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WO2006001806A2 - Methode de thermometrie non invasive dans laquelle sont utilises des conjugues polypeptidiques semblables a l'elastine - Google Patents

Methode de thermometrie non invasive dans laquelle sont utilises des conjugues polypeptidiques semblables a l'elastine Download PDF

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Publication number
WO2006001806A2
WO2006001806A2 PCT/US2004/019132 US2004019132W WO2006001806A2 WO 2006001806 A2 WO2006001806 A2 WO 2006001806A2 US 2004019132 W US2004019132 W US 2004019132W WO 2006001806 A2 WO2006001806 A2 WO 2006001806A2
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Prior art keywords
elp
temperature
polypeptide
elastin
spin label
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WO2006001806A3 (fr
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Mark Dewhirst
Matthew Dreher
Ashutosh Chilkoti
Howard Halpern
Gerald Rosen
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Duke University
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Duke University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
    • G01K7/00—Measuring temperature based on the use of electric or magnetic elements directly sensitive to heat ; Power supply therefor, e.g. using thermoelectric elements
    • G01K7/32—Measuring temperature based on the use of electric or magnetic elements directly sensitive to heat ; Power supply therefor, e.g. using thermoelectric elements using change of resonant frequency of a crystal
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
    • G01K7/00—Measuring temperature based on the use of electric or magnetic elements directly sensitive to heat ; Power supply therefor, e.g. using thermoelectric elements
    • G01K7/36—Measuring temperature based on the use of electric or magnetic elements directly sensitive to heat ; Power supply therefor, e.g. using thermoelectric elements using magnetic elements, e.g. magnets, coils
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/48—NMR imaging systems
    • G01R33/4804—Spatially selective measurement of temperature or pH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/60—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using electron paramagnetic resonance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
    • G01K2213/00—Temperature mapping

Definitions

  • the presently disclosed subject matter generally relates to compositions for temperature-sensitive electron paramagnetic resonance spectroscopy and imaging. Also provided are methods for making and using the disclosed compositions.
  • Temperatures in tumors during a hyperthermia treatment typically range from 39-45°C and treatment durations are between 60 and 120 minutes. This type of therapy is distinct from thermal ablation, where temperatures are routinely raised to above 7O 0 C for seconds to minutes and the treatment objective is to kill tumor cells directly with heat.
  • the relevant biological effects of a hyperthermia treatment are both time and temperature dependent, creating an urgent need for accurate thermometry with high temporal and spatial resolution. Reliable and accurate thermometry will allow heating with greater precision while sparing adjacent normal tissue. Acquiring accurate temperature measurements with high spatial and temporal resolution throughout a volume of tissue is challenging.
  • the temperature distributions in solid tumors are non-uniform due to tissue heterogeneity, variation in blood vessel distribution and perfusion, and non ⁇ uniform power deposition imposed by typical hyperthermia applicators.
  • Thermometry catheters containing thermocouple, thermistor, or optical sensors have been employed for in vivo thermometry, but due to their invasive nature, only sparse sampling of a tumor volume is typically obtained, leading to unreliable thermometry despite their high degree of accuracy ( ⁇ 0.1-0.2 0 C).
  • Several non-invasive thermometry methods have been developed that hold promise for providing real-time, three-dimensional temperature distribution data.
  • Nuclear magnetic resonance (NMR) techniques including diffusion weighted echo-planar imaging, proton resonance frequency shift, and spin-lattice decay time, have to date shown the most promise.
  • Other methods include microwave radiometry, electrical impedance, ultrasound, and temperature responsive contrast agents.
  • microwave radiometry electrical impedance
  • ultrasound ultrasound
  • temperature responsive contrast agents include temperature responsive contrast agents.
  • microwave radiometry tissue dependent variations in impedance and hysteresis effects when tissue is damaged from heat (electrical impedance), problems associated with hyperthermia treatment changing the average scatter spacing properties of the tissue (ultrasound), and problems with toxicity, pH, cosolutes, or concentration dependencies (injectable contrast agents).
  • the presently disclosed subject matter addresses these and other needs in the art. Summary
  • the presently disclosed subject matter provides a temperature- sensitive elastin-like polypeptide (ELP) conjugated (e.g., covalently attached) to a spin label (e.g., a nitroxide) and a method of detection by electron paramagnetic resonance (EPR) spectroscopy.
  • ELP temperature- sensitive elastin-like polypeptide
  • EPR electron paramagnetic resonance
  • the presently disclosed subject matter also provides a method of thermometry by spin label/electron paramagnetic resonance (EPR) spectroscopy.
  • the method comprises (a) introducing into a target a temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label; and (b) determining the temperature of the target from an electron paramagnetic resonance spectrum produced by the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label.
  • ELP temperature-sensitive elastin-like polypeptide
  • the presently disclosed subject matter also provides a method for imaging a target in a subject by electron paramagnetic resonance (EPR) spectroscopy.
  • EPR electron paramagnetic resonance
  • the method comprises (a) administering to the subject a temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label under conditions sufficient to allow the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label to become present in the target; (b) heating the target; (c) detecting the presence of the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label in the target by EPR; and (d) creating an image of the target from the detected temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label in the target site of the subject.
  • ELP temperature-sensitive elastin-like polypeptide
  • the presently disclosed subject matter also provides a method of synthesizing a temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label.
  • the method comprises (a) providing an elastin-like polypeptide (ELP); and (b) conjugating the elastin- like polypeptide (ELP) to a spin label.
  • a spin label is a nitroxide.
  • the presently disclosed subject matter also provides a method of delivering a therapeutic composition to a target.
  • the method comprises (a) introducing into the target a therapeutic agent and a temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label, wherein: i) the therapeutic agent comprises a first elastin-like polypeptide (ELP) conjugated to the therapeutic composition; ii) the temperature-sensitive electron paramagnetic resonance (EPR) spin label comprises a second elastin-like polypeptide (ELP) conjugated to a spin label; and iii) the therapeutic agent has a first transition temperature (Tt) that is lower than a second Tt of the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label; and (b) heating the target to a temperature that is between the first Tt and the second Tt, wherein the temperature of the target is confirmed from the electron paramagnetic resonance spectrum of the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label.
  • EPR electron paramagnetic resonance
  • the therapeutic composition comprises a chemotherapeutic agent, a toxin, a radiotherapeutic agent, and combinations thereof.
  • the therapeutic agent and the spin label are conjugated to the same ELP molecule.
  • the Tt of the ELP-therapeutic agent-spin label conjugate is lower than, about equal to, or higher than the target temperature.
  • the chemotherapeutic agent is selected from the group consisting of an anti-tumor drug, a cytokine, an anti-metabolite, an alkylating agent, a hormone, methotrexate, doxorubicin, daunorubicin, cytosine arabinoside, etoposide, 4-fluorouracil, 5- fluorouracil, melphalan, chlorambucil, a nitrogen mustard, cyclophosphamide, cis-platinum, vindesine, vinca alkaloids, mitomycin, bleomycin, purothionin, macromomycin, 1 ,4-benzoquinone derivatives, trenimon, steroids, aminopterin, anthracyclines, demecolcine, etoposide, mithramycin, doxorubicin, daunomycin, vinblastine, neocarzinostatin, macromycin, ⁇ -amanitin, and
  • the toxin is selected from the group consisting of Russell's Viper Venom, activated Factor IX, activated Factor X, thrombin, phospholipase C, cobra venom factor, ricin, ricin A chain, Pseudomonas exotoxin, diphtheria toxin, bovine pancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin A chain, gelonin, saporin, modeccin, viscumin, volkensin, and combinations thereof.
  • the radiotherapeutic agent is selected from 47 Sc, 67 Cu, 90 Y, 109 Pd, 123 I, 125 I, 131 I 1 186 Re, 188 Re, 199 Au, 211 At, 212 Pb, 212 Bi, 32 P, 33 P, 71 Ge, 77 As, 103 Pb, 105 Rh, 111 Ag, 119 Sb, 121 Sn, 131 Cs, 143 Pr, 161 Tb, 177 Lu, 191 Os, 193M Pt, and 197 Hg.
  • the elastin-like polypeptide comprises an amino acid sequence comprising one or more repeats of VPGXG (SEQ ID NO: 1 ), wherein X is a guest residue defined as any amino acid except proline, and X can be the same or different in each repeat of SEQ ID NO: 1.
  • the elastin-like polypeptide comprises an amino acid sequence comprising at least one, in some embodiments five, and in some embodiments ten, repeats of VPGAG VPGGG VPGAG VPGGG VPGAG VPGGG VPGAG VPGGG VPGAG VPGGG VPGAG VPGGG VPGAG VPGGG VPGGG VPGVG VPGVG (SEQ ID NO: 3).
  • the spin label comprises a nitroxide.
  • the spin label comprises a moiety selected from the group consisting of 2,2,6,6-tetramethyl-1- piperidinyloxyl (TEMPO), 2,2,5,5-tetramethypyrrolidiny-i-oxyl (PROXYL), 4,4-dimethyl-oxazolidine-3-oxyl (DOXYL), and derivatives thereof.
  • the spin label is conjugated to the temperature-sensitive ELP via a linker moiety, in some embodiments a lysine residue.
  • the target is present within a vertebrate subject, in some embodiments a mammal, and in some embodiments a human.
  • the target comprises a neoplasm, in some embodiments a neoplasm selected from the group consisting of benign intracranial melanomas, arteriovenous malformation, angioma, macular degeneration, melanoma, adenocarcinoma, malignant glioma, prostatic carcinoma, kidney carcinoma, bladder carcinoma, pancreatic carcinoma, thyroid carcinoma, lung carcinoma, colon carcinoma, rectal carcinoma, brain carcinoma, liver carcinoma, breast carcinoma, ovary carcinoma, solid tumors, solid tumor metastases, angiofibromas, retrolental fibroplasia, hemangiomas, Karposi's sarcoma, and combinations thereof.
