WO2010060497A1 - A nonapeptide with anti-tumour activity - Google Patents
A nonapeptide with anti-tumour activity Download PDFInfo
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- WO2010060497A1 WO2010060497A1 PCT/EP2009/006744 EP2009006744W WO2010060497A1 WO 2010060497 A1 WO2010060497 A1 WO 2010060497A1 EP 2009006744 W EP2009006744 W EP 2009006744W WO 2010060497 A1 WO2010060497 A1 WO 2010060497A1
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- ltx
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- amide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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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
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
Definitions
- the present invention relates to peptides or peptide like molecules and their uses in therapy, in particular as anti-tumour agents and also as adjuvants for vaccines including tumour vaccines.
- Peptides and their derivatives have long been recognised as therapeutically interesting molecules.
- a wide variety of organisms use peptides as part of their host defence mechanism.
- Antimicrobial peptides have been isolated from species as diverse as bacteria and mammals. Generally, these peptides have a net positive charge and a propensity to form amphiphilic ⁇ -helix or ⁇ -sheet structures upon interaction with the outer phospholipid bilayer in bacterial cell membranes. In most cases the detailed molecular mechanisms of the antibiotic action are unknown, although some peptides categorised as class L (lytic) peptides are believed to interact with bacterial cell membranes, forming ion-channels, pores or other structures capable of destabilising the membrane.
- class L (lytic) peptides are believed to interact with bacterial cell membranes, forming ion-channels, pores or other structures capable of destabilising the membrane.
- lytic antimicrobial peptides Some have also been shown to have anti-tumour activity. Eukaryotic cell membranes of tumour cells develop properties similar to the cell membranes of prokaryotes and it has been postulated that this could provide a degree of selectivity of these lytic peptides for tumour cells in vivo. Some anti-tumour activity of peptides having amphiphilic character and a net positive charge has been described in WO 00/12542, WO 01/66147, WO 01/19852 and by Yang et al. in Journal of Peptide Science [2004], 10, pp 37-46 (PMID: 14959890).
- anti-tumour activity requires larger peptides than can be designed for antibacterial uses. Balancing the need for good target cell cytotoxicity and in vivo selectivity presents particular problems in the generation of useful anti-tumour peptides, in part because there remains a high degree of similarity between the cell membrane of tumour and non-transformed cells. Nevertheless, the prevalence of cancer in human and animal populations and its role in mortality means there is a continuing need for new drugs which are effective against tumours. Elimination of a tumour or a reduction in its size or reducing the number of cancer cells circulating in the blood or lymph systems may be beneficial in a variety of ways; reducing pain or discomfort, preventing metastasis, facilitating operative intervention, prolonging life.
- the present inventors During their investigations into the biological activity of peptides, the present inventors have identified a small group of molecules which exhibit particularly good activity against a range of cancer types and good selectivity for cancerous cells over normal cells.
- one molecule (LTX-315) is particularly preferred and therefore according to a first aspect of the present invention there is provided a compound having the formula of SEQ ID NO: 23, or a salt, ester or amide thereof.
- the present invention provides a compound, preferably a peptide, having the following characteristics: a) consisting of 9 amino acids in a linear arrangement; b) of those 9 amino acids, 5 are cationic and 4 have a lipophilic R group; c) at least one of said 9 amino acids is a non-genetically coded amino acid or a modified derivative of a genetically coded amino acid; and optionally d) the lipophilic and cationic residues are arranged such that there are no more than two of either type of residue adjacent to one another; and further optionally e) the molecule comprises two pairs of adjacent cationic amino acids and one or two pairs of adjacent lipophilic residues.
- the cationic amino acids which may be the same or different, are preferably lysine or arginine but may be histidine or any non-genetically coded or modified amino acid carrying a positive charge at pH 7.0.
- Suitable non-genetically coded cationic amino acids and modified cationic amino acids include analogues of lysine, arginine and histidine such as homolysine, ornithine, diaminobutyric acid, diaminopimelic acid, diaminopropionic acid and homoarginine as well as trimethylysine and trimethylomithine, 4-aminopiperidine-4-carboxylic acid, A- amino-i-carbamimidoylpiperidine-4-carboxylic acid and 4-guanidinophenylalanine.
- the lipophilic amino acids i.e. amino acids with a lipophilic R group
- An amino acid with a lipophilic R group is referred to herein as a lipophilic amino acid.
- the lipophilic R group has at least one, preferably two cyclic groups, which may be fused or connected.
- the lipophilic R group may contain hetero atoms such as O, N or S but typically there is no more than one heteroatom, preferably it is nitrogen. This R group will preferably have no more than 2 polar groups, more preferably none or one, most preferably none.
- Tryptophan is a preferred lipophilic amino acid and the molecules preferably comprise 1 to 3, more preferably 2 or 3, most preferably 3 tryptophan residues. Further genetically coded lipophilic amino acids which may be incorporated are phenylalanine and tyrosine.
- one of the lipophilic amino acids is a non-genetically coded amino acid.
- the molecule consists of 3 genetically coded lipophilic amino acids, 5 genetically coded cationic amino acids and 1 non-genetically coded lipophilic amino acid.
- a D amino acid while not strictly genetically coded, is not considered to be a "non-genetically coded amino acid", which should be structurally, not just stereospecifically, different from the 20 genetically coded L amino acids.
- the molecules of the invention may have some or all of the amino acids present in the D form, preferably however all amino acids are in the L form.
- the R group of that amino acid preferably contains no more than 35 non-hydrogen atoms, more preferably no more than 30, most preferably no more than 25 non-hydrogen atoms.
