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WO2018185290A1 - Utilisation d'un liposome encapsulant un composé glucidique en imagerie cest - Google Patents

Utilisation d'un liposome encapsulant un composé glucidique en imagerie cest Download PDF

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Publication number
WO2018185290A1
WO2018185290A1 PCT/EP2018/058869 EP2018058869W WO2018185290A1 WO 2018185290 A1 WO2018185290 A1 WO 2018185290A1 EP 2018058869 W EP2018058869 W EP 2018058869W WO 2018185290 A1 WO2018185290 A1 WO 2018185290A1
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agent
imaging
liposome
glucose
liposomes
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PCT/EP2018/058869
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English (en)
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Xavier Golay
Alethea TABOR
Harriet STORY
Eleni DEMETRIOU
Helen Hailes
Robin BOFINGER
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Ucl Business Plc
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Priority to EP18719072.3A priority Critical patent/EP3606563A1/fr
Publication of WO2018185290A1 publication Critical patent/WO2018185290A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
    • A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates generally to methods and materials for use in magnetic resonance imaging (MRI), and in particular for use in saturation transfer mediated imaging, for example of tumours and the like.
  • MRI magnetic resonance imaging
  • saturation transfer mediated imaging for example of tumours and the like.
  • the current standard method for imaging tumours in vivo is [ 18 F]FDG-PET.
  • the method relies on the administration of [ 18 F]FDG to patients which is relatively expensive and delivers a major radiation dose, which is undesirable for repeated scanning.
  • Saturation transfer mediated imaging methods represent an alternative to [ 18 F]FDG-PET.
  • CEST chemical exchange saturation transfer
  • an off-resonance pulse A variation of the off resonance CEST technique is the use of an off-resonance spin-lock technique, which in practice does not change the outcome.
  • an on-resonance spin-lock technique can be used, providing an image which is Ti p-weighed. By choosing the amplitude of the spin-lock pulse, one can adjust the sensitivity of the technique to a specific exchange rate regime.
  • T2- weighted imaging has also been shown to be usable to detect changes due to an increase in fast exchanging protons such as hydroxyl protons and the like.
  • GlucoCEST is an imaging technique that enables visualisation of natural, non-radioactive glucose 1 .
  • GlucoCEST utilizes two properties of hydroxyl protons: first, when exposed to a magnetic field, the magnetic moments of hydroxyl protons precess at a different frequency as those of bulk tissue water and can therefore be selectively labeled using radiofrequency pulses; second, hydroxyl and water protons undergo exchange, thereby allowing magnetic labeling to be transferred from glucose to water and for glucose to be detected from the change in water signal in the MRI images.
  • the CEST technique thus provides an amplification of detection by using the very large water signal rather than relying on the much smaller signal from glucose. GlucoCEST can therefore provide higher resolution imaging than the existing standard method,
  • US20160095945A1 relates to the use of non-labeled sugars and detection by MRI for assessing tissue perfusion and metabolism.
  • glucose, or related analogues when used as CEST or related saturation transfer mediated imaging methods will interact with the body glucose sensing system.
  • the present inventors have developed a novel series of imaging agents, for use in saturation transfer mediated imaging methods, based on liposome-encapsulated sugar compounds.
  • CEST is used herein in relation to these methods and compositions. Nevertheless it will be understood that, unless context demands otherwise, any disclosure in relation to CEST applies mutatis mutandis to the other saturation transfer mediated imaging methods (T1 rho or T2-weighted imaging). Furthermore, the encapsulation of any of these sugar compounds in the liposome may be referred herein for brevity a “LipoGluco-CEST” but likewise will be understood to refer mutatis mutandis to the other sugar compounds described herein.
  • the methods and materials of the invention can not only improve circulation time of the imaging agents, but also aid in protecting the patient from the effects of administration of the sugar, and avoid triggering an acute insulin response.
  • This allows for high resolution GlucoCEST imaging of tumours, metabolic imaging and "dose painting" for radiotherapy available to all patients, and mitigates the variability in the imaging caused by the glycaemic response.
  • the inventors have further investigated the effects of modifying the physical and chemical properties of the liposomes employed in the methods, for example using kinds of lipid constituents, and have demonstrated, unexpectedly, that lipids comprising n-EG or PEG head groups could be formulated into "shielded" liposomes which nevertheless gave excellent CEST signals.
  • the use of these or other modified lipids additionally provides the ability to achieve enhanced cell-targeting, for example based on targeting peptides.
  • agents of the invention have particular utility in measurements of glucose or other sugar uptake in tumours, either through the enhanced permeability and retention (EPR) effect or via tumour cell targeting on the liposomal surface.
  • EPR enhanced permeability and retention
  • the invention also provides for the use of sugar compound (e.g. glucose analogue) - loaded liposomes to provide simultaneous tumour imaging and chemotherapy by targeting areas of greater tumour metabolism.
  • sugar compound e.g. glucose analogue
  • 2-DG 2-deoxy-D-glucose
  • the results showed it was possible to achieve an increase in signal for 2-DG loaded liposomes when compared to both free 2-DG and glucose.
  • the invention provides an agent for use in a method of saturation transfer mediated imaging, such as chemical exchange saturation transfer (CEST) imaging, in a subject, which agent comprises a liposome encapsulating a sugar compound, as defined herein wherein: the liposome is equal to or between 10 to 500 nm in diameter, and the concentration of sugar compound encapsulated in the liposome equal to or between 5 and 100 mM, and the sugar compound is encapsulated in an aqueous solution of pH between 5 and 8.
  • CEST chemical exchange saturation transfer
  • the method for imaging may comprise administering the agent to the subject and imaging the agent using magnetic resonance imaging as described above.
  • the agent may be administered in an effective amount.
  • the imaging may be performed using convention magnetic resonance imaging and any scanners have appropriate magnetic field strengths e.g. a 1 .5T MRI scanner, a 3.0T MRI scanner, or higher etc.
  • the agent may be used theranostically.
  • the liposome encapsulation can be used to modulate glycaemic response in the subject i.e. the agent may be used for this purpose.
  • Modulate in this context means reducing the effect of the glycaemic response in the subject on the concentration of the sugar compound. For example an acute injection of glucose may lead to transient hyperglycaemia, followed by hypoglycaemia due to the triggered acute insulin response. Modulation here has the effect of reducing variability between different imaging events in a given subject, leading to an enhanced replicability, or between different subjects in a cohort or group, leading to an enhanced reproducibility.
  • the subject may be one known to have abnormal glucose response.
  • the subject may be one known to have a normal glucose response.
  • the liposome encapsulated agent may be used to reduce variability between subjects
  • Liposomal encapsulation of the sugar compound will therefore mean that elderly patients (who are at higher risk of suffering from type 2 diabetes as well as cancer) and patients with Type 1 diabetes will be able to benefit from effective and more reproducible glucoCEST imaging.
  • Other variability in the glycaemic response arising from e.g. presence or not of comorbidities affecting the general metabolism, muscle mass, general health, age, and pharmaceutical use (e.g. of steroids) will also be mitigated.
  • the subject may be over 60 years old, may have Type 1 or Type 2 diabetes, and ⁇ or may be one who is being administered (i.e. prescribed a course of) steroids.
  • the agent may be used in methods in which the subject or subject group is actively selected in relation to their glycaemic response - for example may be a subject or subject group which is known, diagnosed, or believed to have one or more of the aforementioned characteristics.
  • the present invention has particular utility for sugar compounds which are capable of generating a glycaemic response in the subject via triggering a glucose sensing response (see e g. Thorens, B. "Glucose sensing and the pathogenesis of obesity and type 2 diabetes.” International journal of obesity 32 (2008): S62-S71 ; Thorens, B. "Brain glucose sensing and neural regulation of insulin and glucagon secretion.” Diabetes, Obesity and Metabolism 13.s1 (201 1 ): 82-88.).
  • the sugar compound will be one suitable for diaCEST i.e. having labile protons which are exchangeable with bulk water.
  • the sugar compound may be one which is subject to enhanced uptake by tumours, such as glucose which is typically taken up by a glucose transporter (see e.g. Vander Heiden, M. G., Cantley, L. C, & Thompson, C. B. (2009). Understanding the Warburg effect: the metabolic requirements of cell proliferation, science, 324(5930), 1029-1033.).
  • the sugar compound may be a suitable unmodified sugar such as a hexose (e.g. allose, altrose, glucose, mannose, gulose, idose, galactose, talose) or pentose (aldopentoses and ketopentoses).
  • a hexose e.g. allose, altrose, glucose, mannose, gulose, idose, galactose, talose
  • pentose aldopentoses and ketopentoses.
  • a further example is fructose.
  • the sugar compound is D-glucose.
  • Such sugars may be used in the present invention to provide suitable CEST signals for imaging e.g. of tumours.
  • the sugar compound may be a suitable modified sugar.
  • the sugar compound may be a sugar analogue e.g. a glucose analogue.
  • Such analogues may have particular utility in therapeutic or theranostic applications.
  • the sugar compound is a D-glucose analogue.
  • the glucose analogue is L-glucose, or an acetate derivative thereof (e.g. L-glucose pentaacetate).
  • glucose analogue is 2-deoxy-D-glucose (2-DG; see Figure 2).
  • 2-DG has been shown to be an effective radiosensitizer in vitro, 3 and has also been proposed as a very potent, and longer-lasting, GlucoCEST agent, 4 5 .
  • GlucoCEST agent 4 5 .
  • 6 and 2-DG has also been shown to trigger the insulin response in vivo 7 .
  • 2-DG means it can be advantageously employed in the present invention within liposomes for simultaneous imaging for radiotherapy dosing of tumours, targeting of radiation therapy and radiosensitisation.
  • Example glucose analogues are shown below and include, but not limited to L-glucose, 6- DG, 3-OMG, and FDG:
  • sugar compounds for use in the invention may be those which have been identified as having potential use in anti-cancer therapies. These include not just sugar analogues, but also conjugates (see e.g. S. R. Punganru et al., Bioorg Med Chem Lett 26, 2829 (2016); C. Granchi et al., MedChemCommun., 7, 1716 (2016); Y.-L Jiang et al., Chem. Biol. Drug. Des., 86, 1017 (2015). Thus the invention also embraces use of e.g. glucose when conjugated to a further therapeutic moiety.
  • Non-limiting examples are as follows:
  • Examples may also include conjugates of disaccharides, for example:
  • Brartemicin is a trehalose-derived metabolite which may have utility as an inhibitor of cancer cell invasion.
  • Suitable lipids for the preparation of liposomes, and methods for preparing liposomes, are described hereinafter. Many lipids are related to ethanolamine, phosphoethanolamine, choline, phosphocholine, and glycerol, shown below.
  • lipid in question is one of the lipids forming the lipid bilayer of the liposome, either alone or in combination with other lipids.
  • DPPC ,2-dipalmitoyl-sn-glycero-3- phosphocholine
  • the inventors have further formulated liposomes including lipids with PEG at the headgroup (MW 2000 - 4000) or lipids with n-EG units at the head-group (MW 900 - 1500).
  • These "shielded" lipids provide a steric coating on the surface of the membrane to hinder clearing of the particles by the reticuloendothelial system (RES). This prolongs the circulating plasma half-life of the drug.
  • the plasma half-life of the drug can be "tuned” from several hours to days depending on the size of the PEG and fatty acids attached to the lipid anchor. In many "standard” liposomal formulations, this is achieved by including 5-10% of lipids such as PEG2000-DSPE in the formulation (N. Bertrand et al., Adv. Drug Deliv. Rev., 66, 323 (2014)).
  • liposomes bearing large polymeric PEG moieties often have greatly reduced cellular uptake and targeting selectivity (L. Sun et al., Coll. Surf. B Biointerfaces, 135, 56 (2015)).
  • Cationic lipids with smaller n-EG units at the head group have recently been shown to provide formulations with prolonged in vivo circulation and also excellent cellular uptake when they reach the tumour target (M. F. Mohd Mustapa et ai, Bioconj. Chem., 20, 518 (2009); J. B. Wong et al., Mol. BioSyst, 4, 532 (2008); N. Mitchell et al., Biomaterials, 34, 1 179 (2013); see also US 7,598,421 and US 9,399,016)
  • n-EG phospholipids have not been previously reported and their effects on liposome stability and lipoCEST signal have not been studied.
  • the inventors have investigated liposomes which comprises an n-EG lipid based on the DPPC structure, such as 3-( ⁇ hydroxy[(17-hydroxy-4-oxo-6,9,12, 15-tetraoxa-3- azaheptadecyl)oxy]phosphoryl ⁇ oxy)propane-1 ,2-diyl dipalmitate (DPPE-EG4) (Fig 2) DPPE-EG4 has been formulated into Lipo 2DG-CEST reagents with similar CEST contrast to Lipo2DG-CEST reagents formulated with DPPC alone or with the industry standard, PEG2000-DPPC. Unexpectedly, these liposomes gave excellent CEST signals when encapsulating sugar compounds.
  • DPPE-EG4 3-( ⁇ hydroxy[(17-hydroxy-4-oxo-6,9,12, 15-tetraoxa-3- azaheptadecyl)oxy]phosphoryl ⁇ oxy)propane-1 ,2-diyl dipalmitate
  • the liposome may comprise a phospholipid, such as a phosphocholine lipid.
  • the liposome may comprise DPPC.
  • the liposome may comprise 1 ,2-distearoyl-sn-glycero-3- phosphocholine (DSPC).
  • the lipid may comprise n-ethylene glycol (n-EG), polyethylene glycol (PEG) at the head group.
  • the liposome may comprise a mixture of lipids, including n-EG lipids and unmodified lipids (i.e. lipids not comprising n-EG or PEG).
  • the liposome may comprise e.g. up to 50% n-EG lipid, e.g. about 10% to 40%, e.g. about 30% n-EG lipid.
  • the liposome may comprise e.g. 50% or more DPPC e.g. about 90% to 60%, e.g. about 70% DPPC.
  • cell-targeting peptides may optionally be attached to the exterior of the liposomes, via the n-EG or PEG lipids.
