EP2964263A1 - Compositions peptidiques nanoparticulaires - Google Patents

Compositions peptidiques nanoparticulaires

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Publication number
EP2964263A1
EP2964263A1 EP14705556.0A EP14705556A EP2964263A1 EP 2964263 A1 EP2964263 A1 EP 2964263A1 EP 14705556 A EP14705556 A EP 14705556A EP 2964263 A1 EP2964263 A1 EP 2964263A1
Authority
EP
European Patent Office
Prior art keywords
nanoparticle composition
nanoparticle
teriparatide
composition according
core
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP14705556.0A
Other languages
German (de)
English (en)
Inventor
Phillip Williams
Thomas Rademacher
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Midatech Ltd
Original Assignee
Midatech Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB1303771.8A external-priority patent/GB201303771D0/en
Application filed by Midatech Ltd filed Critical Midatech Ltd
Publication of EP2964263A1 publication Critical patent/EP2964263A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle

Definitions

  • the present invention relates to peptide-carrying nanoparticles , particularly for use in medicine, and includes methods for treatment of disorders, e.g., of bone density.
  • the present invention is directed at compositions and products, and methods of making and administering such compositions and products, including for the treatment of mammals and particularly humans.
  • Bioactive agents such as peptides, frequently suffer from poor stability, particularly thermo-stability, which may limit the conditions to which the agents can be subjected during preparation, processing, storage and/or delivery.
  • Medical preparations of peptides for human use are generally formulated with one or more preservatives and/or stabilisers. Moreover, limited
  • gastrointestinal stability typically presents a barrier to effective oral administration of bioactive peptides.
  • WO 2011/154711 describes glyconanoparticles that have a gold core surrounded by a carbohydrate corona and which act as carriers for peptides such as insulin.
  • Teriparatide a recombinant fragment (residues 1-34) of human parathyroid hormone, is used in osteoporosis therapy typically via daily subcutaneous (s.c.) injection.
  • the present invention relates to teriparatide peptide- carrying nanoparticle compositions.
  • the present inventors have found that nanoparticles having a corona of glutathione ligands bind teriparatide (in some cases with a binding capacity of around 15 teriparatide molecules per nanoparticle) .
  • Nanoparticles as defined herein therefor provide a carrier for the formulation and delivery of teriparatide to subjects in need of teriparatide therapeutic treatment .
  • the present invention provides a nanoparticle composition comprising:
  • a core comprising a metal and/or a semiconductor
  • a corona comprising a plurality of ligands covalently linked to the core, wherein said plurality of ligands comprise at least one glutathione;
  • the teriparatide peptide may comprise or consist of an amino acid sequence having at least 70%, 80%, 90%, 95% or 99% amino acid sequence identity with the full-length amino acid sequence set forth as SEQ ID NO: 1. In some cases, the teriparatide peptide comprises or consists of the full-length amino acid sequence
  • SEQ ID NO: 1 is the 34 amino acid sequence of residues 32-65 of the complete 115 amino acid sequence of the human parathyroid hormone polypeptide set forth below as SEQ ID NO: 2 and disclosed under UniProt accession no. P01270, version 136, dated 31 October 2012.
  • MIPAKOMRKVMIVM RICF-LTKSOGKSVKKRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPR DAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ (SEQ ID NO: 2).
  • the 84 amino acid sequence of the mature human parathyroid hormone (residues 32- 115) is shown in italics (SEQ ID NO: 3) .
  • the 34 amino acid sequence of teriparatide (residues 32-65) is shown underlined (SEQ ID NO: 1) .
  • the teriparatide peptide may be selected from the group consisting of:
  • a peptide comprising or consisting of a variant sequence of the full-length amino acid sequence set forth in SEQ ID NO: 1 or 3, wherein said variant differs by addition, deletion, substitution or modification of not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or not more than 10 amino acids from said full-length amino acid sequence set forth in SEQ ID NO: 1 or 3;
  • the teriparatide peptide exhibits biological activity of teriparatide.
