EA051237B1 - LIPOSOMAL COMPOSITIONS - Google Patents

Description

Field of technology

The present invention relates to the field of thermosensitive liposomes and compositions based thereon, preferably for the treatment of cancer, more preferably soft tissue sarcoma.

Prior art

Stimulus-responsive nanocarriers such as thermosensitive liposomes (TSL) have been proposed as drug delivery systems for various active pharmaceutical ingredients. Yatvin et al. described the first thermosensitive liposome formulation that released a hydrophilic active pharmaceutical ingredient upon heating [1], based on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

A lysolipid-based thermosensitive liposome (LTSL) formulation developed by Needham et al. [2] has undergone human clinical trials (Thermodox®). This formulation consists of DPPC, 1-stearoyl-sn-glycero-3-phosphocholine (S-Lyso-PC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Nmethoxy(PEG)-2000 (DSPE-PEG2000). However, this formulation showed poor stability in the bloodstream.

To overcome this stability problem, 1,2-diacyl-sn-glycero-3-phosphorac-oligoglycerol (PGn) was developed. In 2004, a phospholipid composition for temperature-sensitive liposomes DPPC/DSPC/DPPG2 50:20:30 (mol/mol) (DPPG ₂ -TSL ₃₀ %) was described [3]. This composition was repeatedly compared in vitro with the LTSL composition. However, how to achieve long-term storage of these compositions has not been investigated [4,5].

Therefore, there is an unmet need in the art for DPPG2-TSL-based drug delivery systems with suitable stability, enabling industrial-scale production and long-term shelf-life. The challenge in preclinical development of temperature-sensitive liposomal formulations for human use is, in particular, to obtain a stable formulation that allows for long-term shelf-life without compromising the desired instability at temperatures >39°C for heat-induced localized drug delivery to solid tumors in patients. The optimal formulation should withstand industrial-scale production and be stored for long periods without altering critical quality-assurance characteristics that could impact therapeutic efficacy and patient safety.

Description of the invention

Temperature-sensitive liposome.

In a first aspect, the present invention provides a thermosensitive liposome comprising a bilayer and an intraliposomal buffer, wherein the molar concentration of 1,2-dipalmitoyl-snglycero-3-phosphodiglycerol (DPPG2) in said bilayer is at least 15 percent, wherein said thermosensitive liposome contains an active pharmaceutical ingredient, and wherein the molar ratio of said active pharmaceutical ingredient to lipids contained in said bilayer is from 0.05 to no more than 0.3. Such thermosensitive liposomes are referred to herein as liposomes according to or according to the present invention or thermosensitive liposomes according to or according to the present invention.

In the context of the present invention, the molar concentration of a lipid in a bilayer refers to the ratio of the molar amount of said lipid to the total molar amount of all lipids contained in said bilayer, unless explicitly stated otherwise.

A liposome is a spherical system comprising at least one bilayer and an intraliposomal buffer, wherein said intraliposomal buffer is an aqueous solution enclosed within said bilayer (see Fig. 1). As is well known to those skilled in the art, the bilayer comprises two layers of amphiphilic molecules oriented in such a way that the supramolecular structure of the bilayer is characterized by two hydrophilic surfaces separated by a hydrophobic middle layer. The intraliposomal buffer of a liposome is any aqueous solution enclosed within the bilayer contained in said liposome. The extraliposomal buffer is an aqueous solution in which said liposome is dispersed. Obviously, the intraliposomal buffer contacts the concave hydrophilic surface of the bilayer, while the extraliposomal buffer contacts the convex hydrophilic surface of the bilayer.

A liposome can be unilamellar or multilamellar. A unilamellar liposome contains only one bilayer enclosing an intraliposomal buffer, whereas a multilamellar liposome contains more than one bilayer. In a multilamellar liposome, the outer bilayer is the bilayer that encloses the other bilayers contained within the multilamellar liposome. In the context of a multilamellar liposome, the intraliposomal buffer refers to any aqueous solution enclosed within the outer bilayer. Preferably, the bilayers contained within a multilamellar liposome are:

- 1 051237 are in a substantially concentric configuration. When reference is made to the specified bilayer or bilayer in the context of a multilamellar liposome, the outer bilayer is meant.

Lipids are molecules that are soluble in non-polar solvents, such as hydrocarbons. Unless explicitly stated otherwise, lipids herein refer to amphiphilic or hydrophobic molecules contained in a bilayer that are soluble in non-polar solvents. Therefore, the terms "bilayer" and "lipid bilayer" are used interchangeably in the context of the present application. In the context of the present invention, amphiphilic surfactants are lipids. The term "lipid" preferably refers only to small molecules that are soluble in non-polar solvents, wherein the small molecules have a molecular weight of less than 900 Daltons. According to this preferred definition, amphiphilic or hydrophobic proteins with a molecular weight greater than 900 Daltons contained in a bilayer are not lipids.

Clearly, a lipid bilayer can contain non-lipid components. A lipid bilayer can also contain non-amphiphilic molecules. For example, small hydrophobic molecules can be present in the hydrophobic middle layer of the bilayer, or hydrophilic peptides can be bound to one of the hydrophilic surfaces of the bilayer.

The liposome according to the present invention can be obtained by the method according to the present invention, as defined below.

Heat sensitivity.

As is well known to those skilled in the art, the bilayer is not a static structure, and the molecules within it are capable of movement, i.e., shearing and rotating, within the bilayer plane. Membrane fluidity, or mobility, refers to the ease with which this movement of molecules within the bilayer plane can occur.

A bilayer can exist in a liquid phase (liquid crystalline phase), which is characterized by high fluidity, or in a solid phase (gel phase, solid gel phase), which is characterized by low fluidity. The phase in which a bilayer of a particular composition exists in a given environment is determined primarily by temperature and the identity of the molecules contained within the bilayer. In the solid phase, the lipids contained within the bilayer are ordered in a structured manner reminiscent of a crystalline structure, whereas in the liquid phase, the lipids are disordered and can diffuse freely within the plane of the bilayer.

A bilayer of a specific composition, for example, consisting of a specific set of lipids in specific molar ratios, will exist in the solid phase if the temperature is below the gel-liquid phase transition temperature, and will exist in the liquid phase if the temperature is above the gel-liquid phase transition temperature. In the context of the present invention, the gel-liquid phase transition temperature is also referred to as the transition temperature or _Tm . The transition temperature is determined primarily by the identity of the molecules contained in said bilayer and, consequently, the lipids contained in said bilayer.

The lipid transition temperature is defined as the transition temperature of a thermosensitive liposome comprising a bilayer, wherein said bilayer essentially consists of said lipid. In this context, "essentially consists" means that the molar concentration of said lipid in said bilayer is at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100%.

If the temperature is within the gel-liquid phase transition temperature range, the structure of said bilayer contains both freely diffusing lipids and clusters of ordered lipids. In other words, the structure of said bilayer is between the structure of said bilayer at a temperature below the transition temperature and the structure of said bilayer at a temperature above the transition temperature. In the context of the present invention, the transition temperature range of a bilayer is the temperature range at which said bilayer is characterized by such a mixed structure. It is understood that the transition temperature range includes the transition temperature, and the transition temperature range is determined primarily by the lipids contained in said bilayer.

The release of an active pharmaceutical ingredient from a thermosensitive liposome according to the present invention refers to the transfer of said active pharmaceutical ingredient from said thermosensitive liposome to an extraliposomal buffer. In this case, it is understood that said transfer does not mean the transfer of all molecules of said active pharmaceutical ingredient to an extraliposomal buffer, but rather the transfer of a predetermined molar percentage, preferably as defined below, of said active pharmaceutical ingredient to an extraliposomal buffer. Preferably, said active pharmaceutical ingredient is contained in an intraliposomal buffer of said thermosensitive liposome, and the transfer occurs from the intraliposomal buffer to the extraliposomal buffer.

- 2 051237

In a preferred embodiment, releasing the active pharmaceutical ingredient from the thermosensitive liposome means transferring at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% of said active pharmaceutical ingredient from said thermosensitive liposome into an extraliposomal buffer over a given period of time. Preferably, said period of time is 30 minutes, 25 minutes, 20 minutes, 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 285 seconds, 270 seconds, 255 seconds, 240 seconds, 225 seconds, 210 seconds, 195 seconds, 180 seconds, 165 seconds, 150 seconds, 135 seconds, 120 seconds, 105 seconds, 90 seconds, 75 seconds, 60 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds or 5 seconds.

In a more preferred embodiment, releasing the active pharmaceutical ingredient from the thermosensitive liposome means transferring at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% of said active pharmaceutical ingredient from said thermosensitive liposome into an extraliposomal buffer within 10 minutes.

In an even more preferred embodiment, releasing the active pharmaceutical ingredient from the thermosensitive liposome means transferring at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or 100% of said active pharmaceutical ingredient from said thermosensitive liposome into an extraliposomal buffer within 5 minutes.

In a most preferred embodiment, releasing the active pharmaceutical ingredient from the thermosensitive liposome means transferring at least 90% of said active pharmaceutical ingredient from said thermosensitive liposome into an extraliposomal buffer within 5 minutes.

The active pharmaceutical ingredient transport rate quantifies the rate of movement of said active pharmaceutical ingredient through the bilayer of a thermosensitive liposome. Clearly, the transport rate may depend on a set of environmental conditions, such as temperature. Release of the active pharmaceutical ingredient refers to the property of a thermosensitive liposome to exhibit a high rate of active pharmaceutical ingredient transport under a given set of conditions, preferably at a temperature above a predetermined one (see below). For example, the release of doxorubicin from a thermosensitive liposome according to the present invention may only occur at temperatures within the transition temperature of the bilayer contained within said thermosensitive liposome, ensuring heat-selective delivery of the anticancer drug.

As described above, the release of the active pharmaceutical ingredient from the thermosensitive liposome according to the present invention preferably occurs from the intraliposomal buffer to the extraliposomal buffer. In the context of the present invention, loading of the active pharmaceutical ingredient is defined as the transfer of said active pharmaceutical ingredient from the extraliposomal buffer to the intraliposomal buffer. The terms "release" and "loading" are preferably used in relation to hydrophilic, water-soluble active pharmaceutical ingredients.

Preferably, said release and loading occur via diffusion of said active pharmaceutical ingredient from the intraliposomal buffer to the extraliposomal buffer or vice versa, wherein said active pharmaceutical ingredient passes through the bilayer without active transport. It is well known to those skilled in the art that active transport of a molecule through or across a (lipid) bilayer requires transport proteins contained within or associated with said bilayer.

It should be noted that active loading does not imply active transport. In the context of this preferred definition, the rate of transport (via diffusion) depends on the permeability of the bilayer for the active pharmaceutical ingredient. This permeability depends on the characteristics of the active pharmaceutical ingredient, such as size, polarity,

- 3 051237 charge and/or hydrophilicity, and from the bilayer leakiness. Bilayer leakiness is the permeability inherent in the bilayer. Preferably, an increase or decrease in bilayer leakiness is defined in this application as an increase or decrease in the permeability of said bilayer, respectively, for a given active pharmaceutical ingredient with a given charge. More preferably, said active pharmaceutical ingredient is an antineoplastic agent, and preferably, said antineoplastic agent is selected from the group consisting of irinotecan, an irinotecan derivative, or a pharmaceutically acceptable salt thereof, and doxorubicin, a doxorubicin derivative, or a pharmaceutically acceptable salt thereof. Most preferably, said active pharmaceutical ingredient is doxorubicin, a doxorubicin derivative, or a pharmaceutically acceptable salt thereof.

The looseness of the bilayer contained in the thermosensitive liposome according to the present invention is an important parameter for the use of said thermosensitive liposome in pharmaceutical applications. For example, the thermosensitive liposome according to the present invention, containing doxorubicin as an active pharmaceutical ingredient, can be used as a drug delivery system if the looseness of the bilayer contained in said thermosensitive liposome ensures the release of said doxorubicin in the body of a patient in need thereof.

A temperature-sensitive liposome is defined in this application as a liposome containing an active pharmaceutical ingredient, wherein said active pharmaceutical ingredient is not released at or below a specific temperature, and wherein said active pharmaceutical ingredient can be released by raising the temperature slightly above the specified temperature. Preferably, the specified temperature is human body temperature. Preferably, the specified temperature is from 36°C to no more than 40°C, or from 36°C to no more than 39°C, or from 36°C to no more than 38°C, or from 36.5°C to no more than 37.5°C. In this context, slightly higher means at least 1°C higher, or at least 1.1°C higher, or at least 1.2°C higher, or at least 1.3°C higher, or at least 1.4°C higher, or at least 1.5°C higher, or at least 1.6°C higher, or at least 1.7°C higher, or at least 1.8°C higher, or at least 1.9°C higher, or at least 2°C higher, or at least 2.1°C higher, or at least 2.2°C higher, or at least 2.3°C higher, or at least 2.4°C higher, or at least 2.5°C higher, or at least 2.6°C higher, or at least 2.7°C higher, or at least 2.8°C higher, or at least 2.9°C higher, or at least 3°C higher, or at least 3.1°C higher, or at least 3.2°C higher, or at least 3.3°C higher, or at least 3.4°C higher, or at least 3.5°C higher, or at least 3.6°C higher, or at least 3.7°C higher, or at least 3.8°C higher, or at least 3.9°C higher, or at least 4°C higher, or at least 4.1°C higher, or at least 4.2°C higher, or at least 4.3°C higher, or at least 4.4°C higher, or at least 4.5°C higher, or at least 4.6°C higher, or at least 4.7°C higher, or at least 4.8°C higher, or at least 4.9°C higher, or at least 5°C higher.

In a preferred embodiment, a temperature-sensitive liposome is defined as a liposome wherein said active pharmaceutical ingredient is not released at or below human body temperature and wherein said active pharmaceutical ingredient can be released by increasing the temperature above 39°C, above 39.1°C, above 39.2°C, above 39.3°C, above 39.4°C, above 39.5°C, above 39.6°C, above 39.7°C, above 39.8°C, above 39.9°C, above 40°C, above 40.1°C, above 40.2°C, above 40.3°C, above 40.4°C, above 40.5°C, above 40.6°C, above 40.7°C, above 40.8°C, above 40.9°C, above 41°C, above 41.1°C, above 41.2°C, above 41.3°C, above 41.4°C, above 41.5°C, above 41.6°C, above 41.7°C, above 41.8°C, above 41.9°C, above 42°C, above 42.1°C, above 42.2°C, above 42.3°C, above 42.4°C, above 42.5°C, above 42.6°C, above 42.7°C, above 42.8°C, above 42.9°C, above 43°C, above 43.1°C, above 43.2°C, above 43.3°C, above 43.4°C, above 43.5°C, above 43.6°C, above 43.7°C, above 43.8°C, above 43.9°C or above 44°C.

In a more preferred embodiment, a temperature-sensitive liposome is defined as a liposome wherein said active pharmaceutical ingredient is not released at a temperature of from 36.5°C to no more than 37.5°C, and wherein said active pharmaceutical ingredient can be released by increasing the temperature above 40°C.

Without being limited by this theory, the transfer of the active pharmaceutical ingredient from the thermosensitive liposome induced by a slight increase in temperature may be due to a transition of the bilayer contained in said thermosensitive liposome from a solid phase to a gel phase. In this context, the transition temperature of said bilayer is slightly above or below the temperature at which said active pharmaceutical ingredient is not released. Above the transition temperature, said active pharmaceutical ingredient may be released by slight heating, as defined above. Within the temperature range

At the transition temperature, the bilayer contains both regions in the solid phase and regions in the liquid phase. Lipid packing defects arise at the interface between these phases, leading to a locally higher diffusion rate of the active pharmaceutical ingredient. In other words, temperature-induced transport of the active pharmaceutical ingredient in a temperature-sensitive liposome can occur through openings in the lipid bilayer that arise within the bilayer transition temperature due to packing defects. Bilayer leakiness is higher within the transition temperature than when the bilayer is entirely in the solid phase.

Active pharmaceutical ingredient.

The active pharmaceutical ingredient contained in the thermosensitive liposome according to the present invention is, as understood by those skilled in the art, a molecule that is biologically or clinically active and responsible for the therapeutic effect (i.e., pharmacodynamic effect) associated with said thermosensitive liposome or with a composition containing said thermosensitive liposome. In this context, the thermosensitive liposome according to the present invention can be provided as a drug delivery system for said active pharmaceutical ingredient. Preferably, said therapeutic effect is the treatment, alleviation, delay, cure, and/or prevention of cancer, more preferably soft tissue sarcoma.

The purity and concentration of the active pharmaceutical ingredients contained in the thermosensitive liposome according to the present invention can be determined by HPLC as described in Example 1.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said active pharmaceutical ingredient is contained in said intraliposomal buffer.

In another preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein at least 70%, or at least 80%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% of said active pharmaceutical ingredient is contained in said intraliposomal buffer.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said active pharmaceutical ingredient is hydrophilic and/or water-soluble. Preferably, water-soluble means a solubility of at least 100 mg/L, 200 mg/L, 300 mg/L, 400 mg/L, 500 mg/L, 600 mg/L, 700 mg/L, 800 mg/L, 900 mg/L, 1000 mg/L, 1500 mg/L, 2000 mg/L, 2500 mg/L, 3000 mg/L, 3500 mg/L, 4000 mg/L, 4500 mg/L, 5000 mg/L, 10000 g/L, 20000 g/L, 30000 g/L, 40000 g/L or at least 50000 g/L in water at 25°C.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said active pharmaceutical ingredient is an antineoplastic agent, wherein preferably said antineoplastic agent is contained in an intraliposomal buffer contained in said thermosensitive liposome.

In a more preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein said active pharmaceutical ingredient is selected from the group consisting of anthracyclines such as doxorubicin, daunorubicin, idarubicin, epirubicin, aclarubicin, amrubicin, pirarubicin, valrubicin and zorubicin; anthracenediones such as mitoxantrone and pixantrone; antitumor antibiotics such as mitomycin and bleomycin; vinca alkaloids such as vinblastine, vincristine and vinorelbine; alkylating agents such as cyclophosphamide and mechlorethamine hydrochloride; Camptothecins such as topotecan, irinotecan (CPT-11), lurtothecan, 9-aminocamptothecin, 9-nitrocamptothecin and 10-hydroxycamptothecin; Purine and pyrimidine derivatives such as 5-fluorouracil, gemcitabine (2',2'-difluoro-2'deoxycytidine, dFdC), floxuridine (FUDR), cytarabine (cytosine arabinoside), 6-azauracil (6-AU); Oxazaphosphorines such as cyclophosphamide, ifosfamide and trofosfamide; Taxanes such as paclitaxel and docetaxel; Podophyllotoxin derivatives such as etopside and teniposide; Platinum-based compounds such as cisplatin, carboplatin, oxaliplatin, nedaplatin; Methotrexate; tyrosine kinase inhibitors such as imatinib, gefitinib, erlotinib, sunitinib, adavosertib and lapatinib; and cytarabines such as cytosine arabinoside.

In an even more preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said active pharmaceutical ingredient is selected from the group consisting of doxorubicin, daunorubicin, mitoxantrone, idarubicin, epirubicin, aclarubicin, amrubicin, pirarubicin, valrubicin, zorubicin, pixantrone,

- 5 051237 mitomycin, bleomycin, vinblastine, vincristine, vinorelbine, cyclophosphamide, mechlorethamine hydrochloride, topotecan, irinotecan, lurtotecan, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 5-fluorouracil, gemcitabine, floxuridine, cytarabine, 6-azauracil, cyclophosphamide, ifosfamide, trofosfamide, paclitaxel, docetaxel, etopside, teniposide, cisplatin, carboplatin, oxaliplatin, nedaplatin, methotrexate, imatinib, gefitinib, erlotinib, sunitinib, adavosertib, lapatinib and cytosine arabinoside.

In a most preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said active pharmaceutical ingredient is selected from the group consisting of doxorubicin, irinotecan, gemcitabine and pharmaceutically acceptable salts thereof, more preferably said active pharmaceutical ingredient is selected from the group consisting of doxorubicin, irinotecan and pharmaceutically acceptable salts thereof, most preferably said active pharmaceutical ingredient is doxorubicin.

In the context of the present invention, an active pharmaceutical ingredient refers to the neutral form, all pharmaceutically acceptable zwitterionic forms, and all pharmaceutically acceptable salts of said active pharmaceutical ingredient. For example, the use of the term doxorubicin herein refers to at least neutral doxorubicin and doxorubicin hydrochloride. As another example, the use of the term irinotecan herein refers to at least neutral irinotecan and irinotecan hydrochloride.

The thermosensitive liposome according to the present invention may contain more than one active pharmaceutical ingredient. Preferably, the thermosensitive liposome according to the present invention comprises doxorubicin or a pharmaceutically acceptable salt thereof, and/or irinotecan or a pharmaceutically acceptable salt thereof, and/or gemcitabine or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein said active pharmaceutical ingredient is doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof to the lipids contained in said bilayer is from 0.06 to at most 0.10, preferably from 0.07 to at most 0.09. Preferably, there is provided a thermosensitive liposome according to this embodiment, wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to at most 150 nanometers; and/or wherein said intraliposomal buffer is characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8; and/or wherein said bilayer does not contain cholesterol or a derivative thereof; and/or wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and/or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), more preferably wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayer is from 0.45 to at most 0.65, preferably from 0.45 to at most 0.55, and/or wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayer is from 0.15 to at most 0.25, and/or wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35; and/or wherein said 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV).

In the context of the present invention, the doxorubicin derivative is preferably an anthracycline such as daunorubicin, mitoxantrone, idarubicin, epirubicin, aclarubicin, amrubicin, pirarubicin, valrubicin or zorubicin, or an anthracenedione such as mitoxantrone or pixantrone, most preferably the doxorubicin derivative is an anthracycline. In light of this, doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof is preferably an anthracycline or anthracenedione or a pharmaceutically acceptable salt thereof, more preferably an anthracycline or a pharmaceutically acceptable salt thereof. Obviously, doxorubicin is considered an anthracycline.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said active pharmaceutical ingredient is irinotecan, an irinotecan derivative, or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of irinotecan, said irinotecan derivative, or said pharmaceutically acceptable salt thereof, to the lipids contained in said bilayer is at least 0.18,

- 6 051237 preferably at least 0.20. Preferably, a thermosensitive liposome according to this embodiment is provided, wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to at most 150 nanometers; and/or wherein said intraliposomal buffer is characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8; and/or wherein said bilayer does not contain cholesterol or a derivative thereof; and/or wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and/or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), more preferably wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayer is from 0.45 to at most 0.65, preferably from 0.45 to at most 0.55, and/or wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayer is from 0.15 to at most 0.25, and/or wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is from 0.15 to at most 0.35, preferably from 0.25 to at most 0.35; and/or wherein said 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV), wherein the molar ratio of irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and lipids contained in said bilayer is less than 0.3.

In the context of the present invention, the irinotecan derivative is preferably a camptothecin, such as topotecan, lurtothecan, 9-aminocamptothecin, 9-nitrocamptothecin, or 10-hydroxycamptothecin. In light of this, irinotecan, an irinotecan derivative, or a pharmaceutically acceptable salt thereof is preferably camptothecin or a pharmaceutically acceptable salt thereof. Irinotecan is obviously considered to be a camptothecin.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein said active pharmaceutical ingredient is gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof to the lipids contained in said bilayer is at least 0.12, preferably at least 0.15. Preferably, there is provided a thermosensitive liposome according to this embodiment, wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to at most 150 nanometers; and/or wherein said intraliposomal buffer is characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8; and/or wherein said bilayer does not contain cholesterol or a derivative thereof; and/or wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and/or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), more preferably wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayer is from 0.45 to at most 0.65, preferably from 0.45 to at most 0.55, and/or wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayer is from 0.15 to at most 0.25, and/or wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35; and/or wherein said 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV), wherein preferably the molar ratio of gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and lipids contained in said bilayer is less than 0.3.

In the context of the present invention, the gemcitabine derivative is preferably a purine or a pyrimidine derivative, such as 5-fluorouracil (5FU), floxuridine (FUDR), cytarabine (cytosine arabinoside) or 6-azauracil (6-AU), more preferably a pyrimidine derivative. In light of this, gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof is preferably a purine, a pyrimidine derivative or a pharmaceutically acceptable salt thereof, more preferably a pyrimidine derivative or a pharmaceutically acceptable salt thereof. Obviously, gemcitabine is considered to be a pyrimidine derivative.

Diameter and zeta potential of thermosensitive liposomes.

The thermosensitive liposome according to the present invention, characterized by a given diameter, is a thermosensitive liposome with a particle size based on area that is 0.95 times larger than the said diameter and 1.05 times smaller than the said diameter, which

- 7 051237 is preferably 0.975 times greater than the specified diameter and 1.025 times less than the specified diameter, which is even more preferably 0.99 times greater than the specified diameter and 1.001 times less than the specified diameter, and most preferably the specified particle size on an area basis is equal to the specified diameter.

The presence of a thermosensitive liposome of the present invention with a given diameter in a population of thermosensitive liposomes can be determined by dynamic light scattering. Preferably, a thermosensitive liposome of the present invention with a given diameter refers to the average diameter z determined by dynamic light scattering. Example 1 describes such a method, using a DLS instrument (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). This instrument was calibrated using a Nanosphere™ size standard (125 nm, Thermo Fisher Scientific, Waltham, MA, USA).

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said thermosensitive liposome is characterized by a diameter of at least 100 nanometers.

In another preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to at most 250 nanometers, or from 100 nanometers to at most 200 nanometers, or from 100 nanometers to at most 150 nanometers, or from 110 nanometers to at most 140 nanometers, or from 115 nanometers to at most 135 nanometers, or from 120 nanometers to at most 130 nanometers.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to no more than 200 nanometers, wherein the molar ratio of doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and lipids contained in said bilayer is from 0.06 to no more than 0.10, and wherein said intraliposomal buffer is characterized by a pH of from 5 to no more than 8.

