WO2016062781A1 - Composés pour l'imagerie de cellules pancréatiques bêta - Google Patents

Composés pour l'imagerie de cellules pancréatiques bêta Download PDF

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WO2016062781A1
WO2016062781A1 PCT/EP2015/074409 EP2015074409W WO2016062781A1 WO 2016062781 A1 WO2016062781 A1 WO 2016062781A1 EP 2015074409 W EP2015074409 W EP 2015074409W WO 2016062781 A1 WO2016062781 A1 WO 2016062781A1
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arg
beta cell
compound according
cell mass
cells
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PCT/EP2015/074409
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English (en)
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Nelson KHOO
Bradley THATCHER
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N-Tigen Insights Ab
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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
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0056—Peptides, proteins, polyamino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • 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
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • 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
    • A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal

Definitions

  • the present invention relates to compounds and methods for imaging pancreatic beta-cells and determination of pancreatic beta-cell mass (BCM).
  • the compounds and methods provides live cell staining for pancreatic islets and beta cells in vitro or in vivo for diagnosis and monitoring the efficacy of treatments for pancreatic beta cell disorders, e.g. Type 1 and Type 2 diabetes.
  • Diabetes is one of the major socioeconomic healthcare problems facing our future generations.
  • the prevalence of diabetes now is 382 million worldwide, projected to reach 592 million in 2035 (http://www.idf.org/di.abetesatlas ).
  • Today, 2.5%-15% of the national annual healthcare budgets are related to diabetes care (including treatment of complications such as kidney failure, retinopathy, diabetic foot etc.), however, the burden may rise up to 40% in high-prevalence countries in the coming years.
  • Diabetes mellitus represents a group of metabolic diseases resulting from an absolute or relative deficiency in insulin secretion by ⁇ -cells residing in the islets of Langerhans, leading to plasma glucose elevation above normal.
  • Type 1 Diabetes is a result of inflammatory destruction by lymphocyte infiltration that selectively destroys of ⁇ -cells in the islets of Langerhans and is undetected until the presentation of hyperglycaemia.
  • TID Type 1 Diabetes
  • HLA major histocompatibility complex
  • TID is being successfully treated using pancreas transplantation, and researchers may soon be able to introduce healthy, functioning isolated pancreatic islet cells into patients from stem cells or induced pluripotent stem cells. Insulin secretory capacity can be measured, but it is a poor evaluative proxy of beta cell mass in the organ or of the transplantation of islet of Langerhans into patients.
  • Type 2 diabetes results from multiple metabolic perturbations i.e. obesity, insulin resistance in peripheral tissues, leading to gradual beta cell failure (and eventually complete loss of BCM) partly due to chronic metabolic stress. Loss of sensitivity to insulin (resistance) is an early factor in the course of T2D. BCM initially increases to compensate for the rising insulin demand for years before clinical diagnosis of diabetes in type 2 patient. Thus, the eventual primary defect in T2D is beta-cell failure as the exhausted BCM and insulin production can no longer maintain control and blood glucose rise. Those at risk for type 2 diabetes can be identified through family history and measurements of insulin resistance.
  • Beta cell dysfunction and reduction of pancreatic BCM are central events in the pathogenesis of both Type 1 and Type 2 diabetes. Because characterization of beta cells is only possible in pancreatic specimens obtained at autopsy, only incomplete information about the course of inflammation in diabetes or the natural history of loss of BCM as well as beta cell function, turnover and cell lifetime is available.
  • Imaging modalities can be broadly divided into nuclear and non-nuclear (usually optical) techniques. These techniques and their potential for islet/ ⁇ - cell imaging are briefly summarized below, including the imaging modalities suggested to be used in this application (for detailed reviews see e.g. Ahlgren, U., and Gotthardt, M. (2010). Approaches for imaging islets: recent advances and future prospects.
  • the ideal biomarker should allow measurements by a minimally invasive technology enabling repeated examinations over time, should identify the early stages of decreased
  • BCM should provide information on progression of beta cell loss and eventual responses to agents aiming to arrest or revert ⁇ -cell loss in diabetes.
  • a good tool or marker as a imaging tool requires the availability of marker conjugated to fluorescence or other optical imaging reagent, nanoparticles or radioligands that satisfy these conditions: good tissue uptake to allow high signal-to-noise ratios, high target affinity to allow high target (specific) signal, low nonspecific signal, and reversible specific binding for the kinetic model- based quantification.
