WO2019022610A1 - Kit and method for microarterial imaging and radiotherapy - Google Patents
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- A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
- A61K51/1244—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles
- A61K51/1251—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles micro- or nanospheres, micro- or nanobeads, micro- or nanocapsules
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- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
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- A61K51/1268—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules host-guest, closed hollow molecules, inclusion complexes, e.g. with cyclodextrins, clathrates, cavitates, fullerenes
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Definitions
- the present invention relates to materials and methods for that support loco-regional therapeutic treatment based on image guidance.
- the present invention provides, in general, compositions comprising a pre-targeting and/or diagnostic vector component, and a second component that selectively binds to the pre-targeting vector component, and is able to elicit a therapeutic response. This combination can be used for in vivo parenteral local administration and target tissue specific induction of a therapeutic response.
- Hepatocellular carcinoma is a major worldwide health problem and provides a typical medical application for the proposed local intervention strategy. It is the most common primary malignancy of the liver and the third most common cause of cancer-related mortality. The incidence of primary liver cancer is increasing in several developed countries, including the United States, and the increase will likely continue for some decades.
- Radio-embolization therapy provides an alternative more targeted means of selective in-situ radiation. Radio- embolization is a therapy method that is often applied for primary liver tumours and metastases that are untreatable via surgery or chemotherapy.
- microspheres or microparticles containing therapeutic radioisotopes are delivered intra-hepatically where they provide localized radiation that is believed to cause damage to the diseased cells.
- therapeutic radioisotopes v-emitters such as yttrium-90 or holmium-166
- v-emitters such as yttrium-90 or holmium-166
- difficulties in confining the microparticles to the target fragments of the liver may result in hepato-pulmonary shunting. Shunting results in the displacement of the therapeutic effect to non-target organs, which may include highly sensitive tissue such as the lungs. This yields ineffective dose distribution and has been described as inducing seriously toxic adverse effects in a number of cases.
- SPECT Single-photon emission computed tomography
- SPECT-CT single-slice CT measurement
- a scout dose of holmium-166 comprising microspheres, or optionally other visualization modalities such as magnetic resonance imaging (M I), or ultrasound (US), may be applied, respectively.
- M I magnetic resonance imaging
- US ultrasound
- the pre-interventional imaging also helps provide a dosimetric distribution model for the therapeutic isotopes, and an accurate three-dimensional visualisation of the tissues targeted.
- the therapeutic microparticles are typically applied.
- Post-therapy SPECT/CT, MRI, and/or positron emission tomography (PET) is then applied to relate the delivery of the therapeutic portion to the distribution defined in the scout scans.
- the present invention provides a composition for use in vivo parenterallocal administration, comprising microparticles as pre-targeting vectors.
- These particles comprise one or more pendent reactive moieties that are able to form a high affinity interaction with a complementary reactive moiety residing on a therapeutic secondary component.
- This selective interaction can comprise of non-covalent and/or covalent bonds.
- one aspect of the present invention relates to a complementary component in vivo parenteral topical administration and for forming the selective non-covalent high affinity interaction with a complementary host moiety, and/or the selective covalent bond with a host compound having a complementary functionality to the pre-targeting vector component according to the invention; wherein the secondary component comprises a complementary guest functionality, and at least a first diagnostic and/or therapeutic agent.
- a still further aspect of the invention relates to a kit for forming a diagnostic and/or therapeutic occlusion at a target site within a body, the kit comprising: a vector component; and a complementary component functionalised for selectively attaching to the vector component in vivo.
- a still further aspect of the invention relates to a method of treating diseased tissue in vivo, the method comprising the steps of: (a) providing an administration medium comprising microparticles of a pre-targeting vector component, and (b) delivering a diagnostically and/or therapeutically effective amount of the a pre-targeting vector component into the tissue of interest; and (c) subjecting the target organ/tissue to a diagnosis step to determine if significant shunting occurs to healthy tissues; and (d) injecting intravenously or locally a therapeutically effective amount of a complementary secondary component comprising a diagnostic or preferably therapeutic agent, thereby producing a therapeutically active matrix in the subject tissue.
- a still further aspect of the invention relates to a method of locally treating a disease, comprising administering to a target area of a patient in need thereof the kit suitable for diagnosing and treating the disease.
- Fig. 1 schematically illustrates the pre-targeting concept according to the invention, in a preferred radioembolization embodiment, by the chemical and functional steps involved.
- A) Representation of the different chemical functionalities and components.
- B) A pre-targeting (guest) agent 99m Tc-MAA-Ad displays either pulmonary or hepatic accumulation depending on the route of administration, intravenous (i.v.) or local.
- C) Following i.v. administration, as a targeted (host) agent, 99m Tc-Cy50.5CD10PIBMA39, a prominent distribution to the kidneys, liver and stomach is observed.
- D) Subsequent to i.v.
- Model I administration of MAA-Ad
- accumulation of 99m Tc-Cy50.5CD10PIBMA39 is seen in the lungs
- Model II liver uptake of 99m Tc-Cy50.5CD10PIBMA39 is most predominant.
- Organs are marked as (1) lungs, (2,) liver, (3) kidneys, (4) stomach, and (5) urinary bladder.
- Fig. 2 shows a fluorescence confocal microscopy-based evaluation of Cy50.5CD10PIBMA39 (Cy5) binding to MAA-Ad (top) and non-functionalized MAA (bottom).
- the MAA-Ad localized particles reveal a higher degree of staining compared to MAA alone, indicated by the higher Cy5-related fuorescence intensities (in red).
- Fig. 3 shows the influence of MAA(-Ad) on the uptake of 99m Tc-
- Cy5o.5CDioPIBMA39 in the lungs and liver (Table 1). A) Uptake in the lungs increased when MAA-Ad was administered i.v. (Method I). B) Increasing uptake in the liver was seen when MAA-Ad was administered locally (Method II).
- Fig. 4 discloses fluorescence confocal microscopy images of a Cy5-Polystyrene- Ad (PS-Ad) as mixroparticulate guest, for a non-covalent functionalization test, indicating the host guest inclusion complexes also work well when employing polymer beads.
- Fig. 5 discloses fluorescence microscopic images (a) of a MAA Cy5 signal covalently bound to MAA using "Click" chemistry, versus a bright-field image, indicating that covalent selective binding via "click” chemistry also works instead of an inclusion complex.
- Fig. 6 discloses fluorescence microscopic images showing the biodistribution of 99m Tc-labeled cyclodextrin polymers in mice with adamantane (Ad)-functionalized macro- aggregates (MAA) administrated into the lungs.
- Fig. 6 (a) 99m Tc-Cy5o.
- Fig. 7 discloses the level of serum binding of radiolabeled Cy5o.5CDgPIBMA 3 9 over 20 h at 37 °C.
- Fig. 8 discloses A) In vitro host-guest complexation between guest vectors MAA-Ad or MAA with host vector m ln-Cy5o.5CD 9 PIBMA 3 9 for lh at 37 °C. Data are expressed as the mean ⁇ SD of the percentage of binding. B) In vitro stability determined at 37 °C of the host-guest complexation between MAA-Ad with m ln-Cy5o.5CD 9 PIBMA39 in PBS or FCS and 99m Tc-MAA-Ad with Cy5o.5CD 9 PIBMA 3 9 in PBS or FCS as well. Values are expressed as the % of radioactivity associated with the washed pellet being representative for the host-guest complex.
- Fig. 9 discloses A) time-related (2, 12, 20 and 44 h) dual-isotope SPECT imaging of A) intrasplenic administration of guest-vector 99m Tc-MAA-Ad and B) I.V. administered host- vector m ln-Cy5o.5CDgPIBMA 3 9. Organs are marked as (1) lungs, (2) liver, (3) spleen, and (4) kidneys. The scale bars indicate the intensity of radioactivity expressed as arbitrary units.
- Fig. 10 discloses the bio-distribution of pre-targeting the liver with locally administered guest vector: 99m Tc-MAA-Ad at various intervals. Data (expressed as the mean ⁇ SD of the percentage of the injected dose per gram tissue (%ID/g) of 3 observations were calculated based on the radioactive counts measured in indicated tissues at 2, 12, and 20h post-injection. Values of 99m Tc-activity calculated at 44h p.i. were unreliable as the radioactivity counts are very low.
- Fig 11 shows A) dual-isotope-SPECT imaging of mice pre-targeted with intrasplenic administration of guest vector 99m Tc-MAA-Ad at 12h p.i.. Mice, pre-targeted with either non-functionalized 99m Tc-MAA or PBS are used for comparison. Organs are marked as (1) lungs, (2) liver, (3) spleen, and (4) kidneys. B) Bio-distribution of host vector m ln- Cy5o.5CD 9 PIBMA 3 9 12h after I.V. administration c. C) Uptake of m ln-Cy5o.5CD 9 PIBMA 3 9 in the liver determined by ex-vivo fluorescence imaging at equal settings. The scale bar indicates the intensity of fluorescence expresses as photons/sec/cm 2 .
- Fig. 12 shows the time-dependent uptake of m ln-Cy5o.5CD9PIBMA39 in various tissues (liver, blood, kidney and joints) determined by radioactivity calculations in ROI's on the scintigrams. Data are expressed as the radioactivity per mm 3 on the scintigrams in regions of interest (ROI's) drawn over various tissues.
- ROI's regions of interest
- Fig. 13 shows in A) bio-distribution of I.V. administered m ln-Cy5o.5CD 9 PIBMA 3 9
- FIG. 13 shows in B) dynamic hepatic uptake of I.V. administered 111 ln-Cy5o.5CDgPIBMA39. Data are expressed as the mean ⁇ SD ratios of the %ID/g in liver and blood measured at 2, 12, 20, and 44h post-injection of m ln-Cy5o.5CD9PIBMA3 9 .
- the present invention provides materials and methods for locally diagnosing and/or generating a therapeutic response to diseased or infected cells/tissue. It more specifically relates to physically anchor, or otherwise to permit an intra-arterially supplied pre-targeting vector to remain in contact with the diseased or disease bearing region and not be immediately eliminated or washed out from that region; and to measure and diagnose the stability of the vectors positioning at the desired location, and eventually, to generate or solicit a therapeutic response with a suitable agent that selectively binds to the vector.
- the present method uses the natural filtering effect of the target tissue, such as the liver, to accumulate the particles.
- the present invention is advantageously based on the principle of first locating microparticles that acts a "vector" compound in the target area, and subsequently functionalisation of the thus located microparticles, by coupling "agent" compounds with a given functionality by a highly selective affinity interaction with the vector compounds.
- Such interactions are often described as host-guest interactions between a host compound and a guest compound, wherein the recognition and localisation may be achieved by host-guest interaction relying on physical affinity or, selective covalent chemical bonding.
- adio-embolization refers to locally administering radioactive materials to patients with cancer as a form of therapy.
- the radioactive materials have been incorporated into microspheres of regular size for injection into the arterial blood supply of the target organ or tumour.
- SIRT Selective Internal Radiation Therapy
- the main application of SIRT has been to treat cancers in the liver, but may expand based on the present materials and methods.
- the present method and components allow to create a source of localized radioactive microparticles locally from a micro-particulate vector components, and by subsequently anchoring radioactive isotopes to these vectors.
- the present invention now allows to log the microparticles in place first, and then to foresee them with a radioactive component, thereby simplifying the preparation and administration greatly.
- the term "radio-embolization” in particular refers to providing a microparticle first at the desired location, and then locking the radiation source to the particle, thereby resulting in a highly targeted and effective 2-component "Selective Internal Radiation Therapy "(SIRT).
- “Chemo-embolization” refers to locally administering chemically or biologically active materials to patients with a disease, such as cancer, an infection, an autoimmune illness or the like, as a form of therapy, whereby the chemically or biologically active materials are coupled to microspheres accumulated locally to induce a 2-component "selective internal chemotherapy".
- the present invention thus makes use of at least two components, the so-called host and the guest component, whereby one is first locally accumulated, and the second component is then introduced into the patient, to selectively couple to the component already in place.
- the host and the guest components are functionalised to provide complementary functionality.
- complementary functionality herein refers to a highly selective binding chemistry, wherein two or more complementarily functionalised partner molecules are likely to react, or bind in a predetermined reaction pathway.
- the host-guest recognition may advantageously be done using selective physical interactions, such as those provided in supramolecular host-guest inclusion complexes.
- inclusion complexes are for instance an adamantane (Ad) as host moiety, and a cyclodextrin (CD) as guest moiety.
- a host guest recognition may also be done by selective covalent chemistry, e.g. chemical bonding as for instance using "Click" chemistry between azide and alkyne moieties, whereby the two reactants advantageously are a "host” moiety bound to the vector compound, and a guest moiety bound to the agent, whereby the host and guest will react to form a covalent bond when exposed to each other under appropriate conditions.
- the present invention both compositions and method, have the clear benefit of providing a first allowing to introduce a pre-targeting vector that in itself may not need to have any strong therapeutic or diagnostic effect, to verify the accurate location, and stability of the positioning of the vector at this location, and then to modify the vector in situ using a secondary component functionalized with one or more desired activities.
- This not only allows to remove steps that are usually employed in radio-embolization treatments at the moment, but also reduces a number of invasive or otherwise cumbersome treatment steps.
- so far difficult to use radioisotopes may be employed e.g. 177 Lu and alpha-emitters.
- As well as other activities may be coupled with the vector that so far were not accessible or possible.
- the present method also allows to use functional compounds that were previously not useful, and does not require a specific radio-activation step of the microparticles themselves, but instead permits the use of readily available standard materials.
- the pre-targeting vector preferably comprises a multitude of microparticles that are dimensioned and configured to ultimately get lodged in the target tissue, and thus provide a local target for a secondary component. Preferably these may be dimensioned to pass through larger blood vessels, such as arteries and veins, but eventually get lodged in small vessels and extravasations, in particular if intravenous administration is preferred.
- Microparticles refers to particles or structures that are of a suitable size and dimension, and of suitable composition to be injected into the desired target area.
- the pre-targeting vector preferably comprises of microparticles which accumulate in cells (e.g. macrophages), in the interstitial cell fluid or in the microvascular bed, or diffuse in the lymphatic network of tissue wherein they have been injected.
- cells e.g. macrophages
- the pre-targeting vector preferably comprises of microparticles which accumulate in cells (e.g. macrophages), in the interstitial cell fluid or in the microvascular bed, or diffuse in the lymphatic network of tissue wherein they have been injected.
- the average particle diameter of the particles in the present invention depends on the desired target area, and to some extent also on the way of administration, e.g. intravascular versus direct injection into the target tissue area.
- intravascular administration considering the diameter of a blood vessel as target site of an embolus, is preferably between 1 and 1000 ⁇ , more preferably between 2 and 300 ⁇ .
- these microparticles are approximately 1-150 ⁇ in maximum diameter, and more preferably in the range of 5-70 ⁇ , and perhaps even 10-40 ⁇ .
- the particles preferably have a weight average particle size distribution width wherein more than 99% of all the particles are below average particle size of 100 ⁇ , more preferably in the range of average particle size of 50 ⁇ .
- the “distribution width of the particle diameter” refers to the range of particle size that would contain more than 99% of all the particles.
- the particle size of the microparticles of the present invention can be measured by a light scattering method or (electron) microscopy, more preferably by a laser diffraction method, wherein the particle size is reported as a volume equivalent particle diameter. Alternatively, the size may be determined by visible light, or electron microscopy, as appropriate.
- Suitable microparticles may include synthetic organic polymeric particles or inorganic glass or metal (e.g. FeO) beads, but also may include naturally occurring and purified compounds such as polypeptides or the like.
- the microparticles may be organic, or inorganic, or can comprise of mixtures or can consist of combined organic/inorganic materials.
- Suitable organic vector components include polymeric components, whether biologically originating, or synthetic.
- Examples of useful synthetic polymers formed by chemical cross-linking include polyethylene glycol (hereinafter, "PEG”), polypropylene glycol (hereinafter, “PPG”), polyvinyl alcohol (hereinafter, “PVA”), polystyrene (“PS”), polyacrylic acid, hydroxyethyl acrylate, polyhydroxy ethyl methacrylate, polyvinyl pyrrolidone, carboxymethyl cellulose, dextrans or other sugar-based polymers; homopolymers or (block) copolymer or the water- soluble polymer of the water-soluble polymer selected from the group consisting of hydroxymethyl cellulose and hydroxyethyl cellulose, a- hydroxy acids, the a- hydroxy acid cyclic dimer, a block copolymer of a monomer selected from the group consisting of hydroxy dicarboxylic acids and cyclic esters may be employed.
- PEG polyethylene glycol
- PPG polypropylene glycol
- PVA polyvin
- Particularly useful materials for the vector include hydrogel beads, such as those made from polyhydroxy polymers such as polyvinyl alcohol (PVA) or copolymers of vinyl alcohol, which may be readily tagged with the host moiety by reaction of pendent hydroxyl moieties within the polymer network, using for instance activating agents such as carbonyldiimidazole.
- PVA polyvinyl alcohol
- particles that are prepared by emulsion or dispersion polymerisation may be employed.
- Suitable inorganic vector components include metal particles, such iron, gold, or silver particles, glass beads, porous Silica beads, refractory beads and the like.
- the shape of the particles of the present invention may be essentially any shape found useful to lodge the microparticles at the desired site.
- Non-spherical particles like MAA or carbon nanotubes would provide a higher degree of accumulation due to their shape, and may remain in place more firmly.
- essentially spherical particles may also be employed for a better flow in intravascular administration, or combinations thereof.
- the term "essentially spherical” as used herein refers to a generally spherical shape having a maximum diameter/minimum diameter ratio of from about 1.0 to about 2.0, more preferably from about 1.0 to about 1.5, and most preferably from about 1.0 to about 1.2. This definition is intended to include true spherical shapes and ellipsoidal shapes, along with any other shapes that are encompassed within the maximum diameter/minimum diameter ratio. This shape is assumed in situ and in absence of shear forces, whereas during the administration, the shape may change under pressure and shear.
- surface means the exterior portion of a microparticle that is accessible to the complementary “guest” component. This may be the surface in the case of non-porous particles, or the accessible surface including pores and voids in the case of a porous particle, or combinations thereof.
- the term "interior portion,” as used herein, means any portion of the particle that is interior to the surface.
- the vector particles according to the invention are solid or highly viscous polymeric particles, and not composed of micelles or cell walls with a fluid interior at the temperature and conditions of the administration.
- the pre-targeting vector microparticles according to the present invention are typically host-tagged particles, preferably carrying pendent host tags or host moieties at their outer surface, or at least in such manner that the host moieties are accessible to the complementary guest component, e.g. where a porous particle is employed, such that the entire particle is rendered active for forming the host-guest complex or covalently bonded component.
- the host and guest functionality may be reversed, e.g. a pre-targeting vector may carry a guest molecule or moiety, where the modification of the secondary component may be negatively affected by carrying a host due to e.g. pharmacokinetics, or renal excretion.
- the pre-targeting vector microparticles according to the present invention preferably also comprises an imaging label, e.g., a diagnostic and/or a detectable label.
- an imaging label e.g., a diagnostic and/or a detectable label.
- the optionally encapsulated imaging label e.g., a diagnostic label and/or a detectable label may be or comprise an imaging label itself, e.g., as a detectable label. This advantageously permits to determine if and when the pre-targeting vector microparticles are in the desired location, and of any loss occurs due to blood flow or degradation that may negatively impact a subsequent treatment.
- the secondary compounds according to the present invention have a smaller size than the pre-targeting vectors and can be tuned for their pharmacokinetics. They may be organic or inorganic, and are prefarbly based on synthetic, or naturally occurring compounds, or combinations thereof.
- the particulate materials may be formed from monomers or preferably of polymeric materials such as, amino acid sequences, polylactic acid, polyglycolic acid, polycaprolactone, polystyrene, polyolefins, polyesters, polyurethanes, polyacrylates and combinations of these polymers, and homo-or copolymers, and blends thereof.
- the particulate materials may further comprise suitable binding agents such as gelatin, polyethylene glycol, polyvinyl alcohol, glycerin, (poly)saccharides, DTPA, DOTO, NOTA other hydrophilic materials, and combinations of these.
- suitable gelatins may include bovine collagen, porcine collagen, ovine collagen, equine collagen, synthetic collagen, agar, synthetic gelatin, and combinations of these. It is note that for covalent, preferably click-chemistry modifications, comparatively short and small components may be employed.
- Examples of useful synthetic polymers formed by chemical cross-linking include polyethylene glycol (hereinafter, "PEG”), polypropylene glycol (hereinafter, “PPG”), polyvinyl alcohol (hereinafter, "PVA”), polyacrylic acid, hydroxyethyl acrylate, polyhydroxy ethyl methacrylate, polyvinyl pyrrolidone, carboxymethyl cellulose, dextrans or other sugar- based polymers; homopolymers or (block) copolymer or the water-soluble polymer of the water-soluble polymer selected from the group consisting of hydroxymethyl cellulose and hydroxyethyl cellulose, a- hydroxy acids, the a- hydroxy acid cyclic dimer, a block copolymer of a monomer selected from the group consisting of hydroxy dicarboxylic acids and cyclic esters may be employed.
