PEPTIDES AND USE THEREOF

Abstract

The present invention relates to a cyclic peptide comprising an active region comprising the amino acid sequence X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6 or a salt, derivative, prodrug or mimetic thereof. X.sup.2 and X.sup.5 are arginine; and either: X.sup.1 is (7-methoxy-coumarin-4-yl)-Ala-OH (Dac) and X.sup.3, X.sup.4 and X.sup.6 are any amino acid; or X.sup.3 is sarcosine (Sar) and X.sup.1, X.sup.4 and X.sup.6 are any amino acid; or X.sup.4 is 5,5-dimethylproline (dmPro) or 3-amino-3-(2-naphthyl)propionic acid (Nap) and X.sup.1, X.sup.3 and X.sup.6 are any amino acid; or X.sup.6 is Nap and X.sup.1, X.sup.3 and X.sup.4 are any amino acid.

The present invention further relates to a pharmaceutical composition comprising the cyclic peptide and the peptide for use in medicine, and particularly cancer.

Claims

1. A cyclic peptide comprising an active region which comprises the amino acid sequence X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6 or a salt, derivative, prodrug or mimetic thereof, wherein: X.sup.2 and X.sup.5 are arginine; and wherein either: X.sup.1 is (7-methoxy-coumarin-4-yl)-Ala-OH (Dac) and X.sup.3, X.sup.4 and X.sup.6 are any amino acid; or X.sup.3 is sarcosine (Sar) and X.sup.1, X.sup.4 and X.sup.6 are any amino acid; or X.sup.4 is 5,5-dimethylproline (dmPro) or 3-amino-3-(2-naphthyl)propionic acid (Nap) and X.sup.1, X.sup.3 and X.sup.6 are any amino acid; or X.sup.6 is Nap and X.sup.1, X.sup.3 and X.sup.4 are any amino acid.

2. The peptide according to claim 1 wherein: X.sup.2 and X.sup.5 are arginine; and wherein either: X.sup.1 is Dac and X.sup.3, X.sup.4 and X.sup.6 are any non-polar amino acid; or X.sup.3 is Sar and X.sup.1, X.sup.4 and X.sup.6 are any non-polar amino acid; or X.sup.4 is dmPro or Nap and X.sup.1, X.sup.3 and X.sup.6 are any non-polar amino acid; or X.sup.6 is Nap and X.sup.1, X.sup.3 and X.sup.4 are any non-polar amino acid.

3. The peptide according to claim 1 wherein X.sup.4 is Nap.

4. The peptide according to claim 1 wherein the active region of the peptide is between 4 and 10 amino acids in length, preferably between 6 and 10 amino acids in length, preferably 6 amino acids in length.

5. The peptide according to claim 1 wherein the active region of the peptide comprises the amino acid sequence: Dac-Arg-Sar-dmPro-Arg-Nap (SEQ ID NO:4) or Dac-Arg-Sar-Nap-Arg-Nap (SEQ ID NO:5).

6. The peptide according to claim 1 wherein the active region of the peptide consists of the amino acid sequence: Dac-Arg-Sar-dmPro-Arg-Nap (SEQ ID NO:4) or Dac-Arg-Sar-Nap-Arg-Nap (SEQ ID NO:5).

7. The peptide according to claim 1 wherein the peptide comprises the amino acid sequence: Y.sup.1A.sup.1X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6A.sup.2Y.sup.2 or a salt, derivative, prodrug or mimetic thereof, wherein: A.sup.1 and A.sup.2 may be present or absent, wherein when A.sup.1 and A.sup.2 are present they comprise one or more amino acids; and Y.sup.1 and Y.sup.2 are cysteine.

8. The peptide according to claim 1 wherein the peptide comprises the amino acid sequence Cys-Dac-Arg-Sar-dmPro-Arg-Nap-Cys (SEQ ID NO:6) or Cys-Dac-Arg-Sar-Nap-Arg-Nap-Cys (SEQ ID NO:7).

