Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1999-12-20
2001-12-11
Fonda, Kathleen Kebler (Department: 1623)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S021000
Reexamination Certificate
active
06329351
ABSTRACT:
The object of the present invention is the use of the sulphaminoheparosansulphates as anti-metastatic agents.
The metastasis is a process consisting of the detachment of cancer cells from the site of the primary cancer, the dissemination in the blood flow, the adhesion to the vascular walls, and the migration and growth in extra-vascular spaces. Said phenomena, and in particular the adhesion to the vascular walls, seem to be regulated by the endogenous heparan sulfate (HS) polysaccharide. Some anticoagulant drugs, among which the heparin (HEP), which shows structural analogies with the heparan sulfate, have been tested as potential anti-metastatic agents. (I. Vlodavsky et al.: “Modulation of neovascularization and metastasis by species of heparin”, in: “Heparin and Related Polysaccharides” (D. A. Lane et al., Eds.), Plenum Press, New York 1992, 317-327). The heparin among said drugs is particularly active as anti-metastatic, but its high anticoagulant activity implies haemorrhagic risks, whereby the search for heparin-like substances having reduced anticoagulant activity is particularly interesting. (D. J. Tyrrell et al.: “Therapeutic uses of heparin beyond its traditional role as an anticoagulant”, TIPS 16, 198-204, 1995).
With the present invention we have found that some semi-synthetic heparan sulfates belonging to the sulphaminoheparosansulphates (SAHS) class (B. Casu et al.,: “Heparin-like compounds prepared by chemical modification of capsular polysaccharide from
E. coli
K5”, Carbohydr. Res. 263, 271-284 (1994)), surprisingly carry on an “in vivo” anti-metastatic activity comparable to the heparin one, even if having an “in vivo” anticoagulant activity an order of magnitude lower than the heparin one.
More particularly, we have found that only the SAHS obtainable from the
Escherichia coli
K5 polysaccharide by deacetilation and subsequent sulfation with the sulphuric anhydride/trimethylamine adduct carried out at 0° C., for times ranging from 0.25 to 2 hours and using a reactant/polysaccharide ratio (SO
3
equivalents/available OH groups equivalents) equal to 5 (named SAHS-B), having molecular weight ranging from 5,000 to 40,000, show anti-metastatic activity comparable to a typical heparin one, while SAHS prepared according to other experimental conditions have anti-metastatic activity notably lower either than the heparin one or than the SAHS-B one.
Moreover we have also found that fractions of SAHS-B having a molecular weight lower than 5,000 keep a significant anti-metastatic activity (also greater than the one of the corresponding heparins having low molecular weight).
Therefore the semi-synthetic SAHS-B heparosansulfates look as anti-metastatic drugs having a reduced haemorrhagic risk.
For the purpose, the SAHS-B will be formulated in suitable pharmaceutical compositions, using conventional techniques and excipients. Such compositions may be administered for the prevention or the therapy of metastases in doses which will obviously depend from several factors but which will be generally ranging from 1 to 1,000 mg of SAHS-B one or more times a day.
The SAHS have been obtained as previously described (B. Casu et al., 1994, loc. cit.; PCT/EP94/01660) from the K5 polysaccharide, which is a constituent of the cell membrane of the Escherichia coli K5 strain. In particular, the K5 polysaccharide has been selectively N-deacetilated and N-sulfated, and then O-sulfated as summarily described in the following scheme, obtaining the SAHS of different kind SAHS-B, SAHS-C, SAHS-A.
Scheme
The first step consists of the N-deacetilation by hydrazinelysis of the K5 polysaccharide (K5-PS). The obtained product (heparosan, AH) is N-sulfated with the sulphuric anhydride/trimethylamine adduct (TMA/SO
3
), with the achievement of the sulphaminoheparosan (SAH). The numbers near the arrows show in order the reaction temperatures (° C.), the reactant/polysaccharide ratios (SO
3
equivalents/equivalents of available hydroxyl groups), and the reaction times (hours).
The anti-metastatic activity of the SAHS, heparin and other reference sulfated polysaccharides has been tested using the method of the colonization to the lung of B16B16 melanoma cells. (N. Caselia et al., Thromb. Haem. 73,964 (1995)). Such a method, lending itself particularly to test the effect of the drugs with inhibitory activity on the cancer haematic dissemination, consists of the evaluation of the number of the cancer colonies which form in the lung after the injection of murine melanoma cells by intravenous way in the mouse. B16B16 melanoma cells have been used. The cells have been cultured in DME with 10% of fetal bovine serum in a CO
2
(5%) incubator in humidity conditions and at 37° C. The cells have been divided two times a week, treating them with 0.25% trypsin/0.05% EDTA. The polysaccharides to test have been dissolved in physiological solution or in phosphate (PBS) buffer, at the proper dilution, and used on the spot. B16B16 melanoma cells, diluted in PBS (10
5
cells/0.1 ml/mouse) have been injected into a side vein of the tail of C57B16 mice having an average weight equal to 20 g, in a final volume equal to 0.2 ml/mouse. The mice have been sacrificed 12-16 days after the injection of the cancer cells; the lungs have been taken and fixed in a Buoin solution for the count of the superficial metastatic nodules, which are pointed out as black masses on a yellow ground. Then the ratio between the number of lung nodules in the treated mice and in the control ones has been estimated. Each experiment has been carried out on a minimum number of five mice, more frequently on 8-10 mice. The inhibition percentages of the metastases discovered in several experiments have been reported in the individual Examples and in Table 1.
REFERENCES:
patent: 5550116 (1996-08-01), Lormeau et al.
patent: 9429352 (1994-12-01), None
TIPS, Jun. 1995 (vol. 16) 199-203.
Heparin and Related Polysaccharides, Lane et al. (1992) 317-327.
Carbohydrate Research 263 (1994) 271-284.
Naggi Annamaria
Torri Giangiacomo
Fonda Kathleen Kebler
Hedman & Costigan ,P.C.
Istituto Scientifico Di Chimica E Biochimica “G. Ronzoni”
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