KIT FOR INHALED CHEMOTHERAPY, AND TREATMENT OF LUNG CANCER WITH SAID KIT

Abstract

A kit for an inhaled chemotherapy includes a series of single hermetically packaged therapeutic doses of a dry powder for inhalation. The dry powder includes at least one anti-neoplastic agent chosen from the group that includes platinum drugs with a lipid matrix and a PEGylated excipient and a series of disposable dry powder inhaler devices. The use and a method of treatment of cancer lungs with said kit is also described.

Claims

1. A kit for an inhaled chemotherapy comprising: a) a series of single hermetically packaged therapeutic doses of a dry powder for inhalation, the dry powder containing at least one anti-neoplastic agent with a lipid matrix and optionally one or more excipient, such as a PEGylated excipient and/or a carrier; b) a series of disposable dry powder inhaler devices, wherein i) each single hermetically packaged therapeutic dose having a close state and an open state, ii) each disposable dry powder inhaler device having a close position and optionally an open position, iii) each single hermetically packaged therapeutic dose being provided for being accommodated in said close state in one disposable dry powder inhaler device of said series of disposable dry powder inhaler devices, and iv) each single disposable dry powder inhaler device is provided to be in said close position before or once each single hermetically packaged therapeutic dose become in said open state.

2. The kit for an inhaled chemotherapy according to claim 1, wherein said single hermetically packaged therapeutic dose is one of a capsule and a blister.

3. The kit for a inhaled chemotherapy according to claim 1, wherein the lipid matrix is a matrix of pure or a mixture of triglyceride.

4. The kit for an inhaled chemotherapy according to claim 1, wherein the PEGylated excipient is derived from one of vitamin E and phospholipids.

5. The kit for a inhaled chemotherapy according to claim 1, wherein each disposable dry powder inhaler device comprises a dispersion chamber in fluid connection with an inhalation channel and connected to a loading chamber configured to accommodate said single hermetically packaged therapeutic dose inside said disposable dry powder inhaler device, said loading chamber comprising activation means having an activated position and a rest position, said activated position being configured to open said single hermetically packaged therapeutic dose and locking means configured to prevent extraction of the single hermetically packaged therapeutic dose once said activation means have been moved to said activated position.

6. The kit for a inhaled chemotherapy according to claim 1, wherein each disposable dry powder inhaler device comprises a dispersion chamber in fluid connection with an inhalation channel and connected to a loading chamber configured to accommodate said single hermetically packaged therapeutic dose inside said disposable dry powder inhaler device, said loading chamber comprising activation means having an activated position and a rest position, said activated position being configured to open said single hermetically packaged therapeutic dose and locking means having an open position and a lock position configured to prevent extraction of the single hermetically packaged therapeutic dose, said activation means being provided to be able to be moved to said activated position only when said locking means are in said lock position.

7. The kit for a inhaled chemotherapy according to claim 1, wherein each disposable dry powder inhaler device comprises a dispersion chamber in fluid connection with an inhalation channel and connected to a loading chamber configured to accommodate said single hermetically packaged therapeutic dose inside said disposable dry powder inhaler device, said loading chamber comprising activation means having an activated position and a rest position, said disposable dry powder inhaler device comprising closing means configured to isolate an outside environment from an inside of the disposable dry powder inhaler device, in particular by closing the inhalation channel.

8. The kit for an inhaled chemotherapy according to claim 1, wherein said disposable dry powder inhaler device is ready to use and comprises a dispersion chamber in fluid connection with an inhalation channel and connected to a loading chamber comprising said single hermetically packaged therapeutic dose inside said disposable dry powder inhaler device.

9. The kit for a inhaled chemotherapy according to claim 1, wherein said single hermetically packaged therapeutic dose comprises a therapeutic dose in the form of a dry and fine powder having a geometric particle size distribution (PSD) d.sub.50 lower than or equal to 30 μm and a geometric particle size distribution (PSD) d.sub.90 lower than or equal to 60 μm.

10. The kit for an inhaled chemotherapy according claim 1, wherein each single hermetically packaged therapeutic dose comprises a total powder weight content between 2 to 100 mg.

11. The kit for an inhaled chemotherapy according to claim 1, wherein each single hermetically packaged therapeutic dose comprises between 0.1 and 90 mg of said anti-neoplastic agent.

12. The kit for an inhaled chemotherapy according to claim 1, wherein each single hermetically packaged therapeutic dose comprises from 0.1 to 99.9 mg of said lipid matrix.

13. The kit for an inhaled chemotherapy according to claim 1, wherein each single hermetically packaged therapeutic dose comprises from 0.01 to 5 mg of PEGylated excipient.

14. The kit for an inhaled chemotherapy according to claim 1, configured for a polytherapy.

15. The kit for an inhaled chemotherapy according to claim 14, wherein said polytherapy comprises one therapy chosen from the group consisting of intravenous infusion or oral chemotherapy, immunotherapy, targeted therapy, hormonotherapy, tumor ablative surgery, ablative surgery for removing a part of or a full organ bearing a tumor, a curative surgery, a radiotherapy and their combination and at least one chemotherapy by inhalation as additional therapy.

16. The kit for an inhaled chemotherapy according to claim 15, wherein said therapy comprises at least intravenous infusion or oral chemotherapy or an immunotherapy administered for a series of cycles, each cycle comprising an administration period and an off-period, said at least one chemotherapy by inhalation being provided to be administered in a period before said therapy and/or during said off-period.

17. The kit for an inhaled chemotherapy according to claim 16, wherein said administration period extends over less than one day, and where said off-period extends over a period of time from 2 to 5 weeks.

18. The kit for a inhaled chemotherapy according to claim 14, wherein said at least one chemotherapy by inhalation is administered at a rate of at least 1 single hermetically packaged therapeutic dose of anti-neoplastic agent/week of period before said therapy and/or during said off-period.

19. The kit for an inhaled chemotherapy according to claim 14, wherein said polytherapy comprises at least tumor ablative surgery, ablative surgery for removing a part of or a full organ bearing a tumor, a curative surgery, or a radiotherapy followed by a recovery period, said at least one chemotherapy by inhalation being administered on a period before, during or after said therapy.

20. The kit for a inhaled chemotherapy according to claim 19, wherein said at least one chemotherapy by inhalation is administered at a rate of at least 1 single hermetically packaged therapeutic dose of anti-neoplastic agent/week of period before, during or after said therapy.

