Canadian Patents Database / Patent 2016948 Summary
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|(12) Patent Application:||(11) CA 2016948|
|(54) English Title:||POLYSACCHARIDE MIXTURE WITH IMMUNE STIMULATING AND ANTI-PROLIFERATING EFFECT, METHOD OF PRODUCTION AND MEDICINES CONTAINING THE SUBSTANCES|
|(54) French Title:||MELANGE DE POLYSACCHARIDES AVANT UN EFFET IMMUNO-STIMULANT ET ANTI-PROLIFERATION, METHODE DE PRODUCTION ET MEDICAMENTS LE CONTENANT|
- Bibliographic Data
- Representative Drawing
- Admin Status
- Owners on Record
|(52) Canadian Patent Classification (CPC):||
|(51) International Patent Classification (IPC):||
|(72) Inventors :||
|(73) Owners :||
|(71) Applicants :|
|(74) Agent:||MALCOLM JOHNSTON & ASSOCIATES|
|(74) Associate agent:|
|(22) Filed Date:||1990-05-16|
|(41) Open to Public Inspection:||1990-11-16|
|(30) Availability of licence:||N/A|
|(30) Language of filing:||English|
|(30) Application Priority Data:|
The invention relates to new polysaccharides with immune stimulating
and antiproliferating effect from plants of the family Nerium
oleander. One of the polysaccharides is made up of .alpha.(1?4) bonded
D-galacturonic acid units and shows a molecular weight of from 30,000
to 40,000 D. Additionally, a lower molecular polysaccharide mixture
of approximately 3000 to 12000 D, having three lesser fractions of
varying molecular weights and content, was isolated.
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR PRIVILEGE
IS CLAIMED ARE DEFINED AS FOLLOWS:
1. Polysaccharide of the general formula:
in which the residual R demonstrates an average methylization
degree of approximately 90% and otherwise indicates,
independently of each other, hydrogen, alkali or earth alkalation
and which has a molecular weight of 30,000 to 40,000 D,
demonstrated through HPLC-gelpermeation chromatography.
2. Polysaccharide having a molecular weight of approximately 10000
to 12000 D, determined by HP-GPC, containing the sugars rhamnose,
arabinose, galactose and glucose in a molar relationship of 1.1 :
4.4 : 3.2 : 1.0, where glucose = 1Ø
3. Polysaccharide having a molecular weight of approximately 5000 to
6000 D, determined by HP-GPC, containing the sugars rhamnose,
arabinose, galactose and glucose in a molar relationship of 0.4 :
3.9 : 2.0 : 1.0, where glucose = 1Ø
4. Polysaccharide having a molecular weight of approximately 2500 to
3500 D, determined by HP-GPC, containing the sugars rhamnose,
arabinose, xylose, galactose and glucose in a molar relationship
of 2.0 : 0.9 : 0.3 : 0.9 : 1.0, where glucose = 1Ø
5. Method for producing a polysaccharide of claims 1 to 3,
characterized in that
(a) parts of the Nerium oleander tree are cooked in water for 2
to 4, preferably 2.5 to 3, hours;
(b) the solid particles are filtered off, the extract is cooled
at room temperature and filtered, the filtrate is dialyzed
against water with a molecular weight limit of approximately
(c) the dialysis product is lyophilized, the lyophilized
material is treated with alcohol and the undissolved parts
are separated off;
(d) polysaccharides having molecular weights of approximately
3500 to 12000 D are isolated from the liquid using gel
6. Method for isolating a polysaccharide according to claim 5, in
which for step (b) the polysaccharide is separated out of the
filtrate, the precipitate is filtered off and dissolved in a
dissolving liquid and, if necessary, lyophilized, and then high
and low molecular fractions are obtained through gel filtration
and an acetic polysaccharide is obtained from the high molecular
fraction by ion exchange chromatography and elution with a salt
7. Method according to claim 5 or 6, in which the solid pieces are
cooked in approximately 5 times as much distilled water.
8. Method according to claim 6, in which the polysaccharide in step
(b) is precipitated through addition of ethanol (96%) in a 1:1
9. Method according to claim 8, in which the ethanol precipitation
is repeated twice.
