WO2016097753A1 - Peptide de l'extrémité c-terminale de l'acétylcholinestérase cyclique dans le traitement ou la prévention du cancer ou de la métastase - Google Patents

Peptide de l'extrémité c-terminale de l'acétylcholinestérase cyclique dans le traitement ou la prévention du cancer ou de la métastase Download PDF

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Publication number
WO2016097753A1
WO2016097753A1 PCT/GB2015/054068 GB2015054068W WO2016097753A1 WO 2016097753 A1 WO2016097753 A1 WO 2016097753A1 GB 2015054068 W GB2015054068 W GB 2015054068W WO 2016097753 A1 WO2016097753 A1 WO 2016097753A1
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Prior art keywords
analogue
derivative
cyclic polypeptide
cyclic
cells
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PCT/GB2015/054068
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English (en)
Inventor
Susan Adele Greenfield
Christopher PEPPER
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Neuro-Bio Ltd
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Priority claimed from GB1508480.9A external-priority patent/GB2538947A/en
Priority to JP2017531901A priority Critical patent/JP7066409B2/ja
Priority to ES15816833T priority patent/ES2739543T3/es
Priority to KR1020177016763A priority patent/KR102652620B1/ko
Priority to AU2015365606A priority patent/AU2015365606B2/en
Priority to US15/535,020 priority patent/US10441638B2/en
Priority to CA2969990A priority patent/CA2969990A1/fr
Priority to SI201530849T priority patent/SI3233110T1/sl
Application filed by Neuro-Bio Ltd filed Critical Neuro-Bio Ltd
Priority to DK15816833.6T priority patent/DK3233110T3/da
Priority to BR112017012896A priority patent/BR112017012896A2/pt
Priority to EP15816833.6A priority patent/EP3233110B1/fr
Priority to CN201580069324.3A priority patent/CN107106643B/zh
Priority to RU2017120535A priority patent/RU2711161C2/ru
Priority to EP19172010.1A priority patent/EP3613428A1/fr
Priority to MX2017007740A priority patent/MX2017007740A/es
Priority to PL15816833T priority patent/PL3233110T3/pl
Publication of WO2016097753A1 publication Critical patent/WO2016097753A1/fr
Priority to US16/557,877 priority patent/US11033609B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01007—Acetylcholinesterase (3.1.1.7)

Definitions

  • the invention relates to cancers, and in particular to novel compositions, therapies and methods for treating, preventing or ameliorating cancer or metastatic disease.
  • Cancer and malignant tumours form a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body, i.e. metastasis.
  • metastasis a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body.
  • metastasis In 2012, approximately 14 million new cases of cancer occurred globally. There is therefore a need to provide an improved medicament for the treatment of cancer, and metastasis.
  • a cyclic peptide derived from the C-terminus of acetylcholinesterase known as "NBP-14"
  • NBP-14 acetylcholinesterase
  • the inventors investigated the effects of a cyclic peptide derived from the C-terminus of acetylcholinesterase (known as "NBP-14") on various cancer cell lines, as well as primary tumour cells derived from patients and lymphocytes derived from healthy age- matched individuals, and found that it showed modest apoptotic and anti-proliferative activity in each of the cancer cell lines tested.
  • the cyclic peptide is non-toxic in normal cells. Accordingly, the inventors believe that cyclic peptides will have therapeutic benefit in the treatment of cancer, tumours and metastatic disease.
  • a cyclic polypeptide, derivative or analogue thereof for use in treating, ameliorating or preventing cancer or metastatic disease.
  • a method of treating, ameliorating or preventing cancer or metastatic disease in a subject comprising, administering to a subject in need of such treatment, a therapeutically effective amount of a cyclic polypeptide, derivative or analogue thereof.
  • NBP- 14 a cyclic peptide derived from the C-terminus of acetylcholinesterase (known as "NBP- 14") on: (i) primary chronic lymphocytic leukaemia (CLL) samples derived from CLL patients with a range of prognostic markers (MEC-i cells); (ii) in KGia (Acute myeloid leukaemia cell line) and H929 and JJN3 (Multiple myeloma cell lines); and (iii) in MCF7 and MDA-MB-231 (breast cancer cell lines).
