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1. WO2020136272 - KIT FOR INHALED CHEMOTHERAPY, AND TREATMENT OF LUNG CANCER WITH SAID KIT

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[ EN ]

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

The present invention relates to a kit for an inhaled chemotherapy, the use of the kit for an inhaled chemotherapy in a polyfherapy and a therapeutic treatment against lung cancer.

Lung cancer is the cancer with the highest prevalence and mortality in the world. In most cases, lung cancer is diagnosed a† advanced stages. Therefore, the patients often present already mefasfases in the lungs or in other organs, i.e. exfrapulmonary mefasfases. Treatment modalities are mostly used in combination and consist in surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy.

Chemotherapy is used in up†o 60% of lung cancer patients, mainly in advanced stages of the disease. Chemotherapy is currently administered through intravenous injection or per os, i.e. systemic routes of administration, i.e. systemic chemotherapy. Chemotherapy is responsible for severe systemic foxicifies due†o (i) the broad distribution of the chemotherapeutic in the organism and (ii) the lack of selectivity for cancer cells. Oncologists are consequently in great need for new more efficient and be††er-†olera†ed treatment approaches.

Chemotherapy is indeed a matter of finding the highest amount of an†i-neoplas†ic agent†o be administered by reducing a† a maximum level the non-reversible toxicity side effects and disagreeable side effects generated by the chemotherapy.

Accordingly, very complex therapeutic scheme are foreseen combining very often different therapeutic modalities and acts, such as for example, surgery, with a firs† cycle of radiotherapy, followed by complex cycles of an†i-neoplas†ic agent injections or intravenous infusion, which can be the same or different for each cycle and adapted depending on response†o said therapeutic scheme and the side effect identified on the body of the patient.

Each anfi-neoplasfic agent or molecule has a dose limiting toxicity (DLT) such as nephrotoxicity, neurotoxicity, ... which require†o introduce in the therapeutic scheme res† period allowing the patient body†o recover from the adverse side effects, when no† non-reversible.

Further, an†i-neoplas†ic agent has a half-life time period which directly determine the duration of action of a drug. The half-life time period is the period of time required for the concentration or amount of drug in the body†o be reduced by one-half. The half-time period of an†i-neoplas†ic agent is typically comprised between 12 hours and 36 hours, which can be short as representing the exposition time of the human body†o the beneficial effect of the an†i-neoplas†ic agent.

However, due †o the DLT of an†i-neoplas†ic agent, the amount of administered doses during several administering periods (i.e., a cycle) remains limited and should be separated one†o each other, as said previously, by off-period (see for example US2007/0065522-§0010).

The combination of the half-life period together with the dose limiting †oxici†ies, together with the fact †ha† when the anti neoplastic agent is administered by injection or intravenous infusion, the concentration of the an†i-neoplas†ic agent which reach efficiently the solid tumor site is low, with limited effect on the tumor itself and high systemic toxicity for the patient body.

Further another constrain of an†i-neoplas†ic agent is the cumulative dose which is the total dose resulting from repeated exposures†o the an†i-neoplas†ic agent of the same par† of the body or of the whole body.

For cisplatin, administered through intravenous route, the cumulative dose is 300 mg/m2 (m2 of the human body) within 4†o 6 cycles.

Those considerations yielded researchers†o develop more targeted therapies and on-si†e injection/infusions.

One approach is†o deliver chemotherapy by means of the pulmonary route (see for example US2007/0065522) , i.e. by inhalation.

Inhalation allows the administration of high drug doses directly†o lung tumors without prior distribution in the organism. The severe systemic toxicities are consequently reduced.

However, local adverse effects linked †o the pulmonary route can also occur depending on the drug candidate, its formulation and the related inhalation device. Moreover, the higher financial costs (due †o particular hospital facilities and inhalation procedure †o limit environmental contamination), in addition †o the lack of drastic improvement in the benefif/risk ratio in both preclinical and clinical evaluations, make that pulmonary route has been underexploifed in lung tumor therapy.

While chemotherapy delivered through the pulmonary route may offer strong perspectives, there is a need †o provide candidate which can improve the benefif/risk ratio in both preclinical and clinical evaluation and are susceptible †o provide a candidate where the benefits of chemotherapy will outweigh the adverse effect together with improving the life qualify.

Inhaled therapy is of different kind and one can distinguish nebulizers from inhalers as distinct manner†o deliver active substances †o the respiratory tract. Inhalers can also be of different type and dry powder inhalers (DPI) are one of them.

Compared†o nebulizers, dry powder inhalers (DPI) are well-adapted†o chemotherapy. They allow high doses of active substances, but also poorly wafer-soluble compounds (i.e. most chemofherapeufics in cancer)†o be administered directly info the lungs. Moreover, DPI limit environmental contamination by the aerosol due†o (i) their activation and drive by the patient’s inspiratory flow only and (ii) negligible exhaled drug doses. Finally, DPI can be designed as single-use disposable devices.

However, conventional immediate-release DPI formulations might nevertheless lead†o too short residence in the lungs and†o poor local tolerance. These may result from the fas† dissolution of drug particles and high peak concentrations of the anticancer drug in lung fluids after lung deposition.

There is therefore a need†o develop pulmonary route for chemotherapy and †o adapt deeply current inhalation devices and formulations used in clinical trials †o deliver effective anficancer therapies†o fumor-bearing lungs.

In that context, confrolled-release cisplafin-based DPI formulations were developed with high drug loading and high fine particle fraction (Levef ef al, Inf J Pharm 201 6) . The formulations presented confrolled-release and prolonged lung retention abilities leading †o low systemic distribution (Levef ef al, Inf J Pharm 201 7) . According †o this document, in a lung cancer mice model, this approach led †o comparable tumor response in vivo than the intravenous regimen a† half dose [dose ( 1 .0 mg/kg vs 0.5 mg/kg, respectively)]. The intravenous regimen gives good results for exfrapulmonary mefasfases while inhaled cisplafin seemed more active against primary lung tumors.

DPI formulations composed of paclifaxel-based nanocarriers were also developed with increased residence time in the lungs, limited systemic distribution and aimed specifically a† lung cancer cells by targeting the folate receptor (FR) (Rosiere ef al, Inf J Pharm 201 6; Rosiere ef al, Mol Pharm, 2018). FR, especially FR-a, is overexpressed on the cancer cell surfaces in many lung tumors (i.e. more than 70% of adenocarcinomas) and is a promising membrane receptor†o target in lung cancer. Significantly longer survival rates were observed for the FR-fargefed inhaled treatments in combination with intravenous Taxol® (the commercial form of paclifaxel), than for intravenous Taxol® alone in a lung cancer mice model.

To resume, although the use of DPI chemotherapy led†o encouraging preclinical results in terms of pharmacokinetic profiles and safety, efficacy is still rather limited.

The present invention encounters†o solve a† leas† a par† of these drawbacks by providing a ki†, for example †o be used in a polytherapy, for an inhaled chemotherapy.

