Patent Grant
10449160
The present invention pertains to the field of physical sciences. More especially, this invention relates to a system comprising nanoparticles and means to remotely apply a gradient of magnetic field on a tumor region. According to the invention, nanoparticles are injected intravenously in a subject having a tumor. Nanoparticles are injected and after migration, are located at the tumor region. Application of a gradient of magnetic field in the tumor region where the nanoparticles are located, results in the generation of a constraint field on the tumor surroundings allowing the reduction of the volume and histological surfaces of the tumor.
Using implantable nanoparticles in medicine is well-known, and a number of references use nanoparticles for magnetic targeting of drugs. For example EP 2508171 mentions the use of nanoparticles for guiding and accumulating to a target tissue a pharmaceutical composition coated onto said nanoparticles. US 2011223255 (MagForce) describes implantable products containing nanoparticles and their use in medicine, particularly for thermotherapeutic after-treatment, after surgical removal of tumors.
In the treatment of cancer, there is an evident need for new medicinal—and particularly physical—strategies for treating tumors: novel cancer treatment therapies using alternating electric field were attempted to disturb the rapid cell division exhibited by cancer cells. One system was developed by Novocure company and was approved by the US FDA on Apr. 15, 2011 for the treatment of recurrent Glioblastoma Multiforme, a type of malignant brain tumor.
This invention proposes a new medical system and method for the treatment of tumors, implementing an injection device comprising injectable magnetizable nanoparticles and a magnetic field gradient for generating a constraint field onto tumor surroundings, and optionally comprising magnets. The method of this invention involves the injection, in a subject in need thereof, of a number of magnetizable nanoparticles and the application of an external gradient of magnetic field in the tumor region where the nanoparticles are retained.
The Applicant observed that, when the nanoparticles are injected intravenously, they are retained by the neoangiogenic network. It was observed that the randomized distribution of the injected nanoparticles in the neoangiogenic network surrounding the tumor surprisingly formed some kind of a hollow and discontinuous sphere. The tumor is thus located within said sphere.
This configuration of injected nanoparticles in the neoangiogenic network surrounding the tumor, creates conditions such that, when a gradient of magnetic field is applied onto the tumor region, a consequence of this application is the generation of a constraint field, and further of a constraint which is applied on the cells; this constraint leads to a change of the phenotype of the tumor cell to a normal or partially normal cell phenotype. Especially, this physical and/or mechanical constraint allows reducing the volume of a tumor and acts on the two histological surfaces of the tumor exposed to the external gradient of magnetic field.
In other words, the biological effect is obtained by the physical and/or mechanical constraint applied on the tumor cells, said constraint resulting from the constraint field, itself resulting from the application of the gradient of magnetic field onto nanoparticles distributed in the neoangiogenic network surrounding the tumor.
In one embodiment the invention relates to a medical system comprising (1) injectable magnetizable nanoparticles and means for injecting the same, and (2) means for applying a physical and/or mechanical constraint to a tumor associated to a neoangiogenic network where, after having been injected, the particles are retained, said means for applying a physical and/or mechanical constraint being a generator releasing a gradient of magnetic field; and
In one embodiment, this invention also relates to a method of treatment which is performed via the constraint applied on the cells; this constraint is a mechanical constraint. In one embodiment, this invention relates to a method of treatment which is performed via the constraint applied on the cells; this constraint is a contact-free constraint. The original approach of this invention shows that the mechanical constraint results from the force applied locally by the nanoparticles situated within the tumor region. The gradient of magnetic field directs the constraint field to the interior of the sphere, i.e. to the tumor cells. In the present invention, no movement of the particles is observed.
In particular, the invention relates to a method for treating tumors by physical and/or mechanical means.
In one embodiment, the method of the invention further comprises a pharmaceutical agent. In one embodiment, the method of the invention does not comprise a pharmaceutical agent. In one embodiment, the method of the invention is not a pharmaceutical method.
In the present invention, the following terms have the following meanings:
This invention relates to a medical system comprising (i) an injection device comprising injectable magnetizable nanoparticles 1 and means for injecting the same, and (ii) means for applying a physical and/or mechanical constraint to a tumor associated to a neoangiogenic network 7 where, after having been injected, the particles 1 are retained.
By definition, it is understood by a neoangiogenic network, new blood vessels developing in the proximal area of the tumor while the stroma refers to the tissue which forms the structure of an organ. The stroma does not represent a vascularization network. Thus, the stroma is not a neoangiogenic network.
Consequently, the present invention relates to a medical system for treating by physical and/or mechanical means tumor cells, wherein said means comprise magnetic particles specifically retained in the neoangiogenic network.