- Preferred non-genetically coded amino acids include: 2-amino-3-(biphenyl-4- yl)propanoic acid (biphenylalanine), 2-amino-3,3-diphenylpropanoic acid
- the compounds of the invention have one of formulae I to V listed below, in which C represents a cationic amino acid as defined above and L represents a lipophilic amino acid as defined above.
- the amino acids being covalently linked, preferably by peptide bonds resulting in a true peptide or by other linkages resulting in a peptidomimetic.
- the free amino or carboxy terminals of these molecules may be modified, the carboxy terminus is preferably modified to remove the negative charge, most preferably the carboxy terminus is amidated, this amide group may be substituted.
- CCLLCCLLC (I) SEQ ID NO: 1
- a peptidomimetic is typically characterised by retaining the polarity, three dimensional size and functionality (bioactivity) of its peptide equivalent but wherein the peptide bonds have been replaced, often by more stable linkages.
- 'stable' is meant more resistant to enzymatic degradation by hydrolytic enzymes.
- the bond which replaces the amide bond conserves many of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, possibility for hydrogen bonding etc. Chapter 14 of "Drug Design and Development", Krogsgaard, Larsen, Liljefors and Madsen (Eds) 1996, Horwood Acad.
- Suitable amide bond surrogates include the following groups: N-alkylation (Schmidt, R. et al., Int. J. Peptide Protein Res., 1995, 46,47), retro-inverse amide (Chorev, M and Goodman, M., Ace. Chem. Res, 1993, 26, 266), thioamide (Sherman D.
- the peptidomimetic compounds of the present invention may have 9 identifiable sub- units which are approximately equivalent in size and function to the 9 cationic and lipophilic amino acids.
- the term 'amino acid' may thus conveniently be used herein to refer to the equivalent sub-units of a peptidomimetic compound.
- peptidomimetics may have groups equivalent to the R groups of amino acids and discussion herein of suitable R groups and of N and C terminal modifying groups applies, mutatis mutandis, to peptidomimetic compounds.
- peptidomimetics may involve the replacement of larger structural moieties with di- or tripeptidomimetic structures and in this case, mimetic moieties involving the peptide bond, such as azole-derived mimetics may be used as dipeptide replacements.
- mimetic moieties involving the peptide bond such as azole-derived mimetics may be used as dipeptide replacements.
- Peptidomimetics and thus peptidomimetic backbones wherein just the amide bonds have been replaced as discussed above are, however, preferred.
- Suitable peptidomimetics include reduced peptides where the amide bond has been reduced to a methylene amine by treatment with a reducing agent e.g. borane or a hydride re?gent such as lithium aluminium-hydride. Such a reduction has the added advantage of increasing the overall cationicity of the molecule.
- a reducing agent e.g. borane or a hydride re?gent such as lithium aluminium-hydride.
- peptidomimetics include peptoids formed, for example, by the stepwise synthesis of amide-functionalised polyglycines.
- peptoids formed, for example, by the stepwise synthesis of amide-functionalised polyglycines.
- Some peptidomimetic backbones will be readily available from their peptide precursors, such as peptides which have been permethylated, suitable methods are described by Ostresh, J. M. et al. in Proc. Natl.
- Preferred peptidomimetic backbones include polyesters, polyamines and derivatives thereof as well as substituted alkanes and alkenes.
- the peptidomimetics will preferably have N and C termini which may be modified as discussed herein.
- ⁇ and ⁇ amino acids as well as ⁇ amino acids are included within the term 'amino acids', as are N-substituted glycines.
- the compounds of the invention include beta peptides and depsipeptides.
- the compounds of the invention incorporate at least one, and preferably one, non-genetically coded amino acid.
- this residue is denoted L 1
- preferred compounds are represented by the following formulae: CCL'LCCLLC (I I ) (SEQ ID NO: 6) CCLLCCLL 1 C (I") (SEQ ID NO: 7) CCLL 1 CCLLC (I 111 ) (SEQ ID NO: 8) LCCLL 1 CCLC (II I ) (SEQ ID NO: 9)
- peptides of formula I and II are especially preferred.
- the peptide is not LTX-302 (SEQ ID NO: 11) (below).
- Ath is 2-amino-3-(anthracen-9-yl)propanoic acid
- Phe(4,4'Bip) is 2-amino-3-[1 ,1':4',1 "-terphenyl-4-yl]propionic acid
- the present invention provides a compound having a formula selected from the group consisting of: SEQ ID NOs: 10 and 12-42, or a salt, ester or amide thereof.
- the molecules are preferably peptides and preferably have a modified, particularly an amidated, C-terminus.
- Amidated peptides may themselves be in salt form and acetate forms are preferred.
- Suitable physiologically acceptable salts are well known in the art and include salts of inorganic or organic acids, and include trifluoracetate as well as acetate and salts formed with HCI.
- the molecules described herein are amphipathic in nature, their 2° structure, which may or may not tend towards the formation of an ⁇ -helix, provides an amphipathic molecule in physiological conditions.
- the compounds of the invention in particular the compounds of formulae I to V, especially the peptides of Table 1 , for use in therapy, particularly for use as an anti-tumour or anticancer agent, the terms are used synonymously herein.
- the compounds exhibit anti-tumour activity; in particular they exert a cytotoxic effect through a direct membrane-affecting mechanism. These molecules are lytic, destabilising or even perforating the cell membrane. This offers a distinct therapeutic advantage over agents which act on or interact with proteinaceous components of the target cells, e.g. cell surface receptors. While mutations may result in new forms of the target proteins leading to chemotherapeutic resistance, it is much less likely that radical changes to the lipid membranes could occur to prevent the cytotoxic effect.
