from the spirit or scope of the invention as set forth herein.

Claims (20)

1. A blood cell protective composition obtained from plasma comprising a polypeptide fraction capable of inhibiting mechanical damage to cells and having the following characteristics heat-stable up to about 100 0 C; inhibitory mechanical cell damage at about pH 5.5 to inhibitory activity unaffected by ribonuclease and/or deoxyribonuclease but partially lost to trypsin; molecular weight about 6 to 100 Kdaltons; denatured by ethanol precipitation; acid labile at pH below about 1.2; and trichloroactic acid-precipitable and protectively active upon redissolution.

2. A blood cell protective composition according to claim 1 obtained by a method comprising separating plasma from blood; heating the plasma to about 90 to 100 0 C to form a heat-labile precipitate and a heat-stable supernatant; separating the heat-labile precipitate from the heat-stable supernatant; separating a peptide fraction of molecular weight about 6 to 100 Kdaltons from the heat-stable supernate; and freeze-drying the peptide fraction.

3. A blood cell protective composition according to claim 1 or 2 being substantially devoid of a blood component selected from the group consisting of albumin, complement, gamma-globulins, haptoglobins, ceruloplasmin and alpha-2-macroglobulin.

4. A cell protective solution, comprising about 0.01 to 500 mg/ml of the composition of claim 1; and a pharmaceutically-acceptable liquid carrier.

A cell protective solution according to claim 4, wherein the carrier comprises an electrolyte balance saline solution. 39

6. A cell protective solution according to claim 4 or WDN 27 further comprising about 5 to 50 mg/ml albumin.

7. A method of preparing a polypeptide fraction capable of inhibiting mechanical damage to blood cells, and having the following characteristics: heat-stable up to about 100 0 C; inhibitory of mechanical cell damage at about pH to inhibitory activity unaffected by ribonuclease and/or deoxyribonuclease but partially lost to trypsin; molecular weight about 6 tc 100 Kdaltons; denatured by ethanol precipitation; acid labile at pH below about 1.2; and trichloroacetic acid-precipitable and protectively active upon redissolution comprising separating plasma from blood; heating the plasma to about 90 to 100 0 C to form a heat-labile precipitate and a heat-stable supernate; separating the heat-labile precipitate from the heat-stable supernate; and separating a polypeptide fraction of molecular weight about 6 to 100 Kdaltons from the heat-stable supernate.

8. A method according to claim 7, wherein the polypeptide fraction is separated by filtering the supernate through a membrane having a molecular weight cut-off of about 6 to 8 Kdaltons and a membrane having a molecular weight cut-off of about 100 Kdaltons.

9. A method according to claim 7 or 8, further S 30 comprising freeze-drying the peptide fraction.

An in vivo method of preventing mechanical damage to cells, comprising intravenously administering to a subject in need of the treatment an anti-lytic amount of the protective solution according to claim 4, 5 or 6.

11. An in vivo method according to claim 10, wherein the administration of the cell protective solution is repeated at an interval effective to maintain a concentration of the peptide fraction in plasma higher than about 0.5 mg/ml. 28

12. An in vivo method according to claim 10 or 11 wherein the subject is afflicted with a hemolytic anemia.

13. An in vivo method according to claim 12, wherein the hemolytic anemia is selected from the group consisting of anemias associated with sepsis, heart surgery, atherosclerosis, hemodialysis, hemolytic-uremic syndrome and combinations thereof.

14. An in vivo method according to claim 13, wherein the anemia associated with heart surgery is selected from the group consisting of prosthetic heart valve surgery, and surgery utilizing a heart-lung bypass machine; and the anemia associated with atherosclerosis comprises elderly anemia.

An in vivo method according to any one of claims to 14, wherein the patient is administered about 0.01 g to 20 g of the peptide fraction per day.

16. An in vivo method according to claim 10, wherein the subject is afflicted with dysentery associated with Shigella dysenteriae.

17. An in vivo method a-cording to any one of claims to 16, wherein the subject is a human.

18. A blood cell protective composition, according to claim 1, substantially as hereinbefore described with reference to any one of the examples.

19. A method according to claim 7, substantially as hereinbefore described with reference to any one of the examples. An in vivo method according to claim 30 substantially as hereinbefore described with reference to any one of the examples. DATED:

20 January 1994 PHILLIPS ORMONDE FITZPATRICK Attorneys for: TEXAS TECH UNIVERSITY HEALTH SCIENCES CENTER 39 5420N 29