Field of the Invention
[0001] The invention concerns a new lipoteichoic acid (in the following LTA-T), a pharmaceutical
composition comprising it, optionally together with a monokine and/or hyaluronidase,
a method of treating cancer comprising administration of an antitumor effective amount
thereof, a method of producing the new compound and the new pharmaceutical composition,
two degradation products of the new LTA-T and their use, and the new Streptococcus
strain from which the new compound can be isolated.
Background of the Invention
[0002] Lipoteichoic acids (LTAs) are a group of amphipathic substances found in the cell
wall of grain-positive bacteria extending from the outer cell membrane through the
cell wall to the surface. The main group of LTAs consists of a hydro-philic poly(glycerophosphate)
backbone and a hydrophobic glycolipid moiety. The hydrophilic backbone may be substituted
with alanine, hexoses and hexosamines. The glycolipids described so far were mainly
dihexosylglycerols and some trihexosylglycerols. Lipoteichoic acids show genus and
species variation in the degree of polymerization of the hydrophilic chain, in the
nature and degree of glycosidic substitution, in the extent of D-alanyl ester substitution,
and in the structure of the lipid moiety (A. J. Wicken et al., Science, 187, 1161
- 1167, (1975), and Microbiology, 360 - 365, (1977); Fischer W., Physiology of lipoteichoic
acids in bacteria. Adv. Microb. Physiol., 29(233); 233-302 (1988), Fischer W., Mannsfeld
T., Hagen G., On the basic structure of poly(glycerophosphate) lipoteichoic acids,
Biochem. Cell Biol., 68 (1): 33-43, (1990).
[0003] LTAs have been reported as having antitumor activity (EP 135 820; USP 4,678,773;
A. Yamamoto et. al. 1985, Br. J. Cancer, 51, 739 - 742; and H. Usami et. al., Br.
J. Cancer, 1988, 57, 70 - 73).
[0004] LTAs were isolated from e. g. Lactobacillus helveticus (NCIB 8025), Lactobacillus
fermenti (NCTC 6991), Streptococcus faecalis, 39, Streptococcus lactis (ATCC 9936),
Streptococcus mutans, AHT (A. J. Wicken et al, 1975), and Streptococcus pyogenes SV
strain (ATCC 21059) (EP 135 820, USP 4,678,773, H. Usami et. al. 1985).
[0005] A glycerophosphogalactofuranosylglycerol has been described by J. H. Veerkamp and
F. W. van Schalk, (1974), Biochim et Biophys. Acta, 348, pages 370-387. The isolation
from
Bifidobacterium bifidum var. pennsylvanicus, however did not result in pure compounds. An amount of 10 to 20 % of the corresponding
galactopyranosyl compounds was still present. The structure of the major compounds
from this bacterium, was later proposed by Werner Fischer, Eur. J. Biochem, 165, 639-646
(1987). It is a galactofuran (8-15 units) linked via glucon (6-9 units) to D-galactopyranosyldiacylglycerol.
[0006] K. K:Brown disclose in WO 94/20115 the use of hyaluronic acid for the treatment of
cancer whereby also lipoteichoic acids may be used in combination therewith.
[0007] A streptococcal acid glycoprotein (SAGP) with antitumor activity was isolated by
M. Kanaoka et. al., Jp. J. Cancer Res. (Gann), 78, 1409 - 1414, (1987) from the low
virulent strain Streptococcus pyogenes Su ATCC 21060. OK-432, a cell preparation from
said strain, has found clinically use as an antitumor agent. However, in the meantime
it was withdrawn from the market.
[0008] The LTAs described up to now carried more than one monosaccharide in the glyceroglycolipid
anchor. Different glycolipid structures have been described by Fischer et al. 1988
and 1990.
[0009] An LTA with a monohexosyldiacylglyceroglycolipid as lipid anchor has not been described
so far.
Object of the Invention
[0010] It is an object of the invention to provide a purified new LTA with a strong antitumor
activity.
[0011] It is a further object to provide pharmaceutical preparations comprising this new
LTA, optionally in combination with a monokine and/or hyaluronidase.
[0012] It is a further object to provide a method of producing a pharmaceutical preparation
for treating cancer comprising administration of an antitumor effective amount of
the new LTA to a patient optionally in combination with a monokine and/or hyaluronidase.
[0013] It is a further object to provide a method of producing the new LTA and the new pharmaceutical
preparation.
[0014] It is a further object to provide two degradation products of the new LTA and their
use.
[0015] It is a further object to provide a new Streptococcus strain from which the new LTA
can be isolated and a method for its proliferation.
Detailed Description of the Invention
[0016] The invention concerns a new purified lipoteichoic acid (LTA-T) isolatable from the
new Streptococcus sp strain DSM 8747 containig a beta-galactofuranosyl(1-3)glycerol-di-ester.
[0017] A first new LTA found is designated as LTA-T. It consists of a defined compound as
it is shown in Formula I, with a microheterogeneity of chain length and fatty acid
composition as it is given in the table on page 4. This microheterogeneity is a typical
feature of lipid macroamphiphiles [Fischer W. (1993), Molecular analysis of lipid
macroamphiphiles by hydrophobic interaction chromatography, exemplified with lipoteichoic
acids, Anal. Biochem., 208, 49-56]. The exact composition of the naturally occuring
LTA-T cannot easily be determined. It depends on the conditions of cultivation of
the microorganisms.
[0018] More particularly the invention provides a lipoteichoic acid LTA-T of the Formula
I

wherein R
1 is hydrogen or D-alanyl with a molar ratio to phosphorous of 0.27 to 0.35, and R
2 are the residues of saturated or unsaturated fatty acids with 12, 14, 16 or 18 carbon
atoms and the mean value for n is 9, and salts thereof.
