AU1151092A - Polypeptide fraction affording protection to cells against mechanical damage and assay - Google Patents

Description

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 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 damage to blood cells.

Description of the Background

Hemolytic uremic syndrome (HUS) is defined as the following conditions hemolytic anemia, renal failure, and thro bocytopenia. 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 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.

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-1-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 th deposition of fibronectin along with fibrinogen and platele antigen, in the vasculature of the kidneys. A deficiency o plasma factor that stimulates prostacyclin (PGI₂) activity an inhibitor of platelet aggregation, has been proposed a playing a role in the pathogensis of HUS and may be the basi for the beneficial effect of fresh plasma in the treatment o HUS.

Most patients with HUS show symptoms of malnutrition. Thi has been associated with hypoproteinemia. A role fo hypotonicity of plasma could also occur in patients wit shigellosis associated with hyponatremia. However, a degree o hypotonicity necessary to cause the lysis of red cells in vitr probably does not develop in even severe cases o hyponatremia. A decreased life span of red blood cells occur in anemia associated with various chronic diseases. This ma very well correlate with low concentrations of plasm proteins. A role for antibiotic treatment in eliciting HUS i not supported by the results of these in vitro experiments, bu it is possible that antibiotic treatment releases for exampl toxins, such a lipopolyaccharides from dying bacteria, whic promote intravascular coagulation and, therefore, increase mechanical trauma to red blood cells.

Up to the present time, there exist no effective methods o treating these conditions.

Accordingly, there is still a need for an effective metho of preventing the mechanical damage of cells in the blood tha is applicable to a variety of disease states and/or surgica procedures. DESCRIPTION OF THE INVENTION

This invention relates to a blood cell protectiv composition comprising a polypeptide fraction capable o inhibiting mechanical damage to cells and having the followin characteristics heat-stable up to about 100°C; inhibitory of mechanical cell damage at about pH 5.5 to 7.5 inhibitory activity unaffected by ribonuclease and/o 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 o mechanical cell damage upon redissolution.

This invention also relates to a cell protective solutio or suspension that comprises about 1 to 100 mg/ml of the peptide fraction of th 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°C for a period of time of, e.g., 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 Kdalto molecular weight from the heat-stable supernate.

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

This invention also relates to a method of assessing t degree of protection against mechanical damage afforded cel in a biological sample that comprises placing cells in a medium under conditions effective f maintenance of their structural integrity; adding to the medium particles capable of undergoing a mi mechanical interaction with the cells; adding to the medium a substance to be tested; agitating the medium comprising the cells for a pres period of time and preset conditions; and determining the amount in the medium of a factor associat with the degree of mechanical damage of the cell.

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

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 represents the hemolysis of human red blood cel after incubation at 37°C for 18 hrs in Tris-buffered saline 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 10O°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.

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 5.5 to 7.5; 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°C as either whole blood or plasma. Outdated blood or plasma of more than 6 weeks after donation, may be used as well.

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 activit was shown not to have substantially lost it. The addition o high concentration of ethanol to the polypeptide fractio produces a precipitate which upon redissolution may be shown t have substantially lost most of its cell protective activity Treatment of the polypeptide fraction with a strong acid suc as hydrochloric acid to lower the pH to about 1.0 followed b neutralization back to pH 7.0 was shown to form a precipitate, and the remaining supernate was devoid of cell protectiv activity.

In another preferred embodiment, the cell-protectiv composition of the invention is substantially devoid of one o more blood component(s) such as albumin, complement, gamma-globulins, haptoglobulins, ceruloplasmin, an alpha-2-macroglobulin. These are all plasma components tha may be eliminated from the polypeptide fraction of th invention.

In one embodiment of the invention, the cell-protectiv extract was shown to be free of albumin and gamma globulins b protein electrophoresis. Complement activity was not present because of the prior heating step. Other proteins, e.g., haptoglobins, ceruloplasmin, and alpha-2-macroglobulin, ar 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 ma 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/o polypeptides of molecular weight greater than about 30 Kdalton, and more preferably greater than about 100 Kdalton ma also be eliminated from the fraction of the invention, e.g., b filtration. In a still more preferred embodiment, th polypeptide fraction encompasses polypeptides of about 20 to 8 Kdalton molecular weight.

