WO1995024206A1 - Stroma, method of preparation thereof and its use in treatment of septic shock in mammals - Google Patents

Stroma, method of preparation thereof and its use in treatment of septic shock in mammals Download PDF

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Publication number
WO1995024206A1
WO1995024206A1 PCT/US1995/003215 US9503215W WO9524206A1 WO 1995024206 A1 WO1995024206 A1 WO 1995024206A1 US 9503215 W US9503215 W US 9503215W WO 9524206 A1 WO9524206 A1 WO 9524206A1
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Prior art keywords
stroma
septic shock
treatment
endotoxin
present
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PCT/US1995/003215
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French (fr)
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Livia Blum
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Livia Blum
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Priority to AU19987/95A priority Critical patent/AU1998795A/en
Publication of WO1995024206A1 publication Critical patent/WO1995024206A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14Blood; Artificial blood
    • A61K35/18Erythrocytes

Definitions

  • This invention pertains to stroma which is useful in the treatment of septic shock in mammals.
  • a method of making stroma free of bacterial or viral contamination is presented, as well as pharmaceutical formulations or dosage forms comprising stroma. Methods of using this material in the treatment of septic shock in mammals are also presented.
  • Septic shock or septicemia is characterized by inadequate circulation to the tissues, usually following an infection with a gram-negative bacteria in the bloodstream of a mammal.
  • Subjects suffering from septic shock in general display inflammation, low blood pressure, low urine output, rapid heartbeat, rapid breathing, and fever. These symptoms are the result of diffuse cell and tissue injury caused by the release of endotoxin, the lipopolysaccharide moiety of the bacterial cell wall, into the blood stream.
  • Endotoxins, complex lipopolysaccharides (LPS) are the major component of the outer envelope of gram-negative bacteria, and are responsible for the most serious clinical consequences of gram-negative bacterial infections.
  • Septic shock is most often associated with gram- negative bacterial infections, but can also be associated with gram-positive bacteremia.
  • Escheri chia coli , Klebsiella - Enterobacter , Pseudomonas , and Serratia are the most frequent causative organisms.
  • Most of the bacteria responsible for gram- negative sepsis are normal flora of the gastrointestinal tract which have gained entry into the bloodstream because of an underlying disease of the subject.
  • Predisposing factors to development of septic shock include: diabetes, cirrhosis, leukemia, lymphoma or disseminated carcinoma and the chemotherapeutic or radiation treatment of these malignancies, transplantation and its associated immunosuppression, childbirth, and a variety of surgical procedures.
  • diabetes cirrhosis
  • leukemia a malignancies
  • lymphoma or disseminated carcinoma a malignancies that transplantation and its associated immunosuppression
  • childbirth a variety of surgical procedures.
  • the majority of septic shock patients are hospitalized at the time of the infection for treatment of their primary underlying condition.
  • the incidence of gram-negative bacterial sepsis is increasing and may be as high as 12 cases per 1000 admissions in large urban hospitals. If the sepsis progresses to septic shock, the mortality rate approaches 50% despite aggressive treatment methods.
  • septic shock The basis for treatment of septic shock is currently antibiotic treatment of the bacterial infection, coupled with treatment of the symptoms of the condition. Although certain antibiotics can effectively treat the primary bacterial infection, they are ineffective in removing the endotoxin from the circulation.
  • antibiotics can effectively treat the primary bacterial infection, they are ineffective in removing the endotoxin from the circulation.
  • Patients suffering from septic shock are generally put on respiratory support, given fluids, and treated with drugs which affect the vascular system by increasing circulation, such as dopamine hydrochloride or isoproterenol .
  • blood transfusions, diuretics, and steroids may be administered.
  • each of these measures treat only the symptoms and do not affect the underlying cause of the disease, namely, the circulating endotoxin.
  • the present invention provides a stroma product which effectively and nonreversibly, under physiological conditions, binds endotoxin. This binding allows for removal of endotoxin from the bloodstream, blocking the interaction of the endotoxin with cell surfaces, a step believed to be necessary for the pathogenic effects of the endotoxin.
  • the present invention provides a method of producing this stroma product, where this product is made from erythrocytes, is free from bacterial or viral contaminants, and retains its ability to bind endotoxin.
  • a further aspect of the present invention provides a method of using of the stroma product in the treatment of septic shock in mammals which has resulted from the toxic effects of circulating bacterial endotoxin.
  • a still further aspect of the present invention provides a pharmaceutical formulation or dosage form for administration to a mammal suffering from toxic shock comprising stroma produced pursuant to the present invention and a pharmaceutically acceptable carrier or diluent.