- the compounds of the invention for use in destabilising and/or permeabilising tumour cell membranes.
- destabilising is meant a perturbation of the normal three dimensional lipid bi-layer configuration including but not limited to membrane thinning, increased membrane permeability (typically not involving channels) to water, ions or metabolites etc.
- the invention provides methods of treating tumours, both solid and nonsolid tumours, by administering the various compounds described herein.
- the amount administered should be effective to kill all or a proportion of the target cells or to prevent or reduce their rate of multiplication, or to inhibit metastasis or otherwise to lessen the harmful effect of the tumour on the patient.
- the clinician or patient should observe improvement in one or more of the parameters or symptoms associated with the tumour. Administration may also be prophylactic.
- the patient will typically be a human patient but non-human animals, such as domestic or livestock animals may also be treated. Unlike the majority of agents which have protein targets, the molecules of the present invention can target a wide variety of cancers, as shown in the Examples hereto.
- Preferred cancer targets include lymphomas, leukemias, neuroblastomas and glioblastomas (e.g. from the brain), carcinomas and adenocarcinomas (particularly from the breast, colon, kidney, liver, lung, ovary, pancreas, prostate and skin) and melanomas.
- Breast cancer is an especially preferred target.
- the peptides of the invention may be synthesised in any convenient way. Generally the reactive groups present (for example amino, thiol and/or carboxyl) will be protected during overall synthesis. The final step in the synthesis will thus be the deprotection of a protected derivative of the invention.
- amine protecting groups may include carbobenzyloxy (also designated Z) t-butoxycarbonyl (also designated Boc), 4-methoxy-2,3,6-trimethylbenzene sulphonyl (Mtr) and 9- fluorenylmethoxy-carbonyl (also designated Fmoc). It will be appreciated that when the peptide is built up from the C-terminal end, an amine-protecting group will be present on the ⁇ -amino group of each new residue added and will need to be removed selectively prior to the next coupling step.
- carbobenzyloxy also designated Z
- Boc 4-methoxy-2,3,6-trimethylbenzene sulphonyl
- Fmoc 9- fluorenylmethoxy-carbonyl
- Carboxyl protecting groups which may, for example be employed include readily cleaved ester groups such as benzyl (BzI), p-nitrobenzyl (ONb), or t-butyl (OtBu) groups as well as the coupling groups on solid supports, for example the Rink amide linked to polystyrene.
- Thiol protecting groups include p-methoxybenzyl (Mob), trityl (Trt) and acetamidomethyl (Acm).
- Preferred peptides of the invention may conveniently be prepared using the t-butyloxycarbonyl (Boc) protecting group for the amine side chains of Lys, Om, Dab and Dap as well as for protection of the indole nitrogen of the tryptophan residues.
- Fmoc can be used for protection of the alpha-amino groups.
- peptides containing Arg 2,2,4,6,7-pentamethyldihydrobenzofurane-5-sulfonyl can be used for protection of the guanidine side chain.
- Amine protecting groups such as Boc and carboxyl protecting groups such as tBu may be removed ' simultaneously by acid treatment, for example with trifluoroacetic acid.
- Thiol protecting groups such as Trt may be removed selectively using an oxidation agent such as iodine.
- Formulations comprising one or more compounds of the invention in admixture with a suitable diluent, carrier or excipient constitute a further aspect of the present invention.
- a suitable diluent, carrier or excipient constitute a further aspect of the present invention.
- Such formulations may be for, inter alia, pharmaceutical (including veterinary) purposes.
- Suitable diluents, excipients and carriers are known to the skilled person.
- formulations according to the present invention preferably comprise at least one compound, salt, ester or amide as defined above, together with at least one other therapeutic ingredient.
- the present invention extends to combination products incorporating the compound of the present invention (or a salt, ester or amide thereof) and at least one other therapeutic ingredient.
- compositions according to the invention may be presented, for example, in a form suitable for oral, topical, nasal, parenteral, intravenal, intratumoral, rectal or regional (e.g. isolated limb perfusion) administration.
- Administration is typically by a parenteral route, preferably by injection subcutaneously, intramuscularly, intracapsularly, intraspinaly, intratumouraly or intravenously.
- the active compounds defined herein may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules or sustained release forms. Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms.
- Organ specific carrier systems may also be used.
- Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are then filled into injection vials or ampoules.
- preservation agents such as p-hydroxybenzoates, or stabilizers, such as EDTA.
- stabilizers such as EDTA.
- Preferred formulations are those in which the peptides are dissolved in saline.
- Such formulations being suitable for use in preferred methods of administration, especially local administration, i.e. intratumoural, e.g. by injection or by perfusion/infusion of a preferably isolated (including partial isolation) limb, body region or organ.
- Dosage units containing the active molecules preferably contain 0.1-10 mg, for example 1-5 mg of the anti-tumour agent.
- the pharmaceutical compositions may additionally comprise further active ingredients, including other cytotoxic agents such as other anti-tumour peptides.
- Other active ingredients may include different types of cytokines e.g. IFN- ⁇ , TNF, CSF and growth factors, immunomodulators, chemotherapeutics e.g. cisplatin or antibodies or cancer vaccines.
- the active molecule is present in an amount to achieve a serum level of the bioactive molecule of at least about 5 ⁇ g/ml.
- the serum level need not exceed 500 ⁇ g/ml.
- a preferred serum level is about 100 ⁇ g/ml.
