U.S. patent number 4,514,331 [Application Number 06/509,123] was granted by the patent office on 1985-04-30 for peptide hormones with calcitonin-like activity.
This patent grant is currently assigned to University Patents, Inc.. Invention is credited to Emil T. Kaiser, Gregory Moe.
United States Patent |
4,514,331 |
Kaiser , et al. |
April 30, 1985 |
**Please see images for:
( Certificate of Correction ) ** |
Peptide hormones with calcitonin-like activity
Abstract
Compounds of the formula: ##STR1## wherein R.sub.1 is a moiety
selected from the group consisting of ##STR2## R.sub.2 -R.sub.22
are amino acid moieties wherein R.sub.2 is an optional moiety which
when present is selected from the group consisting of Ser and Gly,
R.sub.8 is Leu, Val, or Ile, R.sub.10 is Gln, Lys, or Gly,
R.sub.11, R.sub.14, and R.sub.20 are each independently selected
from the group consisting of Gln and Lys, R.sub.12 is Leu or Trp,
R.sub.13 is Gln or Ser R.sub.17 is Gln or His, R.sub.21 is Gln or
Thr, R.sub.22 is an optional moiety which when present is selected
from the group consisting of Leu, Tyr, or Phe; X comprises a series
of eight amino acids each independently selected from the group
consisting of Gly, Ser, Thr, Cys, Tyr, Asn, Gln, Asp, Glu, Lys,
Arg, and His, with the proviso that not more than one of said eight
amino acids may be selected from the group consisting of Asp, Glu,
Lys, Arg, and His, and with the proviso that no four or more of
said eight amino acids will spontaneously form helical,
.beta.-sheet, or .beta.-turn conformations; and R.sub.23 is an
amino acid amide selected from the group consisting of proline
amide and glycine amide; the pharmaceutically acceptable salts
thereof, compositions containing said compounds, and a method of
lowering serum calcium levels using said compounds.
Inventors: |
Kaiser; Emil T. (New York,
NY), Moe; Gregory (New York, NY) |
Assignee: |
University Patents, Inc.
(Westport, CT)
|
Family
ID: |
24025364 |
Appl.
No.: |
06/509,123 |
Filed: |
June 29, 1983 |
Current U.S.
Class: |
530/317; 530/307;
930/60; 930/DIG.670; 930/DIG.671; 930/DIG.821 |
Current CPC
Class: |
C07K
14/585 (20130101); A61K 38/00 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/585 (20060101); A61K
38/00 (20060101); C07C 103/52 () |
Field of
Search: |
;260/112.5T |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4086221 |
April 1978 |
Sakakihara et al. |
|
Other References
Calcitonin 1980, Pecile ed., Excerpta Medica, Princeton, N.J.,
1981, pp. 11-24. .
Lehninger, Biochemistry, 2nd Ed., 1975, pp. 73-75. .
Nakamuta et al., Japan J. Pharmacol. 31, 1981, pp. 53-60. .
Morikawa et al., Experientia, vol. 32, No. 9, 1976, pp. 1104-1106.
.
Maier et al., Clinical Endocrinology, 5(Suppl.) 1976, pp.
327s-332s. .
Yamashiro et al., J. Am. Chem. Soc., vol. 100, 1974, pp. 5174-5179.
.
Pietta et al., Chemical Communications, 1970, pp. 650-651. .
MacIntyre et al., Arthritis and Rheumatism, vol. 23, No. 10, 1980,
pp. 1139-1147. .
Pollet et al., J. Biol. Chem., vol. 254, No. 1, 1979, pp. 30-33.
.
Morrisett et al., Biochemistry, vol. 12, No. 7, 1973, pp.
1290-1299. .
Hunter et al., Nature, vol. 194, 1962, pp. 495-496. .
Edelstein et al., J. Lipid Res., vol. 29, 1979, pp. 143-153. .
Chou et al., Ann. Rev. Biochem., vol. 245, No. 9, 1970, pp.
251-276. .
Brewer et al., J. Biol. Chem., vol. 245, No. 9, 1970, pp.
2402-2408. .
The Peptides, vol. 2, Academic Press, N.Y., 1979, pp. 3-284. .
