Field of the Invention

The present invention relates to Precision Therapeutics. This may involve the identification of target and/or the subsequent guidance of a therapeutic agent to target and the final confirmation of therapeutic effect. Identification of target can be carried out with imaging lipid nanoparticles and/or theranostic nanoparticles (capable of therapy and/or diagnostic imaging applications). The present invention relates to pharmaceutical compositions comprising such imaging lipid nanoparticles and/or theranostic nanoparticles, and their use in treatment.

Background to the Invention

Cancer is one of the most important disease areas where cellular imaging could play a major role, in enhancing pre-existing therapies and opening up opportunities for further alternative, more effective therapies. Advances in cancer nanomedicine have provided new opportunities to combine clinically established lipid nanoparticle (LNP) formulations with nanoparticle-based diagnostic imaging.

MRI is a clinical imaging modality that produces 3D opaque images of tissues containing water. Over 40% of clinical imaging world-wide today requires the injection of some form of MRI contrast agent. This is due to the fact that magnetic resonance imaging (MRI) suffers from an inherent lack of sensitivity and often in order to diagnose pathology correctly, a paramagnetic contrast agent is injected intravenously into patients to further enhance the magnetic resonance (MR) signal and hence site of disease.

WO 2011/061541 describes an improved MRI contrast agent comprising gadolinium. The contrast agent involves imaging lipid nanoparticles (imaging LNPs) comprising gadolinium (III) 2-(4,7-bis-carboxymethyl-10-[(/V,/\/-distearylamidomethyl)-/ \/ ^, -amido-methyl]-1 , 4,7,10-tetra- azacyclododec-1-yl)-acetic acid (Gd.DOTA.DSA) that were developed by replacing unsaturated phospholipid 1 ,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with saturated phospholipid 1 ,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) ¹ . In particular, WO 2011/061541 describes an imaging LNP (Gadonano-F), which is a novel gadolinium (III) (Gd ³⁺ )-containing, imaging liposome that was designed to act as a folate receptor (FR)-targeted, positive contrast agent to enable magnetic resonance imaging (MRI) of FR-presenting cancerous lesions (primary and metastatic) including breast, prostate, pancreatic, bone, lung, intestinal, renal, and ovarian cancers (see also Figure 1). Nanotechnology has enabled the development of novel therapeutics and diagnostic strategies such as advances in targeted drug delivery systems, versatile molecular imaging modalities and potential theranostic agents in cancer therapy. Many of the nanotechnology breakthroughs have occurred in cancer therapy, including drug delivery systems based on lipids and image contrast agents in nanoscale dimensions to aid in diagnostic imaging, or image-guided therapy. The liposomal or LNP drug delivery system involving doxorubicin, known as Doxil®, is among the first generation of nanodrugs to bring a therapeutic benefit to cancer patients and demonstrate a decreased cardiotoxicity compared to free doxorubicin ² . However, such liposomal or LNP drug delivery systems for cancer have not necessarily shown the expected therapeutic results. For example, in the case of Doxil®, increases in drug (doxorubicin) concentration in tumours has been poor (approx. 3% of overall dose) and release of drug from encapsulation is slow, not reaching appropriate levels of drug in the tumour for effective therapy. In addition, biological factors such as tumour vascularisation and perfusion can influence cancer cell uptake of Doxil® nanoparticles ² . There is therefore a need for improved delivery of such therapeutic agents, hence the need for Precision Therapeutics approaches.

LNPs are suitably biocompatible and/or biodegradable - they may be well suited to medicinal applications - in targeted drug delivery and in vivo imaging. Structural lipids such as cholesterol and glycerophospholipids are able to comprise the macromolecular assemblies that come to make up cellular membranes and other barriers in nature precisely because they have an unrivalled capability for self-association, driven by weak short range forces and the hydrophobic effect ⁶ . This capacity for self-association can be exploited in the laboratory. One can create self- assembly LNPs (say, 50- 200 nm, approx 100nm in diameter) that conform in structure to the ABCD nanoparticle paradigm (Figure 2). These can enable the functional delivery of a therapeutic agent or active pharmaceutical ingredient (API), to target cells in vivo. The number of lipid variations is large, so too the number and variety of possible LNPs that may be produced for use in biological situations. Therefore, LNPs offer the potential opportunity for tailor-made preparation and production leading in the future even to the possibility of LNP-mediated personalized medicine ^3"5 .

Summary of the invention

The present invention can solve, or at least alleviate, (some of) the problems of the prior art.

In one aspect, the present invention provides a pharmaceutical composition comprising (a combination of):

- imaging lipid nanoparticles (imaging LNPs) ; and

- one or more therapeutic agent(s). The one or more therapeutic agent(s) may be separate from said imaging LNPs and/or entrapped within said imaging LNPs to form theranostic nanoparticles (TNPs).

In one embodiment the imaging LNPs (or TNPs) are thermally insensitive.

In one embodiment, the imaging LNPs (or TNPs) comprise gadolinium (III) 2-(4,7-bis- carboxymethyl-IO-fi/V./V-distearylamidomethy -A/ ^' -amido-methyll-l ^.y.lO-tetra-azacyclododec- 1-yl)-acetic acid (Gd.DOTA.DSA) and a neutral, fully saturated phospholipid component.

In a further aspect, the present invention provides a pharmaceutical composition comprising said imaging LNPs and/or TNPs used in combination with said (additional) therapeutic agent(s) for simultaneous, separate or sequential use in a method of treatment, such as of cancer.

The invention further provides said imaging LNPs (or TNPs) for use in a method of treatment, such as of cancer, which method comprises the administration simultaneously, separately or sequentially to a subject (in need thereof) of said imaging LNPs and/or TNPs with said

(additional) therapeutic agent(s).

The invention further provides said (additional) therapeutic agent(s) for use in a method of treatment, such as of cancer, which method comprises administering simultaneously, separately or sequentially to a subject in need thereof said (additional) therapeutic agent(s) with said imaging LNPs and/or TNPs.

The invention also relates to use of said imaging LNPs and/or TNPs in the manufacture of a medicament, for use in a method of treatment, such as of cancer, which method may comprise administering simultaneously, separately or sequentially to a subject (in need thereof) said imaging LNPs and/or TNPs with said (additional) therapeutic agent(s), and to the use of said (additional) therapeutic agent(s) in the manufacture of a medicament for use in a method of treatment, such as of cancer, which method comprises administering simultaneously, separately or sequentially to a subject (in need thereof) said (additional) therapeutic agent(s) with said imaging LNPs and/or TNPs.

The invention further relates to a method of treatment of cancer comprising:

- administering to a subject (in need thereof) a (therapeutically effective amount of) said pharmaceutical composition;

- imaging and/or detecting the presence of the imaging LNPs (or TNPs) (e.g. of the

pharmaceutical composition) at, in or near target tissue (of interest); and - inducing (image-guided focused) hyperthermia (IgFHT) at the target tissue (of interest, e.g. a tumour or cancerous tissue or organ).

The present invention also relates to a kit comprising:

(a) at least one selected from the group consisting of a package, an instruction and an attached document describing combined use of said imaging LNPs and/or TNPs formulation with said (additional) therapeutic agent(s); and

(b) said pharmaceutical composition, and a kit comprising a set of a formulation comprising said imaging LNPs (or TNPs), plus a formulation comprising said (additional) therapeutic agent(s).

Guidance of a therapeutic agent to target involves image-guided hyperthermia to direct either receptor-targeted imaging lipid nanoparticles or theranostic nanoparticles to target(s).

Confirmation of therapeutic effect is monitored by means of residual imaging LNPs (or TNPs) resident at target(s). Preferred imaging modalities include clinically relevant modalities such as magnetic resonance imaging. Preferred (image-guided) hyperthermia (modality) can comprise (image-guided) focussed ultrasound.

Description of the Figures

Figure 1. Gadonano-F receptor-targeted imaging LNPs for targeted delivery of MRI contrast agents to tumour cells.

Gadonano-F imaging LNPs are prepared as described previously with the indicated lipid components ^{1 , 3} . Double labelling is made possible using gadolinium metallochelating lipid (Gd.DOTA.DSA) for MRI positive contrast imaging and rhodamine fluorescent probe conjugated lipid (DOPE-Rhoda) for fluorescence imaging. Folate association with the Gadonano-F imaging LNP surfaces is made possible using a polyethylene glycol (PEG) lipid (folate-PEG ²⁰⁰⁰ -DSPE), ensuring that Gadonano-F imaging LNPs are set up for FR-specific targeting to tumour cells in vivo. The remaining PEG lipid (PEG ²⁰⁰⁰ -DSPE) ensures that the Gadonano-F receptor-targeted imaging LNPs possess stability in biological fluids (e.g. serum) ^{1 ,3} .

Figure 2. Functional LNPs for therapeutic applications.

In functional LNPs, active pharmaceutical ingredients (APIs) (A) are condensed within functional concentric layers of chemical components designed for delivery into cells and intracellular trafficking (B components, primarily lipids and lipid-related components), biological stability (C stealth/biocompatibility components-typically Polyethylene Glycol [PEG]) and biological targeting to target cells (D components, biological receptor-specific targeting ligands) ⁴ ' ⁵ . Figure 3, FUS-induced hyperthermia in tumours.

Temperatures measured using fine-wire thermocouples implanted s.c. above and below the tumour (with respect to the transducer location). The cold junction is the temperature of the thermocouple electronics and is approximately 2°C above room temperature. Tumour temperatures are measured at 50 ms resolution and FUS acoustic power settings adjusted manually to converge on the target temperature. Representative traces are shown for control group 1 during (a) the first FUS burst to 43°C; (b) the second to 42°C.

Figure 4, In vivo groups and treatment timescales.

Each group consists of 5-6 mice (a) with two IGROV xenograft over each flank, the right side being treated with FUS; (b) group 1 (control; no injection), 2 (TNPs, thermosensitive, containing doxorubicin) and 3 (Doxil) all followed the same twin FUS burst protocol, with an initial accumulation enhancement treatment at ~ 30 min, followed by a FUS burst at 1 h 30; (c) group 4 experienced TNPs (containing doxorubicin) but combined with a 'pre-FUS' protocol, consisting of the accumulation enhancement treatment 30 min pre- and an FUS burst 45 min post- injection. Doxorubicin dosage was 8 mg/mL throughout. Tumour sizes were recorded every 2 days. FUS times indicated are minimums.

Figure 5, Representative example of near infrared fluorescence (NIR) & doxorubicin fluorescence imaging on group 2 TNP treated animals.

Time points are given post-injection and red bars indicate the position of two FUS burst treatments. The NIR XL750 label associated with TNPs (thermosensitive, containing

doxorubicin) was excited at 704 nm and fluorescence emission collected over 740-950 nm. Doxorubicin florescence was excited at 455 nm and imaged from 560-750 nm. The resulting stacks were unmixed and balanced by means of Maestro control software (Calipre Life

Sciences, MA USA; v 3.0.1) accessing known fluorophore spectra, then false colour imaged using Image J (NIH, USA, v 1.49). Intensity data are processed in grey scale: the nearer white the image the higher the intensity of fluorescence and the higher the TNP/doxorubicin concentration in tumour volumes.