  • Frute is defined herein as the washed, incubated fragments of the mammalian erythrocyte cell membrane produced pursuant to the methods of the present invention.
  • mammals are defined herein as any organism having an immune system and susceptible to developing septic shock.
  • “Sepsis” is defined herein as bacterial contamination or infection of the blood. “Treatment” is defined herein as administration of the stroma of the present invention to patients suffering from septic shock and to those at risk for developing septic shock as defined below. "At risk for developing septic shock” is defined herein as those patients suffering from any one of diabetes, cirrhosis, leukemia, lymphoma, disseminated carcinoma and treatment for these malignancies, transplantation and its associated immunosuppression and post-surgical procedures and in the known high risk group for developing septic shock.
  • Endotoxin or “LPS” is defined herein as the liposaccharide moiety of gram-negative bacterial membranes.
  • Washing is defined herein as pelleting by low speed centrifugation (e.g. about 3,000 rpm) for the red blood cells or the stroma of the present invention, and resuspension in aqueous media.
  • the product of the present invention is a preparation of human erythrocyte cell wall, or stroma.
  • the stroma is produced through the lysis of normal erythrocytes in water, preferably under particular temperature and pH conditions.
  • Erythrocytes suitable to produce the stroma of the present invention include any which contain on its surface the lipopolysaccharide or endotoxin receptor, and should preferably be of the same species as the subject being treated.
  • an additional concern is the blood type of the donor erythrocytes used to make the stroma. Preferably, they should be type 0 (the "Universal Donor") , Rh " to assure compatibility with the recipient. If the blood type of the recipient is known prior to production of the stroma, erythrocytes of a compatible blood type can be used.
  • a preferred source for the erythrocytes is donated blood which has passed the 30 day shelf life for transfusion.
  • the stroma is then washed, pH adjusted to between about pH 7.0 and 7.4, resuspended in a 0.15M NaCl solution, and incubated at about 56°C for about 30 minutes.
  • Stroma produced according to the method of the present invention is stable, and can be stored for at least three months, although it is preferable that the preparation be used within 30 days.
  • a preferred method for the production of the stroma of the present invention is described in detail in Example 1 herein below.
  • this method includes osmotic conditions which will lyse any membrane bound cells, and low speed centrifugation, which will not pellet viruses and cause contamination of the pelleted stroma.
  • the result of these steps is a stroma preparation which is free of contaminating cells and organisms.
  • Stroma produced according to the method of the present invention can, without pretreatments, irreversibly bind endotoxin under the conditions present in the bloodstream, i.e. at physiological temperatures, in the presence of human serum, and without the addition of further cofactors.
  • This preparation is nontoxic and does not contain residual bacterial or viral contaminants which may have been present in the initial erythrocyte sample.
  • the stroma should not be immunologically recognized by the body as foreign.
  • the stroma product described above may be used in a method of treating mammals suffering from septic shock.
  • Introduction of the stroma into the bloodstream of a mammal provides sites for the endotoxin to bind.
  • the endotoxin Upon binding to the stroma, the endotoxin is effectively removed from the circulation, as it can no longer bind to the cells of the treated subject.
  • the stroma is being used as a molecular "decoy" to bind irreversibly to any endotoxin (whether bacterially- associated or free) and remove or nullify its adverse biological effects.
  • the blockage of this binding is believed to prevent, or at least diminish, the direct effects of endotoxin binding, such as cell membrane damage by the endotoxin.
  • stroma produced according to the present invention binds endotoxin irreversibly for 24-48 hours at 37 3 C.
  • the present treatment method involves the administration of an amount of the stroma of the present invention effective to treat septic shock to a mammal suffering from or at risk for septic shock. It is important that this treatment be initiated early in the onset of the disease for optimal treatment, although later treatment may have the ability to stop the development of further damage. If susceptibility of a patient to septic shock is suspected, stroma may also be administered prophylactically.
  • the stroma prepared according to the present invention is administered parenterally and preferably intravenously.
  • the effective amount of stroma to be administered to a mammal in need of such treatment is subject to optimization and may differ according to the stage of septic shock, the causative organism (the bacteria) and the underlying disease of the patient.
  • determination of the optimum dosage is considered well within the reach of one of ordinary skill in the art.
  • an effective dosage is one which results in measurable improvement of any one or more of the well known symptoms of septic shock.
  • the minimum effective dosage can be determined by conducting no more than routine experiments, i.e. beginning by administering a relatively low amount of stroma, and monitoring the patient for any reduction in the well-known symptoms of the disease. If no effect is observed, log increases of stroma can be administered until positive effects are noted. It is believed that the stroma produced pursuant to the present invention will be safe and non-toxic as long as type 0, Rh " or a compatible donor's blood is used to produce the stroma.