[0019] LTA-T is a new type of lipoteichoic acid in that it contains a monohexosylglycolipid
moiety. Such monohexosylglycolipid moiety has not been found yet in other organisms
as a part of lipoteichoic acids. This lipidanchor, as shown in Formula II below, is
a beta-galactofuranosyl (1-3) glycerol-di-R
2-ester wherein R
2 are different rests of fatty acids esterified to the two adjacent hydroxy groups
in the glycerol moiety.
[0020] The fatty acid rests R
2 are derived from straight-chain saturated or mono-unsaturated carboxylic acids having
12, 14, 16, or 18 carbon atoms and include the saturated lauric (C-12), myristic (C-14),
palmitic (C-16) and stearic (C-18) acid, and corresponding mono-unsaturated carboxylic
acids with one double bond in 7, 9, 11 or 13 position, respectively. The distribution
is heterogenous and reflects the distribution in whole membrane lipids. Following
aproximative percentages have been found for R
2 for a typical cultivation:
C-12, saturated |
ca. 6.0%; |
C-14, saturated |
ca. 17.0%; |
C-14, mono-unsaturated (position unknown) |
ca. 3.7%; |
C-16, saturated |
ca. 33.0% |
C-16, mono-unsaturated probably in 7-position |
ca. 3.8%; |
C-16 mono-unsaturated in cis-9-position |
ca. 11.3%; |
C-16, mono-unsaturated in cis-11-position |
ca. 2.4%; |
C-18, saturated |
ca. 10.0% |
C-18, mono-unsaturated probably in 9-position |
ca. 3.2% |
C-18, mono-unsaturated in 11-position (cis) |
ca. 8.5% |
C-18, mono-unsaturated probably in 13-position |
ca. 1.1%. |
[0021] The hydrophilic backbone consists of a poly(glycerophosphate) with a mean of 10 glycerophosphate
units. The hydroxygroups at position 2 of the glycerol moieties are free or esterified
by D-alanine. The molar ratio of substitution to phosphorous is 0.27-0.35, corresponding
to 2.7 to 3.5 D-alanine groups per molecule LTA-T. The D-alanine content depends on
the cultivation conditions.
[0022] The free hydroxy groups at the phosphorous atoms are acidic. At pH 4.7 in sodium
acetate buffer and in physiological saline the cation is a sodium ion. LTA-T may form
salts with other positively charged ions, in particular physiologically acceptable
salts, such as alkali metal or alkaline earth metal salts, also heavy metal salts,
such as zinc or iron salts, or primary, secondary, tertiary or quaternary ammonium
salts (acid addition salts). Such other salts are e. g. potassium, calcium, ammonium,
mono-, di-, tri- or tetra-lower alkyl-, e. g. methyl- or ethyl-, or methyl-ethyl,
propyl- or butylammonium salts. Non-physiologically acceptable salts, such as heavy
metall salts, e. g. copper salts, may be used for isolation and purification of LTA-T.
A preferred salt is the sodium salt, when the LTA is purified as described.
[0023] For therapeutical use the amount of the positively charged ions in the pharmaceutical
composition is to be adjusted to result in a physiologically acceptable pH, in particular
around pH 7 or 7.2.
[0024] The invention concerns a method for the preparation of a lipoteichoic acid LTA-T,
characterized in isolating it from Streptococcus sp (DSM 8747) and purifying it by
conventional methods.
[0025] Isolation and purification of LTA-T can be achieved in analogy to Fischer W., Koch
H. U., Haas R. (1983), Improved preparation of lipoteichoic acids, Eur. J. Biochem.,
133: 523-530, or any other method. For example, bacteria cells (DSM 8747) are suspended
in distilled water or preferably a buffer, e. g. citrate buffer of pH 3.0, and disrupted,
e. g. by means of a homogeniser and glass beads, preferably under cooling. The suspension
of the broken cells is adjusted to about pH 4.7, e. g. with sodium bicarbonate. The
aqueous suspension is extracted with phenol at moderately elevated temperature, e.
g. up to about 68° C. The water phase is separated and several times dialysed, e.
g. against sodium acetate buffer of pH 4.7, with a diaphragma having a molecular weight
cut off of 10 - 12 kD. The remaining clear solution is concentrated in an ultrafiltration
device with a PM 10 membrane and insoluble material, such as polysaccharides, removed
by centrifugation.
[0026] The crude extract is further freed from undesired material, such as proteins, nucleic
acids and polysaccharides, e. g. by hydrophobic interaction chromatography (HIC),
e. g. by loading in a solution of propanol/sodiumacetate pH 4.7 on an octyl-Sepharose
column. The LTA-T is eluted, e.g with a linear gradient of propanol in sodium acetate
pH 4.7. The effluents are monitored by a colorimetric determination of organic phosphorus
according to Schnitger H., Papenberg K., Ganae E., Czok R., Bücher T., Adam H. (1959),
Chromatographie phosphathaltiger Metabolite eines menschlichen Leberpunktats, Biochem.
Zentralblatt, 332; 167187. LTA-T is eluted at a propanol concentration of about 30-38%.
The LTA-T containing fractions are dialysed against a buffer, e. g. sodiumacetate
pH 4.7, and concentrated by ultrafiltration with a PM 10 membrane or completely dried
in vacuum. The purified LTA-T or the concentrated solution thereof is stored at -20°C.
