Collagen related peptide

Abstract

The invention relates to a compound of formula (I), which is a synthetic collagen related peptide, in the form of a double cross-linked trimer. The invention extends to a composition comprising a compound of formula (II) and formula (III), where formula (II) is a synthetic collagen related peptide, in the form of a double cross-linked trimer, and formula (III) is a synthetic collagen related peptide, in the form of a double cross-linked dimer. The invention also extends to a method of producing the compound of formula (II). The invention also extends to medical uses of the compound and the composition, including treating a skin condition, and methods of cosmetic treatment using the compound and the composition.

Description

The relates to a synthetic collagen related peptide, in the form of a double cross-linked trimer. The collagen related peptide may be used, for example, in promoting the release of bioactive materials from platelets. The use of PRP (Platelet-Rich Plasma) is well known in various clinical solutions, including the treatment of skin problems. The release of bioactive materials, such as growth factors, cytokines and chemokines, from platelets in PRP can be promoted by platelet agonists. Platelet agonists bind to and activate platelet glycoprotein VI (GPVI) (see Watson, S. P., Auger, J. M., McCarty, O. J., & Pearce, A. C. (2005) GPVI and integrin alphallb beta3 signaling in platelets. J Thromb Haemost, 3, 1752-62), initiating a well-defined set of signaling events that lead to internal Ca2+ rise, shape change, extrusion of granules' contents and platelet aggregation (see Sharda, A., & Flaumenhaft, R. (2018) The life cycle of platelet granules. FlOOORes, 7, 236). Collagen is a well-known strong platelet agonist and is widely used in platelet research. For example, collagen I, one of the principal vessel wall species responsible for platelet activation after injury, can be extracted from animal tendons. However, there are limitations associated with the use of animal collagen. In particular, animal collagen is poorly defined, and it is difficult to achieve a reproducible batch-to-batch composition. CRP-XL is a synthetic collagen-related peptide (CRP), a Gly-Pro-Hyp (GPO) polymer, with the sequence {GC(scaffold)0[GPO]ioGC(scaffold)OG}3, that adopts a triple-helical conformation that is essential for activating GPVI (see Smethurst, P. A., Onley, D. J., Jarvis, G. E., O'Connor, M. N., Knight, C. G., Herr, A. B.,... &. Farndale, R. W. (2007). Structural basis for the platelet-collagen interaction: the smallest motif within collagen that recognizes and activates platelet Glycoprotein VI contains two glycine-proline-hydroxyproline triplets. J Biol Chern, 282(2), 1296-1304). This peptide will bind GPVI but will not elicit activation of platelets unless cross-linked. The cross-linking is achieved via the cysteine residues and the N-terminus by usage of the heterobifunctional cross-linking agent, / V-succinimidyl 3-(2-pyridyldithio)propionate. However, the cross-linking process is difficult to control, leading to variable potency and stability in solution or suspension. Hence each batch needs to be prepared, crosslinked and calibrated individually, meaning that it is not suitable as a stable therapeutic or diagnostic reagent. A reliable and scalable manufacturing route to a synthetic collagen-like peptide that ensures a consistent cross-linking process and high batch-to-batch consistency is therefore desired. The usage of 1,3,5-tribromomesitylene (T3) as a cross-linking linking molecule in a synthetic triple helical collagen peptide has been described by Sang et al. (Sang, Y., Huskens, D., Wichapong, K., de Laat, B., Nicolaes, G. A. F., & Roest, M. (2019). A Synthetic Triple Helical Collagen Peptide as a New Agonist for Flow Cytometric Measurement of GPVI-Specific Platelet Activation. Thromb Haemost, 119(12), 20052013). The product showed equal or even better biological results and better stability than CRP-XL. However, scaled manufacture of a synthetic collagen related peptide comprising T3 as a linking molecule is non-trivial. The method described by Sang et al. uses repeated HPLC purification steps throughout the synthesis, making it unsuitable for scale-up. The present invention arose from the inventors' work in attempting to address the problems associated with the prior art. In accordance with a first aspect of the invention, there is provided a compound of Formula I: R3 is OH or NH2; a point of bonding to a carbon atom which is also bonded to the R2 group; L2 is a linker; and nl and n2 are each independently 0 or an integer between 1 and 5; or a cosmetically or pharmaceutically acceptable salt or solvate thereof. L2 may be , wherein X1 is a phenyl, a 6 membered heterocycle optionally substituted with one or more oxo groups, a 6 membered heteroaryl, cyclohexyl or N; and L8 is absent or is CO; L9 is absent or is a C1-3 alkyl; L10 is absent or is C=O, C(OH), *-NHCO-, *-NHSO-, *-NHSO2- or *-OC(O)-, where an asterisk indicates a point of attachment to L9, or in embodiments where L9 is absent to L8 or X1; and L11 is absent or is a C1-3 alkyl. Preferably, at least one of L8 to L11 is present. In embodiments where it is present, L9 may be -CH2- or -CH2CH2-. Preferably, in embodiments where L8 is CO, L9 is -CH2- or -CH2CH2-. L9 may be -CH2-. Preferably, in embodiments where L8 is absent, L9 may be absent, -CH2- or -CH2CH2-. In embodiments where it is present, L11 may be -CH2- or -CH2CH2-. Preferably, in embodiments where L10 is C(OH) then L11 is -CH2-. Preferably, in embodiments where L10 is C=0, *-NHCO-, *-NHSO-, *-NHS02- or *-0C(0)- then L11 is -CH2- or -CH2CH2-. In some embodiments, L2 is nl and n2 may each independently be an integer between 1 and 3. Preferably, nl and n2 are each 1 or 2. In some embodiments, nl and n2 are each 1. Accordingly, the compound of formula I may be a compound of formula la: 10 The compound of formula I may be a compound of formula lb: (li) The compound of formula la may be a compound of formula lai: (lai) It may be appreciated that the compound of formula I may alternatively be represented as: [GC(cross-linker)0[GPO]ioGC(cross-linker)OG]3 15 SEQ ID No. 1 In accordance with a second aspect, there is provided a composition comprising a compound of formula II and a compound of formula III: (II) h2n wherein R4 is R5 is           OH or -COR3; R3 is OH or NH2; and OH L1 is OH                                           , where an asterisk indicates 10 a point of bonding to a carbon atom which is also bonded to the R4 group; L2 is a linker; L3 is a linker; and nl and n2 are each independently 0 or an integer between 1 and 5; or a cosmetically or pharmaceutically acceptable salt or solvate thereof; 15 wherein the molar ratio of the compound of formula II to the compound of formula Illa is at least 2.3:1. R4 may be L2 may be nl and n2 may be as defined in relation to the first aspect. Accordingly, the compound of formula II may be a compound of formula I, as defined in relation to the first aspect. Preferably, the compound of formula II is a compound of formula la, as defined in relation to the first aspect. I L9 V I 13 I 9 y1 I 9 J 11 L3 may be L l l l s wherein X1 and L8 to L11 are as defined in relation to the first aspect; L12 is absent or is C=O, *-NHCO-, *-NHSO-, *-NHSO2- or *-OC(O)-; L13 is absent or is a C1-3 alkyl; and R21 is a leaving group, L13 may be -CH2- or -CH2CH2-. In some embodiments, L12 is C=O, *-NHCO-, *-NHSO-, *-NHS02- or *-OC(O)-; L13 is -CH2- and R21 is a leaving group. L10 may be C=O, *-NHCO-, *-NHSO-, *-NHS02- or *-OC(O)-. L10 may be the same as L12. L11 may be -CH2-. In some embodiments, L12 is C=O, *-NHCO-, *-NHSO-, *-NHS02- or *-OC(O)-; L13 is absent and R21 is . L10 may be C=O, *-NHCO-, *-NHSO-, *-NHSO2- or *-OC(O)-. L10 may be the same as L12. L11 may be -CH2CH2-. In some embodiments, L10 is C(OH) and L11 is -CH2-. Preferably, L12 is absent. .0 Preferably, L13 is absent. Preferably, R21 is . A LG JUL % L3 may be                  r where LG is a leaving group. LG may a halide. Preferably, LG is chlorine or bromine. LG may be bromine. Preferably, the molar ratio of the compound of formula II to the compound of formula Illa is at least 2.5:1, more preferably at least 2.8:1. The composition may also comprise a monomer. The monomer may have formula (Illb): Preferably, the molar ratio of the compound of formula II to the compound of formula Illb is at least 13:1, more preferably at least 15:1, at least 20:1, at least 25:1 or at least 30:1, and most preferably at least 35:1, at least 36:1 or at least 37:1. In some embodiments, the molar ratio of the compound of formula II to the compound of formula Illb is at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1, at least 50:1, at least 52:1, at least 54:1, or at least 55:1. WO 2025 / 133607                                   PCT / GB2024 / 053160 - 10 - Advantageously, the composition of the second aspect is stable. The composition may be a freeze dried solid. In some embodiments, after a period of storage, the molar ratio of the compound of formula II to the compound of formula Illa is at least 2.5:1, or at least 2.8:1. In some embodiments, after a period of storage, the molar ratio of the compound of formula II to the compound of formula Illb is at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1, at least 50:1, at least 51:1, or at least 52:1. The composition may be stored at a storage temperature of between -50 and -520 °C, between -40 and 10 °C, between -35 and 0 °C, between -30 and 0 °C, between -25 and -10 °C or between -22 and -15 °C. The storage temperature may be about -20 °C. The period of storage may be at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 20 months, at least 22 months or at least 24 months. In accordance with a third aspect, there is provided a method of producing a compound of formula II: (II) , wherein L1, L2, R4, R5, nl and n2 are as defined in the second aspect; the method comprising: - providing a support with a plurality of peptide monomers disposed thereon, wherein the support and plurality of monomers are represent by formula (IV): (IV) wherein, S1 is a first support; 5 COO*- or -CON(H)*-; where an asterisk indicates a point of bonding to S1; where an asterisk 10 indicates a point of bonding to a carbon atom which is also bonded to the L4 group; PG1, PG2 and PG3 are each a protecting group; and n is an integer of at least 3; - contacting the support and plurality of monomers of formula (IV) and a first cross-linking reagent to provide a mono cross-linked trimer disposed on the first support, which is represented by formula (V): PG2 i PG2 (V) wherein L2 is as defined in the second aspect; and - removing protecting group PG1 from the mono cross-linked trimer to thereby 5 deprotect N-termini of the mono cross-linked trimer, contacting the mono cross-linked trimer and a support attachment reagent to thereby dispose a support attachment group onto each of the N-termini, cleaving the mono cross-linked trimer from the first support, removing protecting groups PG2 from the mono cross-linked trimer and contacting the mono cross-linked trimer and a second support to immobilize it 10 thereon, wherein the mono cross-linked trimer immobilized on the second support is represented by formula (VI): (VI) wherein L6 is a linker; and 15 S2 is a second support; - contacting the immobilized cross-linked trimer of formula (VI) and a second cross-linking reagent to provide a double cross-linked trimer; and cleaving the double-cross-linked trimer from the second support to provide the compound of Formula (II); OPG3 OPG3 and / or L4 wherein if L5 is method further comprises removing protecting group PG3. The first support is preferably a solid support. The first support may be a solid support suitable for peptide synthesis. Preferably, the first support is a resin. Preferably, the resin is suitable for solid phase peptide synthesis (SPPS). More preferably, the first support is a resin suitable for Fmoc SPPS. The first support may comprise polystyrene. The first support may comprise amide groups. The first support may be or comprise a rink amide resin. Still more preferably, the first support is an Fmoc-Rink Amide aminomethyl-polystyrene resin. Providing the support with the plurality of peptide monomers disposed thereon may comprise synthesizing the peptide monomers, by solid phase peptide synthesis (SPPS) or solution phase synthesis (SPS). Preferably, the peptide monomers are synthesized via solid phase peptide synthesis. Most preferably, the peptide monomers are synthesized via Fmoc SPPS. Preferably, the method comprises synthesizing the plurality of peptide monomers by SPPS on the first solid support. Synthesizing the plurality of peptide monomers by SPPS on the first support may comprise synthesizing a plurality of peptide monomers on the first support represented by formula (VII): (VII) , wherein PG4 is a protecting group. The method may comprise removing the protecting group PG4 to provide the solid support with the plurality of peptide monomers of formula (IV) disposed thereon. PG4 is preferably an orthogonal protecting group to PG2. PG4 is preferably an orthogonal protecting group to PG1. PG4 may be an orthogonal protecting group to PG3. If protecting groups are orthogonal it may be understood to mean that the protecting groups may be removed independently, i.e. one of the protecting groups may be removed while leaving the other in place. Removal of a protecting group may be understood to mean that that protecting group is removed in a deprotection step. Preferably, the protecting group is replaced by a hydrogen atom. PG4 may be any suitable cysteine protecting group. Preferably, PG4 is a trityl protecting group (Trt). Removing the protecting group PG4 may comprise contacting the plurality of peptide monomers on the first solid support of formula (VII) with a PG4 deprotection solution. The PG4 deprotection solution may comprise a solvent. Preferably, the solvent is an organic solvent. More preferably, the solvent is DCM (dichloromethane). Preferably, the PG4 deprotection solution further comprises an acid. Preferably, the acid is a strong acid. More preferably, the acid is TFA (trifluoracetic acid). The PG4 deprotection solution may comprise at least 1 vol%, at least 2 vol%, at least 4 vol% or at least 5 vol% acid. The PG4 deprotection solution may comprise between 1 and 9 vol%, between 2 and 8 vol%, between 3 and 7 vol% or