Proteins, vol. 2, 3rd ed. Academic Press, N.Y., 1976, pp. 105-253.
.
Behrens et al., Ann. Rev. Biochem., 38, 83-112 (1969). .
Noda et al., J. Biochem., 79, 353-359 (1976)..
|
Primary Examiner: Phillips; Delbert R.
Assistant Examiner: Moezie; F. T.
Attorney, Agent or Firm: Shimei; Barbara A.
Claims
We claim:
1. The compound of the formula: ##STR9## and the pharmaceutically
acceptable salts thereof.
2. A method of lowering serum plasma calcium levels in warm-blooded
animals comprising administering to said warm-blooded animal a
serum plasma calcium-lowering effective amount of a compound
according to claim 1.
Description
The invention described herein was made in the course of work under
a grant from the National Institutes of Health.
This invention relates to novel peptide hormones which exhibit
calcitonin-like activity, to the pharmaceutically acceptable
non-toxic salts thereof, to compositions containing said hormones,
and to methods of lowering serum calcium levels by the
administration of said hormones.
Calcitonin is a peptide hormone with a molecular weight of
approximately 3,500 daltons which is produced by the parafollicular
cells; these cells are scattered throughout the thyroid in mammals
but in lower animals constitute a distinct organ, the
ultimobranchial body. The hormone regulates serum calcium
concentrations by opposing the bone and renal effects of
parathyroid hormone and inhibiting bone resorption of calcium,
resulting in hypocalcemia, hypophosphatemia, and decreased urinary
calcium concentrations. Calcitonin is therefore used in the
treatment of Paget's Disease, hyperparathyroidism, idiopathic
hypercalcemia of infancy, osteolytic bone metastases, and to
counteract the osteolytic effect of overdoses of vitamins A and
D.
Calcitonins from at least seven different species, and the two
isohormones of salmon calcitonin, have been sequenced and
characterized biologically and a number of synthetic analogs have
been studied, but few clear correlations between structure and
function have been made. The common form of the hormone consists of
32 amino acids with a disulfide bridge between cysteine residues at
positions 1 and 7 and prolinamide at the carboxy terminus.
Otherwise the structures of the various calcitonins differ markedly
from each other; human calcitonin differs from porcine calcitonin
at 18 of the 32 residues. It is generally recognized that the
cysteines at positions 1 and 7 taken together may be replaced by
2-aminooctanedioic acid, resulting in the analogous structure
wherein the disulfide bridge of the cysteines has been replaced by
an ethylene bridge. For a general review, see Behrens and Grinnan,
Ann. Rev. Biochem. 38:83 (1969); Foster et al., "Calcitonin" in
Clinics in Endocrinology and Metabolism [I. MacIntyre, ed.] (W. B.
Saunders, Philadelphia, 1972) pp. 93-124.
Because of its therapeutic value, calcitonin is in great demand. Of
the nine or more known calcitonins, only three, salmon, porcine,
and human, are commercially available. Porcine calcitonin is
isolated and purified at great expense from pork glands, whereas
salmon and human calcitonin are primarily synthesized in vitro.
Salmon calcitonin is the most active of the known calcitonins, and
porcine is the most active known mammalian calcitonin. However,
because foreign calcitonins tend to trigger an antigenic response
and because human calcitonin is only weakly active there is a need
for improved synthetic alternate peptide hormones with
calcitonin-like activity. There is also a need to understand the
elements required for activity so that these compounds can be
modified to introduce desired pharmaceutical characteristics, such
as increased half-life or oral activity, without losing
efficacy.