  • a neoplasm selected from the group consisting of benign intracranial melanomas, arteriovenous malformation, angioma, macular degeneration, melanoma, adenocarcinoma, malignant glioma, prostatic carcinoma, kidney carcinoma, bladder carcinoma, pancreatic carcinoma, thyroid carcinoma, lung carcinoma, colon carcinoma, rec
  • the methods further comprise exposing the neoplasm to a therapeutic dose of ionizing radiation.
  • the transition temperature (Tt) of the temperature-sensitive elastin-like polypeptide (ELP) conjugated to the spin label is at least 30°C, in some embodiments at least 40 0 C, in some embodiments at least 45°C, in some embodiments at least 50 0 C, and in some embodiments at least 60 0 C. Accordingly, it is an object of the presently claimed subject matter to provide temperature-sensitive elastin-like polypeptides (ELPs) conjugated to a spin label and methods of making and using the same. These and other objects are achieved in whole or in part by the presently claimed subject matter.
  • Figure 1 depicts an ELP-nitroxide conjugation scheme.
  • the conjugate (i.e., the product), was purified by inverse transition cycling (ITC) and size exclusion chromatography.
  • Figures 2A and 2B depict the thermal properties of an ELP- conjugated nitroxide.
  • a characteristic turbidity profile is shown, and the first derivative of the turbidity profile with respect to temperature is shown in the top panel.
  • the transition temperature (Tt) of the ELP- conjugated nitroxide determined from the data presented in Figure 2A, is shown in addition to the Tts for the parent unconjugated ELP.
  • the Tt of the ELP was reduced by about 0.7°C by conjugation to the nitroxide.
  • Figure 3B depicts ELP-conjugated nitroxide at 37°C (80 ⁇ M).
  • Figure 3C depicts ELP-conjugated nitroxide at 55°C (80 ⁇ M).
  • Figure 3D depicts free nitroxide at 19 0 C.
  • EPR spectra were recorded on a Varian E-12 spectrometer operating at 9.41 GHz, using 25 mW of power. The time constant and dwell time were set equal to 0.1 second, with 512 points per spectrum. Modulation amplitude was 1.6 Gauss peak-to-peak. The dashed lines are included to better visualize the differences in line width for LLW3 and are all equal distances apart for the spectra shown in Figures 3A-3C.
  • Figure 4 depicts Lorentzian line widths (LLW) of the ELP-nitroxide at 80 ⁇ M.
  • the high field line (LLW3) is broadened and the width is sensitive to temperature.
  • the Tt corresponds to the intersection of these two thermal response regions where the lower temperature region would consist of soluble ELP and the higher temperature region consists of aggregated or transitioned ELP.
  • Figure 5 depicts Lorentzian line width 3 (LLW3) of the ELP-nitroxide conjugate as a function of temperature.
  • the LLW3 narrows as the ELP approaches its Tt and then broadens for temperatures greater than Tt.
  • the first thermal response region is relatively independent of ELP concentration, while both the Tt and second thermal response region are dependent on ELP concentration.
  • Figures 6A-6C depict the sequences used to construct and express exemplary ELPs.
  • Figure 6A depicts the nucleic acid and encoded amino acid sequences (SEQ ID NOs: 4 and 5, respectively) of the sense strand of an ELP [V 5 A 2 G 3 -I O] monomer.
  • Figure 6B depicts that nucleic acid and encoded amino acid sequences (SEQ ID NOs: 6 and 7, respectively) of the sense strand of an ELP [ViA 8 G 7 -I e] monomer.
  • Figure 6C depicts the nucleic acid and encoded amino acid sequences (SEQ ID NOs: 8 and 9, respectively) of the cloning region of a modified pET25 expression vector.
  • SEQ ID NOs: 8 and 9 the restriction enzyme sites that are used in recursive directional ligation (RDL) are shown, with the recognition sequences in bold, the cleavage sites indicated with arrows, and the cohesive ends underlined.
  • RDL recursive directional ligation
  • SEQ ID NO: 2 is the amino acid sequence of a representative ELP polypeptide unit, wherein the guest residues are valine, alanine, and glycine in a 5:2:3 ratio.
  • SEQ ID NO: 3 is the amino acid sequence of a representative ELP polypeptide unit, wherein the guest residues are valine, alanine, and glycine in a 1 :8:7 ratio.
  • SEQ ID NOs: 4 and 5 are the nucleic acid and amino acid sequences, respectively, of the ELP [V 5 A 2 G 3 -I O] monomer used in recursive directional ligation of some ELPs.
  • SEQ ID NOs: 6 and 7 are the nucleic acid and amino acid sequences, respectively of the ELP [V 1 A 8 G 7 -Ie] monomer used in recursive directional ligation of some ELPs.
  • SEQ ID NOs: 8 and 9 are the nucleic acid and amino acid sequences, respectively of cloning sites of a modified pET25 vector.
  • the presently disclosed subject matter generally relates to methods and compositions for accurate, non-invasive thermometry.
  • the methods involve the use of a temperature-sensitive elastin- like polypeptide (ELP) conjugated (for example, covalently attached) to a spin label and EPR spectroscopy and imaging for determining the temperature of a target.
  • ELP temperature-sensitive elastin- like polypeptide
  • Electron paramagnetic resonance (EPR) spectroscopy is a magnetic resonance technique similar to nuclear magnetic resonance (NMR) but unlike NMR, in which energy is absorbed by nuclei with net magnetic moments that are prepared in a magnetic field, in EPR the energy is absorbed by the magnetic moments of unpaired electrons. Unpaired electrons with measurable spectral lines are extremely rare in biologic systems. Thus, the in vivo background signal is very low for EPR spectroscopy and imaging.
  • An exogenous paramagnetic species (also known as an EPR probe, a spin probe, or a spin label) can be injected into animals to obtain spectra and images.
  • EPR probe also known as an EPR probe, a spin probe, or a spin label
  • probes can also be designed to respond to specific aspects of the tissue environment.
  • EPR spectroscopy and imaging was chosen for the development of a new non-invasive thermometry modality because it has the potential to provide significant improvement over prior methods, with sub-centimeter spatial resolution, high temperature resolution ( ⁇ 1.0°C), and its intrinsically high signal to noise ratio.
  • the challenge in using EPR spectroscopy for in vivo thermal imaging is to render an EPR spin probe sensitive to the surrounding thermal environment.
  • a spin label for example, a nitroxide
  • a temperature responsive polypeptide for example, an elastin-like polypeptide
  • the temperature-dependent spectral line width exhibited by a spin label conjugated to a thermally responsive polypeptide forms a basis of this new thermometry modality.
  • an elastin-like polypeptide ELP
  • ELPs are a class of temperature responsive biopolymers that are derived from a structural motif found in mammalian elastin. This family of polypeptides comprises polymers of the pentapeptide Val-Pro-Gly-Xaa-Gly (SEQ ID NO: 1 ), where the "guest residue" Xaa is any amino acid except Pro. ELPs undergo an inverse temperature phase transition, also known as a lower critical solution temperature transition, in aqueous solution in response to an increase in solution temperature. ELPs are soluble at temperatures below their transition temperature (Tt), but become insoluble and aggregate at temperatures above their Tt.
  • Tt transition temperature
  • the inverse temperature transition is fully reversible, so that an aggregated ELP redissolves in aqueous solution when the temperature is decreased below its Tt.
  • the feasibility of using ELP/spin label conjugates for non-invasive thermometry is disclosed herein. Also disclosed herein is that structural changes in the ELP in response to temperature affect the EPR spectral lines of the spin label, allowing for accurate temperature determinations.
  • Hyperthermia has shown promise in the treatment of cancer, both as a primary therapy and as an adjuvant to treatment with radiation and/or chemotherapy. The relevant biological effects of hyperthermia treatment are both time and temperature dependent, creating a need for accurate thermometry.
  • thermometry modality that combines a temperature responsive biopolymer, the elastin-like polypeptide (ELP), to which a spin label is conjugated (e.g., covalently attached) to produce a temperature-sensitive ELP-conjugated spin label.
  • EPR electron paramagnetic resonance
  • the temperature-sensitive elastin-like polypeptide (ELP) conjugated spin label has temperature dependent EPR spectral line widths that are relatively concentration independent with an accuracy of ⁇ 0.3°C (80 ⁇ M).
  • amino acid and “amino acid residue” are used interchangeably and refer to any of the twenty naturally occurring amino acids. An amino acid is formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are in some embodiments in the "L" isomeric form.
  • residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • abbreviations for amino acid residues are shown in tabular form presented hereinabove. It is noted that all amino acid residue sequences represented herein by formulae have a left-to-right orientation in the conventional direction of amino terminus to carboxy terminus.
  • the phrases "amino acid” and “amino acid residue” are broadly defined to include modified and unusual amino acids.
  • nucleic acid molecule refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar properties as the reference natural nucleic acid.
  • nucleic acid molecule or “nucleotide sequence” can also be used in place of "gene", “cDNA”, or "mRNA”. Nucleic acids can be derived from any source, including any organism. Additionally, nucleic acids can be synthesized using techniques known in the art. The term “isolated”, as used in the context of a nucleic acid molecule, indicates that the nucleic acid molecule exists apart from its native environment and is not a product of nature.
  • An isolated DNA molecule can exist in a purified form or can exist in a non-native environment such as a recombinant host cell.