  • phosholipids which are polyethyleneglycol- modified phosphatidylethanolamines of Formula 1 , and their uses in the methods and compositions described herein:
  • n is an integer from 1 to 10;
  • X is -OH, -NH 2 , or -Q;
  • -Q is -L 1 -A or -L 2 -Pep; -L 1 - is a linking group;
  • -A is a reactive conjugating group
  • -Pep is a peptide group.
  • -R FA1 is a linear or branched saturated C9-19 alkyl group
  • -R FA2 is a linear or branched saturated C9-19 alkyl group.
  • -R FA1 is a linear or branched saturated Cn-17 alkyl group
  • -R FA2 is a linear or branched saturated Cn-17 alkyl group.
  • -R FA1 is a linear saturated Cn-17 alkyl group
  • -R FA2 is a linear saturated Cn-17 alkyl group.
  • -R FA1 is a linear saturated Cn , C13, C15, or C17 alkyl group
  • -R FA2 is a linear saturated Cn , C13, C15, or C17 alkyl group.
  • -R FA1 and -R FA2 are the same.
  • -R FA1 and -R FA2 are different.
  • n is an integer from 1 to 8.
  • n is an integer from 1 to 6.
  • n is an integer from 2 to 10.
  • n is an integer from 2 to 8.
  • n is an integer from 2 to 6.
  • X is -OH.
  • the polyethyleneglycol-modified phosphatidylethanolamine has the following formula:
  • phosphatidylethanolamine has the following formula (wherein each of -R FA1 and -R FA2 is a linear saturated C15 alkyl group (as found in palmitic acid); n is 4; and X is -OH), conveniently denoted "DPPE-EG4-OH”):
  • X is -NH2. In one embodiment, X is -Q.
  • -Q if present, is -L 1 -A.
  • the reactive conjugating group, -A is suitable for reaction with a sulfhydryl group (-SH), for example, as found in peptides, for example as a cysteine residue. This is described in more detail below.
  • -SH sulfhydryl group
  • the liposomes described herein are equal to or between 10 to 500 nm in diameter, and the concentration of sugar compound encapsulated in the liposome is equal to or between 5 and 100 mM, and the sugar compound is encapsulated in an aqueous solution of pH equal to or between 5 and 8.
  • the liposomes described herein are equal to or between 50 and 300 nm in diameter e.g. 150 nm to 250 nm e.g. about 200 nm in diameter.
  • the liposomes described herein the concentration of sugar compound encapsulated in the liposome equal to or between 15 and 60 mM, e.g.
  • the sugar compound is encapsulated in an aqueous solution of pH between pH 5.7 to 6.2 e.g. pH 5.8 to 6.0.
  • the inventors have shown that under physiologically relevant conditions (20% PBS, pH 5.8 (mimicking the acidic microenvironment of the tumors), 37 °C) liposomes having these properties are stable for at least 24 hr with minimal (4%) leakage of the sugar.
  • the liposome-encapsulated LipoGluco-CEST reagent can produce a larger CEST contrast than free glucose
  • the Lipo2DG-CEST reagent can produce a larger CEST contrast than free 2-DG
  • the table shows the CEST signal (herein defined as the standard measure of the asymmetry in the magnetization transfer ratio MTR aS ym) from glucose and 2-DG liposomes and the free sugar controls with equal overall concentration at pH 5.8 and 20 °C or 37 °C. Signal is expressed as the average percentage water suppression caused by hydroxyl group saturation in the range 0-3.75 ppm.
  • the CEST imaging methods and uses of the present invention may be for the purpose of metabolic imaging.
  • the CEST imaging methods and uses of the present invention may be for the purpose of tumour imaging.
  • the CEST imaging methods and uses of the present invention may be for the purpose of perfusion imaging.
  • the CEST imaging methods and uses of the present invention may be for the purpose of imaging of the glucose transport intracellularly.
  • the CEST imaging methods and uses of the present invention may be for the purpose of imaging of the glycolytic rate in an individual.
  • Tumour imaging may be for the purpose of distinguishing tumour types, or for "dose painting", defined as the process to use the parts of tumours with the largest metabolism as targets for radiotherapeutic interventions.
  • the agents of the present invention have particular utility in tumour imaging and theranostics.
  • the extracellular tumour microenvironment is more acidic than normal tissue and blood (6.0 - 7.0, vs 7.4) when the sugar compound (e.g. 2-DG) liposomes reach the tumour, the CEST signal may be significantly enhanced.
  • the agents of the invention may be used to distinguish tumour types with differing metabolic characteristics and pathophysiologies - for example based on differential tumour glucose accumulation e.g. in colorectal cancers.
  • Liposomal agents of the present invention may be expected to preferentially accumulate in tumor tissue over normal tissues via the enhanced permeability and retention (EPR) effect (see e.g. Maeda, Hiroshi, et al.
  • EPR enhanced permeability and retention
  • tumour selectivity can be enhanced by the presence of cell-targeting peptides to the exterior of the liposomes (abbreviated to "pep” in some of the formulae hereinafter).
  • lipids including a reactive conjugating group for example present on the n-EG or PEG group.
  • X may be -Q.
  • -Q if present, is -L 1 -A.
  • the reactive conjugating group, -A is suitable for reaction with a sulfhydryl group (-SH), for example, as found in peptides, for example as a Cys residue, which may optionally be at the terminus of a cell- targeting peptide.
  • -SH sulfhydryl group
  • Examples of such reactive conjugated groups include: maleimide groups; iodoacetyl groups; pyridyldithiol groups; vinylsulfone groups; and thiosulfonate groups. Examples of conjugation reactions using such groups are shown below.
  • the group -L 1 -A comprises a maleimide group, and has the following formula:
  • phosphatidylethanolamine has the following formula:
  • phosphatidylethanolamine has the following formula (wherein each of -R FA1 and -R FA2 is a linear saturated C15 alkyl group (as found in palmitic acid); n is 4; and X is -L 1 -A as described above):
  • the reactive conjugating group, -A is suitable for reaction with an amino group (-NH2), for example, as found in peptides.
  • -NH2 amino group
  • reactive conjugated groups include: N-hydroxysuccinimide (NHS) ester groups; Sulflo-N-hydroxysuccinimide (NHS) ester groups; pentafluorophenyl ester groups; and hydroxymethylphosphine groups. Examples of conjugation reactions using such groups are shown below.
  • the reactive conjugating group, -A is suitable for reaction with a hydroxyl group (-OH), for example, as found in peptides.
  • Examples of such reactive conjugated groups include: isocyanate groups.
  • An example of a conjugation reaction using such groups is shown below.
  • -Q if present, is -L 2 -Pep.
  • the group -L 2 -Pep comprises a group derived from a maleimide group, and has the following formula:
  • phosphatidylethanolamine has the following formula:
  • phosphatidylethanolamine has the following formula (wherein each of -R FA1 and -R FA2 is a linear saturated C15 alkyl group (as found in palmitic acid); n is 4; and X is -L 2 -P as described above):
  • tumour targeting peptide may be any known in the art, and may be selected by those skilled in the art according to the required tumour target in which the invention is to be applied.