10. Method according to one of claims 6 to 9, in which the acetic
polysaccharide is isolated by elution with 0.1 molar NaCl
11. Pharmaceutical product containing at least one polysaccharide
according to one of claims 1 to 4 or a polysaccharide obtained by
one of the methods of claims 5 to 10 in combination with
acceptable pharmaceutical carriers and/or auxiliary materials.
POLYSACCHARIDE MIXII)RE WIT~I IMMt~NE STlM[lIATING AND ANTI-PR(~
ME;THOD OF PRODUC~IOW AND MEDIC~IES ~C~TAINING THE SUBSTANOES
The invention relates to a polysaccharide with immune stimulat~ng
effect, a method for its production, as well as medicines
containing this polysaccharide.
Immune stimulation as a therapeutic concept has long been known
in medicine. In general, the term comprehends prescription of
substances, which themselves have only small if any antigenic
effect, but which are capable of inducing the body's own defense
mechanism in an unspecific way. It is known today that a
plurality of substancès are capable of stimulating the immune
r¢sponse, including especially various minerals, for example
AL(OH)3, MgSO~, beryllium, plant oils with or without addition of
microbacterium, as well as a number of plant hormonal groups guch
as alkaloids, Sesgui- and diterpene bonds, quinones and -~
polysaccharides. The whole process of ~mmune stimulation is
described in detail, for example, by Chedid, L., et al.
"Immunstimulation", Publisher: Springer, Heidelberg/New York,
1980: Heidelberger, M., "Structure and Immunological Specificity
o~ Polysaccharides"; Fortschritte der chem. org. Naturst. 42, 288
(1982):~Drews, J., "Immunpharamacologyn, Springer Verlag, 1986, ;~;
G. Dannhardt, Therapeutikon 11, November 1988, 653;
~Immunopotentionn:~Ciba~Foundation symposium, Elsevier, Amsterdam
1973,~"Immunopharamaco}ogy of Infectious Diseasesn, Ed. J.A.
Ma~de,~ Alan R. ~iss, New York 1987.
Up until today, in most cases the exact operation of the immune
stimulating sùbstances cannot be definitively explainedl. In
general, these substances show, among others, an effect on the
function~and/or proliferation of immune response capable cells,
however }eaving no memory reaction. This means, that
macrophages, granulocytes, as well as T and B-lymphocytes are ;~
primary targets for the immune stimulating 6ubstances. The
effect can occur directly or indirectly, for example, throuqh the
compIement system or lymphocytes, through production of kinin, `~
for example, interferon, interleukin, tumor-necrosi6-factor,
colony stimulating factor and others, as well as a proliferation
of macro and microphagocytes. Since non-specific and specific ~ -
defense mechanisms interrelate, cascaded effects and simultaneous
influencing of multiple defense mechanisms can be anticipated.
In medicine, preferred uses for immune stimulation include
especially the therapy of mixed infections, chronically-
persisting, chemotherapeutic-resisting bacterial and viral
infections, the therapy of opportunistic infections in high risk
patients, the therapy of malignant illnesses and to a certain
extent also the therapy of autoimmune diseases. Immune
stimulation could also be used during Zytostatica-therapy to
partially compensate for the associated immune suppression.
It is known that various polysaccharides, especially mushrooms
and higher plants, are capable of enhancing the activity of the
immune system. Of the best known immune stimulants of this type
are (alpha)-1,3/1,6-glucose Lentina and Schizophylla, compare for
example "Immunpharmacology of Infectious Diseases", Publisher:
From EP-A 0 246 069, a watery extract of plants of the family
Nerium with proliferation inhibiting characteristics are known.
It is a goal of the present invention to make available a new ~-
polysaccharide with immune stimulating effect, a method for its
production, and a medicine containing this polysaccharide.