The polypeptide fraction of the composition of th invention may be prepared by the method described below However, the present invention also covers composition comprising the active component of the composition which ar prepared by other methods and have have the ability to inhibi mechanical damage inflicted to cells.

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

In a particularly preferred embodiment of the invention the cell-protective solution comprises about 1 to 200 mg/m cell-protective composition.

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

The cell-protective solution of the invention finds its us in in vivo and in vitro methods. In one embodiment, it i provided herein an in vivo method of inhibiting mechanica damage to cells comprising the intravenous administration o the solution of this invention to a patient in need of th treatment. Accordingly, the protective composition and th cell-protective solution of the invention must be free of toxicity and particles of any blood transmissible diseases suc as AIDS, and various other viruses and bacteria. Testing an 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 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 polypeptid fraction such as the one described above that is capable o inhibiting mechanical damage to cells, the method comprising separating plasma from blood; heating the plasma to about 90 to 100°C for a period o time of, e.g., about 15 to 30 min or longer, to form heat-labile precipitate and a heat-stable supernate; separating the heat-labile precipitate from the heat-stabl supernate; and separating a polypeptide fraction of about 6 to 100 Kdalto molecular weight from the heat-stable supernate.

The separation of plasma from mammalian blood, includin human blood may be conducted as is known in the art. Briefly the separation of plasma from blood may be conducted b centrifuging anticoagulated blood at about 1,500 x g for abou 15 min. The clear upper plasma layer may be removed e.g., 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 o the blood, it may be heated by itself to a temperature of abou 85 to 100°C, and more preferably to about 90 to 100°C for period of time, e.g., of about 10 to 60 min, and mor preferably about 15 to 30 min, to separate heat-precipitabl components. These precipatible components include albumi beta globulins, gamma globulins, and complement, among others.

The heat-labile precipitate may be separated from thi supernate by implementing technology which is known in t art. By means of example, the separation may be conducted centrifugation or filtration. Of these, preferable i centrifugation because no component is generally lost adhesion to a filter and no extraneous material is added b elution from the filter. However, other means of separatin the precipitate from the supernate may also be utilized whic are known in the art.

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

The separation of the specified molecular weigh polypeptide fraction may be conducted as is known in the art. By means of example, dialysis may be conducted with a membran sieving molecules of less than about 6 Kdaltons, and mor preferably less than about 8 Kdaltons. In a separate step, ultrafiltration may be conducted with a membrane with a cut-of molecular weight of 300 Kdalton, and more preferably about 10 Kdalton. However, other methods may also be utilized such a gel filtration, ion exchange, and column chromatography, amon others.

Once the desired molecular weight range polypeptid fraction is separated from the remaining supernate, it may, I >_

optionally, be freeze-dried, particularly, when it is intende for storage. The freeze-dried polypeptide fraction may b stored in a sealed container as is known in the art at temperature of about -60 to 4°C, and particularly belo -20°C. The freeze-dried polypeptide fraction has been shown t have a shelf life of at least about 3 months at a temperatur of about 4°C. The freeze-dried polypeptide fraction may b refrigerated and/or stored at freezing temperatures, as well.

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

Also provided herein is a method of preventing mechanica damage to cells that comprises the intravenous administratio to a subject in need of the treatment of an anti-lytic amoun of the polypeptide fraction of the invention, optionally in th form of the cell protective solution of the invention. Th intravenous administration of the polypeptide fraction of thi invention is recommended since the polypeptides and/or protei in the fraction would be hydrolyzed in the gastrointestina tract when administered orally or by other routes, such as b rectal suppository.

The cell protective solution of the invention may b administered to a subject at a concentration of 10 to 500 m polypeptide fraction/ml solution, and more preferably about 5 to 100 mg polypeptide in the fraction/ml solution.

When administered to a subject in need of on-going cel protection from mechanical damage, the cell protective solutio may be repeatedly administered at intervals effective t maintain a concentration of the polypeptide fraction in th 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 of 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.

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, th regime for the administration of the polypeptide fraction o the invention and/or the cell protective solution describe herein is similar to what was described above.

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

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

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

Once the cells are placed in the medium, various similarl prepared samples may be run side-by-side. One will remai without the addition of a substance to be tested and on without the particles whereas a third one, as well as othe test samples if there is more than one substance to be tested will have the particles and the substance(s) to be tested adde to the medium under standardized conditions. Typically, th test may be conducted at room temperature or at a temperatur suitable for the maintenance and growth of the cell on whic the substance is to be tested. However, all different sample are to be run under similar conditions.