  • isolated indicates that the polypeptide exists apart from its native environment and is not a product of nature.
  • An isolated polypeptide can exist in a purified form or can exist in a non-native environment such as, for example, in a recombinant host cell.
  • gene refers broadly to any segment of DNA associated with a biological function.
  • a gene encompasses sequences including, but not limited to a coding sequence, a promoter region, a transcriptional regulatory sequence, a non-expressed DNA segment that is a specific recognition sequence for regulatory proteins, a non-expressed DNA segment that contributes to gene expression, a DNA segment designed to have desired parameters, or combinations thereof.
  • a gene can be obtained by a variety of methods, including cloning from a biological sample, synthesis based on known or predicted sequence information, and recombinant derivation of an existing sequence.
  • the term "gene expression” generally refers to the cellular processes by which a biologically active polypeptide is produced from a DNA sequence.
  • operatively linked refers to a promoter region that is connected to a nucleotide sequence in such a way that the transcription of that nucleotide sequence is controlled and regulated by that promoter region. Similarly, a nucleotide sequence is said to be under the "transcriptional control" of a promoter to which it is operatively linked. Techniques for operatively linking a promoter region to a nucleotide sequence are known in the art.
  • heterologous gene refers to a sequence that originates from a source foreign to an intended host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example by mutagenesis or by isolation from native transcriptional regulatory sequences.
  • the terms also include non-naturally occurring multiple copies of a naturally occurring nucleotide sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid wherein the element is not ordinarily found.
  • expression construct refers to a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operatively linked to the nucleotide sequence of interest which is operatively linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the construct comprising the nucleotide sequence of interest can be chimeric.
  • the construct can also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • Nucleic acids of the presently disclosed subject matter can be cloned, synthesized, recombinantly altered, mutagenized, or combinations thereof.
  • Standard recombinant DNA and molecular cloning techniques used to isolate nucleic acids are known in the art. Exemplary, non-limiting methods are described by Silhavy et al., 1984 (Experiments with Gene Fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America); Ausubel et al., 1989 (Current Protocols in Molecular Biology. Wiley, New York, New York, United States of America); Glover and Hames, 1995 (DNA Cloning : A Practical Approach.
  • polypeptide means any polymer comprising any of the 20 protein amino acids, or amino acid analogs, regardless of its size or function.
  • protein is often used in reference to relatively large polypeptides and “peptide” is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies.
  • polypeptide refers to peptides, polypeptides and proteins, unless otherwise noted.
  • protein polypeptide
  • polypeptide and peptide are used interchangeably.
  • the polypeptides employed in accordance with the presently disclosed subject matter include, but are not limited to a therapeutic polypeptide as defined herein below; a polypeptide substantially identical to a therapeutic polypeptide as defined herein below; a polypeptide fragment of a therapeutic polypeptide as defined herein below (in one embodiment biologically functional fragments), fusion proteins comprising a therapeutic polypeptide as defined herein below, biologically functional analogs thereof, and polypeptides that cross-react with an antibody that specifically recognizes a therapeutic polypeptide as defined herein below.
  • polypeptides employed in accordance with the presently disclosed subject matter include, but are not limited to isolated polypeptides, polypeptide fragments, fusion proteins comprising the disclosed amino acid sequences, biologically functional analogs, and polypeptides that cross-react with an antibody that specifically recognizes a disclosed polypeptide.
  • the presently disclosed subject matter also encompasses recombinant production of the disclosed polypeptides. Briefly, a nucleic acid sequence encoding a polypeptide is cloned into an expression construct and the expression construct is introduced into a host organism, where it is recombinantly produced.
  • the presently disclosed subject matter provides temperature-sensitive elastin-like polypeptides (ELP) conjugated to a spin label.
  • ELP- conjugated spin label As used herein, the phrases "temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label", “temperature-sensitive ELP- conjugated spin label”, and “ELP/spin label conjugates” are used interchangeably and refer to a paramagnetic molecule (i.e. a spin label) that can be detected by EPR spectroscopy and/or imaging and is characterized by an EPR spectrum that changes with temperature.
  • the ELP is conjugated (for example, covalently attached) to a nitroxide.
  • the conjugates are also referred to as "ELP-conjugated spin labels".
  • IHA Elastin-like Polypeptides ELPs
  • a temperature-sensitive ELP-conjugated spin label comprises an elastin-like polypeptide (ELP) conjugated to a spin label.
  • elastin-like peptide As used herein, the terms “elastin-like peptide”, “elastin-like polypeptide”, and “elastin-like protein” are used interchangeably and refer to polypeptides comprising polymers of the pentapeptide Val-Pro-Gly-Xaa-Gly (SEQ ID NO: 1 ). ELPs undergo an inverse temperature phase transition, such that below their transition temperature (Tt) they are soluble in aqueous solution. Above their Tt, however, ELPs undergo a sharp phase transition ( ⁇ 2°C) during which they hydrophobically collapse and aggregate. This phase transition is fully reversible, so that the aggregated ELP redissolves in aqueous solution once the temperature is decreased below the Tt.
  • Tt transition temperature
  • ⁇ 2°C phase transition
  • an ELP is also referred to herein as a “thermally responsive polypeptide” and/or a “temperature-sensitive polypeptide”.
  • the term “transition temperature” or “Tt” refers to the temperature above which a polymer that undergoes an inverse temperature transition (for example, an ELP) is insoluble in an aqueous system (e.g., water, physiological saline solution, blood, or serum), and below which such a polymer is soluble in the aqueous system.
  • Representative Tts for in vitro applications include O 0 C 1 1O 0 C, 15°C, 20°C to 6O 0 C, and 6O 0 C to 100°C, including all temperatures in between.
  • representative Tts include 35°C to 65°C inclusive, including, but not limited to 35-40°C, 40-45°C, 45-5O 0 C, 50-55°C, and 55-60 0 C.
  • One of skill in the art can employ an ELP having a certain Tt based upon such parameters as what temperatures the ELP is to be exposed to and whether it would be desirable for the ELP to remain soluble or become insoluble under specific conditions.
  • the Tt of a given ELP is affected by several variables, one of which is the "guest residue" (Xaa of SEQ ID NO: 1 ). Generally, as the hydrophobicity of the guest residue increases, the Tt decreases.
  • ELPs that comprise polymers of SEQ ID NO: 1
  • the Tt of the ELP decreases.
  • ELPs can be synthesized with different Tts based upon the mole fraction of different residues chosen as the guest residue.
  • the relative hydrophobicities and hydrophilities of the naturally occurring amino acids are known, as well as the general effect on Tt that can be expected when a given amino acid is present as the guest residue.
  • ELPs can employ combinations of valine, alanine, and glycine at each guest residue position.
  • Another parameter that can affect Tt is the length of the ELP. Generally, a longer ELP will have a lower Tt than a shorter ELP with the same mole fraction of various guest residues. Thus, another way to influence the Tt of a given ELP is to lengthen or shorten it. For a given mole fraction of individual guest residues, the Tt can be varied over 2O 0 C or more depending on the length of the ELP.
  • ELP ELP with only VaI, Ala, and GIy guest residues in a ratio of 1 :8:7, respectively (referred to herein also as "ELP [V 1 AeG 7 -Z], where Z is the number of pentapeptide repeats present in the ELP), a 128 amino acid ELP has a Tt of about 77 0 C (25 ⁇ M in PBS), a 160 amino acid ELP has a Tt of about 71 0 C, a 256 amino acid ELP has a Tt of about 63 0 C, and a 320 amino acid ELP has a Tt of about 60 0 C.
  • an ELP is synthesized with a transition temperature (Tt) of in some embodiments at least 3O 0 C, in some embodiments at least 35 0 C, in some embodiments at least 40 0 C, in some embodiments at least 45°C, in some embodiments at least 5O 0 C, in some embodiments at least 55 0 C, and in some embodiments at least 60°C.
  • Tt transition temperature
  • ELPs comprise an amino acid sequence comprising one or more repeats of VPGXG (SEQ ID NO: 1 ), wherein X is a guest residue defined as any amino acid except proline, and X can be the same or different in each repeat of SEQ ID NO: 1.
  • an ELP comprises at least ten, twenty, thirty, forty, fifty, sixty, or more repeats of VPGXG (SEQ ID NO: 1 ), wherein the guest residue is valine, alanine, and glycine in a 5:2:3 ratio.
  • a representative ELP of this nature is a polypeptide with the amino acid sequence VPGVG VPGGG VPGAG VPGVG VPGVG VPGVG VPGGG VPGAG VPGGG VPGVG (SEQ ID NO: 2), or a polypeptide with one, two, three, four, five, six, or more repeats of SEQ ID NO: 2.
  • an ELP comprises at least sixteen, thirty-two, forty-eight, sixty-four, or more repeats of VPGXG (SEQ ID NO: 1 ), wherein the guest residue is valine, alanine, and glycine in a 1 :8:7 ratio.
  • a representative ELP of this nature is a polypeptide with the amino acid sequence VPGAG VPGGG VPGAG VPGGG VPGAG VPGGG VPGAG VPGGG VPGAG VPGGG VPGAG VPGGG VPGGG VPGGG VPGGG VPGVG VPGVG (SEQ ID NO: 3), or a polypeptide with one, two, three, four, five, six, or more repeats of SEQ ID NO: 3.
  • Methods of synthesizing ELPs include, but are not limited to synthesis using recombinant expression vectors encoding ELPs.
  • a representative recombinant DNA synthesis scheme is the recursive directional ligation (RDL) technique.
  • the RDL technique is outlined in Example 1 below.