between 4 and 6 vol% acid. Preferably, the PG4 deprotection solution further comprises a cation scavenger. Preferably, the cation scavenger is TIS (triisopropylsilane). The PG4 deprotection solution may comprise at least 1 vol%, at least 2 vol%, at least 4 vol% or at least 5 vol% cation scavenger. The PG4 deprotection solution may comprise between 1 and 9 vol%, between 2 and 8 vol%, between 3 and 7 vol% or between 4 and 6 vol% cation scavenger. Preferably, the PG4 deprotection solution comprises about 5 vol% cation scavenger. Removing the protecting group PG4 may comprise contacting the plurality of peptide monomers on the first solid support of formula (VII) and the PG4 deprotection solution for at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes or at least 5 minutes. Removing the protecting group PG4 may comprise repeating contacting the plurality of peptide monomers on the first solid support of formula (VII) and the PG4 deprotection solution. The plurality of peptide monomers on the first solid support of formula (VII) may be contacted at least once, at least twice, at least three times, at least four times at least five time or at least six times with the PG4 deprotection solution. Preferably, removing the protecting group PG4 comprises contacting the plurality of peptide monomers on the first solid support of formula (VII) and the PG4 deprotection solution for at least 5 minutes and at least 6 times. Removing the protecting group PG4 may comprise contacting the plurality of peptide monomers on the first solid support of formula (VII) with at least 10 pL, at least 50 pL, at least 75 pL or at least 100 pL of the PG4 deprotection solution per pmol peptide. Preferably, removing the protecting group PG4 comprises contacting the plurality of peptide monomers on the first solid support of formula (VII) with at least 100 pL of the PG4 deprotection solution per pmol peptide. The concentration of the PG2 PG4R6%)n2 peptide may be understood to be the concentration of the group 'ni PG2 PG4 R»SQ2 which is present. It may be understood that the group                   is bonded to S1. The concentration may be an assumed concentration. PG3 may be any suitable hydroxyl protecting group. PG3 and PG1 may be orthogonal. PG3 and PG2 may be orthogonal. PG3 may be t-butyl. OPG3 OPG3 and the method comprises removing protecting group PG3. 10 Protecting group PG3 may be removed before, after or at the same time as removing protecting group PG4. Protecting group PG3 may be removed prior to contacting the support and plurality of monomers of formula (IV) with a cross-linking reagent. Accordingly, providing a support with a plurality of peptide monomers disposed 15 thereon may comprise: providing a support with a plurality of peptide monomers of formula (IVa): (IVa) OPG3 OPG3 - removing protecting group PG3 to provide a support with a plurality of peptide monomers of formula (IVb): 10 (Ivb) The protecting group PG1 may be any suitable amine protecting group. Preferably, PG1 is an amine protecting group selected from the group including Alloc, Fmoc, Dde and ivDde. Most preferably, PG1 is Fmoc. The protecting group PG2 may be any suitable cysteine protecting group. Preferably, PG2 is Dpm, Acm, StBu or Gly. Most preferably, PG2 is Dpm. Preferably, the protecting groups PG1 and PG2 are orthogonal. Preferably, R6 is It may be appreciated that n may be 3 or any integer greater than 3. The first cross-linking reagent may be a compound of formula (VIII): R21 L13 X12 L9 I 13      । 9 v1 I 9 J 13 'r21 (VIII) , wherein X1, L8, L9, L12, L13 and R21 are as defined in relation to the first and second aspects. The first cross-linking reagent may be a compound of formula (Villa) to (VUIq): (Ville) (VHIf) (VUIg) (VUIk) (VIIII) , wherein LG is a leaving group. Preferably, the leaving group is a halide atom. The leaving group may be Br or Cl. In some embodiments, the leaving group is Br. In some embodiments, the first cross linking reagent is Contacting the support and plurality of monomers of formula (IV) and the first crosslinking reagent may comprise contacting the support and plurality of monomers of formula (IV) with a cross-linking solution comprising the cross-linking reagent. The method may comprise contacting the support and plurality of monomers of formula (IV) with the first cross-linking reagent in a relative molar ratio of crosslinking reagent to peptide of between 0.2:3 and 5:3, between 0.4:3 and 2:3, between 0.6:3 and 1.6:3, between 0.7:3 and 1.5:3, between 0.8:3 and 1.4:3, between 0.9:3 and 1.3:3 or between 1.0:3 and 1.2:3. Preferably, the relative molar ratio of cross linking reagent to peptide is about 1.1:3. The peptide may understand to be the group PG2                                            PG2 r6%)                               r6%) K '1 'n2                                                                      Y '1'n2 ' 'n1             . It may be understood that the group 'is bonded to S1. The concentration of the peptide may be an assumed concentration. Contacting the support and plurality of monomers of formula (IV) with the first crosslinking reagent may comprise contacting the support and plurality of monomers of formula (IV) with a first cross-linking solution comprising the first cross-linking reagent. The first cross-linking solution may further comprise a solvent. Preferably, the solvent comprises an organic solvent and / or water. The organic solvent may be or comprise a polar organic solvent. The polar organic solvent may be or comprise MeCN. In some embodiments, the solvent is a combination of the organic solvent and water. The first cross-linking solution may comprise the organic solvent and water in a volumetric ratio of between 20:80 and 80:20, between 30:70 and 70:30, between 40:60 and 60:40, between 45:55 and 55:45. The first cross-linking solution may further comprise an alkaline agent. Preferably, the alkaline agent is a weak alkali. Preferably, the alkaline agent is NaHCOs. The first cross-linking solution may have a pH at 20°C of between 7.0 and 9.5, between 7.5 and 9.0, between 8.0 and 8.5, between 8.1 and 8.4 or between 8.2 and 8.4. Preferably, the first cross-linking solution has a pH of about 8.3 at 20°C. The support and plurality of monomers of formula (IV) and the first cross-linking reagent may be contacted for at least 1 hour, at least 5 hours, at least 10 hours, at least 30 hours, at least 60 hours or at least 90 hours. Preferably, support and plurality of monomers of formula (IV) and the first cross-linking reagent are contacted for at least 60 hours. Preferably, the method comprises stirring the support and plurality of monomers of formula (IV) and the first cross-linking reagent while they are contacted. Prior to contacting the support and plurality of monomers of formula (IV) and the first cross-linking reagent the method may comprise swelling the first support. The first support may be swollen using any known technique. For instance, the first support may be swollen by contacting the support and plurality of monomers of formula (IV) with an organic solvent. The organic solvent may be a polar organic solvent. The polar organic solvent may be DMSO. The first support and the organic solvent may be contacted for at least 1 minute, at least 5 minutes, at least 10 minutes or at least 15 minutes. The first support and the organic solvent may be contacted for between 1 minute and 12 hours, between 5 minutes and 1 hour, between 10 and 30 minutes or between 14 and 20 minutes. Preferably, prior to contacting the support and plurality of monomers of formula (IV) and the first cross-linking reagent the method comprises contacting the support and plurality of monomers of formula (IV) and a pre-cross-linking solution. The method may comprise contacting the support and plurality of monomers of formula (IV) and the pre-cross-linking solution subsequent to swelling the first support. The pre-cross-linking solution may comprise a base. Preferably, the base is a non-nucleophilic base. Still more preferably, the base is DIEA (N, N-diisopropylethylamine). The pre-cross-linking solution may further comprise a solvent. Preferably, the solvent is an organic solvent. More preferably, the solvent is a polar organic solvent. Most preferably, the solvent is DMSO. The pre-cross-linking solution may comprise the solvent and the base in a volume ratio of between 80:20 and 99.9:0.1, between 90:10 and 99.5:0.5, between 95:5 and 99:1 or between 96:4 and 98:2. Preferably, the pre-cross-linking solution comprises the solvent and the base in a volume ratio of about 97:3. The pre-cross-linking solution may further comprise a denaturation agent. The denaturation agent may be selected or configured to prevent unwanted aggregation. Preferably, the denaturation agent is urea. The denaturation agent may be provided at a concentration of at least 0.1 M, at least 0.5 M, at least 1 M, at least 2 M, at least 3 M, at least 4 M or at least 5M. The denaturation agent may be provided at a concentration between 0.1 and 50 M, between 0.5 and 25 M, between 1 and 20M, between 2 and 15 M, between 3 and 10 M, between 4 and 7M or between 5 and 6M. The method may comprise washing the mono cross-linked trimer disposed on the first support. Washing the mono cross-linked trimer disposed on the first support may comprise contacting the mono cross-linked trimer disposed on the first supportand water at least once, at least twice, at least three times, at least four times, at least five times or at least six times. Washing the mono cross-linked trimer disposed on the first support may further comprise contacting the mono cross-linked trimer disposed on the first support and an organic solvent at least once, at least twice, at least three times, at least four time, at least five times or at least six times. Preferably, the organic solvent is DMF and / or DCM. Preferably, washing the mono cross-linked trimer disposed on the first support comprises contacting the mono cross-linked trimer disposed on the first support with DMF at least three times and with DCM at least three times. It may be appreciated that the steps of removing protecting group PG1 from the mono cross-linked trimer to thereby deprotect N-termini of the mono cross-linked trimer, contacting the mono cross-linked trimer with a support attachment reagent to thereby dispose a support attachment group onto each of the N-termini, cleaving the mono cross-linked trimer from the first support, removing protecting groups PG2 from the mono cross-linked trimer and contacting the mono cross-linked trimer with a second support to immobilize it thereon may be undertaken concurrently or separately. If two or more of the steps are undertaken separately, it may be appreciated that these steps may be undertaken in different orders. In some embodiments, the method comprises: - removing protecting group PG1 from the mono cross-linked trimer to thereby provide a mono-cross-linked trimer of formula (IX): (IX) , wherein S1, L2, L4, L5 and PG2 are as defined above; and H H2N^fNy R7 is 0 or NH2; and subsequently - contacting the mono cross-linked trimer of formula (IX) and a support attachment reagent to provide a mono-cross-linked trimer of formula (X): PG2 1 PG2 (X) , wherein S1, L2, L4, L5 and PG2 are as defined above; and R8 is or NHR9; and R9 is a support attachment group. R9 may be -U-O-NR^R11, wherein L7 is a linker and R10 and R11 are each H or PG5, wherein PG5 is a protecting group. Removing protecting groups PG1 may comprise contacting the mono cross-linked trimer of formula (V) and a PG1 deprotection solution. The PG1 deprotection solution may comprise a base. Preferably, the base is a strong organic base. More preferably, the base is piperidine. The PG1 deprotection solution may comprise at least 5 vol%, at least 10 vol%, at least 15 vol%, at least 16 vol%, at least 18 vol%, or at least 20 vol% base. The PG1 deprotection solution may comprise between 5 and 25 vol%, between 10 and 20 vol%, between 15 and 25 vol% or between 19 and 21 vol% base. Preferably, the PG1 deprotection solution comprises about 20 vol% base. The PG1 deprotection solution may further comprise a solvent. Preferably, the solvent is or comprises an organic solvent. Preferably, the solvent is or comprises DMF (dimethylformamide). Removing protecting groups PG1 may comprise contacting the mono cross-linked trimer of formula (V) and the PG1 deprotection solution at least once or at least twice. Removing protecting groups PG1 may comprise contacting the mono cross-linked trimer of formula (V) and the PG1 deprotection solution for at least 1 minute, at least 5 minutes or at least 8 minutes. Preferably, the method comprises contacting the mono cross-linked trimer of formula (V) and the PG1 deprotection solution for between 1 minute and 1 hour, between 2 minutes and 30 minutes or between 5 minutes and 10 minutes. The support attachment reagent may be a compound as described in WO 2020 / 043747 Al, the contents of which is incorporated herein by reference. Accordingly, the support attachment reagent may be a compound of formula (XI): X-Tb-Va-U-Y-Z (XI) , wherein „                 R11 PG5 I PG5 A, A N. A O n A JI r10 n N ji X is R10" '° , 'Ox\ R12 ,   R12 orR137; each R10 and R11 is independently from each other selected from H or PG5, wherein at least R10 or R11 is PG5; R12 is selected from H or PG5; R13 is selected from H, C1-12 alkyl or C6-12 aryl, wherein the aldehyde or keto group may be protected by an acid labile protecting group; PG5 is an acid labile amine protecting group; T is a linear or branched spacer comprising at least one of the moieties -C1-12 alkylene, (-C2H4O-)1-12, -C(=O)-, -C(=0)-JR14-, -JR14-C(=0)-, -JR14-, phenylene, 5- or 6membered heteroarylene, wherein J is CH or N, R14 is selected from H, Ci-4 alkyl, -C1-6 alkyl-NH2, -C1-6 alkyl-NHPG6, -C1-6 alkylene-NPG62, -R17, -C1-6 alkyl-NH-R17, -C1-6 alkyl-NH-R17'; PG6 is an acid labile amine protecting group; R17 is a blocking agent that is able to react with an aldehyde moiety; b is 0 or 1, V is an electron-withdrawing moiety selected from -NR18-C(=O)-, -C(=O)-NR18-, -S(=O)-, -NR19-(CH2)P-, -piperazinylene-(CH2)P-, pyridinylene, pyrimidinylene, q / ^N-(CH2)P4-(U) pyrazinylene, pyridazinylene,                         , -C(=O)-, -C(=O)-O-, , wherein R18 is selected from H and Ci^alky, R19 is selected from H and Ci-4, p is 0, 1 or 2, a is 0 or 1, wherein the sum of a and b is 1 or 2, U is a phenylene or a five- or six-membered heteroarylene moiety, that