EP3487542A1 - Agents d'imagerie ciblant la protéine zéro de la myéline - Google Patents
Agents d'imagerie ciblant la protéine zéro de la myélineInfo
- Publication number
- EP3487542A1 EP3487542A1 EP17754818.7A EP17754818A EP3487542A1 EP 3487542 A1 EP3487542 A1 EP 3487542A1 EP 17754818 A EP17754818 A EP 17754818A EP 3487542 A1 EP3487542 A1 EP 3487542A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- val
- peptide
- thr
- label
- gly
- 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.)
- Pending
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
Definitions
- the invention relates to the field of imaging and image guided interventions.
- the invention relates to agents for imaging of nerve tissue of the peripheral nervous system and uses thereof.
- Fluorescence guidance either alone or in combination with other modalities has already been shown to be of great value in hybrid tracer- based combined pre- and intraoperative imaging.
- intraoperative guidance towards the lesions e.g. tumor lesions or possibly diseased sentinel lymph nodes
- fluorescence guidance is provided using fluorescence guidance.
- Imaging agents that specifically highlight nerve tissue could be used in a comparable approach; fluorescence-based illumination of peripheral nerve structures within the surgical field has the potential to make surgically- induced complications obsolete.
- Fluorescence-guidance may not only be used to prevent accidental nerve injury, it can also empower surgeons to focus their attention specifically on the preservation of nerves during e.g. complex orthopedic, cardiologic, or oncologic interventions.
- Gibbs-Strauss et al. 2011 describe the use of non-targeted fluorescent dyes for staining of nerves.
- the dyes thus do not contain a targeting moiety, the staining is not nerve specific and staining also occurs in the central nervous system and adipose tissue.
- WO2009029936 describes the use of probes containing a fluorescent stilbene derivatives for imaging of myelin. However, the probes contain only a dye and again no targeting moiety. Next to showing uptake in adipose tissue, the probes enter the brain and selectively localize in myelinated regions. The dyes thus also stain the central nervous system and are further not nerve specific.
- MBP myelination determining myelination.
- a disadvantage of these agents is that MBP is located intracellularly and therefore requires imaging agents that non- specifically enter and accumulate in all cells in order to stain their target. Again these agents are fluorescent dyes without a specific targeting moiety that also enter into the CNS. Moreover, the agents have prominent binding to adipose tissue while binding less to nerve and are thus not nerve specific.
- the invention therefore provides a compound comprising a peptide and a detectable imaging label, wherein said peptide comprises a sequence that has at least 80% sequence identity with 2-100 consecutive amino acids of the extracellular domain of myelin protein-zero (P0), said extracellular domain consisting of the sequence depicted in figure 1.
- the invention provides a composition comprising a compound according to the invention.
- the invention further provides a compound according to the invention for use as an imaging or diagnostic agent.
- the invention provides a compound according to the invention for use in imaging of a neuron, Swann cell, an axon, a nerve or nerve tissue, preferably of the peripheral nervous system.
- the invention provides a compound according to the invention for use in detecting myelin in an individual.
- the invention provides a method for imaging a neuron, a Schwann cell, a nerve or nerve tissue, the method comprising contacting said neuron, nerve or nerve tissue with a compound according to the invention.
- the invention provides the use of a compound according to the invention as an imaging agent or diagnostic agent.
- the invention provides a method for imaging a neuron, a Schwann cell, a nerve or nerve tissue, the method comprising contacting a neuron, a Schwann cell, an axon, a nerve or nerve tissue with a compound according to the invention.
- the invention provides a compound according to the invention for use in determining whether an individual is suffering from a myehn related disorder or from nerve tissue damage, preferably wherein said disorder is a demyelinating disorder.
- the invention provides a method for determining whether an individual is suffering from a myelin related disorder or from nerve tissue damage comprising administering the compound of the invention to an individual or to a sample of the individual and detecting the presence, amount or distribution of said detectable label.
- the method preferably further comprises comparing the presence, amount or distribution of said detectable label with a reference value.
- the invention provides a compound according to the invention for use in image-guided surgery.
- the invention provides a method for image- guided surgery, the method comprising administering a compound according to the invention to an individual in need thereof prior to surgery and/or during surgery, visualizing said detectable imaging label and performing said surgery.
- the invention provides a use of a compound according to the invention for the preparation of a pharmaceutical composition for use in image-guided surgery.
- the invention provides a method for imaging a neuron, a Swann cell, a nerve or nerve tissue, the method comprising contacting said neuron, Swann cell, nerve or nerve tissue with a compound according to the invention.
- said method is an in vitro method.
- said method is an in vivo method.
- the invention is based on the finding that peptides derived from the extracellular domain of myelin protein zero (P0) are particularly suitable for the specific targeting and imaging of nerve tissue in the peripheral nervous system.
- P0 myelin protein zero
- imaging agents that are both specific for nerve tissue over other types of tissue, such as adipose tissue, and specific for nerve tissue of the peripheral nervous system over that of the central nervous system were not known.
- the peptides used in the imaging agents of the invention have the ability to interact with P0 of the myelin sheath that covers the axons of neurons. Since P0 is unique to the peripheral nervous system and constitutes up to 80% of the myelin -related protein content it was found to be an ideal target for peripheral nervous system specificity.
- the invention provides a compound comprising a peptide and a detectable imaging label, wherein said peptide comprises a sequence that has at least 80% sequence identity with 2-100 consecutive amino acids of the extracellular domain of myelin protein-zero (P0).
- a "compound comprising a peptide and a detectable imaging label, wherein said peptide comprises a sequence that has at least 80% sequence identity with 2-100 consecutive amino acids of the extracellular domain of myelin protein-zero (P0)" is herein also referred to as a compound according to the invention.
- Said peptide comprising a sequence that has at least 80% sequence identity with 2-100 consecutive amino acids of the extracellular domain of P0 is herein also referred to as a peptide of a compound according to the invention.
- amino acids are denoted by single-letter or three-letter symbols. These single-letter symbols and three-letter symbols are well known to the person skilled in the art and have the following meaning: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (lie) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gin) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, Y
- peptide refers to a peptide or polypeptide that comprise multiple amino acids.
- the terms “peptide” and “polypeptide” are used interchangeably.
- the smallest peptide in a compound according to the invention has a length of 2 amino acids.
- the amino acid sequence or variant thereof can be part of a larger peptide, i.e. of a peptide that has been N terminally and/or C-terminally extended by a one or more additional amino acids.
- the amino acid sequence or variant thereof of a peptide of the invention may also be N-terminally and/or C-terminally modified, preferably by comprising an N- and/or C-terminal elongating group.
