Calcitonin derivatives

The present invention relates to compounds represented by formula (I): ##STR1## wherein Z represents Gly or Cys; X represents an (.alpha.-amino acid residue; Y represents a natural calcitonin moiety, a partial natural calcitonin peptide moiety, or a natural calcitonin-like peptide moiety; m represents an integer of 5-8, .alpha.-amino acid residues represented by X being the same or different; and n represents an integer of 0-3; provided that when m is 5, the sequence of 4 C-terminal residues of --(X).sub.m -- is different from the sequence of the third to sixth amino acids of natural calcitonin, and pharmaceutically acceptable salts thereof.

TECHNICAL FIELD 
The present invention relates to calcitonin derivatives that have a 
biological activity, in which a cyclic peptide having a particular 
structure is bonded to calcitonin, a partial calcitonin peptide or an 
analogue thereof having an amino acid sequence necessary for the 
expression of a calcitonin-like biological activity optionally via a 
spacer. 
The peptides of the present invention have higher activity and/or stability 
than calcitonin, partial calcitonin peptides, or analogues thereof. 
BACKGROUND ART 
So far, calcitonin, derived from eel, salmon, human beings, pigs, fowl, 
cattle, sheep, rats and stingray, has been known as natural calcitonin. 
Calcitonin peptides of such various origins are all polypeptides 
consisting of 32 amino acids that have the common characteristics, that 
is, the first and the seventh amino acids are L-cysteine, mercapto groups 
of these two amino acids form a disulfide bond and the carboxyl terminals 
are prolinamide. 
The disulfide bond of these various calcitonins is expected to be unstable 
in a solution. To solve this problem, calcitonin derivatives were known to 
be prepared in which the first amino acid cysteine was deleted, the 
seventh amino acid cysteine was replaced by .alpha.-amino acid having a 
kind of lower carboxyalkylene group, and the side-chain carboxyl group of 
this amino acid and .alpha.-amino group of the second amino acid were 
combined to form an amide bond (Japanese Published Unexamined Patent 
Application Nos. 128993/76, 59688/78, and 112099/86). Particularly, the 
analogues based on the eel calcitonin sequence have been provided for 
practicable use as therapeutic agents against bone Paget disease, 
hypercalcemia, and osteoporosis. Analogues of this type in which single 
bonds in alkylene chain are partially replaced by double bond(s) or triple 
bond(s) are also known (WO 93/15106). As calcitonin analogues prepared for 
the same purpose, there are also known polypeptides in which the first 
amino acid is replaced by glycine or .beta.-alanine, the seventh amino 
acid is replaced by aspartic acid or glutamic acid, and an amide bond is 
formed between the .alpha.-amino group of the former and the side-chain 
carboxyl group of the latter, and peptides in which alkylene groups are 
partially replaced by phenylene group in addition to the above 
modifications (Japanese Published Unexamined Patent Application Nos. 
262595/90 and 178993/91). 
Further, many kinds of calcitonin analogues prepared for the purpose of 
improving physiological activities of natural calcitonin have been 
reported [e.g., Endocrinology, vol. 117, p. 801 (1985), Eur. J. Biochem., 
vol. 159, p. 125 (1986), Biochem. Biophys, Res. Commun., vol. 152, p. 203 
(1988), Endocrinology, vol. 127, p. 163 (1990)]. 
DISCLOSURE OF THE INVENTION 
The present invention relates to compounds represented by formula (I): 
##STR2## 
wherein Z represents Gly or Cys; X represents an .alpha.-amino acid 
residue; Y represents a natural calcitonin moiety, a partial natural 
calcitonin peptide moiety, or a natural calcitonin-like peptide moiety; m 
represents an integer of 5-8 .alpha.-amino acid residues represented by X 
being the same or different; and n represents an integer of 0-3; provided 
that when m is 5, the sequence of 4 C-terminal residues of --(X).sub.m -- 
is different from the sequence of the third to sixth amino acids of 
natural calcitonin, and pharmaceutically acceptable salts thereof. 
Hereinafter, peptide compounds represented by formula (I) are referred to 
as Compounds (I). 