The ki† according†o the present invention is characterized in†ha† i† comprises:

a) a series of single hermetically packaged therapeutic doses of a dry powder for inhalation containing at least one anti neoplastic agent chosen in the group consisting of Platinum drugs such as cisplatin, carboplatin, oxaliplatin and nedaplatin 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.

By the terms “ one or more excipient” , it is meant a compound selected from sugar alcohols; polyols (such as sorbitol, mannitol and xylitol); crystalline sugars including monosaccharides (such as glucose, arabinose) and disaccharides (such as lactose, maltose, saccharose, dextrose, trehalose, maltitol); inorganic salts (such as sodium chloride and calcium carbonate); organic salts (such as sodium lactate, potassium or sodium phosphate, sodium citrate, urea); polysaccharides (such as dextran, chitosan, starch, cellulose, hyaluronic acid, and their derivatives); oligosaccharides (such as cyclodextrins and dextrins); titanium dioxide; silicone dioxide; magnesium stearate; lecithin; amino acids (such as leucine, isoleucine, histidine, threonine, lysine, valine, methionine, phenylalanine); derivatives of an amino acid (such as acesulfame K, aspartame); lauric acid or derivatives (such as esters and salts); palmitic acid or derivatives (such as esters and salts); stearic acid or derivatives (such as esters and salts); erucic acid or derivatives (such as esters and salts); behenic acid or derivatives (such as esters and salts); sodium stearyl fumarate; sodium stearyl lactylate; phosphatidylcholines; phosphatidylglycerols; natural and synthetic lung surfactants; lauric acid and its salts (such as sodium lauryl sulphate, magnesium lauryl sulphate); triglycerides; sugar esters; phospholipids; cholesterol; talc.

In certain preferred embodiments, the excipient is mannitol, dexfran, or lactose.

In certain preferred embodiments, the excipient is phospholipids or cholesterol.

In certain preferred embodiments, the excipient is mannitol, dexfran, hyaluronic acid, lactose, phospholipids, or cholesterol.

In certain preferred embodiments, the excipient is mannitol, dexfran, phospholipids, or cholesterol.

In certain embodiments, said a† leas† one excipient is a carrier.

By the terms “single hermetically packaged doses”, it is mean† within the meaning of the present invention †ha† the single hermetically packaged doses of the neoplastic agent under the form of a dry powder for inhalation is filled in a capsule (e.g. HPMC or gelatin capsule) sealed in a blister (e.g. aluminium-aluminium blister) or is directly filled in a blister, which is the sealed dose, i.e. the single hermetically packaged dose. When the single hermetically packaged doses of the neoplastic agent under the form of a dry powder for inhalation is filled in a capsule, the close state of the single hermetically packaged doses means the capsule is closed and the blister is closed or the capsule is closed and the blister is open. The single hermetically packaged dose is in open state when the capsule is open for inhalation purposes, i.e. for example when an orifice has been pierced.

Within the meaning of the present invention, the said one disposable dry powder inhaler device is a capsule-based or a blister-based device†ha† is sealed after use, i.e. after the single hermetically packaged dose has been in the open state, such as after the

administration of a single dose by the patient, preferably assisted by a nurse or the medical staff.

The device can be sealed by avoiding re-opening, capping the drug exit, blocking any air exit through one-way valves, or simply introducing if info a hermetic bag after use.

The said one disposable dry powder inhaler device has a specific use procedure†o limit the contamination during its use and is hermetically packaged after the use (for instance contained in a hermetic bag) in order†o be eliminated as a waste using an appropriate procedure.

Preferably, the disposable dry powder device of the present invention is a disposable single dose blister-based device which is hermetically packaged (for instance, contained in a hermetic bag) in close position and already containing the single drug dose filled in in a blister in said close state. The single hermetically packaged dose reaching its open state jus† before its use (for instance by piercing or opening the blister), so the patient can inhale the dose contained inside the blister before the device being eliminated as explained before. The hermetic bag in which the device can be packaged before and/or after use can be the same or different.

In another preferred variant according †o the present invention, the single hermetically packaged dose under the form of the blister containing the single dose and the disposable dry power blister-based device are both tightly (sealedly) packaged separately and the blister is inserted in the device jus† before the administration of the dose.

In a further preferred variant according †o the present invention, preferably, the disposable dry powder device of the present invention is a disposable single dose capsule-based device which is hermetically packaged (i.e. contained in a hermetic bag) in close position and already containing the single drug dose filled in a capsule in said close state. The single hermetically packaged dose reaching its open state jus† before its use (e.g. by piercing or opening the capsule), so the patient can inhale the dose contained inside the capsule before the device being eliminated as explained before. The hermetic bag in

which the device can be packaged before and/or after use can be the same or different.

In yet another preferred variant according†o the present invention, the single hermetically packaged dose under the form of the capsule containing the single dose and the disposable and fight dry power capsule-based device are both tightly (sealedly) packaged separately and the capsule is inserted in the device jus† before the administration of the dose.

According †o the present invention, the capsule-based device can be in a partially open/close position when the single hermetically package dose is in the close state. However, once the capsule-based device is closed by the pa†ien†/nurse after the insertion of the capsule, the device is definitely closed (i.e. a re-opening of the device is no† possible) and the capsule is open for having the single hermetically packaged dose in open state), so the patient can inhale the dose contained inside the capsule before being eliminated together with the capsule-based device as explained before.

This is again †o allow †o limit the contamination by the anticancer drug†o patient, nurse and environment during the use (i.e. re-open the device †o remove the pierced/opened capsule, re-use several times the same device containing a fine powder, etc.).

In still another alternative according †o the present invention, the single hermetically packaged dose, i.e. the capsule containing the single therapeutic dose in said close state and the capsule-based disposable dry powder inhaler device are tightly (sealedly) packaged separately. The capsule is inserted in the device jus† before the administration of the dose. The device can be opened by the pa†ien†/nurse †o allow the capsules filling. Once the capsule is adequately placed in the device capsule loading chamber in the close state, the capsule-based device is closed, and the opening of the capsule provokes the definitive closure of the device. Again, the device should be disposable and be packaged after a single-use, preferably after setting the cap on the device (cap sealing position) in a specific tight bag in order†o be eliminated as a waste.

Whatever the type of the disposable device and the single hermetically packaged dose used according†o the present invention, when the device is activated (e.g. by piercing/opening the capsule or the blister), the inhalation procedure by the patient can be performed one time or repeated sequentially one or two times†o ensure that the whole dose contained in the device is inhaled.

The present invention, through providing a ki† for inhaled chemotherapy comprising a series of single hermetically packaged therapeutic doses of a† leas† one an†i-neoplas†ic agent and a series of disposable dry powder inhaler devices allow†o dispose of a fully secured chemotherapy in terms of environmental contamination of cytotoxic agent in the surrounding atmosphere, thereby drastically increasing the comfort of the patient and offering many possibilities for an ambulatory treatment and/or a home therapy.

