WO2003097068A1

WO2003097068A1 - Use of peroxovanadium compounds as tumour cell growth suppressor

Info

Publication number: WO2003097068A1
Application number: PCT/CA2003/000738
Authority: WO
Inventors: Robert Faure; Pierre Savard; Charles Doillon
Original assignee: Universite Laval
Priority date: 2002-05-17
Filing date: 2003-05-15
Publication date: 2003-11-27
Also published as: AU2003233285A1; CA2386759A1; CA2525672A1; EP1511493A1

Abstract

The present invention relates to the use of peroxovanadium compounds as antitumorigenic agents. These compounds have been found to be suitable, at low dosages, as antitumorigenic agents in the treatment of cancer, such as breast cancer and prostate cancer. It has been found that even at low dosages, these compounds can prevent or arrest further tumour growth.

Description

TITLE OF THE INVENTION

Use of Peroxovanadium Compounds as Tumour Cell Growth Suppressor

FIELD OF THE INVENTION

This invention relates to the use of low dosage levels of peroxovanadium compounds, such as potassium bisperoxo(1,10-phenanthroline)oxovanadate [bpV(phen)], potassium bisperoxo(pyridine-2-carboxylato)oxovanadate [bpV(pic)] and potassium bisperoxo(2,2'-bipyridyl) oxovanadate [bpV(bipy)], for preventing and suppressing tumour growth in a mammal.

BACKGROUND OF THE INVENTION

Synthetic peroxovanadium (pV) compounds are structurally versatile molecules which are potent inhibitors of phosphotyrosyl phosphatases (PTPs) (1). These compounds contain one oxo Iigand, one or two peroxo groups, one ancillary Iigand, all coordinated to vanadium. They are stable in aqueous solution at physiological pH when shielded from light. Their mode of action lies in the modulation of the activity of cellular transduction pathways involved in the progression of pathological conditions. Their effects are transitory and disappear within a few days after administration(2),

Phosphotyrosine phosphatases (PTPs) are enzymes which remove phosphates from tyrosine residues of proteins. They are involved in several cell functions regulating proliferation, differentiation and metabolism. Their number is estimated at about 100 in the human genome (3). These enzymes function by engulfing in their catalytic site phosphates located on the tyrosine residues of target proteins. The mechanisms underlying the inhibition of PTPs and the specificity of peroxo-anionic compounds towards inhibition of PTPs have been characterized. The inhibiting potential of PTPs by pVs is a 100 to a 1000 times more powerful than that of oxovanadate (1). When compared to known inhibitors of PTPs such as orthovanadate, molybdate, tungstate and zinc, the increased inhibiting potential of pV can be explained by the presence of the peroxide groups, which have the ability to irreversibly oxidize an essential conserved cysteine residue located in the catalytic domain of practically all PTPs (4).

The possibility of manipulating the ancillary ligands of pVs is important in regulating potency and specificity (1). The ancillary ligands are more or less hydrophilic or hydrophobic and provide the molecule with a specific mode of action and distribution for the different PTPs. These ligands allow for the specific targeting of certain PTPs.

International Patent Publication WO 95/19177 teaches the use of vanadate compounds for the treatment of proliferative disorders, metastasis and drug- resistant tumours. This publication does not describe peroxovanadate compounds. The publication further shows that an anti-tumour effect is observed at dosages of vanadate higher than 5 mM. It is admitted that a concentration of vanadate compound of 1.3 mM or lower has no apparent anti- tumour effect.

Montesano et al. (5) teach, on the contrary, that vanadate compounds cause endothelial cells to proliferateot anti-angiogenic.

US Patent 5,716,981 (Hunter et al.) mentions the use of vanadium compounds, namely oxovanadate, orthovanadate and vanadyl compounds, in anti-angiogenic applications.