21. A use of the kit for an inhaled chemotherapy according to claim 1, in a polytherapy.

22. The use of the kit for an inhaled chemotherapy according to claim 21, wherein said polytherapy comprises one therapy chosen from the group consisting of intravenous infusion or oral chemotherapy, immunotherapy, targeted therapy, hormonotherapy, tumor ablative surgery, ablative surgery for removing a part of or a full organ bearing a tumor, a curative surgery, a radiotherapy and their combination and at least one chemotherapy by inhalation as additional therapy.

23. The use of the kit for an inhaled chemotherapy according to claim 22, wherein said therapy comprises at least intravenous infusion or oral chemotherapy or an immunotherapy administered for a series of cycles, each cycle comprising an administration period and an off-period, said chemotherapy by inhalation being administered during period before said therapy and/or during said off-period.

24. The use of the kit for an inhaled chemotherapy according to claim 23, wherein said administration period extends over less than one day, and where said off-period extends over a period of time from 2 to 5 weeks.

25. The use of the kit for a inhaled chemotherapy according to claim 23, at a rate of at least 1 single hermetically packaged therapeutic dose of anti-neoplastic agent/week of period before said therapy and/or during said off-period.

26. The use of the kit for an inhaled chemotherapy according to claim 21, wherein said polytherapy comprises at least tumor ablative surgery, ablative surgery for removing a part of or a full organ bearing a tumor, a curative surgery, or a radiotherapy followed by a recovery period, said at least one chemotherapy by inhalation being provided to be administered in a period before, during or after said therapy.

27. The use of the kit for a inhaled chemotherapy according to claim 26, wherein said chemotherapy by inhalation is provided to be administered at a rate of at least 1 single hermetically packaged therapeutic dose of anti-neoplastic agent/week.

28. A therapeutic treatment against lung cancer comprising the steps of administering at least one inhaled chemotherapy in a polytherapy comprising at least one therapy chosen from the group consisting of intravenous infusion or oral chemotherapy, immunotherapy, targeted therapy, hormonotherapy, tumor ablative surgery, ablative surgery for removing a part of or a full organ bearing a tumor, a curative surgery, a radiotherapy and their combination said at least one inhaled chemotherapy being an additional therapy.

29. The therapeutic treatment against lung cancer according to claim 28, wherein said therapy comprises at least intravenous infusion or oral chemotherapy or an immunotherapy provided to be administered for a series of cycles, each cycle comprising an administration period and an off-period, said chemotherapy by inhalation being provided to be administered during a period before said therapy and/or during said off-period.

30. The therapeutic treatment against lung cancer according to claim 29, wherein said administration period extends over less than one day, and where said off-period extends over a period of time from 2 to 5 weeks.

31. The therapeutic treatment against lung cancer according to claim 29, wherein said at least one chemotherapy by inhalation is administered at a rate of at least 1 single hermetically packaged therapeutic dose of anti-neoplastic agent/week of period before said therapy and/or during said off-period.

32. The therapeutic treatment against lung cancer according to claim 28, wherein said polytherapy comprises at least tumor ablative surgery, ablative surgery for removing a part of or a full organ bearing a tumor, a curative surgery, or a radiotherapy followed by a recovery period, said at least one chemotherapy by inhalation being provided to be administered in a period before, during or after said therapy.

33. The therapeutic treatment against lung cancer according to claim 32, wherein said at least one inhaled chemotherapy is administered at a rate of at least 1 single hermetically packaged therapeutic dose of anti-neoplastic agent/week of period before, during or after said therapy.

34. The therapeutic treatment against lung cancer according to claim 28, wherein said lung cancer is any lung tumor.

35. The therapeutic treatment against lung cancer according to claim 28, wherein said lung cancer is a small cell lung cancer.

36. The therapeutic treatment against lung cancer according to claim 28, wherein said lung cancer is a non-small cell lung cancer.

Description

[0105] Other characteristics and advantages of the present invention will be derived from the non-limitative following description, and by making reference to the drawings and the examples.

[0106] In the drawings, FIG. 1 shows the tumor growth for the two cisplatin-treated groups, compared with the untreated control group of example 3. Data are presented as the mean ratio of bioluminescence signals related to day 3 post graft ±SEM.

[0107] FIG. 2 shows the survival rates (p<0.05, Log-rank Test) for the two cisplatin-treated groups, compared with the untreated control group of example 3.

[0108] FIG. 3 shows the tumor growth in the chest and in the lungs for cisplatin IV compared with cisplatin DPI from example 3. Data are presented as mean bioluminescence signal ±SEM.

[0109] FIG. 4 shows the tumor growth for the two cisplatin-treated groups, compared with the untreated control group of example 4. Data are presented as the mean ratio of bioluminescence signals related to day 3 post graft ±SEM.

[0110] FIG. 5 shows the survival rates (p<0.05, Log-rank Test) for the two cisplatin-treated groups, compared with untreated the untreated control group of example 4.

[0111] FIG. 6 shows the results of bioluminescence assays of tumor growth for example 5.

[0112] FIG. 7 shows the survival rates (p<0.05, Log-rank Test) for the groups of example 5.

[0113] FIG. 8 shows the results of bioluminescence assays of tumor growth for example 6.

[0114] FIG. 9 presents the pictures obtained in bioluminescence at days 7 and 38 for the 2 mice remaining alive at day 38, one mouse in the anti-PD1 monotherapy group and one in the combination group of example 6.

[0115] FIG. 10 shows the results of bioluminescence assays of tumor growth for example 9.

[0116] FIG. 11 shows the survival rates (p<0.05, Log-rank Test) for the groups of example 9.

[0117] FIG. 12 shows the cisplatin powder fraction responsible for contamination from example 10.

