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Title:
IMAGING AGENTS TARGETING MYELIN PROTEIN ZERO
Document Type and Number:
WIPO Patent Application WO/2018/016960
Kind Code:
A1
Abstract:
The present invention relates to compounds comprising a peptide and a detectable imaging label, their use as imaging and diagnostic agents and their use in imaging of a neuron, an axon, a nerve or nerve tissue, preferably of the peripheral nervous system, in image-guided surgery and for determining whether an individual is suffering from a myelin related disorder or from nerve tissue damage.

Inventors:
VAN LEEUWEN FIJS WILLEM BERNHARD (NL)
BUCKLE TESSA (NL)
VAN WILLIGEN DANNY MICHEL (NL)
VAN DER WAL STEFFEN (NL)
Application Number:
PCT/NL2017/050497
Publication Date:
January 25, 2018
Filing Date:
July 21, 2017
Export Citation:
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Assignee:
ACADEMISCH ZIEKENHUIS LEIDEN (NL)
International Classes:
A61K49/00; C07K14/47
Domestic Patent References:
WO2005053751A12005-06-16
WO2010121023A22010-10-21
WO2009029936A12009-03-05
Foreign References:
US8658129B22014-02-25
Other References:
EKICI ARIF B ET AL: "Tracing Myelin Protein Zero (P0) in vivo by construction of P0-GFP fusion proteins", BMC CELL BIOLOGY, BIOMED CENTRAL, LONDON, GB, vol. 3, no. 1, 26 November 2002 (2002-11-26), pages 29, XP021014072, ISSN: 1471-2121, DOI: 10.1186/1471-2121-3-29
D'URSO D ET AL: "Protein zero of peripheral nerve myelin: Biosynthesis, membrane insertion, and evidence for homotypic interaction", NEURON, CELL PRESS, US, vol. 4, no. 3, 1 March 1990 (1990-03-01), pages 449 - 460, XP027463248, ISSN: 0896-6273, [retrieved on 19900301], DOI: 10.1016/0896-6273(90)90057-M
ZHIGANG LIU ET AL: "Crystal structure of the extracellular domain of human myelin protein zero", PROTEINS: STRUCTURE, FUNCTION, AND BIOINFORMATICS, vol. 80, no. 1, 4 January 2012 (2012-01-04), US, pages 307 - 313, XP055334419, ISSN: 0887-3585, DOI: 10.1002/prot.23164
GIBBS-STRAUSS SUMMER L ET AL: "Nerve-highlighting fluorescent contrast agents for image-guided surgery", MOLECULAR IMAGING, DECKER PUBLISHING, vol. 10, no. 2, 1 April 2011 (2011-04-01), pages 91 - 101, XP008156846, ISSN: 1536-0121, [retrieved on 20110301]
EICHBERG J.: "Myelin P 0 : New Knowledge and New Roles*", NEUROCHEMICAL RESEARCH, vol. 27, no. 11, 1 November 2002 (2002-11-01) - 2002, pages 1331 - 1340, XP055334732, Retrieved from the Internet
MERRIFIELD, J. AM. CHEM. SOC., vol. 85, 1963, pages 2149 - 2156
ATHERTON ET AL.: "Solid Phase Peptide Synthesis", 1989, IRL PRESS
BUNSCHOTEN ET AL., BIOCONJUGATE CHEM, vol. 27, 2016, pages 1253 - 1258
KOOPMAN ET AL., BIOORG MED CHEM, vol. 21, 2013, pages 553
WHITNEY ET AL., NAT BIOTECHNOL, vol. 29, 2011, pages 352 - 356
GIBBS-STRAUSS SL ET AL.: "Nerve-highlighting fluorescent contrast agents for image-guided surgery", MOL IMAGING, vol. 10, no. 2, 2011, pages 91 - 101, XP008156846
HASSE ET AL., MOLL CELL NEUROSCI, vol. 27, 2004, pages 370 - 378
HAACK; MUTTER, TETRAHEDRON LETTERS, vol. 33, 1992, pages 1589
KUIL ET AL., BIOCONJ CHEM, vol. 22, 2011, pages 859
MAKOWSKA ET AL., J NEUROL NEUROSURG PSYCHIATRY, vol. 79, 2008, pages 664 - 671
MUJUMDAR ET AL., BIOCONJUGATE CHEM, vol. 4, 1993, pages 105
NICLOU ET AL., MOL CELL NEUROSCI, vol. 24, 2003, pages 902 - 912
WHITNEY MA ET AL., NAT BIOTECHNOL., vol. 29, no. 4, 2011, pages 352 - 356
WHITE ET AL., J. PEPTIDE SCI., vol. 10, 2004, pages 18 - 26
Attorney, Agent or Firm:
JANSEN, C.M. (NL)
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Claims:
Claims

1. 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.

2. The compound according to claim 1, wherein said peptide consists of a sequence that has at least 80% 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. 3. The compound according to claim 1 or 2, wherein said peptide 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 substitutions. 4. The compound according to any one of claims 1-3, wherein said peptide is cyclized, and/or is N-terminally and/or C-terminally modified, preferably by N -terminal acylation, such as by acetic acid and/or by C- terminal amidation. 5. The compound according to any one of claims 1-4 wherein said peptide comprises a sequence that has at least 80% sequence identity with 5-50 consecutive amino acids of amino acids 1-85 or 95-125 of said sequence.

6. The compound according to any one of claims 1-5 wherein said peptide is selected from the group consisting of: ne-Val-Val-Tyr-Thr-Asp-Arg-Glu-Val-ffis-Gly-Ala-Val-Gly-Ser-Arg-Val-Thr- Leu-His-Cys-Ser-Phe-Trp-Ser,

Pro-Glu-Gly-Gly-Arg-Asp-Ala-Ile-Ser-Ile-Phe-His-Tyr-Ala-Lys-Gly-Gln-Pro- Tyr-Ile-Asp-Glu-Val-Gly-Thr-Cys,

Asp-Glu-Val-Gly-Thr-Phe-Lys-Glu-Ai-g-Ile-Gln-Trp-Val-Gly-Asp-Pro-Arg- Trp-Lys-Asp-Gly-Ser-Ile-Val-Ile-Cys,

Gly-Ser-Ile-Val-Ile-His-Asn-Leu-Asp-Tyr-Ser-Asp-Asn-Gly-Thr-Plie-Tlir- Cys-Asp-Val-Lys-Asn-Pro-Pro-Asp,

Thr-Phe-Thr-Ala-Asp-Val-Lys-Asn-Pro-Pro-Asp-Ile-Val-Gly-Lys-Thr-Ser- Gln-Val-Thr-Leu-Tyr-Val-Phe-Glu-Lys-Cys,

Lys-Asn-Pro-PiO-Asp-Ile-Val-Gly-Lys-Thr-Ser-Gln-Val-Thr-Leu-Tyr-Val- Phe-Glu-Lys-Val-Pro-Thr-Arg-Tyr-Cys,

Gly-Lys-Thr-Ser-Gln-Val-Thr-Leu-Tyr-Val-Phe-Glu-Lys-Val-Pro-Thr-Arg- Tyr-Cys, and multimers thereof.

