Background of the Invention Calcitonin is a 32-residue peptide hormone which is produced in the C-cells of the thyroid of mammals and is involved in calcium regulation and bone dynamics (Silverman, S. L. , 1997. Am J Med Sci. 313,13-16). Calcitonin causes a rapid but short-lived drop in the level of calcium and phosphate in the blood by promoting the incorporation of these ions into bone.

Human calcitonin has a high tendency to aggregate both in vivo and in vitro, even at low concentrations (Sletten, K. et al., 1976, J Exp Med. 143, 993-998 ; Silver, M. M. et al., 1988, J.

Histochem Cytochem. 36, 1031-1036 ; Siebel, P et al., 1970, Helv Chim Acta. 53,2135-2150 ; Arvinte, T. et al., 1993, J Biol Chem.

268,6415-6422). This constitutes a serious problem in the production, processing and administration of human calcitonin, since aggregate calcitonin is not physiologically active and can potentially generate cytotoxicity or an undesired immune response, as reported for other aggregated therapeutic polypeptides (Braun, A. et al., 1997, Pharm Res 14, 1472-8 ; Curatolo, L. et al., 1997, Cytokine 9,734-9 ; Brange, J. et al., 1997, J Pharm Sci 86,517-25).

Salmon calcitonin (sCT) is currently used in preference to human calcitonin for the treatment of hypercalcaemia and bone disorders such as osteoporosis and Paget's Disease. Salmon calcitonin is less prone to aggregation than the human form, although it has a lower activity (when aggregation of the human form is prevented) (Cudd, A. et al., 1995, J Pharm Sci. 84,717-719). Because salmon calcitonin shares low sequence identity with human calcitonin (16 differences in 32 residues or 50% sequence identity), long-term treatment with the salmon variant may lead to resistance or allergy through the generation of antibodies to the foreign peptide (Levy, F. et al., 1988, J Clin Endocrinol & Metabol. 67, 541-545 ; Muff, R. et al., 1991, Osteoporos Int. 1, 72-75; Grauer, A. et al., 1995, Exp Clin Endocrinol Diabets. 103, 345-351). When resistance occurs, the dose of salmon calcitonin must be increased in order for treatment to be effective.

Eventually, resistance may render the treatment totally ineffective. Additionally to hypocalcaemic activity, calcitonin variants of different origin also show anorectic responses in animals (Gaggi R, et al. , 1985, Pharmacol Res Commun. 17,209- 15).

There are a number of reports of modifications to the human calcitonin sequence to reduce its self-aggregation activity (GB0109438. 2, US 3,798, 203, US 3,910, 872, WO 02/083734, Udal et al Biol. Pharm. Bull. (1999) 22 (3), 244-252). However, it would be desirable to reduce the self-aggregation activity of human calcitonin still further in order to provide effective therapies.

In particular, lower self-aggregation activity simplifies the production and manipulation of calcitonin and allows formulations which are not be possible with aggregation-prone molecules (i. e. aerosol, oral, etc. ). Side effects linked to aggregation are also reduced (e. g. generation of allergies or immune responses, low efficacy, etc), and lower doses of the drug may be used to achieve the same response.

Summary of the Invention The present invention provides for calcitonin peptides whose self-aggregation activity is lower than both native human calcitonin (hCT) and previously described modified human calcitonin peptides.

Preferred calcitonin peptides of the invention may also have improved receptor-binding characteristics relative to native human calcitonin, making them especially suitable for therapeutic applications.

In one aspect, the invention provides a calcitonin (CT) peptide comprising the amino acid sequence; CGN LST CML GX1Y TQD FX2K FX3T FPX4 TAXI GX6G AP wherein Xi to X6 are independently proline (P), glycine (G), a polar amino acid selected from the group consisting of S, T, Q and N, or a charged residue selected from the group consisting of R, K, D H and E.

Other aspects of the invention relate to nucleic acid encoding calcitonin peptides as described above, methods of production and

therapeutic applications of calcitonin peptides as described above.

Description of the Figures Figure 1 compares the aggregation propensities exhibited by the designed calcitonin variants in comparison with that of hCT.

Aggregation is measured by monitoring the evolution of turbidity with time.

Figure 2 shows the effect of CT peptides on [125I] hCT binding and cAMP accumulation in T47D cells.

Figure 3 shows the effect of CT peptides on persistent cAMP accumulation in T47D cells.

Figure 4 shows the hypocalcaemic activity of the engineered calcitonin variants on rats at different concentrations.

Table 1 shows the amount of soluble peptide remaining in solution at the end of the aggregation measurements in PBS, at a peptide concentration of 10 mg/mL and 37°C.

Table 2 shows the potencies of CT peptides in [12-1,] hCT binding inhibition and cAMP stimulation. Results are means SEM. Binding and cAMP experiments were performed 3 and 6-times, respectively.

Description of the Sequences SEQ ID NO: 1 is the amino acid sequence of human calcitonin (hCT).

'SEQ ID NO: 2 is the amino acid sequence of salmon calcitonin (sCT).

SEQ ID NO: 3 is the amino acid sequence of calcitonin peptides of the invention.

SEQ ID NOs: 4-30 are examples of modified polypeptides according to embodiments of the invention.