  • the present invention also provides pharmaceutical formulations or dosage forms comprising stroma produced pursuant to the methods of the present invention and a pharmaceutically acceptable carrier or diluent.
  • pharmaceutically acceptable carriers or diluents of the present invention include 0.15M NaCl, 5% dextrose, and glycine phosphate, pH 7.4. It will be understood to those of ordinary skill in the art that the pharmaceutical formulations or dosage forms of the present invention need not constitute an effective amount of the stroma of the present invention to treat septic shock as such effective amount can be attained by administration of a plurality of such pharmaceutical formulations or dosage forms.
  • the methods of preparing the stroma of the present invention as described herein can, with some modifications, be used to prepare cell walls from normal human cells. Such modifications include removing contaminating DNA. Such DNA would be present because, unlike human red blood cells, eukaryotic cells contain nuclei. Removal of such substances is well within the ambit of those of ordinary skill in the art.
  • rbc preferably from blood group 0, Rh-negative, but not limited to O-Rh-negative as long as the rules that apply to rbc used for transfusions are observed.
  • Stroma will not replace the function of rbc. Stroma could serve as a prophylactic agent for the protection of rbc to prevent loss through destruction during gram-negative bacterial sepsis which could lead to septic shock.
  • Stroma was prepared as set forth below:
  • One unit (450-500 ml) of blood from a healthy donor's red blood cells were separated from plasma and other cellular components of blood producing a packed rbc pellet. From the packed rbc pellet, 50 ml was withdrawn and distributed in 10 ml amounts in 100 ml capacity centrifuge tubes.
  • the rbc suspensions produced above was centrifuged at 2°C for 30 minutes at 3,000 r.p.m. in a fixed angle rotor in an International centrifuge.
  • Steps 5, 6 and 7 were repeated three more times.
  • test tubes 10. The contents of the five test tubes were pooled by pouring them into a 1,000 ml flask.
  • the centrifuge tubes were centrifuged for 60 minutes at 2-C at 3,000 r.p.m. 15. The supernatant was removed by aspiration and discarded.
  • the washed stroma was pooled with the addition of
  • the pH of the pooled stroma was adjusted to 7.0 to 7.4+0.05 with 0.IN NaOH.
  • the NaCl concentration was adjusted with 2M NaCl to 0.15M by deducting the NaOH contribution to Na concentration when the final pH was between 7.0 to 7.4+.0.05 and the total volume was brought to 100 ml with additional deionized distilled H 2 0.
  • the stroma obtained as above may be reprecipitated by repeating steps 9-20.
  • the stroma which is at pH 7.0-7.4 and at an ionic strength of 0.15M, is diluted 10-fold with cold, deionized, distilled H 2 0 (i.e., for each one volume of stroma, nine volumes of cold deionized, distilled H 2 0 is added, step 9 above) .
  • the pH of this mixture is adjusted to pH 5.2-5.4 with 0.IN acetic acid and incubated at 2°C for 18 hours, etc.
  • EXAMPLE 2 Schwartzman Test to Determine Efficacy The following in vivo test can be used to determine the ability of the present treatment to remove endotoxin from the bloodstream.
  • the Schwartzman phenomenon is a well-known in vivo test for determining the presence or absence of active endotoxin in a sample (Schwartzmann, G. , Phenomenon of Local Tissue .Reactivity, Paul B. Hoeber, Inc., 1937; Apitz, K. , Virchow. Arch. Path . Anat . 289 :46, 1933; Thomas, H. , Ann. Rev. Physiol. 16:467, 1954) . Similar techniques to the one set forth below have been described in Stetson, CA. , Jr., J. Exp . Med. lCL:421-430, 1951 and Stetson, C.A., Jr., J " . Exp. Med. £4:347-358, 1951. I- The Schwartzman Test
  • E. coli endotoxin can be obtained directly from the bacteria.
  • the endotoxin will be phenol extracted before use in this assay.
  • E. coli endotoxin can be obtained from numerous commercial sources such as Sigma Chemical Co. (St. Louis, MO) , Difco Laboratories (Detroit, MI) or Calbichem (San Diego, CA) .
  • the commercially available endotoxin is dissolved in aqueous media (e.g. PBS, 0.15M NaCl, etc.) before use.
  • Test (A) no pretreatment
  • Test (C) incubation for 30 minutes at 37°C.
