Modulation of the physical interaction between platelets and the cell surface effecting cell proliferation

Abstract

The growth and/or proliferation of mammalian cells are modulated by modulating the physical interaction between platelets (thrombocytes) and the surface of the cells. Sulfated polysaccharides, preferably glycosaminoglycans, can be used as a medicament for the inhibition of the physical interaction between the cell surface and platelets in the treatment of a medical disorder associated with unwanted cell growth and/or proliferation. The physical interaction between platelets (thrombocytes) and the surface of the cells can be modulated in vitro in order to modulate cell proliferation. Inhibition of the interaction between the cell surface and platelets can inhibit cell growth, and enhancement of the interaction between platelets and the surface of the cell can enhance cell growth.

Claims

1. A method of treating cancer in a subject in need thereof comprising administering a pharmaceutical composition comprising a soluble sulfated polysaccharide to said subject, wherein said soluble sulfated polysaccharide does not comprise the terminal pentasaccharide of Heparin and has a degree of sulfation of greater than or equal to 1, and wherein proliferation of cancerous cells in the subject is reduced by said soluble sulfated polysaccharide inhibiting a physical interaction between cancer cell surfaces and platelets.

2. The method according to claim 1, wherein said subject is a human.

3. The method according to claim 1, wherein the degree of sulfation of said polysaccharide is >1.2.

4. The method according to claim 1, wherein the degree of sulfation of said polysaccharide is >1.4.

5. The method according to claim 1, wherein the soluble sulfated polysaccharide exhibits an average molecular weight of about 5000 to about 12000 daltons.

6. The method according to claim 1, wherein the soluble sulfated polysaccharide is pentosan polysulfate (PPS).

7. The method according to claim 1, wherein the soluble sulfated polysaccharide is dextran sulfate (DXS).

8. The method according to claim 1, wherein said soluble sulfated polysaccharide is a sulfated alginate.

9. The method according to claim 1, wherein said soluble sulfated polysaccharide is a sulfated fucoidan.

10. The method according to claim 1, wherein said soluble sulfated polysaccharide is locally administered in proximity to a tumor.

Description

FIGURES

(1) The invention is further described by the figures. These are not intended to limit the scope of the invention.

(2) FIG. 1. Selective binding of thrombocytes to the surfaces of growing HeLa cells. (A) Culture 2 days after passage. Platelets shown as dark spots. (B) Enlarged view demonstrates cell surface interaction.

(3) FIG. 2. HeLa growth experiment. Cell division and growth are still found after seven days in serum-free culture medium when the cells are co-incubated once a day for 30 minutes with platelets and are subsequently washed and further cultured with serum-free culture medium. Unstained, living cells, nearly confluent after 7 days in culture are shown. Arrows: metaphase of mitosis. Adherent platelets shown as light spots.

(4) FIG. 3. Growing cells bind platelets. Here human primary skin fibroblasts in the first passage are shown. Platelets are shown as light spots.

(5) FIG. 4. Aging culture of skin fibroblasts. Poor platelet binding is shown. Platelets are shown as dark spots.

(6) FIG. 5. Untreated HeLa cells, clone S3. Platelets are shown as dark spots.

(7) FIG. 6. HeLa cells, clone S3, treated with 1 U/mL Enoxaparin. Platelets are shown as dark spots.

(8) FIG. 7. HeLa cells, clone S3, treated with 1 U/mL Dalteparin. Platelets are shown as dark spots.

(9) FIG. 8. HeLa cells, clone S3, treated with 1 U/mL Tinzaparin. Platelets are shown as dark spots.

(10) FIG. 9. LMW Heparins inhibit platelets from binding to growing cells. Quantification of microscopic analysis. The 0-value relates to cells with no addition of thrombocytes to the cell culture. The positive control shows platelet binding without addition of LMW Heparins. Error bars relate to standard deviation over experiments performed in triplicate.

(11) FIG. 10. HeLa cells, clone S3, treated with 0.1 ug/mL PPS. Platelets are shown as dark spots.