In one embodiment, the medical system is not a cell construct or an implant.
In one embodiment, the medical system does not comprise a pharmaceutical agent for treating the tumor; in particular the nanoparticles are not used as drug carriers.
In one embodiment, the medical system is not for use as an imaging or a contrast system.
Tumor
In one embodiment, the tumor is associated with an angiogenic network 7. In another embodiment, the tumor is a locally advanced tumor. In one embodiment, the tumor is a malignant epithelial tumor. In one embodiment, the malignant tumor is pancreas cancer. In one embodiment, the malignant tumor is liver cancer. In another embodiment, the malignant tumor is breast cancer. In one embodiment, the tumor is within an animal or a human body.
Means for Applying a Mechanical and/or Physical Constraint
In one embodiment, the means for applying a mechanical and/or physical constraint is a generator releasing a gradient of magnetic field 2. In one embodiment, the magnetic field generator is controlled to produce a gradient of magnetic field 2. In on embodiment, the generator comprises an electromagnet 9. In another embodiment, the generator is a coil, preferably a moving coil, more preferably a Helmotz coil in a tumble configuration.
In one embodiment, the means for applying a mechanical and/or physical constraint further comprise at least one magnet, permanent magnet and/or electromagnet; preferably said means further comprise more than one external magnet, permanent magnet and/or electromagnet; more preferably more than one external magnet, permanent magnet and/or electromagnet are localized at various preferably opposite sides of the tumor.
In a very preferred embodiment, means for applying a mechanical and/or physical constraint (such as for example external magnets) are located outside the tumor, preferably located outside the body of the subject. In one embodiment, the means are moving, preferably turning around the tumor region. In one embodiment, the means are located at a distance at the tumor ranging from 0.1 to 100 mm; preferably 1 to 10 mm.
In one embodiment, the magnetic field is a variable or constant external field, having a frequency varying or ranging from 0.1 Hz to 1 TeraHz, preferably from 1 Hz to 1 MHz, more preferably from 10 Hz to 500 kHz.
In one embodiment, the magnetic field ranges from 0.1 μT to 50 T, preferably from 0.01 mT to 5 T, more preferably from 0.1 mT to 700 mT.
In one embodiment, the magnetic field is moving, preferably turning around the tumor region.
In one embodiment, the magnetic field is not an alternative field. In one embodiment, the magnetic field is not an alternative current electric field.
In one embodiment, the gradient of magnetic field 2 varies in intensity, frequency and/or in direction. Advantageously, the gradient of magnetic field 2 is applied in a way such that the constraint on the tumor varies in intensity, direction and frequency. One skilled in the art shall modify the intensity, frequency and/or in direction of the gradient of magnetic field, depending on the location of the tumor and its size and possibly on other biological data.
In one embodiment, the gradient of magnetic field 2 ranges from 0.001 to 10 T/cm, preferably from 0.01 to 1 T/cm, more preferably from 0.1 to 0.5 T/cm; even more preferably around 0.4 T/cm.
In one embodiment, the frequencies of the gradient of field 2 may be varied to search and reach a constraint field having the resonance frequency, or harmonics thereof, of the cytoskeleton and/or of cells walls and/or of the surrounding soft and/or hard tissues. Advantageously, the frequency of the gradient of field may be adapted so that the constraint field triggers the resonance frequency of the continuous assembly formed by the tumor cells components in the tumor.
In one embodiment, the system comprises means to vary the direction of the magnetic field or the direction of the generator, in order to direct the field.
Magnetizable Nanoparticles
In one embodiment, the particles or nanoparticles 1 comprises, are composed of or consists of any magnetizable material, preferably, iron oxide or manganese, more preferably magnetite (Fe3O4), maghemite ([gamma]-Fe2O3) or ferumoxides, i.e. a dextran-coated colloidal iron oxide, or mixtures thereof.
By particle, it is understood in the present invention, one unitary particle or an assembly of nanoparticles.
In one embodiment, the particles or nanoparticles 1 have a size or a hydrodynamic volume such that they cannot be uptaken by the tumor cells.
In one embodiment, the particles or nanoparticles 1 have a size or a hydrodynamic volume such that they are retained by the neoangiogenic network 7 surrounding the tumor and remain at the periphery of the tumor without entering into the tumor or the tumor cells.
In one embodiment, the particles or nanoparticles 1 have a mean diameter ranging from 30 to 1000 nm, preferably 40 to 800 nm, more preferably 60 to 300 nm.
In a very preferred embodiment, the particles or nanoparticles have a mean diameter higher than 100 nm in order to avoid capture by the cell tumors and/or organs; preferably about 200 nm.