Figure 6, NIR fluorescence brightness profiles for group 2 TNP treated animals

These profiles were calculated in ImageJ using manually placed equal-area ROIs, the y-axis is zeroed against a upper-right shoulder muscle reading taken at the earliest time point after injection (1-5 min). Each point indicates the mean brightness across the ROI, the error bars show 1 S.D. · indicate right-side tumours (FUS) while□ indicate left side (no FUS). Figure 7, Relative tumour volumes for groups 1-4

Data were measured by calliper every 2-3 days and calibrated as an N-fold increase over the initial size. Each bar represents the mean volume across the group with the data binned over 2 days, (a) is for the left (no FUS) tumours; (b) is for the right (FUS treated); (c) is a replot of (a) showing groups 1 and 3 data alone (no FUS) tumours; (d) is a replot of (b) showing groups 1 and 3 data alone (FUS treated). The error bars indicate 1 SE.

Detailed description of the invention

The present invention relates to a pharmaceutical composition comprising (a combination of):

- imaging lipid nanoparticles (imaging LNPs); and

- one or more therapeutic agent(s).

The one or more therapeutic agent(s) may be separate from said imaging LNPs. Alternatively, one or more therapeutic agent(s) may be entrapped, encased or present within said imaging LNPs, e.g. to form theranostic nanoparticles (TNPs).

The pharmaceutical composition may comprise only TNPs (i.e. containing no separately formulated additional therapeutic agent). Alternatively, the pharmaceutical composition may comprise TNPs (i.e. encapsulating one or more therapeutic agent(s)), and may further comprise one or more additional therapeutic agent(s), separate from said TNPs. Examples of possible pharmaceutical compositions according to the present invention include (but are not limited to) the following:

(i) Imaging LNPs and one or more separate therapeutic agent(s);

(ii) TNPs (i.e. encapsulating one or more therapeutic agent(s));

(iii) TNPs and one or more additional separate therapeutic agent(s); or

(iv) A combination of imaging LNPs and TNPs and optional (separate) therapeutic

agent(s).

As described herein, the lipid nanoparticle component of the TNP is the same as that of the imaging LNP. Thus discussion herein relating to preferred formulations of the imaging LNP also applies to the TNP.

Imaging lipid nanoparticles (imaging LNPs)

Imaging LNPs are lipid nanoparticles that comprise an imaging agent. This can be an agent which is suitable for imaging in any imaging modality or technique, such as a clinical imaging technique, for example fluorescence imaging, magnetic resonance imaging (MRI), electron microscopy and image processing, electron spin resonance, radio imaging, positron emission tomography (PET) or single-photon emission computed tomography (SPECT) or near infrared fluorescence (NIRF). The imaging agent may preferably be an imaging lipid.

The LNP (or TNP) of the present invention may comprise one or more further imaging agents, such as an imaging lipid selected from fluorescent lipids, nuclear magnetic resonance imaging lipids, electron microscopy and image processing lipids, electron spin resonance lipids and radioimaging lipids. The imaging agent may also be a positron emission tomography (PET) or single-photon emission computed tomography (SPECT) imaging agent. Suitable and preferred lipids in each of these classes are given below.

Fluorescent lipids

Examples of fluorescent lipids are 1 ,2-dioleoyl-sn-glycero-3-Phosphoethanolamine-/\/-(5- dimethylamino-1-naphthalenesulfonyl, 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-/V-(1- pyrenesulfonyl),1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine-/\/-(carboxyflu orescein), 1- oleoyl-2-[6-[(7-nitro-2-1 ,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phospho-L -serine, 25-{/V-[(7-nitrobenz-2-oxa-1 ,3-diazol-4-yl)-methyl]amino}-27-norcholesterol, -oleoyl-2-[6-[(7- nitro-2-1 ,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphoet hanolamine and 1 ,2- dioleoyl-sn-glycero-3-phosphoethanolamine-A/-(lissamine rhodamine B sulfonyl). Fluorescent lipids of interest preferentially include near infra-red fluorescent lipids such as ΛΓ -Xenol_ight750- A/,A/-distearylamidomethylamine (XL750.DSA) or DSA derivatives of is IRDye™ 800CW, ATTO680™ or DyLight 680™.

Magnetic resonance imaging

A suitable MRI lipid may comprise, for example, a paramagnetic metal suitable for MRI, e.g. chelated to a head group, preferably an optionally substituted poly(aminocarboxylate) group, such as DTPA or DOTA. The head group (preferably poly(aminocarboxylate)) chelate may be used as it is, or alternatively may be conjugated to one or more hydrophobic hydrocarbon chains via a linker.

The paramagnetic metal may be, for example, Gd, or radiometals such as ⁶⁴ Cu. Preferably, the paramagnetic metal is gadolinium (Gd).

Examples of such lipids are Gd-DTPA, Gd.DOTA, GdHPD03A, Gd-DTPA-bis(stearylamide) (Gd-BSA); Gd-DTPA-bis(myrisitylamide) (GdDTPA-BMA); 1 ,2-dimyristoyl-sn-glycero-3- phosphoethanolaminediethylene-triamine- pentaacetate : Gd ³⁺ (DMPEDTPA:Gd ³⁺ ); D35-1.2- dihexanoyl-sn-glycero-3-phosphocholine; gadolinium (III) 2-{4,7-bis-carboxymethyl-10-[(/V, Λ/- distearylamidomethyl-A/ ^' -amido-methyll-I ^J.IO-tetra-azacyclododec-l-y^-acetic acid

(Gd.DOTA.DSA); gadolinium (III) 2-(1-[(/V,/V-distearyl-amidomethyl)- /\f-amidomethyl]-4,7,7-tris- carboxymethyl-1 ,4,7-triaza-sept-1-yl) acetic acid (Gd.DTPA.DSA); gadolinium (III) 1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid mono(/V ¹ -cholesteryloxy-3-carbonyl-1 ,2- diaminoethane)amide (Gd.DOTA.Chol); gadolinium (III) 1 , 4,7,10-tetraazacyclododecane- 1 ,4,7,10-tetraacetic acid mono(/V ¹ - distearoylphosphatidylethanolamine)amide

(Gd.DOTA.DSPE) and gadolinium (III) diethylene triamine 1 ,1 ,4,7,10-penta(acetic acid)-10- acetic acid mono(/V ¹ -cholesteryloxy-3-carbonyl-1 ,2-diaminoethane)amide (Gd.DTPA.Chol).

Electron microscopy and image processing

An example of a suitable lipid to be included in imaging LNPs (or TNPs) for this purpose is 1 ,2- dioleoyl-SA7-glycero-3-{[/V(5-amino-1-carboxypentyl) iminodiacetic acid]succinyl}-(nickel salt).

Electron Spin Resonance

An example of a suitable lipid to be included in imaging LNPs (or TNPs) for this purpose is 1 ,2- diacyl-SA7-glycero-3-phosphotempocholine,1-palmitoyl-2-stear oyl-(n-DOXYL)-sn-glycero-3- phospho-choline.

Radioimaging

An example of a suitable lipid to be included in imaging LNPs (or TNPs) is (99m)Tc-DTPA- bis(stearylamide); (99m)Tc-DTPA-bis(myrisitylamide).

PET/SPECT

Suitable PET/SPECT radiometals can be incorporated into lipids for inclusion in imaging LNP (or TNP) bilayers, and include ⁸⁹ Z; ⁶⁴ Cu; ⁶⁸ Ga; ¹²⁴ l and ⁸⁶ Y. For example, these agents can provide signal when chelated to a head group (preferably an optionally substituted poly- (aminocarboxylate) group), such as replacing the gadolinium ion in the lipid Gd.DOTA.DSA used to incorporate gadolinium into imaging LNPs (or TNPs) for MRI purposes.

Near infrared fluorescence (NIRF) imaging label

A NIRF imaging agent (such as XenoLight750™, IRDye™ 800CW, TTO680™ or DyLight 680™) may be conjugated to a lipid (such as a lipid comprising saturated lipid)methylamine, for example comprising /V,/V-distearylamidomethylamine (DSA)) for inclusion in imaging LNP (or TNP) bilayers.

LNPs can be thermally insensitive, in that their lipid composition is such that nanoparticles are stable at 37°C and stable to increases in temperature up to at least 47°C (e.g. Doxil®). LNPs can also be thermosensitive, in that their lipid composition is such that nanoparticles are stable at 37°C and unstable in structure to increases in temperature up to at least 47°C, resulting in temperature sensitive drug release (e.g. ThermoDox®, Celsion), In one preferred embodiment the imaging LNPs (or TNPs) of the present invention are thermally insensitive, e.g. such that they do not undergo a phase transition at a particular temperature, and are not thermosensitive. Thus, for example, the thermally insensitive imaging LNPs (or TNPs) of the present invention do not undergo a phase transition at a particular temperature from a bilayer form to an inverted hexagonal form, or an(other) inversion of their normal or usual topology. In addition (or alternatively), the thermally insensitive imaging LNPs (or TNPs) of the present invention do not undergo a phase transition at a particular temperature that

corresponds to a shift from a lamellar state, such as to another fluid mesophase, e.g, another lamellar state or a cubic state. In one embodiment, the thermally insensitive imaging LNPs (or TNPs) of the present invention do not undergo a phase transition at a particular temperature that results in lower stability of the LNPs (or TNPs), such that the lipid bilayer is disrupted, and/or (if present) any contents of the LNPs (or TNPs) are released.

The temperature range at which the thermally insensitive imaging LNPs (or TNPs) of the present invention are stable (for example at which they do not undergo a phase transition as described above) suitably corresponds to the temperatures typically reached during application of hyperthermia (e.g. applied image-guided focused hyperthermia (IgFHT)). Thus, for example, the thermally insensitive imaging LNPs (or TNPs) typically do not undergo such a phase transition under the hyperthermia conditions employed in methods (e.g. to produce IgFHT) according to the present invention as discussed below. Preferably, the thermally insensitive imaging LNPs (or TNPs) may be stable at temperatures of up to at least about 45 °C, e.g. from about 39 to about 45 °C, preferably up to at least about 47°C, e.g from about 39 to about 47 °C.

At lower temperatures, it is desirable to avoid a phase transition from a liquid fluid state to a solid state. Thus the thermally insensitive imaging LNPs (or TNPs) are preferably stable at ambient/room temperature (e.g. the temperature at which they are formulated); more preferably they are also stable at refrigeration temperatures such as may be used during storage, e.g. at greater than or equal to about 8 °C. Thus in a further preferred embodiment, the thermally insensitive imaging LNPs (or TNPs) may be stable at temperatures of from about 0 (e.g. from about 5, or from about 20) to about 45 °C, preferably from about 0 (e.g. from about 15, or from about 20) to about 47 °C.

The imaging agent of the thermally insensitive imaging LNPs (or TNPs) of the present invention may be present inside the LNPs (or TNP)s and/or attached to the membrane of (or otherwise associated with) the LNPs (or TNPs). In one embodiment, the imaging agent is an imaging lipid, preferably attached to (or otherwise associated with) the thermally insensitive imaging LNPs (or TNPs). In one embodiment, the LNPs (or TNPs) of the present invention comprise imaging LNPs described as in WO 2011/061541 (herein incorporated by reference). Said imaging LNPs comprise Gd.DOTA.DSA and further comprise a neutral and/or fully saturated phospholipid component.

