Peptide conjugates comprising blood brain barrier penetrating oligopeptides for use in therapeutic and diagnostic methods

Abstract

The present invention relates to peptide conjugates that comprise a plant-derived oligopeptide capable of crossing the blood-brain barrier (BBB) and a therapeutic or diagnostic agent. The BBB-penetrating oligopeptide is an oligopeptide according to Formula I, Asp–R2–Gly–Leu–R5–R6–R7–Leu–Gly–R10–R11–R12, wherein R2 represents Arg, Lys, Cyt, D-Arg or Orn; R5 represents Phe, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, Orn, 4-aminophenylalanine or 3-phenylpropionate; R6 represents Pro, Leu, Glu or Lys; R7 represents Phe or Trp; R10, R11 and R12 each independently represent Lys, Arg or Orn; or an oligopeptide according to Formula II, Glu–R2'–R3'–Gly–R5'–R6'–Glu–R8'–R9'–R10'–Glu–R12'–Leu–Pro–Gly, wherein R2', R3', R8', R10' and R12' each independently represent Lys, Arg or Orn; R5' represents Phe, Ile, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, 4-aminophenylalanine, or 3-phenylpropionate; R6' represents Met, Leu, Ile, Asn, norleucin, D-norleucin, seleno- methionine or D / L-2-hydroxy-(4-methylseleno)butanoic acid; and R9' represents Leu or Ile. The conjugates comprising a BBB-penetrating oligopeptide compound and a therapeutic agent can be used in treatments for diseases of the central nervous system (CNS). Furthermore, conjugates with a BBB-penetrating oligopeptide compound and a diagnostic agent can be used in both in vivo and in vitro diagnostic methods.

Description

[0001] PEPTIDE CONJUGATES COMPRISING BLOOD BRAIN BARRIER PENETRATING OLIGOPEPTIDES FOR USE IN THERAPEUTIC AND DIAGNOSTIC METHODS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to peptide conjugates that comprise a plant-derived oligopeptide capable of crossing the blood-brain barrier (BBB) and a therapeutic or diagnostic agent. The conjugates comprising a BBB-penetrating oligopeptide compound and a therapeutic agent can be used in treatments for diseases of the central nervous system (CNS). Furthermore, conjugates with a BBB-penetrating oligopeptide compound and a diagnostic agent (diagnostic probe) can be used in both in vivo and in vitro diagnostic methods.

[0004] BACKGROUND

[0005] The development of peptides as drug-delivery agents is a significant area of research and a critical aspect of modern pharmaceutical science. Peptides are known for being versatile molecules that can be linked to drugs. US patent US10653801B2 describes short-chain sequences of targeting peptides used for diagnostic and cancer therapy purposes.

[0006] Reversible opening of biological barriers is one of the ways to increase drug delivery to the systemic circulation orthe CNS. Bocsik et al. (Bocsik et al., J Pharm Sci. 2016 Feb;105(2):754- 765) investigate the opening effects of different cell -penetrating peptides (CPPs) on both cells of the epithelial barrier and a model of the BBB.

[0007] Kalmar's thesis (LC-MS investigation of lysosomal metabolism of peptide-drug conjugates (Chemist MSc Thesis, ELTE, Research Group of Peptide Chemistry, 2019) and other papers present several examples of the delivery peptides used in cancer therapy. In these works, peptide-drug conjugates were investigated, and they consisted of peptide targeting units and daunomycin as a delivered cytostatic drug (Gomena J, et al., Eur J Med Chem. 2024 Nov 5;277: 116767; Mezo G, et al. Int J Mol Sci. 2024 Feb 3;25(3): 1864; Szabo I, et al. Int J Mol Sci. 2024 Jan 16;25(2): 1095).

[0008] Furthermore, Baranyai et al, described that cell-penetrating and tandem peptides can deliver therapeutic drugs cross in vitro simple HUVEC BBB barrier model, these peptides have limitations caused by their cytotoxicity over 25 pM (Baranyai Z, et al. J Med Chem. 2021 Mar 25;64(6):2982-3005).

[0009] Peptides carrying imaging agents / probes / radiopahrmacores etc. are described in these papers: In vivo imaging of tumor hypoxia and angiogenesis with positron emission tomography (PET) is playing an increasingly important role in the diagnosis of tumors. In addition, more effective antitumor treatments can be planned by using new, specific radiopharmaceuticals that detect angiogenesis and hypoxia in malignant tumors (Kis A, et al., Biomed Res Int. 2020 Aug 7;2020:4952372).

[0010] Selection of the appropriate 68Ga -labelled NGR-peptide based radiopharmaceutical (e.g.,68Ga-NOTA- and NODAGA-c(NGR) with the highest binding affinity in this study) is critical for the precise detection of tumors neo-angiogenesis and for monitoring the efficacy of anti cancer therapy (Denes N, et al., Appl Radiat Isot. 2021 Aug; 174: 109778.; Kis A,et al, Int J Pharm. 2020 Nov 15;589: 119881).