- extracellular domain of myelin protein - zero and "extracellular domain of P0” refers to the part of myelin protein- zero (P0) having the amino acid sequence as depicted in figure 1. Hence, the extracellular domain of P0 has the sequence
- a peptide in a compound according to the invention, comprises a sequence that has at least 80% sequence identity with 2-100 consecutive amino acids of the extracellular domain of P0 as defined herein.
- said peptide comprises 2-75 consecutive amino acids of said sequence, more preferably 2-50 consecutive amino acids of said sequence, more preferably 2- 35 consecutive amino acids of said sequence, more preferably 2-30
- said peptide comprises 3-75 consecutive amino acids of said sequence, more preferably 4-50 consecutive amino acids of said sequence, more preferably 5- 50 consecutive amino acids of said sequence, more preferably 5-35 consecutive amino acids of said sequence, more preferably 6-30 consecutive amino acids, more preferably 7-30 consecutive amino acids of said sequence.
- a further preferred peptide comprises 10-75 consecutive amino acids of said sequence, more preferred 12-50 consecutive amino acids of said sequence, more preferred 15-30 consecutive amino acids of said sequence.
- a peptide according to the invention compiises a sequence that has at least 80% sequence identity with 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35
- % sequence identity is defined herein as the percentage of residues in an amino acid sequence that is identical with the residues in a reference sequence after aligning the two sequences over their whole length. Methods and computer programs for the alignment are well known in the art (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT). One computer program which may be used or adapted for purposes of determining whether a candidate sequence falls within this definition is "Align 2", authored by Genentech, Inc., which was filed with user documentation in the United States
- a peptide may be present in a compound according to the invention as a monomelic peptide or as a multimeric peptide, such as a dimeric peptide, a trimeric peptide, a tetrameric peptide, a pentameric peptide or a hexmeric peptide. It is shown that a multimeric peptide has improved interaction with the peripheral nervous system.
- a peptide in a compound according to the invention comprises a sequence that has at least 80% sequence identity with 2-100 consecutive amino acids of the extracellular domain of P0 as defined herein. Hence, a limited number of amino acid substitutions as compared to the sequence of the extracellular domain of P0 as defined herein is allowed. Indeed, for a peptide that has at least 80% sequence identity with such sequence, up to 20% variation in sequence in a peptide according to the invention as compared to the corresponding sequence of the extracellular domain of P0 is allowed.
- a peptide according to the invention comprises a sequence that has at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity, most preferred 100% sequence identity with 2- 100 consecutive amino acids of the extracellular domain of P0. More preferably, said peptide has such sequence identity with 3-75 consecutive amino acids, more preferably with 4-40 consecutive amino acids, more preferably 5-50 consecutive amino acids, more preferably 7-35 consecutive amino acids, more preferably 10-75 consecutive amino acids, more preferably 12-50 consecutive amino acids, more preferably 15-30 consecutive amino acids of the extracellular domain of PO.
- a peptide according to the invention comprises a sequence that has at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably 100%>, sequence identity with 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 consecutive amino acids of said extracellular domain.
- a peptide according to the invention comprises one or more substitutions independently selected from the group consisting of substitution of one amino acid by alanine and one or more conservative amino acid
- the up to 20% variation in sequence preferably up to 15% variation, more preferably up to 10% variation, more preferably up to 5% variation in a peptide according to the invention as compared to the corresponding sequence of the extracellular domain of P0 is preferably the result of a conservative amino acid substitution and/or a substitution of any amino acid with alanine.
- a peptide according to the invention comprises at most 2 substitutions of any amino acid with alanine, more preferably at most 1 substitution of an amino acid with alanine.
- a “conservative amino acid substitution” as used herein is a substitution in which an amino acid is substituted by another amino acid having a side chain with similar chemical properties, in particular charge or hydrophobicity.
- a person skilled in the art is well aware of suitable conservative amino acid substitutions for each amino acid. The following five groups each list amino acids that are preferred conservative amino acid substitutions.
- a conservative amino acid substitution as defined herein preferably means substitution of one of the amino acids of these groups 1), 2), 3) 4) or 5) with another amino acid from the same group.
- a peptide according to the invention comprises at most 2 conservative amino acid substitutions as defined herein, more preferably at most one.
- a peptide according to the invention therefore comprises one or more substitutions independently selected from the group consisting of:
- a peptide according to the invention preferably comprises a sequence that has at least 80% sequence identity with 2-50, preferably 2-35, consecutive amino acids of amino acids 1-85 or 95-125 of said sequence.
- a peptide according to the invention comprises a sequence that has at least 80% sequence identity with 2-30, consecutive amino acids of amino acids 1-25, 41-85 or 95-125 of said sequence.
- a peptide according to the invention comprises a sequence that has at least 80% sequence identity with 2-30 consecutive amino acids of amino acids 1-25, 61-85 or 95-125 of said sequence.
- a peptide according to the invention comprises a sequence that has at least 80% sequence identity with 2-30 consecutive amino acids of amino acids 1-25 or 95-125 of said sequence.
- a peptide according to the invention comprises at least 2 amino acids. Such peptide may contain up to 100 amino acids. However, smaller peptides are preferred.
- a preferred peptide according to the invention is therefore 5-50 amino acids in length, more preferably 5-45 amino acids, more preferably 5-40 amino acids, more preferably 5-35 amino acids, more preferably 5-30 amino acids, more preferably 7-35 amino acids, more preferably 10-35 amino acids, more preferably 15-25 amino acids, more preferably 20-35 amino acids.
- a particularly preferred peptide has 20-30 amino acids, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.
- a peptide according to the invention may further have an N -terminal, C- terminal modification and/or an internal modification.
- Non-limiting examples of N-terminal modification are addition of an acetyl-, hexanoyl-, decanoyl-, myristoyl-, NH-(CH2-CH2-0)n -CO- or propionyl-residue.
- Non-limiting examples of C-terminal modification are addition of amide-, NH- (CH2-CH2-0)n-CO-amide- and one or two amino-hexanoyl groups.
- a preferred N-terminal modification is acetylation.
- a preferred C-terminal modification is amidation.
- a preferred internal modification is cyclization or incorporation of a fluorescent amino acid.
- a compound according to the invention comprising a peptides according to the invention which comprises an N-terminal modification, a C-terminal modification and/or an internal modification.
- a peptide according to the invention consists of a sequence that has at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, with 2- 100 consecutive amino acids of said extracellular domain of P0 optionally having 1 to 3 additional amino acid C-terminally and or 1 to 3 additional amino acids N- terminally.
- said peptide optionally has 1 additional amino acid C-terminally and/or N-terminally.
- said peptide has an additional cysteine residue C-terminally.
- a cysteine is for instance incorporated in order to attach a thiol-reactive detectable imaging label to the peptide.
- Said percentage sequence identity preferably refers to percentage sequence identity with amino acids 1-85 or 95- 125 of the extracellular domain of PO, more preferably with amino acids 1-25, 41- 85 or 95-125 of said domain.
