WO2020254430A1

WO2020254430A1 - Medical device for applying plasma

Info

Publication number: WO2020254430A1
Application number: PCT/EP2020/066802
Authority: WO
Inventors: Laura FOUASSIER; Thierry Dufour; Florian JUDEE; Sylvain PLEDEL; Javier VAQUERO
Original assignee: INSERM (Institut National de la Santé et de la Recherche Médicale); Sorbonne Universite
Priority date: 2019-06-17
Filing date: 2020-06-17
Publication date: 2020-12-24

Abstract

The present invention relates to medical device configured for applying a plasma jet on a target, wherein the medical device comprises a tip configured to homogenize and enlarge the plasma in contact with the target. Based on a study of porcine and murine models, the inventors have clearly demonstrated that cold plasma drive to anti-tumor effects without causing deleterious consequences (burnings). In order to enhance this effect, the inventors further developed a removable tip which in more to homogeneize and enlarge plasma ensures a better control of its physico- chemical properties over treatment time. In another aspect, the invention relates to method of treatment of cholangiocarcinoma comprising irradiating a tumor with a cold plasma jet. In yet another aspect, the invention relates to method for manufacturing a removable tip according to the present invention.

Description

MEDICAL DEVICE FOR APPLYING PLASMA

The present invention relates to the field of cancer therapy and more specifically to the field of plasma therapy.

Cold atmospheric plasmas are weakly ionized gases containing energetic and chemical transient species (electrons, ions, metastables, radicals) while presenting radiation, gas flowing and electromagnetic field properties . In laboratory, they can be easily generated by supplying electrical power to a device containing either one or two electrodes. The electrode connected to the high voltage power supply is commonly referred as the exciting (or powered) electrode while the second optional electrode is referred as the counter electrode and is usually brought to the ground. As a result, a non-linear and high magnitude electric field can be generated to create electrical discharges that partially ionize the gas into cold plasma. Of the most commonly cold plasma devices used in laboratories, the dielectric barrier discharge (DBD) appears ubiquitous owing to its low manufacturing and implementation costs, as well as its great versatility in regard of the many diversified applications like ozone generation, incoherent excimer UV radiation, air purification, surface modifications, etc.

In these DBDs, one electrical insulating layer, typically a dielectric material like quartz or alumina is utilized as a barrier to prevent arcing from plasma current. Whatever their 1 or 2 electrode(s) configurations, two types of DBD can be distinguished: (i) the non-flowing DBDs where the powered electrode is enwrapped by an insulator material and where plasma remains confined in the interelectrode region (ii) gas-flowing DBDs where plasma is located in this region as well as further away : in the post-electrode region. There, the ionized gas is referred as a plume to design its emissive properties although long lifetime radicals can propagate much further away in the post discharge without emitting any radiation. The gas-flowing DBDs are more commonly referred as plasma jets and more specifically as atmospheric pressure plasma jets (APPJ) if they operate in ambient air. One can distinguish two types of APPI configurations: (i) APPJ devices with a single metal electrode biased to the exciting potential (typically high voltage) while the counter-electrode is the biological target (grounded or floating potential) exposed to plasma, (ii) APPJ devices with two metal electrodes (exciting electrode and counter electrode) while the biological target can eventually play a role of third electrode. Among the ‘two-electrodes APPJ’ successfully applied upon in vivo experiments, the plasma gun (PG) is composed of an outer ring electrode and an inner pin electrode directly in contact with the gas/plasma. Another configuration of interest, referred in this article as plasma Tesla jet (PTJ), presents two distant ring electrodes located on the outer tube.

Due to its metastable nature at atmospheric pressure, applying cold plasma uniformly on a large targeted area (typically a few square centimeters) and over a long period time is often uneasy and drives to non-homogeneous treatment of this target. Such instabilities are particularly true in the case of two-electrode DBDs due to the lack of parallelism between the two electrodes as well as to boundary effects . In the case of an APPJ, the plasma plume interacts with the ambient air and generates radicals as well as long lifetime reactive species . Owing to the relative humidity in the ambient air, the gaseous densities of these latter species is rarely controlled. Moreover, the chemical composition of the ambient air being essentially limited by a N2/O2 ratio close to 3.7, the gaseous chemistry operating in the plasma plume remains limited. Surprisingly, in the field of plasma medicine, reactive species are often considered as one of the main reason of therapeutic effects demonstrated upon in vitro and in vivo experiments.

Due to the difficulties inherent to its application, therapeutic use of cold plasma is a field which remains still largely unexplored.

Since plasma jet devices have already successfully demonstrated antitumor effects, there is a need to provide technological incrementations to enlarge and homogenize the plasma plume upon its interaction with a tumor, with the aim to cover it entirely.

