Methods for identifying blood brain barrier peptide shuttles

Abstract

This invention relates to a method of identifying a Blood Brain Barrier peptide shuttle (BBBpS) comprising determining a set of physicochemical properties of a test peptide. The set of physicochemical properties may comprise Molecular weight, Extinction coefficient, Hydrophobic nature, Isoelectric point, Net charge at pH 7, Charge, Average hydrophobicity, Hydrophobicity at pH 7, and Hydrophilic residues ratio. If each of these physicochemical properties falls within a reference range then the test peptide is identified as a BBB peptide shuttle. Methods of identifying producing and using BBB peptide shuttles are provided.

Description

Field of the Invention The invention relates to blood-brain barrier peptide shuttles (BBBpS) and methods for the identification of BBBpS. Background to the Invention Interest in peptide-based biotechnological products is significantly increasing thanks to improvements in their function and / or in vivo stability 12. Peptides are now routinely used in therapeutic / diagnostic protocols and are currently gaining a foothold in drug-delivery strategies for their ability to ferry payloads, such as proteins, nucleic acids, drugs, or nanoparticles into cells 3 5. Cell-penetrating peptides (CPPs) are relatively short peptides that internalize into cells without membrane damage 6. They have been successfully applied as intracellular carriers for, among others, proteins, nucleic acids, pharmaceuticals, and nanoparticles. Most CPPs are hydrophobic and positively charged at physiological pH 7 8. While their detailed internalization mechanisms remain unclear, even controversial, there is consensus that CPP physicochemical properties and payload, as well as cell type and uptake conditions, have all a bearing on the mode of action 5. Some CPPs can traverse cellular barriers. Among them, those able to traverse the blood-brain barrier (BBB) and access the brain, appropriately named BBB peptide shuttles (BBBpS)4, are particularly relevant in neuropharmacology. The first BBBpS described was the HIV trans-activator of transduction (TAT) peptide 9. Subseguently, other peptides, such as SynB, penetratin, Angiopep-2, dNP2, TP10, MiniAp-4, and PepH3 have been investigated and have shown good translocation properties The BBB is a highly selective semipermeable border of endothelial cells that prevents solutes in the blood from non-selectively crossing into the extracellular fluid of the central nervous system (CNS). The BBB typically blocks most peptides from passing through it. It remains unclear why not all CPPs traverse cellular barriers and what turns a CPP into a BBBpS. Summary of the Invention The present invention relates to the finding that peptides that are capable of translocation across the BBB are characterised by a specific set of physicochemical properties. This allows the design and synthesis of BBBpS for example for use in drug conjugates. A first aspect of the invention provides a method of identifying a BBBpS comprising; determining a set of physicochemical properties of a test peptide, determining whether each of the physicochemical properties in the set falls within a reference range, and identifying the test peptide as a BBBpS if each physicochemical property in the set falls within the reference range. The set of physicochemical properties may comprise Molecular weight, Extinction coefficient, Hydrophobic nature, Isoelectric point, Net charge at pH 7, Charge, Average hydrophobicity, Hydrophobicity at pH 7, and Hydrophilic residues ratio. Preferred reference ranges for the physicochemical properties in the set include; Molecular weight 852-3065 g.mol'1, Extinction coefficient 0.0-7786 MTcm1, Hydrophobic nature 18-50%, Isoelectric point 8.0-13.3, Net charge at pH -1.0-4.7, Charge 0.0-6.0, Average hydrophobicity -0.6 to 0.8, Hydrophobicity at pH 7, 5.5 to 34.6, and Hydrophilic residues ratio 17 to 56.0%. A method of the first aspect may further comprise synthesising a peptide identified as a BBBpS. A second aspect of the invention provides a computer program product comprising instructions which, when the program is executed by a computer, cause the computer to perform the method of the first aspect. A third aspect of the invention provides a computer system comprising a processor, wherein the processor is configured to execute the method of the first aspect. A fourth aspect of the invention provides a BBBpS identified by a method of the first aspect. A fifth aspect of the invention provides a BBBpS selected from the group consisting of: BBBpS_1 - KYKGAIIGNIK-amide (SEQ ID NO: 1), BBBpS_2 - KYRSGAITIGY-amide (SEQ ID NO: 2), BBBpS_3 - EEGRLYMRYYSPTTRRYG-amide (SEQ ID NO: 3), BBBpS_4   - APWHLSSQYSRT-amide (SEQ ID NO: 4), BBBpS_5   - HIQLSPFSQSWR-amide (SEQ ID NO: 5), BBBpS_6 - YTQDFNKFHTFPQTAIGVGAP-amide (SEQ ID NO: 6), BBBpS_7 - LKTLTETLKELTKTLTEL-amide (SEQ ID NO: 7), BBBpS_8 - YKEATSTFTNITYRGT-amide (SEQ ID NO: 8), BBBpS_9 - NRPDSAQFWLHH-amide (SEQ ID NO: 9), BBBpS_10 - RPKPQQFFGLM-amide (SEQ ID NO: 10), BpAYG21 - AAYGILEHAKYKAHELIGYAA-amide (SEQ ID NO: 14) BpAYK21, AAYKILEHAKYKAHELIKYAA-amide (SEQ ID NO: 15) BpARY17, AYILEHAKYKAHELIYA-amide (SEQ ID NO: 16) BpARH17, AYILEHAKHKAHELIYA-amide (SEQ ID NO: 17) 5 BpYRH17, AYIKEHYKHKYHEKIYA-amide (SEQ ID NO: 18) BpRYI15, KYILEHKYKHELIYK-amide (SEQ ID NO: 19) BpAYG11, AAYGILEHAKY-amide (SEQ ID NO: 20) BpAYK11, AAYKILEHAKY-amide, (SEQ ID NO: 21) BpARY9, AYILEHAKY-amide, (SEQ ID NO: 22) 10 BpARH9, AYILEHAKH-amide, (SEQ ID NO: 23) BpAYG21_1 YAYGILEHARYRAHELIGYAA-amide (SEQ ID NO: 24), BpAYG21_2 CAYGILEHARYRAHELIGYAA-amide (SEQ ID NO: 25) BpAYG21_1 AAYKIFEHARYRAHELIKYAA-amide (SEQ ID NO: 26) BpAYG21_2 AAYKITEHARYRAHELIKYAA-amide (SEQ ID NO: 27) 15 BpAYG21_3 AAYKIREHARYRAHELIKYAA-amide (SEQ ID NO: 28) BpARY17_1 AYILRHAKYKAHELCYA-amide (SEQ ID NO: 29) BpARH17_1 AYILRHAKHKAHELCYA-amide (SEQ ID NO: 30) BpYRH17_1 AYIKRHYKHKYHEKCYA-amide (SEQ ID NO: 31) BpYRH17_2 AYIKPHYKHKYHEKCYA-amide (SEQ ID NO: 32) 20 BpYRH17_3 AYIKRHYKHKYHEKRYA-amide (SEQ ID NO: 33) BpYRH17_4 AYIKRHYKHKYHEKGYA-amide (SEQ ID NO: 34) BpYRH17_5 AYIKTHYKHKYHEKCYA-amide (SEQ ID NO: 35) BpYRH17_6 AYIKRHYKHKYHEKTYA-amide (SEQ ID NO: 36) BpYRH17_7 AYIKRHYKHKYHEKVYA-amide (SEQ ID NO: 37) 25 BpYRH17_8 AYIKRHYKHKYHEKYYA-amide (SEQ ID NO: 38) BpYRH17_9 AYlKWHYKHKYHEKCYA-amide (SEQ ID NO: 39) BpYRH17_10 AYIKPHYKHKYHEKRYA-amide (SEQ ID NO: 40) BpYRH17_11 AYIKYHYKHKYHEKCYA-amide (SEQ ID NO: 41) BpYRH17_12 AYlKGHYKHKYHEKCYA-amide (SEQ ID NO: 42) 30 BpYRH17_13 AYIKRHYKHKYHEKWYA-amide (SEQ ID NO: 43) BpRYI15_1 KYILRHKYKHELRYK-amide (SEQ ID NO: 44) BpRYI15_2 KYILRHKYKHELYYK-amide (SEQ ID NO: 45) BpRYI15_3 KYILRHKYKHELCYK-amide (SEQ ID NO: 46) BpRYI15_4 KYILRHKYKHELVYK-amide (SEQ ID NO: 47) 35 BpRYI15_5 KYILRHKYKHELTYK-amide (SEQ ID NO: 48) BpRYI15_6 KYILPHKYKHELYYK-amide (SEQ ID NO: 49) BpRYI15_7 KYILPHKYKHELCYK-amide (SEQ ID NO: 50) BpRYI15_8 KYILPHKYKHELRYK-amide (SEQ ID NO: 51) BpRYI15_9 KYILPHKYKHELVYK-amide (SEQ ID NO: 52) 40 BpRYI15_10 KYILRHKYKHELPYK-amide (SEQ ID NO: 53) BpAYG11_1 AAYGILVHAKY-amide (SEQ ID NO: 54) BpAYG11_2 AAYGILTHAKY-amide (SEQ ID NO: 55) BpAYG11_3 AAYGILYHAKY-amide (SEQ ID NO: 56) BpAYG11_4 AAYGILCHAKY-amide (SEQ ID NO: 57) 45 BpAYG11_5 AAYGILRHAKY-amide (SEQ ID NO: 58) BpAYK11_1 AAYKRLEHAKY-amide (SEQ ID NO: 59) BpARY9_1 AYILRHAKY-amide (SEQ ID NO: 60) BpARY9_2 AYILPHAKY-amide (SEQ ID NO: 61) BpARY9_3 AYILTHAKY-amide (SEQ ID NO: 62) BpARY9_4 AYILVHAKY-amide (SEQ ID NO: 63) BpARY9_5 AYILYHAKY-amide (SEQ ID NO: 64) BpARH9_1 AYILRHAKH-amide (SEQ ID NO: 65) BpARH9_2 AYILPHAKH-amide (SEQ ID NO: 66) BpARH9_3 AYILTHAKH-amide (SEQ ID NO: 67) BpARH9_4 AYILYHAKH-amide (SEQ ID NO: 68), and BpARH9_5 AYILVHAKH-amide (SEQ ID NO: 69) A sixth aspect of the invention provides a drug conjugate comprising a therapeutic agent conjugated to a BBBpS of the fourth or fifth aspects. A seventh aspect of the invention provides a method of making a drug conjugate comprising conjugating a therapeutic agent to a BBBpS of the fourth or fifth aspects. A therapeutic agent of the sixth or seventh aspects may be a therapeutic agent for treatment of a brain disease. An eighth aspect of the invention provides a pharmaceutical composition comprising a BBBpS of the fourth or fifth aspects or a drug conjugate of the sixth aspect and a pharmaceutically acceptable carrier. A ninth aspect of the invention provides a method of treating or preventing a brain disease in a subject, comprising administering to the subject, a BBBpS of the fourth or fifth aspects ora drug conjugate of the sixth aspect. A tenth aspect of the invention provides a BBBpS of the fourth or fifth aspects or a drug conjugate of the sixth aspect for use in a method of treating or preventing a brain disease in a subject, for example a method of the ninth aspect. An eleventh aspect of the invention provides the use of a BBBpS of the fourth or fifth aspects or a drug conjugate of the sixth aspect in the manufacture of a medicament for use in a method of treating or preventing a brain disease in a subject, for example a method of the ninth aspect. Other aspects and embodiments of the invention are described in more detail below. Brief Description of the Figures Figure 1 shows a flow diagram for the process of identifying peptides as BBBpS. Figure 2 shows a graph showing the correlation between the theoretical translocation predicted for each peptide compared to the experimentally observed values. Figure 3A shows a graph showing the level of translocation of each peptide. Figure 3B shows a graph showing the tightness of the BBB when exposed to each peptide, as a measure of permeability to fluorescent dextran. Figure 4 shows a set of graphs showing how the cell viability of various cell lines with increasing concentration of each peptide, effectively showing the relative toxicity of each peptide. Figures 5 and 6 each show a series of graphs showing the level of internalisation ability of each peptide into various cell lines. Detailed Description The present invention relates to methods for the identification of BBBpS. The methods comprise determining a set of physicochemical properties of a test peptide and determining whether each of the physicochemical properties in the set falls within a reference range. The test peptide is identified as a BBBpS if the physicochemical properties in the set fall within the reference ranges. A BBBpS is a peptide able to cross the blood-brain barrier (BBB) and access brain tissue. BBBpS may therefore be useful as vectors for the delivery of therapeutic agents to the brain and CNS. The ability of BBBpS to translocate across the BBB may be determined in vitro or in vivo, for example using assays described below. A test peptide may be any peptide whose ability to cross the BBB is to be assessed or predicted. A peptide is a short chain of amino acids linked by peptide bonds. A peptide suitable for use as described herein may comprise 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more,10 or more,11 or more, or 12 or more amino acids, preferably between 7 and 22 amino acids. Suitable test peptides include cell-penetrating peptides (CPPs). ACPP is a peptide that is known to cross cell membranes. However, many CPPs cannot cross the BBB. Methods described herein may be useful in determining if a CPP is capable of crossing the BBB and acting as a BBBpS. Other suitable test peptides include peptides that are not CPPs. Test peptides may include conjugates in which the peptide is bound to another substance of interest such as a nucleic acid, a small molecule, a protein, an antibody, a nanoparticle, a medicament, or a reporter. Physicochemical properties are physical or chemical parameters that are displayed by a test peptide. A set of specific physicochemical properties are shown herein to be a key determinant of ability to cross the BBB. The physicochemical properties in the set may include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more or most preferably all nine of (i) molecular weight, (ii) extinction coefficient, (iii) hydrophobic nature, (iv) isoelectric point, (v) net charge at pH 7, (vi) charge, (vii) average