9. The peptide according to claim 1 wherein the peptide consists of the amino acid sequence Cys-Dac-Arg-Sar-dmPro-Arg-Nap-Cys (SEQ ID NO:6) or Cys-Dac-Arg-Sar-Nap-Arg-Nap-Cys (SEQ ID NO:7).

10. The peptide according to claim 7 wherein the peptide is cyclised by reacting two thiol groups in the N- and C-terminal cysteine residues with a di-benzyl bromide linker.

11. A pharmaceutical composition comprising the peptide described in claim 1 and a pharmaceutical acceptable carrier, diluent or excipient.

12. The pharmaceutical composition of claim 11 wherein the composition comprises a further therapeutic agent.

13. The peptide of claim 1 or the pharmaceutical composition of claim 11 for use in medicine.

14. The peptide of claim 1 or pharmaceutical composition of claim 11 for use in the treatment of cancer.

15. The peptide or pharmaceutical composition for use of claim 14 wherein the compound or composition is administered with a further therapeutic agent.

16. The compound or pharmaceutical composition for use of claim 14 wherein the compound or composition is to be used in a treatment regime further comprising the use of radiation therapy and/or surgery.

17. The compound or pharmaceutical composition for use of claim 14 wherein the cancer comprises one or more of breast cancer, prostate cancer, colorectal cancer, bladder cancer, ovarian cancer, endometrial cancer, cervical cancer, head and neck cancer, stomach cancer, pancreatic cancer, oesophagus cancer, small cell lung cancer, non-small cell lung cancer, melanoma, neuroblastoma, leukaemia, lymphoma, sarcoma or glioma.

Description

[0104] The present invention will now be further described with reference to the following figures which show:

[0105] FIG. 1: Human non-small cell lung cancer cell survival following treatment with Hilra-025, with and without Nanocin Pro™;

[0106] FIG. 2: H460 lung cancer cell survival curves following peptide exposure;

[0107] FIG. 3: NCI-H460 cell viability assays comparing the effect of a glycosylated peptide (HILRa-Glu-01) and an unglycosylated peptide (HILRa-CL-17); and

[0108] FIG. 4: Distribution of DAC peptide Cys-[Pro-Arg-Gly-Pro-Arg-Pro-DAC-Trp-Trp-Arg-Arg-Trp-Tp-Arg-Arg-Trp-Trp] in the presence of Nanocin Pro™.

EXAMPLES

[0109] The present invention will now be described in further detail with reference to the following examples, which are provided for illustration only.

Example 1: Increased Internalisation Improves Potency of Cyclic Amphiphilic Peptides (FIGS. 1 and 4)

[0110] Poor uptake of drugs into cells is a common problem that blocks the drug development progress of many promising peptides and proteins. A nanoparticle reagent (Nanocin Pro™, Tecrea Limited) is available, which enables improved internalisation of biomolecules including peptides.

[0111] The present inventor tested the cytotoxicity of previously described cyclic peptides with and without Nanocin Pro™. Proliferation assays were performed to examine the effect of HilRa linear Peptide-025 on the viability NCI-H460 cells. Assays were performed in the presence and absence of the peptide transfection reagent Nanocin-PRO at concentrations of 0.1 μl, 0.2 μl and 0.5 μl.

[0112] The following protocol was used: [0113] 1) NCI-H460 cells were grown in Ham's F12 media supplemented with 10% FBS. [0114] 2) Cells were harvested and seeded into 96-well plates at 500 cells/well. [0115] 3) Peptides were diluted from DMSO stock solutions in 9-point doubling dilutions from a top concentration of 500 μM. Final DMSO concentration was constant at 1% (v/v). [0116] 4) In line with the supplied Nanocin-Pro protocol, diluted peptides were incubated with Nanocin-Pro in media for 20 minutes prior to addition to cells. Nanocin-Pro was used at a final assay concentrations of 0.2 μl/well. [0117] 5) Cells were grown in the presence of peptide for 96 hours at 37° C., 5% CO.sub.2 in a humidified atmosphere. [0118] 6) After 96 h, Alamar blue 10% (v/v) was added, incubated for a further 4 h and fluorescent product detected using a BMG FLUOstar plate reader. [0119] 7) Media only background readings were subtracted before data was analysed using a 4-parameter logistic equation in GraphPad Prism.