[0011] A new, combined HER2 binding conjugate is obtained that interacts with HER2- overexpressing cells with high affinity and specificity. The Her-2 binding specific peptides and their highly specific conjugate can be used for diagnostical or drug-targeting purposes in Her- 2 expressing tumors (Biri-Kovacs B, et al., Biomolecules. 2020 Jan 25; 10(2): 183).

[0012] CNS diseases are among the most difficult to treat, mainly because most drugs are unable to cross the BBB and reach the brain tissue in therapeutically effective concentrations. There are more than 150 peptides in clinical development, and the field of peptide carriers and shuttles for brain delivery has significantly evolved, with several candidates in clinical trials (Lau JL and Dunn MK, Bioorg Med Chem. 2018 Jun l;26(10):2700-2707). One primary focus of these studies is on CPPs. CPPs possess beneficial properties, including relatively low immunogenicity and biocompatibility. They are positively charged and composed of fewer than 30 amino acid residues.

[0013] CPPs have been used to deliver both small molecules and proteins to the brain, with applications in diverse therapeutic areas such as brain cancers, neurodegenerative diseases, and imaging (Ayo A and Laakkonnen P, Pharmaceutics. 2021 Apr 2; 13(4):481.; Parrasia S, et al., Mol Pharm. 2022 Nov 7;19(11):3700-3729). The development of drugs for treating brain- related diseases is hampered by the difficulty of delivering them through the BBB. Most peptides have unfavorable physicochemical properties for unrestricted permeation. Therefore, the discovery and introduction of novel BBB -shuttle peptides are in high demand (Yoneda Y, et al., Bioorg Med Chem Lett. 2008 Mar 1;18(5): 1632-1636., 1632-1636.; Banks WA, Peptides. 2015 Oct; 72: 16-19; Oiler-Salvia B, et al., Chem Soc Rev. 2016 Aug 22;45(17):4690-707).

[0014] US patent No. US 9 993 564 B2 describes an invention that comprises a BBB shuttle, comprising a brain effector entity and a brain-targeting peptide. This system utilizes engineered peptides that can selectively bind to receptors on the BBB, facilitating the transport of drugs into the brain. Bioactive peptides of plant origin, are known as phyto-peptides. They can act as potential drug carriers or shuttles. Interest in these biomolecules is increasing, and recently, a manually curated database of plant-originated peptides with various functions and therapeutic activities has been compiled (Das D, et al., Sci Rep. 2020 Feb 10; 10(l):2194). The lysine-rich segments of these peptides, called K-segments, contribute to chaperone activity and support cell viability under stress conditions (Kovacs D, et al., Plant Physiol. 2008 May;147(l):381-90.; Graether SP and Boddington KF, Front Plant Sci. 2014 Oct 31;5 :576). In various plant dehydrins (DHN1, Lti30), K-segments have been identified as the interaction sites with anionic lipid bilayers (Koag M. C, et al., Plant Physiol. 2009 Jul;150(3): 1503-14.; Eriksson SK, et al., Plant Cell. 2011 Jun;23(6):2391-404).

[0015] The article by Hara et al. (Hara et al., J Plant Physiol. 2017 Mar:210: 18-23) presents data on the cryoprotective role of the ERD-C (E K K G I L E K I K E K L P G) peptide and some of its variants. The publication describes dehydrins, which are group II late embryogenesis abundant proteins that accumulate in plants during exposure to abiotic stresses, including low temperature. They contain conserved lysine-rich K-segments which are related to their cryoprotection activity. However, the specific sequence features that contribute to cryoprotection have yet to be determined. The mutation of the isolated sequence demonstrated that hydrophobic amino acids are clearly involved in preventing cryoinactivation, cryoaggregation, and cryodenaturation of the enzyme lactate dehydrogenase. The study provides no evidence of drug delivery properties, nor does it discuss possibilities for BBB penetration.

[0016] In a recent study, Sebak et al. (Sebak F, et al ACS Omega. 2021 Dec 6;6(50):34470-34484) describe the synthesis of novel CPPs, z.e., ERD-A, ERD-B, and ERD-C peptides and publish experimental data on their structural characterization and cellular internalization. These systems are considered potential candidates for delivering small -molecule organic drugs into cells as well. These lysine-rich peptides are derived from the plant dehydrin ERD14. Peptides were conventionally labeled with carboxyfluorescein (Cf), to track their internalization into cells. The study compares the effect of (5)-Cf and (6)-Cf conjugates on the structural properties of the peptides. The results show that the peptides enter cells through energy -dependent pathways with localization in different cellular compartments (e.g., cytosol, lysosomes, and vesicles). Specific residues are of great importance, such as aromatic side chains and proline residues, which are found to influence the internalization and efficacy of the peptides. Plant-derived peptides show potential advantages over animal -derived peptides, including reduced allergenicity and cytotoxicity. (Allergenicity depends on the peptide sequence and structure, as well as its interaction with the immune system, particularly the presence of B -cell epitopes that trigger IgE-mediated responses.)