7. The compound according to any one of claims 1-6, wherein said compound comprises a detectable label selected from the group consisting of a fluorescent label, a luminescent label, a (radio)isotope label and a paramagnetic label.

8. The compound according to any one of claims 1-7 wherein one or more amino acid of said 2-100 consecutive amino acids is replaced by said detectable imaging label or wherein said detectable label is located between two amino acids of said 2-100 consecutive amino acids.

9. A compound according to any one of claims 1-8 for use as an imaging or diagnostic agent.

10. A compound according to any one of claims 1-8 for use in imaging of a neuron, Swann cell, an axon, a nerve or nerve tissue, preferably of the peripheral nervous system. 11. A compound according to any one of claims 1-8 for use in image- guided surgery.

12. The compound for use according to claim 11, wherein said use comprises administering said compound to an individual prior to surgery and/or during surgery, visualizing said detectable imaging label and performing said surgery.

13. A compound according to any one of claims 1-8 for use in detecting myelin in an individual.

14. A method for determining whether an individual is suffering from a myehn related disorder or from nerve tissue damage comprising administering the compound according to any one of claims 1-8 to the individual or to a sample of the individual and detecting the presence, amount or distribution of said detectable label, preferably wherein said disorder is a demyelinating disorder, 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.

15. 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 any one of claims 1-8.

Description:
Title: Imaging agents targeting myelin protein zero Field of the invention

The invention relates to the field of imaging and image guided interventions. In particular, the invention relates to agents for imaging of nerve tissue of the peripheral nervous system and uses thereof.

Background of the invention

Patients with (surgically-induced) nerve injury suffer from a wide variety of complaints. Depending on the anatomy wherein the surgery was performed, the functionality of crucial organs can even be compromised.

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. Here, intraoperative guidance towards the lesions (e.g. tumor lesions or possibly diseased sentinel lymph nodes) of interest 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.

Research on such (fluorescent) imaging agents is currently ongoing, but has to date not yet resulted in agents that can be clinically applied, mainly as the currently available agents lack a nerve specific target or the specificity for the peripheral nervous system. The latter is of the upmost importance when realizing that staining of the central nervous system may result in highly unwanted systemic side effects. Whitney et al 2011 and WO 2010/121023 describe fluorescently labelled peptides selected using phage display that stain nerves. However, the target of the peptides is not known and therefore it is difficult to predict their function in humans. Further, the peptides also stain adipose tissue, reducing specificity for nervous tissue and decreasing the in vivo visibility of the nerve.

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. Using a similar approach 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.

US 8,658, 129 describes agents capable of binding to myehn basic protein (MBP) for detection of myehn associated neuropathy and

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.

Hence, there exists a need in the art for improved imaging agents for nerve tissue, in particular imaging agent that specifically target surface markers expressed in the peripheral nervous system. Summary of the invention

It is an object of the present invention to provide imaging agents that specifically stain nerve tissue. In particular, it is an object to provide imaging agents that are specific for nerve tissue of the peripheral nervous system. It is a further object of the present invention to provide methods for detecting nerve tissue and for image-guided surgery.

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.

In a further aspect, 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.

In a further aspect, 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.

In a further aspect, the invention provides a compound according to the invention for use in detecting myelin in an individual.

In a further aspect, 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.

In a further aspect, the invention provides the use of a compound according to the invention as an imaging agent or diagnostic agent.

In a further aspect, 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. In a further aspect, 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.

In a further aspect, 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.

In a further aspect, the invention provides a compound according to the invention for use in image-guided surgery.

In a further aspect, 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.

In a further aspect, 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.

In a further aspect, 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. In one embodiment said method is an in vitro method. In another embodiment, said method is an in vivo method.

Detailed description

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. Until the present invention, 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. Without being bound to theory, it is believed that 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.

Accordingly, in a first aspect 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.

In amino acid sequences as defined or indicated herein 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 (Tyr) is tyrosine. As used herein a "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. However, 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.

As used herein the term "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

IWYTDREVHGAVGSRVTLHCSFWSSEWVSDDISFTWRYQPEGGRDAISI FHYAKGQPYIDEVGTFKERIQWVGDPRWKDGSIVIHNLDYSDNGTFTCD VKNPPDIVGKTSQVTLYVFEKVPTRY.

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. Preferably, 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

consecutive amino acids of said sequence. In a further embodiment, 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. For instance, 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

consecutive amino acids of said extracellular domain.

The percentage of identity of an amino acid sequence or the term

"% 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

Copyright Office, Washington, D.C. 20559, on Dec. 10, 1991.

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.

Preferably, 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. For instance, 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.

For instance, conservative amino acid substitutions, and substitutions of any amino acid with alanine can be made in the peptide without losing the ability of the compound of the invention to interact with PO in the myelin sheath of axons. Therefore, in a preferred embodiment, 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

substitution. Hence, 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.

In one embodiment, at most 10% of the amino acids of a peptide according to the invention are substituted with an alanine. In a preferred embodiment 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

substitutions for one another:

1) serine, threonine asparagine, glutamine;

2) aspartic acid, glutamic acid;

3) histidine, arginine, lysine;

4) isoleucine, leucine, methionine, alanine, valine, and

5) phenylalanine, tyrosine, tryptophan.

Hence 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. In a preferred

embodiment, a peptide according to the invention comprises at most 2 conservative amino acid substitutions as defined herein, more preferably at most one.