  • MRI Magnetic Resonance Imaging
  • PET Positron Emission Tomography
  • SPECT Single Positron Emission Computer Tomography
  • absorption or fluorescence spectroscopy make it likely that a clinical exam to monitor beta cell number, mass, function, or lymphocyte infiltration can soon be established. This would allow high-risk individuals to be monitored prior to onset of diabetes; patients could be monitored over the course of their disease to determine the exact stage of their disease; and it would also allow monitoring responses to therapy.
  • the root of the issue is the minute size range of the islets and the spatial distribution of the islets representing a total of ⁇ 2 % of the adult pancreas.
  • the islets themselves are heterogeneous composed of ⁇ - (insulin producing) cells, a (glucagon producing) cells, ⁇ (somatostatin producing) cells, ⁇ (ghrelin producing) cells and PP
  • contrast agent uptake as well as means to corroborate and calibrate the noninvasive read out.
  • Non-invasive imaging of an organ A demanding predicament for non-invasive imaging assessments is the very limited availability of biomarkers that could be used for monitoring specific intracellular targets, functional processes or events following systemic administration.
  • the requirements of successful contrast agents are especially strict in diabetes imaging. They must: i) be highly specific, ii) be stable, iii) yield high signal intensity, iv) have low background noise, v) be non-toxic and vi) have excellent distribution kinetics to the specific cells or host.
  • these reagents should ideally harmonize with currently existing imaging platforms and permit the simultaneous detection of different cell types/substructures (i.e., not scaffold restricted). Whereas some recent progress has been made in biomarker development for imaging beta- cells (see e.g.
  • T1D Type 1 diabetes
  • the inventors disclose herein that visualization and staining of insulin expression in ⁇ -cells by use of a new class of imaging compounds provides such a method for non-invasive measurements of ⁇ -cells in islets in vitro and in vivo. More particularly, the inventors disclose that the new class of imaging compounds can be used to image the endocrine pancreas in vivo using fluorescence and that can be modified with this radioligand to allow PET imaging to visualize BCM in a host.
  • the present invention provides compounds comprising:
  • the compounds can be used in methods for in vivo determination of pancreatic beta cell mass. Determination of pancreatic beta cell mass can be used in methods for diagnosis, prognosis and therapy of beta cell disorders, such as diabetes. LEGENDS TO FIGURES
  • FIG. 1 A. Live Islet Staining. Representative dispersed pancreas stained with compound 1. Bright islets are present with exocrine tissue with nominal background. Single islet shown that allows use in sorting or FACs analysis (shown in B. Flow Cytometry). B. Flow Cytometry
  • Cytometry Analysis comparing staining of live beta cells with compound 1 to staining with standard reagent (anti-insulin antibody) used in the field of permeabilized and fixed beta cells. Staining with both reagents on aliquots of the same preparation show identical population percentage.
  • Figure 2 A. Optical Projection Tomography. Representative of a pancreas following intravenous injection of compound 1 and visualized in OPT ex vivo. Islets are clearly visible with nominal background of tissue auto fluorescence.
  • Compounds stained beta cells in the representative islets from mouse and human organs Compounds stained beta cells in the representative islets from mouse and human organs.
  • FIG. 1 A. Systemic injection targets beta cells not glucagon or somatostatin.
  • cryosection pancreas showing an islet after injection of compound 1 counterstained with markers for somatostatin, glucagon and pancreatic polypeptide cells present in an islet, showing specificity of the compound 1.
  • FIG. 4 A. Viability after staining. Representative islets from mouse and human pancreas were stained with compound 1 and compound 2 and compared to control islets not stained with compounds. The bar graphs show that compound 1 and compound 2 did not affect viability of islets.
  • a first aspect of the present invention provides compounds comprising:
  • the therapeutic moiety is a therapeutic moiety with efficacy in the treatment of a pancreatic beta cell disorder.
  • the peptide B can have a length of 5 to 15 amino acids, such as a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
  • the insulin binding amino acid sequence of peptide B is selected from the peptide sequences Cys-Val-Glu-Glu-Ala-Ser (SEQ ID NO: 1), Cys-Ile-Lys-Lys-Pro-Ala (SEQ ID NO:2), Leu-Val-Glu-Ala-Leu-Tyr-Leu (SEQ IDNO:3), and Arg-Gly-Phe-Phe-Tyr-Thr (SEQ ID NO:4).
  • the peptide P can have a length of 5 to 15 amino acids, such as a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
  • the peptide P can comprise the sequence of essentially any known cell penetrating peptide in the art.
  • the peptide P comprises an amino acid sequences consisting of L-Arg and D-Arg amino acid residues.
  • the sequence can comprise a mixture of L-Arg and D-Arg amino acid residues.
  • the sequence can be 4 to 10 amino acids long.
  • the peptide P comprises an amino acid sequences selected from (L-Arg)n and (D-Arg)n wherein n is an integer from 4 to 10, preferably n is 8.
  • the peptide P preferably comprises the amino acid sequence L-Arg- L-Arg- L-Arg- L-Arg- L-Arg- L-Arg- L-Arg- L-Arg (SEQ ID NO:5), or D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg-D-Arg.
  • the peptides B and P may comprise any of the naturally occurring amino acids.
  • the peptides may further comprise non-naturally occurring amino acids.
  • the non-naturally occurring amino acids can be selected from e.g.
  • caged amino acids e.g. S-o-nitrobenzyl-cystein and O- o-nitrobenzyl-tyrosine, D-amino acids, homo amino acids, N-methyl amino acids, alpha- methyl amino acids, beta ( ⁇ 3 and ⁇ 2 ), (homo) amino acids, gamma amino acids, helix/turn stabilizing motifs, Proline and Pyruvic acid derivatives, 3 -substituted Alanine
  • L comprises biotin
  • L comprises a chelating agent.
  • the chelating agent can be selected from diethylenetriamine pentaacetic acid-nateglinide, diethylenetriamine pentaacetic acid- glipizide, diethylenetriamine pentaacetic acid-glyburide, or diethylenetriamine pentaacetic acid-glimepiride.
  • L comprises a radioligand.
  • the radioligand is bound to a polypeptide component of the compound via a chelating agent.
  • Suitable chelating agents include both macrocyclics and acyclic chelators, such as derivatives of 1,4,7,10- tetraazacyclododecane-l,4,7,10,tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic avid (DTP A), derivatives of S-2-(4-Isothiocyanatobenzyl)- l,4,7-triazacyclononane-l,4,7-triacetic acid (NOT A) and derivatives of 1,4,8,11- tetraazacyclodocedan-l,4,8,l l-tetraacetic acid (TETA), derivatives of 3,6,9,15- Tetraazabicyclo [9.3.1] -pentadeca- 1 ( 15), 11 , 13 -triene
  • the radioligand can comprise a radioactive isotope selected from the group consisting of: fluorine- 18, carbon-11, nitrogen-13, zirconium-89, copper-60, copper-61, copper-62, copper- 63, copper-64, gallium-67, gallium-68, technetium-99m, indium-I l l, iodine-123, xenon-133, holmium-166, rhenium- 187, rhenium- 187, and thallium-201 or other isotopes suitable for PET and/or SPECT imaging.
  • L comprises a fluorescent label.
  • the fluorescent label can be a fluorescent dye. A large number of fluorescent dyes are known in the art. It is anticipated that L can comprise essentially any known fluorescent dye.
  • the fluorescent label can be selected from e.g. cyanine, fluorescein, rhodamine, Alexa Fluor dyes, Dylight Fluor dyes, Atto Dyes, Tracy Dyes, BODIPY Dyes, Seta Dyes, SeTau Dyes, Sulfo Cy dyes, HiLyte Fluor dues, FluoProbe dyes.
  • L comprises a therapeutic agent with efficacy in the treatment of a pancreatic beta cell disorder.
  • the therapeutic agent can selected from both biologicals and chemical drugs.
  • the therapeutic agent can be selected from such examples: Glucagon-like peptide-! (GLP-1), incretin, Glitazones, Dipeptidyl-peptidase IV (DPP4), verapamil, 1- Azakenpaullone, a GSK-3P inhibitor, GKA50, a glucokinase activator, PSN632408, a GPR119 agonist, Bay K 8644, an L-type calcium channel activator, 5-Iodotubercidin, an adenosine kinase inhibitor, but not to exclude others.
  • B is a peptide comprising the insulin binding amino acid sequences Cys-Val-Glu-Glu- Ala-Ser (SEQ ID NO: 1,
  • P is a peptide comprising the cell penetrating amino acid sequence Arg-Arg-Arg-Arg-
  • (c) L comprises the fluorescent dye Cy5 or FITC.
  • the compounds of the invention comprise a recombinant polypeptide.