hydrophobicity, (viii) hydrophobicity at pH 7, and (ix) hydrophilic residues ratio. The physicochemical properties in the set may be determined for a test peptide by any suitable technique. For example, a method may comprise measuring, predicting, calculating or ascertaining the set of physicochemical properties of the test peptide. In some embodiments, physicochemical properties may be predicted or determined from the amino acid sequence of the test peptide. For each property in the set, a value may be determined. The value of the property may be compared to a reference range. If the values of the physicochemical properties in the set for a test peptide fall within the reference ranges, then the test peptide is identified as a BBBpS. For example, a method may comprise determining whether the physicochemical properties in the set for the test peptide fall within the reference range. Molecular weight (MW) is the weight in grams of one mole of a test peptide. Suitable methods for measuring molecular weight are well known in the art and include mass spectrometry or gel electrophoresis. Molecular weight may also be determined from the known molecular weights of the individual amino acids in the test peptide, or the known atomic weights of the individual atoms of the test peptide. For example, the molecular weight M of a peptide may be estimated by calculating; 1 1 i where N, are the number, and M, the average residue molecular weights, of the amino acids. Mn + Me are added to the total in order to account for the termini: H at the N-terminus and OH at the C-terminus. Of course, if the termini are modified, these additions are replaced by those of the modifiers. Exemplary values for molecular weights used in the Innovagen Peptide Calculator36 are: A, Ala 71.07793 C, Cys 103.1454 D, Asp 115.0873 E, Glu 129.1139 F, Phe 147.1734 G, Gly 57.05138 H, His 137.1394 I, He 113.1576 K, Lys 128.1724 L, Leu 113.1576 M, Met 131.1985 N, Asn 114.1028 P, Pro T, Thr 97.11508 101.1039 Q, Gin V, Vai 128.1293 99.13103 R, Arg W, Trp 156.1861 186.2095 S, Ser 87.07733 Y, Tyr 163.1728 H 1.00797 OH 17.00738 phos-Ser 167.0573 Acetyl 43.04453 Amide 16.0228 phos-Thr 181.0838 Biotin 227.3056 phos-Tyr 243.1528 A suitable reference range for molecular weight for the identification of BBBpSs as described herein may be 500 - 5000 gmol1, 750 - 4000 gmol1, 850 to 3100 gmol1 or preferably 852 - 3065 gmol1. Extinction coefficient is a measure of the level of light absorbance of a substance in solution. Extinction coefficient may be measured at or near 280nm, which is in the ultra-violet (UV) part of the spectrum. Extinction coefficient is typically reported in units of M-1 cm'1. Suitable methods for measuring extinction coefficient are well known in the art and include UV spectroscopy. In some embodiments, extinction coefficient may be predicted for a test peptide from the known absorbance of the individual amino acids in the peptide sequence. For example, the molar extinction coefficient e at 280 nm of a peptide can be estimated by calculating: & =      + flfSf + where nw, ny, and nc are the number of Tryptophans (W), Tyrosines (Y) and Cystines (i.e. disulphide bonds, but here denoted C) in the sequence. Exemplary values for the molar extinction coefficients, used in Innovagen's Peptide Calculator36 are: ew= 5690 M'1cm'1 ey = 1280 M'1cm'1 ec = 120 M'1cm'1 These values are derived from Gill and von Hippel37. Non-natural amino acids, modifications, and labels may add to the extinction coefficient, and may need to be considered separately. A suitable reference range for extinction coefficient for the identification of BBBpSs as described herein may be 0 - 8000 M'1cm-1, 0-7800 M'1cm-1, or preferably 0-7786 M'1cm-1. Hydrophobic nature is the percentage of hydrophobic amino acids in the test peptide compared to the total number of amino acids in the peptide. This is determined based on the amino acid sequence of the peptide and the number of hydrophobic amino acids in the sequence. Hydrophobic amino acids include glycine , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan. A suitable reference range for hydrophobic nature for the identification of BBBpSs as described herein may be 10 - 60%, 15-55%, or preferably 19 - 50%. Isoelectric point (pl) is the measured or predicted pH at which the net charge of a test peptide is zero (i.e. the test peptide has no net electrical charge). Suitable methods for determining the isoelectric point are well known in the art. Innovagen's Peptide Calculator36 first calculates the net charge for pH 7.0. If the charge is > 0 the next pH to check is 7 + 3.5, if the charge is < 0 then pH 7 - 3.5 is checked. This is repeated, using increments / decrements half the size of the previous, until the charge found equals 0, or is sufficiently close to 0. Discussion of the calculation of net charge is given below. A suitable reference range for isoelectric point for the identification of BBBpSs as described herein may be 7.0 - 14.0, 8.0 - 13.5 or preferably 8.0 -13.3. Net charge at pH 7 is the overall charge of a test peptide at pH 7. Suitable methods for determining the net charge at pH 7 are well known in the art and include capillary electrophoresis. Net charge may also be predicted based on the pKa values of the amino acids that make up the test peptide. The net charge of the amino acids can be calculated at pH 7 from their pKa values. By combining the net charges of these amino acids, the overall net charge of the test peptide at pH 7 can be calculated. The net charge Z of a peptide at a certain pH can also be estimated by calculating; where Ni is the number of arginine, lysine, and histidine residues, and pKai is the pKa values, of the N-terminus and the side chains of Arginine, Lysine, and Histidine. Similarly, Nj is the number of aspartic acid, glutamic acid, cysteine, and tyrosine amino acids. pKaj is the pKa values of the C-terminus and the Aspartic Acid, Glutamic Acid, Cysteine, Tyrosine amino acids. Innovagen's Peptide Calculator36 calculates the net charge for all pH values of 0.1 to 14 in increments of 0.1, and plots these producing a titration curve. The resulting net charge depends on what pKa values the algorithm uses. Innovagen's Peptide Property Calculator36 uses the values taken from the CRC Handbook of Chemistry and Physics, 87th edition38. A suitable reference range for net charge at pH 7 for the identification of BBBpSs as described herein may be -2.0 to 5.5, -1.5 to 5.0, or preferably -1.0 to 4.7. In the present application, “charge” refers to the average charge of the peptide between pH 1 and 14. This is calculated in the same way as at pH 7, instead at each integer pH between 1 and 14, and then an average is taken. A suitable reference range for charge for the identification of BBBpSs as described herein may be 0.0 to 7.0 or preferably 0.0 to 6.0. Average hydrophobicity refers to a property determined by calculating a moving average of the water solubility of the amino acids in a peptide sequence, using the techniques as described in Hopp & Woods39. A suitable reference range for average hydrophobicity for the identification of BBBpSs as described herein may be -1 0 to 1.0, -0.8 to 0.9 or preferably -0.6 to 0.8. A suitable reference range for hydrophobicity at pH 7.0 for the identification of BBBpSs as described herein may be 5.0 to 35.0 or 5.4 to 34.6. Hydrophilic residues ratio is the percentage of hydrophilic amino acids in the test peptide compared to the total number of amino acids in the peptide. This is determined based on the amino acid sequence of the peptide and the number of hydrophilic amino acids in the sequence. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, threonine, and tyrosine. A suitable reference range for the hydrophilic residues ratio for the identification of BBBpSs as described herein may be 15 to 60%, preferably 17.0 to 56.0%. Test peptides that have a set of physicochemical properties that all fall within the reference ranges may be identified as BBBpSs i,e. peptides that art able to cross the BBB. In some embodiments, a method described herein may be a computer-based method. For example a computer may be programmed or adapted to perform a method described herein. Also disclosed herein is a computer system comprising a processor configured to execute a method of identifying a BBBpS as described herein and a computer program product comprising instructions which, when the program is executed by a computer, cause the computer to perform the method of identifying a BBBpS as described herein. The computer program product may be software or may be stored in computer memory. Once a test peptide has been identified as a BBBpS, the peptide may be synthesised or recombinantly expressed and optionally subjected further modification and / or further in-vitro or in vivo testing or other uses. In some embodiments, the BBBpS may be synthesised. Suitable methods are known in the art and include solid-phase peptide synthesis, liquid phase peptide synthesis, native chemical ligation, and enzymatic peptide synthesis. For example, peptides are typically synthesized by solid phase synthesis in a stepwise fashion from the C terminus to the N terminus. In an initial step, an N protected amino acid is covalently attached to an insoluble solid support via its carbonyl group. Suitable groups for N protecting the amino acid include 9-fluorenylmethyloxycarbonyl group (Fmoc) and t-butyloxycarbonyl (Boc). Following covalent attachment of the N protected amino acid, the N protecting group is removed and the deprotected NH2 group of the attached amino acid is reacted with the carboxylic acid group of the next N protected amino acid to generate a nascent peptide comprising 2 amino acids that is covalently attached to the solid phase. This process is repeated until the complete peptide sequence is built up on the solid phase. In some embodiments, protecting groups may be employed to prevent functional groups in the side chains of amino acids from reacting with an incoming N protected amino acids. These side chain protecting groups may be present throughout the synthesis of the peptide and may be removed in a final deprotection step. Methods of solid phase peptide synthesis are well-established in the art (see for example Coin et al Nature Protocols 2, 3247-3256 (2007) Stawikowski (2002) Curr Protoc Protein Sci. 2002 Unit-18.1. oi:10.1002 / 0471140864. ps1801s26; Chan and White; Fmoc Solid Phase Peptide Synthesis - A Practical Approach. Oxford University Press, 2000; Stewart, J. M.; Young, J. D. Solid-Phase Peptide Synthesis (2nd ed.), Pierce Chemical Co., Rockford, IL, 1984; Atherton, E.; Sheppard, R. C., SolidPhase Peptide Synthesis: A Practical Approach. Oxford University Press: New York City, 1989; M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); J. H. Jones, The Chemical Synthesis of Peptides. Oxford University Press, Oxford 1991; in Applied Biosystems 430A User’s Manual, ABI Inc., Foster City, California; G. A. Grant, (Ed.) Synthetic Peptides, A User’s Guide. W. H. Freeman & Co., New York 1992, and G.B. Fields, (Ed.) Solid-Phase Peptide Synthesis (Methods in Enzymology Vol. 289). Academic Press, New York and London 1997).. In other embodiments, the BBBpS may be recombinantly expressed. Peptides may be recombinantly expressed by introducing a nucleic acid encoding the peptide into a cell, which directs the cell to produce the peptide by transcription and / or translation of the nucleic acid. The cell can be cultured and the peptide harvested from the culture. Molecular biology techniques suitable for the producing peptides according to the invention in cells are well known in the art, such as those set out in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989 The peptide may be expressed from a nucleotide sequence. A BBBpS identified by a method described herein may have a set of physicochemical properties that fall within the reference ranges set out herein. For example, the BBBpS may have a molecular weight of 852-3065 g.mol1, an extinction coefficient of 0.0-7786 M Lcm1, a hydrophobic nature of 18-50%, an isoelectric point of 8.0-13.3, a net charge at pH of -1.0-4.7, a charge of 0.0-6.0, an average hydrophobicity of -0.6 to 0.8, a hydrophobicity at pH 7 of 5.5 to 34.6, and a hydrophilic residues ratio of 17 to 56.0%. Suitable BBBpSs may comprise 5 to 25 amino acids, 6 to 24 amino acids, 7 to 23 amino acids, 8 to 22 amino acids, 9 to 21 amino acids, or 10 to 20 amino acids. For example, a BBBpS may comprise the amino acid sequence of any one of SEQ ID Nos: 1 to 10 (BBBpS_1-10) as shown in Table 6 or may be a variant thereof. A variant of a reference sequence set out herein, such as a reference BBBpS sequence may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. Particular amino acid sequence variants may differ from a reference sequence shown herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids. A BBBpS having a variant of a reference sequence set out herein may have a set of physicochemical properties that fall within the reference ranges set out above. Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147'. 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used. Sequence comparison may be made over the full-length of the relevant sequence described herein. In some embodiments, a BBBpS may be conjugated to a therapeutic agent to produce a drug conjugate. A drug conjugate comprising a therapeutic agent and a BBBpS may be able to cross the BBB more effectively that the therapeutic agent alone. In some embodiments, a BBBpS may have therapeutic activity without being conjugated to another molecule. Preferably, a BBBpS is conjugated to a therapeutic agent by a covalent linkage. Suitable techniques for conjugating a BBBpS to a therapeutic agent are well known in the art and include, for example, amide bond formation between peptides, thiol chemistry, click chemistry, biorthogonal chemistry, or disulfide bond formation. Peptide therapeutic agents can be conjugated to the BBBpS by using recombinant DNA technology to conjugate nucleic acid sequences encoding each of the BBBpS and the therapeutic agent, such that when the conjugated nucleic acid is expressed, a peptide is produced which is a conjugate of the BBBpS and the therapeutic agent. In some embodiments, the therapeutic agent may be a therapeutic peptide. The drug conjugate may be a fusion protein comprising the therapeutic peptide and the BBBpS. The BBBpS and the therapeutic agent may be connected directly or via a linker. Suitable linkers are well known in the art. Suitable linkers are known in the art and include PEG, aminohexanoic acid, and aminocaprotic acid. The therapeutic agent may be suitable for the treatment of a brain disease. Conjugation to a BBBpS may increase the ability of the agent to cross the BBB, thereby increasing its efficacy. For example, suitable therapeutic agents may include any one of antidepressants, antipsychotics, mood stabilisers, anti-anxiety medications, stimulants, antiepileptic drugs, cholinesterase inhibitors, dopamine agonists, dopamine reuptake inhibitors, nonsteroidal anti-inflammatory drugs, antimetabolites, anticonvulsants, benzodiazepines, nootropics, beta blockers, antiviral drugs, chemotherapeutic drugs, antibodies, nutrients, antibiotics, immunosuppressants, serotonin-norepinephrine reuptake inhibitors, analgesics, selective serotonin reuptake inhibitors, NMDA receptor antagonists, corticosteroids, thrombolytics, vasodilators, and neuroprotective agents A BBBpS or drug conjugate comprising a BBBpS as described above may be administered alone or may be formulated into a pharmaceutical composition. A pharmaceutical composition is a formulation comprising one or more active agents and one or more pharmaceutically acceptable excipients. The pharmaceutical composition may be capable of eliciting a therapeutic effect. A suitable pharmaceutical composition for use as described herein may comprise an agent described above and a pharmaceutically acceptable excipient. For example, a pharmaceutical composition may comprise a BBBpS or drug conjugate comprising a BBBpS, as described herein, and a pharmaceutically acceptable excipient. The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable excipients and carriers include, without limitation, water, saline, buffered saline, phosphate buffer, alcoholic / aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants, and excipients may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH-adjusting agents may also be employed, and include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid (e.g., citrates), ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, phthalic acid, Tris, trimethylamine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, lactated Ringer's, or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents (e.g., EGTA; EDTA), inert gases, and the like may also be provided in the pharmaceutical carriers. The pharmaceutical compositions described herein are not limited by the selection of the carrier. The preparation of these pharmaceutically-acceptable compositions, from the above-described components, having appropriate pH, isotonicity, stability and other conventional characteristics, is within the skill of the art. Suitable carriers, excipients, etc. may be found in standard pharmaceutical texts, for example, Remington’s Pharmaceutical Sciences and The Handbook of Pharmaceutical Excipients, 4th edit., eds. R. C. Rowe et al, APhA Publications, 2003. The pharmaceutical compositions and formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the agent with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers. Formulations may for example be in the form of liquids or solutions. Pharmaceutical compositions described herein may be produced in various forms, depending upon the route of administration. The pharmaceutical compositions may be prepared for administration to subjects in the form of, for example, liquids, powders, aerosols, tablets, capsules, enteric-coated tablets or capsules, or suppositories. Pharmaceutical compositions may also be in the form of suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. Pharmaceutical compositions may be made in the form of sterile aqueous solutions or dispersions, suitable for injectable use, or made in lyophilized forms using freeze-drying techniques. Lyophilized pharmaceutical compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g., saline or HEPES, with or without adjuvant. Pharmaceutical compositions can also be made in the form of suspensions or emulsions. The precise nature of the carrier or other material will depend on the route of administration, which may be any convenient route, for example by oral administration or by injection, e.g. cutaneous, subcutaneous, or intravenous. Preferably, the agent is administered systemically, e.g. intravenously. The pharmaceutical compositions comprising the active compounds may be formulated in a dosage unit formulation that is appropriate for the intended route of administration. Pharmaceutical compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections immediately prior to use. Methods of determining the most effective means and dosage of administration are well known in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician. Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Multiple doses of the composition may be administered, for example 2, 3, 4, 5 or more than 5 doses may be administered. The administration of the composition may continue for sustained periods of time. For example, treatment with the composition may be continued for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month or at least 2 months. Treatment with the composition may be continued for as long as is necessary to reduce symptoms. A BBBpS or drug conjugate comprising a BBBpS or a pharmaceutical composition comprising such a BBBpS or drug conjugate may be useful in treating a brain disease in a patient. The brain disease may be a disease or a disorder of the brain or CNS. Suitable diseases include Alzheimer’s disease, dementia, brain cancer, epilepsy, seizure disorders, mental disorders, Parkinson’s disease, movement disorders, stroke, transient ischemic attack, cancer, infections (e.g. from bacteria, viruses, fungi, or parasites). Mental disorders may include anxiety disorders, mood disorders, psychotic disorders, eating disorders, impulse control disorders, addition disorders and personality disorders. Treatment may be any treatment and therapy, whether of a human or an animal (e.g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, improving or ameliorating one or more symptoms of brain disease. An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), murine (e.g. a mouse), canine (e.g. a dog), feline (e.g. a cat), equine (e.g. a horse), a primate, simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon), ora human. In some preferred embodiments, the individual is a human. In other preferred embodiments, nonhuman mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or leporid) may be employed. An individual with a brain disease may display at least one identifiable sign, symptom, or laboratory finding that is sufficient to make a diagnosis of brain disease in accordance with clinical standards known in the art. Examples of such clinical standards can be found in textbooks of medicine. It will be appreciated that appropriate dosages of an agent can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular agent, the route of administration, the time of administration, the rate of loss or inactivation of the agent, the duration of the treatment, other drugs, compounds, and / or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The dosage of agent and the route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of injury which achieve the desired effect without causing substantial harmful or deleterious side-effects. In some embodiments, the agent may be administered at a dosage that is effective in reducing MYLIP or CD9 expression or activity at the injury site. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and / or progression of a disease being treated. Appropriate doses of therapeutic polypeptides are well known in the art (Ledermann J.A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Specific dosages may be indicated herein or in the Physician's Desk Reference (2003) as appropriate for the type of medicament being administered may be used. A therapeutically effective amount or suitable dose of a agent described herein may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. Treatment may comprise the administration of a therapeutically effective amount of the agent or pharmaceutical composition to the individual. “Therapeutically effective amount" relates to the amount of a agent or pharmaceutical composition that is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit / risk ratio. For example, a suitable amount of a agent or pharmaceutical composition for administration to an individual may be an amount that generates a therapeutic effect in the individual. A therapeutic effect may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. In some embodiments, a treatment as described herein may have a duration of up to 3 weeks, up to 6 weeks, up to 3 months, up to 6 months or up to 12 months. The treatment schedule for an individual may be dependent on the pharmocokinetic and pharmacodynamic properties of the agent, the route of administration and the nature of the condition being treated. Treatment may be in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Treatment may be periodic, and the period between administrations may be about 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 96 hours or more, or one week or more. Suitable formulations and routes of administration are described above and may be readily determined by a physician for any individual patient. In other embodiments, a BBBpS or conjugate as described herein may be administered in combination with one or more other therapies, either simultaneously or sequentially dependent upon the circumstances of the individual to be treated. When the therapeutic agents are used in combination with additional therapeutic agents, the compounds may be administered either sequentially or simultaneously by any convenient route. When a therapeutic agent is used in combination with an additional therapeutic agent active against the same disease, the dose of each agent in the combination may differ from that when the therapeutic agents are used alone. Appropriate doses will be readily appreciated by those skilled in the art. Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of’ and the aspects and embodiments described above with the term “comprising” replaced by the term ’’consisting essentially of’. It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and / or optional features either singly or together with any of the other aspects, unless the context demands otherwise. Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention. All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes. The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Experimental We created a database with - to our best knowledge - most known BBBpS (71 entries) and, from the amino acid residues sequence of those peptides, defined nine relevant physicochemical parameters and established the central boundaries (i.e., comprising 60% of data) for each property. Using a second database with most known CPPs (521 entries), we have identified CPPs with physicochemical properties matching the BBBpS hallmarks and applying an ordinary least squares (OLS) method, ranked CPPs for potential BBBpS activity and selected the ten best BBBpS candidates. These ten best BBBpS leads were next produced and tested in an in vitro BBB model to validate experimentally the methodology with nine out ten peptide showing moderate-to-high translocation. We then selected the four best performers and made in vivo biodistribution studies to assess their brain uptake in a mice model. Results confirmed that all peptides had high brain penetration (>0.5% ID / g tissue) compared to others Taken together, the in vitro and in vivo data showed good correlation with the predicted ranking, confirming the robustness and reliability of our methodology. Methods 1. Database design and data collection CPPs and BBBpS are published over a wide variety of journals. The search was focused on 33 journals providing a broad coverage of peptide science areas, from academic scientific research to health applications. To identify papers relevant for the BBBpS and CPP databases, the search engine was trained at each journal website for keywords. Specifically, for the CPP database, the keywords “peptides”, “cell-penetrating peptides”, “CPP”, “internalization”, “membrane”, “uptake”, and / or “penetration” were chosen, while for the BBBpS database the keywords were “peptides”, “blood-brain barrier”, “BBB”, “BBB peptide shuttle”, “trans-BBB peptide”, “translocation”, “brain”, “endothelial”, and / or “uptake”. In addition, ten recent reviews (from 2016 onwards) were examined to complement the databases 4,5,12,29-35 The information exported to the database was the peptide name (if available), sequence, main cargoes reported, cellular model, pathologies addressed, and proposed internalization / translocation mechanism. 2. Identifying CPPs as potential BBBpS The selection of potential new BBBpS from CPPs followed a multi-step strategy (Fig. 1). After compiling both peptide databases, nine physicochemical parameters were determined for each entry using either online tools 17 or information from the literature: a) molecular weight (g.mol1), b) UV-Vis extinction coefficient (MTcm1), c) hydrophobic percentage (%), d) isoelectric point, e) net charge (pH 7.0), f) charge, g) average hydrophobicity, h) hydrophobicity (pH 7.0), and i) hydrophilic residues ratio (%)■ For each of these properties in the BBBpS database, a 60% inclusion range, i.e., an interval reasonably representative of the property value distribution was defined and applied as cutoffs to identify potential BBBpS in the larger CPP database. Peptides with all parameters within the defined cutoffs were then ranked using an OLS method: TV                        ,                 , 2 $ _ V-1 Pi,CPP — Yi,BBBpS / BBBpS . Eq. 1 where, N is the number of parameters, Pi,cpp is the value of parameter i for a specific CPP, and Pi,bbbps and oiBBBps are respectively the average value and the standard deviation of parameter i in the BBBpS database. In our case, the a-i parameters defined above imply N=9. According to Eq. 1, the best BBBpS candidates in the CPP database will be those with minimal sums of squares (S). The ten CPPs with lowest S values were thus selected to be experimentally tested as BBBpS candidates. Likewise, the three CPPs with the highest S values among those outside the 60% intervals were selected as negative controls (non-BBBpS). For simplicity, the BBBpS candidates are hereafter referred to as BBBpS_X (X = 1,2, ..., 10), and the three non-BBBpS as non-BBBpS_Y (Y = 1,2, 3). These peptides are identified in Table 1. Numbering is assigned alphabetically according to peptide names hence does not involve a best / worst BBBpS candidate ranking. An example of the application of the method to the “net charge at pH 7” parameter is shown in Fig. 1. 3. Chemicals and materials Fmoc-protected amino acids, Fmoc-Rink amide (MBHA) resin, 2-(1H-benzotriazol-1-yl)-1, 1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and A / -hydroxybenzotriazole (HOBt) were from Iris Biotech (Marktredwitz, Germany). HPLC-grade acetonitrile (ACN), and peptide-synthesis grade N,N-dimethylformamide (DMF), dichloromethane (DCM), A / ,A / -diisopropylethylamine (DIEA), N,N-diisopropylcarbodiimide (DIPCI), trifluoroacetic acid (TFA), and triisopropylsilane (TIS) were from Carlo Erba-SDS (Sabadell, Spain). 3,6-dioxa-1,8-octanedithiol (DODT), 5(6)-carboxyfluorescein (CF), and tetramethylrhodamine isothiocyanate-4 KDa dextran (TRITC-Dx4) were from Sigma-Aldrich (Spain). Dulbecco’s modified Eagle medium (DMEM), DMEM / Ham’s F-12 (DMEM:F12), DMEM:F12 without phenol-red, trypsin-EDTA, attachment factor protein solution (AF), fetal bovine serum (FBS), and penicillin-streptomycin antibiotic solution (Pen / Strep) were from Gibco / Thermo Fischer (USA). Minimum essential medium Eagle (EMEM), and endothelial cell growth supplement (ECGS) were from Sigma-Aldrich (Spain). CellTiter-Blue® cell viability reagent was from Promega (Spain). 4. Peptides The peptide sequences in Table 1 were assembled in a Prelude synthesizer (Gyros Protein Technologies, USA) running Fmoc (FastMoc) solid-phase peptide synthesis (SPPS) protocols at 0.1 mmol scale on a Fmoc-Rink-amide ChemMatrix resin. Non-labeled versions of the peptides were obtained upon acidolytic (TFA) deprotection and cleavage of the respective peptide-resins, followed by semi-preparative reverse phase HPLC purification and analytical documentation by HPLC and MS as described 2’19. Fluoro- and / or radiolabeled versions of each peptide (Table 1) were also made as required, by coupling either 5(6)-carboxyfluorescein (CF)19 or the 67Ga chelating unit NODA-Ga(tBu)3 1120 at the N-terminus of the corresponding peptide-resin, following optimized protocols . The CF- and NODA-Ga labeled peptides were obtained after TFA treatment of the corresponding peptide-resins, and purified and characterized similarly to the free versions above. For radiolabeling with 67Ga3+, a fraction of 67GaCh (0.5 Ml, 156 MBq) eluted from a Sep-Pak® Classic Silica cartridge (690 mg, 55105 pm, Waters™) was adjusted to pH 5.5 by 0.5 mL OF 0.4 M sodium acetate buffer pH 5.5. An aliquot of this solution (190 pL, 28-32 MBq) was added to the purified NODA-Ga labeled peptide (10 pL, 0.75 mM), and the mixture was incubated for 30 min at r.t. The radiochemical purity of the 67Ga-peptides was evaluated by analytical RP-HPLC as described 11’20. 5. Cell culture Human cerebral microvasculature endothelial cells (HEBC-5i, ATCC® CRL-3245™), human fibroblast (Hs68, ATCC® CRL-1635™), human epithelial cells (HeLa, ATCC® CCL-2™), human breast cancer cells (MDA-MB-231, ATCC® HTB-26™), and human embryonic kidney cells (HEK-293, ATCC® CRL-1573™) were purchased from American Type Culture Collection (Manassas, VA). Hs68, HeLa, and MDA-MB-231 cells were cultured as a monolayer in DMEM supplemented with 10% FBS, and 1% pen / strep, according to manufacturer’s instructions. HEK-293 cells were cultured as a monolayer in EMEM supplemented with 10% FBS, and 1% pen / strep, according to manufacturer’s instructions. HBEC-5i cells were cultured as a monolayer on AF-coated T-flasks in DMEM:F12 supplemented with 10% FBS, 1% pen / strep, and 40.0 pg / mL ECGS, according to manufacturer’s instructions. All cells were grown in a humidified atmosphere of 5% CO2 at 37°C (MCO-18AIC (UV), Sanyo, Japan) with the medium changed every other day. 6. Translocation across a human endothelial cell line The translocation capacity of all CF-peptides was evaluated using an in vitro HBEC-5i cell model, as previously described 19. Briefly, HBEC-5i cells were carefully harvested with trypsin-EDTAand seeded at 8.000 cells / well in AF pre-coated tissue culture inserts (transparent polyester (PET) membrane with 1.0 pm pores) for 24-well plates (BD Falcon, USA). Throughout 8 days, medium was changed every other day. On the day of the experiment, cells were washed twice with 1X PBS (137.0 mM NaCI, 2.7 mM KCI, 10.0 mM Na2HPO4, and 1.8 mM KH2PO4), and once with DMEM:F12 without phenol red. Then, CF-peptides (5.0 pM, in DMEM:F12 without phenol red) were added to the apical side of the in vitro BBB model and incubated for 24 h. Finally, samples from the apical and basolateral side were collected and fluorescence intensity analyzed using a Varioskan™ LUX multimode microplate reader (Thermo Fisher, Spain). The percentage (%) of translocation was calculated using the following equation: (P' _p        \ —-----------I x 100 ^peptide — Medium / Eq. 2 where Fi, Fceiis, Fpeptideand FMedium denote respectively the fluorescence intensity recovered, that of untreated cells, that of total peptide initially added to the transwell apical side, and that of the medium. Experiments were performed in triplicates on different days using three independently grown cell cultures. 7. In vitro BBB model integrity assay After the translocation assay, an in vitro BBB integrity assay was performed. Herein, cells were washed twice with 1X PBS and once with DMEM:F12 without phenol red. Then, previously diluted TRITC-Dx4 was added to the apical side and incubated for 2h. TRITC-Dx4 was diluted in DMEM:F12 without phenol red to an absorbance below 0.1. Finally, samples from the apical and basolateral side were collected and fluorescence intensity analyzed using a Varioskan™ LUX multimode microplate reader. The percentage of TRITC-Dx4 recovered was determined using the following equation: TRITC_Dx4 Permeability (%) = (-----—----) x 100 \Ftritc-Dx4 — ^Medium' Eq. 3 where Fi, Fceiis, Ftritc-dx4 and FMedium are defined as above. The integrity of the in vitro BBB model is indirectly proportional to the percentage of TRITX-Dx4 recovered and was determined using the following equation: Integrity(%) = 100 — TRITCDx4Permeability (%) Eq. 4 8. Cytotoxicity towards a panel of human cell lines Peptide cytotoxicity was determined using the CellTiter-Blue® cell viability assay, following a described protocol19. Briefly, all cell lines were carefully harvested with trypsin-EDTAand seeded 10.000 - 20.000 cells / 100 pL into 96-well clear flat-bottomed polystyrene plates (Corning, USA) for 24h. After medium removal, cells were washed twice with 1X PBS, and 100 pL of previously diluted peptides (range from 0.05 - 100.0 pM) in the respective medium were added to cells. Then, after 24h, cells were washed twice with 1X PBS and 20 pL of CellTiter-Blue® Reagent (diluted in 100 pL of medium) was added to each well and incubated for 3h in culturing conditions. The fluorescence intensity was measured using Varioskan™ LUX multimode microplate reader. IC50 values were determined using GraphPad Prism 7.0 software using a log(inhibitor) versus normalized response. Experiments were performed in triplicates on different days using three independently grown cell cultures. 