is bound to at least one of the moieties V, Wq and En and that may optionally be substituted by a Ci-6 alkyl, and preferably by a Ci-3 alkyl, wherein W is selected from -N3, -NO2, -N=N-phenyl, -S(=O)-R20, -S-S-R20, -O-CH2-N3,-N=N- R20 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, C1-6alkyl or -(CH2)P-NMe2, with p being 1, 2, 3 or 4; E is an electron withdrawing group under acidic conditions; n being is an integer between 0 and 4, and q is an integer between 0 and 4, wherein the sum of n and q is equal or lower than 4; and in case of U being a phenyl moiety and Y being -(CH2)m-O-C(=O)-, the sum of Hammett constants of V, W, E under acidic conditions is larger than 0.45, and wherein W is in ortho or para position in relation to Y; Y is -(CH2)m-C(=O)- or -(CH2)m-O-C(=O)- with m being 1 , 2 or 3, and Z is an electron-withdrawing leaving group. „                 R11 PG5 r11                I   u Hu i h PG5        A I^A N A m R10 N D , U| v ■ R10" VG         A         p12        r12 Preferably, Xis            , O , R or R More preferably, X is R11 i ,N Most preferably, X is R10 Preferably, R10 and R11 are both PG5. PG5 may be Boc. T preferably comprises 1 to 5 of the moieties -C1-12 alkylene-, (-C2H4O-)i-i2, -C(=O)-, -C(=0)-JR14-, -JR14-C(=0)-, -JR14-, phenylene and / or 5- or 6-membered heteroarylene. Preferably, J is N. Preferably, T is a spacer selected from: -C1-12 alkylene-, preferably C1-6alkylene, more preferably C1-3 alkyl; -R15-C(=O)-; -R15-C(=O)-NR14-R16-; -R15-C(=O)-NR14-; -C(=O)-NR14-R16-; -R15-NR14-C(=O)-R16-; -R15-NR14-R15-NR14-C(=O)-R16-; -R15-C(=O)-NR14-R15-NR14,-C(=O)-R16-; -R15-NR14-; -R15-NR14-R16-; -R15-NR14-R15-NR14-R16-; -R15-C(=O)-NR14-R15'-NR14'-R16-; -R15-C(=O)-O-R16-; -C(=O)-O-R16-; -R15-phenylene-R16-; -R15-phenylene-; -phenylene-R16-; -phenylene-; -R15-pyrroylene; -R15-pyrazoylene; -R15-imidazoylene; -R15-piperazinylene-; -R15-pyridinylene; -R15-pyrimidinylene; -R15-pyrazinylene; -R15-pyridazinylene; -R15-pyrroylene-R16-; -R15-pyrazoylene-R16-; -R15-imidazoylene-R16-; -R15-piperazinylene-R16-; -R15-pyridinylene-R16-; -R15-pyrimidinylene-R16- ; -R15-pyrazinyl-R16-; -R15-pyridazinylene-R16-; -pyrroylene-R16-; -pyrazoylene-R16-; -imidazoylene-R16; -piparazinylene-R16-; -pyridinylene-R16-; -pyrimidinylene-R16-; -pyrazinylene-R16-; -pyridazinyl-R16-; pyrroylene; pyrazoylene; imidazoylene; piperazinyenel; pyridinylene; pyrimidinylene; pyrazinylene; or pyridazinylene, wherein R15, R15' and R16 are independently selected from -C1-12 alkylene- or (-C2H4O-)i-i2, and R4' is selected from H, Ci-4 alkyl, -C1-6 alkyl-NH2 , -C1-6 alkyl-NHPG6, -C1-6 alkylene-NPG62, -R17, -C1-6 alkyl-NH-R17, -C1-6 alkyl-NH-R17'. Preferably, R15, R15' and R16 are independently C1-6 alkylene, more preferably C1-3 alkylene. Preferably, R4 and R4' are independently selected from H and C1-2 alkyl. More preferably, R4 is H. In a preferred embodiment, T is -R15-C(=O)-NR14-R16-. More preferably, T is -CH2-C( = O)-NHCH2CH2-. WO 2025 / 133607                                   PCT / GB2024 / 053160 - 28 - R17 is preferably selected from cysteinyl, threoninyl, 2-mercaptoethanol, cysteamine, ethandithiole, hydroxylamine, O-methylhydroxylamine, N- methylhydroxylamine, dithiothreitol, hydrazine, in particular cysteinyl and N-methylhydroxylamine, more preferably cysteinyl, wherein amine and / or thiol moieties of the blocking agent may be protected by an independently selected acid labile amine protecting group (preferably Boc), and / or an acid labile thiol protecting group (preferably trityl). Preferably, b is 1. Preferably, V is -NR18-C(=O)-, -C(=O)-NR18-, -S(=O)-, -NR19-(CH2)P-, -piperazinylene-(CH2)P-, -pyridinylene- or pyrimidinylene. R18 is preferably from H and Ci-2 alkyl, and more preferably R18 is H. R19 is preferably from H and C1-2 alkyl, and more preferably R19 is methyl. More preferably, V is -NH-C(=O)-, -C(=O)-NH-, -N-(CH3)-, -piperazinylene-(CH2)P-, pyridinylene or pyrimidinylene. Most preferably, V is -NH-C(=O)-. p is preferably 0 or 1. U is preferably a phenylene or a six-membered heteroarylene moiety, and more preferably a phenylene. Preferably, W is selected from N3, -S-S-R20, -O-CH2-N3, and / or -N = N-R8. Most preferably, W is N3. Preferably, E is a halogen. The halogen may be bromine. R20 is preferably pyridyl or C1-6 alkyl. Preferably, n is an integer between 0 and 2, more particularly 0 or 1. Preferably, n is 1. Preferably, q is an integer between 0 and 2, more particularly 0 and 1. Preferably, q is 1. Preferably, m is 1 or 2, more particularly 1. Accordingly, Y is preferably -CH2OC(O)-. Accordingly, the compound of formula (XI) may be (Boc)2N Accordingly, R9 may be -Y-U-Va-Tb-X, where Y, U, V, T, X, a and b are as defined above. X may be -O-NR^R11. Accordingly, L7 may be -Y-U-Va-Tb-. Accordingly, R9 Contacting the mono cross-linked trimer and the support attachment reagent may comprise contacting the mono cross-linked trimer and the support attachment reagent such that there are at least 1 mole of the support attachment reagent for every 1 mole of the mono cross-linked trimer, more preferably at least 2 moles or at least 3 moles of the support attachment reagent for every 1 mole of the mono cross-linked trimer, and most preferably at least 3.5 or at least 3.8 moles of the support attachment reagent for every 1 mole of the mono cross-linked trimer. Contacting the mono cross-linked trimer and the support attachment reagent may comprise contacting the mono cross-linked trimer and the support attachment reagent at a molar ratio of between 1:2 and 1:20, between 1:2.5 and 1:5.5, between 1:3 and 1:5, between 1:3 and 1:4 or between 1:3.8 and 1:4.2 mono cross-linked trimer to support attachment reagent. Contacting the mono cross-linked trimer and the support attachment reagent may comprise contacting the mono cross-linked trimer and a support attachment solution, wherein the support attachment solution comprises the support attachment reagent. The support attachment solution may further comprise a coupling additive. Preferably, the coupling additive is oxyma, HOBt or HOAt. The support attachment solution may comprise at least 1 mole of the coupling additive per mole of mono cross-linked trimer, more preferably at least 2 moles, at least 3 moles or at least 4 moles of the coupling additive per mole of mono cross-linked trimer, and most preferably at least 4.5 moles, at least 5 moles, at least 5.5 moles or at least 5.8 moles of the coupling additive per mole of mono cross-linked trimer. The support attachment solution may comprise between 4 and 20 moles, between 4.5 and 15 moles, between 5 and 10 moles, between 5.5 and 7 moles or between 5.8 and 6.2 moles of the coupling additive per mole of the mono cross-linked trimer. The support attachment solution may further comprise a base. Preferably, the base is a non-nucleophilic base. More preferably, the base is DIEA (N, N-Diisopropylethylamine). The support attachment solution may comprise at least 1 mole of the base per mole of mono cross-linked trimer, more preferably at least 2 moles, at least 3 moles or at least 4 moles of the base per mole of mono cross-linked trimer, and most preferably at least 4.5 moles, at least 5 moles, at least 5.5 moles or at least 5.8 moles of the base per mole of mono cross-linked trimer. The support attachment solution may comprise between 4 and 20 moles, between 4.5 and 15 moles, between 5 and 10 moles, between 5.5 and 7 moles or between 5.8 and 6.2 moles of the base per mole of the mono cross-linked trimer. The support attachment solution may further comprise a solvent. Preferably the solvent is an organic solvent, more preferably a polar organic solvent. Most preferably, the solvent is DMF. Preferably, the method comprises agitating the mono cross-linked trimer and the support attachment reagent while contacting them. The method may comprise contacting the mono cross-linked trimer and the support attachment reagent for at least 30 minutes, at least 1 hour, at least 1.5 hours, or at least 2 hours. The method may comprise conducting a capping step. The method may comprise conducting the capping step subsequent to deprotecting N-termini of the mono crosslinked trimer. The method may comprise conducting the capping step subsequent to disposing a support attachment group onto each of the N-termini. The method may comprise conducting the capping step prior to cleaving the mono cross-linked trimer from the first support. The capping step may comprise contacting the monocrosslinked trimer and a first capping reagent. The capping step may comprise contacting the mono-crosslinked trimer and a second capping agent. In some WO 2025 / 133607                                   PCT / GB2024 / 053160 - 31 -embodiments, the capping step comprises contacting the mono-crosslinked trimer and a first capping reagent and subsequently contacting the mono-crosslinked trimer and a second capping agent. The first capping agent may be a compound of formula XIV: 0 (XIV) , Wherein LG1 is a leaving group. LG1 may be a halide. The halide may be bromine. Contacting the mono-crosslinked trimer and the first capping reagent may comprise contacting the mono-crosslinked trimer with a first capping reagent solution, wherein the first capping reagent solution comprises the first capping reagent. The first capping reagent solution may comprise the first capping reagent at a concentration of at least 0.01 M, at least 0.1 M, at least 0.2 M, at least 0.4 M, at least 0.6 M or at least 0.8 M. The first capping reagent solution may comprise the first capping reagent at a concentration of between 0.01 and 10 M, between 0.1 and 5 M, between 0.2 and 3 M, between 0.4 and 2 M, between 0.6 and 1.5 M or between 0.8 and 1.2 M. The first capping reagent solution may further comprise a solvent. Preferably, the solvent is or comprises an organic solvent. Preferably, the solvent is a polar solvent. Preferably, the solvent is or comprises DMF (dimethylformamide) and / or NMP (N-methyl-2-pyrrolidone). Preferably, the first capping reagent solution comprises a base. Preferably, the base is a non-nucleophilic base. The base may be DIEA (N,N-diisopropylethylamine). The method may comprise contacting the mono-crosslinked trimer and the first capping reagent for at least 30 seconds, at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 12 minutes or at least 14 minutes. The method may comprise contacting the mono-crosslinked trimer and the first capping reagent for between 30 seconds and 6 hours, between 1 minute and an hour, between 5 and 45 minutes, between 10 and 30 minutes, between 12 and 20 minutes or between 14 minutes and 16 minutes. Between contacting the mono-crosslinked trimer and a first capping reagent and contacting the mono-crosslinked trimer and a second capping agent, the method may comprise washing the mono-crosslinked trimer. Washing the mono-crosslinked trimer may be as defined above. The second capping reagent may be an amino acid. The amino acid may be cysteine. The amino acid may be L-cysteine. Contacting the mono-crosslinked trimer and the second capping reagent may comprise contacting the mono-crosslinked trimer with a second capping reagent solution, wherein the second capping reagent solution comprises the second capping reagent. The second capping reagent solution may comprise the second capping reagent at a concentration of at least 0.01 w%, at least 0.1 w%, at least 0.5 w%, at least 1 w%, at least 1.5 w% or at least 2 w%. The second capping reagent solution may comprise the second capping reagent at a concentration of between 0.01 and 25 w%, between 0.1 and 10 w%, between 0.5 and 5 w%, between 1 and 3 w%, between 1.5 and 2.5 w% or between 1.8 and 2.2 w%. The first capping reagent solution may further comprise a solvent. Preferably, the solvent is or comprises water. The second capping reagent solution may comprise a base. The base may be a weak base. The base may be NaHCOs (sodium hydrogen carbonate). The second capping reagent solution may comprise the base at a concentration of at least 0.01 M per pmol assumed peptide concentration, at least 0.1 M per pmol assumed peptide concentration, at least 0.2 M per pmol assumed peptide concentration, at least 0.4 M per pmol assumed peptide concentration or at least 0.5 M per pmol assumed peptide concentration. The second capping reagent solution may comprise the base at a concentration of between 0.01 and 5 M per pmol assumed peptide concentration, between 0.1 and 2 M per pmol assumed peptide concentration, between 0.2 and 1 M per pmol assumed peptide concentration, between 0.4 and 0.8 M per pmol assumed peptide concentration or between 0.5 and 0.7 M per pmol assumed peptide concentration. The assumed peptide concentration may be as defined above. Subsequent to contacting the mono-crosslinked trimer and a second capping agent, the method may comprise washing the mono-crosslinked trimer. Washing the monocrosslinked trimer may be as defined above. The method may comprise cleaving the mono cross-linked trimer from the first support and subsequently removing protecting groups PG2 from the mono crosslinked trimer. However, in a preferred embodiment, the method comprises simultaneously cleaving the mono cross-linked trimer from the first support and removing protecting group PG2 from the mono cross-linked trimer. Protecting group PG2 may be orthogonal to protecting group PG5. However, in a preferred embodiment, protecting group PG2 is not orthogonal to protecting group PG5. Accordingly, removing protecting groups PG2 from the mono cross-linked trimer may also comprise removing protecting group PG5 from the mono cross-linked trimer. Accordingly, the method may comprise simultaneously cleaving the mono cross-linked trimer from the first support and removing protecting groups PG2 and PG5 from the mono cross-linked trimer. The method may comprise contacting the mono cross-linked trimer and a cleavage and / or deprotection solution to thereby cleave the mono cross-linked trimer from the first support and / or remove protecting group PG2 and / or PG5. The method may comprise contacting the mono cross-linked trimer and the cleavage and / or deprotection solution for at least 30 minutes, at least 1 hour, at least 1.5 hours, or at least 2 hours. The cleavage and / or deprotection solution may comprise a solvent. Preferably, the solvent is water. The cleavage and / or deprotection solution may comprise at least 1 vol%, at least 2 vol%, at least 3 vol%, at least 3.5 vol%, or at least 4 vol% solvent. The cleavage