In the definition of formula (I), the .alpha.-amino acid residue means a 
residue of natural amino acids such as glycine, L- or D-alanine, 
asparagine, aspartic acid, arginine, cysteine, glutamine, glutamic acid, 
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, 
proline, serine, threonine, tryptophan, tyrosine, and valine, or a residue 
of non-natural amino acids such as .beta.-alanine, .gamma.-aminobutyric 
acid, aminobenzoic acid, L- or D-hydroxyproline, norvaline, and 
.beta.-2-naphthylalanine. 
Examples of --(X).sub.m -- in formula (I) are -X.sup.1 -Trp-X.sup.2 
-Gly-Thr-Ala-X.sup.3 -(SEQ ID NO: 35) (wherein X.sup.1 represents Asn or 
Asp; X.sup.2 represents His or Lys; and X.sup.3 represents Pro or Ala), 
-Ser-Ala-Ala-Val-Tyr-Phe-(SEQ ID NO: 36), -Phe-Ile-Gly-Trp-Gly-Asn-(SEQ ID 
NO: 37), -Tyr-Pro-Trp-Trp-Asn-Tyr-Arg-(SEQ ID NO: 38), and 
-Leu-Gly-Val-Gly-Ser-X.sup.4 -Asn-(SEQ ID NO: 39) (wherein X.sup.4 
represents Cys, Ala or Ser). 
The natural calcitonin moiety represented by Y means a peptide moiety 
having a natural calcitonin-like physiological activity. The partial 
natural calcitonin peptide moiety or natural calcitonin-like peptide 
moiety represented by Y means a peptide moiety whose amino acid sequence 
has at least more than 20% homology to at least one amino acid sequence of 
natural calcitonin moiety, and the moiety represented by the following 
formula (II) may be given as an example: 
##STR3## 
wherein P.sup.1 represents a single bond, Cys-Gly-Asn-Leu-Ser-Thr-Cys (SEQ 
ID NO: 40), Ser-Gly-Asn-Leu-Ser-Thr-Ser (SEQ ID NO: 41), 
Cys-Ser-Asn-Leu-Ser-Thr-Cys (SEQ ID NO: 42) or Ser-Ser-Asn-Leu-Ser-Thr-Ser 
(SEQ ID NO: 43); P.sup.2 represents Val, Met, Gly or a single bond; 
P.sup.3 represents Gly-Lys, Ala-Ala, Gly-Thr or Gly-Ser; P.sup.4 
represents Leu or Tyr; P.sup.5 represents Ser-Gln-Glu, Ala-Ala-Ala, 
Thr-Gln-Asp, Thr-Glu-Val or Thr-Gla-Val; P.sup.6 represents Leu or Phe; 
P.sup.7 represents His-Lys, Ala-Ala, Asn-Lys or Ala-Lys; P.sup.8 
represents Gln, Ala or His; P.sup.9 represents Thr, Ala, Glu or Gla; 
P.sup.10 represents Tyr, Phe or Leu; P.sup.11 represents Pro or Hyp; 
P.sup.12 represents Arg, Gln, Lys or D-Arg; P.sup.13 represents Thr or 
Ser; P.sup.14 represents Asn, Gln or Ala; P.sup.15 represents Thr or Ile; 
P.sup.16 represents Gly or .beta.-Ala; P.sup.17 represents Ser, Val or 
Ala; P.sup.18 represents Thr or Ala; and P.sup.19 represents amino group 
or a group represented by the following formula (III). In the amino acid 
sequence represented by formula (II), at least one amino acid may be 
deleted, inserted or substituted. 
##STR4## 
The pharmaceutically acceptable salts of Compounds (I) include acid 
addition salts, metal salts, and organic base addition salts. Examples of 
the pharmaceutically acceptable acid addition salts are inorganic acid 
addition salts such as hydrochloride, sulfate, and phosphate, and organic 
acid addition salts such as acetate, maleate, fumarate, tartrate, and 
citrate. Examples of the pharmaceutically acceptable metal salts are 
alkali metal salts such as sodium salt and potassium salt, alkaline earth 
metal salts such as magnesium salt and calcium salt, aluminum salt, and 
zinc salt. Examples of the pharmaceutically acceptable organic base 
addition salts are salts with primary amines such as methylamine, 
ethylamine, and aniline, secondary amines such as dimethylamine, 
diethylamine, pyrrolidine, piperidine, morpholine, and piperazine, and 
tertiary amines such as trimethylamine, triethylamine, 
N,N-dimethylaniline, and pyridine, and ammonium salts. 