9. Internalization across human cell lines The ability of peptides to cross cellular membranes was evaluated using a previously described protocol with minor alterations 20. Briefly, cells were harvested with trypsin-EDTAand seeded at 50,000 cells / 500 pL into 24-well clear flat-bottomed polystyrene plates (Corning, USA) and incubated in their respective medium for 24h. After medium removal, cells were washed twice with 1X PBS, and 5.0 pM of CF-peptides predissolved in the respective medium were added. After 24h, cells were washed twice with 1X PBS, harvested, and washed again twice with 1X PBS. The fluorescence intensity of 10,000 cells, measured with a BD LSRFortessa X-20 flow cytometer (BD Biosciences, USA), defines the ratio between the mean fluorescence of a sample and that of untreated cells. Experiments were performed in triplicates on different days using three independently grown cell cultures. 10.     Biodistribution All animal experiments were performed in compliance with national and EU legislation for good practices on laboratory animal science. The animals were housed in a temperature and humidity-controlled environment with a 12h light / 12h dark schedule. Biodistribution of radiolabeled peptides was performed on CD1 mice. Animals were intravenously injected into the tail vein with the 67Ga-peptide diluted in 100 pL of saline. The mice were euthanized by cervical dislocation at 2 min and 1h after injection. The dose administered and the radioactivity in the euthanized animals was measured using a dose calibrator (Carpintec CRC-15W, Ramsey, USA). The difference between the radioactivity in the injected and the euthanized animals was assumed to be due to excretion. Brain and tissues of interest were dissected, washed, and weighed, and their radioactivity was measured using a y counter (Hidex AMG, Hidex, Turku, Finland). The uptake in the brain and tissues of interest was calculated and expressed as a percentage of injected radioactivity dose per gram of tissue (% ID / g). 11.      Statistical analysis Quantitative data were processed using Excel 2013 (Microsoft, USA) and the GraphPad Prism version 7.0 software (USA). Medians, means, and standard deviations are shown in figures. Pairwise significances were calculated using one-way ANOVA followed by Tukey’s or Dunnett’s multiple comparison test, and nonparametric Mann-Whitney, or unpaired t-tests. Results 1. Selection of BBBpS and non-BBBpS The BBBpS and GPP databases built from literature searches had respectively 71 and 521 entries. Theoretical values for each physicochemical parameter were calculated from the amino acid sequence. These values for BBBpS 1-10 and nonBBBpS 1-3 are shown in Table 2. Cutoff values, defining symmetrical intervals enclosing 60% of each BBBpS parameter (relative to the mean), are shown in Table 5. Fig. 1 shows an example for the “net charge at pH 7.0” criterion. In this case, the average net charge is 2.1 and 60% of BBBpS within the -1.0 to 4.7 interval. Applying similar criteria to the CPP database (Fig. 1), a subset of 14 CPPs with parameters (within cutoff limits) matching those in the BBBpS selection was identified. The peptides selected in both databases were then ranked using the OLS method and the ten best-ranking BBBpS candidates and three non-BBBpS negative controls were synthesized and experimentally tested for their ability to transverse the BBB model (Table 1). Results were compared to PepH3, a well-tested BBBpS standard. 2. Translocation across an in vitro BBB model The relative fluorescence intensity of samples from apical and basolateral compartments in the in vitro BBB model are shown in Table 3 and Fig. 3A. For PepH3 a translocation of 54.0±2.2% was achieved after 24h incubation. As for the BBBpS studied, translocation levels above 30% were observed, except for BBBpS_7 (20.3±5.1%). Interestingly, some of the BBBpS selected had similar or higher BBB permeability than PepH3, e.g., BBBpS_1 (61.4±2.3%), BBBpS_2 (41.4±3.9%), BBBpS_5 (53.0±4.7%), and BBBpS_9 (46.0±4.3%). Importantly, these results agreed with the predicted OLS ranking of BBBpS (Fig. 2), i.e., peptides with low S values displayed high translocation ability and, conversely, those with high S values showed moderate / low translocation. Also, and in tune with the experimental design, all non-BBBpS had low translocation efficiency, e.g., for non-BBBpS_2 gave 2.6±1.2% and for non-BBBpS_3 gave 11.1±2.6%, and for non-BBBpS_1 gave 20.0±3.5%. These results altogether corroborate the negative correlation between translocation capacity and S (see Equation 1 in methods below and Fig. 2). Finally, it must be noted that the peptide concentration (5.0 pM) used in all translocation assays is well below the IC50 toxicity values determined on a panel of human cell lines (see section d) below -Toxicity towards a human cell line panel). 3. In vitro BBB model barrier integrity None of the peptides increased paracellular permeability in the in vitro BBB model (HBEC-5i integrity > 90.0% - Fig. 3B). The difference between all peptides and control was not statistically significant (p>0.05). 4. Toxicity towards a human cell line panel In vitro peptide cytotoxicity was studied on a panel of cell lines commonly used in preclinical studies to assess translocation (HBEC-5i), toxicity (HeLa, MDA-MB-231, and Hs68), and transfection (HEK-293). The CellTiter-Blue® assay gave IC50 values above 100.0 pM for all peptides (Table 3 and Fig. 4), with the only exception of non-BBBpS_2, with a minimum IC50 of 14.7±1.4 pM in HEK-293 cells and a maximum of 39.4±1.8 pM in HeLa cells. 5. Internalization into human cell lines The capacity of all peptides to traverse cell membranes and internalize in the cytoplasm was evaluated on the same cell panel used for the cytotoxicity tests. Flow cytometry data (Table 3 and Figs. 5 and 6) report the relative fluorescence intensity (RFI) measured on each cell line upon treatment with CF-peptide (5.0 pM) in comparison with untreated cells. Selected peptides underwent moderate (8.0 < RFI < 200.0) to high internalization (RFI > 200.0) regardless of cell type, with HEK-293 and MDA-MB-231 respectively displaying slightly higher and lower internalization than the rest of the panel. The potential BBBpS candidates revealed in all cases an ability to accumulate inside cells. BBBpS_2, BBBpS_5, and BBBpS_7 showed the highest internalization (RFI > 20.0), with no particular selectivity, while BBBpS_3 had some selectivity towards HEK-293 cells (RFI > 40.0). BBBpS_4, BBBpS_6, and BBBpS_10 had the lowest uptakes (RFI < 10.0). The three negative control peptides were internalized successfully (RFI > 150.0), confirming their CPP nature, non-BBBpS_1 and non-BBBpS_2 being the most efficient and showing selectivity towards HBEC-5i and HEK-293 cells. 6. Biodistribution The most promising BBBpS, namely BBBpS_1, BBBpS_2, BBBpS_5, and BBBpS_9, were selected for / n vivo biodistribution studies in healthy mice using 67Ga-radiolabed peptide derivatives. A non_BBBpS, non-BBBpS_2, was used as a negative control to validate the capacity to discriminate potential BBBpS from non_BBBpS. The biodistribution profile of 67Ga-BBBpS_1,67Ga-BBBpS_2, 67Ga-BBBpS_5,67Ga-BBBpS_9, and 67Ga-non-BBBpS_2 including brain uptake is shown in Table 4. The results show that brain penetration of BBBpS was fast, with brain uptake > 0.5% ID / g after 2 min in all 67Ga-labeled BBBpS tested. Importantly, the brain uptake for 67Ga-BBBpS_1 (0.66±0.16% ID / g), 67Ga-BBBpS_2 (0.52±0.23% ID / g), 67Ga-BBBpS_5 (0.74±0.01% ID / g), and 67Ga-BBBpS_9 (0.61±0.11% ID / g) at 2 min postinjection is, respectively, 2.4-, 1.9-, 2.7-, and 2.3-fold higher than the brain accumulation of 67Ga-non-BBBpS_2 (0.27±0.04% ID / g) at 2 min post-injection. Brought together, meta-analysis, in vitro and in vivo data suggest that a CPP is not necessarily a good BBBpS. Moreover, all BBBpS have fast clearance from blood primarily through renal excretion (high kidney uptake at 2 min followed by rapid elimination at 1 h time point) and rapid brain washout (<0.1% ID / g after 1h post-injection), in accordance with fast radioactivity elimination from most organs. In addition, they all present high excretion rate (>75 % at 1h post-injection). Unlike the 67Ga-BBBpS tested, the biodistribution profile of the 67Ga- non-BBBpS_2 is different. It has also a fast blood clearance and an important radioactivity fraction is eliminated via the urinary pathway, as suggested by the kidney uptake. However, high liver uptake and retention was found (19.8±0.7 and 22.8±10.1% ID / g at 2 min and 1h post injection, respectively) indicating an important contribution of the hepatobiliary tract on the elimination of this radiolabeled peptide. In agreement with this finding, the total radioactivity excretion was much lower, 13.5±5.1% ID / g at 1h after administration. Overall, the results show clearly that all BBBpS display a high brain uptake, and the non-BBBpS is very different, considering not only brain penetration but also the rest of the biodistribution. A multi-step methodology was devised to shed light on the physicochemical characteristics enabling BBBpS to traverse cell barriers. To this end, databases were generated for all published BBBpS and CPPs (71 and 521 entries, respectively). The size difference between both databases suggests that research on BBBpS lags behind that of CPPs. In the former database physicochemical properties easily evaluated from the peptide sequence 17 were included and it was found that, overall, BBBpS have: i) a small size (average molar mass of 2046 g.mol-1), ii) none or few aromatic residues (average molar absorptivity of 3790 MLcm1 at 280 nm, corresponding to 1-2 Tyr or 0-1 Trp residues, Hi) a slightly hydrophobic nature (x = 35% - mean content in hydrophobic residues), and iv) a slightly cationic charge (average net charge of +2). The following step was identifying, within the large CPP family, peptides most likely to perform as BBBpS. To this end, the 521 entries of the CPP database were screened against nine physicochemical parameters for which statistically relevant cutoffs had previously been defined within the BBBpS set (Fig. 1). The result was a subgroup of 14 CPPs with presumable BBBpS potential. This selection involved the exclusion of all other 504 entries (-98%) in the CPP database, which strongly suggests that the overlap between CPP and BBBpS families is indeed small. Consequently, brain-targeting drug delivery strategies based on “chemical intuition” assumptions of alleged CPP-BBBpS equivalence are ill-founded and likely to be proven unrealistic. Ten peptides were synthesized and then their activity was tested in vitro (Table 1), selected among the 14 BBBpS entries by their lowest S score in an OLS ranking 18. An in vitro model with a HBEC-5i cell monolayer was used to assess the BBB translocation of the peptides 19’20. In this quick and robust molecular screen the fluorescence intensity measured on both the apical and basolateral compartments of the device is readily converted into the rate of cellular barrier crossing by the peptide under study. Most BBBpS candidates tested positive in this in vitro BBB model, with four of them, namely, BBBpS_1, BBBpS_2, BBBpS_5, and BBBpS_9 being highly active (Table 3 and Fig. 3), similar to PepH3, a well-characterized and efficacious BBBpS 1119-22. Conversely, non-BBBpS displayed poor endothelial cell barrier translocating abilities, thus further validating the methodology adopted. Importantly, the experimental results show a high correlation with the S values obtained in the prior OLS ranking. Peptides with lower S scores corresponding to the most active BBBpS and those (non-BBBpS) with high S values being unable to effective cross the BBB (Fig. 2). In conclusion, while all peptides tested perform well as CPP, only the ones predicted as BBBpS perform well in traversing a BBB cell model, which further confirms that both conditions are independent from each other. Moreover, the BBBpS perform slightly worst as CPP, which may relate to their ability to enter endothelial cells. Peptide ability to internalize a cell line panel commonly applied in preclinical toxicity studies was also evaluated (Table 3 and Figs. 5 and 6). Results