[0118] In the drawings, the same reference numbers have been allocated to the same or analog element

EXAMPLES

Example 1.—Preparation of Cisplatin Dry Powder Formulation No 1

[0119] Briefly, raw cisplatin microcrystals from bulk powder (Shanghai Jinhe Bio-technology Co., Ltd., Shanghai, PRC) were first suspended in 50 mL isopropanol to reach a 5% w/v suspension and reduced in size by high-speed (10 min at 24 000 rpm) (X620 motor and a T10 dispersing shaft, Ingenieurbüro CAT M. Zipperer GmbH, Staufen, Germany) and high-pressure homogenization (EmulsiFlex-C5 high-pressure homogenizer, Avestin Inc., Ottawa, Canada) at 5 000 psi then 10 000 psi over 10 pre-milling cycles each, then at 20 000 psi over 20 milling cycles. A heat exchanger was connected to the homogenizing valve and maintained at −15° C. using an F32-MA cooling circulator (Julabo GmbH, Seelbach, Germany). An aliquot was removed from the suspension at the end of the process to measure the particle size distribution (PSD) of cisplatin microcrystals by laser diffraction (see below).

[0120] Then, tristearin (Tokyo Chemical Company, Tokyo, Japan) and TPGS (Sigma-Aldrich, St-Louis, USA) solubilized in heated isopropanol were added to the microcrystal suspension to obtain a final concentration of 1.0% w/v of cisplatin and 1.0% w/v of tristearin/TPGS (99:1 w/w) mixture and spray dried with a Mini-Spray Dryer B-290 (Buchi Labortechnik AG, Flawil, Switzerland) to obtain a DPI formulation for human use.

[0121] The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70° C., 0.7 mm nozzle, 1.5 mm nozzle-cap, compressed air at 800 L/min and drying air flow 35 ma/h. The apparatus was equipped with a B-296 dehumidifier (Buchi Labortechnik AG) to maintain the relative humidity at 50% HR during spray-drying.

[0122] The geometrical PSD of bulk cisplatin, cisplatin microparticles from the size-reduction process, and cisplatin dry powder formulation, was measured as suspended and individualized particles. This was done using a Mastersizer 3000 laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK) connected to a Hydro MV dispenser equipped with a 40 W ultrasonic probe (Malvern Instruments Ltd.). Measurements of bulk cisplatin and aliquots of cisplatin microparticles from the size-reduction process were performed in cisplatin-saturated isopropanol. The cisplatin dry powder formulation was previously dispersed, vortexed and measured in cisplatin-saturated NaCl 0.9% aqueous solution containing 0.1% w/v Poloxamer 407 (BASF, Ludwigshafen, Germany). The PSD was expressed as the volume median diameter d.sub.50 (for which 50% of particles lie under the expressed diameter) and the volume mean diameter D[4.3]. The results are presented in table 1.

TABLE-US-00001 TABLE 1 Volume median diameter d.sub.50 (for which 50% of particles lie under the expressed diameter), and the volume mean diameter D[4,3] of raw cisplatin, cisplatin microparticles from the size-reduction process, and cisplatin dry powder formulation according to Example 1. Data are expressed as the mean ± SD, n = 3. D.sub.50 (μm) D[4,3] (μm) Raw cisplatin 16.5 ± 3.6  18.9 ± 4.3  Cisplatin microparticles 0.89 ± 0.01 1.0 ± 0.1 Cisplatin dry powder formulation 4.2 ± 0.2 4.4 ± 0.1 according to Example 1

Example 2.—Preparation of Cisplatin Dry Powder Formulation No 2

[0123] Cisplatin (Umicore, Hanau-Wolfgang, Germany) was first suspended in 50 mL ethanol to reach a concentration of 5% w/v and reduced in size by high-speed (10 min at 24 000 rpm) (X620 motor and a T10 dispersing shaft, Ingenieurbüro CAT M. Zipperer GmbH, Staufen, Germany) and high-pressure homogenization using an EmulsiFlex-C3 high-pressure homogenizer (Avestin Inc., Ottawa, Canada) for 40 cycles at 20,000 PSI. These cycles were conducted by recirculating the processed suspension directly into the sample tank (closed loop). Because HPH causes a sample temperature increase, all operations were carried out using a heat exchanger placed ahead of the homogenizing valve, with the sample temperature maintained at 15±1° C.

[0124] An aliquot was removed from the suspension at the end of the process to measure the PSD of cisplatin microcrystals by laser diffraction (see below).

[0125] Then, castor oil hydrogenated (BASF, Ludwigshafen, Germany) and TPGS (Sigma-Aldrich, St-Louis, USA) solubilized in heated isopropanol were added to the microcrystal suspension to obtain a final concentration of 2.0% w/v of cisplatin and 2.0% w/v of castor oil hydrogenated/TPGS (99:1 w/w) mixture and spray dried with a Mini-Spray Dryer B-290 (Buchi Labortechnik AG, Flawil, Switzerland) to obtain a DPI formulation for human use. The operating parameters used during spray-drying were as follows: feed rate 3.0 g/min, inlet temperature 70° C., 0.7 mm nozzle, 1.5 mm nozzle-cap, compressed air at 800 L/min and drying air flow 35 m.sup.3/h. The apparatus was equipped with a B-296 dehumidifier (Buchi Labortechnik AG) to maintain the relative humidity at 50% HR during spray-drying.

[0126] The geometrical PSD of bulk cisplatin, cisplatin microparticles from the size-reduction process, and cisplatin dry powder formulation, was measured as suspended and individualized particles as described in the Example 1 and are presented in table 2.

TABLE-US-00002 TABLE 2 Volume median diameter d.sub.50 (for which 50% of particles lie under the expressed diameter) and the volume mean diameter D[4,3] of raw cisplatin, cisplatin microparticles from the size-reduction process, and cisplatin dry powder formulation according to Example 2. Data are expressed as the mean ± SD, n = 3. D.sub.50 (μm) D[4,3] (μm) Raw cisplatin 6.3 ± 0.1 7.1 ± 0.2 Cisplatin microparticles 0.68 ± 0.01 0.94 ± 0.01 Cisplatin dry powder formulation 2.240 ± 0.001 2.87 ± 0.01 according to Example 2

Example 3.—Cisplatin Administration to Mice Model 1: Cisplatin as a Monotherapv

[0127] All the animal studies were approved by ULB's ethical committee from the Faculty of Medicine (protocol 424N and 585N) and Center for Microscopy and Molecular Imaging (protocol CMMI 2017 01).

[0128] Luna Cancer Mice Model.