To that end, a first aspect of the present invention relates to a medical device configured for exposing a target tumor to a plasma jet, wherein the medical device comprises a tip configured to enlarge and homogenize the plasma jet in contact with the exposed target. Obviously, this interestingly allows improving the quality, the versability and the reproducibility of the plasma therapy.

Typical medical devices used to generate cold plasma include dielectric barrier devices. For instance, such device can comprise a dielectric tube in which at least one gas such as helium is injected. Two distant metallic rings are maintained around the tube and a voltage is applied on them so as to create an electric field in the tube, located between the electrodes. The potential difference is chosen so as to ionize the gas flowing inside the tube and thus to generate a flowing cold plasma which propagates outside the tube for a short distance, forming a so-called“plasma jet”, which can also be referred to as a plasma plume when considering the plasma which goes beyond the electrodes.

In order to enlarge and to homogenize the plasma jet, the invention provides a tip which can be set up between the tube’s end and the tumor tissue to be treated.

Preferably, the target tumor has an external location. As a matter of fact, although cold plasma can penetrate skin to a certain extent, or be applied through surgery, the invention is easier to use on external tissues, whether lesions, tumors or inflammated. As a consequence, exemplary target tumors of choice for the present invention comprise melanoma and ectopic tumors such as cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a heterogeneous group of aggressive malignancies that can emerge at every point of the biliary tree from the canals of Tiering into the liver to the main bile duct. CCA is the second most frequent type of primary liver cancer and -3% of all gastrointestinal neoplasia. Cholangiocarcinoma are generally asymptomatic in early stages, they are diagnosed when the disease has already metastasized, drastically complicating their therapeutic treatment options. Surgical resection is the only effective therapy, but it can only be applied in 20% of patients and the 5 year survival rate remains as low as 15%-40%. Most of the patients who cannot benefit from surgery undergo a palliative treatment with a combination of gemcitabine and oxaliplatin platinum salt (GEMOX), the only chemotherapy validated for advanced unresectable CCA (Valle et al 2010). In case of tumor progression after this first line of treatment, there is no other treatment approved to date. Tumor size and other features (anatomical location, vascular and lymph node invasion and metastasis) condition the potential surgical and/or radiological options but chances of recurrence are very high. Owing to these limitations, the emergence of new therapeutic options is eagerly needed.

Preferably, the tip comprises a hollow cavity configured to be in contact with the target tumor. This cavity can be of any shape and allows confining the plasma plume from the ambient atmosphere, thus preventing the plasma jet to dissipate or to interact with the oxygen of the atmosphere without control, thus allowing the plasma to be homogenously applied to the treated zone.

The shape of the cavity can be designed and engineered to encompass the size of the tumor, taking into account its topography and improve the effectiveness of the exposure. Indeed, the effectiveness depends both on the exposure time and on the electrical power of the plasma. Although the electrical power cannot be easily measured with high accuracy, it is largely correlated with the gas partial pressure per surface of exposure and to the plasma, both of which can be controlled by the shape of the tip.

Preferably, the cavity is a cone-shaped, a truncated cone-shaped or a gradual curve-shape cavity. Such shapes will allow smoothly increasing or decreasing the section of tumor tissue exposed to the plasma.

As such, the cavity can present a sectional area which increases outward; such cavity will be referred to as A-shaped cavity and allows increasing the surface of exposure. The term outward is to be understood as meaning from the plasma generation device toward the lesion to be exposed.

Alternatively, the cavity presents a sectional area which decreases outward; such cavity will be referred to as V-shaped cavity and allows concentrating the plasma when the surface of exposure can be decreased. The term outward is to be understood as meaning from the plasma generation device toward the lesion to be exposed.

The tip can be made of any material which does not conduct electrical charges and preferably biocompatible such as polymers, most of 3D-pintable materials, etc.

Preferably, the cold plasma is to present a gas temperature lower than 40°C to not induce drying effects of the tissues although these effects are time-dependant and although thermal effects might be intended for other applications where hyperthermia could play a therapeutic role. In all cases, the gas temperature cannot exceed the temperature at which the insulator material constituting the tip starts to induce a change of its state (typically vitreous transition temperature, melting temperature, etc.)·

Preferably, the tip is removable. As a matter of fact, depending on the size and on the location of the target tumors, it may be necessary or interesting to opt for a different tip. Furthermore, a removable tip couldbe interestingly installed on pre existing medical devices.

The tip can comprise lateral micro-holes, preferably lateral micro-holes presenting a diameter comprised between 0.1 mm to 10 mm with a typic size of 1 mm. These holes can be drilled to ensure two functions: first, when the tip is in perfect contact with the target (no possible gas leakage), the surpressure of gaseous plasma contained into the cavity can be evacuated through these holes; second, offering the opportunity to connect through these holes captors that will monitor parameters of interest in real time (e.g. chemical composition, gas temperature, etc.).