  • Preferred lipid-peptide conjugates are those bearing peptide sequences such as
  • YHWYGYTPQNVI, LARLLT and CAEYLR which are known to bind to the EGFR receptor which is overexpressed on several types of tumor cells (see e.g. Z. Li et al., FASEB J 19, 1978 (2005); S. Song et al., FASEB J 23, 1396 (2009); C.-Y. Han et al. Int. J. Nanomedicine S, 1541 (2013)).
  • the peptide group (sometimes referred to as -Pep herein) may be selected from
  • GRP 78 SNTRVAP 1 Bombesin receptors D-Tyr-Gln-Trp-Ala-Val-bAla-His-Phe-Nle-NH2 2,3
  • FCFWKTCT-ol Oletide and other analogues
  • any of these may include an additional Cys residue at the N- terminus to facilitate conjugation to the lipid (e.g. via a maleimide).
  • Lipids comprising cell targeting peptides may optionally be additional to any of the phospholipids or shielded lipids described above.
  • lipid-peptide conjugates may be those shown below in Formula 2 or Formula 3. Any of these conjugates may be e co-formulated with mixtures of DPPC and n-EG- or PEG- lipids to afford cell-targeted shielded liposomes.
  • Liposomal agents of the present invention may be employed as theranostic agents. As explained above liposomes can be localized at the tumour via the EPR effect, and optionally other cell targeting agents.
  • 2DG is a highly effective glycolytic inhibitor agent, and has been shown to be an effective radiosensitizer in vitro, through disruption of the thiol metabolism (X. Lin et al., Cancer Res., 63, 3413 (2003). 2DG has also been proposed as a very potent, and longer-lasting, GlucoCEST agent (F. A. Nasrallah et al., J. Cereb. Blood Flow Metab., 33, 1270 (2013); M. Rivlin et al., Sci. Rep., 3, 3045 (2013).
  • 2-DG may therefore have utility for "dose painting" in which CEST imaging of 2-DG is used to define areas within the tumour which may require a higher radiation dose, related to tumour metabolism, and the tumor is also radiosensitized by the administration of 2DG.
  • 2DG itself cannot be used as a radiosensitizing agent in humans due to its toxicity: 2DG would be barely detectable by GlucoCEST at the levels needed to avoid severe secondary effects (L.E. Raez, et al., Cancer Chemother Pharmacol 71 , 523 (2013)).
  • liposomal delivery can mitigate toxicity arising from off-target effects.
  • 2-DG loaded liposomes can be employed as theranostic agents for the simultaneous imaging and treatment of tumors.
  • An example use is adjuvant therapy, in which the agents are employed for radiosensitisation prior to radiotherapy.
  • Example cancers which usually involve radiotherapy as part of their standard of care include colorectal cancer, and head and neck squamous cell carcinoma.
  • the combination of the characteristics of the agents of the invention thus allow for high levels of sensitivity and resolution, thereby permitting dose painting to be performed during radiotherapy planning.
  • the agents may also include other anticancer agents co- encapsulated with the sugar compound e.g. along with the 2-DG or glucose.
  • the agents may not include any other anticancer agents co-encapsulated with the sugar compound.
  • the agent is used in an effective amount.
  • An "effective amount”, as used herein in relation to diagnostics or theranostics, is an amount being fit for the purpose intended. Thus for diagnostics this will be sufficient to generate a CEST signal. For theranostics this will be sufficient to show benefit to the individual, optionally in combination with other therapies as described herein.
  • the actual amount administered, and rate and time- course of administration, will depend on the subject and the intervention in hand.
  • a CEST imaging agent comprising: a liposome, encapsulating a sugar compound, which is 2-DG. Also provided is use of this 2-DG agent as a theranostic, which is optionally targeted to the tumour, as described above. For example for radiosensitization prior to radiotherapy.
  • n-EG phospholipids of Formula 1 above examples include DPPE- EG4-OH as described herein. As shown in the Examples, these n-EG phospho lipids can provide stabilised liposomes should provide prolonged in vivo circulation and with improved tumor targeting in vivo, compared to liposomes formulated with small amounts of DSPE-PEG2000, while at the same time providing a lipoCEST signal comparable to liposomes formulated without any form of shielding (such as 100% DPPC liposomes).
  • lipid-peptide conjugates such as those shown in Formula 2 or Formula 3 above.
  • liposomes comprising these n-EG phospholipids and ⁇ or lipid-peptide conjugates.
  • an agent which comprises a liposome encapsulating a sugar compound, which compound is selected from glucose or a glucose analogue, wherein: the liposome is equal to or between 10 to 500 nm in diameter, and the concentration of sugar compound encapsulated in the liposome equal to or between 5 and 100 mM, and the glucose is encapsulated in an aqueous solution of pH between 5 and 8, in a method of saturation transfer mediated imaging, such as chemical exchange saturation transfer (CEST) imaging, in a subject, which is optionally theranostic imaging, in a subject.
  • CEST chemical exchange saturation transfer
  • a method for saturation transfer mediated imaging such as chemical exchange saturation transfer (CEST) imaging, in a subject, which is optionally theranostic imaging, in a subject, which method comprises administering an agent of the invention to the subject and imaging the agent in the subject using magnetic resonance imaging.
  • CEST chemical exchange saturation transfer
  • a liposome to modulate a glycaemic response in a subject to a reagent for saturation transfer mediated imaging, such as chemical exchange saturation transfer (CEST) imaging, in a subject, which reagent is a sugar compound selected from glucose or a glucose analogue, said liposome being used to encapsulate said sugar compound.
  • CEST chemical exchange saturation transfer
  • the liposome is as described herein e.g. equal to or between 10 to 500 nm in diameter, and the concentration of sugar compound encapsulated in the liposome equal to or between 5 and 100 mM, and the sugar compound is encapsulated in an aqueous solution of pH between 5 and 8.
  • the present invention utilises liposomes to deliver sugar compounds for imaging.
  • Liposomes and methods of preparing them are described herein.
  • liposomes and methods of preparing them are known in the art, and are summarised (by way of non-limiting example) in WO2014/124006 or WO2016036735.
  • liposomes are vesicles or particles which possess a lipid bilayer enclosing an aqueous compartment.
  • the sugar compound is encapsulated within the aqueous compartment.
  • the lipids may be natural or synthetic lipids.
  • Liposomes composed of natural phospholipids are biologically inert and weakly immunogenic, and they possess low intrinsic toxicity. Liposomes can be classified according to their lamellarity (uni-, oligo-, and multi-lamellar vesicles), size (small, intermediate, or large) and preparation method (such as reverse phase evaporation vesicles, VETs).
  • Unilamellar vesicles comprise one lipid bilayer and generally have diameters of 50-250 nm. They contain a large aqueous core and are the preferred type of liposome used herein.
  • SUV refers to a small unilamellar liposome vesicle (SUV) having a single lipid bilayer.
  • the vesicle-forming lipids preferably have two hydrocarbon chains, typically acyl chains, and a head group, either polar or nonpolar.
  • the hydrocarbon chains may be saturated or have varying degrees of unsaturation. For the present invention saturated chains are preferred.