Surprisingly, Applicant has found that from the leaves and
branches of the Nerium oleander tree a polysaccharide with immune
stimulating activity can be isolated. - - ~
In contrast to previously known ~entina- and Schizophylla-
polysaccharides, one of the inventive polysaccharides is composed
essentially exclusively of alpha (1- >43 galacturonic acid units
; - . , . : ,.
having approximately 90% of its carboxyl groups methylated. The
molecular weight of this polysaccharide was identified with
30,000 to 40,000 D, preferably with approximately 35,000 D, HPLC-
It was further discovered that a polysaccharide mixture, obtained
as an intermediate product, has not only a general
immunestimulating activity but also demonstrates a cell
proliferation inhibiting activity. This polysaccharide mixture,
obtained through precipitation of-the polysaccharide of the
plant, contains essentially the above-described alpha (1->4)
galacturonics as well as additional galacturonics of varying ~
molecular weights and oligosaccharides. ~ ~ -
Additionally, a low molecular polysaccharide mixture was ;~
isolated, the molecular weight of which was determined with -~
approximately 2500 to 12000 D in HPLC-gel permeation
~hromatography. This mixture thus definitely falls below the
molecular weight (approx. 20000D) of the previously known -~
polysaccharide with immune stimulating effect. This
polysaccharide mixture, obtained from the watery extract of the -~
Nerium oleander plant, contains three lesser fractions of varying
molecular weights and content.
The immune stimulating effect of the inventive polysaccharide was ;~
tested through in vitro methods structured to measure the
capability of the mononuclear system as well as the stimulation
of T and B-lymphocytes.
Through these examinations it was surprisingly found that the ;`~
inventive polysaccharide is specially capable of enhancing the
secretion of tumor-necrosis-factor. The tumor-necrosis-factor is
formed in macrophages during induction and released to the blood.
It plays an important role during tumor resistance.
4 ``- -
In other tests the inventive polysaccharide as well demonstrated
immune stimulating activity.
Branches and leaves of the oleander tree (Nerium oleander) are
suitable as starting materials for the isolation of the inventive
polysaccharide. Leaves are preferably used.
The method of isolating the inventive polysaccharide essentially
comprises cooking the crushed starting material with ;-
approximately 5 times the volume of distilled water, for 2 to 4,
preferably 2.5 to 3 hours, followed by filtration of the solid
particles, cooling of the extract to room temperature and
The high molecular polysaccharide can be obtained from the
extract as an intermediary product through precipitation with
alcohol, over complex-formation with heavy metal salts or -~
quaternary ammonia salts. Preferably, the polysaccharide is
precipitated with ethanol which is added in a ratio from 3:1 to
1:3, preferably 1:1. After the solution has set for at least 12
hours, it is filtered, the precipitate is dissolved in distilled
water, and the precipitation is repeated twice. The final -
precipitate, which again is dissolved in water, can be
lyophilized if necessary.
The raw fraction can be separated into high and low molecular
weight portions through a customary gelchromatography procedure,
for example through use of a Biogel- P-60 column.
From the high molecular weight fraction the inventive
polysaccharide is isolated through ion exchange chromatography,
for example through use of a DEAE-Sepharose C 6-B column,
through elution with for example a 0.1 molar NaCl gradient at a
NaCl concentration of approximately 0.2 molar.
The method of isolating the inventive low molecular
polysaccharide, that is the mixture, consist essentially of
lyophilization of the filtrate obtained according to the above-
described extraction step, dissolving of the freeze dried
material in distilled H20 and dialyzing against distilled H20
(exclusion point of approximately loooo D). The freeze dried
dialysi~ product is then treated with alcohol and the precipitate
i8 further separated by known methods of gelchromatography -~
following centrifugation. ~ ~;
Isolation and characterization of the hiqh molecular ;~
polvsaccharide from Nerium oleander leaves ;
The yield of polysaccharide varies according to the source and
time of harvest of the starting material.
All the steps described below were conducted at 4-C, unlesæ ;~
otherwise stated. ;
The leaves of the Nerium oleander tree were crushed and cooked in
the approximately 5-fold volume distilled water for 3 hours. The
solid particles were filtered off and following cooling to room
temperature the solution was again filtered. The filtrate was
mixed with ethanol (96%) in a 1:1 ratio. This solution was
allowed to set for 12 hours, the formed gelsuspension was
filtered off and the remainder taken up in distilled water. This ~ -
solution was again mixed with ethanol (96%) in a 1:1 ratio.