  • CLL primary chronic lymphocytic leukaemia
  • MEC-i cells prognostic markers
  • KGia Acute myeloid leukaemia cell line
  • H929 and JJN3 Multiple myeloma cell lines
  • MCF7 and MDA-MB-231 breast cancer cell lines
  • the inventors have surprisingly shown that the cyclic peptide, NBP-14, showed apoptotic effects in each of the cell lines tested at concentrations >0. ⁇ . Moreover, MCF7 cells showed increased sensitivity to NBP-14. The cyclic peptide, NBP-14, showed evidence of anti-proliferative activity in MDA-MB-231 cells, and similar effects were also observed in JJN3 cells, KGia cells, MEC-i cells and H929 cells with concentrations of peptide >0. ⁇ . Advantageously, they have also shown that the cyclic peptide is non-toxic in normal cells at the same concentrations.
  • the cancer which is treated may be leukaemia.
  • the cancer may be lymphocytic leukaemia or chronic lymphocytic leukaemia (CLL).
  • CLL chronic lymphocytic leukaemia
  • the cancer may be myeloid leukaemia, or acute myeloid leukaemia.
  • the cancer may be multiple myeloma.
  • the cancer may be breast cancer.
  • the cyclic polypeptide, derivative or analogue thereof is for use in treating, ameliorating or preventing metastatic disease.
  • Cyclic polypeptides are peptide chains whose N- and C-termini are themselves linked together with a peptide bond that forms a circular chain of amino acids, as shown in Figure 8B.
  • derivative or analogue thereof can mean a polypeptide within which amino acid residues are replaced by residues (whether natural amino acids, non-natural amino acids or amino acid mimics) with similar side chains or peptide backbone properties. Additionally, the terminals of such peptides may be protected by N- and C- terminal protecting groups with similar properties to acetyl or amide groups. Derivatives and analogues of peptides according to the invention may also include those that increase the peptide's half-life in vivo.
  • a derivative or analogue of the peptides of the invention may include peptoid and retropeptoid derivatives of the peptides, peptide-peptoid hybrids and D-amino acid derivatives of the peptides.
  • Peptoids, or poly-N-substituted glycines are a class of peptidomimetics whose side chains are appended to the nitrogen atom of the peptide backbone, rather than to the alpha-carbons, as they are in amino acids.
  • Peptoid derivatives of the peptides of the invention may be readily designed from knowledge of the structure of the peptide.
  • Retropeptoids in which all amino acids are replaced by peptoid residues in reversed order are also suitable derivatives in accordance with the invention.
  • a retropeptoid is expected to bind in the opposite direction in the ligand-binding groove, as compared to a peptide or peptoid-peptide hybrid containing one peptoid residue.
  • the side chains of the peptoid residues are able point in the same direction as the side chains in the original peptide.
  • the cyclic polypeptide, derivative or analogue thereof comprises or consists of an amino acid sequence derived from the C-terminus of acetylcholinesterase (AChE), or a truncation thereof.
  • the inventors were very surprised to observe that the cyclic AChE-derived polypeptides of the invention selectively target tumour cells rather than normal tissue.
  • the term "derived from” can mean an amino acid sequence, which is a derivative or a modification of an amino acid sequence that is present in, or forms, the C-terminus of AChE, and portion thereof.
  • truncation thereof can mean the cyclic polypeptide derived from AChE is reduced in size by the removal of amino acids.
  • the reduction of amino acids maybe achieved by removal of residues from the C- or N-terminal of the peptide prior to cyclisation into the cyclic polypeptide of the invention, or may be achieved by deletion of one or more amino acids from within the core of the peptide prior to cyclisation.
  • Acetylcholinesterase is a serine protease that hydrolyses acetylcholine, and will be well- known to the skilled person.
  • the major form of acetylcholinesterase which is found in the brain is known as tailed acetylcholinesterase (T-AChE).
  • T-AChE tailed acetylcholinesterase
  • the cyclic polypeptide, derivative or analogue thereof comprises an amino acid sequence derived from the C-terminus of tailed acetylcholinesterase (T-AChE), or a truncation thereof.
  • the protein sequence of one embodiment of human tailed acetylcholinesterase (Gen Bank: AAA68151.1) is 614 amino acids in length, and is provided herein as SEQ ID No:i, as follows:
  • the cyclic polypeptide, derivative or analogue thereof comprises or consists of an amino acid sequence derived from the C-terminus of
  • acetylcholinesterase or a truncation thereof, wherein the acetylcholinesterase comprises an amino acid sequence substantially as set out in SEQ ID No:i, preferably excluding the 31 amino acids at the N-terminal.
  • the cyclic polypeptide, derivative or analogue thereof comprises or consists of an amino acid sequence derived from the last 300, 200, 100 or 50 amino acids forming the C-terminus of acetylcholinesterase, or a truncation thereof, most preferably wherein the acetylcholinesterase comprises or consists of an amino acid sequence substantially as set out in SEQ ID No:i.