In a preferred embodiment, a peptide according to the invention therefore comprises one or more substitutions independently selected from the group consisting of:

- substitution of one amino acid by alanine;

- substitution of: an amino acid selected from the group consisting of serine, threonine asparagine and glutamine by another amino acid selected from said group;

an amino acid selected from the group consisting of aspartic acid and glutamic acid by another amino acid selected from said group;

an amino acid selected from the group consisting of histidine, arginine and lysine by another amino acid selected from said group; an amino acid selected from the group consisting of isoleucine, leucine, methionine, alanine and valine by another amino acid selected from said group;

an amino acid selected from the group consisting of phenylalanine, tyrosine and tryptophan by another amino acid selected from said group. 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. In a further preferred embodiment, 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. In a further preferred embodiment, 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. In a further preferred embodiment, 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. Provided is therefore 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.

In a further particularly preferred embodiment, 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. Preferably, said peptide optionally has 1 additional amino acid C-terminally and/or N-terminally. In a particularly preferred embodiment, 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. Particularly preferred are compounds comprising a peptide comprising the sequence of peptide PO-6, PO-7 or PO-8 as depicted in Table 1. It is further preferred that 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.

Table 1. Amino acid sequences of preferred peptides.

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

Table 2. More preferably, 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. It is further preferred that 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.

Table 2. Amino acid sequences of preferred peptides.

Yet 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. As an alternative, or in addition, 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.

Table 3. Amino acid sequences of further preferred peptides.

In one embodiment, 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. Hence, in such embodiment, a peptide of the invention preferably comprises at least one amino acid substitution in the amino acid sequence as defined herein.

In a preferred embodiment, a compound according to the invention consists of a peptide according to the invention and a detectable imaging label as described herein. In a particularly preferred embodiment, 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. More preferably 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. For instance, 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. Here, 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. Preferred, but non-limiting examples of 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(¾r(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)-Thr(w(Me,Me)Pro)-OH, Fmoc-Glu(OtBu)-

Ser(nj(Me,Me)Pro)-OH, Fmoc-Glu(OtBu)-Thr(nj(Me,Me)Pro)-OH, Fmoc-Gly- Ser(¾rMe, Mepro)-OH, Fmoc-Gly-Thr( (Me,Me)Pro)-OH, Fmoc-Ile- Ser( (Me,Me)Pro)-OH, Fmoc-Ile-Thr(¾r(Me,Me)Pro)-OH, Fmoc-Leu- Ser(¾r(Me,Me)Pro)-OH, Fmoc-Leu-Thr( (Me,Me)Pro)-OH, Fmoc-Lys(Boc)- Ser(nj(Me,Me)Pro)-OH, Fmoc-Lys(Boc)-Thr( (Me,Me)Pro)-OH, Fmoc-Phe- Ser( (Me,Me)Pro)-OH, Fmoc-Phe-Thr(¾f(Me,Me)Pro)-OH, Fmoc-Ser(tBu)- Ser( (Me,Me)Pro)-OH, Fmoc-Ser(tBu)-Thr(¾f(Me,Me)Pro)-OH, Fmoc- Trp(Boc)-Ser( (Me,Me)Pro)-OH, Fmoc-Trp(Boc)-Thr(nj(Me,Me)Pro)-OH, Fmoc-Tyr(tBu)-Ser(¾f(Me,Me)Pro)-OH, Fmoc-Tyr(tBu)-Thr(ψ(Me,Me)PI )- OH, Fmoc-Val-Ser(nj(Me,Me)Pro)-OH and Fmoc-Val-Thr(nj(Me,Me)Pro)-OH.

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. Preferred, but non-limiting examples of 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. Hence, 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. Preferred, but non- limiting examples of suitable isoacyl dipeptides that can be incorporated into a peptide according to the invention are: Boc-L-Ser(Fmoc-L-Ala)-OH, Boc-L-Ser[Fmoc-L-Arg(Pbf)]-OH, Boc-L-Ser[Fmoc-L-Asn(Trt)]-OH, Boc-L- Ser[Fmoc-L-Asp(tBu)]-OH, Boc-L-Ser[Fmoc-L-Cys(Trt)]-OH, Boc-L- Ser[Fmoc-L-Gln(Trt)]-OH, Boc-L-Ser[Fmoc-L-Glu(tBu)]-OH, Boc-L- Ser(Fmoc-Gly)-OH, Boc-L-Ser[Fmoc-L-His(Trt)]-OH, Boc-L-Ser(Fmoc-L-Ile)- OH, Boc-L-Ser(Fmoc-L-Leu)-OH, Boc-L-Ser[Fmoc-L-Lys(Boc)]-OH, Boc-L- Ser(Fmoc-L-Met)-OH, Boc-L-Ser(Fmoc-L-Phe)-OH, Boc-L-Ser[Fmoc-L- Ser(tBu)]-OH, Boc-L-Ser[Fmoc-L-Thr(tBu)]-OH, Boc-L-Ser[Fmoc-L- Trp(Boc)]-OH, Boc-L-Ser[Fmoc-L-Tyr(tBu)]-OH, Boc-L-Ser(Fmoc-L-Val)-OH, Boc-L-Thr(Fmoc-L-Ala)-OH, Boc-L-Thr[Fmoc-L-Arg(Pbf)])-OH, Boc-L- Thr[Fmoc-L-Asn(Trt)]-OH, Boc-L-Thr[Fmoc-L-Asp(tBu)]-OH, Boc-L- Thr[Fmoc-L-Cys(Trt)]-OH, Boc-L-Thr[Fmoc-L-Gln(Trt)]-OH, Boc-L- Thr[Fmoc-L-Glu(tBu)]-OH, Boc-L-Thr(Fmoc-Gly)-OH, Boc-L-Thr[Fmoc-L- His(Trt)]-OH, Boc-L-Thr(Fmoc-L-Ile)-OH, Boc-L-Thr(Fmoc-L-Leu)-OH, Boc- L-Thr[Fmoc-L-Lys(Boc)]-OH, Boc-L-Thr(Fmoc-L-Met)-OH, Boc-L-Thr(Fmoc- L-Phe)-OH, Boc-L-Thr[Fmoc-L-Ser(tBu)]-OH, Boc-L-Thr[Fmoc-L-Thr(tBu)]- OH, Boc-L-Thr[Fmoc-L-Trp(Boc)]-OH and Boc-L-Thr(Fmoc-L-Val)-OH.

Alternatively, 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. Preferably 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. Examples of 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. Examples of yeast cells are Saccharomyc.es and Pichia. Examples of 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. Examples of 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.