It has now been discovered that compounds of the formula (I):
##STR3## wherein R.sub.1 is a moiety selected from the group
consisting of ##STR4## R.sub.2 -R.sub.22 are amino acid moieties
wherein R.sub.2 is an optional moiety which when present is
selected from the group consisting of Ser and Gly,
R.sub.8 is Leu, Val, or Ile,
R.sub.10 is Gln, Lys, or Gly,
R.sub.11, R.sub.14, and R.sub.20 are each independently selected
from the group consisting of Gln and Lys,
R.sub.12 is Leu or Trp,
R.sub.13 is Gln or Ser,
R.sub.17 is Gln or His,
R.sub.21 is Gln or Thr,
R.sub.22 is an optional moiety which when present is selected from
the group consisting of Leu, Tyr, or Phe;
X comprises a series of eight amino acids each independently
selected from the group consisting of Gly, Ser, Thr, Cys, Tyr, Asn,
Gln, Asp, Glu, Lys, Arg, and His, with the proviso that not more
than one of said eight amino acids may be selected from the group
consisting of Asp, Glu, Lys, Arg, and His, and with the proviso
that no four or more of said eight amino acids will spontaneously
form helical, .beta.-sheet, or .beta.-turn configurations; and
R.sub.23 is an amino acid amide selected from the group consisting
of proline amide and glycine amide
have calcitonin-like activity in vivo. It has also been discovered
that in addition to the 7-amino acid sequence at the amino end of
the peptide with cysteine residues at positions 1 and 7 linked by a
disulfide bridge (or with 2-aminooctanedioic acid replacing these
two cysteines), which sequence can be designated as Section 1 of
the peptide hormone, the following features are essential to
activity:
(2) A 15-amino acid sequence at positions 8-22, which sequence
spontaneously forms an amphiphilic helix characterized in that the
hydrophilic amino acid residues are segregated along one side of
the vertical axis of the helix while the hydrophobic amino acid
residues are segregated along the opposite side of the vertical
axis of the helix. Residues are considered hydrophobic if their
hydrophobicity parameter as defined by Edelstein, C., F. J. Kezdy,
A. M. Scanu and B. L. Shen, J. Lipid Res. 20:148 (1979), is greater
than or equal to 0.5 and hydrophilic if the parameter is less than
0.5. The average .alpha.-helicity parameter, <P.alpha.>, as
described by Chou, P. Y. and G. D. Fasman, Ann. Rev. Biochem.
47:251-76 (1978) must be greater than 1.03, and no more than half
of the hydrophilic amino acid residues may be charged at pH
6.0-7.0.
(3) A 10-amino acid sequence at positions 23-32 (carboxy end of the
peptide) having a proline residue at position 23 and an amino acid
amide residue at position 32. These 10 amino acid residues are
hydrophilic and no more than one may be charged at pH 6.0-7.0. They
are selected to form a "random chain" so that no four or more of
said 10 amino acids will spontaneously assume a helical,
.beta.-sheet, or .beta.-turn configuration according to the
empirical predictive parameters defined by Chou, P. Y. and G. D.
Gasman, Ann. Rev. Biochem. 47:251-76 (1978).
These characteristics of the helix are more readily visualized when
the compounds of the present invention are depicted in the
following form: ##STR5## ##SPC1## wherein R.sub.1, R.sub.2-22, X,
and R.sub.23 are as previously defined. The hydrophilic amino acid
residues in the helix are marked by an asterisk (*); the unmarked
residues are hydrophobic. From this depiction it can easily be seen
that the hydrophobic and hydrophilic residues are segregated on
opposite sides of the helix. It is believed that this configuration
is necessary for the interaction of the hormone with its specific
receptor sites.
As used hereinabove and below, the three-letter abbreviations for
the amino acid residues are those commonly used and accepted by
persons in the peptide art; see, e.g., Lehninger, Albert L.,
Biochemistry, 2nd Ed. (Worth Publishers, Inc., New York, 1975), pp.
73-75. All amino acids and their derivatives are in the L-form.
Preferably, R.sub.8 is Leu; R.sub.10 is Gln or Lys; R.sub.13,
R.sub.17, and R.sub.21 are each Gln; and R.sub.22 is an optional
moiety which when present is selected from the group consisting of
Leu and Tyr. Particularly preferred are compounds wherein R.sub.1
is --S--S--. Most particularly preferred is the compound of the
formula (II), which has been designated "MCT-I": ##STR6##
The basic amino acid residues (lysine, arginine, and histidine) of
the compounds of Formula I may be in the form of their
acid-addition salts. The hydrochloride, acetate, phosphate,
citrate, fumarate, maleate, succinate, pamoate, and sulfate
acid-addition salts are preferred. Particularly preferred are the
acetate and hydrochloride salts. It is to be understood that for
the purposes of this invention, the acid-addition salts of the
hormone of Formula I are equivalent to the parent free peptide.
The compounds of Formula I may be synthesized by methods well-known
to those skilled in the art of peptide synthesis, e.g. solution
phase synthesis (see Finn, F. M. and K. Hofmann, in Proteins, Vol.
2, 3rd Ed., H. Neurath and R. L. Hill, eds. (Academic Press, New
York, 1976), pp 105-253), or solid phase synthesis (see Barany, G.
and R. B. Merrifield, in The Peptides, Vol. 2, E. Gross and J.
Meienhofer, eds. (Academic Press, New York, 1979) pp. 3-284).
Preferably these compounds are synthesized by the solid phase
method on a benzhydrylamine-substituted polystyrene resin
crosslinked with 1% divinylbenzene, see Pietta, P. G. and G. R.
Marshall, J. Chem. Soc. D: 650-651 (1970). The .alpha.-amino group
of the carboxy-terminal amino acid (AA.sub.32) is first shielded
with a selectively cleavable N-Terminal protecting group.
Preferably, this group is t-butoxycarbonyl (Boc). Amino acids with
the N.sup..alpha. -Boc shielding group in place are commercially
available from Bachem Inc., Marina Del Rey, Calif. The blocked
amino acid (N.sup..alpha. -Boc-AA.sub.32) is then coupled to the
resin using N-hydroxybenzotriazole (HOBt) in conjunction with
dicyclohexylcarbodiimide (DCC) as condensing agents. The
N.sup..alpha. -Boc group is subsequently removed by treatment with
a strong anhydrous organic acid, preferably trifluoroacetic acid
neat or about 25-75% (50% preferred) in methylene chloride, at
about 20.degree.-30.degree. C. for about 30-60 minutes. The
reaction mixture is then neutralized with a hindered organic base,
e.g. diisopropylethylamine or N-methylmorpholine, preferably about
2-10% diisopropylethylamine in methylene chloride at about
20.degree.-30.degree. C. for about 2-6 minutes. The amino acid of
position 31 (AA.sub.31) is then added to the N-terminal amine of
AA.sub.32 by reaction with the symmetric anhydride or active ester
of N.sup..alpha. -Boc-AA.sub.31 in the presence of methylene
chloride at about 20.degree.-30.degree. C. for about 20-60 minutes,
followed by removal of the N.sup..alpha. -Boc blocking group of
AA.sub.31 by treatment with about 25-75% (50% preferred)
trifluoroacetic acid in methylene chloride at about
20.degree.-30.degree. C. for about 30-60 minutes. In a similar
manner, the remaining amino acid residues are added in sequence and
the peptide chain is built up from the C-terminal end. See
Yamashiro, D. and C. H. Li, J. Am. Chem. Soc. 100:5174 (1978).
If R.sub.1 is to be ##STR7## the C-2 amino group and the C-8
carboxyl group of 2-aminooctanedioic acid are first protected; when
the growing peptide chain has reached the point where AA.sub.8
(R.sub.8) is in position, the C-1 carboxy on the shielded
2-aminooctanedioic acid is bonded to the .alpha.-amine moiety of
AA.sub.8. The .alpha.-carboxyl group of AA.sub.6 is then added to
the C-2 amine of the 2-aminooctanedioic acid; AA.sub.5 is added to
AA.sub.6, and so on through AA.sub.2. The C-8 carboxyl group on the
2-aminooctanedioic acid is then deprotected to allow it to react
with the .alpha.-amino moiety of AA.sub.2. Thus the two halves of
2-aminooctanedioic acid each function as a separate amino acid at
positions 1 and 7, linked through an ethylene bridge. For details
on methods of incorporating 2-aminooctanedioic acid in the proper
positions, see Morikawa, T. et al., Experientia 32:1104-1106
(1976).
It is understood by those skilled in the art that certain amino
acids contain reactive side groups which must be shielded during
the coupling reaction. Thus the N-guanidinium moiety of
N.sup..alpha. -BocArg is tosylated to yield N.sup..alpha.
-BocArg(N.sup.g -Tos). The thiol group of N.sup..alpha. -BocCys is
protected by a 4-methoxybenzyl moiety to yield N.sup..alpha.
