JPS6257166B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention The object of the present invention is a method for purifying trehalose dimycolate (TDM). TDM is
It is an isolate of bacteria that, in combination with the cell wall skeleton (CWS), forms a composition that is effective in suppressing and degrading tumor cells. The combination of cell wall scaffolds and TDM is known in the art ( Biologically Active Components from Mycobacterial Cell Walls, Suppression and Regression of the Pathome in Strain-2 Guinea Pigs; Mayer et al., J. National Cancer Inst. Volume 52, No. 1, January 1974; and Mycobacterial Cell Wall Components in Tumor Suppression and Regression; Livi et al., National
Cancer Inst. Monograph No. 39, pages 115-120,
The cell wall skeleton is essentially a cell wall with many of the proteins and lipids normally found in the removed cell wall. It is a polymeric mycolic acid arabinogalactan mucobeptide containing remnants of tocohalose mycolate (“P3”) and undigested (degraded) Mycobacterium tuberculosis proteins. The cell wall skeleton is obtained from mycobacteria, including M.
smegmatis, M.phlei , Nocardiarubra , Nocardia
asteroides, diphtheriae , Corynebacterium
parvum, M. kansasii , Mycobacterium tuberculosis (strains H37RV and Ayoma B), and M. bovis strain BCG.
Additionally, cell wall scaffolds can also be obtained from non-mycobacteria, such as E. coli , M. bangs , and Coxiella type . The process of generating cell wall scaffolds is time consuming. M. bovis , strain BCG (Bacillus carmetu-geran), is grown and collected. The resulting whole cell residue is separated using a cell fractionator [Livi Cell Fractionator (Solvelle, model RF-1)] that disrupts the cells and separates the cell wall or outer envelope from cytoplasmic impurities.
Processed by. A purified cell wall skeleton is obtained by subjecting the resulting cell wall to a series of solvent extractions and enzymatic treatments (eg, trypsin and/or chymotrypsin). Second component trehalose dimycolate (TDM)
from those containing mycobacteria, e.g.
M.avium , M.phlei , Mycobacterium tuberculosis (strains H37RV and
Ayoma B), M.bovis BCG, M.smegmatis ,
M.kansasii , Nocardia rubra , M.bovinis and
Obtained from B. diphtheriae . Bacteria such as M.avium are grown, harvested, and then heat sterilized. The cell mass is extracted with several solvents and the active, solvent soluble fraction is extracted. This extraction is further purified by a series of solvent extractions to provide crude TDM ( biologically active components from mycobacterial cell walls. I. Isolation and composition of the cell wall skeleton and component _P3 : Asma, et al.
J.National Cancer Inst.Volume 52, pages 95-101
(see 1974). Crude TDM is purified by centrifugal microparticulate silica gel chromatography to obtain purified TDM, as revealed by Azuma et al. CWS and TDM generated as described above
can be used as an oil droplet emulsion to obtain an anti-tumor composition suitable for injection (Immunotherapy with non-viable microbial components: Livi et al.: Annals of
New Yovk Academy of Science, vol. 227, 228~
238, September 20, 1976). The prior art regarding emulsions suffers from major disadvantages. Remaining impurities in purified TDM severely affect the efficacy of the composition limiting its effectiveness in treating tumors. Furthermore
Prior art attempts to purify TDM generally involve expensive, time-consuming, and repeated elutions using high-speed (high-pressure) silica gel columns. Applicants have demonstrated that the use of low pressure columns with silica gel particles having sizes from approximately 15 to 63 microns is surprisingly effective in removing crude TDM.
It has been shown that virtually all impurities are removed from the Therefore, it is an object of the present invention to provide a method for purifying crude TDM. Another object of the present invention is to provide a purified TDM product that can be used in combination with CWS to produce an oil-in-salt emulsifier that is effective as an anti-tumor agent. Invention The present invention is directed to a method for substantially removing all impurities that are normally associated with crude TDM. The method comprises dissolving crude TDM in a solvent and then applying the solution to a low pressure silica gel column with particle size from about 15 to 63 microns. The pressures used in the column are usually about 10 and 300 psi, preferably about 30 and 80 psi. A wide variety of non-polar solvents are used to dissolve crude TDM. Preferred solvents include, for example, chloroform, ether, hexane, notanol, ethanol, tetrahydrofuran, petroleum ether, heptane, methylene chloride, ligroin, propanol, butanol, ethyl acetate, benzene, toluene, acetic acid and the like, including combinations thereof. Can be mentioned. The purified TDM fractions are combined and the solvent is distilled off. The resulting product is substantially free of detectable impurities (ie, purity equal to or greater than 99.9%). By using the method described above, highly purified TDM products are obtained without the need for repeated purification steps. The products can be effectively combined with CWS in a conventional manner to produce potent anti-tumor compositions. It goes without saying that the following examples are for illustrative purposes only, and the scope of the invention is not limited or in any way redefined by these examples. Nor. Example 1 Preparation of Crude TDM 600 g (water content) of M. phlei whole cells, previously heat sterilized, were stirred overnight in 95% ethanol 4 using a magnetic stirrer and filtered using Whatman No. 1 filter paper.