[0027] The bacteria cells (DSM 8747) are obtained by culturing in a conventional manner
in a complex medium, e. g. Todd Hewitt broth or Tryptic Soy broth, at 37° C and a
pH of about 7.2 under stirring and without aeration. At the end of the logarithmic
growth phase the cells are harvested, e. g. by centrifugation, suspended in a convenient
buffer, e. g. a citrate buffer of pH 3, in which they can be stored at low temperature,
e. g. at -20° C for further use.
[0028] The invention concerns further a pharmaceutical preparation comprising a lipoteichoic
acid LTA-T or a physiologically acceptable salt thereof, optionally in combination
with a monokine and/or hyaluronidase.
[0029] Monokines are for example interferons, such as of the alpha group, e. g. interferon
alpha 2b, or interferon gamma, cytokines, are for example inter-leukins, e. g. interleukin-1-alpha,
-1-beta, -1-ra, -2, -3, -4, -5, -6 -7 or -8, tumornekrose-factors, e. g. TNF-alpha
or -beta, or TGF-beta-1, -beta-2, -beta-3, -beta-5 and -alpha.
[0030] Hyaluronidase is any commercially available one, e.g. Permease®.
[0031] The pharmaceutical preparations are of conventional manner.
[0032] The LTA-T or the pharmaceutical combinations of the present invention are administered
orally or parenterally to achieve the therapeutic effect in any of the usual pharmaceutical
forms. These include solid and liquid unit oral dosage forms such as tablets, capsules,
powders, suspensions, solutions and syrups, transdermal plasters, inhalable formulations,
and the like, including sustained release preparations, and fluid injectable forms,
such as sterile solutions and suspensions. The term dosage form as used in this specification
and the claims refer to physically discrete units to be administered in single or
multiple dosage to humans or warmblooded animals, each unit containing a predetermined
quantity of active material in association with the required diluent, carrier or vehicle.
The quantity of active material is that calculated to produce the desired therapeutic
effect upon administration of one or more of such units.
[0033] Powders are prepared by comminuting the compound to a suitably fine size and mixing
with a similarly comminuted diluent pharmaceutical carrier, such as an edible carbohydrate
material as for example, starch. Sweetening ,flavoring, preservative, dispersing and
coloring agents can also be added. Powders are advantageously applied by inhaling
and are for this purpose filled into inhalers. Such inhalers for dry powders are known
in the art.
[0034] Capsules are made by preparing a powder as described above and filling formed gelatin
sheaths. A lubricant, such as talc, magnesium stearate and calcium stearate can be
added to the powder mixture as an adjuvant before the filling operation. A glidant
such as colloidal silica may be added to improve flow properties. A disintegrating
or solubilizing agent may be added to improve the availability of the medicament when
the capsule is ingested.
[0035] Tablets are made by preparing a powder mixture, granulating or slugging, adding a
lubricant and disintegrant and pressing into the desired form. A powder mixture is
prepared by mixing the compound, suitably comminuted, with a diluent or base such
as starch, sucrose, kaolin, dicalcium phosphate and the like. The powder mixture can
be granulated by wetting with a binder such as syrup, starch paste, acacia mucilage
or solutions of cellulosic or polymeric materials and forcing through a screen. As
an alternative to granulating, the powder mixture can be run through the tablet machine
and the resulting imperfectly formed slugs broken into granules. The granules can
be lubricated to prevent sticking to the tablet forming dies by means of the addition
of stearic acid, a stearate salt, talc or mineral oil. The lubricated mixture is then
pressed into tablets. The medicaments can also be combined with free flowing inert
carriers and compressed into tablets directly without going through the granulating
or slugging steps. A protective coating consisting of a sealing coat of shellac, a
coating of sugar or polymeric material and polish coating of wax can be provided.
The coating can be resistant in the stomach and the active ingredients to be released
in the intestine. Dyestuffs can be added to these coatings to distinguish different
unit dosages.
[0036] Oral fluids such as syrups and elixirs can be prepared in unit dosage form so that
a given quantitiy, e.g. a teaspoonful, contains a predetermined amount of the compound.
Syrups can be prepared by dissolving the active compound in a suitably flavored aqueous
sucrose solution, while elixirs are prepared through the use of a non-toxic alcoholic,
e.g. ethanolic, vehicle. Suspensions and emulsions can be formulated by dispersing
the medicament in a non-toxic vehicle.
[0037] For parenteral administration, fluid unit dosage forms can be prepared by suspending
or dissolving a measured amount of the active material in a non-toxic liquid vehicle
suitable for injection such as an aqueous, alcoholic, e.g. ethanolic, or oleaginous
medium. Such fluid dosage unit forms may contain solubilizers, such as a polyethyleneglycol,
stabilizers, and buffers, such as a citric acid/sodium citrate buffer, to provide
the desired osmotic pressure. Alternatively a measured amount of the active material
is placed in a vial and the vial and its contents are sterilized and sealed. An accompanying
vial or vehicle can be provided for mixing prior to administration. Solutions can
also be specifically prepared for inhalation and applied by means of an inhaler. Inhalers
for fluids are known in the art.
[0038] For transdermal application powders or syrups may be manufactured into suitable transdermal
plasters. Such plasters are known in the art.
[0039] If combinations of LTA-T with a monokine and/or hyaluronidase are envisaged such
combinations may be used separately and simultaneously or consecutively, or otherwise
formulated together in one pharmaceutical preparation according to the methods described
above.
[0040] The invention concerns further a method of producing a pharmaceutical preparation
comprising LTA-T or a physiologically acceptable salt thereof and optionally a monokine
and/or hyaluronidase by a conventional method.