[0017] The atomic level characterization of the isolated ERD-A, ERD-B and ERD-C peptides has been performed and nuclear magnetic resonance (NMR) parameters were deposited in the Biological Magnetic Resonance Data Bank under BRMB Entry 51040 (2021-07-29) for ERD- A, Entry 51039 (2021-07-29) for ERD-B, Entry 51041 (2021-07-29) for ERD-C.

[0018] The special cells of the BBB at the level of the brain microvasculature hamper the entry of the potential neuropharmaceuticals, especially biopharmacons, peptide or protein drugs into the CNS by restricted para- and transcellular mechanisms. Several clinical trials ended with failure because of the low penetration of the biologic drugs across the BBB (Gosselet F et al., Neurochem Int. 2021 Mar; 144: 104952). Therefore, the general ability of peptides to internalize across cell membranes does not imply that they are also capable of crossing the highly complex and strictly regulated.

[0019] The currently available oligopeptide compounds used for drug delivery across the BBB do not possess ideal biophysical properties, particularly in terms of solubility, toxicity, and efficient drug delivery. Therefore, we seek biomolecules with improved performance compared to standard biomolecules, which (a) cross the BBB more efficiently even in larger amounts; (b) have no, or reduced cytotoxicity, and (c) are capable of drug delivery.

[0020] Surprisingly, it has been discovered that plant-derived oligopeptides, ERD-A, ERD-B, their variants and their conjugates to a therapeutic or diagnostic agent capable of crossing the BBB. Coupling therapeutic or diagnostic agents to these oligopeptides allows fortheir delivery across BBB.

[0021] SUMMARY OF THE INVENTION

[0022] Therefore, the present invention relates to a peptide conjugate for use in the treatment of a disease of the central nervous system (CNS), comprising a blood-brain barrier (BBB) penetrating oligopeptide and a therapeutic agent for the treatment of a disease of the CNS, which BBB -penetrating oligopeptide is an oligopeptide according to Formula I,

[0023] Asp-R2-Gly-Leu-R5-R6-R7-Leu-Gly-R10-Rl 1-R12 (Formula I) wherein

[0024] R2 represents Arg, Lys, Cyt, D-Arg or Om; R5 represents Phe, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, Orn, 4-aminophenylalanine or 3 -phenylpropionate;

[0025] R6 represents Pro, Leu, Glu or Lys;

[0026] R7 represents Phe or Trp;

[0027] RIO, R11 and R12 each independently represent Lys, Arg or Orn; or an oligopeptide according to Formula II,

[0028] Glu-R2’-R3’-Gly-R5’-R6’-Glu-R8’-R9’-R10’-Glu-R12’-Leu-Pro-Gly (Formula II), wherein

[0029] R2’, R3’, R8’, RIO’ and R12’ each independently represent Lys, Arg or Orn;

[0030] R5’ represents Phe, He, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, 4-aminophenylalanine, or 3 -phenylpropionate;

[0031] R6’ represents Met, Leu, He, Asn, norleucin, D-norleucin, selenomethionine or D / L-2-hydroxy-(4-methylseleno)butanoic acid;

[0032] R9’ represents Leu or He.

[0033] Preferably the oligopeptide is an oligopeptide according to Formula I, wherein

[0034] R2 represents Arg;

[0035] R5 represents Phe or Trp;

[0036] R6 represents Asp or Pro;

[0037] R7 represents Phe or Trp;

[0038] RIO, Rl l and R12 represent Lys; or an oligopeptide according to Formula II, wherein

[0039] R2’, R3’, R8’, RIO’ and R12’ represent Lys;

[0040] R5’ represents Phe; and

[0041] R6’ represents Met; preferably the oligopeptide is

[0042] Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A; SEQ ID NO: 1),

[0043] Asp-Arg-Gly-Leu-Phe-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A D6P; SEQ ID NO:2), Asp-Arg-Gly-Leu-Trp-Asp-Trp-Leu-Gly-Lys-Lys-Lys (ERD-A F5,7W; SEQ ID NO:3), or

[0044] Glu-Lys-Lys-Gly-Phe-Met-Glu-Lys-Leu-Lys-Glu-Lys-Leu-Pro-Gly (ERD-B; SEQ ID NO:4). The present invention also relates to a pharmaceutical composition for use in the treatment of a disease of CNS comprising the peptide conjugate and a pharmaceutically acceptable excipient.

[0045] The pharmaceutical composition may be formulated for administration via parenteral, including intrathecal, subcutaneous, intravenous or intramuscular administration.