In a more preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to no more than 150 nanometers, wherein the molar ratio of doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and lipids contained in said bilayer is from 0.07 to no more than 0.09, and wherein said intraliposomal buffer is characterized by a pH of from 6.4 to no more than 8.0.

In a most preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to no more than 150 nanometers. Preferably, a thermosensitive liposome according to this embodiment is provided, wherein said active pharmaceutical ingredient is selected from the group consisting of doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof, irinotecan, an irinotecan derivative or a pharmaceutically acceptable salt thereof, and gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof, wherein more preferably said active pharmaceutical ingredient is selected from the group consisting of doxorubicin or a pharmaceutically acceptable salt thereof, irinotecan or a pharmaceutically acceptable salt thereof, and gemcitabine or a pharmaceutically acceptable salt thereof, and wherein said active pharmaceutical ingredient is contained in an intraliposomal buffer, more preferably wherein the molar ratio of doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is from 0.06 to at most 0.10, preferably from 0.07 to no more than 0.09, wherein the molar ratio of irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.18, preferably at least 0.20, wherein the molar ratio of gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.12, preferably at least 0.15; and/or wherein said intraliposomal buffer is characterized by a pH of from 5 to no more than 8, preferably from 6 to no more than 8; and/or wherein said bilayer does not contain cholesterol or a derivative thereof; and/or wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and/or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), more preferably wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayer is from 0.45 to no more than 0.65, preferably from 0.45 to no more than 0.55, and/or

- 8 051237 wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayer is from 0.15 to no more than 0.25, and/or wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35; and/or wherein said 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV).

The thermosensitive liposome according to the present invention, characterized by a zeta potential of a given value, is a thermosensitive liposome with a zeta potential that is 0.95 times greater than the specified value and 1.05 times less than the specified value, which is preferably 0.975 times greater than the specified value and 1.025 times less than the specified value, which is even more preferably 0.99 times greater than the specified value and 1.001 times less than the specified value, and most preferably the specified particle size based on area is equal to the specified value.

The presence of a thermosensitive liposome according to the present invention, characterized by a given zeta potential, in a population of thermosensitive liposomes can be determined using a DLS instrument (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK) after diluting the samples in saline.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, characterized by a zeta potential of from -40 mV to not more than -10 mV, preferably from -35 mV to not more than -20 mV.

pH of the intraliposomal buffer.

In a preferred embodiment, a temperature-sensitive liposome according to the present invention is provided, wherein said intraliposomal buffer is characterized by a pH of from 5.0 to at most 8.0, from 5.5 to at most 8.0, from 6.0 to at most 8.0, from 6.5 to at most 8.0, from 7.0 to at most 8.0, or from 7.0 to at most 7.5. In the context of the present application, the pH can be measured using a pH meter calibrated using a reference standard solution of known pH in a two-point calibration.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said intraliposomal buffer is characterized by a pH from 5.0 to 8.0, and wherein the molar ratio of doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and lipids contained in said bilayer is from 0.06 to no more than 0.10, or wherein the molar ratio of irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and lipids contained in said bilayer is at least 0.18, or wherein the molar ratio of gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and lipids in said bilayer is at least 0.12.

In a more preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said intraliposomal buffer is characterized by a pH from 6.0 to 8.0, and wherein the molar ratio of doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is from 0.07 to no more than 0.09, or wherein the molar ratio of irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.20, or wherein the molar ratio of gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.15.

In a more preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said intraliposomal buffer is characterized by a pH of from 6.0 to no more than 8.0. Preferably, a thermosensitive liposome according to this embodiment is provided, wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to no more than 150 nanometers; and/or wherein said active pharmaceutical ingredient is selected from the group consisting of doxorubicin, said doxorubicin derivative or a pharmaceutically acceptable salt thereof, irinotecan, an irinotecan derivative or a pharmaceutically acceptable salt thereof, and gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof, wherein more preferably said active pharmaceutical ingredient is selected from the group consisting of doxorubicin or a pharmaceutically acceptable salt thereof, irinotecan or a pharmaceutically acceptable salt thereof, and gemcitabine or a pharmaceutically acceptable salt thereof, and wherein said active pharmaceutical ingredient is contained in an intraliposomal buffer, more preferably wherein the molar ratio of doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is from 0.06 to no more than 0.10, preferably from 0.07 to no more than 0.09,

- 9 051237 wherein the molar ratio of irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.18, preferably at least 0.20, wherein the molar ratio of gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.12, preferably at least 0.15; and/or wherein said bilayer does not contain cholesterol or a derivative thereof; and/or wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and/or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), more preferably wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayer is from 0.45 to at most 0.65, preferably from 0.45 to at most 0.55, and/or wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayer is from 0.15 to at most 0.25, and/or wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35; and/or wherein said 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV).

Lipid composition of the bilayer.

The composition of the bilayer contained in the thermosensitive liposome according to the present invention determines the temperature-induced release and stability characteristics of said thermosensitive liposome and, therefore, determines the pharmacokinetic properties of said thermosensitive liposome. Both the identity of the lipids contained in said bilayer and their ratios are important in this regard. As described in Example 1, the bilayer composition can be determined using TLC, and the purity and concentration of the lipids contained in the bilayer can be determined using HPLC.

The thermosensitive liposome according to the present invention has a bilayer containing

DPPG2. Without being limited by this theory, the presence of DPPG2, which contains a highly hydrated diglycerol group, in said bilayer sterically inhibits interactions with blood components through both electrostatic and hydrophobic interactions, thereby rendering said thermosensitive liposome according to the present invention non-toxic, as defined below.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein the molar concentration of said DPPG2 in said bilayer is at least 15%, or at least 16%, or at least 17%, or at least 18%, or at least 19%, or at least 20%, or at least 21%, or at least 22%, or at least 23%, or at least 24%, or at least 25%, or at least 26%, or at least 27%, or at least 28%, or at least 29%, or at least 30%, or at least 31%, or at least 32%, or at least 33%, or at least 34%, or at least 35%.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein the molar concentration of DPPG2 in said bilayer is less than 55%, or less than 50%, or less than 45%, or less than 40%, or less than 35%, or less than 30%, or less than 25%, or less than 54%, or less than 49%, or less than 44%, or less than 39%, or less than 34%, or less than 29%, or less than 24%, or less than 53%, or less than 48%, or less than 43%, or less than 38%, or less than 33%, or less than 28%, or less than 23%, or less than 52%, or less than 47%, or less than 42%, or less than 37%, or less than 32%, or less than 27%, or less than 22%, or less than 51%, or less than 46%, or less than 41%, or less than 36%, or less than 31%, or less than 26%, or less than 21%, or less than 20%, or less than 19%, or less than 18%, or less than 17%, or less than 16%.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein the concentration of said DPPG2 in said bilayer is at least 15%, or from 15% to at most 55%, or from 15% to at most 54%, or from 15% to at most 53%, or from 15% to at most 52%, or from 15% to at most 51%, or from 15% to at most 50%, or from 15% to at most 49%, or from 15% to at most 48%, or from 15% to at most 47%, or from 15% to at most 46%, or from 15% to at most 45%, or from 15% to at most 44%, or from 15% to at most 43%, or from 15% to at most 42%, or from 15% to no more than 41%, or from 15% to no more than 40%, or from 15% to no more than 39%, or from 15% to no more than 38%, or from 15% to no more than 37%, or from 15% to no more than 36%, or from 15% to no more than 35%, or from 15% to no more than 34%, or from 15% to no more than 33%, or from 15% to no more than 32%, or from 15% to no more than 31%, or from 15% to no more than 30%, or from 15% to no more than 29%, or from 15% to no more than 28%, or from 15% to no more than 27%, or from 15% to no more than 26%, or from 15% to no more than 25%, or from 15% to no more than 24%, or from 15% to no more than 23%, or from 15% to no more than 22%, or from 15% to no

- 10 051237 more than 21%, or from 15% to no more than 20%, or from 15% to no more than 19%, or from 15% to no more than 18%, or from 15% to no more than 17%, or from 15% to no more than 16%.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein the concentration of said DPPG2 in said bilayer is from 15% to at most 40%, from 16% to at most 40%, from 17% to at most 40%, from 18% to at most 40%, from 19% to at most 40%, from 20% to at most 40%, from 21% to at most 40%, from 22% to at most 40%, from 23% to at most 40%, from 24% to at most 40%, from 25% to at most 40%, from 26% to at most 40%, from 27% to at most 40%, from 28% to at most 40%, from 29% to at most 40%, from 30% to at most 40%, from 15% to no more than 35%, from 16% to no more than 35%, from 17% to no more than 35%, from 18% to no more than 35%, from 19% to no more than 35%, from 20% to no more than 35%, from 21% to no more than 35%, from 22% to no more than 35%, from 23% to no more than 35%, from 24% to no more than 35%, from 25% to no more than 35%, from 26% to no more than 35%, from 27% to no more than 35%, from 28% to no more than 35%, from 29% to no more than 35%, from 30% to no more than 35%.

The bilayer contained in the thermosensitive liposome according to the present invention preferably comprises phospholipids, more preferably glycerophospholipids.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said bilayer comprises 1,2-diacyl-sn-glycero-3-phosphatidylethanolamine (PE), 1,2-diacyl-sn-glycero-3-phosphatidylcholine (PC), 1,2-diacyl-sn-glycero-3-phosphatidylserine (PS), 1,2-diacyl-sn-glycero-3-phosphatidylinositol (PI), 1,2-diacyl-sn-glycero-3-phosphatidylinositol phosphate (PIP), 1,2-diacyl-sn-glycero-3-phosphatidylinositol biphosphate (PIP2), 1,2-diacyl-sn-glycero-3-phosphatidylinositol triphosphate (PIP3), 1,2-diacyl-sn-glycero-3-phosphatidylglycerol (PG), 1,2-diacyl-sn-glycero-3-phosphatidyldiglycerol (PG2), 1,2-diacyl-sn-glycero-3-phosphatidyltriglycerol (PG3) and/or 1,2-diacyl-sn-glycero-3-phosphatidyltetraglycerol (PG4). As explained above, the thermosensitive liposome according to the present invention comprises 1,2-dipalmitoyl-sn-glycero-3-phosphatidyldiglycerol (DPPG2), a specific type of PG2 according to the definition.

In a more preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein 1,2-diacyl-sn-glycero-3-phosphatidylcholine (PC), 1,2-diacyl-sn-glycero-3-phosphatidylethanolamine (PE) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidyldiglycerol (DPPG2) having a total concentration of at least 50 mol.%, at least 60 mol.%, at least 70 mol.%, at least 80 mol.%, at least 90 mol.%, at least 91 mol.%, at least 92 mol.%, at least 93 mol.%, at least 94 mol.%, at least 95 mol.%, at least 96 mol.%, at least 97 mol.%, at least 98 mol.% or at least 99 mol.%, are contained in said bilayer.

In a more preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein 1,2-diacyl-sn-glycero-3-phosphatidylcholine (PC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidyldiglycerol (DPPG2) have a total concentration of at least 50 mol.%, at least 60 mol.%, at least 70 mol.%, at least 80 mol.%, at least 90 mol.%, at least 91 mol.%, at least 92 mol.%, at least 93 mol.%, at least 94 mol.%, at least 95 mol.%, at least 96 mol.%, at least 97 mol.%, at least 98 mol.% or at least 99 mol.% in said bilayer.

The (glycero)phospholipids described herein may be covalently attached to polyethyleneglycol (PEG), glycerol, or polyglycerol to increase the molecular weight and molecular size of the hydrophilic portion of said (glycero)phospholipids. In the case of PEG, such a (glycero)phospholipid is indicated as PEGylated. For example, DSPE-PEG2000 is Nmethoxy(PEG)-2000-1,2-diacyl-sn-glycero-3-phosphatidylethanolamine (PE). Without being limited by this theory, the presence of PEGylated (glycero)phospholipids in the temperature-sensitive liposome has a similar effect to the presence of DPPG2 described above, namely, steric inhibition of interactions with a blood component.

A preferred embodiment of the present invention provides a thermosensitive liposome wherein said bilayer is PEG-free. In other words, said bilayer does not contain PEGylated lipids.

An important advantage of using DPPG2 over PEGylated lipids is the significantly lower modification of the terminal group of the phospholipid anchor (74 Da for each glycerol unit compared to approximately 2000 Da). As a result, 1,2-dipalmitoyl-snglycero-3-phosphodiglycerol (DPPG2) forms lamellar rather than micellar structures and can thus be incorporated into the temperature-sensitive liposome in an amount of no more than 70 mol%. On the other hand, the incorporation of DSPE-PEG2000 is limited because it functions as a surfactant at a critical micellar concentration of 0.5-1.0 μM and, at higher concentrations, impairs the stability of the membrane bilayer.

In the (glycero)phospholipids mentioned above, preferred acyl groups are C14-C22 acyl groups, more preferably C18-20 acyl groups. Preferably

- 11 051237 the acyl group is selected from the group consisting of arachidyl, arachlyl, behenyl, brassidyl, ceryl, cetyl, elaidyl, erucyl, gadoleyl, geddyl, isostearyl, lauryl, lignoceryl, montanyl, myricyl, myristyl, oleyl, palmitoleyl, palmitoyl, petroselinyl and stearyl. More preferably, the acyl groups are selected from the group consisting of stearyl and palmitoyl. Most preferably, the acyl group is palmitoyl.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE), 1,2-distearyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE), N-methoxy(PEG)-2000-1,2-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE-PEG2ooo) and/or 1,2-dipalmitoyl-sn-glycero-3-phosphatidyldiglycerol (DPPG2).

In a preferred embodiment, a thermosensitive liposome is provided according to the present invention, wherein 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE), 1,2-distearyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE), Nmethoxy(PEG)-2000-1,2-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE-PEG2000) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidyldiglycerol (DPPG2) have a total concentration of at least 50 mol.%, at least 60 mol.%, at least 70 mol.%, at least 80 mol.%, at least 90 mol.%, at least 91 mol.%, at least 92 mol.%, at least 93 mol.%, at least 94 mol.%, at least 95 mol.%, at least 96 mol.%, at least 97 mol.%, at least 98 mol.% or at least 99 mol.% in said bilayer. In the context of this embodiment, it is understood that not all lipids mentioned above may, but do not have to, be contained in said thermosensitive liposome.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE), N-methoxy(PEG)-2000-1,2-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE-PEG2000) and/or 1,2-dipalmitoyl-sn-glycero-3-phosphatidyldiglycerol (DPPG2).

In a preferred embodiment, a thermosensitive liposome is provided according to the present invention, wherein 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearyl-sn-glycero-3-phosphatidylcholine (DSPC), 1,2-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE), N-methoxy(PEG)-2000-1,2-distearyl-sn-glycero-3-phosphatidylethanolamine (DSPE-PEG ₂₀₀₀ ) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidyldiglycerol (DPPG2) have a total concentration of at least 50 mol.%, at least 60 mol.%, at least 70 mol.%, at least 80 mol.%, at least 90 mol.%, at least 91 mol.%, at least 92 mol.%, at least 93 mol.%, at least 94 mol.%, at least 95 mol.%, at least 96 mol.%, at least 97 mol.%, at least 98 mol.% or at least 99 mol.% in said bilayer. In the context of this embodiment, it is understood that not all lipids mentioned above may, but do not have to, be contained in said thermosensitive liposome.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and/or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). One skilled in the art will understand that DPPG2 refers to all compounds represented by formula (I); DPPC refers to all compounds represented by formula (II); DSPC refers to all compounds represented by formula (III).

- 12 051237

Formula (I)

Formula (II)

Formula (III)

Unless explicitly stated otherwise, the Lewis structure or formula, trivial name, or systematic name of a molecule is intended to encompass all tautomers, i.e., structural isomers that readily interconvert, and stereoisomers of these molecules in the context of the present invention. For example, formula (I) contains three chiral carbon centers. The stereoconfiguration of one of these chiral carbon centers has been precisely determined and should be interpreted as such. The stereoconfiguration of the other two chiral carbon centers has not been determined. Thus, all epimers with respect to these two chiral carbon centers are described by formula (I). It should also be noted that the Lewis structure or formula, trivial name, or systematic name of a molecule is intended to encompass all protonation and salt states unless explicitly stated otherwise. For example, formula (I) also describes DPPG ₂ , where one of the oxygen atoms covalently attached to phosphorus is negatively charged.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said 1,2-dipalmitoyl-8n-glycero-3-phosphodiglycerol (DPPGJ) is represented by formula (IV):

γονο οΧΌ γΌΗ ¹⁴ ° ^{n he}

A 14

Formula (IV).

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein 1,2-dipalmitoyl-8p-glycero-3-phosphatidylcholine (DPPC), 1,2-distearyl-8p-glycero-3-phosphatidylcholine (DSPC) and 1,2-dipalmitoyl-8p-glycero-3-phosphatidyl diglycerides (DPPGJ) have a total concentration of at least 50 mol %, at least 60 mol %, at least 70 mol %, at least 80 mol %, at least 90 mol %, at least 91 mol %, at least 92 mol %, at least 93 mol %, at least 94 mol %, at least 95 mol %, at least 96 mol %, at least 97 mol %, at least 98 mol %, or at least at least 99 mol%, or at least 99.5 mol%, or at least 99.6 mol%, or at least 99.7 mol%, or at least 99.8 mol%, or at least 99.9 mol% in said bilayer. In the context of this embodiment, it is understood that not all lipids mentioned above may, but do not have to, be contained in said thermosensitive liposome.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein said bilayer consists of 1,2-dipalmitoyl-8n-glycero-3-phosphatidylcholine (DPPC), 1,2-distearyl-8n-glycero-3-phosphatidylcholine (DSPC) and 1,2-dipalmitoyl sn-glycero-3-phosphatidyl diglyceride (DPPGj).

The purity of the thermosensitive liposome according to the present invention is the total concentration of 1,2-dipalmitoyl-8p-glycero-3-phosphatidylcholine (DPPC), 1,2-distearyl-8p-glycero-3-phosphatidylcholine (DSPC) and 1,2-dipalmitoyl-8p-glycero-3-phosphatidyldiglycerol

- 13 051237 (DPPG2) in the specified bilayer. Thus, in a preferred embodiment, a thermosensitive liposome according to the present invention is provided with a purity of at least 50 mol%, at least 60 mol%, at least 70 mol%, at least 80 mol%, at least 90 mol%, at least 91 mol%, at least 92 mol%, at least 93 mol%, at least 94 mol%, at least 95 mol%, at least 96 mol%, at least 97 mol%, at least 98 mol%, or at least 99 mol%, or at least 99.5 mol%, or at least 99.6 mol%, or at least 99.7 mol%, or at least 99.8 mol%, or at least 99.9 mol%, or 100 mol%.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearyl-sn-glycero-3-phosphatidylcholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidyldiglycerol (DPPG2) together constitute at least 95% of the number of molecules contained in said bilayer, and wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to no more than 150 nanometers; and/or wherein said active pharmaceutical ingredient is selected from the group consisting of doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof, irinotecan, an irinotecan derivative or a pharmaceutically acceptable salt thereof, and gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof, and wherein said active pharmaceutical ingredient is contained in an intraliposomal buffer, more preferably wherein the molar ratio of doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is from 0.06 to at most 0.10, preferably from 0.07 to at most 0.09, wherein the molar ratio of irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.18, preferably at least 0.20, wherein the molar ratio of gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and lipids contained in said bilayer is at least 0.12, preferably at least 0.15, and/or wherein said intraliposomal buffer is characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8; and/or wherein said bilayer does not contain cholesterol or a derivative thereof; and/or wherein said 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV).

Clearly, if the total concentration of a lipid group in a bilayer is given in mol%, then that bilayer may, but does not necessarily, contain all lipid types from that lipid group. For example, a bilayer in which the concentration of each of DPPC and DSPC is 45 mol% can be described as a bilayer in which the total concentration of DPPC, DSPC, and DPPG2 is at least 90 mol%.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and/or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayer is from 0.45 to at most 0.65, preferably from 0.45 to at most 0.55; and/or wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayer is from 0.15 to at most 0.25; and/or wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayer is from 0.45 to at most 0.65, preferably from 0.45 to at most 0.55; and/or wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayer is from 0.15 to 0.25; and wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is from 0.15 to at most 0.35, preferably from 0.25 to at most 0.35, preferably wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to at most 150 nanometers; and/or wherein said active pharmaceutical ingredient is selected from the group consisting of doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof, irinotecan, an irinotecan derivative or a pharmaceutically acceptable salt thereof, and gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof, wherein more preferably said active pharmaceutical ingredient is selected from the group consisting of doxorubicin or its

- 14 051237 pharmaceutically acceptable salt, irinotecan or a pharmaceutically acceptable salt thereof, and irinotecan or a pharmaceutically acceptable salt thereof, and wherein said active pharmaceutical ingredient is contained in an intraliposomal buffer, more preferably wherein the molar ratio of doxorubicin, said doxorubicin derivative, or said pharmaceutically acceptable salt thereof, to the lipids contained in said bilayer is from 0.06 to at most 0.10, preferably from 0.07 to at most 0.09, wherein the molar ratio of irinotecan, said irinotecan derivative, or said pharmaceutically acceptable salt thereof, to the lipids contained in said bilayer is at least 0.18, preferably at least 0.20, wherein the molar ratio of gemcitabine, said gemcitabine derivative, or said pharmaceutically acceptable salt thereof, to the lipids contained in said bilayer, is at least 0.12, preferably at least 0.15, and/or wherein said intraliposomal buffer is characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8; and/or wherein said bilayer does not contain cholesterol or a derivative thereof; and/or wherein said 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV).

DPPC is the main component in the temperature-sensitive liposome according to the preferred embodiment above, as its transition temperature (41.4°C) is slightly above body temperature. Undesirable drug leakage at body temperature, as defined below, can be reduced by mixing DPPC with small amounts of DSPC, which has a transition temperature of 54.9°C.

A lysolipid is an amphiphilic molecule that is a product of lipid hydrolysis. For example, 1-palmitoyl-sn-glycero-3-phosphatidylcholine, obtained by hydrolysis of the 2-ester bond in DPPC, is a lysolipid. Preferably, the lysolipid is monoacyl-sn-glycero-3-phosphatidylcholine, monoacyl-sn-glycero-3-phosphatidylglycerol, or monoacyl-sn-glycero-3-phosphatidyldiglycerol.

Without being limited by this theory, lysolipids influence the transition temperature of the bilayer contained in the thermosensitive liposome according to the present invention, thereby influencing the heat-induced release of the active pharmaceutical ingredient contained in said thermosensitive liposome. This was demonstrated using the thermosensitive liposome according to the present invention in Example 5. Thus, in the thermosensitive liposome according to the present invention, a low concentration of lysolipids is preferred, preferably less than 5 mol.%, less than 4 mol.%, less than 3 mol.%, less than 2 mol.%, less than 1 mol. %, less than 1 mol.%, less than 0.9 mol.%, less than 0.8 mol.%, less than 0.7 mol.%, less than 0.6 mol.%, less than 0.5 mol.%, less than 0.4 mol.%, less than 0.3 mol.%, less than 0.2 mol.% or less than 0.1 mol.%.

DPPGn phospholipids, such as DPPG2, are more prone to degradation than standard phospholipids such as DPPC and DSPC. This increases the challenge of stabilizing the thermosensitive liposome and composition of the present invention compared to other formulations, such as LTSL. The stability of the thermosensitive liposome and composition of the present invention is discussed below. It should also be noted that the active pharmaceutical ingredient contained in said thermosensitive liposome or composition can promote or induce the formation of lysolipids.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein the concentration of lysolipids contained in the bilayer contained in said thermosensitive liposome is less than 5 mol.%, less than 4 mol.%, less than 3 mol.%, less than 2 mol.%, less than 1 mol.%, less than 1 mol.%, less than 0.9 mol.%, less than 0.8 mol.%, less than 0.7 mol.%, less than 0.6 mol.%, less than 0.5 mol.%, less than 0.4 mol.%, less than 0.3 mol.%, less than 0.2 mol.% or less than 0.1 mol.%.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein the concentration of lysolipids contained in the bilayer contained in said thermosensitive liposome is less than 2 mol%, preferably less than 1 mol%, and wherein 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearyl-sn-glycero-3-phosphatidylcholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG2) in combination constitute at least 95% of the number of molecules contained in said bilayer; and/or wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to no more than 150 nanometers; and/or

- 15 051237 wherein said active pharmaceutical ingredient is selected from the group consisting of doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof, irinotecan, an irinotecan derivative or a pharmaceutically acceptable salt thereof, and gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof, wherein more preferably said active pharmaceutical ingredient is selected from the group consisting of doxorubicin or a pharmaceutically acceptable salt thereof, gemcitabine or a pharmaceutically acceptable salt thereof, and irinotecan or a pharmaceutically acceptable salt thereof, and wherein said active pharmaceutical ingredient is contained in an intraliposomal buffer, more preferably wherein the molar ratio of doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and lipids contained in said bilayer is from 0.06 to no more than 0.10, preferably from 0.07 to no more than 0.09, wherein the molar ratio of irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.18, preferably at least 0.20, wherein the molar ratio of gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.12, preferably at least 0.15, and/or wherein said intraliposomal buffer is characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8; and/or wherein said bilayer does not contain cholesterol or a derivative thereof; and/or wherein said 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV).

A preferred embodiment of the present invention provides a thermosensitive liposome wherein the bilayer contained in said thermosensitive liposome is cholesterol-free. In the context of the present invention, a cholesterol-free bilayer means a bilayer that does not contain a detectable amount of cholesterol or a derivative thereof. In this context, a cholesterol derivative preferably means a cholesterol ester.