- a compound according to the invention preferably comprises a peptide as depicted in any one of Tables 1-3, and/or depicted in Figure IB).
- a preferred compound according to the invention comprises a peptide comprising an amino acid sequence of peptides PO-1, PO-3, PO-4, PO-5, PO-6 PO-7, or PO-8 as depicted in Table 1. More preferably, said compound comprises a peptide comprising an amino acid sequence of peptides PO-1, PO-4, PO-6, PO-7 or PO-8 as depicted in Table 1, even more preferably of peptides PO-1, PO-6, PO-7 or PO-8 as depicted in Table 1.
- a compound according to the invention comprises a peptide consisting of the sequence of the peptides PO-1, PO-3, PO-4, PO-6, PO-7 or PO-8, preferably peptide PO-1, PO-4, P0-6,P0-7 or PO-8, more preferably peptide PO-1, PO-6, PO-7 or PO-8.
- Such compound further preferably consists of such peptide and a detectable imaging label.
- a further preferred compound according to the invention comprises a peptide comprising an amino acid sequence of peptides PO-9, PO-10, PO- 11, PO- 12, PO- 13 P0-14, PO-15, PO-16 or PO- 17, as depicted in
- said compound comprises a peptide comprising an amino acid sequence of peptides PO-9, PO- 10, PO-11, PO-13, P0-14 or PO- 17, as depicted in Table 2, even more preferably of peptides PO-9, PO-10, PO- 11, PO-13 or P0- 14, as depicted in Table 2.
- Particularly preferred are compounds comprising a peptide comprising the sequence of peptide PO- 10, PO-11, or P0-14, as depicted in Table 2.
- a compound according to the invention comprises a peptide consisting of the sequence of the peptides PO-9, PO-10, PO-11, PO-12, PO- 13 P0-14, PO-15 or PO-17, as depicted in Table 2, preferably peptide PO-9, PO-10, PO- 11, PO- 13 or P0-14, more preferably peptide PO-10, PO-11, or PO-14.
- Such compound further preferably consists of such peptide and a detectable imaging label.
- a further preferred compound according to the invention comprises a peptide comprising an amino acid sequence of peptides PO-18 (OPD2), OPD3, OPD4 and/or OPD5, as depicted in Table 3, and/or OPDl as depicted in Figure 1.
- Said compound comprises a dimer as an inverted repeat. The binding of a dimer is enhanced, when compared to a monomer. For example, binding of PO-18 to P0 was enhanced, when compared to peptide PO-7.
- a compound according to the invention comprises a climer as a direct repeat, or a multinier such as a trimer or tetramer as direct repeats.
- a compound of the invention comprises a peptide that does not occur as such in nature, i.e. a peptide of the invention is a non-naturally occurring peptide.
- Non-naturally occurring as used herein means that the peptide is not found in nature in that form, preferably that the amino acid sequence of the peptide is not found in nature.
- a peptide of the invention preferably comprises at least one amino acid substitution in the amino acid sequence as defined herein.
- a compound according to the invention consists of a peptide according to the invention and a detectable imaging label as described herein.
- said peptide consists of a sequence that has at least 80% sequence identity with 2-100 consecutive amino acids, more preferably 3-75 consecutive amino acids of said sequence, more preferably 5-50 consecutive amino acids of said sequence, more preferably 7-35 consecutive amino acids of said extracellular domain of P0, optionally having 1 to 3 additional amino acid C-terminally and/or 1 to 3 additional amino acids N-terminally.
- a compound consists of a peptide consisting of a sequence that has at least 80% sequence identity, or at least 85% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, with amino acids 1-85 or 95-125 of the extracellular domain of P0 as defined herein.
- Peptides according to the invention can be prepared by various methods.
- a peptide can be synthesized by commonly used solid-phase synthesis methods, e.g. methods that involve t-BOC or FMOC protection of alpha-amino groups well known in the art.
- amino acids are sequentially added to a growing chain of amino acids.
- Such methods are for instance described in Merrifield (1963), J. Am. Chem. Soc. 85: 2149-2156 ; and Atherton et al., "Solid Phase Peptide Synthesis," IRL Press, London, (1989).
- a suitable solid-phase peptide synthesis is further described in the Examples herein. Sohd-phase synthesis methods are particularly suitable for synthesis of peptides or relatively short length, such as peptides with a length of up to about 70 amino acids in large-scale production.
- Substitution of 2 amino acids during the synthesis procedure in a peptide according to the invention with a pseudoproline (oxazolidine) dipeptide, a dimethoxybenzyl dipeptide or an isoacyl dipeptide are typically used in order to optimize the synthetic procedure of peptides and improve quahty and yield of synthetic peptides, in particular to increase purity of peptides and/or to minimize aggregation.
- Pseudoproline (oxazolidine) dipeptides, dimethoxybenzyl dipeptides and isoacyl dipeptide are well known in the art and are commercially available.
- Pseudoproline di e tides are particularly suitable to replace serine, threonine or cysteine and an amino acid directly adjacent to said serine, threonine or cysteine, in particular an amino acid directly preceding said serine, threonine or cysteine.
- Suitable pseudoproline dipeptides that can be incorporated into a peptide according to the invention are: Fmoc-Ala-Ser(n (Me,Me)Pro)-OH, Fmoc-Ala- Thr(nj(Me,Me)Pro)-OH, Fmoc-Asn(Trt)-Ser(nj(Me,Me)Pro)-OH, Fmoc- Asn(Trt)-Thr( (Me,Me)Pro)-OH, Fmoc-Asp(OtBu)-Ser(3 ⁇ 4r(Me,Me)Pro)-OH, Fmoc-Asp(OtBu)-Thr(nj(Me,Me)Pro)-OH, Fmoc-Gln(Trt)-Ser(nj(Me,Me)Pro)- OH, Fmoc-Gln(Trt)-Ser(nj(Me,Me)
- Dimethoxybenzyl (dmb) dipeptides are particularly suitable to replace glycine and an amino acid directly adjacent thereto, in particular glycine and an amino acid directly preceding said glycine.
- suitable dmb dipeptides that can be incorporated into a peptide according to the invention are: Fmoc-Asp(OtBu)-(Dmb)Gly- OH, Fmoc-Gly-(Dmb)Gly-OH, Fmoc-Ile-(Dmb)Gly-OH, Fmoc-Leu-(Dmb)Gly- OH, Fmoc-Val-(Dmb)Gly-OH, Fmoc-L-Glu(tBu)-DmbGly-OH, Fmoc-L- Lys(Boc)-DmbGly-OH and Fmoc-L-Ser(tBu)-DmbGly-OH
- Isoacyl dipeptides are used to prepare peptides in which the peptide chain is attached to the side chain oxygen atom of serine or threonine residues instead of the alpha nitrogen atom.