  • the cyclic polypeptide, derivative or analogue thereof preferably comprises or consists of an amino acid sequence derived from the last 40 amino acids forming the C-terminus of acetylcholinesterase, or a truncation thereof.
  • the cyclic polypeptide, derivative or analogue thereof comprises or consists of between 8 and 40 amino acid residues, more preferably between 10 and 30 amino acids, and most preferably between 12 and 20 amino acids.
  • the inventor has prepared three peptide sequences that are derived from the C-terminus of AChE, and which are referred to herein as T30, T14 and T15, where the number corresponds to the amino acid number.
  • T30 (which corresponds to the last 30 amino acid residues of SEQ ID No:i) is provided herein as SEQ ID No:2, as follows: -
  • SEQ ID No: 2 The amino acid sequence of T14 (which corresponds to the 14 amino acid residues located towards the end of SEQ ID No:i, and lacks the final 15 amino acids found in T30) is provided herein as SEQ ID No:3, as follows: -
  • T15 (which corresponds to the last 15 amino acid residues of SEQ ID No:i) is provided herein as SEQ ID No:4, as follows: -
  • any of the sequences represented as SEQ ID No: 2-4 can be readily cyclised or cyclated to form a cyclic polypeptide of the first aspect.
  • cyclization of peptides can be achieved by side-chain-to-side-chain, side-chain-to- backbone, or head-to-tail (C-terminus to N-terminus) cyclization techniques.
  • head-to-tail cyclization is the preferred method by which the cyclic polypeptides are produced.
  • the cyclic polypeptides may be synthesised using either classical solution-phase linear peptide cyclization or resin-based cyclization.
  • the polypeptide is produced using a cyclization cleavage approach, in which the cyclic polypeptide is synthesized by cyclization after step-wise linear peptide synthesis.
  • An advantage of this method is that the side-chain does not need to be anchored, making the approach more general.
  • resultant samples of cyclic peptides can be analysed by MALDI-TOF MS.
  • a preferred polypeptide according to the invention comprises or consists of cyclic SEQ ID No: 2, 3 or 4, or a functional variant or fragment thereof.
  • cyclated SEQ ID No: 3 i.e. referred to herein as "cyclated T14", “CT14” or “NBP-14"
  • CT14 cancer cell lines tested compared to healthy cells
  • NBP-14 non-toxic in the normal, non-cancerous cells.
  • a most preferred cyclic polypeptide of the first aspect comprises or consists of cyclic SEQ ID No:3, or a functional variant or fragment thereof.
  • the cyclic polypeptide according to the invention maybe used in a medicament, which may be used as a monotherapy (i.e. use of the cyclic polypeptide, derivative or analogue thereof alone), for treating, ameliorating, or preventing cancer or metastasis.
  • the cyclic polypeptide according to the invention maybe used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing cancer.
  • the cyclic polypeptide according to the invention maybe combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
  • the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment.
  • the vehicle of medicaments according to the invention should be one which is well -tolerated by the subject to whom it is given, and preferably enables delivery of the cyclic polypeptide across the blood-brain barrier when treating brain tumours.
  • Cyclic polypeptides according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with cyclic polypeptides used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).
  • medicaments according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. For example, the medicament may be injected at least adjacent the brain.
  • Injections maybe intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).
  • amount of the cyclic polypeptide that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the cyclic polypeptide and whether it is being used as a monotherapy or in a combined therapy.
  • the frequency of administration will also be influenced by the half-life of the cyclic polypeptide within the subject being treated.
  • Optimal dosages to be administered maybe determined by those skilled in the art, and will vary with the particular cyclic polypeptide in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the cancer or metastasis. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
  • a daily dose of between o.oo ⁇ g/kg of body weight and lomg/kg of body weight, or between o.o ⁇ g/kg of body weight and lmg/kg of body weight, of the cyclic polypeptide according to the invention may be used for treating, ameliorating, or preventing cancer or metastasis, depending upon which cyclic polypeptide is used.
  • the cyclic polypeptide may be administered before, during or after onset of cancer. Daily doses maybe given as a single administration (e.g. a single daily injection or inhalation of a nasal spray). Alternatively, the cyclic polypeptide may require administration twice or more times during a day. As an example, cyclic polypeptides may be administered as two (or more depending upon the severity of the cancer or metastasis being treated) daily doses of between 0.07 ⁇ g and 700 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.
  • a slow release device maybe used to provide optimal doses of cyclic polypeptide according to the invention to a patient without the need to administer repeated doses.
  • Known procedures such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the cyclic polypeptide according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of
  • an anti-cancer or anti- metastatic pharmaceutical composition comprising a therapeutically effective amount of the cyclic polypeptide, derivative or analogue thereof according to the first aspect, and optionally a pharmaceutically acceptable vehicle.