Detailed Description of the Invention As described above, a calcitonin (CT) peptide of the invention may comprise or consist of the amino acid sequence; CGN LST CML GX1Y TQD FX2K FX3T FPX4 TAX5 GX6G AP wherein X1 to X6 are independently proline (P), glycine (G), a polar amino acid selected from the group consisting of serine (S), threonine (T), glutamine (Q) and asparagine (N), or a charged residue selected from the group consisting of arginine (R), lysine (K), aspartic acid (D), histidine (H) and glutamic acid (E).

In some embodiments, each of residues X1 to X6 are independently an amino acid residue selected from the group consisting of P, T, S, R, K, H, D and E.

In some embodiments, each of residues X1, X2 and X4 are independently P, R, K, D or E, residue X3 is independently P, R,

K, H, D or E and each of residues X5 and X5 are independently S or T.

Residue Xi may be independently H or T, preferably S or P, more preferably E or K or most preferably D or R. In some preferred embodiments, residue Xi is independently R.

Residue X2 may be independently H or T, preferably S or P, more preferably E or K or most preferably D or R. In some preferred embodiments, residue X2 is independently R.

Residue X3 may be independently T, preferably S or P, more preferably E or K or most preferably D, H or R. In some preferred embodiments, residue X3 is independently H or R.

Residue X4 may be independently H or T, preferably S or P, more preferably E or K or most preferably D or R. In some preferred embodiments, residue X4 is independently R.

Residue X6 may be independently H, preferably P, more preferably E or K or most preferably S, T, D or R. In some preferred embodiments, residue X6 is independently S.

Residue X6 may be independently H, preferably P, more preferably E or K or most preferably S, T, D or R. In some preferred embodiments, residue X6 is independently T.

Examples of preferred calcitonin peptides-of the invention are calcitonin peptides 5P (SEQ ID NO : 4) and 6S (SEQ ID NO : 5).

Other preferred calcitonin peptides of the invention are shown in SEQ ID NOS 6 to 30.

Calcitonin peptides as described herein have reduced self- aggregation activity compared to unmodified human calcitonin (hCT) i. e. they have increased solubility and/or a reduced propensity or tendency to self-aggregate in aqueous solution.

Self-aggregation activity may be determined by any suitable method, for example by monitoring the turbidity of a peptide solution over time. The self-aggregation activity of a modified calcitonin peptide may be assessed relative to unmodified hCT.

A calcitonin peptide as described herein possesses all or part of the calcitonin activity of unmodified hCT. Some preferred calcitonin peptides may have increased activity relative to unmodified hCT. Calcitonin activity can be readily monitored using conventional methods, for example by in vitro cellular assays (Zimmerman, U. et al, 1997, J. Endocrinol. 155,423-431 ; Miyamoto, K. I. et al, 1998, Jpn. J. Pharmacol 76,193-198), or using animal models (Maier, R. , 1975, Horm Metab Res. 7: 511-4; McKee, C. et al. , 1998, Nat. Biotechnol. 16,647 ; BBRC. 267,362- 367; Wang, Y. et al. , 2003, BBRC. 306,582-589).

In preferred embodiments, a calcitonin peptide as described herein may show increased or improved receptor binding relative to unmodified hCT, thereby increasing the duration and/or persistence of the calcitonin activity.

A calcitonin peptide of the invention may comprise one or more non-naturally occurring amino acids, including the D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha- aminoisobutyric acid, 4-aminobutyric acid (4Abu), 2-aminobutyric acid (Abu), 6-aminohexanoic acid (epsilon-Ahx), 2-aminoisobutyric acid (Aib), 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine (bAla), fluoro-amino acids, designer amino acids such as beta-methyl amino acids, C-alpha- methyl amino. acids, N-alpha-methyl amino acids, and amino acid analogs in general.

A calcitonin peptide may have one or more peptidyl--C (O) NR-- linkages (bonds) replaced by a non-peptidyl linkage such as a-- CH2-carbamate linkage (--CH2OC (0) NR--), a phosphonate linkage, a -CH2-sulfonamide (-CH2--S (O) 2NR--) linkage, a urea (--NHC (O) NH--) linkage, a--CH2-secondary amine linkage, or an alkylated peptidyl linkage (--C (O) NR--) wherein R is C1-C4 alkyl.

A calcitonin peptide may have the N-terminus derivatized to a-- NRR1 group, to a--NRC (0) R group, to a--NRC (O) OR group, to a-- NRS (0) 2R group, to a--NHC (0) NHR group where R and R1 are hydrogen or C1-C4 alkyl with the proviso that R and R1 are not both hydrogen and/or a C terminus is derivatized to--C (O) R2 where R2 is selected from the group consisting of C1-C4 alkoxy, and--NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and C1-C4 alkyl.

A calcitonin peptide of the invention may be in the form of a pharmaceutically acceptable salt. Examples of the salts with pharmaceutically acceptable acids are those with mineral acids, such as for example the hydrochloride, hydrobromide, sulfate, phosphate, borate, hydrogensulfate, dihydrogenphosphate or nitrate, and those with organic acids, such as for example the acetate, oxalate, tartrate, succinate, maleate, fumarate, gluconate, citrate, pamoate, malate, ascorbate, benzoate, p- toluenesulfonate or naphtalenesulfonate. Examples of the salts with pharmaceutically acceptable bases are those with alkali or alkaline earth metals such as'sodium, potassium, calcium or magnesium, and those with organic bases such as amines, trometamol and N-methylglutamine.