After allowing the solution to set for at least 12 hours, the
gelsuspension was filtered off, the remainder taken up in
distilled water and lyophilized. In this way, a raw extract was
obtained as an intermediary product. ~
6 ~ `
. . .,: :
To produce the inventive polysaccharide, the high molecular -
substances were carefully separated from the low molecular
substances. In that regard the raw extract was dissolved in
water, the solution centrifuged and the precipitate placed on a
biogel P-60-column and eluded with distilled water whereby two
fractions were obtained. The first fraction contained a
polysaccharide mixture of molecular weight range of 17,000 to
120,000 D. The second fraction (MG approximately ~ 10,000 D)
contained lower molecular weight poly and oligosaccharides and ~ - -
The inventive polysaccharide was obtained through chromatography
of the high molecular weight fraction in an ion exchangP DEAE
Sepharose CL 6 B column. For this the high molecular weight
fraction was dissolved in water and placed on the column filled ;-
with DEE Sepharose and eluded with 0.1 molar NaCl. The
inventive polysaccharide eluded at approximately 0.2 molar NaCl.
It shows a positive optical rotation of 38.12- (1 mg./ml. H~0,
The following structure was determined for the inventive
in which the residue R demonstrates an average methylization
degree of approximately 90% and otherwise indicates, independent
of each other, hydrogen, alkali or earth alkalation. ~-
The structure of the inventive polysaccharide was further
examined through methanol determination and '3C-NMR-spectroscopy.
During these examinations it was found that the polysaccharide ~ '
contains only D-galacturonic acid units which are approximately ;
90% methylated. -
The permethylization analysis demonstrated the bond relationships
of this galacturonic acid ~Hakomori, S.I., J. Biochem. (Tokyo)
55, 205 (1964)).
The GC-MS results of the permethylization analysis showed that
the polysaccharide chain is constructed of ~ >4) bonded D-
galacturonic acid units, the carboxyl groups of which are ~ ;
methylated at approximately 90%. Other than ~ >4) glycolic ~ ~
bonds, no other bond relationships could be determined. That ~ -
means: the polysaccharide chain is constructed linearly and ; -
contains no branching.
For further analysis of these polysaccharides the molecular
weight was next determined.
Determination of the molecular weight occurred through HPLC-gel
permeation chromatography (HP-GPC). The HP-GPC-System used~
~-Bondagel E 125 + 5 Bondagel E 500 (Fa. Waters).
Buffersystem- 0.5 molar phosphate buffer, pH6
For comparative substances Dextran T10, T40, T70, TllO, T2000 and
glucose were used. ~
` : , ~ , ~; ;'
:,,, ': ~
''"'"' '" ~'"
8 ; ~
. .: . - , . .
': '` "'~ l .; ' '' : ' ' ` ' ' ' ` ' :`:
Using this method the inventive polysaccharide was determined to
have a molecular weight of 30,000 to 40,000 D, preferably of
approximately 35,000 D.
However, it must be pointed out, that the molecular weight may
vary according to the methods of analysis used.
The uranic acid content of the inventive polysaccharide was
determined using the carbazole test (Bitter T. and Muir, H.M.,
Analyt. Biochem. 4, 330 (1962)
Uranic Aaid Content in Percentaae
SamDle , ,. ~ ::
1. Test 2. Test
Polysaccharide 99,9 100
To determine the content of the inventive polysaccharide, the
polysaccharide was mixed with TFA and heated for two hours in a
121-C dry oven. After removal of the TFA a thin layer
chromatography W8S conducted.
Adsorbent: Kieselgel GF ~5~-ready plates for the NAN0-DC(HPTLC)
20 x 20 cm (Fa. Merck). -~ ~
~ 'Running system: n-Butanol-Acetone-acetic acid-water
Detection: Anilindiphenylaminophosphoric acid.
Results showed that only D-galacturonic acid can be demonstrated
The ~(1 >4) bonds of the D-galactruonic acid was additionally
examined with pectinase. The polysaccharide was dissolved in
distilled water and incubated with pectinase for 3 hours at room
temperature. Then, the liberated galactruonic acid was
demonstrated through thin layer chromatography.
