Note: Descriptions are shown in the official language in which they were submitted.
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U-Wp-26~1-(2)
B~CKGROUND OF THE INV~NTION
The present invention is directed to a pyridine-soluble
extract of a microorganism which! when`combined with cell wall
skeleton (CWS) and trehalose dimycolate (TDM~ provides a
pharmaceutical composition possessing anti-tumor properties.
Bacteria such as CorYnebacteri-um parvum have been the
subject of experimental work to isolate and characterize the
component responsible for inducing inhibition of tumor growth
[see, for example, nti Tumor Activity and Lymphoreticular
Stimulation Properties of Fractions Isolated from ~. pa_vum;
Cantrell, et al, Cancer Research 39, pgs. 3554-3563 (September,
1979)3. Apart from anti-tumor activity, C. parvum has shown to
be a potent stimulator of the lymphoretlcular sy~tem resultlng
in undesirable increases in spleen and liver weights and
blastogenesis. Appll`icant has discovered that a pyridine-soluble
extract of a microorganism possesses potent anti-tumor
properties~without the undesirable~toxic effects associated with
the prior art products.
Cell wall skeleton is essentially cell wall which has had
much of the protein and lipids normally found in the cell wall
removed. It is a polymeric mycolic acid arabinogalactan muco-
peptid~ contalning remnants of trehalose mycolates ("P3") and
undigest~d tubercu~oproteins. Ce~l wall skeleton is obtained
from any microorganism including, ~ut not limited to,
M.smegmatis, M~phlei, Nocardia rubra, Nocardia asteroides,
Corynebacterium d_phtheria, Corynebacterium_parvum, kansasiî,
.
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M tuberculosis (S~rain ll 37 RV and Ayoma J3~, and M.bovis S~rain
BCG. Additionally, cell wall skeleton may be obtained from such
other organisms as E.coli, B.abortus NS _oxiella burnettii.
Cell wall skeleton may be produced by first growing and
harvesting bacteria such as M.bovis strain BCG (Bacillus
Calmette - Guerin). The resulting whole cell residue is
processed through a cell fractionator [Ribi Cell Fractionator
(Sorvall, Model RF-1)] which disrupts the cells, separating the
outer envelope or cell wall from the protoplasmic irnpurities.
The resulting cell walls are then subjected to a series of
solvent extractions and enzymatic treatments (e.g., trypsin
and/or chymotrypsin) to give purified cell wall skeleton.
Trehalose dimycolates (TDM), may be obtained from the
organisms such as, for example, M.avium, M.phlei, M.tuberculosis
(Strain H 37 RV and Ayoma B), M.bovis BCG, M.smegmatis,
M.kansasii, Nocardia rubra, M.bovinls and orynebacterium
diphtheriae.
Bacteria such as M~avium are grown, harvested and then
heat killed. The cell mass is then extracted with several
solvents and then an active, solvent soluble, fraction is
extracted. This extract is further purified by a series of
solvent extractions to provide crude TDM (see biolo~ically
active components from mycobacterial cell walls. i. isolation
and composition of cell wall skeleton and component p3: Asuma,
et al, Journal of the National Cancer Institute, Volume 52, pgs.
95-101, 1974). As disclosed in Aæuma er al, crude TDM may then
be further purified by centri-fugal microparticulate silica gel
chromatography to give purified TDM.
It is, therefore, an obiect of the present invention to
provide a pharmaceutical composition containing a pyridine-
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soluble extract of a microorganism in combination with cell wallskeleton and trehalose dimycolate.
It is another object of the invention to provide a method
of treating tumors in warm blooded animals and humans using the
composition containing ~he pyridine-soluble extract of a micro-
organism, cell wall skeleton and trehalose dimycalate.