and / or deprotection solution may comprise between 1 and 7 vol%, between 2 and 6 vol%, between 3 and 5 vol% or between 3.5 and 4.5 vol% solvent. Preferably, the cleavage and / or deprotection solution comprises about 4 vol% solvent. The cleavage and / or deprotection solution may further comprise an acid. Preferably, the acid is TFA. The cleavage and / or deprotection solution may comprise at least 50 vol%, at least 60 vol%, at least 70 vol%, at least 80 vol%, at least 85 vol%, at least 90 vol% or at least 94 vol% acid. The cleavage and / or deprotection solution may comprise between 90 and 98 vol%, between 91 and 97 vol%, between 92 and 96 vol% or between 93 and 95 vol% acid. Preferably, the cleavage and / or deprotection solution comprises about 94 vol% acid. The cleavage and / or deprotection solution may further comprise a cation scavenger. Preferably, the cation scavenger is TIS (triisopropylsilane). The cleavage and / or deprotection solution may comprise at least 0.5 vol%, at least 1 vol%, at least 1.5 vol% or at least 2 vol% cation scavenger. The cleavage and / or deprotection solution may comprise between 0.5 and 4.5 vol%, between 1 and 3 vol% or between 1.5 and 2.5 vol% cation scavenger. Preferably, the cleavage and / or deprotection solution comprises about 2 vol% cation scavenger. Preferably, the cleavage solution comprises acid, water, cation scavenger in a vol% ratio of 94: 4 :2 (acid: water: cation scavenger). The method may further comprise purifying the cleaved mono-cross-linked trimer. Purifying the cleaved mono-cross-linked trimer may comprise contacting the cleaved mono-cross-linked trimer with a cold solvent, separating precipitated mono-crosslinked trimer from the solvent and drying the mono-cross-linked trimer. The temperature of the cold solvent may be less than 0°C, less than -10°C, less than -15°C, less than -18°C or less than -20°C. Preferably, the temperature of the solvent is less than -20°C. Preferably, the solvent is an organic solvent. More preferably the solvent is EtzO. The cleaved mono-cross-linked trimer may be a compound of formula (XII): PG2 i The second support is preferably a solid support. The second support may be or comprise a polymer. The polymer may be or comprise agarose, hydroxylated poly(methyl acrylate), poly(glycidyl acrylate), poly(glycidyl methacrylate), polylysine, polyethylene glycol, polyamide, polyacrylamide, polystyrene or copolymers of those. The solid support may comprise agarose beads or hydroxylated poly(methyl acrylate) beads. Preferably, the solid support comprises poly(methyl acrylate) beads. The second support, S2, preferably comprises a plurality of aldehyde groups. Accordingly, the polymer may be modified to comprise aldehyde groups. The second support, S2, may be or comprise aldehyde modified agarose or aldehyde modified hydroxylated poly(methyl acrylate). The second support may be represented by formula (XIII): , wherein S2 is a support; and m is an integer of at least 3. The method may comprise dissolving the cleaved mono-cross-linked trimer in a solvent to provide a mono-cross-linked timer solution and subsequently contacting the mono-cross-linked timer solution and the second support, S2. Preferably, the solvent comprises water. The solvent may comprise at least 10 vol%, at least 20 vol%, at least 30 vol%, at least 40 vol%, at least 45 vol% or at least 50 vol% water. The solvent may comprise between 10 and 90 vol%, between 20 and 80 vol%, between 30 and 70 vol%, between 40 and 60 vol% or between 45 and 55 vol% water. Preferably, the solvent comprises about 50 vol% water. The solvent may further comprise an acid. Preferably, the solvent comprises TFA. The solvent may comprise at least 10 vol%, at least 20 vol%, at least 30 vol%, at least 40 vol%, at least 45 vol% or at least 50 vol% acid. The solvent may comprise between 10 and 90 vol%, between 20 and 80 vol%, between 30 and 70 vol%, between 40 and 60 vol% or between 45 and 55 vol% acid. Preferably, the solvent comprises about 50 vol% acid. The method may comprise contacting the mono-cross-linked trimer and the second support for at least 10 minutes, at least 30 minutes, at least an hour, at least 2 hours, at least 4 hours, at least 8 hours or at least 16 hours. Contacting the immobilized cross-linked trimer of formula (VI) and the second crosslinking reagent may comprise contacting the immobilized cross-linked trimer of formula (VI) and a second cross-linking solution, wherein the second cross-linking solution comprises the second cross-linking reagent. The second cross-linking reagent may be the same as the first cross-linking reagent, and may be as defined above. The second cross-linking solution may be the same as the first cross-linking solution, and may be as defined above. The method may comprise contacting the immobilized cross-linked trimer of formula (VI) and the second cross-linking reagent in a relative molar ratio of cross-linking reagent to immobilized cross-linked trimer of formula (VI) of between 0.2:1 and 5:1, between 0.4:1 and 2:1, between 0.6:1 and 1.6:1, between 0.7:1 and 1.5:1, between 0.8:1 and 1.4:1, between 0.9:1 and 1.3:1 or between 1.0:1 and 1.2:1. Preferably, the relative molar ratio of cross-linking reagent to immobilized cross-linked trimer of formula (VI) is about 1.1:1. The concentration of the immobilized cross-linked trimer of formula (VI) may be an assumed concentration. The method of contacting the immobilized cross-linked trimer of formula (VI) and the second cross-linking reagent may be, mutatis mutandis, the same as the method of contacting the support and plurality of monomers of formula (IV) and the first crosslinking reagent, as defined above. The mono cross-linked trimer immobilized on the second support may be represented by formula (Via): HS' / n2 wherein L8 is -NH- or O , where an asterisk indicates a point of attachment to L7. The method may comprise reducing the support attachment group. The method may comprise reducing the support attachment group subsequent to contacting the mono cross-linked trimer and the second support. The method may comprise reducing the support attachment group prior to contacting the immobilized cross-linked trimer of formula (VI) and the second cross-linking reagent. Alternatively, the method may comprise reducing the support attachment group subsequently to contacting the immobilized cross-linked trimer of formula (VI) and the second cross-linking reagent. The method may comprise reducing the support attachment group prior to cleaving the double-cross-linked trimer from the second support. Reducing the support attachment group may comprise reducing the group W within the support attachment group. For instance, in embodiments where W is N3 in the support attachment group, reducing the support attachment group may comprise reducing the N3 group to an NH2 group. Preferably, reducing the support attachment group makes the support attachment group sensitive to acid. Reducing the support attachment group may comprise contacting the immobilized cross-linked trimer of formula (VI) or the double-cross-linked trimer and a reducing agent. The reducing agent may be DTT (dithiothreitol). The method may comprise contacting the immobilized cross-linked trimer of formula (VI) or the double-cross-linked trimer and a reducing solution, wherein the reducing solution comprises the reducing agent. The reducing solution may comprise the reducing agent at a concentration of at least 0.01M, at least 0.1 M, at least 0.2 M, at least 0.4 M, at least 0.5 M or at least 0.6 M. The reducing solution may comprise the reducing agent at a concentration of between 0.01 and 10 M, between 0.1 and 5 M, between 0.2 and 2 M, between 0.4 and 1 M, between 0.5 and 0.8 M or between 0.6 and 0.7 M. The reducing solution may further comprise a solvent. The solvent may comprise water and / or an organic solvent. In some embodiments, the reducing solution may comprise water and an organic solvent. The organic solvent may comprise an alcohol. The alcohol may be ethanol. The reducing solution may comprise a base. The base may be a weak base. The base may be NaHCOs (sodium hydrogen carbonate). The reducing solution may comprise the base at a concentration of at least 0.01 M, at least 0.1 M, at least 0.2 M or at least 0.3 M. The reducing solution may comprise the base at a concentration of between 0.01 and 5 M, between 0.1 and 2 M per, between 0.2 and 1 M, between 0.3 and 0.5 M. The method may comprise contacting the immobilized cross-linked trimer of formula (VI) or the double-cross-linked trimer and the reducing agent for at least 30 second, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes or at least 25 minutes. The method may comprise contacting the immobilized cross-linked trimer of formula (VI) or the double-cross-linked trimer and the reducing agent for between 30 seconds and 24 hours, between 1 minutes and 6 hours, between 5 minutes and 2 hours, between 10 and 60 minutes, between 20 and 45 minutes or between 25 and 35 minutes. Cleaving the double-cross-linked trimer from the second support may comprise contacting the double cross-linked trimer and a cleaving solution. The cleaving solution may comprise or consist of an acid. The acid may be a carboxylic acid. The acid may be acetic acid or trifluoroacetic acid (TFA). The cleaving solution may comprise at least 5 vol% acid, at least 10 vol% acid, at least 20 vol% acid, at least 30 vol% acid, at least at least 40 vol% acid, at least 50 vol% acid, at least 60 vol% acid, at least 70 vol% acid, at least 80 vol% acid, at least 90 vol% acid or at least 95 vol% acid. The cleaving solution may comprise between 10 and 99.99 vol% acid, between 20 and 99.9 vol% acid, 40 and 99.5 vol% acid, 60 and 99 vol% acid, 80 and 98 vol% acid, between 90 and 97 vol% acid or between 94 and 96 vol% acid. The cleaving solution may comprise water. The cleaving solution may comprise at least 0.5 vol% water, at least 1 vol % water, at least 2 vol% water, at least 3 vol% water or at least 4 vol% water. The cleaving solution may comprise between 0.01 and 90 vol% water, between 0.1 and 80 vol% water, between 0.5 and 60 vol% water, between 1 and 40 vol% water, between 2 and 20 vol% water, between 3 and 10 vol% water or between 4 and 6 vol% water. The double cross-linked trimer and the cleaving solution may be contacted for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes or at least 40 minutes. The double cross-linked trimer and the cleaving solution may be contacted for between 1 minutes and 24 hours, between 5 minutes and 12 hours, between 10 minutes and 6 hours, between 20 minutes and 2 hours, between 30 and 60 minutes or between 40 and 50 minutes. The method may further comprise purifying the cleaved double-cross-linked trimer. Purifying the cleaved double-cross-linked trimer may comprise contacting the cleaved double-cross-linked trimer with a cold solvent; separating precipitated double-crosslinked trimer from the solvent; and drying the double-cross-linked trimer. The temperature of the solvent may be less than 0°C, less than -10°C, less than -15°C, less than -18°C or less than -20°C. Preferably, the temperature of the solvent is less than -20°C. Preferably, the solvent is an organic solvent. More preferably, the solvent is EtzO. Preferably, separating the precipitated double-cross-linked trimer from the solvent is achieved by centrifugation. The method may comprise freeze drying the resultant composition. In accordance with a fourth aspect, there is provided a composition comprising the compound of the first aspect, or a cosmetically or pharmaceutically acceptable salt or solvate thereof, or the composition of the second aspect and a carrier. In accordance with a fifth aspect, there is provided a composition comprising the compound of the first aspect, or a cosmetically or pharmaceutically acceptable salt or solvate thereof, or the composition of the second aspect and platelet rich plasma (PRP). The composition of the fifth aspect may further comprise a carrier. The carrier may be a pharmaceutically acceptable carrier and / or a cosmetically acceptable carrier. The compositions of the fourth and fifth aspects having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal. In accordance with a sixth aspect, there is provided a method of providing the composition of the fifth aspect, the method comprising contacting the compound of the first aspect or the composition of the second aspect and platelet rich plasma (PRP). The method may comprise contacting the composition of the fourth aspect and PRP. The method may comprise contacting the compound of the first aspect or the composition of the second aspect and PRP less than 4 hours prior to intended use of the composition. Preferably, the method comprises contacting the compound of the first aspect or the composition of the second aspect and PRP less than 3 hours, less than 2 hours or less than 1 hour prior to intended use of the composition. Most preferably, the method comprises contacting the compound of the first aspect or the composition of the second aspect and PRP less than 30 minutes, less than 20 minutes or less than 10 minutes prior to the intended use of the composition. Advantageously, the method ensures that the PRP may be activated and release growth factors before use, but the growth factors do not degrade before they are used. In accordance with a seventh aspect, there is provided the composition of the fifth aspect for use as a medicament. In accordance with an eighth aspect, there is provided the composition of the fifth aspect for use in the treatment of a skin condition. In accordance with a ninth aspect, there is provided a method of treating, preventing or ameliorating a skin condition, the method comprises administering to a subject in need of such treatment a therapeutically effective amount of the composition of the fifth aspect. The term "preventing" may be understood to mean reducing the likelihood of the patient developing a skin condition. In accordance with a tenth aspect, there is provided a method of cosmetic treatment, the method comprises administering to a subject a cosmetically effective amount of the composition of the fifth aspect. The method of the ninth aspect may be conducted once or twice a day. The method of the sixth aspect may be conducted before the method of the ninth or tenth aspect. The method of the sixth aspect may be conducted less than 4 hours before the method of the ninth or tenth aspect. The method