  • Suitable methods for the production of such recombinant polypeptides are well known in the art, such as expression in prokaryotic or eukaryotic hosts cells (for example, see Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor, New York, the relevant disclosures in which document are hereby incorporated by reference).
  • the compounds of the invention comprise a synthetic polypeptide.
  • Suitable methods for the production of such synthetic polypeptides are well known in the art. (for example see Merrifield, 1963, Solid Phase Peptide Synthesis. I. The Synthesis of a
  • peptides B and P may be covalently joined, for example as a fusion peptide.
  • detectable label L may be joined to peptide B and/or P non-covalently, for example via a chelator agent (see above).
  • Another aspect of the present invention provides a pharmaceutical formulation comprising a compound as defined above and a pharmaceutically-acceptable diluent, carrier or excipient.
  • pharmaceutically acceptable we mean a non-toxic material that does not decrease the effectiveness of the activity of the compound of the invention.
  • Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's
  • buffer is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilising pH.
  • buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole,
  • diluent is intended to mean an aqueous or non-aqueous solution with the purpose of diluting the compound in the pharmaceutical preparation.
  • the diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).
  • adjuvant is intended to mean any compound added to the formulation to increase the biological effect of the compound of the invention.
  • the adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition.
  • the adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly( vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.
  • cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly( vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.
  • the excipient may be one or more of carbohydrates, polymers, lipids and minerals.
  • carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrines, which are added to the composition, e.g., for facilitating lyophilisation.
  • polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers,
  • polyvinylpyrrolidone all of different molecular weight, which are added to the composition, e.g., for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation.
  • lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers.
  • minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.
  • the compounds of the invention may be formulated into any type of pharmaceutical composition known in the art to be suitable for the delivery thereof.
  • compositions of the invention may include ions and a defined pH for potentiation of action of the active antibody polypeptide.
  • the compositions may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc.
  • the pharmaceutical compositions according to the invention may be administered via any suitable route known to those skilled in the art.
  • possible routes of administration include parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar, buccal, oral, parenteral, and rectal.
  • Infusion may be a desired route because of the potentially high cytotoxicity of the
  • the pharmaceutical compositions are administered by injection, for example, intravenously, intracerebroventricularly, intraarticularly, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially,
  • intramuscularly or subcutaneously may be administered by infusion techniques.
  • a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • aqueous solutions should be suitably buffered (for example, to a pH of from 3 to 9), if necessary.
  • suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Another aspect of the present invention provides methods for determining the beta cell mass in the pancreas of a subject.
  • the method comprises administering to the subject an effective amount of a compound of the invention.
  • the compound comprises an insulin binding peptide sequence and a label.
  • the label is a fluorescent label or a radioligand.
  • the method further comprises obtaining one or more image(s) of the test subject's pancreas; and quantitatively analyzing the computerized image(s) in order to determine the beta cell mass in the pancreas of the test subject.
  • the computerized image can be obtained using optical projection tomography (OPT) or near infrared OPT (NIR-OPT), when the label is a fluorescent label.
  • OPT optical projection tomography
  • NIR-OPT near infrared OPT
  • the computerized image can be obtained using positron emission tomography (PET) when the label is a radioligand.
  • PET positron emission tomography
  • the method can be used to determine the beta cell mass in any mammalian subject.
  • the subject is preferably human.
  • the method can be used for diagnosing a pancreatic beta cell disorder in a subject by determining the beta cell mass in the pancreas of the test subject; and comparing the beta cell mass with a baseline measure of beta cell mass, where decreased beta cell mass or increased beta cell mass compared to the baseline measure is associated with a pancreatic beta cell disorder. More specifically, a decreased beta cell mass compared to the baseline measure is associated with the development of diabetes.
  • the diabetes can be diabetes type 1 or diabetes type 2.
  • the method can further be used for assessing the prognosis of a subject at risk for developing diabetes by periodically determining the beta cell mass in the pancreas of the test subject; and comparing the periodically determined beta cell mass with a baseline measurement of beta cell mass, where decreased beta cell mass allows the monitoring of the progression from a pre-diabetic condition to a diabetic condition.
  • the method can further be used for managing and/or monitoring the effect of treatment of subject suffering from a pancreatic beta cell disorder by periodically determining the beta cell mass in the pancreas of the test subject; and comparing the periodically determined beta cell mass with a baseline measurement of beta cell mass established for the host.