(12) FIG. 11. Inhibition of platelet binding to growing cells by the sulfated GAGs PPS and DXS. Quantification of microscopic analysis. Error bars relate to standard deviation over experiments performed in triplicate. Inhibition of platelet binding to growing cells by the sulfated GAG's PPS and DXS amounts to roughly 75% by 0.1 ppm of PPS or DXS and to more than 95% by 1 ppm.

(13) FIG. 12. Comparison between highly and lowly sulphated GAGs on platelet binding to growing cells. Danaparoid (Orgaran) (squares) exhibits an inhibitory effect on the interaction between platelets and the cell surface, although to a lower extent than the highly sulphated PPS (diamonds).

EXAMPLES

(14) The invention is further described by the following examples. These are not intended to limit the scope of the invention. The experimental examples relate to qualitative and quantitative microscopic analysis of various cell types and the adherence of platelets to the surface of said cells. Various Glycosaminoglycans and their effect on platelet binding have been assessed.

(15) The methods used in the following examples are described below. The process outlined herein for the determination of platelet interaction with cells is suitable for the examination of whether, and in which concentration, a test substance is able to prevent the platelets binding to the surface of a growing cell. Furthermore, the method can be used as a bioassay for determining the concentration of substances, which can inhibit platelet binding to cells.

Summary of the Examples

(16) Example 1: Microscopic analysis of HeLa cells and skin fibroblasts with respect to platelet binding to the surface of cells (FIGS. 1 to 4).

(17) Example 2: Analysis of low molecular weight (LMW) heparins with respect to inhibition of platelet binding to the surface of growing cells (FIGS. 5 to 9)

(18) Example 3: Analysis of fondaparinux and danaparoid with respect to inhibition of platelet binding to the surface of growing cells.

(19) Example 4: Analysis of sulfated GAGs pentosan polysulfate (PPS) and dextranpolysulfate (DXS) with respect to inhibition of platelet binding to the surface of growing cells (FIGS. 10 and 11).

(20) Example 5: Comparative analysis of GAGs on platelet binding between highly and lowly sulfated GAGs (FIG. 12).

Detailed Description of the Examples

Example 1

(21) As shown in FIG. 1, it was confirmed that HeLa cells have a specific binding ability for platelets. The binding of platelets to HeLa cells was 20 to 100 times higher than that on the surface of the cell culture plates. Platelets were prepared as described below and applied to cells in culture as described.

(22) FIG. 2 demonstrates that serum free medium may be used for HeLa culture if platelets are provided to the cells for 30 minutes daily. The short duration of daily platelet contact to the cells leads to slightly reduced proliferation. This experiment shows that platelets are able to sustain proliferation of cells, substituting the serum supplement otherwise needed in vitro.

(23) FIGS. 3 and 4 demonstrate that skin fibroblasts bind platelets on their surface dependent on their growth phase. Fibroblasts that are proliferating in culture bind significantly more platelets than those in older cultures. Primary human skin fibroblasts NHDF bind platelets in the first logarithmic growth phase, similar to the examined HeLa cells, but to a somewhat lower extent than in HeLa cells. The concomitant decreased proliferation with decreased platelet binding of NHDF shows that platelets are also of importance in the proliferation of normal primary cells. The higher percentage of cells in the G0 phase is associated with down-regulation of the number of platelet receptors. This indicates that the platelet itself can initiate the transduction of proliferative stimulus. This can be a crucial phenomenon in malignant transformation. This could be similar to some cells entering the cell cycle by inducing platelet receptors, which are not always present. Platelet binding therefore leads to proliferation-inducing signal transduction.

Example 2

(24) It could also be shown that enoxaparin, dalteparin and tinzaparin, corresponding to 0.1 U/mL, almost completely inhibited the binding of platelets to the HeLa cells (FIGS. 5 to 9). Even in a concentration of 0.01 U/mL a significant reduction of platelet binding to the cells took place.

Example 3

(25) However, fondaparinux, a synthetic pentasaccharide corresponding to the five monomeric sugar units that can be isolated after either chemical or enzymatic cleavage of the polymeric glycosaminoglycan heparin, was unable to significantly inhibit platelet binding to HeLa cells. Furthermore, treatment with danaparoid, an anticoagulant chemically distinct from heparin (also known as Orgaran), showed a significantly lower effect on inhibiting the interaction between platelets and the cell surface.