  • said teriparatide peptide of any one of (i)-(iv) may exhibit at least 50% of the activity of the
  • teriparatide peptide of SEQ ID NO: 1 or at least 50% of the activity of the peptide of SEQ ID NO: 3 in an in vitro or in vivo bioassay of teriparatide activity.
  • the teriparatide activity may comprise one or more activities selected from the group
  • PTH receptor agonist activity consisting of: PTH receptor agonist activity; modification of the osteoblast/osteoclast bone formation/resorption balance; enhancement of kidney calcium and/or magnesium reabsorption; regulation of plasma calcium and/or phosphate concentration; and enhancement of intestinal calcium absorption.
  • Parathyroid hormone increases serum calcium, partially accomplishing this by increasing bone resorption. Thus, chronically elevated PTH will deplete bone stores.
  • intermittent exposure to PTH has been found to activate osteoblasts and have an anabolic effect. The mechanism of this anabolic effect is unknown but clinical studies have confirmed that treatment with teriparatide such as once-daily injections of teriparatide, has the net effect of stimulating new bone formation leading to increased bone mineral density and improves bone mineral density and bone mineral content in patients with osteoporosis (Teriparatide: A Review
  • the teriparatide peptide exhibits the ability, e.g. upon intermittent administration to a mammalian subject, to produce a net positive effect on bone
  • the nanoparticles in accordance with the present invention may be provided with a variety of numbers of ligands forming the corona.
  • the corona comprises at least 5, 10, 20 or at least 50 ligands per core, e.g. between about 10 to about 1000 ligands per core.
  • the nanoparticle compositions in accordance with any aspect of the present invention may comprise at least 5, 10, 15, 20 or at least 50 glutathione ligands per core.
  • the number of teriparatide peptide molecules bound per core is not particularly limited. For certain applications, it may be desirable to employ as few as 1, 2, 3 or 4 teriparatide peptides per core, while in other cases the nanoparticle of the invention may comprise at least 5, 10, 15, 20 or at least 50 or more teriparatide peptide molecules bound per core.
  • the at least one teriparatide peptide may be bound to the corona of the nanoparticle in a reversible manner.
  • the teriparatide peptide may be bound to the corona such that at least a fraction of the bound teriparatide peptide is released from the nanoparticle upon contacting the nanoparticle with a physiological solution.
  • said ligands comprise glutathione alone or in conjunction with other species of ligand, e.g., combinations of glutathione and carbohydrate ligands (including glucose-containing ligands) are specifically contemplated herein.
  • the nanoparticle comprises at least 10, at least 20, at least 30, at least 40 or at least 50 ligands which are (i) glutathione ligands; or (ii) both glutathione ligands and ligands other than glutathione, such as carbohydrate-containing ligands.
  • the diameter of the core of the nanoparticle is in the range 1 nm to 5 nm.
  • the diameter of the nanoparticle including its ligands is in the range 2 nm to 50 nm, optionally 3 nm to 30 nm, or 4 nm to 20 nm, or 5 nm to 15 nm.
  • the core comprises a metal selected from the group consisting of: Au, Ag, Cu, Pt, Pd, Fe, Co, Gd and Zn, or any combination thereof.
  • the core is magnetic
  • the core comprises a semiconductor
  • semiconductor may comprise metal atoms, such as cadmium. Alternatively or additionally, the semiconductor may comprise non- metal atoms. Organic semiconductors are specifically contemplated herein. Preferred semiconductors, in accordance with the present invention, may be selected from the group consisting of: cadmium selenide, cadmium sulphide, cadmium tellurium and zinc sulphide.
  • the core is capable of acting as a quantum dot.
  • the composition in accordance with the first aspect of the invention comprises a plurality, e.g., 100, 1000, 100000, or more, of said nanoparticles , wherein at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the nanoparticles in said
  • composition have at least one teriparatide peptide bound.
