Note: Descriptions are shown in the official language in which they were submitted.
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BACKGROUND OF THE INVENTION
The subject matter of the present invention is directed to
a method of purifying crude trehalose dimycolates (TDM). TDM is
an isolate of bacteria and when combined with cell wall skeleton
(CWS) forms a composition which ~s effective in obtaining
suppression and regression of tumor cells.
The combination of cell wall skeleton and TDM is known in
the art (See Biologically Active Components from Mycobacterial
Cell Walls. II. -Suppression and Re2ression 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 Reqression; Ribi,
et al, National Cancer Institute Monograph No. 39, pages
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
obtained from any mycobacteria including, but limited to,
M.smegmatis, M.phlei, Nocardia rubra, Nocardia astero;des,
Corynebacterium diphtheriae, Corvnebacterium parvum,
M.kansasii, M.tuberculosis (Strain H 37 RV and Ayoma B3, and
M.bovis Strain BC~ Additionally, cell wall skeleton may be
obtained from such non-mycobacteria as E.coli, B.abortus and
Coxiella burnettiiO
The process of producing cell wall skeleton is time
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consuming. The bacteria such as M.bovis, Strain BC~ (bacillus
Calmette-Guerin) is grown and harvested. The resulting whole
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 ~he protoplasmic
impurities. The resulting cell walls are then subjected to a
series of solvent extractions and enzymatic treatments (e.gO,
trypsin and/or chymotrypsin) to give purified cell wall
skeleton.
The second component trehalose dimycolates (TDM) may be
obtained from any mycobacteria including, for example, M.avium,
M.phlei, M.tuberculosis (Strain ~1 37 RV and Ayoma B), M.bovis
BCG, M.sme~matis, M.kansasii, Nocardia rubra, M. bovinis and
Corynebacterium_diphtheriae.
Bacteria, such as M.avium, are grown, harvested and then
heat killed. The cell mass is 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 TD~ (See Biolo~ically Active
Components from Mycobacterial Cell Walls. I. Isolation and
Composition of Cell Wall Skeleton_and Component P3; Azuma, et
al, Journ~l oE the National Cancer Institute, Volume 52, pages
95-101, 1974). As disclosed in Azuma, et al, crude TDM may then
be further purified by centri~ugal microparticulate silica gel
chromotography to g~ve purified TDM.
CWS and ~DM produced as described above can be used as an
oil droplet emulsion to obtain an anti-tumor composition
suitable for injection (See Immuno herapy with Non-viable
Microbial Components; Ribi, et al; Annals of the New York
Academy of Science, Volume 227, pages 228-238, September 2~th,
1976).
The prior art emulsions, however, suffer from a major
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disadvantage. Impurities remaining in the purified TDM
seriously affect the potency of the composition limiting its
effectiveness in treating tumors. Prior art attempt~ at further
purifying TDM have generally involved repeated time consuming
costly elutions through high pressure silica gel columns.
Applicants have discovered that the use of a low pressure (i.e.,
between about 10 and 300 psi) column with silica gel particles
having a size between about 15 and 63 microns surprisingly and
effectively removes virtually all impurities from crude TDM.
It is thereEore an object of the invention to provide a
process for purifying crude TDM. It is another object of the
invention to provide a purified TDM product which can be
combined with CWS to produce an oil in saline emulsion which is
effective as an anti-tumor agent.
THE INVENTION
The present invention is directed to a process for
purifying TDM to eliminate virtually all impurities normally
associated with crude TDM. The process compri~es dissolving the
crude TDM in a solvent and then subjecting the solution to a low
pressure isilica gel column having a particle size of between
about 15 and 63 microns~ The pressure employed in the column is
normally betwe~n about 10 and 300 psi, preferably between about
30 and 80 psi.
A wide variety of non-polar solvents may be used to
dissolve the crude TDM. The preferred solvents include, for
example, chloroform, ether, hexane~ methanol, ethanol,
tetrahydrofuran, petroleum ether, heptane, methylene chloride,
ligroin, propanol, butanol, ethyl acetate, ben~ene, toluene,
acetic acid and the like including combinations thereof.
Fractions oE the purified TDM are combined and the solvent
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removed. The resulting product has virtually ns detectable
impurities (i.e., purity equal to or greater than 99.9 percent)~
By employing the process described above, a highly pure TDM
product is obtained without the need for repetitious purifying
steps. The product can be ef~ectively combined with CWS in a
conventional manner to produce a potent anti-tumor composition.
The following examples are for illustrati~e purposes only
and are not intended to limit or in any way redefine the
invention as claimed in the claims appended hereto.