of the sixth aspect may be conducted less than 3 hours, less than 2 hours or less than 1 hour before the method of the ninth or tenth aspect. Most preferably, the method of the sixth aspect is conducted less than 30 minutes, less than 20 minutes or less than 10 minutes before the method of the ninth or tenth aspect. Cosmetically and pharmaceutically acceptable salts include any salt of a compound of formula (I) provided herein which retains its biological properties and which is not toxic or otherwise undesirable for cosmetic or pharmaceutical use. The cosmetically or pharmaceutically acceptable salt may be derived from a variety of organic and inorganic counter-ions well known in the art. The cosmetically or pharmaceutically acceptable salt may comprise an acid addition salt formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic, camphoric, camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic, glucoheptonic, 3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid and the like acids. Alternatively, the pharmaceutically acceptable salt may comprise a base addition salt formed when an acidic proton present in the parent compound is either replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, an aluminium ion, alkali metal or alkaline earth metal hydroxides, such as sodium, potassium, calcium, magnesium, aluminium, lithium, zinc, and barium hydroxide, or coordinates with an organic base, such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like. A cosmetically or pharmaceutically acceptable solvate refers to a compound of formula (I) provided herein, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate. It will be appreciated that the composition of the fifth aspect may be used in a medicament which may be used in a monotherapy. Alternatively, the composition of the fifth aspect may be used as an adjunct to, or in combination with, known therapies. The composition of the fifth aspect may be administered by inhalation (e.g. intranasally). The composition of the fifth aspect may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion). Alternatively, the composition of the fifth aspect may be administered orally. In a preferred embodiment, the composition of the fifth aspect is formulated for topical use. For instance, creams or ointments may be applied to the skin. The composition of the fifth aspect may be administered during or after onset of the skin condition to be treated. Daily doses may be given as a single administration. Alternatively, the composition of the fifth aspect may be given two or more times during a day. A "subject" may be a vertebrate, mammal, or domestic animal. Hence, the composition of the fifth aspect may be used to treat any mammal, for example livestock (e.g. a horse), pets (e.g. a dog), or may be used in other veterinary applications. Most preferably, however, the subject is a human being. A "therapeutically effective amount" of the composition of the fifth aspect is any amount which, when administered to a subject, is the amount needed to treat the skin condition. A "cosmetically or pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating cosmetic or pharmaceutical compositions. In one embodiment, the cosmetically or pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid cosmetically or pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents. The vehicle may also be an encapsulating material. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like. However, the cosmetically or pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The liquid vehicle can contain other suitable cosmetic or pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant. All features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which:- Figure 1 shows an optimized synthetic route to a double cross-linked collagen-related peptide trimer, CRP-A, shown on a simplified molecular level; Figure 2 shows the structure of CRP-A; Figure 3 shows a) UPLC-UV / vis chromatogram (at 210 nm) of PEC purified doublecross-linked CRP from feasibility synthesis using CRP-StBu monomers; b) Maldi-MS spectrum of PEC purified double-cross-linked CRP from feasibility synthesis using CRP-StBu monomers; c) UPLC-UV / vis chromatogram (at 210 nm) of PEC purified doublecross-linked CRP, synthesized from CRP-Dpm monomers, after heating to 90°C for 1015 min; d) Maldi MS spectrum of PEC purified double-cross-linked CRP, synthesized from CRP-Dpm monomers, after heating to 90°C for 10-15 min; e) UPLC-UV / vis chromatogram (at 210 nm) of commercial double-cross-linked CRP-XL; and f) Maldi-MS spectrum of commercial double-cross-linked CRP-XL; Figure 4 shows concentrations of two growth factors, EGF (Epidermal growth factor) and PDGF-BB (Platelet Derived growth factor BB), measured in blood samples from two different donors after treatment with CPR-A, CRP-XL and CaCl2. *p<0.05, **p<0.01 and ***p<0.001; Figure 5 shows platelet activation in two PRP samples after treatment with CRP-A (condition 1), CRP-XL (condition 2), CaCl2 (condition 3), a negative control and a positive control. P-selectin expression was used as the measure of platelet activation and quantified by CD62P+ staining; Figure 6 shows platelet activation in whole blood samples after treatment with different batches of CRP-A and CRP-XL; Figure 7 shows platelet aggregation in PRP samples after treatment with different batches of CRP-A and CRP-XL; Figure 8 shows platelet activation in whole blood samples treated with different batches of CRP-A with and without the specific inhibitor glenzocimab; WO 2025 / 133607                                   PCT / GB2024 / 053160 - 45 - Figure 9 shows platelet activation in whole blood samples treated with different batches of CRP-A before and after 3 hours storage at room temperature; Figure 10 shows platelet activation, measured by concentration of the growth factor PDGF-BB released, of CRP-A after 6 months storage in different conditions. W = water, Ac = acetic acid, *p<0.05, **p<0.01 and ***p<0.001; Figure 11 shows a) RP-UPLC results for CRP-A after 6 months storage in different conditions; and b) RP-UPLC results for CRP-A dissolved in water after 2 and 6 months at different storage temperatures; Figure 12 provides SE-UPLC results for CRP-A after 6 months storage in different conditions; Figure 13 provides SE-UPLC results for CRP-A and commercial CRP-XL under chaotropic and reductive conditions; Figure 14 shows SEC-UPLC-UV / Vis results for CRP-A before (top) and after (below) two years storage; and Figure 15 shows RP-UPLC-UV / Vis results for CRP-A before (top) and after (below) two years storage. Examples Example 1: CRP-A synthesis Collagen-related peptide (CRP) has the following sequence: GCO-(GPO)io-GCOG SEQ ID No. 2 CRP will only elicit activation of platelets when cross-linked. Accordingly, the inventors sought a reliable manufacturing route that ensured consistent cross-linking of the CRP monomers and enabled a high batch-to-batch consistency. PEC (peptide easy clean) technology was identified as a means of achieving consistent purification and completion of the second cross-linking. It applies a catch and release methodology based on traceless cleavable linker molecules (PEC-linkers) and activated filter materials. Figure 1 shows the optimized synthetic route identified by the inventors. First the inventors synthesized monomeric CRP-Dpm: Fmoc-GC(Trt)0[GPO]ioGC(Dpm)OG-resin In the above sequence, O is L-hydroxyproline protected with tBu. This was synthesized using solid phase peptide synthesis (SPPS). The N-terminal is protected with a fluorenylmethoxycarbonyl (Fmoc) protecting group and the C-terminal is bound to the resin. The cysteine residue in the sequence which is closest to the C-terminal / the resin is protected with a diphenylmethyl (Dpm) protecting group. Additionally, the cysteine residue in the sequence which is closest to the N-terminal / furthest from the resin is protected with a trityl (Trt) protecting group. Subsequently, the inventors selectively deprotected the Cys(Trt) residue, while leaving the Dpm protecting group in place. N-terminal cross-linking was then achieved with 1,3,5-tribromomesitylene (T3) to provide a mono-crosslinked trimer. Fmoc deprotection was then carried out, and a PEC Linker RC+ (tert-butyl (2-((2-(3-azido-2-bromo-6-((((4-nitrophenoxy)carbonyl)oxy)methyl)benzamido)ethyl)amino)-2-oxoethoxy)(tertbutoxycarbonyl)carbamate) was installed on the N-terminus. The mono-crosslinked trimer was then cleaved from the SPPS resin and dpm group was deprotected. The mono-cross-linked trimer was immobilized on PEC resin beads (aldehyde modified agarose). Finally, a second (C-terminal) cross-linking was carried out to provide a double cross-linked trimer, which is then was released from the PEC beads to provide CRP-A, the sequence of which is provided below. [GC(cross-linker)0[GPO]ioGC(cross-linker)OG]3 SEQ ID No. 4 In the above sequence, "cross-linker" indicates that the cysteine group is bonded to a cross-linker with the following structure: The structure of CRP-A is shown schematically in Figure 2. Example 2: Optimization of selective L-cvsteine(Trt) deprotection Different conditions for selective trityl (Trt) deprotection of CRP-Acm monomers on SPPS resin were tested and are shown in Table 1. Table 1. Tested conditions for selective Trt deprotections and their outcomes Condition for selective Trt deprotection Outcome A: 5x1 min with DCM / TFA / TIS (95:2.5:2.5) Incomplete Trt deprotection B: 6x5 min with DCM / TFA / TIS (90:5:5) Complete Trt deprotection C: 5x5 min with DCM / TFA / TIS (87.5:10:2.5) Complete Trt deprotection, higher TFA content can lead to unwanted peptide release or tBu deprotection Six times treatment of CRP monomers on SPPS resin with DCM / TFA / TIS (90:5:5) for 5 min each was identified as the best condition. Example 3: Optimization of N-terminal cross-linking of CRP monomers with T3 The N-terminal cross-linking of selectively trityl deprotected CRP-Acm monomers on SPPS resin was tested using different conditions (Table 2). To identify the optimum conditions, after cross-linking, the peptide was cleaved from SPPS resin with 10 TFA / Water / TIS (95:2.5:2.5) for 1 h and analyzed via MALDI-MS. The ratio of trimer to monomer and dimer in the MALDI-MS spectra was assessed to judge the quality of the experimental outcome. Table 2. Tested conditions for N-terminal cross-linking of CRP-Acm monomers and 15                                their respective outcomes Condition for N-terminal crosslinking of CRP-Acm on SPPS resin Outcome A: 8 M aq. Urea / MeCN / DMF / 0.2 M aq. NH4HCO3 (1:1:1:0.66) + 0.33 eq. T3 in MeCN (0.014 M), RT, 30 min stirring Only very small amount of CRP trimer and CRP dimer B: 8 M aq. Urea / MeCN / DMF / 0.2 M aq. NH4HCO3 (1:1:1:0.66) + 3 eq. T3 in MeCN (0.12 M), RT, 30 min stirring Slightly higher ratio of CRP trimer to CRP monomer / dimer, but also large amount of CRP monomer bound to T3 scaffold C: 8 M aq. Urea / MeCN / DMF / 0.2 M aq. NH4HCO3 (1:1:1:0.66) + 30 eq. T3 in MeCN (0.6 M), RT, 30 min stirring Slightly higher ratio of CRP trimer to CRP monomer / dimer, but also very large amount of CRP monomer bound to T3 scaffold D: 8 M aq. Urea / MeCN / DMF / 0.2 M aq. NH4HCO3 (1:1:1:0.66) + 3 eq. T3 in MeCN (0.12 M), RT, 16 h stirring Higher ratio of CRP trimer to CRP monomer / dimer, but also large amount of CRP monomer bound to T3 scaffold E: 6 M Urea in DMSO / DIEA (97:3) + 3 eq. T3 in MeCN (0.12 M), RT, 30 min stirring Significantly higher ratio of CRP trimer to CRP monomer / dimer, but also medium amount of CRP monomer bound to T3 scaffold F: 6 M Urea in DMSO / DIEA (97:3) + 0.33 eq. T3 in MeCN (0.12 M), RT, 6090 h stirring Highest ratio of CRP trimer to CRP monomer / dimer, significantly lower amount of CRP monomer bound to T3 scaffold Condition F (Table 2) was identified as best condition according to the MALDI-MS spectra. After further optimization, the optimum conditions identified for the 1st and 2nd crosslinking steps were found to use 1.1 eq T3 in a 1:1 volumetric ratio of acetonitrile : 0.6 M aqueous NaHCOs pH 8.3 at 20°C. The amount of T3 used was based on the assumption that the concentration of the peptide is 20 pmol. Example 4: Optimisation of starting monomer CRP-Acm, CRP-Gly, CRP-StBu and CRP-Dpm were considers as alternative monomers which could be used as the starting monomer in the above method. These monomers had the following sequences: Fmoc-GC(Trt)0[GPO]ioGC(Dpm)OG-resin SEQ ID No. 5 Fmoc-GC(Trt)0[GPO]ioGC(Acm)OG-resin SEQ ID No. 6 Fmoc-GC(Trt)0[GPO]ioGC(StBu)OG-resin SEQ ID No. 7 Fmoc-GC(Trt)0[GPO]ioGGOG-resin In the above sequences O is L-hydroxyproline to provide CRP-Dpm (SEQ ID No. 5), CRP-Acm (SEQ ID No. 6), CRP-StBu (SEQ ID No. 7) or CRP-Gly (SEQ ID No. 8). CRP-A was produced from the CRP-StBu monomer, as described in the methods section. The UPLC-UV / vis chromatogram and the MALDI-MS spectrum for the product are shown in Figures 3a) and 3b) respectively. The inventors found that isolation of the desired double-cross-linked trimer was difficult. The use of Fmoc-L-Cys(StBu)-OH as a building block was considered to have had a negative impact and led to a low yield, since it only allows for cross-linking after reduction of PEC-linker RC+. Consequently, Fmoc-L-Cys(Dpm)-OH was tried as an alternative building block. CRP-A was produced from the CRP-Dpm monomer, as described in the methods section. The UPLC-UV / vis chromatogram and the MALDI-MS spectrum for the product of this synthesis after heating to 90°C for 10-15 min, are shown in Figures 3c) and 3d), respectively. Comparison between the UPLC-UV-vis chromatograms 3a) and 3c) shows that starting from the monomer CRP-Dpm gave an improved purity. Additionally, using the CRP-Dpm monomer gave a 650% increase in yield compared to using the CRP-StBu monomer. Comparison of the MALDI-MS spectrum for CRP-A (Figure 3d) with the MALDI-MS spectrum for a commercial sample of CRP-XL (Figure 3f), shows that CRP-A contains a higher ratio of desired trimer to undesired dimer / monomer and fewer side products to CRP-XL. According to size exclusion chromatography with detection based on UV-Absorption the molar ratio of the monomer:dimer:trimer for CRP-XL is 1:5.4:12. Accordingly, this means that the molar ratio of the dimer:trimer 1:2.22. Conversely, for CRP-A the molar ratio of the monomer:trimer 1: >20. Example 5: Optimization of dpm deprotection Optimized dpm deprotection conditions: peptide 4h with TFA / water / TIS / thioanisol 92.5:2.5:2.5:2.5. In