[0120] HILRa-025 showed an LD.sub.50 of −4.7 M against NCI-460, in medium alone and −6.5 M in the presence of Nanocin 2 μl. The concentration of Nanocin-Pro used, reduced cell viability by 36% in DMSO control wells compared to control wells in the absence of Nanocin-Pro. No peptide precipitation was observed for any peptides.

[0121] The results in FIG. 1 show that Nanocin Pro™ enhances the cytotoxicity of peptide Hilra-025, a cyclic peptide composed of a ‘warhead’ and ‘backbone’ as described in WO/2016/020437. This indicates that the potency of previously described cyclic peptides (for example those disclosed in WO12016/020437) which are not, alone, potent enough to be used in the clinic, have the potential of much higher specific activity if they could be better internalised.

[0122] HILRa-025 peptide without Dac incorporated showed auto-fluorescent properties above concentrations of 10 μM. The auto-fluorescent signal was predominantly located in the nucleus (data not shown). At lower concentrations of HILRa-025-Dac and CNTRL-Dac (6 μM in the absence of Nanocin-Pro, or 3 μM in the presence of Nanocin-Pro), clear imaging of DAC-peptide within the cell was achieved. The distribution was distinctly different to that seen at higher concentrations; being throughout the nucleus and cytoplasm, and showing a punctate appearance particularly in the cytoplasm (FIG. 4). Compared to controls treated with DMSO, cells treated with HILRa peptide had an appearance consistent with a state of autophagy. The punctate cytoplasmic localisation of DAC-peptide appeared to co-localise with autophagic vesicles. HILRa-025-DAC and CNTRL-DAC showed indistinguishable patterns of distribution within the cell. The presence or absence of Nanocin-PRO also did not cause a noticeable change in peptide localisation.

Example 2: Cyclisation of Linear Peptides Using CLIPS Technology

[0123] Synthesis and biological testing of peptides described herein was carried out by commercial research companies:

[0124] Peptide cyclisation was via the CLIPS method synthesised commercially by PEPSCAN PRESTO Zuiderslusveg 2, 8243 RC LELYSTAD, PO Box 2098, 8203 AB LELYSTAD

[0125] Biological assays were carried out by: HORIZON DISCOVERY SERVICES, 8100 CAMBRIDGE RESEARCH PARK, UNITED KINGDOM, CB25 9TL

[0126] Earlier experiments (Warenius et al, 2011 Selective anticancer activity of a hexapeptide with sequence homology to a non-kinase domain of Cyclin Dependent Kinase 4. Mol Cancer. 2011; 10: 72-88) in which the warhead was combined in a cyclic cassette with an amphiphilic polymer produced optimal results when the number of residues in the cassette was close to fifteen. This suggests that conformational constraint is likely to play a part in the activity of the warhead.

[0127] As an expert in constrained peptides, Pepscan has developed a proprietary, highly versatile constraining technology, called CLIPS (Chemical Linkage of Peptides onto Scaffolds; WO2004/077062). CLIPS technology involves the cyclization of linear peptides via reaction of thiol-functionalities of the cysteines with a small rigid entity. This anchor reacts exclusively with thiols and attaches to the peptide via covalent bonds. The CLIPS cyclization technology is unique for its versatility and ease of application. The cyclization reaction lasts no longer than 30 min, runs at room temperature and does not require any sort of catalysis.

[0128] The inventor undertook testing of the effect of various peptides which were cyclised using CLIPS technology on the killing of human non-small cell lung cancer cells (NCIH-460) in vitro. For each of the peptides, between two and four isomer fractions were received, giving a total of 12 peptide entities tested.

[0129] The following warhead lengths, obtained by adding flanking sarcosine residues, were tested:

TABLE-US-00010 TABLE 1 Optimising Length of Warhead-including Linear Peptide for CLIPS % inhibition at 1 mM 0.2 μl Nanocin-PRO No Peptide Sequence per well Nanocin-PRO HILRa-CL-01 Ac-CPRGPRPC-NH2 12.9 0.0 HILRa-CL-02 Ac-C[Sar]PRGPRP[Sar]C-NH2 2.0 0.0 HILRa-CL-03 Ac-C[Sar][Sar]PRGPRP[Sar][Sar]C-NH2 9.5 0.0 HILRa-CL-04 Ac-CPKGPRPC-NH2 8.5 0.0 HILRa-CL-05 Ac-C[Sar]PKGPRP[Sar]C-NH2 1.1 0.0 HILRa-CL-06 Ac-C[Sar][Sar]PKGPRP[Sar][Sar]C-NH2 6.1 0.0 HILRa-CL-07 Ac-CPRGPKPC-NH2 1.5 0.0 HILRa-CL-08 Ac-C[Sar]PRGPKP[Sar]C-NH2 3.2 0.0 HILRa-CL-09 Ac-C[Sar][Sar]PRGPKP[Sar][Sar]C-NH2 3.0 1.1 HILRa-CL-10 Ac-CPKGPKPC-NH2 0.8 2.2 HILRa-CL-11 Ac-C[Sar]PKGPKP[Sar]C-NH2 1.2 0.0 HILRa-CL-12 Ac-C[Sar][Sar]PKGPKP[Sar][Sar]C-NH2 0.8 0.0 Paclitaxel — IC.sub.50 = 7.4 nM —

[0130] Although the ‘clipped’ linear peptides all showed very low killing of human non-small cell lung cancer in vitro at a concentration of 1 mM, using Nanocin Pro™ it was still possible to see that HILRa-CL-01 [Ac-(Pro-Arg-Gly-Pro-Arg-Pro)-NH2] was the optimal pre-clipped linear peptide warhead length (6 amino acids) when flanked by a cysteine residue at both the N- and C-termini. Interestingly, other 6 amino acid warheads (HILRa-CL-04, HILRa-CL-7 and HILRa-CL-10) in which the arginine residues at positions 2 and 5 were modified, showed varying activities. This data indicates that an arginine residue is required at least at position 5 in order to exert some cell killing effect. Substituting both anionic arginines by cationic homocysteic acids completely removed the cell killing effect (see for example HILRa-CL-16 A, B and C in FIG. 2). The data obtained also surprisingly indicates that an optimum warhead length of 6 amino acids results in optimal cancer cell cytotoxicity.

Example 3: Modification of Warhead Sequences

[0131] Using Nanocin Pro™, the ‘clipped’ Pro-Arg-Gly-Pro-Arg-Pro warhead could be demonstrated to cause just over 10% lethality of human non-small cell lung cancer cells at the very high concentration of 1.0 mM (see table 1).

[0132] IC.sub.50 values obtained and % inhibition at the top concentration used in the assay (300 NM) are shown in the table below. Values in brackets are approximate due to extrapolated or incomplete dose response curves.

[0133] Cumulative experience from past experiments underlined the role of the two charged amino acids in cytotoxic activity but these charged groups gave the molecule a hydrophilicity that was likely to impair cell uptake and bind poorly in a putative hydrophobic binding site. All amino acids in the Pro-Arg-Gly-Pro-Arg-Pro site except the arginines were thus replaced by highly non-polar residues, chosen from a range of non-naturally occurring peptide molecules likely to provide resistance to peptidolysis.