[0046] The present invention also encompasses a peptide conjugate for use in an in vivo method of diagnosis of a disease of CNS, comprising a BBB -penetrating oligopeptide and a diagnostic agent specific for said disease, which BBB -penetrating oligopeptide is an oligopeptide according to Formula I,

[0047] Asp-R2-Gly-Leu-R5-R6-R7-Leu-Gly-R10-Rl 1-R12 (Formula I) wherein

[0048] R2 represents Arg, Lys, Cyt, D-Arg or Om;

[0049] R5 represents Phe, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, Orn, 4-aminophenylalanine or 3 -phenylpropionate;

[0050] R6 represents Pro, Leu, Glu or Lys;

[0051] R7 represents Phe or Trp;

[0052] RIO, R11 and R12 each independently represent Lys, Arg or Orn; or an oligopeptide according to Formula II,

[0053] Glu-R2’-R3’-Gly-R5’-R6’-Glu-R8’-R9’-R10’-Glu-R12’-Leu-Pro-Gly (Formula II), wherein

[0054] R2’, R3’, R8’, RIO’ and R12’ each independently represent Lys, Arg or Orn;

[0055] R5’ represents Phe, He, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, 4-aminophenylalanine, or 3 -phenylpropionate;

[0056] R6’ represents Met, Leu, He, Asn, norleucin, D-norleucin, selenomethionine or D / L-2-hydroxy-(4-methylseleno)butanoic acid;

[0057] R9’ represents Leu or He.

[0058] Preferably, the oligopeptide is an oligopeptide according to Formula I, wherein

[0059] R2 represents Arg;

[0060] R5 represents Phe or Trp;

[0061] R6 represents Asp or Pro;

[0062] R7 represents Phe or Trp;

[0063] RIO, Rl l and R12 each independently represent Lys; or an oligopeptide according to Formula II, wherein

[0064] R2’, R3’, R8’, RIO’ and R12’ represent Lys; R5’ represents Phe;

[0065] R6’ represents Met; and

[0066] R9’ represents Leu; preferably the oligopeptide is

[0067] Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A; SEQ ID NO: 1), Asp-Arg-Gly-Leu-Phe-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A D6P; SEQ ID NO:2), Asp-Arg-Gly-Leu-Trp-Asp-Trp-Leu-Gly-Lys-Lys-Lys (ERD-A F5,7W; SEQ ID NO:3), or

[0068] Glu-Lys-Lys-Gly-Phe-Met-Glu-Lys-Leu-Lys-Glu-Lys-Leu-Pro-Gly (ERD-B; SEQ ID NO:4).

[0069] Another aspect of the invention is a diagnostic composition for use in an in vivo method of diagnosis of a disease of CNS comprising the peptide conjugate as defined above and a diagnostically suitable excipient.

[0070] Furthermore, the invention relates to the use of the peptide conjugate comprising a BBB- penetrating oligopeptide compound as defined above and a diagnostic agent in an in vitro diagnostic method that requires the peptide conjugate to cross the blood-brain barrier for detecting a molecular entity.

[0071] BRIEF DESCRIPTION OF THE FIGURE

[0072] Figure 1: The effect of (A) ERD-A, (C) ERD-B, their carboxyfluorescein (Cf) conjugates (B) Cf-ERD-A, (D) Cf-ERD-B, and modified ERDA peptides (E) ERDA-D6P and (F) ERDA-F5- 7W on the viability of endothelial cells after 24 hours treatments. Values presented are means ± SD and are given as normalized cell index. Statistical analysis: One-way ANOVA followed by Bonferroni posttest, *p <0.05; **p <0.01; ****p <0.0001 compared to control group, n=6- 12. ns: not significant; Control: medium treated group.

[0073] Abbreviations

[0074] C or Cys Cysteine

[0075] D or Asp Aspartic acid

[0076] E or Glu Glutamic acid

[0077] F or Phe Phenylalanine

[0078] G or Gly Glycine

[0079] H or His Histidine I or He Isoleucine

[0080] K or Lys Lysine L or Leu Leucine

[0081] M or Met Methionine

[0082] N or Asn Asparagine P or Pro Proline

[0083] Q or Gin Glutamine R or Arg Arginine W or Trp Tryptophan Y or Tyr Tyrosine Cit Citrulline

[0084] Orn Ornithine

[0085] ERD14 is a 185-residue plant stress protein from Arabidopsis thaliana that is intrinsically disordered and functions as a chaperone, preventing heat-induced aggregation or inactivation of various substrates.

[0086] ERD-A: arbitrarily selected Lys-rich region of ERD14 protein, provided that its amino acid sequence is: Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (D R G L F D F L G K K K) (SEQ ID NO:1).