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

The agitation of the medium comprising the cells and th other ingredients is conducted for a preset period of time an under similar conditions. Typically, the agitation i conducted at about 100 to 200 oscillations/minute, and mor preferably about 150 to 180 oscillations/minute at temperature of, e.g., about 35 to 39°C.

Once the preset period of time for the test has bee completed, agitation may be stopped and the amount of a factor that is associated with the degree of mechanical damage to th 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 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 show in the examples. The determination of hemoglobin in the mediu may be conducted by measuring the optical density (O.D.) of th various samples containing the cells at about 412 nm Similarly, for other types of cells, a parameter can be foun which correlates with the degree of damage inflicted to them Leukocyte esterase may be measured for the measurement o damage to white blood cells and cytokines such as interleukin- may be assayed for infected macrophages.

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

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

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 l,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 7.5 (TBS), plasma, or other tested solution. After mixing, the tubes were incubated at 37°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 of mechanical hemolysis. The tubes were placed in a shaking water bath at a 45° 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 hemoglobin. The per cent protection of tested solutio against mechanical hemolysis was expressed as the O.D. of r blood cells shaken in TBS minus the O.D. of cells shaken in t test solution, divided by the O.D. of cells shaken in TB times 100.

Example 2: Preparation of protein solutions

Human plasma and dilutions of plasma in TBS were made fr blood obtained from normal volunteers in heparin to a fin concentration of 50 units/ml. Autologous plasma was used wi RBC in hemolytic assays. Plasma was heated in a water bath 56°C for 20 min to inactivate complement. Plasma was dialyz against normal saline in a dialysis tube with MW cut-o 6,000-8,000 (Spectra/por, Los Angeles, CA) . Purified hum plasma proteins were obtained to test their effect in t protection against mechanical hemolysis after dissolving in T 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 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°C and lyophilizing the dialysand. The total protein concentration was measured by the biuret method (Doumas, A.T., Bayse, D.D., Carter, R.J., Peters, T., and Schaffer, R., "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, C.B., "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 a Ostwald viscometer (Lowe,G.D.O. , and Barbenel,J.C. , "Plasma an blood viscosity", in Clinical Blood Rheology, Vol. I., Low G.D.O., ed., CRC Press, Boca Raton, pp.18-20 (1988)). Result were expressed as relative viscosity, which was the tim required by a liquid to pass two separate marks divided by th time required to travel the same distance by deionized water The time for each solution was recorded as the mean of tw consecutive times that agreed within 3 sec of each other Viscosities of solutions of dextran with an average MW 72,00 (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 fo 18 hrs at 37°C resulted in a sharp increase in hemoglobi release to about 30% (see, Figure 1).

Shaking human plasma instead of TBS in the presence o beads protected the red cells against hemolysis. Seria dilutions of plasma in TBS showed a dose-dependent protectio that was detectable down to 6% plasma (see, Figure 2) .

Solutions of plasma in saline were prepared and compare with solutions of dextran in saline for viscosity an protection against mechanical hemolysis. Although dextra solutions provided some protection against hemolysis, th amounts of protection afforded the cells were much less tha that of plasma in concentrations with comparable viscosities These data are shown in Table 1 below. 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 s Protection Tested Viscosity , Mechanical

Hemolysis*+

Plasma, whole Plasma 50% in .15M NaCl Plasma 25% in .15M NaCl Dextran 3% in .15M NaCl Dextran 1.5% in .15M Nal Dextran 0.75% in .15M NaCl Heat-stable extract of FFP

20 mg/ml in .15M NaCl Tris-buffered .135M NaCl

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 Tris-Buffered 0.135M

NaCl

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 after heating occurred as may be determined from Table 2 below. 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 + 5

Human Plasma

(56°C for 20 min) 74 ± 2

Human plasma

(dialysis against saline) 59 ± 4

Human serum albumin

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 3 above).

Immune serum globulin in concentrations that occur in plasma showed very weak protective activity, and the plasma proteins haptoglobin, ceruloplasmin, and alρha-2-macroglobulin gave no protection.

Example 5: 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 15 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 protective 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.