  • the nanoparticle composition comprises a carrier, such as solution, a polymer, a powder, or a cream, in which the nanoparticles and bound teriparatide peptides are suspended.
  • the composition may be in the form of a patch or film for delivery to or across skin, mouth, vagina, rectum or in the form of a spray for delivery into the mouth, nose, lungs or the rectum or vagina.
  • the composition may be in an associated form, a suspension or contained together in a single package, container or carrier.
  • the composition may take the form of one or more doses (e.g. a defined quantity of teriparatide peptide or
  • teriparatide peptide activity units such as in the form of a therapeutic dose or defined number of doses.
  • the nanoparticle composition further comprises at least one permeation enhancer that is non-covalently or covalently bound to said core and/or or said corona.
  • at least one permeation enhancer that is non-covalently or covalently bound to said core and/or or said corona.
  • permeation enhancers may be advantageously bound to the nanoparticle without displacing any significant active peptide, such as the amylin peptide as defined herein.
  • said permeation enhancer is selected from an alkyl-D-maltoside (e.g.
  • tetradecyl-D-maltoside dodecyl-p-D- maltoside, hexyl- -D-maltoside , octyl- -D-maltoside, nonyl- ⁇ - ⁇ - maltoside, decyl- -D-maltoside , undecyl-p-D-maltoside , tridecyl- ⁇ - ⁇ - maltoside, or hexadecyl- -D-maltoside ) and lysalbinic acid.
  • said permeation enhancer e.g. tetradecyl-D- maltoside, dodecyl- -D-maltoside and/or lysalbinic acid is non- covalently bound to said corona.
  • the present invention provides a nanoparticle composition as defined in accordance with the first aspect, for use in medicine.
  • the present invention provides a nanoparticle composition as defined in accordance with the first aspect, for use in a method of therape " .tic or prophylactic treatment of osteoporosis in a mammalian subject
  • the present invention provides use of a
  • nanoparticle composition as defined in accordance with the fir aspect in the preparation of a medicament for therapeutic or prophylactic treatment of osteoporosis in a mammalian subject.
  • the present invention provides a method of therapeutic or prophylactic treatment of osteoporosis in a mammalian subject, the method comprising administering a therapeutically or prophylactically effective amount of a nanoparticle composition as defined in accordance with the first aspect to the subject in need of said treatment.
  • the present invention provides a method of increasing bone mineral density in a mammalian subject, the method comprising administering an effective amount of a nanoparticle composition as defined in accordance with the first aspect to the sub ect .
  • the subject may be a human, a companion animal (e.g. a dog or cat), a laboratory animal (e.g. a mouse, rat, rabbit, pig or non-human primate), a domestic or farm animal (e.g. a pig, cow, horse or sheep) .
  • the subject is a human.
  • the subject is female, e.g. a human female such as a post-menopausal woman .
  • the subject may in certain cases have a disorder that results in abnormally lowered bone mineral density (e.g. lower than normal considering the subject's age and/or gender) .
  • a disorder that results in abnormally lowered bone mineral density e.g. lower than normal considering the subject's age and/or gender
  • specifically contemplated herein is a subjects having, or being at risk of developing, osteoporosis.
  • the subject may or may not have previously been diagnosed with osteoporosis.
  • the subject may have been identified as being at risk of developing osteoporosis (e.g. by virtue of the subject's gender, age,
  • the subject may, in some cases, be following a course of treatment for osteoporosis.
  • the subject may be taking, or have been advised to take, teriparatide, a bisphosphonate medication, hormone replacement therapy, calcium, vitamin D and/or vitamin K.
  • the nanoparticle composition may be administered or for administration with (i.e. simultaneously, separately or
  • one or more therapeutic agents for the control of bone mineral density for example, a bisphosphonate medication, hormone replacement therapy, calcium, vitamin D, menatetrenone and/or vitamin K.
  • the nanoparticle composition may be administered or for administration by any suitable route.