Amphiphilic molecules contained in the bilayer contained in the thermosensitive liposome according to the present invention have a tendency to form lamellar or micellar structures. An amphiphilic molecule with a tendency to form micellar structures thermodynamically promotes the formation of micelles or micellar structures in suspension. An amphiphilic molecule with a tendency to form lamellar structures thermodynamically promotes the formation of lamellar structures or liposomes in suspension. In the context of the present invention, an amphiphilic molecule with a high tendency to form micellar structures is called a surfactant. For example, most lysolipids and DSPE-PEG2000 are considered surfactants in the context of the present invention.

Without being limited by this theory, since the thermosensitive liposome according to the present invention is a lamellar structure, the formation of said thermosensitive liposome is thermodynamically unfavorable, i.e., said thermosensitive liposome is destabilized due to the high concentration of surfactants.

In a preferred embodiment, a thermosensitive liposome according to the present invention is provided, wherein the concentration of surfactants is less than 5 mol.%, less than 4 mol.%, less than 3 mol.%, less than 2 mol.%, less than 1 mol.%, less than 1 mol.%, less than 0.9 mol.%, less than 0.8 mol.%, less than 0.7 mol.%, less than 0.6 mol.%, less than 0.5 mol.%, less than 0.4 mol.%, less than 0.3 mol.%, less than 0.2 mol.% or less than 0.1 mol.%.

Preferred thermosensitive liposomes.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein said active pharmaceutical ingredient is doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is from 0.06 to at most 0.10, preferably from 0.07 to at most 0.09, wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to at most 150 nanometers; and wherein said intraliposomal buffer is characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8; and wherein preferably said bilayer does not contain cholesterol or a derivative thereof; and wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),

- 16 051237 wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayer is from 0.45 to no more than 0.65, preferably from 0.45 to no more than 0.55, and wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayer is from 0.15 to no more than 0.25, and wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35; and wherein preferably said 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV).

Preferably, the thermosensitive liposome according to the above embodiment is contained in a suitable drug delivery system, more preferably for use in the treatment of cancer.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein said active pharmaceutical ingredient is irinotecan, an irinotecan derivative or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.18, preferably at least 0.20, wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to at most 150 nanometers; and wherein said intraliposomal buffer is characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8; and wherein preferably said bilayer does not contain cholesterol or a derivative thereof; and wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayer is from 0.45 to at most 0.65, preferably from 0.45 to at most 0.55, and wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayer is from 0.15 to at most 0.25, and wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35; and where preferably said 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV).

Preferably, the thermosensitive liposome according to the above embodiment is contained in a suitable drug delivery system, more preferably for use in the treatment of cancer.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, wherein said active pharmaceutical ingredient is gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.12, preferably at least 0.15, wherein said thermosensitive liposome is characterized by a diameter of from 100 nanometers to at most 150 nanometers; and wherein said intraliposomal buffer is characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8; and wherein preferably said bilayer does not contain cholesterol or a derivative thereof; and wherein said bilayer comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayer is from 0.45 to at most 0.65, preferably from 0.45 to at most 0.55, and wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayer is from 0.15 to at most 0.25, and wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35; and where preferably said 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV).

Preferably, the thermosensitive liposome according to the above embodiment is contained in a suitable drug delivery system, more preferably for use in the treatment of cancer.

- 17 051237

Additional thermosensitive liposomes.

The present invention further provides a thermosensitive liposome comprising a bilayer and an intraliposomal buffer, wherein the molar concentration of 1,2-dipalmitoyl-snglycero-3-phosphodiglycerol (DPPG2) in said bilayer is at least 15 percent, wherein said thermosensitive liposome comprises an active pharmaceutical ingredient, wherein said intraliposomal buffer is characterized by a pH of from 5 to no more than 8, preferably from 6 to no more than 8. Such thermosensitive liposomes are referred to in this application as additional liposomes according to or according to the present invention or additional thermosensitive liposomes according to or according to the present invention.

All preferred embodiments and definitions described above in the context of the thermosensitive liposomes of the present invention apply to additional thermosensitive liposomes of the present invention.

In a preferred embodiment, an additional thermosensitive liposome according to the present invention is provided, which is also a thermosensitive liposome according to the present invention.

In a preferred embodiment, an additional thermosensitive liposome according to the present invention is provided, wherein the molar ratio of said active pharmaceutical ingredient and lipids contained in said bilayer is from 0.05 to no more than 0.3.

In a preferred embodiment, an additional thermosensitive liposome according to the present invention is provided, wherein said active pharmaceutical ingredient is doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof, wherein preferably the molar ratio of said doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is from 0.06 to no more than 0.10, more preferably from 0.07 to no more than 0.09.

In a preferred embodiment, an additional thermosensitive liposome according to the present invention is provided, wherein said active pharmaceutical ingredient is irinotecan, an irinotecan derivative or a pharmaceutically acceptable salt thereof, wherein preferably the molar ratio of said irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.18, more preferably at least 0.20.

In a preferred embodiment, there is provided an additional thermosensitive liposome according to the present invention, wherein said active pharmaceutical ingredient is gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof, wherein preferably the molar ratio of said gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.12, more preferably at least 0.15. Preparation of liposomes

Liposomes containing an active pharmaceutical ingredient, such as the thermosensitive liposomes of the present invention or additional thermosensitive liposomes of the present invention, can be prepared using a wide range of different techniques, such as lipid film hydration, ethanol incorporation, and extrusion methods, known to those skilled in the art. Example 1 describes the preparation of thermosensitive liposomes according to the present invention.

Preferably, the liposome can be obtained by a method comprising:

a) obtaining an unloaded liposome, wherein said unloaded liposome comprises a bilayer with the same lipid composition as said liposome, and wherein said unloaded liposome does not contain an active pharmaceutical ingredient contained in said liposome, or wherein the concentration of said active pharmaceutical ingredient in the intraliposomal buffer of said unloaded liposome is low, as defined below;

ac) preferably extruding said unloaded liposome to obtain a unilamellar liposome and

b) loading said unloaded liposome into a loading buffer.

In the preparation of a liposome containing an active pharmaceutical ingredient in an intraliposomal buffer according to the above method, such as the thermosensitive liposome according to the present invention described in Example 1, a corresponding unloaded liposome is first prepared that does not contain said active pharmaceutical ingredient in said intraliposomal buffer or contains a low concentration thereof (unloaded liposome). Then, the concentration of said active pharmaceutical ingredient in said intraliposomal buffer is increased during loading. A low concentration preferably means a concentration that is less than 0.1 times, or less than 0.01 times, or

- 18 051237 less than 0.001 times, or less than 0.0001 times lower than the concentration of the said active pharmaceutical ingredient in the intraliposomal buffer of the final liposome. The absence of an active pharmaceutical ingredient preferably means the absence of a detectable amount of the said active pharmaceutical ingredient. In this document, the concentration of the said active pharmaceutical ingredient can be determined by high-performance liquid chromatography (HPLC), as shown in Example 7.

The loading process, carried out in step b) of the above method, involves transferring the active pharmaceutical ingredient from the extraliposomal buffer to the intraliposomal buffer, whereby said active pharmaceutical ingredient passes through the bilayer. In this sense, loading (of the active pharmaceutical ingredient) can be envisaged as the reverse process of release (of the active pharmaceutical ingredient). The extraliposomal buffer during loading is also referred to as the loading buffer in the context of the present invention.

In one aspect of the present invention, there is provided a method for producing a liposome containing an active pharmaceutical ingredient, wherein the method comprises steps a) and b) described above, and wherein said loading buffer is characterized by a salt concentration of at least 66 mM and an osmolarity of at least 250 mOsmol/kg. More preferably, said loading buffer is characterized by a salt concentration of at least 66 mM and an osmolarity of from 250 mOsmol/kg to no more than 350 mOsmol/kg. Preferably, said liposome in said preferred method is a thermosensitive liposome according to the present invention or an additional thermosensitive liposome according to the present invention.

Example 6 shows that the thermosensitive liposomes of the present invention can be prepared by the preferred method described above using a loading buffer with a salt concentration of 66 mM and an osmolarity of 294 mOsmol/kg or 300 mOsmol/kg. In particular, Example 6 shows that loading buffers with high salt concentrations and osmolarities result in shorter loading times (20 minutes for buffer A with an osmolarity of 300 mOsmol/kg without trehalose, 90 minutes for buffer C with an osmolarity of 294 mOsmol/kg and 8.9% (w/v) trehalose).

In a further aspect of the present invention, there is provided a method for producing a liposome containing an active pharmaceutical ingredient, wherein the method comprises steps a) and b) as described above, wherein said method comprises exchanging a buffer, wherein said method comprises using a loading buffer and using a storage buffer, and wherein said storage buffer is characterized by a salt concentration of less than 100 mM and an osmolarity of more than 300 mOsmol/kg. More preferably, said storage buffer is characterized by a salt concentration of less than 100 mM and an osmolarity of from 300 mOsmol/kg to no more than 450 mOsmol/kg.

The aqueous solution in contact with the bilayer contained in the liposome according to the present invention constitutes an extraliposomal buffer. In the context of (pharmaceutical) compositions, said extraliposomal buffer is referred to as a storage buffer if said thermosensitive liposome is dispersed in said extraliposomal buffer.

Example 9 shows that a method that utilizes a buffer replacement with a low salt concentration storage buffer results in lower leakage of the contained active pharmaceutical ingredient and higher dispersion stability.

The method according to the above aspects of the present invention is referred to as the method of the present invention in the context of this application. It should be noted that the definitions and embodiments provided above in the context of thermosensitive liposomes can be applied, with appropriate modifications, to liposomes in general, as mentioned in the context of the method of the present invention. For example, the diameter of a thermosensitive liposome is clearly defined in the same way as the diameter of the liposome for which the production method is described herein.

It will be understood by one skilled in the art that the composition according to the present invention or an additional composition according to the present invention can also be obtained by means of the method according to the present invention or by means of a method comprising the method according to the present invention.

Any preference or embodiment relating to a thermosensitive liposome according to the present invention or a composition comprising a thermosensitive liposome according to the present invention may be combined with a method according to the present invention, it being understood that these preferences and embodiments also disclose corresponding methods according to the present invention for producing such thermosensitive liposomes or compositions.

- 19 051237

From the context of the method according to the present invention, it is understood that the concentration regarding the lipids contained in the bilayer may refer either to the lipids contained in the bilayer of said unloaded liposome or to the bilayer of said liposome, since the lipid compositions of these bilayers are the same.

In a preferred embodiment, a method according to the present invention is provided, wherein the loading buffer is characterized by a salt concentration of at least 66 mM and an osmolarity of 250 mOsmol/kg to no more than 350 mOsmol/kg, and said storage buffer is characterized by a salt concentration of less than 100 mM and an osmolarity of more than 300 mOsmol/kg. Most preferably, said loading buffer is characterized by a salt concentration of 66 mM to no more than 120 mM and an osmolarity of 250 mOsmol/kg to no more than 350 mOsmol/kg, and said storage buffer is characterized by a salt concentration of less than 100 mM and an osmolarity of 390 mOsmol/kg.

In a preferred embodiment, there is provided a method according to the present invention, wherein said liposome comprises 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2), wherein preferably the molar concentration of DPPG2 in the bilayer of said liposome is from 0.10 to at most 0.95, from 0.10 to at most 0.90, from 0.10 to at most 0.85, from 0.10 to at most 0.80, from 0.10 to at most 0.75, from 0.10 to at most 0.70, from 0.10 to at most 0.65, from 0.10 to at most 0.60, from 0.10 to at most 0.55, from 0.10 to at most 0.50, from 0.10 to at most 0.45, from 0.10 up to no more than 0.40, from 0.15 to no more than 0.35 or from 0.20 to no more than 0.30.

In a more preferred embodiment, the method according to the present invention is provided, wherein said liposome is a thermosensitive liposome. In an even more preferred embodiment, the method according to the present invention is provided, wherein said liposome is a thermosensitive liposome according to the present invention or an additional thermosensitive liposome according to the present invention. In a most preferred embodiment, the method according to the present invention is provided, wherein said liposome is a thermosensitive liposome according to the present invention.

In a preferred embodiment, a method according to the present invention is provided, wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayer contained in said unloaded liposome is from 0.45 to at most 0.65, preferably from 0.45 to at most 0.55; and/or wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayer contained in said unloaded liposome is from 0.15 to at most 0.25; and/or wherein the molar concentration of 1,2-dipalmitoyl-snglycero-3-phosphodiglycerol (DPPG2) in said bilayer contained in said unloaded liposome is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35.

In a preferred embodiment, the method according to the present invention is provided, wherein said unloaded liposome has a diameter of from 100 nanometers to no more than 200 nanometers, preferably from 100 nanometers to no more than 150 nanometers.

In a preferred embodiment, there is provided a method according to the present invention, wherein said active pharmaceutical ingredient is doxorubicin, a doxorubicin derivative, or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of doxorubicin, said doxorubicin derivative, or said pharmaceutically acceptable salt thereof, to the lipids contained in said bilayer is from 0.06 to at most 0.10, preferably from 0.07 to at most 0.09; or wherein said active pharmaceutical ingredient is irinotecan, an irinotecan derivative, or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of irinotecan, said irinotecan derivative, or said pharmaceutically acceptable salt thereof, to the lipids contained in said bilayer is at least 0.18, preferably at least 0.20; or wherein said active pharmaceutical ingredient is gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.12, preferably at least 0.15.

Preferably, the method according to the present invention does not include the addition of cholesterol or a derivative thereof.

In a preferred embodiment, a method according to the present invention is provided, wherein step a) included in said method comprises:

- 20 051237 aa) obtaining a solution of lipids in an organic solvent with the same molar ratio as the lipids in the bilayer of said liposome; and ab) mixing said lipid solution with an aqueous solution, wherein said aqueous solution is characterized by essentially the same composition as the intraliposomal buffer contained in said liposome, in the absence of said active pharmaceutical ingredient, wherein preferably said aqueous solution is characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8, and wherein said mixing is preferably carried out by introducing an organic solvent, and wherein said mixing results in the formation of an unloaded liposome; and ac) preferably extruding said unloaded liposome to obtain a unilamellar liposome.

In a preferred embodiment, a method according to the present invention is provided, wherein step a) included in said method comprises steps aa), ab) and ac) defined above, and wherein said organic solvent used in step aa) is selected from the group of organic solvents capable of dissolving lipids, wherein preferably said organic solvent is selected from the group consisting of a chloroform/methanol 9:1 (v/v) mixture, chloroform, methanol, isopropanol, ethanol and mixtures thereof.

In a preferred embodiment, a method according to the present invention is provided, wherein step a) included in said method comprises steps aa), ab) and ac) defined above, and wherein said aqueous solution used in step ab) comprises a buffer selected from the group consisting of a buffer based on (NH^SO^, a buffer based on (NH ₄ ) ₂ HPO ₄ , a phosphate buffer and a buffer based on HBS.

In a preferred embodiment, a method according to the present invention is provided, wherein step a) included in said method comprises steps aa), ab) and ac) defined above, and wherein said extrusion is carried out at temperatures at which the lipids are in a liquid phase state.

Said loading of the unloaded liposome in step b) of the method according to the present invention results in the transfer of said active pharmaceutical ingredient into the intraliposomal buffer and is described in more detail below.

Preferably, said loading is carried out at a temperature of from 35°C to 39°C, more preferably from 36°C to 38°C, most preferably from 37°C to 38°C.

The method according to the present invention preferably further comprises filtration steps.

The preferred method of preparation described above is exemplified in Fig. 2, where step (ab) involves the introduction of ethanol. It should be noted that Fig. 2 shows two buffer exchanges. The first involves replacing the extraliposomal buffer at the end of step (ab) with loading buffer. The second involves replacing the loading buffer with storage buffer.

Preferably, the method according to the present invention comprises buffer exchange. Buffer exchange is defined as the complete or partial replacement of an extraliposomal buffer with a new extraliposomal buffer. It should be understood that the replacement may involve adjusting the concentration of a component in said buffer, adding a component to said buffer, and/or removing a component from said buffer. A preferred buffer exchange method is selected from the group consisting of flow-through filtration (TFF), chromatography, and centrifugation, preferably flow-through filtration.

Passive loading is loading under the influence of a concentration gradient of said active pharmaceutical ingredient through said bilayer. With respect to the thermosensitive liposome according to the present invention, at a temperature above the transition temperature of said thermosensitive liposome, a high ratio of the concentration of said active pharmaceutical ingredient in the extraliposomal buffer to the concentration of said active pharmaceutical ingredient in the intraliposomal buffer leads to transfer through said bilayer, which is in the liquid phase. A high ratio preferably means at least 10, or at least 100, or at least 1000, or at least 10,000. Preferably, at the beginning of passive loading, a detectable amount of said active pharmaceutical ingredient is absent in said intraliposomal buffer. In this case, the concentration of said active pharmaceutical ingredient is determined by HPLC. After the required amount of active pharmaceutical ingredient has been transferred into the intraliposomal buffer, the temperature is lowered, and said bilayer transitions back to the gel phase. Since the permeability of the bilayer is lower in the gel phase, the active pharmaceutical ingredient of the intraliposomal buffer will not easily pass into

- 21 051237 extra-liposomal buffer, even if the concentration of the active pharmaceutical ingredient in the extra-liposomal buffer decreases.

Active loading is loading under the influence of an ion gradient in the bilayer. This can be an ammonium gradient, a proton gradient (i.e., a pH gradient), or an EDTA gradient, for example. For example, doxorubicin, irinotecan, and gemcitabine, all of which are water-soluble active pharmaceutical ingredients, can be transported into the intraliposomal buffer in the temperature-sensitive liposome of the present invention via active loading. This is described in detail in Example 1.

Preferably, the method according to the present invention comprises d) storing said loaded liposome in a storage buffer for 1 to no more than 5 weeks at a temperature of about 5°C, 1 to no more than 10 weeks at a temperature of about 5°C, 1 to no more than 15 weeks at a temperature of about 5°C, 1 to no more than 20 weeks at a temperature of about 5°C, or 1 to no more than 20 weeks at a temperature of about 5°C, or 1 to no more than 16 months at a temperature of about -20°C, or 12 to no more than 16 months at a temperature of about -20°C. More preferably, said loaded liposome is stable during said storage, as defined below.

Preferably, the method according to the present invention comprises adding at least one excipient that can further contribute to improving the delivery of said liposome and/or composition containing said composition to and/or into a tissue and/or cell.

In a preferred embodiment, there is provided a method for producing a liposome, preferably a thermosensitive liposome, more preferably a thermosensitive liposome according to the present invention or a further thermosensitive liposome according to the present invention, most preferably a thermosensitive liposome according to the present invention, comprising:

a) obtaining an unloaded liposome, wherein said unloaded liposome comprises a bilayer with the same lipid composition as said liposome and wherein said unloaded liposome does not contain an active pharmaceutical ingredient contained in said liposome, wherein said a) obtaining said unloaded liposome comprises:

aa) obtaining a solution of lipids in an organic solvent with the same molar ratio as the lipids in the bilayer of said liposome, wherein preferably said organic solvent is a 9:1 (v/v) chloroform/methanol mixture, chloroform or ethanol; and ab) mixing said lipid solution with an aqueous solution, wherein said aqueous solution is characterized by essentially the same composition as an intraliposomal buffer contained in said liposome, in the absence of said active pharmaceutical ingredient, wherein preferably said aqueous solution is characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8, and wherein preferably said aqueous solution comprises a buffer selected from the group consisting of a (NH ₄ ) ₂ SO ₄ based buffer, a (NH ₄ ) ₂ HPO ₄ based buffer, a phosphate buffer and an HBS based buffer, and wherein said mixing is preferably carried out by introducing an organic solvent, and wherein said mixing results in the formation of an unloaded liposome; and ac) preferably extruding said unloaded liposome,

b) providing loading of said unloaded liposome with an active pharmaceutical ingredient by active loading using a loading buffer to form a loaded liposome, wherein said loading buffer is characterized by a salt concentration of from 66 mM to no more than 120 mM and an osmolarity of from 250 mOsmol/kg to no more than 350 mOsmol/kg, wherein said loading is preferably carried out at a temperature of from 35°C to 39°C, more preferably from 36°C to 38°C, most preferably from 37°C to 38°C;

c) replacing said loading buffer with a storage buffer, wherein said storage buffer has a salt concentration of less than 100 mM and an osmolarity of greater than 300 mOsmol/kg,

d) preferably storing said loaded liposome in said storage buffer for 1 to no more than 20 weeks at a temperature of about 5°C, or 1 to no more than 20 weeks at a temperature of about 5°C, or 1 to no more than 16 months at a temperature of about -20°C, or 12 to no more than 16 months at a temperature of about -20°C.

In a preferred embodiment, a method according to the previous embodiment is provided, wherein the liposome obtained at the end of step c) is stable upon said storage in step d), wherein the storage stability or storage is defined below.

- 22 051237

In a more preferred embodiment, a method according to the previous embodiment is provided, wherein preferably said storage in step d) is from 12 to no more than 16 months at a temperature of approximately -20°C, wherein the liposome obtained at the end of step c) is storage stable, wherein storage stability means the following:

the concentration of the active pharmaceutical ingredient contained in said liposome obtained at the end of step c) does not change by more than 15% upon storage; and/or the diameter of said liposome obtained at the end of step c) does not change by more than 30% upon storage; and/or the bilayer concentration of lysolipids contained in said liposome obtained at the end of step c) does not increase by more than 5% upon storage; and/or the polydispersity of said thermosensitive liposome or the polydispersity of said liposome obtained at the end of step c) does not increase above 0.5 upon storage; and/or if said liposome obtained at the end of step c) is characterized by highly selective delivery upon heat treatment, then said liposome continues to be characterized by highly selective delivery upon heat treatment after storage; and/or if said liposome obtained at the end of step c) is a suitable drug delivery system, then said liposome still is a suitable drug delivery system after storage.

Composition.

In one aspect, the present invention provides a composition comprising a thermosensitive liposome according to the present invention, wherein preferably said composition comprises at least one excipient that can further contribute to improving the targeting or delivery of said composition, or said thermosensitive liposome, or said active pharmaceutical ingredient to or into a tissue or cell. The preferred tissue or cell is a tumor or a tumor cell. The compositions described herein are referred to herein as compositions according to or according to the present invention. A composition according to the present invention may comprise one or more thermosensitive liposomes according to the present invention. In the context of the present invention, the excipient may be a separate molecule, but it may also be a conjugated moiety.

In a preferred embodiment, said composition is intended for use as a medicament, preferably for use in the treatment, alleviation, delay, cure and/or prevention of cancer. Said composition is thus a pharmaceutical composition. A pharmaceutical composition typically comprises a pharmaceutically acceptable carrier, diluent and/or excipient. In a preferred embodiment, the composition of the present invention comprises a thermosensitive liposome as defined herein and further comprises a pharmaceutically acceptable formulation, filler, preservative, solubilizer, carrier, diluent, excipient, salt, adjuvant and/or solvent. Such pharmaceutically acceptable carrier, filler, preservative, solubilizer, diluent, salt, adjuvant, solvent and/or excipient can be found, for example, in Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000.

The thermosensitive liposome of the present invention may comprise at least one ionogenic group. The ionogenic group may be basic or acidic and may be charged or neutral. The ionogenic group may be present as an ion pair with a corresponding counterion that bears the opposite charge(s). Examples of cationic counterions include sodium, potassium, cesium, tris, lithium, calcium, magnesium, trialkylammonium, triethylammonium, and tetraalkylammonium. Examples of anionic counterions include chloride, bromide, iodide, lactate, mesylate, besylate, triflate, acetate, trifluoroacetate, dichloroacetate, tartrate, lactate, and citrate.

The pharmaceutical composition according to the present invention may contain an auxiliary agent for improving the stability, solubility, absorption, bioavailability, activity, pharmacokinetics, pharmacodynamics, cellular uptake and intracellular trafficking of the said active pharmaceutical ingredient.

The composition according to the present invention can be administered at an effective concentration, a specified number of times, to an animal, preferably a mammal. Administration can occur by local, systemic and/or parenteral routes, for example, intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular, intraocular, nasal, urogenital, intradermal, cutaneous, enteral, intravitreal, intracavernous, intracerebral, intrathecal, epidural or oral routes.

- 23 051237

The compounds contained in the composition according to the present invention may also be provided separately, for example, to ensure sequential administration of the active components of the composition according to the present invention. In such a case, the composition according to the present invention is a combination of compounds comprising at least a thermosensitive liposome according to the present invention and at least one excipient.

As defined above, the aqueous solution in contact with the bilayer contained in the thermosensitive liposome according to the present invention constitutes an extraliposomal buffer. In the context of (pharmaceutical) compositions, said extraliposomal buffer is referred to as a storage buffer if said thermosensitive liposome is dispersed in said extraliposomal buffer.

In a preferred embodiment, a composition according to the present invention is provided, comprising a storage buffer, wherein said storage buffer is characterized by a salt concentration of less than 120 mM, or less than 110 mM, or less than 100 mM, or less than 90 mM, or less than 80 mM, or less than 70 mM, or less than 60 mM, or less than 50 mM, or less than 40 mM, or less than 30 mM, or less than 20 mM, or less than 10 mM.

Example 9 shows that the composition of the present invention, characterized by a low salt concentration, results in lower leakage of the active pharmaceutical ingredient contained therein and higher dispersion stability.

In another preferred embodiment, there is provided a composition according to the present invention comprising a storage buffer, wherein the osmolarity of said storage buffer is greater than 150 mOsmol/kg, or greater than 200 mOsmol/kg, or greater than 250 mOsmol/kg, or greater than 300 mOsmol/kg, or greater than 350 mOsmol/kg, or greater than 400 mOsmol/kg. Preferably, the osmolarity of said storage buffer is less than 450 mOsmol/kg, more preferably less than 410 mOsmol/kg. In a most preferred embodiment, a composition according to the present invention is provided, comprising a storage buffer, wherein the osmolarity of said storage buffer is from 200 mOsmol/kg to no more than 450 mOsmol/kg, or wherein the osmolarity of said storage buffer is from 300 mOsmol/kg to no more than 450 mOsmol/kg, or wherein the osmolarity of said storage buffer is from 200 mOsmol/kg to no more than 410 mOsmol/kg, or wherein the osmolarity of said storage buffer is from 300 mOsmol/kg to no more than 410 mOsmol/kg.