International Patent Publication WO 01/12180 teaches the use of peroxovanadium compounds for preventing angio-genesis, restenosis and the production of endothelins in mammals. However, the publication teaches away from the disclosure that peroxovanadium compounds are effective in preventing or arresting the growth of tumour cells. International Patent Publication WO 00/57860 teaches the use of peroxovanadium compounds as antineoplastic agents for the treatment of cancer. However, this publication teaches that the peroxovanadium compounds are cytotoxic to several human tumour types and cause cell death through apoptosis. However, the main drawback of such use of peroxovanadium compounds is that they must be administered in large quantities of 15-20 mg/kg of body weight per day, or more. This is shown in Figure 8 of the publication. In such large quantities, the side-effect of the administration of the drugs are deleterious. Indeed, transition metals such as vanadium are known to be toxic (2).

In contrast, the use described in the present invention is aimed at obtaining a cytostatic effect meaning that tumour cells remain alive but stop dividing so as to prevent or arrest the growth of an invading tumour.

SUMMARY OF THE INVENTION

Surprisingly, it has been found that peroxovanadium compounds can obtain a cytostatic effect at dosage levels much lower than those proposed in International Patent Publication WO 00/57860 thereby minimising side-effects of the drug while improving the therapeutic outcomes of patients.

Thus, the present invention provides the use of peroxovanadium compounds, at low dosages, as antitumorigenic agents, for example in the treatment of cancer, such as breast cancer and prostate cancer. It has been found that a low dosages, these compounds can prevent or arrest further tumour growth.

Preferably the dosage levels will be 0.001 to less than 15 per mg of body weight per day and most preferably 1 to 10 mg/kg of body weight/day. In serum concentration, the dosage levels are preferably between 1 and 50 μM.

Preferably, the molecules also contain an ancillary Iigand, which includes any molecule capable of binding the transition metal atom (usually, through bonds involving oxygen and nitrogen). Phenanthroline, picolinic acid, bipyridine, oxalic acid, 4,7-dimethyl-phenanthroline and peptides are examples of such ligands.

Peroxo vanadate complexes include complexes such as the following: methavanadate (VO3^"), orthovanadate (VO4³"), salts thereof, vanadyl compounds (VO²⁺) like vanadyl acetyl acetonate and vanadyl sulfate. Most preferred peroxides comprise the following: t-butylhydroperoxide, benzoyl peroxide, m-chloroperoxibenzoic acid, cumene hydroperoxide, peracetic acid, hydroperoxiloneic acid, ethyl peroxide, pyridine peroxide and hydrogen peroxide.

The general structure of the compounds of the present invention is the following:

wherein:

Y is oxygen or hydroxyl;

Z and Z' are independently selected from oxygen and peroxide and at least one of them is peroxide; and

L and L' are any group which can donate an electron pair.

In a preferred embodiment, Y is oxygen, Z and Z' are peroxide and L and L' are the nitrogen atoms of 1 ,10-phenanthroline.

In another preferred embodiment, Y is oxygen, Z and Z' are peroxide and L and L' are nitrogen or oxygen atoms of picolinic acid. In yet another preferred embodiment, Y is oxygen, Z and Σ are peroxide and L and L' are nitrogen atoms of 2,2'-bipyridine.

In one specific embodiment, the present invention relates to the inhibiting action on tumour growth of bpV(phen), demonstrating efficiency in vivo. In addition it is shown that bpV(phen) has also the capacity to inhibit the migration of tumour cells in vitro.

Other objects, advantages and features of the present invention will become apparent upon reading the following non-restrictive description of preferred embodiments thereof, with references to the accompanying drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE PRESENT INVENTION

This invention will now be described by referring to specific embodiments and the appended Figures, which purpose is to illustrate the invention rather than to limit its scope.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Peroxovanadium compounds inhibit the proliferation of endothelial cells. Human umbilical vein endothelial cells (HUVECs) were extracted with collagenase-controlled digestion. Pure HUVECs were used before the fourth passage (trypsin-EDTA at each passage). The cells were analyzed for their capacity to incorporate di-acetyl LDL and to be labelled with factor Vlll-related antigen. Endothelial cells were plated at a density of 2500 cells/cm2 in a sterile plate coated with gelatin. Cells were cultured in complete medium (M199: heparin (90mg/ml) or L-glutamine (2mM), bicarbonate, FBS (20%) and ECGS (100mg/ml) for 24 hours to ensure cell adhesion. Then, cells were washed 3 times with PBS and culture medium was added according to experimental conditions. The last PBS wash was considered as time t=0.. Cell proliferation was evaluated with the amount of DNA present in the petri dishes. Each experiment was performed in triplicate. The culture medium was changed daily. After 96 hours in culture, cells were lysed with Na-Citrate-SDS solution and incubated with Hoescht 33358. Samples were read at 365 nm with a spectrofluorometer. The results show a dose-response inhibition of endothelial cell proliferation with the pV compounds. The approximate IC₅₀ is 2mM for bpV(phen) and 3.5 mM for bpV(pic).