  • a sense oligonucleotide encoding a polymer of SEQ ID NO: 1 is synthesized (for example, using conventional oligonucleotide synthesis techniques), wherein the synthesized oligonucleotide encodes a polymer of SEQ ID NO: 1 having different guest residues.
  • An exemplary oligonucleotide encodes one of SEQ ID NOs: 2 and 3.
  • a 100% complementary antisense oligonucleotide is also synthesized and hybridized to the sense oligonucleotide.
  • the now double stranded molecule is cloned into an expression vector (for example, a bacterial expression vector), and the ELP is expressed and purified using standard techniques.
  • multiple copies of the double stranded molecule are cloned into the expression vector, resulting in ELPs that are repeats of the encoded sequence.
  • ELPs that are repeats of the encoded sequence.
  • other molecules that undergo inverse phase transitions can be used to produce temperature-sensitive reagents for EPR spectroscopy and imaging including, but not limited to PEG and PoIy-NIPAAm. III.B. Paramagnetic Species/Spin Labels
  • an ELP must be conjugated (for example, covalently attached) to a paramagnetic species, also referred to as a spin label.
  • any spin label that can be used in EPR spectroscopy can be used in conjunction with an ELP to produce a temperature-sensitive spin label.
  • Representative paramagnetic species are characterized by the presence of nitroxides.
  • the spin label comprises a moiety selected from the group consisting of 3-[[(2,5- dioxo-1-pyrrolidinyl)oxy]carbonyl]-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl, 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO), 2,2,5,5- tetramethylpyrroldinyl-1 -oxyl (PROXYL), 4,4-dimethyl-oxazolidine-3-oxyl (DOXYL), and derivatives thereof.
  • Spin labels can be synthesized using techniques known in the arts of synthetic organic and inorganic chemistry, or can be purchased from suppliers such as Aldrich Chemical Company (Milwaukee, Wisconsin, United States of America).
  • III. C. Methods of Synthesizing Temperature-sensitive ELP Conjugates The presently disclosed subject matter also provides methods for synthesizing temperature-sensitive elastin-like polypeptides (ELP) conjugated to a spin label.
  • ELP temperature-sensitive elastin-like polypeptides
  • the methods comprise (a) providing an elastin-like polypeptide (ELP); and (b) conjugating to the elastin-like polypeptide (ELP) a spin label.
  • ELP and the spin label can be conjugated to each other in any way, including directly attaching the spin label to the ELP, for example by reacting the spin label or a spin label precursor with a side chain or the N- terminal amine or C-terminal carboxyl of the ELP. Methods for conjugating various molecules to proteins are known in the art.
  • the spin label can be conjugated to one or more of the lysines using techniques that are known to the skilled artisan.
  • a linker molecule can be added to the ELP to facilitate conjugation of the spin label.
  • a linker is a peptide linker, which can be added to one or both termini of the ELP.
  • the linker comprises a lysine residue.
  • the spin label can be conjugated to one or more of the lysines using techniques that are known to the skilled artisan. [V.
  • Thermometry Methods also provides methods of thermometry that employ the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label disclosed herein.
  • the methods comprise (a) introducing into a target a temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label; and (b) determining the temperature of the target from an electron paramagnetic resonance spectrum produced by the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label.
  • the term "target” refers to a site for which knowledge of its temperature is desirable.
  • the instant method can be used to determine the temperature of a target present within a subject, for example, a target tissue.
  • a target is a tumor or a neoplasm, particularly a tumor or neoplasm for which hyperthermic treatment might be desirable.
  • hyperthermia is a common adjuvant to radiation and chemotherapy, and has shown promise in the treatment of cancer.
  • the relevant biological effects of a hyperthermia treatment are both time and temperature dependent, and thus the ability to carefully monitor the temperature of a target comprising a tumor or neoplasm can be important for ensuring that the treatment is maximally effective.
  • the instant method comprises determining the temperature of the target from the EPR spectrum produced by the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label.
  • ELP temperature-sensitive elastin-like polypeptide
  • Lorentzian line widths in particular the high field line width (LLW3), can be used to determine the temperature of a target as shown in Figures 4 and 5.
  • the ELP-conjugated spin label is administered intravenously and allowed to circulate throughout the body including the target.
  • a device thereafter applies hyperthermic treatment, such as radio frequency (RF) phased arrays or ultrasound, while collecting thermometry data through a coupled EPR imager.
  • RF radio frequency
  • thermometry modality might involve the use of real-time, intratumoral temperature measurements to modify power deposition during treatment, thereby optimizing temperatures. Additionally, knowledge of the temperature distribution during treatment is likely to have prognostic importance, as has been demonstrated in numerous studies using invasive thermometry methods.
  • the methods comprise (a) administering to the subject a temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label under conditions sufficient to allow the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label to become present in the target; (b) heating the target; (c) detecting the presence of the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label by EPR spectroscopy; and (d) creating an EPR image of the target from the detected temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label in the target.
  • ELP temperature-sensitive elastin-like polypeptide
  • the presently disclosed subject matter employs heating (for example, by employing hyperthermia treatment) of a target.
  • hyperthermia treatment comprises an increase in the temperature of the target to 39°C-43°C, in some embodiments to about 42°C-45°C.
  • thermal treatment can be accomplished by any one of several methods. Most methods involve delivery of energy into the body using microwaves, ultrasound, radiofrequency, or radiant heat (infrared). Methods for delivery of hyperthermia are roughly broken down into the following categories: whole body hyperthermia, local hyperthermia, regional hyperthermia, and interstitial hyperthermia. Power can be delivered from external sources or from interstitial (implanted) sources.
  • the distribution of temperatures achieved during hyperthermia treatment is variable, depending on the method used to deliver the treatment and the nature of the target.
  • the most uniform temperatures are achieved with whole body hyperthermia, but this method does not achieve a differential temperature between the tumor and the surrounding normal tissue. Typical variations throughout the body are about 0.5 0 C.
  • uniform hyperthermia can nevertheless provoke a tumor-specific response.
  • heating of normal tissue in addition to heating of tumor tissue, is acceptable.
  • local provision of heat is employed and can be accomplished by the techniques described below.
  • the temperature distributions that are achieved with methods of restricted heating are more variable.
  • a method for heat treatment can be selected for a particular application, and parameters of the method (e.g., device, temperature, duration, etc.) can be tailored to achieve defined temperature ranges. See e.g., Thrall et a/., 2000 ("Using units of CEM 43 degrees C T90, local hyperthermia thermal dose can be delivered as prescribed", lntl J Hyperthermia 16(5):415-28). Using such methods, tumors of the extremities and other sites, including ovary, brain, breast, prostate, and head and neck, can be heated to a temperature range that is adequate for hyperthermia treatment.
  • Total body hyperthermia can be used for hyperthermia treatment of any body region, in particular those tissues that are not amenable to local heating (e.g., lung).
  • the temperature-sensitive elastin-like polypeptide (ELP) conjugates of the presently disclosed subject matter can be used to image a target by EPR imaging.
  • ELP elastin-like polypeptide
  • any suitable method for heat provision can be used in accordance with the methods of the presently disclosed subject matter and that the method for heat provision is not a limitation of the method.
  • Total Body Hyperthermia Total body hyperthermia is most commonly administered using radiant heating devices. These chamber type devices surround the entire body and emit infrared energy from their surface, which is absorbed in the superficial skin layers.
  • Local hyperthermia refers to heating tumors or other targets that are typically less than 4-5 cm from the body surface if an external heating device is employed. Targets that fit into this category would be skin lesions, such as melanoma, squamous cell carcinoma, basal cell carcinoma, neck nodes resulting from head and neck cancer, and chest wall recurrences following breast cancer. Radiofrequency, microwave, and ultrasound applicators have been developed that are specifically designed for heating these types of targets. Typically, the hyperthermia applicator is positioned immediately on top of the target, and power is applied directly to the skin surface overlying the target. With these types of targets, focusing of power is usually not necessary, since power is being controlled by the location of the heating device and its proximity to the surface of the tumor.
  • Regional hyperthermia refers to delivery of power to large portions of the body, such as a portion of the body where a target (e.g. a tumor) resides. Examples of targets that fit into this category include tumors of the pelvis and abdomen, such as cancers of the rectum, cervix, prostate, and ovary. Other examples include large tumors of the extremities, such as sarcomas and primary breast cancers.
  • Radiofrequency, microwave, and ultrasound methods are also used for this type of heating, but require devices specifically designed for a particular application.
  • one method for heating the pelvis or abdomen uses a phased microwave array. This device delivers power from several antennas that are positioned circumferentially around the body. Power can be steered toward the region of interest by varying the phase and amplitude of each of the antennas in the array.
  • ultrasound devices can be designed to focus energy directly into deep lesions by using multiple beams that enter into the body from several positions.
  • Interstitial/lntraluminal Hyperthermia This type of hyperthermia is usually used to deliver local hyperthermia, wherein the heating device is placed directly into the target. Radiofrequency, microwave, and ultrasound devices have been designed for this type of application.
  • Targets that are located in hollow organs, such as the bladder, esophagus, and rectum, are amenable to intraluminal hyperthermia if the tumor is less than a few cm in size. Interstitial heating has been successfully applied to tumors of the brain, prostate, cervix, and other sites that are accessible for interstitial implants.
  • V.B. Creating an Image Once temperature data are acquired for a target (for example, a tumor), an EPR image can be created by constructing a two-dimensional or three-dimensional temperature map from the acquired data. Methods for creating an image using EPR are known to one of ordinary skill in the art.
  • EPR imaging has conventionally been carried out by continuous wave (CW) method using a standard EPR spectrometer, where the frequency of radiation is held constant, while the magnetic field is swept.
