WO2024220548A2 - Irm moléculaire utilisant des micelles ciblant ccr2 pour la détection de métastases de nœud lymphatique - Google Patents

Irm moléculaire utilisant des micelles ciblant ccr2 pour la détection de métastases de nœud lymphatique Download PDF

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WO2024220548A2
WO2024220548A2 PCT/US2024/024999 US2024024999W WO2024220548A2 WO 2024220548 A2 WO2024220548 A2 WO 2024220548A2 US 2024024999 W US2024024999 W US 2024024999W WO 2024220548 A2 WO2024220548 A2 WO 2024220548A2
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mri contrast
delivery system
mri
mcp1
nanoparticles
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WO2024220548A3 (fr
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Eun Ji Paige CHUNG YOO
Noah Tom TRAC
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University of Southern California USC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1866—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid

Definitions

  • the present invention is related to targeted micelles for the detection of lymph node metastases. BACKGROUND Nearly 20 million patients are diagnosed with cancer each year globally.
  • LN metastasis Given that an estimated 90% of solid cancers metastasize through the lymph nodes (LNs), the accurate detection of LN metastasis is critical in cancer staging, treatment, and patient outcome. (2) For example, melanoma progression from regional LN to distant metastasis is associated with a 55% decrease in patient 5-year survival, demonstrating the importance of the detection and treatment of LN metastasis. (3) In addition, early detection of metastatic LN recurrence has been reported to increase patient survival from 14% to 36% by identifying patients that would benefit from adjuvant chemo- and immunotherapies. (4) Despite the clear significance of metastatic LN detection in patient outcomes, LN biopsies remain the clinical standard for detecting LN metastasis during initial cancer screening.
  • PET/CT Positron emission tomography/computed tomography
  • NIRF Near- infrared fluorescence
  • US Ultrasonography
  • MRI Magnetic resonance imaging
  • MRI is limited by low sensitivity ( ⁇ 35%), as traditional gadolinium contrast agents cannot differentiate healthy from metastatic LNs due to nonspecific accumulation.
  • MRI scans are used to visualize LN size and shape to determine metastasis.
  • these physical characteristics are poor diagnostic factors for LNs with micrometastases ( ⁇ 1 cm), resulting in misdiagnosis rates as high as 60%.
  • the safety of clinical gadolinium contrast agents has been of concern, as they have been demonstrated to accumulate in the brain, bones, and liver.
  • nanomedicine approaches have the potential to develop contrast agents with superior sensitivity and safety over gadolinium contrast agents.
  • peptide amphiphile micelles are nanoparticles self-assembled from amphiphilic monomers comprised of a bioactive peptide headgroup and a hydrophobic lipid tail.
  • the headgroup can be engineered to incorporate other payloads, such as gadolinium (Gd) chelates, to enable multimodal functionality, including therapy, imaging, and molecular targeting.
  • Gd gadolinium
  • PAMs are synthesized using poly(ethylene glycol) (PEG) as a linker, which mitigates nanoparticle clearance by the mononuclear phagocyte system (MPS).
  • PAMs have also been demonstrated to be biocompatible and cleared within 7 days through renal filtration.
  • an MRI contrast delivery system includes nanoparticles that are each equipped with targeting peptides for CCR2 receptors and an MRI contrast moiety. These components are conjugated to the nanoparticles, which are made from hydrophobic lipids. This design enables the nanoparticles to "hitchhike" on monocytes, leveraging their natural migration pathways to efficiently target metastatic lymph nodes (LNs).
  • a diagnostic agent is designed for use in magnetic resonance imaging (MRI) to detect lymph node (LN) metastasis.
  • This agent includes nanoparticles that incorporate gadolinium (Gd) and a peptide derived from the C ⁇ C chemokine receptor type 2 (CCR2)-binding motif of monocyte chemotactic protein 1 (MCP1).
  • Gd gadolinium
  • CCR2 C ⁇ C chemokine receptor type 2
  • MCP1 monocyte chemotactic protein 1
  • a gadolinium-loaded micelle platform targeted to tumor cells for the detection of lymph node metastases in cancer.
  • micelles chelated with gadolinium ions and functionalized with a 23-residue peptide derived from the binding motif of the MCP1 protein MCP1-Gd micelles
  • MCP1-Gd micelles MCP1-Gd micelles
  • CCR2 C-C chemokine receptor 2
  • MCP1-Gd micelles the interactions between MCP1-Gd micelles and monocytes are characterized in vitro, and the influence of circulating monocytes on MCP1-Gd accumulation in the lymph nodes will be investigated using in vivo models of metastatic melanoma. 2022-067-01
  • techniques are provided for the accurate detection of cancer metastasis in lymph nodes which is crucial for patient treatment and prognosis.
  • the standard clinical lymph node biopsy while informative, often causes chronic pain and lymphedema while suffering from sampling bias.
  • Conventional MRI does not provide the needed sensitivity and specificity for diagnosing metastases in normal-sized lymph nodes.