  • the nanoparticle composition may be administered or for administration via a route selected from the group consisting of: intravenous (i.v.)/ intramuscular (i.m.)/ intradermal (i.d.), intraperitoneal or subcutaneous (s.c.) injection or infusion; buccal; sublabial;
  • sublingual by inhalation; via one or more mucosal membranes;
  • the present invention provides an article of manufacture comprising:
  • nanoparticle composition as defined in accordance with the first aspect of the invention
  • the insert and/or label provide instructions, dosage and/or administration information relating to the use of the nanoparticle composition in a method of treatment of a disorder of bone density.
  • the disorder may be osteoporosis.
  • the present invention provides a process for producing a nanoparticle composition as defined in accordance with the first aspect of the invention, the process comprising:
  • a nanoparticle comprising a core comprising a metal and/or a semiconductor and a corona comprising a plurality of ligands covalently linked to the core, wherein said ligands comprise glutathione;
  • the process comprises an earlier step of producing the nanoparticle, said earlier step comprising: combining a solution comprising glutathione with a solution comprising a core-forming material (e.g. gold III chloride) and with a reducing agent (e.g. sodium borohydride) , thereby causing the nanoparticle to self- assemble .
  • a solution comprising glutathione with a solution comprising a core-forming material (e.g. gold III chloride) and with a reducing agent (e.g. sodium borohydride) , thereby causing the nanoparticle to self- assemble .
  • a core-forming material e.g. gold III chloride
  • a reducing agent e.g. sodium borohydride
  • Figure 1 shows Forteo ( teriparatide ) binding under varying GSH NP concentrations.
  • the apparent downward trend of GSH NP may be explained by higher interference of the NPs on the BCA assay.
  • GSH NP showed apparent lower NP Forteo binding.
  • Figure 2 shows the near identical binding capacity of GSHNP for Forteo (teriparatide) regardless of the presence of zinc ions, when corrected for actual NP in the pellet.
  • Figure 3 shows Forteo (teriparatide) binding to variable ratio C2- Glucose and glutathione (GSH) ligand NPs.
  • Figure 4 shows a binding curve with variable/excess GSHNP and a lower level of Forteo (teriparatide) .
  • Figure 5 shows a variable pH binding curve of Forteo (teriparatide) to GSHNPs.
  • nanoparticle refers to a particle having a nanomeric scale, and is not intended to convey any specific shape limitation.
  • nanoparticle encompasses nanospheres, nanotubes, nanoboxes, nanoclusters , nanorods and the like.
  • the nanoparticles and/or nanoparticle cores contemplated herein have a generally polyhedral or spherical geometry .
  • Nanoparticles comprising a plurality of carbohydrate-containing ligands have been described in, for example, WO 2002/032404, WO 2004/108165, WO 2005/116226, WO 2006/037979, WO 2007/015105, WO 2007/122388, WO 2005/091704 (the entire contents of each of which is expressly incorporated herein by reference) and such nanoparticles may find use in accordance with the present invention.
  • nanoparticles /nanoparticle cores are specifically contemplated for use as nanoparticles /nanoparticle cores in
  • corona refers to a layer or coating, which may partially or completely cover the exposed surface of the
  • the corona includes a plurality of ligands which generally include at least one carbohydrate moiety, one surfactant moiety and/or one glutathione moiety.
  • the corona may be considered to be an organic layer that surrounds or partially surrounds the metallic core.
  • the corona provides and/or participates in passivating the core of the
  • the corona may include a sufficiently complete coating layer substantially to stabilise the semiconductor or metal-containing core.
  • certain nanoparticles having cores e.g., that include a metal oxide-containing inner core coated with a noble metal may include a corona that only partially coats the core surface.
  • the corona facilitates solubility, such as water solubility, of the nanoparticles of the present invention.
