Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~0"8~4
Hitherto, calcitonins have been administered
pharmacologically in the form of solutions, optionally
in the presence of gelatin to prolong their activity. It
has now been found that it is more advantageous to ad-
S minister calcitonins in the form of suspensions of the
sparingly soluble free base in a liquid. The base can be
suspended in aqueous medium or in oil. The suspensions
can be administered intramuscularly or subcutaneously.
The present invention therefore provides calcitonin
preparations with prolonged action which contain a cal-
citonin with hypocalcemic activity and an isoelectric
point in the range of app. pH 5 to 9, in the form of a
suspension of the sparingly soluble free base in a liquid,
and a process for their manufacture by methods known per se.
The suspensions of the present invention have a
substantially prolonged action. It has been established
that, in the test on rats, 1 mg/kg of human calcitonin
(Calcitonin M) in the form of the free base has a hypo-
calcemic action of app. 40 hours duration, whereas
Calcitonin M in salt form, for example as acet~te, dis-
solved in water, has an action of 8 hours. In the presence
of geLatin (16%), the activity of 1 mg/kg of Calcitonin M
(as hydrochloride) is app. 12 hours.
_ 2 ~
.
~0~44
The preparations are ob~ained in a manner known
per se, for example by suspending the calcitonin base,
if appropriate already in concentrated aqueous suspension,
in the isoelectric range in aqueous isotonic solutions.
The oil suspensions are obtained, for example, by suspen-
ding the anhydrous base in the desired oily medium.
The term "isoelectric range" is used, because in
the compounds of this invention the isoelectric point is
not sharply defined, but extends over a wide pH range,
cf. Kort~m, Lehrbuch der Elektrochemie, Verlag Chemie,
Weinheim, 1966, p. 336. By isoelectric range is meant the
pH range of pI ~ app. 1 pH unit
A calcitonin wi~h an isoelectric point in the range
of app. 5 to 9 is primarily Calcitonin M or one of the
derivatives thereof, and any one of the various analogs.
Such calcitonins are described, for example, in Belgian
patents 737,890, 740,256, 757,786, and in German Offen-
legungsschrift 2,413,106. Analogs are above all those
which, instead of containing methionine8, contain an
~-lower alkyl-~-amino-acetic acid, in particular one where-
in the lower alkyl group has 2 to 4 carbon atoms a especially
valine, also norvaline, leucinea isoleucine, norleucine or
~-aminobutyric acid. Further preferred replacing amino
1~8444
acids, which can also be present in 8-position besides
the replacing amino acids referred to, are L-lysinell,
L~leucine , L-leucinel6, L-leucinel9, L-tyrosine 2,
L-asparagine , and L-threonine 7. Derivatives are, above
all, corresponding desaminol-peptides and N~-acyl deri-
vatives.
Acyl groups for the acylation of the N~-amino group
are the radicals of carboxylic acids, such as aliphatic,
aromatic, araliphatic, heterocyclic and heterocyclyl-ali-
phatic carboxylic acids, in particular of lower univalent
or bivalent alkane or alkene acids, primarily lower alkane
acids containing 1 to 4 carbon atoms, such as formic acid,
ace~ic acid, propionic acid, butyric acid, also acrylic acid,
succinic acid, of alicyclic carboxylic acids, such as
cycloalkylcarboxylic acids, for example unsubstituted and
substituted benzoic acid or phthalic acid; of unsubstituted
and aryl-substituted aryl-lower alkyl- or alkenylcarboxylic
acids, ~or example phenylacetic acid; of unsubstituted
or substituted univalent or bivalent 5- ~o 6-membered
heterocyclic acids containing nitrogen, sulphur and/or
oxygen as heteroatoms, such as pyridinecarboxylic acids,
thiophenecarboxylic acids; or of heterocyclyl-lower alkane
acids, such as pyridylacetic acid, imidazolylacetic acid,
.
~l~'a8444
wherein the substi~uents of the rings are, for example,
halogen atoms, nitro groups, lower alkyl or lower alkoxy
groups or lower carbalkoxy groups. Further exa~ples of
acyl radicals are above all acyl radicals of amino acids,
in particular of ~ amino acids, for example glycyl-
L-leucyl, L-pyroglutamyl, and acyl radicals which are
derived from carbonic acid or thiocarbonic acid or the
esters or amides thereof, for example lower alkyloxy-
carbonyl groups, such as ethoxycarbonyl, tert. butyloxy-
carbonyl, and benzyloxycarbonyl, carbamoyl and thiocarbamoyl,
which are unsubstituted or substituted as stated above,
and N-substituted carbamoyl and thiocarbamoyl, for example
N-lower alkylcarbamoyl, N-phenyl-carbamoyl, N-phenyl-thio-
carbamoyl
Human calcitonin has an isoelectric range of pH 7 to 8.