  • CW continuous wave
  • gradient currents are turned on to do one-dimensional, two-dimensional, or three-dimensional EPR imaging or spectral-spatial work.
  • Using an apparatus that measures electron paramagnetic resonance a real-time image of a macroscopic object, including living tissue, can be obtained.
  • Several EPRI apparatuses are commercially available, including hardware and software components available from the EPR Division of Bruker BioSpin Corporation (Billerica, Massachusetts, United States of America).
  • EPRI provides the capability to obtain multi-dimensional images (including spectral-spatial images) for diagnosis or research.
  • the application for extrinsically introduced nitroxide has demonstrated utility as a relatively low resolution in vivo EPR imaging agent.
  • a radio frequency Fourier transform EPR spectrometer for detecting free radical species and for in vivo imaging has been constructed.
  • the methods comprise (a) introducing into the target a therapeutic agent and a temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label, wherein: i) the therapeutic agent comprises a first elastin-like polypeptide (ELP) conjugated to a therapeutic composition; ii) the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label comprises a second elastin-like polypeptide (ELP); and iii) the therapeutic agent has a first transition temperature (Tt) that is lower than a second Tt of the temperature-sensitive elastin-like polypeptide (ELP) conjugated to a spin label; and (b) heating the target to a temperature that is between the first Tt and the second Tt, wherein the temperature of the target is confirmed from the
  • the methods comprise (a) introducing into the target an elastin-like polypeptide conjugated to both a therapeutic agent and an electron paramagnetic resonance (EPR) spin label; and (b) heating the target, wherein the temperature of the target is confirmed from the electron paramagnetic resonance spectrum of the electron paramagnetic resonance (EPR) spin label.
  • EPR electron paramagnetic resonance
  • targeted delivery can be accomplished using at least two general approaches.
  • the ELP is conjugated to both the spin probe and a therapeutic agent.
  • the therapy, biodistribution, and temperature distribution can all be seamlessly combined into one molecule.
  • the Tt can either be above the hyperthermic temperature or between the body temperature and the hyperthermic temperature.
  • thermometry When the Tt is greater than the hyperthermic temperature passive targeting combined with non-invasive thermometry result. If the Tt is between body temperature and the hyperthermic temperature, active targeting results, but the thermometry can potentially be complicated since there might be two temperatures for each line width. This could be overcome by confining the analysis to temperatures above about 39°C. However, despite the potential loss in accuracy, hot and cold spots could be readily identified to more homogeneously heat the target.
  • two or more separately labeled ELPs are employed where one is conjugated to the spin probe and has a high Tt (for example, greater than 50°C), and one or more other ELPs are conjugated to one or more therapeutic compositions.
  • a therapeutic agent comprises an ELP conjugated to a therapeutic composition (also referred to herein as an "active agent").
  • therapeutic composition refers to a polypeptide (referred to herein as a "therapeutic polypeptide") or other molecule than when introduced into a target results in a therapeutically beneficial effect.
  • Representative therapeutic compositions comprise chemotherapeutic agents, toxins, radiotherapeutics, and combinations thereof. Each agent is loaded in a total amount effective to accomplish the desired result in the target, whether the desired result be imaging the target or treating the target.
  • Chemotherapeutics useful as active agents are typically small chemical entities produced by chemical synthesis. Chemotherapeutics include cytotoxic and cytostatic drugs. Chemotherapeutics can include those that have other effects on cells including, but not limited to reversal of a transformed state to a differentiated state or those that inhibit cell replication.
  • chemotherapeutic agents include, but are not limited to anti-tumor drugs, cytokines, anti-metabolites, alkylating agents, hormones, and the like. Additional examples of chemotherapeutics include common cytotoxic or cytostatic drugs such as, for example, methotrexate (amethoptehn), doxorubicin (adrimycin), daunorubicin, cytosine arabinoside, etoposide, 4- fluorouracil, 5-fluorouracil, melphalan, chlorambucil, and other nitrogen mustards (e.g. cyclophosphamide), cis-platinum, vindesine (and other vinca alkaloids), mitomycin and bleomycin.
  • methotrexate amethoptehn
  • doxorubicin doxorubicin
  • daunorubicin daunorubicin
  • cytosine arabinoside etoposide
  • chemotherapeutics include, but are not limited to purothionin (barley flour oligopeptide), macromomycin, 1 ,4- benzoquinone derivatives, trenimon, steroids, aminopterin, anthracyclines, demecolcine, etoposide, mithramycin, daunomycin, vinblastine, neocarzinostatin, macromycin, ⁇ -amanitin, and the like.
  • purothionin barley flour oligopeptide
  • macromomycin 1 ,4- benzoquinone derivatives
  • trenimon steroids
  • aminopterin anthracyclines
  • demecolcine demecolcine
  • etoposide mithramycin
  • daunomycin vinblastine
  • neocarzinostatin macromycin
  • ⁇ -amanitin and the like.
  • the use of combinations of chemotherapeutic agents is also provided in accordance with the presently disclosed subject matter.
  • the chemotherapeutic agent is selected from the group consisting of an anti ⁇ tumor drug, a cytokine, an anti-metabolite, an alkylating agent, a hormone, methotrexate, doxorubicin, daunorubicin, cytosine arabinoside, etoposide, 4- fluorouracil, 5-fluorouracil, melphalan, chlorambucil, a nitrogen mustard, cyclophosphamide, cis-platinum, vindesine, vinca alkaloids, mitomycin, bleomycin, purothionin, macromomycin, 1 ,4-benzoquinone derivatives, trenimon, steroids, aminopterin, anthracyclines, demecolcine, etoposide, mithramycin, doxorubicin, daunomycin, vinblastine, neocarzinostatin, macromycin, ⁇ -amanitin, and combinations thereof.
  • Toxins can also be employed as active agents.
  • a toxin When a toxin is loaded onto a thermally responsive polypeptide (for example, an ELP), the toxin-loaded thermally responsive polypeptide can be delivered to a target by way of exposure of the target to hyperthermia. Once delivered, the toxin moiety can kill cells in the target.
  • thermally responsive polypeptide for example, an ELP
  • the toxin-loaded thermally responsive polypeptide can be delivered to a target by way of exposure of the target to hyperthermia. Once delivered, the toxin moiety can kill cells in the target.
  • Toxins are generally complex toxic products of various organisms including bacteria, plants, etc.
  • Exemplary toxins include, but are not limited to coagulants such as Russell's Viper Venom, activated Factor IX, activated Factor X, and thrombin; and cell surface lytic agents such as phospholipase C, (Flickinger and Trost, 1976) and cobra venom factor (CVF; Vogel and Muller-Eberhard, 1981 ) which should lyse neoplastic cells directly.
  • coagulants such as Russell's Viper Venom, activated Factor IX, activated Factor X, and thrombin
  • cell surface lytic agents such as phospholipase C, (Flickinger and Trost, 1976) and cobra venom factor (CVF; Vogel and Muller-Eberhard, 1981 ) which should lyse neoplastic cells directly.
  • toxins include, but are not limited to ricin, ricin A chain (ricin toxin), Pseudomonas exotoxin (PE), diphtheria toxin (DT), bovine pancreatic ribonuclease (BPR), pokeweed antiviral protein (PAP), abrin, abrin A chain (abrin toxin), gelonin (GEL), saporin (SAP), modeccin, viscumin, and volkensin.
  • ricin ricin A chain
  • PE Pseudomonas exotoxin
  • DT diphtheria toxin
  • BPR bovine pancreatic ribonuclease
  • PAP pokeweed antiviral protein
  • abrin abrin
  • abrin A chain abrin A chain
  • GEL gelonin
  • SAP saporin
  • the toxin is selected from the group consisting of Russell's Viper Venom, activated Factor IX, activated Factor X, thrombin, phospholipase C, cobra venom factor, ricin, ricin A chain, Pseudomonas exotoxin, diphtheria toxin, bovine pancreatic ribonuclease, pokeweed antiviral protein, abrin, abrin A chain, gelonin, saporin, modeccin, viscumin, volkensin, and combinations thereof.
  • Radiotherapeutic agents can also be employed as active agents.
  • radiotherapeutic agents include, but are not limited to 47 Sc, 67 Cu, 90 Y, 109 Pd, 123 I, 125 I, 131 I 1 111 In, 186 Re, 188 Re, 199 Au, 211 At, 212 Pb, and 212 Bi.
  • Other radiotherapeutic agents that can be employed include 32 P and 33 P, 71 Ge, 77 As, 103 Pb, 105 Rh, 111 Ag, 119 Sb, 121 Sn, 131 Cs 1 143 Pr, 161 Tb, 177 Lu, 191 Os, 1931 ⁇ Pt 1 197 Hg, all beta negative and/or auger emitters.
  • radiotherapeutic agents include 90 Y, 131 I 1 211 At, and 212 Pb/ 212 Bi.
  • the radiotherapeutic agent is selected from the group consisting Of 47 Sc, 67 Cu, 90 Y, 109 Pd, 123 I, 125 I, 131 I, 186 Re, 188 Re, 199 Au, 211 At, 212 Pb, 212 Bi, 32 P, 33 P, 71 Ge, 77 As, 103 Pb, 105 Rh, 111 Ag, 119 Sb, 121 Sn, 131 Cs, 143 Pr, 161 Tb, 177 Lu, 191 Os, 193M Pt, and 197 Hg.
  • the target is a tumor or neoplasm.
