CELL PENETRATING PEPTIDES

Abstract

The present invention relates to peptides, in particular cell penetrating peptides, of 40 amino acid residues or less comprising at least one directly glycosylated amino residue and one or more arginine rich arm domains, and to conjugates of such cell penetrating peptides with a therapeutic molecule. The present invention further relates to the use of the peptides or conjugates in methods of treatment or as a medicament, especially in the treatment of genetic disorders of the central nervous system.

Claims

1. A peptide comprising at least one directly glycosylated amino acid residue and one or more arginine-rich arm domains, wherein the total length of the peptide is 40 amino acid residues or less.

2. A peptide according to claim 1, wherein the at least one directly glycosylated amino acid residue is O-linked glycosylated, N-linked glycosylated or S-linked glycosylated.

3. A peptide according to claim 1, wherein at least one directly glycosylated amino acid residue is glycosylated at a functional group present in the amino acid side chain selected from an OH, NH.sub.2, NH.sub.3 and SH.

4. A peptide according to claim 1, wherein the at least one directly glycosylated amino acid residue is selected from a glycosylated serine, cysteine, threonine, asparagine, glutamine, aminoproline, hydroxyproline, tyrosine, lysine, and amino acid analogues thereof, preferably the at least one directly glycosylated amino acid residue is a glycosylated serine.

5-7. (canceled)

8. A peptide according to claim 1, wherein the at least one directly glycosylated amino acid residue is glycosylated with a sugar selected from: glucose, allose, altrose, idose, gulose, talose, xylose, lactose, mannose, galactose, mannoseamine, glucosamine, galactosamine, N-acetylgalactosamine, D-2-Acetylamino Glucose, N-acetylglucosamine, lactose, maltose, isomaltose, trehalose and sialic acid, preferably the at least one amino acid residue is directly glucosylated.

9-10. (canceled)

11. A peptide according to claim 8, wherein the at least one amino acid residue is directly glucosylated with β-D glucose, preferably the at least one amino acid residue is a β-D glucosyl serine.

12. (canceled)

13. A peptide according to claim 1, wherein the arginine-rich arm domains comprise a combined total of between 5-10 Arginine residues.

14. A peptide according to claim 1, wherein the arginine-rich arm domains comprise no more than 3 contiguous Arginine residues.

15. A peptide according to claim 1, wherein the arginine-rich arm domains comprise a length of between 1-12 amino acid residues, preferably wherein the amino acid residues are selected from the group consisting of: arginine, alanine, beta-alanine, histidine, proline, glycine, cysteine, tryptophan, hydroxyproline, aminohexanoic acid, 3-azetidine-carboxylic acid (Az), 1-(amino)cyclohexanecarboxylic acid (Cy), amino acid analogues thereof, and any other non-natural amino acid.

16. (canceled)

17. A peptide according to claim 1, wherein each arginine-rich arm domain is selected from the following sequences: RXRRBRRXR (SEQ ID NO.81), RXRBRXR (SEQ ID NO.82), RXRRBRR (SEQ ID NO.83), RBRXR (SEQ ID NO.84), RBRRBRRBR (SEQ ID NO.85), RBRBRBR (SEQ ID NO.86), RGRRGRRGR (SEQ ID NO.87), RGRGRGR (SEQ ID NO.88), RPRRPRRPR (SEQ ID NO.89), RPRPRPR (SEQ ID NO.90), RHypRRHypRRHypR (SEQ ID NO.91), RHypRHypRHypR (SEQ ID NO.92), RARRARRAR (SEQ ID NO.93), RARARAR (SEQ ID NO.94), RCy*RRCy*RRCy*R (SEQ ID NO.95), RCy*RCy*RCy*R (SEQ ID NO.96), RRBRRBR (SEQ ID NO.97), RBRRBR (SEQ ID NO.98), RRBR (SEQ ID NO.99), RBR, R, RBRBR (SEQ ID NO.100), RBRBRR (SEQ ID NO.101), RBRRR (SEQ ID NO.102), RRRR (SEQ ID NO.103), RBRRBRRR (SEQ ID NO.104, RBRRRRR (SEQ ID NO.105), RRRRRR (SEQ ID NO.106), RRBRR (SEQ ID NO.107), RGRR (SEQ ID NO.108), GRRGR (SEQ ID NO.109), RGGRBRGGR (SEQ ID NO.110), RXRRBRRXRRXRBRXR (SEQ ID NO.113), RXRR (SEQ ID NO.114, RRXR (SEQ ID NO.115), RXR, RRBRBRXR (SEQ ID NO.117), RRBRRBRBRXR (SEQ ID NO.118), RXRRBRRBR (SEQ ID NO.119), RXRRBRRBRBR (SEQ ID NO.120), RXRRBR (SEQ ID NO.121), RXRBRR (SEQ ID NO.122), HXHRBRRXR (SEQ ID NO.123), RXHBHXR (SEQ ID NO.124), RR, RXRXR (SEQ ID NO.125), BRBRBR (SEQ ID NO.127), BRKBRKRBBR (SEQ ID NO.128), BRKBRKRBBRK (SEQ ID NO.129), RAzRRAzRR (SEQ ID NO.130), RAzRAzR (SEQ ID NO.131), and RXRBR (SEQ ID NO.132).

18. (canceled)

19. A peptide according to claim 1, wherein each of the arginine-rich arm domains present in the peptide is separated from any other arginine rich arm domain present in the peptide by a directly glycosylated amino acid residue.

20. A peptide according to claim 1, wherein the peptide comprises a first arginine-rich arm domain selected from the following sequences: RXRRBRRXR (SEQ ID NO.81), RXRRBRR (SEQ ID NO.83), RBRBR (SEQ ID NO.100), and RXRXR (SEQ ID NO.125), optionally wherein the peptide further comprises a second arginine-rich arm domain selected from the following sequences: RXRBRXR (SEQ ID NO.82), RBRXR (SEQ ID NO.84), RBRRBR (SEQ ID NO.98), RXRBR (SEQ ID NO.132), RBRBR (SEQ ID NO.100), and RXRXR (SEQ ID NO.125).

21. (canceled)

22. A peptide according to claim 1, wherein the peptide comprises one or more hydrophobic core domains.

23. A peptide according to claim 22, wherein the each hydrophobic core domain comprises between 1-4 hydrophobic amino acid residues, preferably wherein the amino acid residues are selected from glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and amino acid analogues thereof.

24-25. (canceled)

26. A peptide according to claim 22, wherein each hydrophobic core domain is contiguous with the at least one directly glycosylated amino acid residue, preferably wherein each hydrophobic core domain contiguous with the directly glycosylated amino acid residue is positioned between two flanking arginine-rich arm domains.

27. (canceled)

28. A peptide according to claim 22, wherein each hydrophobic core domain is selected from the following sequences: GFTGPL (SEQ ID NO.133), QFL, Z, ZL, F, FL, FQILY (SEQ ID NO.134), FQ, WF, QF, FQ, and YQFLI (SEQ ID NO.135).

