LIPOPROTEINS CONTAINING PLATINUM COMPLEXES FOR THE TREATMENT OF CANCER

Abstract

The present invention relates to lipoproteins containing platinum complex. The invention also relates to a kit comprising said lipoproteins. In particular, the present invention relates to the use of said platinum-complex-bearing lipoproteins for the specific targeting of macrophages and tumour cells in the treatment of cancer.

Claims

1. A lipoprotein wherein the lipoprotein is a low-density lipoprotein (LDL) charged with platinum complex, or the lipoprotein is a high-density lipoprotein (HDL) charged with platinum complex, or the lipoprotein is an oxidised low-density lipoprotein charged with platinum complex, or the lipoprotein is an acetylated low-density lipoprotein charged with platinum complex.

2-8. (canceled)

9. Kit wherein the kit comprises: a high-density lipoprotein (HDL) charged with platinum complex or an oxidised or acetylated low-density lipoprotein charged with platinum complex or a mixture thereof, and a low-density lipoprotein (LDL) charged with platinum complex.

10-12. (canceled)

13. Lipoprotein according to claim 1, wherein the platinum complex is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, tetraplatin, iproplatin, satraplatin, nedaplatin, lobaplatin, picoplatin and ProLindac (polymer-platinate-DACH AP5346).

14. (canceled)

15. Lipoprotein according to claim 1, wherein the platinum complex is cisplatin.

16. Kit according to claim 9, wherein the platinum complex is selected from the group consisting of cisplatin, carboplatin, oxaliplatin, tetraplatin, iproplatin, satraplatin, nedaplatin, lobaplatin, picoplatin and ProLindac (polymer-platinate-DACH AP5346).

17. Kit according to claim 9, wherein the platinum complex is cisplatin.

18. Method for the treatment of a disease, wherein said method comprises the administration to a patient of a therapeutically effective quantity of the lipoprotein according to claim 1.

19. Method according to claim 18, wherein the disease is cancer.

20. Method according to claim 18, wherein the low-density lipoprotein (LDL) charged with platinum complex induces tumour cell death by apoptosis.

21. Method according to claim 18, wherein the low-density lipoprotein (LDL) charged with platinum complex is used in combination with an oxidised or acetylated low-density lipoprotein charged with platinum complex.

22. Method according to claim 18, wherein: the high-density lipoprotein (HDL) charged with platinum complex activates macrophages, or the oxidised low-density lipoprotein charged with platinum complex activates macrophages, or the acetylated low-density lipoprotein charged with platinum complex activates macrophages.

23. Method according to claim 18, wherein the high-density lipoprotein (HDL) charged with platinum complex is used in combination with a low-density lipoprotein (LDL) charged with platinum complex.

24. Method for the treatment of cancer which comprises the step of using the kit according to claim 9.

Description

FIGURES

[0058] FIG. 1: Study of cisplatin vectorisation in LDLs and HDLs

[0059] FIG. 1A: Cisplatin vectorisation

[0060] FIG. 1B: Evaluation of cisplatin exchanges between charged LDLs/HDLs and native LDLs/HDLs

[0061] FIG. 2: Effect of cisplatin vectorisation on cancer cells and on macrophages

[0062] FIG. 2A: Effect of cisplatin vectorisation on tumour cells

[0063] FIG. 2B: Effect of cisplatin vectorisation on macrophages

[0064] FIG. 2C: Effect of cisplatin vectorisation on macrophages (with oxidised LDL)

[0065] FIG. 3: Study of potency of LDLs charged with cisplatin and HDLs charged with cisplatin on cancer cells and on macrophages in tumour extracts

[0066] FIG. 4: Cisplatin vectorisation by LDLsenhancement of tumour efficacyin vivo