A calcitonin peptide may be produced by any method of peptide synthesis known in the art, for example by chemical synthesis or recombinant in vitro or in vivo expression.

Calcitonin peptides may be generated wholly or partly by chemical synthesis. A calcitonin peptide as described herein can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A.-Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); Applied Biosystems 430A Users Manual, ABI Inc. , Foster City, California; Roberge, J. Y. et al.

(1995) Science 269: 202-204 ; Caruthers, M. H. et al. (1980) Nucl.

Acids Res. Symp. Ser. 215-223; Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232 ; and Merrifield J. (. 1963) J. Am. Chem.

Soc. 85: 2149-2154), or they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e. g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.

Automated synthesis may be performed, for example, using the ABI 431 A Peptide Synthesizer (Applied Biosystems).

A calcitonin peptide may have an amino terminal (N-terminal) capping group (e. g. an acetyl group) and/or a carboxy terminal (C-terminal) capping group, (e. g. an amide group) to protect the terminal residue from undesirable chemical reactions during use . or to permit further conjugations or manipulations of the peptide.

A newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e. g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, W H Freeman and Co. , New York, N. Y. ) or other comparable techniques available in the art. The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e. g. the Edman degradation procedure).

Recombinant in vivo or in vitro production of a calcitonin peptide may be achieved by the expression of a nucleic acid that

comprises an encoding nucleotide sequence using conventional recombinant techniques.

Another aspect of the invention provides an isolated nucleic acid encoding a calcitonin peptide as described herein.

Nucleic acid may be double-or single-stranded, cDNA or genomic DNA, or RNA. The nucleic acid may be wholly or partially synthetic, depending on design. The nucleotide codons that encode each amino acid are well known in the art and a skilled person is readily able to identify and/or design appropriate nucleic acid coding sequence encoding any peptide sequence. Naturally, the skilled person will understand that where the nucleic acid includes RNA, reference to the sequence shown should be construed as reference to the RNA equivalent, with U substituted for T.

Nucleic acid may be produced recombinantly, synthetically, or by any means available to those of skill in the art and may also be cloned using standard techniques. For example, nucleic acid may be cloned into any convenient vector. Nucleic acid is typically provided in isolated and/or purified form.

Nucleic acids may be produced by synthetic means, involving a stepwise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this, using automated techniques, are well known. Synthesised nucleic acids may be ligated together to produce longer nucleic acid molecules.

Alternatively, a nucleic acid may be produced by PCR or other recombinant technique.

In some embodiments, a nucleic acid encoding a calcitonin peptide as described herein may be obtained by site-directed mutagenesis of a polynucleotide comprising the unmodified sequence. This technique may be performed in any convenient manner, for example, by PCR. A primer may be used which comprises a nucleotide sequence substantially identical to a region of the unmodified sequence in which mutation is desired, other than changes at the specific nucleotide residues which will bring about the desired alteration in the encoded amino acid sequence. Use of this primer in PCR introduces the desired changes to the nucleotide sequence of the amplified product. Other sequence changes may be included in order to introduce restriction enzyme recognition sites to facilitate cloning, or to further alter or modify the property or function of the encoded calcitonin peptide.

A nucleic acid modified as described above may be sequenced to confirm the nucleotide sequence. Sequencing techniques are well known in the art (Molecular Cloning : a Laboratory Manual: 3rd edition, Sambrook et al, 2001, Cold Spring Harbor Laboratory Press) A nucleic acid as described above may be operably linked to a regulatory sequence which is capable of directing the expression of the coding sequence by the host cell.

The term"operably linked"refers to. a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence"operably

linked"to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

A nucleic acid encoding a calcitonin peptide as described above may be operably linked to a regulatory element and/or comprised in a vector.

Such vectors may be transformed into a suitable host cell as described above to provide for expression of a polypeptide or polypeptide fragment of the invention. A process for preparing polypeptide may comprise cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression of the polypeptide, and recovering the expressed peptide.

A vector may be, for example, a plasmid, virus or phage vector provided with an origin of replication, optionally a promoter for the expression of the said nucleic acid and optionally. a regulator of the promoter. A vector may contain one or more selectable marker genes, for example, an ampicillin resistance gene in the case of a bacterial plasmid, a neomycin resistance gene for a mammalian vector, or a kanamycin resistance gene for a plant vector. A vector may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.

The vector may also be adapted to be used in vivo, for example in a method of gene therapy.

Those skilled in the art can construct vectors and design protocols for recombinant gene expression, for example in a microbial or mammalian cells. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook et al, 2001, Cold Spring Harbor Laboratory Press and Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992.

A vector may be introduced into a host cell using conventional techniques including calcium phosphate precipitation, DEAE- dextran transfection, electroporation, particle bombardment or Agrobacterium tumefaciens-mediated techniques. Expression from the host cell may be transient. Stable host cell transformation may be achieved by integration of a nucleic acid as described herein into a genome of the host cell. Typically, the transformed genome is nuclear, although transformation of other genomes may be desired, for example the mitochondrial genome of eukaryotic cells, or a plastidic genome of plant cells.