- Such serum levels may be achieved by incorporating the bioactive molecule in a composition to be administered systemically at a dose of from 1 to about 10 mg/kg. In general, the molecule(s) need not be administered at a dose exceeding 100 mg/kg.
- Also provided according to the present invention is a compound, salt, ester or amide according to the present invention in combination with an at least one vaccine.
- Preferred vaccines include, but are not limited to, anti cancer vaccines containing at least one protein and/or peptide with amino acid sequences corresponding to immunogenic sequence(s) from tumour associated antigen(s) (TAA), preferred TAAs, but not limited to, are telomerase, survivin oncogenic p21 ras, abl, gip, gsp, ret, terk, and antivirus vaccines containing at least one protein and/or peptide with amino acid sequence(s) corresponding to immunogenic sequences from viral protein(s).
- TAA tumour associated antigen
- preferred TAAs are telomerase, survivin oncogenic p21 ras, abl, gip, gsp, ret, terk, and antivirus vaccines containing at least one protein and/or peptide with amino acid sequence(s) corresponding to immunogenic sequences from viral protein(s).
- tumour lysate can be used.
- Examples of preferred vaccines include vaccines, peptides, peptide fragments and immunogens taught by WO 92/14756, WO 00/66153 (NO 309798), WO 00/02581 , WO 02/051994, WO 02/070679, WO 02/094312, WO 99/58552, and WO 99/58564.
- Also provided according to the present invention is the use of a compound, salt, ester or amide or combination according to the present invention in the manufacture of a medicament, particularly in the manufacture of a medicament for the treatment of cancer and/or in the manufacture of a vaccine.
- the medicament is for the treatment of multidrug resistant (MDR) tumours.
- MDR multidrug resistant
- a pharmaceutical pack comprising: (i) an at least one vaccine; and (ii) a compound, salt, ester or amide according to the present invention.
- the at least one vaccine and the compound, salt, ester or amide can for administration separately (for example with a time delay of about, at least, or no more than 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 45, 60, 90 or 120 minutes).
- the pharmaceutical pack can of course also comprise instructions for administration.
- a method of treatment of a tumour comprising the step of administering a pharmaceutically effective amount of a compound, salt, ester or amide or combination according to the present invention to a patient in need of same.
- Also provided according to the present invention is a method of vaccination, comprising the step of administering a pharmaceutically effective amount of a compound, salt, ester or amide or combination according to the present invention to a patient.
- Also provided according to the present invention is a method of manufacture of a medicament, comprising admixing a compound having the formula of SEQ ID NO: 23 (LTX-315) or a salt, ester or amide thereof with a pharmaceutically acceptable carrier, diluent or excipient.
- Also provided according to the present invention is a method of manufacture of a medicament, comprising admixing a compound having the formula of SEQ ID NO: 23 (LTX-315) or a salt, ester or amide thereof with tumour cells.
- Figure 1 is a graph showing the percentage of red blood cell death in a series of experiments to test peptide LTX-315 at varying concentrations.
- X-axis shows peptide concentration ( ⁇ g/ml).
- Y- axis shows % cell death;
- Figure 2 shows tumour growth in mice re-inoculated with murine A20 B cell lymphoma cells compared with growth in the control animals from the initial study.
- Diamonds indicate controls from primary studies.
- Solid squares indicate re-inoculated mice;
- Figure 3 shows tumour growth in individual mice re-inoculated with murine A20 B cell lymphoma cells having been initially treated with LTX-315.
- Squares indicate Mouse 1.
- Triangles (base at bottom) indicate Mouse 2.
- Triangles (base at top) indicate Mouse 3.
- Diamonds indicate Mouse 4;
- Figure 4 shows tumour growth in mice re-inoculated with murine CT26WT colon carcinoma cells compared with growth in the control animals.
- Diamonds indicate controls from primary studies.
- Solid squares indicate re-inoculated mice;
- Figure 5 shows tumour growth in individual mice re-inoculated with murine CT26WT colon carcinoma cells having been initially treated with LTX-315.
- Small squares indicate Mouse 1. Small triangles (base at bottom) indicate Mouse 2. Small triangles (base at top) indicate Mouse 3. Small diamonds indicate Mouse 4; Circles indicate Mouse 5. Large squares indicate Mouse 6. Large triangles (base at bottom) indicate Mouse 7. Large triangles (base at top) indicate Mouse 8. Large diamonds indicate Mouse 9;
- Example 2 that LTX-315 is the most potent of the 5 tested compounds in an in vitro cytotoxic activity study against a panel of 10 lymphoma cell lines.
- Example 3 that LTX 315 has a mean EC 50 value greater than 1200 ⁇ g/ml (833 ⁇ M) against human red blood cells.
- Example 4 that the anti-tumour activity of LTX-315 resulted in a complete tumour response in 3 of 7 treated mice for the Group receiving the optimal dose (Group 1) in an investigation into the effect of LTX-315 at different dose levels on a murine A20 B-cell lymphoma in mice.
- Example 5 that four different LTX-315 treatment regimes demonstrated a strong anti tumour effect against murine CT26WT (multidrug resistant) tumours.
- Example 6 that LTX-315 has a broad spectrum of activity against various multidrug resistant cancer cell lines and, significantly, a much weaker cytotoxic effect on normal human cells.
- Example 7 that complete tumour regression following initial treatment of solid murine tumours with LTX-315 resulted in a form of endogenous long- term protection against growth of the same tumours following re- inoculation.
- Example 8 that treatment with LTX-315 may confer long term protection against specific tumours by eliciting an immune response.
- Example 9 that an anti A20 cell immune response have been induced by the injection of the cocktail of LTX-315 and lysed A20 cells.
