Note: Descriptions are shown in the official language in which they were submitted.
~L202~101~311
BACKGROUND OF THE INVENTION
The subject matter of the present invention is directed
to a stable, therapeutically efective composition containing
cell wall skeleton (CWS~ and purified trehalose dimycolates
(TDM). Both substances are isolates of bacteria and when used
together as a composition, are effective in obtaining suppres-
sion and regression of tumor cells. The present invention also
includes a method of preparing the composition as well as use
of the composition in treating cancerous tumors and as an ad-
juvant.
~ he comoination of CWS and TDM is known in the art (SeeBiologically Active Compounds from Mycobacterial Cell Walls.
II. Suppression and Regression of Strain-2 Guinea Pig Hepatoma;
Meyer et al, Journal of the National Cancer Institute, Volume
52, No. 1, January, 1974; and Mycobacterial Cell Wall Components
in Tumor Suppression and Regression; Ribi et al, National Can-
cer Institute Monograph No. 39, pgs. 115-120, October, 1972)
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
mucopeptide containing remnants of trehalose mycolates ("P3")
and undigested tuberculoproteins. Cell wall skeleton is ob-
tained from any mycobacteria including, but not limited to,
M.smegmatis, M.phlei, M. avium, Nocardia rubra, Nocardia
asteroides, Corynebacterium diphtheriae, Corynebacterium
parvum, M. bovinis, M. Kansasii, M. Tuberculosis (Strain H 37
RV and Ayoma B), and M. Bovis Strain BCG. Additionally, cell
wall skeleton may be obtained from such non-mycobacteria as
E.coli, B.abor-tus and Coxiella Burnettii.
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The process of producing cell wall skeleton is time
consuming. The bacteria such as M. Bovis Strain BCG (Bacillus
Calmette - Guerin) is grown and harvested. The resulting ~-hole
cell residue is processed through a cell fractionator [Ribi Cell
Fractionator (Sorvall, Model RF-l)] which disrupts the cells,
separating the outer envelope or cell wall from the protoplasmic
impurities. 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.
The second component of the instant composition, tr~halose
dimycolates (TDM), may be obtained from any mycobacteria as, for
example, _. avium, M. phlei, M. tuberculosis (Strain H 37 RV and
Ayoma B), M. bovis BCG, M. smegmatis, M._Kansasii, Nocardia
rubra, M. bovinis and Corynebacterium diphtheriae.
Bacteria such as M. avium is 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 Biologically Active Components from
~cobacte~ial Cell Walls. I. Isolation and Composition of Cell
Wall Skeleton and Component P3; Azuma, et al, Journal of the
National Cancer Institute, Volume 52, pgs. 95-101, 1974). As
disclosed in A~uma, et al, crude TDM may then be further purified
by centrifu~al microparticulate silica gel chromatography to give
purified TDM.
CWS and TDM produced as described above have been combined
in an oil droplet emulsion. The non-iiving components are
ground with a small amount of mineral oil and emulsified in
saline to produce an anti-tumor composition suitable for
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injection (See Immunotherapy with Non-viable Micro~ial
Components, Ribi, et al; Annals of the National Academy o~
Siences, Volume 227, pgs. 228-238, September 20th, 1976).
However, the prior art oil in saline emulsions con-
taining CWS and TDM suffer from a major disadvantage. The emul-
sion has a relatively short shelf life at room temperature and,
therefore, must be used shortly after preparation to produce
the desired results.
It is well-known in the art that lyophilizing a
pharmaceutical preparation can extend shelf life considerably
(see, for example, USP 3,932,943; USP 3,594,471; and USP
4,134,21~). To be successul, the lyophilized product must be
able to be reconstituted at a later time without any loss in
potency, that is, with the same potency as the pre-lyophilized
product. However, the prior art CWS-TDM oil in saline emul-
sions have not been effectively lyophilized. Applicants have
discovered that if these emulsions are not stabilized without
delay, as, for example, by lyophili~ation, they will begin a
process of degradation resulting in a significant percent of
oil droplets becoming uncoated. The uncoated material is not
active in tumor regression. The therapeutically effective emul-
sion of the present invention is stabilized by a lyophilization
procedure; other stabilization procedures can also be utilized.
It is therefore an object of the invention to provide a
stable CWS-TDM composition which may be effectively recon-
stituted to produce an effective anti-tumor preparation having
a superior shelf life that is, a shelf life o~ a year and even
longer. It is another object of the invention to provide a
CWS-TDM composition having a large number of coated oil drop
lets which are very effective in tumor regression, without side
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effects.
It is still another object of the invention to provide
a method of treating various cancer tumors with a stable and
potent CWS-TDM composition. It is another object of the
invention to employ the CWS-TDM composition as an adjuvant as
for example, to increase the immune response to immunogens
including, for example, microorganisms r proteins, carbohydrates,
allergens, viruses and the like.
THE INVENTION
The present invention is directed to a stable composition
comprising CWS and TDM in which the active materials are coated
on oil droplets. The composition may be effectively
reconstituted in an aqueous solution with the same potency as in
the pre-lyophilized state.
The process of producing the instant composition comprises
mixing CWS and TDM for a time sufficient to form a uniEorm
suspension. If desirable, the TDM may be dissolved in a
suitable solvent known to those skilled in the art. For
example, such solvents include chloroform, ether, methanol,
ethanol, combinations thereof and the like. The weight r~tio of
C~S to TDM is in the range of between about 1.0 and 6:1,
preferably between about 2.5 and 3.0:1.