Alternatively, stable transformation may be achieved using replicable autonomous vectors. The expression vector may contain a selectable marker and/or such a selectable marker may be co- transfected with the expression vector and stable transfected cells may be selected.

A further embodiment of the invention provides a host cell comprising (i. e. transformed or transfected with) a vector for the replication and expression of a nucleic acid encoding a calcitonin peptide as described above. A suitable cell may, for example, be bacterial, yeast, plant, insect, or mammalian.

Suitable cells include cells in which the abovementioned vectors may be expressed. These include microbial cells such as bacteria such as E. coli, mammalian cells such as CHO cells, COS7 cells, P388 cells, HepG2 cells, KB cells, EL4 cells or Hela cells, insect cells, yeast such as Saccharomyces or plant cells, typically of crop plants such as wheat, maize or oil-seed rape.

Baculovirus or vaccinia expression systems may be used.

Cells may be cultured under standard conditions. Commercially available media for cell culture are widely available and can be used in accordance with manufacturer's instructions.

A calcitonin peptide which is expressed from encoding nucleic acid in host cells may be recovered by any technique known in the art. This may lead to isolation and purification of the calcitonin peptide.

Another aspect of the invention provides a method of producing a calcitonin peptide which comprises: (a) causing expression from nucleic acid which encodes a calcitonin peptide in a suitable expression system to produce the polypeptide recombinantly; or producing a synthetic calcitonin peptide, and, optionally ;

(b) testing the recombinantly produced polypeptide for calcitonin activity and/or self aggregation activity.

A calcitonin polypeptide as described herein may be manufactured and/or used in preparation, i. e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, e. g. for the treatment of a bone condition or other purpose as discussed elsewhere herein.

An aspect of the invention provides a calcitonin peptide or nucleic acid encoding a calcitonin peptide, as described above, for use in a method of treatment of the human or animal body.

Such a method may comprise administering to such a subject a therapeutically-effective amount of a calcitonin peptide or nucleic acid, preferably in the form of a pharmaceutical composition.

The term"treatment"as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e. g. in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i. e. prophylaxis) is also included.

The term"therapeutically-effective amount"as used herein, pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.

The invention further encompasses the use of a calcitonin peptide or nucleic acid encoding a calcitonin peptide as described above in the manufacture of a medicament for the treatment of a condition selected from the group consisting of Paget's disease of bone (osteitis deformans), osteoporosis, hypercalcaemia (i. e. high blood calcium levels), and pain.

The use of salmon calcitonin to treat such conditions is well established in the art.

Hypercalcaemia may be due to tumoral osteolysis secondary to breast, lung, kidney, genito-urinary (renal, cervix, uterus, ovary) cancer and other malignancies, osteolysis induced by myeloma, primary hyperparathyroidism, neurological complications, increased bone turnover reflected in elevated alkaline phosphatase and hydroxyproline secretion, progressive extension of bone lesions or incomplete or repeated fractures.

Pain may include pain associated with advanced metastatic cancer, in particular advanced metastatic bone cancer.

The invention further provides a method of treating a bone condition or other disorder as described above, for example

Paget's disease of bone (osteitis deformans), osteoporosis, hypercalcaemia, or pain, the method comprising; administering a calcitonin polypeptide or nucleic acid encoding a calcitonin peptide as described herein to an individual in need thereof.

The anorectic effect of calcitonin is well documented. The invention further provides a method of reducing appetite in an individual, for example in the treatment of obesity, comprising; administering a calcitonin polypeptide or nucleic acid encoding a calcitonin peptide as described herein to an individual in need thereof.

In a related aspect, the invention provides the use of a calcitonin peptide or nucleic acid encoding a calcitonin peptide as described above in the manufacture of a medicament for the reduction of appetite in an individual, for example in the treatment of obesity.

Another aspect of the invention provides a pharmaceutical composition comprising a calcitonin peptide as described herein and a pharmaceutically acceptable excipient, vehicle or carrier.

A pharmaceutical composition may be used to treat a bone condition or other disorder as described above.

A pharmaceutical composition may be produced by a method comprising ;

synthesising or producing a calcitonin peptide as described herein and admixing the calcitonin peptide with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials.

The precise nature of the excipient, vehicle or carrier will depend on the route of administration, which may be oral, or by injection, e. g. cutaneous, subcutaneous, intramuscular or intravenous, or intranasally.

The term"pharmaceutically acceptable"as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e. g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable"in the sense of being compatible with the other ingredients of the formulation.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the calcitonin peptide or encoding nucleic acid with the carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association calcitonin peptide or encoding nucleic acid with liquid carriers or finely

divided solid carriers or both, and then if necessary shaping the product.

Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

Formulations suitable for oral administration (e. g. by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of calcitonin peptide ; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

A tablet may be made by conventional means, e. g. , compression or moulding, optionally with one or more accessory ingredients.

Compressed tablets may be prepared by compressing in a suitable machine the calcitonin peptide in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e. g. povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose) ; fillers or diluents (e. g. lactose, microcrystalline cellulose, calcium hydrogen phosphate) ; lubricants (e. g. magnesium stearate, talc, silica) ; disintegrants (e. g. sodium starch glycolate, cross-linked povidone, cross- linked sodium carboxymethyl cellulose) ; surface-active or

dispersing or wetting agents (e. g. sodium lauryl sulfate); and preservatives (e. g. methyl p-hydroxybenzoate, propyl p- hydroxybenzoate, sorbic acid). Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered calcitonin peptide moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.