-BocCys(S-4-MeO-Bzl). N.sup..alpha. -Boc-Lys is converted to
N.sup..alpha. -BocLys(N.sup..epsilon. -2-ClZ) wherein the
.alpha.-amino of lysine is protected by a 2-chlorobenzyloxycarbonyl
moiety. N.sup..alpha. -BocSer(OBzl) and N.sup..alpha. -BocThr(OBzl)
are formed from N.sup..alpha. -BocSer and N.sup..alpha. -BocThr,
respectively; the hydroxy groups of serine and threonine are
converted to an ether linkage with the benzyl moiety. The indole
nitrogen of N.sup..alpha. -BocTrp is formylated for protection to
yield N.sup..alpha. -BocTrp(N.sup.in -For). These shielded amino
acids may be prepared according to methods given in Barany, G. and
R. B. Merrifield, in The Peptides, Vol. 2, E. Gross and J.
Meienhofer, eds. (Academic Press, New York, 1979) pp. 169-250, or
they may be obtained commercially from Bachem Inc., Marina Del Rey,
Calif.
The shielded amino acid residues are converted to their symmetrical
anhydrides by reaction with dicyclohexylcarbodiimide in methylene
chloride in a ratio of 2 molar equivalents of amino acid per molar
equivalent of DCC at about 5.degree.-10.degree. C. for about 15
minutes. The resulting product is suitable for use without further
isolation and purification. Alternatively, the shielded amino acids
are converted to their active esters by reaction with HOBt and DCC
in a ratio of 1:1:1 molar equivalents.
The completed peptide is cleaved from the resin with simultaneous
removal of all protecting groups except the N.sup.in -formyl by
treatment with anhydrous liquid hydrofluoric acid:anisole (7-9:1,
v/v) at 0.degree. C. for about 30-60 min. One of the advantages of
the benzhydrylamine-substituted polystyrene resin used is that the
carboxy-terminal amino acid residue (AA.sub.32) is spontaneously
yielded in its amino acid amide form upon cleavage. Crude peptide
is removed from the resin by washing with 5-20% acetic acid. Ten
percent acetic acid is preferred.
The crude peptide is then preferably lyophilized. During synthesis,
the cysteine residues may have oxidized. The thiol groups are
reduced to their free form by treatment with a reducing agent such
as excess dithiothreitol or .beta.-mercaptoethanol in a mild
physiological buffer such as sodium phosphate or carbonate, tris,
MOPS, etc. 0.05M sodium phosphate, pH 7.0, is preferred.
If R.sub.1 is to be ##STR8## the peptide solution is diluted in the
same buffer as above to a volume of about 5 liters and a solution
of 0.02M K.sub.3 Fe(CN).sub.6 (oxidizing agent) is added slowly
with stirring at 20.degree.-30.degree. C. to induce the formation
of the disulfide bridge between the cysteine residues at positions
1 and 7.
The peptide is then concentrated and purified by procedures
well-known to those skilled in the art, e.g. by molecular sieving,
ion exchange chromatography, HPLC, evaporation, lyophilization,
etc., and the N.sup.in -formyl group is removed. Preferably, the
peptide is concentrated and purified by absorption on an ion
exchange column such as CM-Sephadex C-25.sup.(R) (Pharmacia Fine
Chemicals, Piscataway, N.J.), followed by elution with a linear
salt gradient, e.g. 0.0 to 0.3M NaCl in the same buffer used to
form the bridge moiety. The peptide elutes in about 2.8M NaCl and
is further purified by HPLC using a linear gradient of from about
20-50% acetonitrile 50% in 0.2M sodium phosphate buffer, pH 2.5.
The resulting solution is desalted and the N.sup.in -formyl
protecting group is removed quantitatively by treatment with a
nucleophilic species in aqueous solution, e.g. piperidine sodium
hydroxide or hydrazine, preferably 0.5M aqueous piperidine, at
0.degree. C. for about 20 minutes. The deprotective reaction is
terminated by addition of acid, preferably acetic acid.