  • MCP1-Gd micelles specifically bind to CCR2-expressing cells and produce a 55% higher T1-weighted MR signal in metastatic lymph nodes than in healthy ones. Further in vivo studies indicated that circulating CCR2+ monocytes help transport and accumulate these micelles in the lymph nodes, underscoring their potential as a contrast-enhancing tool in MRI for detecting lymph node metastases.
  • the techniques described herein relate to the MRI contrast delivery system wherein the nanoparticles are configured to hitchhike on monocytes for targeted delivery to metastatic lymph nodes, thereby enhancing MRI signal specificity and sensitivity.
  • the techniques described herein relate to a method for detecting lymph node (LN) metastasis in a subject using magnetic resonance imaging (MRI).
  • the method involves: administering to the subject a composition that includes micelles incorporating gadolinium (Gd) and peptides derived from the CCR2-binding motif of MCP1; allowing for the migration of monocytes hitchhiked by the micelles to metastatic LNs; and conducting MRI to detect the enhanced signal from the metastatic LNs due to the accumulation of the gadolinium-incorporating micelles or fragments therefrom.
  • Gd gadolinium
  • MCP1 CCR2-binding motif of MCP1
  • FIGURE 1 MCP1-Gd micelles accumulate in metastatic lymph nodes (MLNs) through CCR2-binding to cancer cells and monocyte hitchhiking.
  • MCP1-Gd and scrMCP1-Gd are biocompatible with SVEC4–10 lymphatic endothelial cells and WEHI-274.1 monocytes after 48 h.
  • N 6.
  • (C) Immunofluorescent staining of sectioned LNs indicates greater CCR2 expression (green) in metastatic LNs (MLNs) than control LNs (CLNs). Sections were counterstained with DAPI (blue). Scale bar 100 ⁇ m.
  • (D) Flow cytometric analysis indicates greater CCR2 expression in MLNs than CLNs. *p ⁇ 0.05. N 4.
  • rMLN metastatic LN recurrence
  • FIGURES 7A and 7B Micelle size influences in vivo lymph node accumulation.
  • A) Size measurements of micelles synthesized using different PEG lengths. *p ⁇ 0.05. N 3.
  • B) In vivo inguinal lymph node accumulation of different-sized Cy7-labeled micelles 3 h post-injection indicates that smaller micelle size trends toward favorable lymph node accumulation. *p ⁇ 0.05. N 4.
  • FIGURE 8. Transmission electron micrograph of scrMCP1-Gd confirms formation of spherical micelles ⁇ 12 nm in diameter.
  • FIGURE 9 R1 relaxivity of scrMCP1-Gd micelles (1.78 mM-1s-1) is similar to that of the clinical contrast agent Magnevist (3.20 mM -1 s -1 ).
  • FIGURE 12. Transwell assay evaluating monocyte migration past a lymphatic endothelial monolayer confirms monocyte migration when stimulated with a chemoattractant (FBS).
  • FBS chemoattractant
  • Ri where i is an integer
  • Ri include hydrogen, alkyl, lower alkyl, C1-6 alkyl, C 6-10 aryl, C 6-10 heteroaryl, -NO 2 , -NH 2 , -N(R’R”), -N(R’R”R”’) + L-, Cl, F, Br, -CF 3 , -CCl 3 , -CN, - SO3H, -PO3H2, -COOH, -CO2R’, -COR’, -CHO, -OH, -OR’, -O-M + , -SO3-M + , -PO3-M + , -COO-M + , - CF2H, -CF2R’, -CFH2, and -CFR’R” where R’, R” and R”’ are C1-10 alkyl or C6-18 aryl groups; single letters (e.g., "n" or "o") are 1, 2, 3, 4, or
  • integer ranges explicitly include all intervening integers.
  • the integer range 1-10 explicitly includes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the range 1 to 100 includes 1, 2, 3, 4. . . .97, 98, 99, 100.
  • intervening numbers that are increments of the difference between the upper limit and the lower limit divided by 10 can be taken as alternative upper or lower limits. For example, if the range is 1.1. to 2.1 the following numbers 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 can be selected as lower or upper limits.
  • concentrations, temperature, and reaction conditions e.g.
  • concentrations, temperature, and reaction conditions e.g., pressure, pH, etc.
  • concentrations, temperature, and reaction conditions e.g., pH, etc.
  • concentrations, temperature, and reaction conditions e.g., pH, etc.
  • concentrations, temperature, and reaction conditions can be practiced with plus or minus 10 percent of the values indicated rounded to three significant figures of the value provided in the examples.
  • concentrations, temperature, and reaction conditions e.g., pressure, pH, flow rates, etc.
  • concentrations, temperature, and reaction conditions can be practiced with plus or minus 50 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples.
  • concentrations, temperature, and reaction conditions e.g., pressure, pH, flow rates, etc.
  • concentrations, temperature, and reaction conditions can be practiced with plus or minus 30 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples.
  • concentrations, temperature, and reaction conditions can be practiced with plus 2022-067-01 or minus 10 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples.
  • concentrations, temperature, and reaction conditions e.g., pressure, pH, flow rates, etc.