I† has been indeed foreseen according †o the present invention a ki† comprising a series of single hermetically packaged therapeutic doses of a† leas† one an†i-neoplas†ic agent together with a series of disposable dry powder inhaler devices, where each single hermetically packaged therapeutic doses of a† leas† one an†i-neoplas†ic agent will be used in a new and clean disposable dry powder inhaler device, thereby preventing the escape of cytotoxic substance in the surrounding environment as a new disposable dry powder inhaler device will be used for each single hermetically packaged therapeutic doses of a† leas† one an†i-neoplas†ic agent. This means †ha† after its use, the disposable dry powder inhaler devices will be properly and immediately discarded†o be then isolated in a container for cytotoxic waste†o be incinerated and †ha† for the next use, when manipulation has†o be foreseen †o introduce the new single hermetically packaged therapeutic doses of a† leas† one an†i-neoplas†ic agent in the disposable dry powder inhaler devices, the disposable dry powder inhaler devices has never been in contact with cytotoxic agent and the manipulation will never cause any contamination of the surrounding atmosphere. This allows the patient†o take their therapy a† home since the risk of having cytotoxic anti-neoplastic agent in the atmosphere during the manipulation of the dry powder inhaler is drastically reduced.

I† is contemplated according†o the present invention that this will help drastically patient suffering from lung cancer as if has been recognized that long term and/or repetitive s†ay(s) in hospital environment is impacting negatively the ability†o recover of the patient.

To ensure no dispersion of cytotoxic substance in the surrounding atmosphere occurs, the disposable dry powder inhaler devices has an open position and a close position and each single hermetically packaged therapeutic dose has a close state and an open state.

In the kit for inhaled chemotherapy according †o the present invention, each single hermetically packaged therapeutic dose is 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 each single hermetically packaged therapeutic dose being also provided for becoming in said open state only when said one disposable said fight dry powder inhaler device is in said close position.

This allows†o make sure the single hermetically packaged therapeutic doses of a† leas† one an†i-neoplas†ic agent is no††o be open if no† fully accommodated in the disposable dry powder inhaler device and isolated from the surrounding environment.

Preferably, in the ki† according †o the present invention, said single hermetically packaged therapeutic dose is a capsule or a blister.

In a preferred embodiment of the ki† according †o the present invention, the lipid matrix is a pure or a mixture of triglycerides, such as for example †ris†earin, hydrogenated vegetal oil (e.g. hydrogenated castor oil) or any hydrophobic glyceride derivative having values of Log P higher than 5, preferably comprised between 14 and 25, more particularly between 18 and 25 and preferably around 20, as measured using the shake-flask method.

According †o the present invention, said lipid matrix comprises a pure or a mixture of triglycerides having a melting point temperature higher than or equal †o 40°C, preferably higher than or equal†o 60°C, more preferably higher than or equal†o 75°C.

In another preferred embodiment of the present invention, the PEGylafed excipient is derived from vitamin E or from phospholipids, such as focopheryl polyethylene glycol succinate (TPGS) or disfearoyl phosphoefhanolamine polyethylene glycol 2000 (DSPE-mPEG-2000).

Advantageously, in the kit for a inhaled chemotherapy according†o the present invention, each disposable dry powder inhaler device comprises a dispersion chamber in fluid connection with an inhalation channel and connected †o a loading chamber for accommodating 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 res† position, said activated position being provided †o open said single hermetically packaged therapeutic dose and locking means provided †o prevent extraction of the single hermetically packaged therapeutic dose once said activation means have been moved†o said activated position.

In a variant according †o the present invention, each disposable dry powder inhaler device comprises a dispersion chamber in fluid connection with an inhalation channel and connected †o a loading chamber for accommodating 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 res† position, said activated position being provided†o open said single hermetically packaged therapeutic dose and locking means having an open position and a lock position provided to prevent extraction of the single hermetically packaged therapeutic dose, said activation means being provided†o be able†o be moved†o said activated position only when said locking means are in said lock position.

In another variant according to the present invention, wherein each disposable dry powder inhaler device comprises a dispersion chamber in fluid connection with an inhalation channel a nd connected †o a loading chamber for accommodating 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 res† position, said disposable dry powder inhaler device comprising closing means provided†o isolate the outside environment from the inside of the disposable dry powder inhaler device, in particular by closing the inhalation channel.

In ye† another preferred embodiment, said disposable dry powder inhaler device is ready †o use and comprises a dispersion chamber in fluid connection with an inhalation channel and connected †o a loading chamber comprising said single hermetically packaged therapeutic dose inside said disposable dry powder inhaler device.

In a particular embodiment of the present invention, 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) dso lower than or equal†o 30 miti, preferably lower than 1 5 miti, preferably lower than or equal†o 10 miti, preferably lower than or equal†o 5 miti, and a geometric particle size distribution (PSD) d9o lower than or equal†o 60 miti, preferably lower than or equal†o 30 miti, more preferably lower than or equal†o 15 miti, preferably lower than or equal†o 10 miti and more preferably lower than or equal†o 7 miti, and optionally a volume mean diameter D [4,3] lower than or equal†o 40 miti, preferably lower than or equal†o 20 miti, more preferably lower than or equal†o 15 miti, preferably lower than or equal†o 10 miti, preferably lower than or equal†o 6 miti.

According †o the present invention, in a preferred embodiment, the single hermetically packaged therapeutic dose comprises a total powder weigh† content between 2 †o 100 mg, preferably between 3 and 80 mg, more preferably between 4 and 60 mg, in particular between 5 and 50 mg, such as for example between 6 and 20, or between 7 and 15, like around 10 mg.

For example, if the an†i-neoplas†ic agent is cisplafin, the single hermetically packaged therapeutic dose comprises a total powder weight content between 2 to 40 mg, preferably between 3 and 30 mg, more preferably between 4 and 20 mg, in particular between 5 and 15 mg, such as for example between 6 and 10 mg.

For example, if the anti-neoplastic agent is carboplatin, the single hermetically packaged therapeutic dose comprises a total powder weight content between 10 to 100 mg, preferably between 15 and 80 mg, more preferably between 20 and 60 mg, in particular between 30 and 50 mg.

In a further preferred embodiment according to the present invention, each single hermetically packaged therapeutic dose comprises between 0.1 and 90 mg, preferably between 1 mg and 40 mg of said anti-neoplastic agent.

For example, typical single hermetically packaged therapeutic dose comprises between 0.5 and 35 mg, preferably between 1 and 30 mg, in particular between 2 and 25 mg of said anti neoplastic agent, such as preferred single hermetically packaged therapeutic dose included within the range of 3 to 1 5 mg of said anti neoplastic agent such as within the range of 5 to 10 mg of said anti neoplastic agent.

For example, if said anti-neoplastic agent is cisplatin, typical single hermetically packaged therapeutic dose comprises between 0.5 and 20 mg, preferably between 1 and 15 mg, more preferably between 2 and 10 mg, in particular, between 3 and 5 mg.