  • the invention also provides in a fourth aspect, a process for making the anti-cancer or anti-metastatic pharmaceutical composition according to the third aspect, the process comprising combining a therapeutically effective amount of the cyclic polypeptide, derivative or analogue thereof according to the first aspect, with a pharmaceutically acceptable vehicle.
  • the cyclic polypeptide, derivative or analogue thereof preferably comprises or consists of Cyclic T14 (i.e. NBP-14) as disclosed herein, i.e. SEQ ID No:3.
  • a "subject” maybe a vertebrate, mammal, or domestic animal. Hence, medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or maybe used in other veterinary applications. Most preferably, however, the subject is a human being.
  • a "therapeutically effective amount" of cyclic polypeptide is any amount which, when administered to a subject, is the amount of active agent that is needed to treat the cancer or metastasis, or produce the desired effect.
  • the cyclic polypeptide, derivative or analogue thereof may be used as an adjuvant for the treatment of solid or metastatic tumours, for example with chemotherapy or radiotherapy. This means that lower doses and exposure times of chemotherapy and/ or radiotherapy are required.
  • the therapeutically effective amount of cyclic polypeptide used maybe from about 0.001 mg to about 800 mg, and preferably from about 0.01 mg to about 500 mg.
  • a "pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
  • the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet.
  • the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
  • Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.
  • the cyclic polypeptide and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
  • the cyclic polypeptide used according to the invention can also be administered orally either in liquid or solid composition form.
  • compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
  • forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
  • nucleic acid or peptide or variant, derivative or analogue thereof which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof.
  • the terms "substantially the amino acid/nucleotide/peptide sequence”, “functional variant” and “functional fragment”, can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequence, “functional variant” and “functional fragment”, can be a sequence that has at least 40% sequence identity with the amino
  • amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
  • sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
  • amino acids referred to amino acids
  • acid/ polynucleotide/ polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.
  • the skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value.
  • the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.
  • the method used to align the sequences for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison
  • the parameters used by the alignment method for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.
  • percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
  • acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs.
  • a substantially similar nucleotide sequence will be encoded by a sequence, which hybridizes to DNA sequences or their complements under stringent conditions.
  • stringent conditions we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/o.i% SDS at approximately 20-65°C.
  • a substantially similar polypeptide may differ by at least l, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID No: 1-4.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
  • Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
  • Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
  • the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
  • Figure l is a comparison of the cytotoxic effects of T15 (SEQ ID No:4), T30 (SEQ ID No: 2), and one embodiment of a cyclic polypeptide according to the invention, i.e. NBP-14 (SEQ ID No:3), in the breast cancer cell lines (A) MCF7 and (B) MDA-MB-231. All assays were carried out in duplicate and are presented as mean ( ⁇ SD) of three independent experiments;
  • Figure 2 shows a comparison of the cytotoxic effects of T15, T30, NBP-14 and Ara-C in the KGia cell line. All assays were carried out in duplicate and are presented as mean ( ⁇ SD) of three independent experiments;
  • Figure 3 shows a comparison of the cytotoxic effects of T15, T30, NBP-14 and fludarabine in (A) H929 and (B) MEC-i cell lines. All assays were carried out in duplicate and are presented as mean ( ⁇ SD) of three independent experiments;
  • Figure 4A shows a comparison of the cytotoxic effects of T15, T30, NBP-14 in primary CLL cells.
  • Figure 4(B) shows the effect of the anti-CD20 monoclonal antibody
  • Figure 5 shows a comparison of the effects of T15, T30, and NBP-14 peptides in normal B- and T-lymphocytes. All assays were carried out in duplicate and data are presented as mean ( ⁇ SD) of three independent experiments;
  • Figure 6 shows a comparison of the anti-proliferative effects of T15, T30 and NBP-14 in (A) MDA-MB-231 cells and (B) MCF7 cells. All assays were carried out in duplicate and data are presented as mean ( ⁇ SD) of three independent experiments;
  • Figure 7 shows a comparison of the anti-proliferative effects of T15, T30 and NBP-14 in (A) KGia cells and (B) MEC-i cells and (C) H929 cells. All assays were carried out in duplicate and data are presented as mean ( ⁇ SD) of three independent experiments;
  • Figure 8A shows the sequence of NBP-14 with the terminal Alanine (A) and Lysine (K) residues forming the cyclisation sites.