Figure 2: Illustration of the antitumour activity of bpV(phen) in vitro using PC3 prostate cancer cells. Collagen gels containing the PC3 cells were prepared according to the method of Esdale and Bard (1972) (reference 10). Briefly, stock collagen solution (3.5mg/ml in acetic acid 0.02N; Rat tail) was added to a mixture composed of culture medium (5x), fetal bovine serum (FBS), bicarbonate (0.26M), and was neutralized with 0.1 N NaOH. A cell suspension (1.42 x 10⁶ cells/ml) was mixed into the collagen-medium mixture to obtain a final concentration of 2.5x10⁵ cells/ml.

A DMEM medium, having a normal concentration of glucose and without phenol red, was used. After gelification (within 1h) of the collagen mixture containing the cells, the gels were removed from their culture wells (mould) and interwoven into a receptor hole prepared in a fibrin gel, as previously described (6). The fibrin gel was made from a 0.3 % fibrinogen solution in Hank's balanced salt solution. Fibrin was allowed to polymerize with thrombin (stock solution at 1.75mg/ml) at a ratio of 1 :003 v/v fibrin to thrombin. The collagen-fibrin complexes were then covered with serum-supplemented medium according to the cell types. An inhibitor of plasminogen activator (Trasylol, Parke Davies) was added into the medium at 10 μl/ml (100U/ml). Cell behavior was periodically monitored over 15 days of culture.

In this model, collagen is believed to mimic the tumour stroma, and fibrin is well recognized as the primary matrix for cancer cell expansion and migration (7). bpV(phen) was used at 2, 5 and 10 μM; the compound was renewed daily over a 2 week period. Phase contrast microscopic observations and micrographs were taken after 15 days, and samples were prepared for histological sections. Histological sections were stained with periodic acid Shiff stain to enhance the matrix contrast.

Figure 3: Illustration of the antitumour activity of bpV(phen) in vitro using ZR-75 breast cancer cells. Experiments were done as described for the figure 2 except that DMEM medium having a high glucose concentration and without phenol red was used. 10% FBS was used instead of 5% FBS. The media was supplemented with 10^"9 M estradiol (final concentration).

Figure 4: figures 4A and 4B, illustrate the antitumour activity of bpV(phen) in vivo.

Figure 5: In vitro antitumour activity of lymphocytes pretreated with bpV(phen). PC-3 cancer cells embedded in a collagen gel, grew as a "primary tumour". Some cells migrated from the primary tumour toward the fibrin gel, forming front edges that can be quantified. Small clumps of cells progressively appear in the fibrin gel and we assume that these cell extension are representative of the invasive potential of the cancer cells. In the control panel (left), PC-3 cells migrated slightly from the primary tumour and formed extensive secondary tumours in the fibrin gel. In the presence of bpV(phen)-treated lymphocytes no secondary tumour was observed and there was no migration front. In addition, the primary tumours appeared less dense than in the control.

Definitions

In order to provide a clear and consistent understanding of terms used in the present description, a number of definitions are provided hereinbelow.

Unless defined otherwise, the scientific and technological terms and nomenclature used herein have the same meaning as commonly understood by a person of ordinary skill to which this invention pertains. For the purposes of the present application, the term "animal" is meant to signify human beings, primates, domestic animals (such as horses, cows, pigs, goats, sheep, cats, dogs, guinea pigs, mice, birds, fish etc.).

From the specification and appended claims, the term "therapeutic agent" should be taken in a broad sense so as to also include a combination of at least two such therapeutic agents.