  • Nanoparticles are small particles, e.g. clusters of metal or semiconductor atoms, that can be used as a substrate for
  • the nanoparticles have cores having mean diameters between 0.5 and 50nm, more preferably between 0.5 and lOnm, more preferably between 0.5 and 5nm, more preferably between 0.5 and 3nm and still more preferably between 0.5 and 2.5nm.
  • the overall mean diameter of the particles is between 2.0 and 20 nm, more preferably between 3 and 10 nm and most preferably between 4 and 5 nm.
  • the mean diameter can be measured using techniques well known in the art such as transmission electron microscopy.
  • the core material can be a metal and/or semiconductor (said
  • semiconductor optionally comprising metal atoms or being an organic semiconductor
  • the core material is a metal selected from Au, Fe or Cu.
  • Nanoparticle cores may also be formed from alloys including Au/Fe, Au/Cu, Au/Gd, Au/Fe/Cu, Au/Fe/Gd and Au/Fe/Cu/Gd, and may be used in the present invention.
  • Preferred core materials are Au and Fe, with the most preferred material being Au.
  • nanoparticles preferably comprise between about 100 and 500 atoms (e.g. gold atoms) to provide core diameters in the nanometre range.
  • Other particularly useful core materials are doped with one or more atoms that are NMR active, allowing the nanoparticles to be detected using NMR, both in vitro and in vivo.
  • NMR active atoms include Mn +2 , Gd°, Eu' 2 , Cu +2 , V +2 , Co +2 , Ni + , Fe +2 , Fe '3 and
  • Nanoparticle cores comprising semiconductor compounds can be detected as nanometre scale semiconductor crystals are capable of acting as quantum dots, that is they can absorb light thereby exciting electrons in the materials to higher energy levels, subsequently releasing photons of light at frequencies
  • An example of a semiconductor core material is cadmium selenide, cadmium sulphide, cadmium tellurium. Also included are the zinc compounds such as zinc sulphide.
  • the core of the nanoparticles may be magnetic and comprise magnetic metal atoms, optionally in combination with passive metal atoms.
  • the passive metal may be gold, platinum, silver or copper
  • the magnetic metal may be iron or gadolinium.
  • the passive metal is gold and the magnetic metal is iron.
  • the ratio of passive metal atoms to magnetic metal atoms in the core is between about 5:0.1 and about 2:5. More preferably, the ratio is between about 5:0.1 and about 5:1.
  • passive metals refers to metals which do not show magnetic properties and are chemically stable to oxidation.
  • the passive metals may be diamagnetic or superparamagnetic.
  • such nanoparticles are superparamagnetic.
  • nanoparticles which have cores comprising a paramagnetic metal include those comprising Mn +2 , Gd ⁇ 3 , Eu +2 , Cu +2 , V +2 , Co" 2 , Ni t2 , Fe +2 , Fe +3 and lanthanides '1"3 .
  • magnétique nanoparticles may be formed from materials such as MnFe (spinel ferrite) or CoFe (cobalt ferrite) can be formed into nanoparticles (magnetic fluid, with or without the addition of a further core material as defined above. Examples of the self- assembly attachment chemistry for producing such nanoparticles is given in Biotechnol. Prog., 19:1095-100 (2003), J. Am. Chem. Soc. 125:9828-33 (2003), J. Colloid Interface Sci. 255:293-8 (2002) .
  • the nanoparticle or its ligand comprises a detectable label.
  • the label may be an element of the core of the nanoparticle or the ligand.
  • the label may be detectable because of an intrinsic property of that element of the nanoparticle or by being linked, conjugated or associated with a further moiety that is detectable.
  • Preferred examples of labels include a label which is a fluorescent group, a radionuclide, a magnetic label or a dye.
  • Fluorescent groups include fluorescein, rhodamine or tetramethyl rhodamine, Texas-Red, Cy3, Cy5, etc., and may be detected by excitation of the fluorescent label and detection of the emitted light using Raman scattering spectroscopy (Y.C. Cao, R. Jin, C. A. Mirkin, Science 2002, 297: 1536-1539) .