Liquid pharmaceutical compositions for oral administration generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.

Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

Formulations suitable for topical administration (e. g. transdermal, intranasal, ocular, buccal, and sublingual) may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol, or oil. Alternatively, a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active compounds and optionally one or more excipients or diluents.

Formulations for topical administration via the skin (i. e. transdermal administration) may include ointments, creams, and emulsions. When formulated in an ointment, the active compound may optionally be employed with either a paraffinic or a water- miscible ointment base. Alternatively, the active compounds may be formulated in a cream with an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i. e. , an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1, 3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. Topical formulations may desirably include a compound which enhances absorption or penetration of the active compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.

When formulated as a topical emulsion, the oily phase may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.

Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier (s) with or without stabiliser (s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.

Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono-or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required.

Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.

Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

Formulations suitable for parenteral administration (e. g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous

isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient ; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.

Typically, the concentration of the calcitonin peptide in the solution is from about 1 ng/ml to about 10 ug/ml, for example from about 10 ng/ml to about 1 ug/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.

Examples of techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.

(ed), 1980.

Administration is preferably in a"therapeutically effective amount", this being sufficient to show benefit to the individual.

It will be appreciated that appropriate dosages of the active compounds, and compositions comprising the active compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular peptide, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side- effects.

Administration in vivo can be effected in one dose, continuously or intermittently (e. g. in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose

level and pattern being selected by the treating physician. In general, a suitable dose of calcitonin peptide may be in the range of about 100 ug to about 250 mg per kilogram body weight of the subject per day.

Other aspects of the invention relate to the methods of reducing the self-aggregation activity of human calcitonin by replacing particular residues within the human calcitonin sequence.

A method of reducing the self aggregation activity of a human calcitonin peptide may comprise; producing a calcitonin peptide having the sequence of human calcitonin with substitutions of the residues at positions 11, 17,20, 24,27 and 29 in the human calcitonin sequence with a residue selected from the group consisting of P, G, S, T, Q, N, R, K, D, H and E.

Preferably, each of the residues at positions 11,17, 24,27 and 29 in the human calcitonin sequence is independently substituted with a residue selected from the group consisting of P, G, S, T, Q, N, R, K, D, and E, and the residue at position 20 in the human calcitonin sequence is independently substituted with a residue selected from the group consisting of P, G, S, T, Q, N, R, K, D, H and E, Preferably, the residues at positions 11,17 and 24 in the human calcitonin sequence are independently substituted with a residue selected from the group consisting of P, S, R, K, D, H, T and E, preferably with a residue selected from the group consisting of

P, S, R, K, D or E, more preferably with a residue selected from the group consisting of R, K, D or E, or most preferably with a residue selected from the group consisting of D or R.

Preferably, the residue at position 20 is independently substituted with a residue selected from the group consisting of P, S, R, K, D, H, T and E, preferably with a residue selected from the group consisting of P, S, R, K, H, D or E, more preferably with a residue selected from the group consisting of R, K, H, D or E, or most preferably with a residue selected from the group consisting of H, D or R.

Preferably, the residue at position 29 is independently substituted with a residue selected from the group consisting of P, S, R, K, D, H, T and E, preferably with a residue selected from the group consisting of P, S, R, K, T, D or E, more preferably with a residue selected from the group consisting of S, T, D or R. In some preferred embodiments, the residue at position 29 in the human calcitonin sequence may be independently substituted with S.

Preferably, the residue at position 27 is independently substituted with a residue selected from the group consisting of P, S, R, K, D, H, T and E, preferably with a residue selected from the group consisting of P, S, R, K, T, D or E, more preferably with a residue selected from the group consisting of S, T, D or R. In some preferred embodiments, the residue at position 27 in the human calcitonin sequence may be independently substituted with T.

The residue at position 11 in the human calcitonin sequence may be independently H or T, preferably S or P, more preferably E or K or most preferably D or R. In some preferred embodiments, the residue at position 11 in the human calcitonin sequence may be independently substituted with R.

The residue at position 17 in the human calcitonin sequence may be independently H or T, preferably S or P, more preferably E or K or most preferably D or R. In some preferred embodiments, the residue at position 17 in the human calcitonin sequence may be independently substituted with R.

The residue at position 24 in the human calcitonin sequence may be independently H or T, preferably S or P, more preferably E or K or most preferably D or R. In some preferred embodiments, the residue at position 24 in the human calcitonin sequence may be independently substituted with R.

The residue at position 20 in the human calcitonin sequence may be independently T, preferably S or P, more preferably E or K or most preferably H, D or R. In some preferred embodiments, the residue at position 20 in the human calcitonin sequence may be independently substituted with H or R.

The self aggregation activity of the peptide having said modified sequence may be determined, for example by determining changes in turbidity of an aqueous solution of the modified peptide over time.

Methods which involve replacing particular residues within the human calcitonin sequence as described above may also be used to increase the level and/or duration of activity of human calcitonin.

Peptides of the invention may be produced by chemical synthesis or by recombinant expression from an encoding nucleic acid.

Methods for the production of peptides are well known in the art and are described in more detail above.

When encoding nucleic acid is employed, residues may be substituted by site directed mutagenesis of the appropriate residues of the encoding nucleic acid using conventional techniques.

All documents mentioned in this specification are hereby incorporated herein by reference.

It will be understand that the invention encompasses each and every combination and sub-combination of the features of the invention which are described above.

Aspects of the present invention will now be illustrated with reference to the accompanying figures described above and experimental exemplification, by way of example and not limitation. Further aspects and embodiments will be apparent to those of ordinary skill in the art.

Experimental 1. Calcitonin Peptides Naturally occurring Calcitonin sequences are as follows: CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP Human SEQ ID NO 1 CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP Salmon SEQ ID NO 2 The following peptides were synthesised and compared to Human and Salmon calcitonin.

CGNLSTCMLGRYTQDFRKFHTFPRTATGSGAP 5P SEQ ID NO 4 CGNLSTCMLGRYTQDFRKFRTFPRTATGSGAP 6S SEQ ID NO 5 Peptides were compared with hCT, sCT and 6chR (Zurdo & Dobson, WO 02/083734, PCT/GB02/01778).

All peptides were synthesised with a cyclic N-terminus (disulfide bridge between Cys residues 1 and 7) and C-terminus amidation.

2. Aggregation studies Aggregation analysis was carried out as reported previously (Arvinte, T. et al., 1993, J. Biol. Chem, 268,6415-6422) using PBS 5mM sodium phosphate, 145 mM NaCl, pH 7.2. (sodium phosphate saline buffer) pH 7.2 and adding a minor amount of sodium azide to prevent bacterial growth (0.1%). Samples were always incubated at 37°C. Aggregation was monitored by measuring turbidity at 340 nm.

The self-aggregation activity of hCT, sCT and 6chR compared to 5P and 6S variants when dissolved at a peptide concentration of 2

mg/mL is shown in figure 1. The aggregation behaviour of all the variants was tested at 37°C and 50°C in PBS (phosphate saline buffer: 10 mM potassium phosphate, 150 mM NaCl) pH 7.2. The graphs indicate clearly that the two calcitonin peptides 5P and 6S aggregate at a much lower rate than the other forms (hCT, sCT and 6chR). Moreover, the two variants 5P and 6S also show a much lower degree of aggregation than any of the other peptides tested. Table 1 reflects the percentage of soluble peptide recovered after the kinetics of aggregation reaches a stationary phase. Both 5P and 6S show a much higher recovery than any of the other three forms of calcitonin tested (hCT, sCT and 6chR) 3. [l25I] hCT binding inhibition and cAMP stimulation of Calcitonin peptides in T47D cells Methods T47D cells were grown in Dulbecco's modified Eagle's medium containing 4.5 g/1 glucose and Ham's F12 medium (1 : 1) supplemented with 2mM L-glutamine, 100 nM dexamethasone and 10% FCS in 5% CO2, 95% air. For binding studies and cAMP measurements the cells were seeded into 24-well plates at a density of 100'000 cells/well and allowed to get confluent.

Lyophilized aliqouts of peptides were dissolved with PBS containing 20 mg/ml D-mannitol to a stock concentration of 100 M and used immediately. Fresh stocks were prepared for each experiment.

Human CT was iodinated with the use of a modified chloramine T method and purified by HPLC as described previously [Hunter, W.

M. et al (1962) Nature 194, 495-498 ; Hussain, A. A. et al (1995) Anal. Biochem. 224, 221-226; Zimmermann, U. et al (1997) J.

Endocrinol. 155, 423-431].

For binding studies, cell culture medium without FCS and dexamethasone but supplemented with 0. 1% BSA was used (binding medium). The cells were incubated for 5 h at 15°C with 200 1 binding medium with 1700 Bq [125I] hCT in the absence and presence of unlabeled peptides. After aspiration of the incubation medium the cells were washed once with 500 pl cold binding medium, lysed with 500 1 0. 5% SDS and lysates counted in a y-counter.

For measurement of cAMP accumulation the cells were incubated in 200 j-t, l binding medium supplemented with lmM isobutylmethylxanthine for 15 min at 37°C in the absence and presence of peptides. After aspiration of the medium the cAMP was extracted from the monolayers with 500 pl ice cold 95% ethanol, pH 3 for 1 h at 15°C. Extracts were then evaporated in a SpeedVac concentrator, reconstituted and cAMP determined by RIA as described [Moran, J. et al (1978) Proc. Natl. Acad. Sci. USA 75, 3984-3988].

For the determination of the reversibility of the interaction of peptides with CT receptors a three-phase incubation procedure was applied. First, the cells were incubated for 15 min at 37°C in 200 all binding medium with 100 nM unlabeled peptides or left untreated. Then, the cells were washed three times with 500 pl binding medium and incubated for 6 h at 37°C with two washing steps every 2 h, resulting in a total of nine washing steps. Then

the cells were incubated for 15 min at 37°C in 200 pl binding medium containing 1 mM isobutylmethylxanthine with or without 100 nM unlabeled peptides. cAMP was then determined as described.

This resulted in three groups of treatments, cells stimulated without pretreatment, unstimulated cells after pretreatment and cells stimulated after pretreatment.

Results As described above, T47D cells were incubated for 5 h at 15°C with 1700 Bq [l25I] hCT in the absence (control) or presence of hCT (EI), sCT (o), 6CH-R (g), 6S (0), and 5P (0) as shown in Figure 2.