  • Varying concentrations of stroma (produced as described above) and LPS will be mixed together in the presence and absence of human serum. Human red blood cells are then added as indicators. The reactions are incubated at 37°C for 24-48 hours. Included in the reactions are a negative control (no LPS added) and a positive control (no stroma added) . The reactions are monitored by testing the red blood cells for susceptability to lysis.

Abstract

Disclosed herein are methods of treating septic shock in mammals comprising administration of stroma to mammals in need of such treatment, methods for producing the stroma and pharmaceutical formulations comprising stroma produced pursuant to the present invention.

Description

STROMA, METHOD OF PREPARATION THEREOF AND ITS USE IN TREATMENT OF SEPTIC SHOCK IN MAMMALS
FIELD OF THE INVENTION
This invention pertains to stroma which is useful in the treatment of septic shock in mammals. A method of making stroma free of bacterial or viral contamination is presented, as well as pharmaceutical formulations or dosage forms comprising stroma. Methods of using this material in the treatment of septic shock in mammals are also presented. BACKGROUND OF THE INVENTION
Septic shock or septicemia is characterized by inadequate circulation to the tissues, usually following an infection with a gram-negative bacteria in the bloodstream of a mammal. Subjects suffering from septic shock in general display inflammation, low blood pressure, low urine output, rapid heartbeat, rapid breathing, and fever. These symptoms are the result of diffuse cell and tissue injury caused by the release of endotoxin, the lipopolysaccharide moiety of the bacterial cell wall, into the blood stream. Endotoxins, complex lipopolysaccharides (LPS) , are the major component of the outer envelope of gram-negative bacteria, and are responsible for the most serious clinical consequences of gram-negative bacterial infections. As little as subnanogram to nanogram per ml concentrations of these agents elicit in the host many cellular and extracellular responses, including the production and release of a diverse network of mediators such as activated complement components, cytokines (e.g., tumor necrosis factor, interleukins) , arachadonate metabolites, etc. (Morrison, D.C. and Ryan, J.L. , Ann. Rev. Med. 38:417-432, 1987) .
Septic shock is most often associated with gram- negative bacterial infections, but can also be associated with gram-positive bacteremia. Escheri chia coli , Klebsiella - Enterobacter , Pseudomonas , and Serratia are the most frequent causative organisms. Most of the bacteria responsible for gram- negative sepsis are normal flora of the gastrointestinal tract which have gained entry into the bloodstream because of an underlying disease of the subject. Predisposing factors to development of septic shock include: diabetes, cirrhosis, leukemia, lymphoma or disseminated carcinoma and the chemotherapeutic or radiation treatment of these malignancies, transplantation and its associated immunosuppression, childbirth, and a variety of surgical procedures. As such, the majority of septic shock patients are hospitalized at the time of the infection for treatment of their primary underlying condition. The incidence of gram-negative bacterial sepsis is increasing and may be as high as 12 cases per 1000 admissions in large urban hospitals. If the sepsis progresses to septic shock, the mortality rate approaches 50% despite aggressive treatment methods.
The basis for treatment of septic shock is currently antibiotic treatment of the bacterial infection, coupled with treatment of the symptoms of the condition. Although certain antibiotics can effectively treat the primary bacterial infection, they are ineffective in removing the endotoxin from the circulation.- Patients suffering from septic shock are generally put on respiratory support, given fluids, and treated with drugs which affect the vascular system by increasing circulation, such as dopamine hydrochloride or isoproterenol . In addition, blood transfusions, diuretics, and steroids may be administered. However, each of these measures treat only the symptoms and do not affect the underlying cause of the disease, namely, the circulating endotoxin. Accordingly, recent efforts have been made to develop a septic shock treatment based on the removal of the endotoxin from the bloodstream. A monoclonal antibody specific for endotoxin (HA-1A) has been developed as a treatment modality for septic shock. However, in clinical trials this treatment proved ineffective. Related monoclonal products (E5 and T88) are currently being tested, but results have not yet been obtained. U.S. Patent No. 5,171,739 discloses the treatment of septic shock by administering Bactericidal/Permeability-Increasing protein (BPI) , which is said to not only kill the bacteria but also neutralize the endotoxin. Other treatment methods include those which block the action of cytokines which produce the inflammation associated with sepsis, such as interleukin-1 and tissue necrosis factor. However, it remains that there is currently no effective treatment reported in the art which removes the causative factor of septic shock, the bacterial endotoxin, from the bloodstream.
