U.S. patent application number 14/019674 was filed with the patent office on 2014-01-02 for formulations with anti-tumour action.
This patent application is currently assigned to GENTIUM SPA. The applicant listed for this patent is GENTIUM SPA. Invention is credited to Laura Iris FERRO, Massimo IACOBELLI, Paul RICHARDSON.
Application Number | 20140005256 14/019674 |
Document ID | / |
Family ID | 37064585 |
Filed Date | 2014-01-02 |
United States Patent
Application |
20140005256 |
Kind Code |
A1 |
FERRO; Laura Iris ; et
al. |
January 2, 2014 |
FORMULATIONS WITH ANTI-TUMOUR ACTION
Abstract
Formulations with anti-tumour action containing defibrotide and
at least another active ingredient as active agents.
Inventors: |
FERRO; Laura Iris; (Milano,
IT) ; IACOBELLI; Massimo; (Milano, IT) ;
RICHARDSON; Paul; (Wellesley, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENTIUM SPA |
Villa Guardia |
|
IT |
|
|
Assignee: |
GENTIUM SPA
Villa Guardia
IT
|
Family ID: |
37064585 |
Appl. No.: |
14/019674 |
Filed: |
September 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11366243 |
Mar 2, 2006 |
8551967 |
|
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14019674 |
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PCT/EP04/09723 |
Aug 27, 2004 |
|
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11366243 |
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Current U.S.
Class: |
514/44R |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 45/06 20130101; A61K 31/337 20130101; A61K 31/665 20130101;
A61K 31/665 20130101; A61K 31/17 20130101; A61K 31/337 20130101;
A61K 31/195 20130101; A61P 35/00 20180101; A61K 31/7088 20130101;
A61K 31/17 20130101; A61K 31/711 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 31/195 20130101; A61K 31/7088
20130101 |
Class at
Publication: |
514/44.R |
International
Class: |
A61K 31/711 20060101
A61K031/711; A61K 45/06 20060101 A61K045/06 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 5, 2003 |
IT |
MI2003A001714 |
Aug 27, 2004 |
EP |
PCT/EP04/09723 |
Claims
1. A formulation containing, as active agents, defibrotide and at
least another active ingredient with anti-tumour action.
2. A formulation according to claim 1, which is an aqueous
solution.
3. A formulation according to claim 1, which contains customary
excipients and/or adjuvants.
4. A formulation according to claim 1, wherein the other active
ingredient with anti-tumour action is selected from paclitaxel,
monocrotaline, BCNU, and/or cyclophosphamide.
5. A formulation according to claim 1, constituted by two distinct
formulations that can be administered separately, one containing
defibrotide and the other containing the other active ingredient
with anti-tumour action.
6. A formulation according to claim 1, as a combined preparation
for simultaneous, separate, or sequential administration.
7. A formulation according to claim 1, wherein defibrotide is
obtained by extraction from animal and/or vegetable tissues.
8. A formulation according to claim 1, wherein defibrotide is
obtained synthetically.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of and claims priority to
U.S. patent application Ser. No. 11/366,243, filed Mar. 2, 2006,
which is a continuation-in-part of and claims priority to prior
International Application No. PCT/EP2004/009723, filed Aug. 27,
2004, which claims priority to Italian Application No.
MI2003A001714, filed Sep. 5, 2003, and U.S. Provisional Application
No. 60/539,344, filed Jan. 28, 2004, the disclosures of all of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The subject of the present invention is a formulation for
treating a tumor-affected mammal by administering to said mammal an
effective amount of defibrotide.
BACKGROUND OF THE INVENTION
[0003] The term defibrotide (hereinafter DF) normally identifies a
polydeoxyribonucleotide that is obtained by extraction from animal
and/or vegetable tissues (1, 2) but which might also be obtained
synthetically; the polydeoxyribonucleotide is normally used in the
form of an alkali-metal salt, generally a sodium salt, and
generally has a molecular weight of about 15-30 kDa (CAS Registry
Number: 83712-60-1).