For administration to humans, the prescribing medical professional will ultimately determine the appropriate form and dosage for a given patient, and this can be expected to vary according to the chosen therapeutic regimen, the response and condition of the patient as well as the severity of the disease.

Compositions within the scope of the present invention should contain the active agent (e.g. compound) in an amount effective to achieve the desired therapeutic effect while avoiding adverse side effects. Typically, the compounds of the present invention can be administered to mammals (e.g. humans) in doses ranging from 0.001 to less than 15 per kg of body weight per day of the mammal which is treated and most preferably 1 to 10 mg/kg body weight/day. Pharmaceutically acceptable preparations and salts of the active agent are within the scope of the present invention and are well known in the art

(Remington's Pharmaceutical Science, 16th Ed., Mack Ed.). The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient. Typically, 0.001 to less than 15 mg/kg/day will be administered to the mammal.

A) PEROXOVANADIUM COMPOUNDS AND THE INHIBITION OF TUMOUR GROWTH

Methods and cells

ZR-75: hormone-dependent cancer (ductal carcinoma) with oestrogen receptors (ATCC, USA is depository) PC-3: adenocarcinoma (grade IV) with bone metastasis (ATCC, USA is depository).

ZR-75-1 human breast cancer: ZR-75-1 human breast cancer cells obtained from the American Tissue Culture Collection (Rockville, MD) were cultured in phenol red-free RPMI 1640. The cells were supplemented with 2mM L- glutamine, 1 mM Na-pyruvate, 100 IU penicillin/ml, 100μg streptomycin/ml, and 10% (v/v) fetal bovine serum and incubated under a humidified atmosphere comprised of 95% air and 5% CO₂ at 37°C.

Female homozygous HSD nu/nu athymic mice (50 days old) were obtained from Harlan Sprague Dawley Inc. (Indianapolis, IN). Five mice were housed per vinyl cage, which was equipped with air filter lids and kept in laminar air flow hoods under pathogen-limiting conditions. The photoperiod was composed of a period of 14h of light and a period of 10h of darkness. Cages, bedding, and food (Agway Pro-Lab R-M-H diet #4018) were autoclaved prior to use. Water was acidified to pH of 2.8, autoclaved, and provided ad libitum. Bilateral ovariectomy (OVX) was performed on all animals one week prior to cell inoculation, under 2.5% isoflurane anesthesia mixed with oxygen. Simultaneously, an oestrogen (E₂) implant was inserted subcutaneously to stimulate initial tumour growth and appearance. E₂ implants were prepared in 1-cm long silastic tubing (inside diameter, 0.062 inch; outside diameter, 0.095 inch) containing 0.5 cm of estradiol/cholesterol diluted at a ratio of 1 :10 (w:w). One week after the ovariectomy, 2.0 x 10⁶ ZR-75-1 cells, in their logarithmic growth phase, were harvested with 0.083% pancreatin/0.3 mM EDTA and inoculated s.c. in 0.1 ml of RPMI 1640 culture medium containing 30% of Matrigel, from each flank of each animal through a 2.5-cm-long 20-gauge needle. Four weeks after ZR-75-1 cell inoculation, the E₂ implants were replaced in all animals by estrone-containing implants (E-i: cholesterol; 1 :25 w:w) (7). Treatments consisting of increased doses of bpV(phen) versus control were started 5 weeks after cell inoculation. Mice bearing tumours of an average area of 15 mm² were randomly assigned to 3 groups, each group containing more than 15 mice. OVX animals first received the most potent natural estrogen, to initiate cell proliferation and the development of tumours. Thereafter, the E₂ implants were replaced by Ei implants as a model for post-menopausal women in which Ei is the main circulating estrogen that is converted into E₂ in peripheral tissues.

On day 0 of the experiment (5 weeks after inoculation), the E releasing implants were removed from the animals in Group 1 only. All mice received a daily administration of bpV(phen) over a period of 42 days (i.p., 100μl, in a 2 blind manner). Groups 1 and 2 received PBS, Group 3 received 2.5 mg/Kg bpV(phen).