- Test substances LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329 (see Table 1) provided in powder form.
- Triton X-1Q0 was used as positive control, supplied by Oncodes/gn (Dijon, France) from Sigma (Saint Quentin Fallavier, France).
- Triton X-100 was obtained by dilution using culture medium.
- Cancer cell lines and culture media were purchased and provided by Oncodesign.
- Tumor cells were grown as adherent monolayers or as suspensions at 37 0 C in a humidified atmosphere (5% CO 2 , 95% air).
- the culture medium was RPMI 1640 containing 2 mM L-glutamine (Lonza, Belgium) and supplemented with 10% fetal bovine serum (FBS, Lonza).
- FBS fetal bovine serum
- the adherent cells were detached from the culture flask by a 5-minute treatment with trypsin-versene (Lonza), diluted in Hanks ' medium without calcium or magnesium (Lonza) and neutralized by addition of complete culture medium. Cells were counted in a hemocytometer and their viability was assessed by 0.25% trypan blue exclusion.
- Mycoplasma detection was performed using the MycoAlert (RTM) Mycoplasma Detection Kit (Lonza) in accordance with the manufacturer's instructions. All tested cells were found to be negative for mycoplasma contamination.
- Tumor cells were plated in 96-well flat-bottom microtitration plates (Nunc, Dutscher, Brumath, France) and incubated at 37°C for 24 hours before treatment in 190 ⁇ l of drug-free culture medium supplemented or not with 10% FBS for adherent or suspension growing cell lines, respectively.
- the adherent cell lines were washed once with 200 ⁇ l FBS-free culture medium before treatment. Tumor cells were incubated for 4 hours with 10 concentrations of compounds in % dilution step with a top dose of 400 ⁇ M (range 4x10 "4 to 4x10 '10 M) 1 with 1 % (final concentration) Triton X-100 as positive control and FBS-free culture medium as negative control. The cells (190 ⁇ l) were incubated in a 200 ⁇ l final volume of FBS-free culture medium containing test substances at 37°C under 5% CO 2 .
- MTS novel tetrazolium compound
- PMS phenazine methosulfate
- IC 50 determination o The dose response inhibition of proliferation (IC) was expressed as follows:
- OD values are the mean of 4 experimental measurements.
- o IC 50 drug concentration to obtain a 50% inhibition of cell proliferation.
- the dose-response curves were plotted using XLFit 3 (IDBS, United Kingdom).
- the IC 50 determination values were calculated using the XLFit 3 software from semi-log curves. Individual IC 50 determination values as well as mean and SD values were generated.
- IC 50compoundA Sensitive cell line
- Resistance index was calculated for each compound for each couple of sensitive and resistant cell lines. Individual resistance index was calculated when IC 50 values of both sensitive and corresponding resistant cell lines were determined within same experiment. In addition, resistance index was also calculated ratio of mean IC 50 values obtained during three independent experiments.
- Mean IC 50 value for LTX-302 compound obtained on the 37 tumor cell lines was 12.05 ⁇ 4.27 ⁇ M with a median value of 11.70 ⁇ M.
- Mean IC 50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- LTX-302 compound Activity of LTX-302 compound seemed to be slightly decreased by acquired resistance towards doxorubicin as exhibited by the Rl values of both HL-60/ADR and MCF-7/mdr cell lines (1.31 and 1.23 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-302 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a Rl value of 0.33 for IGROV-1/CDDP cell line.
- LTX-313 All thirty seven (37) human tumor cell lines tested were sensitive to LTX-313 compound with IC 50 values ranging from 4.01 ⁇ 0.39 ⁇ M to 18.49 ⁇ 4.86 ⁇ M for RPMI 8226 and U- 87 MG cell lines, respectively.
- Mean IC 50 value for LTX-313 compound obtained on the 37 tumor cell lines was 9.60 ⁇ 3.73 ⁇ M with a median value of 8.83 ⁇ M.
- Mean IC 50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- Activity of LTX-313 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the Rl values of CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines (0.76, 1.16 and 1.24 for CCRF-CEM/VLB, HL-60/ADR and MCF- 7/mdr cell lines, respectively). On the contrary, activity of LTX-313 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a Rl value of 0.49 for IGROV-1/CDDP cell line.
- Mean IC 50 value for LTX-315 compound obtained on the 37 tumor cell lines was 3.63 ⁇ 1.45 ⁇ M with a median value of 3.27 ⁇ M.
- Mean IC 50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- LTX-315 compound Activity of LTX-315 compound seemed to be slightly decreased by acquired resistance towards doxorubicin as exhibited by the Rl values of HL-60/ADR and MCF-7/mdr cell lines (1.45 and 1.12 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-315 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a Rl value of 0.50 for IGROV-1/CDDP cell line.
- All thirty seven (37) human tumor cell lines tested were sensitive to LTX-320 compound with IC 50 values ranging from 3.46 ⁇ 0.22 ⁇ M to 16.64 ⁇ 3.15 ⁇ M for T-47D and Hep G2 cell lines, respectively.
- Mean IC 50 value for LTX-320 compound obtained on the 37 tumor cell lines was 7.58 ⁇ 2.79 ⁇ M with a median value of 6.92 ⁇ M.
- Mean IC 50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- LTX-320 compound Activity of LTX-320 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the Rl values of HL-60/ADR and MCF-7/mdr cell lines (0.90 and 1.19 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-320 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a Rl value of 0.49 for IGROV-1/CDDP cell line.