[0087] ERD-B: arbitrarily selected Lys-rich region of ERD14 protein, provided that its amino acid sequence is: Glu-Lys-Lys-Gly-Phe-Met-Glu-Lys-Leu-Lys-Glu-Lys-Leu-Pro-Gly (E K K G F M E K L K E K L P G) (SEQ ID NO:4).

[0088] Based on the recent study by Sebak et al. (Sebak F, et al ACS Omega. 2021 Dec 6;6(50):34470-34484), in our investigations we focused on Lys-rich CPP peptides from plant dehydrin ERD14. We chose to investigate the two Lys-rich region, denoted ERD-A and ERD- B, and synthesized and characterized their derivatives. We conducted biophysical, structural, and cellular uptake analyses of ERD segments comprising 12-15 amino acid residues and their derivatives, followed by an assessment of their ability to penetrate the BBB.

[0089] These oligopeptide compounds of the invention can be rapidly synthetized using established solid-phase peptide synthesis techniques, and they are readily obtainable in large quantities.

[0090] ERD-A and ERD-B, their synthesized derivatives are usually relatively chemically stable and tolerate the more severe conditions applied during modification and conjugation. The peptide sequence can be modified with amino acids or groups that do not interfere with their penetration abilities across BBB / selective binding abilities to human cells, but positively affect the physicochemical properties of the entire conjugate. This process allows for the enhancement of the water solubility of the drug or conjugates and stability within the bloodstream. Modifications of amino acids can effectively delay their elimination through the kidneys or reduce their accumulation in the kidneys. Additionally, peptide-drug conjugates, due to their small size, typically do not elicit an immune response.

[0091] To optimize their intracellular traffic, the amino acid sequence of plant-derived cationic peptides should be adjusted for the intended purpose. Therefore, variants of the ERD-A and -B peptide containing the specified substitutions in accordance with the invention can be synthesized.

[0092] The ERD-A and -B variants may contain amino acid substitutions or mutations that do not significantly impact their biological properties, such as BBB -penetrating activity. These conservative amino acid substitutions are well understood.

[0093] However, the disclosed variants preferably contain mutations that confer enhanced efficacy and / or improved physicochemical properties to facilitate BBB penetration compared to the parent peptides, making them especially suitable for therapeutic and diagnostic uses.

[0094] The variants of oligopeptide according of Formula I may contain 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions, preferably not more than 3 substitutions.

[0095] The variants of oligopeptide according of Formula II may contain 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions, preferably not more than 3 substitutions.

[0096] The following was taken into account when variants of the ERD-A and ERD-B peptides were designed.

[0097] We introduce mutations that affect the total molecular charge (Lys, K; Glu, E; Asp, D), influence membrane binding and BBB -penetrating ability, and also mutations that impact the length and polarity of the charged amino acid residues (Leu, L; Glu, E). Pro increases conformational heterogeneity and prevents the formation of secondary structural propensities. Substitutions with lysine (Lys, K) and leucine (Leu, L) generally enhance membrane binding and cell penetration, whereas substitutions with glutamic acid (Glu, E) and aspartic acid (Asp, D) tend to reduce these properties.

[0098] The mutations can fine-tune the strength of membrane interaction by utilizing various amino acid residues with different aromatic side chains. Arginine (Arg, R), lysine (Lys, K) hydrophilic, polar residues also contribute through increased charge, while tryptophan (Trp, W) tyrosine (Tyr, Y), phenylalanine (Phe, F) enhance hydrophobicity. Substitutions with arginine Arg (R), Lys (K), Trp (W), Tyr (Y), Phe (F) generally enhance the membrane-binding and cellpenetrating properties of peptides.

[0099] The oligopeptides must maintain a constant positive charge. Arginine (Arg, R) keeps its zwitterion structure over a broader pH range than lysine (Lys, K). Therefore, an R / K substitution can enhance the cellular uptake of the peptide conjugate, as well as its selectivity and solubility.

[0100] Since tryptophan (Trp, W) among the amino acids with aromatic side chains tends to interact more strongly with the membrane, it can be preferable to substitute phenylalanine for tryptophan (F— W). This is indirectly demonstrated by the better penetration activity of the Trp (W) variants (see Table 1, ERD-A F5,7W).

[0101] Proline (Pro, P) provides greater structural heterogeneity due to the cis / trans isomerism. The presence of the amphipathic Pro instead of D (Asp) increases cellular uptake and selectivity (see Table 1, ERD-AD6P).

[0102] Substituting to any amino acid to cysteine (Cys, C), histidine (His, H), Isoleucine (He, I), methionine (Met, M), or ornithine (Orn) can enhance stability and uptake. For His, this effect is particularly pronounced under mildly acidic conditions, whereas for Orn, it becomes evident when substituting for arginine (Arg, R).

[0103] Substitution with glutamine (Gin, Q) generally exerts a neutral to slightly negative impact on membrane binding and cell-penetrating capabilities of a peptide, while enhancing its aqueous solubility.