[0134] To ascertain whether anionic groups could act in the warhead in a similar way to the cationic guanidium groups of the arginines, homocysteic acids were substituted at the arginine sites. The HILRa peptides were synthesised by standard automated peptide-synthesis using both normal and unusual amino acid precursors:

Dac=(Fmoc-β-(7-methoxy-coumarin-4-yl)-Ala-OH B-37407) BACHEM
Hca=Homocysteic acid Fmoc-4-(neopentyloxysulfonyl)-Abu-OH (S)-2-(Fmoc-amino)-4-neopentyloxysulfonyl-butyric acid. CAS 220951-81-5 Santa Cruz Biotechnology Inc
Sar=Sarcosine (Fmoc precursor widely available commercially)
Nap=Naphthalene Fmoc-(R,S)-3-amino-3-(2-Naphthyl)propionic acid CAS NO 269078-81-1 Santa Cruz Biotechnology Inc.
dmPro=dimethyl proline (R,S)-Boc-3,3-dimethyl-proline CAS No 143979-40-2 Polypeptide.com

[0135] The following protocol was used: [0136] 1) NCI-H460 cells were grown in Ham's F12 media supplemented with 10% FBS. [0137] 2) Cells were harvested and seeded into 96-well plates at 500 cells per well. [0138] 3) Peptides were diluted 1:2 from DMSO stock solutions to generate a 9-point dose range from a top concentration of 300 μM. Final DMSO concentration was constant at 1% (v/v). [0139] 4) In line with the supplied Nanocin-PRO protocol, diluted peptides were incubated with Nanocin-PRO in media for 20 minutes prior to addition to cells. Nanocin-PRO was used at a final assay concentration of 0.2 μl per well. [0140] 5) Cells were grown in the presence of peptide for 96 hours at 37° C., 5% CO.sub.2 in a humidified atmosphere. [0141] 6) After 96 h, Alamar blue 10% (v/v) was added, incubated for a further 4 h and fluorescent product detected using a BMG FLUOstar plate reader. [0142] 7) Media only background readings were subtracted before data was analysed using a 4-parameter logistic equation in GraphPad Prism.

[0143] The naphthalene precursor in the automated peptide synthesis used here, was Fmoc-(R,S)-3-amino-3-(2-Naphthyl)propionic acid) which is a mixture of sterereoisomers. Thus, after synthesis, purification and ‘clipping’ of the novel peptides each of the 4 warheads (as shown in Table 2 below) presented a mixture of stereoisomers (A, B and C) which were selectively purified and tested individually.

[0144] Viability assays of the CLIP cyclised peptides were performed to examine the effect of four HILRa clipped peptides on the viability of NCIH460 cells (up to three isomers for each of the peptides). Six isomers had no cytotoxic effect (data not shown). Results for HILRa 16A, 16B, 16C and HILRa 17A, 17B and 17C are depicted in FIG. 2.

[0145] As shown in Table 2 and FIG. 2, HILRa-CL-14 A, B and 16 A, B, C which contained homocysteic acid (Hca) had low detectable cancer cell-necrotic activity. Only the arginine-containing warheads HILRa-CL-15 A, B, C (SEQ ID No:6) and HILRA-CL-17 A, B, C (SEQ ID No:7) showed strong cytotoxic activity.

[0146] Assays were performed in the presence and absence of the peptide transfection reagent Nanocin-PRO. HILRa-CL-14 and HILRa-CL-16 (all isomers) showed negligible activity against NCIH460 cells. HILRa-CL-15 and HILRa-CL17 (all isomers) showed activity in the assay, with HILRa-CL-17 showing greater potency than HILRa-CL-15, and both peptides exhibiting greater potency in the presence of Nanocin-PRO. The presence of Nanocin-PRO reduced cell viability by 29% in DMSO control wells compared to control wells in the absence of Nanocin-PRO.