The change in the isoelectric range when replacing some
amino acids by others can be determined experimentally,
for example by electrophoresis, or can be calculated. The
isoelectric point of the peptides can be calculated, for
example, according to J. Greenstein and M. Winitz,
"Chemistry of the Amino Acids", vol. 1, ed. ~ohn Wiley &
Sons Inc., New York, London, lg61, p. 4~2 ff
The calcitonin base can be prepared advantageously
by the process of German Offenlegungsschrift 2,457~470,
~)98~4
i.e. the free base can be precipitated, for example,
from a salt of the peptide with an acid or a base by
adding a base or an acid (the free base is also defined
in this Offenlegungsschrift as the "inner salt" of the
peptide). The base can also be precipitated with ion
exchangers or with buffers or salt mixtures, such as
phosphates, citrates or glycine, instead of with bases
or acids. For example, the calcitonin base can be pre-
cipitated from a solution of a salt of the calcitonin
with an acid, for example with acetic acid3 by adding
sodium hydroxide solution or ammonia until the pH which
corresponds to the isoelectric range is attained. The
pH increases during the precipitation of the peptide by
app. one pH unit and then remains constant.
To obtain the preparations of the present invention
it is possible to use the suspensions of the peptide base
prepared by these methods direct, by diluting them, for
example, with a further suspending agent, for example an
aqueous suspending agent, such as one of the cited buffers
or salt mixtures, and bringing them into the desired
form, for example ampoules. A preerred method of obtainin~
the preparations in the form of ampoules or vials consists
in freezing the concentrated suspension, which contains
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~Q~84~4
the peptide base which has been precipitated, for example,
from a corresponding salt solution, with or without further
dilution by adding a further suspending agent, in an
appropriate lyophilisation apparatus and lyophilising it,
and resuspending the dry lyophilisate in the desired sus-
pending agent and then filling ampoules and vials with the
resultant suspension The lyophilisation is most preferably
carried out direct in the ampoules or vials, as is also
the reconstitution of the desired suspension. The suspension
required for obtaining the preparations can also be pre-
pared by mixing a salt of the calcitonin base whose iso-
electric point is in the range of pH app. 5 to 9 with a
buffer of the cited pl~, or adding an acid or a base until
this pH is attained, and using the resultant suspension
direct. Finally, the preparations can be obtained by
separating the pure base, which has been precipitated by
the methods referred to, from the aqueous phase by known
methods, for example those described in the German Offen-
legungsschrift referred to hereinabove, and subsequently
suspending them in the desired liquid.
The particle size and the degree of agglomeration
of the suspensions obtainable by the described methods
depend on the chosen concentrations of the starting solu-
lQ~84~4
tions~ the tempera~ure, the precipitation conditions of
the peptide bases, for example stirring speed, and on
the nature of the precipitant. By observing certain con-
ditions, for example those described in the illustrative
~xamples, it is possible to obtain suspensions which can
easily be made homogeneous by shaking with the advantageous
particle size of app. 1 to 100 ~, in par~icular of 10
to 50 lu.
The aqueous suspensions can contain substances which
increase the viscosity, such as sodium carboxymethyl
cellulose, carboxymethyl cellulose, dextrane, polyvinyl
pyrrolidone, gelatin and the like. If desired, the suspen-
sions can be stabilised by the customary wetting agents
for injection preparations or suitable preservatives can
be added to them.
Suspensions in oil contain as oily components the
customary vegetable, synthetic or semi-synthetic oils for
injection purposes. As such particular mention may be made
of liquid fatty acid esters which contain as acid component
a long-chain fatty acid with ~ to 22, in pflrticular 12 to
22 carbon atoms, for example lauric acid, tridecyl acid,
myristic acid, pentadecylic acid, palmitic acid, margaric
acid, stearic acid, arachinic acid, behenic acid, or cor-
~Q98~
responding unsaturated acids, for example oleic acid,
elaidic acid, erucic acid, brasidic acid or linolic acid.
The alcohol component contains a maximum of 6 carbon atoms
and is a monohydric or polyhydric alcohol, for example a
monohydric, dihydric or trihydric alcohol, for example
`methanol, ethanol, propanol, butanol or pentanol or isomers
thereof, principally, llowever, glycol or glycerol. As fatty
acid esters there are therefore to be mentioned for example:
ethyl oleate, isopropyl myristate, isopropyl palmitate,
"Labrafil M 2735" (polyoxyethylene glycerol trioleate,
produc~ of Gattefossé, Paris), "Miglyol 812" (triglyceride
of saturated fatty acids having a chain length of 8 to 12
carbon atoms, product of Chemische Werke Witten/Ruhr,
Germany), and especially vegetable oils, such as cotton
seed oil, almond oil, olive oil, castor oi~, sesame oil,
soya bean oil, above all ground nut oil.
The suspensions contain 0.1 to 5 mg/ml of the
calcitonin base. The amount normally administered by in-
jection is 1 to 2 ml once to seven times per week and con-
tains preferably 0 2 mg to 2 mg of calcitonin base, in
particular Calcitonin M base.
The injection preparations are obtained in conven-
tional manner under antimicrobial conditions, likewise the
.
~ ~radem~rl~5
_ g _
1~84~4
filling and sealing oE the ampoules and vials.