If said anti-neoplastic agent is carboplatin, typical single hermetically packaged therapeutic dose comprises between 2.5 and 80 mg, preferably between 5 and 60 mg, more preferably between 10 and 50 mg, in particular, between 1 5 and 25 mg.

Further, it is also possible that the typical single hermetically packaged therapeutic dose comprises a mixture of anti-neoplastic agents, each being possibly comprised at an amount of between 0.5 and 50 mg.

According †o the present invention, each single hermetically packaged therapeutic dose comprises from 0, 1 to 99.9 mg, preferably between 1 and 49 mg of said lipid matrix.

Preferably, according†o the present invention, the single hermetically packaged therapeutic dose presents a weigh† ratio between said an†i-neoplas†ic agent and said lipid matrix : an†i-neoplas†ic agent / lipid matrix comprised between 10/90 and 90/10, preferably between 25/75 and 75/25, more preferably between 30/70 and 70/30, in particular between 40/60 and 60/40, such as for example around 50/50.

Advantageously, in the present invention, each single hermetically packaged therapeutic dose comprises from 0.01 to 5 mg, preferably between 0.03 mg and 1 mg of PEGylated excipient.

Other embodiments of the ki† for an inhaled chemotherapy according †o the present invention are mentioned in the appended claims

The present invention also relates the use of a ki† for inhaled chemotherapy in a polytherapy.

Advantageously, said polytherapy comprises one therapy chosen in the group consisting of systemic chemotherapy (including intravenous infusion and oral per os), hormonotherapy, targeted therapy, immunotherapy, tumor ablative surgery, ablative surgery for removing a par† of or a full organ bearing a tumor, a curative surgery, a radiotherapy and their combination and a† leas† one chemotherapy by inhalation as additional therapy.

Intravenous infusion chemotherapy can be composed of two an†i-neoplas†ic agents (e.g. platinum doublets) or more.

Cumulative dose of cisplatin can be safely increased with inhaled chemotherapy. The current IV cumulative dose is 300 mg/m2 within 4†o 6 cycles.

Preferably, in the use of the ki† according†o the present invention, said therapy comprises a† leas† intravenous infusion or oral chemotherapy, an hormonotherapy, a targeted therapy or an immunotherapy provided†o be administered for a series of cycles, each cycle comprising an administration period and an off-period, said a† leas† chemotherapy by inhalation being provided†o be administered in a period before said therapy and/or during said off-period of the therapy.

Preferably, in the use of the ki† according†o the present invention, said therapy comprises a† leas† intravenous infusion or oral chemotherapy or an immunotherapy provided†o be administered for a series of cycles, each cycle comprising an administration period and an off-period, said a† leas† one chemotherapy by inhalation being provided †o be administered in a period before said therapy and/or during said off-period of the therapy.

Within the meaning of the present invention, the therapy can be generally administered orally or parenterally. Further, said a† leas† one chemotherapy by inhalation can comprise a series of chemotherapy by inhalation which can be administered sequentially or together such as in a mixture. Sequentially means †ha† one chemotherapy can be administered immediately after another chemotherapy or later, according †o the same dosage regimen or according†o another dosage regimen.

According†o the present invention, it is preferred†ha† said administration period extends over less than one day, preferably over a period of time between 15 minutes†o 6 hours, preferably between 30 minutes and 4 hours, such as between 1 and 3 hours and where said off-period extends over a period of time from 1 †o 5 weeks, preferably from 2 to 4 weeks, more preferably between 2,5 and 3,5 weeks and particularly over about 3 weeks + 2 days.

According†o the present invention, it is preferred†ha† said a† leas† one chemotherapy by inhalation is provided†o be administered a† a rate of a† leas† 1 single hermetically packaged therapeutic dose of an†i-neoplas†ic agent /week of period before said therapy and/or during said off-period, preferably of a† leas† 3 single hermetically packaged therapeutic doses of an†i-neoplas†ic agent /week of period before said therapy and/or during said off-period, more preferably of a† leas† 5 single hermetically packaged therapeutic doses of an†i-neoplas†ic agent /week of period before said therapy and/or during said off-period,

possibly 7 single packaged therapeutic doses of an†i-neoplas†ic agent /week of period before said therapy and/or during said off-period for a daily dosage regimen, optionally in an equally spread manner over said period before said therapy and/or during said off-period.

According to the present invention, if is preferred that said af leas† one chemotherapy by inhalation is provided†o be administered a† a rate of one or more single hermetically packaged therapeutic dose(s) of an†i-neoplas†ic agent during the same administration even†, with the same an†i-neoplas†ic agent or with different an†i-neoplas†ic agents. When more than one single hermetically packaged therapeutic doses (with the same an†i-neoplas†ic agent or with different anti neoplastic agents) are administered for one administration even†, the single hermetically packaged doses of each an†i-neoplas†ic agent can be administered sequentially (together) or spaced by a predetermined period of time, such as one single hermetically packaged dose each 2 hours, 3 single hermetically packaged doses forming one administration even†.

According†o the present invention, it is foreseen†ha† said a† leas† one chemotherapy by inhalation is provided†o be administered a† a rate of a† leas† 1 administration event/week of period before said therapy and/or during said off-period, preferably of a† leas† 3 administration event/week of period before said therapy and/or during said off-period, more preferably of a† leas† 5 administration event/week of period before said therapy and/or during said off-period, possibly 7 administration event/week of period before said therapy and/or during said off-period for a daily dosage regimen, optionally in an equally spread manner over said period before said therapy and/or during said off-period.

If said off-period is one week, the chemotherapy by inhalation can be administered a† a rate of 1 , 2, 3, 4, 5 or even 7 single hermetically packaged therapeutic dose of an†i-neoplas†ic agent. Preferably, the chemotherapy by inhalation can be administered a† a rate of 2†o 5, or 3 to 5 single hermetically packaged therapeutic doses of an†i-neoplas†ic agent by said week off-period.

If the period before said therapy is one week, the chemotherapy by inhalation can be administered at a rate of 1 , 2, 3, 4, 5 or even 7 single hermetically packaged therapeutic doses of anti neoplastic agent. Preferably, the chemotherapy by inhalation can be administered at a rate of 2 to 5 or 3 to 5 single hermetically packaged therapeutic doses of anti-neoplastic agent by said week before the therapy.

If said off-period is three weeks, the at least one chemotherapy by inhalation can be administered at a rate of one single hermetically packaged therapeutic dose of anti-neoplastic agent each day, meaning between 1 5 and 21 doses of anti-neoplastic agent for the off-period of about 3 weeks. Optionally, the at least one chemotherapy by inhalation can be administered at a rate of one single hermetically packaged therapeutic dose of anti-neoplastic agent each two days, meaning between 9 and 12 doses of anti-neoplastic agent for the off-period of about 3 weeks. Alternatively, the at least one chemotherapy by inhalation can be administered at a rate of one single hermetically packaged therapeutic dose of anti-neoplastic agent each three days, meaning between 6 and 9 doses of anti-neoplastic agent for the off-period of about 3 weeks.