particular, it was noted that the optimum conditions used a high concentration of TFA and relatively long reaction times. Example 6: Optimization of immobilization of mono-cross-linked CRP-A (SO396) Different conditions for the immobilization of mono-cross-linked CRP-A were investigated. The crude mono-cross-linked CRP-A comprising the PEC-linker was dissolved in a solvent and immobilized over 16 hours on beads. The solvents, beads and / or temperatures used were varied, as set out in Table 3. Table 3. Tested conditions for immobilization of mono-cross-linked CRP-A Condition Solvent Beads Temperature A HFIP Agarose (standard) RT B DMSO / citric acid buffer +GdmCI (9:1, pH 3.5) Agarose (standard) RT C DMSO / aniline buffer (9:1, pH 3.5) Agarose (standard) RT D HFIP / citric acid buffer +GdmCI (9:1, pH 3.5) PEC-Polymethacrylate (PMA) beads RT E TFA / water (1:1) PEC-Polymethacrylate (PMA) beads RT F HFIP / citric acid buffer +GdmCI (9:1, pH 3.5) PEC-Polymethacrylate (PMA) beads 50°C The results of the optimization tests are provided in Table 4, below. Table 4. Results of optimization tests for immobilization of mono-cross-linked CRP-A Condition Immobilization Efficiency Final weight after full process [mg] Trimer Dimer Monomer A 63.4% 1.96 ~5 -8 1 B 60.7% 3.44 ~7 -8 1 C 58.7% 4.61 ~7 ~9 1 D 52.0% 1.70 ~11 -6 1 E 80.2% 4.45 ~16 -8 1 F 56.1% 3.37 ~13 -6 1 The immobilization efficiency was calculated using the following equation: Amount of peptide in solution after immobilization Amount of peptide in solution before addition to PEC resin The amount of peptide in solution was determined with Ellman's test. The relative ratios of the trimer, dimer and monomer were calculated based upon the respective peak heights in the size exclusion chromatography (SEC) chromatographs for the products. This study found that conditions E gave the best immobilisation efficiency and also had the highest trimer to dimer ratio. The method was repeated with conditions E using a large batch. This produced a product with a ratio of ~38:~13:1 trimer:dimer:monomer and 49.2 mg of product. Example 7: Biological efficacy - concentration of growth factors in activated samples The ability of CRP-A to activate platelets to release of growth factors compared with commercial CRP-XL and CaCl2 was tested on blood samples from two different donors (donor #1 and donor #2). Platelet activation ability was determined by measuring the concentration of the growth factors PDGF-BB (platelet derived growth factor BB) and EGF (epidermal growth factor) in blood samples after treatment with CRP-A / other activators. The results are shown in Figure 4. Promisingly, the results showed that, for both donors, CRP-A and commercial CRP-XL, caused the release of similar levels of EGF and PDGF-BB. The concentrations of PDGF-BB measured in the donor samples after treatment with CRP-A were found to be significantly higher than the concentrations of PDGF-BB in the donor samples after treatment with the commonly used platelet activator CaCl2. Example 8: Biological efficacy - P-selectin expression on platelets Further comparison testing between CRP-A and CRP-XL was carried out by measuring P-selectin (CD62P) expression on platelets from PRP. P-selectin is a platelet surface marker commonly used to measure platelet activation. Two PRP samples (PRP #3 and PRP #4) were prepared from whole blood samples collected just before analysis. Each PRP was quantified and 2 x 106 platelets were distributed in the staining tubes. The PRPs were then activated by 3 different conditions (condition 1: treated with CRP-A, condition 2: treated with CRP-XL and condition 3: treated with CaCl2). A positive control was treated with TRAP-6 (thrombin receptor activator). An sample with no treatment was provided as a negative control. Staining with anti-P-selectin antibody was then performed following a defined standard procedure. Analysis was performed using isotype control staining and flow cytometry. Figure 5 shows the percentage of CD62P (P-selectin) positive platelets detected after treatment. The results again showed that CRP-A (condition 1) and CRP-XL (condition 2) caused similarly high levels of platelet activation, like those caused by TRAP6. CaCl2 (condition 3) caused no activation for PRP #3 and only mild activation for PRP #4, with some intraindividual heterogeneity for PRP #4. The tubes treated by CaCh displayed a more gelatinous appearance than conditions 1 and 2. Example 9: Batch-to-batch consistency of CRP-A Further activation experiments were carried out using different batches of CRR-A, to test batch-to-batch consistency. The effect of 3 different batches of CRP-A on platelet activation in whole blood (WB) samples was tested. The results, shown in Figure 6, show that CRP-A activates P-selectin expression on platelets consistently from batch to batch. To further confirm the batch-to-batch consistency of platelet activation by CRP-A, 3 different batches of CRP-A were evaluated in terms of their impact on platelet aggregation in PRP and compared with CRP-XL. The results are presented in Figure 7 and show that CRP-A causes similar levels of platelet aggregation to CRP-XL regardless of the batch. The ECso values for CRP-XL and the three different batches of CRP-A are presented in table 5. These EC50 values are the concentration of agonist required to induce 50% of maximal platelet aggregation. Table 5. Mean EC50 in terms of platelet aggregation for CRP-XL and three different batches of CRP-A. CRP Mean EC50 (pg / mL) CRP-XL 0.06 CRP-A (SO396) 0.07 CRP-A (2022-3) 0.06 CRP-A (2022-4) 0.04 Example 10: Elucidating the activation pathway Experiments were conducted to assess the specific inhibitory effect of glenzocimab, a specific anti-GPVI monoclonal antibody, on CRP-A-induced platelet activation. GPVI is a platelet receptor that binds specifically to collagen, triggering platelet activation. To demonstrate the specificity of the pathway of activation by CRP-A, the impact of CRP-A on platelet activation was tested in the presence glenzocimab. The thrombin activator TRAP was used as a control of platelet activation via another pathway. The results of these experiments are shown in Figure 8. The concentration response curves of CRP-A-induced platelet activation was completely inhibited by glenzocimab at 20.0 |jg / mL. This indicates that CRP-A is a potent agonist specific to the GPVI platelet receptor which triggers platelet activation. Example 11: Stability The platelet activation ability of 3 different batches of CRP-A was tested on whole blood samples. The different batches of CRP-A were tested immediately after they were reconstituted and again after 3 hours storage at room temperature. Platelet activation was measured via P-selectin expression. The results, shown in Figure 9, illustrate that, once reconstituted in solution, CRP-A remains active after at least 3 hours at room temperature. The platelet activation ability of CRP-A stored in different conditions (lyophilized, in water, and in acetic acid, each at room temperature, -4 °C and -20 °C) was tested monthly over 6 months in order to better understand the long-term stability and therefore efficacy of CRP-A as a platelet activator. Platelet activation ability was determined by measuring the concentration of platelet derived growth factor BB (PDGF-BB) in blood samples treated with CRP-A. The results of the tests after 6 months are shown in Figure 10. It is noted that all samples were able to activate platelets, indicating that it is stable. The purity of CRP-A was tested regularly by reversed-phased ultra-performance liquid chromatography (RP-UPLC) over a period of 6 months. Figure Ila) shows the results for CRP-A after 6 months storage in different conditions and Figure 11b) the results for CRP-A dissolved in water after storage for 2 and 6 months at selected temperatures. The RP-UPLC results showed that only minor amounts of side products were produced over the storage period. The side products may be generated by unreacted and partly hydrolysed T3. Promisingly, the UV- purity of a sample dissolved in water and stored at room temperature decreased by only 2.1% over six months, see Figure 11b). Size-exclusion UPLC (SE-UPLC) was used to confirm the stability of CRP-A over 6 months. Figure 12 shows the results for CRP-A after 6 months storage in different conditions. The SE-UPLC results confirmed that the trimer-dimer and multimer-dimer ratios do not decrease over time, since no monomer becomes visible. Surprisingly, it was found that the ratio of the trimer to the dimer actually increases over time. This effect was most significant for the samples dissolved in 0.01 M AcOH and stored at RT and -4°C. This change in ratio may be a consequence of aggregation and selfassembly of the cross-linked CRP-A over time. Example 12: Comparison of CRP-A with CRP-XL in chaotropic conditions Experiments were conducted to compare the behaviour of CRP-A with that of CRP-XL under chaotropic conditions for 1 h at 40 °C and chaotropic and reductive conditions for 1 h at 40 °C. Figure 12 shows the SE-UPLC results of these investigations. This shows that under chaotropic conditions CRP-A mainly aggregates and under reductive conditions these aggregates are stable. In contrast, under chaotropic conditions CRP-XL mainly undergoes chemical polymerisation and under reductive conditions the product of this polymerisation is not stable. Figure 13 shows that under reductive and denaturing conditions there is no trimer left for CRP-XL. Conversely, CRP-A is stable under these conditions. Example 13: 2-vears-stabilitv measurement of CRP-A The stability of CRP-A over two years was assessed by comparison of four different parameters (appearance, chemical identification, UV-purity and trimer / monomer ratio), each measured before and after > 2 years of storage. The CRP-A material was stored for the full duration (> 2 years) as a freeze-dried solid in a freezer (-20 °C). Appearance was evaluated by inspection of the colour and texture of the material. It was found that the colour and texture of the material was the same after two years. In particular, it remained a white solid powder. Chemical identification of the material was determined using size-exclusion (SEC) ultra-performance liquid-chromatography (UPLC) chromatograms, and in particular using the peak retention times. As shown in Figure 14, the trimer peak retention times in the SEC chromatograms before and after 24 months were the same. Advantageously, this showed that the CRP-A contained the same components after two years' storage. The CRP-A trimer / monomer ratio was also determined using SEC UPLC chromatograms at a wavelength of 210 nm, and in particular using the peak areas. Advantageously, it was found that the trimer to monomer ratio was substantially the same, and decreased only slightly from 55:1 to 52:1. Additionally, the ratio of trimer / dimer increased, due to self-assembly of the CRP-A. UV-purity was determined from reversed-phase (RP) UPLC chromatograms at a wavelength of 210 nm, and in particular using the peak areas. These chromatograms are shown in Figure 15. It was found that the UV-purity slightly decreased only from 99.5% to 97.1 %. Methodology Amino Acids, Solvents and Chemicals were obtained from Carl ROTH, Carbolution, TCI and Sigma-Aldrich and were used as received. If not noted otherwise, water was purified with a Mili-Q Ultra Pure Plus Water Purification System from Merck. The peptide synthesis was carried out under contract by PurePep Chorus from Gyros Protein Technologies. The PEC-linker RC+ and PEC resin beads used were produced by Belyntic, and are commercially available products. SPPS synthesis Automated SPPS was done with an Intavis MultiPep RSI synthesizer in different scales. Fmoc-Rink Amide aminomethyl-polystyrene resin from IRIS Biotech was used (100200 mesh, 1% DVB) as solid support for the SPPS. CRP was synthesized in monomeric form using the following general sequence: GC(Trt)Q[GPO]ioGXOG (O = L-Hydroxyproline, X = L-Cysteine(Dpm), L-Cysteine(Acm), L-Cysteine(StBu) or Glycine). For C (L-Cysteine) different building blocks were used to enable orthogonal deprotection on the thiol groups of the Cysteine sidechains: Fmoc-L-Cysteine(Trt)-OH, Fmoc-L-Cysteine(Dpm)-OH, Fmoc-L-Cysteine(StBu)-OH or Fmoc-L-Cysteine(Acm)-OH. CRP-Acm was synthesized on a 25 pmol scale using the following SPPS protocol: Cycles 1-14 (lx Amino Acid Coupling): 1. Fmoc-Deprotection (2x 400 pL Piperidine / DMF (1:4) for 5 min & 8 min); 2. 5x Wash (EtOAc / DMSO (8:2)); 3. Amino Acid Coupling (90 pL DIC (1.5 M in DMF), 90 pL Oxyma (1 M in DMF), 15 pL NMP, 180 pL Derivative (0.5 M in DMF) for 45 min); 4. Capping (400 pL Pyridine &. Acetic anhydride (2 M in DMF) for 5 min); and 5. 3x Wash (EtOAc / DMSO (8:2)). Cycles 1537 (2x Amino Acid Coupling): 1. Fmoc-Deprotection (2x 400 pL Piperidine / DMF (1:4) for 6 min & 9 min); 2. 5x Wash (EtOAc / DMSO (8:2)); 3. First Amino Acid Coupling (90 pL DIC (1.5 M in DMF), 90 pL Oxyma (1 M in DMF), 15 pL NMP, 180 pL Derivative (0.5 M in DMF) for 45 min); 4. 2x Wash (EtOAc / DMSO (8:2)); 5. Second Amino Acid Coupling (175 pL HATU (0.5 M in DMF), 66 pL DIEA (50% in NMP), 15 pL NMP, 180 pL Derivative (0.5 M in DMF) for 45 min); 6. Capping (400 pL Pyridine8i Acetic anhydride(2 M in DMF) for 5 min); and 7. 3x Wash (EtOAc / DMSO (8:2)). CRP-Gly and CRP-StBu were synthesized on a 50 |jmol scale using the following SPPS protocol: Cycles 1-5 (lx Amino Acid Coupling): 1. Fmoc Deprotection (2x 600 pL Piperidine / DMF (1:4) for 5 min & 8 min); 2. 5x Wash (EtOAc / DMSO (8:2)); 3. Amino Acid Coupling (150 pL DIC (1.5 M in DMF), 150 pL Oxyma (1 M in DMF), 10 pL NMP, 300 pL Derivative (0.5 M in DMF) for 45 min); 4. Capping (600 pL Pyridine 8i Acetic anhydride (2 M in DMF) for 5 min); and 5. 3x Wash (EtOAc / DMSO (8:2)). Cycles 6-37 (2x Amino Acid Coupling): 1. Fmoc Deprotection (2x 600 pL Piperidine / DMF (1:4) for 6 min & 9 min); 2. 5x Wash (EtOAc / DMSO (8:2)); 3. First Amino Acid Coupling (150 pL DIC (1.5 M in DMF), 150 pL Oxyma (1 M in DMF), 10 pL NMP, 300 pL Derivative (0.5 M in DMF) for 45 min); 4. lx Wash (EtOAc / DMSO (8:2)); 5. Second Amino Acid Coupling (290 pL HATU (0.5 M in DMF), 110 pL DIEA (50% in NMP), 10 pL NMP, 300 pL Derivative (0.5 M in DMF) for 45 min); 6. Capping (600 pL Pyridine & Acetic anhydride (2 M in DMF) for 5 min); and 7. 