[0067] FIG. 4A: Progression of tumour size over time

[0068] FIG. 5: Cisplatin vectorisation by LDLsreduction of toxicity in vivo

[0069] FIG. 5A: Effect of cisplatin vectorisation by LDLstumour volume

[0070] FIG. 5B: Effect of cisplatin vectorisation by LDLsweight loss

EXAMPLES

Preparation of Lipoproteins Charged with Cisplatin

[0071] Low-density lipoproteins and high-density lipoproteins were isolated from plasma from healthy donors by a potassium bromide (KBr)-differential density gradient ultracentrifugation separation technique (Redgrave technique, 1975). After extraction, the lipoproteins were adjusted to a cholesterol concentration of 1 mM. 100 l of a cisplatin solution (at 10 mg/ml, in physiological saline solution) was then added for an expected final concentration of 1 mg/ml. In order to enable the binding of cisplatin with the lipoproteins and eliminate the unbound fraction of cisplatin, the samples were incubated for 3 hours at 37 C., then subjected to two successive dialyses (against 1000 times the volume of phosphate buffered saline (PBS), Cutoff 7000 Da) of 1 hour and 18 hours respectively. After dialysis, the cisplatin concentration was determined by graphite furnace absorption spectrometry (GF-AAS) (FIG. 1A). As demonstrated in FIG. 1A, the cisplatin concentration in the LDLs is 0.3 mg/mL of final solution, i.e. the solution obtained by adding lipoproteins in a phosphate buffered saline (PBS) solution containing cisplatin. The cisplatin concentration in the HDLs is 0.5 mg/mL of said final solution. These concentrations are measured for a cholesterol concentration of 1 mmol/mL of said final solution.

[0072] Thus, over 30% of the initial cisplatin concentration was vectorised in purified HDL and LDL fractions.

Stability study of Lipoproteins Charged with Cisplatin (FIG. 1B)

[0073] LDLs containing vectorised cisplatin (LDL-Cis) were incubated for 18 hours at 37 C. with native HDLs (HDL 0). Similarly, HDLs containing vectorised cisplatin (HDL-Cis) were therefore incubated for 18 hours at 37 C. with native LDLs (LDL 0).

[0074] After incubation, these lipoprotein fractions were extracted with a potassium bromide (KBr)-differential density gradient ultracentrifugation separation technique. The quantity of cisplatin bound to the different fractions was then determined by graphite furnace absorption spectrometry (GF-AAS).

[0075] As demonstrated in FIG. 1B, after 18 hours of incubation, 0.13 mg and 0.26 mg of cisplatin per mL of said final solution were still present respectively in the LDL-Cis and HDL-Cis fractions. On the other hand, no trace of cisplatin was detected in the native LDL and HDL fractions (FIG. 1B).

[0076] These results demonstrate therefore that binding of cisplatin with the lipoproteins is stable. Indeed, after 18 hours of incubation, approximately 50% of the initially vectorised cisplatin was still present in the lipoprotein fractions, and no exchange of cisplatin with other lipoprotein classes occurred.

In Vitro Study of Effects of Cisplatin Vectorisation by HDL and LDL Lipoproteins on Adenocarcinoma Cells or Macrophages in Culture (FIG. 2A)

[0077] Adenocarcinoma cells and macrophages are mostly detected in colon tumours.

[0078] For this test, SW480 colorectal cancer lines were treated for 48 hours with native LDLs (LDL 0), native HDLs (HDL 0), non-vectorised cisplatin, LDL-Cis or HDL-Cis (final cisplatin concentration: 25 M). The cell viability was then evaluated by flow cytometry. As per FIG. 2A, the non-vectorised cisplatin induced a 41% cancer cell mortality. On the other hand, the native LDLs and HDLs did not induce any effect on the cancer cells. Moreover, the HDL-Cis induced a 37% cancer cell mortality, which is comparable to the effect of non-vectorised cisplatin. On the other hand, the LDL-Cis induced a 58% mortality of the SW480 cells, i.e. a much superior effect to that obtained for non-vectorised cisplatin (FIG. 2A).

Study of the Impact of Cisplatin Vectorisation on ROS Production (FIG. 2B)

[0079] After 7 days of culture in human M-CSF from Miltenyi, Biotec. (Macrophage Colony-Stimulating Factor) at 100 ng/ml, human macrophages were differentiated, from monocytes, into M2 alternative phenotype macrophages (protumoral). These macrophages were then stimulated for 2 hours with native LDLs (LDL 0), native HDLs (HDL 0), non-vectorised cisplatin, LDL-Cis or HDL-Cis (final cisplatin concentration: 25 M). The production of reactive oxygen species (ROS, representative of an anti-tumour action) by the macrophages was then determined by flow cytometry after labelling with Dihydroethidium (DHE). This test demonstrates therefore that the use of non-vectorised cisplatin makes it possible to increase the basal ROS production from 8.2% to 18.2% by macrophages compared with the control sample (CTL). Furthermore, the native HDLs, native LDLs and the LDL-Cis had no effect on ROS production by the macrophages. On the other hand, the HDL-Cis induce 26.8% macrophage activation, i.e. an approximately 50% more effective effect than non-vectorised cisplatin (FIG. 2B).