  • Figure 8B shows the cyclic NBP-14 peptide in which the terminal Alanine and Lysine residues are linked together;
  • Figure 9 shows the comparison of the anti-migratory dose-responses induced by NBP- 14 peptide in MDA-MB-231, MCF7, JJN3 and KGia cancer cell lines. All data are presented as mean ( ⁇ SD) of three independent experiments. *; P ⁇ 0.05;
  • FIG 10 shows the comparison of the anti-migratory effects of T15, T30, NBP-14 in the breast cancer cell lines (A) MCF7 and (B) MDA-MB-231. All data are presented as mean ( ⁇ SD) of five independent experiments;
  • FIG 11 shows the comparison of the anti-migratory effects of T15, T30, NBP-14 in the KGia cell line. All data are presented as mean ( ⁇ SD) of five independent
  • FIG. 12 shows the comparison of the anti-migratory effects of T15, T30, NBP-14 in the JJN3 cell line. All data are presented as mean ( ⁇ SD) of five independent
  • Figure 13 shows the comparison of the cytotoxic effects of T15, T30, NBP-14 in primary CLL cells. All assays were carried out in duplicate and data are presented as mean ( ⁇ SD) of ten independent experiments;
  • Figure 14 shows the comparison of the anti-migratory effects of T15, T30 and NBP-14 peptides on normal B-cells. All assays were carried out in duplicate and data are presented as mean ( ⁇ SD) of five independent experiments;
  • Figure 15 shows the comparison of the effects of NBP-14 peptides in primary CLL cells and normal B-lymphocytes. All assays were carried out in duplicate and data are presented as mean ( ⁇ SD) of three independent experiments; Figure 16 shows the correlation between baseline migration and the percentage decrease in migration induced by NBP-14;
  • Figure 17 shows the amount of baseline migration in a variety of cell lines in the absence of NBP-14, including MDA-MB-231, CLL cells, Normal B-cells, MEC-i, JJN3, KGia, MCF7 and H929 cells; and
  • Figure 18 shows the percentage decrease in migration induced by ⁇ NBP-14 in a variety of cell lines, including MDA-MB-231, CLL cells, Normal B-cells, MEC-i, JJN3, KGia, MCF7 and H929 cells.
  • the inventors have generated a number of linear and cyclic peptides based on the C- terminus of acetylcholinesterase known as T15, T30 and NBP-14 peptides and evaluated their effects in a number of cell lines and primary leukaemia cells derived from patients.
  • SEQ ID No: 3 is referred to herein as "cyclated T14", “CT14” or “NBP-14”, and is a cyclic peptide with an amino acid sequence derived from the C-terminus of Tailed acetylcholinesterase.
  • AML acute myeloid leukaemia
  • the multiple myeloma (MM) cell line H929, the two breast cancer cell lines (MCF7 and MDA-MB-231), the MEC-i cells and the primary chronic lymphocytic leukaemia cells were maintained in RPMI medium supplemented with 100 units/ml penicillin, lOO g/ml streptomycin and 10% foetal calf serum.
  • the media used contained acetylcholine but after the initial set of experiments an additional ⁇ of acetylcholine was added to the culture media.
  • Cells were subsequently aliquoted (10 6 cells/ml) into 24-well plates and were incubated at 37°C in a humidified 5% carbon dioxide atmosphere for 72h in the presence of the peptides (T15, T30, NBP-14 and the combination of T30+NBP-14) at concentrations between o.inM and ⁇ . In addition, control cultures were carried out to which no peptide was added. Cells were
  • Cultured cells were harvested by centrifugation and were then counted using a Vi-Cell XR cell viability counter. The number of viable cells in each culture was then expressed as a percentage of the viable cells in the control cultures (no peptide).
  • Example 1 - Cyclic T14 i.e. "NBP-14"
  • T-AChE The 'tailed' acetylcholinesterase
  • the amino acid sequence of the linear peptide, T14 is AEFHRWSSYMVHWK [SEQ ID No:3].
  • the amino acid sequence of the linear peptide, T30 is
  • T15 corresponds to the last 15 amino acid residues of SEQ ID No:i, i.e. NQFDHYSKQDRCSDL [SEQ ID No: 4].
  • the AChE C-terminal peptide "T14"' has been identified as being the salient part of the AChE molecule responsible for its range of non-hydrolytic actions.
  • the synthetic 14 amino acids peptide analogue (i.e. "T14"), and subsequently the larger, more stable, and more potent amino acid sequence in which it is embedded (i.e. "T30”) display actions comparable to those reported for 'non-cholinergic' AChE.
  • FIG. 8A there is shown the 14 amino acid long cyclic T14 peptide (i.e. "NBP-14").