The invention also relates to a method of treatment of cholangiocarcinoma comprising irradiating a tumor with a cold plasma jet.

The tumor can e.g. be exposed to the cold plasma jet for a duration comprised between a few seconds to several minutes.

The plasma jet plume is preferably confined so as to prevent ionization of ambient air, although some lateral holes can be drilled into the tip to enable the plasma gas to be released in the air and therefore to prevent any surpressure or transient plasma phenomena propagating at counter-flow.

The method according to the present invention preferably comprises using a medical device according to the invention.

The present invention also relates to a removable tip for a medical device according to the invention.

The present invention also relates to a method for manufacturing a removable tip according to the present invention. The invention can be better understood at the reading of the detailed examples below, which constitute non-limitative embodiments of the present invention and at the examining of the annexed drawing, on which:

Figure 1 shows different medical devices according to the invention exposing an ex- vivo target to a plasma jet,

Figure 2 is an infrared photography of a plasma jet device treating a target tumor, and

Figure 3 is a flowchart representing the volumic growth of tumors with and without plasma treatment.

It is understood that the described embodiments are not restrictive and that it is possible to make improvements to the invention without leaving the framework thereof.

In case of the present embodiment, a cold plasma operating at atmospheric pressure was generated in a dielectric barrier device in which a flow of helium was flowing at a rate of 1 L per minute. In order to ionize the flowing helium, the dielectric barrier device was powered by a AC high- voltage electric generator. The pulses presented an amplitude of 8000V and were delivered at a frequency of 10kHz for a duty cycle of 14% .

Post-mortem porcine skin samples presenting a dimension comprised between 3.5 and 4.5 cm² were used for the sake of this embodiment. The exposure time was set to 2 minutes .

The samples were placed on a metal plate connected to a resistance and a capacitor mounted in parallel so as to mimic the electrical response of the animal model under plasma exposure.

If no tip was used, the diameter of the plasma plume in contact with the targeted tissue was lower than 2mm. Adding a tip with either an A-shaped or a V-shaped cavity allowed to significantly increase the diameter of the plume upon its contact with the target.

The A-shaped tip allowed to triple the diameter whereas the V-shaped tip allowed to multiply it by five. Depending on the topology and size of the targeted tumor, it is possible to engineer a tip which can cover the whole tumor volume so as to provide a homogenous treatment. Without any tip, the plasma must be moved above the tumor so as to effectively scan the whole region to be treated. Such step is inconvenient, potentially dangerous, and renders the treatment heterogeneous.

Figure 1 shows a cold plasma jet generated with an A-shaped tip ( l a, lb, l c) and with a V-shaped tip ( Id, le, If) and applied to a target sample at different distances. Fig l a and Id correspond to a gap of 10 mm between the extremity of the tip and the target sample, fig lb and le correspond to a gap of 5mm and fig l c and If correspond to a gap lower than 1 mm.

It shows that in order to obtain an optimal effect, the tip should be applied right on the target sample. Otherwise, the homogenization and spreading of the plasma plume do not occur in the tip cavity whilst the plasma jet keeps propagating in a thin ray that focuses on the sample tissue.

Figure 2 is an infrared photography which allows mapping the temperature of the plasma jet. Figure 2a corresponds to a set-up without any tip, figure 2b to a set-up with an A-shaped tip, and figure 2c to a set-up with a V-shaped tip.

These pictures clearly show that the gas temperature of plasma stays well under 30°C, thus preventing any burns of the target sample and authorizing treatment times potentially larger than a few minutes.

The current therapeutic effect of the plasma has been evidenced even without any tip in a subcutaneous xenograft tumor model performed with EGI- 1 cholangiocarcinoma cells.

A few days after injection of tumor cells under mice skin, the proliferation of these cells has been high enough to create a tumor exhibiting a measurable volume that mimicks human tumor (Vaquero et al 2018). As shown in Figure 3, when tumor volumes reach an approximate volume as high as 200 mm³, two successive PG treatments are achieved at days 13 and 20 for an exposure time of 1 min, repetition frequency of 1 kHz and D_Cycie=14%. The tumor volumes remain the same versus time whatever the control and plasma groups. Then, after leaving a 14 d refractory period, the tumors are treated using the PTJ at days 34 and 41 with the same aforementioned plasma conditions. However in that latter case, there was a strong reduction of tumor growth in the plasma group compared with the control group. At day 48, tumor volumes remain limited to 1250 mm³ for mice belonging to the plasma group versus 1730 mm³ for mice belonging to the control group. Besides, the p-value between these two tumor volumes is lower than 0.05, hence underlining the significant therapeutic effect of the plasma therapy. Experiments have not been carried out further to respect ethical protocols limiting tumor volume to less than 2000 mm³ for the two groups.