TABLE-US-00011 TABLE 2 Cancer Cell Toxicity of Different Warhead Amino-acid Combinations No 0.2 μl Nanocin-PRO Nanocin-PRO per well Peptide or % inhibition % inhibition Compound Peptide sequence IC.sub.50 at 300 μM IC.sub.50 at 300 μM HILRa CL-14-A Ac-CDacHcaSarProHcaNapC-NH2 n/a 10.5 n/a 9.9 HILRa CL-14-B n/a 5.8 n/a 6.8 HILRa CL-15-A Ac-C[Dac]R[Sar][dmPro]R[Nap]C-NH2 n/a 22.7 62.8 μM 84.5 HILRa CL-15-B n/a 20.2 81.3 μM 87.5 HILRa CL-15-C n/a 23.8 47.3 μM 88.5 HILRa CL-15-D n/a 25.2 48.7 μM 89.1 HILRa CL-16-A Ac-CDacHcaSarNapHcaNapC-NH2 n/a 9.9 n/a 0.0 HILRa CL-16-B n/a 14.1 n/a 0.0 HILRa CL-16-C n/a 26.9 n/a 0.5 HILRa CL-17-A Ac-C[Dac]R[Sar][Nap]R[Nap]C-NH2  213 μM 90.3 5.38 μM 98.7 HILRa CL-17-B  130 μM 99.2 (2.10 μM) 99.3 HILRa CL-17-C 92.2 μM 99.6 (0.88 μM) 99.4 Paclitaxel n/a 4.95 nM n/a n/a n/a

[0147] Surprisingly, the substitution of dmPro in HILRa CL-15 with Nap in HILRa CL-17 resulted in a significant increase in inhibition which could not have been predicted. These experiments indicate that peptide warheads can have high cancer cell-necrotic activity which is dependent on the warhead sequence. The present inventor has surprisingly identified a number of peptide warheads which can be utilised to specifically target cancer cells for killing.

Example 4: NCI-H460 Cell Viability Assays Comparing the Effect of a Glycosylated Peptide (HILRa-Glu-01) and an Unglycosylated Peptide (HILRa-CL-17)

[0148] The inventor next undertook testing to determine whether the cytotoxicity of the peptides of the invention could be increased by glycosylation of the peptides. NCI-H460 cell viability assays were performed to compare the effect of a glycosylated clipped peptide (HILRa-Glu-01) versus the un-glycosylated version (HILRa-CL-17, HILRa-CL-17 was used in this study as a 1:1:1 mix of the three isomers HILRa-CL-17A, HILRa-CL-17B and HILRa-CL-17C).

[0149] Assays were performed in the presence and absence of the peptide transfection agent Nanocin-Pro.

[0150] The following protocol was used: [0151] 1) NCI-H460 cells were grown in Ham's F12 media supplemented with 10% FBS. [0152] 2) Cells were harvested and seeded into 96-well plates at 500 cells per well. [0153] 3) HILRa peptides were diluted 1:2 from DMSO stock solutions to generate a 9-point dose range from a top concentration of 300 μM. Final DMSO concentration was constant at 1% (v/v). [0154] 4) In line with the supplied Nanocin-Pro protocol, diluted peptides were incubated with Nanocin-Pro in media for 20 minutes prior to addition to cells. Nanocin-Pro was used at a final assay concentration of 0.2 μl per well. [0155] 5) Cells were grown in the presence of peptide for 96 hours at 37° C., 5% CO2 in a humidified atmosphere. [0156] 6) After 96 h, Alamar blue 10% (v/v) was added, incubated for a further 4 h and fluorescent product detected using a BMG FLUOstar plate reader. [0157] 7) Media only background readings were subtracted before data was analysed using a 4-parameter logistic equation in GraphPad Prism.

[0158] The glycosylated peptide (HILRa-Glu-01) showed slightly lower activity in the assay compared to the un-glycosylated version (PRO CL-17) (FIG. 3). Both peptides showed much higher activity in the presence of Nanocin-PRO. The presence of Nanocin-Pro reduced cell viability in DMSO control wells by 16%.

[0159] It will be appreciated that numerous modifications to the above described peptides, pharmaceutical compositions, methods and uses may be made without departing from the scope of the invention as defined in the appended claims. For example, although in the example shown above the peptides showing very high cytotoxic activity have specific amino acid sequences, namely, Dac-Arg-Sar-dmPro-Arg-Nap and Dac-Arg-Sar-Nap-Arg-Nap, it will be appreciated that variations to these sequences can be made without departing from the scope of the invention as defined by the claims.