show both candidate BBBpS and non-BBBpS to have internalization capacity, consistent with their primary CPP nature. Interestingly, however, non-BBBpS internalize more efficiently than BBBpS candidates, regardless of cell line. Overall, peptides internalize more in HEK-293 than in other lines, which is not surprising as CPPs are commonly designed and applied for transfection 23 and HEK-293 is extensively used for protein expression 2425. In contrast, the lowest internalization was observed in MDA-MB-231 cells, which are triple-negative breast cancer metastatic cells able to colonize the brain. These cells have a unique membrane composition 26, which is a factor affecting peptide internalization. For all BBBpS, the in vivo data showed a rapid (up to 2 min. after injection) and efficient (>0.5% ID / g) brain penetration, followed by a quick brain washout (<0.1% ID / g after 1 h), and clearance from all organs (Table 4). Since 0.1% ID / g has been accepted as a good BBB crossing 27, our BBBpS demonstrated a potent brain targeting, achieving even higher values than the ones obtain for known brain-targeted molecules in the literature. For instance, TAT, penetratin, synB1 and others range from 0.2 - 0.9% ID / g 28. In addition, the selected BBBpS present high excretion rate (>75% ID / g at 1h postinjection), which is an important feature to avoid toxicity associated to accumulation in the main organs. This characteristic ensures that all BBBpS not only can be used as an active carrier to the brain, but can also be an active shuttle in-and-out the brain. For non-BBBpS_2, our results demonstrate a lower brain accumulation (0.27±0.04% ID / g after 2 min post-injection), followed by a more slowly brain washout (0.13±0.03% ID / g after 1 h), and high accumulation in the liver (22.8±10.1% ID / g after 1h). The different effects observed with BBBpS and CPPs support the claim that that both families are not strictly correlated. The internalization capacity of CPPs can be exploited to shuttle payloads across membranes into the cytoplasm or the nucleus while, in using BBBpS for brain delivery, researchers may expect endothelial barrier translocation. Thus, instead of cargo delivery to a primary target cell, a BBBpS can be expected to transcytose an endothelial cell and deliver a given payload at a target site in the brain parenchyma. These complementary behaviors will no doubt continue to be further explored by researchers aiming at more efficient delivery systems for therapeutic purposes. Although the main physicochemical characteristics underlying BBBpS activity remain elusive, intuitive approaches naively tend to assume all CPPs as potential BBBpS, in a perspective where BBB translocation is viewed as a sequential crossing of cellular membranes. In these experiments, a quantitative methodology was devised and applied that combines meta-analysis and statistical reasoning, and demonstrate that, based on intrinsic structural features, i.e., small size, few aromatic residues, and slightly hydrophobic and slightly cationic nature, very few CPPs are indeed BBBpS, in practice demonstrating that both peptide families should better be separately viewed. In addition, this work has identified four BBBpS with high translocation abilities in vitro and high brain penetration in vivo. References 1 Cavaco, M., Andreu, D. & Castanho, M. A. R. B. The Challenge of Peptide Proteolytic Stability Studies: Scarce Data, Difficult Readability, and the Need for Harmonization" doi:10.1002 / ange .202006372 (2020). 2 Cavaco, M., Valle, J., Flores, I., Andreu, D. & Castanho A.R.B., M. Estimating peptide half-life in serum from tunable, sequence-related physicochemical properties. Clinical and Translational Science, doi: 10.1111 / cts. 12985 (2021). 3 Zou, L.-L., Ma, J.-L., Wang, T., Yang, T.-B. & Liu, C.-B. Cell-Penetrating Peptide-Mediated Therapeutic Molecule Delivery into the Central Nervous System. Curr Neuropharmacol 11, 197-208, doi:10.2174 / 1570159X11311020006 (2013). 4 Oiler-Salvia, B., Sanchez-Navarro, M., Giralt, E. &Teixido, M. Blood-brain barrier shuttle peptides: an emerging paradigm for brain delivery. Chern Soc Rev 45, 4690-4707, doi:10.1039 / C6CS00076B (2016). 5 Gallo, M., Defaus, S. & Andreu, D. 1988-2018: Thirty years of drug smuggling at the nano scale. Challenges and opportunities of cell-penetrating peptides in biomedical research. Archives of Biochemistry and Biophysics 661,74-86, doi:https: / / doi.org / 10.1016 / j.abb.2018.11.010 (2019). 6 Stalmans, S. et al. Cell-Penetrating Peptides Selectively Cross the Blood-Brain Barrier In Vivo. PloSone 10, e0139652-e0139652, doi:10.1371 / journal.pone.0139652 (2015). 7 Patel, S. G. et al. Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines. Scientific Reports 9, 6298, doi: 10.1038 / S41598-019-42456-8 (2019). 8 Xie, J. et al. Cell-Penetrating Peptides in Diagnosis and Treatment of Human Diseases: From Preclinical Research to Clinical Application. Frontiers in Pharmacology 11, 697 (2020). 9 Frankel, A. D. & Pabo, C. O. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189-1193, doi:https: / / doi.org / 10.1016 / 0092-8674(88)90263-2 (1988). 10 Oiler-Salvia, B. et al. MiniAp-4: A Venom-Inspired Peptidomimetic for Brain Delivery. Angewandte Chemie International Edition 55, 572-575, doi:10.1002 / anie.201508445 (2016). 11 Neves, V. et al. Novel Peptides Derived from Dengue Virus Capsid Protein Translocate Reversibly the Blood-Brain Barrier through a Receptor-Free Mechanism. ACS Chemical Biology 12, 1257-1268, doi:10.1021 / acschembio.7b00087 (2017). 12 Kristensen, M. & Brodin, B. Routes for Drug Translocation Across the Blood-Brain Barrier: Exploiting Peptides as Delivery Vectors. Journal of Pharmaceutical Sciences 106, 2326-2334, doi:10.1016 / j.xphs.2017.04.080 (2017). 13 Cavaco, M., Gaspar, D., Arb Castanho, M. & Neves, V. Antibodies for the Treatment of Brain Metastases, a Dream ora Reality? Pharmaceutics 12, 62, doi:10.3390 / pharmaceutics12010062 (2020). 14 Cavaco, M., Castanho, M. A. R. B. & Neves, V. Peptibodies: An elegant solution for a longstanding problem. Peptide Science 110, e23095, doi:10.1002 / bip.23095 (2018). Zou, H. Identifying blood-brain barrier peptides by using amino acids physicochemical properties and features fusion method. Peptide Science 114, e24247, doi:https: / / doi.org / 10.1002 / pep2.24247 (2022). Chen, X. et al. BBPpredict: A Web Service for Identifying Blood-Brain Barrier Penetrating Peptides. Frontiers in Genetics 13 (2022). bioSYNTHESIS. Vol. ver 3.1 https: / / www.biosyn.com / peptidepropertycalculator / PeptidePropertyCalculator.aspx (2013). Lau, L.-S., Ng, C.-F., Cheah, S.-P. & Choong, C.-K. in Environmental Kuznets Curve (EKC) (eds Burcu Ozcan & llhan Ozturk) 101-113 (Academic Press, 2019). Cavaco, M. et al. To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models. Frontiers in Bioengineering and Biotechnology 8, 1059 (2020). Marco Calvinho, C. et al. DPepH3, an Improved Peptide Shuttle for Receptor-independent Transport Across the Blood-Brain Barrier. Current Pharmaceutical Design 26, 1-12, doi:http: / / dx.doi.org / 10.2174 / 1381612826666200213094556 (2020). Neves-Coelho, S., Eleuterio, R. P, Enguita, F. J., Neves, V. & Castanho, M. A. R. B. A New Noncanonical Anionic Peptide That Translocates a Cellular Blood-Brain Barrier Model. Molecules (Basel, Switzerland) 22, 1753, doi:10.3390 / molecules22101753 (2017). Corte-Real, S. et al. Antibody molecules and peptide delivery systems for use in alzheimer's disease and related disorders. (2016). Gagat, M., Zielihska, W. & Grzanka, A. Cell-penetrating peptides and their utility in genome function modifications (Review). Int J Mol Med 40, 1615-1623, doi:10.3892 / ijmm.2017.3172 (2017). Ooi, A., Wong, A., Esau, L., Lemtiri-Chlieh, F. & Gehring, C. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization. Frontiers in Physiology 7, 300 (2016). Thomas, P. & Smart, T. G. HEK293 cell line: A vehicle for the expression of recombinant proteins. Journal of Pharmacological and Toxicological Methods 51, 187-200, doi:https: / / doi.org / 10.1016 / j.vascn.2004.08.014 (2005). He, M., Guo, S. & Li, Z. In situ characterizing membrane lipid phenotype of breast cancer cells using mass spectrometry profiling. Scientific Reports 5, 11298, doi:10.1038 / srep11298 (2015). Lalatsa, A., Schatzlein, A. G. & Uchegbu, I. F. Strategies To Deliver Peptide Drugs to the Brain. Molecular Pharmaceutics 11, 1081-1093, doi:10.1021 / mp400680d (2014). Sarko, D. et al. The Pharmacokinetics of Cell-Penetrating Peptides. Molecular Pharmaceutics 7, 2224-2231, doi:10.1021 / mp100223d (2010). Dissanayake, S., Denny, W. A., Gamage, S. & Sarojini, V. Recent developments in anticancer drug delivery using cell penetrating and tumor targeting peptides. Journal of Controlled Release 250, 62-76, doi:https: / / doi.org / 10.1016 / j.jconrel.2017.02.006 (2017). 30 Xu, J. et al. Cell-penetrating peptide: a means of breaking through the physiological barriers of different tissues and organs. Journal of Controlled Release 309, 106-124, doi:https: / / doi.org / 10.1016 / j.jconrel.2019.07.020 (2019). 31 Bolhassani, A., Jafarzade, B. S. & Mardani, G. In vitro and in vivo delivery of therapeutic proteins using cell penetrating peptides. Peptides 87, 50-63, doi:https: / / doi.org / 10.1016 / j.peptides.2016.11.011 (2017). 32 Singh, T., Murthy, A. S. N., Yang, H.-J. & Im, J. Versatility of cell-penetrating peptides for intracellular delivery of siRNA. Drug delivery 25, 1996-2006, doi: 10.1080 / 10717544.2018.1543366 (2018). 33 Kalafatovic, D. & Giralt, E. Cell-Penetrating Peptides: Design Strategies beyond Primary Structure and Amphipathicity. Molecules (Basel, Switzerland) 22, 1929, doi:10.3390 / molecules22111929 (2017). 34 McCully, M., Sanchez-Navarro, M., Teixido, M. & Giralt, E. Peptide Mediated Brain Delivery of Nano- and Submicroparticles: A Synergistic Approach. Current pharmaceutical design 24, 1366-1376, doi:10.2174 / 1381612824666171201115126 (2018). 35 Ghosh, D., Peng, X., Leal, J. & Mohanty, R. P. Peptides as drug delivery vehicles across biological barriers. Journal of Pharmaceutical Investigation 48, 89-111, doi:10.1007 / s40005-017-0374-0 (2018). 36 Innovagen Peptide Calculator (PepCalc) - https: / / pepcalc.com / ppc.php 37 Stanley C. Gill, Peter H. von Hippel. Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry, Volume 182, Issue 2, 1989, Pages 319-326, ISSN 0003-2697, https: / / doi.Org / 10.1016 / 0003-2697(89)90602-7 38. CRC Handbook of Chemistry and Physics, 87th Edition, David R. Lide (editor) - ISBN 0849304873 39 Thomas P. Hopp, Kenneth R. Woods. A computer program for predicting protein antigenic determinants. Molecular Immunology, Volume 20, Issue 4, 1983, Pages 483-489, https: / / d0i.0rg / l 0.1016 / 0161-5890(83)90029-9. Table 1. Sequence and analytical data for the peptides Peptide Code Amino Acid Sequence Theoretical mass (Da)a Experimental mass (Da)b HPLC tR (min) Purity (%)c A KYKGAIIGNIK-amide 1203.57 1203.49 4.99 96.54 CF-A BBBpS_1 CF-KYKGAIIGNIK-amide 1561.89 1560.79 7.05 96.02 67Ga-A 67Ga-KYKGAIIGNIK-amide N.A N.A 11.75 >95 B KYRSGAITIGY-amide 1227.49 1227.43 5.65 97.29 CF-B BBBpS_2 CF-KYRSGAITIGY-amide 1585.81 1584.73 7.62 94.63 67Ga-B 67Ga-KYRSGAITIGY-amide N.A N.A 11.95 >95 CPP(II) BBBpS_3 EEGRLYMRYYSPTTRRYG-amide 2297.66 2297.58 5.60 97.06 CF-CPP(II) CF-EEGRLYMRYYSPTTRRYG-amide 2655.98 2654.88 7.95 90.79 CTP BBBpS_4 APWHLSSQYSRT-amide 1431.63 1431.57 5.51 95.66 CF-CTP CF-APWHLSSQYSRT-amide 1789.95 1788.87 8.45 93.68 HAP-2 HIQLSPFSQSWR-amide 1484.75 1484.68 7.14 94.53 CF-HAP-2 BBBpS_5 CF-H IQLSPFSQSWR-amide 1843.07 1841.98 9.50 93.70 67Ga-HAP-2 67Ga-HIQLSPFSQSWR-amide N.A N.A 12.97 >95 hCR(12-32) CF-hCR(12- BBBpS_6 YTQDFNKFHTFPQTAIGVGAP-amide 2338.72 2338.61 6.78 97.46 CF-YTQDFNKFHTFPQTAIGVGAP-amide 2697.04 2695.91 8.79 94.18 32) KLA13 BBBpS_7 LKTLTETLKELTKTLTEL-amide 2074.61 207.49 10.05 95.95 CF-KLA13 CF-LKTLTETLKELTKTLTEL-amide 2432.93 2431.79 13.51 98.50 Peptide b3-1 CF-Peptide BBBpS_8 YKEATSTFTNITYRGT-amide 1852.11 1852.03 5.87 86.69 CF-YKEATSTFTNITYRGT-amide 2210.43 2209.33 8.63 95.32 b3-1 Peptide third BBBpS_9 NRPDSAQFWLHH-amide 1506.71 1506.65 6.13 93.08 WO 2025 / 109110                                   PCT / EP2024 / 083182 Peptide Code Amino Acid Sequence Theoretical mass (Da)a Experimental mass (Da)b HPLC tR (min) Purity (%)c CF-Peptide third 67Ga-Peptide third CF-NRPDSAQFWLHH-amide 67Ga-NRPDSAQFWLHH-amide 1865.03 N.A 1863.95 N.A 8.01 12.25 96.68 >95 SPA BBBpS 10 RPKPQQFFGLM-amide 1347.71 1347.65 7.58 95.23 CF-SPA P1746c27 non- BBBpS_1 CF-RPKPQQFFGLM-amide KKKKQPPKPKKPKTQEKKKKQPPKPKR-amide OF- 1706.03 3262.23 1704.95 3262.08 9.25 2.77 95.05 98.57 CF-P1746c27 KKKKQPPKPKKPKTQEKKKKQPPKPKR-amide 3620.55 3619.38 4.23 98.04 Tl non-BBBpS KWCFRVCYRGICYRRCR-amide 2267.85 2267.81 6.81 94.63 CF-TI _2 CF-KWCFRVCYRGICYRRCR-amide 2626.17 2625.11 8.11 87.61 67Ga-TI 67Ga-KWCFRVCYRGICYRRCR-amide N.A N.A 12.99 99.0 YDEGE non-BBBpS YDEEGGGE-amide 853.84 853.80 2.60 95.71 CF-YDEGE _3 CF-YDEEGGGE-amide 1212.16 1211.10 7.89 98.39 Calculated using GPMAW version 8.10 bFrom the mass spectrum c Estimated by HPLC peak integration of UV chromatogram (free and CF-labelled versions) or radio-chromatogram (67Ga-labelled versions) OF, 5(6)-carboxyfluorescein 67Ga, NODA-GA Gallium 67 chelate N.A., not applicable WO 2025 / 109110                                   PCT / EP2024 / 083182 Physicochemical properties Code Sequence Molecular weight (g / mol) Extinction coefficient (M-1.cm-1) Hydro-phobic (%) Isoelectric point Net charge (pH 7) Charge Average hydrophobicity Hydrophobicity (pH 7) Ratio hydrophilic residues (%) BBBpS_1 KYKGAIIGNIK 1203.57 1280 45.45 10.98 2 3 0.1 26.45 36 BBBpS_2 KYRSGAITIGY 1227.49 2560 45.45 10.74 1 2 -0.3 30.55 27 BBBpS_3 EEGRLYMRYY SPTTRRYG 2298.64 5120 33.33 9.81 3 3 0.3 15.72 39 BBBpS_4 APWHLSSQYS RT 1432.61 6970 33.33 10.02 2.11 3 -0.4 19.75 42 BBBpS_5 HIQLSPFSQSW R 1485.73 5690 33.33 11.18 2.11 3 -0.5 25.5 50 BBBpS_6 YTQDFNKFHTF PQTAIGVGAP 2339.7 1280 38.1 7.92 1.11 2 -0.4 20.33 24 BBBpS_7 LKTLTETLKELT KTLTEL 2075.59 0 33.33 9.8 1 1 0.3 28.67 33 BBBpS_8 YKEATSTFTNIT YRGT 1853.09 2560 31.25 9.52 2 2 -0.1 19.62 31 BBBpS_9 NRPDSAQFWL HH 1507.69 5690 33.33 8.13 1.22 3 -0.2 15 42 BBBpS_10 RPKPQQFFGL M 1348.69 0 36.36 11.6 3 3 -0.2 19.91 36 non- BBBpS_1 KKKKQPPKPKK PKTQEKKKKQ PPKPKR 3263.21 0 0 11.77 15 15 1.8 -26.15 70 non- BBBpS_2 KWCFRVCYRG ICYRRCR 2268.83 8730 58.82 10.18 6.63 7 0 35.18 35 non- BBBpS 3 YDEEGGGE 854.82 1280 12.5 2.79 -4 -4 1.2 -10.62 50 WO 2025 / 109110                                   PCT / EP2024 / 083182 Table 3. Summary of the cell-based assays. Peptide Cytotoxicity (pM)a HBEC-5i HeLa Hs68 MDA-MB-231 HEK- 293 IC50 IC50 IC50 IC50 IC50 BBBpS_1 >100.0 >100.0 >100.0 >100.0 >100.0 BBBpS_2 >100.0 >100.0 >100.0 >100.0 >100.0 BBBpS_3 >100.0 >100.0 >100.0 >100.0 >100.0 BBBpS_4 >100.0 >100.0 >100.0 >100.0 >100.0 BBBpS_5 >100.0 >100.0 >100.0 >100.0 >100.0 BBBpS_6 >100.0 >100.0 >100.0 >100.0 >100.0 BBBpS_7 >100.0 >100.0 >100.0 >100.0 >100.0 BBBpS_8 >100.0 >100.0 >100.0 >100.0 >100.0 BBBpS_9 >100.0 >100.0 >100.0 >100.0 >100.0 BBBpS_10 >100.0 >100.0 >100.0 >100.0 >100.0 non-BBBpS _1 >100.0 >100.0 >100.0 >100.0 >100.0 non-BBBpS _2 20.7 ±1.0 39.4 ± 1.8 26.4 ± 2.9 26.9 ± 3.0 14.7 ± 1.4 non-BBBpS _3 >100.0 >100.0 >100.0 >100.0 >100.0 adetermined using CellTiter-Blue® assay bevaluated by fluorescence intensity using flow cytometry cevaluated by fluorescence intensity using a plate reader IC50, concentration that causes cell death in 50% of cells Internalization (relative fluorescence intensity)13 Translocation (%)c HBEC-5i He La Hs68 MDA-MB- 231 HEK293 HBEC-5i 19.6 ±0.4 11.9 ±0.0 10.3 ±0.7 6.9 ±1.2 27.5 ±6.6 61.4 ±2.3 67.5 ±7.9 18.3 ±1.5 50.7 ±6.9 23.5 ±5.6 35.5 ±6.5 41.4 ±3.9 18.7 ±0.7 13.4 ±0.3 9.8± 1.0 8.4 ±0.9 43.8 ± 10.6 35.7 ± 3.6 8.9 ±0.3 8.9 ± 1.1 6.5 ±0.5 6.4 ±0.9 8.1 ±2.5 33.4 ±3.5 15.4 ± 1.3 23.2 ± 5.4 8.8 ±0.5 17.3 ±3.7 24.5 ±4.2 53.0 ±4.7 8.0 ±0.9 7.9 ±0.1 6.7 ±0.9 5.7 ±1.2 8.7 ±4.0 35.6 ±3.9 36.6 ±4.5 83.5 ±27.8 32.9 ±3.3 26.4 ±4.5 28.0 ±7.2 20.3 ± 5.0 13.0 ±0.2 21.8±2.7 11.0 ± 1.4 12.4 ±0.6 13.4 ±0.7 35.4 ±8.5 13.8 ±0.5 16.6 ±0.1 11.6±0.7 6.8 ±0.7 23.3 ±3.0 46.0 ± 4.3 10.7 ±0.9 9.4 ±0.0 6.9 ±0.7 8.8 ±0.3 13.5±2.1 38.4 ±5.3 1577.7 ±251.1 174.8 ±60.3 225.3 ± 17.3 219.8 ±14.9 895.0 ±45.6 20.0 ±3.5 656.7 ± 52.6 268.3 ± 9.0 307.1 ±27.2 237.4 ±36.9 462.4 ± 65.9 2.6 ±1.2 212.8 ±24.3 187.0 ± 15.1 254.0 ± 29.2 157.0 ±28.5 269.0 ±61.8 11.1 ±2.6 WO 2025 / 109110                                   PCT / EP2024 / 083182 Table 4. Biodistribution Profiles of the 67Ga-labeled Peptides3. Organ 67Ga-BBBpS_1 67Ga- BBBpS_2 67Ga- BBBpS_5 67Ga- BBBpS_9 67Ga-non-BBBpS_2 2 min 1h 2 min 1h 2 min 1h 2 min 1h 2 min 1h Blood 12.6 + 2.1 0.5 + 0.3 13.4 + 3.8 1.4 + 0.9 10.7 + 2.1 1.2 + 0.3 11.6 + 1.5 0.8 + 0.4 9.3 + 6.3 0.9 + 0.6 Liver 4.1+0.8 0.3 + 0.2 3.5+1.2 0.6 + 0.2 5.2 + 1.1 1.0 + 0.4 4.7 + 0.4 2.6 + 0.3 19.8 + 0.7 22.8 + 10.1 Intestine 1.7 + 0.3 0.2 + 0.1 1.5 + 0.2 0.4 + 0.0 2.2 + 0.2 0.4 + 0.2 1.9 + 0.2 0.2 + 0.0 2.7 + 0.7 1.6 + 0.5 Spleen 2.0 + 0.2 0.2 + 0.1 1.9 + 0.6 0.5 + 0.2 2.2 + 0.1 0.3 + 0.1 2.6 + 0.3 0.9 + 0.2 5.6 + 0.4 3.7 + 1.8 Heart 3.4 + 1.0 0.3 + 0.1 3.1+0.7 0.8 + 0.1 3.8 + 0.8 0.5 + 0.0 3.3 + 0.1 0.3 + 0.0 6.0 + 0.8 1.7 + 0.1 Lung 6.1+0.7 2.5 + 0.3 6.0 + 1.2 1.7 + 0.4 7.8 + 2.8 1.2 + 0.2 5.4 + 0.6 0.7 + 0.1 7.6+1.5 2.4 + 0.7 Kidney 33.8 + 3.8 2.4 + 1.8 18.1+2.1 1.4 + 0.6 22.0 + 3.6 1.1+0.6 30.0 + 3.7 3.1+0.3 19.9 + 0.4 5.4 + 0.8 Muscle 2.4 + 0.2 0.3 + 0.1 2.0 + 0.4 0.7 + 0.1 3.1+0.8 0.5 + 0.2 2.6 + 0.3 0.2 + 0.1 1.8 + 0.5 1.2 + 0.4 Bone 3.4 + 0.3 0.3 + 0.1 3.1+0.1 0.8 + 0.5 3.8 + 0.5 0.6 + 0.4 3.4 + 0.3 0.5 + 0.1 3.7 + 0.5 1.6 + 0.1 Stomach 1.4 + 0.5 0.1+0.0 1.0 + 0.1 0.2 + 0.1 2.6 + 0.0 0.3 + 0.4 1.5 + 0.2 0.3 + 0.1 2.1 + 0.6 1.7 + 0.6 Brain 0.66 + 0.16 0.04 + 0.02 0.52 + 0.23 0.09 + 0.03 0.74 + 0.01 0.05 + 0.01 0.61 + 0.11 0.05 + 0.01 0.27 + 0.04 0.13 + 0.03 Excretion (% ID) - 79.3 ±3.3 - 77.7 ±2.6 - 78.3 ±4.7 - 82.0 ±6.1 - 13.5 ±5.1 aTissue distribution of 67Ga-BBB_1,67Ga-BBB_2,67Ga-BBB_5,67Ga-BBB_9, and 67Ga-non-BBBpS_2 at 2 min and 1h post injection via tail vein in CD1 mice. Results are expressed as the average of percentage of injected dose (ID) per gram of tissue (%ID / g tissue; mean ± SD), n=3. WO 2025 / 109110                                   PCT / EP2024 / 083182 Table 5. Limits selected for each parameter using the BBB peptide shuttle database. Physicochemical property Limits Minimum Maximum Molecular weight (g.mol-1) 852.0 3065.0 Extinction coefficient (M Lcm1) 0.0 7786.0 Hydrophobic nature (%) 18.8 50.0 Isoelectric point 8.0 13.3 Net charge (pH 7.0) -1.0 4.7 Charge 0.0 6.0 Average hydrophobicity -0.6 0.8 Hydrophobicity (pH 7.0) 5.4 34.6 Hydrophilic residues ratio (%) 17.0 56.0 Table 6 BBBpS_1 KYKGAIIGNIK SEQ ID NO: 1 BBBpS_2 KYRSGAITIGY SEQ ID NO: 2 BBBpS_3 EEGRLYMRYYSPTTRRYG SEQ ID NO: 3 BBBpS_4 APWHLSSQYSRT SEQ ID NO: 4 BBBpS_5 HIQLSPFSQSWR SEQ ID NO: 5 BBBpS_6 YTQDFNKFHTFPQTAIGVGAP SEQ ID NO: 6 BBBpS_7 LKTLTETLKELTKTLTEL SEQ ID NO: 7 BBBpS_8 YKEATSTFTNITYRGT SEQ ID NO: 8 BBBpS_9 NRPDSAQFWLHH SEQ ID NO: 9 BBBpS_10 RPKPQQFFGLM SEQ ID NO: 10 non-BBBpS_1 KKKKQPPKPKKPKTQEKKKKQPPKPKR SEQ ID NO: 11 non-BBBpS_2 KWCFRVCYRGICYRRCR SEQ ID NO: 12 non-BBBpS_3 YDEEGGGE SEQ ID NO: 13 Table 7 Peptide Sequence BpAYG21 AAYGILEHAKYKAHELIGYAA SEQ ID NO: 14 BpAYK21 AAYKILEHAKYKAHELIKYAA SEQ ID NO: 15 BpARY17 AYILEHAKYKAHELIYA SEQ ID NO: 16 BpARH17 AYILEHAKHKAHELIYA SEQ ID NO: 17 BpYRH17 AYIKEHYKHKYHEKIYA SEQ ID NO: 18 BpRYI15 KYILEHKYKHELIYK SEQ ID NO: 19 BpAYG11 AAYGILEHAKY SEQ ID NO: 20 BpAYK11 AAYKILEHAKY SEQ ID NO: 21 BpARY9 AYILEHAKY SEQ ID NO: 22 BpARH9 AYILEHAKH SEQ ID NO: 23 Table 8 Analytical data for the peptides Peptide Modfication Theoretical mass (Da)a Experimental mass (Da)b HPLC tR (min) Purity (%)c BpAYG21 unmodified 2290.00 2288.20 7.08 97.9 5(6)-carboxyfluroescein 2648.32 3006.64 9.02 95.6 BpAYK21 unmodified 2432.90 2430.51 6.86 97.0 5(6)-carboxyfluroescein 2790.13 3148.45 8.80 96.6 BpARY17 unmodified 2030.00 2032.25 6.28 96.9 5(6)-carboxyfluroescein 2388.51 2746.83 8.22 97.1 BpARH17 unmodified 2007.00 2006.12 5.88 97.4 5(6)-carboxyfluroescein 2365.23 2723.55 7.82 98.0 BpYRH17 unmodified 2322.00 2220.30 4.28 97.4 5(6)-carboxyfluroescein 2463.06 2821.38 6.22 95.1 BpRYI15 unmodified 2105.46 2004.21 4.91 92.1 5(6)-carboxyfluroescein 2463.63 2821.95 6.95 95.1 BpAYG11 unmodified 1236.00 1234.60 5.54 94.7 5(6)-carboxyfluroescein 1594.23 1952.55 7.58 92.6 BpAYK11 unmodified 1305.00 1305.65 4.98 96.8 5(6)-carboxyfluroescein 1663.62 2021.94 6.92 95.7 BpARY9 unmodified 1107.00 1106.50 5.02 98.2 5(6)-carboxyfluroescein 1465.92 1824.24 6.96 97.2 BpARH9 unmodified 1180.00 1080.55 4.05 93.5 5(6)-carboxyfluroescein 1538.36 1896.68 5.99 95.1 Calculated using GPMAW version 8.10 bFrom the mass spectrum c Estimated by HPLC peak integration of UV chromatogram (free and CF-labelled versions) Table 9. Physicochemical properties of peptides Peptide Physicochemical properties Molecular weight (g / mol) Extinction coefficient (M'1.cnr1) Hydrophobic (%) Isoelectric point Net charge (pH 7) Charg e Average hydro-phobicit y Ratio hydrophilic residues (%) BpAYG21 2290.00 2840 48 9,36 1,18 2,0 -0,29 19 BpAYK21 2432.90 3840 48 10,18 3,18 4,2 0,00 29 BpARY17 2030.00 3840 47 9,36 1,18 2,0 -0,30 24 BpARH17 2007.00 2560 47 9,53 1,27 2,0 -0,19 24 BpYRH17 2322.00 5120 24 10,22 3,27 4,0 0,16 35 BpRYI15 2105.46 3840 27 10,55 3,18 4,0 0,19 40 BpAYG11 1236.00 2560 45 9,53 1,09 2,0 -0,38 18 BpAYK11 1305.00 2560 45 10,1 2,09 3,0 -0,11 27 BpARY9 1107.00 2560 44 9,53 1,09 3,0 -0,41 22 BpARH9 1180.00 1280 44 9,85 1,18 2,0 -0,21 22 Table 10. Summary of the cell-based assays. Peptide Cytotoxicity (p.M)a Translocation (%)b Stability (min)c HBEC-5i SHSY5Y IC50 IC50 BpAYG21 >100 >100 51.4±2.6 62.5 BpAYK21 >100 >100 49.6±2.7 187.2 BpARY17 >100 >100 51.4±0.7 110.4 BpARH17 >100 >100 53.4±1.7 288.1 BpYRH17 >100 >100 49.8±1.8 299.9 BpRYI15 >100 >100 49.6±2.4 18.7 BpAYG11 >100 >100 49.5±3.3 135.4 BpAYK11 >100 >100 49.7±2.7 55.1 BpARY9 >100 >100 49.9±2.3 222.6 BpARH9 >100 >100 47.9±0.9 22.4 adetermined using CellTiter-Blue® assay bevaluated by fluorescence intensity using flow cytometry cevaluated by AUC analysis using an HPLC IC50, concentration that causes cell death in 50% of cells Table 11. Sequence of variant peptides Peptide Sequence Peptide modified Sequence BpAYG21 AAYGILEHAKYKAHELIGYAA BpAYG21_1 YAYGILEHARYRAHELIGYAA SEQ ID NO: 24 BpAYG21_2 CAYGILEHARYRAHELIGYAA SEQ ID NO: 25 BpAYK21 AAYKILEHAKYKAHELIKYAA BpAYG21_1 AAYKIFEHARYRAHELIKYAA SEQ ID NO: 26 BpAYG21_2 AAYKITEHARYRAHELIKYAA SEQ ID NO: 27 BpAYG21_3 AAYKIREHARYRAHELIKYAA SEQ ID NO: 28 BpARY17 AYILEHAKYKAHELIYA BpARY17_1 AYILRHAKYKAHELCYA SEQ ID NO: 29 BpARH17 AYILEHAKHKAH ELIYA BpARH17_1 AYILRHAKHKAHELCYA SEQ ID NO: 30 BpYRH17 AYIKEHYKHKYHEKIYA BpYRH17_1 AYIKRHYKHKYHEKCYA SEQ ID NO: 31 BpYRH17_2 AYIKRHYKHKYHEKCYA SEQ ID NO: 32 BpYRH17_3 AYIKRHYKHKYHEKRYA SEQ ID NO: 33 BpYRH17_4 AYIKRHYKHKYHEKCYA SEQ ID NO: 34 BpYRH17_5 AYIKTHYKHKYHEKCYA SEQ ID NO: 35 BpYRH17_6 AYIKRHYKHKYHEKTYA SEQ ID NO: 36 BpYRH17_7 AYIKRHYKHKYHEKVYA SEQ ID NO: 37 BpYRH17_8 AYIKRHYKHKYHEKYYA SEQ ID NO: 38 BpYRH17_9 AYIKWHYKHKYHEKCYA SEQ ID NO: 39 BpYRH17_10 AYIKRHYKHKYHEKRYA SEQ ID NO: 40 BpYRH17_11 AYIKYHYKHKYHEKCYA SEQ ID NO: 41 BpYRH17_12 AYIKGHYKHKYHEKCYA SEQ ID NO: 42 BpYRH17_13 AYIKRHYKHKYHEKWYA SEQ ID NO: 43 BpRYI15 KYILEHKYKHELIYK BpRYI15_1 KYILRHKYKHELRYK SEQ ID NO: 44 BpRYI15_2 KYILRHKYKHELYYK SEQ ID NO: 45 BpRYI15_3 KYILRHKYKHELCYK SEQ ID NO: 46 BpRYI15_4 KYILRHKYKHELVYK SEQ ID NO: 47 BpRYI15_5 KYILRHKYKHELTYK SEQ ID NO: 48 BpRYI15_6 KYILRHKYKHELYYK SEQ ID NO: 49 BpRYI15_7 KYILRHKYKHELCYK SEQ ID NO: 50 BpRYI15_8 KYILPHKYKHELRYK SEQ ID NO: 51 BpRYI15_9 KYILPHKYKHELVYK SEQ ID NO: 52 BpRYI15_10 KYILRHKYKHELPYK SEQ ID NO: 53 BpAYG11 AAYGILEHAKY BpAYG11_1 AAYGILVHAKY SEQ ID NO: 54 BpAYG11_2 AAYGILTHAKY SEQ ID NO: 55 BpAYG11_3 AAYGILYHAKY SEQ ID NO: 56 BpAYG11_4 AAYGILCHAKY SEQ ID NO: 57 BpAYG11_5 AAYGILRHAKY SEQ ID NO: 58 BpAYK11 AAYKILEHAKY BpAYK11_1 AAYKRLEHAKY SEQ ID NO: 59 BpARY9 AYILEHAKY BpARY9_1 AYILRHAKY SEQ ID NO: 60 BpARY9_2 AYILPHAKY SEQ ID NO: 61 BpARY9_3 AYILTHAKY SEQ ID NO: 62 BpARY9_4 AYILVHAKY SEQ ID NO: 63 BpARY9_5 AYILYHAKY SEQ ID NO: 64 BpARH9 AYILEHAKH BpARH9_1 AYILRHAKH SEQ ID NO: 65 BpARH9_2 AYILPHAKH SEQ ID NO: 66 BpARH9_3 AYILTHAKH SEQ ID NO: 67 BpARH9_4 AYILYHAKH SEQ ID NO: 68 BpARH9_5 AYILVHAKH SEQ ID NO: 69