- PEG polyethylene glycol
- PPG polypropylene glycol
- PVA polyvinyl alcohol
- PEG polyethylene glyco
- Partially water-soluble polymers may be used, wherein typically biocompatibility is higher than for hydrophobic polymers. Also, due to the presence of hydroxyl groups, PEG, PPG, PVA, poly hydroxyethyl acrylate, poly hydroxyethyl methacrylate (hereinafter, "poly-HEMA”) permits easy functionalization.
- poly-HEMA poly hydroxyethyl methacrylate
- the secondary components are in any case preferably storage stable, and susceptible to nucleophilic substitution reactions, which may be used to functionalize the particles controlled manner to provide the guest moiety for inclusion complexes.
- the latter are preferably covalently bound, preferably at the particle surface, as appropriate, depending on the activation chemistry selected.
- the weight average molecular weight of useful polymers is preferably 200 or more. Further, for a discharge ex vivo to be facilitated by the living body, it is preferably 50,000 or less.
- the weight average molecular weig of the vector component may be suitably selected such that the desired pharmacokinetics and biological half-life are achieved.
- the weight average molecular weight of the polymers employed may be advantageously determined by gel permeation chromatography.
- compositions and the molecular weight depends also on the clearance from the body. Rapid and complete clearance of non-bound secondary components is critical. For example resorption of a radioisotopes in the kidneys may result in kidney failure as side effect from therapy, and hence fast clearance is desired.
- Useful secondary components may include bio-resorbable components, which can be broken down by the metabolism, and excreted in a suitable time period to be removed alongside with the embolized tissue, depending on the desired duration of the vector component in the tissue.
- microparticles it is necessary to obtain the desired number of microparticles to be injected into the vascular system and/or tissue of a patient, in order to achieve a sufficiently high amount of diagnostic and/or therapeutic agents to be present. While the number of particles which may be injected or otherwise delivered will vary depending upon the patient's blood pressure, blood flow rate, metabolism, body weight, organ weight, etc., it has been found that approximately 200 to 2,500,000 or more particles suspended in a biologically safe solution and delivered to the patient may suffice, although larger quantities of particles may be necessary and desirable depending on various circumstances and conditions of use, e.g., tumour size, etc.
- the pre-targeting vector microparticle material is associated with at least one host moiety, while the secondary vector material is associated with at least one guest moiety.
- the pre-targeting vector is covalently linked to a host molecule, in which case the guest-modified pre-targeting vector comprises one or more host functional groups.
- such a pre-targeting vector has been linked to a first guest molecule and thereby comprises one or more host functional groups, is also called the " host -modified micro-particle" or a "pre-target vector”.
- the secondary vector material is also linked to an at least one guest moiety, thus forming the "guest -modified secondary vector"
- the modification of the vector molecules to comprise the host or guest moiety may be done by any suitable method that allows to link the gust moiety to the vector surface.
- the linking of the guest or host molecule to the vector or secondary component is preferably done by conventional conjugation techniques known to the person skilled in the art.
- This guest molecule which is part of the guest-modified microparticle, then binds (non- )covalently to a host functional group residing on a preferably multivalent host structure.
- this interaction takes place by a one-to-one interaction between a guest functional group in the guest molecule and a host functional group in the host structure.
- a higher load of guest molecules may result in a better complexation ratio, while too high of a load may prevent interactions by sterical hindrance.
- multivalent guests are preferred for non-covalent interactions, which may comprise multiple binding sites in one molecule, or microparticles that comprise separately multiple binding sites. The same applies mutatis mutandis to the host molecule, and the host-modified secondary vector.
- guest and host refer to two different, but complementary, binding partners that non-covalently, or covalently interact with each other.
- guest moiety or “group” means the part or moiety of a monomer of the guest molecule, which enables the binding to a complementary host functional group.
- a “guest molecule” is in turn a molecule that comprises one or more guest functional groups, where a monovalent guest molecule comprises one guest functional group and a multivalent guest molecule comprises at least two guest functional groups.
- the term "host functional group” means the part or moiety of a monomer of the host molecule, which enables the binding to a complementary guest functional group.
- a "host molecule” is in turn a molecule that comprises one or more host functional groups, where a monovalent host molecule comprises one host functional group and a multivalent host molecule comprises at least two host functional groups.
- one guest moiety interacts with one host moiety.
- non-covalent host-guest pairs the interaction between the guest and the host may be reversible, and is determined by the affinity strength as expressed by dissociation constants.
- a guest molecule does not normally interact with another guest molecule.
- non-covalent guest-host interaction include beta-cyclodextrin- adamantane, beta-cyclodextrin-ferrocene, gamma-cyclodextrin-pyrene, cucurbituril- viologen, and/or a Ni(NTA)-His tag.
- covalent interaction include an Azide (N 3 )- alkyne interaction as a host-guest interaction.
- DBCO dibenzocyclooctyne group
- BCN dibenzocyclooctyne group
- DBCO groups will preferentially and spontaneously label molecules containing azide groups (-N 3 ).
- the DBCO group does not react with amines or hydroxyls naturally present in many biomolecules.
- the reaction of the DBCO group with the azide group is significantly faster than with sulfhydryl groups, making this a highly selective reaction.
- inclusion complex refers to any material wherein the host compound absorbs or embeds a guest compound to form a complex.
- the guest compound may be embedded in a cavity formed by the host compound.
- inclusion material is used as synonyms in this application.
- the guest-host components are each readily available, and enable to perform the methods of the present invention on a relatively large scale and/or in low cost devices.
- guest-host molecule interactions includes the non- covalent binding between respective guest and host functional groups.
- hydrophobic interactions such as lipophilic interactions, are being used instead of interactions that are based on charge.
- the functionality may be reversed, i.e. using the host molecule as the molecule that is linked to the vector, but as used herein, the host molecule or host structure is not linked to the vector directly, primarily via the guest molecule.
- the vector particle preferably may comprise a multivalent host structure, comprising multiple host functional groups, some of the host functional groups may be (non-) covalently bound to a guest molecule, while others remain free.
- Preferred, essentially non-covalent host compounds are cyclodextrins, with adamantane moieties acting as host molecules.
- Cyclodextrins are cyclic polysaccharides containing naturally occurring D(+)-glucopyranose units in an a-(l,4) linkage.
- the most common cyclodextrins are alpha (a)-cyclodextrins, beta )-cyclodextrins and gamma (y)- cyclodextrins which contain, respectively, six, seven or eight glucopyranose units.
- the cyclic nature of a cyclodextrin forms a torus or donut-like shape having an inner apolar or hydrophobic cavity, the secondary hydroxyl groups situated on one side of the cyclodextrin torus and the primary hydroxyl groups situated on the other.
- beta- cyclodextrin is used, which is the best binding partner for adamantane.
- the cyclodextrin may contain additional groups, such as an amine to attach it to a scaffold, one or more thiols to bind the cyclodextrin to a gold surface, or hydroxypropyl groups to increase solubility and biocompatibility.
- cyclodextrin family most likely alpha and gamma
- Other members of the cyclodextrin family can also be used for host-guest interaction, although different guests have to be introduced to achieve this. Accordingly, a good, but not the only, example of supramolecular host-guest interactions that can be applied in the invention is the non-covalent interaction between adamantane (as the guest molecule) and ⁇ -cyclodextrin (as the host molecule).
- the side on which the secondary hydroxyl groups are located has a wider diameter than the side on which the primary hydroxyl groups are located.
- the hydrophobic nature of the cyclodextrin inner cavity allows for the inclusion of a variety of compounds. (Comprehensive Supramolecular Chemistry, Volume 3, J.L. Atwood et al., eds., Pergamon Press (1996); T. Cserhati, Analytical Biochemistry, 225:328-332 (1995); Husain et al., Applied Spectroscopy, 46:652-658 (1992); F 2 665 169).
- Various cyclodextrin containing polymers and methods of their preparation are also known in the art.
- Cyclodextrin-based polymers have been used for therapeutic applications (Kandoth et al. Two-photon fluorescence imaging and bimodal phototherapy of epidermal cancer cells with biocompatible self-assembled polymer nanoparticles. Biomacromolecules 2014 (15):1768-1776) and imaging agents (Yan et al. Poly beta-cyclodextrin inclusion- induced formation of two-photon fluorescent nanomicelles for biomedical imaging. Chemical Communications 2014 (50):8398-8401) and they showed excellent biocompatibility.
- Adamantane is a lipophilic small molecule that may be attached to a vector, or conjugated to a microparticle that can be physically lodged in the target tissue.
- the adamantane structure combines rigidity with the ability to form diamondoid structures, and offers high binding affinities with cyclodextrins.
- the guest molecule may be adamantane, whereas the host molecule is preferably then a cyclodextrin that non-covalently interacts with adamantane. Both compounds are relatively cheap and easy to produce in controlled settings and non-toxic.
- the guest moiety may be connected to the microparticle by a "scaffold", i.e. a binding unit comprising a spacer molecule or otherwise suitable molecular structure.
- the scaffold may be intended to ensure that the guest moiety is presented to the incoming host moiety, and/or may permit to modify the microparticle easily.
- several guest moieties or guest functional groups may be interconnected through a scaffold molecule to form a multivalent guest structure.
- multivalent refers to a number of host or guest molecules, or functional groups thereof, that are part of the same molecule or structure. Multivalent interactions contain at least two functional groups of the same type (e.g. at least two guest functional groups, or at least two host functional groups) bound to each other through a backbone (or scaffold) that allows the multimerization of the matching guest or host molecule.
- the upper limit in multivalency depends on the purpose of the layer-by-layer manufacturing of guest-host molecule interactions, as disclosed herein. Without wishing to be bound to any particular theory, it is believed that multivalency enhances the affinity, and thus improves the binding, as monomeric guest molecules tend to show a significantly lower non-covalent interaction.
- a “multivalent host structure” or a “multivalent guest structure” is a structure comprising at least two host functional groups or guest functional groups, respectively. It can in principle be a dimer or polymer of suitable host or guest monomers, but typically the host or guest molecules have been attached or engrafted onto a polymer of a different type that allows for the attachment of the host or guest molecules. This is preferably used for non-covalent interactions, thereby increasing the binding strength.
- a "multivalent host structure” or “multivalent guest structure” preferably comprises a scaffold or linker structure onto which the at least two host molecules or at least two guest molecules have been attached or engrafted resulting in that the scaffold structure comprises at least two host functional groups or at least two guest functional groups.
- the scaffold structure can be anything that allows attachment of the host or guest molecules of choice.
- the scaffold structure may of course also be part of, or integrally form the microparticle.
- the scaffold molecule is a polypeptide comprising less than about 30, such as, e.g. less than about 25, less than about 20, less than about 15, less than about 10, less than about 5 or less than about 6 amino acids.
- oligo peptide such as, e.g. a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
- the repeat unit of the poly or oligepeptide is ⁇ -alanine.
- Suitable host components are components that are modified to selectively link up or react with the pre-targeting vector comprising the guest moiety.
- the selectivity of this linkage preferably allows to introduce the host component intravenously, rather than into the tissue, as the host component then automatically binds to the vector.
- a moiety with the worst pharmacokinetics is defined as guest and will be part of the pre-targeting microparticles. This means its influence on the targeting is minimal.
- the complementary host is then the moiety with the most favourable pharmacokinetics e.g. the least non-specific interactions.
- the secondary, host component may for instance be simply introduced into the blood stream, as it will bind selectively to the guest once encountered.
- a dosage for particular treatment not at the reactor, as presently done for each treatment separately, but simply by calculating and administrating the amount of suitably modified host components which can be done at a patient treatment site, e.g. a hospital pharmacy.
- the host structure or secondary component thus may comprise a targeting moiety or molecule coupled to the particle, and the targeting moiety that in this manner helps deliver the bioactive agent and/or the imaging agent to a desired, i.e. vector location in a patient.
- An exemplary method for preparing a host structure may include providing host nanoparticles comprising a pharmaceutically acceptable polymer and coupling, e.g., by coating, covalent linkage, or co-localization, to the surface of the nanoparticles the host moiety for coupling to the guest, and an imaging agent, a detectable label, or otherwise targeting moiety.
- the method may further include one or more of forming a particle suspension, passing the particle suspension through a filter, removing impurities from the particle suspension, centrifugation to pellet the particles, dialyzing the particle suspension, and/or adjusting the pH of the particle suspension.
- the method may also include quenching the covalent linking reaction.
- Suitable host components may include organic or inorganics components or mixtures thereof.
- the host components can be selected from monomers- polymers and nanoparticles.
- Suitable polymers for example can be selected from poly(isobutylene-alt-maleic anhydride) (PIBMA), PAMAM, poly-acrylic acid, polysaccharides, polypeptides and oligopeptides.
- PIBMA poly(isobutylene-alt-maleic anhydride)
- PAMAM poly-acrylic acid
- polysaccharides polypeptides and oligopeptides.
- Some such polymers, such a PIBMA has the further advantage that it prevents interaction with the immune system and thereby also functions as a cloaking group.
- the selected structure is non-toxic.
- eliciting of an immune response may be a means to induce a therapeutic effect.
- Suitable polymeric host components may comprise a pharmaceutically acceptable polymer core and a one or more bioactive agents and/or imaging agents.
- the secondary component may comprise a pharmaceutically acceptable polymer core, and one or more bioactive agents, such as a drug or medicament encapsulated in the core.
- the complementary component comprises multiple host moieties, thereby increasing the affinity for the guest functional groups of the guest- modified vector.
- the host structure according to the present invention is multivalent, i.e. comprises at least two host functional groups.
- the first multivalent host structure comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9 or at least 10 host functional groups.
- the host comprises a scaffold structure, such as a polymer comprising as least about 5, such as at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35 or at least about 40 monomer repeat units.
- a preferred second component vector molecule may comprise multiple host sites, to generate multivalent structures containing either the host functional group of cyclodextrin adamantane.
- a preferred example is poly-isobutylene-alt-maleic anhydride (PIBMA), which can be easily functionalized, is non-toxic, and presents a multitude of negative charges after functionalization which increases water-solubility and provide cloaking.
- PIBMA poly-isobutylene-alt-maleic anhydride
- PAMAM polymers containing anhydrides
- nanoparticles e.g. quantum dots
- poly- acrylic acid polysaccharides
- a benefit of the subject process is that various compounds may be employed as diagnostic or therapeutic labels, and may include chelates, a diagnostic, a therapeutic agent, or any combination thereof, using a simple pre-targeting platform.
- the second, complementary component may comprise a therapeutic agent or an imaging agent, or combinations thereof, e.g., it may include a diagnostic agent and/or a detectable label.
- Immunogenic Agents refers to a substance which is capable, under appropriate conditions, of inducing a specific immune response and of reacting with the products of that response (e.g., a specific antibody, specifically sensitized T- lymphocytes or both), while the term “immunogenic” relates to a reaction triggered by the presence of the immunogen.
- Both the pre-targeting vector component and the secondary component may also carry various labels or agents other than host or guest moieties.
- the one or more bioactive agents are covalently or non-covalently associated with the secondary component.
- a nucleic acid or protein included in the particle may comprise an imaging agent itself, e.g., a detectable label that may be attached to DNA or a protein.
- the secondary component may comprise an imaging agent that is separate from a nucleic acid or a protein, e.g., encapsulated in the core or disposed on or coupled to the surface.
- the component may comprise one or more targeting moieties or molecules coupled to the component and/or the protein or nucleic acid, and the targeting moiety can help deliver the nucleic acid, the protein, and/or the therapeutic, imaging, and/or diagnostic agent.
- the component may comprise an imaging agent, e.g., a diagnostic agent and/or a detectable label.
- the optionally encapsulated bioactive agent may be or comprise an imaging agent itself, e.g., a detectable label may be attached to a therapeutic agent.
- the particle may comprise an imaging agent that is separate from the bioactive agent.
- a plurality of radioisotopes, bioactive agents, such as bioactive agents or contrast agents may be loaded onto a single host component, or spread over the guest and host component.
- the potentially radioactive isotope may be an isotope possessing a high absorption cross-section to neutrons, protons, electrons, or high energy photons.
- the potentially radioactive isotope has a high absorption cross-section to neutrons, and is selected from the group consisting of 10 B, 149 SAM, 157 Gd, and 155 Gd or any combination thereof, thereby providing a target area for fast neutron therapy.
- isotopes may also be conjugated to the complementary compound, e.g. allowing the use of 177 Lu , 99m Tc , m ln isotopes, or combination thereof, for instance.
- a magnetic resonance contrast agent may be employed, as for instance those selected from Manganese Oxide, perfluorocarbons, Feridex, Gadolinium, Combidex, Bang Magnetic Particles, Gd-DTPA, Gadolinium And Manganese Derivatives, Superparamagnetic Iron Oxide Particles, gadopentetate dimeglumine, Gd- DOTA, Gd-DTPA- BMA, Gd-HP-D03A, Gd-DTPA-BMEA, Gd-D03A-butrol, Gd-BOPTA, Mn-DPDP, Gd-EOB-DTPA, Gd-BOPTA, AMI-25, SH U 555A, gadoflourine-M, AMI-227, EP-2104R, P947, Gd-DTPA mesophorphryn, SH U 555 C, NC- 100150, MS-325, gadoflourine-M, gadomelitolm manganese chloride, ferric amon
- a negative contrast agent may be employed, as for instance those based on superparamagnetic iron oxides: SPIO (Superparamagnetic Iron Oxide), typically employed for liver tropism, and USPIO (Ultrasmall Superparamagnetic Iron Oxide) typically for ganglion tropism.
- SPIO Superparamagnetic Iron Oxide
- USPIO Ultrasmall Superparamagnetic Iron Oxide
- a contrast agent that can act as a Raman spectroscopy reporter, thereby allowing detection of the component with Raman spectroscopy.
- a contrast agent that can act as a fluorescence spectroscopy reporter, thereby allowing detection of the component with fluorescence spectroscopy.
- the agent may comprise a photoacoustic contrast agent, thereby allowing detection of the component acoustically.
- bioactive agents may be attached to the complementary component, as well as compounds that enhance the interaction with other cells e.g. immune cells, thereby triggering for instance an enhanced and targeted immune response.
- Bioactive agents according to the invention include but are not limited to a nucleic acid, DNA (e.g., a gene therapy vector or plasmid), an RNA (e.g., an mRNA, the transcript of an RNAi construct, or an siRNA), a small molecule, a peptidomimetic, a protein, peptide, lipid, surfactant and combinations thereof.
- RNA e.g., an mRNA, the transcript of an RNAi construct, or an siRNA
- therapeutic agents include but are not limited to a nucleic acid, a nucleic acid analog, a small molecule, a peptidomimetic, a protein, peptide, lipid, or surfactant, and combinations thereof.
- the imaging agent further comprises a detectable label.
- combinations of the above agents may be present, either on a single secondary component, or a mixture of differently functionalised secondary components.
- the vector components according to the present invention are administered locally, parenterally and/or topically, i.e. they are injected or infused into a particular area, and/or into a particular organ or tissue in a patient body.
- Examples include intrahepatic injections or infusions, intrapancreatic injections, or functionally, generally, intratumoral injections; intradermal and/or peritumoral injections or infusions.
- the components, in particular the pre- targeting vector are therefore chosen and designed in among other factors size, size distribution, compressibility, water content, flowability, deformation creep and/or stability, as well as optimal pharmacokinetics, e.g. rapid clearance and minimal background of non- complexed components, such that they can be infused or injected, and that they are dimensioned and functionalised to show minimal movement once they are distributed inside the relevant organ, with minimal or no shunting or otherwise bypassing occurring.
- the delivery of the vector material via injection or implantation provides a means to effectively mechanically target the biomaterial to a specific site or location, thereby localizing the biomaterial and minimizing systemic side effects.
- the administration of the vector material via implant or needle based injection is minimally invasive and usually can be performed on an outpatient basis, resulting in a lower cost than other surgical forms.
- "delivering” comprises positioning a delivery device, e.g. a syringe or infusion catheter in proximity to a target region of a blood vessel, or directly into a target tissue not via vasculature, and ejecting the microparticles from the delivery device such that the particles are positioned in the target region.
- a delivery device e.g. a syringe or infusion catheter in proximity to a target region of a blood vessel, or directly into a target tissue not via vasculature
- the complementary secondary vector components preferably are administered intravenously, or more generally intravascularly, since the components are accumulated at the pre-targeting vector component location, or likely excreted if not bound to the vector. This permits to reduce the number of steps in a treatment and diagnosis, which reduces the negative effect on a patient.
- the complementary secondary component may be injected locally, e.g. by inserting a cannula into the desired region, and injecting the material into the vicinity of the vector component.
- Administration Medium e.g. by inserting a cannula into the desired region, and injecting the material into the vicinity of the vector component.