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. Examples of 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. A compound according to the invention comprises, in addition to a peptide derived from the extracellular domain of P0, a detectable imaging label. As used herein the term "detectably 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. Such 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

pharmacokinetic of the peptides.

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

paramagnetic label. 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. Particularly preferred 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. Most preferred is a fluorescent label. Said fluorescent or fluorescent hybrid 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. Atto465, Atto488, Atto495, Atto550, Atto647), cyanine (Cy) dyes, including 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-ylidene)hepta- l,3,5-trien-l-yl]-3H-indolium-5- sulfonate), Alexa Fluor (e.g. Alexa Fluor 647, Alexa488, Alexa532, Alexa546, Alexa594, Alexa633, Alexa647), Bodipy (e.g. Bodipy® FL), Dylight (e.g.

DyLight 488, DyLight 550), Trp (Tryptophan), Lucifer Yellow (ethylene diamine or 6-Amino-2-(2-amino-ethyl)-l,3-dioxo-2,3-dihydro-lH- benzo[de]isoquinoline-5,8-chsulfonic acid) and derivatives thereof. Such labels can also occur in (bio)nan op articles that will be conjugated to the targeting peptide. Alternatively, 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. In a particularly preferred embodiment, a compound according to the invention comprises a peptide according to the invention and a trisulfonated Cy5 as the detectable imaging label. Such trisulfonated Cy5 is shown in the Examples herein and described in Mujumdar et al., 1993, which is incorporated by reference herein. Uniquely the cyanine dye (and its Cy3 and Cy7 analogues), allow for easy tuning of the pharmacokinetic properties as was recently exemplified by Bunschoten et al., 2016 (Bunschoten et al., 2016. Bioconjugate Chem 27: 1253-1258). For multispectral applications fluorescein will be most preferred. Hence, preferred labels include fluorescein or a Cy dye, preferably C3, C5 or C7.

In one embodiment, the detectable imaging label, preferably a label comprising a fluorescent moiety, is bound directly to the peptide according to the invention using known techniques in conjugation

chemistry. Such labels can be inserted at the distal ends of the peptides sequences using known conjugation techniques e.g. click-chemistry.

Alternatively, 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. For instance, the label can be incorporated at the C- or N-terminus or at Cys or Lys side chains of the peptide.

In another embodiment, 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.

Examples of such 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. In yet another, preferred, embodiment, 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.

In another preferred embodiment, 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

introducing a fluorescent dye in a peptide during synthesis. 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. For instance, a fluorescent label is detected by exciting the fluorophore with the appropriate wavelength of light and detecting the fluorescence. Such 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.

Further provided is a composition comprising a compound according to the invention. Said composition further preferably comprises at least one carrier, diluent and/or excipient. In one preferred embodiment, the composition is a pharmaceutical composition which further comprises a pharmaceutically acceptable carrier, diluent and/or excipient. By

"pharmaceutically acceptable" it is meant that the auxiliary, carrier, diluent or excipient must be compatible with the other ingredients of the

formulation and not deleterious to the recipient thereof. 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. In particular, compounds according to the invention are useful as imaging agent, both in vitro and in vivo, and as diagnostic agents.

In one aspect the invention therefore provides a compound according to the invention for use as an imaging or diagnostic agent.

Further provided is the use of a compound according to the invention as an imaging agent or diagnostic agent.

As described herein before, it is believed that 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. Hence, 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 (PNS) as used herein 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.

Also provided is therefore 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. In one preferred embodiment, the labeling of a neuron, a Schwann cell, an axon, a nerve or nerve tissue, preferably of the peripheral nervous system, occurs in vivo. In another preferred embodiment, 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 for instance encompasses labelhng of nerve tissue in tissue samples of an individual, e.g. for research purposes. Provided is therefore a method for imaging a neuron, a Schwann cell, a nerve or nerve tissue, the method comprising contacting a Schwann cell, an axon, a nerve or nerve tissue with a compound according to the invention. In one embodiment, said method is an in vitro method, and said contacting occurs for instance in a sample obtained from an individual. In another embodiment, 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. In one embodiment, said method comprises determining whether a neuron, a Schwann cell, axon, nerve or nerve tissue is present in said sample. In another

embodiment, said method comprises determining the amount of label, e.g. fluorescent label, in a sample obtained from an individual.

In vitro or in vivo use as imaging agent for instance encompasses labelhng of nerve tissue in an individual or in tissue samples of an

individual for diagnostic purposes. Provided is therefore the use of a compound according to the invention as a diagnostic agent. 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. In another embodiment, 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.

As used herein, 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.

As used herein, "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. Preferably, said disorder is therefore a demyelinating disorder. Examples of 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.

"Nerve 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.

Further provided is 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. Also provided is a method for detecting myelin in an individual, the method comprising:

- administering a compound according to the invention to said individual and

- detecting the presence of said detectable label. 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. Also provided is 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

fluorescence intensity of a reference, for instance a reference as described above. Similarly, 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. However, 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.

Provided is therefore a compound according to the invention for use in image-guided surgery. Said use preferably comprises administering said compound to an individual prior to surgery and/or during surgery, visuahzing said detectable imaging label and performing said surgery. Also provided is 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. Further provided is the use of a compound according to the invention for the preparation of a pharmaceutical composition for use in image-guided surgery. Said administering 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). As is known to a skilled person, these technologies are being used for radio- guided surgery of sentinel lymph nodes and can easily be adapted, if necessary, for detection of compounds according to the invention. The use of 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.

It is noted that fluorescence will be a most relevant label for surgical guidance. Hence, fluorescence guided surgery (FGS) 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. 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

NIRDye800CW, ZW800, ICG, Cy5.5 etc. To make nerve visualization compatible with fluorescence guidance towards e.g. tumor tissue or lymph node visible fluorescent dyes (400-600 nm range) may also be employed as imaging labels. A preferred example herein is fluorescein (FITC) and analogues thereof.

Bimodal or hybrid labels may be applied wherein both the radio and fluorescent label are attached to the same peptide sequence.

Features may be described herein as part of the same or separate aspects or embodiments of the present invention for the purpose of clarity and a concise description. It will be appreciated by the skilled person that the scope of the invention may include embodiments having combinations of all or some of the features described herein as part of the same or separate embodiments.