[0129] The orthotopic intrapulmonary implantation procedure was performed using an adaption of the protocol described previously (Wauthoz et al., Nanomedicine 2015). Briefly, under anesthesia, 6 to 7-week-old BALB/c mice were grafted with 1.4 million M109-HiFR-LUC2 cells in 20 μL of a 50:50 v/v mix of folate-free RPMI/Matrigel® injected intercostally after incision of the skin and dissection of subcutaneous muscles. The different tissue incisions were stitched with a 3-0 silk (Ethicon®, St-Stevens-Woluwe, Belgium). The mice were closely observed until complete recovery. The therapeutic response of the M109 model to cisplatin was evaluated by in vivo optical imaging (bioluminescence) and survival analysis. Bioluminescence is directly proportional to the number of living M109-HiFR-LUC2 tumor cells. Bioluminescence was performed up to day 19 post tumor cell engraftment using a Photonlmager RT (Biospace Lab, France) using the conventional supplier's protocol. Mice were weighted three times a week and sacrificed if their weight loss exceeded 20% from the beginning of the experiment or 15% from the last measurement or if they were suffocating. In these cases, the mouse death was notified as occurred the day after.

[0130] Three groups of 6-8 mice were compared: (i) untreated negative control, (ii) mice injected intravenously (IV) with 1.5 mg/kg cisplatin in a one-week cycle (one administration per cycle in weeks 1 and 3, week 2 is a week-off), (iii) mice treated with cisplatin DPI prepared in example 1 at a dose of 0.5 mg/kg cisplatin in a one-week cycle (three administrations per cycle in weeks 1 and 3, week 2 is a week-off) using an endotracheal device adapted to mice (Dry Powder Insufflator™ model DP4-M®, Penn-Century, Philadelphia, USA) and using the protocol described by Levet et al (Levet et al., Int J Pharm 2017). Treatments were administered from day 3 post tumor cell engraftment.

[0131] As it can be seen in FIG. 1, a decrease in tumor growth for the two cisplatin-treated groups, compared with the untreated control group was observed, but the differences were not statistically significant (p>0.05, 1-way ANOVA). Data are presented as the mean ratio of bioluminescence signals related to day 3 post graft ±SEM.

[0132] As it can be seen on FIG. 2, a significant increase in survival rates (p<0.05, Log-rank Test) for the two cisplatin-treated groups, compared with the untreated control group was observed. No significant differences between cisplatin DPI and cisplatin IV is observed (p>0.05, Log-rank Test).

[0133] An additional experiment was performed evaluating cisplatin DPI according to example 1 vs cisplatin IV on the same lung cancer mice model.

[0134] The mice were euthanized on day 19 post tumor cell engraftment to perform an ex vivo bioluminescence analysis to analyze tumor localization in the resected lungs (as considered as the primary lung tumors) vs in the chest cavity (as considered as loco-regional metastases).

[0135] As it can be seen in FIG. 3, a decrease of tumor growth in the chest for cisplatin IV compared with cisplatin DPI was observed but the tumor growth in the lungs was similar. Data are presented as mean bioluminescence signal ±SEM.

Example 4.—Combination of Cisplatin DPI with Systemic Cisplatin Administration to Mice Model

[0136] The protocol described in example 3 has been also performed with further groups. Three groups of 6-10 mice were compared:

[0137] (i) untreated negative control,

[0138] (ii) mice injected IV with 1.5 mg/kg cisplatin in a one-week cycle for two cycles (one administration per cycle in weeks 1 and 3, week 2 is a week-off),

[0139] (iii) mice treated with a combination of cisplatin DPI according to example 1 at a dose of 0.5 mg/kg cisplatin and cisplatin IV at 1.5 mg/kg in a one-week cycle (three administrations per cycle in weeks 1 and 3, week 2 is a week-off).

[0140] As it can be seen in FIG. 4, a decrease in tumor growth for the two cisplatin-treated groups compared with the untreated control group was observed. This decrease was significant for the combination (iii) (p<0.05, 1-way ANOVA) but not for the cisplatin IV (ii) (p>0.05, 1-way ANOVA). Data are presented as the mean ratio of bioluminescence signals related to day 3 post graft ±SEM.

[0141] As it can be seen in FIG. 5, a significant increase in survival rates (p<0.05, Log-rank Test) for the two cisplatin-treated groups compared with the untreated control group was observed. Significant increase in survival rates (p<0.05, Log-rank Test) for the combination compared with cisplatin IV was also observed.

Example 5.—Combination of Cisplatin DPI with Systemic Cisplatin and Paclitaxel as Systemic Platinum Doublets (Pt Doublets) Administration to Mice Model

[0142] The aim of the example is to demonstrate an additive/synergistic effect between cisplatin Dry Powder Inhalation (CIS DPI) and conventional systemic chemotherapy, i.e. platinum-based doublets (Pt doublets), widely used in stage III and IV non-small cell lung cancer (NSCLC) patients who are currently treated by chemotherapy as first, second and following line treatments.

[0143] Two main parameters were carefully followed during the study: [0144] The tumour growth over time by bioluminescence (in vivo optical imaging, the bioluminescence signal is directly proportional to the number of living M109 tumour cells in vivo). Bioluminescence analysis was performed two times a week, from day 3 (randomization) to day 38. [0145] Survival analysis, including Kaplan Meier survival curve and median survival (corresponding to the time where 50% mice died of their lung tumours and/or metastases).

[0146] The protocol described in example 3 has been reproduced, except that survival rates were also determined (no euthanasia on day 19 and no ex vivo bioluminescence analysis), the treatment started on day 6 according to the scheme table 3 below and that three groups of 4-15 mice were compared:

[0147] (i) untreated negative controls which were injected iv with saline,

[0148] (ii) mice injected IV with a platinum doublet composed of 1.5 mg/kg cisplatin and 10 mg/kg paclitaxel in a one-week cycle (a single IV injection per cycle) for 2 consecutive cycles,

[0149] (iii) mice treated with a combination of CIS DPI at a dose of 0.5 mg/kg cisplatin (from example 2) and the platinum doublet in a one-week cycle (3 CIS DPI administrations and a single Pt doublets IV injection per cycle) for 2 consecutive cycles.