The present invention is described in detail below. 
The abbreviations for amino acids and their protecting groups used herein 
follow the recommendations by IU-IUB Joint Commission on Biochemical 
Nomenclature [Eur. J. Biochem., vol. 138, p. 9 (1984)]. 
The abbreviations for amino acids and their protecting groups are as 
follows, unless otherwise specified. 
Gly; Glycine 
Ala; L-Alanine 
.beta.-Ala; .beta.-Alanine 
Thr; L-Threonine 
Pro; L-Proline 
Hyp; Trans-4-hydroxy-L-proline 
Asp; L-Aspartic acid 
Asn; L-Asparagine 
Asx; L-Aspartic acid or L-asparagine 
Glu; L-Glutamic acid 
Gln; L-Glutamine 
Gla; .gamma.-Carboxy-L-glutamic acid 
Glx; L-Glutamic acid, L-glutamine or .gamma.-carboxy-L-glutamic acid 
His; L-Histidine 
Trp; L-Tryptophan 
Val; L-Valine 
Leu; L-Leucine 
Ser; L-Serine 
Met; L-Methionine 
Cys; L-Cysteine 
Ile; L-Isoleucine 
Phe; L-Phenylalanine 
Tyr; L-Tyrosine 
Lys; L-Lysine 
Arg; L-Arginine 
D-Arg; D-Arginine 
Fmoc; 9-Fluorenylmethyloxycarbonyl 
t-Bu; t-Butyl 
Trt; Trityl 
Bzl; Benzyl 
Bzl(NO.sub.2); p-Nitrobenzyl 
Pmc; 2,2,5,7,8-Pentamethylchroman-6-sulfonyl 
Boc; t-Butyloxycarbonyl 
The abbreviations for side-chain-protected amino acids are as follows. 
Fmoc-Asp-OBzl (NO.sub.2); N.sup..alpha. 
-9-Fluorenylmethyloxycarbonyl-L-aspartic acid p-nitrobenzyl ester 
Fmoc-Asp (Ot-Bu)-OBzl (NO.sub.2); .beta.-t-Butyl .alpha.-p-nitrobenzyl 
N.sup..alpha. -9-fluorenylmethyloxycarbonyl-L-aspartate 
Fmoc-Asp(Ot-Bu)-OH; N.sup..alpha. -9-Fluorenylmethyloxycarbonyl-L-aspartic 
acid .beta.-t-butyl ester 
Fmoc-Glu(Ot-Bu)-OH; N.sup..alpha. -9-Fluorenylmethyloxycarbonyl-L-glutamic 
acid .gamma.-t-butyl ester 
Fmoc-Gla(Ot-Bu).sub.2 -OH; y,y-Di-t-butyl N.sup..alpha. 
-9-fluorenylmethyloxycarbonyl-.gamma.-carboxy-L-glutamate 
Fmoc-His(Trt)-OH; N.sup..alpha. -9-Fluorenylmethyloxycarbonyl-N.sup.im 
-trityl-L-histidine 
Fmoc-Thr(t-Bu)-OH; N.sup..alpha. 
-9-Fluorenylmethyloxycarbonyl-O.beta.-t-butyl-L-threonine 
Fmoc-Ser(t-Bu)-OH; N.sup..alpha. 
-9-Fluorenylmethyloxycarbonyl-O-t-butyl-L-serine 
Fmoc-Tyr(t-Bu)-OH; N.sup..alpha. 
-9-Fluorenylmethyloxycarbonyl-O-t-butyl-L-tyrosine 
Fmoc-Hyp(t-Bu)-OH; N.sup..alpha. 
-9-Fluorenylmethyloxycarbonyl-O-t-butyl-trans-4-hydroxy-L-proline 
Fmoc-Lys(Boc)-OH; N.sup..alpha. 
-9-Fluorenylmethyloxycarbonyl-N.sup..epsilon. -t 
-butyloxycarbonyl-L-lysine 
Fmoc-Asn(Trt)-OH; N.sup..alpha. -9-Fluorenylmethyloxycarbonyl-N.sup..gamma. 