29-34. (canceled)

35. A peptide according to claim 1, wherein the peptide is selected from the following sequences: RXRRBRRXRS*FLRXRBRXR (SEQ ID NO.14), RXRRBRRFS*RBRXR (SEQ ID NO.17), RXRRBRRZS*RBRXR (SEQ ID NO.73), RXRRBRRFS1*RBRXR (SEQ ID NO.74), RBRBRS*RBRBR (SEQ ID NO.70) and RXRXRS*RXRXR (SEQ ID NO.71); wherein Z represents 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, and wherein S.sup.1* represents D-serine glycosylated with D-Glucose sugar.

36. A conjugate comprising the peptide according to claim 1 covalently linked to a therapeutic molecule, optionally wherein the therapeutic molecule is selected from: a nucleic acid, peptide nucleic acid, antisense oligonucleotide (such as PNA, PMO), short interfering RNA, micro RNA, peptide, cyclic peptide, protein, pharmaceutical and drug, preferably the therapeutic molecule is an antisense oligonucleotide.

37-39. (canceled)

40. A method of treating a disease in a subject comprising administering a conjugate according to claim 36 to the subject in a therapeutically effective amount, optionally wherein the disease is (a) of the central nervous system; or (b) is selected from: Duchenne Muscular Dystrophy (DMD), Bucher Muscular Dystrophy (BMD), Menkes disease, Beta-thalassemia, dementia, Parkinson's Disease, Spinal Muscular Atrophy (SMA), myotonic dystrophy (DM), Huntington's Disease, Hutchinson-Gilford Progeria Syndrome, Ataxia-telangiectasia, and cancer.

41. (canceled)

42. A method of treating a disease according to claim 40, wherein the disease is caused by splicing deficiencies.

43-50. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0315] Certain embodiments of the present invention will now be described with reference to the following figures and tables in which:

[0316] FIG. 1: Shows the structure of Beta D-glucosyl serine residue;

[0317] FIG. 2: Shows a series of graphs showing increase in full length SMN2 transcript expression analysed via qPCR in three skeletal muscles (tibialis anterior (TA), quadriceps (Quad) and gastrocnemius (Gastro)), three brain compartments (cortex, brainstem, and cerebellum) and three spinal cord compartments (cervical, thoracic, and lumbar) following intravenous delivery of peptide 17-PMO conjugate and peptide 14-PMO conjugate in SMA adult mice at a dose of 2×15 mg/kg given approximately 48 hours apart. Tissues were harvested 7 days post final administration. Expression levels shown are normalised to saline treated controls, represented by dashed line). Data represented as mean±SD (*P≤0.05; **P≤0.005; ***P≤0.0005; ****P≤0.00005 by Student's t test in comparison to saline treated controls);

[0318] FIG. 3: Cell screening of glucosylated peptide-PMO conjugates for SMN2 exon 7 inclusion in human SMA patient fibroblast cell culture for peptides 1-16 by semi quantitative PCR at 50 nM concentration;

[0319] FIG. 4: Cell screening of glucosylated peptide-PMO conjugates for SMN2 exon 7 inclusion in human SMA patient fibroblast cell culture for peptides 3-54 by qPCR at different concentrations (667 nM and 2 μM);

[0320] FIG. 5: Cell screening of glucosylated peptide-PMO conjugates for SMN2 exon 7 inclusion in human SMA patient fibroblast cell culture for peptides 55-72 by qPCR at different concentrations (667 nM and 2 μM);

[0321] FIG. 6: Cell screening of glycosylated peptide-PMO conjugates for SMN2 exon 7 inclusion in human SMA patient fibroblast cell culture for peptides 73-80 at different concentrations (4 μM, 2 μM, 1 μM, 500 nM, 250 nM);

[0322] FIG. 7: Shows a series of graphs showing increase in full length SMN2 transcript expression analysed via qPCR in three skeletal muscles (tibialis anterior (TA), quadriceps (Quad) and gastrocnemius (Gastro)), three brain compartments (cortex, brainstem, and cerebellum) and three spinal cord compartments (cervical, thoracic, and lumbar) following intravenous delivery of peptide 17-PMO conjugate and currently available peptide 6-PMO conjugate. Expression levels shown are normalised to saline treated controls, represented by dashed line). Data represented as mean±SD (*P≤0.05; **P≤0.005; ***P≤0.0005; ****P≤0.00005 by Student's t test in comparison to saline treated controls);

[0323] FIG. 8: Shows urinary KIM-1 and Lipocalin-2 (NGAL) levels normalised to creatinine, two and seven days post-administration of 25 mg/kg single dose of currently available peptide 6 conjugate (Pip8b4-PMO) or peptide 17 conjugate (DPEP 5.17-PMO);

[0324] Table 1: Shows the sequences of the peptides tested in vivo incorporating SEQ ID NO.s 1-7 and 14 and 17 with additional N and C terminal modifications, including peptide and SEQ ID NO.s 1-7 of currently available peptides, and peptide and SEQ ID NO.s 14 and 17 of the invention;

[0325] Table 2: Shows the sequences of peptides screened in vitro in SMA patient fibroblasts incorporating SEQ ID NO.s 1-80 with additional N and C terminal modifications, including peptide and SEQ ID NO.s 1-7 of currently available peptides, and peptide and SEQ ID NO.s 8-80 of the invention;

[0326] Table 3: Shows quantitative PCR data for levels of full-length SMN2 transcripts generated in adult SMA mice treated with doses of 2×15 mg/kg of PMO conjugates with the peptides listed given approximately 48 hours apart. Tissues were harvested 7 days post final administration and RNA collected for qPCR analysis. Data represented as mean expression level (*P≤0.05; **P≤0.005; ***P≤0.0005; ****P≤0.00005 by Student's t test in comparison to saline treated controls). P values for liver samples all exceeded P≤0.00005 (not represented in table).

[0327] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0328] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or 27 process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

[0329] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

EXAMPLES

[0330] 1. Peptides Comprising a Beta D-Glucosyl Serine Residue.

[0331] The present inventors set out to find short peptides that, when conjugated to an oligonucleotide therapeutic, might lead to significant and effective cell penetration and activity in all or most spinal cord and brain compartments.

[0332] Surprisingly, the present inventors discovered that inclusion of a directly glycosylated amino acid (for example: S*, FIG. 1) into short arginine rich peptide carriers, and in addition systemic injection into adult SMA mice results in exon inclusion in 6 brain and spinal cord compartments as well in skeletal muscles. This is demonstrated in the present examples with the synthesis of a series of arginine rich peptides having one or more β-D-glucosyl serine residues, and in some cases additional hydrophobic domains.