3x Wash (EtOAc / DMSO (8:2)). Where used above, the term "derivative" refers to the amino acid building block. Selected Trt-deprotection Per pmol assumed peptide amount, 100 pL of the mixtures described in Table 1 (above) were added. After the last repetition, the resin was washed 3x with DCM. To ensure quantitative deprotection, capping with 100 pL of a 1 M solution of 2-bromoacetic acid (2-BrAcOH) in DMF / NMP / DIEA (75:12.5:12.5) per pmol assumed peptide amount was done for 15 min following the Trt deprotection. After cleavage from SPPS resin and peptide global deprotection for 1 h with 100 pL TFA / water / TIS (97.5:2.5) per pmol assumed peptide amount, the ratio of free CRP monomers to 2-BrAcOH capped CRP monomers was calculated using UPLC-UV / vis-ESI-MS analysis to identify the optimal conditions for selective deprotection of L-Cysteine(Trt). Optimal conditions for N-terminal cross-linking of CRP monomers with T3 (Example 3, Condition F, Table 2) The air-dried resin with CRP monomers was left swelling in DMSO for 15 min. After filtration of DMSO, 100 pL of a mixture of 6 M Urea in DMSO / DIEA (97:3) per pmol assumed peptide amount was added and subsequently 2.8 pL of a 0.12 M solution of T3 in MeCN per pmol assumed peptide amount (0.33 eq) was added. The resin was left stirring for 60-90 h and thereafter it was washed with 6x Water, 3x DMF and 3x DCM. Subsequently, the peptide was cleaved from SPPS resin with TFA / Water / TIS (95:2.5:2.5) for 1 h. Optimal conditions for cross-linking (Example 3) Crosslinking: After removing the DMF for beads swelling, first 5.2 mL DMF / DIEA (97:3) and second 146 pL of 0.12 M T3 in MeCN (17.52 pmol) were added. The cartridge was left shaking for 65 h. Afterwards including Dpm removal: After first crosslinking on SPPS resin from Friday 16:30 to Monday 9:30 (65 h, LuS0480) or from Tuesday (17:30) to Monday (9:30) (136 h, LuS0477), the resin in all three cartridge reactors was washed 3x with water, 3x with DMF and was first treated for 10 min with 2 mL 20 mM TCEP in water, second for 5 min with 4 mL 1 M BrAcOH in DMF / NMP / DIEA (50:25:25), third with 4 mL 2% w / v l-Cys in 0.6 M aq. NaHCO3 buffer for 15 min and fourth 2x with 2 mL DMF / Pip (4:1) for 7 min each. In between, the resin was washed 3x with water and 3x with DMF each time and after last treatment, it was washed 5x with DMF and left swelling in DMSO / EtOAc (2:8). Meanwhile, PEC-Linker RC+ was prepared as stated in 100 pmol PEC Guide (in DMSO / EtOAc (2:8); double amount for each cartridge) and was installed on N-terminus of the crude mono-crosslinked CRP for 1.5 h + 3x wash with DMF and 3x with DCM + drying. After negative Chloranil-Test, the peptide was cleaved from SPPS resin + peptide global deprotection with 18 mL TFA / PhSMe / Water / TIS (92.5:2.5:2.5:2.5) for 4 h. Synthesis of CRP-A using CRP-StBu as the starting monomer The CRP-StBu monomer was synthesized, the Trt was selectively deprotected and N-terminal was cross-linked on the SPPS resin, as described above. After Fmoc deprotection (also done on the SPPS resin) and swelling the mono-cross-linked CRP on SPPS resin in DMF for 15 min, a PEC-linker RC+ was installed on the N-terminus as follows: the DMF was removed by filtration and 0.7 mL of a mixture of PEC-linker RC+ (150 mg, 0.3 M, 4 eq.) in DMF with oxyma (43 mg, 6 eq.) and DIEA (53 pL, 6 eq.) was added to the resin. After shaking for 2 h, the mixture was filtrated off and the resin was washed 3x with DMF and 3x with DCM. Then the mono-cross-linked and linker modified CRP-StBu was capped with 2-BrAcOH and L-Cys using the following procedure: the SPPS resin was swollen in DMF for 5 min and after filtration of DMF, 100 pL of a 1 M solution of 2-bromoacetic acid in DMF / NMP / DIEA (75:12.5:12.5) per pmol assumed peptide amount was added and the resin was left shaking for 15 min. Subsequently, the resin was washed 3x with DMF. Thereafter, to the resin 100 pL of a 2 w% L-Cysteine solution in 0.6 M aq. NaHCOs per |jmol assumed peptide amount was added and the resin was left shaking for 15 min, followed by washing with 3x water and 3x DMF. The mono-cross-linked and linker modified CRP-StBu was cleaved from SPPS resin with 5 mL TFA / Water / TIS (94:4:2) for 2 h and then precipitated in cold EtzO (-20°C). The ether phase was discarded, and the remaining peptide pellet was dried. The dried crude material was dissolved in 2.5 mL of an aq. sodium citrate buffer (pH 4.5) and it was immobilized on the PEC resin (aldehyde modified agarose) for 90 min. Subsequently, the resin was washed with 3x 0.9 M GdmCI in DMSO, 3x 0.1 M aq. NaCI / EtOH (3:7) and once with water. The PEC-linker RC+ was reduced with a solution of 250 mg DTT in 2.5 mL aq. 0.6 M NaHCOs / EtOH (1:1) for 30 min. After reduction and additional wash steps with water and MeCN, the C-terminal cross-linking was done in 1 mL aq. NaHCOs / DMF / MeCN (1:1:1) with 0.5 mL T3 (7.5 eq., 0.15 M in MeCN) for 20 min. Finally, the double-cross-linked CRP (CRP-A) was released from PEC resin with 1 mL 40% TFA for 1 h and further eluted with 3x 1 mL TFA. After precipitation in EtzO, the peptide was collected by centrifugation and decantation of the organic phase and freeze-dried. 3.0 mg of the purified compound were obtained as a white solid. Synthesis of CRP-A using CRP-Dom as the starting monomer The CRP-Dpm monomer was synthesized, the Trt was selectively deprotected, the N-terminal was cross-linked on the SPPS resin, and capped with 2-BrAcOH and L-Cys (as described above). After Fmoc deprotection and installation of the PEC-linker RC+ on the N-terminus (as above), the mono-cross-linked and linker modified CRP-Dpm was cleaved from the SPPS resin (TFA / EDT / PhSMe / HzO / TIS 83:5:5:5:2) and immobilised on the PEC resin (DMSO, pH 3.5). Free aldehyde groups were blocked with HzNOMe, the second cross-linking performed, the PEC linker reduced and the double crosslinked CRP (CRP-A) released from the PEC resin. Immobilisation Efficiency Test (Example 6) To test the immobilization efficiency, UV / vis chromatography and Ellmans-Test was used. 6 of the 10 different 10 pmol aliquotes from LuS0400 were used. The solvents used are provided in example 6. The peptides were dissolved in 500 pL of each solvent composition. Immobilization was done overnight for 17 h. PMA-Beads: D6550 (old), loading = 93 pmol / g. 5 pL of the samples before and after immobilization were diluted 40x (+ 195 pL) with RB (RB = 100 mM sodium hydrogenphosphate, 1 mM EDTA, pH 8). Then, 780 pL RB and 20 pL 4 mg / mL Ellmans reagent in RB was added. After incubation for > 10 min, the samples UV-absorption was measured vs an RB blank at 412 nm. Furthermore, samples lOOx diluted were measured via UPLC-UV / vis with 7 min method for comparison before and after immobilization. Optimised Large Batch Method (Example 6) 140 pmol mono-crosslinked CRP-A was dissolved in sum in 7 mLTFA / water (1:1) and was added to 4520 mg PMA beads (washed beforehand with 3x water, 3x IB and lx water). Immobilization was done overnight (17 h). Small samples were measured before and after immobilization (lOOx diluted). After immobilization, the PMA beads were washed 3x with water and blocked 15 min with Buffer# 1 / 5% w / v aq. HzNOMe (4:1). The peptide on PMA beads was washed with 3x water and 3x MeCN. Second crosslinking was done by addition of 7560 pL MeCN I 5% w / v aq. NaHCO3 (1:1) and 210 pL 0.12 M T3 in MeCN (25.2 pmol). The cartridges were left shaking over 2 days (x h). Subsequently, the beads were washed (3x 5mL 0.9 M GdmCI in DMSO, 3x 5 mL 0.1 M NaCI in H2O / EtOH (3:7)), reduced with 7 mL 10% w / v DTT in 0.6 M aq. NaHCO3 buffer for 15 min and washed again with water and MeCN. Finally, the peptide was released with 4.2 mL 95% TFA in water for 45 min. From the PMA beads, the peptide was eluted using 2x 4.2 mL 95% TFA and then precipitated with 33 mL ice-cold Et20. After freezing the peptide in the freezer for 30 min (and in liquid nitrogen for 30-60 seconds), it was centrifuged, and the organic phase was removed by decantation. After letting the precipitate dry, it was redissolved in 7 mL water, measured lOx diluted via UPLC-UV / vis and lyophilized overnight. The peptide was then weighed. From the lyophilized peptides, RP-UPLC was recorded from a 0.66 mg / mL solution in 0.01 M AcOH and SE-UPLC from a 0.66 mg / mL solution in aq. PB (pH 7.4) / MeCN (8:2). Platelet activation using different batches of CRP-A in whole blood samples Human blood samples were collected into monovette tubes containing 3.2% trisodium citrate from healthy volunteers, following written informed consent. To prevent platelet aggregation and preserve the platelets as individual cells, EDTA (final concentration of 4.0 mM) was added to the whole blood (WB). The WB was warmed in a dry bath for 10 minutes at 37°C and added to a 96-well round-bottom plates containing CRP-XL and three different batches of CRP-A (S0396, 2022-3 and 2022-4) at the concentrations of 0.0, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0 and 10.0 pg / mL (each condition was completed in duplicates). At 37°C the WB was stimulated with the CRP-A for 5 minutes and then platelet activation marker fixative (PAMFix, Platelet Services Ltd.) was added to the samples to stop stimulation. The fixed WB samples were analysed on the flow cytometer by dual labelling with anti-GPIIIa antibody (CD61-Alexa 647), used as a platelet marker, and with anti-P-selectin antibody (CD62P-FITC) to measure the level of P-selectin expression. Three thousand CD61-positive events were acquired and the % of platelets that expressed P-selectin and corresponding median fluorescence intensity values (MFI) were recorded. The negative logarithm of half maximal effective concentration (pEC50) for CRP-XL and the three different batches of CRP-A were calculated from the line graphs in PRISM software (version 9). Measuring the effect of different batches of CRP-A on platelet aggregation Human blood samples were collected into monovette tubes containing 3.2% trisodium citrate from healthy volunteers, following written informed consent. Platelet rich plasma (PRP) was obtained by centrifugation of whole blood (WB) at 218 x g for 10 minutes and platelet poor plasma (PPP) was obtained by centrifugation of the residual blood at 1859 x g for 10 minutes which was required to set 100% light transmission on the AggRam. Platelet rich plasma was dispensed into cuvettes containing a magnetic stir bar and the cuvettes were placed into the warming wells (37°C) of the AggRAM without agitation. After 2 minutes of warming, the cuvettes were moved to the optical wells of the AggRAM with stirring and light transmission was set to record for one minute to establish the 0% transmission baseline. After one minute of stirring, CRP-XL and three different batches of CRP-A (S0396, 2022-3 and 2022-4) at the concentrations of 0.0, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0 and 10.0 pg / mL were added to the PRP and the aggregation responses were recorded for 6 minutes. Activating platelets in the presence of glenzocimab Human blood samples were collected into monovette tubes containing 3.2% trisodium citrate from healthy volunteers, following written informed consent. To prevent platelet aggregation and preserve the platelets as individual cells, EDTA (final concentration of 4.0 mM) was added to the whole blood (WB). The WB was incubated with either glenzocimab at 20.8 pg / mL (final assay concentration of 20.0 pg / mL) or PBS (vehicle control) in a dry bath for 10 minutes at 37°C. After 10 minutes of incubation the WB was added to a 96-well round-bottom plates containing two different batches of CRP-A (collagen related peptide), 2022-3 and 2022-4, at the concentrations of 0.0, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0 and 10.0 pg / mL and thrombin receptor activating peptide (TRAP) at the concentrations of 0.0, 1.25, 2.5, 5.0, 7.5, 10.0, 15.0 and 20.0 pM (each condition was completed in duplicates). At 37°C the WB was stimulated with 2022-3, 2022-4 and TRAP with and without glenzocimab for 5 minutes and platelet activation marker fixative (PAMFix, Platelet Services Ltd.) was added to the samples to stop stimulation. The fixed WB samples were analysed on the flow cytometer by dual labelling with anti-GPIIIa antibody (CD61-Alexa 647), used as a platelet marker, and with anti-P-selectin antibody (CD62P-FITC) to measure the level of P-selectin expression. Three thousand CD61-positive events were acquired and the % of platelets that expressed P-selectin (% positive) and corresponding median fluorescence intensity values (MFI) were recorded. The raw data was normalised to the % response of the highest CRP or TRAP-induced platelet activation without the inhibitor. Two-year stability tests (Example 13) RP-UPLC chromatograms were measured on a Waters H-Class Acquity UPLC with a Acquity Premier Peptide CSH C18 (50x2.1 mm, particle size 1.7 pm, pore size 13 nm) using a gradient of 0.1 % Trifluoroacetic acid (TFA) in acetonitrile from 0-70% in 0.1 % TFA in water over 7 min. SEC-UPLC chromatograms were measured on the same device with a YMC-Pack Diol-60 column (150x4.6 mm, particle size 3 pm, pore size 6 nm), using 0.1 M phosphate buffer (pH 7.4 ) / acetonitrile (8:2) over 15 min. Analysis Unless otherwise stated, the following analysis methods were used. UV-Chromatograms were recorded on an analytical Acquity H-Class UPLC-UV / vis-ESI-MS system from Waters on a C-18 column (1.7 pM, 2.1 x 500 mm). As mobile phase, mixtures of water (A) and MeCN (B) with 0.1% TFA were used and the sample was measured with a gradient of 0% B to 60% B in 5 min or 0% B to 60% B in 11 min. UV-purity was determined by peak-integration at a wavelength of 210 nm, identity was determined by ESI-MS with Cone-Voltage of 5 or 20 V. Analytical samples were prepared by dissolution of solid material in water / MeCN (7:3) + 0.1% TFA and dilution with water / MeCN (7:3) + 0.1% TFA or saturated aq. GdmCI. MALDI-MS spectra were recorded on a Bruker Autoflex II Smartbeam in linear mode from 3000-15000 Da. For ionization, a smartbeam Laser (220 nm, 200 Hz, 24 kV) was used. For detection, a Time-Of-Flight (TOF) analyzer was used. Analytical samples were prepared by dissolution of solid material in water / MeCN (7:3). Samples were 5 desalted via flash chromatography or by usage of ZipTip sample prep, pipette tips from Merck (0.6 pL, C18 resin). Flash chromatography was done with a Selekt Flash Chromatography Instrument from Biotage on a Biotage Sfar Bio C18 D column (25 g, CV = 41 mL). As mobile phase, 10 mixtures of water (A) and MeCN (B) with 0.1% TFA were used.