The pharmacological effect of the inventive polysaccharide was -
examined through accepted in vitro tests.
Since no specific test methods for the verification of immune
stimulating effect of substances exists to date, in vitro and in ;
vivo methods which measiure the effect of bonds or plant extract -~
on the function and capability of mononucleated systems as well
as the ability to stimulate T- and B-lymphocytes are generally -~
Through these examinations it was determined that the inventive
polysaccharide is especially effective in the tumor-necrosis
factor liberation test.
TNF (Tumor-necrosis-factor) is a protein which, in people,
consist of 157 amino acids. It is synthesized in macrophages
during induction and delivered to the blood. TNF stimulates the -
defense system and kills tumor cells. During the TNF test the ~ -~
substance to be tested is injected into experimental animals.
Then the TNF concentration through necrotic effect on the tumor
cell lines is measured in the blood of the experimental animals
and used as a parameter for the stimulating effect of the test
:, .. . .. .
The tumor-necrosis-factor test (TNF-T) was conducted according to ^
Stim PL, M., Proksch, A., Wagner, H. and Lohmann-Matthes, M.L.,
Infection and Immunity 46, 845 (1984). The results are set forth
in the following table.
... ....... ...
PS 50 256
PS 25 64
PS 12.S 8
PS 6.2 4
PS = Inventive polysaccharide
As can be deduced from the table, the inventive polysaccharide
causes a definite elevation in ~NF concentration, i.e. it induces
the synthesis of TNF in macrophages.
The immune stimulating effect of the raw fraction obtained as an
intermediary product was as well tested using the Tumor-necrosis-
factor liberation test. The results are set forth in the
Polysaccharide ConcentrationTNF Concentration
Raw Fraction (~g) (U/ml)
PS 50 1000 ~ -
The immune stimulating ef~ect of the polysaccharide raw fraction
was additionally examined through the granulocyte test according
to Brandt ~Brandt, L., Scand. J. Haemat. (Supplement) 2 (1967).
During the granulocyte test according to Brandt, the number of ~ ~ -
phagocytic in yeast cells or bacteria are determined through a
granulocyte fraction, derived from human serum, under a
microscope in in vitro tests. The percent elevation of
phagocytes due to the inventive polysaccharide is measured. The -
results are set forth in the following table.
Phagocyte Amounts in Percent by concentration (mg/ml)
Polysaccharide lo-l 10-3 10-' 10-5 1o~6 ~:
Raw Fraction 95 74 70 42 32
As the results in the table show, the polysaccharide mixture
produced as an intermediary prodùct effects a definite elevation
in phagocytes which correlates well with the stimulation data
obtained in the TNF-liberation test.
The intermediary product furthermore has a cell proliferation
inhibiting effect (anti-proliferation effect).
~ ~ . . ..
Isolation and characterization of the low molecular
~olvsaccharide mixture. that is the low molecular polvsacchaFide~
from Nerium oleander leaves
The yield of polysaccharide mixture varies according to the
source and time of harvest of the starting material. All the
steps described below were conducted at 4C, unless otherwise
The leaves of the Nerium oleander tree were crushed and cooked in ; -~;
approximately 5-fulled volume of distilled water for 3 hours.
The solid particles were then filtered off and following cooling -
to room temperature the solution was again filtered. The
filtrate was lyophilized, the precipitate dissolved in distilled
water and dialysized in a dialysis tube (having a molecular
weight upper border of approximately lO,OOOD) against water for 3
24 hour periods. Following each 24 period the dialysis product
was exchanged against distilled water. -
~01~948 ~ -
To obtain the inventive polysaccharide mixture, the dialysis
product was lyophilized, the precipitate mixed with methanol,
shaken and centrifuged for 10 minutes at 3,000 rpm. These steps
were repeated 3 times.