The invention will be explained in more detail in the following, non-limiting examples. Brief description of the drawings

Figure 1: A. Sequence of the extracellular domain of myelin protein zero. B. Peptides used. Indicated are altered amino acid residues (underlined); the positioning of a label (X); the use of pseudoprolines (highlighted); and sequences that were described in Makowski et al., 2008.

Figure 2: 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.

Figure 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.

Figure 5: Evaluation of PO-specificity and binding affinity. A) Concentration dependent fluorescence measurements performed using a specially designed FLIZA set-up showed P0-specific fluorescence in PO-precoated wells (circles) and not in non-precoated wells (triangles). B) Comparison between binding of PO-8 and positive (anti PO-Ab) and negative (NP41) controls showed P0- specific binding for the antibody and not for the non-P0 binding peptide. Flow cytometry-basecl saturation binding experiments revealed the affinity of C) PO-8 and D) PO-6 for P0. Figure 6: Staining of DRG explants. A) Schematic overview of an unstained DRG and its axonal outgrowths. White hght image of a DRG explant at B) lOx and C) 40x times magnification. D) Schematic representation of axonal staining with corresponding images of a DRG explant after staining with NP41-Cy5 at E) lOx and F) 40x magnification. G) Schematic representation of PO-specific staining with corresponding images of a DRG explant after staining with PO-8 at H) lOx and I) 40x magnification. In all images the DRG itself is marked with *, whereas an example of an axonal outgrowth is marked with a white arrow. An example of a stained Schwann cell is marked with a grey arrow. The nucleus of the cells are stained in blue.

Figure 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. A) Multiphoton fluorescence microscopy set-up for direct in vivo evaluation of nerve staining. B) Multiphoton image of the Sciatic nerve after intra-neural injection of PO-8 (5x magnification). C) Confocal image the Sciatic nerve showing the blood flow into the nerve after injection of PO-8 into the femoral artery (lOx magnification). Confocal images of dissected nerve samples showing the distribution of staining within the nerve after injection of PO-8 D) into the femoral artery or E) directly into the Sciatic nerve. In both cases peptide specific red staining (Cy5, in red and examples indicated by arrows) was seen along the course of the axons (GFP, in green and examples indicated by arrows), while the axons were not stained. F) Ex vivo

incubation of the Sciatic nerve with PO-8. Figure 9: Overlay of confocal images onto brightfield images with nucleus in blue, lysosomes in green and staining PO-targeting peptide with Cy5 dye in red. A. Peptide sequence: KVPTRY-NH2 (OP33). B. Peptide sequence:

KNPPDI-NH2 (OP 19).

Examples

Materials and methods All chemicals were obtained from commercial sources and used without further purification. DMF was dried over 4 A molecular sieves for 24 hours prior to use in water sensitive reactions. High-pressure liquid

chromatography (HPLC) was performed on a Waters HPLC system (Waters Chromatography B.V., Etten-Leur, The Netherlands) using a 1525EF pump and a 2489 UV detector. For preparative HPLC, a Maisch Repro Sil-Pur 120 C18-AQ 10 μΜ (250 mm x 20 mm) column (Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany) was used at a flow rate of 12 mL/min. For analytical HPLC a Maisch Repro Sil-Pur C18-AQ 5μΜ (250 mm x 4.6 mm) column was used with a gradient of 0.1% TFA in H20/MeCN 95:5 to 0.1% TFA in H 2 0 MeCN 5:95 in 20 min (1 mL/min). Mass spectrometry was performed on a Bruker microflex MALDI-TOF mass spectrometer.

General peptide synthesis

Peptides PO-1, PO-2, PO-3, P0-4, PO-5 and PO-7 (Figure 2), 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). In 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 ;

Ac-Cys-Ser-Phe-Trp-Ser-Ser-Glu-Trp-Val-Ser-Asp-Asp-Ile-Se r-Phe-Thr-Trp-

Arg-Tyr-Gln-Pro-Glu-Gly-Gly-Arg-NH 2

PO-3 (amino acid 41-65): Ac-PEGGRDAISIFHYAKGQPYIDEVGTC-NH 2 ; Ac-Pro-Glu-Gly-Gly-Arg-Asp-Ala-Ile-Ser-Ile-Phe-His-Tyr-Ala-L ys-Gly-Gln- Pro-Tyr-Ile-Asp-Glu-Val-Gly-Thr-Cys=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-A sp-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-T hr-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-L eu-Tyr-Val- Phe-Glu-Lys-Val-Pro-ThrArg-Tyr-Cys-NH 2 .

After deprotection and cleavage from the resin using a cleavage cocktail containing TFA with 5% water and ethanethiol as scavengers for 3 hours, a drop of triethyl silane was added to scavenge (trityl) cations. The crude peptides were isolated from the cleavage cocktail by precipitation from MTBE/hexanes and purified by preparative reverse phase HPLC

chromatography.

To improve the overall yield of the synthesis, 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- Lvs-Val- Pro-ThrArg-Tyr-Cys-NH 2 ) were synthesized using a pseudoprolines method as first described by Haack and Mutter 1992. Here the corresponding bolded and underlined amino acid residues were substituted by commercially available dipeptides Fmoc-Val-Thr( l F(Me,Me)Pro)-OH and Fmoc-Lys(Boc)- Thr( T F(Me,Me)Pro)-OH without modifications in the synthesis- or

deprotection-protocol.

Pseudoproline dipeptides could be used in the synthesis of the previously described peptides at the designated positions:

H-IWYTDREVHGAVGSRVTLHCSFWS-NH2

Ac-PEGGRDAISIFHYAKGQPYIDEVGTC-NH 2

AC-DEVGTFKERIQWVGDPRWKDGSIVIC-NH2

AC-GSIVIHNLDYSDNGTFTCDVKNPPD-NH2,

wherein the bolded and underlined amino acid residues were substituted by commercially available dipeptides Fmoc-Tyr(tBu)-Thr( T P(Me,Me)Pro)-OH (for YT), Fmoc-Val-Thr(<F(Me,Me)Pro)-OH (for VT), Fmoc-Ile-

Ser F(Me,Me)Pro)-OH (for IS), Fmoc-Gly-Thr(*F(Me,Me)Pro)-OH (for GT), Fmoc-Gly-Ser F(Me,Me)Pro)-OH (for GS), Fmoc-Tyr(tBu)- SerOF(Me,Me)Pro)-OH (for YS) and Fmoc-Phe-Thr F(Me,Me)Pro)-OH (for FT) without modifications in the synthesis- or deprotection-protocol.