[0333] The present inventors synthesised a series of candidate peptides named the ‘D-PEP5’ peptides. The first of which, peptide 14 (Table 1 and 2), comprises 10-Arg flanking arm domain sequences and an S* residue adjacent to a shortened 2-amino acid hydrophobic core of FL, and the second of which, peptide 17, comprises flanking 8-Arg arm domain sequences but has an S* residue adjacent to a single amino acid hydrophobic core of F (Table 1 and 2). A variety of currently available peptides were also synthesised for comparison with the inventive D-PEP5 peptides. These are numbered as peptides 1-7 in table 1 and incorporate the following peptide sequences:

TABLE-US-00004 (SEQ ID NO. 1) RXRRBRRXRYQFLIRXRBRXR (SEQ ID NO. 2) RXRRBRRXRQFLRXRBRXR (SEQ ID NO. 3) RXRRBRRFQILYRBRXR (SEQ ID NO. 4) RXRRBRRYQFLIRBRXR (SEQ ID NO. 5) RXRRBRRQFLRBRXR (SEQ ID NO. 6) RXRRBRRFLRBRXR (SEQ ID NO. 7) RXRRBRFQILYRBRXR

[0334] 2. Increase in FLSMN2 Expression in Skeletal Muscles, Brain Compartments and Spinal Cord Compartments by Intravenous Delivery of Peptide 17-PMO or Peptide 14-PMO in SMO Mice.

[0335] The inventors then tested the in vivo administration of peptides 17 and 14 and the currently available peptides 1-7 described above by conjugating the peptides to an antisense oligonucleotide therapeutic specifically a PMO. The antisense oligonucleotide was specifically directed at treating SMA by increasing full length SMN2 transcript production.

[0336] Smn1tm1Hung/wt; SMN2tg/tg and Smn1wt/wt; SMN2tg/tg mice were treated at 7-8 weeks of age with two administrations given two days (approximately 48 hours) apart of 15 mg/kg peptide 17-PMO, peptide 14-PMO conjugates or saline only (control). Tissues were harvested one week post administration. Quantitative PCR was performed on extracted RNA to analyse the amount of full-length SMN2 transcripts (in relation to total SMN2 transcripts), see FIG. 2. Each treatment groups was normalised to their saline controls, represented by the dashed line. Statistical significance determined by Student's t-test *p≤0.05, **p≤0.005, ***p≤0.0005, ****p≤0.00005.

[0337] Quantitative PCR analysis of brain, spinal cord and skeletal muscle tissues showed a significant increase in the amount of full-length SMN2 (FLSMN2) transcripts in several areas of the brain and spinal cord (FIG. 2). Treated skeletal muscles gave around a 3-fold increase in full-length SMN2 expression.

[0338] Quantitative data for levels of cell penetration of the peptides in vivo, measured by an increase in full-length SMN2 transcripts, are shown in Table 3 for the peptide 14 conjugate and the peptide 17 conjugate when compared with currently available peptide conjugates to the same antisense oligonucleotide. Data could not be obtained for TA skeletal muscle after treatment with peptide 6 conjugate in this experiment. However, the data demonstrate that cell penetration is increased in several compartments of the central nervous system and the skeletal muscle for peptide 14 and peptide 17 of the invention when compared with currently available peptides 1-7.

[0339] Specifically, peptide 6 can be compared with peptide 17 as both share a very similar sequence with the exception that peptide 17 of the invention has a glycosylated serine residue in place of a hydrophobic residue. Peptide 2 can be compared directly with peptide 14 as both share the same sequence with the exception that peptide 14 of the invention has a glycosylated serine residue in place of a glutamine residue and contains an additional linker beta-alanine. Peptide 17 shows increased cell penetration in the cortex, cerebellum, cervical, and thoracic compartments of the CNS, and increases in skeletal muscle penetration compared with peptide 6 of up to 60.2%. Peptide 14 shows increased cell penetration in the cortex, brainstem, cerebellum, thoracic and lumbar compartments of the CNS when compared with peptide 2 of up to 59.7%.

[0340] Further data was obtained to show a direct comparison of peptide 6 which is a currently available carrier peptide (designated Pip8b4) with peptide 17; a carrier peptide of the invention. Two systemic intravenous doses of 15 mg/kg of peptide-PMO conjugates were administered 48 hours apart in adult intermediate SMA mice (non-symptomatic SMN2 transgenic mice) and critical central and peripheral tissues were harvested 7 days post-final administration similar to the method described above. Results are shown in FIG. 7. Levels of exon 7 included transcripts (FLSMN) were assayed via qPCR as explained above. The levels of full length transcript were increased by treatment with peptide 17 for each tissue, exceeding the 1 fold increase threshold of FLSMN in all tissues (central spinal cord and peripheral skeletal muscle and liver). Moreover, peptide 17 of the invention showed equivalent or improved activity in the critical central brain tissues of the cerebellum and cortex when compared with the closest currently available carrier peptide; peptide 6. This indicates that the peptides of the invention are more effective than those that are currently available. This further indicates that the inventive peptides are effective as a therapy.

[0341] 3. Toxicology Profile of Glucosylated Peptide-PMO Conjugates

[0342] The inventors then further tested the toxicology of peptide 17 of the invention (designated DPEP5.17) in comparison with the currently available peptide; peptide 6 (designated Pip8b4).

[0343] Urinary and serum markers of kidney and liver toxicity were measured following a single dose administration of saline or the relevant peptide-PMO conjugate to 8 week old adult C57/BL10 female mice (N=6 per group). A bolus IV (tail vein) injection was administered and urine collected Day 2 and Day 7 after administration. Animals were then scarified prior to necropsy in which kidney, liver, diaphragm, heart, TA, gastric and serum were collected. Urine clinical indicators: KIM-1, NGAL were measured by ELISA (R&D cat #MKM100) with samples diluted to fit within standard curve. Values were normalised to urinary creatinine levels (Harwell) to account for urine protein concentration. Results are shown in FIG. 8.

[0344] Group 1—0.9% Saline

[0345] Group 2—25 mg/kg Pip8b4-PMO SMN

[0346] Group 3—25 mg/kg DPEP5.17-PMO SMN

[0347] By 7 days post administration, the levels of both KIM-1 and NGAL were lower in mice which had received the inventive peptide 17 than those which had received peptide 6. Furthermore, 2 days after administration, the levels of the marker KIM-1 were far lower in mice which had received the inventive peptide 17 than those which had received peptide 6. This indicates that the peptides of the invention have a better toxicology profile than those that are currently available, and therefore a lower toxicity than the currently available peptides. This indicates that the inventive peptides are suitable for use as a therapy.

[0348] 4. Cell Screening of Glucosylated Peptide-PMO Conjugates for Exon Inclusion in Human SMA Patient Fibroblast Cell Culture of Peptides 8-54.

[0349] In order to generate further peptide candidates containing a glycosylated Serine residue for in vivo evaluation, the inventors synthesized a range of similarly glycosylated peptides, conjugated them to the same PMO therapeutic and screened the resultant P-PMO conjugates in human SMA patient fibroblast cell culture, which assesses their ability to enter cell nuclei to give exon inclusion.