In additional separation steps, the precipitate obtained from
centrifugation was dissolved in distilled water, placed on a
Sephadex0-LH-20-column and eluded with distilled water. The
SephadexD-LH-20-column separated the precipitate into numerous
fractions. The first fraction (NOAG-II) contains a mixture of
polysaccharide with molecular weights between approximately 2,500
To isolate the lesser fraction polysaccharide from NOAG-II, this
fraction was dissolved in distilled water, if necessary following
lyophilization, placed on a column filled with Sephadex~-G-50 and
eluded with distilled water. Through this chromatography the
NOAG II was separated into 3 polysaccharide fractions. The
lesser fraction will be hence forth designated as NOAG-III, -~
NOAG-IV, NOAG-V. -
., ,. ~.....
By adding a 15% solution of Trichloracetic acid to the
polysaccharide fractions dissolved in water, 5-7% of the protein
content could be removed.
To characte~ize the inventive polysaccharide mixture the ;~
~ollowing tests were performed.
: .. ~:
1. Qualitative sugar determination:
The fractions were mixed with 2 N TFA for hydrolysis and were
heated in a desiccation chamber for 2 hours at 120-C. Following
the removal of TFA a thin layer chromatography was performed.
Abso-bance: Kieselgel G F254 ready plates for the Nano-DC(~PT~C)
20 X 20 cm tFa. Merck).
Running system: n-Butanol-Acetone-Acetic acid water -
Detection: Anilindiphenylaminophosphoric acid.
The results showed that the following sugars could be determined:
Glucose ! ~', ,' "" `'
2. Quantitative neutral sugar determination:
The lesser polysaccharide fractions were hydrolyzed and then ,
changed to Alditolacetates (BLAKENEY A.B. et al. Carbohydrate
Res. 113(1983)291) and the sugar is determined by gas - :
Instrument: Perkin-Elmer 900
: . ~ . .
Column: Glass, 6 x 2 mm, GP3% SP-23-30 on 100/200 Supelcoport. -~
''. '; ~.,,',-
Temperature: 210C ~ ~
:, . ~ ';;"The following table shows the molar relationship of the neutral
`:, ' ' ' '~
.. ~ .- -
-~ - ' ~-.
. '~ .
Fractions Rhamnose: Arabinose: Xylose: Galactose: Glucose:
NOAG II 0.3 0.2 0.2 0.9 1.0
NOAG III 1.1 4.4 -- 3.2 1.0
NOAG IV 0.4 3.9 -- 2.0 1.0
NOAG V 0.2 0,9 0.3 0.9 1.0
The calculation of the molar relationship is determined with
glucose equalling 1Ø
3. Uranic acid determination:
With the help of the carbazole tests of Bitter, T. and Muir, H.M.
Analytical Biochemistry 4, 330 (1962) the uranic acid content o~
the polysaccharide fractions was determined to be 40 to 70~.
4. Molecular wei~ht determination:
The molecular weights of the isolated polysaccharide were
determined with HPLC-Gelpermeation chromatography (HP-GPC).
~-Porasil GPC 60 A + ~-Bondagel E-500.
Buffer system: ~;
0.05 M phosphate buffer, pH 6.0 with 0.15 M NaCl.
~ . .
Glucose, Maltotriose, Maltoheptaose, Dextransulfate 5,000, ~
Dextran T-10, T-40, T-70, T-110, T-2000. ~ ;
. I I , . .
Polvsaccharide Molecular Weight (D~
NOAG III 10,000 to 12,000
NOAG IV 5,000 to 6,000
NOAG V 2,500 to 3,500
5. Protein content:
The protein content of the separate fractions was determined
accsrding to Lowry O.H., Journal of Biologlcal Chemistry 193
, .. .. .
Through these tests it was shown that the polysaccharide mixture
has a protein content of approximately 6 to 10%. ~ ;
The optical orientation of the substances was determined with a ~ ~ ;
polarimeter (Perkin-Elmer Polarimeter 241) at 20-C.
Wavelength: 589 nm (Na)
Concentration c: 0.1%
Dissolving material: distilled water.