  • vesicle-forming lipids and naturally-occurring vesicle-forming lipids, including the sphingolipids, ether lipids, sterols, phospholipids, particularly the phosphoglycerides, and the glycolipids, such as the cerebrosides and gangliosides.
  • Phosphoglycerides include phospholipids such as phosphatidylcholine,
  • phosphatidylethanolamine phosphatidic acid, phosphatidylinositol, phosphatidylserine phosphatidylglycerol and diphosphatidylglycerol (cardiolipin), where the two hydrocarbon chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation. Lipids containing either saturated and unsaturated fatty acids are widely available to those of skill in the art. Additionally, the two hydrocarbon chains of the lipid may be symmetrical or asymmetrical. The above-described lipids and
  • Exemplary phosphatidylcholines include dilauroyl phophatidylcholine,
  • dimyristoylphophatidylcholine dipalmitoylphophatidylcholine, distearoylphophatidyl- choline, diarachidoylphophatidylcholine, dioleoylphophatidylcholine, dilinoleoyl- phophatidylcholine, dierucoylphophatidylcholine, palmitoyl-oleoyl-phophatidylcholine, egg phosphatidylcholine, myristoyl-palmitoylphosphatidylcholine, palmitoyl-myristoyl- phdsphatidylcholine, myristoyl-stearoylphosphatidylcholine, palmitoyl-stearoyl- phosphatidylcholine, stearoyl-palmitoylphosphatidylcholine, stearoyl-o
  • Symmetric phosphatidylcholines are referred to as l,2-diacyl-sn-glycero-3- phosphocholines.
  • PC phosphatidylcholine
  • phosphatidylcholine 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine is abbreviated herein as "DMPC.”
  • DMPC phosphatidylcholine l,2-dioleoyl-sn-glycero-3-phosphocholine
  • DOPC phosphatidylcholine l,2-dipalmitoyl-sn-glycero-3- phosphocholine
  • saturated acyl groups found in various lipids include groups having the trivial names propionyl, butanoyl, pentanoyl, caproyl, heptanoyl, capryloyl, nonanoyl, capryl, undecanoyl, lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, phytanoyl, heptadecanoyl, stearoyl, nonadecanoyl, arachidoyl, heneicosanoyl, behenoyl, nestisanoyl and lignoceroyl.
  • the corresponding lUPAC names for saturated acyl groups are trianoic, tetranoic, pentanoic, hexanoic, heptanoic, octanoic, nonanoic, decanoic, undecanoic, dodecanoic, tridecanoic, tetradecanoic, pentadecanoic, hexadecanoic, 3,7, 1 1 , 15- tetramethylhexadecanoic, heptadecanoic, octadecanoic, nonadecanoic, eicosanoic, heneicosanoic, docosanoic, trocosanoic and tetracosanoic.
  • Unsaturated acyl groups found in both symmetric and asymmetric phosphatidylcholines include myristoleoyl, palmitoleyl, oleoyl, elaidoyl, linoleoyl, linolenoyl, eicosenoyl and arachidonoyl.
  • the corresponding lUPAC names for unsaturated acyl groups are 9-cis-tetradecanoic, 9-cis- hexadecanoic, 9-cis-octadecanoic, 9-trans-octadecanoic, 9-cis-12-cis-octadecadienoic, 9- cis-12-cis-15-cis-octadecatrienoic, 1 1-cis-eicosenoic and 5 ⁇ cis-8-cis-ll-cis-14-cis- eicosatetraenoic.
  • Exemplary phosphatidylethanolamines include dimyristoyl-phosphatidylethanolamine, dipalmitoyl-phosphatidylethanolamine, distearoyl-phosphatidylethanolamine, dioleoyl- phosphatidylethanolamine and egg phosphatidylethanolamine.
  • Phosphatidylethanolamines may also be referred to under lUPAC naming systems as 1, 2- diacyl-sn-glycero-3-phosphoethanolamines or 1 -acyl-2-acyl-sn-glycero-3 - phosphoethanolamine, depending on whether they are symmetric or assymetric lipids.
  • Exemplary phosphatidic acids include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid and dioleoyi phosphatidic acid.
  • Phosphatidic acids may also be referred to under lUPAC naming systems as 1 ,2-diacyl-sn-glycero-3 -phosphate or l-acyl-2-acyl- sn-glycero-3 -phosphate, depending on whether they are symmetric or assymetric lipids.
  • Exemplary phosphatidylserines include dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, dioleoylphosphatidylserine, distearoyl phosphatidylserine, palmitoyl- oleylphosphatidylserine and brain phosphatidylserine.
  • Phosphatidylserines may also be referred to under lUPAC naming systems as 1 ,2-diacyl- sn-glycero-3-[phospho-L-serine] or 1 -acyl-2-acyl-sn-glycero-3-[phospho-L-serine], depending on whether they are symmetric or assymetric lipids.
  • PS refers to phosphatidylserine.
  • Exemplary phosphatidylglycerols include dilauryloylphosphatidylglycerol,
  • dipalmitoylphosphatidylglycerol distearoylphosphatidylglycerol, dioleoyl- phosphatidylglycerol, dimyristoylphosphatidylglycerol, palmitoyl-oleoyl- phosphatidylglycerol and egg phosphatidylglycerol.
  • Phosphatidylglycerols may also be referred to under lUPAC naming systems as 1, 2- diacyl-sn-glycero-3-[phospho-rac-(l-glycerol)] or 1 -acyl-2-acyl-sn-glycero-3-[phospho-rac- (I -glycerol)], depending on whether they are symmetric or assymetric lipids.
  • the phosphatidylglycerol l,2-dimyristoyl-sn-glycero-3-[phospho-rac-(l-glycerol)] is abbreviated herein as "DMPG”.
  • the phosphatidylglycerol 1 ,2-dipalmitoyl-sn-glycero-3-(phospho-rac-l - glycerol) (sodium salt) is abbreviated herein as "DPPG".
  • Suitable sphingomyelins might include brain sphingomyelin, egg sphingomyelin, dipalmitoyl sphingomyelin, and distearoyl sphingomyelin.
  • Other suitable lipids include glycolipids, sphingolipids, ether lipids, glycolipids such as the cerebrosides and gangliosides, and sterols, such as cholesterol or ergosterol.
  • cholesterol is sometimes abbreviated as "Choi” or "CHOL”.
  • lipids suitable for use in liposomes are known to persons of skill in the art and are cited in a variety of sources, such as 1998 McCutcheon's Detergents and Emulsifiers, 1998 McCutcheon's Functional Materials, both published by McCutcheon Publishing Co., New Jersey, and the Avanti Polar Lipids, Inc. Catalog.
  • the overall surface charge of the liposome can affect the tissue uptake of a liposome.
  • Anionic phospholipids such as phosphatidylserine, phosphatidylinositol, phosphatidic acid, and cardiolipin may be used.
  • Neutral lipids such as dioleoylphosphatidyl
  • ethanolamine may be used to target uptake of liposomes by specific tissues or to increase circulation times of intravenously administered liposomes.
  • Cationic lipids may be used for alteration of liposomal charge, where the cationic lipid can be included as a minor component of the lipid composition or as a major or sole component.