[0104] Substitutions with glycine (Gly, G), asparagine (Asn, N), proline (Pro, P), and citrulline (Cit) generally reduce the membrane-binding and cell-penetrating capabilities of peptides. In the case of proline, this effect is particularly pronounced when the original sequence relies on structured elements such as a-helices or P-sheets. Consequently, its impact is highly position- and context-dependent.

[0105] The designed peptides were synthesized using solid-phase peptide synthesis (see Example 1).

[0106] According to the invention, the ERD-A, ERD-B, and their variants are conjugated to a therapeutic or diagnostic agent. Coupling therapeutic or diagnostic agents to these oligopeptides allows for their delivery across BBB. An active substance, such as a therapeutic or diagnostic agent, can bind to both the N-terminal and the C-terminal of the oligopeptide compound.

[0107] In the context of the present disclosure, the diagnostic agent of the invention comprises a moiety that specifically binds to the target molecule to be detected, for example a marker associated with a CNS disease such as cancer, and a detectable moiety. The detectable moiety may be, for instance, a chelating agent, an imaging agent, a fluorescent label, or another suitable detectable group.

[0108] It is well-known, that cell penetrating peptides are possible to be covalently linked via chemical synthetic routes to a therapeutic cargo. The possible routes and the recently available trends were reviewed by Gayraud et al. (Gayraud F, et al., Molecules. 2021 Mar 13;26(6): 1591) The ability of these oligopeptides to carry normally impermeable molecules in large quantities makes these oligopeptide conjugates formidable tools in the delivery of therapeutic medications or imaging molecules Moreover, these conjugates can reach into usually difficult to penetrate environments, such as the BBB. Recent advances in the field, therapeutic interventions and the need for BBB penetrating conjugates have been reviewed by Pancholi et al. (Zheng F, et al., Front Immunol. 2022 Nov 8; 13:978513; Walrant et al., Comptes Rendus Chimie. 2025 Jan 28(Gl):37-51). Oligopeptide conjugates have been used for efficient and fast delivery of radioactive metal complexes (Polyakov V, et al., Bioconjug Chem. 2000 Nov-Dec;l l(6):762- 71).

[0109] Additional therapeutic and diagnostic agents suitable for use in the present conjugates are disclosed in the prior art references cited above.

[0110] EXAMPLES

[0111] Example 1: Peptide synthesis

[0112] The designed peptides were synthesized using solid-phase peptide synthesis, applying the Fmoc / tBu strategy using a CEM Liberty Blue microwave-assisted, fully automated peptide synthesizer. The crude products were purified using anRP-18 HPLC on a PerfectSil 100 ODS-3 5 pm (250 x 10 mm) column. Analytical HPLC and mass spectrometry were used to identify the purified products. In all cases, the measured masses matched the calculated values, and a purity of at least 97% was observed.

[0113] Example 2: Preparation of a peptide conjugate with carboxyfluorescein (Cf) serving as a model drug (Fluorescent labeling)

[0114] In vitro cell penetration studies require fluorescent labeling. The used the (5)- and (6)- Cf tag (3 molar equivalent, 0.15 mmol, 56 mg) was conjugated manually to the N-terminus of 0.05 mmol of peptide (297 mg of resin), using hydroxybenzotriazole (HOBT, 3 molar equivalent, 0.15 mmol, 22.9 mg) and N, N'-di cyclohexyl carbodiimide (DCC, 3 molar equivalent, 0.15 mmol, 30.9 mg) in dimethylformamide (DMF) using double coupling at room temperature overnight. The Cf peptides were detached from the solid support using trifluoroacetic acid (TFA, 90%) in the presence of water (5%), 1,4-dithiothreitol (DTT, 2.5%), and triisopropyl silane TIS (2.5%).

[0115] Example 3: Biophysical properties, structural characterization Atomic level information from nuclear magnetic resonance (NMR) measurements

[0116] The structural information on the peptides under investigation, in both their native and Cf- labeled forms, was obtained from NMR measurements. All experiments were performed on a Bruker Avance III 700 MHz spectrometer using a Prodigy TCI H&F-C / N-D 5mm probe-head equipped with z-gradient. The resonance assignment was conducted on the basis of 2D homonuclear TOCSY, NOESY, ROESY measurements. Additionally, heteronuclear 2D SOFAST-HMQC and ^^C-HSQC, as well as ^^C-HMBC measurements were performed on natural isotopic abundance.

[0117] The results of NMR measurements show that the amino acid sequence of the oligopeptide is also a key factor: Cf conjugation causes changes in ERD-A and ERD-B, especially in the N- terminal region where aromatic residues are present. Aromatic and hydrophobic interactions between the side chain and the Cf moiety are possible. Although no stable secondary structural elements were identified, most peptides showed weak NOE cross-peaks between Cf and the F5 residue. Regarding pro environments, cis / trans isomerization results in minor forms, creating a more heterogeneous system that may influence cell penetration properties.