[125I] hCT results in Figure 2 are means of three independent experiments. Total [125I] hCT binding was 197 27 Bq/500'000 cells, which amounted to 12 2% of total ligand added (1700 Bq).

Nonspecific binding ranged from 15-25% of total binding. hCT displaced [125I] hCT binding with IC50 of 3220 240 pM (Table 2; Figure 2). sCT, 6CH-R, 6S and 5P were equipotent and about 10- fold more potent than hCT (P < 0.01).

For cAMP stimulation the cells were incubated for 15 min at 37°C in the presence of 1mM isobutylmethylxanthine. Basal cAMP levels as shown in Figure 2 ranged from 2-6 pmol/500'000 cells and maximal cAMP levels from 500-3000 pmol/500'000 cells. sCT, hCT, 6CH-R, 6S and 5P maximally stimulated cAMP accumulation 300-360- fold over basal levels. hCT stimulated cAMP with an EC50 of 231 73 pM (Figure 2, Table 2). sCT, 6CH-R and 6S were equipotent and as a group about 2-fold more potent than hCT. 5P was 2-fold more potent than sCT, 6CH-R, and 6S and 5-fold more potent than hCT (P

< 0.05). The cAMP results shown in Figure 2 are means of six independent experiments.

To assess for the reversibility of the interaction of peptides with the CTR, the T47D cells were pretreated with a maximal concentration (100 nM) of unlabeled peptides for 15 min at 37°C (hatched and closed bars) or left untreated (open bars).

Isobutylmethylxanthine was omitted during this period. Cells were then washed three times and incubated for 6 h at 37°C in the absence of peptides with two washing steps each two hour.

Cells were then stimulated in the presence of isobutylmethylxanthine with 100 nM peptides for 15 min at 37°C (open and closed bars) or left untreated (crossed bars). cAMP levels were then measured. The results shown in Figure 3 for cells without pretreatment (acute: open) and after pretreatment (restimulated: closed), and in pretreated cells but without a second incubation with peptides (recovered: crossed) are means SEM of four independent experiments.

Maximal cAMP levels were the same for all peptides in acutely stimulated cells (Figure 3). In recovered cells prestimulated with hCT, cAMP levels returned to 17 4% of that in acutely stimulated cells (P < 0.01) indicating partial recovery. In contrast, cAMP levels were indistinguishable from acutely stimulated cells after recovery from pretreatment with sCT, 6CH- R, 6S and 5P indicating persistent activation. Levels of cAMP after restimulation of pretreated cells were similar to levels in acutely stimulated cells for all of the peptides, indicating that

no significant receptor downregulation has occurred during the chase period.

4. Hypocalcaemic activity of calcitonin variants in rats The aim of this study was to test the efficacy of calcitonin peptides in a rat hypercalcemia model as a treatment of osteoporosis. Calcitonin has been used in various forms in the treatment of osteoporosis. It results in stronger bones and reduces the risk of fracture. The current therapeutic model is a native sequence from salmon or human calcitonin. Two variants of native calcitonin were tested in this study.

Vehicle preparation Fifty ml of deionised water was added to the falcon tube containing the lyophilised vehicle. It was mixed until all components were totally dissolved. Once reconstituted the composition of the vehicle is: 10 mM Phosphate, 137 mM NaCl, 2.7 mM KC1, 20 mg/mL D-mannitol, pH 7.4.

Preparation of Calcitonin solutions Calcitonin variants where resuspended at a concentration of 2.0 mg/ml in 8.0 mg/ml of D-mannitol in bi-distilled water. Aliquots of 100 ul were lyophilised and kept at-20C until the experiment was performed. To one eppendorf containing human calcitonin and mannitol, 1 mL of vehicle was added, mixed by gentle inversion of the tube until all solid material were fully dissolved. This stock solution has a peptide concentration of 0.2 mg/mL.

This stock solution was diluted to make 3 dosing solutions with concentrations of 0.0001 mg/mL, 0.0003 mg/mL and 0.001 mg/mL.

Final doses administered to the rats were 0.1, 0.3 and 1.0 ug/kg. Doses were established according to the individual weights of the animals.

Animal Management A total of eighty-five (85) male Sprague-Dawley rats, were obtained from Harlan Sprague Dawley with eighty (80) animals being used on study. The spare animals were humanely euthanized at the end of the study. The rats were approximately 140 grams upon receipt and 165 grams at study initiation. Upon receipt, rats were unpacked and placed in cages. Each animal was evaluated for health status and all animals were found in good health. The animals were allowed to acclimatize for one week prior to study activities. The temperature was maintained at 18- 26°C (64-79°F) with a relative humidity of 30-70%. The rooms were maintained on a 12-hour light/dark cycle (light hours being approximately 0600 to 1800). The rats had ad libitum access to regular rodent chow and deionized water.

Experimental Procedures Over a course of three days, the animals underwent timed bleeds and dosing. The animals were briefly sedated by inhalation anesthetic (ether) prior to all blood draws. The blood samples were collected using retro-orbital bleed (SOP ST-AEP009).