  • the cyclic peptide, NBP-14 has been cyclated via the terminal Alanine (A) and Lysine (K) residues, and is shown in Figure 8B.
  • Cyclisation can be achieved by several different means. For example, Genosphere Biotechnologies (France) performed the cyclisation of T14 by transforming the linear peptide into an N-terminal to C- terminal lactam. Cyclisation of T14 to create cyclic NBP-14 brings together both ends, i.e. HWK-AEF.
  • Example 2 The effect of acetylcholinesterase-derived peptides in the MCF7 and MDA- MB-231 cell lines
  • the inventors examined the ability of the acetylcholinesterase-derived peptides (NBP- 14 and/or T30) to induce apoptosis in the two breast cancer cell lines, and the results are shown in Figures lA and lB.
  • the MCF7 cells showed evidence of apoptosis at peptide concentrations above 0.1 ⁇ .
  • the MDA-MB-231 cell line was less sensitive to the effects of the peptides under the same conditions.
  • Example 3 The effect of the acetylcholinesterase-derived peptides in the KGia AML cell line
  • KGia cells were cultured with the peptides for 72h and their apoptotic effects were assessed, and the results are shown in Figure 2.
  • the KGia cells were also cultured with Ara-C, a commonly used cytotoxic agent used for the treatment of AML.
  • the acetylcholinesterase-derived peptides showed some toxicity in KGia cells, and Ara-C showed a dose-response at concentrations above ⁇ . ⁇ .
  • Example 4 The effect of the acetylcholinesterase-derived peptides on HQ2Q and MEC- 1 B-cell lines
  • the acetylcholinesterase-derived peptides showed a small cytotoxic effect in H929 cells and MEC-i cells, and the results are shown in Figure 3A and 3B.
  • the nucleoside analogue fludarabine induced a dose-response in both cell lines. - l8 -
  • Example 5 The effect of acetylcholinesterase-derived peptides in primary CLL cells The inventors next examined the effects of the acetylcholinesterase-derived peptides in primary CLL cells derived from patients, and the results are shown in Figures 4A and 4B.
  • NBP-14 showed evidence of dose-response at concentrations above ⁇ . ⁇ .
  • the inventors next compared this response with a non-genotoxic anti-CD20 monoclonal antibody (Rituximab). Rituximab induced a more pronounced dose-response at clinically used concentrations of the agent when compared to NBP-14.
  • Example 6 The apoptotic effect of acetylcholinesterase-derived peptides in normal B- and T-lymphocytes
  • Example 7 The effect of the acetylcholinesterase-derived peptides on proliferation of the cell lines
  • the inventors next examined the ability of the acetylcholinesterase-derived peptides to induce cytostasis, i.e. to inhibit proliferation in the various cell lines employed in this study.
  • the results are shown in Figure 6A and 6B.
  • the two breast cancer cell lines showed differential responses following incubation with the acetylcholinesterase- derived peptides.
  • the more proliferative cell line MDA-MB-231 showed a significant reduction in proliferation with NBP-14 peptide concentrations above o.iuM when compared with the T15 control peptide. This effect was not as significant in the less proliferative MCF7 cell line.
  • the MDA-MB-231 cell line showed increased proliferation in the presence of sub-nanomolar concentrations of T30 and NBP-14+T30.
  • the KGia cell line, the MEC-i cell line and the H929 cell line all showed reduced proliferation following incubation with concentrations of NBP-14 above ⁇ . ⁇ .
  • the effect of Ara-C (KGia cells) and fludarabine (MEC-i and H929 cells) are shown for comparison.
  • MCF7 cells showed relatively increased sensitivity to NBP-14, it was not preferentially cytotoxic in these cells when compared with the control peptide (T15) and the toxic peptide (T30).
  • NBP-14 showed clear anti-proliferative activity in the migratory cell line, MDA- MB-231 cells. Similar effects were also observed in KGia cells, MEC-i cells and H929 cells with concentrations of peptide >0. ⁇ . The anti-proliferative effects on MCF7 cells were less marked but this is the slowest growing of all the cell lines used in this study.
  • Acetylcholinesterase-derived peptides including NBP-14 exhibit an anti- metastatic effect.
  • cyclic peptides derived from the C-terminus of tailed acetylcholinesterase, and in particular, NBP-14, i.e. SEQ ID N0.3 can be used to treat cancer and prevent metastasis. Accordingly, these cyclic peptides can be used as an adjuvant for the treatment of solid or metastatic tumours with chemotherapy/ radiotherapy. This means that lower doses and exposure times of chemotherapy and/ or radiotherapy are required.