[0041] The invention concerns further a method of producing a pharmaceutical preparation
for treating cancer comprising administration of an antitumor effective amount of
a lipoteichoic acid LTA-T or a physiologically acceptable salt thereof and optionally
a monokine and/or hyaluronidase to a patient suffering from cancer, a tumor or a malignant
cell thereof.
[0042] The following biological effects, determined according to Bhakdi S., Klonisch T.,
Nuber P., Fischer W., Stimulation of monokine production by lipoteichoic acids, Infect.
Immun., 59(12): 4614-4620, (1991), and Keller R., Fischer W., Keist R., Bassetti S.,
Macrophage response to bacteria: induction of marked secretory and cellular activities
by lipoteichoic acids, Infect. Immun., 60(9): 3664-3672,(1992), respectively, were
found 8 hours after induction of monocytes with LTA-T;
Table 1
Amount of LTA-T used for Induction, ug |
Amount of monocytes found 8 h after induction, ng ml-1 |
0.50 |
TNF: 25 |
1.50 |
TNF: 60 |
0.25 |
IL-6: 27 |
2.00 |
IL-6: 30 |
1.00 |
IL-1b: 35 |
4.00 |
IL-1b: 35 |
[0043] The values for induction with the known LTAs of
S. pyogenes and
S. lactia as obtained in the same set of experiments were 2-4 times less than these data.
[0044] The new LTA-T is preferably administered subcutaneously, intravenoulsy or intraperitoneally
in dosage unit form of a pharmaceutical preparation comprising LTA-T or a physiologically
acceptable salt thereof in an amount of from 0.1 to 20 micromol/ml and one or more
pharmaceutical carriers. An antitumor effective amount of LTA-T is e. g. of from about
0.001 to about 20 mg, e. g. from 1 to 20 mg/kg, preferably of from 0.01 to 2 mg/kg,
which is administered to a patient of normal weight once or preferably several times
during the entire period of treatment, as need may be. The amount and mode of administration
depend on the type and severity of the disease, the weight and general condition of
the patient and is to be left to the judgement of the physician. The new LTA-T may
be applied prophylactically in the amounts given hereinbefore.
[0045] If hyaluronidase is used it is applied in amounts of between about 500 and about
5000, preferably about 1000 U USP, and preferably subcutaneously.
[0046] If a monokine is used it is applied in amounts of between about 0.1 x 10
6 and about 20 x 10
6, preferably about 6 x 5 mio units, and preferably subcutaneously.
[0047] Eight patients suffering from various Kinds of tumors/cancer were treated subcutaneously
with a solution having a concentration of 1 micromol/ml LTA-T in physiological saline
in single or repeated administration. Most of the patients obtained also subcutaneously
hyaluronidase and one obtained also an interferon-alpha. The results are compiled
in Example 7, Table 2.
[0048] The invention concerns further the new Streptococcus sp strain DSM 8747 from which
the new LTA-T can be isolated.
[0049] The new bacteria strain was isolated from an erysipelas of a female patient with
a malignant breast carcinoma in complete remission. The strain is a new species within
the genus
streptococcus. It was designated as Streptococcus sp. PT and deposited under the Budapest Treaty
at the Deutsche Sammlung für Mikroorganismen, Braunschweig, Germany, under the deposition
number DSM 8747 on November 25, 1993.
[0050] The strain can be cultured and stored under conventional conditions as described
hereinbefore.
[0051] The invention concerns further the new degradation products of LTA-T and the methods
of their preparation by conventional means, e. g. by alkaline or hydrogen fluoride
(HF) hydrolysis.
[0052] Such new degradation product is for example the deacylated dLTA-T of the Formula
I, wherein R
1 and R
2 are both hydrogen. This compound is obtained by splitting off the fatty acid and
the D-alanyl groups by conventional methods, e. g. by treatment of LTA-T with a base,
e. g. 0.1 m aqueous NaOH at 37° C for about one hour. The formed dLTA-T is separated
and purified according to the method of Folch J, Lees M., Sloan-Stanley, G. M. S.
(1957), A simple method for the isolation and purification of total lipids from animal
tissues, J. Biol. Chem. 226, 497 - 509, by partition between the two phase system
chloroform: methanol:water (1:0.9:0.9).
[0053] Another new degradation product is beta-galactofuranosyl(1-3)glycerol-di-R
2-ester of the Formula II

wherein R
2 is the rest of a saturated or unsaturated fatty acid with 12, 14, 16 or 18 carbon
atoms, a single compound falling under Formula II, and salts thereof.
[0054] A compound of the Formula II is produced from a compound of the Formula I by splitting
the bond between the 6-hydroxy group of the galactofuranosyl group and the phosphoric
acid moiety, e. g. by treatment of LTA-T with 48 % hydrogen fluoride at 2° C for about
36 hours.
[0055] A compound of the formula II, wherein R
2 is hydrogen (deacylated lipid anchor) is produced from a compound of the formula
I, e. g. by treatment with 0.2 M NaOH for 12 hours at 100° C, and subsequent cleavage
of phosphomonoesters by phosphomonoesterase.
[0056] The degradation products are useful as analytical tools for the identification and
characterisation of LTA-T and as starting materials for the preparation of new LTAs
with defined groups R
2, for example by esterification of dLTA-T with specific fatty acids, and for the preparation
of new LTA with a defined hydrophilic group esterified to the 6-hydroxy group of the
galactofuranosyl moiety.
[0057] The following examples describe the invention in more detail. They should however
not be construed as a limitation thereof.