- Mean IC 50 value for LTX-329 compound obtained on the 37 tumor cell lines was 8.17 ⁇ 3.20 ⁇ M wi+h a median value of 7.89 ⁇ M.
- Mean IC 50 value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.
- LTX-329 compound Activity of LTX-329 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the Rl values of CCRF-CEM/VLB, HL-60/ADR and
- MCF-7/mdr cell lines (0.76, 0.80 and 1.07 for CCRF-CEM/VLB, HL-60/ADR and MCF- 7/mdr cell lines, respectively).
- activity of LTX-329 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a Rl value of 0.46 for IGROV-1/CDDP cell line.
- T-47D breast cancer cell line is the most sensitive cell line whatever the LTX compound tested.
- Hematological cancer cell lines are the most sensitive histological type for all five compounds tested, liver and ovarian cancer cell lines being within the most resistant cell lines.
- LTX-315 compound is the most potent compound from the five compounds tested.
- LTX-315 compound is the most potent compound tested with IC 50 values between 1 and 5 micromolar on all 37 human cancer cell lines tested.
- STUDY AIM To determine the concentrations of five novel compounds to obtain a 50% inhibition of proliferation (IC 50 ) against a panel of 10 lymphoma cell lines.
- test substances 2.1.1. Test substances
- Triton X-100 was used as positive control and supplied by Oncodesign (Dijon, France) from Sigma (Saint Quentin Fallavier, France).
- Triton X-100 was obtained by dilution using culture medium.
- Cancer cell lines and culture media were purchased and provided by Oncodesign.
- Mycoplasma detection was performed using the MycoAlert (RTM) Mycoplasma Detection Kit (Lonza) in accordance with the manufacturer's instructions. All tested cells were found to be negative for mycoplasma contamination.
- Tumor cells were plated in 96-well flat-bottom microtitration plates (Nunc, Dutscher, Brumath, France) and incubated at 37°C for 24 hours before treatment in 190 ⁇ l of drug-free and FBS-free culture medium.
- Tumor cells were incubated for 4 hours with 10 concentrations of compounds in % dilution step with a top dose of 400 ⁇ M (range 4x10 "4 to 4x10 "10 M), with 1 % (final concentration) Triton X-100 as positive control and FBS-free culture medium as negative control.
- the cells (190 ⁇ l) were incubated in a 200 ⁇ l final volume of FBS-free culture medium containing test substances at 37°C under 5% CO 2 .
- Dilutions of tested compound as well as distribution to plates containing cells were performed using a Sciclone ALH 3000 liquid handling system (Caliper Life Sciences S. A.). According to automate use, a single range of concentrations was tested whatever the cell lines to be tested. The range was not adapted for each cell line.
- MTS assay The in vitro cytotoxic activity of the test substance was revealed by a MTS assay (Baltorp et al.) using a novel tetrazolium compound (MTS, 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron coupling reagent named PFvIS (phenazine methosulfate).
- MTS novel tetrazolium compound
- PFvIS phenazine methosulfate
- Mean IC 50 value for LTX-302 compound obtained on 10 sensitive cell lines was 8.11 ⁇ 2.44 ⁇ M with a median value of 7.53 ⁇ M.
- Mean IC 50 value for LTX-313 compound obtained on 10 sensitive cell lines was 7.05 ⁇ 3.91 ⁇ M with a median value of 5.89 ⁇ M.
- LTX-315 All ten (10) human lymphoma cell lines tested were sensitive to LTX-315 compound with IC 50 values ranging from 1.15 ⁇ 0.42 ⁇ M to 4.93 ⁇ 1.03 ⁇ M for U-937 and Raji cell lines, respectively.
- Mean IC 50 value for LTX-320 compound obtained on 10 sensitive cell lines was 5.03 ⁇ 2.82 ⁇ M with a median value of 4.84 ⁇ M.
- LTX-329 All ten (10) human lymphoma cell lines tested were sensitive to LTX-329 compound with IC 50 values ranging from 2.46 ⁇ NA ⁇ M to 8.70 ⁇ 1.70 ⁇ M for Hs 445 and Raji cell lines, respectively.
- Mean IC 50 value for LTX-329 compound obtained on 10 sensitive cell lines was 5.76 ⁇ 2.27 ⁇ M with a median value of 5.72 ⁇ M.
- KARPAS-299 and Raji cell lines are the most resistant cell lines whatever the LTX compound tested.
- Hs 445, Ramos and U-937 cell lines are the most sensitive cell lines whatever the LTX compound tested.
- LTX-315 compound is the most potent compound from the five compounds tested.
- LTX-302, LTX-313, LTX-315, LTX-320 and LTX- 329) exhibited cytolytic activity against the 10 human lymphoma cell lines tested with IC 50 values in micromolar range.
- > LTX-315 compound is the most potent compound tested with IC 50 values between 1 and 5 micromolar on all 10 human lymphoma cell lines tested.
- Freshly collected human blood was centrifuged at 1500 rpm for 10 minutes in order to isolate the red blood cells.
- the red blood cells (RBC) were washed three times with PBS [35 mM phosphate buffer with 150 mM NaCI, pH 7.4] by centrifugation at 1500 rpm for 10 minutes, and adjusted to 10% haematocrit with PBS.
- LTX-315 solutions were added to give a final concentration range of the peptide from 1200 ⁇ g/ml to 1 ⁇ g/ml and an RBC concentration of 1%.
- the resulting suspension was incubated with agitation for one hour at 37 0 C.
- the suspension was centrifuged at 4000 rpm for 5 minutes, and the released haemoglobin were monitored by measuring the absorbance of the supernatant at 405 nm.