Altogether, the dermal toxicity test and the xenograft tumor experiments show that the PTJ is as safe as the PG and displays in vivo antitumor properties. The PTJ properties are more interesting since PTJ effect appears the day after PTJ treatment, i.e. at day 35 in the in vivo experiment and that this effect is obtained at a very advanced stage of tumor development.

To understand why one of the two plasma sources validates an anti-tumor effect and not the other, we propose to briefly compare them at the light of the usual cold plasma properties, i.e. radiative, thermal, chemical, electrical and gas flow properties. The gas temperature (neutral species) is always close to 305 K (±6 K) whatever the plasma source (with the exception of the arc regime which cannot be applied on living organisms). Therefore, we assume that for such a low value, the temperature cannot induce antitumor effect owing to the absence of such results with the PG. Regarding flow properties, it has been shown that profiles may significantly differ if the target is changed but not when the PG is replaced with the PTJ. Again, in our own experimental conditions, the impact of flow properties can be considered as negligible to induce antitumor effects. The radiative properties are stronger with PG than with PTJ whereas more chemical species may be produced with PTJ, likely to explain the tumor size reduction. In our in vivo experiments, the tumors were ectopically grafted on mice, i.e. covered by a thin skin layer likely to mitigate the diffusion of exogeneous radicals from plasma. Since reactive species can be delivered several millimeters into tissues (Szili et al 2018), it is important to identify if those detected with PTJ and PG are likely to induce antitumor effects: - The hydroxyl (OH) radical is known as the most electrophilic ROS with high reactivity. It can cause oxidative damage to DNA, proteins and lipids as long as it is produced in their vicinity (Hadi et al 2010, Cadet and Davies 2017). In our research works, even if OH radicals are significantly produced with the two APPI, their therapeutic potential remains questionable in inducing antitumor effects.

- Low concentrations of extracellular singlet oxygen can inactivate catalase on the membrane of tumor cells and thus abrogate the antioxidant activity of one of the central molecules of tumor cells (Riethmiiller et al 2015). Although produced in low amounts with our APPJ, the role of O radical as antitumoral agent must not be underestimated. In the vicinity of inactivated catalase, it could prevent NO from oxidation and prevent ¾0 and peroxynitrite (constantly produced outside of tumor cells) to be decomposed (Bauer 2016). Then, the subsequent protonation of perox ynitrite into peroxynitrous acid can enable the production of intracellular NO2 and hydroxyl radicals. - To the best of the authors knowledge, the H radical is not described in the literature as a candidate likely to induce strong anticancer effects. For these reasons, H radicals can reasonably be considered as playing a negligible role in the demonstrated antitumor effects.

- As reminded by D. Graves (2014), nitric oxide (NO) is a biologically significant molecule that can induce several pivotal effects, e.g. immune modulation of tumor growth, modulation of angiogenesis and inhibition of cell respiration (Janakiram and Rao 2015, Morbidelli et al 2019). Although NO has not been investigated in the preset study, further works could be carried out to generate it on purpose, as selectively as possible. Finally, the plasma electrical properties are also assumed to play an important role in the antitumor effect. Depending on whether PG or PTJ is used on the same target, the resulting V=f(t) profiles could highlight substantial differences (change in voltage polarity, change in pulses duration, ...).

Claims

I. Medical device configured for exposing a target tumor to a plasma jet, wherein the medical device comprises a tip configured to homogenize and enlarge the plasma jet in contact with the exposed target.

2. Medical device according to claim 1, wherein the target tumor has an external location.

3. Medical device according to claim 2, wherein the target tumor is a melanoma or a cholangiocarcinoma.

4. Medical device according to any of claim 1 or 3, wherein the tip comprises a hollow cavity configured to be in contact with the target tumor.

5. Medical device according to claim 4, wherein the hollow cavity is a cone- shaped, a truncated cone-shaped cavity or a gradual curve-shaped cavity.

6. Medical device according to claim 4 or 5, wherein the cavity presents a sectional area which increases outward.

7. Medical device according to claim 4 or 5, wherein the cavity presents a sectional area which decreases outward.

8. Medical device according to any of claims 1 to 7, wherein the cold plasma is to present a temperature inferior to 30°C.

9. Medical device according to claims 1 to 8, wherein the tip comprises lateral micro-holes , preferably lateral micro-holes presenting a diameter comprised between

0.1 mm to 10 mm

10. Medical device according to claims 1 to 9, wherein the tip is removable.

I I . Method of treatment of cholangiocarcinoma comprising irradiating a tumor with a cold plasma jet.

12. Method of treatment according to claim 11, wherein the plasma jet is confined so as to prevent ionization of ambient air.

13. Method of treatment according to claim 11 to 12, wherein the method comprises using a medical device according to any of claim 1 to 10.

14. Removable tip for a medical device according to any of claims 1 to 10.

15. Method for manufacturing a removable tip according to claim 14.