Example 1: Bacterial strain and cultivation
[0058] The gram-positive bacterium
Streptococcus sp. PT, deposited at the Deutsche Sammlung für Mikroorganismen unter No. DSM 8747, was
isolated from a erysipelas of a human patient with a malignant breast carcinoma. It
belongs to the group of
streptococci. 16 S RNA sequencing revealed that this strain cannot be classified in the known groups
of
streptococci. It was designated
Streptococcus sp. PT and has the following growth characteristics:
Morphology: chain forming cocci with 5-40 units, depending on shear forces
Growth optima: pHopt: pH 7.2; Topt: 37°c; microaerophilic growth
[0059] The bacteria are cultivated in Todd Hewitt broth (Difco, USA) to the end of the logarithmic
growth phase. Cultivation conditions are:
working Volume VR |
500 l |
Temperature |
37°C |
pH |
7.2±0.1 |
Aeration rate |
none - 0.05 vvm |
Stirring speed |
500 rpm |
[0060] The culture broth is cooled and the cells harvested immediately by centrifugation.
The cells (400g wet weight per litre) are suspended in 0.1M citrate buffer pH 3.0
and stored at -20°C for further use.
Example 2: Isolation and Purification of lipoteichoic acid LTA-T
[0061] When not mentioned otherwise all steps are accomplished at 4°C.
[0062] A suspension (250 ml) of bacteria cells DSM 8747 in 0.1 M citrate buffer pH 3.0 (400g
wet weight per litre, obtained as described in Example 1) is mixed with an equal volume
of glass beads (Braun Melsungen, Ø 0.17-0.18 mm) and agitated under cooling in a Braun
disintegrator fitted with a CO
2 cooling device for 6 min. The suspension of broken cells is decanted through a glass
filter G1 and the remaining glass beads are washed with 0.1 M of sodium acetate pH
4.7. The combined filtrate and washing fluid is adjusted to pH 4.7 with 1 M NaHCO
3. The crude suspension is extracted in an equal volume of 80:20 (v/v) phenol/water
at 68° C for 1 hour. After cooling, the water phase is separated by centrifugation
at 3000 rpm (1800g) for 30 minutes. The upper water phase is collected and an equal
volume of 0.1 M sodiumacetate buffer pH 4.7 is added to the remaining phenol phase
and extracted, centrifuged and collected as described before. If the water phase is
cloudy, it is extracted again with phenol at room temperature (1/8 (v/v) of the water
volume) for 30 min and centrifuged as before.
[0063] The combined water phases are extensively dialysed against 0.05 M sodiumacetate pH
4.7 (four 5 litre changes for at least 24h) in a Medicell® tubing with a molecular
weight cut off (MWCO) of 10-12 kD.
[0064] The clear solution is concentrated in an Amicon® Ultrafiltration device with a PM
10 membrane (MWCO 10 kDa) and insoluble material (e.g. polysaccharides) is separated
by centrifugation.
[0065] The crude extract solution is freed from proteins, nucleic acids and polysaccharides
by hydrophobic interaction chromatography (HIC). For that purpose the crude LTA preparation
is loaded in 15% propanol in 0.1 M sodiumacetate pH 4.7 on an octyl-Sepharose (Pharmacia
LKB Sweden) column, previously equilibrated with the same buffer-propanol solution.
After separation of nucleic acids, proteins and polysaccharides, the LTA-T is eluted
with a linear gradient of 15-55% (v/v) propanol in 0.1 M sodiumacetate pH 4.7. Each
effluent is monitored by a colorimetric determination of organic phosphorus according
to Schnitger, ibid. The LTA is eluted at a propanol concentration of about 33%. The
LTA containing fractions are collected and dialysed against 0.05 M sodiumacetate pH
4.7 and concentrated down to about 5 micromol phosphorus content/ml by ultrafiltration
in an Amicon® Ultrafiltration device with a PM 10 membrane (MWCO 10 kDa). The concentrated
solution of LTA-T is stored at -20°C.
[0066] This clear solution of LTA-T is free of contaminant proteins (shown by HPLC of amino
acids after acid hydrolysis), nucleic acids (exact Gro/P ratio) and carbohydrates
(no contaminant sugars after acid hydrolysis). It can be evaporated to dryness to
give a powder which is difficult to solubilize again in water for reason of micell
formation. It can be solubilized in a mixture of water and an organic solvent, e.
g. ethanol, or a solubilizer, e. g. polyethyleneglycol.
[0067] The LTA-T can be characterised by its unique lipidanchor after hydrolysis with hydrogen
fluoride, as described in Example 3.
Example 3: Structural Characterisation
[0068] The purified LTA is submitted to HF hydrolysis (48 % HF, 36 h, 2°C) and the hydrophilic
part (products of the backbone) and the hydrophobic part (lipidanchor) separated by
Folch partition [Folch J., Lees M., Sloane-Stanley G. H. S. (1957), A simple method
for the isolation and purification of total lipids from animal tissues, J. Biol. Chem.,
226: 497-509] in chloroform:methanol:water (1:0.9:0.9).
[0069] The two parts are analysed separately. The core of the lipid anchor is analysed after
deacylation as partially methylated alditol acetate by GLC-MS analysis. The typical
fragmentation pattern of 1,2-dimethyl-3-acetyl-glycerol and 2,3,5,6-terra-0-methyl-1,4-di-0-acetyl-galactitol
can be observed. The hydrophilic products are analysed by GLC (gas liquid chromatography)
before and after HCl hydrolysis or alkaline dealanylation. Thereby no sugars are detected.
[0070] For molecular composition the LTA is hydrolysed with 2M HCl for 2,5 hours at 100
°C and afterwards treated with phosphonomonoesterase in order to remove phosphonomonoesters.
