Improvements in Curative and Immunizing Preparations.
Year: 1899

page_text: Date of Application, 1st May, 1899-Accepted, 1st July, 1899
Improvements in Curative and Immunizing Preparations.
I, OSCAR LOEW, Ph.D., of Garret Park, Maryland, in the United States of America,
do hereby declare the nature of this invention and in what manner the same is to
be performed, to be particularly described and ascertained in and by the following
statement:
My present invention relates to curative and immunizing preparations for infectious
and contagious diseases. It consists in a method of treating the cultures of bacillus
pyocyaneus and certain other related bacilli to obtain therefrom an enzym solution,
purified and free of toxin. It further consists in certain other steps of treatment of
said enzym to provide the same in suitable form for subcutaneous or hypodermic in-
jections. The invention thus contemplates extracting the curative properties of
certain bacterial cultures, and preparing the same in a concentrated and purified form,
as a solid and durable body, or further in combination with an animal protein, as
presently described and definitely set forth in the claims.
In certain bacterial cultures, development gradually ceases notwithstanding the
fact, that a sufficient quantity of nourishing material is still present in the liquid.
Different views have been expressed on this point, but I have ascertained that the
true cause is the formation of a soluble ferment or enzym, which becoming gradually
more concentrated by its being continually secreted from the bacteria, finally dis-
solves the bacteria themselves. One kind of enzym may dissolve different kinds of
bacteria. Thus I have observed that 1 c.c. of the concentrated enzymn solution from
bacillus pyocyaneus dissolves millions of bacilli of anthrax, cholera, typhoid fever,
diphtheria and black plague, very easily in 12 to 24 hours. The gonococus and
staphylococcus are also attacked.
I have further ascertained that this dissolving process continues in the animal
body itself, eg. if anthrax bacilli are first injected into a rabbit or other test animal,
and a solution of the enzym is afterwards injected at another part of the body, it
will be observed upon killing and dissecting the animal, after a period of about 24
hours subsequent to injection, that the bacilli, which had penetrated into the liver
and spleen, have undergone more or less disintegration and dissolution. Thus anthrax
in animals can readily be cured by injections of the concentrated enzym of the bac.
pyocyaneus. Observations that filtered cultures of bacillus pyocyaneus can cure
anthrax have also been made by Hüppe and by Bouchard, but these authors did not
ascertain in what the curing property consisted, nor did they ever try to obtain it in
a concentrated purified solution or in form of a dry powder, as I have succeeded in
doing.
Formerly either fresh cultures of bacteria or also the filtrate therefrom, containing all
the products of metabolism and the rest of the organic or inorganic bacterial nutrients
were subcutaneously injected for curative purposes,--or secondly, the serum of
animals treated with certain kinds of bacteria. The first method is unsafe, as
noxious compounds are often present among the products of metabolism, and it is
inconvenient, as on account of the solution being highly diluted, repeated injections
are necessary. The latter method of applying the serum of animals, which have been
subjected to treatment, requires a long time and much labour before the serum has
acquired the necessary properties.