- isoacyl dipeptides are particularly suitable to replace serine or threonine and an amino acid directly adjacent to said serine or threonine, in particular an amino acid directly preceding said serine or threonine.
- Boc-L-Ser(Fmoc-L-Ala)-OH Boc-L-Ser[Fmoc-L-Arg(Pbf)]-OH
- Boc-L- Ser(Fmoc-Gly)-OH Boc-L-Ser[Fmoc-L-His(Trt)]-OH
- Boc-L-Ser(Fmoc-Gly)-OH Boc-L-Ser[Fmoc-L-His(Trt)]-OH
- a peptide of the invention can be prepared using recombinant techniques well known in the art in which a nucleotide sequence encoding the peptide is expressed in host cells.
- a host cell used for the preparation of a peptide according to the invention is for instance a Gram-positive prokaryote, a Gram-negative prokaryote or an eukaryote.
- said host cell is an eukaryotic cell, such as a plant cell, a yeast cell, a mammalian cell or an insect cell, most preferably an insect cell or a mammalian cell.
- suitable host cells include plant cells such as corn cells, rice cells, duckweed cells, tobacco cells (such as BY-2 or NT-1 cells), and potato cells.
- yeast cells are Saccharomyc.es and Pichia.
- insect cells are Spodoptera frugiperda cells, such as Tn5, SF-9 and SF-21 cells, and Drosophila cells, such as Drosophila Schneider 2 (S2) cells.
- mammahan cells that are suitable for expressing a peptide according to the invention include, but are not limited to, African Green Monkey kidney (Vero) cells, baby hamster kidney (such as BHK-21) cells, Human retina cells (for example PerC6 cells), human embryonic kidney cells (such as HEK293 cells), Madin Darby Canine kidney (MDCK) cells, Chicken embryo fibroblasts (CEF), Chicken embryo kidney cells (CEK cells), blastoderm-derived embryonic stem cells (e.g. EB14), mouse embryonic fibroblasts (such as 3T3 cells), Chinese hamster ovary (CHO) cells, and derivatives of these cell types.
- Vero African Green Monkey kidney
- baby hamster kidney such as BHK-21
- Human retina cells for example PerC6 cells
- human embryonic kidney cells such
- Suitable vector for introducing the nucleotide sequence encoding the peptide are derived from an animal virus, examples of which include, but not limited to, vaccinia virus (including attenuated derivatives such as the Modified Vaccinia virus Ankara, MVA), Newcastle Disease virus (NDV), adenovirus or retrovirus.
- suitable promoters for expression of peptides according to the invention in eukaryotic host cells include, but are not hmited to, beta-actin promoter, immunoglobin promoter, 5S RNA promoter, or virus derived promoters such as cytomegalovirus (CMV), Rous sarcoma virus (RSV) and Simian virus 40 (SV40) promoters for mammalian hosts.
- CMV cytomegalovirus
- RSV Simian virus 40
- a compound according to the invention comprises, in addition to a peptide derived from the extracellular domain of P0, a detectable imaging label.
- a detectable imaging label refers to a moiety which allows detection of the compound of the invention, e.g. when present in or bound to a cell or tissue in vitro, in vivo or ex vivo. Such label is preferably capable of generating a signal that is detectable. Any label (or combination thereof) that can be used to image tissue in vivo and or in vitro and that can be attached to a peptide is suitable for use in a compound according to the invention.
- detectable imaging labels are well known in the art and different variants are commercially available. Alternatively tailor made variants can be introduced to improve the overall
- Preferred compounds according to the invention comprises a detectable imaging label selected from the group consisting of a fluorescent label, a luminescent label, a (radio)isotope label, a paramagnetic label, a (bio)nanop article label, a combination of two or more of said labels and a hybrid thereof.
- a combination of detectable imaging labels is preferably a combination of two labels selected from the group consisting of a fluorescent label, a luminescent label, a (radio)isotope label and a paramagnetic label. More preferably, a combination is a combination of a fluorescent label and a (radio)isotope label or a combination of a fluorescent label and a
- a hybrid label is preferably a hybrid fluorescent, (radio)isotope label or a hybrid fluorescent, paramagnetic label.
- Preferred combined labels for combined pre- and intraoperative imaging comprise of both a fluorescent and a (radio)isotope label or a fluorescent and
- paramagnetic label or a hybrid fluorescent, (radio)isotope or hybrid fluorescent, paramagnetic label It is advantageous and preferred that such a label is tunable to the application to allow for multispectral imaging application together with disease specific fluorescently labeled agents.
- a (radio)isotope label is preferably a radioactive label.
- a luminescent label preferably is a label with excitation and emission in the 200-1000nm range.
- a fluorescent label preferably is a fluorescent label, a combination of a fluorescent and (radio)isotope label, a combination of a fluorescent and paramagnetic label, a hybrid fluorescent, (radio)isotope label or a hybrid fluorescent, paramagnetic label.
- a fluorescent label preferably is a label with excitation and emission in the 400nm- 1000nm range.
- Particularly preferred for the surgical guidance application is a fluorescent label with excitation and emission in the 400nm-1000nm range.
- Non-hmiting examples of fluorescent labels that can be included in a compound according to the invention are Abz (Anthranilyl, 2-Aminobenzoyl), N-Me-Abz (N-Methyl- anthranilyl, N-Methyl-2-Aminobenzoyl), FITC (Fluorescein isothiocyanate), 5-FAM (5-Carboxyfluorescein), 6-FAM (6-Carboxyfluorescein), TAMRA (Carboxytetramethyl rhodamine), Mca (7-Methoxycoumarinyl-4-acetyl), AMCA or Amc (Aminomethylcoumarin Acetate), dansyl (5-(Dimethylamino) naphthalene- 1-sulfonyl), EDANS (5-[(2-Aminoethyl)amino] naphthalene- 1- sulfonic acid), Atto (e.g.
- Cy3 (l-(5-carboxypentyl)-3,3-dimethyl-2- ((lE,3E)-3-(l,3,3-trimethylindolin-2-ylidene)prop-l-en- l-yl)-3H-indol-l-ium chloride), Cy5 (l-(5-carboxypentyl)-3,3-dimethyl-2-((lE,3E,5E)-5-(l,3,3- trimethylindolin-2-ylidene)penta- l,3-dienyl)-3H-indohum chloride), including trisulfonated Cy5, and Cy7 (l-(5-carboxypentyl)-2-[7-(l-ethyl-5- sulfo- l,3-dihydro-2H-indol-2-
- fluorescent labels can also occur in (bio)nan op articles that will be conjugated to the targeting peptide.
- inorganic dyes or dyes with a relatively long luminesce lifetime may be used e.g. quantum dots, silver/gold-p articles, or luminescent transition metal complexes.