TABLE-US-00003 TABLE 3 Dose regimen and scheme of administration followed in Example 5 Groups: iii. Platinum ii. Platinum doublet iv + i. Negative control doublet iv CIS DPI Day 0 M109 cell engraftment Day 3 Randomization Day 6 Saline iv 1.5 mg/kg cisplatin 1.5 mg/kg cisplatin iv and 10 mg/kg iv and 10 mg/kg paclitaxel iv paclitaxel iv Day 7 Off-period Off-period 0.5 mg/kg CIS DPI Day 8 Off-period Day 9 0.5 mg/kg CIS DPI Day 10 Off-period Day 11 0.5 mg/kg CIS DPI Day 12 Off-period Day 13 Saline iv 1.5 mg/kg cisplatin 1.5 mg/kg cisplatin iv and 10 mg/kg iv and 10 mg/kg paclitaxel iv paclitaxel iv Day 14 Off-period Off-period 0.5 mg/kg CIS DPI Day 15 Off-period Day 16 0.5 mg/kg CIS DPI Day 17 Off-period Day 18 0.5 mg/kg CIS DPI Day 19 Off-period

[0150] The mice that died during the endotracheal administration procedure are not considered in the analysis—7/15 died during the procedures in the combination group.

[0151] As it can be seen in FIG. 6, the addition of CIS-DPI to the platinum doublet allowed a better control of tumor growth during treatment (until day 18, which corresponded to the last administered dose), with a large majority of responders (see individual curves), which was more discernible than with the platinum doublet: [0152] Response to the platinum doublet: [0153] Most of the mice (7/8, 88%) responded to the platinum doublets. However, this was a temporary response, as observed with the re-growth of the tumor from day 21. [0154] Reduction of the tumor cells (temporary bioluminescence values <100%−tumor size under the day 3 reference, i.e. before treatment) was observed in 4/8 (50%) mice. [0155] Response to the platinum doublet+CIS-DPI [0156] All the mice (8/8, 100%) responded to the combination, with 6/8 (75%) mice having reduction of the tumor cells compared to day 3 reference (values <100%).

[0157] As it can be seen in FIG. 7 and in table 4, the addition of CIS-DPI to the platinum doublet tended to prolong the survival rates (p=0.10, log-rank test), with a median survival increase of 20% compared to the platinum doublet (31 vs 26 days, respectively).

TABLE-US-00004 TABLE 4 Survival rate at different timepoints Mice alive (%) Mice alive (%) Mice alive (%) Group at day 24: at day 28: at day 35: Pt doublet + CIS- 8/8 (100%) 4/8 (50%)  2/8 (25%) DPI Pt doublet 5/8 (62%)  2/8 (25%) 0/8 (0%) negative control 0/4 (0%)  0/4 (0%)  0/4 (0%)

Example 6. Combination of Cisplatin DPI with Systemic Immunotherapy

[0158] The aim of this example is to demonstrate a synergistic effect between CIS DPI and immune checkpoint inhibitors as monotherapy, i.e. not combined with iv chemotherapy, which is the current standard of care in ˜30% stage IV NSCLC (PD-L1 biomarker expression >50%).

[0159] Two main parameters were carefully followed during the study: [0160] The tumour growth over time by bioluminescence (in vivo optical imaging, the bioluminescence signal is directly proportional to the number of living M109 tumour cells in vivo). Bioluminescence analysis was performed two times a week, from day 3 (randomization) to day 38. [0161] Survival analysis, including Kaplan Meier survival curve and median survival (corresponding to the time where 50% mice died of their lung tumours or metastases.

[0162] The protocol described in example 3 has been reproduced, except that survival rates were also determined (no euthanasia on day 19 and no ex vivo bioluminescence analysis), the treatment started on day 3 according to the scheme table 5 below and three groups of 8-16 mice were compared:

[0163] (i) Negative controls, i.e. saline iv injection+Anti-βGAL rat IgG2a intraperitoneal (ip) injection (isotype control of anti-PD1, BioXCell, West Lebanon, N.H., USA)+DPI vehicle (i.e. the powder without cisplatin, only the excipients),

[0164] (ii) mice injected ip with an anti-PD1 antibody (RMP 1-14, BioXCell, West Lebanon, N.H., USA) in a one-week cycle for one cycle according to the scheme below,

[0165] (iii) mice treated with a combination of cisplatin DPI at a dose of 0.5 mg/kg cisplatin (from example 2) and the anti-PD1 antibody IP (RMP 1-14, BioXCell, West Lebanon, N.H., USA) in a one-week cycle for one cycle according to the scheme below.

TABLE-US-00005 TABLE 5 Dose regimen and scheme of administration followed in Example 6 Groups: iii. anti-PD1 ip + i. Negative control ii. anti-PD1 ip CIS DPI Day 0 M109 cell engraftment Day 3 Randomization Randomization Randomization Vehicle DPI 0.5 mg/kg CIS DPI Day 5 Vehicle DPI 0.5 mg/kg CIS DPI Day 7 Isotype 5 mg/kg Anti-PD1 5 mg/kg Anti-PD1 5 mg/kg Vehicle DPI 0.5 mg/kg CIS DPI Day 8 Isotype 5 mg/kg Anti-PD1 5 mg/kg Anti-PD1 5 mg/kg Day 9 Isotype 5 mg/kg Anti-PD1 5 mg/kg Anti-PD1 5 mg/kg Vehicle DPI 0.5 mg/kg CIS DPI Day 10 Isotype 5 mg/kg Anti-PD1 5 mg/kg Anti-PD1 5 mg/kg

[0166] The mice that died during the endotracheal administration procedure were not considered in the analysis—9/16 of negative controls and 4/11 of cisplatin DPI+anti-PD1 died during the procedures.

[0167] The data presented in FIG. 8 demonstrated a potential synergy of cisplatin DPI with immunotherapy. While no reduction of tumor size was observed after 2 administrations of cisplatin DPI without anti-PD1 (i.e. at days 3 and 5), a sharp reduction was observed from day 10 once a PD1 was administrated but only in the combination group, with mean values below 100% at days 14 and 17, indicating a reduction of tumour cell number compared to day 3 (reference tumour size 100%). In contrast, tumour growth of anti-PD1-treated mice followed the negative controls'. [0168] Negative controls: not controlled and continuous tumour growth, with more than 500-fold increase of tumour cell number between day 3 and 21. [0169] Response to the immunotherapy: 3/8 mice (37.5%) responded to anti-PD1 monotherapy but no long responders observed. For these 3 responders, re-growth of the tumour after treatment, from day 14. [0170] Response to the combination of cisplatin DPI with immunotherapy: [0171] All the mice (7/7, 100%) responded to the combination with an initial tumor growth up to ˜day 10 followed by a sharp decrease of the tumor size and a control of tumor growth up to day 17 observed in all the mice. [0172] Indubitable additive effect of cisplatin DPI on anti-PD1 therapy up to day 17, i.e. during the time the treatment is active.