  • the pancreatic beta cell disorder can be an insulinoma or an endocrine tumor, where the decreased ⁇ -cell mass is indicative of an ameliorative therapy.
  • the beta cell disorder can be diabetes, where an increased beta cell mass is indicative of a successful therapy ameliorating the effects of the disease.
  • the compound according to the invention can be administered to a human or animal subject by known procedures including, without limitation, parenteral administration (e.g. intravascular, intravenous, intravenous, intra parenchymatous administration).
  • parenteral administration e.g. intravascular, intravenous, intravenous, intra parenchymatous administration.
  • One preferred method of administration is parenteral administration, by venous or arterial injection.
  • pancreatic beta cell disorder in a subject
  • the invention further provides an in vitro method for detecting, identifying and/or isolating insulin-producing pancreatic beta cells comprising:
  • step (c) determining the quantity of the compound contained in the pancreatic cells wherein pancreatic cells containing the compound are identified as insulin producing pancreatic beta cells. Determining the quantity of the compound contained in the pancreatic cells in step (c) can comprise flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), microscopy, or any combination thereof.
  • the sample of pancreatic cells may be live or fixed. Definitions
  • the term "effective amount” refers to an amount of a compound according to the invention to provide an image of the region of interest using computer tomography, optical projection tomography (OPT) or near infrared OPT (NIR-OPT).
  • OPT optical projection tomography
  • NIR-OPT near infrared OPT
  • the amount of compound that is effective in providing an image will vary depending the particular factors of each case, including the type of radioligand or fluorescent label, the type of subject, the subject' s weight and the method of administration. These amounts can be readily determined by the skilled artisan.
  • pancreatic beta cell associated disorder refers to any disorder or disease characterized by changes in beta cell mass or function including, but not limited to, diabetes, preclinical diabetes and hypoglycemic disorders including insulinoma and pancreatic endocrine tumors.
  • diabetes refers to any disorder of glucose metabolism leading to hyperglycemia and includes both type 1 and type 2 diabetes.
  • baseline measure of BCM refers to a measure of BCM that is compared with a quantitative measure of BCM of the test subject.
  • the baseline measure of BCM can be the BCM of a control subject.
  • control subject refers to a mammal including, without limitation, a cow, dog, mouse, pig, rat, monkey or human that does not have a metabolic disorder, In a preferred embodiment of the invention, the control subject is human.
  • baseline measure of BCM may refer to the BCM of the test subject measured at an earlier point in time.
  • amino acid and any reference to a specific amino acid is generally meant to include naturally occurring proteogenic amino acids as well as non-naturally occurring amino acids such as amino acid analogs.
  • This broad definition for one skilled in the art would know that the reference to an amino acid, unless indicated otherwise specifically, includes, i.e. naturally occurring proteogenic (L) -amino acids, (D) -amino acids, chemically modified amino acids, including amino acid analogs e.g. penicillamine (3-mercapto-D-valine) , naturally occurring non-proteogenic amino acids e.g. norleucine and chemically synthesized compounds that have properties known in the art to be characteristic of an amino acid.
  • Pancreata were removed from the murine hosts and stained with Compound 1. Dispersed pancreata were incubated with the imaging compound for 30 min in serum-free medium. The dispersed pancreata were washed and visualized in a fluorescence microscope. The method of staining by compound 1 allows isolation of live cells. It is different and diverges from the stand antibody staining of the islets.
  • islet cells were stained with monoclonal rat-anti mouse insulin- -allophycocyanin (APC) antibody (both from R&D Systems, MN, USA). Islet cells were incubated in the dark with antibodies for 30 minutes at 4°C and then washed twice again with Permeabilization / Wash Buffer before resuspending them in staining buffer and acquired by flow cytometry on FACS/Calibur® Instrument and data were analyzed using CellQuest® software (both from Becton Dickinson, San Jose, CA, USA).
  • API monoclonal rat-anti mouse insulin- -allophycocyanin
  • the target cell population recognized by compound 1 and anti-insulin antibody (rat anti- mouse allophycocyanin (APC) conjugated, R&D Systems) were determined ( Figure 1). It is shown that the subpopulation of cells recognized in the same single cell preparation of pancreas is ⁇ 50% in a flow cytometry analysis using the two different detection reagents.