Human prostate adenocarcinoma (PC-3) in the athymic mice: Male Balb/c nude (nu/nu) were purchased at 4-6 weeks of age from Charles Rivers Inc. Mice were housed under pathogen free conditions and maintained on a 12-h light/12- h dark cycle with food and water supplied ad libitum. The hormono-independent PC3 human tumour cells were from the American Tissue Cuture Collection. Cells were grown in DMEM in the presence of 5 % foetal bovine serum. Cells were collected at confluence, included in a matrix (1.0 X 10⁶ cells /ml; 30 % Matrigel). An equal volume of the tumour cell suspension was injected s.c. in the right flank of each mouse. After 5 days, a palpable tumour of approximately 5X5 mm was detected in the inoculated animals. Mice with palpable tumours were divided into five groups (18 mice/group) for the treatment study. All mice in each treatment group had tumour of similar size at the start of treatment. For administration to mice, bpV(phen) was dissolved and diluted in phosphate buffered saline (PBS) at pH 7.4. A 5mg/Kg dose of bpV(phen) was administed daily by i.p. for 39 days. Taxol was used as a positive control, at the dose of 20mg/Kg and injected i.p. at every three days. A control group of 10 animals was injected with PBS. The injection volume was kept constant at 100 μl/g body weight. The mice were weighed three times during the experimental period to assess toxicity of the treatment, and the tumours were measured twice weekly using calipers. Tumour volume was calculated from the two-dimensional caliper measurements using the following formula: tumour volume = length X (width)² X 0.53. The treatment period was completed after 39 days, when the PBS treated group of mice had large tumours, requiring that the animals be sacrificed according to the Animal Care Procedures. On the final day of the study, the mice were sacrificed by carbon dioxyde inhalation. The s.c. tumours was removed and weighed.

Statistical analysis: Tumour growth curves are presented in terms of treatment group means and SEs. Statistical significance of treatment effect was assessed by repeated measures ANOVA after applying a power transformation to equalize residual variances and linearize the tumour growth curves.

Results

1. Progression of tumour cells in vitro

The cells embedded in the collagen gel, grew as a "primary tumour". Some cells migrated from the primary tumour towards the fibrin gel, forming front edges. Small clumps of cells were observed in the fibrin gel; in this model they represent "secondary tumours". Their extension and numbers are representative of the invasive potential of the cancer cells.

PC-3: In control experiments, PC-3 cells migrated slightly from the primary tumour and formed extensive secondary tumours in the fibrin gel. In the presence of 2μM bpV(phen), a decrease in the size of the secondary tumour was observed and the migration front from the primary tumour was similar to that seen in the control gels. In the presence of 5 and 10 μM bpV(phen), there was no migration front and there were no secondary tumours in the fibrin gel. In addition, the primary tumours appeared clearer than in the control (Figure 2).

ZR-75-1: In control experiments ZR-75-1 cells migrated into the fibrin as small spheroidal secondary tumours, with a limited and sparsely visible migration front.

The presence of 2μM bpV(phen) restricted the growth of the primary tumour. At 5 and 10μM bpV(phen) there were no secondary tumours and the primary tumour had a lower cell density (Figure 3).

2. Inhibition of tumour progression in vivo

ZR-75-1 human breast cancer: The tumour size in the control group, having not received Ei replacement therapy, did not increase. The tumour size in the animals in the other control groups having received Ei replacement therapy was found to have increased significantly (p<0.05) from 15 to 26 mm² on day 42. The daily administration of bpV(phen) do not resulted in increase of tumour size (p<0.05). The results show that bpV(phen) has the capacity to inhibit the progression of tumours in vivo (Figure 4A).

Human prostate adenocarcinoma (PC-3) in the athymic mice: Daily administration of bpV(phen) caused a significant (p<0.001) 59 % suppression of the final tumour compared with PBS-treated control animals (Figure 4B). No death were observed among the vehicle-treated controls or bpV(phen), and, on average these mice gained 1.5 and 1.7 grams in body weight respectively, relative to their weight at the initiation of the treatment.