  • the nanoparticles may comprise a radionuclide for use in detecting the nanoparticle using the radioactivity emitted by the radionuclide, e.g. by using PET, SPECT, or for therapy, i.e. for killing target cells.
  • radionuclides commonly used in the art that could be readily adapted for use in the present invention include 99m Tc, which exists in a variety of oxidation states although the most stable is Tc0 4 ⁇ ; 32 P or 33 P; 57 Co; 59 Fe; 67 Cu which is often used as Cu 2+ salts; 67 Ga which is commonly used a Ga 3+ salt, e.g.
  • radionuclides as labels and tracers is well known in the art and could readily be adapted by the skilled person for use in the aspects of the present invention.
  • the radionuclides may be employed most easily by doping the cores of the nanoparticles or including them as labels present as part of ligands immobilised on the nanoparticles.
  • the nanoparticles of the present invention can be detected using a number of techniques well known in the art using a label associated with the nanoparticle as indicated above or by employing a property of them.
  • These methods of detecting nanoparticles can range from detecting the aggregation that results when the nanoparticles bind to another species, e.g. by simple visual inspection or by using light scattering ( transmittance of a solution containing the nanoparticles) , to using sophisticated techniques such as transmission electron microscopy (TEM) or atomic force microscopy (AFM) to visualise the nanoparticles.
  • TEM transmission electron microscopy
  • AFM atomic force microscopy
  • a further method of detecting metal particles is to employ plasmon resonance that is the excitation of electrons at the surface of a metal, usually caused by optical radiation.
  • the phenomenon of surface plasmon resonance (SPR) exists at the interface of a metal (such as Ag or Au) and a dielectric material such as air or water. As changes in SPR occur as analytes bind to the ligand immobilised on the surface of a nanoparticle changing the refractive index of the interface.
  • SPR surface plasmon resonance
  • a further advantage of SPR is that it can be used to monitor real time interactions. As mentioned above, if the
  • nanoparticles include or are doped with atoms which are NMR active, then this technique can be used to detect the particles, both in vitro or in vivo, using techniques well known in the art.
  • Nanoparticles can also be detected using a system based on
  • the "teriparatide peptide” may be selected from the group consisting of:
  • a peptide comprising or consisting of an amino acid sequence having at least 70%, 80%, 90%, 95% or 99% amino acid sequence identity to the full-length sequence set forth in SEQ ID NO: 1 or 3;
  • a peptide comprising or consisting of a variant sequence of the full-length amino acid sequence set forth in SEQ ID NO: 1 or 3, wherein said variant differs by addition, deletion, substitution or modification of not more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or not more than 10 amino acids from said full-length amino acid sequence set forth in SEQ ID NO: 1 or 3;
  • a peptide comprising or consisting of a fragment of any one of (i)-(iii), said fragment having a sequence length of at least 15, 20, 25 or 30 amino acids.
  • Sequence identity may be calculated using any suitable method, as would be readily apparent to the skilled person.
  • amino acid sequence identity between a candidate sequence and a reference sequence e.g. the sequence of SEQ ID NO: 1
  • said teriparatide peptide of any one of (i)-(iv) exhibits biological activity of teriparatide.
  • said teriparatide peptide of any one of (i)-(iv) may exhibit at least 50% of the activity of the teriparatide peptide of SEQ ID NO: 1 or at least 50% of the activity of the teriparatide peptide of SEQ ID NO: 3 in an in vitro or in vivo bioassay of teriparatide activity.
  • the teriparatide activity may comprise PTH receptor agonist activity; modification of the osteoblast/osteoclast bone formation/resorption balance; enhancement of kidney calcium and/or magnesium reabsorption; regulation of plasma calcium and/or
  • phosphate concentration enhancement of conversion of 25 (OH) D3 to 1, 25 (OH) 2 vitamin D3; and/or enhancement of intestinal calcium absorption.
  • teriparatide and “Forteo” (RTM) are used interchangeably.