- a compound according to the invention comprises a peptide according to the invention and a trisulfonated Cy5 as the detectable imaging label.
- a trisulfonated Cy5 is shown in the Examples herein and described in Mujumdar et al., 1993, which is incorporated by reference herein.
- fluorescein will be most preferred.
- preferred labels include fluorescein or a Cy dye, preferably C3, C5 or C7.
- the detectable imaging label preferably a label comprising a fluorescent moiety
- the detectable imaging label is bound directly to the peptide according to the invention using known techniques in conjugation
- Such labels can be inserted at the distal ends of the peptides sequences using known conjugation techniques e.g. click-chemistry.
- these labels may be introduced at any place in the peptide sequence, either as attachment to an amino acid e.g. lysine or as linking part in the amino acid sequence.
- the label can be incorporated at the C- or N-terminus or at Cys or Lys side chains of the peptide.
- the detectable imaging label is attached to the peptide via a linker using know techniques in conjugation chemistry.
- a linker is for example suitable when using a (bio)nanop article label.
- linkers are carbon linkers, peptide linkers and polyether linkers. These linker have functional groups, such as amide, alkyl halide and carboxylic acids, which can be used to form bond with both the label and the peptide. Linkers can also be dendritic in nature allowing the grafting of one ore more imaging labels to one ore more peptide sequences.
- the detectable imaging label is included in the peptide during synthesis thereof, preferably by replacing one or more amino acids with the label or by inserting the label between two amino acids of the peptide. Therefore, in a preferred embodiment, one or more amino acids of the peptide, preferably one or more of the 2-100 consecutive amino acids, is replaced by the detectable label. Said one or more amino acids that are replaced are preferably at most four amino acids, more preferably at most three amino acids, such as one, two or three amino acids.
- said detectable label is located between two amino acids of the peptide, preferably between two amino acids of said 2-100 consecutive amino acids.
- Methods for including a fluorescent dye in the peptide during synthesis are known in the art. Reference is for instance made to Koopman et al, Bioorg Med Chem 2013, 21, 553, which is incorporated herein by reference, for a description of a method for
- a label that is included in the peptide is preferably a fluorescent label.
- Particularly preferred examples of fluorescent dyes that can be included in a peptide during synthesis are cyanine derivatives containing an amino- and a carboxylic acid moiety.
- Imaging labels can be detected using any suitable method known in the art.
- a fluorescent label is detected by exciting the fluorophore with the appropriate wavelength of light and detecting the fluorescence.
- detection can for instance be done using e.g. a microscope or an endoscope provided with a suitable excitation and emission settings for the fluorescent label used.
- composition comprising a compound according to the invention.
- Said composition further preferably comprises at least one carrier, diluent and/or excipient.
- the composition is a pharmaceutical composition which further comprises a pharmaceutically acceptable carrier, diluent and/or excipient.
- auxiliary, carrier, diluent or excipient must be compatible with the other ingredients of the
- the pharmaceutical composition is preferably a sterile composition for injection.
- Sterile compositions for injection can be formulated according to conventional pharmaceutical practice by dissolving or suspending the peptide of the invention in a vehicle for injection, such as water or a naturally occurring vegetable oil like sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or a synthetic fatty vehicle like ethyl oleate or the like. Buffers, preservatives, antioxidants and the like may also be incorporated.
- a compound according to the invention can be advantageously used in both therapeutic and nontherapeutic applications.
- compounds according to the invention are useful as imaging agent, both in vitro and in vivo, and as diagnostic agents.
- the invention therefore provides a compound according to the invention for use as an imaging or diagnostic agent.
- a compound according to the invention as an imaging agent or diagnostic agent.
- the compounds of the invention are capable of interacting with myelin protein zero.
- P0 is a glycoprotein which is a major structural component of the myelin sheath that surrounds the axon of nerve cells.
- the compounds of the invention are particularly suitable for imaging and/or detecting P0 and/or myelin or any tissue comprising P0 and/or myelin.
- the target of a compound according to the invention is preferably a neuron, a Schwann cell, an axon, a nerve or nerve tissue or a part or component thereof, in particular a neuron, a Schwann cell, an axon, a nerve or nerve tissue or a part or component thereof of the peripheral nervous system.
- the peripheral nervous system refers to the part of the nervous system that consists of the nerves and neurons outside the brain and spinal cord.
- Schwann cells are the cells that surround the axons of the peripheral nerves and that form the myelin sheath of myelinated nerve fibers.
- Said neuron, Schwann cell, axon, nerve or nerve tissue can be any nerve of the peripheral nervous system, including, but not limited to, sensory nerves or neurons, motor nerves or neurons, nerves, axons, neurons or tissue of the autonomic nervous system, the sympathetic nervous system and the parasympathetic nervous system, the brachial plexus, lumbosacral plexus and cervical plexus.
- Said part or component preferably comprises PO.
- a compound according to the invention for use in imaging of a neuron, a Schwann cell, an axon, a nerve or nerve tissue, preferably of the peripheral nervous system.
- the labeling of a neuron, a Schwann cell, an axon, a nerve or nerve tissue, preferably of the peripheral nervous system occurs in vivo.
- the labeling of a neuron, a Schwann cell, an axon, a nerve or nerve tissue, preferably of the peripheral nervous system occurs in vitro or ex vivo.
- In vitro or ex vivo use as imaging agent encompasses labelhng of nerve tissue in tissue samples of an individual, e.g. for research purposes.
- a method for imaging a neuron, a Schwann cell, a nerve or nerve tissue comprising contacting a Schwann cell, an axon, a nerve or nerve tissue with a compound according to the invention.
- said method is an in vitro method, and said contacting occurs for instance in a sample obtained from an individual.
- said method is an in vivo method.
- Said neuron, Schwann cell, axon, nerve or nerve tissue is preferably present in a sample obtained from an individual or present in an individual.
- said method comprises determining whether a neuron, a Schwann cell, axon, nerve or nerve tissue is present in said sample.
- said method comprises determining the amount of label, e.g. fluorescent label, in a sample obtained from an individual.
- label e.g. fluorescent label
- a compound according to the invention is particularly suitable for diagnosis of myelin related disorder or from nerve tissue damage. Further provided is therefore a compound according to the invention for use in determining whether an individual is suffering from a myelin related disorder or from nerve tissue damage. Also provided is a method for determining whether an individual is suffering from a myehn related disorder or from nerve tissue damage. Said method or use preferably comprises administering the compound of the invention to an individual, detecting the presence, amount or distribution of said detectable label and comparing the presence, amount or distribution of said detectable label with a reference value.
- said method or use comprises administering the compound of the invention to a sample of an individual, detecting the presence, amount or distribution of said detectable label and comparing the presence, amount or distribution of said detectable label with a reference value.