- PBS was used as negative control and assumed to cause no haemolysis.
- 0.1% Triton was used as positive control and assumed to cause complete haemolysis.
- reaction mixtures Components of reaction mixtures: LTX-315, 10 % Triton X-100, PBS and RBC (10% haematocrit)
- % Haemolysis [(A 405 LTX-315 - A 405 PBS)/( A 405 0,1 % Triton X-100 - A 405 PBS)] x 100
- LTX-315 concentration corresponding to 50% haemolysis (EC 50 ) was determined from a dose-response curve.
- the aim of the study was to investigate the effect of LTX-315 at different dose levels on a murine A20 B-cell lymphoma in mice.
- the administration took place by intratumoural injection of LTX-315 dissolved in sterile saline.
- mice Female mice were inoculated subcutaneously in the abdomen with 5 million murine A20 cells (ATCC, LGC Promochem AB, Middlesex, England) in a volume of 50 ⁇ l. The mice were divided into four groups (see Table 5 below for details). The intratumoural treatment was initiated when the tumours had reached the desired size of approximately 5 mm in diameter (minimum of 20 mm 2 ).
- LTX-315 Three dose levels of LTX-315, 1 mg (Group 1), 0.5 mg (Group 2) and 0.25 mg (Group 3) per injection, were investigated. The volume was 50 ⁇ l for all injections. LTX-315 was dissolved in sterile 0.9% NaCI water solution. This vehicle was used as control (Group 4). All four groups received three injections.
- mice were monitored during the study by measuring the tumours and weighing the animals regularly. The mice were followed until the maximum tumour burden of 125 mm 2 was reached, or until serious adverse events occurred (i.e. wound formation upon repeated treatments during the follow up period), then the mice were sacrificed. A calliper was used for tumour size measurements and weighing and physical examination were used as health control.
- the degree of tumour response in the different treatment groups is summarised in Table 7 below.
- LTX-315 dissolved in sterile saline (0.9% NaCI in sterile water).
- mice Each of a total of 40 female mice was inoculated with five million murine CT26WT cells (ATCC, LGC Promochem AB, Boras, Sweden) subcutaneously on the abdomen surface in a volume of 50 ⁇ l.
- the mice were divided into five groups, 8 mice in each group.
- the tumours reached the desired size of 20 mm 2 the treatment by intra tumoural injection was initiated.
- Group one was treated solely on day 1 , Group two on day 1 and 2, Group three on day 1 and 3 and Group four on day 1 , 2 and 3. All daily treatments were one single injection of 1.0 mg LTX-315 dissolved in 50 ⁇ l (20 mg/ml).
- Group five was treated with the 50 ⁇ l of vehicle for LTX-315 (Group 5).
- mice were monitored during the study by measuring the tumours (digital calliper) and weighing the animals regularly. The mice were followed until the maximum tumour burden of 125 mm 2 was reached, or until serious adverse events occurred (i.e. wound formation due to repeated injections), then the mice were sacrificed. Weighing and physical examination were used as health controls. Animals: Specific pathogen-free female Balb/c mice, 6-8 weeks old, supplied form
- the treatment was started when the tumours had reached the desired size of a minimum of 20 mm 2 and animals were sacrificed when the tumours reached the maximum tumour burden of 125 mm 2 . End of study was defined as day 17 when six out of eight control animals (Group 5) were sacrificed.
- necrosis at the tumour site was seen in all treatment groups. Generally, animals in Group 2, 3 and 4 showed more necrosis, wound and crust formation than the animals in Group 1 that were given only one injection of LTX-315. Group 4 animals, which were given three injections, showed the most necrosis, wound and crust formation. The difference in necrosis between Group 1 and Group 4 was quite large but the animals given the highest number of treatments seemed to cope well. No toxic or other adverse effects besides local necrotic tissue and wound formation were observed in either of the treated groups of animals.
- the amount of necrosis, wound and crust formation was proportional to the number of LTX-315 treatments given.
- Example 6 LTX-315 activity against sensitive and multidrug-resistant cancer cells and normal human cells
- Example 8 Immunological effects of LTX-315 in a murine A20 B-cell lymphoma model. An in vivo adoptive spleen cell transfer pilot study.
- Naive receiver mice were irradiated and divided into 2 groups. Group 1 received isolated splenocytes from cured mice, whereas group 2 received isolated splenocytes from naive mice. Freshly prepared cells were injected (20 x 106 per 100 ⁇ l) via the tail vein. Twenty four hours later receiver mice were inoculated with 5 million murine A20 B-cell lymphoma cells on the abdominal surface as described above. Tumour size and body weight were monitored until the maximum tumour burden of ⁇ 125mm 2 was reached, or a serious adverse events occurred (i.e. wound formation due to tumour tissue necrosis) at which point mice were sacrificed.
- the data provides evidence for an adaptive immune response in the animals that received splenocytes from animals that previously showed complete regression of A20-B lymphoma tumours following treatment with LTX-315.
- This data suggests that treatment with LTX-315 may confer long term protection against specific tumours by eliciting an immune response.
- the objective of the study was to investigate the anti-cancer effect of prophylactic vaccination with A20 lymphoma cells lysed by 10 mg/ml LTX-315: (i) alone; and
- LTX-315 LTX-315 dissolved in growth media containing A20 lymphoma cells.
- the cell-LTX-315 "cocktail" was left for 30 min prior to injection in order to assure complete lysis of the cancer cells.
- Group 1 mice were injected subcutaneously on the abdomen surface with 50 ⁇ l of a "cocktail" of ten million murine A20 cells (ATCC, LGC Promochem AB, Boras, Sweden) and 10 mg/ml LTX-315 (“A20 lysate”).