B) THE USE OF INHIBITORS OF PROTEIN TYROSINE PHOSPHATASES (PTP) FOR ANTI-TUMOUR IMMUNOTHERAPY

Lymphocytes with anti-tumour activity can be isolated from patients and grown in vitro for use in cell-tranfer therapies (8). The incubation of immune cells with the PTP inhibitor bpV(phen) augments their activation state (9). Therefore, the re-administration of bpV(phen)-activated immune cells to cancer patients may enhance the immune response towards tumour cells. The results described below demonstrate the efficacy of a method in which a peroxometallic compound (bpV(phen) is used ex vivo on autologous immune cells in order to stimulate the potency of these cells and once returned into blood circulation of cancer patients fight invasion malignant cells. Method

An in vitro cancer invasion system that has been previously designed was used. Briefly, prostate cancer cells (PC-3; American Type Culture Collection, Rockville MD) were grown in DMEM medium with 5 % fetal bovine serum, 2 mM L-glutamine, and antibiotics. They were, incubated under a humidified atmosphere of 95 % air/5% CO₂ at 37^°C. Collagen gels containing the PC-3 cells were prepared according the method of Esdale and Bard (10). The cell- embedded collagen gels were laid down onto a layer of fibrin gel, and anchored by a second layer of fibrin gel. The top of the collagen gel was not fully covered with fibrin gel in order to allow direct contacts between the cancer cells in collagen and the splenocytes. The latter were directly seeded onto the top layer of the collagen and fibrin gel. Prior to the molding of the cancer invasion system, leucocytes were isolated from spleen of either healthy mice or mice bearing PC- 3 tumours. They were treated in vitro with bpV(phen) (25 M) for 24 hr. Thereafter, treated cells were washed, counted and seeded (10⁶ cells per gel) on the cancer cells-embedded gels. Untreated leukocytes seeded on the top of the cancer invasion system (same concentration) were used as a control experiment. Medium was renewed periodically. During the whole experiment, most leucocytes remained on the top of the gels, and have a normal morphology. Cell behavior was periodically observed for 7 days of culture, then recorded (by photography).

Results

Clumps of PC3 cells progressively appeared in the fibrin gel representing the invasive potential of the cancer cells. In the control 3D culture system, PC-3 cells migrated slightly from the primary site and formed extensive secondary tumours in the fibrin gel as described in previous studies (11 , 12). In contrast to this, in the presence of bpV(phen)-treated leucocytes, neither secondary tumours nor migration front was observed. In addition, the primary tumours appeared less dense than in the control, indicating a smaller number of growing cells (Figure 5). It is to be understood that the use of bpV's as a therapeutic agent my be alone or in combination with other active ingredient such as interieukins or chemokines.

It is also to be understood that the bpV dosage compositions of the present invention may be used as vaccines for eliciting an immuno response from a mammal.

Although the present invention has been described by way of preferred embodiments thereof, these embodiments can be modified at will, within the scope of the appended claims, without departing from the spirit and nature of the subject invention.

LIST OF REFERENCES

1. Posner, B.I., Faure, R., Burgess, J.W., Bevan, A.P., Lachance, D., Zhan- Sun, G., Fantus, I.G., Ng, J.B., Hall, D.A., Lum, B.S. & Shaver, A. (1994). Peroxovanadium compounds: a new class of potent phosphotyrosine- phosphatase inhibitors which are insulin mimetics. J. Biol. Chem. 269: 4596-4604.

2. Faure, R., Vincent, M., Dufour, M., Shaver, A. & Posner, B. (1995). Arrest at the G2/M transition of the cell-cycle by protein-tyrosine phosphatase inhibition: Studies on a neuronal and a glial cell line. J. Cell. Biochem. 58:

389-401.

3. Tonks, N.K. and B.G. Heel Combinatorial control of the specificity of _. PTPs. Current opinion in cell Biology 2001 13(2): 182-195.

4. Huyer et al. (1997)

5. Montesano, R., Pepper, M.S., Belin, D., Vassalli, J.-D. & Orci, L. (1988). Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases. J. Cell. Physiol. 134: 460-466.