Production of extracellular part ofPO.

Transformation of BL21/DE3 bacteria with a S11IEG3 plasmid containing the open reading frame of the extracellular part op P0 (PO-ex) was performed via a previously described method. (Hasse et al. 2004). Single colonies were grown over night (o/n) in 3 ml LB containing 100 mg/ml ampicillin. Hereafter 2.5 ml o/n culture was added to 250 ml LB containing ampicillin and grown at 37°C. When ODeoo = 0.7-0.8 (2-3 hrs)ld was reached, the culture was cooled down to RT in cold water with ice. 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. When 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). Hereafter the pellet was at -20°C for later use.

Prior to the initiation of PO-ex production a vial containing bacteria was thawed and re-suspended in 4 ml PBS pH 8 + ImM DTT + protease inhibitor before being sonicated on ice (lOx 10 sec). 1% Triton X-100 and 100 mg lysozyme were added after the bacteria suspension was dissolved in 1 ml PBS. The suspension was then incubated for 1- 2 hrs at 4°C until the cells were lysed and the suspension became viscous. DNase (100 μΐ of a 100 u/μΐ solution) was added to the bacterial suspension and incubated for 15-30 minutes before being spinned down at 8000 rpm for 30 min at 4°C. 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. 3 ml of a 20 mM Reduced Glutathione (out of a freshly made 100 mM stock) in 50mM Tris pH 8.0 was then added to the beads and incubated o/n at 4°C. The mixture was then centrifuged 1500 rpm for 10 min, where after the supernatant containing PO-ex was obtained , yielding approximately 900 pg of POex, determined using standard BCA assay. Syn thesis of the thiol-reactive trisulfonated Cyo-dye

Trisulfonated Cy5 was synthesized according to the method described previously by Mujumdar et al. 1993. This dye (25 mg, 30 μηιοΐ) was dissolved in dry DMF (400 μΙ_). together with PyBOP (17.3 mg, 33 μηιοΐ) and aminoethyl maleimide trifluoroacetate (9 mg, 34 μιηοΐ). Subsequently, N- methyl morpholine (15 μΤ», 136 μιηοΐ) 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 μΐ ^ ) 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 μη οΐ) was obtained. MALDI-TOF mass spectrometry: Calculated 887.3, found 887.7. The thiol-reactive dye has the following structure:

Fluorescent labeling of the PO-based peptides

To enable optical detection all peptides were labeled on the available cysteine residue with the thiol reactive Cy5 dye, resulting in the fluorescent peptides PO-1, PO-3, P0-4, PO-5, PO-6, PO-7 and PO-8. Peptide labeling was performed in phosphate buffer (100 μΜ, pH 7.4, 3 mg peptide/mL). In case the peptide was poorly soluble in phosphate buffer alone a few drops of DMF were added. The reaction mixture was then shaken for 2 hours before being acidified with acetic acid and purified using preparative reverse phase HPLC. Lyophilisation yielded the peptide conjugates as fluffy light blue solids. Stock solutions of the fluorescently labeled peptides were prepared by dissolving the conjugates in a small amount of ethanol and diluting with PBS. The resulting concentration of the conjugates was determined via absorption measurements at 650 nm using an extinction coefficient of 2.5 x 10 5 as reported by Mujumdar (Mujumclar et al. 1993). MALDI-TOF analysis of the labeled peptides resulted in the following masses and confirmed the identity of the peptides:

PO-1: found 3706.8, calculated 3703.68

PO-3: found 3754.1, calculated 3750.63

P0-4: found 3966.1, calculated 3960.81

PO-5: found 3651.6, calculated 3648.53

PO-6: found 3928.4, calculated 3927.82

PO-7: found 3914.8, calculated 3909.86

PO-8: found 3910.7, calculated 3909.86 An tibody and PO-ex labeling

General

For all protein labelings, a 5.1 mM stock solution of Cy5-OSu in DMSO was used. Amicon centrifugal filters (Merck Millipore, Billerica, MA, USA) were used for purification. The degree of labeling was estimated via absorption measurements at 650 nm using an Ultrospec 3000 absorption spectrometer (Pfizer/Pharmacia Biotech, Capelle a/d IJssel, Netherlands) and extinction coefficient of 2.5 x 10 5 as reported by Mujumdar (Mujumdar et al. 1993). Rabbit polyclonal anti-myelin antibody was purchased from Abeam

(Cambridge, UK), Rabbit polyclonal anti-SlOO antibody was purchased from Dako (Heverlee, Belgium), Rabbit polyclonal POAb (H60) antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Anti-Myelin-Cy5

100 pg (3.85 nmol) of anti-myelin antibody was concentrated using a 10K Amicon and washed two times with 100 μΐ of 0.1M phosphate buffer pH 8.4. After collecting the concentrate, another 100 μΐ of buffer and 7.6 μΐ (38.46 nmol) of Cy5-OSu stock solution were added. After agitating for 3.5 hours at room temperature the mixture was filtered over a fresh 10K Amicon filter and washed with PBS until a colorless filtrate was observed. The

concentrate (30 μΐ) was collected and supplemented with 70 μΐ PBS. The degree of labeling was 2.60 dyes/protein. The conjugate was refrigerated prior to use.

Anti-Sl00-Cy5

500 pg (3.33 nmol) of Anti-SlOO was concentrated using a 3K Amicon and washed two times with 100 μΐ of 0.1M phosphate buffer pH 8.4. After collecting the concentrate, another 100 μΐ of buffer and 18 μΐ (90.93 nmol) of Cy5-OSu were added. After agitating for 3.5 hours at room temperature the mixture was filtered over a fresh 3K Amicon filter and washed with PBS until a colorless filtrate was observed. The concentrate (74 μΐ) was supplemented with 426 μΐ of PBS. The degree of labehng was 15.00 dyes/protein. The conjugate was refrigerated prior to use. P0ex-Cy5

100 pg (2.50 nmol) of pOex was concentrated using a 10K Amicon

centrifugonal filter and washed two times with 100 μΐ of 0.1M phosphate buffer pH 8.4. After collecting the concentrate, another 100 μΐ of buffer and 2.5 μΐ (12.50 nmol) of Cy5-OSu stock solution were added. After agitating for 3 hours at room temperature the mixture was filtered over a fresh 10K Amicon filter and washed with PBS until a colorless filtrate was observed. The concentrate (30 μΐ) was collected and supplemented with 70 μΐ PBS. The degree of labeling was 0.64 dyes/protein. The conjugate was refrigerated prior to use.