  • concentrations, temperature, and reaction conditions can be practiced with plus 2022-067-01 or minus 10 percent of the values indicated rounded to or truncated to two significant figures of the value provided in the examples.
  • the term "conservative substitutions” describes the process of replacing one or more amino acids in a polypeptide with others that possess similar chemical and physical properties, thereby preserving the protein's structural integrity and functionality. Hydrophobic, nonpolar amino acids such as leucine, isoleucine, and valine are often exchanged for each other, with methionine also being a suitable substitute due to its hydrophobic nature.
  • aromatic amino acids phenylalanine, tyrosine, and tryptophan can replace each other, although tyrosine is sometimes considered separately because of its polar hydroxyl group.
  • the group of polar, uncharged amino acids includes serine, threonine, asparagine, glutamine, and occasionally cysteine, which is included because of its reactive thiol group.
  • lysine, arginine, and histidine share similarities, though histidine is notable for its pH-sensitive properties.
  • Acidic amino acids, such as aspartic acid and glutamic acid are typically interchangeable.
  • Special cases include glycine, recognized for its small size, and proline, known for its ring structure that influences protein folding; these are less commonly substituted but can be replaced with alanine or other small, non-critical amino acids in certain regions of the protein.
  • percent identity i.e., percent sequence identity
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the sequences are aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection.
  • the length of a first sequence aligned for comparison purposes is at least 80% of the length of a second sequence and, in some embodiments, is at least 90%, 95%, or 100%.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical 2022-067-01 positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • BLAST GenBank URL: www.ncbi.nlm.nih.gov/cgi-bin/BLAST/, using default parameters: Program: BLASTN; Database: nr; Expect 10; filter: default; Alignment: pairwise; Query genetic Codes: Standard(1)), BLAST2 (EMBL URL: http://www.embl- heidelberg.de/Services/index.html using default parameters: Matrix BLOSUM62; Filter: default, echofilter: on, Expect:10, cutoff: default; Strand: both; Descriptions: 50, Alignments: 50), or FASTA, search, using default parameters.
  • sequences differ in conservative substitutions the percent identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. In any peptide sequence provided herein, sequences with at least 70%, 80%, 90%, 95%,98%, or 99% identity can be alternatively used.
  • nanoparticle refers to a particle having at least one dimension less than or equal to 100 nm.
  • an MRI contrast delivery system for imaging metastasis and in particular, lymph node metastasis.
  • the MRI contrast delivery system 10 includes a plurality of nanoparticles 12 wherein each nanoparticle includes one or more targeting peptides 14 that target the CCR2 receptor conjugated thereto and a magnetic resonance contrast moiety conjugated thereto.
  • the magnetic resonance contrast moiety allows for the enhancement of magnetic resonance imaging.
  • the nanoparticles are configured to hitchhike on monocytes for 2022-067-01 targeted delivery to metastatic LNs, thereby enhancing MRI signal specificity and sensitivity.
  • the nanoparticles e.g., micelles
  • an MRI contrast delivery system for imaging metastasis in a subject, and in particular, for imaging lymph node metastasis in a subject is provided.
  • the MRI contrast delivery system includes a plurality of nanoparticles wherein each nanoparticle includes CCR2 receptor-targeting peptides conjugated thereto and an MRI contrast moiety conjugated thereto.
  • the plurality of nanoparticles includes nanoparticles selected from the group consisting of micelles, liposomes, lipid nanoparticles, and combinations thereof.
  • the plurality of nanoparticles includes a plurality of single-layer micelles, also known as unilamellar micelles.
  • the nanoparticles have an average diameter of about 5 to 25 nm.
  • the nanoparticles have an average diameter of at least 2, 3, 5, 8, or 10 nm and an average diameter of at most 50, 40, 30, 25, 20, or 15 nm.
  • the CCR2 receptor-targeting peptide is a fragment of a MCP1.
  • the CCR2 receptor-targeting peptide is a fragment of a peptide having sequence QPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDS MDHLDKQTQTPKT (SEQ ID NO: 1).
  • fragments include at least 10, 11, 12, 13, 14, 15, 16 or 17 contiguous amino acids from SEQ ID NO: 1 and at most 40, 35, 30, 28, 25, 23, 20 or 18 contiguous amino acids from SEQ ID NO: 1.
  • the CCR2 receptor-targeting peptide is a peptide having CYNFTNRKISVQRLASYRRITSSK (SEQ ID NO: 2) or a peptide thereof with 1 to 7 conservative substitutions.
  • the peptide having SEQ ID NO: 2 is derived from human MCP1.
  • the CCR2 receptor-targeting peptides include a fragment of SEQ ID NO: 2 having 5 to 18 contiguous amino acids or a peptide thereof with 1 to 7 conservative substitutions.
  • the CCR2 receptor-targeting peptide is MCP1 with at least 70, 80, 90, 95, 98, or 99 percent identity to SEQ ID NO: 1).
  • Examples of the peptide with conservated substitution include but are not limited to, 2022-067-01 CFNFTNRKISVQRLASYRRITSSK (SEQ ID NO: 3), CYNFTNRRISVQRLASYRRITTSK (SEQ ID NO: 4), CYNFTNRRISVQRLAAYRRITTSK (SEQ ID NO: 5), CYQFTNRKISVQRLASYRRITSSK (SEQ ID NO: 6), CYNFTNRKLSVQRLASYRRILSSK (SEQ ID NO: 7), or CFNFTNRRISVQRLAFYRRITTSK (SEQ ID NO: 8).
  • the CCR2 receptor-targeting peptides and the MRI contrast moiety are independently connected to the nanoparticles by reaction with a functional group.
  • the functional group that can be used for linking includes amines, carboxylic acids, NHS esters, acid anhydrides, or unsaturated imides (e.g., maleimide).
  • the functional group for the MRI contrast moiety includes a chelating moiety.
  • the chemistry described herein is related to the functionalization of nanoparticles, particularly for attaching targeting peptides and MRI contrast moieties through various types of linkages. This functionalization is key in the design of targeted imaging agents, where both specificity and imaging contrast are crucial.
  • Various methods are employed for attaching molecules, such as peptides, to nanoparticles, which is crucial for developing sophisticated diagnostic tools.
  • One common method involves forming robust amide bonds between amines and carboxylic acids by activating the acid with EDC (1-ethyl-3-(3- dimethylaminopropyl)carbodiimide, which creates a reactive O-acylisourea intermediate that couples with an amine.
  • EDC 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide
  • NHS esters also form amide bonds with amines under mild conditions and are particularly efficient for labeling peptides with functional groups such as dyes, drugs, or contrast agents, reacting specifically with primary amines.
  • Another method utilizes acid anhydrides, which can react with amines to produce one or two amide bonds, introducing carboxylic acid functionalities that can be further modified. Additionally, maleimide groups are used for coupling with thiols, forming stable thioether bonds specifically with cysteine residues in peptides, a process that preserves the biological activity of peptides.
  • the choice of linking chemistry depends on the functionality present on both the nanoparticle and the molecule to be attached (peptide or contrast agent).
  • chelating moieties are derived from chelators like DOTA (1,4,7,10- tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) and DTPA (diethylenetriaminepentaacetic acid), 2022-067-01 which are used to securely hold metal ions such as gadolinium, attaching to nanoparticles through any of the aforementioned methods depending on the functional groups involved.
  • the MRI contrast moiety includes gadolinium, and in particular, gadolinium cations (e.g., Gd(III) or Gd 3+ ).
  • the MRI contrast moiety includes iron oxide.
  • the MRI contrast moiety includes manganese, and in particular, manganese cations (e.g., Mn(II) or Mn 2+ ).
  • the plurality of nanoparticles can include a plurality of micelles.
  • each micelle can include a plurality of targeting peptide-conjugated amphiphiles, a plurality of MRI contrast moiety-conjugated amphiphiles, and an optional plurality of non-targeted amphiphiles.
  • the plurality of targeting peptide-conjugated amphiphiles includes amphiphiles having a first phospholipid conjugated to a CCR2 receptor-targeting peptide with a first linking group.
  • the plurality of contrast moiety-conjugated amphiphiles includes amphiphiles having a second phospholipid conjugated to an MRI contrast moiety with a second linking group, and the optional plurality of non-targeted amphiphiles includes amphiphiles having a third phospholipid conjugated to a capping moiety with a third linking group.
  • the first linking group, the second linking group, and the third linking group are each independently a polyethylene glycol having a weight average molecular weight from about 500 to 10000 Daltons.
  • the first phospholipid, the second phospholipid, and the third phospholipid are each independently selected from the group consisting of phosphatidic acids, phosphatidyl inositols, phosphatidyl cholines, phosphatidyl ethanolamines, phosphatidyl serines, phosphatidyl glycerols, and any combinations thereof.
  • the first phospholipid, the second phospholipid, and the third phospholipid are each independently selected from the group consisting of phosphatidylglycerol, lecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, N-(2,3-di(9-(Z)-octadecenyloxy))- prop-1-yl-N,N,N-trimethylammonium chloride, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylinositol, cephalin, cardiolipin, cerebrosides, dicetylphosphate, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, palmitoyl-oleoyl- phosphat
  • the MRI contrast delivery system further includes a pharmaceutically acceptable carrier (e.g., saline).
  • a pharmaceutically acceptable carrier e.g., saline
  • This carrier serves multiple purposes: it acts as a solvent or diluent for the MRI contrast agents, ensuring they are in a suitable form for administration, and it aids in the dispersion and stabilization of the nanoparticles or other contrast media throughout the administration process.
  • Saline being isotonic with body fluids, is particularly favored as it minimizes irritation and is compatible with biological tissues, making it an ideal choice for intravenous injections.
  • the use of a pharmaceutically acceptable carrier like saline is crucial for maintaining the bioavailability and optimal pharmacokinetic properties of the contrast agents, ensuring they reach their target tissues or organs effectively without precipitating or degrading prematurely.
  • a chemotherapeutic agent is conjugated to the nanoparticles or encapsulated by the nanoparticles.