Study of the Impact of Cisplatin Vectorisation on ROS Production for Oxidised LDLs Charged with Cisplatin (FIG. 2C)

[0080] The same protocol was repeated so as to compare the effect between the HDL-Cis and the oxidised LDL lipoproteins charged with cisplatin (LDLox+Cis) (see FIG. 2C). Thus, the LDLox+Cis induce superior macrophage activation to that induced by the HDL-Cis. The oxidised LDLs were obtained by incubating native

[0081] LDLs (cholesterol, 1 mM) for 24 hours at 37 C. in the presence of copper sulphate (5 M). After oxidation, the oxidised LDLs are dialysed in a PBS buffer.

Specific Targeting

[0082] The above tests make it possible therefore to demonstrate that, in vitro, charged LDLs appear to have an effect only on cancer cells, whereas charged HDLs have an effect only on macrophages. The vectorisation of cisplatin by HDLs makes it possible to increase the efficacy of the treatment by almost 50% compared to non-vectorised cisplatin. Similarly, cisplatin vectorisation by LDLs makes it possible to increase the efficacy of the treatment by almost 50% compared to non-vectorised cisplatin.

[0083] In a further test, tumours obtained from an ectopic (subcutaneous) allograft model and CT-26 colon tumours in BALB-C mice were isolated and placed in contact with LDL or HDL type fluorescent lipoproteins (bodipy) (FIG. 3). As shown in FIG. 3, the LDLs are preferentially taken up by the tumour cells whereas HDLs are for their part mostly taken up by macrophages.

In Vivo Tests

[0084] In order to verify the in vitro and ex vivo results above for an in vivo model, the ectopic (subcutaneous) allograft model of CT-26 colon tumours in BALB-C mice was used. As shown by FIG. 4A, after 25 days of treatment, the mice treated by 1.5 mg/kg LDL-Cis exhibit much less developed tumours compared with the control group (CLT) and with the non-vectorised 1.5. mg/kg cisplatin group. Furthermore, as proven by the histological tumour analysis (not shown), vectorisation enhances the production of radical species (DHE) and induces more apoptosis (caspase-3 cleavage) compared to the non-vectorised cisplatin group. These experiments therefore demonstrated that the vectorisation of a cytotoxic agent enhanced the anti-tumour efficacy thereof.

Study of Nephrotoxicity and Other Side-Effects for Vectorised Cisplatin

[0085] The purpose of this test is to verify that the vectorisation of cisplatin indeed makes it possible to reduce systemic and renal toxicity compared to non-vectorised cisplatin. For this test, cisplatin was administered at a dose of 20 mg/kg for 3 days to our mouse model described above, i.e. the ectopic (subcutaneous) allograft model of CT-26 colon tumours in BALB-C mice (validated cisplatin-induced nephrotoxicity protocol).

[0086] As demonstrated in FIG. 5B, non-vectorised cisplatin induces, on one hand, weight loss of almost 15% compared to the control sample (CTL). Moreover, as proven by the histological analyses (not shown), non-vectorised cisplatin induces a high level of nephrotoxicity which is characterised by deepithelialisations, the presence of hyaline bodies and necrosis and apoptosis phenomena. In comparison, no weight loss or signs of nephrotoxicity were observed for the LDL-vectorised cisplatin group (see FIG. 5B). Thus, the vectorisation of cisplatin makes it possible to reduce the toxicity associated with the use thereof compared to non-vectorised cisplatin. Moreover, cisplatin vectorised by LDLs induces apoptosis of the cells within the tumour but not that of renal cells (histological analysis not shown). The use of vectorised cisplatin therefore makes it possible to do away with the side-effects associated with the use of non-vectorised cisplatin.