  • Suitable cationic lipids typically have a lipophilic moiety, such as a sterol, an acyl or diacyl chain, and where the lipid has an overall net positive charge.
  • the head group of the lipid carries the positive charge.
  • Liposomes having a more rigid lipid bilayer, or a liquid crystalline bilayer are achieved by incorporation of a relatively rigid lipid.
  • the rigidity of the lipid bilayer correlates with the phase transition temperature of the lipids present in the bilayer.
  • Phase transition temperature is the temperature at which the lipid changes physical state and shifts from an ordered gel phase to a disordered liquid crystalline phase.
  • Several factors affect the phase transition temperature of a lipid including hydrocarbon chain length and degree of unsaturation, charge and headgroup species of the lipid. Lipid having a relatively high phase transition temperature will produce a more rigid bilayer.
  • Other lipid components, such as cholesterol, are also known to contribute to membrane rigidity in lipid bilayer structures.
  • Cholesterol is widely used by those of skill in the art to manipulate the fluidity, elasticity and permeability of the lipid bilayer. It is thought to function by filling in gaps in the lipid bilayer. In contrast, lipid fluidity is achieved by incorporation of a relatively fluid lipid, typically one having a lower phase transition temperature. Phase transition temperatures of many lipids are tabulated in a variety of sources, such as Avanti Polar Lipids catalogue and Lipidat by Martin Caffrey, CRC Press.
  • Liposomes are preferably made from endogenous phospholipids such as dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG), phosphatidyl serine, phosphatidyl choline, dioleoyphosphatidyl choline [DOPC], cholesterol and cardiolipin, or DPPC as described hereinbefore.
  • DMPC dimyristoyl phosphatidylcholine
  • DMPG dimyristoyl phosphatidylglycerol
  • DOPC dioleoyphosphatidyl choline
  • cholesterol and cardiolipin or DPPC as described hereinbefore.
  • MPS mononuclear phagocyte system
  • SUVs possess the disadvantage of low aqueous entrapment volume, and the use of charged liposomes can be toxic. These are overcome by coating the surface of the liposomes with inert molecules to form a spatial barrier.
  • poly-(ethylene glycol) (PEG) has been widely used as polymeric steric stabilizer or "shield”.
  • Surface modification of liposomes with PEG can be achieved in several ways: by physically adsorbing the polymer onto the surface of the vesicles, by incorporating the PEG-lipid conjugate during liposome preparation, or by covalently attaching reactive groups onto the surface of preformed liposomes.
  • PEG- distearoylphosphatidylethanolamine [DSPE] a cross-linked lipid
  • DSPE PEG- distearoylphosphatidylethanolamine
  • PEGylated liposomes The behaviour of PEGylated liposomes depends on the characteristics and properties of the specific PEG linked to the surface. The molecular mass of the polymer, as well as the graft density, determine the degree of surface coverage and the distance between graft sites. The most evident characteristic of PEG-grafted liposomes (PEGylated-liposomes) is their circulation longevity, regardless of surface charge or the inclusion of stabilizing agent such as cholesterol.
  • liposomes described herein can be prepared by a variety of techniques known in the art - see e.g. Liposome Technology, Vols. 1 , 2 and 3, Gregory Gregoriadis, ed., CRC Press, Inc.
  • the method selected is dependent on a variety of factors, such as: (1 ) the physicochemical characteristics of the material to be entrapped and those of the liposomal ingredients; (2) the nature of the medium in which the lipid vesicles are dispersed; (3) the effective concentration of the entrapped substance and its potential toxicity; (4) additional processes involved during application/delivery of the vesicles; (5) optimum size, polydispersity and shelf-life of the vesicles for the intended application; and (6) batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
  • liposomes can be created by sonicating phosphatidylcholine rich
  • liposomal particles are prepared using a conventional thin film hydration and extrusion method.
  • the lipid, PEG-conjugated lipid, and stabilizer, if present, are dissolved in an organic solvent (e.g., chloroform) at pre-determined molar ratios.
  • an organic solvent e.g., chloroform
  • a small proportion of a labeled-lipid, such as rhodamine labeled PE (Rho-PE) can be added to the mixture to enable visualization of the liposomal particles via fluorescence microscopy.
  • the mixture is placed in a rotavap with reduced atmosphere pressure to evaporate the organic solvent.
  • the resulting lipid film is hydrated, such as with phosphate buffered saline (PBS), while agitated using a water bath sonicator to form multi-lamellar vehicles (MLV).
  • PBS phosphate buffered saline
  • MLV multi-lamellar vehicles
  • the suspension is subsequently extruded through polycarbonate filters with pore sizes of 400 nm and 200 nm to generate unilamellar vehicles.
  • Fig 1 two volunteers blood glucose level changes under an hyperglycemic clamp. In a), a 15 mmol plateau was easily reached while it was impossible to get the glucose level > 10 mmol glucose in b).
  • Fig 2 - schematic of liposomal contrast agents and the chemical structures of 2-DG, glucose, DPPC and the novel PEG lipid DPPE-EG4.
  • L4 0.5 M glucose
  • L5 1 M glucose
  • L6 2 M glucose.
  • Fig 4 - this shows Z spectra (a,b) and MRTasym curves (c,d) for liposome samples with overall glucose concentrations of 55.5 mM (L9), 44.6 mM (L8) and 22.6 mM (L7).
  • Spectra were acquired with a saturation pulse frequency of 1.5 ⁇ (a,c) or 8.0 ⁇ (b,d) at room temperature with a Bo of 9.4 T.
  • Fig 5 - this shows Z spectra (a and c) and MTRasym curves (c and d) showing the pH dependence of CEST signal magnitude produced by glucose loaded liposomes L10-L13 at 25 °C (a and b) and 37 °C (c and d).
  • Fig 7 - shows the MTRasym spectra at 1.5 ⁇ and pH 6 for 2-DG liposomes, (a): 20°C, (b): 37 °C, as described in the Examples herein.
  • Fig 8 - Figure 8 shows the MTRasym spectra at 1.5 ⁇ and 5.0 ⁇ for 2-DG liposomes at pH 7 at 20°C and 37° with various PBS concentration.
  • Fig 10 - shows the MTRasym spectra at pH 6 or 7 for 2-DG liposomes at 20°C and 37°C using DPPC or DSPC lipids.
  • Figs 16A and 16B - this summarise the data in Figure 15 for the liposomes (A) and control sample (B).
  • Fig 17 - this shows the results of the assessment of release over time from the liposomes, as described in the Examples herein.
  • Fig 18 - shows the Z- and MTR asy m spectra for four sugars and their chemical structures.
  • Fig 24 MTRasymmetry values for 2-DG liposomes at a power of 1 .5 ⁇ and 5.0 ⁇ and temperatures of 20 °C and 37 °C a) with varying PBS concentrations at pH 6 and pH 7 b) at pH 7 with varying liposome diameter and with overall 2-DG concentrations of 39 mM or 14 mM.
  • Lipo-2DG caused more cell death than the same concentration of free 2-DG, both with and without a radiation dose of 4 Gy.