- Group 2 (“vaccine + adjuvant”) mice were treated as per Group 1 , but in addition were given 25 ⁇ l of 20 mg/ml LTX- 315 subcutaneously at the site of vaccination 5 minutes prior to the A20 lysate injection.
- Group 3 (“control”) mice received no treatment. Six weeks after the treatment, all mice were inoculated with 5 million viable A20 B-cell lymphoma cells subcutaneously on the abdomen surface in a volume of 50 ⁇ l.
- mice were monitored during the study by measuring the tumour size and weighing the animals regularly. The mice were followed until the maximum tumour burden of ⁇ 130 mm 2 was reached, at which point the mice were sacrificed.
- Test substance Murine A20 cells lysed by LTX-315 (Lot 1013687), and LTX-315 (Lot 1013687).
- Test substance preparation 10x10 6 A20 cells were added to a 50 ⁇ l 10mg/ml LTX- 315/vehicle ("A20 lysate"). The test substance was ready for use 30 minutes after mixing. LTX-315 alone was dissolved in 0.9 % NaCI in sterile H 2 O
- the results show that the tumours developed more slowly in both LTX-315/A20-lysate treatment Groups as compared to the control Group.
- the median survival of Group 1 was 28 days, 33 days for Group 2, and 25 days for the control group (Group 3). Increase in median survival was 12% for Group 1 and 35% for Group 2 as compared to the control group (Group 3).
- the data indicate a prolonged survival of the treated groups compared to the untreated control group.
- 50% of the animals in Group 2 were still alive while 37.5% of the animals in Group 1 were still alive.
- End of study was defined as day 60.
- a total of 3 of the 16 treated animals had a complete regression of an initially developing tumour and were tumour free.
- 25% of animals from Group 1 were observed to be tumour free.
- LTX-315 may have a dual use by lysing the tumour cells and inducing release of danger signals from normal cells at the injection site.
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WO2016091490A1 (en) * | 2014-12-11 | 2016-06-16 | Lytix Biopharma As | Chemotherapeutic combinations of cationic antimicrobial peptides and chemotherapeutics |
WO2016091487A1 (en) | 2014-12-11 | 2016-06-16 | Lytix Biopharma As | Immune checkpoint inhibitor combinations |
WO2017134175A1 (en) | 2016-02-02 | 2017-08-10 | Lytix Biopharma As | Methods of generating populations of tumour-infiltrating t cells |
US11123398B2 (en) | 2014-12-11 | 2021-09-21 | Lytix Biopharma As | Chemotherapeutic combinations of cationic antimicrobial peptides and chemotherapeutics |
WO2024133580A1 (en) | 2022-12-20 | 2024-06-27 | Lytix Biopharma As | Medical products containing an aqueous formulation of a peptide |
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GB0821616D0 (en) * | 2008-11-26 | 2008-12-31 | Lytix Biopharma As | Compounds |
EP3951006A4 (en) | 2019-04-04 | 2023-01-11 | Adeka Corporation | Raw material for forming thin film, method for producing thin film, and scandium compound |
CN111150833B (en) * | 2020-03-16 | 2022-11-08 | 中国科学院微生物研究所 | Application of LTX-315 in preparation of product for inhibiting coronavirus |
CN113549129B (en) * | 2021-06-10 | 2024-03-26 | 青岛大学 | D-configuration anti-tumor peptide and preparation method and application thereof |
WO2023197129A1 (en) * | 2022-04-12 | 2023-10-19 | Rise Biopharmaceuticals Inc. | Oncolytic peptide and use thereof |
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WO2015118028A1 (en) * | 2014-02-04 | 2015-08-13 | University of Tromsø | Anti-lymphoma peptides |
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AU2015359884B2 (en) * | 2014-12-11 | 2021-10-28 | Lytix Biopharma As | Chemotherapeutic combinations of cationic antimicrobial peptides and chemotherapeutics |
WO2017134175A1 (en) | 2016-02-02 | 2017-08-10 | Lytix Biopharma As | Methods of generating populations of tumour-infiltrating t cells |
WO2024133580A1 (en) | 2022-12-20 | 2024-06-27 | Lytix Biopharma As | Medical products containing an aqueous formulation of a peptide |
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CN102232082B (en) | 2015-05-27 |
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ES2733129T3 (en) | 2019-11-27 |
US20110318364A1 (en) | 2011-12-29 |
JP2012509914A (en) | 2012-04-26 |
KR20140084355A (en) | 2014-07-04 |
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CA2743986C (en) | 2016-10-11 |
AU2009319454B2 (en) | 2013-01-17 |
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US20160176923A1 (en) | 2016-06-23 |
US9241968B2 (en) | 2016-01-26 |
CA2743986A1 (en) | 2010-06-03 |
JP2016138112A (en) | 2016-08-04 |
EP2402364A2 (en) | 2012-01-04 |
TR201909522T4 (en) | 2019-07-22 |
RU2503685C2 (en) | 2014-01-10 |
BRPI0922832A2 (en) | 2018-11-06 |
GB0821616D0 (en) | 2008-12-31 |
KR101445910B1 (en) | 2014-10-15 |
EP2361258A1 (en) | 2011-08-31 |
CN102232082A (en) | 2011-11-02 |
SG171823A1 (en) | 2011-07-28 |
AU2009319454A1 (en) | 2011-07-21 |
KR20110112303A (en) | 2011-10-12 |
BRPI0922832B1 (en) | 2021-08-24 |
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