[0004] DF is used mainly on account of its antithrombotic activity
(3), although it can be used in other applications such as, for
example, the treatment of acute renal insufficiency (4) and the
treatment of acute myocardial ischaemia (5). DF is also used in the
treatment of emergency clinical conditions, for example, for
suppressing the toxicity correlated with high doses of chemotherapy
regimens, in particular, the hepatic veno-occlusive syndrome (10,
11); DF has been shown to have protective action towards apoptosis
induced by fludarabine and towards the alloactivation of
endothelial and epithelial cells, without also altering the
antileukaemic effects of fludarabine (12); pre-clinical data also
exists on the protective effects of DF that have been achieved in a
model of endothelial damage mediated by lipopolysaccharide
(13).
[0005] A method of producing DF that can produce a product which
has uniform and well-defined physical/chemical characteristics and
which is also free of possible undesirable side effects is
described in United States patents (6, 7).
[0006] Within the purposes of the present invention DF is either of
extractive or of synthetic origin.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 is a graph of the cytotoxicity of cultured mouse
EMT-6 mammary carcinoma cells exposed to 4HC, with and without
DF;
[0008] FIG. 2a is a chart of in vivo tumor growth delay in mouse
mammary carcinoma 13762 exposed to DF and other agents;
[0009] FIG. 2b is a graph of tumor volume in mouse mammary
carcinoma 13762 exposed to DF and other agents.
DESCRIPTION OF THE INVENTION
[0010] In the following study, DF was examined in combination with
antiblastic cytotoxic agents in a model of mouse EMT-6 mammary
carcinoma cells and in bovine endothelial cells, in cell cultures
and in an experimental model in which rats carrying tumours
subjected to high doses of chemotherapy were used.
[0011] Exposure to DF at a concentration of 50 .mu.g/ml, either
before and during, or during and after the exposure of mouse EMT-6
mammary carcinoma cells in culture with
4-hydroperoxycyclo-phosphamide (4HC) considerably increases the
cytotoxicity of 4HC to the extent of bringing about an increment of
2 logarithmic units in the killing of the tumour cells at 4HC
concentrations of between 50 and 250 .mu.mol (see FIG. 1). Exposure
to DF at concentrations of 50 .mu.g/ml also leads to an increase in
the cytotoxicity of thiotepa with a clear difference based on the
method of exposure. In particular, exposure of EMT-6 cells to DF
before and during exposure to thiotepa increases cytotoxicity
towards the tumour cells by two logarithmic units for thiotepa
concentrations of between 100 and 250 .mu.mol. An interesting datum
which emerges is that the exposure of EMT-6 cells to DF during and
after exposure to thiotepa leads to an increase in cytotoxicity,
although to a lesser extent, showing an increase of between 0.5 and
1 logarithmic unit in the cytotoxicity of thiotepa. A similar
result has been observed with carboplatin; however, exposure to DF
before and during or during and after exposure to melphalan did not
show any significant effect on the cytotoxicity of melphalan
towards mouse EMT-6 mammary carcinoma cells in culture.
[0012] On the other hand, although it was demonstrated that the
cytotoxicity of these antiblastic alkylating agents (AA) alone
towards bovine endothelial cells in culture was similar to that
observed in EMT-6 mammary carcinoma cells, no increase in
cytotoxicity was shown when this type of cell culture model was
exposed to AAs in association with DF at a concentration of 50
.mu.g/ml.
[0013] The hepatotoxin monocrotaline and the AA carmustine (BCNU),
alone or in association with DF, were tested in vivo in an
experimental model which used rats carrying mammary carcinoma
13762. In this experimental model, no additional toxicity was shown
in the animals when they were exposed to these agents together with
DF, but a significant tumour growth delay (TGD) was observed (see
Table 1 and FIGS. 2a and 2b).
TABLE-US-00001 TABLE 1 Tumour growth delay in rats carrying mammary
carcinoma 13762 after treatment with monocrotaline or BCNU, alone
or in association with defibrotide (DF). The tumour was implanted
on day 0 and the chemotherapy was administered on day 8 and day 18.
Days to reach Treatment Group 500 mm.sup.3 TGD (days) p Value
Controls 14.6 .+-. 0.8 -- -- Monocrotaline (350 mg/kg) 15.6 .+-.
1.0 1.0 0.435 ip days 8 & 18 DF (200 mg/kg) iv 16.1 .+-. 0.6
1.5 0.134 twice per day, days 8-26 + Monocrotaline DF (200 mg/kg)
iv 18.2 .+-. 1.5 3.6 0.034 twice per day, days 10-26 +
Monocrotaline BCNU (150 mg/kg) ip 18.0 .+-. 2.5 3.4 0.195 days 8
& 18 DF (200 mg/kg) iv 19.7 .+-. 1.5 5.1 0.003 twice per day,
days 8-26 + BCNU DF (200 mg/kg) iv 21.3 .+-. 1.6 6.7 0.0002 twice
per day, days 10-26 + BCNU
[0014] These studies have been reproduced with the use of
monocrotaline, BCNU, and cyclophosphamide (CTX), alone or in
combination with DF, in the same experimental model. In comparison
with the control, a significant tumour growth delay (TGD) was
observed with the use of DF alone (p<0.05); this delay was
particularly significant when DF was associated with CTX and BCNU
(p<0.04) and was notably greater than that obtained by the
individual use of each agent. Unexpectedly, when DF was used alone,
at first it delayed the growth of the tumour but afterwards tumour
growth became normal again. Moreover, when DF was used in
combination with an AA, the tumour regrowth became rapid as soon as
the co-administration of DF ceased. This data suggests not only an
additional anti-tumour effect of DF but also a direct antiblastic
activity of DF itself.
[0015] A reduction in tumour growth (TGD) and in the number of
pulmonary metastases was also observed in mice carrying Lewis
pulmonary carcinoma when DF was added to treatment with paclitaxel,
whether or not it was associated with carboplatin and in comparison
with cytotoxic therapy alone, but without showing an obvious
increase in toxicity (data not presented). The mechanism underlying
these effects remains to be explained, but it is possible that the
anti-adhesive properties of DF are involved, given the role of cell
adhesion in the mechanisms implicated in drug resistance (8,
9).
[0016] It was also tested whether DF has in vivo activity in a
murine model of human multiple myeloma (MM). Sixty male SCID/NOD
mice (6-8 weeks old) were irradiated (450 rads) and, 24 hrs later,
injected s.c. with 5.times.10 6 MM-1S human MM cells. Upon
formation of palpable tumors, mice were randomly assigned to 6
cohorts (10 mice each) receiving a) vehicle; b) DF (i.v. 450 mg/kg
b.i.d); c) melphalan (MEL) 2.5 mg/kg i.p. once weekly; d)
cyclophosphamide (CTX) 50 mg/kg i.p., on days 8, 10, 12, 20, 22 and
24; e) and f) combinations of DF (300 mg/kg i.v.) with MEL or CTX,
respectively. Mice were monitored q3 days for body weight,
potential toxicity, and electronic caliper-based tumor volumes.
[0017] DF, either as single agent or in combination with MEL or
CTX, was well tolerated without hemorrhagic complications or body
weight loss (P>0.05) in all groups. The major endpoints for
efficacy were a) tumor volume changes and b) overall survival
(time-to-sacrifice, performed when tumor diameters>2 cm). DF
treatment resulted in significantly lower tumor volumes than in
control mice (P<0.05 for all comparisons by analysis of variance
and post-hoc tests); in combination with MEL or CTX it induced
significantly lower tumor volumes than the respective single-agent
cytotoxic chemotherapy (P<0.05 for all comparisons).