Example 1 - Preparation of Crude TDM
600 g (moist weight) of whole cells of M.Phlei which had
previously been heat killed were stirred overnight on a magnetic
stirrer in 4 liters of 95 percent ethanol and then vacuum
filtered through a 27 cm Buchner funnel, fitted with a 24.0 cm
Whatman No. l filter paper, into a 2 liter filter flask. The
ethanol solution was removed. The cell residue was resu~pended
in a 2 liter flask with 1500 ml of a 1:1 ethyl ether-ethanol
solution and stirred overnight on a magnetic stirrer and then
filtered as described above. The ether-ethanol solution was
removed and a second ethyl ether-ethanol extraction and
filtration was then performed. After removal of the
ether-ethanol solution, the cell residue was resuspended in 1500
ml of a 2:1 chloroform-methanol solution and stirred overnight
on a magnetic stirrer and filtered through a Buchner funnel or
centrifuged at 10,000 rpm for 30 seconds. The chloroform-
methanol solution was removed and the extractlon and filtration
process repeated twice. The cell residue was air dried and
bottled. The three chloroEorm-methanol solutions were combined
and evaporated on a Buchi Ro~o-vapor in a tared round bottom
flask. The weight of the chloroform-methanol residue was 13.0 g.
The residue was dissolved in 500 ml of a 2.1
chloroform-methanol solution and stirred Eor 1 hour on a
magnetic stirrer and then filtered through a sintered glass
Buchner ~unnel tcoarse, 300 ml). The solvent was evaporated on
a Buchi Roto-vapor in a tared round bottom flask to provide a
residue weighing 12.5 grams.
The resulting residue was resuspended in 400 ml oE ethyl
ether and stirred on a magnetic stirrer for 1 hour. The
suspension was then centrifuged in 2 screw-cap centrifuge
bottles in a GSA Rotor at 5000 rpm for 30 minutes. The ether
soluble ~raction was then decanted. Both the ether soluble and
ether insoluble fractions were preserved.
The ether insoluble material was disssolved in a 200 ml
2:1 chloroform-methanol solution and filtered through a Buchner
funnel.
The ether soluble fraction was evaporated on a Buchi Roto-
vapor in a tared round bottom flask and an ether soluble residue
was obtained. This residue was dissolved in 300 ml of ether and
precipitated into 900 ml of methanol. The precipitate was
filtered through a Buchner funnel using Whatman No. 1 filter
paper and combined with the ~:1 chloroform-methanol solutlon
containing the ether insoluble material described above. The
resulting solution was evaporated on a Buchi Roto-vapor in a
tared round bottom flask to obtain 6.5 grams of a residue.
The ras~due was dissolved in 200 ml o~ a 2:1 chloroform-
methanol solution and poure~ into a 500 ml separatory funnel.
This solution was then added drop-wise into a 2 liter flask, ~n
a magnetic stirrer, containing 600 ml of acetone. The resulting
precipitate was filtered into a Buchner funnel, air dried and
placed in a tared bottle to obtain 4.5 9 of crude TDM.
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Exa~ple 2 - Purification of Crude ~DM
2 grams of crude TDM as obtained in Example 1 were
dissolved in 2 ml of a 10:1 chloroform-methanol solution and
then drawn onto a 5 ml sample loop. The remainder o~ the loop
was filled with solvent. The solution was pumped onto a silica
gel 60 column (25 X 1000 mm) having a particle size of from
about 15 to 63 microns, using an Isco Model 132 pump. The
column was eluted with 800 ml of chloroform, followed by 1200 ml
of a 98:2 chloroform-methanol solution at a rate of 4 ml/minute.
(The flow rate is critical as it has been found that resolution
is less when the flow rate is unduly increased. Consequently~
elutlon is generally affected at a flow rate varying between
about 0.1 ml to 20 mls./minute and normally at a rate between
about 2 and 5 mIs./minute.) The effluent from the column was
then connected to a fraction collector and fractions of 8 ml per
tube were collected while the column was being eluted with 3500
ml of a 96:4 chloroform-methanol solution. The tubes containing
purified TDM were determined by spotting aliquots of the various
fractions on TLC plates (silica gel F-254, 5XlOcm, 0.25mm thick)
using a 10:1 chloroform-methanol solution as an eluant and
comparing these fractions with pure TDM previously isolated.
Visualization of TLC plates were produced by spraying plates
with 10 percent (w/v) of phosphomolybdic acid in ethanol.
Fractions containing purified TDM were combined and solvent
evaporated on a Buchi Roto-vapor in a tared round bottom flask~
358 mg of purified TDM were obtained.