Phosphorus, glycerol, galactose and alanine are obtained in a ratio of 1:1.05:0.11:0,27
indicating the proposed structure given by Formula I. [According to the methods described
in Fischer W. (1988), Physiology of lipoteichoic acids in bacteria, Adv, Microb. Physiol.,
29 (233): 233-302].
[0071] NMR analysis of the deacylated compound of LTA-T (dLTA-T) allows a definite structural
characterisation. The NMR spectrum is shown in Figure 1. The identification of the
peaks are listed in the following Table 2:
Table 2
C-atom |
dLTA |
|
ppm |
|
ppm |
beta-Gl |
108.24 |
Gro Al |
63.45 |
X1 |
67.14 |
G2 |
81.83 |
A2 |
71.16 |
X2 |
70.40 |
G3 |
77.61 |
A3 |
69.50 |
X3 |
67.00 |
G4 |
83.82 |
D1 |
67.37 |
|
|
G5 |
70.50 |
D2 |
71.62 |
|
|
G6 |
67.27 |
D3 |
63.04 |
|
|
[0072] Summing up, the characteristics of this LTA for distinction of other LTAS are the
following:
- beta-Galf-(1-3)diacylglycerol as lipid anchor
- non-glycosylated, linear, unbranched GroP-chain
- mean chain length of 10 GroP units
- lipid pattern
Example 4: Preparation of Deacyl-LTA-T (dLTA-T)
[0073] LTA-T is submitted to mild alcaline hydrolysis (0.1M NaOH, 1 h, 37°C). The solution
is adjusted to pH 3 with HCl and the fatty acids are extracted four times with petroleumether:
chloroform (4:1). The water solution is neutralized with NaOH and extensively dialysed
against water in a tubing with a cut off of 2 kD. The product in the retentate is
LTA-T without alanine esters and without fatty acids and is called dLTA-T.
Example 5: Preparation of beta-Galactofuranosyl(1-3)glycerol-di-R2-ester
[0074] The lipidanchor beta-galactofuranosyl(1-3)glycerol-di-R
2-ester of the Formula II can be isolated as it is outlined in Example 3 after HF hydrolysis.
[0075] Since galactofuranosyl-beta-1-3-glycerol is also found as a part of the membrane
lipids, it can be isolated from whole lipid preparations.
[0076] The lipids are isolated by the method of Bligh-Dyer [Bligh E. G., Dyer W. J. (1959,
A rapid method of total lipid extraction and purification, Can J. Biochem Physiol,
37: 9111-9117] and the crude lipid extract is first fractionated on an anion exchange
column (DEAE Cellulose) and further purified on silicagel. Elution is made with different
mixtures of chloroform:acetone. Final purification is made by preparative TLC on silicagel
plates [Kates M. (1986) Techniques in lipidology. In: Laboratory techniques in biochemistry
and molecular biology. Work T.S., Work E. (eds.), North-Holland publishing company,
Amsterdam].
Example 6: Pharmaceutical Formulation
[0077] The purified LTA-T in 0.05M sodiumacetate pH 4.7 is dialysed extensively against
physiological NaCl solution (0.9%) and the volume is adjusted to 1 µmol LTA-T (based
on the phosphorus content) with physiological saline. After filtration of the solution
through a filter membrane (Millipore 0.22µm), 1 ml aliquots of the filtrate are placed
in sterilized vials under sterile conditions. These vials contain 1 micromol/ml LTA-T
phosphorus and are used subcutaneously for therapeutical purposes.
Example 7: Results of Clinical Treatments
[0078] Eight patients suffering from various kinds of tumors/cancer were treated subcutaneously
with a solution having a concentration of 1 micromol/ml LTA-T in physiological saline
in single or repeated administration. Most of the patients obtained also subcutaneously
hyaluronidase and one obtained also an interferon-alpha. The results are compiled
in Table 3:
Table 3
Patient |
Type of Tumor |
Treatment |
Result |
M.F., *1952 |
Malignant fibrous Histiocytoma. Removed surgically 3/91. Local recurrence 5/92. Incomplete
excision 11/92. |
LTA-T
s.c. at the site of the tumor. Total dose 8 micromol phosphate1): 1-2/93 |
CR (>10 months) |
H.R., *1909 |
Cancer of the Prostate, diagnosed 6/92 |
7/92: LTA-T, 1 micromol P in combination with Hyaluronidase2) 1000 NFU s.c. 9/93: LTA-T, 2 micromol P incombination with Hyaluronidase 1000 NFU
s.c. |
PR (PSA: 6/92: 108 mcg/l, 9/92: 63 mcg/l) |
L.B., *1943 |
Colon carcinoma pT3 pN2 G2, Resection 1/90 |
LTA-T
3/90: 8 micromol P in combination with Hyaluronidase 1000 NFU s.c.