Appropriate neutral, fully saturated phospholipids suitable for use in the construction of

Gd.DOTA.DSA containing imaging LNPs (or TNPs) of the present invention are typically 1 ,2- di(Ci2-C2o saturated lipid)-sn-glycero-3-phosphocholines, wherein the saturated lipid groups can be the same or different from each other. More preferred examples include 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) or 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids. 1 ,2-distearoyl-sn-glycero-3-phospocholine (DSPC) is most preferred. Typically, the amount of fully saturated phospholipid component in said imaging LNPs (or TNPs) is from about 32 to about 34mol% of the total LNP (or TNP) formulation, and most preferably it is about 33mol %. Typically, the amount of Gd.DOTA.DSA component in said imaging LNPs (or TNPs) is from about 29 to about 31mol% of the total LNP (or TNP) formulation, and most preferably it is about 30m ol %.

Typically, the said imaging LNPs (or TNPs) have a size (e.g. average particle size at 10X dilution in phosphate buffer solution) of about 100, 80 or 50 nm or less. By carefully

nanoengineering of imaging LNPs (or TNPs) in this way to ensure that their size remains below about 100 nm, this size range is considered optimal, e.g. for the accumulation of imaging LNPs (or TNPs) in solid tumours due to the characteristics of tumour tissue.

Cholesterol may preferably be incorporated into the formulation since this lipid induces diverse effects on the liposomal bilayer. Cholesterol has been shown to increase the head group spacing in liposome formulations. It can stabilise the resulting bilayer membrane. Here, cholesterol presence in the imaging LNP (or TNP) formulation can control membrane permeability, say of both fluid and rigid bilayers. It may induce conformational ordering of the lipid. In addition, cholesterol can reduce serum-induced aggregation as a direct result of its neutral charge. Typically, the amount of cholesterol component in said imaging LNPs (or TNPs) is from about 29 to about 31mol% of the total LNP (or TNP) formulation, and most preferably it is about 30mol %.

In order to prolong the circulation time of the imaging LNPs (or TNPs), for example to ensure maximum tumour exposure, polyethylene glycol (PEG) may also be included, e.g. anchored, in or into the liposome bilayer, for example using a polyethylene glycol-phospholipid tethered construct. Examples of preferred polyethylene glycol-phospholipids for use in the imaging LNPs (or TNPs) of the invention include (ω-methoxy-polyethylene glycol 2000)-/V-carboxy-1 ,2- distearoyl-sn-glycero-3-phospocholine (PEG ²⁰⁰⁰ -DSPE).

Imaging LNPs (or TNPs) bearing a surface decorated with or comprising the neutral hydrophilic PEG polymer may benefit from prolonged circulation times with half lives reported from 2 to 24 h in rodents, and as high as 45 h in human ⁷ . Surface-grafted PEG coating of imaging LNPs (or TNPs) can help reduce uptake by liver cells by reducing plasma protein binding ³ . These LNPs (or TNPs) are also referred to as sterically stabilised. The PEG layer may sterically inhibit both electrostatic and hydrophobic interactions of plasma components with the liposome bilayer of target cells. Typically, the amount of polyethylene glycol-phospholipid component in said imaging LNPs (or TNPs) is from about 5 to about 8 mol% of the total LNP (or TNP) formulation, and most preferably it is about 7mol %.

For in vivo purposes, fully saturated phospholipids with neutral head groups are incorporated in the imaging LNP (or TNP) formulation. As described above, these include, but are not limited to; 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1 ,2-dimyristoyl-sn-glycero-3- phosphocholine (DM PC) lipids. The utilisation of neutral lipids (in addition to the incorporation of between 5-10 mol% of a PEGylated lipid, may provide steric stabilisation and/or protection, (e.g. from blood plasma proteins such as opsonin), and may lead to the reduction of Kupffer cell uptake. It is thought that stabilisation may occur by the formation of highly hydrated shields of polymer molecules, e.g. around the LNP (or TNP) surface. Due to this "shielding" characteristic, these types of imaging LNPs (or TNPs), may be referred to as having "stealth" characteristics. In a further embodiment of the present invention, the imaging LNPs (or TNPs) of the present invention may further incorporate a (receptor) targeting agent, for example comprising an antibody, diabody, nanobody, aptamer or peptide. For example, the targeting agent may be selected from folic acid (folate); antibodies (such as Trastuzumab or cetuximab); peptides (such as octreotide, LHRH antagonists or uPAR specific peptides); transferrin; mannose and galactose for asialoglycoprotein receptors; and aptamers.

In one embodiment, imaging LNPs (or TNPs) of the present invention may comprise a tumour specific-targeting agent, typically a ligand for a receptor that is over-expressed in target tissue cells, e.g. relative to the expression of said receptors in the cells of normal (non-tumourous) tissue of mammals.

One example of a targeting agent is one that comprises a folate comprising moiety, e.g. a folate-phospholipid. In preferred examples of the present invention, the tumour-targeting agent is a folate-polyethylene glycol-phospholipid compound. More preferably the compound is (folate-/V-co-polyethylene glycol 2000)-/V-carboxy-1 ,2-distearoyl-sn-glycero-3-phospho- ethanolamine (folate-PEG ²⁰⁰⁰ -DSPE).

Typically, the amount of the folate moiety present in the imaging LNPs (or TNPs) is about 1 to about 3 mol% of the total LNP (or TNP) formulation.

As an example of a targeting agents, folate is a good example of a cancer targeting moiety; as folate-based targeting systems can present an effective means of selectively delivering imaging agents to tumours ⁸ . It has been shown that aggressive or undifferentiated tumours at an advanced stage can have an increased folate receptor (FR) density, indicating that cancer therapy could benefit from the broad approach that FR mediated drug delivery offers ⁹ . The FR is over-expressed in several cancer types, such as brain, kidney, lung and breast cancers and in particular, in epithelial carcinomas such as ovarian cancers ¹⁰ . The FR ligand, folate (or folic acid), is a vitamin that is used for the biosynthesis of nucleotides and is utilized in high levels to meet the needs of proliferating cancer cells ¹¹ .

In addition to numerous drug delivery efforts, folate-receptor targeted technology has been successfully applied to radio-imaging of therapeutic agents ¹² , fluorescence imaging of cancer cells ¹³ , MRI contrast agents ¹⁴ , and gadolinium liposomes ¹⁵ . Choi et al., have demonstrated the use of folate-receptor targeted iron oxide nanoparticles for the imaging of induced KB tumours and showed these particles to have a 38% signal intensity increase compared to controls ¹⁶ . Successful tumour MRI with a non-targeted bimodal liposomal contrast agent was shown recently, whereby bimodal paramagnetic and fluorescent imaging LNPs of ~ 100 nm in size were seen to accumulate in a mouse xenograft model of ovarian cancer ¹⁷ . Imaging LNPs (or TNPs) are able to accumulate within tumour tissue due to the widely reported enhanced permeability and retention effect (EPR) which relies on the passive accumulation of colloidal macromolecules of ~ 40 kDa and above in tumours ¹⁸ . The EPR effect arises due to aberrant tumour endothelium, which as a result of its "leakiness" allows the penetration of nanoparticles into tumour tissue. Imaging LNP (or TNP) accumulation in tumour tissue could be improved through the use of receptor targeting moieties that are either post-conjugated to the surface of liposomes, or are attached to lipids that become incorporated within the lipid bilayer. Since FR binding affinity (Kd = 0.1 nM) does not appear to be affected when its ligand, folate is conjugated to an imaging agent or therapeutic moiety via its γ-carboxyl ¹⁹ , a folate ligand tethered onto the distal end of a lipidic PEG amphiphile allows for the development of a FR targeted imaging LNPs (or TNPs).

The human nasopharyngeal KB carcinoma cell line is considered to have the highest level of FR expression, yet the number of cases for this cancer are low in comparison to ovarian cancer which has the highest frequency (>90 % of cases) ²⁰ . In particular, the a-FR isoform that is a glycosyl phosphatidylinositol (GPI)-anchored membrane protein is highly expressed in ovarian carcinoma ²¹ . Additionally, the a-FR isoform has also been shown to have specific biomarker value, aiding in the identification of metastatic tumour site origin ²² . Therefore, the present inventors were interested in using this receptor in order to test the efficacy of FR targeted imaging LNPs (or TNPs) for the imaging of tumours using MRI. Folate-based drug delivery has been studied extensively ²³ , however, the rate-enhancing effect of imaging LNP accumulation in tumours due to folate-receptor targeting has not been studied dynamically in real-time to a great extent. Effective tumour signal enhancement was anticipated since the FR is expressed in significantly lower amounts in normal tissue, limited mainly to kidney tubuli, lung epithelium, and placenta tissue ²⁴ . To assess the value of the addition of a targeting agent on the rate and extent of accumulation of imaging LNPs in solid tumours, in the present invention FR targeted imaging LNPs were formulated and compared to non-targeted imaging LNPs by both MRI and fluorescence microscopy. The present inventors observed low toxicity, excellent targeted MR signal enhancement and, after rapid accumulation in the tumour initially, a quick and natural clearance of the contrast agents from the body thereafter. Said imaging LNPs described here presenting folate ligand are also known as Gadonano-F. Those imaging LNPs without folate ligand are known as Gadonano.

Said imaging LNPs (or TNPs) of the present invention have a function in terms of precision therapeutics to identify the extent and position of targets, in particular cancerous lesions (primary and secondary metastatic), by clinical imaging, in particular by MRI, but this could be adapted to cover other potential clinical imaging techniques, such as fluorescence imaging, magnetic resonance imaging (MRI), electron microscopy and image processing, electrons spin resonance, radio imaging, positron emission tomography (PET) or single-photon emission computed tomography (SPECT) or near infrared fluorescence (NIRF). Suitable imaging agents (preferably imaging lipids) for use in such techniques are as discussed above.

Identification of extent and position of targets allows these to be addressed by image-guided focussed hyperthermia as discussed below, and especially by imaged-guided focused ultrasound (IgFUS), including most especially magnetic resonance guided focused ultrasound (MRgFUS). Application of MRgFUS to target tissues is intended to guide therapeutic agent concurrently in the blood pool, from the blood pool to target tissue subject to IgFUS irradiation.

Imaging LNPs (or TNPs) of this invention preferably comprise but are not limited to

Gd.DOTA.DSA, cholesterol, DSPC and/or PEG ²⁰⁰⁰ -DSPE, preferably Gd.DOTA.DSA, cholesterol, DSPC and PEG ²⁰⁰⁰ -DSPE, wherein Gd.DOTA.DSA, cholesterol, DSPC and

PEG ²⁰⁰⁰ -DSPE are present most preferably in a ratio of about 30:33:30:7 mol% respectively. These imaging LNPs (or TNPs) may also comprise a receptor-targeting agent (such as a folate), Such receptor-targeted imaging LNPs (or TNPs) comprise but are not limited to

Gd.DOTA.DSA, cholesterol, DSPC, PEG ²⁰⁰⁰ -DSPE and/or folate-PEG ²⁰⁰⁰ -DSPE, preferably Gd.DOTA.DSA, cholesterol, DSPC, PEG ²⁰⁰⁰ -DSPE and folate-PEG ²⁰⁰⁰ -DSPE, wherein

Gd.DOTA.DSA, cholesterol, DSPC, PEG ²⁰⁰⁰ -DSPE and folate-PEG ²⁰⁰⁰ -DSPE are present most preferably in a ratio of about 30:33:30:5.5:1.5 mol% respectively.