  • Example 1 variability of glucose response
  • adjunct therapies such as e.g. steroids, itself affecting the blood glucose level and the patient's insulin response;
  • Table 1 shows the formulation parameters and measurements for DPPC liposomes encapsulating glucose, L4-L13.
  • Table 1 Table showing the formulation parameters and measurements for DPPC liposomes L4-L13. Characterisation: the liposomes were sized by DLS before, during and after extrusion.
  • Samples for DLS were prepared by taking a 5-20 ⁇ aliquot of the stock liposome solution and diluting with H2O to give a final volume of 1 mL and a final concentration of approximately 0.2 mM. DLS measurements were taken at 25 °C in triplicate using clear 1 mL zeta potential cuvettes. The mean and standard deviation of three measurements is reported for each liposomal sample in Table 1 .
  • Liposome sample volume was slightly increased for more concentrated formulations.
  • SB1 , SB2 and RB measurement of A340 could be carried out immediately after mixing of the components in a measuring cuvette. However for T1 and T2, 15 minutes was allowed to ensure completion of the enzymatic cascade. The mg glucose/mL was calculated using Equation 1 and converted into glucose concentration (mM).
  • Table 2 Table showing the volume of glucose assay reagent, liposome sample, deionised water and Triton used for each measurement of A340 taken during the Glucose HK Assay ® . Volumes stated were used for 30 mM liposome samples encapsulating 0.5 M glucose.
  • L4 0.5 M glucose
  • L5 1 M glucose
  • L6 2 M glucose.
  • This experiment showed us that glucose solutions as concentrated as 1 M or more concentrated cannot be successfully encapsulated inside the DPPC lipid bilayer (see the exterior glucose concentrations reported in Table 1 , exterior concentration values higher than 5 mM are considered significant). Therefore all further formulations were carried out encapsulating 0.5 M glucose.
  • Figure 4 shows Z spectra (a,b) and MRTasym curves (c,d) for liposome samples with overall glucose concentrations of 55.5 mM (L9), 44.6 mM (L8) and 22.6 mM (L7). Spectra were acquired with a saturation pulse frequency of 1.5 ⁇ (a,c) or 8.0 ⁇ (b,d) at room temperature with a Bo of 9.4 T. The pH and glucose concentration of each liposomal sample is shown in Table 1 .
  • CEST suppression is expressed as an average of the percentage reduction in water signal caused by presaturation across the range 0-4.5 ppm.
  • FIG. 5 shows Z spectra (a and c) and MTRasym curves (c and d) showing the pH dependence of CEST signal magnitude produced by glucose loaded liposomes L10-L13 at 25 °C (a and b) and 37 °C (c and d).
  • Table 4 shows the CEST suppression of the water peak for glucose liposomes L10-L13 measured at both 25°C and 37 °C.
  • CEST suppression is expressed as an average of the percentage reduction in water signal caused by presaturation across the range 0-3.75 ppm.
  • the Glucose GO Assay ® (supplied by Sigma-Aldrich) was used to measure the exterior and overall 2-DG concentration of 2-DG encapsulating liposome samples. As the assay was designed to measure glucose concentration, a calibration curve must be constructed with 2-DG. An intact liposome sample (T1 ) was measured to obtain an exterior 2-DG concentration and a disrupted liposome sample with Triton-X100 (T2) was used to measure the overall 2-DG concentration in the same way that glucose liposomes are measured with The Glucose HK Assay ® .
  • Glucose and 2-DG liposomes both gave better signal than the corresponding controls. Glucose and 2-DG liposomes give greater signal at 37 °C than at room temp. The best signal in this experiment was from 2-DG liposomes at 37 °C.
  • Example 4 - optimisation of parameters for CEST detection of 2-DG liposomes 4. 1 Varying PBS at pH 6 and pH 7
  • Table 7 shows the formulation parameters and measurements for DPPC liposomes (30 mM) at pH 6 encapsulating 2-DG with varying PBS concentration.
  • Figure 7 shows the MTR asy m spectra at 1.5 ⁇ and pH 6 for 2-DG liposomes, (a): 20°C, (b): 37 °C.
  • Table 8 shows the formulation parameters and measurements for DPPC liposomes (30 mM) at pH 7 encapsulating 2-DG with varying PBS concentration.
  • Table 8 Figure 8 shows the MTRasym spectra at 1.5 ⁇ and 5.0 ⁇ for 2-DG liposomes at pH 7 at 20°C and 37°C.
  • Table 9 shows the formulation parameters and measurements for DPPC and DSPC liposomes (30 mM) at pH 6 or 7 encapsulating 2-DG with 20% PBS.
  • Table 9 Figure 10 shows the MTRasym spectra at pH 6 or 7 for 2-DG liposomes at 20°C and 37°C using DPPC or DSPC lipids.
  • Figure 1 1 summarises the results. As can be seen, DPPC liposomes were generally better than DSPC liposomes across different temperatures and powers.
  • DSPC liposomes may provide more stable and robust encapsulation.
  • Table 10 shows the formulation parameters and measurements for DPPC and DSPC liposomes (30 mM) at pH 6 or 7 encapsulating 2-DG with 20% PBS. A control was made up with 35 mM free 2-DG in 0.25 M NaCI and 20% PBS at pH 7.
  • Figure 12 shows the MTRasym spectra obtained at 37 oC and both 1 .5 ⁇ and 5.0 ⁇ .
  • Figure 13 summarises the results.
  • Fig 14 shows that 0.5 Mol% Rh-DHPE lipid can be incorporated into the liposomes for future fluorescent labelling studies without detrimental effects on 2-DG encapsulation or resultant CEST signal.
  • Example 5 investigation of liposome diameter for CEST detection of 2-DG liposomes
  • Table 1 1 shows the formulation parameters and measurements for liposomes of different diameters.
  • the liposomes were differentially diluted down to have comparable overall 2-DG concentrations. The parameters are shown in Table 12. Measurements taken post diluted are shown in bold.
  • Figure 14 summarises the CEST results from before and after dilution of the 2-DG liposomes at various temperature and saturation power. CEST suppression is expressed as an average of the percentage reduction in water signal caused by presaturation across the range 0-3.5 ppm.
  • Results shows how the diameter of 2-DG encapsulating liposomes (bilayer comprising 100% DPPC, in 20% PBS at pH 7) affects the CEST signal for diameters of 120 nm, 150 nm and 200 nm.
  • Liposomes were formulated as described in Section 2.1 and the slightly enhanced CEST signal seen for the 200 nm liposomes is assumed to be due to more efficient 2-DG encapsulation in the larger aqueous interior volume. Liposomes were differentially diluted to give overall 2-DG concentrations of 14 mM (to offset differences in encapsulation) and this trend of larger diameter giving larger CEST signal is no longer apparent.
  • no large difference in CEST was caused by varying liposome diameter in the range 120-200 nm. Liposomes can therefore be kept in this range, as this is also a good size for the EPR effect (passive targeting of liposomes to tumors).
  • DPPC liposomes (30 mM) encapsulating 2-DG (0.5 M) at pH 7 were sized to 200 nm and dialysed into 0.25 M NaCI solution with 20% PBS. The formulation parameters are shown in Table 13. The liposomes were scanned at 25, 28, 31 , 34 and 37 °C. A separate aliquot of liposomes was used for each temperature as some leakage occurs during scanning for the higher temperatures. A control was made up of 35 mM free 2-DG at pH 7 in 20% PBS.