There are reports in the literature of a lipopolysaccharide receptor isolated from the surface of human erythrocytes (Springer et al. , Biochemistry 13 (7) :1379-1389, 1974) which was said to bind to LPS, and thus could potentially be used therapeutically. However, the method of production of the isolated receptor renders it unacceptable for clinical use, and involves pretreatments such as boiling and enzymatic exposure. Further, this publication discloses that the interaction of the receptor with endotoxin is reversible, an unacceptable situation in a clinical treatment. Finally, the reported binding experiments were not done in the presence of human serum and thus may have little applicability for prediction of receptor function in an in vivo environment.
Therefore, what is needed in the art are effective treatments for septic shock which are capable of removing or sequestering bacterial endotoxin from the bloodstream of patients at risk for or suffering from septic shock caused by gra - negative bacterial endotoxin. SUMMARY OF THE INVENTION
Accordingly, in one aspect the present invention provides a stroma product which effectively and nonreversibly, under physiological conditions, binds endotoxin. This binding allows for removal of endotoxin from the bloodstream, blocking the interaction of the endotoxin with cell surfaces, a step believed to be necessary for the pathogenic effects of the endotoxin.
In another aspect the present invention provides a method of producing this stroma product, where this product is made from erythrocytes, is free from bacterial or viral contaminants, and retains its ability to bind endotoxin.
A further aspect of the present invention provides a method of using of the stroma product in the treatment of septic shock in mammals which has resulted from the toxic effects of circulating bacterial endotoxin.
A still further aspect of the present invention provides a pharmaceutical formulation or dosage form for administration to a mammal suffering from toxic shock comprising stroma produced pursuant to the present invention and a pharmaceutically acceptable carrier or diluent.
Other aspects and advantages of this invention will be apparent to those of ordinary skill in the art from the following description and claims. DETAILED DESCRIPTION OF THE INVENTION
In the present specification, the following terms are defined below.
"Stroma" is defined herein as the washed, incubated fragments of the mammalian erythrocyte cell membrane produced pursuant to the methods of the present invention.
"Mammals" are defined herein as any organism having an immune system and susceptible to developing septic shock.
"Sepsis" is defined herein as bacterial contamination or infection of the blood. "Treatment" is defined herein as administration of the stroma of the present invention to patients suffering from septic shock and to those at risk for developing septic shock as defined below. "At risk for developing septic shock" is defined herein as those patients suffering from any one of diabetes, cirrhosis, leukemia, lymphoma, disseminated carcinoma and treatment for these malignancies, transplantation and its associated immunosuppression and post-surgical procedures and in the known high risk group for developing septic shock.
"Endotoxin" or "LPS" is defined herein as the liposaccharide moiety of gram-negative bacterial membranes.
"Washed" or "washing" is defined herein as pelleting by low speed centrifugation (e.g. about 3,000 rpm) for the red blood cells or the stroma of the present invention, and resuspension in aqueous media.
The product of the present invention is a preparation of human erythrocyte cell wall, or stroma. The stroma is produced through the lysis of normal erythrocytes in water, preferably under particular temperature and pH conditions. Erythrocytes suitable to produce the stroma of the present invention include any which contain on its surface the lipopolysaccharide or endotoxin receptor, and should preferably be of the same species as the subject being treated. In humans, an additional concern is the blood type of the donor erythrocytes used to make the stroma. Preferably, they should be type 0 (the "Universal Donor") , Rh" to assure compatibility with the recipient. If the blood type of the recipient is known prior to production of the stroma, erythrocytes of a compatible blood type can be used. A preferred source for the erythrocytes is donated blood which has passed the 30 day shelf life for transfusion.
After lysis of the red blood cells, the cell membranes are centrifuged, washed, adjusted to between about pH 5.2 and about 5.4 and incubated at 2=C for 18 hours. The stroma is then washed, pH adjusted to between about pH 7.0 and 7.4, resuspended in a 0.15M NaCl solution, and incubated at about 56°C for about 30 minutes. In a particularly preferred embodiment of the present invention the stroma is brought to between pH 5.0 and 5.4, further washed, incubated at about 2°C for about 18 hours, resuspended in 0.15M NaCl and the pH re-adjusted to 7.0 - 7.4 ± 0.05. In either case, the stroma can then be used or stored at between about 2°C and about 4=C.
Stroma produced according to the method of the present invention is stable, and can be stored for at least three months, although it is preferable that the preparation be used within 30 days. A preferred method for the production of the stroma of the present invention is described in detail in Example 1 herein below. In the present method, there are several steps which bring about the elimination of any viral or bacterial contamination which may be present in the initial erythrocyte sample. Specifically, this method includes osmotic conditions which will lyse any membrane bound cells, and low speed centrifugation, which will not pellet viruses and cause contamination of the pelleted stroma. The result of these steps is a stroma preparation which is free of contaminating cells and organisms.