  • FIG. 1 A shows that compound 1 stains beta cells in the pancreatic islets ( ⁇ -cells; shows disaggregated pancreas (mixed dispersed pancreas)). The islets stained brightly but the exocrine tissue interspersed show nominal signal. Furthermore, the pool of islets were trypsinized, separated into identical aliquots and stained with N-timerTM and the standard anti-insulin antibody conjugated with Allophycocyanin (APC). Flow cytometry analysis using compound 1 was compared and found to detect the same percentage of ⁇ -cells (approx. ⁇ 50%) ( Figure IB). Also note cells populations reflect the health of the cells as the antibody stained cells were dead following permeabilization and fixation. In contrast, compound 1 stained cells remained viable.
  • Example 2 In situ staining of beta cells in host pancreas.
  • Compound 1 was administered to mice (6-8 weeks, B6, Taconic) by intravenous injection via the tail vein.
  • the pancreata were removed for cryo section and for optical projection tomography (OPT) two hours later.
  • OPT optical projection tomography
  • the pancreata showed specific targeting of insulin- producing beta cells and did not stain exocrine tissue in the pancreata. Furthermore, staining was not observed in other tissues except for the liver and kidney where the administered compound are cleared from the circulation.
  • pancreas from B6 mice were dissected, cleaned of residual fat tissue and immediately submersed into freshly made 4% paraformaldehyde (Sigma P6148) in PBS for 3 h at 4°C, washed in PBS stepwise transferred to 100% methanol (MeOH) and stored at -20°C.
  • DAPI global nuclei stain
  • islets were obtained from mouse and human pancreata ex vivo. These islets were incubated with compound land compound 2 in vitro for 30 mins and washed three times to remove excess compound. The islets were visualized using a fluorescent microscope. Compound 2 was able to stain human beta cells as well with the same specificity. Both compounds stained human and murine islets in vitro. The stainings were observed to be specific and islets remain viable.
  • Example 3 Staining of pancreas in vitro Specificity Islets obtained from mouse pancreas were processed ex vivo. These islets were counterstained with Somatostatin and Glucagon and demonstrated that compound 1 do not stain these populations of cells found within the islets of Langerhans ( Figure 2).
  • Min6 cells were incubated with the compound 2 for 2 h at 37°C in complete medium.
  • the cells were then imaged using a LEICA SP2 confocal microscope with the following settings: 63x 1.2NA water immersion lens, excitation 488nm, detection 505-525 nm for FITC plus Brightfield imaging to demonstrate cellular integrity. (Figure 3).
  • the image is a single confocal slice.
  • the scale bar represents ⁇ . A pancreas was removed and cryosections were prepared after intravenous injection of compound 1.
  • the representative islet in the photomicrograph demonstrates the staining of insulin positive beta cells only by compound 1 and counterstained with antibodies to glucagon, somatostatin and polypeptide of pancreas (PP) clearly demonstrate specificity of the compound 1 for insulin producing cells.
  • Compound 1 was also used to stain Min 6 cell line known to express insulin in granules and is located inside the cell membrane. Punctate granules can be observed in the cytoplasm surrounding the nucleus. A matching Brightfield image was provided to demonstrate viable live cells.
  • Islets of Langerhans stained with compound 1 or compound 2 were also analyzed to determine if staining interfered with the expected function of the cells and the release of insulin from the beta cells derived from the islets of Langerhans. Briefly, human islets were stained with 0.8 ⁇ compound 1 or compound 2 for 1 h, wash once with PBS and cultured for 5 days. On day 5, pancreatic ⁇ -cells incubated with Newport Green acetate stain

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Abstract

La présente invention concerne des composés et des méthodes pour l'imagerie de cellules pancréatiques bêta et la détermination de la masse de cellules pancréatiques bêta (BCM). Lesdits composés comprennent un peptide comportant des séquences d'acides aminés de liaison à l'insuline, un peptide de cellule comportant une séquence d'acides aminés de pénétration, et un ligand, par exemple un ligand radioactif ou un ligand fluorescent. Lesdites méthodes consistent à colorer les cellules vivantes d'îlots et de cellules bêta pancréatiques in vitro ou in vivo pour le diagnostic et la surveillance de l'efficacité de traitements de troubles des cellules pancréatiques bêta, par exemple le diabète de type 1 et de type 2.
PCT/EP2015/074409 2014-10-21 2015-10-21 Composés pour l'imagerie de cellules pancréatiques bêta WO2016062781A1 (fr)

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GB1418692.8 2014-10-21

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WO2016062781A1 true WO2016062781A1 (fr) 2016-04-28

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