[0350] These further peptides are shown in Table 2, as peptides 8-80. Such a cellular screen provides candidates that are competent for entering cells and effecting SMN2 exon inclusion of an attached PMO. Some important conclusions could be reached as to what changes affected exon inclusion activity in cells. In addition, P-PMO conjugates were checked by MALDI-TOF spectrometry for their serum stability in mouse serum.

[0351] In the first screen (FIG. 3, D-PEP5 peptides 8-16 and FIG. 4, D-PEP5 peptides 14, and 17-54), a study of 10-Arginine peptides related to the first DPEP5 peptide number 14 was undertaken. It was found that replacement of all X residues by B (peptide 18) had no significant effect on cell activity. Replacement of S*FL by S in the core region (peptide 44) had no significant effect on cell activity. Replacement by S*F resulted in only a slightly reduced activity (peptide 43). The key result is that a glycosylated residue and core domain sequence such as S*FL could be placed in a variety of positions and contexts in the sequence without a large loss in cell activity (peptides 33, 34, 36, and 37). S*FL placed close to the C-terminus (peptide 48) was almost as active as the first peptide, peptide 14.

[0352] It was found that exon inclusion activity is generally reduced as the number of Arginine residues is reduced in the peptide. For example, the 9-Arginine sequences peptide 24 and peptide 29 were less active than the first peptide 14, 8-Arginine sequences less active than these and the 7-Arginine peptide 26 slightly less active only.

[0353] 5. Cell Screening of Glucosylated Peptide-PMO Conjugates for Exon Inclusion in Human SMA Patient Fibroblast Cell Culture of D-PEP5 Peptides 55-72

[0354] Following the screen of D-PEP5 peptides 8-54, a second screen in cells was carried out (FIG. 5) following synthesis of still further glucosylated D-PEP5 peptides 55-72 as shown in (Table 2). For the 8-Arginine peptides, there was no significant alteration in activity levels when FS* (peptides 56 and 57) was replaced by S* (peptides 58 and 59). Double S* (peptide 67) and Triple S* (peptide 68) peptides retained good activity in cells. Amongst the 6-Arginine peptides, by and large these had acceptable activity as PMO conjugates. However, P-PMO conjugates with peptide 70 and peptide 71 showed remarkably good activity.

[0355] 6. Cell Screening of Glycosylated Peptide-PMO Conjugates for Exon Inclusion in Human SMA Patient Fibroblast Cell Culture of D-Pep5 Peptides 73-80.

[0356] Following the screen of the D-PEP5 peptides 8-72 a third screen in cells was carried out (FIG. 6) following synthesis of still further glycosylated peptides 73-80 as shown in (Table 2). For the 8-Arginine peptides, there was no significant alteration in activity levels when S* was replaced by different glycosylated serine and asparagine moieties N*, and S.sup.2*-S.sup.6* (peptide 75 to peptide 79). Improved activity was seen for peptide 74 carrying a glycosylated unnatural D-serine residue and thereby preventing protease induced cleavage of the peptide. Further stabilisation of the peptide by introducing a unnatural amino acid (peptide 73, Z=Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) within the hydrophobic core-region greatly improved alteration in activity levels and even showed good activity at low doses.

[0357] The three in vitro screens performed evaluate the ability of the inventive peptides to enter cells and effect SMN2 exon 7 inclusion in human SMA patient fibroblast cell culture and is a pre-requisite before in vivo evaluation.

[0358] In summary, the present inventors have synthesised and demonstrated the improved effectiveness of a series of peptides having one or more directly glucosylated amino acid residues present within an arginine rich structure in penetrating into compartments of the CNS and muscular system for use as carriers for therapeutic molecules with a lower toxicity.

[0359] Materials and Methods

[0360] Reagents and General Methods

[0361] 9-Fluorenylmethoxycarbonyl (Fmoc) protected L-amino acids, and the Fmoc-β-Ala-OH preloaded Wang resin (0.19 mmol g.sup.−1) were obtained from Merck (Hohenbrunn, Germany). HPLC grade acetonitrile, methanol and synthesis grade N-methyl-2-pyrrolidone (NMP) were from Fisher Scientific (Loughborough, UK). Peptide synthesis grade N,N-dimethylformamide (DMF), benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium (PyBOP) and diethyl ether were obtained AGTC Bioproducts (Yorkshire, UK). Piperidine and trifluoroacetic acid (TFA) were obtained from Alfa Aesar (Heysham, England). PMO was purchased from Gene Tools Inc. (Philomath, USA). Fetal bovine serum, mouse serum and Superscript III Platinium One-Step qRT-PCR Kit and Platinum PCR SuperMix High Fidelity were obtained from Thermofisher Scientific (Waltham, US). iScript cDNA Synthesis Kit was obtained from Biorad (Hercules US). All other reagents were obtained from Sigma-Aldrich (St. Louis, Mo., USA) unless otherwise stated. MALDI-TOF mass spectrometry was carried out using a Voyager DE Pro BioSpectrometry workstation. A stock solution of 10 mg mL.sup.−1 of α-cyano-4-hydroxycinnamic acid or sinapinic acid in 60% acetonitrile in water containing 0.1% TFA was used as matrix. Fmoc-L-Ser(Ac.sub.4-β-D-Glc)-OH=S*, Fmoc-D-Ser(Ac.sub.4-β-D-Glc)-OH=S.sup.1*, Fmoc-L-Ser(Ac.sub.4-β-L-Glc)-OH=S.sup.2*, Fmoc-L-Ser(Ac.sub.4-β-D-Gal)-OH=S.sup.6*, Fmoc-L-Ser(Ac.sub.4-α-D-Man)-OH=S.sup.3*, 2-N-Fmoc-4-N-[Ac.sub.4-β-D-Glc)-L-Asn-OH=N*, Fmoc-L-Ser(β-D-Lac(Ac).sub.7)-OH=S.sup.4*, were synthesized as previously described (15-18).

[0362] Synthesis of Peptides on 100 μMol Scale

[0363] Peptides were synthesized on a 100 μmol scale using a CEM Liberty™ microwave Peptide Synthesizer (Buckingham, UK) and Fmoc chemistry following manufacturer's recommendations. The side chain protecting groups used were labile to trifluoroacetic acid treatment and the peptide was synthesized using a 5-fold excess of Fmoc-protected amino acids (0.25 mmol) that were activated using PyBOP (5-fold excess) in the presence of DIPEA or with DIC|Oxyma. Piperidine (20% v/v in DMF) was used to remove N-Fmoc protecting groups. The coupling was carried out once at 75° C. for 5 min at 60-watt microwave power except for arginine and the glycosylated amino acid residues, which were coupled twice each.