Stroma produced according to the method of the present invention can, without pretreatments, irreversibly bind endotoxin under the conditions present in the bloodstream, i.e. at physiological temperatures, in the presence of human serum, and without the addition of further cofactors. This preparation is nontoxic and does not contain residual bacterial or viral contaminants which may have been present in the initial erythrocyte sample. Subject to the guidelines known by those of ordinary skill in the art and used for other blood product transfusions, the stroma should not be immunologically recognized by the body as foreign.
The stroma product described above may be used in a method of treating mammals suffering from septic shock. Introduction of the stroma into the bloodstream of a mammal provides sites for the endotoxin to bind. Upon binding to the stroma, the endotoxin is effectively removed from the circulation, as it can no longer bind to the cells of the treated subject. Thus, the stroma is being used as a molecular "decoy" to bind irreversibly to any endotoxin (whether bacterially- associated or free) and remove or nullify its adverse biological effects. Without being bound by theory, the blockage of this binding is believed to prevent, or at least diminish, the direct effects of endotoxin binding, such as cell membrane damage by the endotoxin. This blockage is also believed to reduce secondary effects of endotoxin binding, such as the extracellular release of lysosomal enzymes and cytokines from leukocytes, the activation of the complement cascade which can lead to hemolysis (lysis of the erythrocytes of the patient) , and tissue injury due to lack of oxygen, which occurs upon damage to the circulating cells and the capillaries. Any reduction in the deleterious biological effects of endotoxin pursuant to the administration of the stroma produced according to the present invention is within the scope of the invention. As shown below in Example 3, stroma produced according to. the method of the present invention binds endotoxin irreversibly for 24-48 hours at 373C. This is a most unexpected finding and demonstrates the efficacy of the use of stroma to treat endotoxin-mediated diseases, such as septic shock. The present treatment method involves the administration of an amount of the stroma of the present invention effective to treat septic shock to a mammal suffering from or at risk for septic shock. It is important that this treatment be initiated early in the onset of the disease for optimal treatment, although later treatment may have the ability to stop the development of further damage. If susceptibility of a patient to septic shock is suspected, stroma may also be administered prophylactically. The stroma prepared according to the present invention is administered parenterally and preferably intravenously. As is evident to one of ordinary skill, the effective amount of stroma to be administered to a mammal in need of such treatment is subject to optimization and may differ according to the stage of septic shock, the causative organism (the bacteria) and the underlying disease of the patient. However, determination of the optimum dosage is considered well within the reach of one of ordinary skill in the art. Essentially, an effective dosage is one which results in measurable improvement of any one or more of the well known symptoms of septic shock. The minimum effective dosage can be determined by conducting no more than routine experiments, i.e. beginning by administering a relatively low amount of stroma, and monitoring the patient for any reduction in the well-known symptoms of the disease. If no effect is observed, log increases of stroma can be administered until positive effects are noted. It is believed that the stroma produced pursuant to the present invention will be safe and non-toxic as long as type 0, Rh" or a compatible donor's blood is used to produce the stroma.
In an alternate embodiment, the present invention also provides pharmaceutical formulations or dosage forms comprising stroma produced pursuant to the methods of the present invention and a pharmaceutically acceptable carrier or diluent. Non- limiting examples of the pharmaceutically acceptable carriers or diluents of the present invention include 0.15M NaCl, 5% dextrose, and glycine phosphate, pH 7.4. It will be understood to those of ordinary skill in the art that the pharmaceutical formulations or dosage forms of the present invention need not constitute an effective amount of the stroma of the present invention to treat septic shock as such effective amount can be attained by administration of a plurality of such pharmaceutical formulations or dosage forms.
The methods of preparing the stroma of the present invention as described herein can, with some modifications, be used to prepare cell walls from normal human cells. Such modifications include removing contaminating DNA. Such DNA would be present because, unlike human red blood cells, eukaryotic cells contain nuclei. Removal of such substances is well within the ambit of those of ordinary skill in the art.
The present invention is further described below in light of the following examples. However, these examples should be considered exemplary and are not limiting to the scope of the invention. EXAMPLE 1: Preparation of Stroma
Stroma was prepared from normal human blood cells
(rbc) , preferably from blood group 0, Rh-negative, but not limited to O-Rh-negative as long as the rules that apply to rbc used for transfusions are observed. Stroma will not replace the function of rbc. Stroma could serve as a prophylactic agent for the protection of rbc to prevent loss through destruction during gram-negative bacterial sepsis which could lead to septic shock. Stroma was prepared as set forth below:
1. One unit (450-500 ml) of blood from a healthy donor's red blood cells were separated from plasma and other cellular components of blood producing a packed rbc pellet. From the packed rbc pellet, 50 ml was withdrawn and distributed in 10 ml amounts in 100 ml capacity centrifuge tubes.