- compositions according to the present invention are particularly useful for embolizing liver tissue.
- the microparticles can be used as component dispersed in an appropriate dispersion medium.
- the administration medium of the components may be a buffer/ serum solution, and may further comprise injection dispersing agent such as polyoxyethylene sorbitan fatty acid ester or carboxymethyl cellulose, such as methyl paraben or propyl paraben preservatives, sodium chloride, preservatives used in tonicity agents or injections, such as mannitol or glucose, stabilizer, solubilizing agents or excipients.
- injection dispersing agent such as polyoxyethylene sorbitan fatty acid ester or carboxymethyl cellulose, such as methyl paraben or propyl paraben preservatives, sodium chloride, preservatives used in tonicity agents or injections, such as mannitol or glucose, stabilizer, solubilizing agents or excipients.
- the present invention thus preferably relates to a complementary component in vivo parenteral topical administration and for forming the selective non-covalent high affinity interaction with a complementary host moiety, and/or the selective covalent bond with a host compound having a complementary functionality of the vector component according to the invention; wherein the component comprises a complementary guest functionality, and at least a first diagnostic and/or therapeutic agent.
- the agent is selected from the group consisting of: a diagnostic agent, an imaging agent, a contrast agent, and a therapeutic agent, preferably a radioactive isotope or a chemotherapeutic agent.
- the agent is selected from the group consisting of one or more: anticancer agents, antibiotics, antihistamines, hormones, steroids, therapeutic proteins, biocompatible materials, imaging agents and contrast agents.
- the diagnostic agent is selected from the group consisting of magnetic resonance contrast agents, radioopaque contrast agents, ultrasound contrast agents, and nuclear medicine imaging contrast agents, more preferably, wherein the radioactive isotope is selected from the group consisting of: Y-90, P-32, P-33, Sc- 47, Cu-64, Cu-67, As-77, Ph-105, Pd-109, Ag-111, Pr-143, Sm-153, Tb-161, Ho-166, Lu-177, Re-186, Re- 188, Re-189, lr-194, Au-199, Pb-212, 1-131, Ac-225, Ra-223, Ra-224, and Bi-213.
- the radioactive isotope is selected from the group consisting of: Y-90, P-32, P-33, Sc- 47, Cu-64, Cu-67, As-77, Ph-105, Pd-109, Ag-111, Pr-143, Sm-153, Tb-161, Ho-166, Lu-177, Re-186, Re- 188
- the present invention also relates to a kit for forming an optionally bioresorbable diagnostic and/or therapeutic occlusion at a target site within a body, the kit comprising:
- the complementary component is functionalised for selectively attaching to the vector component in vivo.
- the complementary component comprises a therapeutic agent for delivery to the targeted target area.
- the present invention also relates to a method, and to a compound or kit of compounds for use in treating a condition in vivo in a patient, the method comprising the steps of: (a) providing an administration medium comprising microparticles of a pre-targeting vector component, and (b) delivering a diagnostically and/or therapeutically effective amount of the a pre-targeting vector component into the target tissue, preferably into the tumour or tumour bearing tissue patient; and (c) subjecting the target tissue to a diagnosis step to determine if significant shunting occurs; and (d) administering a therapeutically effective amount of a complementary component comprising a therapeutic agent to the patient, thereby producing a therapeutically active matrix in the subject tissue.
- (b) comprises delivering the pre-targeting vector in a target region of a blood vessel.
- "delivering” further comprises positioning a delivery device in proximity to a target region of the blood vessel, and ejecting the pre-targeting vector particle(s) from the delivery device such that the particles are positioned in the target region.
- the target area is selected from one or more of liver, pancreas, thyroid, heart, peripheral nerve scaffold, breast, bladder, cartilage, bone, tendon, ligament, blood vessel, skin, lymph nodes, spinal cord, and/or tumour tissue
- the present invention also relates to a method, and compound of use for topically treating a disease, comprising administering to a target area of a patient in need thereof a kit according to the invention suitable for diagnosing and treating the disease.
- a kit according to the invention suitable for diagnosing and treating the disease.
- a particularly useful example is employed as a vector component, i.e. a host- modified Technetium 99 m Tc macro aggregated albumin (99 m Tc-MAA), in an injectable form, comprising a sterile aqueous suspension of Technetium-99 m ( 99m Tc) labelled to human albumin aggregate particles.
- 99m Tc-MAA a host- modified Technetium 99 m Tc macro aggregated albumin
- MAA scintigraphy may be used to assess lung or digestive shunting prior to therapy, based on tumoural targeting and dosimetry. Computed tomography (CT) may then be performed during the angiography to aid in digestive vascularization recognition, as well as perfused tissue and tumour targeting evaluation.
- CT Computed tomography
- MAA scintigraphy may not only be used for lung-shunt evaluation, but can also aid in digestive shunt recognition and dosimetric evaluation and the marking of diseased tissue for radio-occult lesion localization.
- Cy5-(S03)Sulfonate-(S03)CO-TFP was synthesized according a procedure previously described in S. J. Spa, A. Bunschoten, M. T. M. Rood, R. J. B. Peters, A. J. Koster and F. W. B. van Leeuwen, Eur. J. Inorg. Chem., 2015, 2015, 4603-4610.
- Cy5-(S03)Sulfonate-(S03)Amine was synthesized according to the method disclosed in M.T. Rood, S. J. Spa, M. M. Welling, J. B. Ten Hove, D. M. van Willigen, T. Buckle, A. H. Velders and F. W. B. van Leeuwen, Sci. Rep., 2017, 7, 39908.
- the polymer was first dialyzed against H 2 0 for 1 day, then against 100 mM phosphate buffer pH 9.0 for 24 h, and subsequently against H2O for another 5 days, while refreshing the dialysis medium daily.
- the solution was lyophilized to give a blue powder (87 mg, 5 ⁇ ) and was stored at -20°C. Before usage, a small amount was aliquoted in PBS at 1 mg/mL concentration and stored ( ⁇ one month) at 7°C.
- 1 H NMR see previous reported literature.
- Macro-aggregates from albumin (TechneScan ® , MAA) were obtained from Mallinckrodt Medical B.V., Petten, The Netherlands. Lyophilized albumin macro-aggregates (2 mg) were dissolved in 1 mL of saline (0.9% NaCI, sterile and pyrogen-free, B. Braun Medical Supplies, Inc., Oss, The Netherlands) and portions of 0.1 mL (containing 0.2 mg MAA) were stored in Eppendorf tubes at -20 °C until further use. For functionalization, one portion was defrosted and 20 SL of Ad-TFP (10 mg/mL DMSO) was added.
- the solution was washed 2 times with phosphate buffered saline (PBS) by 2 centrifugation steps (3 min x 1,200 rpm).
- PBS phosphate buffered saline
- the obtained MAA-Ad was diluted in 1 mL of PBS to 0.2 mg/ml.
- the MAA functionalization was also performed with Cy5-TFP according the same procedure as with Ad-TFP. Subsequently, the absorbance at 650 nM of the MAA-Cy5 constructs was measured using a NanoDrop Spectrophotometer (Thermo Fisher Scientific Inc. Wilmington, DE, USA).
- the number of Cy5/MAA aggregates was then ca lculated by dividing the calculated Cy5 concentration by the known MAA concentration, resulting in a ratio of 3.07( ⁇ 0.24) x 10 8 Cy5/MAA particle on average.
- Ad-TFP reacts in a similar fashion as Cy5-TFP, it was estimated that the ratio of Ad/MAA would be in the same order of magnitude.
- Radiolabeling of Cy5o.5CDi 0 PIBMA 3 9 was performed as follows: to 10 0L of Cy5o.sCDioPIBMA 3 9 (1 mg/mL PBS), 4 ⁇ of SnCl2.2H 2 0 (0.44 mg/mL saline, Technescan PYP, Mallinckrodt Medical B.V.), and 100 ⁇ of a freshly eluted 99m Tc-Na-pertechnetate solution (500 MBq/mL, Mallinckrodt Medical B.V.) were added and the mixture was gently stirred in a shaking water bath for 1 h at 37 °C, as described in M. M. Welling, A. Paulusma-Annema, H. S.
- mice All in vivo studies were performed using 2-3 month old Swiss mice (20-25 g, CrhOFl strain, Charles River Laboratories, USA). All animal studies were approved by the institutional Animal Ethics Committee (DEC permit 12160) of the Leiden University Medical Center. All mice were kept under specific pathogen-free conditions in the animal housing facility of the LUMC. Food and water were given ad libitum.
- SPECT imaging was performed as follows: at 2 h after injection of 99m Tc-labeled compounds mice were placed and fixed onto a dedicated positioned bed of a three-headed U-SPECT-2 (MILabs, Utrecht, The Netherlands) under continuous 1-2% isoflurane anesthesia. Radioactivity counts from total body scans or selected regions of interest (ROI) were acquired for 60 min using a 0.6 mm mouse multi-pinhole collimator in list mode data. For reconstruction from list mode data, the photo peak energy window was centered at 140 keV with a window width of 20%. Side windows of 5% were applied to correct for scatter and down scatter corrections.
- ROI selected regions of interest
- the image was reconstructed using 24 Pixel based Ordered Subset Expectation Maximization iterations (POSEM) with 4 subsets, 0.2 mm isotropic voxel size and with decay and triple energy scatter correction integrated into the reconstruction with a post filter setting of 0.25 mm, as described in W. Branderhorst, B. Vastenhouw and F. J. Beekman, Phys. Med. Biol., 2010, 55, 2023-2034.
- Volume-rendered images were generated from 2-4 mm slices and analyzed using Matlab R2014a software (version 8.3.0.532, MathWorks 8 Natick, MA). Images were generated from maximum intensity protocols (MIP) adjusting the color scale threshold to optimal depiction of the tissues of interest, as set out in M. N.
- MIP maximum intensity protocols
- mice were euthanized by an intraperitoneal injection of 0.25 mL Euthasol (ASTfarma, Oudewater, The Netherlands).
- organs were collected from mice and counted for radioactivity in a dose-calibrator (VDC 101, Veenstra Instruments, Joure, the Netherlands) or a gamma counter (Wizard2 2470 automatic gamma scintillation counter, Perkin Elmer). After collecting and counting all tissues together with the remaining activity in the carcass, the total amount of remaining radioactivity in the animal was counted and, after correction for physical decay, the urinary excretion expressed as the percentage of the total injected dose (%ID) was calculated. Radioactivity counts in tissues were expressed as the percentage of the total injected dose of radioactivity per gram tissue (%ID/g).
- Fig. 1A An embolization setup in the liver was performed to mimic the clinical setup for liver radio- embolization (Fig. 1A, Model II).
- animals were anesthetized by intraperitoneal injection of a mixture containing Hypnorm (Vetapharma, Leeds, United Kingdom), dormicum (Roche, Basel, Switzerland), and water (1:1:2).
- Hypnorm Vetapharma, Leeds, United Kingdom
- dormicum Roche, Basel, Switzerland
- water 1:1:2
- the abdominal cavity was incised for 0.5 cm and the spleen was exposed outside the mouse.
- Figure 1 shows the SPECT imaging of (Model I, comparison without pre-targeting ) 99m Tc-labeled Ad-MAA (for mapping biodistribution of MAA-Ad) or (Model II, according to the invention) pre-targeting with unlabeled MAA-Ad or (Model C) none followed by intravenous 9 m Tc-Cy5o.5CDioPIBMA 3 9.
- SPECT imaging is performed 2 h thereafter to visualize radioactivity in the (1) lungs, (2) liver, (3) kidneys, (4) stomach, and (5) urinary bladder.
- Figure 2 then illustrates the binding of 99m Tc-Cy5o.5CDi 0 PIBMA 3 9 to MAA-Ad and MAA, quantified by radioactivity and expressed as a percentage of the total amount of radioactivity ( 99m Tc-Cy5o.sCDioPIBMA 3 9) added.
- the data in Figure 2 indicates a 5.7 times higher binding to MAA functionalized with the Ad guest moiety, as compared to non-functionalized MAA. Significance of difference is marked with ** (P ⁇ 0.01).
- Table 1 shows the resultant measurements
- Table 1 shows the bio-distribution of 99m Tc-Cy5o.5CDioPIBMA39 following injection of:
- the data is expressed as the mean + SD of the percentage of the injected dose per gram tissue (%ID/g) of 5 observations)), and is calculated from radioactivity counts in various tissues at 2 h post-injection of the tracer.
- Table 2 shows the biodistribution of 99m Tc-labeled cyclodextrin polymers in mice with adamantane (Ad)-functionalized macro- aggregates (MAA) administrated into the lungs.
- Data (expressed as the mean ⁇ sd of the percentage of the injected dose per gram tissue of at least 4 observations) are calculated from radioactivity counts in various tissues (%ID/g) of various tissues at 2 hr post-injection of the tracer.
- * P ⁇ 0.05 compared to none or non-functionalized MAA.
- Example 2 was repeated, however using polystyrene beads as pre-targeting vector material.
- Cy5-Polystyrene-Ad(PS Ad) was prepared, and incubated with Cy31.5CD72PIBMA389.
- the Polystyrene particles were visualized by excitation at 633 nm and the emission was collected at 650 to 600 nm.
- the Cy31.5CD72PIBMA398 was visualized by excitation at 514 nm laser and the signal was collected at 550- 600 nm.
- the MAA was then resuspended in 100 ⁇ Phosphate buffer (0.1 M, pH 8.4) and 5.7 ⁇ (28 ⁇ g, 40 nmol) of a Cy5-(S03)azide-(S03)COOH solution in H20 (5 mg/mL) was added. After stirring for 2h at RT the particles were washed again by pelleting (3 min, 13.4 rcf) until the filtrate was no longer blue.
- the functionalized MAA was resuspended in 100 ⁇ H20 and a drop of the solution was added to a petri dish. After the particles were settled down they were imaged using a SP5 Leica microscope.
- Cy5o, 5 CD9PIBMA39 with indium-Ill m ln-Cy5o, 5 CD9PIBMA39
- the host-vector, Cy5o.sCDgPIBMA39 which contains an abundance of freely available -COOH moieties, was radiolabeled with indium-Ill.
- Cy5o.5CD 9 PIBMA 3 9 (1 mg/mL PBS) was added 25-150 ⁇ of an acidic solution of m lnCI 3 (370 MBq/mL, Mallinckrodt Medical, Petten, The Netherlands). This mixture was gently shaken in the dark for 1 h at 37 °C. Thereafter, the pH was adjusted to 7.5 in PBS.
- the radiochemical purity of m ln-Cy5o.5CD9PIBMA39 was determined at 1 and 20 h by instant thin layer chromatography (ITLC) on 1x7 cm ITLC-SG paper strips (Agilent Technologies, USA) with 0.25 M NH 4 -acetate (pH 5) as mobile phase.
- ITLC instant thin layer chromatography
- FCS fetal calf serum
- FCS fetal calf serum
- the decay corrected radioactivity of the pellet and supernatant was measured in a dose-calibrator. Following correction for background activity, the host-guest interaction was expressed as the percentage of the total amount of radioactivity (% binding).
- % binding For stability measurements, either 0.1 mL MAA-Ad (0.1 mg/mL) with 0.1 mL m ln- Cy5o.
- the supramolecular host-guest interactions in the [ 99m Tc-MAA-Ad- m ln-Cy5o.5CD9PIBMA 3 9] complex can be monitored in vivo using dual-isotope multiplexing up to 20 h after administration due to the physical short half-life of technetium-99m.
- In vivo studies were performed using 2-4-month-old Swiss mice (20-35 g, CrhOFl strain, Charles River Laboratories, USA). All animal studies were approved by the institutional Animal Ethics Committee (DEC permit 12160) of the Leiden University Medical Center. Mice were kept under specific pathogen-free conditions in the animal housing facility of the LUMC. Food and water were provided ad libitum.
- liver radioembolization setup of the liver was used to mimic the clinical setup for liver radioembolization according to previously described procedures (pa SJ, Welling MM, van Oosterom MN, Rietbergen DDD, Burgmans MC, Verboom W, et al. A Supramolecular approach for liver radioembolization. Theranostics. 2018;8(9):2377-86 and Kasuya H, Kuruppu DK, Donahue JM, Choi EW, Kawasaki H, Tanabe KK. Mouse models of subcutaneous spleen reservoir for multiple portal venous injections to treat liver malignancies. Cancer Res. 2005;65(9):3823-7).
- ROI's drawn over the jugular veins were taken as a representative of the blood values. After imaging, mice were euthanized and the organs were removed and weighed to determine the percentage of injected dose per gram tissue (%ID/g). Blood samples obtained at various intervals of sacrifice, were used to determine the clearance from the blood fraction (expressed as the pharmacological half-life ti/ 2 ) was calculated using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego CA, USA). Dual-isotope SPECT imaging facilitated in vivo monitoring of host-guest interactions between m ln-Cy5o. 5 CD 9 PIBMA39 and 99m Tc-MAA-Ad.
- Fig 9A demonstrates time-related (2, 12, 20 and 44 h) dual-isotope SPECT imaging of A) intrasplenic administration of guest-vector 99m Tc-MAA-Ad and B) I.V. administered host-vector m ln-Cy5o.5CD9PIBMA39. Organs are marked as (1) lungs, (2) liver, (3) spleen, and (4) kidneys. The scale bars indicate the intensity of radioactivity expressed as arbitrary units. At 20 h p.i.
- Fig 10 indicates the bio-distribution of pre-targeting the liver with locally administered guest vector: 99m Tc-MAA- Ad at various intervals.
- Data expressed as the mean ⁇ SD of the percentage of the injected dose per gram tissue (%ID/g) of 3 observations were calculated based on the radioactive counts measured in indicated tissues at 2, 12, and 20h post-injection. Values of 99m Tc-activity calculated at 44h p.i. were unreliable as the radioactivity counts are very low.
- Fig 9B shows the distribution of the secondary host-vector ( m ln-Cy5o.5CD9PIBMA 3 9) in mice pre-targeted with 99m Tc-MAA-Ad at 2, 12, 20 or 44 h p.i. Dual-isotope imaging was possible up to 20 h p.i.
- Fig 11 shows A) dual-isotope-SPECT imaging of mice pre-targeted with intrasplenic administration of guest vector 9m Tc-MAA-Ad at 12h p.i..
- mice pre-targeted with either non-functionalized 99m Tc-MAA or PBS are used for comparison. Organs are marked as (1) lungs, (2) liver, (3) spleen, and (4) kidneys.
- Muscle 2 4.0 ⁇ 1.9** 2.0 + 0.4 2.2 + 0.4
- Liver-to-blood ratios were determined from the biodistribution data and were compared to 99m Tc-MAA (control) and PBS (for the regular clearance of the secondary vector). At all intervals liver-to-blood ratios for mice pre-targeted with 99m Tc-MAA-Ad were highest. At 12- 20 h p.i. they were significantly (p ⁇ 0.01) higher than those pre-targeted with 99m Tc-MAA or PBS, as shown in Fig 13B: Dynamic hepatic uptake of I.V. administered m ln- Cy5o.5CDgPIBMA39.
- Data are expressed as the mean + SD ratios of the %ID/g in liver and blood measured at 2, 12, 20, and 44h post-injection of m ln-Cy5o.5CD9PIBMA 3 9.
- the significance of difference (p ⁇ 0.01) of hepatic uptake of 111 ln-Cy5o.5CDgPIBMA39 in mice pre-targeted with 99m Tc-MAA-Ad (blue bars) is indicated with * compared to 99m Tc-MAA (control clearance, red bars), or # compared to PBS (regular distribution, green bars) according to Student's T-test.
- * compared to 99m Tc-MAA control clearance, red bars
- PBS regular distribution, green bars
- liver-to-blood ratios for m ln-Cy5o.5CD9PIBMA39 are increased compared to the earlier intervals which is indicative for clearance of proteolytic or metabolic products and in Table 4: Dynamic hepatic uptake of I.V. administered m ln- Cy5o.5CDgPIBMA39.
- Data expressed as the mean ⁇ SD liver-to-blood ratios calculated between the percentages of the injected dose per gram tissue (%ID/g) of liver and blood (of 3-6 observations) based on the radioactive counts measured in these tissues at 2, 12, 20, and 44h post-injection of m ln-Cy5o.5CD9PIBMA39 after hepatic pre-targeting.
- liver-to-blood ratio between both groups of 99m Tc-MAA-Ad or 99m Tc-MAA pre-targeted mice.
- m ln-Cy5o.5CD 9 PIBMA39 rapidly assembles with 99m Tc-MAA-Ad in the liver (Table 3) Up to 20 h p.i., circulating m ln-Cy5o.5CD9PIBMA39 increased uptake in the liver to values of -27 %ID/g). Also in kidneys and spleen the values for m ln-Cy5o.5CD9PIBMA 3 9 increased in time.
- the observed intestinal uptake of m ln-isotopes is shown in Table 5 illustrate the uptake of I.V. administered m ln-Cy5o.5CD9PIBMA39 in scavenging tissues, excretion rate and clearance.