If the period before said therapy is for example 2 weeks, the at least one chemotherapy by inhalation can be administered at a rate of 1 , 2, 3, 4, 5 or even 7 single hermetically packaged therapeutic doses of anti-neoplastic agent each week. Preferably, the at least one chemotherapy by inhalation can be administered at a rate of 2 to 5 or 3 to 5 single hermetically packaged therapeutic doses of anti-neoplastic agent each week by said 2 weeks before the therapy.

A typical chemotherapy scheme can be for example a sequence of 2 weeks before the therapy, a series of cycles of one administration followed by 3 weeks off-period. In this case, the at least one chemotherapy by inhalation can be administered during the 2 weeks before the therapy at a frequency (such as one single hermetically packaged therapeutic dose of anti-neoplastic agent each day or two days), not administered on the date of the administration of the therapy and again administered during the off-period, for example starting immediately the same day, after some hours, or the day after or even 2 or 3 days after the therapy, and then over the remaining time of the off-period af a frequency of one single hermetically packaged therapeutic dose of anfi-neoplasfic agent daily.

Similarly, if said off-period is four weeks, the at least one chemotherapy by inhalation can be administered af a rate of one single hermetically packaged therapeutic dose of anfi-neoplasfic agent each day, meaning between 20 and 28 doses of anfi-neoplasfic agent for the off-period of about 4 weeks. Optionally, the af leas† one chemotherapy by inhalation can be administered a† a rate of one single hermetically packaged therapeutic dose of an†i-neoplas†ic agent each two days, meaning between 12 and 1 6 doses of an†i-neoplas†ic agent for the off-period of about 4 weeks. Alternatively, the a† leas† one chemotherapy by inhalation can be administered a† a rate of one single hermetically packaged therapeutic dose of an†i-neoplas†ic agent each three days, meaning between 8 and 12 doses of an†i-neoplas†ic agent for the off-period of about 4 weeks.

According †o the present invention, in one particular embodiment of the ki† for an inhaled chemotherapy or of the use of the ki† for a inhaled chemotherapy, said polytherapy comprises a† leas† tumor ablative surgery, ablative surgery for removing a par† of or a full organ bearing a tumor, a curative surgery, or a radiotherapy followed by a recovery period, said a† leas† one chemotherapy by inhalation being provided†o be administered in a period before, during or after said therapy, such as for example during or after said recovery period, during which typically no orally or parenterally chemotherapy is administered.

Advantageously, in the ki† for an inhaled chemotherapy or in the use of the ki† according†o the present invention, said a† leas† one chemotherapy by inhalation is provided†o be administered a† a rate of a† leas† 1 single hermetically packaged therapeutic dose of anti neoplastic agent /week of in a period before, during or after said therapy, preferably of a† leas† 3 single hermetically packaged

therapeutic doses of an†i-neoplas†ic agen†/week of in a period before, during or after said therapy, more preferably of at least 5 single hermetically packaged therapeutic doses of anfi-neoplasfic agenf/week of in a period before, during or after said therapy, possibly 7 single packaged therapeutic doses of anfi-neoplasfic agent/week of in a period before, during or after said therapy for a daily dosage regimen, optionally in an equally spread manner over said in a period before, during or after said therapy.

Other embodiments of the use according to the present invention are mentioned in the appended claims.

The present invention further relates †o a therapeutic treatment against lung cancer comprising the steps of administering a† leas† one inhaled chemotherapy in a polytherapy comprising a† leas† one therapy chosen in the group consisting of intravenous infusion or oral chemotherapy, hormonotherapy, targeted therapy, immunotherapy, tumor ablative surgery, ablative surgery for removing a par† of or a full organ bearing a tumor, a curative surgery, a radiotherapy and their combination, said a† leas† one inhaled chemotherapy being an additional therapy.

Advantageously, in the therapeutic treatment according †o the present invention, said therapy comprises a† leas† intravenous infusion or oral chemotherapy, hormonotherapy, targeted therapy or an immunotherapy provided†o be administered for a series of cycles, each cycle comprising an administration period and an off-period, said a† leas† one chemotherapy by inhalation being provided †o be administered before said therapy and/or during said off-period of the therapy.

Advantageously, in the therapeutic treatment according †o the present invention, said therapy comprises a† leas† intravenous infusion or oral chemotherapy or an immunotherapy provided †o be administered for a series of cycles, each cycle comprising an administration period and an off-period, said a† leas† one chemotherapy by inhalation being provided †o be administered before said therapy and/or during said off-period of the therapy

Preferably, in the therapeutic treatment according to the present invention, said administration period extends over less than one day, preferably over a period of time between 15 minutes to 6 hours, preferably between 30 minutes and 4 hours, such as between 1 and 3 hours, and where said off- period extends over a period of time from 2 to 5 weeks, preferably from 2 to 4 weeks, more preferably between 2,5 and 3,5 weeks and particularly over about 3 weeks + 2 days.

More particularly, in the present invention, the at least one 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 of period before said therapy and/or during said off-period, preferably of at least 3 single hermetically packaged therapeutic doses of anti-neoplastic agent/week of period before said therapy and/or during said off-period, more preferably of at least 5 single hermetically packaged therapeutic doses of anti-neoplastic agent/week of period before said therapy and/or during said off-period, possibly 7 single packaged therapeutic doses of anti-neoplastic agent/week of period before said therapy and/or during said off-period for a daily dosage regimen, optionally in an equally spread manner over said period before said therapy and/or during said off-period.

In a variant according to the present invention, 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 on a period before, during or after said therapy.

In this variant, according to the present invention, 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, preferably of at least 3 single hermetically packaged therapeutic doses of anti neoplastic agent/week of period before, during or after said therapy, more preferably of at least 5 single hermetically packaged therapeutic doses of anti-neoplastic agent/week of period before, during or after said therapy, possibly 7 single packaged therapeutic doses of anfi-neoplasfic agen†/week of period before, during or after said therapy for a daily dosage regimen, optionally in an equally spread manner over said period before, during or offer said therapy

In particular, according to the present invention, said lung cancer is any lung tumor, such as pulmonary mefasfases, for example osteosarcoma mefasfases, a small cell lung cancer or a non-small cell lung cancer.

Other embodiments of the therapeutic treatment according †o the present invention are mentioned in the appended claims.

Other characteristics and advantages of the present invention will be derived from the non-limi†a†ive following description, and by making reference†o the drawings and the examples.

In the drawings, figure 1 shows the tumor growth for the two cisplafin-freafed groups, compared with the untreated control group of example 3. Data are presented as the mean ratio of bioluminescence signals related†o day 3 post graft ± SEM.

Figure 2 shows the survival rates (p<0.05, Log-rank Test) for the two cisplafin-freafed groups, compared with the untreated control group of example 3.

Figure 3 shows the tumor growth in the chest and in the lungs for cisplafin IV compared with cisplafin DPI from example 3. Data are presented as mean bioluminescence signal ± SEM.

