Novel polysaccharide extracts and method of use

Novel polysaccharide extracts of microbial bodies of Haemophilus influenza having an apparent molecular weight greater than 200,000 and constituted principally of galactose, glucose and mannose having remarkable antigenic and immunostimulating activity with very good tolerance in warm-blooded animals and a process for their preparation.

STATE OF THE ART 
Polysaccharide extracts of certain strains of Haemophilus influenza are 
described in the literature such as the article published in Journal of 
Immunology, Vol. 107, No. 4 (October 1971), p. 1070-1080 which describes 
the preparation of such polysaccharides from bacterial capsules of a 
serotype b Haemophilus influenza strain. According to the publication, the 
majority of the effects caused by Haemophilus influenza, in man as well as 
the infant, and notably in the case of meningitis is due to the presence 
of a toxin of a polysaccharide nature present in the capsules of serotype 
b Heaemophilus influenza. The object of the publication was to prepare a 
capsular extract and then to eliminate from the extract endotoxins and 
pyrogenic substances by deproteinization in order to discover a remedy for 
the said reactions. 
To effect such a preparation, the authors sought to prepare in a 
predominate manner the capsular form of Haemophilus influenze in order to 
isolate the desired product. Schematically, the procedure was to treat 
with formaldehyde followed by precipitation with ethanol and then elution 
from a column. According to the indications reported in the article, the 
obtained polysaccharide was constituted by a polyribose phosphate 
possessing glycosidic bonds between the C.sub.1 and C.sub.4 atoms in the 
furanoside form of D-ribose and the product lacked toxic properties while 
maintaining antigenic properties and specific immunisant properties 
against Haemophilus influenza. 
OBJECTS OF THE INVENTION 
It is a object of the invention to provide novel polysaccharide extracts 
from microbial bodies of Haemophilus influenza and to provide a novel 
process for their preparation. 
It is another object of the invention to provide novel antigenic and 
immunostimulating compositions and to provide a novel method of treating 
Haemophilus influenza infections in warm-blooded animals. 
These and other objects and advantages of the invention will become obvious 
from the following detailed description. 
THE INVENTION 
The novel polysaccharide extracts of the invention are comprised of 
polysaccharide extracts of microbial bodies of Haemophilus influenza 
having an apparent molecular weight greater than 200,000 and constituted 
pricipally of galactose, glucose and mannose having remarkable antigenic 
and immunostimulating activity with very good tolerance in warm-blooded 
animals. 
The apparent molecular weight is the molecular weight determined by means 
of a standarized porous gel using known macromolecular solutions. Examples 
of standarized porous gels acting as a molecular sieve are the commercial 
gels sold under the trademark Sephadex such as Sephadex G 200 gels and 
agarose gels (Sepharose 4B). 
The polysaccharide expression consitutes principally galactose, glucose and 
mannose signifying that the polysaccharides contain about 40 to 50% of 
oses. Among the polysaccharides of the invention are notably 
polysaccharides characterized in that it contains also a small amount of 
osamines which is constituted by glucosamine. 
The amount of osamines determined by the Elson Morgan method is on the 
order of 2% for the polysaccharides of the invention and the amount of the 
neutral oses such as galactose, glucose and mannose is on the order of 
44.5% determined by the corrected Orcinol method. The products contain 
traces of migrant sugars by layer chromatography at the level of pentoses 
but do not contain heptoses or uronic acids or diaminopimelic acid. The 
absence of diaminopimelic acid from the polysaccharides indicates that 
there is no contamination by peptido-glycanes of the membrane cell wall. 
Among the preferred polysaccharides of the invention are those obtained 
from the Haemophilus influenza strains deposited at Pasteur Institute in 
Paris under No. 52,151 and No. 5,481, especially No. 52,151. The strains 
belong to serotype a. 
The novel process of the invention for the preparation of the 
polysaccharides comprises cultivating a microbial strain of Haemophilus 
influenza on a solid or liquid medium, harvesting the microbial bodies 
after complete development, extracting the washed microbial bodies with 
phenol, recovering the phenol phase and effecting a return to the aqueous 
phase to obtain an impure product, subjecting the latter to careful 
hydrolysis to obtain a purified product and removing combined fatty acids 
to obtain the desired polysaccharide. 