- Urine & bladder 2 5.9 ⁇ 0.8 6.2 ⁇ 0.9 7.5 ⁇ 1.5
- Thyroid gland 2 8.0 ⁇ 2.9** 6.8 ⁇ 1.7 5.8 ⁇ 0.7
- Salivary gland 2 5.8 ⁇ 2.1** 4.3 ⁇ 0.8 4.5 ⁇ 0.8
- Dual-labeled Cy5o.sCD9PIBMA39 was also equipped with a Cy5 fluorophore to perform confocal microscopy and allows to perform macroscopic and eventually microscopic evaluation of the fluorescent signal of CV50.5CD9PIBMA39 in excised tissues from mice using a preclinical S Spectrum imaging system (Caliper Life Science, Hopkinton, MA). Images of the Cy5-dye were acquired following excitation at 640 nm, and light was collected > 680 nm (acquisition time 5 s). Quantitative analysis of the fluorescence in the tissues (photons/sec/cm 2 ) was performed using the Living Image software from xenogeny v 3.2 (Caliper LS) at equal image adjustment settings.
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Abstract
The present invention provides, in general, compositions comprising a microparticle-based pre-targeting vector comprising a diagnostic and/or therapeutic functionality, and a second component that selectively binds to the pre-targeting vector component. This secondary component carries a diagnostic or preferably a therapeutic functionality, for in vivo parenteral topical administration and target tissue specific delivery. The invention also features compositions comprising secondary compounds that can be coupled with a label, for instance a diagnostic or therapeutic label and that selectively bind to the pre-targeting vector, and methods for its use in treating patient.
Description
Kit and Method for Microarterial Imaging and Radiotherapy
Field of the Invention
The present invention relates to materials and methods for that support loco-regional therapeutic treatment based on image guidance. The present invention provides, in general, compositions comprising a pre-targeting and/or diagnostic vector component, and a second component that selectively binds to the pre-targeting vector component, and is able to elicit a therapeutic response. This combination can be used for in vivo parenteral local administration and target tissue specific induction of a therapeutic response.
Background of the Invention
Hepatocellular carcinoma (HCC) is a major worldwide health problem and provides a typical medical application for the proposed local intervention strategy. It is the most common primary malignancy of the liver and the third most common cause of cancer-related mortality. The incidence of primary liver cancer is increasing in several developed countries, including the United States, and the increase will likely continue for some decades.
About 90% of patients with HCC are untreatable via surgery, or poorly responsive to chemotherapy. While externally delivered conventional radiotherapy, typically using a gamma radiation source can cause hepatic tumours to regress or even be destroyed, the dose of radiation required to destroy hepatic tumours far exceeds the tolerance of the normal liver cells immediately adjacent to the tumour. Hence, direct radiation therapy is difficult to use because of targeting and radiation dose limitations. Radio-embolization therapy provides an alternative more targeted means of selective in-situ radiation. Radio- embolization is a therapy method that is often applied for primary liver tumours and metastases that are untreatable via surgery or chemotherapy. During the therapy currently microspheres or microparticles containing therapeutic radioisotopes (v-emitters such as yttrium-90 or holmium-166) are delivered intra-hepatically where they provide localized radiation that is believed to cause damage to the diseased cells. In spite of the clinical benefits of this local therapy, difficulties in confining the microparticles to the target fragments of the liver may result in hepato-pulmonary shunting.
Shunting results in the displacement of the therapeutic effect to non-target organs, which may include highly sensitive tissue such as the lungs. This yields ineffective dose distribution and has been described as inducing seriously toxic adverse effects in a number of cases.
To predict the likely occurrence of shunting during radioembolization procedures, patients are typically subjected to a pre-treatment diagnosis, generally referred to as a scout scan. During this scan procedure, a non-toxic diagnostic marker, typically microparticulate technetium-99-labelled macro-albumin aggregates (MAA)) is applied that mimics the behaviour of the 90Yttrium therapeutic microparticles prior to treatment. The distribution of this diagnostic marker is typically documented by Single-photon emission computed tomography (SPECT), a nuclear medicine tomographic imaging technique using gamma rays, often coupled with a single-slice CT measurement (SPECT-CT). For other diagnostic markers e.g. a scout dose of holmium-166 comprising microspheres, or optionally other visualization modalities such as magnetic resonance imaging (M I), or ultrasound (US), may be applied, respectively. Besides providing a quantitative insight in the degree of shunting, the pre-interventional imaging also helps provide a dosimetric distribution model for the therapeutic isotopes, and an accurate three-dimensional visualisation of the tissues targeted.
After validation and/or optimization of the microparticle delivery using one or multiple scout scans, the therapeutic microparticles are typically applied. Post-therapy SPECT/CT, MRI, and/or positron emission tomography (PET) is then applied to relate the delivery of the therapeutic portion to the distribution defined in the scout scans.
Unfortunately here discrepancies in particle composition and pharmacokinetics between the diagnostic microparticles and their therapeutic counterparts results in discrepancies between the diagnostic and therapeutic procedures. Also, there is a time lag between the two treatments, as well as the fact that during the both treatments, a new injection has to be performed. As result, shunting still occurs during a significant portion of the therapeutic interventions (up to 25%). At that point, preventive measures no longer can be taken.
In Mark T.M. Rood et al, Obtaining control of cell surface functionalizations via Pre-targeting and Supramolecular host guest interactions' Scientific Reports, part 7, 6
January 2017 (2017-01-06), pages 39908-1 - 39908-11, XP055421250, DOI: 10.1038/srep39908 a scientific investigation is described of how membrane -receptor (pre)targeting could be combined with supramolecular host-guest interactions based on β- cyclodextrin (CD) and adamantane (Ad). In vitro testing was used to study the interaction.
To create an increased level of control a technique is needed that supports selective local response to therapy, improves the logistics of local therapy delivery and/or reduces the chance of toxic side effects.
Summary of the Invention
It is therefore an object of the present invention to provide a method of local pre-targeted therapy delivery based on microparticles.
Accordingly, in a first aspect, the present invention provides a composition for use in vivo parenterallocal administration, comprising microparticles as pre-targeting vectors.
These particles comprise one or more pendent reactive moieties that are able to form a high affinity interaction with a complementary reactive moiety residing on a therapeutic secondary component. This selective interaction can comprise of non-covalent and/or covalent bonds.
Accordingly, one aspect of the present invention relates to a complementary component in vivo parenteral topical administration and for forming the selective non-covalent high affinity interaction with a complementary host moiety, and/or the selective covalent bond with a host compound having a complementary functionality to the pre-targeting vector component according to the invention; wherein the secondary component comprises a complementary guest functionality, and at least a first diagnostic and/or therapeutic agent.
A still further aspect of the invention relates to a kit for forming a diagnostic and/or therapeutic occlusion at a target site within a body, the kit comprising: a vector component; and a complementary component functionalised for selectively attaching to the vector component in vivo.
A still further aspect of the invention relates to a method of treating diseased tissue in vivo, the method comprising the steps of: (a) providing an administration medium comprising microparticles of a pre-targeting vector component, and (b) delivering a diagnostically and/or therapeutically effective amount of the a pre-targeting vector component into the tissue of interest; and (c) subjecting the target organ/tissue to a diagnosis
step to determine if significant shunting occurs to healthy tissues; and (d) injecting intravenously or locally a therapeutically effective amount of a complementary secondary component comprising a diagnostic or preferably therapeutic agent, thereby producing a therapeutically active matrix in the subject tissue.
A still further aspect of the invention relates to a method of locally treating a disease, comprising administering to a target area of a patient in need thereof the kit suitable for diagnosing and treating the disease.
Description of the Figures
Fig. 1 schematically illustrates the pre-targeting concept according to the invention, in a preferred radioembolization embodiment, by the chemical and functional steps involved. A) Representation of the different chemical functionalities and components. B) A pre-targeting (guest) agent 99mTc-MAA-Ad displays either pulmonary or hepatic accumulation depending on the route of administration, intravenous (i.v.) or local. C) Following i.v. administration, as a targeted (host) agent, 99mTc-Cy50.5CD10PIBMA39, a prominent distribution to the kidneys, liver and stomach is observed. D) Subsequent to i.v. administration (Model I) of MAA-Ad, accumulation of 99mTc-Cy50.5CD10PIBMA39 is seen in the lungs, whereas following local administration of MAA-Ad into the liver (Model II), liver uptake of 99mTc-Cy50.5CD10PIBMA39 is most predominant. Organs are marked as (1) lungs, (2,) liver, (3) kidneys, (4) stomach, and (5) urinary bladder.
Fig. 2 shows a fluorescence confocal microscopy-based evaluation of Cy50.5CD10PIBMA39 (Cy5) binding to MAA-Ad (top) and non-functionalized MAA (bottom). The MAA-Ad localized particles (brightfield) reveal a higher degree of staining compared to MAA alone, indicated by the higher Cy5-related fuorescence intensities (in red).
Fig. 3 shows the influence of MAA(-Ad) on the uptake of 99mTc-
Cy5o.5CDioPIBMA39 in the lungs and liver (Table 1). A) Uptake in the lungs increased when MAA-Ad was administered i.v. (Method I). B) Increasing uptake in the liver was seen when MAA-Ad was administered locally (Method II).
Fig. 4 discloses fluorescence confocal microscopy images of a Cy5-Polystyrene- Ad (PS-Ad) as mixroparticulate guest, for a non-covalent functionalization test, indicating the host guest inclusion complexes also work well when employing polymer beads.
Fig. 5 discloses fluorescence microscopic images (a) of a MAA Cy5 signal covalently bound to MAA using "Click" chemistry, versus a bright-field image, indicating that covalent selective binding via "click" chemistry also works instead of an inclusion complex.
Fig. 6 discloses fluorescence microscopic images showing the biodistribution of 99mTc-labeled cyclodextrin polymers in mice with adamantane (Ad)-functionalized macro- aggregates (MAA) administrated into the lungs. Fig. 6 (a) 99mTc-Cy5o.5CDioPiBMA3g 2 hours post injection, Ad-MAA in lungs of mice, (b) 99mTc-Cy5i.5CD72PIBMA389 2 hours post injection, Ad- MAA in lungs of mice, (c) 99mTc-Cy3o.sCDioPIBMA392 hours post injection without Ad-MAA in lungs of mice, and (d) 99mTc-Cy3i.5CD72PIBMA389 2 hours post injection without Ad-MAA in lungs of mice, illustrating the benefit of multiple binding sites.
Fig. 7 discloses the level of serum binding of radiolabeled Cy5o.5CDgPIBMA39 over 20 h at 37 °C.
Fig. 8 discloses A) In vitro host-guest complexation between guest vectors MAA-Ad or MAA with host vector mln-Cy5o.5CD9PIBMA39 for lh at 37 °C. Data are expressed as the mean ± SD of the percentage of binding. B) In vitro stability determined at 37 °C of the host-guest complexation between MAA-Ad with mln-Cy5o.5CD9PIBMA39 in PBS or FCS and 99mTc-MAA-Ad with Cy5o.5CD9PIBMA39 in PBS or FCS as well. Values are expressed as the % of radioactivity associated with the washed pellet being representative for the host-guest complex.
Fig. 9 discloses A) time-related (2, 12, 20 and 44 h) dual-isotope SPECT imaging of A) intrasplenic administration of guest-vector 99mTc-MAA-Ad and B) I.V. administered host- vector mln-Cy5o.5CDgPIBMA39. Organs are marked as (1) lungs, (2) liver, (3) spleen, and (4) kidneys. The scale bars indicate the intensity of radioactivity expressed as arbitrary units.
Fig. 10 discloses the bio-distribution of pre-targeting the liver with locally administered guest vector: 99mTc-MAA-Ad at various intervals. Data (expressed as the mean ± SD of the percentage of the injected dose per gram tissue (%ID/g) of 3 observations were calculated based on the radioactive counts measured in indicated tissues at 2, 12, and 20h post-injection. Values of 99mTc-activity calculated at 44h p.i. were unreliable as the radioactivity counts are very low.
Fig 11 shows A) dual-isotope-SPECT imaging of mice pre-targeted with intrasplenic administration of guest vector 99mTc-MAA-Ad at 12h p.i.. Mice, pre-targeted with
either non-functionalized 99mTc-MAA or PBS are used for comparison. Organs are marked as (1) lungs, (2) liver, (3) spleen, and (4) kidneys. B) Bio-distribution of host vector mln- Cy5o.5CD9PIBMA39 12h after I.V. administration c. C) Uptake of mln-Cy5o.5CD9PIBMA39 in the liver determined by ex-vivo fluorescence imaging at equal settings. The scale bar indicates the intensity of fluorescence expresses as photons/sec/cm2.
Fig. 12 shows the time-dependent uptake of mln-Cy5o.5CD9PIBMA39 in various tissues (liver, blood, kidney and joints) determined by radioactivity calculations in ROI's on the scintigrams. Data are expressed as the radioactivity per mm3 on the scintigrams in regions of interest (ROI's) drawn over various tissues.
Fig. 13 shows in A) bio-distribution of I.V. administered mln-Cy5o.5CD9PIBMA39
(host molecule) 12h after hepatic pre-targeting with guest vectors 99mTc-MAA-Ad (blue bars), 9 mTc-MAA (red bars) or PBS (green bars). The significance of difference (p<0.01) is indicated with * according to Student's T-test. Fig. 13 shows in B) dynamic hepatic uptake of I.V. administered 111ln-Cy5o.5CDgPIBMA39. Data are expressed as the mean ± SD ratios of the %ID/g in liver and blood measured at 2, 12, 20, and 44h post-injection of mln-Cy5o.5CD9PIBMA39. The significance of difference (p<0.01) of hepatic uptake of inln-Cy5o.sCD9PIBMA39 in mice pre-targeted with 99mTc-MAA-Ad (blue bars) is indicated with * compared to 99mTc-MAA (control clearance, red bars), or # compared to PBS (regular distribution, green bars) according to Student's T-test. For all pre-targeting settings at 44h p.i. liver-to-blood ratios for mln- CV50.5CD9PIBMA39 are increased compared to the earlier intervals which is indicative for clearance of proteolytic or metabolic products.
Detailed Description of the Invention
The present invention provides materials and methods for locally diagnosing and/or generating a therapeutic response to diseased or infected cells/tissue. It more specifically relates to physically anchor, or otherwise to permit an intra-arterially supplied pre-targeting vector to remain in contact with the diseased or disease bearing region and not be immediately eliminated or washed out from that region; and to measure and diagnose the stability of the vectors positioning at the desired location, and eventually, to generate or solicit a therapeutic response with a suitable agent that selectively binds to the vector. By choosing the appropriate
diameter and shape, the present method uses the natural filtering effect of the target tissue, such as the liver, to accumulate the particles.
The present invention is advantageously based on the principle of first locating microparticles that acts a "vector" compound in the target area, and subsequently functionalisation of the thus located microparticles, by coupling "agent" compounds with a given functionality by a highly selective affinity interaction with the vector compounds. Such interactions are often described as host-guest interactions between a host compound and a guest compound, wherein the recognition and localisation may be achieved by host-guest interaction relying on physical affinity or, selective covalent chemical bonding.
Definitions
Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
" adio-embolization" refers to locally administering radioactive materials to patients with cancer as a form of therapy. In traditional radio-embolization, the radioactive materials have been incorporated into microspheres of regular size for injection into the arterial blood supply of the target organ or tumour. When these radioactive particles or microspheres are administered into the blood supply of the target organ, local accumulation of the microspheres induces "Selective Internal Radiation Therapy "(SIRT). Generally, the main application of SIRT has been to treat cancers in the liver, but may expand based on the present materials and methods. The present method and components allow to create a source of localized radioactive microparticles locally from a micro-particulate vector components, and by subsequently anchoring radioactive isotopes to these vectors. The present invention now allows to log the microparticles in place first, and then to foresee them with a radioactive component, thereby simplifying the preparation and administration greatly. Herein, thus, the term "radio-embolization" in particular refers to providing a microparticle first at the desired location, and then locking the radiation source to the particle, thereby resulting in a highly targeted and effective 2-component "Selective Internal Radiation Therapy "(SIRT).
"Chemo-embolization" refers to locally administering chemically or biologically active materials to patients with a disease, such as cancer, an infection, an autoimmune illness or the like, as a form of therapy, whereby the chemically or biologically active materials are coupled to microspheres accumulated locally to induce a 2-component "selective internal chemotherapy".
The present invention thus makes use of at least two components, the so- called host and the guest component, whereby one is first locally accumulated, and the second component is then introduced into the patient, to selectively couple to the component already in place. The host and the guest components are functionalised to provide complementary functionality. The term "complementary functionality" herein refers to a highly selective binding chemistry, wherein two or more complementarily functionalised partner molecules are likely to react, or bind in a predetermined reaction pathway.
As stated, the host-guest recognition may advantageously be done using selective physical interactions, such as those provided in supramolecular host-guest inclusion complexes. A preferred example for such inclusion complexes are for instance an adamantane (Ad) as host moiety, and a cyclodextrin (CD) as guest moiety. Alternatively, or additionally, a host guest recognition may also be done by selective covalent chemistry, e.g. chemical bonding as for instance using "Click" chemistry between azide and alkyne moieties, whereby the two reactants advantageously are a "host" moiety bound to the vector compound, and a guest moiety bound to the agent, whereby the host and guest will react to form a covalent bond when exposed to each other under appropriate conditions.
The present invention, both compositions and method, have the clear benefit of providing a first allowing to introduce a pre-targeting vector that in itself may not need to have any strong therapeutic or diagnostic effect, to verify the accurate location, and stability of the positioning of the vector at this location, and then to modify the vector in situ using a secondary component functionalized with one or more desired activities. This not only allows to remove steps that are usually employed in radio-embolization treatments at the moment, but also reduces a number of invasive or otherwise cumbersome treatment steps. At the same time, so far difficult to use radioisotopes may be employed e.g. 177Lu and alpha-emitters. As well as other activities may be coupled with the vector that so far were not accessible or possible.
Lastly, the present method also allows to use functional compounds that were previously not useful, and does not require a specific radio-activation step of the microparticles themselves, but instead permits the use of readily available standard materials. Micro-particulate Pre-targeting Vector
The pre-targeting vector preferably comprises a multitude of microparticles that are dimensioned and configured to ultimately get lodged in the target tissue, and thus provide a local target for a secondary component. Preferably these may be dimensioned to pass through larger blood vessels, such as arteries and veins, but eventually get lodged in small vessels and extravasations, in particular if intravenous administration is preferred. "Microparticles" refers to particles or structures that are of a suitable size and dimension, and of suitable composition to be injected into the desired target area.
The pre-targeting vector preferably comprises of microparticles which accumulate in cells (e.g. macrophages), in the interstitial cell fluid or in the microvascular bed, or diffuse in the lymphatic network of tissue wherein they have been injected.
The average particle diameter of the particles in the present invention depends on the desired target area, and to some extent also on the way of administration, e.g. intravascular versus direct injection into the target tissue area. In particular for intravascular administration, considering the diameter of a blood vessel as target site of an embolus, is preferably between 1 and 1000 μιη, more preferably between 2 and 300 μιη. In a preferred embodiment, these microparticles are approximately 1-150 μιη in maximum diameter, and more preferably in the range of 5-70 μιη, and perhaps even 10-40 μιη.
Furthermore, the particles preferably have a weight average particle size distribution width wherein more than 99% of all the particles are below average particle size of 100 μηι, more preferably in the range of average particle size of 50μηι.
Here, the "distribution width of the particle diameter" refers to the range of particle size that would contain more than 99% of all the particles. The particle size of the microparticles of the present invention can be measured by a light scattering method or (electron) microscopy, more preferably by a laser diffraction method, wherein the particle size is reported as a volume equivalent particle diameter. Alternatively, the size may be determined by visible light, or electron microscopy, as appropriate.
Suitable microparticles may include synthetic organic polymeric particles or inorganic glass or metal (e.g. FeO) beads, but also may include naturally occurring and purified compounds such as polypeptides or the like. The microparticles may be organic, or inorganic, or can comprise of mixtures or can consist of combined organic/inorganic materials.
Suitable organic vector components include polymeric components, whether biologically originating, or synthetic.
Examples of useful synthetic polymers formed by chemical cross-linking include polyethylene glycol (hereinafter, "PEG"), polypropylene glycol (hereinafter, "PPG"), polyvinyl alcohol (hereinafter, "PVA"), polystyrene ("PS"), polyacrylic acid, hydroxyethyl acrylate, polyhydroxy ethyl methacrylate, polyvinyl pyrrolidone, carboxymethyl cellulose, dextrans or other sugar-based polymers; homopolymers or (block) copolymer or the water- soluble polymer of the water-soluble polymer selected from the group consisting of hydroxymethyl cellulose and hydroxyethyl cellulose, a- hydroxy acids, the a- hydroxy acid cyclic dimer, a block copolymer of a monomer selected from the group consisting of hydroxy dicarboxylic acids and cyclic esters may be employed.