Kaplan-Meier survival analyses showed that DF administration,
either as single agent or in combination with cytotoxic
chemotherapy (MEL or CTX), was associated with statistically
significant prolongation of overall survival, in comparison to
vehicle-treated control group or MEL- or CTX-treated groups,
respectively (P<0.001 for all comparisons, log-rank test).
Interestingly, the in vitro studies have not shown a significant
direct in vitro cytotoxic effect of DF against MM cells, suggesting
that the observed in vivo activity may be due to effect(s) on
interactions of MM cells with their local microenvironment.
[0018] These promising results demonstrate that DF does confer
tumor protection in this MM chemotherapy model and constitutes the
first proof-of-principle that DF not only has in vivo anti-tumor
activity against MM but also enhances responses to cytotoxic
treatment. This study suggests that the anti-MM activity of DF is
possibly due to its effects on MM cell interactions with their
microenvironment and provides a framework for future clinical
trials of DF in combination with other agents for the treatment of
MM and other neoplasias.
[0019] A method for treating a tumor-affected mammal, preferably a
human, by administration of an effective amount of DF is therefore
an object of the present invention. DF may be administered in
combination with at least another active ingredient with
anti-tumour action. The other active ingredient with anti-tumour
action may be selected from paclitaxel, monocrotaline, BCNU,
melphalan and/or cyclophosphamide.
[0020] Further objects of the invention are represented by the
formulations containing DF and at least one other active ingredient
with anti-tumour action; the formulations will preferably be in the
form of aqueous solutions and, even more preferably, suitable for
intravenous administration, and may contain the excipients and
coadjuvants known in the art.
[0021] For the purposes of the present invention, the term
defibrotide (DF) should thus be understood as any oligonucleotide
and/or polynucleotide produced by extraction from animal and/or
vegetable tissues, in particular, from mammalian organs.
Preferably, the DF will be produced in accordance with the method
described in United States patents (6, 7) which are incorporated
herein by reference.
BIBLIOGRAPHY
[0022] 1. U.S. Pat. No. 3,770,720 [0023] 2. U.S. Pat. No. 3,899,481
[0024] 3. U.S. Pat. No. 3,829,567 [0025] 4. U.S. Pat. No. 4,694,134
[0026] 5. U.S. Pat. No. 4,693,995 [0027] 6. U.S. Pat. No. 4,985,552
[0028] 7. U.S. Pat. No. 5,223,609 [0029] 8. Carlo-Stella, C., Di
Nicola, M., Magni M., et al., Defibrotide in Combination with
Granulocyte Colony-stimulating Factor Significantly Enhances the
Mobilization of Primitive and Committed Peripheral Blood Progenitor
Cells in Mice. Cancer Research, 2002, 62:6152-6157 (Nov. 1, 2002).
[0030] 9. Hazlehurst, L., Damiano, J., Buyuksal, I., Pledger, W.
J., Dalton, W. S., Adhesion to fibronectin via b1 integrins
regulates p27 kip1 levels and contributes to cell adhesion mediated
drug resistance (CAM-DR). Oncogene, 2000; 19:4319-4327. [0031] 10.
Richardson, P. G., Elias, A. D., Krishnan, A., et al. Treatment of
severe veno-occlusive disease with defibrotide: compassionate use
results in response without significant toxicity in a high-risk
population. Blood, 1998; 92: 737-44. [0032] 11. Richardson, P.,
Murakami, C., Jin, Z., et al., Multi-institutional use of
defibrotide in 88 patients after stem cell transplantation with
severe veno-occlusive disease and multi-system organ failure:
response without significant toxicity in a high risk population and
factors predictive of outcome. Blood, 2002; 100(13):4337-4343.
[0033] 12. Eissner, G., Multhoff, G., Gerbitz, A., et al.,
Fludarabine induces apoptosis, activation, and allogenicity in
human endothelial and epithelial cells: protective effect of
defibrotide. Blood, 2002; 100:334-340. [0034] 13. Falanga, A.,
Vignoli, A., Marchetti, M., Barbui, T., Defibrotide reduces
procoagulant activity and increases fibrinolytic properties of
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