The Haemophilus influenza strains are preferably cultivated in a stirred 
liquid medium under aerobic conditions. The culture medium used is that 
usually used for such strains and may for example contain meat extracts, 
caesin peptone, yeast autolysates, soya papainic peptone sugars, mineral 
elements, hemin, coenzyme I and distilled water. 
To obtain the maximum yield of polysaccharides, the germs are recovered 
after complete development of microbial bodies or after about 6 hours at 
37.degree. C. and the microbial bodies are then washed. The phenolic 
extraction of the washed microbial bodies is effected with an aqueous 
phenolic solution at a temperature of about 65.degree. C. The purification 
of the raw product consists of precipitation of nucleic acids with 
Streptomycin sulfate. 
The Streptomycin sulfate present in the surnagent is advantageously 
eliminated by dialysis with a porous membrane which can be in the form of 
a shell of hollow fibers such as Hollow fiber H.sub.1 DP.sub.10 fibers 
possessing a threshold retention of substances with a molecular weight 
greater than 10,000. The purified product is preferably subjected to 
lyophilisis. The careful hydrolysis is effected by heating in an aqueous 
organic solvent in the presence of an ion exchange resin. 
The careful hydrolysis of the purified product is controlled by the 
Schwartzman reaction which is evidenced in rabbits by a non-specific local 
hypersensibility shown by an alteration of the endothelium of cutaneous 
vessels and a leucocytary infiltration of the wall of small vessels. The 
careful hydrolysis is preferably effected in refluxing aqueous chloroform 
in the presence of an ion exchange resin such as Dowex 50 W.times.8, 200 
to 400 mesh H.sup.+ No. 41631. The careful hydrolysis is arrested when the 
Schwartzman becomes negative which under the test conditions becomes 
negative in about 15 hours. The resulting product is then preferably 
lyophilized. 
The novel antigenic and immunostimulant compositions of the invention 
having very good tolerance are comprised of an antigenically and an 
immunostimulantly effective amount of a polysaccharide of the invention 
and an inert pharmaceutical carrier or excipient. The compositions may be 
in the form of tablets, dragees, gelules, granules, suppositories or 
injectable solutions or suspensions. 
Examples of suitable excipients are talc, arabic gum, lactose, starch, 
magnesium stearate, cacao butter, aqueous or non-aqueous vehicles, fatty 
bodies of animal or vegetable origin, paraffinic derivatives, glycols, 
preservatives and diverse wetting agents, dispersants and emulsifiers. 
The compositions are useful for the treatment or prevention of Haemophilus 
influenza infections, in the treatment or prevention of respiratory 
affections, of bronchitis and chronic bronchitis. 
The novel method of treating or preventing Haemophilus influenza infections 
in warm-blooded animals, including humans, comprises administering to 
warm-blooded animals an antigenically and immunostimulatingly effective 
amount of a polysaccharide of the invention. The products may be 
administered orally, rectally, parenterally or locally. For example, an 
effective daily dose is 0.5 to 10 .mu.g/kg when administered perlingually.

In the following examples there are described several preferred embodiments 
to illustrate the invention. However, it should be understood that the 
invention is not intended to be limited to the specific embodiments. 