[0173] Moreover, in the combination group, 1/7 (14%) long responder was observed with no trace of any tumor, neither primary lung tumor nor locoregional nor systemic metastases (FIG. 9). In contrast with the anti-PD1 monotherapy group, no long responder was observed (FIG. 9).

[0174] As presented in table 6, the addition of cisplatin DPI to anti-PD1 immunotherapy tend to prolong survival rates, in particular before day 27, with median survivals of 23 days and 27 days for antiPD1 and anti-PD1+cisplatin DPI, respectively.

TABLE-US-00006 TABLE 6 Survival rate at different timepoints Mice alive (%) Mice alive (%) Mice alive (%) Group at day 17: at day 21: at day 24: Anti-DPI +  7/7 (100%) 6/7 (86%) 5/7 (71%) CIS-DPI Anti-DPI 6/8 (75%) 5/8 (62%) 3/7 (43%) monotherapy negative 4/7 (57%) 1/7 (14%) 0/7 (0%)  control

Example 7. Combination of Cisplatin DPI with Systemic Chemotherapy and Systemic Immunotherapy

[0175] The protocol described in example 3 has been reproduced, except that three groups of 6-10 mice were compared:

[0176] (i) untreated negative control,

[0177] (ii) mice injected intraperitoneally (IP) with an anti-PD1 antibody (RMP 1-14, Bioceros, Utrecht, The Netherlands) in a two-week cycle (four administrations in weeks 1, week 2 is a week-off),

[0178] (iii) mice treated with a combination of cisplatin DPI at a dose of 0.5 mg/kg cisplatin and cisplatin IV at 1.5 mg/kg in a three-week cycle (three administration in weeks 1 and 3, week 2 is a week-off) and injected intraperitoneally (IP) with an anti-PD1 antibody (RMP 1-14, Bioceros, Utrecht, The Netherlands) in a two-week cycle (four administrations in weeks 1, week 2 is a week-off).

[0179] A decrease in tumor growth with the combination group (iii) compared with the untreated control group (i) and with group (ii) was observed as well as an increase in survival rates for the combination group (iii) compared with the untreated control group and group (ii) was observed.

Example 8. Combination of Cisplatin DPI with Systemic Chemotherapy and Systemic Immunotherapy

[0180] The protocol described in example 3 has been reproduced, except that three groups of 6-10 mice were compared:

[0181] (iv) untreated negative control,

[0182] (v) mice injected intraperitoneally (IP) with an anti-PD1 antibody (RMP 1-14, Bioceros, Utrecht, The Netherlands) in a two-week cycle (four administrations in weeks 1, week 2 is a week-off),

[0183] (vi) mice treated with a combination of cisplatin DPI at a dose of 0.5 mg/kg cisplatin and carboplatin IV at a cytotoxic dose in a three-week cycle (three administration in weeks 1 and 3, week 2 is a week-off) and injected intraperitoneally (IP) with an anti-PD1 antibody (RMP 1-14, Bioceros, Utrecht, The Netherlands) in a two-week cycle (four administrations in weeks 1, week 2 is a week-off).

[0184] A decrease in tumor growth with the combination group (iii) compared with the untreated control group and with group (ii) was observed as well as an increase in survival rates for the combination group (iii) compared with the untreated control group and group (ii) was observed.

Example 9.—Combination of Cisplatin DPI with Systemic Platinum Doublets and Systemic Immunotherapy

[0185] The aim of this example is to demonstrate a synergistic effect between cisplatin DPI and immune checkpoint inhibitors (i.e. anti-PD1 immunotherapy), when combined with iv chemotherapy (i.e. systemic platinum doublet), which is the current standard of care in ˜60% stage IV NSCLC (PD-L1 biomarker expression <50%)

[0186] Two main parameters were carefully followed during the study: [0187] The tumor growth over time by bioluminescence (in vivo optical imaging, the bioluminescence signal is directly proportional to the number of living M109 tumor cells in vivo). Bioluminescence analysis was performed two times a week, from day 3 (randomization) to day 38. [0188] Survival analysis, including Kaplan Meier survival curve and median survival (corresponding to the time where 50% mice died of their lung tumours or metastases.

[0189] The protocol described in example 3 has been reproduced, except that except that survival rates were also determined (no euthanasia on day 19 and no ex vivo bioluminescence analysis), the treatment started on day 6 according to the scheme table 7 below and that four groups of 8-24 mice were compared:

[0190] (i) Negative controls, i.e. saline iv injection+Anti-$GAL rat IgG2a ip injection (isotype control of anti-PD1, BioXCell, West Lebanon, N.H., USA)+DPI vehicle (i.e. the powder without cisplatin, only the excipients),

[0191] (ii) mice injected ip with an anti-PD1 antibody (RMP 1-14, BioXCell, West Lebanon, N.H., USA) and with a platinum doublet iv composed of 1.5 mg/kg cisplatin and 10 mg/kg paclitaxel in a one-week cycle for one cycle according to the scheme below,

[0192] (iii) mice treated with a combination of cisplatin DPI at a dose of 0.5 mg/kg cisplatin (from example 2) and the anti-PD1 antibody ip (RMP 1-14, BioXCell, West Lebanon, N.H., USA) and 10 mg/kg paclitaxel iv (i.e. Taxol) in a one-week cycle for one cycle according to the scheme below.

[0193] (iv) mice treated with a combination of cisplatin DPI at a dose of 0.5 mg/kg cisplatin (from example 2) and the anti-PD1 antibody ip (RMP 1-14, BioXCell, West Lebanon, N.H., USA) and platinum doublet iv composed of 1.5 mg/kg cisplatin and 10 mg/kg paclitaxel in a one-week cycle for one cycle.