[0364] Histidine and cysteine residues were coupled once at 50° C. for 5 min at 60-watt microwave power. Each deprotection reaction was carried out at 75° C. twice, once for 30 sec and then for 3 min at 35-watt microwave power. Once synthesis was complete, the resin was washed with DMF (3×50 mL) and the N-terminus of the solid phase bound peptide was acetylated with acetic anhydride in the presence of DIPEA. The peptide was cleaved from the solid support by treatment with a cleavage cocktail consisting of trifluoroacetic acid (TFA): 3,6-dioxa-1,8-octanedithiol (DODT): H.sub.2O: triisopropylsilane (TIPS) (94%: 2.5%: 2.5%: 1%, 10 mL) or trifluoroacetic acid (TFA): H.sub.2O: m-cresol: triisopropylsilane (TIPS) (94%: 2.5%: 2.5%: 1%, 1 mL) or trifluoroacetic acid (TFA): H.sub.2O: triisopropylsilane (TIPS) (96.5%: 2.5%: 1%, 1 mL) for 2 h at room temperature for 2-3 h at room temperature. Excess TFA was removed by blowing N2 through the peptide solution. The cleaved peptide was precipitated via the addition of ice-cold diethyl ether and centrifuged at 3000 rpm for 5 min. The peptide pellet was washed in ice-cold diethyl ether thrice. The crude peptide was dissolved in water, analyzed and purified by RP-HPLC on Phenomenex Jupiter column (21.2×250 mm, C18, 10 μm) at a flow rate of 20 mL/min with the following gradient (A: 0.1% TFA, B: 90% CH.sub.3CN, 0.1% TFA) 0-2 min 5% B 2-35 min 5%-60% B 35-40 min 60%-90% B used. The fractions containing the desired peptide were combined and lyophilized to give the product as a white solid.

[0365] Synthesis of a Library of Peptide Variants on 5 μMol Scale

[0366] Each peptide was prepared on a 5 μmal scale using an Intavis Parallel Peptide Synthesizer using Fmoc-Gly-HMP-Tentagel resin (0.2 mmol g.sup.−1) or Fmoc-β-Ala-Wang Chemmatrix resin (0.3 mmol g.sup.−1) by applying standard Fmoc chemistry and following manufacturer's recommendations. Double coupling steps were used with a PyBOP/NMM coupling mixture followed by acetic anhydride capping after each step. The peptides were cleaved from the solid support by treatment with a cleavage cocktail consisting of trifluoroacetic acid (TFA): 3,6-dioxa-1,8-octanedithiol (DODT): H.sub.2O: triisopropylsilane (TIPS) (94%: 2.5%: 2.5%: 1%, 1 mL) or trifluoroacetic acid (TFA): H.sub.2O: m-cresol: triisopropylsilane (TIPS) (94%: 2.5%: 2.5%: 1%, 1 mL) or trifluoroacetic acid (TFA): H.sub.2O: triisopropylsilane (TIPS) (96.5%: 2.5%: 1%, 1 mL) for 2 h at room temperature. After peptide release, excess TFA was removed by blowing N.sub.2 gas into the TFA solution. The crude peptide was precipitated by the addition of cold diethyl ether (12 mL) and centrifuged at 2500 rpm for 5 min. The crude peptide pellet was washed thrice by cold diethyl ether (3×12 mL). The crude peptide was dissolved in 1500 μL H.sub.2O: CH.sub.3CN mixture and purified by RP-HPLC using a Phenomenex Jupiter column (10×250 mm, C18, 10 mm) at a flow rate 5 mL/min with the following gradient (A: 0.1% TFA, B: 90% CH.sub.3CN, 0.1% TFA) 0-2 min 5% B 2-35 min 5%-60% B 35-40 min 60%-90% B. The fractions containing the desired peptide were combined and lyophilized to yield the peptide as a white solid (see Table 1 for yields).

[0367] Synthesis of PMO-Peptide Conjugates

[0368] The following PMO antisense sequences targeting the human SMN2-gene were used.

[0369] ISS-N1-20 mer: ATT CAC TTT CAT AAT GCT GG (SEQ ID NO.147)

[0370] ISS-N1-25 mer: GTA AGA TTC ACT TTC ATA ATG CTG G (SEQ ID NO.148)

[0371] The peptide was conjugated to the 3′-end of the PMO through its C-terminal carboxyl group. This was achieved using 2.5 and 2 equivalents of HBTU and HOAt in NMP respectively in the presence of 2.5 equivalents of DIPEA and 2.5 fold excess of peptide over PMO dissolved in DMSO was used.

TABLE-US-00005 PMO Peptide-COOH HBTU HOAt DIPEA 10 mM 100 mM 300 mM 300 mM 1 eq. 2.5 eq. 5.75 eq. 5.75 eq. 5.75 eq. 100 nmol 250 nmol 575 nmol 575 nmol 575 nmol 10 μl 2.5 μl 1.92 μl 1.92 μl 0.11 μl

[0372] To a solution of peptide (250 nmol) in N-methylpyrrolidone (NMP, 2.5 μL) were added HBTU (1.92 μL of 0.3 M in NMP), HOAt in (1.92 μL of 0.3 M NMP), DIPEA (0.1 μL) and PMO (10 μL of 10 mM in DMSO). The mixture was left for 2-3 h at 40° C. and the sugar protecting groups were globally deprotected by the addition of 10 μl hydrazine hydrate. After 10 min the deprotection reaction was quenched by the addition of ice cold 5% AcOH (1000 μL). This solution was then purified by Ion exchange chromatography using a Resource S HR-16|100 column at a flow rate 6 mL/min with the following gradient (A: 25 mM phosphate buffer pH 7 with 25% ACN, B: 25 mM phosphate buffer pH 7 with 25% CH.sub.3CN and 1M NaCl) 0-2 min 0% B 2-20 min 0%-75% B 20-23 min 100% B, 23-28 100% A. The fractions containing the desired compound were desalted (Amicon 15 Ultracel, MWCO 3 kDa, EMD Millipore) and lyophilized.

[0373] Mouse Serum Stability Experiments

[0374] 10 nmol of lyophilized P-PMO was dissolved in 100 μl mouse serum and incubated at 37° C. for different time periods. Samples were diluted with 300 μl guanidinium-HCl solution (10 ml of 1M guanidinium-HCl containing 1 tablet complete mini protease inhibitor cocktail (Roche, Basel, Switzerland) and with 600 μl ice cold acetonitrile and centrifuged at 14.000 rpm for 3 min. The supernatant was collected and analysed by MALDI TOF MS and ion exchange chromatography.

[0375] Cell Culture

[0376] GM03813 patient fibroblast cells were cultured in T75 flasks at 37° C., under 5% CO.sub.2 in Dulbecco's modified Eagle's medium (DMEM with Glutamax, Thermofisher) supplemented with 10% heat-inactivated fetal bovine serum (FBS Gold, PAA laboratories), 1% penicillin-streptomycin-neomycin antibiotic mixture (PSN, Gibco).