- Said individual is preferably suffering from or suspected of suffering from a myelin related disorder or from nerve tissue damage, preferably a demyelinating disorder.
- Said sample preferably comprises a nerve, an axon, a neuron, or nerve tissue of said individual.
- an "individual” is a human or an animal. Individuals include, but are not limited to, mammals such as humans, pigs, ferrets, monkeys, rabbits, cats, dogs, cows and horses. In a preferred embodiment of the invention a subject is an animal with P0 expression on the nerves. In a particularly preferred embodiment the subject is a human.
- a myelin related disorder refers to any disorder that is characterized by or associated with changes in the level or amount of myelin or myelination, in particular disorder characterized by or associated with degradation of myelin and demyelination.
- said disorder is therefore a demyelinating disorder.
- demyelinating disorders are multiple sclerosis (MS), Tay-Sachs disease, Niemann-Pick disease, Gaucher disease, Hurler syndrome, Guillain-Barre syndrome, Charcot Marie Tooth and Dejerine-Sottas disease.
- Nerv tissue damage refers to any damage that has been caused or is suspected to have been caused in an individual.
- causes of damage to nerve tissue are, for instance, disease, chemically induced toxicity, and trauma, including falls and accidents, including fractures resulting thereof.
- a compound according to the invention for use in detecting myelin in an individual.
- Said individual is preferably suffering from or suspected of suffering from a demyelinating disorder.
- a method for detecting myelin in an individual comprising:
- Said method further preferably comprises determining a level of myelination in said individual by comparing the presence of said detectable imaging label with a reference value, thereby determining a level of myelination in said individual.
- the method further preferably comprises determining the amount or relative amount of said detectable imaging label and/or the amount of fluorescent staining present in nerve tissue of the peripheral nervous system of said individual. Such method is particularly suitable for detecting the present of myelin in an individual suffering from or suspected of suffering from a myelin related disorder or from nerve tissue damage.
- Said disorder is preferably a demyelinating disorder, more preferably selected from the group consisting of multiple sclerosis, Tay-Sachs disease, Niemann-Pick disease, Gaucher disease, Hurler syndrome, Guillain-Barre syndrome, Charcot Marie Tooth and Dejerine-Sottas disease.
- a demyelinating disorder more preferably selected from the group consisting of multiple sclerosis, Tay-Sachs disease, Niemann-Pick disease, Gaucher disease, Hurler syndrome, Guillain-Barre syndrome, Charcot Marie Tooth and Dejerine-Sottas disease.
- a method for detecting myelin in a sample the method contacting said neuron, Swann cell, nerve or nerve tissue with a compound according to the invention and detecting the presence, amount or distribution of said detectable label.
- the method preferably further comprises comparing the presence, amount or distribution of said detectable label with a reference value.
- Said sample is preferably a sample from an individual, such as an individual suffering from or suspected of suffering from a myehn related disorder or from nerve tissue damage.
- Said sample further preferably comprises a neuron, Schwann cell, axon, nerve or nerve tissue.
- a “reference value” as used herein is for instance a sample of tissue of a healthy individual, which tissue is identical of the same type as the tissue present in the sample of the individual suffering from or suspected of suffering from a demyelinating disorder. For instance, if the tissue of said individual is or comprises nerve tissue, the reference sample comprises nerve tissue and if the tissue of said individual is or comprises hver tissue, the reference sample comprises liver tissue.
- the detectable imaging label for use in diagnosis, including intraoperative diagnosis is preferably a fluorescent label, a combination of a fluorescent and a (radio)isotope label or a combination fluorescent and paramagnetic label, most preferably a fluorescent label.
- Determining the amount or relative amount of a fluorescent label preferably comprises determining the fluorescence intensity in said individual with the
- comparing the presence of a fluorescent label with a reference value preferably comprises determining the fluorescence intensity in said individual and the fluorescence intensity in a reference sample, e.g. a reference sample as described herein.
- a compound according to the invention is further particularly suitable for image-guided surgery.
- Image-guided surgery refers to a surgical procedure where, e.g., a tissue of interest is becomes visible in order to assist in or guide a surgical procedure.
- Imaging agents that specifically highlight nerve tissue of the peripheral nervous system can be used to reduce or even prevent surgically -induced complications, in particular nerve tissue injury.
- image-guidance may not only prevent accidental nerve injury, it can also empower surgeons to focus their attention specifically on the preservation of nerves during complex orthopedic, cardiologic, or oncologic interventions.
- a compound according to the invention for use in image-guided surgery preferably comprises administering said compound to an individual prior to surgery and/or during surgery, visuahzing said detectable imaging label and performing said surgery.
- a method for image-guided surgery the method comprising administering a compound according to the invention to an individual in need thereof prior to surgery and/or during surgery, visualizing said detectable imaging label and performing said surgery.
- a compound according to the invention for the preparation of a pharmaceutical composition for use in image-guided surgery can be systemically or locally.
- Said detectable imaging label is preferably a fluorescent label, a combination of a fluorescent and a radioactive label or a combination fluorescent and paramagnetic label, most preferably a fluorescent label.
- Said surgery can any type of surgery, including, but not hmited to orthopedic, cardiologic, or oncologic surgery.
- Imaging may be accomplished using, for example, scintigraphy, single-photon emission computed tomography (SPECT), including SPECT- computed tomography (SPECT-CT), positron emission tomography (PET), gamma probe, mobile gamma camera or freehand SPECT (intraoperative).
- SPECT single-photon emission computed tomography
- PET positron emission tomography
- gamma probe gamma probe
- mobile gamma camera freehand SPECT (intraoperative).
- radiolabels resides, for example, in assessment of the tracers pharmacokinetics (related to toxicity), and/or in validation of targeting to ensure that fluorescent labelhng of the nerves can be used to guide surgery.
- fluorescence guided surgery is a preferred medical imaging technique used to detect fluorescently labelled nerve structures during surgery.
- FGS is performed using, for example, halogen lamps, xenon-lamps, light-emitting diodes, or laser diodes, for excitation of the fluorescent label, while digital cameras, such as charge-coupled device (CCD) or complementary metal oxide semiconductor (CMOS), are used to produce the final image.
- CCD charge-coupled device
- CMOS complementary metal oxide semiconductor
- Preferred FGS is using far red or near infra-red wavelengths (600-1000nm range), employing, for example cyanine dyes based on Cy5 and Cy7 structures as dye candidates. This covers
- FITC fluorescein
- Bimodal or hybrid labels may be applied wherein both the radio and fluorescent label are attached to the same peptide sequence.
- Figure 1 A. Sequence of the extracellular domain of myelin protein zero.