Therapeutic agents

Therapeutic agents of the present invention may be a drug, compound or analogue thereof, particularly a small molecular weight compound, or a biopharmaceutical agent. For example, the agent may be a known drug or compound or an analogue thereof. In one embodiment, the therapeutic agent is a drug or biopharmaceutical agent.

Examples of therapeutic agents include, but are not limited to, anti-inflammatory agents; anticancer and anti-tumour agents; anti-microbial and anti-viral agents, including antibiotics; antiparasitic agents; vasodilators; bronchodilators, anti-allergic and anti-asthmatic agents; peptides, proteins, glycoproteins, and lipoproteins; carbohydrates; receptors; growth factors; hormones and steroids; neurotransmitters; analgesics and anaesthetics; narcotics; catalysts and enzymes; vaccines or genetic material. Additional examples of therapeutic agents include a nucleic acid or a polynucleotide (which may be single or double-stranded), for example DNA, RNA, mRNA, siRNA or antisense olignucleotides. These may be naturally occurring or synthetic. Further examples of therapeutic agents include an antibody, for example, a polyclonal antibody, a monoclonal antibody or a monoclonal humanised antibody.

In a preferred embodiment, the therapeutic agent is an anti-cancer agent, an anti-inflammatory agent, an antibody or an antibiotic.

Suitable drugs include, but are not limited to hydrophilic drugs, hydrophobic drugs, and water- insoluble drugs. A hydrophilic drug or other active agent is readily dissolved in water. A hydrophobic drug or other active agent has a low affinity for water, and does not readily dissolve in aqueous solutions. The dissolution of hydrophobic drugs or other active agents in water, however, is not impossible, and can be achieved under certain conditions that are known to those skilled in the art. Hydrophobic drugs or other active agents typically are dissolved in non- polar (e.g., lipophilic) solvents. Organic solvents can be used to dissolve water-insoluble drugs or other active agents. Hydrophilic active agents may be included in the interior of the said LNPs such that the bilayer creates a diffusion barrier preventing it from diffusing throughout the body. In one embodiment, the therapeutic agents are preferably anticancer agents - such as chemotherapeutic agents - in that they are capable of inducing (either directly or indirectly) cancer cell or tumour cell cytotoxicity. Examples of such anticancer agents include, but are not limited to, mitoxantrone (as described in WO02/32400), taxanes (as described in WO01/70220 and WO00/01366), paclitaxel, camptothecin, camptothecin derivatives (as described in

WO02/058622 and WO04/017940), topotecan, gemcitabine (as described in WO04/017944), vinorelbine (as described in WO03/018018), vinblastine, anthracyclines, adria, adriamycin (doxorubicin), adriamycine, capecitabine, docetaxel, didanosine (ddl), stavudine (d4T), antisense oligonucleotides - such as c- raf antisense oligonucleotide (RafAON) (as described in US6, 126,965 and US6,559,129), antibodies - such as herceptin, immunotoxins, hydroxyurea, melphalan, chlormethine, extramustinephosphate, uramustine, ifosfamide, mannomustine, trifosfamide, streptozotocin, mitobronitol, mitoxantrone, methotrexate, 5-fluorouracil, cytarabine, tegafur, idoxide, taxol, daunomycin, daunorubicin, bleomycin, amphotericin (e. g., amphotericin B), carboplatin, cisplatin, BCNU, vincristine, camptothecin, mitomycin, etopside, histermine dihydrochloride, tamoxifen, Cytoxan, leucovorin, oxaliplatin, irinotecan (as described in

WO03/030864), 5-irinotecan, raltitrexed, epirubicin, anastrozole, proleukin, sulindac, EKI-569, erthroxylaceae, cerubidine, cytokines - such as interleukins (e.g. interleukin-2), ribozymes, interferons, oligonucleotides, and functional derivatives of the foregoing.

Preferably, the anticancer drug is selected as defined by the American Cancer Society as alkylating agents (e.g. nitrogen mustards etc), antimetabolites (e.g. 5-FU), anti tumour antimetabolites (e.g. doxorubicin), topoisomerase inhibitors (e.g. topotecan), mitotic inhibitors (e.g. taxanes), corticosteroids (e.g. dexamethasone), miscellaneous (e.g. bortezomid), targeted therapeutics (e.g. gleevec), differentiating agents (e.g. retinioids), hormone therapy (e.g.

tamoxifen), immunotherapy (e.g. therapeutic antibodies or interleukin-2), paclitaxel, microRNA- 122, asparaginase, vincristine, cisplatin, cisplatin/NDDP, oxaliplatin and TUSC2/FUS1 or a pharmaceutically acceptable salt thereof, or solvate. In particular, the anticancer drug may be doxorubicin or a pharmacologically acceptable salt thereof or a solvate thereof.

Thus in a preferred embodiment, said one or more therapeutic agents or additional therapeutic agents comprise an anticancer agent such as an alkylating reagent, antimetabolite, anti-tumour antibiotic, topoisomerase inhibitor, mitotic inhibitor, corticosteroid, targeted therapeutic, differentiating agent, hormone therapy, immunotherapeutic or inhibitor of protein translation, preferably nitrogen mustards, platinum based drug, 5-fluouracil, doxorubicin, topotecan, taxanes, vincristine, dexamethasone, bortezomid, Gleevec, retinoid, tamoxifen, antibody or microRNAs, asparaginase or TUSC2/FUS1 , or a pharmaceutically acceptable salt thereof, or a solvate thereof. Preferably, said one or more therapeutic agents or additional therapeutic agents comprise topotecan, doxorubicin, paclitaxel, microRNA-122, asparaginase, vincristine, cisplatin, cisplatin/NDDP, oxaliplatin and TUSC2/FUS1 , or a pharmaceutically acceptable salt thereof, or a solvate thereof.

Said one or more therapeutic agents or additional therapeutic agents may also (or alternatively) comprise a small molecule anti-inflammatory agent or antibiotic.

In another embodiment, the therapeutic agents could be nephrotoxic, such as cyclosporins and amphotericin B, or cardiotoxic, such as amphotericin B and paclitaxel. Additional examples of drugs which may be delivered include but are not limited to, prochlorperzine edisylate, ferrous sulfate, aminocaproic acid, mecamylamine hydrochloride, procainamide hydrochloride, amphetamine sulfate, methamphetamine hydrochloride, benzamphetamine hydrochloride, isoproterenol sulfate, phenmetrazine hydrochloride, bethanechol chloride, methacholine chloride, pilocarpine hydrochloride, atropine sulfate, scopolamine bromide, isopropamide iodide, tridihexethyl chloride, phenformin hydrochloride, methylphenidate hydrochloride, theophylline cholinate, cephalexin hydrochloride, diphenidol, meclizine hydrochloride, prochlorperazine maleate, phenoxybenzamine, thiethylperzine maleate, anisindone, diphenadione erythrityl tetranitrate, digoxin, isoflurophate, acetazolamide, methazolamide, bendroflumethiazide, chloropromaide, tolazamide, chlormadinone acetate, phenaglycodol, allopurinol, aluminum aspirin, methotrexate, acetyl sulfisoxazole, erythromycin, hydrocortisone, hydrocorticosterone acetate, cortisone acetate, dexamethasone and its derivatives such as betamethasone, triamcinolone, methyltestosterone, 17-S-estradiol, ethinyl estradiol, ethinyl estradiol 3-methyl ether, prednisolone, 17a-hydroxyprogesterone acetate, 19-norprogesterone, norgestrel, norethindrone, norethisterone, norethiederone, progesterone, norgesterone, norethynodrel, aspirin, indomethacin, naproxen, fenoprofen, indoprofen, nitroglycerin, isosorbide dinitrate, propranolol, timolol, atenolol, alprenolol, cimetidine, clonidine, imipramine, levodopa, chlorpromazine, methyldopa, dihydroxyphenylalanine, theophylline, calcium gluconate, ketoprofen, ibuprofen, cephalexin, haloperidol, zomepirac, ferrous lactate, vincamine, diazepam, phenoxybenzamine, diltiazem, milrinone, mandol, quanbenz, hydrochlorothiazide, ranitidine, flurbiprofen, fenufen, fluprofen, tolmetin, alclofenac, mefenamic, flufenamic, difuinal, nimodipine, nitrendipine, nisoldipine, nicardipine, felodipine, lidoflazine, tiapamil, gallopamil, amlodipine, mioflazine, lisinolpril, enalapril, enalaprilat captopril, ramipril, famotidine, nizatidine, sucralfate, etintidine, tetratolol, minoxidil, chlordiazepoxide, diazepam, amitriptyline, and imipramine. Further examples are proteins and peptides which include, but are not limited to, bone morphogenic proteins, insulin, heparin, colchicine, glucagon, thyroid stimulating hormone, parathyroid and pituitary hormones, calcitonin, renin, prolactin, corticotrophin, thyrotropic hormone, follicle stimulating hormone, chorionic gonadotropin, gonadotropin releasing hormone, somatotropins (e.g., bovine somatotropin, porcine somatotropin, etc.), oxytocin, vasopressin, GRF, somatostatin, lypressin, pancreozymin, luteinizing hormone, LHRH, LHRH agonists and antagonists, leuprolide, interferons, interleukins, growth hormones (e.g. human growth hormone and its derivatives such as methione-human growth hormone and des-phenylalanine human growth hormone, bovine growth hormone, porcine growth hormone, insulin-like growth hormone, etc.), fertility inhibitors such as the prostaglandins, fertility promoters, growth factors such as insulin-like growth factor, coagulation factors, pancreas hormone releasing factor, analogues and derivatives of these compounds, and pharmaceutically acceptable salts of these compounds, or their analogues or derivatives.

The term "derivative" or "derivatised" as used herein includes chemical modification of an agent. Illustrative of such chemical modifications would be replacement of hydrogen by a halo group, an alkyl group, an acyl group or an amino group.

Further examples of suitable drugs include disease modifying antirheumatoid agents

(DMARDs).

Examples of biopharmaceutical agents include peptides, RNA interference (RNAi) effectors and anti-tumour necrosis factor a (TNFa) agents.

In a preferred embodiment, therapeutic agent(s) used in conjunction with said imaging LNPs, or additional therapeutic agent(s) used in conjunction with said TNPs, can be pre-formulated nanodrugs or comprise macromolecular biological agents. Those (additional) therapeutic agents that are pre-formulated nanodrugs may be co-administered with said imaging LNPs (or TNPs), or they could be administered before or after said imaging LNPs (or TNPs).

Liposomal doxorubicin, or Doxil®, is among the first generation of nanodrugs to bring a therapeutic benefit to cancer patients and demonstrate a decreased cardiotoxicity compared to free doxorubicin ² . It is noteworthy to list the current FDA approved marketed nanoparticles and some clinically relevant liposomal formulations, which are currently under investigation in clinical trials or FDA approval for cancer therapy (Tables 1 and 2). Newer systems are being developed continuously that may make use in clinic of tumour specific-targeting agent(s) ^25"29 .

Table 1 Nanodrugs approved by one or more regulatory bodies; note that as of 2015,

Thermodox is not currently approved.

Table 2 Clinically relevant liposomal formulations

Thus in particular, said nanodrug may be selected from Doxil®, Myocet®, ThermoDox®, Abraxane®, Rexin-G®, Oncaspar®, Alkaloid Marqibo®, Alkylating LiPlaCis Aroplatin®, SPI-77, Lipoplatin® and Lipoxal. In a preferred embodiment, said nanodrug is Doxil®.