  • Example 8 The effects of NBP-14 on migration in cancer cell lines and primary CLL samples
  • JJN3 Multiple myeloma cell line
  • MDA-MB-231 and MCF-7 using transwell assays.
  • MDA-MB-231, KGia, and MEC-i cells are highly migratory cancer cell lines.
  • JJN3, CLL and MCF-7 are less migratory cancer cell lines.
  • B-lymphocytes are normal, non-cancerous cells. Rationale
  • the acute myeloid leukaemia (AML) KGia cell line was maintained in RPMI medium (Invitrogen) supplemented with 100 units/ml penicillin, lOO g/ml streptomycin and 5% foetal calf serum.
  • the multiple myeloma (MM) cell line JJN3, the two breast cancer cell lines (MCF7 and MDA-MB-231), the primary chronic lymphocytic leukaemia cells and normal B-lymphocytes were maintained in RPMI medium supplemented with 100 units/ml penicillin, lOO g/ ml streptomycin and 5% foetal calf serum.
  • ⁇ of acetylcholine was added to the culture media to ensure that the availability of acetylcholine was not a limiting factor in these experiments.
  • In vitro migration assays were performed by using 6.0 ⁇ m pore size transwell migration plates (Costar, Corning, N.Y.). A total of 10 6 CLL cells in 500 ⁇ 1 of RPMI media were added to the upper chamber of the transwell insert, loong/ ml of CXCL12 was added to the baso-lateral chamber for all the cell types tested apart from KGia cells. These cells do not express CXCR4 and so are unresponsive to CXCL12. Instead, media containing 10% foetal calf serum was added to the baso-lateral chamber in these experiments.
  • the plates were incubated for 24.I1 at 37 0 C in 5% C0 2 in the presence of the peptides (T15, T30, NBP-14 and the combination of T30+NBP-14) at concentrations between o.inM and iouM.
  • control cultures were carried out to which no peptide was added.
  • Cells were subsequently harvested by centrifugation and were analysed by flow cytometry using an Accuri C6 flow cytometer (BD). None of the conditions tested induced significant cell death in the cultures.
  • Migration of CLL cells was determined by counting cells that migrated to the lower (baso-lateral) chamber of the transwell plate and then expressed as a percentage of the total number of cells initially added to the upper (apical) chamber.
  • Example Q The effect of acetylcholinesterase-derived peptides in the MCF7 and MDA- MB-231 cell lines
  • Example 10 The effect of the acetylcholinesterase-derived peptides in the KGia Acute Myeloid Leukaemia cell line
  • KGia cells were cultured with the peptides for 24.I1 and their effects on migration were assessed.
  • Example 11 The effect of the acetylcholinesterase-derived peptides on the JJN3 Multiple Myeloma cell line
  • Example 13 The effect of acetylcholinesterase-derived peptides in normal B- lymphocytes
  • NBP-14 significantly inhibited the migratory activity of both primary CLL cells and normal B-cells.
  • the inventors plotted the mean baseline percentage migration for each of the cell lines and primary cells tested against the percentage reduction in migration induced by ⁇ NBP-14. Referring to Figure 16, there was clear relationship between the level of baseline migration and the anti-migratory response to NBP-14; high basal migration was associated with a larger percentage decrease in migration. The relationship was even stronger when the normal B-cells were removed from the analysis.
  • Example 16 Comparison of baseline migration between various cell types and prior to exposure to NBP-14
  • NBP-14 showed significant anti-migratory effects in all of the cell lines tested with the exception of MCF7 cells, which showed the lowest basal migration under control (no peptide) conditions; an observation that is in keeping with the known low metastatic potential of these cells. Dose-response analysis revealed that NBP-14 was effective at inhibiting migration at concentrations > ⁇ .
  • control peptide (T15) and the toxic peptide (T30) were made at ⁇ . None of the peptides induced significant cytotoxic effects in the cell lines or the primary malignant and non-malignant B-cells under the conditions tested. Therefore, the reductions in migration observed were not caused by increased cell death in the cultures.
  • NBP-14 has utility as an anti-cancer therapeutic, particularly in those tumours that are prone to metastasis.

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Abstract

L'invention concerne des polypeptides cycliques dérivés de l'extrémité C-terminale de l'acétylcholinestérase à utiliser dans le traitement ou la prévention du cancer ou d'une maladie métastatique.