Claims

1. A method of identifying a BBB peptide shuttle (BBBpS) comprising;determining a set of physicochemical properties of a test peptide, determining whether each of the physicochemical properties in the set falls within a reference range, andidentifying the test peptide as a BBBpS if each physicochemical property in the set falls within the reference range.

2. A method according to claim 1 wherein the set of physicochemical properties comprises Molecular weight, Extinction coefficient, Hydrophobic nature, Isoelectric point, Net charge at pH 7, Charge, Average hydrophobicity, Hydrophobicity at pH 7, and Hydrophilic residues ratio.

3. A method according to claim 2 wherein the reference range for molecular weight is 852-3065 g.mol'1.

4. A method according to claim 2 or 3 wherein the reference range for extinction coefficient is 0.0-7786 M-1.cm1.

5. A method according to any one of claims 2 to 4 wherein the reference range for hydrophobic nature is 18-50%.

6. A method according to any one of claims 2 to 5 wherein the reference range for isoelectric point 8.0-13.3.

7. A method according to any one of claims 2 to 6 wherein the reference range for net charge at pH -1.0-4.7.

8. A method according to any one of claims 2 to 7 wherein the reference range for charge is 0.06.0.

9. A method according to any one of claims 2 to 8 wherein the reference range for average hydrophobicity is -0.6 to 0.8.

10. A method according to any one of claims 2 to 9 wherein the reference range for hydrophobicity at pH 7 is 5.5 to 34.6.

11. A method according to any one of claims 2 to 10 wherein the reference range for hydrophilic residues ratio 17 to 56.0.

12. The method according to any one of claims 1 to 11, wherein the step of determining the set of physicochemical properties is performed by predicting the properties from the amino acid sequence of the test peptide.

13. The method of according to any one of claims 1 to 12, further comprising synthesising the BBBpS.

14. The method according to any one of claims 1 to 12, further comprising conjugating the BBBpS to a therapeutic agent.

15. The method according to any one of claims 13 to 14, further comprising formulating the BBBpS with a pharmaceutically acceptable carrier.

16. A computer system comprising a processor configured to execute a method of identifying a BBBpS according to any one of claims 1 to 12.

17. A computer program product comprising instructions which, when the program is executed by a computer, cause the computer to perform the method according to any one of claims 1 to 12.

18. A BBBpS produced by a method according to claim 12.19     A BBBpS according to claim 18 having a molecular weight of 852-3065 g.mol'1, an extinctioncoefficient of 0.0-7786 M Tcm1, a hydrophobic nature of 18-50%, an isoelectric point of 8.0-13.3, a net charge at pH of -1.0 to 4.7, a charge of 0.0-6.0, an average hydrophobicity of -0.6 to 0.8, a hydrophobicity at pH 7 of 5.5 to 34.6, and a hydrophilic residues ratio of 17 to 56.0%.

20. A BBBpS according to any one of claims 18-19 comprising the amino acid sequence of any one of SEQ ID Nos: 1 to 10 or SEQ ID Nos: 14 to 69.

21. A BBBpS comprising the amino acid sequence of any one of SEQ ID Nos: 1 to 10 or SEQ ID Nos: 14 to 69.

22. A drug conjugate comprising a therapeutic agent conjugated to a BBBpS according to any one of claims 18 to 21.

23. A drug conjugate according to claim 22 wherein the therapeutic agent is selected from any one of antidepressants, antipsychotics, mood stabilisers, anti-anxiety medications, stimulants, antiepileptic drugs, cholinesterase inhibitors, dopamine agonists, dopamine reuptake inhibitors, nonsteroidal anti-inflammatory drugs, antimetabolites, anticonvulsants, benzodiazepines, nootropics, beta blockers, antiviral drugs, immunosuppressants, serotonin-norepinephrine reuptake inhibitors,analgesics, selective serotonin reuptake inhibitors, NMDA receptor antagonists, corticosteroids, thrombolytics, vasodilators, neuroprotective agents, antibiotics, antivirals, anticancer agents, and antibodies.

24. A pharmaceutical composition comprising a BBBpS according to any one of claims 18 to 22 or a drug conjugate according to claim 22 or 23 and a pharmaceutically acceptable carrier.

25. A method of treating or preventing a brain disease in a subject, comprising;administering the agent to the subject, wherein the agent is a BBBpS according to any one of claims 18 to 21 or a drug conjugate according to claim 22 or 23 or a pharmaceutical composition according to claim 24.

26. An agent for use in the treatment or prevention of a brain disease in a subject, comprising administering the agent to the subject, wherein the agent is a BBBpS according to any one of claims 18 to 21 ora drug conjugate according to claim 22 or23 ora pharmaceutical composition according to claim 24.

27. Use of an agent in the manufacture of a medicament for the treatment of a brain disease, wherein the treatment comprises administering the agent to a subject and the agent is a BBBpS according to any one of claims 18 to 21 or a drug conjugate according to claim 22 or 23 or a pharmaceutical composition according to claim 24.

28. The method of claim 24, the agent for use of claim 25, or the use of claim 26 wherein the brain disease is selected from any one of Alzheimer’s disease, dementia, brain cancer, epilepsy, seizure disorders, mental disorders, Parkinson’s disease, movement disorders, stroke, or transient ischemic attack.

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