Another preferred embodiment provides a composition according to the present invention comprising a storage buffer, wherein said storage buffer comprises a cryoprotectant. Preferably, said cryoprotectant is trehalose or sucrose. Most preferably, said cryoprotectant is trehalose.

The inclusion of a cryoprotectant in the composition according to the present invention allows for the storage of said composition at a low temperature to prevent the degradation of the active pharmaceutical ingredient contained in the temperature-sensitive liposome contained in said composition. In a preferred embodiment, a composition according to the present invention is provided, wherein said composition still represents a suitable drug delivery system as defined above after storage at a temperature below 0°C, or below -5°C, or below -10°C, or below -15°C, or below -20°C for at least 30 days.

Example 9 shows that the composition according to the present invention, characterized by high osmolarity and the presence of trehalose as a cryoprotectant, is capable of preventing the degradation of the active pharmaceutical ingredient.

In a preferred embodiment, a composition according to the present invention is provided, wherein the temperature-sensitive liposome contained in said composition is dispersed in a storage buffer, wherein the salt concentration of said storage buffer is less than 100 mM, and/or the osmolarity of said storage buffer is greater than 300 mOsmol/kg. Preferably, the composition according to this embodiment comprises a cryoprotectant.

In a preferred embodiment, there is provided a composition according to the present invention, comprising a plurality of thermosensitive liposomes according to the present invention, dispersed in a storage buffer, wherein the concentration of said thermosensitive liposomes in said storage buffer is from 10 mmol/L to not more than 50 mmol/L, or from 15 mmol/L to not more than 50 mmol/L, or from 20 mmol/L to not more than 50 mmol/L, or from 25 mmol/L to not more than 50 mmol/L, or from 30 mmol/L to not more than 50 mmol/L, or from 35 mmol/L to not more than 45 mmol/L, or from 37.5 mmol/L to not more than 42.5 mmol/L. Preferably, the salt concentration of said storage buffer is less than 100 mM and/or the osmolarity of said storage buffer is more than 300 mOsmol/kg.

- 24 051237

In another preferred embodiment, a composition according to the present invention is provided, wherein the concentration of thermosensitive liposomes according to the present invention is from 35 mmol/L to no more than 45 mmol/L, wherein the polydispersity index (PDI) of said thermosensitive liposomes is less than 0.5, or less than 0.4, or less than 0.3, or less than 0.2, or less than 0.1, or less than 0.09, or less than 0.08, or less than 0.07, or less than 0.06, or less than 0.05. Preferably, the salt concentration of said storage buffer is less than 100 mM, and/or the osmolarity of said storage buffer is more than 300 mOsmol/kg.

In another preferred embodiment, there is provided a composition according to the present invention, wherein the diameter of the thermosensitive liposomes according to the present invention contained in said composition is from 100 nm to no more than 150 nm, wherein the polydispersity index (PDI) of said thermosensitive liposomes is less than 0.5, or less than 0.4, or less than 0.3, or less than 0.2, or less than 0.1, or less than 0.09, or less than 0.08, or less than 0.07, or less than 0.06, or less than 0.05; wherein more preferably the polydispersity of said thermosensitive liposomes is less than 0.1. Preferably, the salt concentration of said storage buffer is less than 100 mM, and/or the osmolarity of said storage buffer is more than 300 mOsmol/kg.

In a preferred embodiment, there is provided a composition according to the present invention comprising a storage buffer, wherein the ratio of the concentration of said active pharmaceutical ingredient in said storage buffer to the concentration of said active pharmaceutical ingredient in an intraliposomal buffer contained in the thermosensitive liposomes contained in said composition is less than 0.1, or less than 0.09, or less than 0.08, or less than 0.07, or less than 0.06, or less than 0.05, or less than 0.04, or less than 0.03, or less than 0.02, or less than 0.01, or less than 0.009, or less than 0.008, or less than 0.007, or less than 0.006, or less than 0.005, or less than 0.004, or less than 0.003, or less than 0.002, or less than 0.001.

In a preferred embodiment, there is provided a composition according to the present invention, wherein the concentration of the thermosensitive liposomes according to the present invention is from 10 mmol/L to no more than 50 mmol/L, preferably from 35 mmol/L to no more than 45 mmol/L; and wherein said composition comprises a storage buffer, wherein the salt concentration of said storage buffer is less than 100 mM, and the osmolarity of said storage buffer is more than 300 mOsmol/kg; and wherein the active pharmaceutical ingredients contained in said thermosensitive liposomes are doxorubicin, a doxorubicin derivative, or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of doxorubicin, said doxorubicin derivative, or said pharmaceutically acceptable salt thereof to the lipids contained in the bilayers contained in said thermosensitive liposomes is from 0.06 to at most 0.10, preferably from 0.07 to at most 0.09; and wherein said thermosensitive liposomes are characterized by a diameter of from 100 nanometers to at most 150 nanometers; and wherein the intraliposomal buffers contained in said thermosensitive liposomes are characterized by a pH of from 5 to at most 8, preferably from 6 to at most 8; and wherein preferably the bilayers contained in said thermosensitive liposomes do not contain cholesterol or a derivative thereof; and wherein the bilayers contained in said thermosensitive liposomes comprise 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayers is from 0.45 to no more than 0.65, preferably from 0.45 to no more than 0.55, and wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayers is from 0.15 to no more than 0.25, and wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayers is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35.

In a more preferred embodiment, a composition according to the above preferred embodiment is provided, wherein the polydispersity index (PDI) of said thermosensitive liposomes is less than 0.5, or less than 0.4, or less than 0.3, or less than 0.2, or less than 0.1, or less than 0.09, or less than 0.08, or less than 0.07, or less than 0.06, or less than 0.05.

Example 10 shows that the composition according to the above embodiment is a suitable drug delivery system.

- 25 051237

In particular, this composition is presented as stable during long-term storage (16 months), with minor doxorubicin leakage and changes in the diameter of the temperature-sensitive liposome when stored at -20°C ± 5°C. The results further indicate that the composition was stable for 11 weeks when stored at 5°C ± 3°C. No signs of phospholipid degradation were observed during 12 months of storage. Furthermore, the composition was stable during six freeze-thaw cycles. Furthermore, doxorubicin accumulation in tumors was demonstrated in vivo through a biodistribution study in Brown Norway rats with subcutaneous administration of BN175 sarcoma cells in the hind limb. Animals treated with the composition demonstrated doxorubicin concentrations in heated tumors that were 15.8 times higher than the concentration in unheated tissue. Treatment of rats with the composition showed significantly improved tumor growth inhibition compared to animals treated with saline (Fig. 13). Moreover, compared to non-liposomal doxorubicin treatment at an equivalent dose, the composition also resulted in significant tumor growth inhibition and increased survival (Fig. 13).

In a preferred embodiment, there is provided a composition according to the present invention, wherein the concentration of the thermosensitive liposomes according to the present invention is from 10 mmol/L to at most 50 mmol/L, preferably from 35 mmol/L to at most 45 mmol/L; and wherein said composition comprises a storage buffer, wherein the salt concentration of said storage buffer is less than 100 mM, and the osmolarity of said storage buffer is more than 300 mOsmol/kg; and wherein the active pharmaceutical ingredients contained in said thermosensitive liposomes are irinotecan, an irinotecan derivative, or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of irinotecan, said irinotecan derivative, or said pharmaceutically acceptable salt thereof, to the lipids contained in the bilayers contained in said thermosensitive liposomes is at least 0.18, preferably at least 0.20; and wherein said thermosensitive liposomes are characterized by a diameter of from 100 nanometers to no more than 150 nanometers; and wherein the intraliposomal buffers contained in said thermosensitive liposomes are characterized by a pH of from 5 to no more than 8, preferably from 6 to no more than 8; and wherein preferably the bilayers contained in said thermosensitive liposomes do not contain cholesterol or a derivative thereof; and wherein the bilayers contained in said thermosensitive liposomes comprise 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayers is from 0.45 to no more than 0.65, preferably from 0.45 to no more than 0.55, and wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayers is from 0.15 to no more than 0.25, and wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayers is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35.

In a more preferred embodiment, a composition according to the above preferred embodiment is provided, wherein the polydispersity index (PDI) of said thermosensitive liposomes is less than 0.5, or less than 0.4, or less than 0.3, or less than 0.2, or less than 0.1, or less than 0.09, or less than 0.08, or less than 0.07, or less than 0.06, or less than 0.05.

In a preferred embodiment, there is provided a composition according to the present invention, wherein the concentration of the thermosensitive liposomes according to the present invention is from 10 mmol/L to at most 50 mmol/L, preferably from 35 mmol/L to at most 45 mmol/L; and wherein said composition comprises a storage buffer, wherein the salt concentration of said storage buffer is less than 100 mM and the osmolarity of said storage buffer is more than 300 mOsmol/kg; and wherein the active pharmaceutical ingredients contained in said thermosensitive liposomes are gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof, and wherein the molar ratio of gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and the lipids contained in the bilayers contained in said thermosensitive liposomes is at least 0.12, preferably at least 0.15; and wherein said thermosensitive liposomes are characterized by a diameter of from 100 nanometers to no more than 150 nanometers; and

- 26 051237 wherein the intraliposomal buffers contained in said thermosensitive liposomes are characterized by a pH of from 5 to no more than 8, preferably from 6 to no more than 8; and wherein preferably the bilayers contained in said thermosensitive liposomes do not contain cholesterol or a derivative thereof; and wherein the bilayers contained in said thermosensitive liposomes comprise 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in said bilayers is from 0.45 to no more than 0.65, preferably from 0.45 to no more than 0.55, and wherein the molar concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) in said bilayers is from 0.15 to no more than 0.25, and wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in the said bilayers is from 0.15 to no more than 0.35, preferably from 0.25 to no more than 0.35.

In a more preferred embodiment, a composition according to the above preferred embodiment is provided, wherein the polydispersity index (PDI) of said thermosensitive liposomes is less than 0.5, or less than 0.4, or less than 0.3, or less than 0.2, or less than 0.1, or less than 0.09, or less than 0.08, or less than 0.07, or less than 0.06, or less than 0.05.

Additional composition.

The present invention further provides a composition comprising a thermosensitive liposome dispersed in a storage buffer, wherein the salt concentration of said storage buffer is less than 100 mM and the osmolarity of said storage buffer is greater than 300 mOsmol/kg; wherein said thermosensitive liposome comprises a bilayer and an intraliposomal buffer; wherein the molar concentration of 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is at least 15 percent; and wherein said thermosensitive liposome comprises an active pharmaceutical ingredient. Such compositions are referred to herein as additional compositions according to or according to the present invention.

All preferred embodiments and definitions described above in the context of the compositions of the present invention or the thermosensitive liposomes of the present invention apply to additional compositions of the present invention.

In a preferred embodiment, an additional composition according to the present invention is provided, which is also a composition according to the present invention.

In a preferred embodiment, an additional composition according to the present invention is provided, wherein said intraliposomal buffer is characterized by a pH of from 5 to no more than 8, preferably from 6 to no more than 8.

In a preferred embodiment, an additional composition according to the present invention is provided, wherein said additional composition contains at least one excipient that can further contribute to improving the delivery of said composition and/or said thermosensitive liposome to and/or into a tissue and/or cell.

In a preferred embodiment, an additional composition according to the present invention is provided, wherein said active pharmaceutical ingredient is doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof, wherein preferably the molar ratio of said doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is from 0.06 to no more than 0.10, more preferably from 0.07 to no more than 0.09.

In a preferred embodiment, there is provided an additional composition according to the present invention, wherein said active pharmaceutical ingredient is irinotecan, an irinotecan derivative or a pharmaceutically acceptable salt thereof, wherein preferably the molar ratio of said irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.18, more preferably at least 0.20.

In a preferred embodiment, there is provided an additional composition according to the present invention, wherein said active pharmaceutical ingredient is gemcitabine, a gemcitabine derivative or a pharmaceutically acceptable salt thereof, wherein preferably the molar ratio of said gemcitabine, said gemcitabine derivative or said pharmaceutically acceptable salt thereof and the lipids contained in said bilayer is at least 0.12, more preferably at least 0.15.

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Drug delivery system.

A drug delivery system is a composition containing an active pharmaceutical ingredient, wherein other compounds contained in said composition contribute to improving the stability, solubility, absorption, bioavailability, activity, pharmacokinetics, pharmacodynamics, cellular uptake and/or intracellular trafficking of said active pharmaceutical ingredient. Preferably, the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention, or the additional composition according to the present invention is a drug delivery system.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention, wherein said thermosensitive liposome, said additional thermosensitive liposome, said composition, or said additional composition has one or more of the following properties, each of which is defined below:

Low complement activation, no anaphylaxis, no toxicity, long half-life in the bloodstream, no ABC, adequate clearance, adequate biodistribution, highly selective delivery during heat treatment, high storage stability.

A thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention, characterized by all of the above properties, is suitable for use as a drug delivery system, as explained below, and is referred to as a suitable drug delivery system in the context of the present application. In a preferred embodiment, a suitable drug delivery system is provided.

Administration of a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention to an animal may result in adverse reactions such as activation of the complement system and anaphylaxis.

The complement system is part of the non-adaptive immune system, as is known to those skilled in the art. Activation of the complement system, in which granulocytes and primarily neutrophils play a key role, can lead to complement-mediated pseudoallergy (CARPA). Activation of the complement system by the introduction of a drug delivery system is clearly undesirable. As explained in Example 2, complement activation associated with the thermosensitive liposome of the present invention or the composition of the present invention can be determined in vitro using C3a, Bb, and SC5b-9 ELISA kits on human plasma incubated with the thermosensitive liposome. Low complement activation means complement activation that is at least 10 times lower, or at least 9 times lower, or at least 8 times lower, or at least 7 times lower, or at least 6 times lower, or at least 5 times lower, or at least 4 times lower, or at least 3 times lower, or at least 2 times lower compared to the positive control (zymosan), wherein said complement activation is determined as a C3a readout, a Bb readout, or an SC5b-9 readout according to the protocol described in Example 2.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention, wherein said (additional) thermosensitive liposome or (additional) composition is characterized by low complement activation after administration of said (additional) thermosensitive liposome or (additional) composition to an animal.

Anaphylaxis is a serious, rapidly developing allergic reaction, as is known to those skilled in the art. Obviously, anaphylaxis caused by the introduction of a system

- 28 051237 drug delivery, is undesirable. The absence of anaphylaxis or anaphylactic reactions is defined for the (additional) thermosensitive liposome or (additional) composition as the absence of anaphylaxis after administration of said thermosensitive liposome or said composition, determined as specified in Example 2.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention, wherein said (additional) thermosensitive liposome or (additional) composition does not result in anaphylaxis after administration of said thermosensitive liposome or composition to an animal.

Preferably, a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention is defined as non-toxic if the administration of said (additional) thermosensitive liposome or said (additional) composition to an animal is characterized by low complement activation and the absence of anaphylaxis. In a preferred embodiment, a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention is provided that is non-toxic.

A drug delivery system is administered to an animal to deliver an active pharmaceutical ingredient contained in said drug delivery system to a target, which is a cell contained in the body of said animal or a part thereof, or a tissue contained in the body of said animal or a part thereof, or an organ contained in the body of said animal or a part thereof. In the context of the present application, said cell is preferably a tumor cell, and said tissue or a part thereof is preferably a tumor. Delivery of an active pharmaceutical ingredient by a drug delivery system means that the active pharmaceutical ingredient contained in said drug delivery system, which is preferably a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention, is transferred from said drug delivery system to said target.

Regardless of the route of administration, the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention, or the additional composition according to the present invention will preferably circulate in the bloodstream of said animal after administration. In order for said (additional) thermosensitive liposome or said (additional) composition to be delivered to said cell, said tissue, said organ, or said part thereof, it is required that said (additional) thermosensitive liposome or said (additional) composition not be eliminated from the bloodstream after a short period of time. In other words, it is required that said (additional) thermosensitive liposome or said (additional) composition be characterized by a long half-life in the bloodstream and/or not lead to accelerated blood clearance (ABC). Both concepts refer to clearance, which is a pharmacokinetic measure of the volume of blood plasma from which the specified (additional) thermosensitive liposome or the specified (additional) composition is completely removed per unit of time.

Long half-life in the bloodstream means that in vivo the half-life of the said active pharmaceutical ingredient in the bloodstream of an animal exceeds 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145 minutes, 150 minutes, 155 minutes, 160 minutes, 165 minutes, 170 minutes, 175 minutes, 180 minutes, 185 minutes, 190 minutes, 195 minutes, 200 minutes, 205 minutes, 210 minutes, 215 minutes, 220 minutes, 225 minutes, 230 minutes, 235 minutes, or 240 minutes. In the present invention, the animal is preferably a human, a rat, a cat, a dog, or a pig. Preferably, the in vivo half-life of said active pharmaceutical ingredient in the bloodstream of an animal is from 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, 120 minutes, 125 minutes, 130 minutes, 135 minutes, 140 minutes, 145 minutes, 150 minutes, 155 minutes, 160 minutes, 165 minutes, 170 minutes, 175 minutes, 180 minutes, 185

- 29 051237 minutes, 190 minutes, 195 minutes, 200 minutes, 205 minutes, 210 minutes, 215 minutes, 220 minutes, 225 minutes, 23 0 minutes, 23 5 minutes or 240 minutes up to no more than 720 minutes.

In a preferred embodiment, a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention is provided, characterized by a long half-life in the bloodstream.

Accelerated blood clearance (ABC) is a phenomenon in which the first administration of an active pharmaceutical ingredient or drug delivery system to an animal results in significantly lower clearance than subsequent administrations within a given period of time. In other words, in the case of accelerated blood clearance, said subsequent administration is characterized by significantly higher clearance, i.e., accelerated clearance. Without being limited by this theory, ABC can be explained by an immune response in said animal following said initial administration. A subsequent administration is preferably the second, third, fourth, fifth, or sixth administration relative to said initial administration.

The absence of ABC is defined for a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention or an additional composition according to the present invention as the fact that the half-life in the bloodstream of said (additional) thermosensitive liposome or said (additional) composition after the first administration to an animal is no more than 150%, or no more than 140%, or no more than 130%, or no more than 120%, or no more than 110%, or no more than 105%, when calculated on the half-life in the bloodstream of said (additional) thermosensitive liposome or said (additional) composition after the second administration to said animal. Preferably, the absence of ABC is measured by the method indicated in Example 3, since it is well known to a person skilled in the art that the half-life in the bloodstream may depend on the species or strains used.

Preferably, the absence of ABC is defined for a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention or an additional composition according to the present invention as the fact that the half-life in the bloodstream of said (additional) thermosensitive liposome or said (additional) composition after the first administration to an animal is no more than 130% in terms of the half-life in the bloodstream of said (additional) thermosensitive liposome or said (additional) composition after the second administration to said animal, wherein said second administration occurs in less than 30 days, or less than 29 days, or less than 28 days, or less than 27 days, or less than 26 days, or less than 25 days, or less than 24 days, or less than 23 days, or less than 22 days, or less than 21 days, or less than 20 days, or less than 19 days, or less than 18 days, or less than 17 days, or less than 16 days, or less than 15 days, or less than 14 days, or less than 13 days, or less than 12 days, or less than 11 days, or less than 10 days, or less than 9 days, or less than 8 days, or less than 7 days, or less than 6 days, or less than 5 days, or less than 4 days, or less than 3 days, or less than 2 days, or less than 1 day after the said first administration.

More preferably, the absence of ABC is defined for a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention or an additional composition according to the present invention as the fact that the half-life in the bloodstream of said (additional) thermosensitive liposome or said (additional) composition after two administrations to an animal is no more than 150%, or no more than 140%, or no more than 130%, or no more than 120%, or no more than 110%, or no more than 105% in terms of the half-life in the bloodstream of said (additional) thermosensitive liposome or said (additional) composition after the third administration to said animal.

Most preferably, the absence of ABC is defined for a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention as the fact that the half-life in the bloodstream of said (additional) thermosensitive liposome or said (additional) composition after the first administration to an animal is no more than 130% in terms of the half-life in the bloodstream of said (additional) thermosensitive liposome or said (additional) composition after the third administration to said animal, wherein said third administration occurs in less than 30 days, or less than 29 days, or less than 28 days, or

- 30 051237 less than 27 days, or less than 26 days, or less than 25 days, or less than 24 days, or less than 23 days, or less than 22 days, or less than 21 days, or less than 20 days, or less than 19 days, or less than 18 days, or less than 17 days, or less than 16 days, or less than 15 days, or less than 14 days, or less than 13 days, or less than 12 days, or less than 11 days, or less than 10 days, or less than 9 days, or less than 8 days, or less than 7 days, or less than 6 days, or less than 5 days, or less than 4 days, or less than 3 days, or less than 2 days, or less than 1 day after the said first administration.

Most preferably, the absence of ABC is defined for a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention or an additional composition according to the present invention as the fact that the half-life in the bloodstream of said (additional) thermosensitive liposome or said (additional) composition after the first administration to an animal is no more than 130% in terms of the half-life in the bloodstream of said (additional) thermosensitive liposome or said (additional) composition after the second, third, fourth, fifth or sixth administration to said animal, wherein said second, third, fourth, fifth or sixth administration occurs less than 30 days after said first administration.

In a preferred embodiment, a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention is provided, characterized by the absence of ABC.

Preferably, a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention is determined to have suitable clearance properties if the administration of said (additional) thermosensitive liposome or said (additional) composition to an animal is characterized by a long half-life in the bloodstream and the absence of ABC. In a preferred embodiment, a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention is provided having suitable clearance properties.

The delivery of the active pharmaceutical ingredient by the drug delivery system is preferably selective, meaning that the rate of transfer of said active pharmaceutical ingredient from said drug delivery system to said target is significantly higher than the rate of transfer of said active pharmaceutical ingredient from said drug delivery system to the corresponding cell, tissue, organ, or part thereof, wherein said corresponding cell, tissue, organ, or part thereof are of the same type but are not part of said target, and/or is higher than the rate of transfer to a cell, tissue, organ, or part thereof located in the area adjacent to said target. For example, selective delivery to a sarcoma target means that the rate of transfer of the active pharmaceutical ingredient to a heated sarcoma is higher than the rate of transfer to fat cells, muscle cells, or tissues located in the adjacent area that are not part of the sarcoma and that have not been heated. In this document, heating defines the target. In the context of this application, this is defined as selective drug delivery. It should be understood that the rate of transfer to inappropriate cells, tissues, organs, or parts thereof that are of a different type and/or not located in the vicinity of the target may be higher than the rate of transfer to the target.

Selective delivery of a medicinal product results in local accumulation of said active pharmaceutical ingredient in said target, wherein the concentration of said active pharmaceutical ingredient in said target is significantly higher than the concentration of said active pharmaceutical ingredient in the corresponding cell, tissue, organ or part thereof, wherein said corresponding cell, tissue, organ or part thereof are of the same type, but are not part of said target, wherein preferably said corresponding cell, tissue, organ or part thereof are in the adjacent region to said target. In the context of this preferred definition, significantly higher preferably means at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 110% higher, at least 120% higher, at least 130% higher, at least 140% higher, at least 150% higher, at least 160% higher, at least 170% higher, at least 180% higher, at least 190% higher, or at least

- 31 051237 at least 200% higher, or at least 300% higher, or at least 400% higher, or at least 500% higher, or at least 600% higher, or at least 700% higher, or at least 800% higher, or at least 900% higher, or at least 1000% higher, or at least 1100% higher, or at least 1200% higher, or at least 1300% higher, or at least 1400% higher, or at least 1500% higher, or at least 1600% higher, or at least 1700% higher, or at least 1800% higher, or at least 1900% higher, or at least 2000% higher, or at least 2500% higher, or at least 3000% higher, or at least 3500% higher, or at least 4000% higher, or at least 4500% higher, or at least 5000% higher. Even more preferably, significantly higher in this context means higher by an amount of 10% higher, or higher by an amount of 20%, or higher by an amount of 30%, or higher by an amount of 40%, or higher by an amount of 50%, or higher by an amount of 60%, or higher by an amount of 70%, or higher by an amount of 80%, or higher by an amount of 90%, or higher by an amount of 100%, or higher by an amount of 110%, or higher by an amount of 120%, or higher by an amount of 130%, or higher by an amount of 140%, or higher by an amount of 150%, or higher by an amount of 160%, or higher by an amount of 170%, or higher by an amount of 180%, or higher by an amount of 190%, or higher by an amount of 200%, or higher by an amount from 300%, or higher by an amount from 400%, or higher by an amount from 500%, or higher by an amount from 600%, or higher by an amount from 700%, or higher by an amount from 800%, or higher by an amount from 900%, or higher by an amount from 1000%, or higher by an amount from 1100%, or higher by an amount from 1200%, or higher by an amount from 1300%, or higher by an amount from 1400%, or higher by an amount from 1500%, or higher by an amount from 1600%, or higher by an amount from 1700%, or higher by an amount from 1800%, or higher by an amount from 1900%, or higher by an amount from 2000%, or higher by an amount from 2500%, or higher by an amount from 3000%, or higher by an amount from 3500%, or higher by at least 4000%, or higher by an amount from 4500%, or higher by an amount from 5000% to not more than 10000%.

For the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention or the additional composition according to the present invention, said selective delivery of the drug can be achieved by locally heating said target contained in the body of said animal, after the administration of said (additional) thermosensitive liposome or said (additional) composition to said animal. It was explained above that the release of the active pharmaceutical ingredient contained in said (additional) thermosensitive liposome or said (additional) composition can occur at temperatures slightly above the body temperature of said animal. Thus, without being limited by this theory, the transfer of said active pharmaceutical ingredient from said (additional) thermosensitive liposome or (additional) composition can be carried out only to those cells, tissues, organs or parts thereof that are locally heated to a temperature slightly above the body temperature of said animal.