PCT/GB2015/054068 2014-12-19 2015-12-18 Peptide de l'extrémité c-terminale de l'acétylcholinestérase cyclique dans le traitement ou la prévention du cancer ou de la métastase WO2016097753A1 (fr)

Priority Applications (16)

Application Number Priority Date Filing Date Title
DK15816833.6T DK3233110T3 (da) 2014-12-19 2015-12-18 Cyklisk acetylcholinesterase-c-terminalt peptid i behandling eller forebyggelse af cancer eller metastase
BR112017012896A BR112017012896A2 (pt) 2014-12-19 2015-12-18 polipeptídeo cíclico, derivado ou análogo do mesmo, composição farmacêutica, processo para produzir a composição farmacêutica
ES15816833T ES2739543T3 (es) 2014-12-19 2015-12-18 Péptido de terminal C de acetilcolinesterasa cíclico en el tratamiento o prevención de cáncer o metástasis
EP15816833.6A EP3233110B1 (fr) 2014-12-19 2015-12-18 Peptide de l'extrémité c-terminale de l'acétylcholinestérase cyclique dans le traitement ou la prévention du cancer ou de la métastase
US15/535,020 US10441638B2 (en) 2014-12-19 2015-12-18 Cyclic acetylcholinesterase c-terminal peptide in the treatment or prevention of cancer or metastasis
CA2969990A CA2969990A1 (fr) 2014-12-19 2015-12-18 Peptide de l'extremite c-terminale de l'acetylcholinesterase cyclique dans le traitement ou la prevention du cancer ou de la metastase
SI201530849T SI3233110T1 (sl) 2014-12-19 2015-12-18 Ciklični acetilholinesterazni c-terminalni peptid pri zdrvaljenju ali preprečevanju raka ali metastaz
JP2017531901A JP7066409B2 (ja) 2014-12-19 2015-12-18 癌または転移性疾患の治療または予防における環状アセチルコリンエステラーゼｃ末端ペプチド
PL15816833T PL3233110T3 (pl) 2014-12-19 2015-12-18 Cykliczny peptyd z C-końca acetylocholinoesterazy w leczeniu lub zapobieganiu nowotworowi złośliwemu lub przerzutowi
KR1020177016763A KR102652620B1 (ko) 2014-12-19 2015-12-18 암 또는 전이의 치료 또는 예방에서의 고리형 아세틸콜린에스터라제 c-말단 펩타이드
AU2015365606A AU2015365606B2 (en) 2014-12-19 2015-12-18 Cyclic acetylcholinesterase C-terminal peptide in the treatment or prevention of cancer or metastasis
CN201580069324.3A CN107106643B (zh) 2014-12-19 2015-12-18 在癌症或癌转移的治疗或预防中的环状乙酰胆碱酯酶c端肽
RU2017120535A RU2711161C2 (ru) 2014-12-19 2015-12-18 Циклический с-концевой пептид ацетилхолинэстеразы в лечении или предупреждении рака или метастазирования
EP19172010.1A EP3613428A1 (fr) 2014-12-19 2015-12-18 Peptide cyclique c-terminal de l'acetylcholinesterase pour traiter le cancer et les metastases
MX2017007740A MX2017007740A (es) 2014-12-19 2015-12-18 Peptido c-terminal de acetilcolinesterasa ciclico en el tratamiento o la prevencion de cancer o metastasis.
US16/557,877 US11033609B2 (en) 2014-12-19 2019-08-30 Cyclic acetylcholinesterase C-terminal peptide in the treatment or prevention of cancer or metastasis

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GB1422715.1 2014-12-19
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GB1508480.9 2015-05-18
GB1508480.9A GB2538947A (en) 2014-12-19 2015-05-18 Cancer

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US16/557,877 Continuation US11033609B2 (en) 2014-12-19 2019-08-30 Cyclic acetylcholinesterase C-terminal peptide in the treatment or prevention of cancer or metastasis

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WO2021198686A1 (fr) * 2020-04-01 2021-10-07 Neuro-Bio Ltd Peptides cycliques provenant de l'extrémité c-terminale de l'acétylcholinestérase pour le traitement de troubles cutanés et pour une utilisation cosmétique
WO2021198684A1 (fr) * 2020-04-01 2021-10-07 Neuro-Bio Ltd Polypeptides dérivés de l'extrémité c-terminale de l'acétylcholinestérase destinés à être utilisés dans les affections cutanées

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WO2021198686A1 (fr) * 2020-04-01 2021-10-07 Neuro-Bio Ltd Peptides cycliques provenant de l'extrémité c-terminale de l'acétylcholinestérase pour le traitement de troubles cutanés et pour une utilisation cosmétique
WO2021198684A1 (fr) * 2020-04-01 2021-10-07 Neuro-Bio Ltd Polypeptides dérivés de l'extrémité c-terminale de l'acétylcholinestérase destinés à être utilisés dans les affections cutanées

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