(26) When danaparoid is applied at 1.25 U/mL, which is about 5 times the upper limit of the therapeutic range, no significant difference is found between the control measurements (15.9+/7.7 platelets/cell) and the danaparoid treatment (13.2 platelets/cell).

(27) Heparins, and some other anticoagulants, have the disadvantage that they have an overly strong anticoagulant effect and can cause serious bleeding complications, which are mainly attributed to the aforementioned inhibition of coagulation factor Xa. The most important factor in the anticoagulant effect of heparin is a sulfated pentasaccharide, which inactivates the coagulation factor Xa. However, the aforementioned pentasaccharide does not inhibit platelet binding to growing cells.

(28) Interestingly, danaparoid exhibits a lower degree of sulfation (typically 0.4 to 0.6) in comparison to Heparin (typically 1 to 2). Also significantly less effective as inhibitors of platelet binding were other low sulfated chondroitin sulfate GAGs, such as dermatan sulfate and heparan sulfate.

Example 4

(29) Pentosan polysulfate (PPS) is a vegetable product and is manufactured as the sodium salt or calcium salt. PPS typically has less than 1/10 of the anticoagulant activity of heparin and has been used for the treatment of interstitial cystitis and used in veterinary medicine for joint disease, in particular for forms of arthritis. Dextran polysulfate (DXS) is also used to treat joint pain, usually in combination with other substances.

(30) As shown in FIGS. 10 and 11, both PPS and DXS exhibit a reproducible inhibition of the physical interaction between platelets and the surface of proliferating cells.

Example 5

(31) As demonstrated in FIG. 12, treatment of the cell cultures with danaparoid in direct comparison to PPS shows the distinct difference between these two molecules with respect to their disruption of platelet binding to the surfaces of cells.

(32) Methods:

(33) The process for evaluating the platelet binding of growing cells involves the isolation of platelets from peripheral blood with two subsequent washes and cell sedimentation. These washes include a partial activation of the platelets, which is relevant for their interaction with Heparin. The incubation with the cells under investigation takes place under conditions in which full activation of platelets is approached, which would lead to lumps forming in the platelets. This would hinder analysis. Therefore, a low concentration of EDTA is added to the assay. The quantification of platelets and the calculation of the surface portions of cells and background is carried out using appropriate software, namely by macros in the open source program ImageJ.

(34) Platelet Isolation Procedure:

(35) Various methods are available for platelet isolation. The following method is preferred.

(36) Experimentation by the inventor has shown that the activation of platelets, which ultimately leads to a viscous metamorphosis of the platelets as a result, can take place in two stages. The first stage involves no significant morphological change in the platelets, but does lead to a slight change in the appearance of their outer surface. These platelets are referred to as partially activated platelets. This change is important for the adhesion test as described herein, in order to optimize the ability to bind receptors on the cell surface.

(37) First centrifuge blood samples for 7 minutes at 190G in a centrifuge without a brake. This separates plasma and erythrocytes. During centrifugation a 10 ml tube with 1 ml of PBS-EDTA is prepared. The platelet-rich plasma is pipetted into the prepared tubes. An EDTA-PBS refill is carried out if necessary. The final mixture should be 1:1 EDTA-PBS and plasma.

(38) The platelet-containing plasma is centrifuged to separate the platelets from the plasma. The centrifugation is carried out at 265-275G. The duration depends on the size of the tube used (for example 16 mm diameter100 mm length; centrifugation for 15 minutes at 270G).

(39) The resulting sediment is quite loose. The supernatant is discarded. The platelet sediment, with very little buffer mixture, is allowed to stand for 3 minutes and is loosened by gentle shaking. The platelets are resuspended in the following buffer mixture:

(40) 2 mL of 1:1 v/v Hank's Balanced Salt Solution (HBSS) with Ca and Mg, and a buffer solution without Ca and Mg. The latter buffer solution can either be HBSS without Ca and Mg or for example Dulbecco's Phosphate Buffered Saline Solution (PBS), which has been produced without Ca and Mg. The aforementioned buffer mixture is treated with 0.02% EDTA.

