Underivatized, aqueous soluble beta (1-3) glucan, composition and method of making same

Abstract

The present invention relates to neutral, aqueous soluble β-glucans which exert potent and specific immunological effects without stimulating the production of certain cytokines, to preparations containing the novel β-glucans, and to a novel manufacturing process therefor. The neutral, aqueous soluble β-glucan preparation has a high affinity for the β-glucan receptor of human monocytes and retains two primary biological (or immunological) activities, (1) the enhancement of microbicidal activity of phagocytic cells, and (2) monocyte, neutrophil and platelet hemopoietic activity. Unlike soluble glucans described in the prior art, the neutral, aqueous soluble β-glucan of this invention neither induces nor primes IL-1β, and TNFα production in vitro and in vivo. Safe and efficacious preparations of neutral, aqueous soluble β-glucan of the present invention can be used in therapeutic and/or prophylactic treatment regimens of humans and animals to enhance their immune response, without stimulating the production of certain biochemical mediators (e.g., IL-1β, TNFα) that can cause detrimental side effects, such as fever and inflammation.

1 . A neutral, aqueous soluble β-glucan preparation which enhances host defense mechanisms to infection and does not induce an inflammatory response.  
     
     
         2 . A neutral, aqueous soluble β-glucan preparation of  claim 1  in which the host is a human.  
     
     
         3 . A neutral, aqueous soluble β-glucan preparation of  claim 1  which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with a human peripheral blood mononuclear cell culture of about 5×10 6  cells/ml, results in a less than 2-fold increase in interleukin-1β and tumor necrosis factor-α synthesis over levels obtained following an otherwise identical incubation with a buffered solution lacking the β-glucan component.  
     
     
         4 . A neutral, aqueous soluble β-glucan preparation of  claim 1  which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with an endotoxin-stimulated human peripheral blood mononuclear cell culture of about 5×10 6  cells/ml, results in a less than 2-fold increase in interleukin-1β and tumor necrosis factor-α synthesis over levels obtained with endotoxin stimulation alone.  
     
     
         5 . A β-glucan preparation of  claim 4  in which the human peripheral blood mononuclear cells are stimulated with Escherichia coli lipopolysaccharide endotoxin at a concentration of about 1 ng/ml.  
     
     
         6 . A neutral, aqueous soluble β-glucan preparation consisting essentially of a molecularspecies which migrates as a single peak when analyzed by gel permeation chromatography, the molecular species being characterized by a triple helical conformation.  
     
     
         7 . A neutral, aguecus soluble β-glucan of  claim 6  wherein the molecular species binds specifically to the β-glucan receptor of human monocytes.  
     
     
         8 . A neutral, agueous soluble β-glucan having a triple helical conformation which when mixed at a concentration of 1 mg/ml with aniline blue forms a fluorescent complex in 25 mM NaOE and which loses about 50% of its 25 mM NaOH fluorescence in 150 mM NaOH.  
     
     
         9 . A method for producing a neutral, aqueous soluble β-glucan preparation, comprising: 
 a) treating a suspension of insoluble β-glucan with an organic acid under conditions sufficient to dissolve the organic acid-soluble portion of the β-glucan;  
 b) treating the organic acid-soluble β-glucan with alkali under conditions sufficient to denature the native conformation of the soluble β-glucan;  
 c) neutralizing the denatured soluble β-glucan under conditions sufficient to re-anneal the soluble β-glucan; and  
 d) purifying the re-annealed soluble β-glucan to obtain a neutrals aqueous soluble β-glucan having a triple helical conformation which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with an endotoxin stimulated human peripheral blood mononuclear cell culture of about 5×10 6  cells/ml, results in a less than 2-fold increase in interleukin-1β and tumor necrosis factor-α synthesis over levels obtained with endotoxin stimulation alone.  
 
     
     
         10 . A method of  claim 9  wherein the insoluble β-glucan is a whole glucan particle.  
     
     
         11 . A method of  claim 9  wherein step a) is performed at a pH of from about 1 to about 5 and a temperature of from about 20 to about 100° C.  
     
     
         12 . A method of  claim 9  wherein the organic acid is acetic acid or formic acid.  
     
     
         13 . The method of  claim 9  wherein step (b) is performed at a pH of from about 7 to about 14 and a temperature of from about 40 to about 121° C.  
     