It is understood that the body temperature of an animal, preferably a human, is relative to the normal body temperature of an animal of the same species that has not developed a fever.

Selective delivery by heat treatment is defined herein for a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention or an additional composition according to the present invention as the release of an active pharmaceutical ingredient contained in said (additional) thermosensitive liposome or (additional) composition to said target, wherein said (additional) thermosensitive liposome or (additional) composition is administered to said animal for the first time, wherein said target is then locally heated, wherein said local heating causes said release, which results in a higher concentration of said active pharmaceutical ingredient in said target than the concentration of said active pharmaceutical ingredient in a corresponding cell, tissue, organ or part thereof, wherein said corresponding cell, tissue, organ or part thereof is of the same type, but is not part of said target, wherein preferably said corresponding cell, tissue, organ or part thereof is in adjacent area to the specified target.

Where delivery or release of the active pharmaceutical ingredient is described as caused, activated, induced, or mediated by (local) heating or (local) thermal treatment of the specified target, it should be understood that this means that delivery or release of the active pharmaceutical ingredient is significantly higher in and near the target due to the elevated temperature resulting from the thermal treatment. This does not mean that in other cells, tissues, organs, or parts thereof contained in the body of the animal to which the system is administered

- 32 051237 drug delivery, delivery, or release does not occur at all, and does not mean that only minor delivery or release occurs. In the context of the present invention, heating and heat treatment can be used interchangeably. In and around the target refers to those parts of the said animal whose temperature increases, as defined above, after heating the target compared to the temperature before applying said heating.

The heat treatment preferably includes a light source such as a near infrared (NIR) lamp or laser, a water bath, a liquid recirculation device, a microwave device, radiofrequency ablation and/or high intensity focused ultrasound.

A preferred heat treatment comprises heating said target for 5 minutes, or 6 minutes, or 7 minutes, or 8 minutes, or 9 minutes, or 10 minutes, or 11 minutes, or 12 minutes, or 13 minutes, or 14 minutes, or 15 minutes, or 16 minutes, or 17 minutes, or 18 minutes, or 19 minutes, or 20 minutes, or 21 minutes, or 22 minutes, or 23 minutes, or 24 minutes, or 25 minutes, or 26 minutes, or 27 minutes, or 28 minutes, or 29 minutes, or 30 minutes, or 31 minutes, or 32 minutes, or 33 minutes, or 34 minutes, or 35 minutes, or 36 minutes, or 37 minutes, or 38 minutes, or 39 minutes, or 40 minutes, or 41 minutes, or 42 minutes, or 43 minutes, or 44 minutes, or 45 minutes, or 46 minutes, or 47 minutes, or 48 minutes, or 49 minutes, or 50 minutes, or 51 minutes, or 52 minutes, or 53 minutes, or 54 minutes, or 55 minutes, or 56 minutes, or 57 minutes, or 58 minutes, or 59 minutes, or 60 minutes, or 65 minutes, or 70 minutes, or 75 minutes, or 80 minutes, or 85 minutes, or 90 minutes, or 95 minutes, or 100 minutes, or 105 minutes, or 110 minutes, or 115 minutes, or 120 minutes. Preferably, said preferred heat treatment comprises heating said target to a temperature of 41°C.

A preferred heat treatment comprises heating said target to a temperature of 40.0°C, or 40.1°C, or 40.2°C, or 40.3°C, or 40.4°C, or 40.5°C, or 40.6°C, or 40.7°C, or 40.8°C, or 40.9°C, or 41.0°C, or 41.1°C, or 41.2°C, or 41.3°C, or 41.4°C, or 41.5°C, or 41.6°C, or 41.7°C, or 41.8°C, or 41.9°C, or 42.0°C, or 42.1°C, or 42.2°C, or 42.3°C, or 42.4°C, or 42.5°C, or 42.6°C, or 42.7°C, or 42.8°C, or 42.9°C, or 43.0°C. In this context, a temperature approximately equal to a set temperature means a temperature that is higher than the set temperature by minus 0.5°C and lower than the set temperature by plus 0.5°C, preferably a temperature that is higher than the set temperature by minus 0.2°C and lower than the set temperature by plus 0.2°C, more preferably a temperature that is higher than the set temperature by minus 0.1°C and lower than the set temperature by plus 0.1°C. Preferably, said preferred heat treatment comprises heating said target for 60 minutes.

A more preferred heat treatment comprises heating said target for 5 minutes, or 10 minutes, or 15 minutes, or 20 minutes, or 25 minutes, or 30 minutes at a temperature of 40°C. Another more preferred heat treatment comprises heating said target for 5 minutes, or 10 minutes, or 15 minutes, or 20 minutes, or 25 minutes, or 30 minutes, or 35 minutes, or 40 minutes, or 45 minutes, or 50 minutes, or 55 minutes, or 60 minutes at a temperature of 40.0°C.

A more preferred heat treatment comprises heating said target for 5 minutes, or 10 minutes, or 15 minutes, or 20 minutes, or 25 minutes, or 30 minutes at a temperature of 40°C. Another more preferred heat treatment comprises heating said target for 5 minutes, or 10 minutes, or 15 minutes, or 20 minutes, or 25 minutes, or 30 minutes, or 35 minutes, or 40 minutes, or 45 minutes, or 50 minutes, or 55 minutes, or 60 minutes at a temperature of 41.0°C.

A more preferred heat treatment comprises heating said target for 5 minutes, or 10 minutes, or 15 minutes, or 20 minutes, or 25 minutes, or 30 minutes at a temperature of 40°C. Another more preferred heat treatment comprises heating said target for 5 minutes, or 10 minutes, or 15 minutes, or 20 minutes, or 25 minutes, or 30 minutes, or 35 minutes, or 40 minutes, or 45 minutes, or 50 minutes, or 55 minutes, or 60 minutes at a temperature of 42.0°C.

A more preferred heat treatment includes heating said target for 5 minutes, or 10 minutes, or 15 minutes, or 20 minutes, or 25 minutes, or 30 minutes at a temperature of 40°C. Another more preferred heat treatment includes heating said target for 5 minutes, or 10 minutes, or 15 minutes, or 20 minutes, or 25 minutes, or 30 minutes, or 35 minutes, or 40 minutes, or 45 minutes, or 50 minutes, or 55 minutes, or 60 minutes at a temperature of 43.0°C.

The thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention or the additional composition according to the present invention has or is characterized by a suitable biodistribution if, after the administration of said (additional) thermosensitive liposome or (additional) composition to an animal, the active pharmaceutical ingredient contained therein is delivered selectively by heat treatment, as defined above, to said target. Preferably, said target is a tumor cell or tumor, and said cell, tissue or organ not contained in said

- 33 051237 targets include the heart, liver, spleen, kidney, lung, or muscle, not subject to heat treatment. Suitable biodistribution can be determined as described in Example 4.

In a preferred embodiment, a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention is provided, characterized by suitable biodistribution.

The selective delivery of the active pharmaceutical ingredient from the drug delivery system during heat treatment is preferably high, which means that a greater portion of the active pharmaceutical ingredient contained in the drug delivery system is transferred to the target than to the corresponding cell, tissue, organ or part thereof, where the corresponding cell, tissue, organ or part thereof are of the same type, but are not part of the target, where preferably the cell, tissue, organ or part thereof are in the adjacent region to the target, after the administration of the drug delivery system to the animal. In the context of this preferred definition, more preferably means at least 10% more, at least 20% more, at least 30% more, at least 40% more, at least 50% more, at least 60% more, at least 70% more, at least 80% more, at least 90% more, at least 100% more, at least 110% more, at least 120% more, at least 130% more, at least 140% more, at least 150% more, at least 160% more, at least 170% more, at least 180% more, at least 190% more, or at least 200% more, or at least 300% more, or at least 400% more, or at least 500% more, or at least 600% more, or at least 700% more, or at least 800% more, or at least 900% more, or at least 1000% more, or at least 1100% more, or at least 1200% more, or at least 1300% more, or at least 1400% more, or at least 1500% more, or at least 1600% more, or at least 1700% more, or at least 1800% more, or at least 1900% more, or at least 2000% more, or at least 2500% more, or at least 3000% more, or at least 3500% more, or at least 4000% more, or at least 4500% more, or at least 5000% more. Even more preferably, more means more by an amount from 200% to not more than 10,000%, more by an amount from 300% to not more than 10,000%, more by an amount from 400% to not more than 10,000%, more by an amount from 500% to not more than 10,000%, more by an amount from 600% to not more than 10,000%, more by an amount from 700% to not more than 10,000%, more by an amount from 800% to not more than 10,000%, more by an amount from 900% to not more than 10,000%, more by an amount from 1,000% to not more than 10,000%, more by an amount from 1,500% to not more than 10,000%, more by an amount from 2,000% to not more than 10,000%, more by an amount from 2,500% to not more than 10,000%, by an amount from 3,000% to no more than 10,000%, by an amount from 3,500% to no more than 10,000%, by an amount from 4,000% to no more than 10,000%, by an amount from 4,500% to no more than 10,000%, or by an amount from 5,000% to no more than 10,000%.

Preferably, the drug delivery system is a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention. In this context, the terms delivery (of a drug) (active pharmaceutical ingredient), release (of an active pharmaceutical ingredient), transfer (of an active pharmaceutical ingredient) from said drug delivery system, (additional) thermosensitive liposome, or (additional) compositions may be used interchangeably.

A drug delivery system, which is a pharmaceutical composition, is preferably stable upon storage. If the drug delivery system is a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention, stability means the following:

the concentration of the active pharmaceutical ingredient contained in said (additional) thermosensitive liposome or said (additional) composition does not change by more than 15% upon storage, preferably by no more than 10%, most preferably by no more than 5%, relative to the concentration of the active pharmaceutical ingredient before storage; and/or the diameter of said (additional) thermosensitive liposome or the diameter of said (additional) thermosensitive liposome contained in said composition does not

- 34 051237 changes by more than 30% upon storage, preferably by no more than 20%, most preferably by no more than 10%, relative to the diameter of said (additional) thermosensitive liposomes before storage; and/or the bilayer concentration of lysolipids contained in said (additional) thermosensitive liposome or said (additional) composition does not increase by more than 5%, more preferably more than 2%, most preferably more than 1% upon storage; and/or the polydispersity of said (additional) thermosensitive liposome or the polydispersity of said (additional) thermosensitive liposome contained in said (additional) composition does not increase by more than 0.5, preferably by more than 0.3, more preferably by more than 0.2 upon storage, and/or if said (additional) thermosensitive liposome or said (additional) composition is characterized by highly selective delivery upon heat treatment before storage, then said (additional) thermosensitive liposome or said (additional) composition is still characterized by highly selective delivery upon heat treatment after storage; and/or if said (additional) thermosensitive liposome or said (additional) composition is a suitable drug delivery system before storage, then said (additional) thermosensitive liposome or said (additional) composition continues to be a suitable drug delivery system after storage.

The concentration of the active pharmaceutical ingredient and lysolipids can be determined using HPLC. Diameter and polydispersity can be determined using dynamic light scattering. Highly selective delivery and suitable drug delivery systems are described above.

In the context of the present application, storage of a drug delivery system, a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention is the period from the production of said drug delivery system, (additional) thermosensitive liposome, or (additional) composition until its administration to an animal. In this case, it should be understood that storage stability may imply that said drug delivery system, (additional) thermosensitive liposome, or (additional) composition is stored under a specific set of storage conditions, such as a low temperature.

Preferably, the storage lasts for at least 1 day, or for at least 2 days, or for at least 3 days, or for at least 4 days, or for at least 5 days, or for at least 6 days, or for at least 7 days, or for at least 8 days, or for at least 9 days, or for at least 10 days, or for at least 11 days, or for at least 12 days, or for at least 13 days, or for at least 14 days, or for at least 15 days, or for at least 16 days, or for at least 17 days, or for at least 18 days, or for at least 19 days, or for at least 20 days, or for at least 21 days, or for at least 22 days, or for at least 23 days, or for at least 24 days, or for at least 25 days, or for at least 26 days, or for at least 27 days, or for at least 28 days, or for at least 29 days, or for at least 30 days, or for at least 5 weeks, or for at least 6 weeks, or for at least 7 weeks, or for at least 8 weeks, or for at least 9 weeks, or for at least 10 weeks, or for at least 11 weeks, or for at least 12 weeks, or for at least 13 weeks, or for at least 14 weeks, or for at least 15 weeks, or for at least 16 weeks, or for at least 5 months, or for at least 6 months, or for at least 7 months, or for at least 8 months, or for at least 9 months, or for at least 10 months, or for at least 11 months, or for at least 12 months, or for at least 13 months, or for at least 14 months, or for at least 15 months, or for at least 16 months, or for at least 17 months, or for at least 18 months, or for at least 19 months, or for at least 20 months, or for at least 21 months, or for at least 22 months, or for at least 23 months, or for at least 24 months, or for

- 35 051237 for at least 25 months, or for at least 26 months, or for at least 27 months, or for at least 28 months, or for at least 29 months, or for at least 30 months.

Preferably, the storage period lasts from 1 day, or from 2 days, or from 3 days, or from 4 days, or from 5 days, or from 6 days, or from 7 days, or from 8 days, or from 9 days, or from 10 days, or from 11 days, or from 12 days, or from 13 days, or from 14 days, or from 15 days, or from 16 days, or from 17 days, or from 18 days, or from 19 days, or from 20 days, or from 21 days, or from 22 days, or from 23 days, or from 24 days, or from 25 days, or from 26 days, or from 27 days, or from 28 days, or from 29 days, or from 30 days, or from 5 weeks, or from 6 weeks, or from 7 weeks, or from 8 weeks, or from 9 weeks, or from 10 weeks, or from 11 weeks, or from 12 weeks, or from 13 weeks, or from 14 weeks, or from 15 weeks, or from 16 weeks, or from 5 months, or from 6 months, or from 7 months, or from 8 months, or from 9 months, or from 10 months, or from 11 months, or from 12 months, or from 13 months, or from 14 months, or from 15 months, or from 16 months, or from 17 months, or from 18 months, or from 19 months, or from 20 months, or from 21 months, or from 22 months, or from 23 months, or from 24 months, or from 25 months, or from 26 months, or from 27 months, or from 28 months, or from 29 months, or from 30 months to not more than 5 years.

Preferably, storage is carried out at a temperature of about 25°C, or about 20°C, or about 15°C, or about 10°C, or about 5°C, or about 0°C, or about -5°C, or about -10°C, or about -15°C, or about -20°C. In this context, a temperature approximately equal to a given temperature means a temperature above the given temperature minus 5°C and below the given temperature plus 5°C, preferably a temperature above the given temperature minus 2°C and below the given temperature plus 2°C, more preferably a temperature above the given temperature minus 1°C and below the given temperature plus 1°C. Preferably, a temperature of about -20°C means a temperature from -15°C to no more than -25°C.

If storage occurs at temperatures below 0°C, stability also implies that the stability conditions are met during and after freezing and thawing of the drug delivery system, (additional) thermosensitive liposome according to the present invention, or (additional) composition according to the present invention. This property is also referred to as freeze-thaw stability and may encompass multiple freeze-thaw cycles.

Preferably, the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention or the additional composition according to the present invention is stable after at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20 freeze-thaw cycles.

The stability of a dispersion based on a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention or an additional composition according to the present invention means that the diameter of said (additional) thermosensitive liposome or the diameter of said (additional) thermosensitive liposome contained in said (additional) composition does not change by more than 30% upon storage, preferably by more than 20%, most preferably by more than 10%, relative to the diameter of said (additional) thermosensitive liposomes before storage.

Leakage of the active pharmaceutical ingredient from the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention, or the additional composition according to the present invention is a transfer from said (additional) thermosensitive liposome or from an (additional) thermosensitive liposome contained in said (additional) composition during storage, which leads to a decrease in the concentration of the active pharmaceutical ingredient therein. Preferably, the decrease in the concentration of the active pharmaceutical ingredient due to leakage is no more than 15% during storage, more preferably no more than 10%, most preferably no more than 5%, relative to the total concentration of the active pharmaceutical ingredient before storage.

The degradation of an active pharmaceutical ingredient in a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention or an additional composition according to the present invention is a decrease in the concentration of said active pharmaceutical ingredient in said (additional) thermosensitive liposome or said (additional) composition in the absence of transfer of said active pharmaceutical ingredient through the bilayer of said (additional) thermosensitive liposome or through the bilayer of a thermosensitive liposome contained in said

- 36 051237 (additional) composition. In other words, decomposition includes a chemical transformation in which the active pharmaceutical ingredient is converted into a molecule with no, reduced, different, or adverse pharmaceutical effect. Preferably, the decrease in the concentration of the active pharmaceutical ingredient due to decomposition is no more than 15% during storage, more preferably no more than 10%, and most preferably no more than 5%, relative to the concentration of the active pharmaceutical ingredient before storage.

The term "on storage" preferably means at least 4 weeks at a temperature of approximately 5°C or at least 12 months at a temperature of approximately -20°C. More preferably, "on storage" preferably means from 4 weeks to no more than 20 weeks at a temperature of approximately 5°C or from 12 months to no more than 16 months at a temperature of approximately -20°C.

In a preferred embodiment, there is provided a thermosensitive liposome, an additional thermosensitive liposome, a composition or an additional composition according to the present invention, which is stable when stored for at least 4 weeks at a temperature of about 5°C, or for at least 12 months at a temperature of about -20°C, or for from 4 weeks to at most 20 weeks at a temperature of about 5°C, or for from 12 months to at most 16 months at a temperature of about -20°C, wherein the concentration of the active pharmaceutical ingredient contained in said (additional) thermosensitive liposome or said (additional) composition does not change by more than 15% upon storage, preferably by no more than 10%, most preferably by no more than 5%, relative to the concentration of the active pharmaceutical ingredient before storage; and the diameter of said (additional) thermosensitive liposome or the diameter of said (additional) thermosensitive liposome contained in said composition does not change by more than 30% upon storage, preferably by more than 20%, most preferably by more than 10%, relative to the diameter of said (additional) thermosensitive liposomes before storage; and the bilayer concentration of lysolipids contained in said (additional) thermosensitive liposome or said (additional) composition does not increase above 5%, more preferably above 2%, most preferably above 1%, upon storage; and the polydispersity of said (additional) thermosensitive liposome or the polydispersity of said (additional) thermosensitive liposome contained in said composition does not increase by more than 0.5, preferably more than 0.3, more preferably more than 0.2 upon storage, and if said (additional) thermosensitive liposome or said (additional) composition is characterized by highly selective delivery upon heat treatment before storage, then said (additional) thermosensitive liposome or said (additional) composition is still characterized by highly selective delivery upon heat treatment after storage; and if said (additional) thermosensitive liposome or said (additional) composition is a suitable drug delivery system before storage, then said (additional) thermosensitive liposome or said (additional) composition is still a suitable drug delivery system after storage.

The thermosensitive liposome, additional thermosensitive liposome, composition or additional composition according to this embodiment is referred to as a highly storage-stable or (additional) thermosensitive liposome or (additional) composition characterized by high storage stability in the context of the present invention.

In a preferred embodiment, there is provided a thermosensitive liposome, an additional thermosensitive liposome, a composition, or an additional composition according to the present invention, which is stable upon storage for from 1 to at most 20 weeks at a temperature of about 5°C, or from 1 to at most 16 months at a temperature of about -20°C, or from 12 to at most 16 months at a temperature of about -20°C, wherein the concentration of the active pharmaceutical ingredient contained in said (additional) thermosensitive liposome or said (additional) composition does not change by more than 15% upon storage, and/or wherein the diameter of said (additional) thermosensitive liposome or the diameter of said (additional) thermosensitive liposome contained in said composition does not change by more than 30% upon storage, wherein the bilayer concentration of the lysolipids contained in said (additional) thermosensitive liposome or said

- 37 051237 (additional) composition, does not increase by more than 5%, and/or where the polydispersity of said thermosensitive liposome or the polydispersity of said (additional) thermosensitive liposome contained in said (additional) composition does not increase by more than 0.5. Preferably, if said (additional) thermosensitive liposome or said (additional) composition is characterized by highly selective delivery upon heat treatment before storage, then said (additional) thermosensitive liposome or said (additional) composition is still characterized by highly selective delivery upon heat treatment after storage, and/or where, if said (additional) thermosensitive liposome or said (additional) composition is a suitable drug delivery system before storage, then said (additional) thermosensitive liposome or said (additional) composition is still a suitable drug delivery system after storage.

In contrast to the thermosensitive liposomes mentioned in prior art document [4] and [5], the examples below clearly show that the thermosensitive liposome according to the present invention is stable upon storage. In particular, example 7 shows that at least an intraliposomal AP1:lipid ratio of 0.05-0.3, characteristic of the thermosensitive liposome according to the present invention, is associated with storage stability for at least 4 weeks at 2-8°C. In contrast, [4] states that storage of TSL with encapsulated 300 mM citrate, pH 4, at 4°C for weeks resulted in hydrolysis of phospholipids and formation of lysolipids, while [5] noted that no Lyso-PC formation was detected during the first 20 minutes after loading, but later, after 60 minutes, the content increased by 1.1%±1.2%.

In addition, several preferred aspects and embodiments of the thermosensitive liposomes of the present invention, as described above, are associated with even greater storage stability:

Example 5 shows that using an intraliposomal pH in the range of 6.0 to 7.4 ensured the stability of the phospholipids for at least 4 weeks at 2-8°C. The corresponding formulation with an intraliposomal pH of 3 showed more than 10% lipid degradation products after 12 weeks of storage at 2-8°C (28.16±1.95% free fatty acids and 14.1±5.1% lysolipids), while for the formulation with pH 6, no change was observed when analyzed after 24 weeks. API leakage during storage also increased after 12 weeks for the formulation with pH 3, while no leakage was detected for the formulation with pH 6. In addition to what was found, Example 7 also shows that a more neutral pH used as an intraliposomal buffer results in a slower degradation of the lipid excipient at 2-8°C than at a more acidic pH.

Example 8 demonstrates storage stability at 2-8°C for a temperature-sensitive liposome diameter ranging from 100 nanometers to no more than 200 nanometers. For example, as the bubble size decreases to an average z value of less than 100 nm, the formulation is prone to degradation of the API and lipid excipient, which, in turn, negatively impacts the temperature-dependent release profile.

Example 9 demonstrates storage stability at 2-8°C using a storage buffer with a salt concentration of no more than 100 mM and an osmolarity of at least 300 mOsmol/kg. For example, reducing the salt concentration in the storage buffer from physiological concentrations (140 mM) to <66 mM stabilized DPPG2-TSL30%-DOX for storage at 2-8°C, resulting in a dispersion that exhibited significantly slower bubble size growth.

Example 9 further demonstrates that the presence of a cryoprotectant such as trehalose improves storage stability. Specifically, the presence of at least 8% (w/v) trehalose is associated with a composition that is stable after at least 6 freeze-thaw cycles.

Furthermore, Example 6 demonstrates that using a salt concentration of no more than 120 mM in the loading buffer accelerates the active loading of doxorubicin and, thus, reduces API and lipid degradation. The total content of API-related impurities in the batch is dependent on the incubation time, with <0.10% area and 4.1% area for 30 min and 90 min, respectively. The total content of lipid-related impurities in the batch is also dependent on the incubation time, with <0.10% area and 0.13% area for 30 min and 90 min, respectively. To achieve formulation shelf life, replacement of the buffer with a storage buffer having a salt concentration of less than 100 mM and an osmolarity of greater than 300 mOsmol/kg is preferred.

Anticancer activity.

The drug delivery system described in the present invention is preferred for delivering an anticancer drug to a tumor, preferably a solid tumor. Preferably, said drug delivery system comprises

- 38 051237 a thermosensitive liposome, and said delivery is induced or facilitated by heating said tumor.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention, wherein said (additional) thermosensitive liposome or (additional) composition exhibits detectable antitumor activity. In the context of the present invention, the antitumor activity is observed exclusively in the tumor or in the tumor cell contained therein, and not in the corresponding healthy, control, reference, non-tumor cell, tissue, or organ. In the context of the present invention, the antitumor activity comprises at least one of the following:

decreasing the viability of said tumor cell, inducing apoptosis in said tumor cell or inducing tumor cell death, inhibiting the proliferation of said tumor cell, suppressing or delaying weight gain, or reducing weight, and/or slowing or suppressing the growth of said tumor.

The manifestation of such detectable antitumor activity is of crucial importance in the present invention in order to be able to treat, alleviate, delay, cure and/or prevent cancer, preferably solid tumors. The terms "antitumor activity" or "effect on a tumor cell" are used interchangeably in the context of the present invention. It is obvious that the use of the terms "antitumor activity" or "effect" and "effect on a tumor cell" with respect to the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention, or the additional composition according to the present invention does not imply that said (additional) thermosensitive liposome or (additional) composition penetrates said tumor or tumor cell. As described above, it is well known that said (additional) thermosensitive liposome or (additional) composition can function as a drug delivery system for an active pharmaceutical ingredient with such antitumor activity.

Evaluation of antitumor activity may be performed in the treated subject periodically, such as every week, every two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or every year.

Thus, increases/decreases in antitumor activity can be assessed periodically, for example, weekly or monthly. Such assessments are preferably performed at multiple time points for a given subject or at one or more time points for a given subject and a healthy control subject. Alternatively, such antitumor activity can be measured by comparing the indicated antitumor activity in a subject's tumor cell with the corresponding activity in a non-tumor cell or a healthy cell of the same subject at a given time point after the start of treatment.

If antitumor activity has been detected at least once, twice or three times, the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention or the additional composition according to the present invention is considered to exhibit detectable antitumor activity.