  • FIG 16A summaries the liposome data.
  • CEST suppression is expressed as an average of the percentage reduction in water signal caused by presaturation across the range 0- 3.5 ppm.
  • Figure 16B summaries the control data.
  • the effect of temperature on CEST signal magnitude is more pronounced for liposome encapsulated 2-DG. This may be due to the induced change in lipid bilayer permeability/flexibility.
  • Example 7 - release over time scanning times
  • Figure 17 shows the release over time profile of 2-DG from DPPC liposomes at 37 degrees.
  • Results show that 21.3% of the encapsulated 2-DG leaks out of DPPC bilayers during the first 4 h which is how long CEST scanning takes.
  • PEG coating of liposomes is often carried out resulting in several advantages.
  • the neutral coating reduces colloidal instability by discouraging aggregation and supressing immunological responses.
  • PEG coating liposomes decreases their rate of removal from the blood by reducing uptake into the reticuloendothelial system (RES) and thereby lengthening their biological half-life.
  • Conventional PEG coating is achieved by formulating liposomes with a small molar percentage (typically 2-8 mol %) of 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine N-[carboxy(polyethyleneglycol)2000] (DSPE-PEG2000).
  • the PEG-2000 chain is long, neutral and hydrophilic causing decreased absorption of serum proteins and opsonins as well as masking the charge of the liposome.
  • Cells have been shown to preferentially take up charged particles and the absorption of serum proteins and opsonins to the liposome surface stimulates interaction with cell walls.
  • PEG-2000 coating of liposomes inhibits cellular internalisation.
  • Mitchell et al. demonstrated that replacing DSPE-PEG2000 with lipids exhibiting short n-EG shielding units significantly enhanced liposome internalisation whilst maintaining similar clearance rates to PEG-2000-stabilised liposomes.
  • Figure 19A shows the Z- and MTRasym spectra obtained from the 4 liposome
  • DPPE-EG4 lipid performed best; liposomes with 30% DPPE-EG4 encapsulated
  • PEG lipid (DPPE-EG4) can be used to form liposomes that entrap 2-DG and enable
  • the maximum overall 2-DG concentration achieved using 30% DPPE-EG-NH2 was 20 mM when formulated at pH 6 which is still significantly lower than encapsulatin with DPPC, 3% DPPE-PEG2000 or 30% DPPE-EG4.
  • Example 1 1 - use of Lipo2-DG-CEST as a theranostic agent for simultaneous tumour imaging and chemotherapy
  • 2-Deoxy-D-glucose (2-DG) is a well-characterized glycolytic inhibitor and has been shown to inhibit tumor growth in vivo [4]. However its use has not been promoted any further due to its inherent toxicity.
  • this Example we investigated whether we could use the sensitivity of GlucoCEST [1] to detect liposome-encapsulated 2-DG. Such a method would allow for high-concentration targeting of the drug to cancer cells via both active and passive targeting, thereby making it less toxic and potentially applicable for
  • Methods 1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes containing 0.5 M glucose or 0.5 M 2-DG were formulated in 20% phosphate buffer saline (PBS) at pH 5.8 via sonication, extruded to achieve an average diameter of -200 nm and dialysed into 0.25 M NaCI solution.
  • PBS phosphate buffer saline
  • the overall sugar concentrations for both liposome samples were enzymatically measured and adjusted to 23 mM. Free sugar controls were made up at 23 mM in 20% PBS at pH 5.8.
  • 2-DG-liposomes with an overall 2-DG concentration of 40 mM and at pH varying between 6 and 7 were prepared at different PBS concentrations of 0%, 5%, 10% and 20 %.
  • 2-DG liposomes were formulated using two different types of lipid (i.e. DPPC, DSPC) at pH 6 and 7.
  • DPPC lipid bilayer composition
  • DSPC lipid bilayer composition
  • Figures 22 and 23 show the Z- and MTRasymmetry spectra for the glucose and 2-DG liposomes and their free sugar controls at power levels of 1 .5 ⁇ and 5.0 ⁇ , respectively.
  • the MTRasymmetry was greater for the liposomal sugar formulations than the respective controls.
  • 2-DG gave a greater CEST contrast than glucose in both liposomal and control samples.
  • Table 12 summarizes the average percentage signal enhancement between 0 and 3.5 ppm for all sample measurements.
  • Table 12 MTRasymmetry from 0.2-3.5 ppm for glucose, 2-DG, glucose and 2-DG- loaded liposomes with overall sugar concentrations of 23 mM at pH 5.8.
  • FIG. 24 displays bar charts of 2-DG liposomes prepared at different PBS concentrations (Fig 24a) and with different diameters (Fig 24b).
  • the CEST signal was found to be larger for a power level of 5 ⁇ at 37 °C when compared to the signal at both 1.5 ⁇ at 20 °C and 5 ⁇ at 20 °C in both experiments.
  • the enhanced CEST detectability of the 2-DG liposomes may be i) due to a difference in the exchange rate of the hydroxyl protons on 2-DG compared to glucose at pH 5.8 and ii) through modification of the global exchange rate, by a combination of exchange through the lipid layer and the chemical exchange itself.
  • Example 12 - liposomal encapsulation of glucose can avoid an insulin response.
  • liposomal encapsulation may be used to make GlucoCEST more accessible to older/obese patients with type II diabetes. Furthermore liposomal encapsulation may be used to make GlucoCEST imaging results more robust and reproducible, as it reduces fluctuations in glucose response seen when injecting glucose directly into different patients.
  • Example 13 further evidence for the use of Lipo-2DG as a theranostic 10 colon and rectal cancer cell lines were screened and their response to free 2DG and liposomally encapsulated 2-DG (Lipo-2DG) was measured. In these experiments neither Lipo-2DG nor free 2-DG appear to act consistently as radiosensitisers in these cell lines.
  • Lipo-2DG was shown to be much more effective at killing RKO colon cancer cells than free 2-DG (see Figure 27). This supports the utility of Lipo-2DG as a theragnostic agent to image cancer cells and deliver a therapeutic dose of an effective anticancer drug.

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Abstract

L'invention concerne des agents destinés à être utilisés dans des méthodes d'imagerie à médiation par transfert de saturation (telle qu'une imagerie CEST) chez un sujet, lesdits agents comprenant un liposome encapsulant un composé glucidique approprié. L'invention concerne de nouvelles formulations de liposomes, qui peuvent être utilisées à des fins diagnostiques et théranostiques. Les liposomes peuvent être protégés et/ou ciblés sur un site d'absorption de glucide tel qu'une tumeur. L'invention est particulièrement utile pour moduler la réponse glycémique au composé glucidique encapsulé chez le sujet.
PCT/EP2018/058869 2017-04-07 2018-04-06 Utilisation d'un liposome encapsulant un composé glucidique en imagerie cest WO2018185290A1 (fr)

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GBGB1705653.2A GB201705653D0 (en) 2017-04-07 2017-04-07 Methods and means for imaging

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