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SUMMARY OF THE INVENTIC)N
,
The present invention relates to pharmaceutical composi-
tions comprising a pyridine-soluble extract of a microorganism
containing between about 7 and 20% by weight of protein, about
10 and 16% by weight of sugar and about 35 to 55~ by weight of
fatty acids in combination with cell wall skeleton (CWS) and
trehalose dimycolate (TDM). The extract preferably contains
about 12~ by weight of each of protein and sugar and about 45%
~y weight of fatty acids.
Any microorganism may be used to obtain the pyridine-
soluble extract includ~ng, for e~ample, M. bovis BCG, M. Phlei,
M. sme~matis, M. kansasii, Nocardia rubra, Corynebacterium
di~htheriae and CorYnebacterium parvum. Corynebacterium parvum
is e~pecially preferred.
Whole cells of the microorganism, preferably in the form
of a paste, are mixed with pyridine. The resulting mixture is
separated to obtain a-supernatant fraction which-contains the
pyridine-soluble extract and a pyridine residue. Optionally,
the pyridine residue may be subjected to repeated separation
procedures as described above using pyridine to remove~further
~uantities of the desired extract.
The pyridine is ~hen removed from the extract and the
dried extract is dialyzed against a suitable liquid such as
distilled water. The absence of whole cell and cell fraymen~
contaminants is confirmed by electron microscopy. The resulting
purified extract may then be lyophilized by known methods to
obtain a stable product.
The pyridine-soluble extract produced in accordance with
this invention may be combined with CWS and TDM to produce a
composition having potent anti tumor activity without stimula-
ting the induction of spleen and liver enlargements. The
c~ncers which may be treated by this composition include animal
tu~or~ such as bovine squamous cell carcinoma, bovine ~ibro-
sarcoma, equine sarcoid, equine melanoma, equine squamous cell
carcinoma, canine mammary tumors, canine adenoma and canine
melanoma and human tumors such as breast tumors, lung tumors,
colon tumors, malignant melanoma, squamous cell carcinomas and
ovarian tumors.
The composition is preferably administered by injection in
a pharmaceutically acceptable medium such as an oil-droplet
emulsion directly into the tumor under conditions more partic-
ularly described below. The aforesaid composition may be
stabilized as for èxample, by a lyophilization procedure and
then reconstituted without loss of potency.
The amount of the pyridine-soluble extract in a single
~n~e~tion for the treatment of animals is hetween about 375 and
2500 microgram~/milliliter. The amount of each of CWS and TDM
is between about 125 and 375 micrograms/milliliter.
The number of milliliters of the blologic injected into
the tumor is determined by the size of the tumor in accordance
with the following table:
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Animal Dosa~e Accordinq to Tumor S~ze
Diameter of Tumor (cm) Amount of Biologic
Injected (ml~
0-l up to 0.5
1-2 0.5 to 2.5
2-3 2.5 to 5
3 5 5 to 10
5-8 10 to 15
greater than 8 15 to 20
The maximum dose per injection is about 40 milligrams for
the pyridine-soluble extract, 40 milligrams for CWS, and 6
milligrams for TDM. The course of treatment comprises up to six
injections administered at abou~ two week intervals.
The present composition in a suitable injection medium
such as an oil-droplet emulsion is administered directly into
human tumors. The amount of the pyridine-soluble extract in a
single injection is between about 200 and S000 micrograms,
preferably be~ween about B00 and 1200 micrograms. The amount of
CWS i8 between about 50 and 2000 micrograms while-the amount of
TDM is between about 50 and lO00 micrograms. The preferred
~ingle dosage level for each of CWS and TDM is between about 475
and 525 microgramsO All of the above-mentioned do~age levels
are ba~ed on a typical 70 kilogram adult patient. The injec-
tions are administered about once every week for up to a total
of 15 injections~
As described above, the composition for treatment of warm
blooded animals and humans may be used in the form of an oil
droplet emulsion. The amount of oil used is in the range of
between about 0.5 and 3.0 percent by volume based on the total
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volwne of the composition. It is preferred to use bet.Jeen abo
0.75 and 1.5 percent by volume of the oil. Rxamples of such
oils include light mineral oil, squalane, squalene, 7-n-hexyl-
octadecane, Conoco ~a trademark) superoil and Drakeol 6 VR (a
trademark) mineral oil (produced by -the Penreco Company, Butler,
Pennsylvania).