Thus, detectable antitumor activity is preferably detected when it is detected for at least one time point. Preferably, such detectable antitumor activity is detected for at least two, three, four, or five time points. In a preferred embodiment, the antitumor activity is assessed in the subject's tumor cells. More preferably, said tumor cells are sarcoma cells or carcinoma cells. In a preferred embodiment, said carcinoma cells are lung carcinoma, hepatocellular carcinoma, or colon carcinoma cells. In a more preferred embodiment, said carcinoma cells are lung carcinoma or hepatocellular carcinoma cells. In a preferred embodiment, said sarcoma cells are angiosarcoma cells, bone sarcoma cells, dermatofibrosarcoma protuberans, epithelioid sarcoma cells, gastrointestinal stromal tumor (GIST), Kaposi's sarcoma cells, leiomyosarcoma cells, liposarcoma cells, malignant peripheral nerve sheath tumors, myxofibrosarcoma cells, pleomorphic sarcoma cells, rhabdomyosarcoma cells, solitary fibrous tumor cells, synovial sarcoma cells, or undifferentiated pleomorphic sarcoma cells.

- 39 051237

When a control thermosensitive liposome is administered, as defined below, the reduction in tumor cell viability or survival may be at least a reduction of at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, or 75%, or more, based on the corresponding tumor cell viability or tumor cell survival.

The induction of apoptosis in tumor cells or the induction of tumor cell death can be at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, or 75%, or more. For example, the induction of apoptosis in tumor cells or the induction of tumor cell death of 50% means that half of the tumor cells treated with the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention, or the additional composition according to the present invention undergo apoptosis or cell death. The viability, or survival, or death of tumor cells can be assessed using techniques known to those skilled in the art. Tumor cell viability or death can be assessed using standard imaging techniques such as MRI, CT, or PET and their derivatives, or by biopsy. Tumor cell viability can be assessed by visualizing lesion expansion at multiple time points. A lesion reduction of 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, or 75%, or greater, relative to lesion expansion at the first time point, observed at least once, will be considered a decrease in tumor cell viability. Tumor cell viability can be assessed by indirect measurement of ATP, such as with the Promega CellTiter-Glo kit. Tumor cell apoptosis can be assessed by measuring caspase-3/7 activity.

Upon administration of a control thermosensitive liposome, as defined below, the inhibition of tumor cell proliferation can be at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, or 75%, or more, relative to the corresponding tumor cell proliferation. Cell proliferation can be assessed using known techniques, such as a standard proliferation assay. Such a proliferation assay can utilize vital dyes, such as Cell Titer Blue (Promega). This involves converting the substrate molecule into a fluorescent molecule via metabolic enzymes. The level of fluorescence then reflects the number of living and metabolically active cells. Alternatively, such a proliferation assay can determine the mitotic index. The mitotic index is based on the number of tumor cells in the proliferative stage compared to the total tumor cell count. Proliferative cells can be labeled using the Ki-67 antibody and immunohistochemical staining. Inhibition of tumor cell proliferation can be observed when the mitotic index decreases by at least 20%, at least 30%, at least 50%, or more (as described by Kearsley J.H. et al., 1990).

In some embodiments, the suppression or reduction in tumor weight, or delay in tumor growth, or inhibition of tumor growth can be at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, or 75%, or more relative to the tumor, when a control temperature-sensitive liposome is administered, as defined below. Tumor weight or tumor growth can be assessed using techniques known to one of skill in the art.

Detection of tumor growth or detection of tumor cell proliferation can be assessed in vivo by measuring changes in glucose utilization using positron emission tomography using the glucose analog 2-[18F]-fluoro-2-deoxy-D-glucose (FDG-PET) or using PET using [18F]-'3-fluoro-'3-deoxy-L-thymidine. An ex vivo alternative is Ki-67 staining of tumor biopsy.

In a preferred embodiment, a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention is provided for use as a medicament.

In a more preferred embodiment, a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention is provided for use in the treatment, alleviation, delay, cure and/or prevention of cancer.

The preferred cancer type is selected from the group consisting of leukemia, Hodgkin's lymphoma, bladder cancer, breast cancer, stomach cancer, lung cancer, ovarian cancer, thyroid cancer, pancreatic cancer, head and neck cancer, prostate cancer, gastrointestinal cancer, soft tissue sarcoma, and multiple myeloma. More preferably, the cancer is a soft tissue sarcoma.

- 40 051237

Preferably, the soft tissue sarcoma is selected from the group consisting of angiosarcoma, bone sarcoma, dermatofibrosarcoma protuberans, epithelioid sarcoma, gastrointestinal stromal tumor (GIST), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumors, myxofibrosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, solitary fibrous tumor, synovial sarcoma, and undifferentiated pleomorphic sarcoma.

In a preferred embodiment, a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention is provided for use in the treatment, alleviation, delay, cure and/or prevention of soft tissue sarcoma.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention or an additional composition according to the present invention for use in the treatment, alleviation, delay, cure and/or prevention of cancer, preferably soft tissue sarcoma, wherein said (additional) thermosensitive liposome or said (additional) composition is characterized by antitumor activity, and wherein said (additional) thermosensitive liposome or said (additional) composition is characterized by one or more of the following properties:

Low complement activation, no anaphylaxis, no toxicity, long half-life in the bloodstream, no ABC, adequate clearance, adequate biodistribution, highly selective delivery during heat treatment, high storage stability.

In a preferred embodiment, there is provided a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention for use in the treatment, alleviation, delay, cure and/or prevention of cancer, preferably soft tissue sarcoma, wherein said (additional) thermosensitive liposome or said (additional) composition is characterized by antitumor activity, and wherein said (additional) thermosensitive liposome or said (additional) composition is a suitable drug delivery system. In other words, the (additional) thermosensitive liposome or (additional) composition according to this embodiment is characterized by antitumor activity, is characterized by appropriate clearance and is highly stable upon storage.

The criteria for assessing therapeutic response are known as the RECIST criteria (Wahl R.L. et al, 2009). In the context of the present invention, the patient may survive and/or be considered to remain healthy for a longer period of time. Alternatively, the disease or condition may have been stopped or delayed. In the context of the present invention, improved quality of life and observed pain relief may mean that the patient may require fewer analgesic medications than at the beginning of treatment. Alternatively, or in combination with taking fewer analgesic medications, the patient may have less constipation than at the beginning of treatment. Less in this context may mean 5% less, 10% less, 20% less, 30% less, 40% less, 50% less, 60% less, 70% less, 80% less, 90% less. The patient may no longer require any analgesic medications. Such improvement in quality of life and observed pain relief may be observed, detected or assessed in a patient after at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months of treatment or more and compared with the quality of life and observed pain relief at the beginning of treatment of said patient.

The delay in the occurrence of metastases and/or tumor cell migration may be at least one week, one month, several months, one year, or longer. The presence of metastases can be assessed using magnetic resonance imaging, CT, or echography, or techniques that detect circulating tumor cells (CTC), free-circulating

- 41 051237

DNA or free circulating RNA. Examples of recent studies include the CellSearch CTC assay (Veridex) and EpCam-based magnetic CTC sorting from peripheral blood.

In some embodiments, tumor growth may be delayed for at least one week, one month, two months, or more. In some embodiments, the occurrence of metastases is delayed for at least one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or more.

Application.

In a further aspect, the use of a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention, as described in the previous sections, for use as a drug or part of a therapy is provided. This document also provides the use of a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention, as described in the previous section, for the manufacture of a drug for the treatment of cancer, preferably soft tissue sarcoma.

Preferably, the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention or the additional composition according to the present invention is intended for use as a drug or part of a therapy for the prevention, delay, cure, alleviation and/or treatment of cancer, preferably soft tissue sarcoma.

In an embodiment of this aspect of the present invention, there is provided a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention or an additional composition according to the present invention for use as a medicament, preferably for the treatment, prevention and/or delay of cancer, preferably soft tissue sarcoma.

Way.

In a further aspect, a method is provided for the prevention, treatment, cure, amelioration and/or delay of a condition or disease as defined in the previous section in an individual, cell, tissue or organ of said individual. The method comprises administering a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention or an additional composition according to the present invention to said individual or subject in need thereof.

Any preference or preferred embodiment disclosing an (additional) thermosensitive liposome according to the present invention, or disclosing an (additional) composition comprising an (additional) thermosensitive liposome according to the present invention, or disclosing a method according to the present invention, may be combined with a method for preventing, treating, curing, alleviating and/or delaying a condition or disease in an individual, in a cell, tissue or organ of said individual, meaning that any such preference or preferred embodiments also disclose a corresponding method for preventing, treating, curing, alleviating and/or delaying a condition or disease.

In an embodiment of this aspect of the present invention, there is provided a method for preventing, treating and/or delaying cancer, preferably soft tissue sarcoma, comprising administering to a subject a thermosensitive liposome according to the present invention, an additional thermosensitive liposome according to the present invention, a composition according to the present invention, or an additional composition according to the present invention.

In a preferred embodiment, the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention or the additional composition according to the present invention is administered to a cell present in an organ or tissue where a tumor is present. Preferably, said organ or tissue contains 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% of tumor cells. The thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention or the additional composition according to the present invention can be targeted to tumor cells, for example, by combining or conjugating a bilayer contained in said thermosensitive liposome with an antibody or

- 42 051237 another tumor-binding fragment. Preferably, said tumor is associated with soft tissue sarcoma.

In a preferred embodiment, the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention, or the additional composition according to the present invention is administered to a cell present in an organ or tissue where a tumor is present, wherein said tumor has not yet metastasized. In another preferred embodiment, the thermosensitive liposome according to the present invention, the additional thermosensitive liposome according to the present invention, the composition according to the present invention, or the additional composition according to the present invention is administered to a cell present in an organ or tissue where a tumor is present, wherein said tumor has metastasized. Preferably, said tumor is associated with soft tissue sarcoma.

In a preferred embodiment, the thermosensitive liposome of the present invention, the additional thermosensitive liposome of the present invention, the composition of the present invention, or the additional composition of the present invention is administered systemically. Alternatively, in another embodiment, the treatment is carried out locally.

In another preferred embodiment, the thermosensitive liposome of the present invention, the additional thermosensitive liposome of the present invention, the composition of the present invention, or the additional composition of the present invention may be administered in combination with standard treatments for a disease or condition associated with cancer, preferably soft tissue sarcoma, such as chemotherapy, radiation therapy, or surgery.

Definitions.

In this document and in the claims, the verb "to comprise" and its conjugations are used in their non-limiting sense to indicate that the elements following the word are included, and elements not specifically mentioned are not excluded. Furthermore, the verb "to consist" may be replaced by "to consist essentially of," meaning that the (thermosensitive) liposome or composition as defined herein may contain additional component(s) in addition to those specifically identified, wherein said additional component(s) do not alter the characteristic feature of the present invention. Furthermore, reference to an element in the singular does not exclude the possibility of the presence of multiple elements, unless the context clearly requires the presence of one and only one of the elements. Thus, the singular generally means at least one.

Each embodiment, as defined herein, may be combined with others unless otherwise indicated. All patent and literature references in this specification are hereby incorporated by reference in their entirety.

In the context of the present invention, the term identical should not be construed so narrowly that only the abundance of isotopes in nature should be taken into account; the term identical should preferably refer only to the molecular structure that will be represented by the structural formula depicted.

Whenever a parameter of a substance is discussed in the context of the present invention, it is understood that, unless otherwise specified, this parameter is determined, measured, or exhibited under physiological conditions. Physiological conditions are known to those skilled in the art and include aqueous solvent systems, atmospheric pressure, pH values from 6 to 8, temperatures in the range from room temperature to about 37°C (from about 20°C to about 40°C), and suitable concentrations of buffer salts or other components. It is understood that charge is often associated with a state of equilibrium. A moiety that is indicated as bearing or having a charge is a moiety that is found more often in a state where it bears or has such a charge than in a state where it does not bear or has no such charge. Thus, an atom that is indicated herein as charged may be uncharged under specific conditions, and a neutral moiety may be charged under specific conditions, as will be understood by those skilled in the art.

In the context of the present invention, a decrease or increase in a parameter to be evaluated means a change of at least 5% relative to the value corresponding to this parameter. More preferably, a decrease or increase in the value means a change of at least 10%, even more preferably of at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it may happen that there is no longer a detectable value associated with the parameter.

The use of a substance as a medicinal product as described in this document may also be interpreted as the use of the said substance in the manufacture of a medicinal product

- 43 051237 of the drug. Similarly, whenever a substance is used for treatment or as a medicinal product, it can also be used to make a medicinal product for treatment.

The word approximately or approximately when used in conjunction with a numerical value (e.g. approximately 10) preferably means that the value may be 0.1% greater or less than the specified value (10).

The word "about" when used in conjunction with a specified temperature preferably means in the range of from the stated specified value minus 3°C to not more than the stated specified value plus 3°C, more preferably in the range of from the stated specified value minus 2°C to not more than the stated specified value plus 2°C, most preferably in the range of the stated specified value minus 1°C to not more than the stated specified value plus 1°C.

The preposition between, when used with integers, refers to a range that includes the specified boundary values. For example, if n represents a value from 1 to 3, n could be 1, 2, or 3. In other words, between X and Y is synonymous with from X to at most Y.

The term "at least" before a list of numbers applies to all numbers in that list, meaning that at least 1, 2, or 3 has the same meaning as at least 1, at least 2, or at least 3. This applies mutatis mutandis to terms such as "from" and "to no more than." For example, "from 1, 2, or 3 to no more than 4" has the same meaning as "from 1 to no more than 4," "from 2 to no more than 4," or "from 3 to no more than 4."

In the context of the present invention, represented by structure X, structures X and with structure X are used interchangeably.

In the context of the present invention, concentration is a molar concentration unless explicitly stated otherwise. A percentage in relation to a concentration in the context of the present invention is a molar percentage unless explicitly stated otherwise. Thus, % means mol% unless explicitly stated otherwise.

The concentration of a compound contained in a bilayer is preferably determined relative to the total number of lipids in said bilayer, unless explicitly stated otherwise.

In particular, the concentration of DPPG2 in the present application refers to the molar concentration of DPPG2 in a bilayer, where preferably said bilayer is contained in the thermosensitive liposome according to the present invention, relative to the total amount of lipids contained in said bilayer.

In the context of the present invention, solutions and/or compositions and/or bilayers having essentially the same lipid composition mean that for each type of lipid, the molar concentration of said lipid differs by less than 10%, 9.5%, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% between said solutions and/or compositions and/or bilayers.

The compounds or compositions of the present invention are preferably intended for use in the methods or applications of the present invention.

In the context of the present invention, the terms cell, cell line, and cell culture may be used interchangeably. All of these terms also include their progeny, which are any or all subsequent generations produced by cell division. Obviously, all progeny may not be identical due to intentional or unintentional mutations.

In the context of the present invention, spherical means approximately or substantially spherical and should not be construed as an absolute geometric property. In particular, liposomes are not limited to perfectly spherical systems, but also include approximately spherical systems.

The molar concentration of a compound in a composition, structure, or (molecular) system is defined as the ratio of the number of molecules of the said compound to the total number of molecules contained in the said composition, said structure, or said system.

The molar ratio of a first compound to a second compound in a composition, structure, or (molecular) system is defined as the ratio of the number of molecules of said first compound contained in said composition, said structure, or said system, to the number of molecules of said second compound in said composition, said structure, or said system.

The animal is preferably a mammal, more preferably selected from the group consisting of a mouse, a rat, a dog and a human, most preferably a human.

Brief description of the drawings

Fig. 1 - schematic representation of a temperature-sensitive liposome (TSL) formed by a membrane bilayer of amphiphilic phospholipid excipients encapsulating an active pharmaceutical ingredient (API).

- 44 051237

Fig. 2 is a schematic representation of a large-scale batch production process for DPPG2TSL ₃₀ %-DOX. In principle, it is also applicable to the encapsulation of other active pharmaceutical ingredients after adjustment of, for example, the intraliposomal buffer, loading buffer, active pharmaceutical ingredient solution, and process parameters.

Fig. 3 - Dynamic light scattering experiments showed that the ζ-potential was significantly increased for TSL with >10 mol% DPPG2 due to protein adsorption.

Fig. 4 - ELISA for in vitro complement activation of different thermosensitive liposome (TSL) formulations in human plasma. C3a (A), Bb (B), and SC5b-9 (C) were used as readout. One-way ANOVA Dunnett's test was performed with 5% PEG TSL as a control set to determine significant differences in complement activation relative to standard TSL formulations. N=3 were tested for each formulation. *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001.

Fig. 5 - optimal content of DPPG2 in DPPC/DSPC/DPPG2 80-x:20:x (mol/mol) with x=10, 20, 30 (mol% _DPPG2 -TSL). N=4 were tested for each composition.

Fig. 6 - Effect of intraliposomal pH in DPPG ₂ -TSL ₃₀ %-DOX on (A) in vitro temperature-dependent DOX release in fetal calf serum (FCS) and (B) lipid degradation during storage at 2-8°C.

Fig. 7 - Effect of intraliposomal pH in DPPG2-TSL ₃₀ %-DOX on the in vitro temperature-dependent release of DOX in fetal calf serum (FCS) during storage of the formulations as frozen (-20°C, A-C) or liquid (2-8°C, DF) dispersions.

Fig. 8. Effect of API:lipid molar ratio in DPPG2-TSL30%-DOX (intraliposomal buffer:ammonium sulfate pH 5.4) on (A) vesicle size (mean z) measured by DLS, (B) PDI measured by DLS, (C) total lipid excipients content (DPPC; DSPC, DPPG2, excluding peak areas of lipid-related degradation products) by HPLC/CAD, (D) total DOX content (excluding peak areas of DOX-related degradation products) by HPLC, (E) background fluorescence as an indicator for DOX leakage (background fluorescence intensity increases as intraliposomal, quenched DOX is released and diluted in the extraliposomal medium), and TDR profile in FCS with DOX release during 5 minutes of incubation at (F) 37°C, (G) 38°C, (H) 39°C, (I) 40°C.

Fig. 9. Effect of API:lipid molar ratio in DPPG2-TSL ₃₀ %-DOX (intraliposomal buffer:ammonium phosphate pH 7.4) on (A) vesicle size (mean z) measured by DLS, (B) PDI measured by DLS, (C) total lipid excipients content (DPPC; DSPC, DPPG2, excluding peak areas of lipid-related degradation products) by HPLC/CAD, (D) total DOX content (excluding peak areas of DOX-related degradation products) by HPLC, (E) background fluorescence as an indicator for DOX leakage (background fluorescence intensity increases when intraliposomal, quenched DOX is released and diluted in the extraliposomal medium), and TDR profile in FCS with DOX release during 5 minutes of incubation at (F) 37°C, (G) 38°C, (H) 39°C, (I) 40°C.

Fig. 10 - Representation of cryo-TEM images of DPPG2-TSL ₃₀ %-DOX loaded with DOX using ammonium phosphate buffer pH 7.4. The bubble size (DLS) was 118.2 nm (PDI 0.120). Cryo-TEM was performed using a Titan EFTEM cryo 300 kV low dose mode microscope. Blot time: 4 s, blot position: -3, grids: Quantifoil 2/2, temperature: 22°C.

Fig. 11. Effect of bubble size and API:lipid molar ratio for DPPG2-TSL30%-DOX (intraliposomal buffer:ammonium phosphate pH 7.4) on (A) total lipid excipients (DPPC; DSPC, DPPG2, excluding peak areas of lipid-related degradation products) by HPLC/CAD, (B) total DOX (excluding peak areas of DOX-related degradation products) by HPLC, (C) background fluorescence as an indicator for DOX leakage (background fluorescence intensity increases as intraliposomal, quenched DOX is released and diluted in the extraliposomal medium), and TDR profile in FCS with DOX release during 5 minutes of incubation at (D) 38°C.

Fig. 12 - Effect of extraliposomal salt and cryoprotectant concentration on the storage stability of DPPG2-TSL30%-DOX as a liquid dispersion: (A) effect of bubble size (mean z), (B) effect on background fluorescence as an indicator of DOX leakage (background fluorescence intensity increases when intraliposomal, quenched DOX is released and diluted in the extraliposomal medium). Freeze-thaw stability of DPPG2-TSL30%DOX: Effect of up to 6 freeze-thaw cycles (FTC) on (C) bubble size (mean z), and (D) background fluorescence as an indicator of DOX leakage.

Fig. 13 - A therapeutic efficacy study in gray rats implanted subcutaneously with a BN175 sarcoma tissue fragment on the left hind limb. Once the tumor was brought to HT (i.e., 41°C) by lamp heating, 0.9% saline (A), non-liposomal DOX (2 mg/kg; B), or DPPG2-TSL30%-DOX batch 1 (2 mg/kg; C) were administered intravenously through a catheter in the tail

- 45 051237 vein and heating was maintained for another hour thereafter. A Kaplan-Meier plot based on tumor volume tripling time showed a significant difference in tumor growth delay between animals treated with saline (0.9%), non-liposomal DOX, and DPPG ₂ -TSL30%-DOX lot 1 (D). Data represented n = 6 per group. Tukey's one-way ANOVA test was performed to determine differences between treatment groups.

Fig. 14. Schematic diagram of the injection of rats for the study of the pharmacokinetics (PK) and accelerated blood clearance (ABC) of carboxyfluorescein (CF)-loaded temperature-sensitive liposomes (TSL). On day 0, rats were injected with the choice formulation with a lipid dose of 5 or 75 μmol/kg, and then a direct PK experiment followed or PK immediately followed the second administration at the indicated 7, 14, 21, or 28 days.

Fig. 15. Pharmacokinetics (PK) and accelerated blood clearance of carboxyfluorescein (CF) formulated in TSLO (A), 5%PEG TSL (B, E), 10%PEG TSL (C), and 30% DPPG ₂ TSL (D, F) in brown rats. In the first set of experiments (A-C), a lipid dose of 5 μmol/kg was used. In the second set of experiments (DF), a lipid dose of 75 μmol/kg was used. PK was performed immediately after the first administration (·), or immediately after the second dose at the indicated 7 days (), 14 days (♦), 21 days (o), or 28 days (V) after the first dose. Rats in the final graphs (G and H) were given an initial dose of 75 μmol/kg 5% PEG TSL (G) or 30% DPPG ₂ TSL (H), followed by an immediate PK experiment (·). The other data sets in these graphs show the PK of CF when rats received the same formulation and dose (i) or 5 μmol/kg 5% PEG TSL (♦) 7 days before the PK experiment. Linear or non-exponential decay curves were fitted to determine CF half-life values (Table 2). N=3 per group.

Links.

[1] M. B. Yatvin, J.N. Weinstein, W.H. Dennis, R. Blumenthal, Design of liposomes for enhanced local release of drugs by hyperthermia, Science 202 (1978) 1290-1293.

[2] D. Needham, G. Anyarambhatla, G. Kong, M.W. Dewhirst, A new temperature-sensitive liposome for use with mild hyperthermia: characterization and testing in a human tumor xenograft model, Cancer Res 60 (2000) 1197-1201.

[3] L.H. Lindner, M.E. Eichhorn, H. Eibl, N. Teichert, M. Schmitt-Sody, R.D. Isseis, M. Dellian, Novel temperature-sensitive liposomes with prolonged circulation time, Clin Cancer Res 10 (2004) 2168-2178. https://doi.org/10.1158/1078-0432.CCR-03-0035.

[4] M. Hossann, M. Wiggenhorn, A. Schwerdt, K. Wachholz, N. Teichert, H. Eibl, R.D. Isseis,

L.H. Lindner, In vitro stability and content release properties of phosphatidylglyceroglycerol containing thermosensitive liposomes. Biochim Biophys Acta 1768 (2007) 2491–9.

https://doi.Org/10.1016/j.bbamem.2007.05.021

[5] S. Limmer, J. Hahn, R. Schmidt, K. Wachholz, A. Zengerle, K. Lechner, H. Eibl, R.D.

Isseis, M. Hossann, L.H. Lindner, Gemcitabine treatment of rat soft tissue sarcoma with phosphatidyldiglycerol-based thermosensitive liposomes, Pharm Res 31 (2014) 2276-86.

https://doi.org/10.1007/sll095-014-1322-6

[6] L. Willerding, S. Limmer, M. Hossann, A. Zengerle, K. Wachholz, T.L.M. ten Hagen, G.A.

Koning, R. Sroka, L.H. Lindner, M. Peller, Method of hyperthermia and tumor size influence effectiveness of doxorubicin release from thermosensitive liposomes in experimental tumors, Journal of Controlled Release 222 (2016) 47-55. https://doi.Org/10.1016/j.jconrel.2015.12.004.

Examples

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Example 1. Obtaining liposomes and determining biophysical characteristics.

Thermosensitive liposomes and their compositions, also known as thermosensitive liposome (TSL) formulations, can be produced using specialized manufacturing methods (e.g., lipid film hydration and extrusion, ethanol incorporation, or other methods). The active pharmaceutical ingredient is loaded using either a passive or active process. Loading with active removal can be achieved using, for example, an ammonium gradient, an acid gradient, or an EDTA gradient.