     
         14 . The method of  claim 9  further comprising the step of purifying the denatured β-glucan prior to step (c) to remove insoluble β-glucans and aggregated soluble β-glucans therefrom.  
     
     
         15 . The method of  claim 9  wherein the purification step is performed using 1,000 to 100,000 dalton nominal molecular weight cut-off ultrafilters.  
     
     
         16 . The method of  claim 9  wherein step (c) is performed at a pH of about 3.5 to 11.0 and at a temperature of from about 50 to 70° C.  
     
     
         17 . The method of  claim 9  wherein the step (d) is performed using a 30,000 to 70,000 nominal molecular weight cut-off ultrafilter and a 100,000 to 500,000,nominal molecular weight cut-off ultrafilter.  
     
     
         18 . A neutral, aqueous soluble β-glucan produced by the method of  claim 9 .  
     
     
         19 . A method for preventing infection in a mammal that is at risk for infection, the method comprising parenterally administering to the mammal a neutral, aqueous soluble β-glucan which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with a human peripheral blood mononuclear cell culture of about 5×10 6  cells/ml, results in a less than 2-fold increase in interleukin-1β and tumor necrosis factor-α synthesis over levels obtained following an otherwise identical incubation with a buffered solution lacking the β-glucan component.  
     
     
         20 . A method of  claim 19  wherein the mammal is at risk for infection as a result of an invasive surgical procedure.  
     
     
         21 . A method for stimulating repair and healing of a wound site on a mammal comprising administering to the wound site, an effective amount of a neutral, aqueous soluble β-glucan which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with a human peripheral blood mononuclear cell culture of about 5×10 6  cells/ml, results in a less than 2-fold increase in interleukin-1β and tumor necrosis factor-α synthesis over levels obtained following an otherwise identical incubation with a buffered solution lacking the β-glucan component.  
     
     
         22 . A method of  claim 21  wherein the β-glucan is topically administered to the wound site or is injected into the wound site.  
     
     
         23 . A method for stimulating platelet proliferation, comprising administering to a mammal a composition comprising a neutral, aqueous soluble β-glucan in a physiologically acceptable vehicle, the neutral, aqueous soluble β-glucan being prepared by: 
 a) treating a suspension of insoluble, β-glucan with an organic acid under conditions sufficient to dissolve the organic acid-soluble portion of the β-glucan;  
 b) treating the organic acid-soluble β-glucan with alkali under conditions sufficient to denature the native conformation of the soluble β-glucan;  
 c) neutralizing the denatured soluble β-glucan under conditions sufficient to re-anneal the soluble β-glucan; and  
 d) purifying the re-annealed soluble β-glucan to obtain a neutral, aqueous soluble β-glucan having a triple helical conformation which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with an endotoxin stimulated human peripheral blood mononuclear cell culture of about 5×10 6  cells/ml, results in a less than 2-fold increase in interleukin-1β and tumor necrosis factor-α synthesis over levels obtained with endotoxin stimulation alone.  
 
     
     
         24 . A pharmaceutical composition comprising a neutral, aqueous soluble β-glucan preparation which enhances host defense mechanisms to infection and does not induce an inflammatory response, the neutral, aqueous soluble β-glucan preparation being solubilized in a pharmaceutically acceptable carrier.  
     
     
         25 . A pharmaceutical composition of  claim 24  which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with a human peripheral blood mononuclear cell culture of about 5×10 6  cells/ml, results in a less than 2-fold increase in interleukin-1β and tumor necrosis factors synthesis over levels obtained following an otherwise identical incubation with a pharmaceutically acceptable carrier lacking the β-glucan component.  
     
     
         26 . A method for treating infection in a mammal that is at risk for infection, the method comprising parenterally administering to the mammal a neutral, aqueous soluble β-glucan which, when incubated for greater than 3 hours at a concentration of about 1 μg/ml with a human peripheral blood mononuclear cell culture of about 5×10 6  cells/ml, results in a less than 2-fold increase in interleukin-1β and tumor necrosis factor-α synthesis over levels obtained following an otherwise identical incubation with a buffered solution lacking the β-glucan component.  
     
     
         27 . A method of  claim 26  wherein the mammal is at risk for infection as a result of an invasive surgical procedure.  
     
     
         28 . The method of  claim 16  wherein step (c) is performed at a ph of about 6.0 to 8.0.