TABLE-US-00007 TABLE 7 Dose regimen and scheme of administration followed in Example 9 Groups: iv. anti-PD1 ii. anti-PD1 iii. anti-PD1 ip + platinum i. Negative ip + platinum ip + Taxol doublet iv + control doublet iv iv + CIS DPI CIS DPI Day 0 M109 cell engraftment Day 3 Randomization Day 6 Saline iv 1.5 mg/kg 10 mg/kg 1.5 mg/kg and Vehicle cisplatin iv paclitaxel iv cisplatin iv DPI and 10 mg/kg and 0.5 mg/kg and 10 mg/kg paclitaxel iv CIS DPI paclitaxel iv and 0.5 mg/kg CIS DPI Day 7 Isotype 5 Anti-PD1 5 Anti-PD1 5 Anti-PD1 5 mg/kg mg/kg mg/kg mg/kg Day 8 Isotype 5 Anti-PD1 5 Anti-PD1 5 Anti-PD1 5 mg/kg and mg/kg mg/kg and 0.5 mg/kg and Vehicle DPI mg/kg CIS DPI 0.5 mg/kg CIS DPI Day 9 Isotype 5 Anti-PD1 5 Anti-PD1 5 Anti-PD1 5 mg/kg mg/kg mg/kg mg/kg Day 10 Isotype 5 Anti-PD1 5 Anti-PD1 5 Anti-PD1 5 mg/kg and mg/kg mg/kg and 0.5 mg/kg and Vehicle DPI mg/kg CIS DPI 0.5 mg/kg CIS DPI

[0194] The mice that died during the endotracheal administration procedure were not considered in the analysis—9/16 of negative controls, 5/14 of anti-PD1/Taxol/cisplatin DPI, and 13/24 of anti-PD1/Pt doublet/cisplatin DPI died during the administration procedure.

[0195] As presented in FIG. 10, Cisplatin DPI contribution in the combination groups (i.e. groups iii and iv) allowed a better control of tumour growth, with a large majority of responders, which was more discernible than with the systemic chemotherapy/immunotherapy (i.e. groups ii). From day 17 (i.e. 7 days after the last treatment administration), the curves separated, with a tumor re-growth observed in the standard of care group (group ii), while tumor cell number kept reducing in both the cisplatin DPI combination groups. [0196] Negative controls: not controlled and continuous tumor growth, with more than 500-fold increase of tumor cell number between day 3 and 21. [0197] Anti-PD1+platinum doublet: 7/13 (54%) mice had temporary bioluminescence values below 100% (which indicates a reduction of tumor cell number compared to before the first treatment administration). [0198] Anti-PD1+Taxol+cisplatin DPI: 7/9 (78%) mice had bioluminescence values below 100% indicating a reduction of tumour cell number compared to day 3. [0199] Anti-PD1+platinum doublet+cisplatin DPI: 9/11 (82%) mice had bioluminescence values below 100% indicating a reduction of tumor cell number compared to day 3.

[0200] As it can be seen in FIG. 11 and in table 8.—, the addition of cisplatin DPI to the systemic anti-PD1/platinum doublet therapy significantly prolong the survival rates (p<0.05, log-rank test), with a median survival increase of 40% compared to the platinum doublet (35 vs 25 days, respectively).

TABLE-US-00008 TABLE 8 Survival rate at different timepoints Mice alive Mice alive Mice alive Group (%) at day 25: (%) at day 32: (%) at day 42: (iv) antiPD1 + Pt 9/11 (82%) 6/11 (56%) 3/11 (27%) doublet + CIS-DPI (iii) antiPD1 +  4/9 (44%)  3/9 (33%)  1/9 (11%) Taxol + CIS-DPI (ii) antiPD1 + Pt 6/13 (46%) 2/13 (15%) 0/13 (0%)  doublet (i) negative control 0/7 (0%) 0/7 (0%) 0/7 (0%)

Example 10. Demonstration of the Absence of Environmental Contamination with the Kit for Inhaled Chemotherapy Described in the Invention (Kit 1), Compared with a Conventional Kit for Inhalation

[0201] The kit of the present invention was compared to a conventional kit in terms of environmental contamination.

[0202] a. Procedure to collect the environmental contamination from the use of a conventional kit composed with an RS.01 dry powder inhaler (RPC Plastiape, Osnago, Italy), a size 3 HPMC capsule (Quali-V-I, Qualicaps, Madrid, Spain) containing 20 mg cisplatin formulation (prepared in Example 2).

[0203] Briefly, a size 3 HPMC capsule (Quali-V-I, Qualicaps) was hand-filled with about 20 mg powder (prepared according to Example 2). The device was then prepared and use as directed in the instructions to the patient.

[0204] All the following preparation steps were performed over a glass crystallizer of about 20 cm diameter in order to collect the powder being at the origin of the environmental contamination. The capsule was placed into a RS.01 dry powder inhaler (RPC Plastiape), the device was closed and the capsule pierced. The device was placed in the apparatus described in the European Pharmacopeia 9.02 for dose collection. This apparatus is capable of quantitatively capturing the delivered dose. To simulate patient's inhalation, a flow rate of 100±5 L/min measured using a DFM3 flow meter (Copley Scientific, Nottingham, UK) was obtained with two HCP5 air pumps (Copley Scientific, Nottingham, UK) connected in series to a TPK critical flow controller (Copley Scientific, Nottingham, UK). A 250 mL beaker was placed around the DPI device during activation of the pumps (i.e. during inhalation), to collect contaminant particles (i.e. air contamination). The critical flow controller was used to ensure a deposition time of 2.4 s at 100 L/min and a critical flow with a P3/P2 ratio <0.5, as required by the European Pharmacopeia 9.02.

[0205] The device was opened, the capsule removed and the device reclosed. The powder fraction responsible for contamination during manipulation (i.e. the powder remaining in the crystallizer, the gloves of the investigator, from the device in open position and outside the capsule) was recovered with extensive washing with DMF and analyzed for its platinum content using the electrothermal atomic absorption spectrometry (ETAAS) validated method described by Levet et al (Levet et al, Int J Pharm 2016).

[0206] b. Procedure to collect the environmental contamination from the use of the kit 1 of the present invention composed with an adapted disposable RS.01 dry powder inhaler (Plastiape), a size 3 HPMC capsule (Quali-V-I, Qualicaps) containing 20 mg cisplatin formulation (prepared in Example 2). The adaptation of the RS.01 device consisted of being not re-openable once the capsule is pierced and was sealed; i.e. isolated from the environment, immediately after inhalation.