(41) The viscous metamorphosis of platelets is dependent on Ca and Mg. The tendency of the platelets to undergo this change should be reduced for the trial. However, when maintained in tissue culture, cells cannot tolerate a one-hour incubation in a completely Ca and Mg-free environment. The aforementioned mixture is sufficient to buffer a conditioning of platelets and contains at the same time sufficient Ca and Mg for metabolic function of the cells in culture.

(42) The platelet suspension has not yet obtained the optimal properties for the adhesion test. They are therefore conditioned by centrifugation for 15 minutes at 265-275 x G. The buffer is discarded and the now noticeably firmer sediment is mixed with 2 mL buffer mixture and allowed to stand for 3 minutes, then loosened by gentle shaking. The centrifugation is then repeated and the platelet count is determined.

(43) Adhesion Experiment (Platelet Binding to Cell Surface):

(44) Virus and mycoplasma free HeLa cells, clone S3, and human skin fibroblasts (NHDF) were purchased from Promo Cell, Heidelberg. Their human origin was confirmed by STR-Analysis. HeLa cells were maintained in Eagles MEM with 10% fetal calf serum. The NHDF were also maintained with 10% fetal calf serum in RPMI 1640. The passaging was performed by trypsin-EDTA treatment. The cell lines were maintained in either 25 ml bottles or in 3 cm plates.

(45) The cells used for the experiment are cultured for 2 or 3 days in standard plates. At the start of the experiment about of the plate surface should be covered with cells. The culture plates are subsequently washed with the buffer mixture described above and filled with further buffer mixture. For example, 30 mm culture dishes are filled with 2 ml 37 C. pre-warmed buffer mixture and 310.sup.7 platelets obtained from the above-mentioned suspension.

(46) The platelet-treated plates are incubated at 37 C. for 60 minutes. In this incubation, the pretreated platelets adhere to proliferating cells. However, cells of G0-phase bind little or none of the pre-treated platelets. This stage of incubation is when the substance to be tested is added to the cell culture buffer, in order to detect whether an effect on platelet binding is observed.

(47) After the one hour incubation, the plates are washed at room temperature with the above mentioned buffer mixture until no free water floating platelets (generally 2 to 4 washes) are present and immediately fixed with glutaraldehyde (1% v/v in H2O).

(48) The evaluation is carried out photographically. For this purpose, the image analysis program ImageJ is applied. The number of cell-bound platelets in relation to the recorded cell number of cells is determined. Additionally, the number of cell-bound platelets in relation to the cell-covered area may be determined.

(49) Photographic Documentation:

(50) Images are produced after the fixation in triplicate per plate. Phase contrast or brightfield 25, 16 or 10 objectives are used. Image analysis is carried out according to SOP IJ. The open source program ImageJ may be used to for differential image subtraction and mask optimization. Subsequently, platelet number, cell number and proportional cell surface are determined by other IJ components.

(51) Cell Culture Experiments for Testing Inhibition of Cell Proliferation:

(52) Additional experimentation shows that administration of sulfated polysaccharides, in particular glycosaminoglycans, leads directly to inhibition of cell proliferation under conditions that mimic the in vivo requirements for cell growth that is dependent on platelet-cell surface interactions. In order to interrogate the effectiveness of PPS and DXS with respect to the inhibition of cell growth, the following experiment may be carried out.

(53) Experiment duration: 5 days; Culture medium: Eagle's MEM without serum addition.

(54) Test approach: PPS or DXS in 1.0, 0.1 g/mL, and control (0 g/mL final concentration).

(55) HeLa cells are cultured from stocks: 10.sup.4 cells/25 cm.sup.2 flask, cell numbers are counted in a Fuchs-Rosenthal chamber. Platelets are prepared as described above.

(56) Platelets and HeLa cells, or other tumor cell lines, either with or without inhibitor, are co-incubated for 30 minutes once every day, for 1 to 4 days. After co-incubation the cells are washed as described above and then allowed to rest under standard culture conditions. Typically 9010.sup.6 platelets are incubated per well per day.

(57) On day 5, cells are washed with HBSS, photographed, trypsinized and the cell number counted as described above. This experimental approach shows that the number of cells, which typically increases via proliferation due to the daily 30 minute incubation with thrombocytes, increases to a reduced extent when either DXS or PPS is co-incubated with the platelets during the 30 minute incubation.