  • the teriparatide peptide is bound to the corona of the nanoparticle .
  • the teriparatide peptide may participate in one or more reversible binding interactions with one or more ligands that provide the corona of the nanoparticle.
  • a portion of the sequence of amino acids may participate in hydrogen bonding, Van der Waals forces and/or electrostatic interactions with one or more ligands (e.g. interacting with one or more glutathione ligands) .
  • the peptide binding may involve adsorption, absorption or other direct or indirect interaction with one or more ligands of the nanoparticle .
  • the teriparatide peptide may be bound such that at least a fraction or portion of the bound teriparatide peptide is released from the nanoparticle upon contacting the nanoparticle with a physiological solution.
  • the teriparatide peptide may be bound to the nanoparticle in a manner such that the teriparatide peptide is stabilised (e.g. thermostabilised) while bound, but is releasable and available in a form that is
  • biologically active for example, releasable such that the
  • teriparatide peptide is detectable by ELISA and/or capable of exerting at least one biological action in an in vitro or in vivo assay system that is characteristic of the free teriparatide peptide) .
  • the teriparatide peptide may be bound to the nanoparticle such that a suspension of the teriparatide-bound nanoparticles gives a positive result in an ELISA for, e.g., (human) teriparatide and/or exerts an effect on a PTH receptor (e.g.
  • nanoparticles and compositions of the invention may be any nanoparticles and compositions of the invention.
  • Parenteral administration includes administration by the following routes: intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraocular, transepithelial , intraperitoneal and topical (including dermal, ocular, rectal, nasal, inhalation and aerosol) , film, patch and rectal systemic routes.
  • the nanoparticles of the invention may be formulated as pharmaceutical compositions that may be in the forms of solid or liquid compositions.
  • Such compositions will generally comprise a carrier of some sort, for example a solid carrier or a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • Physiological saline solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • Such compositions and preparations generally contain at least 0.1 wt% of the compound.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • suitable solutions using, for example, solutions of the compounds or a derivative thereof, e.g. in physiological saline, a dispersion prepared with glycerol, liquid polyethylene glycol or oils.
  • compositions can comprise one or more of a pharmaceutically acceptable excipient, carrier, buffer, stabiliser, isotonicising agent, preservative or anti-oxidant or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • carrier or other material may depend on the route of
  • administration e.g. intraveneously, orally or parenterally.
  • Liquid pharmaceutical compositions are typically formulated to have a pH between about 3.0 and 9.0, more preferably between about 4.5 and 8.5 and still more preferably between about 5.0 and 8.0.
  • the pH of a composition can be maintained by the use of a buffer such as acetate, citrate, phosphate, succinate, Tris or histidine, typically employed in the range from about 1 mM to 50 m .
  • the pH of compositions can otherwise be adjusted by using physiologically acceptable acids or bases.
  • Preservatives are generally included in pharmaceutical compositions to retard microbial growth, extending the shelf life of the
  • compositions and allowing multiple use packaging examples include phenol, meta-cresol, benzyl alcohol, para- hydroxybenzoic acid and its esters, methyl paraben, propyl paraben, benzalconium chloride and benzethonium chloride.
  • Preservatives are typically employed in the range of about 0.1 to 1.0 % (w/v) .
  • the pharmaceutically compositions are given to an individual in a prophylactically effective amount or a
  • therapeutically effective amount (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. Typically, this will be to cause a therapeutically useful activity providing benefit to the individual.
  • amount of the compounds administered, and rate and time- course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g.
  • compositions are preferably administered to patients in dosages of between about 0.01 and lOOmg of active compound per kg of body weight, and more preferably between about 0.5 and lOmg/kg of body weight .
  • dosages between about 0.01 and lOOmg of active compound per kg of body weight, and more preferably between about 0.5 and lOmg/kg of body weight .
  • Gold nanoparticles having a corona of carbohydrate ligands or glutathione ligands were synthesised essentially as described previously (WO 2011/154711; and Lund et al . , 2011, Biomaterials Vol. 32 pp. 9776-9784, the entire contents of which are expressly incorporated herein by reference) .