  • This strategy significantly enhances the delivery mechanisms of cancer treatments.
  • a chemotherapeutic agent is conjugated to nanoparticles, it is typically covalently bonded to the surface or the matrix of the nanoparticles. This approach allows for targeted delivery, where the chemotherapeutic can be released in a controlled manner directly at the tumor site, reducing the overall systemic toxicity and increasing the drug's efficacy against cancer cells.
  • encapsulating the chemotherapeutic agent within the nanoparticles offers another sophisticated method of drug delivery.
  • the drug is physically enclosed within the nanoparticle, which may be composed of lipids, polymers, or other suitable materials that can carry 2022-067-01 the drug molecules.
  • Encapsulation protects the chemotherapeutic agent from the external environment, potentially enhancing its stability and solubility. Moreover, it can provide a sustained release profile, where the drug is gradually released over time, maintaining therapeutic concentrations in the target area for longer periods.
  • Both conjugation and encapsulation methods aim to exploit the unique properties of nanoparticles, such as their small size, large surface area to volume ratio, and the ability to modify their surface with specific ligands. These features can be harnessed to increase the drug's accumulation at the specific site of action while minimizing off-target effects and adverse reactions.
  • a method for imaging lymph node metastasis in a subject includes administering the MRI contrast delivery system described herein to a subject at risk for having lymph node metastasis.
  • MRI images are taken of the subject to determine the presence of lymph node metastasis.
  • the lymph node metastasis includes micro metastasis.
  • the techniques described herein relate to a method for detecting lymph node (LN) metastasis in a subject using magnetic resonance imaging (MRI).
  • the method includes a step of administering to the subject a composition including micelles incorporating gadolinium (Gd) and peptides derived from the CCR2-binding motif of MCP1 and then allowing for the migration of monocytes hitchhiked by the micelles to metastatic LNs.
  • MRI is then conducted to detect the enhanced signal from the metastatic LNs due to the accumulation of the gadolinium-incorporating micelles or fragments therefrom.
  • the method includes a detecting step that quantifies the MRI signal enhancement in the lymph nodes (LNs), thereby providing a measure of metastatic burden.
  • the method includes a step of peptide synthesis using solid-phase peptide synthesis techniques.
  • the method further includes a step of purifying the peptide using high-performance liquid chromatography (HPLC).
  • HPLC high-performance liquid chromatography
  • the method further includes a step of conjugation of the peptide to the lipid tail using a maleimide-functionalized lipid.
  • the method further includes a step of characterizing the micelles using dynamic light scattering to determine their size.
  • a method for manufacturing the diagnostic agent set forth above includes synthesizing the peptide derived from the MCP1 CCR2-binding motif, conjugating the peptide to nanoparticles incorporating gadolinium, and verifying the capability of the resultant micelles to hitchhike on monocytes.
  • CCR2 C–C chemokine receptor 2
  • MCP1 monocyte chemotactic protein 1
  • MCP1-Gd gadolinium
  • MCP1-Gd When injected into in vivo models of LN metastasis and LN recurrence, MCP1-Gd targeted metastatic LNs and enabled MRI detection earlier than with a clinical gadolinium contrast agent standard.
  • LNs lymph nodes
  • MCP1-Gd transport across the endothelial barrier were evaluated with or without migrating monocytes.
  • DNA quantification through the PicoGreen assay confirmed monocyte migration in response to chemoattractant and also demonstrated that MCP1-Gd binding did not inhibit monocyte migration (Figure 12).
  • incubating MCP1-Gd with migrating monocytes increased micelle transport 2-fold compared to micelle-only controls ( Figure 2H, p ⁇ 0.005). Importantly, there was no change in transport when micelles were incubated with nonmigrating monocytes (no chemoattractant).
  • a metastatic lymph node (MetLN) mouse model was developed by implanting luciferase-expressing B16F10-Luc2 cells into the flanks of C57BL/6 mice ( Figure 13). After 14 days, bioluminescence imaging confirmed the presence of metastasis in the ipsilateral inguinal LN (MLN), while no metastasis was detected in the contralateral inguinal LN (CLN, Figures 3B). After confirming the development of the in vivo MetLN model, CCR2 expression in the LNs of MetLN mice were characterized. Immunofluorescent staining of sectioned LNs indicated increased CCR2 expression in MLNs compared to that in CLNs ( Figure 3C).
  • LNs from mice injected with MCP1-Gd or scrMCP1-Gd were sectioned and immunostained for CCR2, as well as LYVE1, which identified lymphatic endothelial cells located in the outer capsule of LNs (Figure 5E).48% of micelle signal was observed in the outer region of the LN in the MCP1-Gd group, increasing to 65% in the scrMCP1-Gd group, indicating that molecular targeting of CCR2 led to increased MCP1-Gd delivery into the LN cortex, where cells of the adaptive immunity are located.