page_text: I have found that the anthrax bacilli exhibit all states of dissolution in the spleen
and other organs of animals that were treated with the said enzym solution, after
infection with anthrax. Thus far no such purified enzym has been obtained free
from toxical properties, or free from an admixture of a toxin.
Certain kinds of bacteria produce more toxin than enzym which at our present state
of science are impossible to separate. My method of preparing non-poisonous enzym
from bacillus pyocyaneus may also be applied with good results to obtain the enzym
from certain other bacterin.
As
The immunization process requires above all, the presence of a bactericidal sub-
stance in the hotly which is not so easily attacked by the destructive metabolism.
the larger portion of the enzym of the bacillus pyocyaneous is destroyed in the
animal, only a small portion is enabled to combine with animal proteins, and forming
thus a combination of a higher molecular order, thereby acquires a higher power of
resistance and is well protected against any destructive forces with which it may
contact. The enzym of the bacillus pyocyaneus is soon destroyed in the animal body,
hence although it is well adapted for curing certain diseases, it is not so well suited
for immunization against these diseases unless the injections are continued for a long
time.
I have found that such an immunizing compound can also be prepared "in vitro"
by mixing fresh blood or fresh spleen or liver with the said concentrated enzym
solution and adding 01 to 0-3 % caustic potash to bring on the chemical condensa-
tion between the two substances. In order to prevent coagulation of the blood 0.2
to 05 potassium oxalate is added directly to the mixture. The preparation thus
obtained can serve not only for curing, but also for immunizing against anthrax and
diphtherin.
The concentrated purified enzym solution is prepared as follows :
The cultures of a non-virulent variety of bacillus pyocyaneus developed for severl
weeks at 30 to 36° C. are first filtered through Berkefeldt or Chamberland filters,
and then neutralized exnetly and evaporated to one-tenth or even less of the original
solution, in vacuo at. 22 to 30° C. This concentrated solution is then subjected to
dialysis for 24 hours, whereby the products of metabolism, nourishing salts as
potassium phosphate, magnesium sulphate and chloride of sodium, and the rest.
of the organic nourishing materials (peptone, sugar, asparagine, kreatine, and leucine)
are removed by exosinosis and the enzym reinains almost wholly behind in the
dialysator.
From this concentrated and purified enzym solution a very durable solid preparation
inny be obtained as follows:
5 to 10% dextrin is dissolved at the ordinary temperature in the said solution and
this is poured directly in absolute alcohol. Hereby the dextrin is precipitated
simultaneously with the enzym and, surrounding this, protects it from the noxious
action of the alcohol. The precipitate thus obtained is separated from the alcohol
and dried in vacuo or over sulphuric acid. It proved still of very powerful property
four months after, curing anthrax easily by subcutaneous injections. In a similar
way also, the immunizing compound above mentioned may be obtained in a dry
and durable form.
A product less durable but still of great value when freshly prepared, can
be obtained by direct precipitation with alcohol, without applying the dextrin.
Having now particularly described and ascertained the nature of the said inven-
tion, and in what manner the same is to be performed, I declare that what I
claim is
1. The herein described method to obtain a cumtive preparation from cultures of
bacillus pyocyaneus, which consists in developing the cultures at a suitable
temperature filtering the same and neutralizing the filtrate; then evaporating it.
in acuo, at a low temperature, to one-tenth more or less, its original volume, and then
subjecting the solution to suitable treatment to remove the nourishing organic
materials present, substantially as hereinhefore set forth.

page_text: 2. The herein-described method of treating cultures of bacillus pyocyaneus to
obtain a concentrated purified enzym solution, which consists in subjecting the
cultures obtained at a temperature of 20 to 36° C. to filtering, neutralizing the same,
evaporating it in vacuo, at a temperature of 22 to 36° C. to one-tenth or less its
original volume, and then subjecting the resulting concentrated solution to dialysis
for a period of 24 hours, more or less, whereby the products of metabolism, nourishing
salts, and other nourishing organic materials present, are removed by exosmosis, and
the enzym remains in pure state, substantially as hereinbefore set forth.
3. The herein-described method to obtain an anti-toxical curative preparation from
cultures of bacillus pyocyaneus, which consists in developing the cultures at a
temperature of 30 to 36° C.; filtering the same and neutralizing the filtrate; then
evaporating it in vacuo at a temperature of 22 to 36° C. to one-tenth or less, its
original volume; subjecting the solution to suitable treatment to remove the
diffusible organic materials present, and combining the remaining product, enzyme,
with a suitable medium whereby the same is rendered durable, substantially as and
for the purpose set forth.
4. The herein-described method of treating cultures of bacillus pyocyaneous, for
the purpose set forth, which consists in developing the cultures of a non-virulent
variety of bacillus pyocyaneus to a temperature of 30 to 36° C. for a period of two to
three weeks, filtering the liquid, neutralizing the same and then evaporating it
in varno, at a low temperature to one-tenth or less its original volume; then subjecting
the solution to suitable treatment to remove all the diffusible organic materials
present, and combining it with an animal protein by means of a diluted solution of
potassa, whereby a durable preparation is obtained, substantially as herein and for
the purpose set forth.
5. The herein-described method of treating a concentrated and purified solution of
enzyme for the purpose set forth, which consists in combining the proteids of fresh
blood with the enzyme in solution, by means of caustic potassa, substantially in the
manner herein before set forth.
Taad 90th day of iil 1800
WM. P. THOMPSON & Co.,
Of 6, Lord Street, Liverpool, Agents for the Applicant.