EXAMPLE 1 
The pH of a nutritive medium consisting of 5 g of meat extracts, 5 g of 
sodium chloride, 5 g of casein peptone, 5 g of yeast autolysate, 3.5 g of 
bipotassium phosphate, 1.5 g of monopotassium phosphate, 20 g of soya 
papainic peptone and sufficient water for a total volume of 1000 ml was 
adjusted to 7.4 to 7.6 and the medium was sterilized. The medium was 
inoculated with a strain of Haemophilus influenza (Institute Pasteur 
Serotype No. 52,151) and then 10 g of glucose, 1 mg of hemin and 1.5 mg of 
coenzyme I were added thereto. The medium was held for 24 hours in an oven 
at 37.degree. C. and the resulting inoculum was added to 10 liters of the 
above nutritive medium containing the same additives and the mixture was 
held at 37.degree. C. in an oven for 16 hours. The resulting preculture 
was used to inoculate 80 liters of the above nutritive medium and then 800 
g of glucose, 120 mg of hemin and 160 mg of coenzyme I were added thereto 
as well as 2.8 liters of a solution of the following composition: 1 liter 
of yeast extract, 400 g of glucose in 1 liter of water and sufficient 
water for a final volume of 10 liters with a pH of 8. The fermenter was 
allowed to stand for 6 hours and was then centrifuged at 30,000 rpm to 
obtain 130 g of moist germs. 
100 g of the moist germs were added to 870 ml of exchanged water heated to 
68.degree. C. and 870 ml of 90% phenol heated to 68.degree. C. The mixture 
was stirred at 2000 rpm and was then allowed to stand at 68.degree. C. for 
30 minutes. The mixture was then held at 4.degree. C. for 12 hours and the 
phenol-water phases were separated by continuous centrifugation in a 
Westphalia centrifuge. The aqueous phase was clarified and the phenol 
phase was extracted again by addition of the sludge from the preceding 
centrifugation step and exchanged water to obtain the same volume as the 
first extraction. The mixture was again stirred for 30 minutes at 
68.degree. C., stood for 12 hours at 4.degree. C. and was centrifuged as 
before. The combined aqueous phases were subjected to dialysis for 4 days 
to eliminate phenol and after the disappearance of phenol, the combined 
aqueous phases were concentrated to a volume of 3.5 liters with an Amicon 
membrane (H.sub.1 DP.sub.10 membrane possessing a threshold retention of 
substances with a molecular weight greater than 10,000). The aqueous phase 
was centrifuged at 4.degree. C. for 20 minutes at 12,000 rpm and was then 
lyophilized to obtain 6 g of a raw product in the form of a floculated 
white powder. 
To purify the product, 240 ml of a solution of 2.5% streptomycin sulfate 
were slowly added over 45 minutes with active stirring to 1200 ml of a 
solution containing 5 mg/ml of the raw product and nucleic acids 
precipitated. The mixture stood for 15 minutes and was then centrifuged at 
4.degree. C. for 20 minutes at 12,000 rpm. The surnageant aqueous phase 
was subjected to dialysis with a porous membrane (hollow fiber shell 
H.sub.1 DP.sub.10) with a threshold retention of substances with a 
molecular weight greater than 10,000 to remove streptomycin sulfate and 
the aqueous phase was lyophilized to obtain 1.3 g of purified product. 
Analysis: %C 40.26 %H 6.84 %N 6.53 content of 9.5% proteins (Lowry 
methanol); 41% of neutral monosaccharides (orcinol method); 35% of neutral 
monosaccharides (corrected orcinol method); 6.8% of uronic acids 
(corrected carbazol); 5.6% of osamines (Elson Morgan method); and 1% of 
pentoses (Bial method). 
For hydrolysis, the 1.3 g of purified product was dissolved in 650 ml of 
water and the solution was stirred at 4.degree. C. for 12 hours and then 
was centrifuged for 20 minutes. The surnageant was added to 650 ml of 
chloroform and 310 g of Dowex resin 50 W.times.8-200-400 mesh H.sup.+ and 
the mixture was drained through a buchner funnel. The mixture was refluxed 
for 15 hours (temperature necessary to obtain a Schwartzman reaction or 
not) and the chloroform was removed by concentration at 40.degree. C. 
under reduced pressure. The aqueous phase was hyophilized to obtain 0.65 g 
of polysaccharide extract of microbial bodies of Haemophilus influenzae. 