  • lymph node (LN) metastasis and recurrence Despite the improved prognosis and survival benefit associated with chemo- and immunotherapy of lymph node (LN) metastasis and recurrence, early detection remains a significant obstacle. Current clinical tools including biopsy and MRI are limited by health risks or lack sensitivity for detecting early metastasis. (63) Targeting nanoparticles to cancer-specific biomarkers as well as to tumor-tropic monocytes has been reported to increase chemotherapy drug delivery to tumor tissues but has yet to be explored in the context of metastatic LN imaging and diagnostics.
  • MCP1-Gd micelles were developed for the imaging and detection of lymph node (LN) metastasis and recurrence through molecular targeting of CCR2, which enables both metastatic cancer cell binding and monocyte hitchhiking for the targeted delivery of gadolinium (Gd) to metastatic LNs.
  • LN lymph node
  • Gd gadolinium
  • MCP1-Gd was found to be biocompatible in vitro and exhibited CCR2-specific binding and hitchhiking onto monocytes, resulting in enhanced transport across lymphatic endothelium.
  • In vivo models of LN metastasis and LN recurrence were developed and utilized to verify MCP1-Gd targeting of metastatic LNs, which enabled earlier cancer detection than conventional MRI.
  • MCP1-Gd accumulation in lymph nodes is facilitated by circulating monocytes, as depletion of these cells in vivo resulted in a significant reduction in micelle targeting to the LNs.
  • monocyte-targeting it is plausible that MCP1-Gd could accumulate 2022-067-01 in tissues or pathologies characterized by high monocyte infiltration that are unrelated to lymph node metastasis, representing both a potential for use in broader diagnostic applications as well as a potential for false positives.
  • DSPE-PEG2000-FITC fluorescein isothiocyanate
  • FITC fluorescein isothiocyanate
  • Diethylenetriamine-N,N,N′′,N′′-tetra-tert- butyl acetate-N′-acetic acid (DTPA-tetra(t-Bu ester)) was purchased from Macrocyclics (Plano, TX, USA).
  • GdCl3 was purchased from Sigma-Aldrich (St. Louis, MO, USA).
  • Antibodies were purchased from Thermo Fisher (Waltham, MA, USA).
  • Cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).
  • MCP1-Gd Micelles MCP1 [CYNFTNRKISVQRLASYRRITSSK] and scrMCP1 [CYNSLVFRIRNSTQRKYRASIST] peptides were synthesized through Fmoc-mediated solid-phase peptide synthesis and cleaved from the resin using a 94:2.5:2.5:1 vol % mixture of trifluoroacetic acid, water, ethanedithiol, and triisopropylsilane.
  • peptides were lyophilized, resuspended in MQ water, and mixed with a 10% molar excess of DSPE-PEG2000-maleimide. The pH of the mixture was then adjusted to 7.2 with 1 M NaOH, incubated for 72 h at room temperature (RT) under constant agitation, purified through HPLC, and characterized via MALDI-TOF-MS.
  • DSPE-PEG2000-Gd amphiphiles were synthesized by first combining DTPA-tetra(t- Bu ester) and DSPE-PEG2000-amine in peptide synthesis-grade dimethylformamide (DMF).
  • Gd3+ ions from GdCl3 were chelated to DSPE-PEG2000-DTPA-tetra in a 0.5 M sodium acetate (pH 5.5) solution for 1 h. Free Gd3+ ions were separated using a desalting column, and successful synthesis of DSPE- PEG2000-Gd was confirmed via MALDI-TOF-MS.
  • DSPE-PEG2000-Cy7 amphiphiles were synthesized by dissolving DSPE-PEG2000- amine in a 0.1 M sodium bicarbonate solution and mixing with a solution containing 3 ⁇ mol equiv of Cy7 NHS ester in peptide synthesis-grade DMF (reaction volume was 90% sodium bicarbonate and 10% DMF). The resultant mixture was protected from light, stirred overnight at RT, and then purified through HPLC before characterization via MALDI-TOF-MS. Micelles were self-assembled through thin-film hydration.
  • MCP1, scrMCP1, DSPE-PEG2000-Gd, DSPE-PEG2000-Cy7, and DSPE-PEG2000-FITC amphiphiles were dissolved in methanol, mixed at desired molar ratios, sonicated, and placed under a gentle nitrogen stream to evaporate the methanol before hydration in MQ water, 1 mM NaCl, or PBS.
  • the micelle formulations and the respective molar ratios used are listed in Table 1.
  • TEM 400-mesh carbon TEM grids (Ted Pella, Redding, CA, USA) were glow-discharged before application of 5 ⁇ L of 30 ⁇ M micelles suspended in MQ water for 5 min. Samples were then wicked to remove excess liquid and washed with MQ water. Grids were stained twice with 2% uranyl acetate for 3 min before washing with MQ water. Grids were dried overnight and then imaged on an FEI Talos F200C microscope (Thermo Fisher).
  • a 9 cm diameter radiofrequency body coil was used (bore size ⁇ 24 mm, up to 600 mT m–1 maximum gradient).
  • T1 and T2 maps were generated through a pixel- by-pixel exponential fitting of signal intensities across the different FA and TE values, respectively.
  • Regions of interest (ROIs) were manually drawn around each concentration of MCP1-Gd micelles that were measured using ImageJ.