It was shown that the polysaccharide has a strong right ~ -~
orientation. Values between 120 and 150 were found. ~-
, . .:
The pharmacological effect of the inventive polysaccharide
mixture was determined using known in vitro tests, whereby it was
discovered that the inventive polysaccharide mixture is
particularly effective in the tumor-necrosis factor liberation ~ ; ~
test . .. .; ,: ,:
Lymphocyte transformation test~
During the lymphocyte transformation test, lymphocyte cultures;~
are mixed with 3H labeled Thymidine (a component of DNA). 3H- ~ ,
Thymidine is taken up by proliferating lymphocytes and utilized
in the structure of DNA. The amount of 3H-Thymidine incorporated
in the DNA is proportional to the lymphocyte proliferation and
;, ~ ' " ,.
can be used as a parameter to determine the stimulating ef~ect o~
The following table shows the average value results of 3
independent measurements and shows a very good stimulation o~ the
lymphocyte proliferation through the polysaccharide mixture NOAG
Lymphocyte stimulation (%) at
Fraction Concentration (ma/ml~
10 1o~2 1o~3 lO 10-5
NOAG II 89 55 38 29 54
The ~rocedure used was as that described in Example 1. -~
The results showed that NOAG II stimulates macrophages to produce
TNF very well in that 1.5 ~g NOAG II parameter median induced
over 500 units per ml TNF. ~
The immune stimulating effect of the polysaccharide mixture NOAG ~-
II was additionally analyzed through a granulocyte test according
to Brandt (Brandt, L. Scand. I. Haemat. (Suppl.) 2, 1967).
- ` : .
The following table shows the results (average values from two ~ ~-
independent measurements). -
F~action Phaaocyte values in % at Concentration ~ma/ml)
0-2 ,~Q-3 10-~ 10-~ 6
NOAG II 70 40 130 21 99 18
As the results in the table show, the polysaccharide mixture NOAG
II effects a definite increase in phagocyte values which
correlates well with the stimulation data obtained in the TNF
.,: - ~. ."- ..,
- - , . ~ ~ ,
,, ~ ,
Chemiluminescence-Test (CL-T): --
With this test the increase in the production of reactive oxygen
bonds with macrophages in granulocytes due to the test substances
is determined. The reactive oxygen bonds are brought to reaction
with amplifiers like Lucigenin which then results in
chemiluminescence. Through measurement of this chemiluminescence
the stimulatory effect of the test substances on the immune -
system can be determined. The following table shows the results
(average values of two independent measurements).
Chemiluminescence Increase in % at Concentration ~ma/ml)
lo-l lo~2 10-3 10' 10-5 10'
NOAG II 7.2 9.3 1.2 24.4 - 7.6 ~ ~;
As can be seen from the table, the polysaccharide mixture NOAG II
indicates activity in this test as well.
The inventive polysaccharide, that is the respective polysaccharide ~ ~;
mixture, can be prescribed separately or as mixtures in a pure - -~ -
substance or in form of a pharmaceutic preparation, however it would
generally be more effective in form of a pharmaceutic preparation.
Preferably the preparation lies in the following ~ormulation:
(1) At least one inventive polysaccharide, that is one polysaccharide
mixture and -
(2) One or more suitable binders, carriers and/or other aid materials
~3) If necessary, other therapeutic agents or adjuvants.
The carriers, binders and/or aid materials must be pharmaceutically
and pharmacologically compatible so that they may be combined with the
other contents of the formulation and have no negative effect on the
organism to be treated.
The formulations include those administrable through parenteral -~
(including subcutaneous, intradermal, intramuscular and intravenous)
or oral dispensation, although the most suitable dispensation method
depends on the condition of the patient.
The production of the formulation occurs utilizing methods generally
known to the art of pharmacy. All methods include the step of mixing
the inventive polysaccharide that is the polysaccharide mixture (i.e.
the hormone) with the carrier, binder and/or help material, whereby
the latter are an additional ingredient. In general, the formulations
are produced by mixing the hormone with the liquid carriers or the
fine particled carriers or both and then turning the obtained product
into the desired dispensation form. The inventive formulation may be
appropriate for oral dispensation by being contained in discrete units
such as capsules or tablets, each containing a predetermined amount of
the inventive hormone. Alternatively, the formulation may be in form
of powders or granules or in a suspension or dissolved state in an
aqueous or non-aqueous liquid or in form of an emulsion, for example,
in form of lipsomes.