2. 50 ml cold 0.15M NaCl was added to each 10 ml rbc in the test tubes. The rbc were suspended with a stirring rod.
3. The rbc suspensions produced above was centrifuged at 2°C for 30 minutes at 3,000 r.p.m. in a fixed angle rotor in an International centrifuge.
4. The supernatant liquid was discarded.
5. 50 ml of fresh cold 0.15M NaCl was added to the pellet. The deposited rbc were resuspended using a stirring rod.
6. The resuspended rbc were centrifuged at 2°C for 30 minutes at 3,000 r.p.m.
7. The supernatant was removed by aspiration and discarded.
8. Steps 5, 6 and 7 were repeated three more times. 9. 50 ml cold deionized distilled H20 was added to each test tube and the rbc were stirred into the H20 with a stirring rod.
10. The contents of the five test tubes were pooled by pouring them into a 1,000 ml flask.
11. 200 ml cold deionized distilled H20 was added to the contents of the flask.
12. The pH of the dissolved rbc obtained above was adjusted to between 5.2 and 5.4 with 0.IN acetic acid. The flask was incubated for 18 hours at 2°C.
13. The contents of the flask was distributed into centrifuge tubes of 100 ml capacity.
14. The centrifuge tubes were centrifuged for 60 minutes at 2-C at 3,000 r.p.m. 15. The supernatant was removed by aspiration and discarded.
16. 50 ml cold deionized distilled H20 was added to the deposited stroma and it was suspended in the H20 with a stirring rod. 17. The suspended stroma was centrifuged at 2:C for 30 to 60 minutes at 3,000 r.p.m. The supernatant was removed by aspiration and discarded.
18. The deposited stroma was washed by repeating steps
15 and 16 four more times. 19. The washed stroma was pooled with the addition of
80 ml cold deionized distilled H20 into a flask.
20. The pH of the pooled stroma was adjusted to 7.0 to 7.4+0.05 with 0.IN NaOH.
21. The NaCl concentration was adjusted with 2M NaCl to 0.15M by deducting the NaOH contribution to Na concentration when the final pH was between 7.0 to 7.4+.0.05 and the total volume was brought to 100 ml with additional deionized distilled H20.
22. The stroma was incubated for 30 minutes at 56°C then stored at 4°C. In a particularly preferred embodiment of the present invention, the stroma obtained as above may be reprecipitated by repeating steps 9-20. For example, the stroma, which is at pH 7.0-7.4 and at an ionic strength of 0.15M, is diluted 10-fold with cold, deionized, distilled H20 (i.e., for each one volume of stroma, nine volumes of cold deionized, distilled H20 is added, step 9 above) . The pH of this mixture is adjusted to pH 5.2-5.4 with 0.IN acetic acid and incubated at 2°C for 18 hours, etc. EXAMPLE 2 : Schwartzman Test to Determine Efficacy The following in vivo test can be used to determine the ability of the present treatment to remove endotoxin from the bloodstream. The Schwartzman phenomenon is a well-known in vivo test for determining the presence or absence of active endotoxin in a sample (Schwartzmann, G. , Phenomenon of Local Tissue .Reactivity, Paul B. Hoeber, Inc., 1937; Apitz, K. , Virchow. Arch. Path . Anat . 289 :46, 1933; Thomas, H. , Ann. Rev. Physiol. 16:467, 1954) . Similar techniques to the one set forth below have been described in Stetson, CA. , Jr., J. Exp . Med. lCL:421-430, 1951 and Stetson, C.A., Jr., J". Exp. Med. £4:347-358, 1951. I- The Schwartzman Test
This is a preliminary test to determine the amount of endotoxin of a smooth strain of E. coli to be used for the purpose of defining the activity of stroma. The endotoxin can be obtained directly from the bacteria. The endotoxin will be phenol extracted before use in this assay. Alternatively, E. coli endotoxin can be obtained from numerous commercial sources such as Sigma Chemical Co. (St. Louis, MO) , Difco Laboratories (Detroit, MI) or Calbichem (San Diego, CA) . The commercially available endotoxin is dissolved in aqueous media (e.g. PBS, 0.15M NaCl, etc.) before use.
(1) The back of a rabbit will be shaved with a clipper and marked with a black felt pen to create a grid on the rabbit's back.