In one embodiment, a macromolecular biological agent might be a targeted antibody therapy that reduces the side effects associated with potent cytotoxic drugs but is limited to antibodies that can modulate a pathway or process that results in cancer cell apoptosis. The conjugation of antibodies to anticancer drugs overcomes this limitation by separating the design requirements of targeting and treatment: the antibody is used to target a molecule that is overexpressed on cancer cells and the drug induces cell death ³⁰ . There are currently only two FDA-approved antibody-drug conjugates for cancer therapy: Brentuximab vedotin (FDA approved in 2001) and Trastuzumab ematansine (FDA approved in 2013).

As described above, the present invention also relates to a pharmaceutical composition comprising TNPs, preferably wherein TNPs are derived from imaging LNPs (or TNPs) that also comprise one or more entrapped therapeutic agents. These entrapped therapeutic agents are more preferentially selected from the anticancer drugs topotecan, doxorubicin, paclitaxel, microRNA-122, asparaginase, vincristine, cisplatin, cisplatin/NDDP, oxaliplatin and

TUSC2/FUS1. These TNPs may be used in conjunction with optional additional therapeutic agents, including agents preformulated as nanodrugs, and/or agents that are macromolecular therapeutic agents, for example all dispersed in a pharmaceutically acceptable carrier, as part of a method of treatment of the human or animal body by therapy. Preferably the nanodrugs are selected from Doxil®, Myocet®, ThermoDox®, Abraxane®, Rexin-G®, Oncaspar®, Alkaloid Marqibo®, Alkylating LiPlaCis Aroplatin®, SPI-77, Lipoplatin® and Lipoxal,

In one embodiment, the above therapeutic agents (in particular the preferred therapeutic agents) are used in conjunction with the preferred imaging LNPs (or TNPs) comprising gadolinium (III) 2-(4,7-bis-carboxymethyl-10-[(/V,/\/-distearylamidomethyl)-/ \/ ^, -amido-methyl]- 1 ,4,7,10-tetra-azacyclododec-1-yl)-acetic acid (Gd.DOTA.DSA) and a neutral, fully saturated phospholipid component, as described above.

Pharmaceutical formulations

The formulation of the imaging LNPs (or TNPs) of the present invention will depend upon factors such as the nature of the therapeutic agents used, and whether a pharmaceutical or veterinary use is intended, etc. The said imaging LNPs (or TNPs) are typically formulated for administration in the present invention with a pharmaceutically acceptable excipient (such as a carrier or diluents). The pharmaceutical carrier or diluent may be, for example, an isotonic solution. Typically, the concentration of the said imaging LNPs (or TNPs) of this invention are 1-50 mg/mL, but not necessarily limited to this range.

Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

Otherwise, for example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tableting, sugar- coating, or film-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g.

propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Formulations for oral administration may be formulated as controlled release formulations, for example they may be formulated for controlled release in the large bowel.

The dose of the said imaging LNPs (or TNPs) may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen.

A physician will be able to determine the required route of administration and dosage for any particular patient. A typical dose is from about 0.01 to 10000 μg, e.g. from about 0.01 to 1000 μg per kg of body weight, according to the age, weight and conditions of the individual to be treated, the type and severity of the condition and the frequency and route of administration. Dosage levels may be, for example, from 10 to 100 mg/m ² (equivalent to drug dose; example Doxil® dose is 50 mg/m ² equivalent to doxorubicin dose).

Medical Uses

In one embodiment, the present invention relates to a pharmaceutical composition as described above, for use in a method of treatment, such as of disease or medical disorder at target sites. The preference is for medical use in a method of treatment of cancer. In one embodiment, the pharmaceutical composition comprises TNPs for use in a method of treatment as described above, having said one or more therapeutic agents entrapped within them. Such as composition may comprise only TNPs (i.e. containing no separately formulated additional therapeutic agent).

Alternatively, the present invention also relates to a pharmaceutical composition comprising imaging LNPs as described above used in combination with one or more (separate) therapeutic agent(s) as described above, preferentially anti-cancer agents, for simultaneous, separate or sequential use in a method of treatment, such as of cancer.

The present invention also relates to a pharmaceutical composition comprising TNPs as described above used in combination with one or more (separate) additional therapeutic agent(s) as described above, preferentially anti-cancer agents, for simultaneous, separate or sequential use in a method of treatment, such as of cancer.

In another embodiment, the present invention relates to imaging LNPs as described above for use in a method of treatment, such as of cancer, which method comprises administering simultaneously, separately or sequentially to a subject (in need thereof) said imaging LNPs and one or more therapeutic agent(s) as described above.

The present invention also relates to TNPs as described above for use in a method of treatment, such as of cancer, which method comprises administering simultaneously, separately or sequentially to a subject (in need thereof) said TNPs and one or more additional therapeutic agent(s) as described above.

In another embodiment, the present invention relates to therapeutic agents as described above for use in a method of treatment of cancer, which method comprises administering

simultaneously, separately or sequentially to a subject (in need thereof) said therapeutic agents and imaging LNPs as described above.

The present invention also relates to additional therapeutic agents as described above for use in a method of treatment of cancer, which method comprises administering simultaneously, separately or sequentially to a subject (in need thereof) said additional therapeutic agents and TNPs as described above.

The present invention also relates to use of TNPs as described above in the manufacture of a medicament for use in a method of treatment, such as of cancer. Such a medicament may comprise only TNPs (i.e. containing no separately formulated additional therapeutic agent). Alternatively, the present invention relates to use of imaging LNPs as described above in the manufacture of a medicament for use in a method of treatment, such as of cancer, which method comprises administering simultaneously, separately or sequentially to a subject (in need thereof) said imaging LNPs and one or more therapeutic agents as described above.

The present invention also relates to use of TNPs as described above in the manufacture of a medicament for use in a method of treatment, such as of cancer, which method comprises administering simultaneously, separately or sequentially to a subject (in need thereof) said TNPs and one or more additional therapeutic agents as described above.

In another embodiment, the present invention relates to use of therapeutic agents as described above in the manufacture of a medicament for use in a method of treatment, such as of cancer, which method comprises administering simultaneously, separately or sequentially to a subject (in need thereof) one or more said therapeutic agents and imaging LNPs as described above.

The present invention also relates to use of additional therapeutic agents as described above in the manufacture of a medicament for use in a method of treatment, such as of cancer, which method comprises administering simultaneously, separately or sequentially to a subject (in need thereof) one or more said additional therapeutic agents and TNPs as described above.

In the methods described above, the therapeutic agents may be co-administered to a subject with imaging LNPs, or administered before or after the LNPs, as appropriate for optimal medical use. Similarly, the additional therapeutic agents may be co-administered to a subject with TNPs, or administered before or after the TNPs, as appropriate for optimal medical use.

In one embodiment, the imaging LNPs (or TNPs) used in the methods described above are the preferred imaging LNPs (or TNPs) comprising gadolinium (III) 2-(4,7-bis-carboxymethyl-10- [(A/./V-distearylamidomethy -A/ ^' -amido-methylj-l ^.y.lO-tetra-azacyclododec-l-y -acetic acid (Gd.DOTA.DSA) and a neutral, fully saturated phospholipid component, as described above.

Tumour tissue and other cancerous lesions can possess an affinity for LNPs (including imaging LNPs and TNPs) and macromolecular agents, termed the enhanced permeability and retention effect (EPR), whereby lipid-based systems or macromolecular agents accumulate in tumour tissue. EPR was first introduced by Maeda et al., ³¹ here; it is believed that tumour properties such as increased angiogenesis, a heterogeneous and destructive vascular infrastructure, impaired lymphatic drainage and a "leaky" endothelial layer are all factors that contribute to the accumulation of LNPs or macromolecular structures within tumour tissue. The mechanism of tumour accumulation of LNPs in tumour tissue has been established as the extravasation of large molecules through the disrupted endothelium lining tumour blood vessels. In addition to complying with the tumour extravasation size threshold, a further reason for liposome size to remain within the 100 nm range for in vivo injections is due to clearance of large LNPs (>150nm) through the liver. Large LNPs (>150nm) are taken up by liver cells which include hepatocytes and Kupffer cells, liposomal particles may accumulate in liver or spleen tissue due to the larger endothelial lining in these organs.

Thus, without wishing to be bound by theory, it is believed that said (additional) therapeutic agents such as anticancer nanodrugs or macromolecular biological agents can be expected to be directed to and taken up at an area of interest (such as a tumour) in the same way as LNPs such as imaging LNPs (or TNPs). In other words, the progress of imaging LNPs (or TNPs) to targets, such as cancer lesions, might be expected to reflect the process of additional therapeutic agents such as anticancer nanodrugs or macromolecular biological agents.

In a preferred embodiment, imaging LNPs (or TNPs) (in particular the preferred imaging LNPs (or TNPs) described above) can be used in a method of treatment known as precision therapeutics, preferably for cancer, wherein the method further comprises induction of hyperthermia, such as at target tissue of interest. This may be for the purposes of stimulating an enhanced permeability and retention effect (EPR), preferably a further enhanced EPR effect, e.g. hyper-permeability and retention (HPR) as discussed further below. In one embodiment, the induction of hyperthermia may be primarily for the purposes of stimulating EPR (preferably HPR), and not to activate thermal sensitivity (e.g. a phase transition as described above) in the LNP (or TNP); indeed, as described above in one preferred embodiment the LNP (or TNP) is thermally insensitive.

Thus in one embodiment, the present invention relates to a pharmaceutical composition for use in a method of treatment as described above, wherein said method comprises (i) administration of said pharmaceutical composition and (ii) induction of hyperthermia at target tissue of interest. In particular, said method may comprise:

- administering to a subject in need thereof a therapeutically effective amount of said pharmaceutical composition;

- imaging and/or detecting the presence of the imaging LNPs (or TNPs) of said pharmaceutical composition at, in or near target tissue of interest, in particular monitoring the identification of target tissue of interest by the arrival or presence of the imaging LNPs (or TNPs) of said pharmaceutical composition at, or in, said target tissue using MRI and/or or other such clinically relevant imaging modality or technique; and - induction of (image-guided focused) hyperthermia (IgFHT) at, in or near the target tissue of interest.

The present invention also relates to imaging LNPs (or TNPs) for use in a method of treatment as described above, wherein said method comprises:

- administering simultaneously, separately or sequentially to a subject in need thereof said imaging LNPs and a therapeutically effective amount of said therapeutic agent(s), or said TNPs and a therapeutically effective amount of said additional therapeutic agent(s);

- imaging and/or detecting the presence of the imaging LNPs (or TNPs) at, in or near target tissue of interest, in particular monitoring the identification of target tissue of interest by the arrival of the imaging LNPs (or TNPs) at or in said target tissue using MRI and/or or other such clinically relevant imaging modality; and

- induction of (image-guided focused) hyperthermia (IgFHT) at the target tissue of interest.

The present invention also relates to therapeutic agent(s) or additional therapeutic agent(s) for use in a method of treatment as described above, wherein said method comprises:

- administering simultaneously, separately or sequentially to a subject in need thereof a therapeutically effective amount of one or more said therapeutic agent(s) and said imaging LNPs, or a therapeutically effective amount of one or more said additional therapeutic agent(s) and said TNPs;

- imaging and/or detecting the presence of the imaging LNPs (or TNPs) at, in or near target tissue of interest, in particular monitoring the identification of target tissue of interest by the arrival of the imaging LNPs (or TNPs) at or in said target tissue using MRI and/or or other such clinically relevant imaging modality; and

- induction of (image-guided focused) hyperthermia (IgFHT) at the target tissue of interest.