  • Oxidized ligand, glutathione (Fluka 49741) was dissolved in 9:1 methanol : water and gold III chloride (Sigma-Aldrich, Poole, UK) added.
  • the organic ligand was used at a fourfold molar excess relative to the gold.
  • the solution was then mixed for 5 min gently on a flat-bed shaker.
  • the nanoparticles were produced by reduction following the rapid addition of a 20 fold molar excess relative to the gold, of freshly made 1 M sodium borohydride (Sigma-Aldrich, Poole, UK) under vigorous vortexing.
  • the samples were vortexed for a total of 30 s followed by a further 1 h gentle mixing on the flat bed shaker.
  • initial purification was by bench centrifugation,
  • concentration of all nanoparticle preparations was determined by a simple colorimetric assay. Briefly 10 ⁇ of nanoparticle sample or 12 mg/ml gold standard (Fluka (Sigma-Aldrich, Poole, UK) ) and blanks were digested with 30 ⁇ of 50:50 water:aqua regia in an ELISA plate for 1 min, this was followed by addition of 150 ⁇ of 2 NaBr, the 405 nm absorbance was then measured immediately, the assay having excellent linearity over the 0-10 ⁇ g range.
  • Example 2 Peptide binding to nanoparticles
  • the present inventors have investigated the ability of the peptide teriparatide to bind nanoparticles .
  • Teriparatide (marketed under the trade name FORTEO (RTM) ) is recombinant human parathyroid hormone (1-34), it has an identical sequence to the 34 N-terminal amino acids (the biologically active region) of the 84-amino acid human parathyroid hormone.
  • Teriparatide has the following sequence:
  • Glutathione nanoparticles were found not only to bind teriparatide, but in the presence of Zn 2+ , increased teriparatide binding was apparently achieved (see Figure 1) .
  • the binding assay tested variable amounts of GSHNP against a fixed amount of Forteo, details of the method are given below.
  • binding/precipitation are of relevance. After mixing GSHNP and teriparatide for a fixed time the samples were centrifuged. If a pellet formed that was considered successful binding due to aggregation of complexes with little net charge, it is however, possible that some teriparatide for example could be bound to GSHNP but that this material fails to spin down as such this would then be defined as non-bound.
  • the glutathione-based NP successfully bound >15 teriparatide peptide molecules per NP at the highest teriparatide/NP ratio. Zn 2 ' addition also gave unusual data initially suggesting it increased
  • the basic binding assay used throughout these following studies was 50 ⁇ 1 teriparatide (at 1.65 or 0.825 mg/ml) in various pH 25 mM potassium phosphate buffers, added to NPs (expressed as Au content) in a total of 200 ⁇ water, test samples mixed and then centrifuged after 30 min and the supernatant assayed for protein content by BCA 560 nm, +/-ve controls included to determine how much material has bound .
  • a binding curve was performed with variable/excess GSHNP and a lower level of teriparatide (see Figure 4) .
  • Figure 4 shows increased teriparatide binding with increasing NP to 60-90 nmole NP Au, then a steady decrease presumably due to

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Abstract

La présente invention concerne des nanoparticules portant des peptides de tériparatide, notamment pour leur utilisation en médecine. La présente invention concerne en outre des procédés de traitement de troubles, par exemple, de la densité osseuse. La composition de nanoparticules comprend une nanoparticule comprenant un cœur comprenant un métal et/ou un semiconducteur ; et une couronne comprenant une pluralité de ligands liés de manière covalente au cœur, ladite pluralité de ligands comprenant au moins un glutathione ; et au moins un peptide de tériparatide qui n'est pas lié de manière covalente à la couronne.
EP14705556.0A 2013-03-04 2014-02-06 Compositions peptidiques nanoparticulaires Withdrawn EP2964263A1 (fr)

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