The hormone may also be used in form of a paste.
The tablets can be produced through pressing or pouring whereby one or
more of the typical supplements can be added as necessary.
Formu}ations to be used for parenteral dispensation include sterile
aqueous or non-aqueous injection solutions, the anti-oxygens, buffers,
bacteriostatiscs and dissolved materials, which make the formulation ~ ;
isotonic in view of humans. Additionally, the inventive -~
polysaccharide that is the polysaccharide mixture may be contained in
form of sterile aqueous or non-aqueous medium suspensions which
contain suspension solutions and thickening agents. The formulations
can be submitted as single or multiple doses for example in form of
ampules or tightly closed flasks and can also be stored in lyophilized
form so that a fluid carrier would simply be added immediately before -
use, for example, injection appropriate water. The injection
solutions and suspensions to be prepared immediately before use can be
''' '' :'
prepared of sterile powders, granules and tablets of the above- ~-
described type. ~ -
The inventive preparations can include other ingredients appropriate
for the formulation in addition to the above named ingredients. For
example, the pharmaceutic preparations to be administered orally may
contain various aroma producing ingredients. ~'',.
The amounts of hormone suitable for each application varies depending -'
on the therapy. Generally a single dose should contain 5% to 95%
active ingredient. This translates for a one time application to for - ~''-';,"'
example 50 ~g to lO0 mg per dose per person parenteral and 1 mg to 500 ~,-mg peroral. This dose, however, will vary within a large boundary
depending on the dispensation method, the condition of the patient and - -
the f ield of therapy. , -
Set forth below are several formulations of the inventive , ,--'
~ .': .-
Tablet Formulation ~
. . ~
,Formulation A hg~ 5~ '~- ;'
(a) active ingredient 250 ~' ' ,
(b) lactose '' 210 -,
(c) PVP 15 -~
(d) sodium starch glycolate 20
(e) magnesium stearate 5 ~ , ,
500 , . .~ '
Eonnulation B (capsule) m~/capsule
active ingredient 250
gelatinized starch 150 , -,
Formulation C mg/capsule
active ingredient 250
microcrystalline cellulose 100
Formulation D ma/capsule :
(a) active inqredient 250 ~-
(b) lactose 143 -
(c) sodium starch glycolate 25
(d) magnesium stearate 2
Formulation E mq/cansule
(a) active ingredient 250 :- -
(b) polyethylene glycol 350 --
Formulation F (in~ection solution)
active ingredient 0.200 g
hydrochloric acid solution0.1 mol/l q.s. to pH 4.0 to 7.0
sodium hydroxide solution0.1 mol/l q.8. to pH 4.0 to 7.0 :~
sterile water q.s. to lOml
active ingredient 0.125 g
~teriIe,~pyrogen-free phosphate buffer with pH 7,
g.s. to 3.00 ml
: :' ~ :~:' . .:
' ' ' ":Y~ ;:, :
.: :-:. ~
;~Oi69~8 ~ ~-
Formulation H --
active ingredient 0.20 g : :-
benzyl alcohol o 1 g
tetrahydrofurfuryl-polyethylan-glycolether 1 45 g
water for injection g.s. to 3.00 ml
Formulation I :
active ingredient 0.2500 g
sorbitol solution 1.5000 g
glycerine 2.0000 g ~ -~
sodium benzoate 0.0050 g . -
flavoring 0.0125 ml
purified water q.s. to 5.0000ml -~
~ . .-
.. .. .
, . .
'' ,: '
'', ~ ':: ~ ` ,
For a clearer understanding of the status of the application/patent presented on this page, the site Disclaimer , as well as the definitions for Patent , Administrative Status , Maintenance Fee and Payment History should be consulted.
|Forecasted Issue Date||Unavailable|
|(41) Open to Public Inspection||1990-11-16|
There is no abandonment history.
|Fee Type||Anniversary Year||Due Date||Amount Paid||Paid Date|
|Current Owners on Record|
|OZEL, HUSEYIN Z.|
|BASER, KEMAL H. C.|
|OZEL, HUSEYIN Z.|
|BASER, KEMAL H. C.|
|Past Owners on Record|