The present invention also relates to use of imaging LNPs, TNPs, therapeutic agent(s) or additional therapeutic agent(s) as described above in the manufacture of a medicament for use in a method of treatment as described above, wherein said method comprises:

- administering simultaneously, separately or sequentially to a subject in need thereof said imaging LNPs and a therapeutically effective amount of one or more said therapeutic agent(s), or said TNPs and optionally a therapeutically effective amount of one or more said additional therapeutic agent(s);

- imaging and/or detecting the presence of the imaging LNPs (or TNPs) at, in or near target tissue of interest, in particular monitoring the identification of target tissue of interest by the arrival of the imaging LNPs (or TNPs) at or in said target tissue using MRI and/or or other such clinically relevant imaging modality; and

- induction of (image-guided focused) hyperthermia (IgFHT) at the target tissue of interest.

The present invention also relates to a method of treatment of cancer comprising:

- administering to subject in need thereof a therapeutically effective amount of a

pharmaceutical composition as described above;

- imaging and/or detecting the presence of the imaging LNPs (or TNPs) of said pharmaceutical composition at, in or near target tissue of interest, in particular monitoring the identification of target tissue of interest by the arrival of imaging LNPs (or TNPs) of the pharmaceutical composition at or in said target tissue using MRI and/or other such clinically relevant imaging modality; and

- induction of (image-guided focussed) hyperthermia (IgFHT) at the target tissue of interest.

The present invention also relates to a method of treatment of cancer comprising:

- administering simultaneously, separately or sequentially to a subject in need thereof imaging LNPs as described above and a therapeutically effective amount of one or more therapeutic agent(s) as described above, or TNPs as described above and a therapeutically effective amount of one or more additional therapeutic agent(s) as described above;

- imaging and/or detecting the presence of the imaging LNPs (or TNPs) at, in or near target tissue of interest, in particular monitoring the identification of target tissue of interest by the arrival of the imaging LNPs (or TNPs) at or in said target tissue using MRI and/or or other such clinically relevant imaging modality; and

- induction of (image-guided focused) hyperthermia (IgFHT) at the target tissue of interest.

Preferably, the above-described methods also comprise a step of confirming therapeutic effects at or in target tissues of interest using MRI or other such clinically relevant imaging modality.

Preferably, the target tissue of interest is one or more primary or metastatic tumour(s).

In particular, the methods of the present invention may comprise administration of imaging LNPs (or TNPs) comprising Gd.DOTA.DSA, cholesterol, DSPC and PEG ²⁰⁰⁰ -DSPE, preferably wherein Gd.DOTA.DSA, cholesterol, DSPC and PEG ²⁰⁰⁰ -DSPE are present in the ratio

30:33:30:7 mol% respectively in said LNP (or TNP) formulation, preferably wherein said LNP (or TNP) further comprises a receptor targeting agent (such as a folate moiety), e.g. comprising Gd.DOTA.DSA, cholesterol, DSPC, PEG ²⁰⁰⁰ -DSPE and folate-PEG ²⁰⁰⁰ -DSPE, preferably wherein Gd.DOTA.DSA, cholesterol, DSPC, PEG ²⁰⁰⁰ -DSPE and folate-PEG ²⁰⁰⁰ -DSPE are present in the ratio 30:33:30:5.5:1.5 mol% respectively in said LNP (or TNP) formulation, simultaneously, separately or sequentially with one or more therapeutic agent(s) selected from topotecan, doxorubicin, paclitaxel, microRNA-122, asparaginase, vincristine, cisplatin, cisplatin/NDDP, oxaliplatin and TUSC2/FUS1 , and/or a nanodrug formulation selected from Doxil®, Myocet®, ThermoDox®, Abraxane®, Rexin-G®, Oncaspar®, Alkaloid Marqibo®, Alkylating LiPlaCis Aroplatin®, SPI-77, Lipoplatin® and Lipoxal, or the methods may comprise administration of a pharmaceutical composition comprising these LNPs (or TNPs) and therapeutic agent(s).

The methods of the present invention can be applied to any type of solid tumour. For example breast cancer can be treated using this method. A patient with breast cancer will be

administered the pharmaceutical composition comprising imaging LNPs (or TNPs) with optional additional therapeutic agent(s). The patient will then be imaged using MRI and the site of the tumour site identified due to accumulation of the imaging LNPs (or TNPs) into the cancerous lesions, which may include the primary tumour and metastases. Hyperthermia will be induced in these target tissues. After such treatment imaging LNPs (or TNPs) remaining in the blood pool will be encouraged to partition into target tissues at enhanced levels from the systemic circulation thereby increasing their effects at these targets. Similarly, said (additional) therapeutic agent(s) that are either preformulated as nanodrugs or are macromolecular biological agent(s) will also partition into target tissues at enhanced levels from the systemic circulation thereby increasing their therapeutic effect as well.

In the method of the present invention, the imaging LNPs (or TNPs) can provide a means to identify the location of primary and or secondary cancerous lesions in a patient with cancer. Use of MRI (and/or other such clinically relevant imaging modalities) enables tracking of imaging LNPs (or TNPs) (and so potentially entrapped therapeutic agent(s)) to the target cancerous lesions. Image-guided focused hyperthermia (IgFHT) can then be induced at the correct or desired location where imaging LNPs (or TNPs) are seen to have accumulated, thus enabling remaining imaging LNPs (or TNPs) with (additional) therapeutic agent(s) to partition out of the blood (pool), and remain resident in target cancerous lesions thereafter.

Preferably IgFHT is applied once targets are made visible under MRI (or any other appropriate medical imaging technique) owing to the effects of said imaging LNPs, and once therapeutic agent(s) or API(s) have been administered to the subject. Administration of therapeutic agent(s) could take place before, during or after the administration of said imaging LNPs to identify targets for IgFHT irradiation.

In one embodiment, one or more therapeutic agent(s) are entrapped within said imaging LNPs, so that said imaging LNPs become theranostic nanoparticles (TNPs) (capable of therapy and diagnostic imaging applications). Accordingly, said TNPs can act in the same manner as said imaging LNPs to make visible targets under medical imaging conditions. At the same time, said TNPs of this invention become a means of targeted delivery of entrapped therapeutic agent(s) to targets, most especially targets subject to IgFHT treatment for guiding therapeutic agent(s) to target (as above). Additional therapeutic agent(s) may be used in conjunction with those entrapped in said TNPs. These additional therapeutic agent(s) may be co-administered with said TNPs or administered before or after the TNPs.

The method of the present invention as described above includes a step of administration and a step or steps of induction of (image-guided focused) hyperthermia (IgFHT). These steps could be performed in any order (or simultaneously). For example, the pharmaceutical composition may be administered first, followed by induction of hyperthermia (e.g IgFHT) (after a time interval), or hyperthermia may be induced at a target area of interest, before administration of a pharmaceutical composition. Thus an effective amount of a pharmaceutical composition as described above comprising imaging LNPs (or TNPs) may thus be administered before, after or concurrently with said (additional) therapeutic agent(s), before or after the induction of hyperthermia. Typically, in the present invention, hyperthermia (e.g IgFHT) is induced after MRI (and/or other such clinically relevant imaging modalities) identification of the location of a target of interest (such as one or more primary or metastatic cancerous lesions) on account of the accumulation of imaging LNPs (or TNPs) at these target locations. Thus in one embodiment, hyperthermia (e.g IgFHT) is preferably induced after administration of the pharmaceutical composition. When imaging LNPs (or TNPs) are administered separately from (additional) therapeutic agent(s), then hyperthermia (e.g IgFHT) is preferably induced after administration of the complete pharmaceutical composition. In an alternative embodiment, hyperthermia (e.g IgFHT) may be induced after administration of the imaging LNPs (or TNPs) but before administration of (additional) therapeutic agent(s), The methods of the present invention may comprise one or more steps of administration of the pharmaceutical compositions, and/or one or more steps of induction of hyperthermia. For example, hyperthermia (e.g IgFHT) may be induced before and after administration of the pharmaceutical composition or several times after such administration.

The intensity and duration of hyperthermia should be sufficient to raise the temperature at the target tissue of interest. For example, the target tissue of interest (such as one or more primary or metastatic tumour(s)) may suitably be raised to a temperature of from about 39°C to about 45 °C, preferably from about 39 °C to about 43 °C, more preferably from about 40 °C to about 42 °C. The temperature at the site of interest may initially be raised to a higher temperature than the indicated ranges, then allowed to cool to this range. The exact regimen used in the methods of the present invention will depend on factors including the type and location of cancer (or tumour) to be treated, the nature of the liposome and anticancer drug and the method employed for induction of hyperthermia. The skilled person will be able to determine an appropriate regimen depending on these circumstances.

The hyperthermia may be generated using a method comprising laser heating, hot water bath, radiofrequency thermal ablation (RFA), microwave hyperthermia and/or ultrasound (US).

Preferably, the hyperthermia (e.g IgFHT) is generated using image guided focused ultrasound (IgFUS), such as magnetic resonance guided focused ultrasound (MRgFUS).

Thus in particular, the methods of the present invention may comprise administration of imaging LNPs (or TNPs) comprising Gd.DOTA.DSA, cholesterol, DSPC and PEG ²⁰⁰⁰ -DSPE, preferably wherein Gd.DOTA.DSA, cholesterol, DSPC and PEG ²⁰⁰⁰ -DSPE are present in the ratio

30:33:30:7 mol% respectively in said LNP (or TNP) formulation, preferably wherein said LNP (or TNP) further comprises a receptor targeting agent (such as a folate moiety), e.g. comprising Gd.DOTA.DSA, cholesterol, DSPC, PEG ²⁰⁰⁰ -DSPE and folate-PEG ²⁰⁰⁰ -DSPE, preferably wherein Gd.DOTA.DSA, cholesterol, DSPC, PEG ²⁰⁰⁰ -DSPE and folate-PEG ²⁰⁰⁰ -DSPE are present in the ratio 30:33:30:5.5:1.5 mol% respectively in said LNP (or TNP) formulation, simultaneously, separately or sequentially with one or more therapeutic agent(s) selected from topotecan, doxorubicin, paclitaxel, microRNA-122, asparaginase, vincristine, cisplatin, cisplatin/NDDP, oxaliplatin and TUSC2/FUS1 , and/or a nanodrug formulation selected from Doxil®, Myocet®, ThermoDox®, Abraxane®, Rexin-G®, Oncaspar®, Alkaloid Marqibo®, Alkylating LiPlaCis Aroplatin®, SPI-77, Lipoplatin® and Lipoxal, or administration of a pharmaceutical composition comprising these LNPs (or TNPs) and therapeutic agent(s), wherein the IgFHT is generated using a method comprising laser heating, hot water bath, radiofrequency thermal ablation (RFA), microwave hyperthermia and/or ultrasound (US), preferably image guided focused ultrasound (IgFUS).