Example 6: Protein Content of

Polypeptide Fraction

The portion of the extract obtained in Example 8 was show to be about 63-86% by weight protein using human serum protei as a standard. The protein content was measured by the biure method using a solution of copper sulfate and measuring OD a 540 mm (Sigma Diagnostics, St. Louis, MO).

Example 7: Characterization of Polypeptide Activity

The hemolytic activity of the extract obtained in Example resisted enzymatic degradation by deoxyribonuclease an ribonuclease under standard conditions. Trypsin treatmen caused a partial loss of protective activity.

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

This indicates that the active principle is neither DNA no RNA and that the protective activity is dependent on protein(s in the extract. Example 8: Trichloroacetic Treatment of Polypeptide Fraction

Treatment of 4.5 ml of the plasma extract of Example 8 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.

Example 9: Alcohol Denaturation of Polypeptide Fraction

Treatment of 2 ml of the plasma extract of Example 5 with 8 ml of 100% ethanol produced a precipitate, which when redissolved substantially lacked protective activity.

This indicates that the active principle is denatured by alcohol.

Example 10: Acid Liability of Polypeptide Fraction

A solution of 100 mg extract in 4 ml of 0.15 M NaCl was treated with hydrochloric acid to reduce the pH to 1.0, and then neutralized to pH 7.0. This treatment 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.

Example 11: Effect of Viscosity of the 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 agains collision of red blood cells with particles. There was a poo correlation between viscosity and protective activities o dilutions of plasma and dextran in isotonic saline, indicatin that viscosity does not appear to be the principal mechanism o protection. Furthermore, a solution containing the heat-stabl extract of plasma of the invention was shown to have a lo relative viscosity but provided significant protection fro hemolysis. In addition to protecting against mechanica hemolysis, plasma also protected red cells against hypotoni lysis. Thus, the components of plasma that protect agains hemolysis may give a surface coating to red cells that serve to repair tears and breaks in the membrane caused by stretchin during exposure to hypotonic solutions or caused by collision with injurious particles. The better protection afforded b plasma than by dextran might be attributed to negativel charged proteins that attach reversibly to red cell membrane and bind sodium ions to give an osmotically active layer tha protects red cells from accumulating excess water.

The invention now being fully described, it will apparen to one of ordinary skill in the art that many changes an modifications can be made thereto without departing from th spirit or scope of the invention as set forth herein.

Claims (20)

1. A 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°C; inhibitory mechanical cell damage at about pH 5.5 to 7.5; 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 protectively active upon redissolution.

2. The cell protective composition of claim 1 obtained by a method comprising separating plasma from blood; heating the plasma to about 90 to 100°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. The cell protective composition of claim 1 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; an a pharmaceutically-acceptable liquid carrier.

5. The cell protective solution of claim 4, wherein the carrier comprises an electrolyte balance sal solution.

6. The cell protective solution of claim 4, furt comprising about 5 to 50 mg/ml albumin.

7. A method of preparing a polypeptide fraction capa of inhibiting mechanical damage to cells, comprising separating plasma from blood; heating the plasma to about 90 to 100°C to form heat-labile precipitate and a heat-stable supernate; separating the heat-labile precipitate from the heat-sta supernate; and separating a polypeptide fraction of molecular weight ab 6 to 100 Kdaltons from the heat-stable supernate.

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

9. The in vivo method of claim 7, further comprising freeze-drying the peptide fraction.

10. 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 of claim 4.

11. The in vivo method of 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.

12. The in vivo method of claim 10, wherein the subject is afflicted with a hemolytic anemia.

13. The method of 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. The in vivo method of 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.

15. The in vivo method of claim 11, wherein the patient is administered about 0.01 g to 20 g of the peptide fraction per day.

16. The in vivo method of claim 10, wherein the subject is afflicted with dysentery associated with Shigella dysenteriae.

17. The in vivo method of claim 10, wherein the subject is a human.

18. An in vitro method of assessing the degree of protection from mechanical damage afforded cells in a biological sample, comprising 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 and under preset conditions; and determining the amount in the medium of a factor associated with the degree of mechanical damage of the cell.

19. The method of claim 18, wherein the cells are red blood cells; and step(s) is conducted by determining the amount of hemoglobin present in the medium.

20. The method of claim 19, wherein the amount of hemoglobin is determined by measuring the absorbance of the medium at about 412 nm.