-trityl-L-asparagine 
Fmoc-Gln(Trt)-OH; N.sup..alpha. -9-Fluorenylmethyloxycarbonyl-N.sup..delta. 
-trityl-L-glutamine 
Fmoc-Arg(Pmc)-OH; N.sup..alpha. -9-Fluorenylmethyloxycarbonyl-N.sup.g 
-2,2,5,7,8-pentamethylchroman-6-sulfonyl-L-arginine 
H-Trp-OBzl; L-Tryptophanbenzylester 
The abbreviations for reaction solvents, reaction reagents, etc. are as 
follows. 
PyBOP; Benzotriazol-1-yloxytrispyrrolidinophosphonium hexafluorophosphate 
HOBt; N-Hydroxybenzotriazole 
NMM; N-Methylmorpholine 
DMF; N,N-Dimethylformamide 
DCM; Dichloromethane 
TFA; Trifluoroacetic acid 
DIEA; Diisopropylethylamine 
Pd/C; Palladium on carbon catalyst 
.alpha.MEM; Minimum medium 
FCS; Fetal calf serum 
BSA; Bovine serum albumin 
HEPES; N-2-Hydroxyethylpiperazine-N'-2-ethansulfonic acid 
PBS; Phosphate-buffered saline 
TRAP; Tartaric acid-resistant acidic phosphatase 
The process for producing Compounds (I) is described below. 
##STR5## 
In the above formulae, Z, X, Y, m and n have the same significances as 
defined above. 
The cyclic peptide moiety of Compound (I) can be obtained by synthesizing a 
partial peptide with appropriately protected side chain by the use of a 
peptide synthesizer described below or according to a conventional 
liquid-phase peptide synthetic method (Fundamentals and Experiments of 
Peptide Synthesis, Nobuo Izumiya et al., Maruzen), and subjecting the 
resulting product to cyclization using a condensing agent such as PyBOP. 
Compound (I) can be obtained by condensing the above cyclic peptide and a 
C-terminal straight chain peptide which is obtained by the use of a 
peptide synthesizer and/or according to a liquid-phase peptide synthetic 
method. 
The synthesis of a peptide by the use of a peptide synthesizer is carried 
out with commercially available peptide synthesizers from Shimadzu 
Corporaten, Applied Biosystems, Inc., U.S.A. (ABI), etc. using an 
appropriately side-chain-protected N.sup..alpha. 
-9-fluorenylmethyloxycarbonyl amino acid according to respective synthesis 
programs. 
Protected amino acids which are starting materials for the synthesis of 
Compound (I) and carrier resins are available from ABI, Shimadzu 
Corporation, Kokusan Chemical Works Co., Ltd., Nova Biochem Co., Watanabe 
Chemical Co., Ltd. and Peptide Institute Co., Ltd. 
Compound (I) thus obtained can be purified by high performance liquid 
chromatography (hereinafter referred to as HPLC) using C-4, C-8, or C-18 
reversed-phase silica gel column, partition, column chromatography using 
adsorption resins, silica gel, chemically-modified silica gel, 
reversed-phase silica gel, alumina, diatomaceous earth, magnesium 
silicate, or ion-exchange resins, gel filtration column chromatography, or 
thin layer chromatography. 
The pharmaceutically acceptable salts of Compound (I) are obtained 
according to an ordinary method. That is, the acid addition salts and 
organic base addition salts of Compound (I) are obtained by dissolving 
Compound (I) in an aqueous solution of the corresponding acid or organic 
base, followed by freeze-drying. The metal salts of Compound (I) are 
obtained by dissolving Compound (I) in an aqueous solution containing the 
corresponding metal ions, followed by purification by gel filtration or 
HPLC. 
Specific examples of Compounds (I) are shown in Table 1. 
TABLE 1 
__________________________________________________________________________ 
Compound No. 
