WO2016091490A1 - Chemotherapeutic combinations of cationic antimicrobial peptides and chemotherapeutics - Google Patents
Chemotherapeutic combinations of cationic antimicrobial peptides and chemotherapeutics Download PDFInfo
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- WO2016091490A1 WO2016091490A1 PCT/EP2015/075754 EP2015075754W WO2016091490A1 WO 2016091490 A1 WO2016091490 A1 WO 2016091490A1 EP 2015075754 W EP2015075754 W EP 2015075754W WO 2016091490 A1 WO2016091490 A1 WO 2016091490A1
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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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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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
Definitions
- the present invention relates to peptides or peptide like molecules and particularly to combined preparations of such peptides with a further agent, and their uses in therapy, in particular as anti-tumour agents.
- 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.
- Genetic and epigenetic alterations that are characteristic of cancers result in antigens that the immune system can recognise and use to differentiate between tumour cells and their healthy equivalents. In principle, this means that the immune system could be a powerful weapon in controlling tumours. However, the reality is that the immune system usually does not provide a strong response to tumour cells. It is of great therapeutic interest to manipulate and therefore harness the immune system in the fight against cancer (Mellman et al. Nature 201 1 , vol. 480, 480-489).
- tumour-specific antigens typically combined with an adjuvant (a substance which is known to cause or enhance an immune response) to the subject.
- adjuvant a substance which is known to cause or enhance an immune response
- cancer vaccines are generally intended to generate or enhance an immune response against an existing cancer, rather than to prevent disease.
- a cancer or tumour vaccine may not require administration of a tumour antigen, the administered product may utilise tumour antigens already present in the body as a result of tumour development and serve to modify the immune response to the existing tumour associated antigens (TAAs).
- TAAs tumour associated antigens
- T cells have a key role in the immune response, which is initiated through antigen recognition by the T cell receptor (TCR), and they coordinate a balance between co-stimulatory and inhibitory signals known as immune checkpoints (Pardoll, Nature 2012, vol. 12, 252-264). Inhibitory signals suppress the immune system which is important for maintenance of self-tolerance and to protect tissues from damage when the immune system is responding to pathogenic infection. However, immune suppression reduces what could otherwise be a helpful response by the body to the development of tumours.
- cytokines other stimulatory molecules such as CpG (stimulating dendritic cells), Toll-like receptor ligands and other molecular adjuvants enhance the immune response.
- Co-stimulatory interactions involving T cells directly can be enhanced using agonistic antibodies to receptors including OX40, CD28, CD27 and CD137. These are all "push"-type approaches to cancer immunotherapy.
- Complementary 'pull' therapies may block or deplete inhibitory cells or molecules and include the use of antagonistic antibodies against what are known as immune checkpoints.
- Immune checkpoints include CTLA-4 and PD-1 and antibodies against these are known in the art; ipilimumab was the first FDA-approved anti-immune checkpoint antibody licensed for the treatment of metastatic melanoma and this blocks cytotoxic T-lymphocyte antigen 4 (CTLA-4) (Naidoo et al. British Journal of Cancer (2014) 1 1 1 , 2214-2219). There are other agents which would be considered classic chemotherapeutics which can reduce immune suppression at sub-cytotoxic doses, these include cyclophosphamide and doxorubicin.
- the present inventors have established that some peptides known to lyse tumour cells through disturbing and permeabilizing the cell membrane, are also highly effective at attacking organelles such as mitochondria and lysosomes and can cause lysis thereof. This may be achieved at low concentrations which do not cause direct lysis of the cell membranes, although loss of cell membrane integrity is seen eventually even on administration of low doses. At higher doses, these molecules can cause lysis of the cell membrane and then of the membranes of organelles.
- the peptides of interest are a sub-set of the group of peptides commonly known as Cationic antimicrobial peptides (CAPs). These are positively charged amphipathic peptides and peptides of this type are found in many species and form part of the innate immune system.
- CAPs Cationic antimicrobial peptides
- the CAP Lactoferricin (LfcinB) is a 25 amino acid peptide which has been shown to have an effect on mitochondria (Eliasen et al. Int. J. Cancer (2006) 1 19, 493-450). It has now surprisingly been found that the much smaller peptide LTX-315, a 9 amino acid peptide (of the type described in WO 2010/060497), also targets the mitochondria. This was unexpected because this small peptide is much more fast acting (causing cell death after 30 minutes of exposure) compared to LfcinB (which is most effective after 24 hours of exposure) and the small peptide acts against a broader spectrum of cell types, which suggests a direct effect on the plasma membrane.
- DAMPs Denssion-associated molecular pattern molecules
- ATP ATP
- Cytochrome C mitochondrial CpG DNA sequences
- mitochondrial formyl peptides mitochondrial formyl peptides
- cathepsins from lysosomes
- HMGB1 from the nucleus
- Lysis of organelles can also result in release of additional tumour-specific antigens (TAAs).
- TAAs tumour-specific antigens
- the present invention provides:
- a compound preferably a peptide, having the following characteristics:
- 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 (e.g. a modified derivative of a genetically coded amino acid); and optionally
- 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
- the molecule comprises two pairs of adjacent cationic amino acids and one or two pairs of adjacent lipophilic residues;
- chemotherapeutic agent for use in the treatment of a tumour by combined, sequential or separate administration with a cytotoxic chemotherapeutic agent that inhibits immune tolerance, wherein the chemotherapeutic agent is administered at a sub-cytotoxic dose.
- the combination therapy proposed herein may, in certain advantageous embodiments, provide a synergistic effect.
- amino acid containing molecules defined above are conveniently referred to herein as the "peptidic compound of the invention", which expression includes all of the peptides and peptidomimetics disclosed herein.
- 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 amino acid carrying a positive charge at pH 7.0.
- Suitable non-genetically coded 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 trimethylornithine, 4-aminopiperidine-4-carboxylic acid, 4- amino-1 -carbamimidoylpiperidine-4-carboxylic acid and 4-guanidinophenylalanine.
- Non-genetically coded amino acids include modified derivatives of genetically coded amino acids and naturally occurring amino acids other than the 20 standard amino acids of the genetic code.
- 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 lipophilic amino acids i.e. amino acids with a lipophilic R group
- the lipophilic amino acids which may be the same or different, all possess an R group with at least 7, preferably at least 8 or 9, more preferably at least 10 non-hydrogen atoms.
- 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.
- 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 (diphenylalanine), 2-amino-3-(anthracen-9-yl)propanoic acid, 2-amino-3-(naphthalen-2- yl)propanoic acid, 2-amino-3-(naphthalen-1 -yl)propanoic acid, 2-amino-3-[1 ,1 ':4',1 "- terphenyl-4-yl]-propionic acid, 2-amino-3-(2,5,7-tri-ie f-butyl-1 H-indol-3-yl)propanoic acid, 2-amino-3-[1 ,1 ':3',1 "-terphenyl-4-yl]-propionic acid, 2-amino-3-
- the peptidic 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, peptides being preferred.
- 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.
- 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., Acc. Chem.
- the peptidomimetic compounds 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 reagent 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 reagent such as lithium aluminium-hydride.
- peptidomimetics include 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. Acad. Sci. USA (1994) 91 , 1 1 138-1 1 142. Strongly basic conditions will favour N- methylation over O-methylation and result in methylation of some or all of the nitrogen atoms in the peptide bonds and the N-terminal nitrogen.
- 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 a amino acids are included within the term 'amino acids', as are N-substituted glycines.
- the peptidic compounds of the invention include beta peptides and depsipeptides.
- the peptidic compounds of the invention incorporate at least one, and preferably one, non-genetically coded amino acid.
- L' preferred compounds are represented by the following formulae:
- Particularly preferred are compounds (preferably peptides) of formula I and II, and of these, compounds (preferably peptides) of formula I" are especially preferred.
- LTX-307 16 K-K-W-Dip-K-K-W-W-K-NH 2
- LTX-308 17 k-k-W-Dip-k-k-W-W-k-NH 2
- Ath is 2-amino-3-(anthracen-9-yl)propanoic acid
- 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 peptidic compounds described herein are amphipathic in nature, their 2° structure, which may or may not tend towards the formation of an a-helix, provides an amphipathic molecule in physiological conditions.
- the combination therapies defined herein are for the treatment of tumours, in particular solid tumours and thus for the treatment of cancer.
- the peptidic compounds of the invention destabilise and/or permeabilise the membranes of tumour cell organelles, e.g. mitochondria, the nucleus or lysomome, in particular the mitochondria.
- tumour cell organelles e.g. mitochondria, the nucleus or lysomome, in particular the mitochondria.
- cytotoxic chemotherapeutic agent is an agent that which, at a maximum tolerated dose (MTD), is effective at killing cells that divide rapidly and thus may be used as an anticancer therapy.
- MTD maximum tolerated dose
- These drugs typically act by interfering with DNA synthesis or producing chemical damage to DNA. Such agents will not be effective in all patients but are prescribed for this ability to kill rapidly dividing cancer cells. Cytotoxicity and chemotherapy are terms that are well known in the field of oncology.
- cytotoxic agents that are discriminately cytotoxic (agents that are able to kill cancer cells specifically) such as monoclonal antibodies.
- the skilled person is aware of the cytotoxic chemotherapeutic agents used in the art and is also able to readily determine whether a putative agent has cytotoxic chemotherapeutic properties.
- the skilled person may for example carry out an anti- proliferative assay, such as an MTS assay described in Example 1 , against rapidly dividing cells.
- these agents are not used for their ability to kill rapidly dividing tumour cells and the dose employed is termed "sub-cytotoxic", i.e. less than cytotoxic.
- Some cytotoxic chemotherapeutic agents inhibit immune tolerance (i.e. reduce the mechanisms of immune suppression). As described in Zheng et al (2015) Cell.
- Immunol., 294, pp 54-9 specific immune cells, effector molecules and signal pathways can act in a process termed immune tolerance, resulting in tumour growth rather than an inhibition in tumour growth.
- Regulatory T cells Teg
- MDSC myeloid-derived suppressor cells
- TAM tumour-associated macrophages
- Cytotoxic chemotherapeutic agents have been reported to disrupt this immune tolerance leading to an immune response against the tumour cells.
- Agents that have been shown to stimulate immune activation include doxorubicin (Alizadeh et al. Cancer Res. (2014) 74(1 ); 104-1 18), paclitaxel (Michels et al. J. Immunotoxicol. (2012) 9(3); 292-300), cyclophosphamide (Heylmann et al. PLOS one, (2013) Vol. 8, Issue 12, e83384), gemcitabine and 5-fluorouracil; the
- chemotherapeutic agent of the invention is preferably one listed here, more preferably cyclophosphamide or doxorubicin.
- chemotherapeutic agents at sub-cytotoxic concentrations can effectively facilitate immune activation with little toxicity and with few of the challenging side effects of conventional chemotherapy.
- the agents can reduce immune tolerance and thus enhance anti-cancer immunity, for example by depleting highly proliferative immunosuppressive cells while sparing less proliferative lymphocytes with a protective function.
- Immunosuppressive cells such as regulatory T cells and MDSCs are targets for these agents, as are cytokines, which may all serve to prevent cytotoxic T lymphocytes and natural killer cells from killing tumour cells.
- a cytotoxic chemotherapeutic agent inhibited immune tolerance e.g. through monitoring for immune tolerance changes such as dendritic cell maturation, an increase in the expression of auxiliary receptors in T cells and a decrease in immunosuppressive cells after administration of the agent in vivo, e.g. in an animal model or clinical study.
- Such monitoring may be carried out, for example, either using immunhistochemistry on isolated tumor tissue or through using flowcytometry on single cells suspensions isolated from tumor tissue.
- the degree of inhibition will be therapeutically significant and may be at least 10% or 20% preferably at least 30% or 40%, more preferably at least 50% of 60% as compared to a suitable control.
- a "sub-cytotoxic dose" of the chemotherapeutic agent is one that is less than the dose usually prescribed to directly kill cancer cells, e.g. less than the MTD for standard chemotherapy.
- these cytotoxic doses are commonly known in the art and may be set out in any regulatory approvals.
- a common dose of cyclophosphamide used in anti-cancer therapy is 40 to 50 mg/kg divided over 2 to 5 days, which equates to 8 mg/kg/day at the lower end of the dosage range. Therefore, a non-cytotoxic dose of cyclophosphamide would be less than 8 mg/kg/day in humans, typically less than 6 or 4 mg/kg/day.
- Preferred sub-cytotoxic doses are between 1 % (e.g. 5%) and 90% of the dose typically used for direct anti-cancer therapy.
- the dose may be, for example less than 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the standard cytotoxic dose.
- Preferably the dose is between 5% and 50%, more preferably between 10% and 40%.
- Suitable sub-cytotoxic doses often referred to simply as 'low dose'; are known and described in the art, e.g. in the art cited herein.
- the non-cytotoxic dose may be still be toxic to non-cancerous rapidly-dividing cells, and such a dose may still cause adverse drug reactions (ADRs) in the subject.
- ADRs adverse drug reactions
- a non-cytotoxic dose may still adversely affect haematopoiesis in a subject.
- a sub- cytotoxic dose may be determined in vivo through gradually increasing the titration of a dose and regular monitoring for effects on cytotoxicity.
- the sub-cytotoxic dose may be administered as a single dose or as multiple doses.
- the non-cytotoxic dose may be administered in blocks, such as once or twice a day for 3-7 days, followed by a break of 3-7 days, followed by a further course of administration. Even if multiple doses are administered, the impact on the body and the classification of the dosage regimen which would be understood by the clinician is still "sub-cytotoxic".
- the uses and methods of the invention employ a cytotoxic chemotherapeutic agent as defined herein administered according to a sub-cytotoxic dosage regimen.
- the sub-cytotoxic dose is a metronomic dose (metronomic dosing is typically intended to be anti-angiogenic).
- a metronomic dose is a non-cytotoxic dose
- the invention provides methods of treating a tumour and a method of treating tumour cells.
- the combination therapy should be effective to kill all or a proportion of the target tumour 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 and this is encompassed by the term "treatment".
- the patient will typically be a human patient but non-human animals, such as domestic or livestock animals may also be treated.
- Cancer targets include sarcomas, lymphomas, leukemias, neuroblastomas and glioblastomas (e.g. from the brain), carcinomas and adenocarcinomas (particularly from the breast, colon, kidney, liver (e.g. hepatocellular carcinoma), lung, ovary, pancreas, prostate and skin) and melanomas.
- Breast, head and neck cancers are preferred targets.
- Melanoma, sarcoma and lymphoma are preferred tumour types for treatment.
- Tumours for treatment are typically solid tumours and may be metastatic lesions that are accessible for transdermal injection.
- the peptides 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. In building up the peptide, one can in principle start either at the C-terminal or the N-terminal although the C-terminal starting procedure is preferred. Methods of peptide synthesis are well known in the art but for the present invention it may be particularly convenient to carry out the synthesis on a solid phase support, such supports being well known in the art. A wide choice of protecting groups for amino acids which are used in the synthesis of peptides are known.
- peptidic compounds including salts, esters or amides thereof
- pharmaceutical formulations i.e. incorporating one or more pharmaceutically acceptable diluents, carriers or excipients.
- the active agents 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, intrapentonealy, intratumouraly, transdermal ⁇ or intravenously.
- administration is preferably intratumoural.
- Particularly preferred are intratumoural injections of the peptidic compound of the invention once a day for several consecutive days, e.g. 2, 3, 4, 5, 6 or 7 days, preferably on 2-4 consecutive days, or at 2, 3, 4, 5, 6 or 7 daily intervals.
- the peptidic compound is preferably administered with or after the chemotherapeutic agent and there are preferably multiple administrations of the peptidic compound, preferably 2-4, e.g. 3 aministrations.
- 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. Simple solutions are preferred.
- 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.
- preservation agents such as p-hydroxybenzoates, or stabilizers, such as EDTA.
- the solutions are then filled into injection vials or ampoules.
- Preferred formulations are those in which the molecules are in saline.
- Such formulations being suitable for use in preferred methods of administration, especially local administration, i.e. intratumoural, e.g. by injection.
- dosage units containing the peptidic molecules preferably contain 0.1 -10 mg, for example 1 -5 mg.
- the formulation 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.
- Also provided according to the present invention is the use of a peptidic compound as defined above in the manufacture of a medicament for the treatment of a tumour, wherein said peptidic compound is co-administered with a chemotherapeutic agent as defined above, at a sub-cytotoxic dose.
- the medicament is for the treatment of multidrug resistant (MDR) tumours.
- MDR multidrug resistant
- a pharmaceutical pack or composition comprising:
- a chemotherapeutic agent as described herein at a sub-cytotoxic dose.
- the components can be for administration separately.
- the pharmaceutical pack can of course also comprise instructions for administration.
- the pack and composition are for use in the treatment of a tumour.
- Also provided according to the present invention is a method of treatment of a tumour, comprising the step of administering a peptidic compound as defined herein and a chemotherapeutic agent as described herein at a sub-cytotoxic dose, together in pharmaceutically effective amounts, to a patient in need of same.
- the peptidic compounds of the invention are able to destabilise mitochondrial membranes and cause release of DAMPs and antigenic material. This can have a powerful positive effect on the immune system's response to cancer cells.
- the immune response is of primary importance, e.g. to treat unidentified secondary tumours, to prevent formation of metastatic tumours, when surgery or other direct intervention is not possible.
- Different cancers are more or less immunogenic and therefore in some scenarios boosting the immune response to cancer is vital.
- the present invention provides a peptidic compound as defined herein for use in the destabilisation of a mitochondrial membrane, wherein said use is in the treatment of a tumour.
- a peptidic compound as defined herein for use in the destabilisation of a mitochondrial membrane, wherein said use is in the treatment of a tumour.
- This can be regarded as an immunotherapeutic use or treatment and the tumour will typically be cancerous.
- Preferred features and embodiments discussed elsewhere in relation to the combination therapies apply, mutatis mutandis, to this aspect.
- the invention provides a composition comprising or consisting of a peptidic compound of the invention and an immunotherapeutic agent.
- the invention provides a method of treating tumours in a patient, said method comprising administration of an effective amount of a peptidic compound of the invention and simultaneous or sequential administration of an effective amount of an immunotherapeutic agent.
- peptidic compound of the invention and an immunotherapeutic agent for use in the treatment of tumours.
- immunotherapeutic agent an agent which modulates the immune response.
- the immunotherapeutic agent enhances the immune response against one or more tumor antigens, for example by suppressing (preferably, tumor antigens), for example by suppressing (preferably, tumor antigens), tumor antigens, for example by suppressing (preferably tumor antigens), tumor antigens, for example by suppressing (preferably tumor antigens), tumor antigens, tumor antigens, for example by suppressing (preferably tumor antigens), for example by suppressing (preferably suppressing).
- the immunotherapeutic agent is preferably cytotoxic T lymphocyte antigen-4 (CTLA-4), an inhibitory receptor expressed on T cells.
- CTLA-4 cytotoxic T lymphocyte antigen-4
- the immunotherapeutic agent e.g. the chemotherapeutic agent is preferably administered prior to or simultaneously with the peptidic compound of the invention, it is most preferred that it is administered prior to the first administration of the peptidic compound of the invention. Preferably, it is administered prior to the peptidic compound of the invention, e.g. at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to the peptidic compound of the invention.
- a single dose is preferred, but multiple doses may be used, which may be e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 days apart.
- Co-administration may be simultaneous or sequential and by the same or different routes of administration, e.g. oral and/or parenteral.
- routes of administration e.g. oral and/or parenteral.
- product containing a peptidic compound of the invention and an immunotherapeutic agent as a combined preparation for separate,
- the inventors have also surprisingly found that the treatment of a tumour with a peptidic compound of the invention in conjunction with an immunotherapeutic agent can induce an adaptive immunity against further tumours.
- the above methods, uses and products (compositions) may therefore optionally extend to the induction of an adaptive immunity against further tumours.
- the invention also provides a peptidic compound of the invention and an immunotherapeutic agent for use in the treatment of tumours in a patient and inducing adaptive immunity against tumour growth, development or establishment in said patient.
- a method of inducing adaptive immunity against tumour growth, development or establishment in a patient comprising administration of an effective amount of a peptidic compound of the invention and simultaneous or sequential administration of an effective amount of an immunotherapeutic agent.
- the invention provides a peptidic compound of the invention and an immunotherapeutic agent for use in inducing an adaptive immunity against tumour growth, development or establishment.
- a peptidic compound of the invention and an immunotherapeutic agent in the manufacture of a medicament for use as a vaccine against tumour growth, development or establishment.
- a product containing a peptidic compound of the invention and an immunotherapeutic agent as a combined preparation for separate, simultaneous or sequential use in inducing an adaptive immunity against tumour growth, development or establishment.
- the invention also provides a method of vaccinating a subject against tumour growth, development or establishment through administration of an effective amount of a peptidic compound of the invention and an immunotherapeutic agent to said patient.
- 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.
- 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
- Figure 6 shows growth of A20 B-cell lymphomas in irradiated mice that received splenocytes from donor mice showing complete tumour regression following treatment with LTX-315 (Group 1 ) or control mice (Group 2) that received splenocytes from naive donor mice.
- Squares indicate Group 1 (mice that received splenocytes from donors showing complete regression).
- Diamonds indicate Group 2 (mice that received splenocytes from naive donors);
- LTX-315 causes rapid cell death in human melanoma cells, (a) In vitro cell killing kinetics of LTX-315 (IC 50 ) against human melanoma cells after designated time points (5, 15, 30, 45, 60, 90, 120 and 180 min) and (b) demonstrating the viability of A375 cells after treatment with different concentrations of LTX-315 for 60min. Results from three experiments are presented for each time point as mean ⁇ SD.
- LTX-315 internalizes and accumulates close to the mitochondria.
- A375 cells treated for 30 minutes with 1 .5 ⁇ fluorescence-labeled LTX-315, and with labeled mitochondria and nucleus. The peptide was internalized and detected in close proximity to the mitochondria.
- FIG. 10 Internalization occurs only in lytic 9-mer compounds such as LTX-315 and not in the non-lytic mock peptide LTX-328.
- LTX-315 was detected in the cytoplasm, while LTX-328 was not internalized.
- FIG. 1 LTX-315 treatment causes ultrastructural changes.
- A&D untreated control cells
- B&E cells treated with 3,5 ⁇
- C &F cells treated with 17 ⁇ .
- Figure 12 ROS generation in LTX-315 induced cell death.
- A375 cells were treated with LTX-315 at different concentrations for 15 minutes.
- carboxy-H2DCFDA was added to the samples and fluorescence was analyzed with a fluorescence plate reader. The experiment was conducted in duplicate, with bars representing mean fluorescence +- S.D.
- FIG. 13 Human melanoma cells treated with LTX-315 release cytochrome-C in the supernatant. Cytochrome-C release in the supernatant after LTX-315 treatment of A375 after designated time points (5, 15, 45 min) were determined by ELISA assay.
- HMGB1 is released in the supernatant after LTX-315 treatment.
- A375 human melanoma cells were treated with 35 ⁇ LTX-315 (top) or LTX- 328 (bottom), and cell lysate (L) and supernatant (S) were analyzed with Western blot, and the LTX-315-treated cells showed a gradual translocation from the cell lysate to the cell supernatant.
- Control cells were treated with media alone, and showed no translocation after 60 minutes.
- Figure 15 Extracellular ATP levels following LTX-315 treatment: A375 cells were treated with LTX-315 for different time point (5, 15, 30, 45, 60 min) at different concentrations or maintained under controlled conditions, and the supernatant was analyzed for the quantification of ATP secretion by luciferase bioluminescence. Quantitative data (mean +- S.D.) for one representative experiment are reported.
- FIG 17 Effect of low dose cyclophosphamide alone (b), LTX-315 alone (c), and the combination of the two therapies (d) on tumor growth of murine A20 lymphomas.
- Palpable lymphoma tumors syngeneic with Balb/c mice were injected i.p. with metronomic cyclophosphamide (day 4) or vehicle (vehicle controls, (a)), and with 1 .0 mg LTX-315 (20 mg/ml) once per day on day 8, 9 and 10 after tumor challenge.
- Example 1 that LTX-315 is the most potent of the 5 tested compounds in an in vitro cytotoxic activity study against a panel of 37 human cancer cell lines.
- 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 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
- 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 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
- Example 10 that treatment with LTX-315 induces hallmarks of immunogenic cell death by mitochondria distortion in human melanoma cells.
- Example 1 1 - that treatment with LTX-315 in combination with CY caused a complete and long-lasting tumor regression in a high proportion of test subjects.
- Test substances LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329 (see Table 1 ) provided in powder form.
- Triton X-100 was used as positive control, supplied by Oncodesign (Dijon, France) from Sigma (Saint Quentin Fallavier, France).
- Triton X-100 was obtained by dilution using culture medium.
- COLO 205 colorectal adenocarcinoma, ascites ATCC metastasis
- Tumor cells were grown as adherent monolayers or as suspensions at 37°C in a humidified atmosphere (5% C0 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 ⁇ 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 ⁇ FBS-free culture medium before treatment.
- Tumor cells were incubated for 4 hours with 10 concentrations of compounds in 1 ⁇ 4 dilution step with a top dose of 400 ⁇ (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 ⁇ ) were incubated in a 200 ⁇ final volume of FBS-free culture medium containing test substances at 37°C under 5% C0 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 novel tetrazolium compound
- PMS phenazine methosulfate
- the dose response inhibition of proliferation was expressed as follows
- the 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 (I DBS, 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. 4.2. Resistance Index (RI)
- 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.
- All thirty seven human tumor cell lines tested were sensitive to LTX-302 compound with IC 50 values ranging from 4.83 ⁇ 0.96 ⁇ to 20.09 ⁇ 4.07 ⁇ for T-47D and Hep G2 cell lines, respectively.
- Mean IC 50 value for LTX-302 compound obtained on the 37 tumor cell lines was 12.05 ⁇ 4.27 ⁇ with a median value of 1 1.70 ⁇ .
- 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.
- Mean IC 50 value for LTX-313 compound obtained on the 37 tumor cell lines was 9.60 ⁇ 3.73 ⁇ with a median value of 8.83 ⁇ .
- 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-CEMA LB, HL- 60/ADR and MCF-7/mdr cell lines (0.76, 1 .16 and 1.24 for CCRF-CEMA LB, 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.
- All thirty seve human tumor cell lines tested were sensitive to LTX-315 compound with IC 50 values ranging from 1.18 ⁇ 0.25 ⁇ to 7.16 ⁇ 0.99 ⁇ for T-47D and SK-OV-3 cell lines, respectively.
- Mean IC 50 value for LTX-315 compound obtained on the 37 tumor cell lines was 3.63 ⁇ 1 .45 ⁇ with a median value of 3.27 ⁇ .
- 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 human tumor cell lines tested were sensitive to LTX-320 compound with IC 50 values ranging from 3.46 ⁇ 0.22 ⁇ to 16.64 ⁇ 3.15 ⁇ 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 ⁇ with a median value of 6.92 ⁇ .
- 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.
- 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-CEMA LB, HL- 60/ADR and MCF-7/mdr cell lines (0.76, 0.80 and 1.07 for CCRF-CEMA LB, HL- 60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, 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. 5.6. General comments
- 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-302, LTX-313, LTX-315, LTX-320 and LTX- 329) exhibited cytolytic activity against 37 human cancer cell lines tested with IC 50 values in micromolar to ten micromolar range.
- 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.
- Test substances LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329 (see Table 1 ) provided in powder form.
- 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.
- Tumor cells were grown as suspensions at 37°C in a humidified atmosphere (5% C0 2 , 95% air). The culture medium for each cell line is described in Table 4 below. For experimental use, 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 ⁇ of drug-free and FBS-free culture medium.
- Tumor cells were incubated for 4 hours with 10 concentrations of compounds in 1 ⁇ 4 dilution step with a top dose of 400 ⁇ (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 ⁇ ) were incubated in a 200 ⁇ final volume of FBS-free culture medium containing test substances at 37°C under 5% C0 2 .
- Three independent experiments were performed, each concentration being issued from quadruplicate. Control cells were treated with vehicle alone. At the end of treatments, the cytotoxic activity was evaluated by a MTS assay (see ⁇ 3.3 below).
- MTS novel tetrazolium compound
- PMS phenazine methosulfate
- Mean IC 50 value for LTX-302 compound obtained on 10 sensitive cell lines was 8.1 1 ⁇ 2.44 ⁇ with a median value of 7.53 ⁇ .
- LTX-313 All ten human lymphoma cell lines tested were sensitive to LTX-313 compound with IC 50 values ranging from 3.21 ⁇ 2.81 ⁇ to 16.08 ⁇ 4.86 ⁇ for Ramos and Raji cell lines, respectively.
- Mean IC 50 value for LTX-313 compound obtained on 10 sensitive cell lines was 7.05 ⁇ 3.91 ⁇ with a median value of 5.89 ⁇ .
- Mean IC 50 value for LTX-315 compound obtained on 10 sensitive cell lines was 3.01 ⁇ 1 .36 ⁇ with a median value of 2.93 ⁇ .
- Mean IC 50 value for LTX-320 compound obtained on 10 sensitive cell lines was 5.03 ⁇ 2.82 ⁇ with a median value of 4.84 ⁇ .
- 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-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.
- the haemolytic activity of the peptide LTX-315 against human red blood cells was measured.
- 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°C.
- LTX-315 concentration corresponding to 50% haemolysis (EC 50 ) was determined from a dose-response curve.
- Figure 1 shows that LTX-315 has a mean value of EC 50 higher than 1200 ⁇ g/ml (833 ⁇ ).
- 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 ⁇ . The mice were divided into four groups (see Table 8 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 ⁇ 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 10 below.
- the administration takes place by intra-tumoural injection of 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 ⁇ .
- 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 ⁇ (20 mg/ml).
- Group five was treated with the 50 ⁇ 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.
- 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 .
- 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.
- 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 ⁇ ) 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.
- Example 9 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:
- 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 ⁇ 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 ⁇ 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.
- mice 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 ⁇ .
- the 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-
- Test substance preparation 10x10 6 A20 cells were added to a 50 ⁇ 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 0
- 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.
- LTX-315 on human melanoma cells.
- the peptide internalized and was shown in association with mitochondria, ultimately leading to a lytic cell death.
- the LTX-315 peptide was designed to treat solid tumors with intratumoral injections through a two-stage mode of action: the first is the collapse of the tumor itself, while the second is the released damage-associated molecular pattern molecules (DAMPs) from the dying tumor cell, which can induce a subsequent immune protection against recurrences and
- DAMPs damage-associated molecular pattern molecules
- LTX-315 and LTX-328 were made on request by Bachem AG (Bubendorf, Switzerland) and Innovagen (Lund, Sweden), respectively.
- LTX-315 Pacific Blue and LTX-328 Pacific Blue were purchased on request from Innovagen (Lund, Sweden) Norud (Troms0, Norway), respectively.
- the A375 cell line A375 (ECACC, 881 13005) is a human malignant melanoma derived from patient material, and was purchased from Public Health England (PHE Culture Collections, Porton Down, Salisbury, UK). Cells were maintained as monolayer cultures in high glucose 4.5% DMEM supplemented with 10% FBS and 1 % L- glutamine, but not as antibiotics (complete media). The cell line was grown in a humidified 5% C0 2 atmosphere at 37°C, and was regularly tested for the presence of mycoplasma with MycoAlert (Lonza).
- LTX-315 In vitro cytotoxicity, MTT-assay The cytotoxic effect of LTX-315 was investigated using the colorimetric MTT viability assay as described in Eliassen et al. (2002), 22(5): pp2703-10.
- the A375 cells were seeded at a concentration of 1 x10 5 cells/ml in a volume of 0.1 ml in 96-well plates, and allowed to adhere in a complete growth media overnight. The media was then removed and the cells were washed twice in serum-free, RPMI-1650 media, before adding LTX-315 dissolved in serum-free RPMI at concentrations ranging from 2.5-300 ⁇ g ml, and incubated for 5-180 minutes.
- Live cell imaging with unlabeled cells - A375 cells were seeded at 10,000 cells/well in a complete media in Nunc Lab-Tec 8-wells chambered covered glass (Sigma) precoated with 25 ⁇ g ml human fibronectin (Sigma) that were allowed to adhere overnight.
- Cells were washed twice with a serum-free RPMI, treated with peptide dissolved in RPMI and investigated using Bright on a Leica TCS SP5 confocal microscope, with a 63X/1.2W objective. The microscope was equipped with an incubation chamber with C0 2 and temperature control.
- Mitotracker - Cells were seeded as for live cell imaging, and treated with Mitotracker CMH2XROS (Invitrogen) at 100 nm for 15 minutes prior to peptide treatment. Cells were treated with 17 ⁇ LTX-315, with negative control serum-free RPMI only. After 60 min of incubation, cells were analyzed using a Zeiss microscope. All confocal imaging experiments were subsequently conducted at least twice with similar results.
- LTX-315 PB exhibited a similar cytotoxic profile as the unlabeled LTX-315 as determined by MTT assay.
- Control cells were treated with unlabeled LTX-315 and also with serum-free RPMI only. After incubation, cells were fixed with 4% paraformaldehyde in PBS, and the wells were covered with Prolong Gold antifade (Invitrogen). Cells were further analyzed by use of a Leica TCS SP5 confocal microscope, with a 693, 1 .2 W objective.
- UV Pacific Blue, GFP and Ds Red were excited using UV, with 488 and 561 lasers, and fluorescence channels were sequentially detected using the following band passes: UV: 420-480nm (with attenuation), 488: 501 - 550nm and 561 : 576-676nm.
- A375 cells were seeded at 1 x10 5 cells per well in 6-well plates and allowed to grow for three days to optimize membrane structures in the culture, and the media was changed on the second day.
- Cells were washed twice in serum-free RPMI before being treated with LTX-315 dissolved in serum-free RPMI at 5, 10 and 25 ⁇ g ml, with serum-free RPMI as a negative control.
- Cells were then washed with PBS twice before fixation for 24 hours in 4°C with 4% formaldehyde and 1 % gluteralaldehyde in a Hepes buffer at pH 7.8.
- Dehydration and post-fixation protocols included incubation in a 5% buffered tannic acid and incubation in a 1 % osmium-reduced ferrocyanide. Ultrathin sections were prepared, and uranyl acetate (5%) and Reynolds's lead citrate were used for staining and contrasting. Samples were examined on a JEOL JEM-1010 transmission electron microscope, and images were taken with an Olympus Morada side-mounted TEM CCD camera (Olympus soft imaging solutions, GmbH, Germany).
- ROS reactive oxygen species
- a DCFDA cellular reactive oxygen species detection assay kit was purchased from abeam®, and A375 cells seeded in a 96-well Costar black clear bottom plate with 20,000 cells per well incubated in 37°C 16 hours prior to DCFDA assay. Cells were washed with a 100 ⁇ -Jwell of pre-warmed PBS one time, and incubated with 20 ⁇ of DCFDA in a buffer solution supplied with the kit at 37°C in a cell culture incubator for 45 min, and then washed again with a buffer solution of 100 L/well.
- the cells were then stimulated with a 100 [ ⁇ Uwe ⁇ LTX-315 peptide dissolved in a buffer solution at concentrations of 17 ⁇ for 30 min, and cells not treated were used as a negative control.
- the fluorescence intensity was determined at an excitation wavelength of 485nm and an emission wavelength of 530 nm on a FLUOstar Galaxy plate reader.
- A375 cells were seeded with 3x10 5 cells/well in 6-well plates in a complete media, and allowed to adhere overnight. Cells were treated with LTX-315 or LTX-328 at 35 ⁇ , and incubated at 37°C and 5% C0 2 for different time points (5, 10, 15, 30, 60 min), and negative controls were serum-free RPMI-1650. Supernatants (S) were collected and centrifuged at 1 ,400g for five minutes, and cell lysates (L) were harvested after washing with PBS twice and then subsequently lysed using a 4X Sample buffer (Invitrogen, number), 0.1 M DTT (Sigma number) and water.
- S Supernatants
- L cell lysates
- A375 cells were seeded as with HMGB1 studies, and treated with 35 ⁇ for different time points (5, 15, 45). Supernatants were collected and concentrated as with HMGB1 studies, and samples from the supernatants were analyzed using a 4.5 hour solid form Cytochrome C- Elisa kit (R&D Systems, USA, #DCTC0) following the manufacturer's description. Shortly thereafter, a 50% diluted sample was analyzed and the optical density was determined using a microplate reader set at 450nm, and this reading was then subtracted from the reading at 540nm. A standard curve was generated for each set of samples assayed. Samples were run in four parallels, and the cytochrome-c released into the supernatant was expressed as a fold over the level of cytochrome-c in the supernatant of untreated cells.
- LTX-315-treated A375 cells was analyzed using an Enliten ATP luciferase assay kit (Promega, USA). Cells were then seeded as with an ROS assay, and treated with LTX-315 in different incubation times, from 1 to 15 minutes with two parallels, which was then conducted three times. Negative controls were untreated A375 cells exposed to serum-free media alone. Samples were diluted at 1 :50 and 1 :100, and analyzed with a Luminoscan RT luminometer according to the
- IC-50 values for the peptide were 30 ⁇ after only five minutes of incubation, and progressed to 14 ⁇ after 90 minutes. Further incubation up to 180 minutes did not offer any additional effect (Figure 8).
- LTX-315 treatment causes rapid cell lysis
- LTX-315 internalizes and targets the mitochondria
- LTX- 315 was labeled with Pacific Blue and incubated with cells at concentrations of 3 ⁇ and 1.5 ⁇ , respectively.
- the labeled LTX-315 rapidly penetrated the plasma membrane and at 1 .5 ⁇ , the peptide showed an accumulation around the
- LTX-315 induces ultra-structural changes in cells
- DAMPs are molecules that are released from intracellular sources during cellular damage. DAMPs can initiate and perpetuate an immune response through binding to Pattern Recognition Receptors (PRRs) on Antigen Presenting Cells (APCs).