Immediately prior to dosing, pre-dose blood was collected. Blood (0.5 ml) was collected into a serum separator tube. Based on treatment group, the stated dose was delivered to the animals

with a single subcutaneous injection. The doses were adjusted for individual animal body weight taken the day before the test. At 20,60 and 120 minutes post-dose, 0.5 mL of blood was collected (at each time point) into a serum separator tube.

The blood samples were centrifuged and the serum separated.

Total serum calcium was analyzed on a COBAS MIRA automated analyzer. The changes of serum calcium over the treatment period were examined using the incremental changes, i. e. each time point is expressed as the difference of serum calcium at each time point from the zero time point of the same animal. Results are presented as mean +/-SD.

Summary of Findings Intact, young male rats were given vehicle or one of 3 different doses (0.1, 0.3 and 1.0 ug/kg) of one calcitonin variant via a subcutaneous injection. The response of the serum calcium to calcitonin was followed immediately prior to the calcitonin administration, and 20,60 and 120 minutes after the calcitonin administration.

6S molecules elicited a rapid hypocalcemic action that was short in duration. At two lower doses of 6S, serum calcium was observed to have a quick rebound over and above the normal levels 60 minutes after its administration. The 5P molecule show a long and sustained response at all time points examined (figure 4).

List of Sequences CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP Human SEQ ID NO 1 CSNLSTCVLGKLSQELHKLQTYPRTNTGSGTP Salmon SEQ ID NO 2 CGNLSTCMLGX1YTQDFX2KFX3TFPX4TAX5GX6GAP Consensus SEQ ID NO 3 Where Xi-g are substituted preferably by a polar (preferably S, T, Q, N), charged residue (preferably R, K, D, E), P or G. With preference of P, T, S, R, K, D or E at positions X5-6 and P, R, K, D or E at positions X1_4.

CGNLSTCMLGRYTQDFRKFHTFPRTATGSGAP 5P SEQ ID NO 4 CGNLSTCMLGRYTQDFRKFRTFPRTATGSGAP 6S SEQ ID NO 5 CGNLSTCMLGDYTQDFDKFHTFPDTADGDGAP SEQ ID NO 6 CGNLSTCMLGRYTQDFDKFHTFPDTADGDGAP SEQ ID NO 7 CGNLSTCMLGRYTQDFDKFHTFPPTADGDGAP SEQ ID NO 8 CGNLSTCMLGRYTQDFDKFHTFPDTADGPGAP SEQ ID NO 9 CGNLSTCMLGRYTQDFRKFHTFPRTARGPGAP SEQ ID NO 10 CGNLSTCMLGDYTQDFNKFHTFPDTADGDGAP SEQ ID NO 11 CGNLSTCMLGRYTQDFNKFHTFPDTADGDGAP SEQ ID NO 12

CGNLSTCMLGTYTQDFDKFHTFPDTADGDGAP SEQ ID NO 13 CGNLSTCMLGDYTQDFNKFHTFPDTARGDGAP SEQ ID NO 14 CGNLSTCMLGDYTQDFNKFHTFPDTADGRGAP SEQ ID NO 15 CGNLSTCMLGRYTQDFDKFHTFPQTADGDGAP SEQ ID NO 16 CGNLSTCMLGDYTQDFRKFHTFPQTADGDGAP SEQ ID NO 17 CGNLSTCMLGDYTQDFDKFHTFPQTADGRGAP SEQ ID NO 18 CGNLSTCMLGDYTQDFDKFHTFPQTARGDGAP SEQ ID NO 19 CGNLSTCMLGDYTQDFNKFHTFPPTADGDGAP SEQ ID NO 20 CGNLSTCMLGDYTQDFNKFHTFPDTAPGDGAP SEQ ID NO 21 CGNLSTCMLGTYTQDFRKFHTFPDTADGDGAP SEQ ID NO 22 CGNLSTCMLGDYTQDFNKFHTFPQTADGDGAP SEQ ID NO 23 CGNLSTCMLGTYTQDFNKFHTFPDTADGDGAP SEQ ID NO 24 CGNLSTCMLGDYTQDFNKFHTFPQTARGDGAP SEQ ID NO 25 CGNLSTCMLGDYTQDFNKFHTFPQTADGRGAP SEQ ID NO 26

CGNLSTCMLGDYTQDFNKFHTFPDTADGVGAP SEQ ID NO 27 CGNLSTCMLGRYTQDFNKFHTFPQTARGRGAP SEQ ID NO 28 CGNLSTCMLGRYTQDFNKFHTFPQTARGDGAP SEQ ID NO 29 CGNLSTCMLGRYTQDFNKFHTFPQTADGRGAP SEQ ID NO 30 CGNLSTCMLGDYTQDFNKFHTFPQTARGRGAP SEQ ID NO 31 CGNLSTCMLGDYTQDFNKFHTFPQTAPGDGAP SEQ ID NO 32 5 Table 1 [125IJCT binding inhibition cAMP stimulation IC50 (pM) EC50 (pM) hCT 3220 ~ 240 231 ~ 73b sCT 224 ~ 25a 131 ~ 18b,c 6CH-R 416 ~ 40a 96 ~ 20b,c 6S 408 ~ 106a 115 ~ 27b,c 5P 154 34a 8 7 5 ap < 0.01 versus hCT ; bp < 0.05 versus 5P. cp < 0.05 versus hCT when treated as a group of equipotent peptides.