3/91: 8 micromol P in combination with Hyaluronidase 1000 NFU s.c.,
12/91: 8 micromol P in combination with Hyaluronidase 1000 NFU s.c.,
11/92: 3 micromol P in combination with Hyaluronidase 1000 NFU s.c.,
9/93: 3 micromol P in combination with Hyaluronidase 1000 NFU s.c. |
CR (>47 months) |
L.K., *1916 |
Colon carcinoma pT4 pN2 G2-3, Resection 4/93 |
LTA-T
5/93: 6 micromol P in combination with Hyaluronidase 1000 NFU s.c. |
CR (>6 months) |
M.R., *1941 |
Breast carcinoma
3/90: Lumpectomy
6/92: Recurrence in regional Lymphnodes and Lung |
LTA-T
10/93: 3 micromol P in combination with Hyaluronidase 1000 NFU and Interferon alpha
2b3), 5 mio U, 6 times s.c. |
PR |
F.M., *1913 |
Lung carcinoma
2/91: Adenocarcinoma right upper lobe |
LTA-T
9/92: 2 micromol P in combination with Hyaluronidase 1000 NFU s.c. |
PR (>14 months) |
A.L., *1922 |
Inflammatory breast carcinoma with bone metastasis 2/92 |
LTA-T
7/92: 9 micromol P in combination with Hyaluronidase 1000 NFU s.c. |
Primary tumor: CR 14 months Bone metastasis: PD (died 9/93) |
C.H., *1921 |
Breast carcinoma with lung and mediastinal metastasis |
LTA-T
7/92: 2 micromol P in combination with Hyaluronidase 1000 NFU s.c. |
PR of mediastinal mass |
1) the dosage is calculated on the amount of phosphate of the LTA-T preparation |
2) the hyaluronidase was obtained from CILAG as Permease® |
3) the interferon-alpha 2b was obtained from ESSEX CHEMIE as Intron H®
Abreviations: CR: complete remission; PR: partial remission;*: birth year; PSA: prostate
specific antigen; U USP: units United States Pharmacopoe; P: phosphate content; |
Deposit of Microorganism:
[0079] The microorganism PT designated as
Streptococcus sp PT, used in this invention was deposited under the Budapest Treaty on November
25, 1993, under the number DSM 8747 at the DSM-DEUTSCHE SAMMLUNG VON MIKROORGANISMEN
UND ZELLKULTUREN GmbH, Mascheroder Weg 1b, D-38124 Braunschweig.
1. A purified lipoteichoic acid (LTA-T) isolatable from the Streptococcus sp PT strain DSM 8747 containing a betagalactofuranosyl(1-3)glycerol-di-ester moiety.
2. A lipoteichoic acid LTA-T according to claim 1 of the Formula I

wherein R
1 is hydrogen or D-alanyl with a molar ratio to phosphorus of 0.27 to 0.35, and R
2 are the residues of saturated or unsaturated fatty acids with 12, 14, 16 or 18 carbon
atoms and the mean value for n is 9, and salts thereof.
3. A lipoteichoic acid according to claim 1, being present in form of a physiologically
acceptable salt, such as derived from positively charged ions, such as alkali metal
or alkaline earth metal ions, or positively charged primary, secondary, tertiary or
quaternary ammonium ions, e. g. sodium, potassium, calcium, zinc, ammonium, mono-,
di-, tri- or tetra-lower alkyl-, e. g. methyl- or ethyl-, or methyl-ethyl-, proyl-
or butyl-ammonium ions, in particular sodium ions.
4. A method for the preparation of a lipoteichoic acid LTA-T according to claim 1, characterized in isolating it from Streptococcus sp (DSM 8747).
5. A pharmaceutical preparation comprising a lipoteichoic acid LTA-T according to claim
1 or a physiologically acceptable salt therof in dosage unit form.
6. A pharmaceutical preparation according to claim 5 in combination with an alpha-interferon.
7. A pharmaceutical preparation according to claim 5 in combination with hyaluronidase.
8. A pharmaceutical preparation according to claim 5 in combination with interferon-alpha
and hyaluronidase.
9. A method of producing a pharmaceutical preparation comprising a lipoteichoic acid
according to claim 1 by mixing the LTA-T with a pharmaceutical carrier.
10. A method of producing a pharmaceutical preparation for treating cancer comprising
administration of an antitumor effective amount of a lipoteichoic acid LTA-T or a
physiologically acceptable salt therof according to claim 1 to a patient suffering
from a tumor or a malignant cell thereof.
11. The Streptococcus sp PT strain DSM 8747.
12. Method for the proliferation of Streptococcus sp PT DSM 8747 characterised in growing said bacteria strain under proliferating conditions.
13. A deacylated dLTA-T of the Formula I according to claim 2, wherein R1 and R2 are both hydrogen, n has the given meaning, or a salt thereof.
14. A beta-galactofuranosyl(1-3)glycerol-di-R
2-ester of the Formula II

wherein R
2 is the rest of a saturated or unsaturated fatty acid with 12, 14, 16 or 18 carbon
atoms, a single compound thereof, and salts thereof.
1. Eine gereinigte Lipoteichonsäure(LTA-T), die aus dem Streptococcus sp PT Stamm DSM 8747 isolierbar ist und eine beta-Galactofuranosyl(1-3)glycerol-di-esterstruktur
enthält.
2. Eine Lipoteichonsäure(LTA-T) gemäss Anspruch 1 der Formul I

worin R
1 Wasserstoff oder D-Alanyl ist mit einem molaren Verhältnis zu Phosphor von 0.27 to
0.35, und R
2 die Reste von gesättigten oder ungesättigten Fettsäuren mit 12, 14, 16 or 18 Kohlenstoffatomen
sind und der Mittelwert für n 9 ist, und Salze davon.
3. Eine Lipoteichonsäure gemäss Anspruch 1, in Form eines physiologisch annehmbaren Salzes,
wie abgeleitet von positiv geladenen Ionen, wie Alkalimetall- oder ErdalkaliemetallIonen,
oder positiv geladenen primären, secundären, tertiären oder quaternären Ammonium-Ionen,
beispielsweise Natrium-, Kalium-, Kalzium-, Zink-, Ammonium-, Mono-, Di-, Tri- oder
Tetra-niederalkyl-, beispielsweise Methyl- oder Ethyl-, oder Methyl-ethyl-, Propyl-
oder Butyl-ammonium-Ionen, insbesondere von Natrium-Ionen.