In one embodiment, the quantity of the particles or nanoparticles 1 loaded in the body is ranging from 0.1 mg to 1000 mg, preferably 1 to 10, more preferably about 5 mg.
In one embodiment, the particles or nanoparticles 1 are not coated.
In one embodiment, the particles or nanoparticles 1 are coated with a non-pharmaceutical composition, preferably said nanoparticles 1 are coated with a hydrophilic coating, more preferably dextran.
In another embodiment, the particles or nanoparticles 1 are coated with a pharmaceutical composition.
In another embodiment, the particles or nanoparticles 1 are coated with at least one biochemical agent; preferably a peptide; more preferably, arginylglycylaspartic acid (RGD).
In another embodiment, the particles or nanoparticles 1 may be helped in their migration towards the tumor region, for example through specific coatings applied onto the nanoparticles 1 prior to injection.
In one embodiment, the particles or nanoparticles 1 are formulated within a suspension, wherein the particles or nanoparticles 1 are suspended in a colloidal or non-colloidal vehicle. In this embodiment, the injection device encompassed in this invention comprises a suspension comprising the magnetizable particles or nanoparticles 1 as described above.
In one embodiment, the particles or nanoparticles 1 are formulated within an emulsion, wherein the particles or nanoparticles 1 are in an emulsion. In this embodiment, the injection device encompassed in this invention comprises an emulsion comprising the magnetizable particles or nanoparticles 1 as described above.
This invention also relates to method for treating tumors by physical and/or mechanical means, especially tumors associated with a neoangiogenic network 7, comprising the steps of:
The nanoparticles 1 used in the method of the invention are as described above. According to the invention, at least 25-60% of the injected particles or nanoparticles 1 are retained in the tumor neoangiogenic network 7. Without willing to be linked by a theory, the Applicant tends to believe that the particles or nanoparticles 1 of the system of the invention, when injected, benefit from the well-known Enhanced Permeability and Retention (EPR) effect, and preferably mainly from the enhanced retention effect, with regard to the neoangiogenic network 7. The EPR effect is the property by which certain sizes of particles or nanoparticles 1 tend to accumulate in tumor regions much more than they do in normal tissues. The EPR effect helps directing the particles or nanoparticles 1 to the tumor region. However, due to the nature and the size of the particles 1 of the invention, they may not enter or contact the tumor cells, and get retained in the neoangiogenic network 7 surrounding the tumor.
In one embodiment, the EPR ratio of the particles or nanoparticles 1 of the invention, i.e. the amount of injected particles or nanoparticles 1 reaching the neoangiogenic network 7 ranges from 25% to 60%. In another embodiment, the EPR ratio ranges from 30% to 40% of the injected particles 1. The EPR ratio may vary, depending on the size of the particles or nanoparticles 1 and/or the location of the tumor region and/or the histological nature of the tumor.
The particles or nanoparticles 1 are retained by the neoangiogenic network 7 surrounding the tumor; in one embodiment, the distribution of the particles or nanoparticles 1 forms a hollow and discontinuous sphere.
According to the invention, the particles or nanoparticles are not retained by or in the stroma.
According to the invention, the particles or nanoparticles 1 may not contact the tumor cells. In one embodiment, the distance between a tumor cell and the particles or nanoparticle 1 is at least 1 (one) micron, preferably 5 to 100 μnm.
In one embodiment, the amount of injected particles or nanoparticles 1 ranges from 100 μg to 10 g, preferably from 500 μg to 2 g.
The means for applying a contact-free constraint is an external generator as described above. By external is meant that the generation of the gradient of magnetic field 2 is located outside the tumor, preferably located outside the body of the subject. In one embodiment, the gradient of magnetic field 2 is applied for a period of time, ranging from 1 minute to 48 hours, preferably 15 minutes to 5 hours, this period of time being repeated over several weeks.
In one embodiment, the subject is an animal, including a human. The subject may be a male or a female. This subject encompasses, within his/her/its body a tumor associated with an angiogenic network 7. In one embodiment, the tumor is a locally advanced tumor. In one embodiment, the tumor is a malignant epithelial tumor. In one embodiment, the malignant tumor is pancreas cancer tumor. In another embodiment, the malignant tumor is breast cancer tumor.
This invention also relates to a method for modifying the cancerous phenotype of cancer cells to normal cells in a target region, the method comprising the steps of:
In particular, the invention also relates to a method for reducing the volume and the surfaces of tumor cells in a target region, the method comprising the steps of:
The present invention is further illustrated by the following examples.