Analysis: %C 40.02 %H 6.53 %N 0.90 content of 1% of proteins (Lowry 
method); 44.5% of neutral oses (corrected orcinol method); 0% of uronic 
acids (corrected carbazol); 2% of osamines (Elson Morgan method); 0.8% of 
pentoses (Bial method); and 0% of heptoses (Dische method). No 
diaminopimelic acid was found. The neutral oses were identified as 
galactose, glucose and mannose by chromatography with cellulose acetate, 
silica gel, paper. Identification of the osamine by electrophoresis showed 
the presence of glucosamine. 
EXAMPLE 2 
Sublingual tablets were prepared containing 25 .mu.g of the polysaccharides 
of Example 1 in sufficient excipient of gum, tragacanth, lactose, talc, 
starch and magnesium stearate to obtain a final weight of 100 mg. 
An injectable solution was prepared containing 25 .mu.g of the 
polysaccharides of Example 1 in sufficient isotonic water to obtain a 
final volume of 2 ml. 
PHARMACOLOGICAL DATA 
A. Determination of antigenic activity 
The antigenic activity of the polysaccharides of Example 1 was determined 
against a type of anti-Haemophilus influenza serum by classic tests used 
in immunology, namely the "ring test", by passive hemagglutination, by 
agglutination of germs, by the technique of Ouchterlony and by 
establishment of a precipitation curve. In these tests, the product of 
Example 1 showed a passive hemagglutination titer of 1/128 and 1/256. 
B. Immunogenic power 
Groups of 6 Fauve de Bourgogne rabbits were used in this test with each 
group receiving intercostally and intraveinously a dose of the product of 
Example 1 at 1, 10 and 100 .mu.g/kg. One series of rabbits received the 
product of Example 1 without adjuvant and another group was immunized with 
the product of Example 1 plus a Freund complete adjuvant. The injection of 
the product was made for 30 days and samples of the blood were taken at 
days 0, 38, 47,74, 76, 101, 105 and 130. The presence of specific 
antibodies was detected beginning the 38th day and increased after the 
adjustment, the titers of hemagglutination found after this immunization 
being given 1/128 and 1/256. The serums were studied by the techniques of 
"ring test", of agglutination and quantitative precipitation. No 
difference was noted between the animals vaccinated with the product of 
Example 1 only and those vaccinated with the product of Example 1 and the 
Freund adjuvant. 
C. Opsonins 
This test determined by a colormetric method the aptitude by leucocytes to 
phagocyter of bacteria. It was assumed at the time of this determination 
that 0% of opsonization is equal to an optic density of 0.050 and that 
100% opsonization is equal to a maximal optic density of 0.250. After 
having effected the calculations, it was found that the percentage of 
opsonization of the animals immunized with the products of Example 1 
varied between 50 and 70% (the control animals showing an opsonization 
percentage of 0%). 
D. Bactericidal power of serum 
The serum of rabbits immunized with the product of Example 1 was contacted 
with different dilutions of a Haemophilus influenza culture. After seeding 
with a gelose medium and culture, the number of living colonies was 
compared to the control rabbits:--rabbit before immunization--day 0 
(positive control=0% mortality of bacteria)--serum of rabbit vaccinated 
with Haemophilus influenza germs (negative control=100% of mortality). 
There was noted a very regular increase in the bactericidal power as a 
function of time: 
______________________________________ 
% Mortality 
Day of bacteria 
______________________________________ 
0 0 
38 0 
47 0 
52 80 
74 75-80 
76 75-80 
101 83 
130 100 
Control anti H-I serum 
100 
______________________________________ 
This test shows the specific activity of the polysaacharide of the 
invention. 
E. Neutralization of local endotoxinic activity 
The product of Example 1 administered to rabbits antagonized by local 
Haemophilus influenza activity neutralized the activity of a product 
releasing a local activity (Schwartzman reaction). 
F. Acute toxicity 
The acute toxicity of the product of Example 1 was determined on groups of 
10 mice and LD.sub.50 (50% lethal dose) by intraperitoneal administration 
and was found to be between 65 and 110 mg/kg. 
Various modifications of the products and processes of the invention may be 
made without departing from the spirit or scope thereof and it is to be 
understood that the invention is intended to be limited only as defined in 
the appended claims.