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Table 1

Examples of buffers and solutions of active pharmaceutical ingredients used to prepare individual DPPG ₂ -TSL preparations

AFY Download Method Intraliposomal buffer Buffer FOR downloading API solution Buffer FOR storage HBS pH 7.4 Passive HBSpH 7.4 Not needed Not applicable HBS pH 7.4 CF Passive IOOmMCF pH 7.2 Not needed See intraliposomal buffer HBS pH 7.4 or 0.9% saline solution DOX (citrate) Active 300 mM citric acid pH 4 HBSpH 7.4 5.7 mg/ml doxorubicin HCl HBS pH 7.4 DOX (ammonium sulfate) Active 240 mM (NH ₄ ) ₂ SO ₄ pH 5.4 HBSpH 7.4 or PBS pH 7.4 5.7 mg/ml doxorubicin HCl 10% (w/v) trehalose, 10.5 mM Na/K phosphate

DOX (ammonium phosphate) Active 300 mM (NH ₄ ) ₂ HPO ₄ pH 7.4 HBSpH 7.4 or PBS pH 7.4 5.7 mg/ml doxorubicin HC1 10% (w/v) trehalose, 10.5 mM Na/K phosphate SRT-11 Active 300 mM (NH ₄ ) ₂ SO ₄ pH 7.4 HBSpH 7.8 20 mg/ml Irinotecan HC1 HBS pH 7.4 dFdC Passive, equilibrium method HBS pH 7.4 HBSpH 7.4 38 mg/ml dFdC, pH 3 or pH 6 (adjusted by adding 600mM HCO3) HBS pH 7.4

Small-scale preparation methods for DPPG ₂ -TSL with encapsulated CF or DOX are in the state of the art. Briefly, all solutions were prepared with deionized and purified water from an ultrapure water system (Milli Q Advantage, Millipore) and then filtered through 0.2 μm before use. Phospholipids in the desired molar ratio (e.g., DPPC/DSPC/DPPG ₂ 50:20:30 for DPPG ₂ -TSL ₂₀ %) were dissolved in chloroform/methanol 9:1 (v/v) in a round-bottomed flask. The solvent was evaporated under vacuum in a rotary evaporator until a fine and uniform

-47051237 lipid film. The lipid film was dried for at least 1 h at 10 mbar/70°C to remove remaining traces of organic solvent. The film was hydrated with the designated intraliposomal buffer/API solution (Table 1) at 60°C for 30 min with shaking. The resulting lipid concentration was 50 mM. Unilamellar vesicles were prepared by 10-fold extrusion at 60°C under _N2 pressure of maximum 20 bar through two polycarbonate filters of the desired particle size (e.g., 200 nm, Whatman, GE Healthcare Europe GmbH, Freiburg, Germany) using a hot-drum extruder (LIPEX™, Northern Lipids Inc. Burnaby, BC, Canada). The dispersion was then cooled to 2-8°C and the buffer was exchanged with loading buffer (Table 1 if active drug or equilibrium loading was performed) or storage buffer (Table 1 if passive loading was performed) using PD10 columns (GE Healthcare). If the API was loaded using the active or equilibrium method, the following steps were performed. The desired loading conditions (e.g., drug/lipid molar ratio and phospholipid concentration) were obtained by mixing the liposome dispersion with loading buffer and API solution (Table 1). The dispersion was heated with shaking (e.g., to 37°C for 60 min for DOX, to 37°C for 45 min for CPT-11, to 60°C for 30 min for dFdC) in an Eppendorf Thermomixer comfort with a 50 ml thermoblock. Traces of unencapsulated API were removed by centrifugation at 75,000 xg (Beckman Coulter Avanti J-26 XP with a JA-25.50 rotor). The supernatant was discarded, and the pellet was carefully resuspended with storage buffer (Table 1).

For the large-scale production of DPPG ₂ -TSL, an adapted method was applied using suitable equipment for handling large dispersion volumes, while the standard preparation steps remained unchanged. The process employed is schematically shown in Fig. 2. Unlike the small-scale process, liposomes were prepared by introducing ethanol rather than by lipid film hydration, and buffer exchange was performed using flow-through filtration (TFF) rather than chromatography or centrifugation, respectively.

Characterization of TSL preparations is well known in the art. Hydrodynamic diameter (mean z), size intensity distribution plot, and zeta potential were determined by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). The instrument was calibrated using a Nanosphere™ size standard (125 nm, Thermo Fisher Scientific, Waltham, MA, USA). Phospholipid composition was quantified by TLC. TLC plates were treated with chloroform/methanol/acetic acid (97.5%)/water 100/60/10/5 (v/v) to achieve separation of phosphatidylcholines (DPPC, DSPC) from DPPG ₂ , lysophosphatidylcholine (Lyso-PC), and lysophosphatidyldiglycerol (Lyso-PG2). A lipid standard containing DPPG ₂ , DPPC, and 1-palmitoyl-sn-glycero-3-phosphocholine (P-Lyso-PC) was used in each TLC run to check the quality of the separation. Phospholipid concentration was measured by phosphate analysis using 1 g/L phosphate solution (Merck KGaA, Darmstadt, Germany) as a reference standard. In vitro temperature-dependent release (TDR) of the API was analyzed in fetal calf serum (FCS) as described previously.

The purity and concentration of DOX, CPT-11, dFdC, and lipid excipients were quantified by HPLC. Free fatty acids were quantified using the endpoint fermentation method with the NEFA kit from DiaSys Diagnostic Systems GmbH (Holzheim, Germany) according to their standard protocol.

Example 2. Increasing the DPPG ₂ content in TSL reduces complement activation. The TSL formulations studied.

Numerous TSL formulations with different lipid compositions have been described to date, but it is unclear which may be optimal for clinical use. A sufficient circulating half-life of TSL after HT treatment is only one important factor for optimal drug delivery to solid tumors. Crucially, these formulations can be administered intravenously to patients without the severe toxicity associated with CARPA. These hypersensitivity reactions can have serious complications during particle infusion, requiring intervention or prophylactic medication. Therefore, several TSL formulations were prepared to study the effect of DSPE-PEG2000 and DPPG2 on complement activation. The lipid formulations were DPPC/DSPC/DSPE-PEG2000 80:20-x:x (mol/mol) x=5, 10, 20, 30 (PEGTSLx%) and DPPC/DSPC/DPPG2 80:20:x (mol/mol) x=5, 10, 20, 30 (DPPG ₂ -TSLx%). The TSL formulation without surface modification, consisting of DPPC/DSPC 80:20 (mol/mol) (TSL ₀ %), served as a control. PEG-TSL ₂₀ % and PEG-TSL ₃₀ % did not show adequate dispersion stability and were excluded from the study. The bubble size ranged from 100 to 150 nm with a PDI <0.10 for all other PEG-TSL and DPPG2-TSL. DPPG2-TSL had a negative surface charge that became more predominant with increasing mol% of DPPG2 (Fig. 3C).

Activation of complement.

Complement activation by TSL formulations was assessed using C3a, Bb, and SC5b-9 ELISA kits (Quidel, San Diego, CA) on human plasma incubated with TSL. 15 μl of TSL

- 48 051237 (25 mM) with encapsulated HBS pH 7.4 was added to 135 μl of citrate-anticoagulated human plasma and incubated at 37°C, 750 rpm for 30 min in a thermoshaker. Incubation was terminated by diluting the samples in the C3a, Bb, or SC5b-9 sample diluent supplied in the respective kits. Samples were loaded into ELISA wells and assayed according to the manufacturer's instructions. 15 μl of 10 mg/ml zymosan A in 0.9% saline was used as a positive control for the assay. After incubation of serum with zymosan, the sample was centrifuged at 800 x g for 10 min, and the supernatant was diluted in sample diluent as described above. Plates were measured for absorbance at 450 nm using an MRX microplate reader (Dynatech Laboratories, Alexandria, VA). TSL ₀ % and PEG-TSL showed no complement activation, whereas incubation with _DPPG2 -TSL ₅ % induced a significant increase in C3a, Bb, and SC5b-9 levels (Fig. 4). Increasing the mol% of DPPG2 to 30 mol% significantly reduced nanoparticle-induced complement activation.

Anionic nanoparticles are known to be potent complement activators and, thus, can induce toxicity after intravenous administration to patients. However, the inclusion of high amounts of anionic DPPG2 in TSL unexpectedly reduced complement activation. This clearly differs from CARPA studies of other negatively charged nanoparticles, which report the opposite. This finding suggests that negatively charged nanoparticles should be carefully evaluated before clinical use, and that not only the negative surface charge itself, but also its magnitude and the specific characteristics of the lipid end group play an important role in CARPA. The low complement activation in human plasma for DPPG2-TSL20% and DPPG2-TSL30% is unique and may indicate that these formulations have a favorable toxicity profile relative to other negatively charged formulations currently used in clinical practice.

Results of a corresponding GLP toxicity study with _DPPG2 -TSL ₃₀ % (Batch 3, Table 3) in dogs confirmed in vitro detection of reduced complement activation. Ten dogs (5 males/5 females) received intravenous infusions of _DPPG2 -TSL ₃₀ % (5.6 mg DPPG2/kg body weight per dose) every three weeks for 12 weeks. No signs of anaphylactic reactions were recorded. This differed from LTSL, where premedication of dogs was necessary to suppress anaphylactic reactions.

Example 3. The DPPG2 content of TSL prevents accelerated clearance from the blood. The TSL formulations studied.

The preparation and composition of the studied liposomal formulations are summarized in Example 2.

Experimental procedure.

Pharmacokinetic (PK) experiments were performed as described in Fig. 14. 1 mL of CF-loaded TSL was administered via the penile vein to BN rats at 5 μmol/kg or 75 μmol/kg lipid doses. 180 μL blood samples were collected in citrate tubes by tail vein incision at t=0 min (before TSL administration), 10 min, 20 min, 30 min, 40 min, 60 min, 4 h, and 8 h and centrifuged at 1,300 x g for 10 min to obtain plasma. Following PK experiments, rats were sacrificed by intracardiac injection of 300 mg/kg pentobarbital, and for the groups of interest, the liver, spleen, and kidneys were excised and stored at -20°C for further use. Plasma samples were analyzed for CF content by fluorometry after incubation in 10% Triton X-100 at 45°C for 15 minutes to ensure complete release of CF. The percentage of injectable dose (% ID) in plasma samples was calculated by comparing the signal from a sample with the signal from 100% of the sample, which represented the maximum expected CF content in the total blood volume of a rat, calculated from its body weight. CF fluorescence was measured in 0.9% NaCl/10 mM Tris buffer pH 8.0 (440:80, v/v) at 493 nm excitation and 513 nm emission.

Accelerated clearance from the blood.

Various published studies have shown that antibody responses to PEG (IgM antibody to PEG) following the first administration of PEGylated liposomes can strongly reduce the pharmacokinetic profile of the next administration, a phenomenon described as accelerated blood clearance (ABC).

CF measurements in the plasma of TSL0-treated rats showed that the lack of surface modification resulted in a negligible circulation time of the encapsulated fluorophore, with a circulating half-life ( _t3 / ₂ ) of a few minutes (Fig. 15A; Table 2). CF encapsulated in 5% PEG TSL and 10% PEG TSL had a significantly longer t1 _/2 with 332±124 min and 155±44 min, respectively (Figs. 15B and 15C; Table 3). However, when additional administration of PEGylated TSL preceded the PK experiment by 7–28 days, t1 _/2 of CF decreased by less than 10 minutes, and this was sustained throughout the 28-day period. Our findings showed that the ABC effect is highly effective and robust in negatively impacting the PK profile for PEGylated TSL formulations, even when anti-PEG IgM antibody levels are low. DPPG2 surface coating was less effective than PEG in improving tx/ ₂ CF, as TSL with 30% DPPG2 had a tx/ ₂ CF of 29±31 minutes (Fig. 15D; Table 2). However, t1/2 CF was not altered by early administration of TSL with 30% DPPG2.

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When the same experiment was repeated at a lipid dose of 75 μmol/kg, ti/2 CF increased for TSL with 5% PEG and TSL with 30% DPPG ₂ to 611 ± 93 minutes and 138 ± 76 minutes, respectively (Figs. 15E and 15F; Table 2). ti/ ₂ CF did not change for TSL with 30% DPPG ₂ when predosed with the same formulation at 75 μmol/kg, which was a similar observation to the previously mentioned results at 5 μmol/kg. In contrast, increasing the dose strongly reduced the ABC effect for TSL with 5% PEG. When rats were first given an injection of 5 μmol/kg TSL with 5% PEG, followed by a PK experiment one week later with 75 μmol/kg TSL with 5% PEG or TSL with 30% _DPPG2 , the ti/ ₂ CF were 41±53 min and 174±53 min, respectively (Figs. 15G and 15H; Table 2). An inverse correlation between liposome dose and ABC is known for long-circulating liposomes with very high membrane stability. For TSL formulations, however, drug leakage is likely the most important factor to consider, also when administered at a higher dose. As described earlier in this section, even relatively low amounts of IgM anti-PEG antibody can be particularly successful in destabilizing the TSL membrane and strongly reduce the ti/ ₂ of the encapsulated compound.

Table 2

Circulating half-life of carboxyfluorescein encapsulated in different TSL formulations

tl/2 0 day (min) tl/2 7 days (min) tl/2 14 days (min) tl/2 21 days (min) tl/2 28 days (min) TSL0 (5 μmol/kg) 3±1 2±0 2±0 2±0 2±1 TSL with 5% PEG (5 µmol/kg) 332±124 3±0 3±0 6±2 8±8 TSL with 5% PEG (10 µmol/kg) 155-44 3±1 3±1 7±3 7±3 TSL with 30% PGz (5 μmol/kg) 29±31 53±31 52±46 21 ±23 42±57 TSL with 5% PEG (75 µmol/kg) 611±93 412±13 602±7 486±43 537±50 TSL with 30% DPPG ₂ (75 μmol/kg) 138±76 106±91 147±69 134±57 120±39 5 (5% PEG) - 75 (5% PEG) 41±53 5 (5% PEG) - 75 (30% DPPG ₂ ) 174±53

Therefore, additional RC studies in rats were performed with PEG-TSL ₅ %, PEG-TSL 1%, or DPPG ₂ -TSL ₃ % encapsulated with CF to evaluate RC parameters. PEG-TSL showed an ABC, as reported in the prior art, whereas administration of DPPG ₂ -TSL ₃ % to rats did not show an ABC. Since the absence of an ABC for nanoparticles coated with polyglycerol moieties has been previously shown, it is reasonable to assume that coating with oligoglycerols, such as DPPG ₂ , also prevents an ABC. Another important factor to consider is the presence of PEG antibodies in previously untreated patients due to frequent exposure to PEG in commonly used products, which may already impair the first cycle of treatment with PEGylated TSL. For these reasons, it is clinically relevant to investigate TSL formulations that do not utilize REOylation, thus making DPPG ₂ -TSL an interesting candidate for clinical development.

Conclusion.

The results of Example 2 showed that the known complement activation of anionic nanoparticles can be suppressed by >10 mol% DPPG ₂ in TSL formulations. As shown in Example 2 and Example 3, together

-50051237 with a longer half-life in the bloodstream, the absence of the ABC phenomenon after repeated injection and the absence of signs of anaphylactic reaction in dogs qualifies the DPPG2-TSL-containing API as a promising candidate for preclinical development.

In conclusion, attractive results were obtained for the thermosensitive liposomes of the present invention, wherein the DPPG2 concentration in the bilayer was at least 15 mol%. Even more attractive results were obtained for the thermosensitive liposomes of the present invention, wherein the DPPG2 concentration in the bilayer was from 15 mol% to 35 mol%.

Example 4. Increasing the DPPG ₂ content of TSL results in superior accumulation of the API at the target site.

The optimal DPPG2 content in DPPC/DSPC/DPPG2 80-x:20:x (mol/mol) with x=10, 20, 30 (DPPG2TSL _x %) to achieve therapeutic efficacy was investigated by biodistribution (BD) and doxorubicin (DOX) tumor enrichment assay in brown rats. DSPC content was set at 20 mol% in all batches to achieve comparable fatty acid composition in the TSL membrane as palmitic acid to stearic acid 80:20 (mol/mol). Three independent DPPG2-TSL formulations differing in DPPG2 content (10, 20, 30 mol%) were prepared using 300 mM citrate pH 4 for active DOX loading. The average Z was 125.1 nm, 120.5 nm, and 114.8 nm for DPPG ₂ -TSLio%, DPPG ₂ -TSL ₂₀ %, and DPPG ₂ -TSL ₃₀ %, respectively. The PDI was <0.07 for all three batches. As expected from the DPPG2 content, the zeta potential decreased from -13.6 mV for DPPG ₂ -TSL ₁₀ %, -22.7 mV for DPPG ₂ -TSL ₂₀ %, to -31.1 mV for DPPG ₂ -TSL ₃₀ %. The lysolipid content was ~1 mol% for each batch, indicating comparable liposome degradation during manufacturing. The final DOX:lipid molar ratios were 0.094, 0.104, and 0.106 for DPPG2-TSLio%, DPPG2TSL ₂₀ %, and DPPG ₂ -TSL ₃₀ %, respectively. In vitro, DOX release in FCS within 5 min at 41°C increased with increasing DPPG2 content: 16.8%, 63.7%, and 93.2% for DPPG2-TSL ₁₀ %, DPPG ₂ -TSL ₂₀ %, and DPPG ₂ -TSL ₃₀ %, respectively. This observation is consistent with the published effect of DPPG2 on the content release rate. The PK profile in brown rats during the first 60 minutes was comparable for all three formulations, showing monoexponential clearance, as known from the prior art (WO9730058, WO2014202680). A BD study was then conducted in BN175 tumor-bearing rats receiving 2 mg/kg DOX intravenously in combination with 60 minutes of tumor hyperthermia (HT) under a lamp (Fig. 5). Tumor volumes were ~1 cm ³ . DPPG2-TSLx% was injected intravenously into the tail vein after reaching the target tumor temperature of 41°C. At the end of HT, a blood sample was collected by cardiac puncture before the animals were sacrificed. After cardiac perfusion, tissue samples were collected. DOX content was quantified by HPLC. DPPG ₂ -TSL ₂₀ % and DPPG ₂ -TSL ₃₀ % achieved significantly higher drug deposition in the tumor than DPPG ₂ -TSL ₁₀ %. There was no difference in DOX content in other tissues except for the spleen, where DOX content was higher for DPPG ₂ -TSL ₁₀ %.

Conclusion: The results show that neither the long-circulating properties of the carrier (as reported, for example, in WO02064116 for DPPG2-TSLio%) alone, nor the in vitro drug release properties alone, are decisive for determining the optimal lipid formulation of DPPG2-TSL that achieves the highest drug concentrations through heat-activated release of the API in heated tumors in vivo. The BD results show that even the slower release of _DPPG2 -TSL ₂₀ % achieves comparable DOX levels as _DPPG2 -TSL ₃₀ %. The long-circulating _DPPG2 -TSL ₁₀ % showed significantly lower accumulation of the API at the target site.

In conclusion, attractive results were obtained for the thermosensitive liposomes of the present invention, wherein the DPPG2 concentration in the bilayer was at least 15 mol%. Even more attractive results were obtained for the thermosensitive liposomes of the present invention, wherein the DPPG2 concentration in the bilayer was from 15 mol% to 35 mol%.

Example 5. Intraliposomal pH influences phospholipid excipient and DPPG2-TSL-API API stability.

The effect of intraliposomal buffer pH on the phospholipid excipient and the stability of the DPPG2-TSL-API was assessed.

DPPG2-TSL3o%-DOX was prepared according to the small-scale method described in Example 1, differing in the pH of the intraliposomal buffer used for active API loading (citric acid pH 4, ammonium sulfate pH 5.4, and ammonium phosphate pH 7.4). The formulations were stored under liquid (2-8°C) and frozen (-20°C) conditions, and biophysical characteristics were tested periodically. Intraliposomal pH showed no effect on the TDR profile of freshly prepared DPPG2-TSL3o%-DOX (Fig. 6A). When stored in the liquid state at 2-8°C, DPPG2-TSL3o%-DOX using an intraliposomal buffer of pH 4 showed rapid degradation of the lipid excipient, with more than 5% lysophosphatidylcholines present after 37 days (Fig. 6B). Unlike cholesterol-containing liposomes, where up to 15% of lipid degradation products do not have a negative effect on membrane permeability, even smaller amounts

- 51 051237 lysophosphatidylcholines (up to ~5%, Fig. 6B) had a marked negative impact on TDR, with impaired API retention at 37°C and markedly increased API release at temperatures >38°C with each day of storage at 2-8°C (Fig. 7D). DPPG ₂ -TSL ₃₀ %-DOX, using an intraliposomal buffer of pH 5.4, showed less rapid degradation, but the rate of hydrolysis increased after 14 days and yielded more than 5% lysophosphatidylcholines after 98 days (Fig. 6B). The TDR profile remained constant for at least 14 days of storage at 2-8°C, but then changed (Fig. 7E). DPPG ₂ -TSL ₃₀ %-DOX, using an intraliposomal buffer of pH 7.4, was the most stable formulation with detectable lysophosphatidylcholine content after 42 days of storage (Fig. 6B). The TDR profile remained constant for at least 14 days of storage at 2-8°C, but then changed (Fig. 7D). The change in the TDR profile was less noticeable compared to DPPG 2 TSL 30%-DOX, using an intraliposomal buffer of pH 5.4.

Preparation of DPPG ₂ -TSL ₃ o% -DOX with intraliposomal buffer with pH values ranging from 6.4 to 7.4 had no effect on the stability of the TSL formulations (data not shown).

The pH of the intraliposomal buffers was also tested for DPPG ₂ -TSL ₃₀ %-dFdC. By adjusting the pH of the dFdC solution to 6-6.5 before loading, no lysolipids were detected after batch receipt. Changing the pH also resulted in a changed TDR profile, with lower release at 41°C and 42°C (22.3±10.5% at 42°C for the pH 6 formulation compared to 45.9±17.6% for the pH 3 formulation). The pH change resulted in a formulation that could be stored at 2-8°C, whereas the published formulation required storage at -20°C. The pH 3 formulation showed more than 10% lipid degradation products after 12 weeks of storage at 2-8°C (28.16±1.95% free fatty acids and 14.1±5.1% lysolipids), while no change was observed for the pH 6 formulation during the 24 weeks analyzed. API leakage during storage also increased after 12 weeks for the pH 3 formulation, while no leakage was detected for the pH 6 formulation.

Conclusion: Intraliposomal pH in the range of 4.0 to 7.4 did not affect the TDR of DPPG ₂ -TSL ₃₀ %, but significantly affected the stability of the lipid excipient and TDR during storage. With increasing amounts of lipid-related impurities, the TDR profile changes, and therefore the formulation can no longer be used in patient treatment. Using an intraliposomal pH in the range of 5.4 to 7.4 ensured the stability of the phospholipids for at least 1 week as a liquid, ensuring no negative effect on the TDR during manufacturing. Using an intraliposomal pH in the range of 6.0 to 7.4 ensured the stability of the phospholipids for at least 4 weeks as a liquid, ensuring no negative effect on the TDR during manufacturing.

In conclusion, attractive results were obtained for the thermosensitive liposomes of the present invention, where the pH of the intraliposomal buffer was from 5.0 to 8.0. Even more attractive results were obtained for the thermosensitive liposomes of the present invention, where the pH of the intraliposomal buffer was from 6.0 to 8.0.

Example 6. Salt concentration in loading buffer accelerates API loading.

Accelerated temperatures at certain stages of the manufacturing process can lead to degradation of lipid excipients or APIs. Since API loading into DPPG2-TSL was performed at 38°C for DOX or 60°C for dFdC, the effects of incubation time and salt concentration in the loading buffer were assessed.

DPPG ₂ -TSL ₃₀ % was prepared according to the large-scale method described in Example 1. Ammonium phosphate pH 7.4 was used as an intraliposomal buffer during liposome production. The buffer was exchanged with separate loading buffers with different salt concentrations. Buffer A: physiological PBS pH 7.4 without trehalose (300 mOsmol/kg). Buffer B: 4.1% (w/v) trehalose, 10.5 mM Na/K phosphate, 66 mM saline (294 mOsmol/kg). Buffer C: 8.9% (w/v) trehalose, 10.5 mM Na/K phosphate, 66 mM saline (294 mOsmol/kg). DOX loading was performed at a designated API:lipid molar ratio of 0.08 at 37-38°C. DOX loading was followed by fluorescence spectroscopy. At selected time points, 20 µL of samples were diluted with 3 mL of HBS pH 7.4. Loading was terminated when the sample fluorescence intensity was <20% of the initial fluorescence intensity without incubation and remained constant. Loading into buffer A, buffer B, and buffer C was completed after 20 min, 45 min, and 90 min, respectively, demonstrating that API loading can be performed in physiological buffers containing high salt concentrations and without cryoprotectant.

DPPG2-TSL30% was then prepared according to the large-scale method described in Example 1. Ammonium phosphate pH 7.4 was used as the intraliposomal buffer during liposome production, and buffer A was used as the loading buffer. DOX loading was performed with a designated API:lipid molar ratio of 0.08 at 37-38°C for 30 minutes and 90 minutes. The total API-related impurities in the batch were dependent on the incubation time, with <0.10% area and 4.1% area at 30 minutes and 90 minutes, respectively. The total lipid-related impurities in the batch

- 52 051237 is also dependent on incubation time, with <0.10% area and 0.13% area at 30 minutes and 90 minutes, respectively.

In conclusion, attractive results were obtained for the thermosensitive liposomes of the present invention, wherein said thermosensitive liposomes were prepared using a loading buffer with a salt concentration of at least 66 mM and an osmolarity of at least 250 mOsmol/kg. Even more attractive results were obtained for the thermosensitive liposomes of the present invention, wherein said thermosensitive liposomes were prepared using a loading buffer with a salt concentration of at least 66 mM and an osmolarity of 250 mOsmol/kg to 350 mOsmol/kg.

Example 7. API:lipid ratio affects the stability of DPPG ₂ -TSL-API.

DPPG ₂ -TSL ₃ o% -DOX was prepared according to the large-scale method described in Example 1, characterized by an API:lipid molar ratio from 0.06 to 0.13. Either ammonium sulfate pH 5.4 or ammonium phosphate pH 7.4 was used as the intraliposomal buffer, respectively. After receipt, lots were stored at 5 ± 3 °C for up to 15 weeks and biophysical characterization was performed, examining vesicle size, PDI, total lipid excipients (DPPC, DSPC, DPPG2, excluding peak areas of lipid-related degradation products) by HPLC/CAD, total DOX (excluding peak areas of DOX-related degradation products) by HPLC, background fluorescence as an indicator of DOX leakage, and TDR profile in FCS.