(2) Into each grid block, graded amounts of endotoxin will be injected subcutaneously. The response to the varying amounts of endotoxin will be denoted by measuring the diameter of the lesion elicited by the endotoxin, which reaches its maximum intensity within 24 hours. Therefore, the animals will be monitored for reactions after 3-4 hours and throughout the 24 hour period. This is a local response.
(3) Upon selecting a size of a lesion which occurs upon response to a known amount of the administered endotoxin, one can determine whether stroma has the ability to inhibit or modify the effect of endotoxin in the rabbit. Stroma diluted with 0.15M NaCl will be used for this purpose and 30 samples will be prepared as shown below:
Test (A) Schwartzman Test
Stroma Endotoxin 0.15M NaCl
1. 0.25ml as selected 0 2 2.. 0 0..2200mmll as selected 0.05
3. 0.10ml as selected 0.15
4. 0.05ml as selected 0.20
5. 0 as selected 0.25
6. 0.25ml 0 0 7 7.. 0 0..2200mmll 0 0.05
8. 0.10ml 0 0.15
9. 0.05ml 0 0.20
10. 0 0 0.25
Three separate treatments of ten samples each will be tested. Test (A) , no pretreatment; Test (B) , incubation for 15 minutes at 37=C; and Test (C) , incubation for 30 minutes at 37°C. SUMMARY
Following 24 hours, the lesions will be recorded by measuring the diameter of the lesions. By using (B) and (C) samples, the uniformity of the respective samples is assured.
EXAMPLE 3 : Stroma Produced Pursuant to the Methods of the Present Invention Irreversibly Binds Endotoxin
Varying concentrations of stroma (produced as described above) and LPS will be mixed together in the presence and absence of human serum. Human red blood cells are then added as indicators. The reactions are incubated at 37°C for 24-48 hours. Included in the reactions are a negative control (no LPS added) and a positive control (no stroma added) . The reactions are monitored by testing the red blood cells for susceptability to lysis.

Claims

WHAT IS CLAIMED IS: 1. A method of producing stroma from mammalian erythrocytes comprising the steps of: a) lysing the erythrocytes to produce stroma, b) washing the stroma, c) adjusting the pH of the stroma of step (b) to between about 5.2 and about 5.4, d) incubating the stroma of step (c) at about 2°C for about 18 hours, e) washing the stroma of step (d) , f) adjusting the pH of the stroma of step (e) to between about 7.0 and about 7.4 and the NaCl concentration to about 0.15M, g) incubating the stroma of step (f) at about 56°C for about 30 minutes, and h) recovering the stroma of step (g) .
2. The method of claim 1 wherein steps (c) - (f) are repeated at least once.
3. The method of claim 1 wherein said erythrocytes are human type 0, Rh negative erythrocytes.
4. Stroma which irreversibly binds endotoxin under physiological conditions produced by the method of claim 1 or 2.
5. A method for treatment of septic shock in a mammal comprising administering to a mammal in need of such treatment an amount of the stroma of claim 1 or 2 effective to treat septic shock.
6. A pharmaceutical formulation or dosage form for treating mammals suffering from or at risk for developing septic shock comprising stroma produced by the method of claim 1 or 2 and a pharmaceutically acceptable carrier or diluent.
7. The pharmaceutical formulation or dosage form of claim 6 wherein said administration is parenteral.
8. The pharmaceutical formulation or dosage form of claim 6 wherein said administration is intravenous.
PCT/US1995/003215 1994-03-10 1995-03-10 Stroma, method of preparation thereof and its use in treatment of septic shock in mammals WO1995024206A1 (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN114053406A (en) * 2021-11-23 2022-02-18 华中科技大学 Multifunctional photo-thermal nano sterilization material and preparation and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3903254A (en) * 1971-07-23 1975-09-02 Upjohn Co Separation of erythrocyte stroma from lysing medium and hemoglobin with acrinol
US4347841A (en) * 1981-03-11 1982-09-07 Human Oltoanyagtermelo Es Kutato Intezet Biological wound covering and method for producing same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3903254A (en) * 1971-07-23 1975-09-02 Upjohn Co Separation of erythrocyte stroma from lysing medium and hemoglobin with acrinol
US4347841A (en) * 1981-03-11 1982-09-07 Human Oltoanyagtermelo Es Kutato Intezet Biological wound covering and method for producing same

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114053406A (en) * 2021-11-23 2022-02-18 华中科技大学 Multifunctional photo-thermal nano sterilization material and preparation and application thereof
CN114053406B (en) * 2021-11-23 2022-12-09 华中科技大学 Multifunctional photo-thermal nano sterilization material and preparation and application thereof

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