In
Objectives
The aims of the study were to analyze 10 human tumors (MDA-MB-231, breast cancer cell line), subcutaneously grafted in mice, and investigate:
Five mice 10 were subcutaneously grafted between skin (West side) 16 and muscle (East side) 15 with human cells of cell line MDA MB 231 (two xenografts per mouse). At the end of the study, all tumors were collected, fixed in 4% formalin and included in paraffin. One of the two xenografts from each mouse 10 was processed for evaluating histopathological features of the tumors (HES staining). Both xenografts samples from each mouse were processed for evaluating tumor cells iron accumulation (Perls special stain). In this study, the ten evaluated MDA-MB-231 xenografts presented comparable histological features, and appeared as undifferentiated carcinomas. Necrotic areas 13 were seen in the central parts of every tumor. They varied from 60% to 80% of the tumor surface. Iron deposits were located in the peri-tumoral areas. They appeared as brown granular deposits on HES stained slides and as dark blue granular deposits in Perls stained slides. No iron deposits were identified in tumor cells, in any tumor of any group.
Minimal deposits were observed in the cytoplasm of macrophages, and the necrotic areas 13.
Results
Tumor Histopathology (HES Staining)
Tumor microscopic features were evaluated on HES stained slides.
In this study, the evaluated MDA-MB-231 xenografts presented comparable histological features, and appeared as undifferentiated carcinomas, without glandular differentiation. These tumors were densely cellular, quite well demarcated and roughly nodular, with infiltrative growth in adjacent subcutaneous tissues, in particular nerves.
Quite numerous inflammatory cells were found in peri-tumoral fibrous stroma, as well as in adjacent tissues in smaller amounts (adipose tissue, skeletal muscle, skin). Those cells mostly consisted in pigment-laden macrophages, with some small lymphocytes and occasional granulocytes. Low numbers of similar inflammatory cells, including pigment-laden macrophages, were also observed in the scant intra-tumoral stroma. Those cells were mainly observed in peripheral parts of the tumor, probably spreading from the peri-tumoral stroma. Pigment-laden macrophages were medium to large-sized cells, and displayed a large cytoplasm which contained large amounts of brown granular pigment, often overlying and hiding round nuclei. Such pigment was occasionally found in the peri-tumoral and intra-tumoral stroma as granular pigment debris.
Tumor Cells Iron Accumulation (Perls Special Stain)
Perls' acid ferrocyanide reaction revealed iron compounds through dark blue labeling. For each tumor, three consecutive sections were evaluated. All three of those sections displayed comparable features.
No blue-stained iron deposits were identified in tumor cells, in any tumor of any group.
Conclusions
The particles 1 were distributed around the tumor and surprisingly formed a hollow and discontinuous sphere.
The conditions for applying a constraint on the tumor seem to be gathered.
Protocol
The aim of the study is to investigate the antitumor efficiency of nanoparticles activated by a magnetic field in the model of subcutaneous MDA-MB-231 human breast tumor bearing mice.
Test Substance
Treatment Doses—Route of Drug Administration
The amount of iron injected with cells is 5 mg. Nanoparticles are administered with tumor cells (at the time of tumor cells injection) to Balb/c Nude mice. The injection volume for the Nanoparticles is 300 μL/mouse/injection.
Animals
Forty five (45) female Balb/c Nude mice, 6-7 week-old and weighing 16-20 g at reception, were obtained from Charles River (France). Animals are observed for at least 7 days in a specific-pathogen-free (SPF) animal care unit before treatment. The animal care unit is authorized by the French ministries of Agriculture and Research (Agreement No. A21231011EA). Animal experiments are performed according to ethical guidelines of animal experimentation and the English guidelines for welfare of animals in experimental neoplasia. All procedures with animals are submitted to the Animal Care and Use Committee of Pharmacy and Medicine University (Dijon).
Submission of Animals to a Magnetic Field
When submitted to the magnetic field using magnet-bearing devices, animals are anaesthetized (with isoflurane) and body temperature of mice is maintained within physiological levels by infrared lamps.
When the tumors reached approximately one centimeter tumors in the treated group (i.e. Right Tumor G3, as explained hereafter), the mice 10 were placed in the gap of two magnets 9 in repulsive mode (see
A gradient of magnetic field 2 is then applied. The gradient applied is about 0.4 Tesla/cm at a depth of 3 mm.
Cancer Cell Line
The cell line that is used is detailed in the table hereafter:
Induction of MDA-MB-231 Tumors in Female Balb/c Nude Mice
Ten millions DA-MB-231 tumor cells resuspended in a volume of 0.3 mL RPMI 1640 medium containing 5 mg of nanoparticles or no nanoparticles are subcutaneously inoculated in the flanks of 45 female SWISS Nude mice, irradiated 24-72 hours before with a γ-source (whole body irradiation, 2 Gy, 60Co, BioMEP Sarl, Bretenière, France).