[0118] Example 4: Cellular internalization, cell viability, intracellular localization

[0119] In our previous research (Sebak F, et al ACS Omega. 2021 Dec 6;6(50):34470-34484), we already characterized the cellular internalization abilities of the compounds under investigation. The proposed peptides are highly water-soluble, making them excellent candidates for delivering lipophilic drug compounds.

[0120] ERD-A is predominantly localized within the cytoplasm, while ERD-B exhibits partial colocalization with lysosomal staining

[0121] The uptake mechanism for ERD-A and ERD-B mainly occur through energy -dependent pathways and can be chemically inhibited. The intracellular localization of these peptides also indicates that, after internalization, they primarily follow endocytic routes toward late endosomes and lysosomes, where they may be degraded within a lysosomal compartment.

[0122] The intracellular fate is highly sensitive to minor changes in the amino acid sequence of plant-derived cationic peptides. This allows fine-tuning of the intracellular trafficking. In conclusion, our findings indicate that all the studied ERD peptides are promising tools for cargo delivery to both vesicular and cytoplasmic targets. They all possess the advantage of being noncytotoxic and having no cytostatic effect.

[0123] Example 5: Characterization of the effect of peptides and their conjugates on the cellular viability of brain endothelial cells

[0124] In our experiments human stem cell-derived brain endothelial cells were grown in endothelial cell culture medium and to enhance the barrier tightness of the model endothelial cells were incubated with a small molecule combination (cARLA; Porkolab G, et al., Pharmaceutics. 2020 Jul 7;12(7):635). Before experiments, ECs grown in 50% brain pericyte- conditioned medium.

[0125] The kinetics of the peptide effect on endothelial cells was monitored by impedance measurement at 10 kHz (RTCA-SP instrument; Agilent, USA), which is a label-free, noninvasive, real-time method. Before experiments, cells were cultured in pericyte conditioned medium for 4 days and monitored every 5 min. After 4 days, cells were treated with ERD-A (100-1000 pg / mL), ERD-B (100-1000 pg / mL), carboxyfluorescein labeled compounds (Cf- ERD-A, Cf-ERD-B; 100-1000 pg / mL) and modified peptides (ERD-A D6P, ERD-A F5,7W; 30-1000 pg / mL), for 24 hours. At the end of the measurements, the normalized cell index values reflect the vitality of the cells. Our results demonstrated that neither the unlabeled nor the fluorescently labeled and the modified peptides had toxic effects on the viability of endothelial cells after 24 hours in the investigated concentration range (Figure 1).

[0126] Example 6: Penetration of peptides and their conjugates across the co-culture model of the BBB

[0127] For permeability studies, we used a contact co-culture model of the BBB, in which brain endothelial cells and brain pericytes are cultured together in a Transwell system (Meszaros M, et al. Cells. 2023 Feb 3;12(3):503). The two cell types were cultured together for 6 days before permeability measurements in a 24-well culture plates.

[0128] The cumulative apparent permeability coefficients (Papp) were calculated as described previously (Bocsik A, et al., JPharm Sci. 2016 Feb; 105(2):754-765) and reflects the penetration of the compounds across the BBB (Table 1). The translocation indicates what percentage of the initial peptide quantity passed through the BBB (Table 1). Table 1: Calculated cumulative apparent permeability coefficients (Papp) of peptides and peptide conjugates.

[0129] Both ERD- A and ERD-B peptides were evaluated for their ability to cross the BBB. The native sequences show better penetration and translocation properties than the literature standard opiorphin molecule.

[0130] Furthermore, rationally designed mutations (D6P and F5,7W) that target variations in hydrophobicity and membrane interaction ability also proved to outperform the standard opiorphin.

[0131] Cf conjugate was chosen as a mimic for a highly hydrophobic, heteroaromatic drug candidate. It was proved, that even with this increased hydrophobicity, the penetration is better than for the reference opiorphin, and also better than the parental, native sequence (ERD-A, ERD-B).

Claims

WHAT IS CLAIMED IS:

1. A peptide conjugate for use in the treatment of a disease of the central nervous system (CNS), comprising a blood-brain barrier (BBB) penetrating oligopeptide and a therapeutic agent for the treatment of a disease of the CNS, which BBB-penetrating oligopeptide is an oligopeptide according to Formula I,Asp-R2-Gly-Leu-R5-R6-R7-Leu-Gly-R10-Rl 1-R12 (Formula I) whereinR2 represents Arg, Lys, Cyt, D-Arg or Orn;R5 represents Phe, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, Orn, 4-aminophenylalanine or 3 -phenylpropionate;R6 represents Pro, Leu, Glu or Lys;R7 represents Phe or Trp;RIO, R11 and R12 each independently represent Lys, Arg or Orn; or an oligopeptide according to Formula II,Glu-R2’-R3’-Gly-R5’-R6’-Glu-R8’-R9’-R10’-Glu-R12’-Leu-Pro-Gly (Formula II), whereinR2’, R3’, R8’, RIO’ and R12’ each independently represent Lys, Arg or Orn;R5’ represents Phe, He, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, 4-aminophenylalanine, or 3 -phenylpropionate;R6’ represents Met, Leu, He, Asn, norleucin, D-norleucin, selenomethionine or D / L-2-hydroxy-(4-methylseleno)butanoic acid;R9’ represents Leu or He.