Sequence 
__________________________________________________________________________ 
Compound 1 
1 (SEQ ID NO:1) 
- Compound 2 
2 (SEQ ID NO:2) 
- Compound 3 
3 (SEQ ID NO:3) 
- Compound 4 
4 (SEQ ID NO:4) 
- Compound 5 
5 (SEQ ID NO:5) 
- Compound 6 
6 (SEQ ID NO:6) 
- Compound 7 
7 (SEQ ID NO:7) 
- Compound 8 
8 (SEQ ID NO:8) 
- Compound 9 
9 (SEQ ID NO:9) 
- Compound 10 
0 (SEQ ID NO:10) 
- Compound 11 
1 (SEQ ID NO:11) 
- Compound 12 
2 (SEQ ID NO:12) 
- Compound 13 
3 (SEQ ID NO:13) 
- Compound 14 
4 (SEQ ID NO:14) 
__________________________________________________________________________ 
G; Gly, A; Ala, N; Asn, Q; Gln, K; Lys, R; Arg, D; Asp, E; Glu, T; Thr, S 
Ser, L; Leu, I; Ile, V; Val, H; His, M; Met, P; Pro, F; Phe, Y; Tyr, W; 
Trp, r; DArg, 
##STR20## 
The biological activity and the stability to protease of Compounds 1-14 ar 
described in the following test examples. 
TEST EXAMPLE 1 
Calcitonin-Like Biological Activity 
1-1 Preparation of Osteoclasts 
Osteoclast-like multinucleated cells which were derived by coculturing 
mouse osteoblasts and bone marrow cells on collagen gel according to the 
method described in Akatsu T. et al., J. Bone Miner. Res., 7, 1297-1306 
(1992) were used as osteoclasts. That is, osteoblasts derived from mouse 
vault of skull (5.times.10.sup.5 cells) were put in a 100 mm dish (IWAKI) 
collagen-coated with Cellmatrix Type I-A (Nitta Gelatin Co., Ltd.) and 
cultured for one day in a CO.sub.2 incubator (37.degree. C., 5% CO.sub.2) 
[medium: .alpha.MEM+10% FCS (both by GIBCO Co., Ltd.)]. After the medium 
was removed, mouse bone marrow cells (6.times.10.sup.6 cells) were put on 
the osteoblasts and 10.sup.-8 M calcitriol (Wako Pure Chemical Industries, 
Ltd.) and 10.sup.-7 M dexamethasone (Sigma Chemical Co.) were added 
thereto. TRAP-positive multinucleate cells (osteoclast-like cells) which 
were obtained 7 days after the inoculation of osteoblasts were used as 
osteoclasts. 
1-2 Culturing of Osteoclasts on Ivory Pieces 
After removal of the medium from the dish, and rinsing with PBS(-), a 
mixture of collagenase and dispase (collagenase: Wako Pure Chemical 
Industries, Ltd., dispase: Godo Shusei Co., Ltd.) was added thereto to 
suspend the cells. The cell suspension was put into a tube and centrifuged 
at 800 rpm for 5 minutes. After removal of the supernatant by suction, the 
cells were suspended again in .alpha.MEM and the suspension was put on 
ivory pieces (diameter: 4 mm, thickness: 20 .mu.m) in 100 .mu.l portions. 
The ivory pieces were left to stand in a CO.sub.2 incubator for 2 hours to 
attach the osteoclasts thereto, and then taken out and gently transferred 
to a 48-well plate (IWAKI) to which a medium containing a test compound 
had been added in advance to determine the bone resorption activity 48 
hours later according to the method described below. The medium containing 
the test compound was prepared by adding the test compound dissolved in 
HEPES buffer to the medium described in 1-1 to give the final 
concentrations shown in Tables 2 and 3. 
1-3 Determination of Bone Resorption Activity by Staining of Ivory Pieces 
and Measurement of Bone Resorption Pit Area 
The ivory pieces taken out of the medium were put into a tube containing 
0.1 N aqueous ammonia and subjected to sonication treatment with a 
sonicator for 20-30 seconds to remove the osteoclasts. After being washed 
with distilled water to remove ammonia, the ivory pieces were dipped in a 
hematoxylin-eosin staining solution to stain resorption pits. The bone 
resorption pit formation rate was calculated based on microphotographs of 
stained ivory pieces according to the following equation by the use of an 
image analyzer. 
##EQU1## 
The bone resorption inhibitory activity of test compounds were calculated 
according to the following equation. 