4. Eine Methode zur Herstelung einer Lipoteichonsäure LTA-T gemäss Anspruch 1, dadurch gekennzeichnet, dass sie aus Streptococcus sp (DSM 8747) isoliert wird.
5. Eine pharmazeutische Darreichungsform umfassend eine Lipoteichonsäure LTA-T gemäss
Anspruch 1 oder ein physiologisch annehmbares Salz davon in Form einer Einheitsdosis.
6. Eine pharmazeutische Darreichungsform gemäss Anspruch 5 in Kombination mit einem alpha-Interferon.
7. Eine pharmazeutische Darreichungsform gemäss Anspruch 5 in Kombination mit Hyaluronidase.
8. Eine pharmazeutische Darreichungsform gemäss Anspruch 5 in Kombination mit einem alpha-Interferon
und Hyaluronidase.
9. Eine Methode zur Herstelung einer pharmazeutischen Darreichungsform umfassend eine
Lipoteichonsäure gemäss Anspruch 1 durch Mischen der LTA-T mit einem pharmazeutischen
Träger.
10. Eine Methode zur Herstelung einer pharmazeutischen Darreichungsform zur Behandlung
von Krebs umfasend Verabreichung einer antitumor wirksamen Menge einer Lipoteichonsäure
LTA-T oder eines physiologisch annehmbaren Salzes davon gemäss Anspruch 1 an einen
Patienten, der unter einem Tumor oder einer krankhaften Zelle davon leidet.
11. Der Streptococcus sp PT Stamm DSM 8747.
12. Methode zur Vermehrung von Streptococcus sp PT DSM 8747, dadurch characterisiert, dass man den genannten Bakterienstamm unter
vermehrenden Bedingungen wachsen lässt.
13. Eine deacylierte dLTA-T der Formel I gemäss Anspruch 2, worin R1 und R2 beide Wasserstoff sind, n die angegebene Bedeuting hat, oder ein Salz davon.
14. Ein beta-Galactofuranosyl(1-3)glycerol-di-R
2-ester der Formel II

worin R
2 der Rest einer gesättigten oder ungesättigten Fettsäuren mit 12, 14, 16 or 18 Kohlenstoffatomen
ist, eine Einzelverbindung davon, und Salze davon.
1. Acide lipotéichoique purifié (désigné par LTA-T), isolable à partir de la souche Streptococcus
sp PT (DSM 8747), qui contient un fragment diester de bétagalactofuranosyl(1-3)glycérol.
2. Acide lipotéichoique LTA-T selon la revendication 1, de formule I

dans laquelle R1 représente un atome d'hydrogène ou un groupe D-alanyle, le rapport
molaire des groupes D-alanyle au phosphore étant de 0,27 à 0,35, les groupes R2 représentent
les restes d'acides gras saturés ou insaturés, ayant 12, 14, 16 ou 18 atomes de carbone,
et la valeur moyenne de n est égale à 9, et les sels de cet acide lipotéichoique.
3. Acide lipotéichoique selon la revendication 1 qui est présent sous la forme d'un sel
physiologiquement acceptable, tel qu'un sel avec des ions positivement chargés, comme
les ions des métaux alcalins ou des métaux alcalino-terreux ou les ions ammonium primaires,
secondaires, tertiaires ou quaternaires, positivement chargés, par exemple les ions
sodium, potassium, calcium, zinc, ammonium, et mono-, di-, tri- ou tétra(alkyl inférieur)ammonium,
par exemple les ions méthyl-, éthyl-, méthyl-éthyl-, propyl- ou butyl-ammonium, et
en particulier les ions sodium.
4. Procédé de préparation d'un acide lipotéichoique LTA-T selon la revendication 1, caractérisé en ce que l'on isole cet acide à partir de Streptococcus sp (DSM 8747).
5. Préparation pharmaceutique comprenant un acide lipotéichoique LTA-T selon la revendication
1 ou un sel physiologiquement acceptable de cet acide, sous la forme d'une dose unitaire.
6. Préparation pharmaceutique selon la revendication 5, en combinaison avec un alpha-interféron.
7. Préparation pharmaceutique selon la revendication 5, en combinaison avec de la hyaluronidase.
8. Préparation pharmaceutique selon la revendication 5, en combinaison avec de l'alpha-interféron
et de la hyaluronidase.
9. Procédé de production d'une préparation pharmaceutique comprenant un acide lipotéichoique
selon la revendication 1, par mélange du LTA-T avec un support pharmaceutique.
10. Procédé de production d'une préparation pharmaceutique destinée au traitement du cancer
comprenant l'administration à un patient souffrant d'une tumeur ou de cellules malignes,
d'une quantité efficace du point de vue de l'effet antitumoral, d'un acide lipotéichoique
LTA-T selon la revendication 1 ou d'un sel physiologiquement acceptable de cet acide.
11. La souche Streptococcus sp PT DSM 8747.
12. Procédé de prolifération de Streptococcus sp PT DSM 8747, caractérisé en ce que l'on fait croître ladite souche bactérienne dans des conditions de prolifération.
13. dLTA-T déacylé répondant à la formule I selon la revendication 2, dans laquelle R1
et R2 représentent tous deux des atomes d'hydrogène et n a la signification indiquée,
ou un sel de ce composé.
14. di-R2-ester de béta-galactofuranosyl(1-3)glycérol de formule II

dans laquelle R2 représente le reste d'un acide gras saturé ou insaturé, ayant 12,
14, 16 ou 18 atomes de carbone, composé unique répondant à cette formule, et sels
de ces composés.