In one embodiment, the methods of the present invention may comprise administration of TNPs of the present invention, preferably wherein the entrapped therapeutic agent(s) are selected from the anticancer drugs topotecan, doxorubicin, paclitaxel, microRNA-122, asparaginase, vincristine, cisplatin, cisplatin/NDDP, oxaliplatin and TUSC2/FUS1 , optionally in conjunction with one or more additional therapeutic agent(s) including agent(s) preformulated as nanodrugs (preferably selected from Doxil®, Myocet®, ThermoDox®, Abraxane®, Rexin-G®, Oncaspar®, Alkaloid Marqibo®, Alkylating LiPlaCis Aroplatin®, SPI-77, Lipoplatin® and Lipoxal), and/or agent(s) that are macromolecular therapeutic agent(s), for example all dispersed in a pharmaceutically acceptable carrier, wherein IgFHT is generated using a method comprising laser heating, hot water bath, radiofrequency thermal ablation (RFA), microwave hyperthermia and/or ultrasound (US), preferably image guided focused ultrasound (IgFUS).

IgFUS and MRgFUS

In the most preferred embodiment of the present invention, the hyperthermia is generated using focused ultrasound (FUS). For example, in the method described above, image guided-focused ultrasound (IgFUS) can enable imaging LNPs, TNPs and (additional) therapeutic agent(s) to partition out of the blood pool into cancerous lesions that are visible to IgFUS application.

Appropriate therapeutic agent(s) are preferably formulated as nanodrugs or macromolecular biological agent(s) to benefit from the effects of IgFUS treatment to assist partition from the blood pool to irradiated target tissues. IgFUS treatment is expected to deliver equivalent therapeutic benefits in cancer therapy for substantially lower doses, potentially minimizing or eliminating side effects.

Hyperthermia

The application of ultrasound (or heating, i.e. hyperthermia) to target tissue, e.g. identified by sites of accumulation of imaging LNPs (or TNPs), particularly receptor-targeted imaging LNPs (or TNPs), can result in the accumulation of therapeutic agent(s) into the target tissue (from the blood-pool) during times of ultrasound application. Ultrasound can applied here as separate bursts or phases of (applied) ultrasound may be for a (short) period of time to a desired site, preferably to cause a temperature increase. The ultrasound is stopped or halted, such as between applications of (continuous) US. However, alternatively, the ultrasound can be fluctuated, between a low and high setting, for example for periods of higher or lower intensity. Hence the intensity may be oscillated as a function of time.

The ultrasound or hyperthermia method may thus comprise the repetition or cycles of application of (continuous) US or heating, e.g. in a cyclical or repetitive manner. Desirably the temperature of the tissue or desired body part is raised or increased to 39°C to 42°C, such as to from 40°C to 41 °C. Suitably, the temperature of the desired site does not exceed 42°C or 43°C.

Preferably the ultrasound application is started after the therapeutic has been administered to the subject, for example 10, 20, 30 or 40 mins after administration, There may then follow an interval, or a period of no, reduced or halted ultrasound. That time interval will depend on the drug administered, the subject, the ultrasound and a variety of factors. However, it is preferably at least 1 or 10 s, or 1 , 2, 3 or 5 mins. Optimally it is at least 10 mins up to 60, 90 or 130 mins, or even up to one or 2 h. This interval is to allow the tissue to cool down for approx 10 mins. After the interval there can be another (e.g. second) application of ultrasound. This can be repeated multiple times. Each cycle or application of FUS comprises a first heating stage, whereby the temperature (of the body part) is increased in a controlled and/or gradual manner, followed by a plateau or maintenance (e.g. second) stage where the temperature can remain substantially constant at steady state temperature e.g. at or around 41 °C, preferably 40-41 °C. Preferably, the heating stage is from about 30s to 90s, preferably about 1 min; the maintenance and or combined maintenance-heating stage time should be about 3 to 5 mins; the interval time in between ultrasound applications can range from one min to 2 h.

Preferably the ultrasound is focussed, in the sense that the ultrasound is placed, focussed or directed at or to a single position, or focussed on a single position or target site that may be altered or reselected as required, in a logical and systematic sequence from one part of the tissue to an adjacent or neighbouring part of the tissue, perhaps in line. The application of focussed ultrasound is continuous and the frequency is at least 10,100 or 1000kHz. Preferably the frequency is from 400, 600 or 800kHz such as up to 1.2, 1.4, 1.6, 1.8 or 2MHz (e.g. about 1 to 1.4MHz). The corresponding duty cycle may be as low as 70% but preferably is at least 95%, 98% or even 99%. The duty may be even as high as (about) 99.9% or higher, delivering on power levels of at least 5 or 10W, preferably up to 20, 30 or 40W.

Kits

The present invention also relates to a kit comprising: (a) at least one selected from the group consisting of a package, an instruction and an attached document describing combined use of imaging LNPs (or TNPs) as described above with one or more (additional) therapeutic agent(s) as described above, and (b) a pharmaceutical composition as described above.

The present invention further relates to a kit comprising a formulation comprising imaging LNPs as described above and a formulation comprising one or more therapeutic agent(s) as described above, and a kit comprising TNPs as described above and a formulation comprising one or more additional therapeutic agent(s) as described above.

The kit of the present invention may be for use a method of treatment of cancer. In particular, the method may further comprise induction of hyperthermia as described above.

The following Examples illustrate the invention:

EXAMPLES

Materials and Methods Imaging LNPs and TNPs

Doxil liposomes were obtained from Avanti Polar Lipids (Alabaster, AL, USA).

1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; 16:0 PC), 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC; 18:0 PC), 1-stearoyl-sn-glycero-3-phosphocholine (MSPC; 18:0 Lyso PC) and (ω-methoxy-polyethylene glycol 2000)-/V-carboxy-1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine (PEG ²⁰⁰⁰ -DSPE) were purchased from Sigma Aldrich (St. Louis, MO, USA) or Avanti Polar Lipids (Alabaster, AL, USA). All lipids were stored in aliquots (10 mg/mL) in either CHC , MeOH/CHC 50:50 (v/v) or MeOH. These lipids were formulated by standard procedures into LNPs (thermosensitive) with the following lipid formulation;

Gd.DOTA.DSA/DPPC/DSPC/MSPC/PEG ²⁰⁰⁰ -DSPE/XL750.DSA, 30:54:5:5:6:0.05

(m/m/m/m/m/m). Lipid stocks were combined in a round bottom flask in proportion to their respective mol% values (total mass of lipid 30-40 mg, as appropriate). The solvent was slowly evaporated in vacuo to ensure a thin and even film formation. This was hydrated in 300 mM ammonium sulphate, pH 4.0 (1 mL) and XL750.DSA freeze/thaw (x 5) by alternately plunging into liquid nitrogen and then hot water to fragment the film. The resulting suspension was sonicated at 60°C then extruded through a 100 nm polycarbonate membrane using a Northern Lipids (Burnaby, Canada) LIPEX extruder heated to 55°C and pressurised to about 10-20 bar. The external buffer was exchanged to sterile 20 mM HEPES pH 7.4 with 5% glucose (w/v) using a PD10 size exclusion column (Amersham, Buckinghamshire, UK). The resulting suspension of imaging LNPs (thermosensitive) was sized using a Nanoseries Nano ZS

(Malvern Instruments, Worcestershire, UK) before incubation with doxorubicin (1 mg/mL aq.) at 38.0°C for 2 h to form doxorubicin-containing TNPs (thermosensitive). This drug-loading step was performed using a Thermocycler (Mastercycler Personal, Eppendorf, Stevenage, UK) in order to provide accurate temperature control. Excess, non-encapsulated drug was removed using a PD10 column loaded with HEPES buffer, giving a clear, yellow/green suspension. The size of the TNPs was recorded using a sample (100 μί) that was then employed to quantify the lipid and doxorubicin concentrations while the remainder was stored at 4°C. Lipid

concentrations were determined using a modified version of the Stewart assay. In brief, TNP samples (50 \JL) were mixed with water (150 \JL) and MeOH:CHCI ₃ , 1 :1 (v/v) (200 \JL) then vortex mixed with vigour giving an emulsion. The sample was centrifuged (4000g; 2 min) to separate fully organic and aqueous layers. Thereafter, an aliquot (70 μί) of the organic layer was combined with Stewart reagent (5 μί, FeC /NhUSCN aq.), and the combination vortex mixed again then centrifuged. Finally, an aliquot (50 μί) was then transferred to a glass 96-well plate (Cayman Chemical, Ann Arbor Ml, USA) and Atss value measured on a plate reader (Infinite 200 Pro, Tecan, Mannedorf, Switzerland) for comparison with known standards. The doxorubicin concentration was measured by HPLC using an Agilent 1 100 HPLC equipped with a cooled sample chamber (8-12°C), a guarded Thermo Hypersil 30x4.6 mm, 5 μηι C18 column, a Diode Array Detector (UV/vis absorbance) and an Agilent 1260 Infinity fluorescence detector. Samples were loaded in deionised water containing 0.1 % trifluroacetic acid and eluted with acetonitrile using the gradient: 0 min 0 %, 1.5 min 0 %, 5 min 50 %, 6 min 50 %, 7 min 0 %, 8.5 min 0 % and a flow rate of 3.5 mL/min. Detection was by absorbance (210 / 254 / 280 nm for proteins and other biologicals; 420 nm for doxorubicin; all using a bandwidth of 8 nm c.p.

reference at 700 nm) and fluorescence (Ex 455 nm; Em 650 nm; PMT-gain 18). Injections were 5-20 μΙ_ depending on the sample concentrations and quantifications were calculated from the fluorescence peak areas after calibration (the detector response was effectively linear over the sensitive range).

Cell culture and generation

IGROV-1 (ovarian cancer) cells were routinely cultured in RPMI-1640 medium supplemented with fetal calf serum (FCS) 10% v/v. When cells reached 80-90 % confluence, they were harvested and prepared for implantation in mice. Post harvesting, cells were washed in saline and counted using a haemocytometer. Accordingly with the cell counting an equal volume of saline containing the cells was mixed with matrigel (Geltrex, Gibco). For the tumor generation, 5x 10 ⁶ cells contained in 50 % matrigel mixture were inoculated subcutaneously on both flanks of 8 weeks old SHO mice (Charles River, Germany). After 2 weeks, the formed tumours on each flank had reached an average diameter of 5-6 mm.

Moderate FUS treatments with TIPS

Accordingly to the different FUS treatments protocols, mice were prepared to receive define FUS by TIPS (Phillips, Netherlands). First, tissue temperature was monitored by 3

thermocouples placed around the tumour. Thereafter, the target tumour was covered by ultrasound gel and the TIPS placed at a distance of 88mm from the target. Each FUS pulse was delivered at a frequency of 1 MHz 99.9 % cycle duty and 10 to 20 W of acoustic power depending on the local temperature variation required (monitored live). Each moderate FUS pulse was seen to increase target tumour tissue temperatures up to a maximum of 42-43°C that was then maintained for a further 2-5 min, the duration of each moderate FUS pulse.

Nanoparticles biodistribution and drug release in vivo

The tumor bearing mice were injected intravenously with Doxil-like TNPs (thermosensitive). Wherein the dose of encapsulated doxorubicin was 8 mg/kg of mouse body weight,

administered by iv. injection in an aliquot (200 μΙ_) of sterile 20 mM HEPES pH 7.4 with 5% glucose (w/v). The injections were performed with anaesthetized mice using a syringe driver holding the syringe connected to a cannula inserted in the tail vein of each mouse. The injection rate used was 400 μΙ/min. Immediately post injection, each anaesthetised animal was placed into the Maestro EX (Caliper US) for imaging. The Maestro settings were adjusted to record doxorubicin (Ex 455 nm and imaged from 560-750 nm) or XL750 (Em 780 nm) signal. Finally, the images were unmixed (multispectral analysis) using the maestro 3.00 software.