The present învention also provides a method of
treating pathological conditions in humans for whom the
administration of a calcitonin, in particular of
Calcitonin M, is indicated, for example hypercalcemic con-
ditions, bone diseases which are characterised by an in-
creased rate of metabolism, for example Paget's disease,
or osteoporosis. The method consists in administering the
calcitonin intramuscularly or subcutaneously in the form
of one of the preparations with prolonged action of the
present invention. Suitable doses, for example in the
treatment with Calcitonin M, are those referred to herein-
above. Preferably the preparations described hereinabove
or in the illustrative Examples are used.
The following Examples will serve to illustrate the
invention.
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Example l
Preparation of aqueous injection su~e~nsions wi.th
. _
calcitonin base
In an atmosphere of nitrogen, the calcitonin base is
precipîtated from a 5% solution of Calcitonin M acetate
at room temperature to 65C, preferably at 40C, by
adding l normal NaOH over a period of 15 to 30 minutes
until a pH of 7 7 is attained. The batch is subsequently
cooled to room temperature in the course of l to 3
minutes and the pH rises to app. 8.2.
To the suspension of the calcitonin base (lO mg per 0.2 ml)
obtained in this way are added, with stirring, NaH2P04,
Na2HP04 and NaCl and distilled water in such amounts as
. to yield a suspension of the composition:
Calcitonin M (base) (100%) 0.2 %
NaH2P04- 2 H20 o 4 %
2 4 2 0.14 %
N~Cl 0 49 %
The above lnjection suspension has a pH of 7, can be
readily rendered homogeneous by shaking and has a particle
...
1~8444
size of 10 ~1 and loose agglomerates of 25 to 50 ~. It is
filled înto ampoules of the composition
Calcitonin M (base) (100%) 2.0 mg
NaH2P04 2 H20 4.0 mg
Na2HP04 12 H20 14.5 mg
NaCl 4.9 mg
distilled watex to1.0 ml
Example 2
Calcitonin M is precipitated with NaOH from the acetate
solution as in Example 1 and the suspension is mixed,
with stirring, with glycine, NaCl, NaOH and distilled
water in such amounts as to yield a suspension of the
compositions
Calcitonin M (base) (100%) 0 1 %
glycine 0.5 ~
NaCl O 7 v~O
NaOH to pH 7.3
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~QQ844~
This suspension is filled into ampoules containing 1 mg
of Calcitonin M and having the composition
Calcitonin M (base) (100%)1.0 mg
glycine . 5.0 mg
NaCl 7.0 mg
NaOH to pH 7.3
distilled water to l.Q ml
Particle size and structure are as indicated in Example 1,
and a homogeneous suspension can be readily reconstituted.
Example 3
Preparation of aqueous injection suspensions with
Calcitonin M
The Calcitonin M base, which has been precipitated as
concentrated suspension (for example, 100 mg/2 ml), from a
solution of the acetate in accordance with the particulars
of Example 1, is frozen and lyophilised in a suitable
lyophilisation apparatus.
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The dry lyophilisa~e is resuspended in sul.table suspending
agents, for example those of Examples 1 and 2, with stir-
ring, and filled for example into ampoules of the com-
position indicated in Examples 1 and 2.
These injection suspensions can be readily reconstituted
in a homogeneous form by shaking and have a particle size
of 10 to 50 ~.
Example 4
A dry lyophilisate of Calcitonin M base as prepared and
described in Example 3 is resuspended in a 5% mannitol
solution with stirring and filled into ampoules of the
composition
Calcitonin M (base) (100%) 1~0 mg
mannitol, free of pyrogens 50.0 mg
distilled water to l.O ml
This suspension has a pH of 8.2, can be readily recon-
stituted in a homogeneous form by shaking and has a particle
siæe of lO to 50 lu.
34~4
Example 5
Ampoules containing Calcitonin M base are prepared in a
manner similar to that described in Example 4, except that
they also contain L-methionine and have the composition
Calcitonin M (base) (100%)1.0 mg
L-methionine 1.0 mg
mannitol, free of pyrogens50.0 mg
NaOH to pH 7.5 to 8
distilled water to 1.0 ml
This suspension has the same particle size as in Example 4
and can also be readily reconstituted in homogeneous form
Example 6
75 mg of Calcitonin M acetate (calculated on the base) are
dissolved in 3 ml of water. To this solution is added
0.3 ml of 1% ammonia to a pH of 7.4. The solution is
stirred until the precipitate has formed and then 12 ~1
of water or phosphate buffer are added. The suspension
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contains 5 mg/ml of Calcitonin M as base.
Example 7
Calcitonin M ~base) (100%) 0.2 mg
NaCl 0.9 mg
distilled water to 1.0 ml
Example 8
Preparation of dry vials with Calcitonin M base
With stirring, 2 ml of a concentrated suspension of
Calcitonin M which is prepared according to the parti-
culars of Example 1 and contains 100 mg of pure base,
is treated with 50 ml of an aqueous solution containing
5% of mannitol and 0.2 % of methionine and the batch is
diluted with 50 ml of distilled water. