Novel MDM2 binding peptides and uses thereof Cross-reference to related application

[0001]. This application claims benefit of, and priority from, U.S. provisional patent application No. 61/223,600, filed on 7 July 2009 the contents of which are hereby incorporated herein by reference.

Field of the Invention

[0002]. The present invention relates to new peptides and uses thereof in treating cancer.

Background Art

[0003]. Cancer is one of the main diseases of current times causing 13% of all deaths globally. New aspects of the genetics of cancer pathogenesis, such as DNA methylation are increasingly recognized as important. While there are several chemicals that can affect rapidly dividing cancer cells most of these are toxic with adverse side effects. The P53 protein is an endogenous protein that induces apoptosis of cells. It is generally activated when cells are under stress. The p53 protein is known to be inactive in many tumors. More than 50 percent of human tumors contain a mutation or deletion of the TP53 gene. Artificially elevating levels of p53 have been shown to cause premature ageing. Restoring endogenous p53 induced apoptosis in tumor cells is desirable in treating cancer but there are no such methods on the market.

[0004]. Upon stress, p53 gets activated and induces pathways that can cause cell cycle arrest, DNA repair, cellular senescence, differentiation and apoptosis (2 and 3), thereby preventing the formation of tumors. The oncoprotein Murine Double Minute Clone 2 (MDM2) is a key regulator of tumor suppressor p53 (1).The p53 protein binds to and is negatively regulated by MDM2 (SEQ ID No. 2). In tumors over expressing MDM2, p53 function can be rescued through the disruption of the MDM2-p53 interactions by small molecules and peptides such as peptide 12-1(33). MDM2 regulates p53 by binding to its transactivation domain inhibiting its transcriptional activity and stimulating its ubiquitination and proteasomal degradation (5). Several tumors have highly expressed M DM2 leading to the destabilization of p53 (6-9). MDMX (SEQ ID No. 3) is a homolog of MDM2 and also negatively regulates p53 (3, and 4). MDMX promotes the ubiquitin ligase activity of M DM2 and also stabilizes p53 (10, 11 , 15). Like MDM2, the N-terminal domain of MDMX also regulates p53 by blocking its N-terminus transactivation domain, but unlike MDM2, MDMX doesn't promote p53's degradation (15, 16). Similar to MDM2, MDMX has also been found to be overexpressed in several cancers (70, 71).Hence the disruption of the interactions between p53 and MDM2/MDMX is a potential therapeutic target in a wide variety of cancers. It is known that MDM2/MDMX also binds to p73 but not p63, the two homologues of p53. Similarly, p73 is not mutated in cancers and can induce apoptosis. This further enhances the need for an antagonist against the MDM2 protein.

[0005]. Structural and biochemical characterization of the p53-MDM2 complexes have shown that the residues Phe19, Trp23 and Leu26 of the transactivation (TA) domain of p53 make extensive van der Waals contacts with MDM2 (Fig. 1A). These three residues are highly conserved and are believed to be implicated in trans- activation (12 and 13). The data describing the p53-MDM2 interactions used in this study has been taken from our previous work (34). It is becoming recognised that dual inhibition of MDM2/MDMX has a higher therapeutic index (40-43) The 19-26 region of p53 is thought to be necessary for binding MDM2 (35,36).

[0006]. The p53 family consists of two other homologues, p63 and p73. All three proteins share a similar architecture, which includes an N-terminal TA domain, a central DNA binding domain (DBD) and a C-terminal Oligomerization domain. Reports have shown that some isoforms of p63 and p73 can bind to p53 responsive elements, transactivate p53-responsive genes and induce apoptosis (17-20). Though p63 and p73 emulate some of the p53 functions, their main functions have been shown to be critical for development and differentiation and, are rarely found to be mutated in human cancers (21-26). Although there is not much overall similarity between the sequences among the family members in the TA domain, the three critical residues Phe19, Trp23 and Leu26 that have been shown to be important for MDM2 binding are well conserved (Table 1). This suggests that MDM2 may bind all 3 family members; however, binding to p63 has not been reported, and given that the majority of the binding energy derives from the packing of the 3 conserved residues, this is surprising (27-32). There is no structural data available either for the TA domains of p63 or p73 or for their interactions with MDM2.

Table 1

Amino acid sequence of the peptides in the TA domain of proteins from the p53 family.

Summary of the Invention

[0007]. The present invention seeks to provide novel peptides and/or methods for the disruption of the MDM2-p53 interaction or MDMX-p53 interaction. This may be useful in treating or slowing cancer cells to ameliorate some of the difficulties with the current treatment of cancer. The invention further seeks to provide in vivo and in vitro methods, for arresting or slowing cell proliferation.

[0008]. We have identified some of the factors necessary for the binding interaction between M DM2 and the p53 family members. We dissect the structural and energetic reasons underlying the interaction between p53 and MDM2. Using this information we have designed a peptide that is intrinsically less helical than p53 and yet has a higher affinity for M DM2 and MDMX that may be able to rescue p53 function in apoptosis by disruption of the MDM2-p53 interaction or MDMX-p53 interaction.

[0009]. Accordingly the first aspect of the invention is a peptide comprising an amino acid sequence of 20 amino acids or less with a phenylalanine in position 3, a serine in position 4, a hydrophobic amino acid in position 6, a Tryptophan in position

7 and Leucine in position 10 wherein the peptide is capable of binding to a MDM2 protein or an MDMX protein.

[00010]. Preferably the hydrophobic amino acid in position 6 is an isoleucine.

[00011]. Preferably the peptide comprises a cationic amino acid at position 8.

[00012]. Preferably the cationic amino acid at position 8 is a lysine.

[00013]. Preferably the peptide comprises the sequence set out in SEQ ID No 1.

[00014]. Preferably the peptide of the invention has a binding affinity (ΔG _bmd ) to

MDM2 or MDMX in the range of -18 Kcal/mol to -46 Kcal/mol.

[00015]. Preferably the peptide of the invention is suitable for use in treating cancer.

[00016]. A further aspect of the invention comprises a method of treating a subject afflicted with cancer comprising administering to the subject a peptide of the invention.

[00017]. Preferably the peptide is suitable for use in the preparation of a medicament for the treatment of cancer.

[00018]. A further aspect of the invention comprises a composition comprising a therapeutically effective amount of a peptide of the invention.

[00019]. Preferably the composition comprises a chemotherapeutic agent.

[00020]. Preferably the composition is suitable for use in treating cancer.

[00021]. Preferably the composition is suitable for use in the preparation of a medicament for the treatment of cancer.

[00022]. A further aspect of the invention comprises a method of disrupting MDM2 interaction with p53 comprising the steps of introducing a peptide of the invention that preferentially binds to MDM2. [00023]. A further aspect of the invention comprises a method of disrupting MDMX interaction with p53 comprising the steps of introducing a peptide of the invention that preferentially binds to MDMX.

[00024]. Throughout this document, unless otherwise indicated to the contrary, the terms "comprising", "consisting of, and the like, are to be construed as non- exhaustive, or in other words, as meaning "including, but not limited to".

Brief Description of the Drawings

[00025]. The invention will be better understood by reference to the following drawings in which:

Figure 1 The structure of the p53-MDM2 complex taken from the crystal structure 1YCR solved at a resolution of 2.6A. MDM2 and p53 are shown in grey and black respectively. The three critical residues, Phe19, Trp23 and Leu26 are shown in sticks.

Figure 1B Root Mean square Deviations (RMSD) of the backbone atoms as a function of time with respect to the starting structures during the MD simulations are shown for MDM2 apo (black), p53 (orange), p63(red),p73(cyan),12-1 (yellow) and pmad(green).

Figure 2A Root mean squared fluctuations (RMSF) of the C-σ atoms during the simulations shown for MDM2 taken from apo (black), p53(orange), p63(red), p73(cyan),12-1 (yellow) and pmad (green) respectively (i). The regions of higher fluctuations of MDM2 are shown in ribbon model (ii).

Figure 2B Temporal evolution of the secondary structure profiles peptides over simulations of 20ns (i for the free peptides) and 10ns (ii for the bound peptides), (a) p53 (b) p63 (c) p73 (d) 12-1 (e) pmad. The secondary structure is colored as follows: purple-σ-helix; blue- 3-10 helix; green-turn; white-random coil.

Figure 3 Residue wise contributions of van der Waals energies of the peptides taken from the simulations of the complexes.

Figure 4 Location of Lys24 of p53 peptide with respect to the electrostatic surface of MDM2. The residues Asp21 , Lys24, Leu 26, Pro27 and Glu28 of p53 are shown in sticks. MDM2 is represented as a electrostatic potential mapped on to the solvent accessible surface. The potentials are colour coded from dark grey (+1 kcal e-1 mol- 1) to light grey (-1 kcal e-1 mol-1). The p53 peptide is shown in black ribbon.

Figure 5 The packing of Pro27 (spheres) of the p53 peptide (shown in black ribbon) against the surface of MDM2.

Figure 6 Three different conformations of Glu17 of pmad peptide. MDM2 is shown in grey ribbon and pmad peptide is shown in black ribbon. The residues Glu17, Lys94 and Gln59 are shown in sticks (A) pmad interacting with Lys94 of σ2' (B) pmad is exposed to solvent (C) pmad interacting with Gln59.

Figure 7 The packing of Pro27 (spheres) of pmad peptide against the MDM2 surface. Three critical residues Phe19, Trp23 and Leu26 are shown in sticks. (A) Packing of Pro27 against the surface of MDM2 (B) the interaction of Pro27 residue with the backbone of Thr26 of MDM2.

Figure 8 The packing against the MDM2 surface of (A) 12-1 peptide (shown in black ribbon) (B) pmad peptide (shown in black ribbon). Three critical residues Phe19, Trp23, Leu26 are shown in sticks. MDM2 surface is shown in grey. Figure 9 Residues of MDMX that contribute to the differential binding of pmad. MDM2 and pmad peptide are shown in ribbon. (A) Glu17 of pmad peptide is not able to make interactions at the N-terminus due the presence of Gln66. Pro95 provides nicer packing near the C-terminus and the presence of Tyτ99 and Arg 103 makes favorable interaction with the peptide backbone and carboxy terminal (B) pmad peptide (shown in black ribbon) packed against the MDMX surface. Three critical residues Phe19, Trp23, Leu26 along with the N-terminus Glu17 and C-terminus Pro29 are shown in sticks. M DM2 surface is shown in light grey.

Detailed Disclosure

[00026]. There is provided a peptide. Peptides are short polymers formed from linking, amino acids with amide bonds. Preferably the peptide is in the range of a 8mer to 20mer, preferably a 13mer or a 12mer having the features that allow the peptide to have high affinity bonding with MDM2 or MDMX. Preferably the peptide has the sequence set out in SEQ ID No 4: GIu, X, Phe, Ser, X, Ne, Trp, X, X, Leu, X, X, X. The peptide in one embodiment preferably comprising the sequence set out in SEQ ID No 1 : GIu, VaI, Phe, Ser, Asp, Me, Trp, Lys, Leu, Leu, GIu, GIn, Pro. The new peptide of SEQ ID No 1. has been named pmad. In pmad, preferably GIuI is attracted by Lys94 of MDM2 which also stabilizes an hydrogen bond between the Glu1 backbone and Gln72 of MDM2; the 2nd state is an intermediate state where Glu1 of pmad is solvated and doesn't make any specific interactions. The 3rd conformation involves a stable packing between the N-terminus of pmad and Met62 of MDM2 and is associated with an increase in the length of the peptide helix by a half turn. In this conformation, preferably the backbone of GIuI forms a hydrogen bond with Ser4 side chain, thus stabilizing the pmad helix. Asp5 is involved in interactions with Lys8 of pmad. Glu11 is involved in a salt bridge with Arg97 of MDM2 and prevents TyMOO of MDM2 from flipping in. Gln12 may form a hydrogen bond largely with the backbone of Lys8 and Leu10 and with Lys51 of MDM2; this gives the C-terminal region of pmad a stable turn-like fold. Pro13 nestles against the aV side of MDM2 in a manner that enables the carboxy terminus to make a charge- charge interaction with Thr27 of MDM2 (Figs 7A ₁ B). Preferably Ile6 provides a local disruption to the packing of the peptide with MDM2. Indeed, although the N-terminal 3 residues and the C-terminal 4 residues of p63 and pmad are the same, the introduction of lie in place of Leu and 3 other changes (ie only a 30% transformation) appears to yield a transformation from a non-binding p63 to the highest affinity binding peptide, pmad to MDM2 or MDMX protein.

[00027]. Without being limited to any particular theory, the new peptide pmad appear to offers a novel approach to peptide design. Here we depart from the stable helix design strategy of a peptide lock and key approach and introduce instability into the peptide through two routes: the hydrophobic collapse and a frustrated landscape. The hydrophobic collapse is introduced via a bulkier hydrophobic residue (Ile6) that nucleates the aggregation of the other hydrophobic residues in the new peptide. The frustration in the conformational landscape is generated by a cation (Lys8) toggling between two anionic fields (Asp5 and Glu11) on either side. The ensuing conflict between these two forces creates instability in the peptide that now enables it to mould into the dynamically undulating surface of MDM2. This enables it to maximize its contacts with MDM2, thereby gaining free energy upon complexation. This gain is not as extensive in the association of p53 and MDM2 because the intrinsic stability of p53 peptides prevents it from surfing the MDM2 binding site as much as pmad does. Hence, a higher affinity between MDM2 (SEQ ID No. 2) or MDMX (SEQ ID No. 3) and the unstable pmad peptide (SEQ ID No. 1 ) exists in comparison to the natural affinity between M DM2 or MDMX and p53 or proteins in the p53 family.

[00028]. The new peptide design results in a dynamic peptide that can exist in different states.

[00029]. It appears that the increased disorder has been introduced by localizing a cationic residue in between two anionic residues, imparting a degree of frustration to the system. In addition, the introduction of a bulkier hydrophobic group towards the centre of the peptide enables the peptide to adapt a bound conformation that on the one hand is most strained, and yet enables the peptide to straddle the largest surface of MDM2, amongst all the peptides. Computations also reveal that this new peptide is a dual inhibitor, binding to MDMX.

[00030]. Peptides of the invention may be synthesized chemically using known techniques such as liquid phase synthesis or solid phase synthesis such as t-BOC or Fmoc, or BOP SPPS or any chemical synthesis method known to those in the art. Alternatively the peptide may be made biologically within a cell or vector designed to use the machinery of translation and/or transcription for peptide synthesis.

[00031]. As described herein the term "Cancer" refers to malignant neoplasm, or a group of cells that display uncontrolled division and growth beyond the normal limits, ie: abnormal proliferation of cells invasion, intrusion on and destruction of adjacent tissues, and sometimes metastasis where the cancer cells have spread to other locations in the body via lymph system or blood. Most cancers form a tumor but some, like leukemia, do not. For the purpose of the invention cancer refers to cells where MDM2 has been upregulated. For example in cancer cells present in lung, breast, gastric, colorectal, liver, prostate, cervical, brain, oral, esophagus, head and neck, lymphoma, leukemia, ovary, bladder, pancreatic, skin, sarcoma or any other cancers known to those skilled in the art.

Method for treating a patient with cancer

[00032]. On the basis of the above, the present invention provides a method for treating a patient with cancer, which comprises the step of: contacting the cells within and around a cancer with a peptide as described above. Desirably, the peptide is provided in a therapeutically effective amount.

[00033]. An alternative form of the present invention resides in the use of the peptide in the manufacture of a medicament for treating a patient with cancer preferably a medicament used in treatment to affect cells over expressing MDM2.

[00034]. "Treatment" and "treat" and synonyms thereof refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a cancer condition. Those in need of such treatment include those already diagnosed with cancer or having cells over expressing MDM2.

[00035]. As used herein a "therapeutically effective amount" of a compound will be an amount of active peptide that is capable of preventing or at least slowing down (lessening) a cancer condition, in particular increasing the average 5 year survival rate of cancer patients. Dosages and administration of an antagonist of the invention in a pharmaceutical composition may be determined by one of ordinary skill in the art of clinical pharmacology or pharmacokinetics. See, for example, Mordenti and Rescigno, (1992) Pharmaceutical Research, 9:17-25; Morenti et al., (1991 ) Pharmaceutical Research, 8:1351-1359; and Mordenti and Chappell, "The use of interspecies scaling in toxicokinetics" in Toxicokinetics and New Drug Development, Yacobi et al. (eds) (Pergamon Press: NY, 1989), pp. 42-96. An effective amount of the peptide to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the mammal. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from about 10 ng/kg to up to 100 mg/kg of the mammal's body weight or more per day, preferably about 1 μg/kg/day to 10 mg/kg/day.

Compositions of the Invention

[00036]. Peptides produced according to the invention can be administered for the treatment of cancer in the form of pharmaceutical compositions.

[00037]. Thus, the present invention also relates to compositions including pharmaceutical compositions comprising a therapeutically effective amount of a peptide that binds to MDM2 with high affinity. As used herein a compound will be therapeutically effective if it is able to affect cancer growth either in vitro or in vivo.

[00038]. Pharmaceutical forms of the invention suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions and or one or more carrier. Alternatively, injectable solutions may be delivered encapsulated in liposomes to assist their transport across cell membrane. Alternatively or in addition such preparations may contain constituents of self-assembling pore structures to facilitate transport across the cellular membrane. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating/destructive action of microorganisms such as, for example, bacteria and fungi.

[00039]. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as, for example, lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Preventing the action of microorganisms in the compositions of the invention is achieved by adding antibacterial and/or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[00040]. Sterile injectable solutions are prepared by incorporating the active peptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, to yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

[00041]. When the active ingredients, in particular small peptides contemplated within the scope of the invention, are suitably protected they may be orally administered, for example, with an inert diluent or with an edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active peptide in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that a dosage unit form contains between about 0.1 μg and 20 g of active compound.

[00042]. The tablets, troches, pills, capsules and the like may also contain binding agents, such as, for example, gum, acacia, corn starch or gelatin. They may also contain an excipient, such as, for example, dicalcium phosphate. They may also contain a disintegrating agent such as, for example, corn starch, potato starch, alginic acid and the like. They may also contain a lubricant such as, for example, magnesium stearate. They may also contain a sweetening agent such a sucrose, lactose or saccharin. They may also contain a flavouring agent such as, for example, peppermint, oil of wintergreen, or cherry flavouring.

[00043]. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. [00044]. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparaben as preservatives, a dye and flavouring such as, for example, cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

[00045]. Pharmaceutically acceptable carriers and/or diluents may also include any and all solvents, dispersion media, coatings, antibacterials and/or antifungals, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated.

[00046]. Supplementary active ingredients can also be incorporated into the compositions. Preferably those supplementary active ingredients are anticancer agents such as chemotherapy agents like, for example; cisplatin, platinum, carboplatin, gemcitabine, paclitaxel, docetaxel, etoposide, vinorelbine, topotecan, or irinotecan; tyrosine kinase inhibitors (e.g., Axitinib, Bosutinib, Cediranib, Dasatinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, Lastaurtinib, Nilotinib, semaxanib, sunitinib, vandetanib, vatalanib or any other suitable tyrosine kinase inhibitor); apoptosis inducing enzymes, for example TNF polypeptides, TRAIL (TRAIL R1 , TRAIL R2) or FasL, Exisulind or other apoptosis inducing enzymes; micro-RNA that initiates apoptosis; or other chemotherapy agents such as those commonly known to a person skilled in the art. Alternatively they may be anticancer treatments such as radiotherapy, chest radiotherapy, surgical resection, the chemotherapy agents mentioned above or any combination of these. [00047]. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.

[00048]. The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 pg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

[00049]. The compounds and compositions may be adapted to be administered to the lungs directly through the airways by inhalation. Compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administrated in standard form using powder inhaler devices or aerosol dispensing devices. Such devices are well known. For administration by inhalation, the powdered formulations typically comprise the active compound together with an inert solid powdered diluents such as lactose or starch. Inhalable dry powder compositions may be presented in capsules and cartridges of gelatin or a like material, or blisters of laminated aluminum foil for use in an inhaler or insufflators. Each capsule or cartridge may generally contain between 20 pg-10 mg of the active compound. Alternatively, the compound of the invention may be presented without excipients.

[00050]. The inhalable compositions may be packaged for unit dose or multi-dose delivery. For example, the compositions can be packaged for multi-dose delivery in a manner analogous to that described in GB 2242134, US6632666, US5860419, US5873360 and US5590 645 (all illustrating the "Diskus" device), or GB2178965, GB2129691 , GB2169265, US4778 054, US4811731 and US5035237 (which illustrate the "Diskhaler" device), or EP 69715 ("Turbuhaler" device), or GB 2064336 and US4353656 ("Rotahaler" device).

[00051]. Spray compositions for topical delivery to the lung by inhalation may be formulated as aqueous solutions or suspensions or as aerosols delivered from pressurised packs, such as a metered dose inhaler (MDI), with the use of a suitable liquefied propellant. The medication in pressurized MDI is most commonly stored in solution in a pressurized canister that contains a propellant, although it may also be a suspension.

[00052]. Aerosol compositions suitable for inhalation can be presented either as suspensions or as solutions and typically contain the active compound and a suitable propellant such as a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and especially 1 ,1 , 1, 2- tetrafluoroethane, 1 ,1 , 1,2, 3,3, 3-heptafluoro-n-propane and mixtures thereof.

[00053]. The aerosol composition may optionally contain additional excipients typically associated with such compositions, for example surfactants such as oleic acid or lecithin and co-solvents such as ethanol. Pressurized formulations will generally be contained within a canister (for example an aluminum canister) closed with a metering valve and fitted into an actuator provided with a mouthpiece. [00054]. Peptides can also be delivered by protein delivery methods known in the art such as transfection, macromolecule delivery vehicles and other methods known to those skilled in the art.

[00055]. The compositions may be for use in treating cancer. Use includes use of a composition of the invention for the preparation of a medicament or a pharmaceutically acceptable composition for the treatment of cancer. The preparation may further comprise a chemotherapeutic agent for the preparation of a medicament for the treatment of cancer.

[00056]. Examples of Specific embodiments

Characterization of the molecular interactions between the p53 family and MDM2

[00057]. Here we develop an understanding of the molecular interactions of the TA domains of the 3 proteins from the p53 family with MDM2 using a combination of molecular modeling and extensive molecular simulations, after appropriately benchmarking against available experimental data. We use the crystallographic data available on the p53-MDM2 interactions and use this as a template to model the interactions of p63 and p73 with MDM2 (where there is no structural data available). We further construct model of a known high affinity peptide, 12-1. In order to understand the molecular basis underlying the known differences between the MDM2-dependent regulations the p53 family members; we carry out a series of mutations. During our investigations, we have identified a novel peptide that displayed a higher affinity for MDM2 than the 3 p53 family members. An in vitro assay was carried out to confirm our findings. The sequence of the new peptide SEQ ID No.1 , pmad, is compared with the sequences that bind to MDM2 in table 2 such as the 3 members of p53 family and peptide 12-1.

Table 2 Amino acid sequence of the TA derived peptides from the p53 family, the 12-1 peptide and the new designed pmad peptide

[00058]. Simulations were carried out using protocols that have been applied before and benchmarked against experimental data (34); the peptide modeling was based on this. The crystal structure of the human p53-MDM2 complex [Protein Data Bank code: 1YCR, 2.6 A (50); Fig. 1 was used as the starting model for our studies. The structure has residues 25-109 of human M DM2 and residues 17-29 of human p53. The structure of the p63 and p73 peptides were designed based on the structure of the p53 peptide (1 YCR) and their side chain conformations were refined using SCWRL (51 ). The protonation states for all the structures were assigned using WHATIF (52). The N- and C- termini of MDM2 were capped with acetyl (ACE) and N-methyl (NME) respectively to keep them neutral; the N-terminus of the p53 peptides was capped with ACE (53). MD simulations were performed using the SANDER module of the AMBER8 (54) package employing the all-atom Cornell force field (55) Simulations were carried out for the complexes of p53-MDM2, p63-MDM2, p73-MDM2, pmad-MDM2,12-1-MDM2 and 20 nano seconds (ns) simulations for the uncomplexed protein (MDM2 apo and p53, p63, p73, pmad, 12-1). Each system was solvated with TIP3P water (56) box, which extended at least 12 A in each direction from the solute. A cut off of 10 A was used for the non-bonded van der Waals interaction. All bonds involving hydrogen atoms were constrained using SHAKE (57). Particle mesh Ewald (PME) (58) was used for the long range electrostatic interactions. After initial minimizations, the systems were gradually heated to 300 K, equilibrated for 100 (pico seconds) (ps) and production run was extended up to 10 ns.

[00059]. The simulations were applied to compute the free energies of complexation of peptides representing a 13 amino acid region of the TA domain of p53, p63 and p73 with MDM2. The data obtained was used to design sets of peptides (8 peptides in total) whose amino acid sequences were based upon partial inter-conversions between the TA of these 3 peptides. We report details only of the 3 family members, a tight binding peptide 12-1 and the tight binding new peptide (pmad) here; the energetics of the complete list is presented in Tables 3 a-f.

Table 3a

The energetic components for the binding of p53 peptide with MDM2

Table 3b

The energetic components for the binding of p63 peptide with MDM2

Table 3c

The energetic components for the binding of p73 peptide with MDM2

Table 3d

The energetic components for the binding of 12-1 peptide with MDM2

Table 3e

The energetic components for the binding of pmad peptide with MDM2

Table 3f

The energetic components for the binding of pmad peptide with MDMX

MDMX

[00060]. The global trends of the deviations from the crystal structures (Fig. 1B) suggest that the simulations are stable. The mobility of MDM2 displays a complex pattern that clearly depends on the sequence of the bound peptide (Fig. 2A). The presence of pmad attenuates the fluctuations the most. The mobility is higher in the σ1 helix (36-46), loop connecting σ2 and βλ (63-73), helices aV (77-89) and σ2' (93- 100).

[00061]. Secondary structure evolution of the free peptides shows that p63 is the least helical amongst the family members; p73 is quite helical while p53, 12-1 and pmad are of comparable helicity, although both 12-1 and pmad are characterized by more disorder. Upon complexation, the structure of p63 remains largely invariant while the other 4 undergo disordering at their C-termini, with pmad again undergoing the largest mobility (Fig. 2B). It is also clear that only p53 retains robust helicity across the 19-26 region; however, there appears to be sufficient helicity in all the other peptides to enable stabilizing interactions of the three critical residues that are needed to keep the peptide stably bound to MDM2. 12-1 , as we see later, is short and so well embedded in MDM2, hence its helicity is quite stable. Pmad undergoes significant disorder at its C-terminus and also gains transient helicity at the N- terminus. All 5 peptides undergo several conformational transitions, particularly among the residues flanking Leu25. The secondary structural variation arises from the complex dynamics of the different peptides that are characterized by differing hydrogen bonding (hereafter referred to as h-bond) patterns. The only conserved h- bond between all 5 peptides is that between the backbone atoms of Phe19 and Trp23. The rest of the h-bonds are dependent upon the local sequence variations and reflect the stability of the secondary structural evolutions.

[00062]. In summary, our simulations show that all peptides except p63 gain significant ordering upon complexation to MDM2.

Free energy of Binding

[00063]. The binding free energy (ΔG _b in _d ) of the p53 peptide (residues 16-27) to MDM2 has been reported to be -6.6 to -7.8 kcal/mol (37) and is similar to that of longer peptide (residues 15-29) (38) Our computed value of ΔG _b ind of the p53 peptide (residues 17-29) to MDM2, at -6.5 kcal/mol (34; and Table 4), is similar. Our methods are robust in that they are close to other computationally derived data.

[00064]. Free energy of binding (ΔG . ) of the peptides to MDM2 was computed using the MM-GBSA (molecular mechanics/Generalized Born surface area) method (59, 60) using the GB module (61) in Amber while the non-polar component is estimated from the solvent accessible surface area using MOLSURF (62) using: ΔG = 0.00542*SASA + 0.92 (14). The energy term was averaged for 5000 solv.πp

frames taken every 2 ps from the simulation. Vibrational entropy was estimated using normal mode analysis (Nmode module of Amber) (64) and averaged over 50 frames of complex and 100 frames of free peptides and MDM2. Secondary structures were computed using VMD, which has DSSP assignments (65), PyMOL (66) and Visual Molecular Dynamics (VMD) were used for visualizations (67).

[00065]. Using this methodology, we compute ΔG _b in _d for p63 at +5.9 kcal/mol, and - 2.8 kcal/mol for p73 respectively (Table 4). This trend also reproduces the experimentally observed fact that the affinity of p53 for M DM2 is higher than for p73 (38) and, p63 is not known to bind to MDM2. Again, in agreement with experimental data (33), we find that 12-1 binds much tighter to MDM2 than does p53. Finally, we find that the tightest binding peptide is pmad (SEQ ID No. 1), with an affinity two-fold higher than that of 12-1 (Table 4). Peptides of the invention have a binding affinity (ΔG _b in _d ) to MDM2 or MDMX in the range of -18 Kcal/mol to -46 Kcal/mol. Preferably peptides of the invention have a binding affinity (ΔG _b i _nd ) to MDM2 or MDMX in the range of -20 Kcal/mol to -46 Kcal/mol. In a particular embodiment the pmad peptide has a binding affinity (ΔG _b ind) to MDM2 in the range of -30 Kcal/mol to -36 Kcal/mol and a binding affinity (ΔG _b in _d ) to MDMX in the range of -30 Kcal/mol to -46 Kcal/mol.

[00066].

Table 4

Computationally derived free energies of binding of the peptides to MDM2

[00067]. The major difference in the affinities of the peptides arises from enthalpy (Tables 3a-e). The electrostatic component dominates in magnitude. This originates in interactions between two charged surfaces. The overall charge on MDM2 is +5 while on p53, p63, p73, 12-1 and pmad peptides are -1 , -3, -3, -1 and -2 respectively. In general this is mirrored in the magnitude of the electrostatic components (the last column in Tables 3 a-e) except for in 12-1 ; this is due to the peptide being short. This is further reflected in the GB term where the systems with the larger electrostatic interactions are the ones where the penalty for burying the charges in highest. The van der Waals energy of p73 is the smallest and originates in the p73 peptide being rich in smaller amino acids, Thr/Ser. Further, the p63 peptide has a very unfavorable internal energy term. This strained conformation arises from a hydrophobic collapse that appears to be caused by the clustering of Phe19, Ile22, Trp23, Leu26 and Pro29, which in turn leads to higher van der Waals interactions. The packing against MDM2 in 12-1 and pmad is the most favorable amongst all the peptides. In pmad, the first half of the simulation is characterized by a hydrophobic collapse as seen in p63 above; this is expected because the residues that are part of this cluster- Phe19, Ile22, Trp23, Leu26 and Pro29 - are conserved between the p63 and pmad (movies of wtp53,pmad at http://web.bii.a- star.edu.sg/~madhumalar/movies/). However this collapse is reversed during the course of the simulation because of the long range attraction between Asp21 and Lys24 which straightens the helix and releases the strain in pmad.

Experimental validation of an embodiment:

[00068]. To test the validity of our computational findings, we carried out florescence based thermal shift assays on the binding of the peptides to MDM2 as described.

Expression and purification of MDM2:

[00069]. DNA encoding residues Gln18 to Asn125 of MDM2 was cloned as a Ndel/BamHI fragment into pET19b (Novagen). Recombinant clones carrying the protein insert were identified by DNA sequencing. Plasmid from these clones was introduced into E. coli BL21(DE3) and protein production in LB medium was induced with IPTG(I mM) when the cell density reached an OD600 between 0.4 and 0.6. Induced cells were grown at room temperature for 5 h, harvested, and then resuspended in buffer A [50 mM Tris pH 8.0, 10 % sucrose]. After cell disruption and centrifugation, supernatant was loaded onto a Ni-nitrilotriacetic acid (NTA) column and Hexa His-tagged MDM2-(18-125) and washed with 5 column volumes of 0.3M NaCI, 5OmM Tris-HCL pH 8.0. MDM2 was eluted with a 1 M imidazole linear gradient. MDM2 was then dialysed into 1OmM Sodium phosphate and 0.05M NaCI. The protein was further purified by cation-exchange chromatography [Pharmacia Mono S 5/5, 1 ml/min, Sodium phospsphate (10 mM), pH 7.0] with a 1M NaCI gradient. Protein concentration was determined using A280 with an extinction

-1 -1

coefficient of 10430 M cm .

Fluorescence based thermal stability assay for peptide binding. [00070]. The thermal shift (68, 69) assay was conducted in the iCycler Real Time Detection System (Bio-Rad, CA). Samples were setup with 10μM of M DM2 in PBS (2.7mM KCL and 137mM NaCI, pH 7.4) with a final volume of 25μl. Peptide binding was analysed by adding 100//M of the peptide under review to the MDM2 protein sample. Peptides were dissolved in DMSO. Final DMSO concentration in MDM2 only and protein:peptide samples were kept at 1%. The plate was heated with a rate of 1°C/min. The fluorescence intensity was measured with Ex/Em: 575/635 nm and Sypro red (Invitrogen) was used as the reporter dye for protein unfolding at a concentration of 2.5x. All thermal stability curves were repeated in triplicate. Peptides were synthesized synthetically in Singapore.

20

[00071]. The fluorescence data was fitted to Eq. (1) to obtain ΔH u , ΔC pu , and T m by nonlinear regression using the program Prism 4.0, Graphpad:

Ft— Fpost (1 )

[00072]. where F t is the fluorescence intensity at temperature T ; T m is the midpoint temperature of the protein-unfolding transition, F pre and F post are the pre-transitional and post-transitional fluorescence intensities, R is the gas constant, ΔH is the enthalpy of protein unfolding, and ΔC is the heat capacity change on protein

TO TO

unfolding. In the absence of ligand, T T , ΔC = ΔC , and ΔH = ΔH and vice m 0 pu pu u u, versa in the presence of ligand. To calculate the ligand-binding affinity at T m for

19

peptide and MDM2 binding, Eq. (2) was used:

[00073]. To compare the binding affinities derived for the two MDM2 interacting peptides from the thermal shift data, the binding affinity at temperature T (K ) must

19

be calculated. K L(T) can be calculated from K L(Tm) using Eq. (3A):

KL(T) = KL(T.) exp

where K is the ligand association constant at temperature T , and ΔH is the van't Hoff enthalpy of binding at temperature T. The value of ΔH was taken to be - 10kcal/mol.

[00074]. It is clear that the pmad peptide has an affinity somewhat higher than that of 12-1 (data not shown). The binding affinity of peptides of the invention to MDM2 or MDMX can be easily tested in this way by a person skilled in the art in addition to any of the other in-silico methods mentioned throughout the specification. In can be quickly and easily established whether a peptide comprising a amino acid sequence of 20 amino acids or less with a phenylalanine in position 3, a serine in position 4, a hydrophobic amino acid in position 6, a Tryptophane in position 7 and Leucine in position 10 is capable of binding to a MDM2 protein or a MDMX protein using the methods described.

[00075]. The dynamics of the p53 family-MDM2 complex shows that the origin of the differential binding between the peptides lies in the (unfavorable intra, inter molecular interactions within the peptide and with MDM2. The first point to note is the van der Waals energies of interactions of each of the residues along the peptides with MDM2 (Fig. 3). This shows that the major contributions arise from the interactions of the 3 key residues, Phe19, Trp23 and Leu26 (33). This reproduction of experimental data further increases the confidence in the robustness of the methods employed here. As one pans along the peptides, at the position 17, GIu in both p53/p63 can interact with Lys70/Lys94, while Thr in p73 is not long enough to interact with either of them; but it gets oriented towards Lys94 and or Gln71. Thr18 in both p53/p73 makes favorable interactions with Asp21/His21 of the peptide as well as with the side chain of Gln72; the equivalent Va118 in p63 is not involved in any interactions. Indeed these accounts for the greater disorder in the N-terminus of p63. The well conserved Phe19 fits well in the hydrophobic cleft in all the three cases. At the 20th position, p53 has Ser whose side chain remains solvated while equivalent GIn and GIu in p63 and p73 are stabilized by Gln59. Asp21 of p53 is stabilized by Lys 24 for extended periods; in p63 and p73, the equivalent residue is His which interact with the N-terminus of the peptide leading to some loss of the packing between MDM2 and this end of the peptide. In p73 interactions between this His and ThM 8 stabilize the system a little. Leu at position 22 of p53/p73 packs well against the M DM2 surface than its bulkier equivalent Ile22 in p63; indeed this destabilization of packing is also transmitted to the N-terminus of p63 and we witness an associated destabilization of helicity at this end. Trp23 packs well in the hydrophobic cleft in all the complexes, including the hydrogen bond between its side chain and the backbone of Leu54. At position 24, a charge reversal takes place across the family. While Lys 24 of p53 points largely towards Asp21 , this is not- possible in p63 as it has anionic Asp at position 24. However the dynamics in this region are quite correlated. The Lys24 in p53 can only either be solvated or point towards the anionic field of Asp21 ; it is pushed towards this by the cationic field of Lys51 of MDM2 which in turn is stabilized by the anionic field created across the backbone carbonyls of Lys24, Leu26 and Pro27 and the side chain of Glu28 occasionally (Fig. 4). In contrast, in p63, Asp 24 points in the direction of Lys51 (doesn't reach it as it is short) and adds to the anionic field. This anionic field now pulls Lys51 side chain as well as the side chain of Gln28 of p63. In p73, Ser24 is only involved in intra peptide interactions that stabilize the helix. At position 25, Leu in p53 seems more favored for packing than its equivalent Phe in p63. In p73, Ser at this position enhances the side chain-backbone interactions, further stabilizing the helix. At position 26, the conserved Leu26 pack well in p53 and p73, but is quite mobile in p63. This latter is correlated with the dynamic nature of Phe25 as well as with the highly flexible C-terminus of p63. At the C-terminus, while Pro27 of p53 nestles in the surface of MDM2 (Fig. 5), Glu27 in p63/p73 makes a stable salt bridge with Arg 97 (and His95 in p73) of MDM2. Glu28 in p53 interacts with Lys51 of MDM2 while GIn of p63 is involved with the anionic field of the region of the peptide mentioned above. Pro28 in p73 is nestled against the surface of MDM2 (this is the surface opposite to where the Pro27 in p53 nestles). The side chain of Gln29 in p53 is largely exposed even as the carboxy terminus interacts briefly with the side chains of TyMOO and Tyr104 and for long periods with Arg97; indeed the orientation of Arg97 is such that its hydrophobic side-chain forms the surface against which Pro27 nestles. In p63, Pro29 only creates a curved peptide but doesn't really interact with anything on MDM2, lending this part high disorder. In p73, with Pro28 stably nestled against MDM2, the side chain of Asp29 is solvated while the carboxy terminus forms a stable salt bridge with Arg97. In all 3 cases, the presence of charge-charge interactions with Arg97 provide a gate that prevents TyMOO (or 104) from flipping in towards the binding cavity.39 In the case of 12-1 , the N-terminal Met (equivalent to position 16 in p53) is always exposed to solvent while Pro17 packs against Tyr22 which in turn packs against the His73 region of MDM2. Arg18 interacts with Asp21 which stabilizes a helical fold of the peptide in this region. Phe19, Trp23 and Leu26 are packed in a manner similar to that seen in the other systems while Met20 adds stability to the complex by packing against the σ1 helix (including Met62). Glu24 is largely exposed although it appears to be involved in long range stabilization with Arg18. Gln28 and this side chain together with the carboxy terminus are stabilized by Lys51 thus giving the peptide a final helical turn. This shortening also enables TyMOO (and TyM 04) to flip in towards the MDM2 cavity, forming interactions with the backbone and the carboxy terminus of Gln28; this reorientation creates a "cosier" fit of the peptide (39)

Characterisation of the novel peptide

[00076]. In the newly designed peptide, pmad, the N-terminus occupies three different states (Figures 6A-C) that are separated by 3-4 kcal/mol (data not shown): in one state, Glu17 is attracted by Lys94 which also stabilizes an h-bond between the Glu17 backbone and Gln72; the 2nd state is an intermediate state where Glu17 is solvated and doesn't make any specific interactions. The 3rd conformation involves a stable packing between the N-terminus and Met62 and is associated with an increase in the length of the peptide helix by a half turn. In this conformation, the backbone of Glu17 h-bonds with Ser20 side chain, thus stabilizing the helix. Asp21 is involved in interactions with Lys24, analogous to p53. Glu27 is involved in a salt bridge with Arg97 and prevents TyMOO from flipping in. Gln28 h-bonds largely with the backbone of Lys24 and Leu26 and with Lys51 ; this gives the C-terminal region a stable turn-like fold. Pro29 nestles against the aV side of MDM2 in a manner that enables the carboxy terminus to make a charge-charge interaction with Thr27 of MDM2 (Figs 7A,B). Ne22 provides a local disruption to the packing of the peptide with MDM2 (as seen in p63). Indeed, although the N-terminal 3 residues and the C- terminal 4 residues of p63 and pmad are the same, the introduction of Ne in place of Leu and 3 other changes (ie only a 30% transformation) appears to yield a transformation from a non-binding p63 to the highest affinity binding between MDM2 and pmad.

[00077]. Overall, we see that in p53 the peptide straddles the side of the MDM2 cleft that contains σ2'. p73 straddles the opposite face, while 12-1 is packed in the most symmetric manner in the cavity (Fig 8A ₁ B). Pmad straddles both faces and its orientation seems to be governed by the bulkier Ne22. This is further reflected in the trend of the surface area based GB term (Tables 3 a-e) where we see that pmad has the most stabilizing contribution followed by 12-1 and p53. [00078]. We computed the binding energy of pmad to MDMX using the structure 3D AB resolved at 1.9A (44) and found that the peptide binds stronger to MDMX than it does to MDM2. At the N-terminus, the conformational flexibility of pmad seen with MDM2 is lost in MDMX because Arg67 in MDM2 is replaced by Gln66 in MDMX. This results in the lack of a driving force on the N-terminus of the peptide towards the aV helix. The Pro95 in MDMX enables a better packed peptide chain than does equivalent His96. In contrast to a stable C-terminus of pmad when complexed with MDM2, in MDMX it is highly mobile as it undergoes conformational transitions with the anionic C-terminus toggling between Lys50 (equivalent to Lys51 in MDM2) and Arg103 (equivalent to TyM 04 in MDM2). Because of the change in the local amino acid composition of the protein near the C-terminus of pmad, in MDMX the peptide can "reach out" towards Arg103 and in the process the curve that is created in the peptide enables the Tyr99 (equivalent TyMOO in MDM2) to point "inwards"(39), making h-bonds with the backbone atoms of Leu26 and Glu27. This is further assisted by the bulkier Met53 (equivalent Leu54) in MDMX preventing the peptide from "embedding into" the pocket as deep as it appears to do in MDM2.

[00079]. Finally, when we compare p53 and pmad, we see that the new peptide is intrinsically less stable than p53. The electrostatics is severely destabilized from - 176 to -54 kcal/mol with a small (-2 kcal/mol) change in packing. This is also seen in the secondary structure plots.

[00080]. Together these results suggest that there are at least two mechanisms for optimal binding of peptides to MDM2 (or MDMX). The first is the classical method (45, 46) whereby the architecture of p53 peptides is modulated in a manner that improves the helicity of the p53 mimics. Because p53 and MDM2 have probably co- evolved, this helix fits well into the M DM2 surface. The necessity of having an amphipathic helix in this region is to perhaps enable efficient sequestration through one face of the helix by the hydrophobic cavity of MDM2, while leaving the other face of the helix open for phosphorylations and binding to coactivators such as p300(47). The design of interrupting peptides with a stable helix recovers some of the free energy that is lost upon complexation of the free peptide. This explains how tight binding peptides such as 12-1 etc have been evolved and fits the paradigm of dynamic 'locks and keys'. This paradigm is characterized by the peptide and the receptor binding through surfaces that share complementary features with the dynamics adding the fine tuning. While these may not be the most populated substates, nevertheless such binding will drive the equilibrium towards such associations.

[00081]. In contrast the new peptide, Pmad (SEQ ID No 1), offers a novel approach to peptide design. Here we depart from the stable helix design strategy and introduce instability into the peptide through two routes: the hydrophobic collapse and a frustrated landscape. The hydrophobic collapse is introduced via a bulkier hydrophobic residue (Ne22) that nucleates the aggregation of the other hydrophobic residues in the peptide. The frustration in the conformational landscape is generated by a cation (Lys24) toggling between two anionic fields (Asp21 and Glu27) on either side. The ensuing conflict between these two forces creates instability in the peptide that now enables it to mould into the dynamically undulating surface of MDM2. This enables it to maximize its contacts with M DM2, thereby gaining free energy upon complexation. This gain is not as extensive in the association of p53 and MDM2 because the intrinsic stability of p53 peptides prevents it from surfing the MDM2 binding site. This is in accord with the paradigm of 'induced fit'. We are currently attempting to amalgamate the two ideas towards designing high affinity peptides.

[0001]. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The invention includes all such variation and modifications. The invention also includes all of the steps, features, formulations and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features. [0002]. Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

[0003]. Any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0004]. The present invention is not to be limited in scope by any of the specific embodiments described herein. These embodiments are intended for the purpose of exemplification only. Functionally equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

[0005]. The invention described herein may include one or more range of values (eg size, concentration etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range which lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

[0006]. Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

[0007]. Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0008]. While the invention has been described with reference to specific methods and embodiments, it will be appreciated that various modifications and changes may be made without departing from the invention.

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