- FIG. 1 Peptide design. Peptides are based on the amino-acid sequence of the extracellular part of the P0 receptor. A. crystal structure of the extracellular part of P0, B. the allocation of the peptide sequences.
- FIG. 3 Staining of P0 expressing cells. Staining of P0 expressing RT4 Schwannoma cells was evaluated using fluorescence confocal imaging.
- Signal of the bound peptide is depicted in red. Staining of the nucleus (in blue) and lysosomes in the cytoplasm of the cells (in green) was used as a reference for localization of the peptide specific membranous signal.
- Figure 4 Comparison of staining intensity using fluorescence confocal microscopy.
- FIG. 5 Evaluation of PO-specificity and binding affinity.
- Flow cytometry-basecl saturation binding experiments revealed the affinity of C) PO-8 and D) PO-6 for P0.
- FIG. 7 Staining of Schwann cells. DRG explants stained with A) PO-ex and B) IMI-PO-8 showed the same spotted staining pattern. C) When zooming in on the stained Schwann cells a comparable staining was seen when using I) PO-8, II) anti-PO antibody and III) PO-ex. IV) No staining was seen in non-PO expressing carcinoma control cells after incubation with P0- 8.
- Figure 8 In vivo P0-based staining of the Sciatic nerve.
- HPLC chromatography
- Peptides PO-1, PO-2, PO-3, P0-4, PO-5 and PO-7 were synthesized in the IHB peptide facility of the LUMC using a standard Fmoc/'Bu solid phase protocol on preloaded Tentagel® S RAM resins (Rapp Polymere GmbH, Tuebingen, Germany).
- peptides PO-3, P0-4 and PO-7 where a cysteine was not part of the (native) peptide sequence, a C-terminal cysteine was added to the amino acid sequence of the particular part of the P0 receptor to enable fluorescent labeling.
- the peptides consisted of the following amino acid sequences (numbering of the amino acids was conducted as previously described by Shy et al):
- PO-1 (amino acid 1-25): H-IWYTDREVHGAVGSRVTLHCSFWS-NH2; H- Ile-Val-Val-Tyr-Thr-Asp-Arg-Glu-Val-His-Gly-Ala-Val-Gly-Ser-Arg-Val-Thr- Leu-His-Cys-Ser-Phe-Trp-Ser-NH2 PO-2 (amino acid 21-45): Ac-CSFWSSEWVSDDISFTWRYQPEGGR-NH 2 ;
- PO-4 (amino acid 61-85): AC-DEVGTFKERIQWVGDPRWKDGSIVIC-NH2; Ac-Asp-Glu-Val-Gly-Thr-Phe-Lys-Glu-Arg-Ile-Gln-Trp-Val-Gly-Asp-Pro-Ai-g- Trp-Lys-Asp-Gly-Ser-Ile-Val-Ile-Cys-NH 2
- PO-5 (amino acid 81- 105): Ac-GSIVfflNLDYSDNGTFTCDVKNPPD-NH 2 ; Ac-Gly-Ser-Ile-Val-Ile-His-Asn-Leu-Asp-Tyr-Ser-Asp-Asn-Gly-Thr-Phe-Thr- Cys-Asp-Val-Lys-Asn-Pro-Pro-Asp-NH 2
- PO-7 (amino acid 101-125): Ac-KNPPDIVGKTSQVTLYVFEKVPTRYC-NH 2 ; Ac-Lys-Asn-Pro-Pro-Asp-Ile-Val-Gly-Lys-Thr-Ser-Gln-Val-Thr-Leu-Tyr-Val- Phe-Glu-Lys-Val-Pro-ThrArg-Tyr-Cys-NH 2 .
- peptide PO-6 and PO-8 (PO-6 (amino acid 95-120): Ac-TFTADVKNPPDIVGKTSQVTLYVFEKC-NH 2 ; Ac- Thr-Phe-Thr-Ala-Asp-Val-Lvs-Asn-Pro-Pro-Asp-Ile-Val-Glv-Lvs-Thr-Ser- Gln-Val-Thr-Leu-Tvr-Val-Phe-Glu-Lvs-Cvs-NH 2 (With the bolded alanine residue replacing the cysteine from native P0) and PO-8 (amino acid 101- 125): AC-KNPPDIVGKTSQVTLYVFEKVPTRYC-NH2: Ac-Lys-Asn-Pro-Pro- Asp-Ile-Val-Glv-Lvs-Thr-Ser-Gln-Val-Thr-Leu-Tvr-Val-Phe-Glu-L
- Pseudoproline dipeptides could be used in the synthesis of the previously described peptides at the designated positions:
- IPTG (1M) with a final concentration of 0.7-1.0 mM was added and the culture was left to grow at 25°C for another 5-6 hrs.
- ODeoo of ca 1.8 - 2.0 was reached, the culture was cooled down again and subsequently spinned down at 4000 rpm for 10 min at 4°C.
- the supernatant was discarded and the pellet was re-suspended in 40 ml PBS (pH 8) and spinned down at 4000 rpm for 10 min (again at 4°C).
- the pellet was at -20°C for later use.
- the obtained pellet re-suspended in 8 ml 50 niM Tris pH 8 +lmM DTT + protease inhibitor, which was spinned down further at 8000 rpm for 30 minutes at 4°C to obtain the inclusion bodies that contain the extracellular part of P0.
- the pellet was again re-suspended, this time in 5-7 ml 5M urea 50 mM Tris pH8 containing 1 mM DTT and protease inhibitor, and incubated on ice for 2-3 hrs before being spinned down at 8000 rpm for 30 min at 4°C.
- the supernatant was transferred to a pre-soaked 70 ml dialysis cassette and diluted with PBS / lOmM Tris pH 8 containing 1 mM DTT and protease inhibitor to a volume of 60 -70 ml and dialysed in 4 ltr 20% glycerol in PBS pH 8 for 2-3 hrs, followed by o/n dialization in PBS / 10 mM Tris pH 8 containing 0.5 mM DTT.
- GST beads were prepared by washing them several times in PBS pH 8.0 containing protease inhibitor. The lysate was divided into two equal samples and centrifuged at 4000 rpm for 20 min before the supernatant was transferred to the tubes with the beads. PO-ex was left to bind o/n at 4°C. Hereafter the beads were spinned down for 20 min at 1500 rpm and the supernatant was carefully removed and transferred to another tube. The beads were washed at least three times with 20 ml PBS pH 8.0 and washed once with 50mM Tris pH 8.
- Trisulfonated Cy5 was synthesized according to the method described previously by Mujumdar et al. 1993. This dye (25 mg, 30 ⁇ ) was dissolved in dry DMF (400 ⁇ _). together with PyBOP (17.3 mg, 33 ⁇ ) and aminoethyl maleimide trifluoroacetate (9 mg, 34 ⁇ ). Subsequently, N- methyl morpholine (15 ⁇ », 136 ⁇ ) was added and the mixture was allowed to stir for 3 hours at room temperature. Afterwards, the reaction mixture was acidified with acetic acid (50 ⁇ ⁇ ) and purification was performed by preparative reverse phase HPLC chromatography.