- PRRs Pattern Recognition Receptors
- APCs Antigen Presenting Cells
- DAMPs are ATP, HMGB1 , Calreticulin, Cytochrome C, mitochondrial DNA and Reactive oxygen species (ROS).
- ROS Reactive oxygen species
- LTX-315 treatment leads to extracellular HMGB1 release
- HMGB1 is a non-histone, chromatin-binding nuclear protein. Once passively released from necrotic cells, HMGB1 is able to trigger the functional maturation of dendritic cells, cytokine stimulation and chemotaxis among several immunopotentiating effects.
- HMGB1 is normally found in the cell nucleus and would be expected in a cell lysate of healthy cells, though not in the culture media (supernatant).
- HMGB1 is normally found in the cell nucleus and would be expected in a cell lysate of healthy cells, though not in the culture media (supernatant).
- Both cell lysate and the cell supernatant of LTX-315- and LTX-328-treated A375 melanoma cells were analyzed using a Western blot. Cells were treated with 35 ⁇ of either LTX-315 or LTX-328, with a gradual translocation from the cell lysate to the supernatant detected in the LTX-315-treated melanoma cells, but not in the cells treated with the mock sequence peptide LTX-328 or a serum-free medium only ( Figure 14).
- LTX-315 treatment causes the production of Reactive Oxygen Species (ROS) in A375 melanoma cells
- LTX-315 labeled with the fluorescent molecule Pacific Blue was internalized within minutes after incubation with A375 melanoma cells, and was distributed in the cytoplasm (Figure 9). At low concentrations, accumulation of the peptide around the mitochondria was evident, whereas at higher concentrations the peptide was more spread within the cytoplasm and accumulated in circular structures closer to the cell membrane ( Figure 10). If the peptide attacks the mitochondrial membrane, a decrease or even a total collapse of the mitochondrial membrane potential would be expected.
- a confocal imaging of cells with the membrane potential-dependent mitochondrial stain Mitotracker CMXh2ROS showed a loss of mitochondrial signal a short time after peptide treatment (data not shown).
- Cytochrome-C is a mitochondrial protein released from the intermembrane space and into the cytosol when the outer mitochondrial membrane is perturbed, and by binding to the apoptotic protease activating factor-1 (Apaf-1 ) it is also a part of the apoptotic cascade that eventually leads to cell death by apoptosis.
- Apaf-1 apoptotic protease activating factor-1
- cytochrome-C is found in the extracellular space, it has been reported to act as a proinflammatory mediator, thus activating NF-kB and inducing cytokine and chemokine production.
- the transition of HMGB1 from the cellular compartment to the extracellular compartment was detected using a western blot ( Figure 14).
- the nuclear protein HMBG1 When the nuclear protein HMBG1 is released into the extracellular fluid, it functions as a DAMP, and can bind to both the PRR TLRs and to the RAGE receptors; the activation of these may lead to a number of inflammatory responses such as the transcription of pro-inflammatory cytokines.
- LTX-315 induces lytic cell death in cancer cells, not only by direct attack on the plasma membrane, but also as a result of an injury to vital intracellular organelles after the internalization of the peptide at concentrations too low to cause an immediate loss of plasma membrane integrity.
- DAMPs may affect the cellular integrity of the damaged cells in several ways, but are also associated with so-called immunogenic cell death.
- DAMPs potent immune stimulatory molecules
- CY cyclophosphamide
- metronomic the drug can lead to enhanced immune responses against a variety of antigens, more specifically through the selective suppression of inhibitory cell subsets including
- CY can also cause dendritic cell activation through the release of HMGB1 and ecto-CRT, which has knock-on effects in terms of pro-inflammatory cytokine production and T cell proliferation.
- a single i.p. injection of 2 mg CY was shown to lead to a reduction in the spleen cells within 24 hours, and with a low-point (50 % drop) on the fourth day after CY administration.
- Relative numbers of CD4+ and CD8+ T cells increased in spleens and LNs after CY administration, whereas the relative number of CD19+ and Tregs decreased sharply.
- LTX-315 (batch number 1037915, correction factor 76.5 %, supplied by Lytix Biopharma).
- Sendoxan cyclophosphamide monohydrate
- Vehicle LTX-315 Saline (0,9% NaCI in sterile H 2 0).
- Vehicle Sendoxan Saline (0,9% NaCI in sterile H 2 0).
- Vehicle control Saline (0,9% NaCI in sterile H 2 0).
- mice Female Balb/c wild-type mice, 5-6 weeks old, were obtained from Charles River, United Kingdom. All mice were housed in cages in a pathogen-free animal facility according to local and European Ethical Committee guidelines.
- Tumor treatment Tumor cells were harvested, washed in RPMI-1640 and injected intradermally (i.d.) into the right side of the abdomen in Balb/c mice (5 ⁇ 10 6
- peptide treatment was initiated using single i.t. injections of LTX-315 (1 .0 mg LTX-315/50 ⁇ saline) once a day for 3 consecutive days, and the vehicle control was saline only (0.9 % NaCI in sterile H 2 0). Tumor size was measured using an electronic caliper and expressed as the area of an ellipse
- A20 lymphomas have been shown to be difficult to treat due to the tumor growth pattern (viscous and undefined tumor) and its metastatic potential.
- LTX-315 induced complete regression of palpable A20 tumors in one of the animals and a partial response in the remainder of the animals, following intratumoral injection.
- Balb/c mice with A20 lymphomas were treated with a single i.p. injection of CY (2 mg/500 ⁇ ) on day 4 and i.t. injections with LTX-315 (1 mg/50 ⁇ ) for three consecutive days ( Figures 17 and 18). Controls were injected i.p. and i.t.
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