Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-01-15
2001-04-10
Lee, Howard C. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C514S025000, C536S017100, C536S116000, C536S117000, C536S120000
Reexamination Certificate
active
06214978
ABSTRACT:
FIELD OF THE INVENTION
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
Lipoteichoic acids (LTAs) are a group of amphipathic substances found in the cell wall of gram-positive bacterial extending from the outer cell membrane through the cell wall to the surface. The main group of LTAs consists of a hydrophilic 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).
LTAs have been reported as having antitumor activity (EP 135 820; U.S. Pat. No. 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).
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, U.S. Pat. No. 4,678,773, H. Usami et. al. 1985).
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.
The LTAs described up to now carried more than one monosaccharide in the glycceroglycolipid anchor. Different glycolipid structures have been described by Fischer et al. 1988 and 1990.
An LTA with a monohexosylidiacylglyceroglycolipid as lipid anchor has not been described so far.
OBJECT OF THE INVENTION
It is an object of the invention to provide a purified new LTA with a strong antitumor activity.
It is a further object to provide pharmaceutical preparations comprising this new LTA, optionally in combination with a monokine and/or hyaluronidase.
It is a further object to provide a method of 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.
It is a further object to provide a method of lowering the blood cholesterol level in a human patient comprising administering a cholesterol lowering amount of LTA to such human patient.
It is a further object to provide a method of producing the new LTA and the new pharmaceutical preparation.
It is a further object to provide two degradation products of the new LTA and their use.
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
The invention concerns a new purified lipoteichoic acid (LTA) isolatable from the new Streptococcus sp strain DSM 8747.
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 3. This microheterogeneity is a typical feature of lipid macroamphilphiles [Fischer W. (1993), Molecular analysis of lipid macroamphilphiles 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.
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 phorphorous 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.
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.
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%.
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.
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, proyl- or butyl- ammonium salts. Non-physiologically acceptable salts, such as heavy metal 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.
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.
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.
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. B
Rothlisberger Peter
Truog Peter
Lee Howard C.
Lunamed AG
Mathews, Collins Shepherd & Gould P.A.
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