2. The peptide conjugate for use in the treatment of a disease of CNS according to claim 1, wherein the oligopeptide is an oligopeptide according to Formula I, whereinR2 represents Arg;R5 represents Phe or Trp;R6 represents Asp or Pro;R7 represents Phe or Trp;RIO, R11 and R12 represent Lys; or an oligopeptide according to Formula II, whereinR2’, R3’, R8’, RIO’ and R12’ represent Lys;R5’ represents Phe; andR6’ represents Met; preferably the oligopeptide isAsp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A; SEQ ID NO: 1),Asp-Arg-Gly-Leu-Phe-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A D6P; SEQ ID NO:2), Asp-Arg-Gly-Leu-Trp-Asp-Trp-Leu-Gly-Lys-Lys-Lys (ERD-A F5,7W; SEQ ID NO:3), orGlu-Lys-Lys-Gly-Phe-Met-Glu-Lys-Leu-Lys-Glu-Lys-Leu-Pro-Gly (ERD-B; SEQ ID NO:4).

3. A pharmaceutical composition for use in the treatment of a disease of CNS comprising: a peptide conjugate according to Claim 1 or 2; and a pharmaceutically acceptable excipient.

4. The pharmaceutical composition for use in the treatment of a disease of CNS according to Claim 3, which is formulated for administration via parenteral, including intrathecal, subcutaneous, intravenous or intramuscular administration.

5. A peptide conjugate for use in an in vivo method of diagnosis of a disease of CNS, comprising a BBB-penetrating oligopeptide and a diagnostic agent specific for said disease, which BBB-penetrating oligopeptide is an oligopeptide according to Formula I, Asp-R2-Gly-Leu-R5-R6-R7-Leu-Gly-R10-Rl 1-R12 (Formula I) whereinR2 represents Arg, Lys, Cyt, D-Arg or Orn;R5 represents Phe, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, Orn, 4-aminophenylalanine or 3 -phenylpropionate;R6 represents Pro, Leu, Glu or Lys;R7 represents Phe or Trp;RIO, R11 and R12 each independently represent Lys, Arg or Orn; or an oligopeptide according to Formula II,Glu-R2’-R3’-Gly-R5’-R6’-Glu-R8’-R9’-R10’-Glu-R12’-Leu-Pro-Gly (Formula II), whereinR2’, R3’, R8’, RIO’ and R12’ each independently represent Lys, Arg or Orn;R5’ represents Phe, He, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, 4-aminophenylalanine, or 3 -phenylpropionate;R6’ represents Met, Leu, He, Asn, norleucin, D-norleucin, selenomethionine or D / L-2-hydroxy-(4-methylseleno)butanoic acid;R9’ represents Leu or He.

6. The peptide conjugate for use in an in vivo method of diagnosis of a disease of CNS according to claim 5, wherein the oligopeptide is an oligopeptide according to Formula I, whereinR2 represents Arg;R5 represents Phe or Trp;R6 represents Asp or Pro;R7 represents Phe or Trp;RIO, Rl l and R12 each independently represent Lys; or an oligopeptide according to Formula II, whereinR2’, R3’, R8’, RIO’ and R12’ represent Lys;R5’ represents Phe;R6’ represents Met; andR9’ represents Leu; preferably the oligopeptide isAsp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A; SEQ ID NO: 1), Asp-Arg-Gly-Leu-Phe-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A D6P; SEQ ID NO:2), Asp-Arg-Gly-Leu-Trp-Asp-Trp-Leu-Gly-Lys-Lys-Lys (ERD-A F5,7W; SEQ ID NO:3), orGlu-Lys-Lys-Gly-Phe-Met-Glu-Lys-Leu-Lys-Glu-Lys-Leu-Pro-Gly (ERD-B; SEQ ID NO:4).

7. A diagnostic composition for use in an in vivo method of diagnosis of a disease of CNS comprising a peptide conjugate according to Claim 5 or 6; and a diagnostically suitable excipient.

8. Use of a peptide conjugate comprising a BBB -penetrating oligopeptide compound as defined in claim 5 or 6 and a diagnostic agent in an in vitro diagnostic method that requires the peptide conjugate to cross the blood-brain barrier for detecting a molecular entity.

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