- the obtained thiol-reactive dye was lyophilised after which a fluffy purple solid (8.1 mg, 8 ⁇ ⁇ ) was obtained.
- MALDI-TOF mass spectrometry Calculated 887.3, found 887.7.
- the thiol-reactive dye has the following structure:
- P0 expressing RT4 D6P2T (rat, ATCC) and MSC80 (human) cells, and non- P0 expressing MDAMB231 mammary tumor cells were grown in Dulbecco's Modified Eagle Medium (Life Technologies, UK) containing penicillin, streptomycin and fetal calf serum (All BD Biosciences) at 37 °C and 5% CO2. Cells were used for evaluation of (P0-specific) staining using fluorescence confocal microscopy and binding affinity using FACS and FLIZA. Animals
- Transgenic B6.Cg-Tg(Thyl-YFP)16Jrs/J mice were obtained from JAX (the Jackson Laboratory) . Mice were bred at the animal facility of the LUMC after approval of the animal ethics committee of the LUMC was obtained (reference # 1611). THY-1 YFP mice express spectral variants of GFP (yellow-YFP) at high levels in motor and sensory neurons. Axons are brightly fluorescent all the way to the terminals where no expression is detectable in non-neural cells. The fluorescent signal in the nerves was used as an internal control for the staining (pattern) of the developed imaging agents. Mice (8-15 weeks old) were used for in vivo evaluation of nerve staining as well as ex vivo evaluation of staining in excised nerve tissue.
- DRG explants were collected from 14 days old THY-1 YFP mouse embryos. Embryos were transferred into HBSS solution immediately after cervical dislocation of a pregnant THY-1 YFP mouse.
- Embryos were dissected in L15 (Leibovitz) medium via a previously described method of dissection of rat embryo's. (Niclou, et al. 2003) Attached nerve roots and other unwanted tissue was removed from the collected DRGs, which were then transferred into DMEM/F12 culture medium, onto the laminin-coated glass coverslips. Experiments were performed according to Dutch law and after approval from the Animal Ethics Committee of the LUMC was obtained (reference # 14042). Evaluation of binding affinity Flow cytometry
- the cells were washed (two times with 300 ⁇ ⁇ of 0.1% BSA in PBS, re-suspended in 300 uL of 0.1% BSA in PBS containing Propidium iodide (PI; 1:500). Fluorescence was measured using a BD FACSCanto flow cytometer (DakoCytomation) with APC-Cy7 settings (635 nm laser and 750 nm long pass filter). Live cells were gated (using the PI signal) on Forward Scatter, Side Scatter and Pulse Width and approximately 20 000 viable cells were analyzed. All experiments were performed in triplicate. The
- Lumitrac 600 96-wells plates were coated with PO-ex (10 ⁇ g/well) and incubated overnight at 4 °C. The coated wells were then blocked with 200 ⁇ casein (2.5 g/100 ml) per well, and incubated for two hours at room
- Cell and DRG samples were analyzed using a Leica SP8 WLL confocal microscope (Leica Microsystems). Samples were co-incubated with Hoechst (lmg/ml, 1:500; for at least 5 minutes) to provide a nuclear stain. In cell samples lysotracker was added (lysotracker green; 2 1/ ⁇ 1). All samples were washed three times with Phosphate Buffered Saline (PBS) solution prior to imaging. Images were acquired following CY5-excitation at 633 nm at 106 and 636 times magnification. Emission was collected between 650-725 nm.
- Hoechst lmg/ml, 1:500; for at least 5 minutes
- Tissue samples were obtained from donor-mice; after cervical dislocation of the donor-mouse the left and right Nervus Ischiadicus was dissected and subsequently excised. Tissue samples were incubated for one hour with a fluorescently labeled version of PO-6, PO-7 or PO-8 (-0.6 ⁇ ). Sonication (20 seconds) was applied to prevent aggregation of the peptides in solution. Together with the intrinsic YFP signal emitted by the nerve samples itself, fluorescently labeled versions of Wheat Germ lectin (WGA) and cholera toxin B (CTB) were used as a reference and non-incubated samples were used as negative control.
- WGA Wheat Germ lectin
- CTB cholera toxin B
- Tissue samples were analyzed using a Leica SP8 WLL confocal microscope (Leica Microsystems). All samples were washed three times with PBS solution prior to imaging. Images were acquired following CY5-excitation at 633 nm at 106 and 636 times magnification. For imaging of the intrinsic YFP signal a 488 nm laser was used for excitation, emission was collected between 510-550 nm. All images were analyzed using Leica Confocal Software (Leica Microsystems).
- mice were killed via cervical dislocation before the start of the imaging session. Hereafter the mouse was placed under a Zeiss 710 NLO upright confocal microscope. Images were collected at prior and during the dissection of the Ischiadic Nerve at a 10 x magnification using a
- Peptides PO-1 and PO-5 could be synthesized fairly easily and with an acceptable yield (15% and 56% respectively). Synthesis of PO-3, P0-4 and P0- 7 resulted in much lower amounts of pure peptide, although still enough peptide remained for initial in vitro assessment (ranging from 5-10% yield). Unfortunately, the synthesis of PO-2 failed repeatedly. This peptide was therefore excluded from further evaluation. Evaluation of the adhesive interface of P0 revealed that a combination of PO-5 and PO-7 could provide a peptide that benefits from parts of the adhesive interface of both PO-5 and PO-7, which resulted in peptide, PO-6. Initial synthesis of PO-6 and PO-7 was complex, but improved considerably when a pseudoproline approach was implemented (White et al. 2004), yields (+/- 60% (12 mg) for both.
- DRG explants obtained from THY- 1 YFP mouse embryos were used to provide an intermediate step between in vitro and in vivo evaluation.
- Fluorescent- derivatives of WGA and CTB, imaging agents that reportedly bind to sugar groups present on cell membranes and the endoneurium provided reference staining of the outline of the axonal outgrowths similar to the schematic representation in Figure 6D. A similar staining pattern was seen when DRG explants were incubated with the non-PO binding peptide NP41 ( Figure 6E-F).
- Intravenous injection tail vein of a relatively low dose of PO-8 (50 ug) did not result in clear staining of the Sciatic nerve at one or 24 hr after injection of the imaging agent.
- Local administration 50ug
- Figure 8B shows a clear example of the staining of two different nerve bundles, with the axons shown in green and the stained Schwann cells in red. After injection of PO-8 into the femoral artery the blood flow into the nerve could be visualized (Figure 8C; in red).
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Abstract
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