Particularly useful materials for the vector include hydrogel beads, such as those made from polyhydroxy polymers such as polyvinyl alcohol (PVA) or copolymers of vinyl alcohol, which may be readily tagged with the host moiety by reaction of pendent hydroxyl moieties within the polymer network, using for instance activating agents such as carbonyldiimidazole. Alternatively, particles that are prepared by emulsion or dispersion polymerisation may be employed.
Suitable inorganic vector components include metal particles, such iron, gold, or silver particles, glass beads, porous Silica beads, refractory beads and the like.
The shape of the particles of the present invention may be essentially any shape found useful to lodge the microparticles at the desired site.
Non-spherical particles like MAA or carbon nanotubes would provide a higher degree of accumulation due to their shape, and may remain in place more firmly. On the other hand, essentially spherical particles may also be employed for a better flow in intravascular administration, or combinations thereof. The term "essentially spherical" as used herein refers to a generally spherical shape having a maximum diameter/minimum diameter ratio of from about 1.0 to about 2.0, more preferably from about 1.0 to about 1.5, and most
preferably from about 1.0 to about 1.2. This definition is intended to include true spherical shapes and ellipsoidal shapes, along with any other shapes that are encompassed within the maximum diameter/minimum diameter ratio. This shape is assumed in situ and in absence of shear forces, whereas during the administration, the shape may change under pressure and shear.
The term "surface," as used herein, means the exterior portion of a microparticle that is accessible to the complementary "guest" component. This may be the surface in the case of non-porous particles, or the accessible surface including pores and voids in the case of a porous particle, or combinations thereof.
The term "interior portion," as used herein, means any portion of the particle that is interior to the surface. Generally, the vector particles according to the invention are solid or highly viscous polymeric particles, and not composed of micelles or cell walls with a fluid interior at the temperature and conditions of the administration.
The pre-targeting vector microparticles according to the present invention are typically host-tagged particles, preferably carrying pendent host tags or host moieties at their outer surface, or at least in such manner that the host moieties are accessible to the complementary guest component, e.g. where a porous particle is employed, such that the entire particle is rendered active for forming the host-guest complex or covalently bonded component. It should be noted that the host and guest functionality may be reversed, e.g. a pre-targeting vector may carry a guest molecule or moiety, where the modification of the secondary component may be negatively affected by carrying a host due to e.g. pharmacokinetics, or renal excretion.
The pre-targeting vector microparticles according to the present invention preferably also comprises an imaging label, e.g., a diagnostic and/or a detectable label.
The optionally encapsulated imaging label, e.g., a diagnostic label and/or a detectable label may be or comprise an imaging label itself, e.g., as a detectable label. This advantageously permits to determine if and when the pre-targeting vector microparticles are in the desired location, and of any loss occurs due to blood flow or degradation that may negatively impact a subsequent treatment.
Secondary Compound Composition
The secondary compounds according to the present invention have a smaller size than the pre-targeting vectors and can be tuned for their pharmacokinetics. They may be organic or inorganic, and are prefarbly based on synthetic, or naturally occurring compounds, or combinations thereof. The particulate materials may be formed from monomers or preferably of polymeric materials such as, amino acid sequences, polylactic acid, polyglycolic acid, polycaprolactone, polystyrene, polyolefins, polyesters, polyurethanes, polyacrylates and combinations of these polymers, and homo-or copolymers, and blends thereof. The particulate materials may further comprise suitable binding agents such as gelatin, polyethylene glycol, polyvinyl alcohol, glycerin, (poly)saccharides, DTPA, DOTO, NOTA other hydrophilic materials, and combinations of these. Suitable gelatins may include bovine collagen, porcine collagen, ovine collagen, equine collagen, synthetic collagen, agar, synthetic gelatin, and combinations of these. It is note that for covalent, preferably click-chemistry modifications, comparatively short and small components may be employed.
Examples of useful synthetic polymers formed by chemical cross-linking include polyethylene glycol (hereinafter, "PEG"), polypropylene glycol (hereinafter, "PPG"), polyvinyl alcohol (hereinafter, "PVA"), polyacrylic acid, hydroxyethyl acrylate, polyhydroxy ethyl methacrylate, polyvinyl pyrrolidone, carboxymethyl cellulose, dextrans or other sugar- based polymers; homopolymers or (block) copolymer or the water-soluble polymer of the water-soluble polymer selected from the group consisting of hydroxymethyl cellulose and hydroxyethyl cellulose, a- hydroxy acids, the a- hydroxy acid cyclic dimer, a block copolymer of a monomer selected from the group consisting of hydroxy dicarboxylic acids and cyclic esters may be employed.
Partially water-soluble polymers may be used, wherein typically biocompatibility is higher than for hydrophobic polymers. Also, due to the presence of hydroxyl groups, PEG, PPG, PVA, poly hydroxyethyl acrylate, poly hydroxyethyl methacrylate (hereinafter, "poly-HEMA") permits easy functionalization.
The secondary components are in any case preferably storage stable, and susceptible to nucleophilic substitution reactions, which may be used to functionalize the particles controlled manner to provide the guest moiety for inclusion complexes. The latter are preferably covalently bound, preferably at the particle surface, as appropriate, depending on the activation chemistry selected.
The weight average molecular weight of useful polymers, is preferably 200 or more. Further, for a discharge ex vivo to be facilitated by the living body, it is preferably 50,000 or less. The weight average molecular weig of the vector component may be suitably selected such that the desired pharmacokinetics and biological half-life are achieved. The weight average molecular weight of the polymers employed may be advantageously determined by gel permeation chromatography. The choice of the composition and the molecular weight depends also on the clearance from the body. Rapid and complete clearance of non-bound secondary components is critical. For example resorption of a radioisotopes in the kidneys may result in kidney failure as side effect from therapy, and hence fast clearance is desired.
Useful secondary components may include bio-resorbable components, which can be broken down by the metabolism, and excreted in a suitable time period to be removed alongside with the embolized tissue, depending on the desired duration of the vector component in the tissue.
Concentration
It is necessary to obtain the desired number of microparticles to be injected into the vascular system and/or tissue of a patient, in order to achieve a sufficiently high amount of diagnostic and/or therapeutic agents to be present. While the number of particles which may be injected or otherwise delivered will vary depending upon the patient's blood pressure, blood flow rate, metabolism, body weight, organ weight, etc., it has been found that approximately 200 to 2,500,000 or more particles suspended in a biologically safe solution and delivered to the patient may suffice, although larger quantities of particles may be necessary and desirable depending on various circumstances and conditions of use, e.g., tumour size, etc.
By knowing the concentration of particles in a given volume of dispersion, one merely needs to withdraw and inject the desired volume of liquid containing the desired number of particles.
Modification of the pre-targeting and secondary targeting vector material
According to the present invention, the pre-targeting vector microparticle material is associated with at least one host moiety, while the secondary vector material is associated with at least one guest moiety.
Preferably, the pre-targeting vector is covalently linked to a host molecule, in which case the guest-modified pre-targeting vector comprises one or more host functional groups. As used herein, such a pre-targeting vector has been linked to a first guest molecule and thereby comprises one or more host functional groups, is also called the " host -modified micro-particle" or a "pre-target vector". The secondary vector material is also linked to an at least one guest moiety, thus forming the "guest -modified secondary vector"
The modification of the vector molecules to comprise the host or guest moiety may be done by any suitable method that allows to link the gust moiety to the vector surface.
The linking of the guest or host molecule to the vector or secondary component is preferably done by conventional conjugation techniques known to the person skilled in the art. This guest molecule which is part of the guest-modified microparticle, then binds (non- )covalently to a host functional group residing on a preferably multivalent host structure. To be more specific, this interaction takes place by a one-to-one interaction between a guest functional group in the guest molecule and a host functional group in the host structure. Obviously, a higher load of guest molecules may result in a better complexation ratio, while too high of a load may prevent interactions by sterical hindrance. Accordingly, multivalent guests are preferred for non-covalent interactions, which may comprise multiple binding sites in one molecule, or microparticles that comprise separately multiple binding sites. The same applies mutatis mutandis to the host molecule, and the host-modified secondary vector.
Guests and hosts
The terms "guest" and "host" as used herein refer to two different, but complementary, binding partners that non-covalently, or covalently interact with each other.
As used herein, the term "guest moiety" or "group" means the part or moiety of a monomer of the guest molecule, which enables the binding to a complementary host functional group.
A "guest molecule" is in turn a molecule that comprises one or more guest functional groups, where a monovalent guest molecule comprises one guest functional group and a multivalent guest molecule comprises at least two guest functional groups. As used herein, the term "host functional group" means the part or moiety of a monomer of the host molecule, which enables the binding to a complementary guest functional group.
A "host molecule" is in turn a molecule that comprises one or more host functional groups, where a monovalent host molecule comprises one host functional group and a multivalent host molecule comprises at least two host functional groups. Preferably, one guest moiety interacts with one host moiety. Where non-covalent host-guest pairs are employed, according to the present invention, the interaction between the guest and the host may be reversible, and is determined by the affinity strength as expressed by dissociation constants. Typically, a guest molecule does not normally interact with another guest molecule. Examples for a non-covalent guest-host interaction include beta-cyclodextrin- adamantane, beta-cyclodextrin-ferrocene, gamma-cyclodextrin-pyrene, cucurbituril- viologen, and/or a Ni(NTA)-His tag. Examples for a covalent interaction include an Azide (N3)- alkyne interaction as a host-guest interaction. For example the dibenzocyclooctyne group (DBCO) , or similar compounds such as BCN allows copper-free "Click" chemistry to be applied to vectors to be used in live organisms. DBCO groups will preferentially and spontaneously label molecules containing azide groups (-N3). Also, within physiological temperature and pH ranges, the DBCO group does not react with amines or hydroxyls naturally present in many biomolecules. Also, the reaction of the DBCO group with the azide group is significantly faster than with sulfhydryl groups, making this a highly selective reaction.
The term "inclusion complex" refers to any material wherein the host compound absorbs or embeds a guest compound to form a complex. For example, the guest compound may be embedded in a cavity formed by the host compound. The terms "inclusion material", "inclusion complex", "inclusion compound" are used as synonyms in this application.
Preferably, the guest-host components are each readily available, and enable to perform the methods of the present invention on a relatively large scale and/or in low cost devices.
As used herein, the term "guest-host molecule interactions" includes the non- covalent binding between respective guest and host functional groups. In a preferred setting, hydrophobic interactions, such as lipophilic interactions, are being used instead of interactions that are based on charge.
The functionality may be reversed, i.e. using the host molecule as the molecule that is linked to the vector, but as used herein, the host molecule or host structure is not
linked to the vector directly, primarily via the guest molecule. The vector particle preferably may comprise a multivalent host structure, comprising multiple host functional groups, some of the host functional groups may be (non-) covalently bound to a guest molecule, while others remain free.
Preferred, essentially non-covalent host compounds are cyclodextrins, with adamantane moieties acting as host molecules. Cyclodextrins are cyclic polysaccharides containing naturally occurring D(+)-glucopyranose units in an a-(l,4) linkage. The most common cyclodextrins are alpha (a)-cyclodextrins, beta )-cyclodextrins and gamma (y)- cyclodextrins which contain, respectively, six, seven or eight glucopyranose units. Structurally, the cyclic nature of a cyclodextrin forms a torus or donut-like shape having an inner apolar or hydrophobic cavity, the secondary hydroxyl groups situated on one side of the cyclodextrin torus and the primary hydroxyl groups situated on the other. Preferably beta- cyclodextrin is used, which is the best binding partner for adamantane. The cyclodextrin may contain additional groups, such as an amine to attach it to a scaffold, one or more thiols to bind the cyclodextrin to a gold surface, or hydroxypropyl groups to increase solubility and biocompatibility. Other members of the cyclodextrin family (most likely alpha and gamma) can also be used for host-guest interaction, although different guests have to be introduced to achieve this. Accordingly, a good, but not the only, example of supramolecular host-guest interactions that can be applied in the invention is the non-covalent interaction between adamantane (as the guest molecule) and β-cyclodextrin (as the host molecule).
The side on which the secondary hydroxyl groups are located has a wider diameter than the side on which the primary hydroxyl groups are located. The hydrophobic nature of the cyclodextrin inner cavity allows for the inclusion of a variety of compounds. (Comprehensive Supramolecular Chemistry, Volume 3, J.L. Atwood et al., eds., Pergamon Press (1996); T. Cserhati, Analytical Biochemistry, 225:328-332 (1995); Husain et al., Applied Spectroscopy, 46:652-658 (1992); F 2 665 169). Various cyclodextrin containing polymers and methods of their preparation are also known in the art. (Comprehensive Supramolecular Chemistry, Volume 3, J.L. Atwood et al., eds., Pergamon Press (1996)). A process for producing a polymer containing immobilized cyclodextrin is described in U.S. Patents 5,608,015, or 5,276,088 describe methods of synthesizing cyclodextrin polymers by either reacting polyvinyl alcohol or cellulose or derivatives thereof with cyclodextrin derivatives or by
copolymerization of a cyclodextrin derivative with vinyl acetate or methyl methacrylate. The resulting cyclodextrin polymer contains a cyclodextrin moiety as a pendant moiety off the main chain of the polymer particle. Cyclodextrin-based polymers have been used for therapeutic applications (Kandoth et al. Two-photon fluorescence imaging and bimodal phototherapy of epidermal cancer cells with biocompatible self-assembled polymer nanoparticles. Biomacromolecules 2014 (15):1768-1776) and imaging agents (Yan et al. Poly beta-cyclodextrin inclusion- induced formation of two-photon fluorescent nanomicelles for biomedical imaging. Chemical Communications 2014 (50):8398-8401) and they showed excellent biocompatibility.
Adamantane is a lipophilic small molecule that may be attached to a vector, or conjugated to a microparticle that can be physically lodged in the target tissue. The adamantane structure combines rigidity with the ability to form diamondoid structures, and offers high binding affinities with cyclodextrins. In one aspect, the guest molecule may be adamantane, whereas the host molecule is preferably then a cyclodextrin that non-covalently interacts with adamantane. Both compounds are relatively cheap and easy to produce in controlled settings and non-toxic.
Scaffolding and Multivalent Guest Structures
The guest moiety may be connected to the microparticle by a "scaffold", i.e. a binding unit comprising a spacer molecule or otherwise suitable molecular structure. The scaffold may be intended to ensure that the guest moiety is presented to the incoming host moiety, and/or may permit to modify the microparticle easily. Also, several guest moieties or guest functional groups may be interconnected through a scaffold molecule to form a multivalent guest structure.
The term "multivalent" as used herein refers to a number of host or guest molecules, or functional groups thereof, that are part of the same molecule or structure. Multivalent interactions contain at least two functional groups of the same type (e.g. at least two guest functional groups, or at least two host functional groups) bound to each other through a backbone (or scaffold) that allows the multimerization of the matching guest or host molecule. The upper limit in multivalency depends on the purpose of the layer-by-layer manufacturing of guest-host molecule interactions, as disclosed herein. Without wishing to
be bound to any particular theory, it is believed that multivalency enhances the affinity, and thus improves the binding, as monomeric guest molecules tend to show a significantly lower non-covalent interaction.
According to the present invention, a "multivalent host structure" or a "multivalent guest structure", is a structure comprising at least two host functional groups or guest functional groups, respectively. It can in principle be a dimer or polymer of suitable host or guest monomers, but typically the host or guest molecules have been attached or engrafted onto a polymer of a different type that allows for the attachment of the host or guest molecules. This is preferably used for non-covalent interactions, thereby increasing the binding strength.
In one preferably non-covalent embodiment of the present invention, a "multivalent host structure" or "multivalent guest structure" preferably comprises a scaffold or linker structure onto which the at least two host molecules or at least two guest molecules have been attached or engrafted resulting in that the scaffold structure comprises at least two host functional groups or at least two guest functional groups. The scaffold structure can be anything that allows attachment of the host or guest molecules of choice. The scaffold structure may of course also be part of, or integrally form the microparticle. In another embodiment, the scaffold molecule is a polypeptide comprising less than about 30, such as, e.g. less than about 25, less than about 20, less than about 15, less than about 10, less than about 5 or less than about 6 amino acids. It may also be an oligo peptide such as, e.g. a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. In one embodiment the repeat unit of the poly or oligepeptide is β-alanine.
Host Components
Suitable host components are components that are modified to selectively link up or react with the pre-targeting vector comprising the guest moiety. The selectivity of this linkage preferably allows to introduce the host component intravenously, rather than into the tissue, as the host component then automatically binds to the vector.
In one preferred embodiment, a moiety with the worst pharmacokinetics, e.g. highest lipophilicity, is defined as guest and will be part of the pre-targeting microparticles. This means its influence on the targeting is minimal.
The complementary host is then the moiety with the most favourable pharmacokinetics e.g. the least non-specific interactions.
In the case of Adamantane -Cyclodextrin complex, this would result in guest- host, as used herein.
For N3-alkyne covalent coupling, this would result in host-guest, meaning the azide should be part of the secondary vector.
Hence once the pre-targeting vector is in place, the secondary, host component may for instance be simply introduced into the blood stream, as it will bind selectively to the guest once encountered.
This not only reduces the steps required to treat and/or diagnose a patient, but also permits the use of various agents, and for instance the use of radioactive isotopes that are presently not used, as presently in SIRT, the microparticles themselves are prepared to contain the radioactive components, and thus the choices are limited to which isotopes can be introduced in the few reactors available for this purpose.
Also, it may permit to determine a dosage for particular treatment not at the reactor, as presently done for each treatment separately, but simply by calculating and administrating the amount of suitably modified host components which can be done at a patient treatment site, e.g. a hospital pharmacy.
Also, several different components may be administered, allowing for a combination therapy.
The host structure or secondary component thus may comprise a targeting moiety or molecule coupled to the particle, and the targeting moiety that in this manner helps deliver the bioactive agent and/or the imaging agent to a desired, i.e. vector location in a patient.
An exemplary method for preparing a host structure may include providing host nanoparticles comprising a pharmaceutically acceptable polymer and coupling, e.g., by coating, covalent linkage, or co-localization, to the surface of the nanoparticles the host moiety for coupling to the guest, and an imaging agent, a detectable label, or otherwise targeting moiety.
The method may further include one or more of forming a particle suspension, passing the particle suspension through a filter, removing impurities from the particle
suspension, centrifugation to pellet the particles, dialyzing the particle suspension, and/or adjusting the pH of the particle suspension. The method may also include quenching the covalent linking reaction.
Suitable host components may include organic or inorganics components or mixtures thereof. For example, the host components can be selected from monomers- polymers and nanoparticles. Suitable polymers for example can be selected from poly(isobutylene-alt-maleic anhydride) (PIBMA), PAMAM, poly-acrylic acid, polysaccharides, polypeptides and oligopeptides. Some such polymers, such a PIBMA, has the further advantage that it prevents interaction with the immune system and thereby also functions as a cloaking group.
For many applications, it is also of importance that the selected structure is non-toxic. For some applications eliciting of an immune response may be a means to induce a therapeutic effect.
Suitable polymeric host components may comprise a pharmaceutically acceptable polymer core and a one or more bioactive agents and/or imaging agents. Preferably, the secondary component may comprise a pharmaceutically acceptable polymer core, and one or more bioactive agents, such as a drug or medicament encapsulated in the core. Multivalent host structure
Preferably, for non-covalent interactions, the complementary component comprises multiple host moieties, thereby increasing the affinity for the guest functional groups of the guest- modified vector.
Preferably, the host structure according to the present invention is multivalent, i.e. comprises at least two host functional groups. In a preferred embodiment, the first multivalent host structure comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 9 or at least 10 host functional groups. In a preferred embodiment, the host comprises a scaffold structure, such as a polymer comprising as least about 5, such as at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35 or at least about 40 monomer repeat units.
A preferred second component vector molecule may comprise multiple host sites, to generate multivalent structures containing either the host functional group of cyclodextrin adamantane. A preferred example is poly-isobutylene-alt-maleic anhydride (PIBMA), which can be easily functionalized, is non-toxic, and presents a multitude of negative charges after functionalization which increases water-solubility and provide cloaking.
Other preferred structures that could be applied in the methods of the present invention to generate multivalent structures comprising guest or host functional groups are other polymers containing anhydrides, PAMAM, nanoparticles (e.g. quantum dots), poly- acrylic acid, and polysaccharides.
Diagnostic/Therapeutic Agents
A benefit of the subject process is that various compounds may be employed as diagnostic or therapeutic labels, and may include chelates, a diagnostic, a therapeutic agent, or any combination thereof, using a simple pre-targeting platform. The second, complementary component may comprise a therapeutic agent or an imaging agent, or combinations thereof, e.g., it may include a diagnostic agent and/or a detectable label.
Immunogenic Agents: The term "immunogen" refers to a substance which is capable, under appropriate conditions, of inducing a specific immune response and of reacting with the products of that response (e.g., a specific antibody, specifically sensitized T- lymphocytes or both), while the term "immunogenic" relates to a reaction triggered by the presence of the immunogen.