[0377] Cytotoxicity

[0378] GM03813 patient fibroblasts were seeded out at 1250 cells|well in 100 μl Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX and 10% fetal bovine serum (FBS) (Life Technologies, Inc.) in 96 well plates, and incubated for 16 hours in a cell culture incubator (37° C., 5% CO.sub.2, 100% rel. humidity). Afterwards the media was removed and cells were washed once with Opti-Mem and treated with different concentrations of PPMO in Opti-Mem in duplicate for 4 hours at 37° C. Subsequently the transfection mixture was replaced by normal culture media and cells were allowed to grow overnight. On the next day 20 μl of MTS Cell Viability assay (Promega) was added to the wells and incubated for 3 hours before measurement at 490 nm were taken. The cell viability percentage was determined by normalizing the average absorbance of triplicate samples to the mean of untreated samples.

[0379] qPCR Analysis of SMN2 Full Length and Δ7 mRNA in Cultured Cells

[0380] GM03813 (Coriell Institute) derived from SMA type I patient fibroblast were seeded out at 2500 cells/well in 100 μl Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX and 5% fetal bovine serum (FBS) (Life Technologies, Inc.) in 96 well plates, and incubated for 16 h in a cell culture incubator (37° C., 5% CO.sub.2, 100% rel. humidity). On the next day cells were then treated with 10 μl P-PMO at different concentrations (in water) in duplicate for 24 hours. After removal of the supernatant, cells were washed once with PBS-buffer and were lysed in lysis buffer (10 mM Tris, 3 mM MgCl.sub.2, 1 mM CaCl.sub.2), 1% Triton X-100, 200 u/ml DNase I and 200 u/ml Proteinase K) for 10 min. Afterwards the lysate were transferred into a 96-well plate (Eppendorf twintec) and incubated at 75° C. for 15 min and subsequently cooled to 4° C. and used immediately. The mRNA levels of SMN2 FL, SMN2 Δ7 and GAPDH were quantified using Taqman-based qRT-PCR (Superscript® III Platinium® One Step qRT-PCR, Thermo Fisher) and SMN2 specific Primers and probes (purchased from IDT Integrated DNA Technologies). (19) SMN2 FL and Δ7 mRNAs were normalized to GAPDH.

[0381] Endpoint qPCR Analysis of SMN2 Full Length and Δ7 mRNA in Cultured Cells

[0382] GM03813 (Coriell Institute) derived from SMA type I patient fibroblasts were seeded out at 1×10.sup.5 cells/well in 2000 μl Dulbecco's modified Eagle's medium (DMEM) with GlutaMAX and 10% fetal bovine serum (FBS) (Life Technologies, Inc.) in 6 well plates, and incubated for 2 days (until cells reach a confluency greater than 90%) in a cell culture incubator (37° C., 5% CO.sub.2, 100% rel. humidity). Afterwards cells were washed once with PBS and Opti-Mem and cells were then treated with 1000 μl of PPMO in Opti-Mem in duplicates for 4 h. The transfection medium was then replaced with DMEM supplemented with 10% fetal bovine serum and 1% PSN and the cells incubated for a further 20 hr at 37° C. Cells were washed with PBS and 0.5 mL of TRI RNA (Sigma) isolation reagent was added to each well. Cells were frozen at −80° C. for 1 h.

[0383] RNA Extraction and Nested RT-PCR Analysis

[0384] Total cellular RNA was extracted using TRI reagent with an extra further precipitation with ethanol. The purified RNA was quantified using a Nanodrop® ND-1000 (Thermo Scientific). The RNA (500 ng) was used as a template for 2 step RT-PCR using iScript cDNA Synthesis Kit (Biorad, Hercules US) and Platinum PCR SuperMix High Fidelity (Thermofisher Scientific (Waltham, US). Primers (Forward: 5′-CTC CCA TAT GTC CAG ATT CTC TT-3′ (SEQ ID NO.149) and Reverse: 5′-CTA CAA CAC CCT TCT CAC AG-3′ (SEQ ID NO.150) were used to amplify full-length (505 bp) and Δ7 SMN2 (451 bp) from cDNA. The products were amplified semi-quantitatively using 30 PCR cycles (94° C. for 30 s, 55° C. for 30 s and 72° C. for 30 s). All PCR products were checked by electrophoresis on 2% agarose gels.

[0385] Data Analysis

[0386] The images of agarose gels were taken on a Molecular Imager ChemiDoc™ XRS.sup.+ imaging system (BioRad, UK) and the images were analysed using Image Lab (V4.1). Microsoft Origin was used to analyse and plot the exon-inclusion assay data, which were expressed as the percentage of Δ7 SMN2 transcript from at least three independent experiments.

[0387] Animal Models

[0388] Experiments were carried out in the Biomedical Sciences Unit, University of Oxford according to procedures authorized by the UK Home Office. Experiments were performed in SMA like mouse strain FVB.Cg-Smn1.sup.tm1HungTg(SMN2)2Hung/J, Jackson Laboratory stock number 5058 (28). The line was maintained and heterozygous mice (Smn1.sup.tm1HUNG/wt; SMN2.sup.tg/tg) were generated as previously described (21). Two doses of 15 mg/kg (given 2 days, about 48 hours apart) were diluted in 0.9% saline (Sigma) and administered via intravenous tail vein at a volume of 5 μl per gram body weight. Tail vein administrations were performed after warming mice at 32° C. Mice were then restrained in approved apparatus and peptide-PMO conjugates IV administered without anaesthetics. Administered mice were allowed to recover in heat box. Saline treated control animals were selected from littermates and handled in the same manner as the treated animals to control for potential changes in SMN expression due to stress. Tissues were harvested 7 days post final-administration. The tissues harvested included: liver, quadriceps, gastric, TA, brain, brainstem, cerebellum, and spinal cord. Spinal cord was divided into cervical, thoracic and lumbar regions. Tissues were snap frozen in liquid nitrogen and stored at −80° C.

[0389] RNA Extraction and cDNA Synthesis

[0390] RNA extraction from tissues was carried out using TRIZOL extraction, however another suitable product is Qiagen RNeasy® Mini Kit (Qiagen #74104), following manufacturer's instructions. One microgram of RNA template was used in a 20 μl reverse transcription reaction using ABI High Capacity cDNA Reverse Transcription Kits (Invitrogen, Carlsbad, Calif.).