Filter under vacuum on a cm Büchner funnel into a 2-filtration flask. Distill the ethanol solution. The cell residue is resuspended in 1500 ml of a 1:1 ethyl ether-ethanol solution in two flasks, stirred overnight with a magnetic stirrer, and then filtered as described above. The ether-ethanol solution is distilled off and a second ethyl ether-ethanol extraction and filtration is performed. After distilling off the ether-ethanol solution, the cell residue was dissolved in a 2:1 chloroform-methanol solution.
Resuspend in 1500 ml, stir with a magnetic stirrer overnight and filter using a Bützner funnel or
Centrifuge for 30 seconds at 10,000 rpm. The chloroform-methanol solution is distilled off and the extraction and filtration steps are repeated twice. The cell residue is dried in the air,
Put it in a bottle. The three chloroform-methanol solutions are combined and evaporated in a tared round bottom flask in a rotary steamer. Chloroform-
The weight of methanol residue is 13.0 g. The residue was dissolved in 500 ml of 2:1 chloroform-methanol solution and stirred with a magnetic stirrer for 1 hour.
It is then filtered using a sintered glass Büchner funnel (crude, 300 ml). The solvent is evaporated in a tared round-bottomed flask using a bush rotary-steamer to obtain a residue of 12.5 g. The residue obtained is resuspended in 400 ml of ethyl ether and stirred for 1 hour with a magnetic stirrer. Place the suspension in two screw cap centrifuge tubes and centrifuge for 30 minutes at 5000 rpm using a GSA rotor. Discard the ether soluble fraction. The ether soluble and ether insoluble fractions are retained. The ether-insoluble material is dissolved in 200 ml of a 2:1 chloroform-methanol solution and filtered using a Büchner funnel. The ether soluble fraction is evaporated in a rotary steamer in a tared round bottom flask to give an ether soluble residue. Ether residue 300
ml and precipitate with 900 ml of methanol.
The precipitate is filtered using a Bückner funnel using Whiteman No. 1 filter paper and combined with a 2:1 chloroform-methanol solution containing the ether-insoluble material described above. The resulting solution is evaporated in a rotary steamer to give 6.5 g of a residue. The residue was dissolved in 2:1 chloroform-methanol solution.
Dissolve in 200ml and put into a 500ml separating funnel. This solution is added dropwise to two flasks equipped with magnetic stirrers containing 600 ml of acetone. The resulting precipitate is filtered on a Büchner funnel, dried in air and placed in a tared bottle, yielding 4.5 g of crude TDM. Example 2 Purification of crude TDM 2 g of crude TDM as obtained in Example 1 was mixed in a 10:1
Dissolve in 2 ml of chloroform-methanol solution,
ml sample - pull loop. The remainder of the loop is filled with solvent. on a silica gel 60 column (25 x 1000 mm) with particle sizes ranging from approximately 15 to 63 microns.
Apply the solution using an Isco 132 type pump. Elute the column with 800 ml of chloroform and 1200 ml of a 98:2 chloroform-methanol solution at a rate of 4 ml/min (flow rate is important because if the flow rate is increased too much, the resolution will be poor).Results Generally, elution is generally about 0.1 ml to 20
Affected with flow rates varying between ml/min, typically 2
ml/min). The elution from the column was connected to a fraction collector, taking 8 ml fractions per tube, while the column
Elute with 3500 ml of 96:4 chloroform-methanol solution. The tubes containing purified TDM were placed on a TLC plate (silica gel F-254, 5 x 10
cm (0.25 mm thickness) and compare those fractions with previously separated purified TDM.
Visualization of the TLC plate is obtained by spraying with 10% (W/U) phosphomolybdic acid in ethanol. The fractions containing purified TDM are combined and the solvent is evaporated in a tared round-bottomed flask in a rotary steamer. 358 mg of purified TDM is obtained.

Claims (1)

Claims: 1. Purification of crude trehalose dimycolate comprises: (a) dissolving the crude trehalose dimycolate in a solvent; and (b) applying the solution to a low pressure silica gel column having a particle size between about 15 and 63 microns. A purification method characterized by applying. 2. Process according to claim 1, characterized in that the pressures are about 10 and 300 psi, preferably about 30 and 80 psi. 3 As a solvent, chloroform, ether, hexane, ethanol, methanol, tetrahydrofuran, petroleum ether, heptane, methylene chloride,
2. Process according to claim 1, characterized in that ligroin, propanol, butanol, ethyl acetate, benzene, toluene, acetic acid or mixtures thereof are used. 4. A method according to any of the preceding claims, characterized in that the trehalose dimycolate is obtained from mycobacteria. 5 As mycobacteria, M. avium, M.
Frey, Mycobacterium tuberculosis (strains H37RV and Ayoma B),
Claim 4, characterized in that M. bovis, BCG, Bacillus pubis, M. kansasii, Nocardia rubra, M. bovinis, or B. diphtheriae are used.
The method described in section. 6. According to any one of claims 1 to 5, the elution is carried out at variable rates of about 0.1 ml and 20 ml/min, preferably about 0.2 ml and 20 ml/min. the method of.