6. Janvier, R., Sourla, A., Koutsilieris, M. & Doillon, C. (1997). Stromal Fibroblasts are required for PC-3 human prostate cancer cells to produce capillary-like formation of endothelial cells in a three-dimensional co- culture system. Anticancer res. 17: 1551-1558.

7. Couillard S, Gutman M, Labrie F, Candos B, Labrie C. (1999). Effect of combined treatment with EM-800 and radiotherapy on the growth of human 212-75-1 breast cancer xenografts in nude mice. Cancer Res.59: 4857-4863.

8. Rosenberg, S.A. Progress in human tumour immunology and immunotherapy. Nature 411 : 380-384. 2001. 9. Olivier, M. Romero-Gallo B. J., St Laurent, R., Racette, N., Stevenson, M., Jacobs, P., Posner, B., Tremblay, M., Faure, R. Modulation of INFg- induced macrophage activation by phosphotyrosine phosphatase inhibition. J. Biol. Chem. 273, 3944-13949 (1998).

10. Esdale, T., Bard, J. Collagen substrata for studies on cell behavior. J. Cell. Biol. 54: 626-637 (1972).

11. Janvier, R., Souria, A., Koutsilieris, M., Doillon, C. Stromal fibroblasts are required for PC-3 human prostate cancer cells to produce capillary -like formation of endothelial cells in a three-dimensional co-culture system. Anticancer Res. 17: 1551 -1557 (1997).

12. Dvorak, H.F., Harvey, V.S., Estrella, P., Brown, L.F., Mc Donagh, J., Dvorak A.M. Fibrin containing gels induce angiogenesis. Implications for stroma generartion and wound healing. Lab Invest. 57: 673-686 (1987).

Claims

WHAT IS CLAIMED IS:

1. The use of the compound of formula:

wherein V is vanadium,

Y is oxygen or hydroxyl,

Z and Z' are independently selected from oxygen and peroxide and at least one of them is peroxide, and L or _.' are any group which can donate one electron pair; in a dosage of from 0.001 to less than 15 mg/kg of body weight/day for preventing or arresting tumour cell growth in a mammal.

2. The use of claim 1 wherein said dosage is from 1 to 10 mg/kg of body weight/day.

3. The use of claim 1 , wherein Z is oxygen and Z' is peroxide.

4. The use of claim 1 , wherein Z and Z' are peroxide.

5. The use of claim 1 wherein said tumour growth consists of cancer.

6. The use of claim 5 wherein said cancer is breast cancer or prostate cancer.

7. The use of any on of the preceeding claims wherein said compound is selected from bpV(phen), bpV(pic), bpV(bipy) and mixtures thereof.

8. The use of claim 7 also comprising the concomittant use of at least one second active ingredient consisting of an interleukin or a chemokine.

9. A pharmaceutical composition consisting of a daily dose of 0.001 to 15 mg/kg of body weight of the compound of formula:

V

wherein V is vanadium, Y is oxygen or hydroxyl,

Z and Z' are independently selected from oxygen and peroxide and at least one of them is peroxide, and L or L' are any group which can donate one electron pair; together with pharmaceutically acceptable excipient(s) for preventing or arresting tumour cell growth in a mammal.

10. The composition of claim 9 wherein said daily dose is from 1 to 10 mg/kg of body weight.

11. The composition of claims 9 or 10 wherein said compound is selected from bpV(phen), bpV(pic), bpV(bipy) and mixtures thereof.

12. A vaccine consisting of a dose of 0.001 to 15 mg/kg of body weight of the compound of formula:

N- Z'

V

wherein V is vanadium, Y is oxygen or hydroxyl, Z and Z' are independently selected from oxygen and peroxide and at least one of them is peroxide, and L or L' are any group which can donate one electron pair; together with pharmaceutically acceptable adjuvant(s) for preventing or arresting tumour cell growth in a mammal.

13. The vaccine of claim 12 wherein said daily dose is from 1 to 10 mg/kg of body weight.

14. The composition of claim 12 or 13 wherein said compound is selected from bpV(phen), bpV(pic), bpV(biby).

15. The use of claim 1 wherein the resulting serum concentration after administration to a mammal is of 1 to 50 μM of the compound described in claim 1.