  • Representative neoplasms that can be targeted by the instant methods are selected from the group consisting of benign intracranial melanomas, arteriovenous malformation, angioma, macular degeneration, melanoma, adenocarcinoma, malignant glioma, prostatic carcinoma, kidney carcinoma, bladder carcinoma, pancreatic carcinoma, thyroid carcinoma, lung carcinoma, colon carcinoma, rectal carcinoma, brain carcinoma, liver carcinoma, breast carcinoma, ovary carcinoma, solid tumors, solid tumor metastases, angiofibromas, retrolental fibroplasia, hemangiomas, Karposi's sarcoma, and combinations thereof.
  • the method further comprises exposing the target (for example, a tumor or neoplasm) to a therapeutic dose of ionizing radiation.
  • ionizing radiation is meant to refer to any radiation where an electron, X-ray, gamma ray, or nuclear particle has sufficient energy to remove an electron or other particle from an atom or molecule, thus producing an ion and a free electron or other particle. Examples of such ionizing radiation include, but are not limited to gamma rays, X-rays, protons, electrons, and alpha particles. Ionizing radiation is commonly used in medical radiotherapy and the specific techniques for such treatment will be apparent to a skilled practitioner in the art.
  • compositions disclosed herein can be used on a target either in vitro (for example, on isolated cells or tissues) or in vivo in a subject.
  • the subject is a human subject, although it is to be understood that the principles of the presently disclosed subject matter indicate that the presently disclosed subject matter is effective with respect to all vertebrate species, including mammals, which are intended to be included in the term "subject".
  • a mammal is understood to include any mammalian species for which employing the compositions and methods disclosed herein is desirable, particularly agricultural and domestic mammalian species.
  • the methods of the presently disclosed subject matter are particularly useful in the treatment of warm-blooded vertebrates.
  • the presently disclosed subject matter concerns mammals and birds. More particularly provided is the treatment of mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economic importance (animals raised on farms for consumption by humans), and/or of social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses.
  • compositions of the presently disclosed subject matter comprise in some embodiments a composition that includes a pharmaceutically acceptable carrier. Any suitable pharmaceutical formulation can be used to prepare the compositions for administration to a subject.
  • suitable formulations can include aqueous and non ⁇ aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostatics, bactericidal antibiotics, and solutes that render the formulation isotonic with the bodily fluids of the subject; and aqueous and non-aqueous sterile suspensions that can include suspending agents and thickening agents.
  • the formulations can be presented in unit-dose or multi- dose containers, for example sealed ampoules and vials, and can be stored in a frozen or freeze-dried (lyophilized) condition requiring only the addition of sterile liquid carrier, for example water for injections, immediately prior to use.
  • Some exemplary ingredients are sodium dodecyl sulfate (SDS) 1 in one example in the range of 0.1 to 10 mg/ml, in another example about 2.0 mg/ml; and/or mannitol or another sugar, for example in the range of 10 to 100 mg/ml, in another example about 30 mg/ml; and/or phosphate-buffered saline (PBS).
  • SDS sodium dodecyl sulfate
  • mannitol or another sugar for example in the range of 10 to 100 mg/ml, in another example about 30 mg/ml
  • PBS phosphate-buffered saline
  • the formulations of this presently disclosed subject matter can include other agents conventional in the art having regard to the type of formulation in question. For example, sterile pyrogen-free aqueous and non ⁇ aqueous solutions can be used. IX.
  • Suitable methods for administration of a composition of the presently disclosed subject matter include, but are not limited to intravenous and intratumoral injection.
  • a composition can be deposited at a site in need of treatment in any other manner, for example by spraying a composition comprising a composition within the pulmonary pathways.
  • the particular mode of administering a composition of the presently disclosed subject matter depends on various factors, including the distribution and abundance of cells to be imaged and/or treated and mechanisms for metabolism or removal of the composition from its site of administration.
  • relatively superficial tumors can be injected intratumorally.
  • internal tumors can be imaged and/or treated following intravenous injection.
  • the method of administration encompasses features for regionalized delivery or accumulation at the site to be imaged and/or treated.
  • a composition is delivered intratumorally.
  • selective delivery of a composition to a target is accomplished by intravenous injection of the composition followed by hyperthermia treatment of the target.
  • compositions of the presently disclosed subject matter can be formulated as an aerosol or coarse spray. Methods for preparation and administration of aerosol or spray formulations can be found, for example, in U.S. Patent Nos. 5,858,784; 6,013,638; 6,022,737; and 6,136,295.
  • X 1 Doses An effective dose of a composition of the presently disclosed subject matter is administered to a subject.
  • An "effective amount" is an amount of the composition sufficient to produce adequate imaging and/or treatment.
  • Actual dosage levels of constituents of the compositions of the presently disclosed subject matter can be varied so as to administer an amount of the composition that is effective to achieve the desired effect for a particular subject and/or target.
  • the selected dosage level will depend upon the activity of the composition and the route of administration.
  • one of ordinary skill in the art can tailor the dosages to an individual subject, taking into account the particular formulation, method of administration to be used with the composition, and nature of the target to be imaged and/or treated.
  • Such adjustments or variations, as well as evaluation of when and how to make such adjustments or variations are well known to those of ordinary skill in the art. Examples The following Examples have been included to illustrate modes of the presently disclosed subject matter.
  • Example 1 Synthesis and Characterization of an ELP Monomer Gene Synthesis. Standard molecular biology protocols were used for DNA manipulation, E. coli culture, and protein expression. ELPs were synthesized using recursive directional ligation (RDL).
  • RDL recursive directional ligation
  • oligonucleotides were annealed by heating an equimolar mixture of the four oligonucleotides (2 ⁇ M each in ligase buffer) to >95°C and then slowly cooling to room temperature to form a double- stranded DNA cassette with EcoR I and HinD III compatible ends.
  • pUC19 was co-digested with EcoR I and HinD III and enzymatically dephosphorylated using calf intestinal alkaline phosphatase (CIP).
  • CIP calf intestinal alkaline phosphatase
  • the linearized pUC19 vector was then purified using a microcentrifuge spin column purification kit and eluted in sterile, deionized water.
  • the annealed oligonucleotides were then ligated to the linearized vector (-200U ligase, -0.1 pmol vector, and ⁇ 1 pmol insert incubated in 20 ⁇ L ligase buffer at 16 0 C for 2 hours). A 10 ⁇ L portion of the ligation mixture was combined with 100 ⁇ L of chemically competent E.
  • coli cells (XL1-Blue strain; Stratagene, La JoIIa, California, United States of America), and the cells were transformed by heat shock (30 minutes at 4°C, followed by 60 seconds at 42 0 C and 5 minutes at 4°C), spread on CIRCLEGROW ® medium (Q-BIOgene, Inc., Carlsbad, California, United States of America) agar plates supplemented with ampicillin (100 ⁇ g/mL), and incubated overnight at 37°C. Colonies were initially screened by blue-white screening and subsequently verified by agarose gel electrophoresis of colony PCR products.
  • ELP [V 5 A 2 G 3 -I O] monomer gene was constructed, a ELP [V 5 A 2 G 3 -2O] gene was synthesized by ligating the 10-mer insert into a vector containing the same 10-mer gene as follows.
  • the vector, containing a copy of the gene of the monomer ELP [V 5 A 2 G 3 -IO] was linearized with PfM I (-10-fold overdigestion for 6 hours), enzymatically dephosphorylated with CIP, and then purified using a microcentrifuge spin column purification kit.
  • a separate sample of vector was doubly digested with PfM I and BgI I to excise the gene encoding ELP [V 5 A 2 G 3 -IO].
  • the reaction products were separated by agarose gel electrophoresis, and the insert was purified using a gel extraction microcentrifuge spin column kit. During purification of the insert from the agarose gel slice, vortexing or heating of the samples above 37°C was avoided to prevent damage to the DNA.
  • the purified insert and the linearized vector were ligated and transformed into XL1-Blue cells using the protocol described above. Transformants were initially screened by colony PCR and/or diagnostic restriction endonuclease (RE) digests, and further confirmed by DNA sequencing.
  • Expression Vector Construction The purified insert and the linearized vector were ligated and transformed into XL1-Blue cells using the protocol described above. Transformants were initially screened by colony PCR and/or diagnostic restriction endonuclease (RE) digests, and further confirmed by DNA sequencing.
  • Expression vectors that are compatible with the ELP genes were constructed by modifying the DNA sequence spanning Nde I to EcoR I of pET-25b(+) (Novagen, Inc., Milwaukee, Wisconsin, United States of America), a commercial T7-lac expression vector, by cassette mutagenesis to incorporate a unique Sf/ I recognition site (see Figure 6C).
  • the modified pET-25b(+) expression vector was digested with Sfi I (-10-fold overdigestion for 6 hours), dephosphorylated, and purified using a microcentrifuge spin column kit.
  • the ELP gene was excised from the pUC19 vector by digestion with PfM I and BgI I 1 and the excised ELP gene was purified by agarose gel extraction following gel electrophoresis.
  • the Sf/ I linearized pET-25b vector and the ELP-encoding gene were ligated and transformed as described above, and plasmids isolated from the resulting transformants were screened by diagnostic RE digest and sequenced.
  • the expression vector adds translation initiation and termination codons, as well as codons for a short leader (Ser-Lys-Gly-Pro-Gly; SEQ ID NO: 9) and trailer (Trp-Pro). Expression.
  • the expression vectors were transformed into the E. coli strain BLR(DE3) (Novagen, Inc.) for expression.
  • starter cultures 250 mL flasks containing 50 imL of medium supplemented with 100 ⁇ g/mL ampicillin
  • DMSO dimethyl sulfoxide
  • Expression cultures (4 L flasks containing 1 L of medium with 100 ⁇ g/mL ampicillin) were inoculated with 2 mL of the resuspended starter culture and incubated with shaking (-300 rpm) at 37 0 C. When the OD 6 oo reached -0.8-1.0 (typically about 3.5 hours post inoculation), expression was induced by the addition of isopropylthio-/?-D-galactoside (IPTG) to a final concentration of 1 mM. The cells were typically harvested 3 hours after induction by centrifugation at 300Og for 20 minutes at 4°C.