P0Ab-Cy5

200 pg (2.99 nmol) of POAb was concentrated using a 10K Amicon

centrifugonal filter and washed two times with 100 μΐ of 0.1M phosphate buffer pH 8.4. After collecting the concentrate, another 200 μΐ of buffer and 1.2 μΐ (5.97 nmol) of Cy5-OSu stock solution were added. After agitating for 2.25 hours at room temperature the mixture was filtered over a fresh 10K Amicon filter and washed with PBS until a colorless filtrate was observed. The concentrate (70 μΐ) was collected and supplemented with 130 μΐ PBS. The degree of labeling was 1.07 dyes/protein. The conjugate was refrigerated prior to use.

Cell lines

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 (THY- 1 YFP) 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.

Culture of dorsal root ganglion (DRG) explants from THY-1 YFP mouse embryos

Glass coverslips were pre-coated o/n with poly-L-lysine (20μ ml). On the day of DRG dissection coverslips were further coated with laminin 40μ#/ηι1 in DMEM/F12 and incubated for 2hr at 37°C. Hereafter the coverslips were rinsed with DMEM/F12 and transferred to 24 wells plate containing 500μ1 of culture medium. Culture medium (DMEM/F12 with Glutamax; 10ml) containing ΙΟΟμΙ N2 supplement (lOOx stock; Sigma 17502), 20ng/ml NGF (20μ1, ^g/ml stock) and ΙΟΟμΙ lx PenStrep.

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

Flow cytometric analysis of the binding affinity of the different peptides was performed using previously described method (Kuil et al 2011). In short: Freshly cultured RT4 cells were trysinized, aliquoted in portions of 300 000 cells, centrifuged (1200 rpm, 5 min, 4 °C) and decanted. For saturation binding experiments the different peptides were added to the cell samples in a range of 0 - 4000 nM in 50 μL· of 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) was added to the cells. After 1 hour of incubation, the cells were washed (two times with 300 μΐ ^ 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

normalized geometric means were fitted with equations in the GraphPad Prism 5 software. The KD values of the evaluated peptides were calculated using the 'Binding - Saturation, One site - Total' nonlinear regression equation, as described previously (Kuil et al. 2011). Evaluation of binding specificity using FLISA

Lumitrac 600 96-wells plates were coated with PO-ex (10 μg/well) and incubated overnight at 4 °C. The coated wells were then blocked with 200 μΐ casein (2.5 g/100 ml) per well, and incubated for two hours at room

temperature. Plates were washed two times with PBS containing 0.05% Tween 20 (Life Technologies Inc.) For saturation binding experiments PO-8 (32 μΜ) was added to the wells in a range of 0 - 4000 nM. A Cy5-labeled version of the anti-PO antibody (three different concentrations used; 300 nM, 66 nM or 17 nM) and a Cy5 labeled version of the nerve staining peptide NP41 (2000 nM; no target known; Whitney et al., 2011. Nat Biotechnol 29: 352-356) were used as controls. After a two hour incubation of the peptides or controls the wells were washed three times with 0.05% Tween 20 in PBS and absorbance of the Cy5 conjugates was measured at 680 nm (excitation 630 nm) using a SpectraMax microplate reader containing a cut-off filter at 665 nm.

Evaluation of the location of staining in cells and DRG explants

Cells were trypsinized and seeded onto 35mm culture dishes (MatTek co) containing a glass insert in such a way that a 60-70% confluency-rate was obtained on the day prior to the imaging experiment. Axonal outgrowths from the DRGs were allowed to grow for 48 hours (at 37°C), prior to incubation with the different imaging agents and subsequent imaging.

Fluorescence confocal imaging was used to evaluate the staining pattern of the different P0 peptides in cells, DRG explant cultures (all n= 3 per peptide or control).

One hour prior to imaging cell/DRG samples were incubated with a fluorescently labeled version of one of the peptides (-0.6 μΜ). Sonication (20 seconds) was applied prior to addition to prevent aggregation of the peptides in solution. Fluorescently labeled versions of an Anti-myelin Ab, Anti-PO Ab, S100 Ab, and PO-ex, axonal staining Wheat Germ lectin (WGA) and cholera toxin B (CTB), as well as the non-P0 staining peptide NP-41 were used as controls. Non-incubated samples and non-P0 expressing cells were used as negative control. 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/ηι1). 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. For visualization of the lysosomes in the cells a 488 nm laser was used for excitation, while emission was collected between 510-550 nm. Hoechst was excited at 405 nm and emission was collected between 409-468 nm. All images were analyzed using Leica Confocal Software (Leica Microsystems).

Ex vivo evaluation staining in nerve tissue

For ex vivo evaluation of the staining pattern of the peptides samples of the Nervous Ischiadicus of THY- 1 YFP mice were used (n=3 per peptide).

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 μΜ). 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.

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).

In vivo evaluation of nerve staining

For evaluation of in vivo staining the imaging agent was administered intravenously ((1.0 mg; either into the tail vain or the femoral vain), intramuscularly (50 μg) or directly into the nerve sheath (20^ig) of THY- 1 TFP mice (n=3 per injection method). Injection into the nerve sheath was performed under general anesthetics (hypnorm/dormicum/water solution (1: 1:2; 5 μΐ/g) via intraperitoneal injection.) Staining of the nerve after injection of the peptide into the nerve sheath was evaluated at different time points (1 hour or 24 hours after injection; n=3 per time point).

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

Spectraphysics Mai Tai Deep See laser with a range between 690-1040nm. ZEN 2011 software was used for collection and evaluation of the images. Identical settings were used for excitation and emission of YFP and CY5 as during the ex vivo evaluation of nerve tissue samples.. Ex vivo and in vivo experiments were performed according to Dutch law and after approval from the Animal Ethics Committee of the LUMC was obtained (reference # 12147). Results

Prior to the PO-binding studies with the fluorescently labeled peptides agents, criteria were set that would lead to the peptide with the highest potential as an imaging agent for the visualization of nerves. Here the focus was placed on the ease of the synthesis of the peptide and the amount of peptide that could be obtained (synthetic yield) as well as their solubility, without clustering in solution. Peptide synthesis and optimization

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.