[0391] SMN mRNA QPCR

[0392] RNA extraction from harvested tissues was carried out using TRIZOL extraction, however another suitable product is Qiagen RNeasy® Mini Kit. One microgram of RNA template was used in a 20 μl reverse transcription reaction using ABI High Capacity cDNA Reverse Transcription Kits (Invitrogen, Carlsbad, Calif.). Synthesized cDNA was diluted 1:5 with ddH2O and used at 20 ng per 20 μl QPCR reaction using Power SYBR® Green Master Mix (Life Technologies). Real time QPCR is performed and analysed on Applied Biosystems® StepOnePlus™ real-time PCR system (Life Technologies). Full length SMN2 transcript (FLSMN2) was amplified using gene-specific primers Exon 6 Fwd: 5′-GCT TTG GGA AGT ATG TTA ATT TCA-3′ (SEQ ID NO.151), Exons 7-8 Rev: 5′-CTA TGC CAG CAT TTC TCC TTA ATT-3′ (SEQ ID NO.152). SMN2 transcripts representing both FL and Δ7 mRNA were amplified using gene specific primers (Exon 2a Fwd: 5′-GCG ATG ATT CTG ACA TTT GG-3′ (SEQ ID NO.153), Exon 2b Rev: 5′-GGA AGC TGC AGT ATT CTT CT-3′ (SEQ ID NO.154). Cycle conditions: 95° C. for 10 minutes holding stage, followed by 40 cycles of 15 seconds at 95° C. and 1 minute at 60° C. The melt curve was determined from 60° C. to 95° C. in 0.6° C. steps. Transcripts were normalized to Polymerase (RNA) II polypeptide J (PolJ) levels. PolJ Forward: 5′-ACCACACTCTGGGGAACATC-3′ (SEQ ID NO.155); PolJ Reverse: 5′-CTCGCTGATGAGGTCTGTGA-3′ (SEQ ID NO.156). ΔΔCt was calculated as the difference between the ΔCt values, determined with the equation (PCR efficiency).sup.−Ct. The PCR efficiency was determined by LinRegPCR software (22,23). One-way ANOVA followed by Tukey's multiple comparisons test was performed using GraphPad Prism version 6.05 for Windows (Graph Pad Software, La Jolla Calif. USA, www.graphpad.com).

REFERENCES

[0393] 1. Singh, N. K., Singh, N. N., Androphy, E. J. and Singh, R.N. (2006) Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron. Mol. Cell Biol., 26, 1333-1346. [0394] 2. Hua, Y., Vickers, T. A., Baker, B.F., Bennett, C. F. and Krainer, A. R. (2007) Enhancement of SMN2 Exon 7 inclusion by antisense oligonucleotides targeting the exon. PLoS Biology, 5, e73. [0395] 3. Disterer, P., Kryczka, A., Liu, Y., Badi, Y. E., Wong, J. J., Owen, J. S. and Khoo, B. (2014) Development of therapeutic splice-switching oligonucleotides. Human Gene Therapy, 25, 587-598. [0396] 4. Hua, Y., Sahashi, K., Rigo, F., Hung, G., G., H., Bennett, C. F. and Krainer, A. R. (2011) Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model. Nature, Δ78, 123-126. [0397] 5. Zhou, H., Janghra, N., Mitrpant, C., Dickinson, R. L., Anthony, K., Price, L., Eperon, I. C., Wilton, S. D., Morgan, J. and Muntoni, F. (2013) A novel morpholino oligomer targeting ISSN-1 improves rescue of severe spinal muscular atrophy transgenic mice. Human Gene Therapy, 24, 331-342. [0398] 6. Farkhani, S. M., Valizadeh, A., Karami, H., Mohammadi, S., Sohrabi, N. and Badrzadeh, F. (2014) Cell penetrating peptides: efficient vectors for delivery of nanoparticles, nanocarriers, therapeutic and diagnostic molecules. Peptides, 57, 78-94. [0399] 7. Kang, T., Gao, X. and Chen, J. (2014) Harnessing the capacity of cell-penetrating peptides for drug delivery to the central nervous system. Curr. Pharm. Biotechnol., 15, 220-230. [0400] 8. Pardridge, W. M. (2012) Drug transport across the blood-brain barrier. J. Cereb. Blood Flow Metab., 32, 1959-1972. [0401] 9. Jearawiriyapaisarn, N., Moulton, H.M., Buckley, B., Roberts, J., Sazani, P., Fucharoen, S., Iversen, P. L. and Kole, R. (2008) Sustained dystrophin expression induced by peptide-conjugated morpholino oligomers in the muscles of mdx mice. Mol. Ther., 16, 1624-1629. [0402] 10. Wu, B., Moulton, H. M., Iversen, P. L., Juang, J., Li, J., Spurney, C. F., Sali, A., Guerron, A. D., Nagaraju, K., Doran, T. et al. (2008) Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modifies morpholino oligomer. Proc. Natl. Acad. Sci. USA, 105, 14814-14819. [0403] 11. Yin, H., Moulton, H.M., Seow, Y., Boyd, C., Boutilier, J., Iversen, P. and Wood, M. J. A. (2008) Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function. Human Molecular Genetics, 17, 3909-3918. [0404] 12. Ivanova, G. D., Arzumanov, A., Abes, R., Yin, H., Wood, M. J. A., Lebleu, B. and Gait, M. J. (2008) Improved cell-penetrating peptide-PNA conjugates for splicing redirection in HeLa cells and exon skipping in mdx mouse muscle. Nucleic Acids Res., 36, 6418-6428. [0405] 13. Yin, H., Moulton, H.M., Betts, C., Merritt, T., Seow, Y., Ashraf, S., Wang, Q., Boutilier, J. and Wood, M. J. A. (2010) Functional rescue of dystrophin-deficient mdx mice by a chimeric peptide-PMO. Mol. Ther., 18, 1822-1827. [0406] 14. Hammond, S. M., Hazell, G., Shabanpoor, F., Saleh, A. F., Bowerman, M., Sleigh, J., Meijboom, K., Tabot, K., Gait, M. J. and Wood, M. J. A. (2016) Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy Proc. Nat. Acad. Sci. USA, 113, doi: 10.1073/pnas.1605731113. [0407] 15. Arsequell, G., Sárries, N. and Valencia, G. (1995) Synthesis of glycosylated hydroxyproline building blocks. Tetrahedron Lett., 36, 7323-7326. [0408] 16. Elofsson, M., Walse, B. and J., K. (1991) Building blocks for glycopeptide synthesis: glycosylation of 3-mercaptopropionic acid and Fmoc amino acids with unprotected carboxyl groups. Tetrahedron Lett., 32, 7613-7616. [0409] 17. Lefever, M. R., Szabó, L. 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TABLE-US-00006 TABLE 1 Peptide number incorporating corresponding SEQ ID. NO. Sequence 1 Ac-RXRRBRRXRYQFLIRXRBRXR-B 2 Ac-RXRRBRRXRQFLRXRBRXR-B 3 Ac-RXRRBRRFQILYRBRXR-B 4 Ac-RXRRBRRYQFLIRBRXR-B 5 Ac-RXRRBRRQFLRBRXR-B 6 Ac-RXRRBRRFLRBRXR-B 7 Ac-RXRRBRFQILYRBRXR-B 14 Ac-RXRRBRRXRS*FLRXRBRXR-BB 17 Ac-RXRRBRR FS*RBRXR-B