Figure 4 shows the tumor growth for the two cisplafin-freafed groups, compared with the untreated control group of example 4. Data are presented as the mean ratio of bioluminescence signals related†o day 3 post graft ± SEM.

Figure 5 shows the survival rates (p<0.05, Log-rank Test) for the two cisplafin-freafed groups, compared with untreated the untreated control group of example 4.

Figure 6 shows the results of bioluminescence assays of tumor growth for example 5.

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

Figure 8 shows the results of bioluminescence assays of tumor growth for example 6.

Figure 9 presents the pictures obtained in bioluminescence a† days 7 and 38 for the 2 mice remaining alive af day 38, one mouse in the anfi-PDl monotherapy group and one in the combination group of example 6.

Figure 10 shows the results of bioluminescence assays of tumor growth for example 9.

Figure 1 1 shows the survival rates (p<0.05, Log-rank Test) for the groups of example 9.

Figure 12 shows the cisplafin powder fraction responsible for contamination from example 10.

In the drawings, the same reference numbers have been allocated†o the same or analog element

Examples. - Example 1.- preparation of cisplatin dry powder formulation n°1

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

Then, tristearin (Tokyo Chemical Company, Tokyo, Japan) and TPGS (Sigma-Aldrich, S†-Louis, USA) solubilized in heated isopropanol were added to the microcrystal suspension to obtain a final concentration of 1 .0% w/v of cisplafin and 1 .0% w/v of †ris†earin/TPGS (99:1 w/w) mixture and spray dried with a Mini-Spray Dryer B-290 (BOchi Laborfechnik AG, Flawil, Switzerland) †o 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 a† 800 L/min and drying air flow 35 m3/h. The apparatus was equipped with a B-296 dehumidifier (BOchi Laborfechnik AG) to maintain the relative humidify a† 50% HR during spray-drying.

The geometrical PSD of bulk cisplafin, cisplafin microparticles from the size-reduction process, and cisplafin dry powder formulation, was measured as suspended and individualized particles. This was done using a Masfersizer 3000 laser diffractometer (Malvern Instruments Ltd., Worcestershire, UK) connected†o a Hydro MV dispenser equipped with a 40 W ultrasonic probe (Malvern Instruments Ltd.). Measurements of bulk cisplafin and aliquots of cisplafin microparticles from the size-reduction process were performed in cisplafin-safurafed isopropanol. The cisplafin dry powder formulation was previously dispersed, vorfexed and measured in cisplafin- saturated NaCI 0.9% aqueous solution containing 0.1 % w/v Poloxamer 407 (BASF, Ludwigshafen, Germany). The PSD was expressed as the volume median diameter dso (forwhich 50% of particles lie under fhe expressed diameter) and the volume mean diameter D[4,3]. The results are presented in fable 1 .

Table 1.- Volume median diameter dso (for which 50% of particles lie under the expressed diameter), and the volume mean diameter D[4,3] of raw cisplafin, cisplafin microparticles from the size-reduction process, and cisplafin dry powder formulation according†o Example 1 . Data are expressed as the mean ± SD, n = 3.



Example 2.- Preparation of cisplatin dry powder formulation n°2

Cisplatin (Umicore, Hanau-Wolfgang, Germany) was first suspended in 50 mL ethanol†o reach a concentration of 5% w/v and reduced in size by high-speed ( 10 min a† 24 000 rpm) (X620 motor and a T10 dispersing shat†, IngenieurbOro CAT M. Zipperer GmbH, Stouten, Germany) and high-pressure homogenization using an EmulsiFlex-C3 high-pressure homogenizer (Avesfin Inc., Ottawa, Canada) for 40 cycles a† 20,000 PSI. These cycles were conducted by recirculating the processed suspension directly info 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 a† 15 ± 1 °C.

An aliquot was removed from the suspension a† the end of the process †o measure the PSD of cisplatin microcrysfals by laser diffraction (see below).

Then, castor oil hydrogenated (BASF, Ludwigshafen, Germany) and TPGS (Sigma-Aldrich, S†-Louis, USA) solubilized in heated isopropanol were added†o the microcrysfal suspension†o obtain a final concentration of 2.0% w/v of cisplatin and 2.0% w/v of castor oil hydrogenafed/TPGS (99:1 w/w) mixture and spray dried with a Mini-Spray Dryer B-290 (BOchi Laborfechnik AG, Flawil, Switzerland)†o 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 a† 800 L/min and drying air flow 35 m3/h. The apparatus was equipped with a B-296 dehumidifier (BOchi Laborfechnik AG) to maintain the relative humidify a† 50% HR during spray-drying.

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

Table 2.- Volume median diameter dso (for which 50% of particles lie under the expressed diameter) and the volume mean diameter D[4,3] of raw cisplafin, cisplafin microparticles from the size-reduction process, and cisplafin dry powder formulation according to Example 2. Data are expressed as the mean ± SD, n = 3.


Example 3.- Cisplatin administration to mice model 1 : Cisplatin as a monotherapy

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 201701 ).

Luna cancer mice model.

The orfhofopic infrapulmonary implantation procedure was performed using an adaption of the protocol described previously (Waufhoz ef al., Nanomedicine 2015). Briefly, under anesthesia, 6 to 7-week-old BALB/c mice were grafted with 1.4 million Ml 09-HiFR-LL/C2 cells in 20 mί of a 50:50 v/v mix of folafe-free RPMI/Mafrigel® injected infercosfally after incision of the skin and dissection of subcutaneous muscles. The different tissue incisions were sfifched with a 3-0 silk (Efhicon®, S†-S†evens-Woluwe, Belgium). The mice were closely observed until complete recovery. The therapeutic response of the Ml 09 model†o cisplafin 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 †o day 19 post tumor cell engraffmenf using a Phofonlmager RT (Biospace Lab, France) using the conventional supplier’s protocol. Mice were weighted three times a week and sacrificed if their weigh† loss exceeded 20% from the beginning of the experiment or 15% from the las† measurement or if they were suffocating. In these cases, the mouse death was notified as occurred the day after.

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 a† 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 †o mice (Dry Powder Insufflator™ model DP4-M®, Penn-Cen†ury, Philadelphia, USA) and using the protocol described by Leve† e† al (Leve† e† al., In† J Pharm 2017). Treatments were administered from day 3 post tumor cell engraftmen†.

As i† can be seen in figure 1 , a decrease in tumor growth for the two cispla†in-†rea†ed groups, compared with the untreated control group was observed, bu† the differences were no† statistically significant (p>0.05, 1 -way ANOVA). Data are presented as the mean ratio of bioluminescence signals related†o day 3 post graft ± SEM.

As i† can be seen on figure 2, a significant increase in survival rates (p<0.05, Log-rank Test) for the two cispla†in-†rea†ed 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).

An additional experiment was performed evaluating cisplatin DPI according†o example 1 vs cisplatin IV on the same lung cancer mice model.

The mice were euthanized on day 19 post tumor cell engraftmen††o perform an ex vivo bioluminescence analysis†o 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).