A light, non-biodegradable oil is then added and the
resulting mixture is homogeni3ed to form a paste-like substance.
The use of a light hydrocarbon non-biodegradable oil is an
essential element of the process since biodegradable oils do not
achieve the objects of the invention. Furthermore, the oil must
be light weight typically having a viscosity of between about 8
and 12 centistokes measured at 100 F. Preferably~ the
viscosity is in the range of between about 10 and 10.6
centistokes.
The amount of oil used in the proccss is in the range of
between about 0.5 and 3.0 pcrcent by volumc based on the total
volume of the composition. It is preferred to use between about
0.75 and 1.5 percent by volume of thè oil. Examples oE such
oils include light mineral oil, squalane, 7-n-hexyloctadecane,
Conoco (a trademark) superoil and Drakeol 6 VR la trademark)
mineral oil (produced by -the Pennreco Company, Butler, Pennsylvania).
The homogeni~ed oil containing mi~t~re i5 then combined
with a detergent ~hich may optionally be dissolved in a saline
solution prior to oixing. The amount of detexgent is typically
between about 0.02 and 0.20 percent by volume and preferably
between about 0.10 and 0.20 percent by volume based on the total
volume of the composition. ~ny common detergent material may be
used including Tween-80 (a trademark), and ~rlacel, ta trademarX)
(pxoduced by the Atlas Chemical Company~.
The mixture resultinc3 from the addition of detergent is
then homogeni~ed -to form a suspension which has a high
percentage of oil drople-ts coated ~ith CWS and TDM as determined
by observation under a microscope.
The novel composition of thc present invention is an
effective a~cnt in the treatmcnt of cancerous tumors in humans
ancl animals and the composi-tion, in thc form of an oil droplet
emulsion, is injected directly into thc -tumor tissue. Cancers
which can be treated includc bovinc fibrosarcoma, equine
sarcoid, equine melanoma, canine melanoMa, bovine ocular
sarcoma, etc. The daily dosage of the composition to a typical
adult patient weic3h1ng about 70 kiloc3rams is sufficient to
provide about 150-300 ~cJ of CWS and 50-100 ~cJ oE TDM. Therapy
may be continued for approximately two -to six months providing a
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total dosage of up to 15 mg of CWS and 15 mg. of TDM.
The instant composi-tion, when used to increase a response
to immunogens, is administered at a dosage of between about 10
and 500 micrograms. CWS and TDM can also be used separately
as adjuvants; each in amounts of about 10 to about 500 micro-
grams.
Description of the Preferred Embodiments
The following examples are for illustrative purposes only
and are not intended to limit or in any way redefine the
invention as claimed i~ the claims appended hereto.
Example 1 - Preparation of CWS-TDM Composition
150 mg of CWS are introduced into a 350 ml tissue homo-
genizer (Bellco). 50 mg of TDM were dissolved in 2.5 ml of a
95:5 chloroform - methanol mixture and then added to the
homogenizer. The resulting CWS-TDM suspension was mixed for
about 15 to 30 seconds and the solvent was evaporated using a
sterile air stream. This was followed by the addition of 2 ml
of sterile Drakeol 6 VR (a trademark) mineral oil ~Pennreco
Company, Butler, Pennsylvania) and the mixture was homogenized
for 1 minute using a drill press while an oil paste consistency
is obtained. 190 ml of 0.2 percent Tween-80 (a trademark) in
saline solution was in-troduced into each homogenizer. Using a
drill press, the mixture was homogenized for about ~ to 5 minutes
until an emulsion was obtained. Microscopic investigation showed
that substantially all of the oil droplets were coated with
CWS and TDM.
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Example 2 - Lyophilizing the CWS-TDM Composition
5 ml of the CWS-TDM oil in saline emulsion (a 1~
oil-in-water emulsion) produced in accordance with the proce-
dure of Example 1 was introduced into a 10 ml Wheaton serum vial.
The vial was frozen in a Revco (a trademark) freezer at a
temperature of -95 C. and lyophilized in a sterile container
on a Labconco freeze dryer. The vial was then capped using
a sterile technique.
Example 3 - Reconstitution of Lyophilized CWS TDM Composition
A vial containing the lyophilized CWS-TDM emulsion
produced in accordance with the procedure of Example 2
containing, for example, 3.75 mg CWS and 1.25 mg TDM in 5 ml.
of a 1% oil droplet emulsion was injected with sterile distilled
water using a No. 20 gauge sterile syringe. The suspension was
then emulsified by repeated aspirations and injections using the
syringe for at least two minutes until the resulting emulsion
gave a cloudy-milky appearance. ~
Example 4 - CWS-TDM Composition - Comparison Data (Animals)
Dose per
Animal (~g) Animals
(guinea pig)* Treated Regression
CWS + TDM 300 + 150 125 76
CWS 300 157 46
CWS + TDM 150 -~ 150 56 63
CWS 150 65 ~3
CWS + TDM 50 + 50 16 19
25 CWS 50 40 10
TDM 150 39 3
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TDM 100 22 0
Oil-tween-saline
controls - 66 0
*Strain 2
The CWS was obtained rom M. bovis.
Example 5 - CWS-TDM Composition - Clinical Test Results
17 adult patients afflicted with metastatic malignant
melanoma were treated with doses of 300 to 1050 ug of CWS
(obtained from M. smegmatis) and 150 to 525 ug ~DM for l to 2
weeks for a total of 8 treatments. This therapy was effective
in 7 patients; of the 7 patients, 6 had complete regression of
at least l injected lesion.