Results

Therapy imaging probe system (TIPS) and moderate FUS treatment regimes

In order to provide FUS in vivo, we developed a preclinical FUS equipment set up with water- filled transducer chamber and thermocouple interface, all under computer control. Individual mice for FUS treatment were located under a therapy imaging probe system (TIPS) and 3 thermocouples (TC1 , TC2 and TC3) were placed around a tumour of interest to closely monitor changes in tissue temperature as a function of the application of short moderate intensity FUS burst. Each such FUS burst (2-5 mins) resulted in local hyperthermia (39-41 °C, up to 42-43°C) in and around the tumour for 5 min post FUS application and with good temperature distribution (Figure 3). From this data set, we deduced that a preclinical FUS burst regime is a reliable, controllable and efficient manner to induce focused hyperthermia in tissue.

Double FUS burst protocol and the effect on drug delivery

In order to investigate the consequences of repeat short moderate intensity FUS treatments, a double FUS protocol was developed (Figure 4) wherein two such short FUS treatments were applied (2 min then 5 min at 42-43°C) 30 and 90 mins post i. v. -administration of theranostic nanoparticles (TNPs) to one of two flank tumours (the other tumour representing a non-FUS control). Prior to the second application, a build up of TNPs was already observed in the FUS treated tumour, this build up continued apace following application of the second application (Figure 5). FUS treatment in this manner is apparently enhancing the enhanced permeability and retention (EPR) mechanism to such an extent that we have induced in effect

hyperpermeability and retention (HPR). Compare the extent of TNP accumulation in tumours, as judged by NIR fluorescence with and without FUS treatment (Figure 6).

Furthermore, when changes in doxorubicin-related fluorescence (Ex 455 nm and imaged from 560-750 nm) were monitored in the tumour with time (Figure 5), substantial drug-related fluorescence was observed post both first and second FUS treatments consistent with local drug release following both treatments. The implication is that TNPs are recruited from the blood pool to the tumour volume awaiting the opportunity for local drug release in the tumour volume. The ability of moderate intensity FUS regime to encourage positive control

accumulation in tumours was also observed to correlate with enhanced persistence of drug signal in the tumour over time (Figure 5) lasting for at least 7 days before clearance in > two weeks.

Therapeutic benefits of moderate FUS treatment regimes and the effect on drug delivery Therapeutic experiments were performed with tumour bearing mice as above (Figure 7). In comparing FUS treated (Figure 7b and 7d) and non-FUS treated (Figure 7a and 7c) tumours, there does appear to be a general if mild anti-tumour effect attributable to FUS irradiation.

Otherwise, results from group 2 (TNP) and 3 (Doxil) data with FUS irradiated tumours attest to the fact that the TNP and Doxil can be encouraged to enter tumour volumes by FUS-induced HPR, and that Doxil is equally effective if not more so as an anti-tumour nanodrug compared to the TNP formulation (thermosensitive, containing doxorubicin) that is itself a comparable nanodrug. Drug effects appear to persist only as long as significant drug is visible in tumour. Thereafter, tumour regrowth begins again albeit at a slower rate. Results from group 2 and 3 data involving tumours that were not FUS irradiated, suggest that given the doses of

doxorubicin administered, even though the majority of each nanodrug must partition into the FUS irradiated tumours, sufficient TNP or Doxil still remains in the blood pool to enter non-FUS treated tumour volumes by normal EPR thereby leading to observed anti-tumour effects. These effects can be expected to attenuate rapidly as doxorubicin doses are lowered.

Discussion

Hyperthermia has shown synergistic effects with neo-adjuvant chemotherapy and radiotherapy in clinic, improving existing treatments. Various methods can be used to induce hyperthermia such as lasers, hot water baths, microwave and radiofrequency applicators ³² . As a clear demonstration of the importance of mild hyperthermia, Li et a/. ³³ have shown significant effects from water bath heating on anti-tumour effects of their formulations. Since every tumour is believed to have a significantly different interstitial fluid flow and/or matrix density, a big challenge remains in the vascular permeation of the tumour in order to improve the drug delivery. Local hyperthermia appears to increase pore sizes in tumour vasculature, decrease steric and hydrodynamic hindrances. This may elevate intratumoral interstitial fluid flow and/or pressure, e.g. in a manner that might facilitate nanoparticle (-125 nm in diameter) or macromolecular biological extravasation. Hyperthermia may also increase local blood perfusion, e.g. in order to modify the pharmacokinetics of an API in the heated volume ³⁴ . Indeed such potential hyperthermia effects in tumours were reported by Kong et a/. ³⁵ . Indeed mild

hyperthermia (41 °C for 1 h) has been reported to generate gaps in the endothelial lining of up to 10 μηι ³³ , for at least 8h. These data supported similar observations by Kong et al. in a previous study ³⁶ .

Mild hyperthermia can enhance local blood plus interstitial fluid flow, thereby promoting the tumour uptake of nanoparticles and macromolecules (of up to 400 nm) ^32,37 Various methods have been introduced to generate hyperthermia such as lasers, hot water baths, plus microwave and radiofrequency applicators with different heat transfer rates. A potentially powerful means to maximize the benefits of hyperthermia is through the application of IgFHT, which is most closely represented by image guided focused ultrasound (IgFUS), in particular that variation known as magnetic resonance guided focused ultrasound (MRgFUS). Image guidance may also assist in overcoming one perceived weakness of FUS treatment, namely drug wash out from the FUS treated tumour exacerbated by premature drug release taking place prior to arrival of drug carrier in the tumour ³⁸ .

Hyperthermia can be induced by pulsed FUS that may reduce extreme tissue heating by allowing the tissue to cool down between US exposures ³⁹ . The increase in temperature can be

3-5°C (hyperthermia) despite the high energy deposited in the tissue. Hyperthermia applied in tumours can increase blood flow and enhance vascular permeability. Studies with canine soft tissue sarcoma, and with human tumour clinical subjects have also demonstrated that hyperthermia improves tumour oxygenation and enhances response of such tumours to radiotherapy or chemoradiotherapy.

The increased blood flow and vascular permeability caused by temperatures such as 42°C may also improve the delivery of chemotherapy drugs, immunotherapeutic agents and genes to tumour cells ⁴⁰ . FUS exposures in pulsed mode lower the rates of energy deposition and generate primarily mechanical effects for enhancing tissue permeability to improve local drug delivery. These pulsed exposures can be modified for low-level hyperthermia as an

enhancement of drug delivery that would lead to better drug deposition and better therapeutic effect ⁴¹ . Mild hyperthermia of 42°C can improve the degree of nanocarrier extravasation as shown by Kong et a/. ^35,36 . The reason that this leads to increased extravasation maybe due to downregulation of VE-cadherin that contributes to vascular integrity as it was shown in HUVEC endothelial cells ⁴² . It is clear that hyperthermia can provide a boost to extravasation and drug deposition in tumours. This should provide an adjuvant effect when nanocarriers are used and accumulate in tumours due to enhanced permeation and retention effect. It would be interesting to investigate the effect of hyperthermia on tumour/tissue drug clearance. FUS can also induce nonthermal effects on tissues. Acoustic cavitation can be induced using microbubbles exposed to US ⁴³ . Acoustic cavitation can be defined as the growth, oscillation, and collapse of gas containing bubbles under the influence of the varying pressure field of sound waves in a fluid and can have an effect on the permeability of a biological tissue ^43"45 . There are two types of acoustic cavitation: noninertial and inertial cavitation. The noninertial (stable) cavitation occurs when bubbles persist for a number of acoustic cycles. In this case the bubble's radius increases and decreases (expands and contracts) according to the applied US frequency. Inertial (transient cavitation) occurs when bubbles grow faster expanding 2- or 3-fold their resonant size, oscillate unstably, and collapse in a single compression half cycle ⁴⁴ . It has been considered that the primary mechanism to affect the structure of intact cells is inertial cavitation that can induce irreversible damage as well as increase cell membrane permeability ⁴⁶ ' ⁴⁷ .

The concept of using liposomes and high intensity focused ultrasound (HIFU) was introduced recently, in 2006 when Frenkel et al. used liposomal doxorubicin (Doxil) in combination with pulsed HIFU exposures in a murine breast cancer tumor model ⁴⁸ . Doxil is a stable liposomal preparation that has no response to increased temperature ⁴⁹ and was developed to minimise doxorubicin's cardiotoxicity, by encapsulating doxorubicin within stealth liposomes. Although Doxil achieves long circulation of doxorubicin with minimum cardiotoxicity it does not rapidly release the drug within the tumour. Pulsed-HIFU exposures were not found to enhance the therapeutic delivery of doxorubicin and did not induce tumour regression. However, a fluorescent dextran showed blood vessels to be dilated as a result of the exposures.

Experiments with polystyrene nanoparticles of similar size to the liposomes showed a greater abundance to be present in the treated tumours ⁴⁸ . Although this study did not achieve or prove a therapeutic advantage of the use of HIFU with temperature stable liposomes it showed clearly that pulsed HIFU induces a substantial increase of permeation of macromolecules and nanoparticles in tumours.

The implication of our data for cancer therapy is as follows. Imaging LNPs, of the type described here, have already been shown to target tumours in vivo leading to strong positive contrast MRI scans of tumour locations. Therefore, once repeated in clinic, imaging LNPs (or TNPs) would provide means for clear target identification of cancerous lesions by MRI (and/or other such clinically relevant imaging modalities). Target lesions so identified can then be subject to image- guided focused hyperthermia (IgFHT) treatment by image-guided focused ultrasound (IgFUS), preferably in the form of magnetic resonance guided focused ultrasound (MRgFUS). Such treatments appropriately given should enable substantial partition of further imaging LNPs (or TNPs) from the blood pool into the IgFHT treated target lesions. Nanodrugs (such as Doxil) and/or macromolecular biological anticancer agents in the blood-pool should behave similarly. This substantial partition effect that appears significantly greater than the EPR effect may be called the hyperpermeability and retention (HPR) effect. Hence, operating in clinic, imaging LNPs, TNPs, nanodrugs and macromolecular biological agents can be expected to experience substantial changes in pharmacokinetic behaviour owing to the HPR effect resulting in the extreme concentration of anticancer agents into FUS-treated lesions thereby allowing for lower effective doses and reduced drug side effects.

The combination of IgFHT and imaging LNPs with appropriate nanodrugs/macromolecular biological agents, or TNPs with or without nanodrugs/macromolecular biological agents, might be called image-guided focused hyperthermia enabled targeted drug delivery (IgFHT enabled TDD) for cancer therapy for which our preferred embodiment is magnetic resonance guided focused ultrasound enabled targeted drug delivery (MRgFUS enabled TDD). However, given that the enhanced accumulation of imaging LNPs (or TNPs) into target lesions also appears to result in persistent labelling of lesions to the extent that therapeutic effects can be monitored for several days (possibly weeks) post administration of therapeutic agents, then we would prefer to describe the whole process as a Precision Therapeutics approach.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and the spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should not be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology or related fields are intended to be within the scope of the following claims.

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