Both the pre-targeting vector component and the secondary component may also carry various labels or agents other than host or guest moieties.
In certain embodiments, the one or more bioactive agents are covalently or non-covalently associated with the secondary component.
For example, a nucleic acid or protein included in the particle may comprise an imaging agent itself, e.g., a detectable label that may be attached to DNA or a protein. Alternatively, the secondary component may comprise an imaging agent that is separate from a nucleic acid or a protein, e.g., encapsulated in the core or disposed on or coupled to the surface. Additionally, the component may comprise one or more targeting moieties or molecules coupled to the component and/or the protein or nucleic acid, and the targeting
moiety can help deliver the nucleic acid, the protein, and/or the therapeutic, imaging, and/or diagnostic agent.
The component may comprise an imaging agent, e.g., a diagnostic agent and/or a detectable label. The optionally encapsulated bioactive agent may be or comprise an imaging agent itself, e.g., a detectable label may be attached to a therapeutic agent. Alternatively, the particle may comprise an imaging agent that is separate from the bioactive agent.
In a preferred embodiment, a plurality of radioisotopes, bioactive agents, such as bioactive agents or contrast agents may be loaded onto a single host component, or spread over the guest and host component.
In one embodiment, the potentially radioactive isotope may be an isotope possessing a high absorption cross-section to neutrons, protons, electrons, or high energy photons. In a related embodiment, the potentially radioactive isotope has a high absorption cross-section to neutrons, and is selected from the group consisting of 10B, 149SAM, 157Gd, and 155Gd or any combination thereof, thereby providing a target area for fast neutron therapy.
Different isotopes may also be conjugated to the complementary compound, e.g. allowing the use of 177Lu , 99mTc , mln isotopes, or combination thereof, for instance.
In one embodiment, a magnetic resonance contrast agent may be employed, as for instance those selected from Manganese Oxide, perfluorocarbons, Feridex, Gadolinium, Combidex, Bang Magnetic Particles, Gd-DTPA, Gadolinium And Manganese Derivatives, Superparamagnetic Iron Oxide Particles, gadopentetate dimeglumine, Gd- DOTA, Gd-DTPA- BMA, Gd-HP-D03A, Gd-DTPA-BMEA, Gd-D03A-butrol, Gd-BOPTA, Mn-DPDP, Gd-EOB-DTPA, Gd-BOPTA, AMI-25, SH U 555A, gadoflourine-M, AMI-227, EP-2104R, P947, Gd-DTPA mesophorphryn, SH U 555 C, NC- 100150, MS-325, gadoflourine-M, gadomelitolm manganese chloride, ferric amonium citrate, and barium sulfate suspensions. In one embodiment, a negative contrast agent may be employed, as for instance those based on superparamagnetic iron oxides: SPIO (Superparamagnetic Iron Oxide), typically employed for liver tropism, and USPIO (Ultrasmall Superparamagnetic Iron Oxide) typically for ganglion tropism.
These particles may predominantly have an effect on magnetic susceptibility (T2), and thus may produce a drop in signal, namely in T2* weighting.
In one embodiment, a contrast agent that can act as a Raman spectroscopy reporter, thereby allowing detection of the component with Raman spectroscopy.
In another embodiment, a contrast agent that can act as a fluorescence spectroscopy reporter, thereby allowing detection of the component with fluorescence spectroscopy.
In another one embodiment, the agent may comprise a photoacoustic contrast agent, thereby allowing detection of the component acoustically.
Also different bioactive agents may be attached to the complementary component, as well as compounds that enhance the interaction with other cells e.g. immune cells, thereby triggering for instance an enhanced and targeted immune response.
Bioactive agents according to the invention include but are not limited to a nucleic acid, DNA (e.g., a gene therapy vector or plasmid), an RNA (e.g., an mRNA, the transcript of an RNAi construct, or an siRNA), a small molecule, a peptidomimetic, a protein, peptide, lipid, surfactant and combinations thereof. Examples of therapeutic agents include but are not limited to a nucleic acid, a nucleic acid analog, a small molecule, a peptidomimetic, a protein, peptide, lipid, or surfactant, and combinations thereof. In certain embodiments, the imaging agent further comprises a detectable label.
In another embodiment, combinations of the above agents may be present, either on a single secondary component, or a mixture of differently functionalised secondary components.
Administration of the components
Administration of the pre-targeting vector
The vector components according to the present invention are administered locally, parenterally and/or topically, i.e. they are injected or infused into a particular area, and/or into a particular organ or tissue in a patient body. Examples include intrahepatic injections or infusions, intrapancreatic injections, or functionally, generally, intratumoral injections; intradermal and/or peritumoral injections or infusions. The components, in particular the pre- targeting vector, are therefore chosen and designed in among other factors size, size distribution, compressibility, water content, flowability, deformation creep and/or stability,
as well as optimal pharmacokinetics, e.g. rapid clearance and minimal background of non- complexed components, such that they can be infused or injected, and that they are dimensioned and functionalised to show minimal movement once they are distributed inside the relevant organ, with minimal or no shunting or otherwise bypassing occurring.
The delivery of the vector material via injection or implantation provides a means to effectively mechanically target the biomaterial to a specific site or location, thereby localizing the biomaterial and minimizing systemic side effects.
Moreover, the administration of the vector material via implant or needle based injection is minimally invasive and usually can be performed on an outpatient basis, resulting in a lower cost than other surgical forms.
Preferably, "delivering" comprises positioning a delivery device, e.g. a syringe or infusion catheter in proximity to a target region of a blood vessel, or directly into a target tissue not via vasculature, and ejecting the microparticles from the delivery device such that the particles are positioned in the target region.
Administration of the secondary Component
The complementary secondary vector components preferably are administered intravenously, or more generally intravascularly, since the components are accumulated at the pre-targeting vector component location, or likely excreted if not bound to the vector. This permits to reduce the number of steps in a treatment and diagnosis, which reduces the negative effect on a patient. Alternatively, the complementary secondary component may be injected locally, e.g. by inserting a cannula into the desired region, and injecting the material into the vicinity of the vector component. Administration Medium
Components according to the present invention are particularly useful for embolizing liver tissue. In this case, the microparticles can be used as component dispersed in an appropriate dispersion medium.
The administration medium of the components may be a buffer/ serum solution, and may further comprise injection dispersing agent such as polyoxyethylene sorbitan fatty acid ester or carboxymethyl cellulose, such as methyl paraben or propyl
paraben preservatives, sodium chloride, preservatives used in tonicity agents or injections, such as mannitol or glucose, stabilizer, solubilizing agents or excipients. The present invention thus preferably relates to a complementary component in vivo parenteral topical administration and for forming the selective non-covalent high affinity interaction with a complementary host moiety, and/or the selective covalent bond with a host compound having a complementary functionality of the vector component according to the invention; wherein the component comprises a complementary guest functionality, and at least a first diagnostic and/or therapeutic agent.
Preferably, the agent is selected from the group consisting of: a diagnostic agent, an imaging agent, a contrast agent, and a therapeutic agent, preferably a radioactive isotope or a chemotherapeutic agent.
Preferably, the agent is selected from the group consisting of one or more: anticancer agents, antibiotics, antihistamines, hormones, steroids, therapeutic proteins, biocompatible materials, imaging agents and contrast agents.
Preferably, the diagnostic agent is selected from the group consisting of magnetic resonance contrast agents, radioopaque contrast agents, ultrasound contrast agents, and nuclear medicine imaging contrast agents, more preferably, wherein the radioactive isotope is selected from the group consisting of: Y-90, P-32, P-33, Sc- 47, Cu-64, Cu-67, As-77, Ph-105, Pd-109, Ag-111, Pr-143, Sm-153, Tb-161, Ho-166, Lu-177, Re-186, Re- 188, Re-189, lr-194, Au-199, Pb-212, 1-131, Ac-225, Ra-223, Ra-224, and Bi-213.
The present invention also relates to a kit for forming an optionally bioresorbable diagnostic and/or therapeutic occlusion at a target site within a body, the kit comprising:
a vector component composition as set out herein above; and - a complementary secondary component as set out herein above, the complementary component being functionalised for selectively attaching to the vector component in vivo. Preferably, the complementary component comprises a therapeutic agent for delivery to the targeted target area.
The present invention also relates to a method, and to a compound or kit of compounds for use in treating a condition in vivo in a patient, the method comprising the steps of: (a) providing an administration medium comprising microparticles of a pre-targeting
vector component, and (b) delivering a diagnostically and/or therapeutically effective amount of the a pre-targeting vector component into the target tissue, preferably into the tumour or tumour bearing tissue patient; and (c) subjecting the target tissue to a diagnosis step to determine if significant shunting occurs; and (d) administering a therapeutically effective amount of a complementary component comprising a therapeutic agent to the patient, thereby producing a therapeutically active matrix in the subject tissue.
Preferably, (b) comprises delivering the pre-targeting vector in a target region of a blood vessel.
Preferably, "delivering" further comprises positioning a delivery device in proximity to a target region of the blood vessel, and ejecting the pre-targeting vector particle(s) from the delivery device such that the particles are positioned in the target region.
Preferably, the target area is selected from one or more of liver, pancreas, thyroid, heart, peripheral nerve scaffold, breast, bladder, cartilage, bone, tendon, ligament, blood vessel, skin, lymph nodes, spinal cord, and/or tumour tissue The present invention also relates to a method, and compound of use for topically treating a disease, comprising administering to a target area of a patient in need thereof a kit according to the invention suitable for diagnosing and treating the disease. The following, non- limiting examples illustrate the invention. Example 1
In this example, a particularly useful example is employed as a vector component, i.e. a host- modified Technetium 99mTc macro aggregated albumin (99mTc-MAA), in an injectable form, comprising a sterile aqueous suspension of Technetium-99m (99mTc) labelled to human albumin aggregate particles. Its use advantageously permits to use for diagnostic purposes SPECT-CT, to visualize the occurrence of shunting, as 99mTc-MAA is a well-known imaging agent. Now modifying the material allows to combine a method in current practice with the method according to the present invention. In this way, clinically Technetium-99m macro- aggregated albumin (MAA) scintigraphy may be used to assess lung or digestive shunting prior to therapy, based on tumoural targeting and dosimetry. Computed tomography (CT) may then be performed during the angiography to aid in digestive vascularization recognition, as well as perfused tissue and tumour targeting evaluation. MAA scintigraphy may not only be used
for lung-shunt evaluation, but can also aid in digestive shunt recognition and dosimetric evaluation and the marking of diseased tissue for radio-occult lesion localization.
All chemicals were obtained from commercial sources and used without further purification. NMR spectra were obtained using a Bruker DPX 300 spectrometer (300 MHz, H NMR) or a Bruker AVANCE III 500 MHz with a TXI gradient probe. All spectra were referenced to residual solvent signal or TMS. HPLC was performed on a Waters system by using a 1525EF pump and a 2489 UV detector. For preparative HPLC a Dr. Maisch GmbH, Reprosil-Pur 120 C18-AQ 10 μιη column was used and a gradient of 0.1 % TFA in H2O/CH3CN (95:5) to 0.1 % TFA in H2O/CH3CN (5:95) in 40 min as employed. For analytical HPLC a Dr. Maisch GmbH, Reprosil-Pur C18-AQ 5 μηι (250x4.6 mm) column was used and a gradient of 0.1 % TFA in H2O/CH3CN (95:5) to 0.1 % TFA in H2O/CH3CN (5:95) in 40 min was employed. MALDI-ToF measurements were performed on a Bruker Microflex. High resolution mass spectra were measured on an Exactive orbitrap high-resolution mass spectrometer (Thermo Fisher Scientific, San Jose, CA) and processed with the use of Thermo Scientific Xcalibur software (V2.1.0.1139). For dialysis Sigma Pur-A-LyzerTM Mega 3,500 units were used.
Synthesis
Adamantane-tetrafluorophenol (Ad-TFP)
1-Adamantanecarboxylic acid (500 mg, 2.8 mmol) and 2,3,5, 6-tetrafluorophenol (718 mg, 4.3 mmol) were dissolved in 10 mL dry DCM and stirred for 10 minutes. Subsequently, N,N- dicyclohexylcarbodiimide (858 mg, 4.3 mmol), dissolved in 5 mL dry DCM, was added dropwise. After stirring for 2 days at RT, the reaction mixture was filtered and the filtrate was concentrated in vacuo. The resulting yellow product was purified by silica column chromatography (DCM:Hexane, 1:1). Pure fractions were pulled and concentrated under vacuo to obtain the product as a white crystalline powder (785 mg, 86%). 1 NMR (300 MHz, CDCI3, 25 °C)= δ 7.06 - 6.89 (m, 1H, CH of TFP), 2.09 (s, 9H, C-CH2-CH and CH2-CH-CH2), 1.78 (s, 6H, CH-CHz-CH). High resolution mass: [Ci7Hi7F402]+ calculated 329.3, found 329.1.
Cv5(S03)Sulfonate-(S03)CO-TFP
Cy5-(S03)Sulfonate-(S03)CO-TFP was synthesized according a procedure previously described in S. J. Spa, A. Bunschoten, M. T. M. Rood, R. J. B. Peters, A. J. Koster and F. W. B. van Leeuwen, Eur. J. Inorg. Chem., 2015, 2015, 4603-4610.
Cv5(S03)Sulfonate-(S03)Amine
Cy5-(S03)Sulfonate-(S03)Amine was synthesized according to the method disclosed in M.T. Rood, S. J. Spa, M. M. Welling, J. B. Ten Hove, D. M. van Willigen, T. Buckle, A. H. Velders and F. W. B. van Leeuwen, Sci. Rep., 2017, 7, 39908.
Cv5o,5CDioPIBMA39
The synthesis of Cy5o.5CDioPIBMA39 was performed as described under the previous item above.
Poly(isobutylene-alt-maleic anhydride) Mw 6,000 (30 mg, 5.0 μηιοΙ, Sigma- Aldrich) and Cy5-(SOs)Sulfonate-(S03)Amine (5.0 mg, 5.6 μιηοΙ) were dissolved in 3 mL dry DMSO and Ν,Ν-diisopropylethylamine (DIPEA, 50 μί, 250 μηιοΙ Sigma-Aldrich) was added. After stirring at 80 °C for 7 h, 6-monodeoxy-6-monoamino^-cyclodextrin (95 mg, 80 μιηοΙ, Cyclodextrin Shop) was added and the reaction mixture was left to stir for another 72 h at 80 °C. After cooling to RT, the polymer was first dialyzed against H20 for 1 day, then against 100 mM phosphate buffer pH 9.0 for 24 h, and subsequently against H2O for another 5 days, while refreshing the dialysis medium daily. The solution was lyophilized to give a blue powder (87 mg, 5 μηιοΙ) and was stored at -20°C. Before usage, a small amount was aliquoted in PBS at 1 mg/mL concentration and stored (< one month) at 7°C. 1H NMR: see previous reported literature.
Functionalization of macro-aggregates with adamantane (MAA-Ad)
Macro-aggregates from albumin (TechneScan®, MAA) were obtained from Mallinckrodt Medical B.V., Petten, The Netherlands. Lyophilized albumin macro-aggregates (2 mg) were dissolved in 1 mL of saline (0.9% NaCI, sterile and pyrogen-free, B. Braun Medical Supplies, Inc., Oss, The Netherlands) and portions of 0.1 mL (containing 0.2 mg MAA) were stored in Eppendorf tubes at -20 °C until further use. For functionalization, one portion was defrosted and 20 SL of Ad-TFP (10 mg/mL DMSO) was added. After agitation in a shaking water bath for
1 h at 37 °C, the solution was washed 2 times with phosphate buffered saline (PBS) by 2 centrifugation steps (3 min x 1,200 rpm). The obtained MAA-Ad was diluted in 1 mL of PBS to 0.2 mg/ml. To estimate the number of Ad conjugated to MAA, the MAA functionalization was also performed with Cy5-TFP according the same procedure as with Ad-TFP. Subsequently, the absorbance at 650 nM of the MAA-Cy5 constructs was measured using a NanoDrop Spectrophotometer (Thermo Fisher Scientific Inc. Wilmington, DE, USA). From the absorbance, the dye concentration was calculated using the law of Lambert Beer (A = ε· I · C) with Ecys = 250 x 103 mol^cnr1. The number of Cy5/MAA aggregates was then ca lculated by dividing the calculated Cy5 concentration by the known MAA concentration, resulting in a ratio of 3.07(±0.24) x 108 Cy5/MAA particle on average. Assuming that Ad-TFP reacts in a similar fashion as Cy5-TFP, it was estimated that the ratio of Ad/MAA would be in the same order of magnitude.
Radiolabeling of CV5O,5CDIQPI BMA39
Radiolabeling of Cy5o.5CDi0PIBMA39 was performed as follows: to 10 0L of Cy5o.sCDioPIBMA39 (1 mg/mL PBS), 4 μΐ of SnCl2.2H20 (0.44 mg/mL saline, Technescan PYP, Mallinckrodt Medical B.V.), and 100 ί of a freshly eluted 99mTc-Na-pertechnetate solution (500 MBq/mL, Mallinckrodt Medical B.V.) were added and the mixture was gently stirred in a shaking water bath for 1 h at 37 °C, as described in M. M. Welling, A. Paulusma-Annema, H. S. Baiter, E. K. J. Pauwels and P. H. Nibbering, Eur. J. Nucl. Med., 2000, 27, 292-301. Subsequently, the labeling yield was estimated over time by ITLC analysis according the following procedure: 2 mL of the reaction mixture was applied on 1x7 cm ITLC-SG paper strips (Agilent Technologies, USA) for 10 min at room temperature with PBS as mobile phase. After 1 h the highest labeling yield of Cy5o.5CDioPIBMA39 with technetium-99m was assessed (49.6±12.8) and the reaction mixture was purified by size exclusion chromatography with sterile PBS as mobile phase using Sephadex™ G-25 (desalting columns PD-10, GE Healthcare Europe GmbH, Freiburg, Germany). Fractions containing 99mTc-Cy5o.5CDi0PIBMA39 were collected and directly applied in the imaging experiments. According the data calculated from the PD-10 purification a labeling yield of 49.2±6.9 was obtained, which was in accordance with the yield estimated by ITLC analysis.
Stability of 99mTc-Cv5o,5CDioPIBMA39
To assess the stability of the radiolabeling, after 24 h the release of radioactivity from PD-10 purified 99mTc-Cy5o.5CDioPIBMA39 was determined with ITLC (according the same method as described for 'Radiolabeling of Cy5o.sCDioPIBMA3g'), and this turned out to be less than 5% of the total radioactivity.
Supramolecular interaction between MAA-Ad and 99mTc-Cv5Q,5CDioPIBMA39
To determine the supramolecular interaction between MAA-Ad and CV50.5CD10PIBMA39 in vitro, 0.1 mL of MAA-Ad in PBS (0.2 mg/mL) and 0.1 mL of 99mTc-Cy5o.5CDioPIBMA39 in PBS (1 mg/mL, 1 MBq) were mixed and the solution was incubated for 1 h in a shaking water bath at 37 °C. Thereafter, the radioactivity of the total amount added and the radioactivity of the pellet after two washing steps with PBS were measured in a dose-calibrator, to determine the amount of binding of 99mTc-Cy5o.5CDi0PIBMA39 to MAA-Ad. After correction for background activity the amount of binding was expressed as the percentage of the total amount of radioactivity (%binding). To assess the effect of the Ad moieties, the same experiment was also performed with non-functionalized MAA and the resulting %binding of 99mTc- Cy5o.sCDioPIBMA39 to MAA and MAA-Ad were compared. As shown in SI Fig. 2, compared to the binding to non-functionalized MAA, a significant (p <0.01) higher binding of 99mTc- Cy5o.sCDioPIBMA39 to MAA-Ad was calculated (using a two tailed student t-Test, n = 4).
The supramocelucal interaction between MAA-Ad and Cy5o.sCDioPIBMA39 was also visualized by confocal microscopy, employing the Cy5 component of the polymer.2 For this purpose, the same experiment was repeated, but this time nonradioactive Cy5o.5CDioPIBMA39 was added to the MAA and MAA-Ad solutions. After washing, 10 0L of MAA (with or without-Ad) Cy5o.5CDi0PIBMA39 solution was pipetted onto culture dishes with glass insert (035mm glass bottom dishes No. 15, poly-d-lysine coated, y-irradiated, MatTek corporation). Images were taken on a Leica SP5 WLL confocal microscope under 63x magnification using Leica Application Suite software.
Cy5 fluorescence was measured with excitation at 633 nm, emission was collected at 650-700 nm.
Example 2: In vivo studies
Animals
All in vivo studies were performed using 2-3 month old Swiss mice (20-25 g, CrhOFl strain, Charles River Laboratories, USA). All animal studies were approved by the institutional Animal Ethics Committee (DEC permit 12160) of the Leiden University Medical Center. All mice were kept under specific pathogen-free conditions in the animal housing facility of the LUMC. Food and water were given ad libitum.