  • R1 maps were generated by taking the inverse of the T1.
  • r1 relaxivity was determined (R1/concentration of Gd) over Gd concentrations of 0.2, 0.4, 0.6, 0.8, and 1.0 mM.
  • Cell Culture SVEC4–10 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L glucose supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (PS). Cells were maintained below 80% confluency, and culture media was renewed every 2–3 days.
  • B16F10-Luc2 cells were cultured in DMEM with 4.5 g/L glucose supplemented with 10% FBS, 1% PS, and 10 ⁇ g/mL blasticidin.
  • NCI-H460 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS and 1% PS. Cells were maintained below 80% confluency, and culture media was renewed every 2–3 days.
  • WEHI-274.1 cells were cultured in DMEM with 4.5 g/L glucose supplemented with 10% FBS, 0.05 mM 2-mercaptoethanol, and 1% PS. Cells were passaged every 2–3 days. All cells were cultured in a humidified incubator set to 37 °C and 5% CO2.
  • PBST peripheral blood mononuclear cells
  • AF594 Thermo Fisher A-11037, 1:500
  • DAPI 2 ⁇ g/mL 4′,6-diamidino-2-phenylindole
  • nonadherent cells For nonadherent cells (WEHI-274.1), 500,000 cells were incubated with 25 ⁇ M Cy7-labeled MCP1-Gd or scrMCP1-Gd in plastic microtubes for 1 h at 37 °C. Cells were then washed with PBS, fixed using 4% PFA, and then spun onto 22 mm ⁇ 22 mm glass coverslips at 500g for 5 min before immunostaining and imaging as described above.
  • In Vitro Micelle Hitchhiking To develop an in vitro model of a lymphatic endothelial barrier, 24-well transwell membrane inserts with 5 ⁇ m pores were coated with 0.1% gelatin for 1 h at RT.
  • transwell systems were incubated at 37 °C for 4 h under the following 3 conditions: (1) MCP1-Gd or scrMCP1- Gd micelles in the top chamber, (2) MCP1-Gd or scrMCP1-Gd micelles and monocytes in the top 2022-067-01 chamber, and (3) MCP1-Gd or scrMCP1-Gd micelles and monocytes in the top chamber and chemoattractant (FBS) in the bottom chamber.
  • FBS chemoattractant
  • mice were injected intraperitoneal (i.p.) with 200 uL of a 15 mg/mL suspension of D-luciferin in sterile PBS, and bioluminescence imaging was performed every 3–5 min to determine the kinetic profile of luciferase activity and optimal time point for dosage (Figure 13).
  • Mice were injected with D-luciferin and euthanized 2 weeks after tumor inoculation, ipsilateral and contralateral inguinal lymph nodes were collected, and bioluminescence imaging was performed to evaluate metastasis.
  • lymph nodes were processed into single-cell suspensions and cultured for 2 weeks in culture media containing 10 ⁇ g/mL blasticidin to determine the presence of B16F10-Luc2 cells within the lymph nodes.
  • Vivo Lymph Node Recurrence Model To simulate lymph node recurrence following surgical resection of a primary tumor, the mouse model described above was first developed, and then the primary tumor was surgically removed 12 days after tumor implantation using a linear incision. Wounds were sutured shut using nonadsorbable nylon sutures. Mice were then euthanized 7 d postsurgery to evaluate the presence of cancer in the LNs through bioluminescent imaging.
  • mice were anesthetized with 2.5% isoflurane at a flow rate of 250 ⁇ L/min, before transfer onto a heated scanner bed with isoflurane levels between 1.5% and 2%. Temperature was monitored and maintained at 37 °C. An electrocardiogram (ECG) was monitored and used for gating by inserting two leads subcutaneously on the anterior side close to each axilla and inserting another lead subcutaneously on the lower right abdomen. A pneumatic pillow was placed underneath the mice for respiration monitoring and gating. The mice were positioned at the magnet bore isocenter by using a motorized system. Temperature, respiration, and ECG were monitored by using SAII equipment and accompanying PC-SAM software.
  • ECG electrocardiogram
  • a bird cage whole mouse body coil with an axial field of view of 60 cm and a 35 mm inner diameter, was used during ECG and respiration-gated cardiac imaging.
  • a gradient echo scan was used to obtain three orthogonal slices for positioning.
  • ImageJ was used to measure T1 signal intensity and calculate signal-to-noise ratios (SNRs).
  • tissues liver, kidneys, spleen, lungs, heart, small intestines, brain, and inguinal lymph nodes
  • OCT optimal cutting temperature
  • frozen tissues were cryosectioned into 10 ⁇ m slices using a CM3050 S Cryostat (Leica, Nussloch, Germany). Tissue sections were stained with hematoxylin and eosin or processed for immunohistochemical (IHC) staining and imaging.
  • OCT optimal cutting temperature
  • the CCR2 antagonist RS102895 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 12.5 mg/mL and then diluted 20 ⁇ in 0.9% saline. Mice were injected i.p. with 200 ⁇ L of the diluted RS102895 solution every 6 h for 18 h (4 total injections) to deplete monocytes. (67) Twenty-four hours after the first RS102895 injection, 100 ⁇ L of 2.2 mM FITC- or Cy7-labeled MCP1- Gd or scrMCP1-Gd were injected via the tail-vein, and mice were euthanized 3 h postinjection.