Labeling of the peptides with a trisulfonated Cy5-derivative resulted in the fluorescent peptides Cy5-P0-1, Cy5-P0-3, Cy5-P0-4, Cy5-P0-5, Cy5-P0-6, Cy5-P0-7 and Cy5P0-8. Conform the homo-typical binding mode of P0, homo-typical interactions between the individual peptides resulted in clustering in solution. The degree of clustering, however, varied between the different peptides. The highest degree of clustering was seen with PO-1 and PO-5, but in all cases sonication was shown to reduce this effect enough for use of the peptides during further evaluation. In vitro evaluation

Incubation of PO-expressing RT4 cells (rat origin) with the PO-1, PO-5, PO-7 and P08 resulted in similar membranous staining patterns, but with different intensities (Figure 3). Almost no staining was seen after incubation with PO-3, PO-4 or PO-5 (Figure 3). Co-incubation of the cells with a nuclear- (Hoechst; in blue) and lysosome-staining agent (lysotracker; in green) gave no overlap. The latter helped to confirm no internalization occurred. Z- stacks revealed that only PO at the solution exposed part of the cell surface was stained.

Unfortunately, the clustering of PO-1 resulted in a lower amount of peptide available for binding and thus, a lower level of binding. Ultimately this lead to the necessity to use higher concentrations of this peptide, in order to achieve a comparable staining intensity compared to P0-8. This shortcoming led to exclusion of PO-1 from further experiments. As the amino acid sequence and staining pattern of P0-7 and PO-8 were identical, further studies were only performed using PO-6 and PO-8.

To ensure that the peptides would also bind human P0, identical

experiments were conducted using the MSC80 cell line, a Schwannoma cell hne of human origin (data not shown). Although the lower expression level of P0 in this cell hne resulted in lower fluorescence intensity levels, identical staining patterns were achieved for PO-6 and PO-8. P0 specificity

Using a FLISA set-up that was especially tailored for evaluation of the P0- binding peptides, the specificity for their intended target P0 was evaluated. Incubation of PO-ex (the extracellular component of P0) pre-coated wells with increasing amounts of the peptides. resulted in an increasing

fluorescence signal Figure 5A and B). In uncoated wells where the same concentration range was added the fluorescence signal did not transcend the background signal (2 AU; Figure 5 A and B, in blue). Identical experiments using PO-6 also showed no signal in the uncoated wells and an increasing in signal in the wells pre-coated with PO-ex, although this increase was only seen at the highest concentrations (2000 and 4000 iiM; data not shown). Control FLIZA experiments using an anti-P0 antibody (115 AU Cy5-P0-Ab; Figure 5B) or non-P0 binding peptide (5 AU at 2000nM of Cy5-NP41; Figure 5B) further underlined the specificity of the peptides (Figure 5B). P0 binding affinity

Quantitative assessment of the affinity of the peptides was performed using flow cytometry-basecl saturation binding experiments (Figure 5C and D). A clear saturation-binding curve could be obtained for PO-8, from which the affinity of the peptide for P0 could be determined (KD = 591 +/- 110 nM). Saturation was not achieved however when the experiment was repeated using PO-6, meaning that, in this case, the KD for this peptide will be higher than luM, which is not very favorable. Combined with the above findings, PO-8 was therefore deemed most suitable for further evaluation. P0 staining in DRG explants

DRG explants obtained from THY- 1 YFP mouse embryos were used to provide an intermediate step between in vitro and in vivo evaluation.

Axonal outgrowths (white arrow) of these gangha (*) could be visualized clearly using standard white-light microscopy at different magnifications (lOx and 40x, Figure 6B-C).

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).

Incubation of the DRG explants with IMI-PO-8 resulted in a more spotted- pattern (red arrow; Figure 6G-I). The stained cells were unevenly

distributed but clearly located along the axons (Figure 6H and I). A similar staining pattern was seen when the DRG explants were incubated with Cy5- PO-ex (Figure 7). Note: The DRG explant used for this staining only showed minor axonal outgrowths in one direction, as such providing an atypical growth pattern.

The staining pattern of PO-8 seen in the DRG explant (Figure 7B), was similar to the staining pattern that was seen in the Schwann cells cultures (see above Figure 3). Especially in the outgrowths of the cells the membrane seemed to be only stained in specific places, hereby covering most, but not all of the cell membrane. Identical staining patterns and locations were seen when RT4 cells were stained with fluorescently labeled versions of the anti- P0 antibody (Figure 7Cii) and PO-ex (Figure 7Ciii). No staining was observed in non PO-expressing MDAMB231 mamma carcinoma cells (Figure 7Civ), underlining the specificity of PO-8 for P0.

In vivo staining of myelin

For in vivo staining of nerves several different administration routes and incubation times were compared for the Sciatic nerve using a fluorescence confocal microscope set-up wherein the whole mouse was placed under the microscopes lens (Figure 8A).

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) however, showed that specific staining occurred, while no fluorescence could be seen in other parts of the animal. Injection of PO-8 into the nerve sheath of the Sciatic nerve provided direct access to the nerve and its surrounding Schwann cells. 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). When zooming in on the nerve PO-8-based staining (in red) was clearly only shown to be located between the axons, while the axons itself (in green) were not stained (Figure 8D); a staining pattern that is in agreement with the location of the Schwann cells within the nerve. Comparable staining patterns were seen in excised nerve tissue samples that were stained in vivo after intraneural injection as well as nerve tissue samples that were ex vivo incubated with PO-8 (Figure 8E).

Qualitative assessment of the binding of the two remaining peptides was performed using fluorescence confocal imaging also showed differences in the binding affinity of PO-8 and PO-6 as after incubation of PO-expressing cells with increasing concentrations of PO-6 or PO-8 differences in the level of staining and signal intensity were seen. Where both peptides showed staining of P0 (comparable to Figure 3), staining with PO-8 was more intense, even at lower concentrations (See Figure 4). Clear staining for PO-8 was already seen at a concentration of 0.5uM, which increased slightly when incubated with 1.0 uM. Here a plateau in the signal intensity seemed to be reached, as no increase was further seen when incubated with higher concentrations (2.0 uM and 3.0 uM). PO-6 however only showed staining of the membrane of the cells at 2.0 uM and 3.0 uM, and at a lower intensity as seen with PO-7 and PO-8. References

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