General SPECT imaging and biodistribution protocol
SPECT imaging was performed as follows: at 2 h after injection of 99mTc-labeled compounds mice were placed and fixed onto a dedicated positioned bed of a three-headed U-SPECT-2 (MILabs, Utrecht, The Netherlands) under continuous 1-2% isoflurane anesthesia. Radioactivity counts from total body scans or selected regions of interest (ROI) were acquired for 60 min using a 0.6 mm mouse multi-pinhole collimator in list mode data. For reconstruction from list mode data, the photo peak energy window was centered at 140 keV with a window width of 20%. Side windows of 5% were applied to correct for scatter and down scatter corrections. The image was reconstructed using 24 Pixel based Ordered Subset Expectation Maximization iterations (POSEM) with 4 subsets, 0.2 mm isotropic voxel size and with decay and triple energy scatter correction integrated into the reconstruction with a post filter setting of 0.25 mm, as described in W. Branderhorst, B. Vastenhouw and F. J. Beekman, Phys. Med. Biol., 2010, 55, 2023-2034. Volume-rendered images were generated from 2-4 mm slices and analyzed using Matlab R2014a software (version 8.3.0.532, MathWorks8 Natick, MA). Images were generated from maximum intensity protocols (MIP) adjusting the color scale threshold to optimal depiction of the tissues of interest, as set out in M. N. van Oosterom, R. Kreuger, T. Buckle, W. A. Mahn, A. Bunschoten, L. Josephson, F. W. B. van Leeuwen and F. J. Beekman, EJNMMI Res., 2014, 4, 56-56.
After imaging, the mice were euthanized by an intraperitoneal injection of 0.25 mL Euthasol (ASTfarma, Oudewater, The Netherlands).
To determine the biodistribution of the tracer, organs were collected from mice and counted for radioactivity in a dose-calibrator (VDC 101, Veenstra Instruments, Joure, the Netherlands) or a gamma counter (Wizard2 2470 automatic gamma scintillation counter,
Perkin Elmer). After collecting and counting all tissues together with the remaining activity in the carcass, the total amount of remaining radioactivity in the animal was counted and, after correction for physical decay, the urinary excretion expressed as the percentage of the total injected dose (%ID) was calculated. Radioactivity counts in tissues were expressed as the percentage of the total injected dose of radioactivity per gram tissue (%ID/g). Additionally, reconstructed images were generated and analyzed using Amide 1.0.2 software, available from Sourceforge, and as disclosed in A. M. Loening and S. S. Gambhir, Mol. Imaging, 2003, 2, 131-137. Mapping the distribution of 99mTc-labeled MAA-Ad in mice via i.v.
administration ( Model I, not according to the invention)
To determine whether MAA embolizations are tolerated by mice and whether MAA-Ad is delivered to the capillaries of the lungs, MAA-Ad was radiolabeled according to the manufacturer's instructions, and 0.1 mL of 99mTc-MAA-Ad in PBS (0.02 mg, 2 mg/mL) was injected into a tail vein of a mouse (Fig. 1A, Model I). At 2 h after injection, the organ distribution of the tracer in mice were imaged using SPECT and quantified with biodistribution studies (see SPECT imaging protocol and biodistribution studies described above, and Fig. 1). Local administration, model II (Example according to the invention)
An embolization setup in the liver was performed to mimic the clinical setup for liver radio- embolization (Fig. 1A, Model II). For this purpose, animals were anesthetized by intraperitoneal injection of a mixture containing Hypnorm (Vetapharma, Leeds, United Kingdom), dormicum (Roche, Basel, Switzerland), and water (1:1:2). After shaving and cleaning with ethanol (70%), the abdominal cavity was incised for 0.5 cm and the spleen was exposed outside the mouse. Of the 99mTc-MAA-Ad solution (2 mg/mL), 100 ί was injected into the spleen using a Myjector U-100 insulin syringe (29G x ½" 0.33 xl2 mm, Terumo Europe, Leuven, Belgium) and after 5 seconds the needle was removed and the spleen was positioned inside the peritoneal cavity. The incision was sutured by 2-4 stitches and the animals were placed under a heating lamp to maintain the body temperature at 37 °C. At 2 h after injection,
the organ distribution of the tracer in mice were imaged using SPECT and quantitated with biodistribution studies as described above (see Fig. 1).
Mapping the distribution of 99mTc-Cv5o.5CDioPIBMA39 in mice
To determine the natural distribution of 99mTc-Cy5o.sCDioPIBMA39 (Fig. 1C), 0.1 mL of PBS containing 99mTc-Cy50.5CDioPI BMA39 (1 mg/mL, 20 MBq) was i.v. injected (see Fig. 2). 2 h after injection SPECT imaging and biodistribution studies were performed as described above.
Mapping the distribution of 99mTc-Cv5o,5CDioPIBMA39 after MAA(-Ad) pre-administration The influence of MAA or MAA-Ad on the distribution of 99mTc-Cy5o.5CDioPIBMA39 was evaluated as follows: first, 0.1 mL containing MAA-Ad or non-functionalized MAA in 0.1 mL (0.02 mg, 2 mg/mL) was injected by either i.v. administration (Fig. ID, Model I) or local administration (Fig. IB, Model II). After 2 h, 0.1 mL of 99mTc-Cy5o.5CDioPIBMA39 in PBS (1 mg/ml, 20 MBq) was i.v. injected (see Fig. 2) and another 2 h later SPECT imaging and bio- distribution studies were performed as described above.
Figure 1 shows the SPECT imaging of (Model I, comparison without pre-targeting ) 99mTc-labeled Ad-MAA (for mapping biodistribution of MAA-Ad) or (Model II, according to the invention) pre-targeting with unlabeled MAA-Ad or (Model C) none followed by intravenous 9 mTc-Cy5o.5CDioPIBMA39. SPECT imaging is performed 2 h thereafter to visualize radioactivity in the (1) lungs, (2) liver, (3) kidneys, (4) stomach, and (5) urinary bladder.
This clearly illustrates the effectiveness and high selectivity that can be obtained for the subject process and materials.
Figure 2 then illustrates the binding of 99mTc-Cy5o.5CDi0PIBMA39 to MAA-Ad and MAA, quantified by radioactivity and expressed as a percentage of the total amount of radioactivity (99mTc-Cy5o.sCDioPIBMA39) added. The data in Figure 2 indicates a 5.7 times higher binding to MAA functionalized with the Ad guest moiety, as compared to non-functionalized MAA. Significance of difference is marked with ** (P < 0.01).
The following table (Table 1) shows the resultant measurements
Table 1 shows the bio-distribution of 99mTc-Cy5o.5CDioPIBMA39 following injection of:
none (reference distribution),
- MAA, or
5 - MAA-Ad and the bio-distribution of 99mTc-MAA-Ad administered via Models I and II.
The data is expressed as the mean + SD of the percentage of the injected dose per gram tissue (%ID/g) of 5 observations)), and is calculated from radioactivity counts in various tissues at 2 h post-injection of the tracer.
N.A.: data not available
Table 2 shows the biodistribution of 99mTc-labeled cyclodextrin polymers in mice with adamantane (Ad)-functionalized macro- aggregates (MAA) administrated into the lungs. Data (expressed as the mean ± sd of the percentage of the injected dose per gram tissue of at least 4 observations) are calculated from radioactivity counts in various tissues (%ID/g) of various tissues at 2 hr post-injection of the tracer.* = P<0.05 compared to none or non-functionalized MAA.
This is also illustrated by Figures 6 (a) to (d), wherein in particular Fig. 6 (a) and (b) shows the benefit of the benefit of multiple binding sites, as compared to the absence of the pre-targeting vector (c) and (d), respectively.
Example 3: Using Polymeric Microparticles
Example 2 was repeated, however using polystyrene beads as pre-targeting vector material. Cy5-Polystyrene-Ad(PS Ad) was prepared, and incubated with Cy31.5CD72PIBMA389.
The resultant distribution of the materials, and the low occurrence of shunting was illustrated by fluorescence spectroscopy (see Figure 3). This shows that any suitably dimensioned particulate materials may be employed.
Ad-CD host guest interaction between Polystyrene(Cy5.5)-Ad particles and Cv31.5CD72PIBMA398 polymer.
To a solution of Polystyrene(Cy5.5)-Ad (90 ■ 106 particles / μί; 1 μιη in size) in 100 μί H20, Cy31.5CD72PIBMA398 was added (0.35 nmol) and the mixture was gently shaken for lh at RT. Subsequently the particles were washed by sequential filtering over 100 kD Amicon filters (2 min, 10 rcf) and recollected in 100 μΐ H20. A drop of the solution was put on a petri dish and the particles were allowed to settle down on the glass before imaging under a SP5 Leica microscope. The Polystyrene particles were visualized by excitation at 633 nm and the emission was collected at 650 to 600 nm. The Cy31.5CD72PIBMA398 was visualized by excitation at 514 nm laser and the signal was collected at 550- 600 nm.
Example 4: Using Click chemistry
MAA functionalization with DBCO and subsequently with Cy5-(S03)azide-(S03)COOH
To a solution of MAA (20 g, 20 mg/mL) in 100 μΐ Phosphate buffer (0.1 M, pH 8.4), 6.4 μΐ (32 μg, 80 nmol) of a dibenzocyclooctyne-N-hydroxysuccinimidyl ester solution in DMSO (5 mg/mL) was added. After stirring overnight at RT the MAA was washed by pelleting (3 min, 13.4 rcf). The MAA was then resuspended in 100 μΐ Phosphate buffer (0.1 M, pH 8.4) and 5.7 μΐ (28 μg, 40 nmol) of a Cy5-(S03)azide-(S03)COOH solution in H20 (5 mg/mL) was added. After stirring for 2h at RT the particles were washed again by pelleting (3 min, 13.4 rcf) until the filtrate was no longer blue. The functionalized MAA was resuspended in 100 μΐ H20 and a drop of the solution was added to a petri dish. After the particles were settled down they were imaged using a SP5 Leica microscope. The MAA was visualized by brightfield and the Cy5-(S03)Azide-(S03)COOH was visualized by excitation at 633 nm and the emission was collected at 650 to 600 nm.
Example 5: In vitro Monitoring of the Host-Guest Complexes Synthesis and analysis
Synthesis and characterization of both adamantane-tetrafluorophenol (Ad-TFP) and β- cyclodextrin-poly(isobutylene-alt-maleic-anhydride) (Cy5o.sCD9PIBMA39, ~18.7 kDa, diameter, ~11.7 nm) were carried out as recently described in Rood MT, Spa SJ, Welling MM, Ten Hove JB, van Willigen DM, Buckle T, et al. Obtaining control of cell surface functionalizations via pre-targeting and supramolecular host guest interactions. Sci Rep. 2017;7:39908 and in Spa SJ, Welling MM, van Oosterom MN, Rietbergen DDD, Burgmans MC, Verboom W, et al. A Supramolecular approach for liver radioembolization. Theranostics. 2018;8(9):2377-86.
Radiolabeling of MAA(-Ad) with technetium-99m and stability testing
Labeling of macro aggregated albumin (MAA) with technetium (99mTc-MAA) and the stability of the 99mTc-chelation was determined in fetal calf serum (FCS, Life Technologies inc. CA) and PBS, after 2, 4, and 20 h. The release of radioactivity was determined with 2 centrifugation (3 min, l,200xg) and washing steps with PBS. Both were performed as described previously in Spa SJ, Welling MM, van Oosterom MN, Rietbergen DDD, Burgmans MC, Verboom W, et al. A Supramolecular approach for liver radioembolization. Theranostics. 2018;8(9):2377-86.
Labeling of Cy5o,5CD9PIBMA39 with indium-Ill (mln-Cy5o,5CD9PIBMA39) and stability testing The host-vector, Cy5o.sCDgPIBMA39, which contains an abundance of freely available -COOH moieties, was radiolabeled with indium-Ill. To 10 E1L of Cy5o.5CD9PIBMA39 (1 mg/mL PBS) was added 25-150 μΐ of an acidic solution of mlnCI3 (370 MBq/mL, Mallinckrodt Medical, Petten, The Netherlands). This mixture was gently shaken in the dark for 1 h at 37 °C. Thereafter, the pH was adjusted to 7.5 in PBS. The radiochemical purity of mln-Cy5o.5CD9PIBMA39 was determined at 1 and 20 h by instant thin layer chromatography (ITLC) on 1x7 cm ITLC-SG paper strips (Agilent Technologies, USA) with 0.25 M NH4-acetate (pH 5) as mobile phase.
The serum stability of the 99mTc-chelation was determined in fetal calf serum (FCS, Life Technologies Inc. CA). After 24 h the release of radioactivity was determined with centrifugation and washing steps as described above. To determine the stability in FCS, mln-
Cy5o.5CD9PIBMA39 was diluted in FCS (2.5 g/mL) and shaken in a water bath at 37 °C for 20h. After 2, 4, and 20h samples of 0.1 mL were taken and the release of radioactivity was assessed by ITLC. For comparison, a similar set-up was performed for mln-Cy5o.5CD9PIBMA3. Labelling of MAA with technetium-99m for 1 h at 37 °C yielded 92.8±3.8% of the total added radioactivity. After challenging 99mTc-MAA in either FCS or PBS for 20 h at 37 °C the release of radioactivity was less than 5%. Labeling of iln to Cy5o.sCD9PIBMA39 for 1 h at 37 °C yielded to 95.6±3.6% of the total added radioactivity determined by instant thin layer chromatography (ITLC). To determine the chelation stability of the latter, mln- Cy5o.5CD9PIBMA39 was challenged in FCS at 37 °C for 20 h. As depicted in Fig 7, over 20 h at 37 °C the amount of indium-Ill activity dissociating from mln-Cy5o.5CD9PIBMA39 was also less than 5%.
In vitro host-guest interactions and complex stability
In vitro evidence for the host-guest complex formation between MAA-Ad and mln- Cy5o.sCDgPIBMA39 was provided by comparing the mln-Cy5o.5CD9PIBMA39 binding to MAA-Ad and non-functionalized MAA (control). Mixtures of 0.1 mL containing either MAA-Ad or MAA (0.1 mg/mL) with 0.1 mL mln-Cy5o.sCD9PI BMA39 (10 g/mL, 1 MBq) were prepared in 0.8 mL PBS and the solutions were incubated for 1 h in a shaking water bath at 37 °C. After 2 rounds of spinning for 5 min at 1,500 x g, the decay corrected radioactivity of the pellet and supernatant was measured in a dose-calibrator. Following correction for background activity, the host-guest interaction was expressed as the percentage of the total amount of radioactivity (% binding). For stability measurements, either 0.1 mL MAA-Ad (0.1 mg/mL) with 0.1 mL mln- Cy5o.5CD9PIBMA39 (10 g/mL, 1 MBq) or 0.1 mL 99 mTc- MAA-Ad (0.1 mg/mL, 1 MBq) with 0.1 mL Cy5o.5CDgPI BMA3g (10 μg/mL) were prepared as described above and after removal of non- complexed materials the complex was diluted in either 0.8 mL PBS or FCS and incubated for 44 h in a shaking water bath at 37 °C. Following incubation durations of 2, 20, and 44 h, 0.1 mL samples were diluted in 1 mL of PBS and centrifuged for 5 min at 1,500 x g. The decay corrected radioactivity of both the pellet and supernatant was measured in a dose-calibrator.
Hereby, the radioactivity of the pellet represented association of CV50.5CD9PIBMA39 to MAA- Ad (expressed as % of binding).
In vitro a near two-fold increase in binding of mln-Cy5o.5CDi0PIBMA39to MAA-Ad (53.8±4.3%) was observed compared to what was achieved with non-Ad-functionalized MAA (29.4±5.1%; p<0.001/ n=8; Fig 8A). This indicates that Ad-CD host-guest interactions influence the complex formation. After 44 h incubation in either PBS or FCS, complex dissociation was found to be in the 10-20% range (see Fig 8B). Example 6: In vivo host-guest complex formation
The supramolecular host-guest interactions in the [99mTc-MAA-Ad-mln-Cy5o.5CD9PIBMA39] complex can be monitored in vivo using dual-isotope multiplexing up to 20 h after administration due to the physical short half-life of technetium-99m. In vivo studies were performed using 2-4-month-old Swiss mice (20-35 g, CrhOFl strain, Charles River Laboratories, USA). All animal studies were approved by the institutional Animal Ethics Committee (DEC permit 12160) of the Leiden University Medical Center. Mice were kept under specific pathogen-free conditions in the animal housing facility of the LUMC. Food and water were provided ad libitum.
Animal model
An embolization setup of the liver was used to mimic the clinical setup for liver radioembolization according to previously described procedures (pa SJ, Welling MM, van Oosterom MN, Rietbergen DDD, Burgmans MC, Verboom W, et al. A Supramolecular approach for liver radioembolization. Theranostics. 2018;8(9):2377-86 and Kasuya H, Kuruppu DK, Donahue JM, Choi EW, Kawasaki H, Tanabe KK. Mouse models of subcutaneous spleen reservoir for multiple portal venous injections to treat liver malignancies. Cancer Res. 2005;65(9):3823-7). 99mTc-MAA-Ad (0.1 mg/mL, 2-5 MBq, n=6) was injected into the spleen of the mice (embolization step). Two h after embolization, a second injection with mln- Cy5o.sCD9PIBMA39 (1 0g, 10 MBq) was administered I.V.. At 2, 12, 20, or 44h the animals were imaged using SPECT and fluorescence imaging and quantified with biodistribution studies (see
SPECT and fluorescence imaging protocols and biodistribution studies described below). Non- functionalized 99mTc-MAA (0.1 mg/mL, 2-5 MBq, n=6), or mere PBS (n=3) served as controls.
General SPECT imaging
Mice were placed and fixed onto a dedicated positioning bed of a three-headed U-SPECT-2 (MILabs, Utrecht, the Netherlands) at various intervals after injection of the secondary vector, while being under continuous 1-2% isoflurane anesthesia. Radioactivity counts (range 0-600 keV) from total body scans were acquired for 30 min. For reconstruction from list mode data, the photo peak energy window was centered at 140 keV (for technetium-99m) or 240 keV (for indium-Ill) with a window width of 20%. Additionally, on the reconstructed images, using AMIDE's Medical Image Data Examiner (http://amide.sourceforge.net) regions of interest (ROI's) were drawn over various tissues to determine the signal intensities herein over time. ROI's drawn over the jugular veins were taken as a representative of the blood values. After imaging, mice were euthanized and the organs were removed and weighed to determine the percentage of injected dose per gram tissue (%ID/g). Blood samples obtained at various intervals of sacrifice, were used to determine the clearance from the blood fraction (expressed as the pharmacological half-life ti/2) was calculated using GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego CA, USA). Dual-isotope SPECT imaging facilitated in vivo monitoring of host-guest interactions between mln-Cy5o.5CD9PIBMA39 and 99mTc-MAA-Ad.
Due to the 6h half-life of 99mTc its distribution could only be reliably monitored up to 20 h p.i. SPECT imaging. Fig 9A demonstrates time-related (2, 12, 20 and 44 h) dual-isotope SPECT imaging of A) intrasplenic administration of guest-vector 99mTc-MAA-Ad and B) I.V. administered host-vector mln-Cy5o.5CD9PIBMA39. Organs are marked as (1) lungs, (2) liver, (3) spleen, and (4) kidneys. The scale bars indicate the intensity of radioactivity expressed as arbitrary units. At 20 h p.i. of the locally administered primary vector (99mTc-MAA-Ad) revealed a profound signal in the spleen (injection side) and in the liver. Biodistribution studies confirmed residual activity in the spleen (injection site; amounting to 80.4±23.2%ID/g, 64.2±5.6%ID/g, and 57.9±8.4%ID/g at 2, 12, and 20 h p.i. respectively) and demonstrated
prolonged diffusion of the radioactive signal to the liver (amounting to 13.2±2.2%ID/g, 36.4±5.3%ID/g, and 43.0±20.2%ID/g at 2, 12, and 20 h p.i. respectively). Fig 10 indicates the bio-distribution of pre-targeting the liver with locally administered guest vector: 99mTc-MAA- Ad at various intervals. Data (expressed as the mean ± SD of the percentage of the injected dose per gram tissue (%ID/g) of 3 observations were calculated based on the radioactive counts measured in indicated tissues at 2, 12, and 20h post-injection. Values of 99mTc-activity calculated at 44h p.i. were unreliable as the radioactivity counts are very low.
A significant uptake of "free" 99mTc in known background tissues such as salivary gland, thyroid, stomach, and intestines is a strong indication of stability of 99mTc-MAA-Ad in vivo.