  • DMSO dimethyl sulfoxide
  • Tissues including the inguinal lymph nodes were collected for ex vivo optical imaging, flow cytometry, or immunofluorescent staining. Control mice were injected with PBS. Flow Cytometry All flow cytometry experiments were run on a CytoFLEX cytometer (Beckman Coulter, Brea, CA, USA) and analyzed using FlowJo.
  • the flow cytometry buffer used was sterile PBS supplemented with 0.1% bovine serum albumin (BSA) and 1 mM ethylenediaminetetraacetic acid (EDTA).
  • BSA bovine serum albumin
  • EDTA ethylenediaminetetraacetic acid
  • Tissue samples were prepared by rough-cutting the tissues with sterile scissors and gently pushing the resultant pieces through 70- ⁇ m cell strainers to yield single-cell suspensions that were then centrifuged at 500g for 5 min at RT. After discarding the supernatant, the cell pellet was resuspended in 100 ⁇ L of flow cytometry buffer and immunostained for CD11b (Miltenyi 130-113- 238, San Diego, CA, 1:50) and CCR2 (Miltenyi 130-119-658, 1:50) 10 min in the dark at 4 °C. Samples were then washed once more with flow cytometry buffer, resuspended at 1 million cells/mL, and analyzed in the flow cytometer.
  • Plasma samples were collected via tail-vein bleed prior to each injection using heparinized capillary tubes. Plasma was collected by spinning blood samples at 1000g for 10 min at 4 °C and collecting the supernatant. Plasma samples were then assayed for anti-PEG IgG and IgM via ELISA according to the manufacturer instructions. Plasma Half-Life and Clearance Blood samples from na ⁇ ve mice or mice injected weekly with 2.2 mM MCP1-Gd (3 prior doses) were collected via the tail-vein 5 min, 1, 3, 6, 12, and 24 h following injection with Cy7- labeled MCP1-Gd and then spun at 1000g for 10 min at 4 °C to obtain plasma.
  • Plasma samples were then analyzed for Cy7 fluorescence using a microplate reader to determine the MCP1-Gd half-life.
  • MCP1-Gd clearance was calculated by dividing the injected dose by the area under the curve (AUC) of the dose-overtime plot.
  • Blood Chemistry Markers of Renal and Liver Health Plasma samples from mice dosed weekly with MCP1-Gd as described above or with PBS were evaluated for hepatic and renal health markers using ALT, AST, BUN, and creatinine kits (Sigma-Aldrich, St. Louis, MO) as directed by manufacturer instructions.
  • Biodistribution was evaluated 3 h after tail-vein injection of 100 ⁇ L of Cy7-labeled MCP1-Gd or Magnevist (450 ⁇ M Gd) via fluorescence imaging or inductively coupled plasma-optical emission spectroscopy (ICP-OES) (Thermo Fisher). Tissues including the liver, kidneys, spleen, lungs, heart, gastrointestinal (GI) tract, brain, and inguinal lymph nodes were dissected following euthanasia and imaged ex vivo for Cy7 fluorescence. Fluorescence data are expressed as % mean radiance. 2022-067-01 Statistical Analysis Data are expressed as mean ⁇ standard deviation (SD).
  • SD standard deviation
  • Verweij, S.L., et al., CCR2 expression on circulating monocytes is associated with arterial wall inflammation assessed by 18F-FDG PET/CT in patients at risk for cardiovascular disease. Cardiovasc Res, 2018. 114(3): p. 468-475. 29. Huang, Y., et al., The effect of size, charge, and peptide ligand length on kidney targeting by small, organic nanoparticles. Bioeng Transl Med, 2020. 5(3): p. e10173. 30. Liu, H., et al., Preparation, characterization, in vivo pharmacokinetics, and biodistribution of polymeric micellar dimethoxycurcumin for tumor targeting. Int J Nanomedicine, 2015.

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Abstract

Un système d'administration de contraste IRM comprend des nanoparticules qui sont chacune équipées de peptides de ciblage pour des récepteurs CCR2 et une fraction de contraste IRM. Ces composants sont conjugués aux nanoparticules, qui sont constituées de lipides hydrophobes. Cette conception permet aux nanoparticules de « faire de l'auto-stop » sur des monocytes, tirant parti de leurs voies de migration naturelles pour cibler efficacement des ganglions lymphatiques métastatiques (LN). Cette approche d'administration ciblée améliore significativement la spécificité et la sensibilité des signaux d'IRM, améliorant l'imagerie de sites métastatiques et aidant potentiellement à un diagnostic et à une évaluation plus précis de la progression du cancer.
PCT/US2024/024999 2023-04-17 2024-04-17 Irm moléculaire utilisant des micelles ciblant ccr2 pour la détection de métastases de nœud lymphatique Ceased WO2024220548A2 (fr)

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