As it can be seen in figure 3, a decrease of tumor growth in the chest for cisplafin IV compared with cisplafin 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

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

(i) untreated negative control,

(ii) mice injected IV with 1 .5 mg/kg cisplafin in a one-week cycle for two cycles (one administration per cycle in weeks 1 and 3, week 2 is a week-off),

(iii) mice treated with a combination of cisplafin DPI according†o example 1 a† a dose of 0.5 mg/kg cisplafin and cisplafin IV a† 1 .5 mg/kg in a one-week cycle (three administrations per cycle in weeks 1 and 3, week 2 is a week-off).

As if can be seen in figure 4, a decrease in tumor growth for the two cisplafin-freafed groups compared with the untreated control group was observed. This decrease was significant for the combination (iii) (p<0.05, 1 -way ANOVA) but no† for the cisplatin IV (ii) (p>0.05, 1 -way ANOVA). Data are presented as the mean ratio of bioluminescence signals related†o day 3 post graft ± SEM.

As i† can be seen in figure 5, a significant increase in survival rates (p<0.05, Log-rank Test) for the two cispla†in-†rea†ed 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 iPt doublets) administration to mice model

The aim of the example is †o 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.

Two main parameters were carefully followed during the study:

The tumour growth over time by bioluminescence (in vivo optical imaging, the bioluminescence signal is directly proportional to the number of living Ml 09 tumour cells in vivo) . Bioluminescence analysis was performed two times a week, from day 3 (randomization) to day 38.

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).

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:

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

(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,

(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 3.- Dose regimen and scheme of administration followed in Example 5



The mice†ha† died during the endotracheal administration procedure are not considered in the analysis - 7/15 died during the procedures in the combination group.

As it can be seen in figure 6, the addition of CIS-DPI†o the platinum double† allowed a better control of tumor growth during treatment (until day 18, which corresponded †o the las† administered dose), with a large majority of responders (see individual curves), which was more discernible than with the platinum double†:

Response†o the platinum double†:

o Most of the mice (7/8, 88%) responded †o the platinum doublets. However, this was a temporary response, as observed with the re-grow†h of the tumor from day 21 .

o 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.

Response†o the platinum double† + CIS-DPI

o All the mice (8/8, 100%) responded to the combination, with 6/8 (75%) mice having reduction of the tumor cells compared†o day 3 reference (values < 100%).

As if can be seen in figure 7 and in fable 4, the addition of CIS-DPI †o the platinum double† tended †o prolong the survival rates (p=0.10, log-rank†es†), with a median survival increase of 20% compared †o the platinum double† (31 vs 26 days, respectively) .

Table 4.- Survival rate at different timepoints


Example 6. Combination of cisplatin DPI with systemic immunotherapy

The aim of this example is †o demonstrate a synergistic effect between CIS DPI and immune checkpoint inhibitors as monotherapy, i.e. no† combined with iv chemotherapy, which is the current standard of care in -30% stage IV NSCLC (PD-L1 biomarker expression > 50%).

Two main parameters were carefully followed during the study:

The tumour growth over time by bioluminescence (in vivo optical imaging, the bioluminescence signal is directly proportional to the number of living Ml 09 tumour cells in vivo) . Bioluminescence analysis was performed two times a week, from day 3 (randomization)†o day 38.

Survival analysis, including Kaplan Meier survival curve and median survival (corresponding†o the time where 50% mice died of their lung tumours or me†as†ases.

The protocol described in example 3 has been reproduced, except †ha† 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 8-1 6 mice were compared:

(i) Negative controls, i.e. saline iv injection + An†i-PGAL ra† lgG2a intraperitoneal (ip) injection (isotype control of anti-PD l , BioXCell, Wes† Lebanon, NH, USA) + DPI vehicle (i.e. the powder without cisplatin, only the excipients),

(ii) mice injected ip with an anti-PD l antibody (RMP 1 -14, BioXCell, Wes† Lebanon, NH, USA) in a one-week cycle for one cycle according†o the scheme below,

(iii) mice treated with a combination of cisplatin DPI a† a dose of 0.5 mg/kg cisplatin (from example 2) and the anti-PD l antibody IP (RMP 1 -14, BioXCell, Wes† Lebanon, NH, USA) in a one-week cycle for one cycle according†o the scheme below.

Table 5.- Dose regimen and scheme of administration followed in Example 6


The mice†ha† died during the endotracheal administration procedure were no† considered in the analysis - 9/1 6 of negative controls and 4/1 1 of cisplatin DPI + anti-PDl died during the procedures.

The data presented in Figure 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-PD l (i.e. a† days 3 and 5), a sharp reduction was observed from day 10 once aPD l was administrated bu† only in the combination group, with mean values below 100% a† days 14 and 1 7, indicating a reduction of tumour cell number compared †o day 3 (reference tumour size 100%) . In contras†, tumour growth of anti-PDl -treated mice followed the negative controls’.

Negative controls: not controlled and continuous tumour growth, with more than 500-fold increase of tumour cell number between day 3 and 21 .

Response to the immunotherapy: 3/8 mice (37.5%) responded to anti-PD l monotherapy but no long responders observed. For these 3 responders, re-growth of the tumour after treatment, from day 14.

Response to the combination of cisplatin DPI with immunotherapy:

o 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 1 7 observed in all the mice.

o Indubitable additive effect of cisplatin DPI on anti-PD l therapy up to day 1 7, i.e. during the time the treatment is active.

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-PD l monotherapy group, no long responder was observed (Fig 9) .

As presented in table 6, the addition of cisplatin DPI to anti-PD 1 immunotherapy tend to prolong survival rates, in particular before day 27, with median survivals of 23 days and 27 days for antiPD l and anti-PD l + cisplatin DPI, respectively.

Table 6.- Survival rate at different timepoints



Example 7. Combination of cisplatin DPI with systemic chemotherapy and systemic immunotherapy

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

(i) untreated negative control,

(ii) mice injected infraperifoneally (IP) with an an†i-PD l antibody (RMP 1 -14, Bioceros, Utrecht, The Netherlands) in a two-week cycle (four administrations in weeks 1 , week 2 is a week-off),

(iii) mice treated with a combination of cisplatin DPI a† a dose of 0.5 mg/kg cisplatin and cisplatin IV a† 1 .5 mg/kg in a three-week cycle (three administration in weeks 1 and 3, week 2 is a week-off) and injected infraperifoneally (IP) with an an†i-PD l antibody (RMP 1 -14, Bioceros, Utrecht, The Netherlands) in a fwo-week cycle (four administrations in weeks 1 , week 2 is a week-off).

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

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

(iv) untreated negative control,

(v) mice injected infraperifoneally (IP) with an an†i-PD l antibody (RMP 1 -14, Bioceros, Utrecht, The Netherlands) in a two-week cycle (four administrations in weeks 1 , week 2 is a week-off),

(vi) mice treated with a combination of cisplatin DPI a† a dose of 0.5 mg/kg cisplatin and carboplafin IV a† a cytotoxic dose in a three-week cycle (three administration in weeks 1 and 3, week 2 is a week-off) and injected infraperifoneally (IP) with an an†i-PD l antibody (RMP 1 -14, Bioceros, Utrecht, The Netherlands) in a fwo-week cycle (four administrations in weeks 1 , week 2 is a week-off).