The homogenized oil containing mixture is then combined with
a detergent which may optionally be dissolved in a saline
solution prior to mixing. The amount of detergent is typically
between about 0.02 and 0.25 ~ercent by ~olume and preferably
between about 0.10 and 0.20 percent, by volume based on the total
volume of the composition. Any common detergent material may be
used including Tween-80 (a -tradernark) and Arlacel (a trademark)
(produced by the A-tlas Chemical Company).
The mixture resulting from the addition of detergent is
then homogenized to form a suspension which has a high percen-
tage of oil droplets coated with the active components as
detexmined by observat,ion under a microscope.
The following examples are for illustrative purposes only
and are not intended to limit or in any way redefine the inven-
tion as claimed in the claims appended hereto.
EXAMPLE I - Preparation of Pyridine-Soluble Ex-tract from
Corynebacterium Parvum
Corynebacterium parvum (P.acnes, Strain 4182) was grown
and harvested at 37C. in NIH thioglycolate broth for between
48 and 72 hours to obtain a whole cell paste. The paste was
washed with 500 mg. of di,stilled waterO 90 grams (wet weight)
of the washed paste was mixed with 200 ml. of neat pyridine and
centrifuged at 1700 x g for one hour at 4C. A pyridine-
soluble extract was removed as a superna-tant fraction. The
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remaining residue was extrac~ed with additional pyridine under
identical conditions as described above. Following filtration,
using Whatman No. 1 paper, the pyridine extracts were pooled and
the solvent was removed by evaporation at 50~ C. in a Buchi
Rotavapor (Brinkmann Instruments, Westbury, New York~. The
dried pyridine extract wa~ extensively dia~yzed against dis~
tilled water and then lyophilized. ~he resul~ing purified
p~ri~ine extract contained about 12% by weight of p~otein, about
1~% by weight of sugar and 45~ by weight of fatty acids. The
ex~ract was examined under an electron microscope and found to
b~ free of contaminat~ng whole cells and cell wall fragments.
The yield of the pyridine-soluble extract was 9~ ~8.1 9.).
EXAMPLE 2 - Pre~aration of Pyridine-Soluble Extract from M.bovis
Strain BCG
M. bovis Strain BCG was grown and harvested in Sautons
medium at 37~ for about 3 to 4 weeks to obtain a washed whole
cell paste. 50 grams ~wet weight) of the washed paste was then
treated in the same manner as Example 1 to produce a yield of
the pyridine-soluble extract of 7% (3.59). The extract
contained 15% by weight of protein, 10~ by weight of sugar and
52% by weight of fat~y acids.
EXAMPLE 3 - Guinea-Pig Line-10 Tumor Tests
Six s~rain 2 guinea pigs having Line-10 tumor growths of
about 9mm. in diameter were injected once with 0.4 ml of a
sterile oil droplet emulsion, i.e., Drakeol 6 VR mineral oil
(Pennsylvania Refining Companyr Butler, Pennsylvania),
containing 300 micrograms of the pyridine-soluble extract
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prepared in accordance with Example 1 and 50 micrograms of each
of cell wall skeleton and trehalose dimycolate, directly into
the tumor tissue.
At the end of three months, the animals were examined and
in 5 of the 6 animals, total regression had occurred.
In a control experiment, six strain 2 guinea pigs having
Line-10 tumor growths of about 9mm. in diameter were injected
once with 0.4 mI of the sterile oil drople~ emulsion described
above without the pyridine extract or cell wall skeleton and
trehalose dimycolate. The injections were made directly into
the tumor tissue. None of the six tumors showed any signs of
regression after three months.
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