  • IPTG isopropylthio-/?-D-galactoside
  • the 1 L cultures could be allowed to grow for about 24 hours without IPTG induction (known as a hyper expression; see also Guda et a/., 1995 ("Hyper expression of an environmentally friendly synthetic polymer gene," Biotechnol. Lett. 17(7), 745-750)). After 24 hours the cells are harvested by centrifugation at 300Og for 20 minutes at 4°C.
  • the harvested cells are resuspended in 35 imL of cold, low ionic strength buffer (typically phosphate- buffered saline (PBS); 137 mM NaCI, 2.7 mM KCI 1 4.2 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , pH 7.3), and lysed by sonic disruption at 4°C (90 seconds of sonication at maximum power, using 10 second pulses separated by 20 seconds with a 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, Pennsylvania, United States of America).
  • PBS phosphate- buffered saline
  • the cell lysate was centrifuged at 20,00Og for 15 minutes at 4°C to remove insoluble cellular matter.
  • Soluble nucleic acids were precipitated by the addition of polyethylenimine (0.5% final concentration, w/v) and removed by centrifugation at 20,00Og for 15 minutes at 4 0 C.
  • ELP Purification ELPs were purified by inverse transition cycling (ITC). Briefly, the ELPs were selectively aggregated by heating the cell lysate (typically 30-45 0 C) and/or by adding NaCI (typically 0.5-2 M). The aggregated protein was separated from solution by centrifugation at 10,000g for 15 minutes at 30-45 0 C. The supernatant, containing soluble contaminants from the lysed E. coli cells, was decanted and discarded.
  • ITC inverse transition cycling
  • the pellet containing the ELP was resolubilized in cold, low ionic strength buffer. Once the ELP was fully resolubilized, the first round of inverse transition cycling was completed with a final centrifugation step at 15,00Og for 10 minutes at 4°C to remove any remaining insoluble contaminants. Typically, two rounds of inverse transition cycling (warm centrifugation, pellet resuspension, and subsequent cold centrifugation) were sequentially performed to purify the ELP. Characterization of the Expressed ELPs. The ELPs were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometry, and UV-vis spectrophotometry.
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • the concentrations of ELP solutions were determined spectrophotometrically using the molar extinction coefficient of Trp at 280 nm (5690 M '1 cm '1 ). SDS- PAGE gels were visualized by copper staining. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was performed by the Duke University Mass Spectrometry Facility in the Department of Chemistry using a PE Biosystems VOYAGER-DETM instrument (Perkin-Elmer Applied Biosystems Inc., Foster City, California, United States of America) equipped with a nitrogen laser (337 nm).
  • MALDI-MS Matrix-assisted laser desorption/ionization mass spectrometry
  • the MALDI-MS samples were prepared in an aqueous 50% acetonitrile solution containing 0.1 % trifluoroacetic acid, using a sinapinic acid matrix.
  • Thermal Characterization To characterize the ELP inverse temperature transition, the OD 350 of ELP solutions (typically 25 ⁇ M ELP in PBS) was monitored as a function of temperature on a Cary 300 UV-visible spectrophotometer equipped with a multicell thermoelectric temperature controller (Varian Instruments, Walnut Creek, California, United States of America). The heating and cooling rates were 1 0 C min "1 . The derivative of the turbidity profile with respect to temperature was numerically calculated, and the Tt was defined as the solution temperature at the maximum of the turbidity gradient.
  • the size of ELP aggregates formed during the inverse temperature transition was characterized as a function of temperature by dynamic light scattering (DLS) using a DynaPro-LSR DLS instrument equipped with a Peltier temperature control unit (Protein Solutions, Charlottesville, Virginia, United States of America).
  • DLS dynamic light scattering
  • a 25 ⁇ M solution of ELP in PBS was centrifuged at 16,00Og for 10 minutes at 4°C to remove air bubbles and insoluble debris, and the cold supernatant was then filtered through a 20 nm Whatman ANODISCTM filter (Whatman, Inc., Clifton, New Jersey, United States of America).
  • Light scattering data (15 measurements, each with a 5 second acquisition time) were collected at 1 °C intervals as the solution was heated from 35 to 60 0 C.
  • 3-carboxy-2,2,5,5-tetramethyl-1- pyrrolidinyloxyl 500 mg, 2.69 millimoles
  • a 50 ml_ tetrahydrofuran (THF) solution was synthesized as described in the literature (Rozantsev, 1970).
  • ⁇ /-hydroxysuccinimide 310 mg, 2.69 millimoles, Aldrich Chemical Company, Milwaukee, Wisconsin, United States of America
  • 4-pyrrolidinopyridine 40 mg, 0.269 mmoles, Aldrich
  • Example 3 Conjugation of the ELP and 3-[[(2,5-dioxo-1-pyrrolidinv ⁇ oxy1carbonyll- 2.2.5, 5-tetramethyl-1-pyrrolidinyloxyl Recombinant synthesis of the ELP from a plasmid-borne synthetic gene in E. coli and its purification by inverse transition cycling (ITC) is described in Example 1 .
  • the Tt of the ELP is controlled by the identity and mole fraction of the guest residue, and the molecular weight (MW) of the ELP.
  • the ELP used herein has a guest residue composition of VaI, Ala, and GIy in a ratio of 5:2:3 and a MW of 59.4 kilodaltons (kDa), corresponding to 150 pentapeptide (Val-Pro-Gly-Xaa-Gly) repeats.
  • a lysine residue was incorporated into the short N-terminal leader sequence of the ELP to enable conjugation of the ELP to 3-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]-2,2,5,5- tetramethyl-1 -pyrrolidinyloxyl.
  • the reaction scheme for conjugation of the nitroxide to the ELP is shown in Figure 1.
  • the ELP- nitroxide conjugate was concentrated and purified by ITC as follows: NaCI was added to the reaction mixture to a final concentration of 1.33 M to aggregate the ELP by depressing its Tt below the solution temperature (the Tt of the ELP is also dependent on cosolutes). This solution was centrifuged (16,00Og) at room temperature for 10 minutes. The supernatant was discarded and the pellet was resuspended in PBS and centrifuged (16,00Og) at 4°C for 5 minutes. The pellet was discarded and the supernatant, which contained the purified ELP-nitroxide conjugate (and unconjugated ELP), was recovered.
  • the ELP-nitroxide conjugate was then further purified by size exclusion chromatography on a PD-10 column (Amersham Biosciences Corp., Piscataway, New Jersey, United States of America) to ensure any remaining free nitroxide was removed.
  • the ELP-conjugated nitroxide was concentrated to a final concentration of about 1 mM by another round of ITC and stored in PBS at -20 0 C until further use.
  • the thermal transition behaviors of the ELP and ELP-nitroxide were characterized by monitoring the extinction of an aqueous solution of ELP or ELP-nitroxide at 350 nm as a function of temperature (1 °C/minute) on a UV- visible spectrophotometer equipped with a multicell thermoelectric temperature controller (Cary 300 Bio, available from Varian Instruments, Palo Alto, California, United States of America).
  • the Tt was defined as the temperature at the maximum in the first derivative of optical density with respect to temperature (dOD/dT).
  • Chelex 100 resin beads Bio- Rad Laboratories, Hercules, California, United States of America
  • the temperature in the flat cell was measured by a non-magnetic thermocouple (Physitemp Inc, Clifton, New Jersey, United States of America) placed inside the flat cell.
  • the flat cell was heated using two lamps situated symmetrically on both sides of the resonator. Both lamps were connected through a VARIAC TM voltage controller and the temperature in the flat cell was controlled by the voltage of the VARIACTM. Typically, the lamps were set to generate a fixed heating rate of 0.2°C/minute and temperature measurements were made every half second.
  • EPR spectra were recorded with a Varian E-12 spectrometer operating at 9.45 GHz using 3 mW of power. The time constant and dwell time were set equal to 0.03 seconds, with 512 points per spectrum.
  • each spectrum took approximately 15 seconds and the temperature change per scan was less than 0.1 0 C, which is less than the nominal accuracy of the thermometer.
  • Modulation amplitude was set to 0.5 Gauss peak-to-peak.
  • the temperature range was from 20 0 C to 63 0 C.
  • Example 5 Data Analysis All EPR spectra were fitted using the algorithms of Robinson et a/., 1999 ("Linewidth analysis of spin labels in liquids. I. Theory and data analysis", J Magn Reson 138 (2): 199-209). Lorentzian line widths (LLW) were obtained for each of the three nitrogen manifolds. These are labeled LLW1 , LLW2, and LLW3, where the high field line is LLW3.
  • the Tt of the ELP-nitroxide was also independently determined by EPR spectroscopy from the LLW versus temperature plot by assuming that the intersection of the two continuous functions was the Tt (see Figure 5).
  • the lower temperature function was fit with a 2 nd order polynomial and the higher temperature function was fit with a linear function only to the initial portion of the curve.
  • the intersection of the two fitted lines was taken as the ELP- conjugated nitroxide's Tt for a given concentration.
  • the ELP-conjugated nitroxide's predictive accuracy of temperature was determined by regression analysis (STATVI EW TM, version 5.0.1 , SAS Institute Inc., Cary, North Carolina, United States of America) on the portion of the LLW versus temperature plots below the Tt.
  • ELP and ELP-conjugated nitroxide had a decreasing logarithmic dependence on concentration, and the Tt of the ELP-conjugated nitroxide was reduced by an average of 0.7 0 C over the concentrations studied as compared to the Tt of the parent ELP.