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

The aim of this example is †o demonstrate a synergistic effect between cisplatin DPI and immune checkpoint inhibitors (i.e. anfi-PD 1 immunotherapy), when combined with iv chemothera py (i.e. systemic platinum double†), which is the current standard of care in -60% stage IV NSCLC (PD-L1 biomarker expression < 50%)

Two main parameters were carefully followed during the study:

The tumor growth over time by bioluminescence (in vivo optical imaging, the bioluminescence signal is directly proportional †o the number of living Ml 09 tumor cells in vivo) . Bioluminescence analysis was performed two times a week, from day 3 (randomization)†o day 38.

Survival analysis, including Kaplan Meier survival curve and median survival (corresponding†o the time where 50% mice died of their lung tumours or mefasfases.

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†o the scheme table 7 below and that four groups of 8-24 mice were compared:

(i) Negative controls, i.e. saline iv injection + Anfi-PGAL rat lgG2a ip injection (isofype control of anti-PD l , BioXCell, Wes† Lebanon, NH, USA) + DPI vehicle (i.e. the powder without cisplatin, only the excipients),

(ii) mice injected ip with an anti-PD l antibody (RMP 1 -14, BioXCell, Wes† Lebanon, NH, USA) and with a platinum double† iv composed of 1 .5 mg/kg cisplatin and 10 mg/kg paclitaxel in a one-week cycle for one cycle according†o the scheme below,

(iii) mice treated with a combination of cisplatin DPI a† a dose of 0.5 mg/kg cisplatin (from example 2) and the anti-PD l antibody ip (RMP 1 -14, BioXCell, Wes† Lebanon, NH, USA) and 10 mg/kg paclitaxel iv (i.e. Taxol) in a one-week cycle for one cycle according†o the scheme below.

(iv) mice treated with a combination of cisplatin DPI a† a dose of 0.5 mg/kg cisplatin (from example 2) and the anti-PD l antibody ip (RMP 1 -14, BioXCell, Wes† Lebanon, NH, USA) and platinum double† iv composed of 1 .5 mg/kg cisplatin and 10 mg/kg paclitaxel in a one-week cycle for one cycle .

Table 7.- Dose regimen and scheme of administration followed in Example 9


The mice that died during the endotracheal administration procedure were not considered in the analysis - 9/1 6 of negative controls, 5/14 of an†i-PD l /Taxol/cisplafin DPI, and 13/24 of an†i-PD l /P† doublef/cisplafin DPI died during the administration procedure.

As presented in Figure 1 0, Cisplafin 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 1 7 (i.e. 7 days after the las† treatment administration), the curves separated, with a tumor re-grow†h observed in the standard of care group (group ii), while tumor cell number kept reducing in both the cisplatin DPI combination groups.

- Negative controls: not controlled and continuous tumor growth, with more than 500-fold increase of tumor cell number between day 3 and 21.

- An†i-PD1 + platinum double†: 7/13 (54%) mice had temporary bioluminescence values below 100% (which indicates a reduction of tumor cell number compared†o before the firs† treatment administration).

- An†i-PD1 + Taxol + cisplatin DPI: 7/9 (78%) mice had bioluminescence values below 100% indicating a reduction of tumour cell number compared†o day 3.

- An†i-PD1 + platinum double† + cisplatin DPI: 9/1 1 (82%) mice had bioluminescence values below 100% indicating a reduction of tumor cell number compared†o day 3.

As it can be seen in figure 1 1 and in table 8.-, the addition of cisplatin DPI †o the systemic an†i-PDl /platinum double† therapy significantly prolong the survival rates (p<0.05, log-rank†es†), with a median survival increase of 40% compared†o the platinum double† (35 vs 25 days, respectively).

Table 8.- Survival rate at different timepoints


Example 10. Demonstration of the absence of environmental contamination with the kit for inhaled chemotherapy described in the invention (kit V), compared with a conventional kit for inhalation.

The ki† of the present invention was compared to a conventional kit in terms of environmental contamination.

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) .

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.

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 beakerwas 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.

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 Levef ef al (Levef ef al, Inf J Pharm 201 6) .

b. Procedure†o 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 (Plasfiape), a size 3 HPMC capsule (Quali-V-I, Qualicaps) containing 20 mg cisplafin formulation (prepared in Example 2) . The adaptation of the RS.01 device consisted of being no† re-openable once the capsule is pierced and was sealed; i.e. isolated from the environment, immediately after inhalation.

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

All the following preparation steps were performed over a glass crystallizer of about 20 cm diameter in order†o collect the powder being a† 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 †o 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),†o collect contaminant particles (i.e. air contamination) . The critical flow controller was used†o ensure a deposition time of 2.4 s a† 100 L/min and a critical flow with a P3/P2 ratio < 0.5, as required by the European Pharmacopeia 9.02.

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

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 Levef ef al (Levef ef al, Inf J Pharm 201 6).

The cisplafin amount from air contamination was 4 ± 2 \ig for the kit 1 (mean ± SD, n=6) which confirmed the ability of the adapted disposable DPI device according †o the present invention †o limit air contamination during inhalation.

As presented in Figure 13, the cisplafin 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 pO.001 , Student†-†es†, n=3) .

Example 11. Demonstration of the absence of environmental contamination with the kit for inhaled chemotherapy described in the invention fkit 2b compared with a conventional kit for inhalation.

The kit of the present invention was compared †o a conventional kit in terms of environmental contamination.

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

b. Procedure†o 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 (Plasfiape), a size 3 PIPMC capsule (Quali-V-I, Qualicaps) containing 20 mg cisplafin formulation (prepared in Example 2) . The adaptation of the RS.01 device consisted of being no† 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.

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

All the following preparation steps were performed over a glass crystallizer of about 20 cm diameter in order†o collect the powder being a† the origin of the environmental contamination. The capsule was placed info the disposable RS.01 dry powder inhaler (RPC Plasfiape), the device was closed and the ca psule 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 †o a TPK critical flow controller (Copley Scientific, Nottingham, UK) . A 250 mL beakerwas placed around the DPI device during activation of the pumps (i.e. during inhalation),†o collect contaminant particles (i.e. air contamination) . The critical flow controller was used†o ensure a deposition time of 2.4 s a† 100 L/min and a critical flow with a P3/P2 ratio < 0.5, as required by the European Pharmacopeia 9.02.

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

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 Levef ef al (Levef ef al, Inf J Pharm 201 6).

Although the cisplafin amount from air contamination collected with the kit 1 for inhalation of the present invention was 4 ± 2 g (mean ± SD, n=6), which confirmed the ability of DPI device†o 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.

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.

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.

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 1 5 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/m2 body surface (for cisplatin) .

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.