Batches of DPPG2-TSL3o%-DOX (ammonium sulfate, pH 5.4) showed comparable change in analytical performance when the API:lipid molar ratio was <0.10 (Fig. 8). In contrast, an API:lipid molar ratio of 0.13 resulted in reduced storage properties, visible in a marked increase in bubble size (Fig. 8A), PDI (Fig. 8B), carrier stability in FCS (DOX release at 37°C, Fig. 8F), and also unexpectedly much faster API degradation (Fig. 8D) after 2 weeks at 5±3°C. There were no detectable differences in lipid degradation (Fig. 8C) and API leakage (Fig. 8E).

DPPG ₂ -TSL ₃₀ %-DOX (ammonium phosphate, pH 7.4) batches showed comparable change in analytical performance when the API:lipid molar ratio was <0.09 (Fig. 9). In contrast, an API:lipid molar ratio of 0.13 resulted in reduced storage properties, visible in a marked increase in bubble size (Fig. 9A), PDI (Fig. 9B), carrier stability in FCS (DOX release at 37°C, Fig. 9F), and also unexpectedly much faster API degradation (Fig. 9D) after 2 weeks at 5±3°C. There were no detectable differences in lipid degradation (Fig. 9C) and API leakage (Fig. 9E). A trend towards destabilization was observed for the batch with an API:lipid molar ratio of 0.10.

When comparing DPPG2-TSL ₃₀ %-DOX (ammonium phosphate, pH 7.4) batches with DPPG2-TSL ₃₀ %-DOX (ammonium sulfate, pH 5.4) having API:lipid molar ratios ranging from 0.06 to 0.10, the latter showed faster degradation of phospholipid excipients and slower degradation of API. The total lipid excipient contents for the ammonium phosphate and ammonium sulfate batches were 94.9±2.0% and 92.4±1.0% of the initial contents after 15 weeks at 5±3°C, respectively. The total API contents for the ammonium phosphate and ammonium sulfate batches were 86.1±2.5% and 91.8±2.4% of the initial contents after 15 weeks at 5±3°C, respectively. Surprisingly, these DPPG ₂ -TSL ₃₀ %-DOX (ammonium phosphate, pH 7.4) lots were more prone to change in TDR profile upon storage compared to DPPG ₂ -TSL30%-DOX (ammonium sulfate, pH 5.4) after 4 weeks at 5±3°C (Fig. 8 FH and Fig. 9 FH), which may be more likely a result of the presence of API-related impurities due to API degradation rather than lipid-related impurities.

In addition, batches were analyzed by cryo-TEM, showing the formation of ring-shaped crystalline structures of the API consisting of fibers wrapped around a bilayer of intraliposomal lipid excipients (Fig. 10).

DPPG ₂ -TSL ₃₀ %-CPT-11 was prepared according to the small-scale method described in Example 1, characterized by an API:lipid molar ratio of 0.078-0.313. Ammonium sulfate pH 7.4 was used as an intraliposomal buffer. After preparation, the API release was analyzed at 37°C after 1 hour. It was shown that with an increase in the API:lipid ratio, the released amount of CPT-11 decreased. The threshold API:lipid molar ratio to have sufficient stability at 37°C was identified as from 0.174 (mol/mol) to 0.21 (mol/mol), with a release of 23.4±0.6% and 10.2±0.2% in FBS, respectively. API:lipid ratios above 0.21 did not result in a greater improvement in release at 37°C than 12.5±3.5%.

Batches of DPPG ₂ -TSL ₃₀ %-CPT-11 with different API:lipid ratios (0.08-0.21) were obtained and stored for up to 4 weeks at 2-8°C. Lysolipids, which are lipid degradation products, were not detected. No change in the mean z-value or PDI, or temperature-dependent release of CPT-11 was observed. To assess whether the analyzed parameters are suitable for determining storage stability, the batches were additionally stored at 4.3-5.3% RT.

- 53 051237 lysolipids were detected after 3 weeks of storage at rt. In the temperature-dependent release of CPT-11, the amount of drug released at 40°C increased, indicating degradation. For example, for 4 weeks of storage of DPPG ₂ -TSL ₃₀ %-CPT-11 (API:lipid ratio 0.174) at 2-8°C, the constant release at 40°C in FBS for 5 min was 48.9±2.5%. However, after another week of storage at rt, the release was 69.7±1.0%.

Conclusion: The intraliposomal API:lipid ratio influences the shelf-life of DPPG ₂ TSL ₃₀ %. The optimal API:lipid ratio is highly dependent on the loaded API. Furthermore, when combined with a specific API:lipid ratio, a more neutral pH used as an intraliposomal buffer is preferable, as the degradation of lipid excipients at 2-8°C is slower than at a more acidic pH, despite the fact that the degradation of the API may be somewhat faster.

In conclusion, attractive results were obtained for the thermosensitive liposome according to the present invention containing doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof as an active pharmaceutical ingredient, wherein the pH of the intraliposomal buffer is from 5.0 to 8.0, and the molar ratio between doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and the lipids contained in the bilayer is from 0.06 to 0.10 for one week, avoiding freezing. Even more attractive results were obtained for the thermosensitive liposome according to the present invention containing doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof as an active pharmaceutical ingredient, wherein the pH of the intraliposomal buffer is from 6.0 to 8.0, and the molar ratio between doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and the lipids contained in the bilayer is from 0.07 to 0.09 for one week, avoiding freezing.

In conclusion, attractive results were obtained for the thermosensitive liposome according to the present invention containing irinotecan, an irinotecan derivative or a pharmaceutically acceptable salt thereof as an active pharmaceutical ingredient, wherein the pH of the intraliposomal buffer is from 5.0 to 8.0, and the molar ratio between irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and the lipids contained in the bilayer is at least 0.18 for one week, avoiding freezing. Even more attractive results were obtained for the thermosensitive liposome according to the present invention containing irinotecan, an irinotecan derivative or a pharmaceutically acceptable salt thereof as an active pharmaceutical ingredient, wherein the pH of the intraliposomal buffer is from 5.0 to 8.0, and the molar ratio between irinotecan, said irinotecan derivative or said pharmaceutically acceptable salt thereof and the lipids contained in the bilayer is at least 0.20 for one week, avoiding freezing.

Example 8. Vial size affects long-term storage stability of DPPG2-TSL-API.

DPPG ₂ -TSL ₃₀ %-DOX was prepared according to the large-scale method described in Example 1, varying the vesicle size from 85 nm to 120 nm. DOX was loaded using an intraliposomal ammonium phosphate buffer, pH 7.4. Two different API:lipid molar ratios (0.06 and 0.08) were further tested. After receipt, lots were stored at 5 ± 3 °C for up to 15 weeks and biophysical characterization was performed, examining vesicle size, PDI, total lipid excipients (DPPC, DSPC, DPPG2, excluding peak areas of lipid-related degradation products) by HPLC/CAD, total DOX (excluding peak areas of DOX-related degradation products) by HPLC, background fluorescence as an indicator of DOX leakage, and the TDR profile in FCS.

Batches with a bubble size of 85 nm showed insufficient dispersion stability with a significant increase in polydispersity after 4 weeks of storage, making DLS measurements of stability at later time points impossible. For all other bubble sizes tested, the average z and PDI increased only gradually during the storage period, indicating sufficient stability when bubble sizes >100 nm were used. Unexpectedly, a decrease in bubble size affected the API stability during storage regardless of the API:lipid molar ratio (Fig. 11B). The API content of DPPG2-TSL30%-DOX with bubble sizes of 85 nm, 100 nm, and 120 nm decreased to 78.2%, 84.1%, and 88.3% after 15 weeks of storage at 5 ± 3°C, respectively. For the total lipid excipient content, increased degradation was visible for 85 nm bubbles for DPPG2-TSL30%-DOX with an API:lipid molar ratio of 0.06 (Fig. IA). The smallest _{DPPG2- TSL30} %-DOX with a bubble size of 85 nm also showed noticeable DOX leakage upon storage, which began after 4 and 9 weeks for formulations with API:lipid molar ratios of 0.06 and 0.08 (Fig. CI), respectively. The effect of decreasing bubble size on the reduced storage ability of DPPG2-TSL30%-DOX was also detectable by increasing

- 54 051237 DOX release at 38°C in FCS when stored for more than 4 weeks at 5±3°C (Fig. 11D). In general, as the bubble size decreases to an average z < 100 nm, the formulation is prone to degradation of the API and lipid excipients, which in turn negatively affects the TDR profile. It is known that decreasing the bubble size in DPPG2-TSL3o% increases water exchange across the lipid excipient bilayer at temperatures <T _{± 100 nm} . This increased water permeability increased the risk for hydrolytic degradation processes in the formulation. If the API is prone to such degradation processes, too small a bubble of DPPG2-TSL should be avoided. Furthermore, one DOX-related impurity generated during long-term storage at 2-8°C in DPPG ₂ -TSL ₃₀ %-DOX is highly lipophilic and may thus be responsible for the change in the TDR profile during storage. Because the DOX crystal is in spatial proximity to the lipid excipient bilayer (Fig. 10), this may form lipid excipient-DOX adducts.

In conclusion, attractive results were obtained for the thermosensitive liposomes according to the present invention containing doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof, wherein the pH of the intraliposomal buffer is from 5.0 to 8.0, wherein the molar ratio between doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and the lipids contained in the bilayer is from 0.06 to 0.10, wherein the diameter of said thermosensitive liposome is from 100 nanometers to 200 nanometers, for one week avoiding freezing. Even more attractive results were obtained for thermosensitive liposomes according to the present invention containing doxorubicin, a doxorubicin derivative or a pharmaceutically acceptable salt thereof, wherein the pH of the intraliposomal buffer is from 6.4 to 8.0, wherein the molar ratio between doxorubicin, said doxorubicin derivative or said pharmaceutically acceptable salt thereof and the lipids contained in the bilayer is from 0.07 to 0.09, wherein the diameter of said thermosensitive liposome is from 100 nanometers to 150 nanometers, for four weeks avoiding freezing.

Example 9. Salt content of storage buffer affects long-term storage stability of DPPG2-TSL-API.

Because DPPG2-TSL30%-DOX is prone to API degradation when stored as a liquid dispersion at 2-8°C, it was decided to store the formulation as a frozen drug product at -20°C. For this purpose, a cryoprotectant was added to the extraliposomal buffer to ensure multiple freeze-thaw cycles (FTC) of the drug product without altering the quality of critical characteristics. Disaccharides, such as trehalose, are optimal cryoprotectants for liposomal formulations. To investigate the effect of additional buffer salts on maintaining a pH near neutrality, isolated DPPG2 _{- TSL30} %-DOX with a vesicle size of ~120 nm and an API:lipid molar ratio of 0.08 was prepared according to the large-scale method described in Example 1.

Reducing the salt concentration in the storage buffer from the physiological concentration (140 mM) to <66 mM stabilized DPPG ₂ -TSL ₃₀ %-DOX for storage at 2-8°C, yielding a dispersion that exhibited a markedly slower increase in bubble size (Fig. 12A). To adjust the osmolarity to the physiological concentration, non-ionic trehalose was added to the storage buffer. The concentration of the buffer salt, Na/K phosphate, was maintained constant (10.5 mM). Also, the API leakage rate during storage was gradually suppressed as the salt concentration was reduced from 240 mM to 0 mM (Fig. 12B).

To achieve freeze-thaw stability, DPPG2-TSL30%-DOX required >8% (w/v) trehalose in the storage buffer. Storage buffer containing 10% (w/v) trehalose and 10.5 mM Na/K phosphate pH 7.4 yielded a liposomal formulation that withstood 6 FTC without detectable change in vesicle size (Fig. 12C) or DOX leakage (Fig. 12D). In addition to trehalose, sucrose was also successfully tested to achieve freeze-thaw stability of DPPG2 _{- TSL30} %-DOX (data not shown).

In conclusion, attractive results were obtained for the thermosensitive liposome according to the present invention, wherein said thermosensitive liposome was dissolved in a storage buffer with a salt concentration of up to 100 mM and an osmolarity of at least 300 mOsmol/kg. Even more attractive results were obtained for the thermosensitive liposome according to the present invention, wherein said thermosensitive liposome was dissolved in a storage buffer with a salt concentration of up to 20 mM and an osmolarity of at least 300 mOsmol/kg.

Example 10. Final composition for DPPG ₂ -TSL ₃₀ %-DOX.

DPPG ₂ -TSL ₃₀ %-DOX, intended for (pre)clinical development, was prepared according to the large-scale preparation method described in Example 1 (Fig. 2). All solutions were prepared with deionized and purified water from an ultrapure water system and then filtered through 0.2 μm before use. Phospholipids in a molar ratio of DPPC/DSPC/DPPG2 50:20:30

- 55 051237 was dissolved in ethanol at 60°C. 300 mM ammonium phosphate buffer was prepared in ultrapure water, the pH was adjusted to 7.4 with phosphoric acid, and the buffer was filtered through 0.2 μm before use. PBS buffer was prepared in ultrapure water, and the pH was adjusted to 7.4 with phosphoric acid, if necessary. The PBS buffer was also filtered through 0.2 μm before use. The ethanolic lipid solution and ammonium phosphate buffer were pumped through two heating coils (60°C), and the streams were connected at 60°C using a tee. The dispersion heated to 60°C was then pumped through an extrusion chamber heated to 60°C, equipped with a plurality of extrusion membranes of the appropriate pore size, to generate liposomes with a vesicle size in the range of 100 to 150 nm. After extrusion, the flow was pumped through a cooling coil cooled to 5°C into an ice-cold collection vessel. A gradient for active DOX loading was generated by exchanging the extraliposomal buffer with PBS pH 7.4 and TFF. The dispersion was then filtered through a 0.2 μm membrane to reduce bioburden. The required API:lipid molar ratio of 0.08 was achieved by mixing the liposomal dispersion with PBS buffer pH 7.4 and an API solution (5.7 mg/mL DOX HCl in ultrapure water). The dispersion was heated with stirring at 37°C for 30 min in a round-bottomed flask equipped with a heating mantle. Traces of unencapsulated DOX were then removed, and the extraliposomal buffer was replaced with 10% (w/v) trehalose, 10.5 mM Na/K phosphate using TFF. Finally, the dispersion was aseptically filtered (0.2 μm). The TSL preparations were characterized as described in Example 1. The turning point was calculated by fitting the TDR profile with a sigmoid function from 37°C to 43°C. Residual solvents were measured by gas chromatography (headspace). In addition, pH, osmolarity, bacterial endotoxin, and sterility were measured using the appropriate methods from the European Pharmacopoeia (EP). Typical analytical results for independent production batches are shown in Table 3.

Batch 1 and Batch 3 showed higher levels of API-related impurities because the DOX loading incubation times were 120 and 90 minutes, respectively, rather than 30 minutes. For Batch 4, optimized HPLC methods with significantly greater specificity and selectivity were used for lipid and DOX content, resulting in the detection of a higher number and level of impurities. TDRI: total drug-related impurities, TLRI: total lipid-related impurities.

Table 3

Typical analytical results of DPPG ₂ -TSL3o%-DOX batches produced by the large-scale production method

Test Party 1 Part 2 Part 3 Part 4 Target Amount Concentration DOX 1 liter low 1 liter high 4 L high 2.5 l average Lipid and API Identity (HPLC) Corresponds to Complies Corresponds to Complies Content (HPLC) 3.15 14.8 14.8 14.9

-56051237

- DPPC (mg/ml) - DSPC (mg/ml) - DPPG ₂ (mg/ml) - Doxorubicin (mg/ml) 1.32 2.10 0.36 6.6 9.2 1.9 6.4 10.2 1.82 6.4 9.8 1.3 TLRI Not found <0.10% area/area ь 0.13% w/w 4.4% area/area TDRI (% area/area) 3.9% <0.10% 4.1% 2.9 Residual EtOH (ppm) wt/wt <500 <500 <500 <300 pH (EP 2.2.3) 7.2 7.4 7.4 7.3 Osmolality (EP 2.2.35) (mOsmol/kg) 499 387 390 402 Particle size (nm) Polydispersity index (PDI) 123 0.098 122.4 0.09 121.5 0.081 130 0.10 Turning point (°C) 40.4 40.9 40.2 40.66 Bacterial endotoxin (EP 2.6.14) < 1.00 IU/ml < 0.88 IU/ml < 0.40 IU/ml <0.5 EU/ml Sterility (EP 2.6.1) Corresponds to Complies Corresponds t Not tested.

The results of a 12-month stability study of DPPG ₂ -TSL ₃ o%-DOX Batch 1 stored at 5°C ± 3°C showed that the stability of the batch was dependent on four different vial materials used to store the dispersion (glass, PP, COP, and COC). Furthermore, the same was true for DPPG ₂ -TSL3o%-DOX Batch 1 stored at -20°C ± 5°C. The results further showed that DPPG ₂ -TSL3o%-DOX Batch 1 was stable for 11 weeks when stored at 5°C ± 3°C. After this time point, DOX degradation was observed, as well as a slight but consistent increase in vial size. No evidence of degradation of the phospholipid excipients was observed during 12 months of storage.

An 18-month (-20°C ± 5°C, Table 4) QA/QC-controlled stability study was conducted with DPPG ₂ -TSL3o%-DOX batch 3. Samples were analyzed using the same methods as described above. DPPG ₂ -TSL3o%-DOX was stable for at least 18 months.

The results of the storage stability study with DPPG ₂ -TSL3o%-DOX batch 3 under ICH condition at -20°C±5°C are shown in Table 4. BF: background fluorescence, TDRI: total drug-related impurities, TLRI: total lipid-related impurities, TP: turning point. The result excluded by QA/QC due to operator deviation in the analysis.

-57051237

Table 4

Results of a storage stability study with DPPG ₂ -TSL:orDOX batch 3 under ICH conditions at -20°C±5°C

Point in time (months) Particle size (nm) PDI TDRI (% area/area) TLRI (% area/area) TR (°C) BF (%) Sterility (EP 2.6.1) 0 121.5 0.08 4.1 0.13 40.2 14.5 Sterile 3 124.4 0.09 _A 0.35 40.6 13.4 But. 6 125.7 0.07 4.4 0.11 40.7 15.1 Sterile 9 124.4 0.07 6.8 0.35 40.2 18.6 But. 12 124.3 0.09 5.9 0.11 40.6 14.9 But. 15 124.6 0.08 4.8 0.24 40.2 20.2 But. 18 125.4 0.07 5.0 0.26 40.2 16.9 But.

Results of the freeze-thaw stability study showed that DPPG ₂ TSL3o%-DOX Batch 1 was stable for 6 freeze-thaw cycles. In addition, different vial materials also had no effect on the freeze-thaw stability of the drug product. The study was repeated with DPPG ₂ -TSL3o%-DOX Batch 4 as a QA/QC control, since a method for measuring non-liposomal (free) DOX using SPE-based sample preparation was available (Table 5). The batch was stable for at least 3 freeze-thaw cycles. DPPG ₂ -TSL3o%-DOX Batch 4 was additionally used for benchtop stability measurements at room temperature as a QA/QC control (Table 6). The batch was stable for at least 8 hours at ambient temperatures.

Table 5

Results of a freeze-thaw stability study with DPPG ₂ -TSL3o%-DOX batch 4 FTC: number of freeze-thaw cycles, i.e.: not determined, TDRI: total drug-related impurities, TLRE: total lipid-related impurities

FTC Particle size (nm) PDI TORI (% area/area) TLRI (% area/area) Free DOX (%) 0 134 0.12 2.8 2.3 6.6 1 126 0.09 2.8 3.1 6.0 2 129 0.09 But. But. BUT. 3 129 0.10 But. But. 9.3

-58051237

Table 6

Benchtop stability results (ability to be stored at ambient temperatures) with DPPG2-TSL ₃ o%-DOX batch 4. a.i.: not determined, TDRI: total drug-related impurities, TLRI: total lipid-related impurities

Moment in time (hours) Particle size (nm) PDI 0 134 0.12 4 127 0.10 8 126 0.10

TDRI (% area/area) TLRI (% area/area) Free DOX (%) 2.8 2.3 6.6 z,o 3.1 4.3 2.8 But. 7.0

Table 7

Biodistribution study in gray rats with BN175 subcutaneous sarcoma on the hind limb, produced with DPPG2-TSL ₃ o%-DOX (small laboratory scale relative to large-scale production method). DOX concentration (ng/mg) in selected organs and tumor tissues

A method for small-scale laboratory production Large-scale production method tumor with NT 47.5±25.4 49.2±4.3 tumor without NT 3.0±0.9 Not measured heart 4.5±0.8 3.7±0.5 liver 13.2±2.0 14.7±2.8 spleen 18.3±2.9 19.9±0.6 kidney 19.6±2.9 16.9±2.5

A BD study was conducted on the binding of DPPG2-TSL ₃ o%-DOX produced by a small laboratory scale and a large-scale production method (batch 1). For the study, 250-300 g gray mice were subcutaneously implanted with a 1 ^mm3 fragment of BN175 sarcoma tumor on each hind limb 7-10 days before the experiment. Once one tumor reached 1 ^cm3 , it was subjected to a 1-hour HT + treatment with DPPG2-TSL ₃ o%-DOX (2 mg DOX/kg body weight). Briefly, a temperature sensor was placed inside the tumor core, followed by positioning the heating lamp to bring the tumor to 41 °C, which took on average up to 20 minutes to reach this target temperature. Once the tumor reached 41 °C, the formulations were administered intravenously through a catheter in the tail vein, and the tumor was heated for an additional hour. Following treatment, tumors, hearts, livers, spleens, and kidneys were excised and DOX concentrations were measured (Table 7) using HPLC. Tissue DOX levels showed no difference in BD profile between both formulations tested. The standard deviation for heated tumors with DPPG2-TSL ₃ o%-DOX prepared by the large-scale method was higher due to the wider range of tumor sizes (expressed as measured tumor weights) examined in the present study. Tumor weights were 651 ± 264 mg versus 661 ± 120 mg, respectively. The underlying effect of tumor size on DOX accumulation efficiency has been shown previously [6]. Animals treated with DPPG2-TSL ₃ o%-DOX prepared by the scaled-up method showed an average ratio of DOX increase between individuals in heated versus unheated tumors of 47.5/3.0 = 15.8. This is in the range for the ratio obtained with state of the art DPPG2-TSL ₃ o%-DOX prepared with 300 mM citrate pH 4 as the intraliposomal buffer, which was a ratio increase in the range of 13 to 17 [6], showing that stabilization of the formulation by changing, for example, excipients, API:lipid ratio, vesicle size (see examples above), did not have a negative impact on the therapeutic efficacy.

Therapeutic studies were conducted with a similar BN175 tumor (5 mm in diameter; any size was acceptable) and the treatment model described above. Unlike the BD study, only one tumor was implanted per hind limb to avoid the need to terminate the experiment due to uncontrolled growth of the untreated tumor. In addition to DPPG2-TSL, ₃ o%-DOX

Claims (7)

In batch 1, animals were treated with a non-liposomal formulation of DOX (a drug product approved for human use) and physiological saline (0.9% NaCl). All animals received an additional 1-hour local HT (41°C) of the tumor as described above for the BD study. Post-treatment, tumors were measured by capillary every other day, and animals were sacrificed when tumors reached a volume of 2.5 ^mm3 or when ulceration was evident. Treatment of rats with DPPG2-TSL ₃ o%-DOX showed significantly improved tumor growth delay relative to saline-treated animals (p<0.01, Fig. 13). Furthermore, compared with non-liposomal DOX treatment at an equivalent dose, DPPG2-TSL ₃ o%-DOX also resulted in a significant tumor growth delay and longer survival (p<0.05, Fig. 13). CLAUSES OF THE INVENTION 1. A composition comprising a thermosensitive liposome for treating, alleviating, delaying, curing and/or preventing cancer, comprising a bilayer and an intraliposomal buffer, wherein the molar concentration of 1,2-dipalmitoyl-8n-glycero-3-phosphodiglycerol (DPPG ₃ ) in said bilayer is at least 15%, wherein said thermosensitive liposome contains doxorubicin or a pharmaceutically acceptable salt thereof, and the molar ratio of doxorubicin, or a pharmaceutically acceptable salt thereof, to lipids contained in said bilayer is from 0.06 to no more than 0.10; wherein said thermosensitive liposome has a diameter from 100 nm to no more than 200 nm, wherein said intraliposomal buffer has a pH from 6 to no more than 8; and wherein said thermosensitive liposome is dispersed in a storage buffer having a salt concentration of less than 100 mM and an osmolarity of more than 300 mOsmol/kg. 2. The composition according to claim 1, comprising a bilayer and an intraliposomal buffer, wherein the molar concentration of 1,2-dipalmitoyl-8p-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is at least 15%, wherein said intraliposomal buffer has a pH of from 6 to no more than 8. 3. The composition according to claim 1 or 2, wherein the concentration of said DPPG2 in said bilayer is from 15 mol.% to no more than 35 mol.%. 4. The composition according to any one of paragraphs 1-3, wherein said bilayer does not contain cholesterol or cholesterol ester. 5. The composition according to any one of paragraphs 1-4, wherein said 1,2-dipalmitoyl-8p-glycero-3-phosphodiglycerol (DPPG2) is represented by formula (IV): Formula (IV). 6. The composition according to any one of paragraphs 1-3, wherein said bilayer further comprises 1,2-dipalmitoyl-8p-glycero-3-phosphocholine (DPPC) and/or 1,2-distearoyl-8p-glycero-3-phosphocholine (DSPC). 7. The composition of claim 6, wherein the molar concentration of 1,2-dipalmitoyl-8p-glycero-3-phosphocholine (DPPC) in said bilayer is from 0.45 to no more than 0.65; and/or wherein the molar concentration of 1,2-distearoyl-8p-glycero-3-phosphocholine (DSPC) in said bilayer is from 0.15 to no more than 0.25; and/or wherein the molar concentration of 1,2-dipalmitoyl-8p-glycero-3-phosphodiglycerol (DPPG2) in said bilayer is from 0.15 to no more than 0.35.