##EQU2## 
A; Bone resorption pit formation rate obtained using no test compound 
B; Bone resorption pit formation rate obtained using a test compound 
The results are shown in Tables 2 and 3. 
TABLE 2 
______________________________________ 
Compound 
concentration Bone resorption inhibitory 
Compound No. (M) activity (% inhibition) 
______________________________________ 
Compound 1 10.sup.-7 61 
Compound 17 10.sup.-7 0 
Compound 2 10.sup.-8 36 
Compound 18 10.sup.-8 15 
Compound 3 10.sup.-6 65 
Compound 19 10.sup.-6 6 
______________________________________ 
TABLE 3 
______________________________________ 
Compound 
concentration Bone resorption inhibitory 
Compound No. (M) activity (% inhibition) 
______________________________________ 
Compound 4 10.sup.-6 29 
Compound 5 10.sup.-6 35 
Compound 6 10.sup.-6 29 
Compound 7 10.sup.-8 50 
Compound 8 10.sup.-6 39 
Compound 9 10.sup.-6 53 
Compound 10 10.sup.-6 27 
Compound 11 10.sup.-7 42 
Compound 12 10.sup.-7 59 
Compound 13 10.sup.-6 35 
Compound 14 10.sup.-6 40 
______________________________________ 
TEST EXAMPLE 2 
Stability to Prolylendopeptidase 
A test compound was dissolved in a PBS(-) buffer (pH 7.2) containing 0.01% 
sodium azide and 0.1 mM calcium chloride to give a concentration of 25 
.mu.g/ml. To the solution was added prolylendopeptidase (Seikagaku 
Corporation) in an amount of 1/50 weight of the test compound. The 
resulting mixture was incubated at 37.degree. C. in a thermostat and 
sampled at intervals. The samples were analyzed by HPLC using a 
reversed-phase column (YMC-Pack ODS-AM 150.times.6 mm I.D.). Elution was 
carried out with a linear concentration gradient using 0-45% acetonitrile 
containing 0.1% TFA for 30 minutes, and the absorbance was measured at 220 
nm. 
From the values obtained at intervals, the residual rate of the test 
compound was calculated as a relative value based on the height of the 
peak for the test compound not treated with prolylendopeptidase, which was 
regarded as 100%. 
The results are shown in FIGS. 1 and 2. As shown in FIG. 1, the residual 
rate of Compound c after 1 hour was 16%, while that of Compound 1 was 32%. 
As shown in FIG. 2, the residual rate of Compound d was 9%, while that of 
Compound 2 was 45%. 
TEST EXAMPLE 3 
Four-weeks-old male SD strain rats (Clea Japan, Inc.) were fasted for 24 
hours before the test and were offered in groups each consisting of 8 
rats. Each rat of the control group was given 0.5 ml of physiological 
saline (Otsuka Pharmaceutical Co., Ltd.) containing 1% BSA (Sigma Chemical 
Co.) by intravenous administration through the tail vein. In the same 
manner, each rat of Compound 9-administered groups was given 0.5 ml of a 
solution of Compound 9 in physiological saline containing 1% BSA. Blood 
was collected from the right femoral artery of a rat 60 minutes after the 
administration and was centrifuged at 4.degree. C. at 3000 rpm for 15 
minutes to obtain a blood serum, and the serum calcium content was 
determined with calcium C-test WAKO (Wako Pure Chemical Industries, Ltd.). 
The results are expressed in terms of value .+-. standard error, and the 
difference was judged to be statistically significant at P &lt;0.05 according 
to the Williams-Wilcoxon test. 
The results are shown in Table 4. The serum calcium content of the Compound 
9-administered group at a concentration of 100 .mu.g/l was significantly 
lowered compared with that of the control group. 
TABLE 4 
______________________________________ 
Serum calcium 
Compound Content 
concentration (.mu.g/ml) N (mg/dl) 
______________________________________ 
0 (Control group) 
8 10.09 .+-. 0.07 
1 8 10.41 .+-. 0.13 
10 8 10.20 .+-. 0.16 
100 8 7.54 .+-. 0.29 ** 
______________________________________ 
**: P &lt; 0.01 (comparison with control group)