  • the decrease in the Tt of ELPs upon conjugation of reporter molecules is a characteristic observation, and might have occurred through a mechanism similar to increasing the mean guest residue's hydrophobicity of the ELP.
  • Example 7 EPR Spectra The room temperature EPR spectrum for ELP-conjugated nitroxide at a concentration of 80 ⁇ M is shown in Figure 3A. The spectrum shows the expected three nitrogen manifolds from the hyperfine splitting induced by the spin 1 nitrogen nucleus.
  • Example 8 Lorentzian Line-width and Predictive Accuracy Figure 4 shows the three Lorentzian line-widths of the ELP- conjugated nitroxide at 80 ⁇ M as a function of temperature. Similar to Figure 3, the high field line (LLW3) was broadened over a range of temperatures. Two different temperature response regions were apparent, one occurring between 2O 0 C to 38°C and the other from 38°C to 6O 0 C. The LLW3 is shown as a function of temperature for concentrations from 10 to 80 ⁇ M in Figure 5. The shape of the temperature response was very similar for all concentrations studied.
  • the intersection of the two temperature response regions was defined as the Tt of the ELP-conjugated nitroxide, and this value was similar to the values predicted from UV-visible spectrophotometry. Therefore, the portion at temperatures below the intersection was attributed to soluble ELP that has not undergone its inverse phase transition and therefore was still highly soluble. This portion of the thermal response curve was best to predict temperature for non-invasive thermometry because it was less sensitive to concentration.
  • the un-transitioned portion of the curves shown in Figure 5 was fit with a 2 nd order polynomial, and the results of the fit are summarized in Table 1 .
  • Example 8 the in vivo application of this technology might differ slightly from the strategy that was employed in the in vitro experiments presented herein.
  • Oxygen gradients are known to exist within tumors, and the presence of oxygen might broaden the spectral lines of the conjugated nitroxide and reduce the ELP-nitroxide conjugate's predictive accuracy.
  • oxygen broadens all spectral lines by the same amount, while temperature affects the spectral lines differently.
  • This alternative strategy is referred to herein as the "difference method".
  • the predictive accuracy of the difference method is similar to the LLW3 method at a single concentration (80 ⁇ M), but slightly less accurate over a range of concentrations.
  • the predictive accuracy can be further improved by determining the in vivo concentration from the intensity of the EPR signal. This can involve developing plausible models of the partitioning of intra and extravascular ELP, and a blood volume MRI to estimate the concentration to an accuracy of approximately 10 - 15%.

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Abstract

La présente invention concerne des compositions comprenant des polypeptides semblables à l'élastine (ELP) thermosensibles combinés avec un marqueur de spin de résonance paramagnétique électronique (EPR). L'invention concerne également des méthodes de synthèse et d'utilisation des conjugués polypeptidiques semblables à l'élastine (ELP) pour l'imagerie et/ou la spectroscopie EPR.
PCT/US2004/019132 2004-06-15 2004-06-15 Methode de thermometrie non invasive dans laquelle sont utilises des conjugues polypeptidiques semblables a l'elastine Ceased WO2006001806A2 (fr)

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WO2008036147A3 (fr) * 2006-07-24 2008-07-03 Univ Duke Administration de médicament avec biopolymères sensibles aux stimuli
WO2007134245A3 (fr) * 2006-05-12 2008-10-23 Wisconsin Alumni Res Found Excipients à base de polymères du type élastine
WO2007090094A3 (fr) * 2006-01-27 2009-04-02 Univ Mississippi Medical Ct Administration thermiquement ciblee de medicaments comme la doxorubicine
US7709227B2 (en) 2006-01-04 2010-05-04 Phasebio Pharmaceuticals, Inc. Multimeric ELP fusion constructs
US8101717B2 (en) 2006-11-13 2012-01-24 The University Of Sydney Use of tropoelastin for repair or restoration of tissue
US8178495B2 (en) 2008-06-27 2012-05-15 Duke University Therapeutic agents comprising a GLP-1 receptor agonist and elastin-like peptide
US8367626B2 (en) 2006-05-12 2013-02-05 Wisconsin Alumni Research Foundation Elastin-like polymer delivery vehicles
US8729018B2 (en) 2005-12-20 2014-05-20 Duke University Therapeutic agents comprising elastic peptides
JP2014513795A (ja) * 2011-04-29 2014-06-05 チルドレンズ ホスピタル アンド リサーチ センター アット オークランド Ｈｄｌの逆コレステロール輸送支持能力を評価するための組成物および方法
US8841414B1 (en) 2006-01-27 2014-09-23 University Of Mississippi Medical Center Targeted delivery of therapeutic peptides by thermally responsive biopolymers
US9682118B1 (en) 2006-01-27 2017-06-20 University Of Mississippi Medical Center Inhibition of metastasis by cell penetrating peptides
US9999789B2 (en) 2010-05-17 2018-06-19 Koninklijke Philips N.V. Temperature distribution determining apparatus
US10258700B2 (en) 2005-12-20 2019-04-16 Duke University Methods and compositions for delivering active agents with enhanced pharmacological properties
CN112955183A (zh) * 2018-09-25 2021-06-11 波尔多聚合技术研究所 多糖和弹性蛋白-状多肽的生物共轭物及其用途

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Title
DREHER M.R. ET AL: 'Nitroxide conjugate of a thermally responsive elastin-like polypeptide for noninvasive thermometry' MED. PHYS. vol. 31, no. 10, 31 October 2004, pages 2755 - 2762 *

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US8841255B2 (en) 2005-12-20 2014-09-23 Duke University Therapeutic agents comprising fusions of vasoactive intestinal peptide and elastic peptides
US10258700B2 (en) 2005-12-20 2019-04-16 Duke University Methods and compositions for delivering active agents with enhanced pharmacological properties
US9328154B2 (en) 2005-12-20 2016-05-03 Duke University Therapeutic agents comprising fusions of growth hormone and elastic peptides
US7709227B2 (en) 2006-01-04 2010-05-04 Phasebio Pharmaceuticals, Inc. Multimeric ELP fusion constructs
US8252740B2 (en) 2006-01-27 2012-08-28 The University Of Mississippi Medical Center Thermally-targeted delivery of medicaments including doxorubicin
JP2009528984A (ja) * 2006-01-27 2009-08-13 ザ・ユニバーシティ・オブ・ミシシッピ・メディカル・センター ドキソルビシンを含む医薬の熱標的化送達
WO2007090094A3 (fr) * 2006-01-27 2009-04-02 Univ Mississippi Medical Ct Administration thermiquement ciblee de medicaments comme la doxorubicine
US8841414B1 (en) 2006-01-27 2014-09-23 University Of Mississippi Medical Center Targeted delivery of therapeutic peptides by thermally responsive biopolymers
US9682118B1 (en) 2006-01-27 2017-06-20 University Of Mississippi Medical Center Inhibition of metastasis by cell penetrating peptides
US8367626B2 (en) 2006-05-12 2013-02-05 Wisconsin Alumni Research Foundation Elastin-like polymer delivery vehicles
WO2007134245A3 (fr) * 2006-05-12 2008-10-23 Wisconsin Alumni Res Found Excipients à base de polymères du type élastine
WO2008036147A3 (fr) * 2006-07-24 2008-07-03 Univ Duke Administration de médicament avec biopolymères sensibles aux stimuli
EP2043689A4 (fr) * 2006-07-24 2013-08-07 Univ Duke Administration de médicament avec biopolymères sensibles aux stimuli
US20100189643A1 (en) * 2006-07-24 2010-07-29 Duke University Drug delivery with stimulus responsive biopolymers
US8101717B2 (en) 2006-11-13 2012-01-24 The University Of Sydney Use of tropoelastin for repair or restoration of tissue
US8178495B2 (en) 2008-06-27 2012-05-15 Duke University Therapeutic agents comprising a GLP-1 receptor agonist and elastin-like peptide
US9200083B2 (en) 2008-06-27 2015-12-01 Duke University Methods of treating diabetes using therapeutic agents comprising a GLP-1 receptor agonist and elastin-like peptides
US9127047B2 (en) 2008-06-27 2015-09-08 Duke University Therapeutic agents comprising insulin and elastin-like peptides
US9821036B2 (en) 2008-06-27 2017-11-21 Duke University Therapeutic agents comprising a GLP-2 peptide and elastin-like peptides
US10596230B2 (en) 2008-06-27 2020-03-24 Duke University Methods of increasing nutrient absorption in the intestine using therapeutic agents comprising GLP-2 and elastin-like peptides
US11103558B2 (en) 2008-06-27 2021-08-31 Duke University Therapeutic agents comprising a BMP-9 peptide and eleastin-like peptides
US9999789B2 (en) 2010-05-17 2018-06-19 Koninklijke Philips N.V. Temperature distribution determining apparatus
JP2014513795A (ja) * 2011-04-29 2014-06-05 チルドレンズ ホスピタル アンド リサーチ センター アット オークランド Ｈｄｌの逆コレステロール輸送支持能力を評価するための組成物および方法
CN112955183A (zh) * 2018-09-25 2021-06-11 波尔多聚合技术研究所 多糖和弹性蛋白-状多肽的生物共轭物及其用途
CN112955183B (zh) * 2018-09-25 2024-04-30 波尔多聚合技术研究所 多糖和弹性蛋白-状多肽的生物共轭物及其用途
US12084488B2 (en) 2018-09-25 2024-09-10 Institut Polytechnique De Bordeaux Bioconjugates of polysaccharides and elastin-like polypeptides and uses thereof

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