TABLE-US-00007 TABLE 2 Peptide number incorporating corresponding SEQ ID. NO. Sequence 1 Ac-RXRRBRRXRYQFLIRXRBRXR-B 2 Ac-RXRRBRRXRQFLRXRBRXR-B 3 Ac-RXRRBRRFQILYRBRXR-B 4 Ac-RXRRBRRYQFLIRBRXR-B 5 Ac-RXRRBRRQFLRBRXR-B 6 Ac-RXRRBRRFLRBRXR-B 7 Ac-RXRRBRFQILYRBRXR-B 8 Ac-RXRRBRRXRQFLRXRBRXRS*-B 9 Ac-RXRRBRRXRQFLRXRS*RXR-B 10 Ac-RXRRS*RRXRQFLRXRBRXR-B 11 Ac-S*RXRRBRRXR QFL RXRBRXR-B 12 Ac-RXRRBRRXR S*QFLS*RXRBRXR-B 13 Ac-RXRRBRRXR S*QFLRXRBRXR-B 14 Ac-RXRRBRRXR S*FLRXRBRXR-BB 15 (Ac-S*BRKBRKRBBR)2K-B 16 Ac-GFTGPLS*BRKBRKRBBR)2K-B 17 Ac-RXRRBRR FS*RBRXR-B 18 Ac-RBRRBRRBR S*FL RBRBRBR-G 19 Ac-RGRRGRRGR S*FL RGRGRGR-G 20 Ac-RPRRPRRPR S*FL RPRPRPR-G 21 Ac-RHypRRHypRRHypRS*FLRHyp RHypRHypR-G Hyp = hydroxy-proline 22 Ac-RARRARRAR S*FL RARARAR-G 23 Ac-RCyRRCyRRCyRS*FLR CyR CyR CyR-G Cy = 1-(amino) cyclohexanecarboxylic acid 24 Ac-RRBRRBRS*FLRBRBRBR-G 25 Ac-RBRRBRS*FLRBRBRBR-G 26 Ac-RRBRS*FLRBRBRBR-G 27 Ac-RBRS*FLRBRBRBR-G 28 Ac-RS*FLRBRBRBR-G 29 Ac-RBRRBRRBRS*FLRBRBR-G 30 Ac-RBRRBRRBRS*FLRBR-G 31 Ac-RBRRBRRBRS*FLR-G 32 Ac-RBRRBRRBRS*FL-G 33 Ac-RBRRBRRBRS*FLRBRBRR-G 34 Ac-RBRRBRRBRS*FLRBRRR-G 35 Ac-RBRRBRRBRS*FLRRRR-G 36 Ac-RBRRBRRRS*FLRBRBRBR-G 37 Ac-RBRRRRRS*FLRBRBRBR-G 38 Ac-RRRRRRS*FLRBRBRBR-G 39 Ac-RBRRBRRRS*FLRRBRR-G 40 Ac-RBRRRRRS*FLRRRR-G 41 Ac-RRRRRRS*FLRRRR-G 42 Ac-RGRRS*GRRGRS*FLRGGRBRGGR-G 43 Ac-RXRRBRRXRS*FRXRBRXR-G 44 Ac-RXRRBRRXRS*RXRBRXR-G 45 Ac-RXRRBRRS*FQILYRBRXR-G 46 Ac-RXRRBRRS*FLRBRXR-G 47 Ac-RXRRBRRXRS*FLRXRBRXRS*FL-G 48 Ac-RXRRBRRXRRXRBRXRS*FL-G 49 Ac-RXRRS*RRXRS*FLRXRS*RXR-G 50 Ac-RXRRBRRXRS*FQRXRBRXR-G 52 Ac-RXRRBRRXRS*WFRXRBRXR-G 53 Ac-RXRRBRRXRS*QFRXRBRXR-G 54 Ac-RXRRBRRXRS*FQRXRBS*YQFLIRXR-G 55 Ac-RXRRBRRS*RBRXR-G 56 Ac-RXRRFS*RRBRBRXR-G 57 Ac-R FS*RRBRRBRBRXR-G 58 Ac-RXRRS*RRBRBRXR-G 59 Ac-RS*RRBRRBRBRXR-G 60 Ac-RXRRBRRBRS*RXR-G 61 Ac-RXRRBRRBRBRS*R-G 62 Ac-RXRRBRFS*RBR-G 63 Ac-RXRRBRS*RBR-G 64 Ac-RXRBRRS*RBR-G 65 Ac-RRBRRS*RBR-G 66 Ac-HXHRBRRXRS*RXHBHXR-G 67 Ac-RXRRBRRS*S*RBRXR-G 68 Ac-RXRRBRRS*S*S* RBRXR-G 69 Ac-RXRRS*RRS*RS*RXR-G 70 Ac-RBRBRS*RBRBR-G 71 Ac-RXRXRS*RXRXR-G 72 Ac-RXRRBS*BRBRBR-G 73 Ac-RXRRBRRZS*RBRXR-B (Z = Tic = 1,2,3,4- tetrahydroisoquinoline-3- carboxylic acid) 74 Ac-RXRRBRRFS.sup.1*RBRXR-B, S.sup.1* = D-Ser[D-Glc] 75 Ac-RXRRBRRFS.sup.2*RBRXR-B, S.sup.2* = L-Ser[L-Glc] 76 Ac-RXRRBRRFS.sup.3*RBRXR-B, S.sup.3* = (L-Ser[D-Man] 77 Ac-RXRRBRRFS.sup.4*RBRXR-B, S.sup.4* = L-Ser[D-Lac] 78 Ac-RXRRBRRFN*RBRXR-B, N* = L-Asn[D-GIcNac] 79 Ac-RXRRBRRFS.sup.6*RBRXR-B, S.sup.6* = L-Ser[Gal]* 80 Ac-RAzRRAzRRZS*RAzRAzR-B (Z = Tic = 1,2,3,4- tetrahydroisoquinoline-3- carboxylic acid, Az = 3-azetidine- carboxylic acid)

TABLE-US-00008 TABLE 3 Peptide No. 1 2 3 4 5 6 7 17 14 Central Nervous System Cortex 1.29** 1.16* 0.93 1.1 1.11 1.13 1.01 1.27* 1.50* Brainstem 1.48*** 1.36*** 0.86 0.98 1.22 1.38** 1.11 1.2 1.97*** Cerebellum 1.25* 1.15* 1.025 0.96 1 1.05 0.97 1.19* 1.26 Cervical 1.50*** 1.78** 0.92 1.14* 1.24* 0.88** 1.26 1.41* 1.23** Thoracic 1.45*** 1.49* 1 1.12 1.23 1.37* 1.33 1.39** 2.38** Lumbar 1.28*** 1.48** 1.1 1 1.25* 1.23* 0.97 1.1 2.23 Skeletal Muscle TA 3.30*** 3.45**** 3.95*** 4.23**** 2.80**** 3.34**** 3.00*** 3.24**** Quad 3.37*** 2.52**** 3.35**** 2.70**** 2.45**** 2.89*** 2.80**** 2.68*** 2.45*** Gastroc 3.03*** 2.98**** 3.4 3.64**** 2.49**** 3.22**** 2.92**** 3.25**** 2.82**** Off Target Liver 2.81 2.91 4.57 3.26 2.73 2.78 3.19 3.78 3.45