As result of the 2.8 d half-life of mln, the time-related uptake of mln-Cy5o.5CD9PIBMA39 in the liver of mice could be studied up to 44 h p.i. Fig 9B shows the distribution of the secondary host-vector (mln-Cy5o.5CD9PIBMA39) in mice pre-targeted with 99mTc-MAA-Ad at 2, 12, 20 or 44 h p.i. Dual-isotope imaging was possible up to 20 h p.i. a nd displayed liver co-localization between 99mTc-MAA-Ad and mln-Cy5o.5CD9PIBMA39 in the liver. The liver accumulation of iln-Cy5o.5CD9PIBMA39 was further studied after pre-targeting with either 99mTc-MAA-Ad, 99mTc-MAA, or PBS (12 h p.i.; see Fig 11B). Fig 11 shows A) dual-isotope-SPECT imaging of mice pre-targeted with intrasplenic administration of guest vector 9mTc-MAA-Ad at 12h p.i.. Mice, pre-targeted with either non-functionalized 99mTc-MAA or PBS are used for comparison. Organs are marked as (1) lungs, (2) liver, (3) spleen, and (4) kidneys. B) Bio-distribution of host vector mln-Cy5o.5CD9PIBMA39 12h after I.V. administration c. C) Uptake of mln- Cy5o.5CDgPIBMA39 in the liver determined by ex-vivo fluorescence imaging at equal settings. The scale bar indicates the intensity of fluorescence expresses as photons/sec/cm2.
Our imaging observations were quantified either by calculating radioactivity counts in ROI's (see Fig 12) or via %ID/g biodistribution studies (see Fig 13A Bio-distribution of I.V. administered mln-Cy5o.5CDgPIBMA39 (host molecule) 12h after hepatic pre-targeting with guest vectors 99mTc-MAA-Ad (blue bars), ""Tc-MAA (red bars) or PBS (green bars). The significance of difference (p<0.01) is indicated with * according to Student's T-test and shown in Table 3: The bio-distribution of I.V. administered mln-Cy5o.5CD9PIBMA39 (host-vector) after
hepatic pre-targeting with guest-vector: 99mTc-MAA-Ad, 99mTc-MAA, or PBS. Data (expressed as the mean ± SD of the percentage of the injected dose per gram tissue (%ID/g) of 3-6 observations) were calculated based on the radioactive counts measured in various tissues at 2, 12, 20, and 44h post-injection of the radioactive tracer. The significance of difference (p<0.01) is indicated with * compared to = 99mTc-MAA (control), or * compared to PBS (mln- CV50.5CD9PIBMA39 reference distribution) according to Student's T-test.
Table 3
Tissue Time Guest: "^n-CySo sCDgPIBMABg
(h p.i.) 99mTc-MAA-Ad 99mTc-MAA PBS
Blood 2 22.5 1 6.4* 18.1 + 2.9* 13.7 + 1.9
12 2.6 1 0.6 4.4 + 1.7 2.5 + 1.0
20 3.5 1 0.8 3.8 + 0.7 3.1 + 1.3
44 1.8 + 0.2 1.6 + 0.1 1.8 + 0.3
Heart 2 10.2 1 4.1** 8.0 1 1.3 7.2 + 1.5
12 5.2 1 0.2* 6.0 + 2.1 3.4 + 0.6
20 4.1 + 0.3* 5.0 + 0.9 3.9 + 1.8
44 8.3 + 2.4* 6.2 + 0.6* 4.5 + 0.1
Lungs 2 10.2 + 2.5** 7.6 1 1.3 8.9 1 1.4
12 3.9 + 1.0* 7.1 + 0.4* 2.6 + 1.3
20 4.2 + 0.5* 5.3 + 0.9 4.6 + 1.8
44 7.3 + 1.1* 5.5 + 1.3 7.6 + 1.0
Liver 2 14.9 + 6.1* 11.4 1 2.7* 7.6 + 2.3
12 25.8 1 2.9** 12.9 + 2.5 11.7 + 2.9
20 27.0 + 1.3** 10.8 + 4.7 8.6 + 3.1
44 26.2 + 2.1** 22.7 + 2.3* 15.7 1 2.8
Spleen 2 9.7 + 3.4* 7.1 + 0.8* 5.5 + 1.4
12 13.5 + 1.6 16.7 + 2.6 9.4 + 2.7
20 11.8 + 1.5* 9.4 1 0.5 10.2 1 2.7
44 17.9 + 5.0** 13.0 + 3.1 11.3 + 0.7
Kidneys 2 20.9 + 7.0** 10.0 + 1.9* 13.0 + 2.3
12 33.5 1 5.8 41.1 + 6.8 31.8 + 6.1
20 29.7 1 5.3 29.2 + 5.8 33.3 1 12.2
44 29.6 + 3.6 29.7 1 3.3* 26.0 + 3.6
Muscle 2 4.0 ± 1.9** 2.0 + 0.4 2.2 + 0.4
12 4.0 1 0.4 3.5 1 1.4 2.9 + 0.7
20 2.2 + 0.6* 3.4 1 0.9 2.6 1 0.6
44 3.9 + 0.4** 3.4 + 0.3* 3.6 + 0.3
Brain 2 0.6 1 0.2** 0.4 + 0.1 0.4 + 0.1
12 0.4 1 0.1* 0.5 + 0.2 0.3 + 0.1
20 0.3 1 0.1 0.5 + 0.2 0.3 + 0.2
44 0.5 + 0.02* 0.5 1 0.04* 0.4 1 0.02
These analyses revealed that up to 20 h p.i. the uptake of mln-Cy5o.5CD9PIBMA39 in the liver increased and again decreased after 44 h p.i. This uptake was nearly 3-fold higher (p<0.01) compared to the liver uptake of mln-Cy5o.5CDgPIBMA39 in control mice injected with 99mTc- MAA (10.8 1 4.7 %ID) or PBS (8.6 + 3.1 %ID). Despite the presence of 99mTc-MAA(-Ad) signal in the spleen, accumulation of iln-Cy5o.5CD9 IBMA39 in this organ was equal to that in PBS controls (Table 3). Whether the uptake of mln-Cy5o.5CD9PIBMA39 in the liver pre-targeted with 99mTc-MAA depends on non-specific interaction with MAA (as we measured in vitro Fig 8B) or because of the clogging of the microvasculature by MAA particles could not be determined.
Liver-to-blood ratios were determined from the biodistribution data and were compared to 99mTc-MAA (control) and PBS (for the regular clearance of the secondary vector). At all intervals liver-to-blood ratios for mice pre-targeted with 99mTc-MAA-Ad were highest. At 12- 20 h p.i. they were significantly (p<0.01) higher than those pre-targeted with 99mTc-MAA or PBS, as shown in Fig 13B: Dynamic hepatic uptake of I.V. administered mln- Cy5o.5CDgPIBMA39. Data are expressed as the mean + SD ratios of the %ID/g in liver and blood measured at 2, 12, 20, and 44h post-injection of mln-Cy5o.5CD9PIBMA39. The significance of difference (p<0.01) of hepatic uptake of 111ln-Cy5o.5CDgPIBMA39 in mice pre-targeted with 99mTc-MAA-Ad (blue bars) is indicated with * compared to 99mTc-MAA (control clearance, red
bars), or # compared to PBS (regular distribution, green bars) according to Student's T-test. For all pre-targeting settings at 44h p.i. liver-to-blood ratios for mln-Cy5o.5CD9PIBMA39 are increased compared to the earlier intervals which is indicative for clearance of proteolytic or metabolic products and in Table 4: Dynamic hepatic uptake of I.V. administered mln- Cy5o.5CDgPIBMA39. Data (expressed as the mean ± SD liver-to-blood ratios calculated between the percentages of the injected dose per gram tissue (%ID/g) of liver and blood (of 3-6 observations) based on the radioactive counts measured in these tissues at 2, 12, 20, and 44h post-injection of mln-Cy5o.5CD9PIBMA39 after hepatic pre-targeting. The significance of difference (p < 0.01) is indicated with * compared to = 99mTc-MAA (control), or * compared to PBS (reference distribution) according to Student's T-test.
Table 4:
At 44h p.i. we found the highest (but not with significant difference) liver-to-blood ratio between both groups of 99mTc-MAA-Ad or 99mTc-MAA pre-targeted mice.
A remarkable observation in the mln-SPECT images from 2 h onwards was the accumulation of activity in the shoulder joints, knee joints, pelvis, kidney, and diffuse uptake in bone (Fig 9B & Fig 11B). In vitro stability studies revealed less than 5% of mln3+ dissociation during 44 h incubation at 37 °C in serum (Fig 7). In vivo, already at 2 h dissociation of mln3+ was observed (Fig 9A and Fig 11A) (20, 21), but bone uptake remained unchanged over time (Fig 9A & 9B). Given the concurrence with known reservoirs for ionic iln3+ (19-21), the isotope is most likely dissociated from the -COOH moieties on the polymer backbone. The background uptake in other tissues such as: blood, heart, lungs, muscle, and brains showed a decrease in uptake of mln radioactivity over time (see Table 3), indicative for the blood clearance of mln-
CV50.5CD9PIBMA39. Another remarkable observation is the shorter blood half-life (ti/2 = 192 min) calculated for inln-Cy5o.5CD9PIBMA39 in mice pre-targeted with 99mTc-MAA-Ad compared to the half-life of mln-Cy5o.5CD9PIBMA39 in mice pre-targeted with 9 mTc-MAA (ti/2 = 306 min) or PBS (ti/2 = 318 min) (Table 5). 2 h after I.V. tracer administration, a significant portion of mln-Cy5o.5CD9PIBMA39 rapidly assembles with 99mTc-MAA-Ad in the liver (Table 3) Up to 20 h p.i., circulating mln-Cy5o.5CD9PIBMA39 increased uptake in the liver to values of -27 %ID/g). Also in kidneys and spleen the values for mln-Cy5o.5CD9PIBMA39 increased in time.
The observed intestinal uptake of mln-isotopes is shown in Table 5 illustrate the uptake of I.V. administered mln-Cy5o.5CD9PIBMA39 in scavenging tissues, excretion rate and clearance. Data (expressed as the mean ± SD of the percentage of the injected dose per gram tissue (%ID/g) of 3-6 observations) were calculated based on the radioactive counts measured in various tissues at 2, 12, 20, a nd 44h post-injection of the radioactive tracer. The significance of difference (p<0.01) is indicated with * compared to = 99mTc-MAA (control), or * compared to PBS (reference distribution) according to Student's T-test.
Table 5
Tissue Time Guest
(h p.i.) 99mTc-MAA-Ad 99mTc-MAA PBS
Urine & bladder 2 5.9 ± 0.8 6.2 ± 0.9 7.5 ± 1.5
12 4.8 ± 1.7 4.3 ± 2.4 3.2 ± 0.6
20 3.6 ± 0.9* 4.7 ± 1.8* 2.5 ± 0.6
44 4.9 ± 0.7* 4.0 ± 0.7 5.6 ± 0.3
Thyroid gland 2 8.0 ± 2.9** 6.8 ± 1.7 5.8 ± 0.7
12 8.2 ± 2.4 5.1 ± 5.2 5.3 ± 2.1
20 6.2 ± 1.1 5.7 ± 2.1 5.8 ± 1.7
44 6.0 ± 2.3 4.5 ± 2.2 5.8 ± 1.6
Salivary gland 2 5.8 ± 2.1** 4.3 ± 0.8 4.5 ± 0.8
12 6.5 ± 1.3* 7.6 ± 1.9 5.2 ± 1.1
20 7.4 ± 0.7* 7.5 ± 1.8 5.7 ± 1.5
44 11.2 ± 0.4 11.3 ± 0.7 11.2 ± 4.6
Stomach 2 2.210.9* 1.910.6 1.7 + 0.6
12 2.3 ±0.7 1.5 + 0.7 1.2 + 0.9
20 2.0 ± 1.0** 1.2 + 0.8 0.911.1
44 5.610.5* 4.510.8* 6.7 + 0.5
Intestines 2 5.112.4** 3.2 + 0.5 2.7 + 0.6
12 5.0 + 2.8 8.1 + 3.6 3.6 + 1.5
20 5.910.7 8.4 + 2.9 4.6 + 3.0
44 11.011.8* 7.7 + 2.6 8.2 + 0.5
Excretion (%ID) 2 4.512.7 4.2 + 6.1 3.6 + 0.7
12 11.119.5* 15.3 + 9.3 21.0+1.7
20 14.311.7* 8.514.6 11.713.6
44 17.712.4 18.8 + 1.2 22.8 + 5.0
Clearance half-life ti/2 (min)
192 306 318
(R20.864) (R20.980) (R20.943)
The data in Table 5 is indicative for hepatically engulfment followed by intra-intestinal secretion. In vitro stability studies revealed less than 5% of mln3+ dissociation during 44 h incubation at 37 °C in serum (Fig 7), following extrapolation of the biodistribution data this related to the in vivo findings (Fig 9B & Table 3). In vivo, already at 2h the accumulation pattern of mln3+was observed (Fig 9Aand Fig HA) (20, 21), but this remained stable (Fig 9A &9B).
Fluorescence imaging protocol
Dual-labeled Cy5o.sCD9PIBMA39was also equipped with a Cy5 fluorophore to perform confocal microscopy and allows to perform macroscopic and eventually microscopic evaluation of the fluorescent signal of CV50.5CD9PIBMA39 in excised tissues from mice using a preclinical S Spectrum imaging system (Caliper Life Science, Hopkinton, MA). Images of the Cy5-dye were acquired following excitation at 640 nm, and light was collected > 680 nm (acquisition time 5 s). Quantitative analysis of the fluorescence in the tissues (photons/sec/cm2) was performed
using the Living Image software from xenogeny v 3.2 (Caliper LS) at equal image adjustment settings.
Ex vivo fluorescence imaging analysis of the biodistribution of mln-Cy5o.5CD9PIBMA39 in various excised tissues of mice pre-targeted with 99mTc-MAA-Ad revealed an intense fluorescence signal in the liver (Fig 11) at all time points after tracer administration (2.0-2.6 x 1010 p/s/cm2/sr) which was higher compared to those for MAA (0.9-1.5 x 1010 p/s/cm2/sr) or PBS (0.06-0.4 x 1010 p/s/cm2/sr) which follows the trend as observed for mln- Cy5o.sCD9PIBMA39. At 44h p.i. all values dropped below 0.6 x 1010 p/s/cm2/sr.
Statistical analysis
All data are presented as mean value (±SD) of 3-6 independent measurements. Statistical analysis for differences between groups in the animal studies were performed by Student's paired T-test with one-tailed distribution. Significance was assigned for p-values < 0.05. All analyses and calculations were performed using Microsoft® Office Excel 2010 and GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, USA).
Claims
1. A composition for use in vivo parenteral local administration, comprising a pre-targeting vector component comprising a microparticle, the microparticle comprising one or more reactive guest moieties able to form a high affinity interaction with a complementary host moiety residing on a secondary component.
2. A composition according to claim 1, wherein the high affinity interaction comprises formation of one or more non-covalent bonds.
3. A composition according to claim 2, wherein the high affinity interaction comprises a supramolecular host-guest interaction in an inclusion affinity complex.
4. The composition according to any one of claims 2 or 3, wherein the host moiety is selected from the group consisting of streptavidin, cyclodextrin, antibodies, antibody fragments, ligands and aptamers, and wherein the guest moiety is a moiety complementary to the respective host, preferably wherein the guest moiety is a multivalent guest moiety.
5. The composition according to claim 4, wherein the guest moiety comprises adamantane groups, and wherein the host moiety includes cyclodextrin groups
6. A composition according to claim 1, wherein the high affinity interaction comprises formation of one or more covalent bonds.
7. The composition according to claim 6, wherein a host moiety is functionalised to form a covalent click connection with a guest moiety.
8. The composition according to claim 6, wherein the host moiety comprises one or more azide groups, and wherein the guest moiety comprises one or more reactive alkyne groups, preferably wherein both groups are suited for copper-free click chemistry.
9. The composition according to any one of claims 1 to 8, wherein the pre-targeting vector component is dimensioned and configured to be able to accumulate in the desired part of the body or organ after administration, and being targeted thereto by the administration route, and/or passive diffusion from a deposition site.
10. The composition according to any one of the previous claims, wherein the composition comprises microparticles capable of lodging themselves in a target tissue in a diagnostically and/or therapeutically effective amount, or in an amount suitable to solicit a diagnostically and/or therapeutically effective response upon exposure to the complementary component.
11. A composition according to any one of claims 1 to 9, wherein the pre-targeting vector further comprises a diagnostic label.
12. A composition according to claim 10, wherein the diagnostic label is selected from the group consisting of: a diagnostic agent, an imaging agent, a contrast agent, preferably a radioactive isotope or radiation excitable agent, preferably wherein the diagnostic label is selected from the group consisting of magnetic resonance contrast labels, radioopaque contrast labels, ultrasound contrast labels, fluorescent contrast labels and (radio)isotope contrast labels, or combinations thereof.
13. The composition according to claim 11, wherein the diagnostic label comprises a source of detectable gamma rays.
14. The composition according to claim 12, wherein the diagnostic label comprises a medical radioisotope, preferably 99mTc.
15. The composition according to any one of the previous claims, wherein the pre-targeting microparticles have an average diameter in the range of from 1 to 1000 μιη.
16. The composition according to any one of the previous claims, wherein at least 90 percent of the pre-targeting microparticles have an average diameter of from 20 to 500 μητ
17. The composition according to any one of the previous claims, wherein the pre-targeting vector comprises one or more polymers.
18. A secondary vector component for intravenous or local administration and for forming a high affinity interaction with a complementary guest moiety of the pre-targeting vector composition according to any one of claims 1 to 17; wherein the secondary component comprises a complementary host functionality, and at least one agent selected from the group consisting of: a diagnostic agent, an imaging agent, a contrast agent, or a therapeutic agent, preferably a radioactive isotope, a chemotherapeutic agent, or a combination or multitude thereof.
19. The component according to claim 18, wherein an agent is selected from the group consisting of one or more: anti-cancer agents, antibiotics, antihistamines, hormones, steroids, immune response activating agents, and therapeutic proteins.
20. The composition according to claim 18, wherein a diagnostic agent is selected from the group consisting of magnetic resonance contrast agents, negative contrast agents, radioopaque contrast agents, ultrasound contrast agents, fluorescent contrast agent and (radio)isotope contrast agents.
21. The component according to claim 19 or 20, wherein the radioactive isotope is selected from the group consisting of: Y-90, P-32, P-33, Sc- 47, Cu-64, Cu-67, As-77, Ph-105, Pd-109, Ag-111,
Pr-143, Sm-153, Tb-161, Ho-166, Lu-177, Re-186, Re-188, Re-189, lr-194, Au-199, Pb-212, I- 131, Ac-225, Ra-223, Ra-224, and Bi-213.
22. A kit for forming a diagnostic and/or therapeutic matrix at a target site within a body, the kit comprising:
a pre-targeting vector component composition according to any one of claims 1 to 17; and
a complementary secondary component according to any one of claims 18 to 21, the complementary component being functionalised with a complementary compound that has a high affinity for the pre-targeting vector component.
23. A kit according to claim 22, wherein the secondary component comprises a therapeutic agent for delivery to the target tissue.
24. A method of treating a condition in vivo in a patient, the method comprising the steps of:
(a) providing an administration medium comprising pre-targeting microparticles with a guest component according any one of claims 1 to 15; and
(b) delivering a diagnostically and/or therapeutically effective amount of the a pre- targeting vector component into the target tissue, preferably into the tumour or tumour bearing tissue; and
(c) subjecting the target tissue to a diagnosis step to determine if significant shunting occurs or if the pre-targeting vector essentially remains in place in the target tissue; and
(d) administering a diagnostically and/or therapeutically effective amount of a complementary secondary component comprising a diagnostic and/or therapeutic agent to the patient, thereby ultimately generating a diagnostic and/or therapeutically active matrix in the subject tissue.
25. The method according to claim 21, wherein (b) comprises delivering the pre-targeting vector in a blood vessel that drains to the target region.
26. The method according to claim 25, wherein step (b) further comprises positioning a delivery device in proximity to a target region of the blood vessel, and ejecting the pre- targeting vector microparticle composition from the delivery device such that the particles are positioned in the target region.
27. The method according to claim 25 or 26, wherein the target area is selected from one or more of tumour, liver, kidney, pancreas, thyroid, heart, brain, nervous system, breast,
bladder, prostate, skin, lymphatics, cartilage, bone, tendon, ligament, blood vessel, spinal cord, Skin, lymph nodes, tumour tissue, and spinal cord.
28. A method of locally treating a disease via a local embolization therapy, comprising administering to a target area of a patient in need thereof a kit according to any one of claims 22 or 23 suitable for diagnosing and treating the disease, in the appropriate order.
29. The method of claim 28, wherein the target area is selected from the group consisting of: liver, kidney, pancreas, thyroid, heart, brain, nervous system, breast, bladder, prostate, skin, lymphatics, cartilage, bone, tendon, ligament, blood vessel, spinal cord, skin, lymph nodes, and tumour tissue.
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