Alternatively, the N.sup.in -formyl group may be removed by methods
given in Barany, G. and R. B. Merrifield, in The Peptides, Vol. 2,
E. Gross and J. Meienhofer, eds. (Academic Press, New York, 1979)
p. 220. The peptide is then once again purified by HPLC, eluting
with about 35% acetonitrite in 0.2M sodium phosphate buffer, pH
2.5.
The acid-addition salts of the basic amino acid residues are
prepared by treatment of the peptide with the appropriate organic
or inorganic acid according to procedures well-known to those
skilled in the art; or the desired salt may be obtained directly by
lyophilization out of the appropriate acid.
The compounds of formula I are useful to lower the serum plasma
calcium level in warm-blooded animals suffering from elevated serum
plasma calcium levels when administered in amounts ranging from
about 0.1 ng. to about 10 ng. per kg. of body weight per day. A
preferred dosage range for optimal results would be from about 0.15
ng. to about 8 ng. per kg. of body weight per day, and such dosage
units are employed so that a total of from about 0.1 mg. to about
0.56 mg. of the active compound for a subject of about 70 kg. of
body weight are administered in a 24-hour period. This dosage
regimen may be adapted to provide the optimum therapeutic response.
For example, several divided doses may be administered daily, or
the dose may be proportionally reduced as indicated by the
exigencies of the therapeutic situation. The compound may be
administered in the form of the free peptide or as a non-toxic
pharmaceutically acceptable salt thereof. The term
"pharmaceutically acceptable salt" refers to those acid-addition
salts of the parent compound which do not significantly adversely
affect the pharmaceutical properties (e.g. toxicity, effectiveness
etc.) of the parent compound, such as are conventionally used in
the pharmaceutical art.
The active compounds may be administered parenterally, e.g. by
subcutaneous, intramuscular, or intravenous injection. Solutions or
suspensions of these active compounds as a pharmaceutically
acceptable salt can be prepared in water suitably mixed with a
surfactant such as hydroxypropylcellulose. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols, and mixtures
thereof in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms. The peptide hormones of the present invention have
a natural tendency to adhere to glass; therefore these preparations
preferably also contain a pharmaceutically acceptable protein such
as gelatin or albumin to competitively inhibit this effect.
The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacterial and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, polyol (e.g., glycerol,
propylene glycol and liquid polyethylene glycol), and suitable
mixtures thereof. Compositions suitable for intramuscular or
subcutaneous injection may also contain minor amounts of salts,
acids, and bases to adjust tonicity and buffer the pH. Suitable
pharmaceutically acceptable buffering and tonicity agents are
readily determinable by persons skilled in the art.
A further understanding of this invention may be had from the
following non-limiting examples. As used hereinabove and below
unless expressly stated to the contrary, all temperatures and
temperature ranges refer to the centigrade system and the terms
ambient or room temperature refer to about 20.degree. C. The term
percent or (%) refers to weight percent and the terms mole and
moles refer to gram moles.
EXAMPLE 1
Synthesis of MCT-I
MCT-I was synthesized by the solid phase method using a
benzhydrylamine-substituted polystyrene resin crosslinked with 1%
divinylbenzene. The C-terminal amino acid BocPro was coupled to the
resin using N-hydroxybenzotriazole (HOBt) and
dicyclohexylcarbodiimide (DCC). Thereafter, symmetric anhydrides of
BocArg (N.sup.g -Tos), BocCys(S-4-MeO-Bzl), BocGly, BocLeu,
BocLys(N.sup..epsilon. -2-ClZ), BocPro, BocSer(OBzl), BocThr(OBzl),
and BocTrp(N.sup.in -For) and a deprotection and coupling program
similar to that employed by Yamashiro, D. and C. H. Li, J. Am.
Chem. Soc. 100:5174 (1978) was used, except for BocAsn, which was
coupled by the HOBt/DCC method. Cleavage from the resin and removal
of all the remaining protecting groups, except for the N.sup.in
-formyl, were accomplished by treatment with anhydrous liquid HF in
the presence of anisole (7:1, v/v) at 0.degree. C. for 45 min.
Crude peptide was removed from the resin by washing with 10% acetic
acid. The residue remaining after lyophilization was treated with
excess dithiothreitol in 0.05M sodium phosphate buffer at pH 7.0.
The intramolecular disulfide bond between cysteine residues 1 and 7
was formed by diluting the peptide solution to a volume of 5 liters
in the same buffer and adding a solution of 0.02M K.sub.3
Fe(CH).sub.6 slowly with stirring. The resultant dilute peptide
solution was concentrated by passing it through a CM-Sephadex C-25
column, followed by a linear gradient of NaCl from 0.0 to 0.3M
employing the same buffer. Fractions from this column were purified
further by loading them directly onto a Waters.TM.C.sub.18
semi-preparative HPLC column and then eluting with a linear
gradient of CH.sub.3 CN from 20% to 50% in 0.2M sodium phosphate
buffer, pH 2.5. After desalting the resultant solution, the
N.sup.in -formyl protecting group was removed quantitatively by
treatment with 0.5M aqueous piperidine at 0.degree. C. for 20
minutes. The deprotective reaction was terminated by the addition
of acetic acid. Final purification was carried out by loading the
reaction mixture directly onto the Waters C.sub.18 semi-preparative
column and eluting with 35% CH.sub.3 CN in the same buffer. The
yield of purified MCT-I after a final desalting step and
lyophilization was 10% based on the original substitution level of
BocPro. The peptide was judged to be pure based on the observation
at 230 nm of a single peak when the peptide was eluted from a
Waters C.sub.18 reversed phase column using a gradient from 20% to
50% CH.sub.3 CN as the eluting solvent and from its amino acid
analysis after hydrolysis with 5.5M HCl.
Amino Acid Analysis: Arg 1.1 (1), Asp 2.01 (2), Cys 2.09 (2), Glu
5.00 (5), Gly 3.02 (3), Leu 7.14 (7), Lys 2.94 (3), Pro 1.63 (2),
Ser 1.61 (2), Thr 3.5 (4).
EXAMPLE 2
Characterization of MCT-I
The circular dichroism (CD) spectra of MCT-I and salmon calcitonin
(designated "SCT-I", available from Armour Pharmaceuticals,
Kankakee, Ill.) from 250 nm to 205 nm show minima at 222 nm and 208
nm characteristic of .alpha.-helical structure. For MCT-I, the mean
residue molar ellipticity at 222 nm, [.theta.].sub.222, was -7,800
deg cm.sup.2 /dmol (10.sup.-4 M peptide, 0.02M sodium phosphate
buffer, 0.16M KCl, pH 7.4), from which the .alpha.-helicity was
estimated to be 30% according to the method of Morrisett, J. D., J.
S. K. Davis, H. J. Pownall, and A. M. Gotto, Biochemistry 12:1290
(1973). The value of [.theta.].sub.222 does not change over a range
of concentration of MCT-1 from 10.sup.-7 M to 10.sup.-4 M, provided
that binding to glass is prevented by pretreatment of the
spectrometer cell with polyethylene glycol (MW 15K-20K). This
suggests strongly that MCT-I remains monomeric over the
concentration range employed, a conclusion supported by the
measurement of a molecular weight of about 4,500 at a concentration
of 10.sup.-4 M MCT-I by means of ultracentrifugation using a
Beckman Spinco Airfuge according to the procedure of Pollet, R. J.,
B. A. Haase, and M. L. Standaert, J. Biol. Chem. 254:30 (1979).
Similarly, the value of [.theta.].sub.222 for solutions of SCT-I
over the same concentration range and under the same conditions
also remains constant at -4,600 deg cm.sup.2 /dmol, leading to an
estimate of 20% .alpha.-helix for this peptide. In 50%
trifluoroethanol, a structure promoting solvent, both MCT-I and
SCT-I were estimated to be 50% .alpha.-helical at a concentration
of 5.times.10.sup.-5 M, as was found by Brewer, H. D. and H.
Edelhoch, J. Biol. Chem. 245:2402 (1970) for porcine calcitonin
(PCT) in 50% 2-chloroethanol.
At the air-water interface, MCT-I and SCT-I form insoluble
monolayers when spread from concentrated solutions in 0.01M HCl.
The force-area (.pi.-A) curves between 5 and 12 dyn/cm are
described by the equation .pi.[A-A.sub..infin. (1-.kappa..pi.)]=nRT
where .kappa. is the compressibility and A.sub..infin. is the
limiting molecular area extrapolated to zero surface pressure. The
parameters calculated for the two peptides were very similar,
.kappa.=0.016 cm/dyn for MCT-I and 0.02 cm/dyn for SCT-I, while
A.sub..infin. =362 .ANG..sup.2 for MCT-I and A.sub..infin. =322
.ANG..sup.2, for SCT I. However, the collapse pressure of 24 dyn/cm
found for the monolayer of MCT-I was much higher than the value of
14 dyn/cm observed for SCT-I.
EXAMPLE 3
In vitro Activity
In order to study the receptor binding properties of MCT-I and
SCT-I, .sup.125 I-SCT-I was prepared by the method of Hunter, W. M.
and F. C. Greenwood, Nature 194:495 (1962). The iodinated hormone
was purified by ion exchange chromatography on SP-Sephadex C-25.TM.
(Pharmacia Fine Chemicals, Piscataway, N.J.). The unreacted
labelling material was first washed from the column with 0.01M
Tris-HCl, 0.1% bovine serum albumin (BSA), pH 7.4 buffer, followed
by elution of the monoiodinated SCT-I with 0.2M NaCl at pH 8 in the
same buffer. Fractions from the single symmetrical peak which was
eluted with this buffer were combined, adjusted to pH 7.5, and
frozen in small aliquots until needed. The specific activity of the
radioiodinated peptide was .about.160 .mu.Ci/.mu.g. Competitive
binding experiments with rat brain homogenates were carried out as
described by Nakamuta H., S. Furukawa, M. Koida, H. Yajima, R. C.
Orlowski, and R. Schlueter, Japan J. Pharmacol. 31:53 (1981). This
method has been shown to given binding curves for calcitonin
analogues comparable to the more commonly used kidney binding
assay, see S. J. Marx, C. J. Woodward, and G. D. Auerbach, Science
178:999 (1972), and the brain tissue is more convenient to prepare
and use. The results are shown in FIG. 1, competitive inhibition of
.sup.125 I-SCT-I binding to brain particulate fraction by SCT-I (O)
and MCT-I (.DELTA.). Each point represents the mean of three
triplicate determinations. The binding curves obtained gave
IC.sub.50 values for SCT-I of about 2.5 nM, in agreement with the
value reported earlier by Nakamura et al., supra, and 17 nM, for
MCT-I which compares with the value of 17 nM found for PCT
(Ibid.)
EXAMPLE 4
In vivo Activity
To assess the biological potency of MCT-I in vivo, 20 male
Sprague-Dawley rats, 3-4 weeks old, were given subcutaneous
injections (0.15 ml/100 g body weight) of SCT-I or MCT-I in 0.9%
saline, 0.1% BSA, pH 4.5 in graded doses or, alternatively, of
saline solution alone. Blood was withdrawn 1 hour after the
injections, and the calcium concentration in the plasma determined
by atomic absorption spectroscopy. The dose-response curve in FIG.
2 summarizes the results for SCT-I (O) and MCT-I (.DELTA.). Each
point represents the difference between the average serum Ca.sup.+2
concentration for rats given only saline and the average for those
given a particular dose of either MCT-I (15 rats per point) or
SCT-I (5 rats per point). As with the binding studies, MCT-I is
about 10-fold less potent than SCT-I, or approximately as active as
PCT, the most potent mammalian analog.
Although the sequence of amino acids in MCT-I differs from that in
SCT-I from positions 8 to 22, the MCT-I reproduced all of the
chemical and biological properties of the salmon calcitonin that
were examined. Like SCT-I, MCT-I was monomeric in aqueous solution.
MCT-I showed somewhat more .alpha.-helical character than the
salmon calcitonin did under these conditions, and at the air-water
interface, an amphiphilic environment, it formed a much more stable
monolayer than did SCT-I. Moreover, MCT-I displaced a specifically
bound ligand from calcitonin receptors in vitro and effected a
potent hypocalcemic response in the rat bioassay. Taken together,
these results provide strong evidence that the region from residues
8 to 22 of the calcitonins has a primarily structural role,
interacting in the amphiphilic .alpha.-helical form with the
amphiphilic environment of the calcitonin receptor.
* * * * *