Claims

1. A compound of Formula I:(I)a point of bonding to a carbon atom which is also bonded to the R2 group; L2 is a linker; andnl and n2 are each independently 0 or an integer between 1 and 5;15 or a cosmetically or pharmaceutically acceptable salt or solvate thereof.

2. The compound according to claim 1, wherein L2 isL10L8, wherein X1 is a phenyl, a 6 membered heterocycleoptionally substituted with one or more oxo groups, a 6 membered heteroaryl, cyclohexyl or N; and L8 is absent or is CO; L9 is absent or is a C1-3 alkyl; L10 is absent or is C=O, C(OH), *-NHCO-, *-NHSO-, *-NHSO2- or *-OC(O)-, where an asterisk indicates a point of attachment to L9, or in embodiments where L9 is absent to L8 or X1; and L11 is absent or is a C1-3 alkyl.3.The compound according to claim 2, wherein L2 is4. The compound according any one of the preceding claims , wherein nl and n2are each independently an integer between 1 and 3.

5. The compound according to any one of the preceding claims, wherein thecompound is a compound of formula lai:(lai)OHOHL1 is6. A composition comprising a compound of formula II and a compound of formula III:(II)h2nwherein R4 isR5 is           OH or -COR3;R3 is OH or NH2; andOHL1 is OH                                           , where an asterisk indicates10 a point of bonding to a carbon atom which is also bonded to the R4 group;L2 is a linker;L3 is a linker; andnl and n2 are each independently 0 or an integer between 1 and 5;or a cosmetically or pharmaceutically acceptable salt or solvate thereof;15 wherein the molar ratio of the compound of formula II to the compound of formula Illa is at least 2.3:1.

7. A method of producing a compound of formula II:(II)5   , wherein L1, L2, R4, R5, nl and n2 are as defined in claim 6;the method comprising:providing a support with a plurality of peptide monomers disposed thereon,wherein the support and plurality of monomers are represent by formula (IV):15 COO*- or -CON(H)*-; where an asterisk indicates a point of bonding to S1;- 67 -OPG3L5 is L1 or OPG3                                        , where an asteriskindicates a point of bonding to a carbon atom which is also bonded to the L4 group;PG1, PG2 and PG3 are each a protecting group; andn is an integer of at least 3;5   - contacting the support and plurality of monomers of formula (IV) and a firstcross-linking reagent to provide a mono cross-linked trimer disposed on the first support, which is represented by formula (V):PG2 iPG2(V) 10 wherein L2 is as defined in the second aspect; and- removing protecting group PG1 from the mono cross-linked trimer to thereby deprotect N-termini of the mono cross-linked trimer, contacting the mono cross-linked trimer and a support attachment reagent to thereby dispose a support attachment group onto each of the N-termini, cleaving the mono cross-linked trimer from the first5 support, removing protecting groups PG2 from the mono cross-linked trimer and contacting the mono cross-linked trimer and a second support to immobilize it thereon, wherein the mono cross-linked trimer immobilized on the second support isrepresented by formula (VI):(VI)wherein L6 is a linker; and S2 is a second support;- contacting the immobilized cross-linked trimer of formula (VI) and a second cross-linking reagent to provide a double cross-linked trimer; and15   - cleaving the double-cross-linked trimer from the second support to provide thecompound of Formula (II);OPG3OPG3and / or L4wherein if L5 ismethod further comprises removing protecting group PG3.

8. The method according to claim 7, wherein the protecting groups PG1 and PG2 are orthogonal.

9. The method of claim 7 or claim 8, wherein the first cross-linking reagent is a compound of formula (VIII):(VII)wherein LG is a leaving group.

10. The method according to any one of claims 7 to 9, wherein the method comprises:- removing protecting group PG1 from the mono cross-linked trimer to thereby provide a mono-cross-linked trimer of formula (IX):PG2 iPG2 (IX), wherein R7 is 0 or NH2; and subsequently- contacting the mono cross-linked trimer of formula (IX) and a support attachment reagent to provide a mono-cross-linked trimer of formula (X):, wherein R8 isPG2 iPG2(X)or NHR9; andR9 is a support attachment group.

11. The method according to claim 10, wherein R9 is -LZ-O-NR^R11, wherein L7 is a linker and R10 and R11 are each H or PG5, wherein PG5 is a protecting group.

12. The method according to any one of claims 7 to 11, wherein the support attachment reagent is a compound of formula (XI):X-Tb-Va-U-Y-Z(XI), wherein„                 R11 PG5I PG5 A, A N. A On A JI r10 n N jiX is R10' "°   ,              R12 ,   R12 orR13V;each R10 and R11 is independently from each other selected from H or PG5, wherein at least R10 or R11 is PG5;R12 is selected from H or PG5;R13 is selected from H, C1-12 alkyl or C6-12 aryl, wherein the aldehyde or keto group may be protected by an acid labile protecting group;PG5 is an acid labile amine protecting group;T is a linear or branched spacer comprising at least one of the moieties -C1-12 alkylene, (-C2H4O-)1-12, -C(=O)-, -C(=0)-JR14-, -JR14-C(=0)-, -JR14-, phenylene, 5- or 6membered heteroarylene, whereinJ is CH or N,R14 is selected from H, Ci-4 alkyl, -Ci-6 alkyl-NH2, -Ci-6 alkyl-NHPG6, -Ci-6 alkylene-NPG62, -R17, -C1-6 alkyl-NH-R17, -C1-6 alkyl-NH-R17';PG6 is an acid labile amine protecting group;R17 is a blocking agent that is able to react with an aldehyde moiety;b is 0 or 1,V is an electron-withdrawing moiety selected from -NR18-C(=O)-, -C(=O)-NR18-,-S(=O)-, -NR19-(CH2)p-, -piperazinylene-(CH2)P-, pyridinylene, pyrimidinylene, q / Ai-(CH2)P4-(U)pyrazinylene, pyridazinylene,                         , -C(=O)-, -C(=O)-O-, , whereinR18 is selected from H and Ci^alky,R19 is selected from H and Ci-4,p is 0, 1 or 2,a is 0 or 1, wherein the sum of a and b is 1 or 2,U is a phenylene or a five- or six-membered heteroarylene moiety, that is bound to at least one of the moieties V, Wq and En and that may optionally be substituted by a C1-6 alkyl, and preferably by a Ci-3 alkyl, whereinW is selected from -N3, -NO2, -N=N-phenyl, -S(=O)-R20, -S-S-R20, -O-CH2-N3,-N=N-R20 is pyridyl, pyrimidinyl, pyrazinyl, pyridazyl, C1-6alkyl or -(CH2)P-NMe2, with p being 1, 2, 3 or 4;E is an electron withdrawing group under acidic conditions;n being is an integer between 0 and 4, and q is an integer between 0 and 4, wherein the sum of n and q is equal or lower than 4; andin case of U being a phenyl moiety and Y being -(CH2)m-O-C(=O)-, the sum of Hammett constants of V, W, E under acidic conditions is larger than 0.45, and wherein W is in ortho or para position in relation to Y;Y is -(CH2)m-C(=O)- or -(CH2)m-O-C(=O)- with m being 1 , 2 or 3, andZ is an electron-withdrawing leaving group.

13. The method of claim 12, wherein the compound of formula (XI) is(Boc)2N14. The method according to any one of claims 7 to 13, wherein the method comprises simultaneously cleaving the mono cross-linked trimer from the first support and removing protecting group PG2 from the mono cross-linked trimer.

15. The method according to any one of claims 7 to 14, wherein the second support is comprises a plurality of aldehyde groups.

16. A composition comprising the compound of any one of claims 1 to 5, or a cosmetically or pharmaceutically acceptable salt or solvate thereof, or the composition of claim 6 and a carrier.

17. A composition comprising the compound of any one of claims 1 to 5, or a cosmetically or pharmaceutically acceptable salt or solvate thereof, or the composition of claim 6 and platelet rich plasma (PRP).

18. The composition of claim 17 for use as a medicament.

19. The composition of claim 17 for use in the treatment of a skin condition.

20. A method of cosmetic treatment, the method comprises administering to a subject a cosmetically effective amount of the composition of claim 17.

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