Tumors cells without the nanoparticles are injected in the left flank of all 45 mice.
Tumors cells with the nanoparticles are injected in the right flank of all 45 mice.
The day of tumor cells injection is considered as the day 0 (D0).
Treatment Schedule
The tumor volume is estimated by the formula:
Tumor volume=½×length×width2
When the mean tumor volume reaches approximately 100-200 mm3, 36 tumor bearing female Balb/c Nude mice are randomized into 3 groups (one group of 8 mice and two groups of 14 mice) according to their individual tumor volumes. The treatment schedule was chosen as follows:
Treatments (i.e. submission to the magnetic field) start on the day after randomization. The table 1 below summarizes the treatment schedule:
Thus the treated group—group 3 right tumor—was compared with three control groups:
Tumors are resected and cut into two pieces. The two pieces will be fixed in 10% neutral buffered formalin.
Results
The treated group (with nanoparticles 1 and with gradient field 2, i.e. Right Tumor G3) was compared with three control groups: There were two statistically significant changes (p<0.05):
On the one hand the overall tumor growth 11/12 is lower in the treated group compared to the three control groups, see
On 59 days after graft (D59), the tumoral volume is much lower in the group Right Tumor G3 (7 mice), than all control groups (33 mice): the median in the treated group is 529.4 mm3 whereas the median in the control group is 1334 mm3 (p=0.014<0.05).
The result on the tumor is shown in
To the Applicant's knowledge, it is the first evidence in vivo action of physical constraint generated by a gradient of magnetic field on tumor growth.
Objectives
The aim of the study is to investigate the reduction of the tumor volume and the surfaces due to nanoparticles activated by a gradient of magnetic field in the model of subcutaneous MDA-MB-231 human breast tumor bearing mice.
Summary
Mice were subcutaneously grafted between skin and muscle with human cells of cell line MDA MB 231. When tumors reached approximately one centimeter tumors in the treated group, the mice were placed in the air-gap of two magnets in a repulsive mode with a particular geometry as shown
Experimental Groups
79 mice having a one-centimeter-sized tumor were distributed in 4 groups:
The day of tumor cells injection is considered as the day 0 (D0). The volume is calculated in mm3.
Results
The treated group (G1′) was compared to the whole untreated group comprising G2′, G3′ and G4′. The table 2 below and
p-Values
The Wilcoxon test for comparing the median values between the treated group (G1′) and the other groups (untreated) gives a p-value equal to 0.0146 (<0.05).
Thus, the treatment of breast tumor cells by magnetic particles under a gradient of magnetic field significantly reduces the tumor volume compared to the untreated groups (G2′, G3′ or G4′).
G1′ Versus Each Untreated Group
Then, the treated group (G1′) was compared to each untreated group (G2′, G3′ or G4′). The table 3 below and
It appears that the treated group (G1′) is the only group to provide the lower tumor volume.
Thus, this example shows that the medical system comprising injectable nanoparticles and a gradient of magnetic field is the best method to reduce a tumor volume by mechanical and/or physical means.
The finer analysis of tumors allowed comparing the West side and the East side. Total surface measurement is available for 51 tumors and the results are shown on Table 4 and
p-Values
A one-way analysis of variance for the West and East surface was performed with log-transformation on the group encompassing both the treated group (G1′) and the control groups (G2′-G4′):
These first results show that the group has a significant global effect both on the West and the East surface of the tumor.
p-Value Comparison Between Untreated Groups
The pairwise comparison using Tukey's adjustment between untreated groups (one-way analysis of variance) led to the results shown in Table 5.
All p-values are higher than 0.05, so there is no significant difference between the three groups untreated as expected.
p-Value Comparison Between the Treated Group (G1′) and an Untreated Group
Finally, a mixed up of this 3 groups is realized and one-way analysis of variance is performed using contrast option shown in Table 6.
Surprisingly, the results show that the surfaces of the treated group are significantly different to untreated groups (mixed up). The surfaces on group treated are lower than on group untreated (estimate of difference is equal to −0.344 log for West side and to −0.485 for East side).
Conclusion
These results demonstrate that the Applicant has discovered a medical system allowing acting on the phenotype of tumor cells, reducing the tumor volume and the surfaces exposed to a mechanical and/or physical constraint implemented by injectable particles and an external gradient of magnetic field.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13176364 | Jul 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/064995 | 7/11/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/004285 | 1/15/2015 | WO | A |
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