[0207] Briefly, a size 3 HPMC capsule (Quali-V-I, Qualicaps) was hand-filled with about 20 mg powder (prepared according to Example 1). The disposable DPI device was then prepared as directed in the instructions to the patient.

[0208] All the following preparation steps were performed over a glass crystallizer of about 20 cm diameter in order to collect the powder being at the origin of the environmental contamination. The capsule was placed into the disposable RS.01 dry powder inhaler (RPC Plastiape), the device was closed and the capsule pierced. The device was placed in the apparatus described in the European Pharmacopeia 9.02 for dose collection. This apparatus is capable of quantitatively capturing the delivered dose. To simulate patient's inhalation, a flow rate of 100±5 L/min measured using a DFM3 flow meter (Copley Scientific, Nottingham, UK) was obtained with two HCP5 air pumps (Copley Scientific, Nottingham, UK) connected in series to a TPK critical flow controller (Copley Scientific, Nottingham, UK). A 250 mL beaker was placed around the DPI device during activation of the pumps (i.e. during inhalation), to collect contaminant particles (i.e. air contamination). The critical flow controller was used to ensure a deposition time of 2.4 s at 100 L/min and a critical flow with a P3/P2 ratio <0.5, as required by the European Pharmacopeia 9.02.

[0209] The disposable DPI device was then immediately sealed, i.e. isolated from the environment.

[0210] The powder fraction responsible for contamination during manipulation (i.e. the powder remaining in the crystallizer, the gloves of the investigator and outside the device) was recovered with extensive washing with DMF and analyzed for its platinum content using the electrothermal atomic absorption spectrometry (ETAAS) validated method described by Levet et al (Levet et al, Int J Pharm 2016).

[0211] The cisplatin amount from air contamination was 4±2 μg for the kit 1 (mean±SD, n=6) which confirmed the ability of the adapted disposable DPI device according to the present invention to limit air contamination during inhalation.

[0212] As presented in FIG. 13, the cisplatin powder fraction responsible for contamination during manipulation collected with the kit 1 of the present invention was significantly lower than with the conventional kit ((***) means p<0.001, Student t-test, n=3).

Example 11. Demonstration of the Absence of Environmental Contamination with the Kit for Inhaled Chemotherapy Described in the Invention (Kit 2), Compared with a Conventional Kit for Inhalation

[0213] The kit of the present invention was compared to a conventional kit in terms of environmental contamination.

[0214] a. The same procedure as described in Example 10 was followed to collect the environmental contamination from the use of a conventional kit composed with an RS.01 dry powder inhaler (RPC Plastiape, Osnago, Italy), a size 3 HPMC capsule (Quali-V-I, Qualicaps, Madrid, Spain) containing 20 mg cisplatin formulation (prepared in Example 2).

[0215] b. Procedure to collect the environmental contamination from the use of the kit 2 of the present invention composed with an adapted disposable RS.01 dry powder inhaler (Plastiape), a size 3 HPMC capsule (Quali-V-I, Qualicaps) containing 20 mg cisplatin formulation (prepared in Example 2). The adaptation of the RS.01 device consisted of being not re-openable once the capsule is pierced, capping the drug exit during capsule piercing and inhalation by blocking any air exit through one-way valves and sealed, i.e. isolated from the environment, immediately after inhalation.

[0216] Briefly, a size 3 HPMC capsule (Quali-V-I, Qualicaps) was hand-filled with about 20 mg powder (prepared according to Example 1). The device was then prepared as directed in the instructions to the patient.

[0217] All the following preparation steps were performed over a glass crystallizer of about 20 cm diameter in order to collect the powder being at the origin of the environmental contamination. The capsule was placed into the disposable RS.01 dry powder inhaler (RPC Plastiape), the device was closed and the capsule pierced. The device was placed in the apparatus described in the European Pharmacopeia 9.02 for dose collection. This apparatus is capable of quantitatively capturing the delivered dose. To simulate patient's inhalation, a flow rate of 100±5 L/min measured using a DFM3 flow meter (Copley Scientific, Nottingham, UK) was obtained with two HCP5 air pumps (Copley Scientific, Nottingham, UK) connected in series to a TPK critical flow controller (Copley Scientific, Nottingham, UK). A 250 mL beaker was placed around the DPI device during activation of the pumps (i.e. during inhalation), to collect contaminant particles (i.e. air contamination). The critical flow controller was used to ensure a deposition time of 2.4 s at 100 L/min and a critical flow with a P3/P2 ratio <0.5, as required by the European Pharmacopeia 9.02.

[0218] The disposable device was then immediately sealed, i.e. isolated from the environment.

[0219] The powder fraction responsible for contamination during manipulation (i.e. the powder remaining in the crystallizer, the gloves of the investigator and outside the device) was recovered with extensive washing with DMF and analyzed for its platinum content using the electrothermal atomic absorption spectrometry (ETAAS) validated method described by Levet et al (Levet et al, Int J Pharm 2016).

[0220] Although the cisplatin amount from air contamination collected with the kit 1 for inhalation of the present invention was 4±2 μg (mean±SD, n=6), which confirmed the ability of DPI device to limit air contamination during inhalation, the cisplatin amount from air contamination with kit 2 of the invention was even much better than the kit 1 as it was below the limit of quantification of the ETAAS method.

[0221] More interestingly, the cisplatin powder fraction responsible for contamination during manipulation collected with the kit 1 and kit 2 of the present invention were both much lower than with the conventional kit.

[0222] It is to be understood that the present invention has been described using a mice model, while being destined to human or mammals. Accordingly, administration scheme, cycles and dose regimen described in the present examples are adapted to mice model and illustrates the human therapeutic plan.

[0223] For example, in mice, the cycle is a one-week cycle illustrating the three week cycle commonly used in chemotherapy in humans. In mice, the number of administration of the single hermetically packaged therapeutic dose per cycle is typically 3, while in human, it is merely foreseen that the number of administration of the single hermetically packaged therapeutic dose per cycle is typically up to 15 to 21. Likewise, in mice, the anti-neoplastic agent in each single hermetically packaged therapeutic dose is typically 0.5 mg/kg (of the animal) while in human, the anti-neoplastic agent in each single hermetically packaged therapeutic dose is typically between 1 and 5 mg/m.sup.2 body surface (for cisplatin).

[0224] It should be understood that the present invention is not limited to the described embodiments and that variations can be applied without going outside of the scope of the appended claims.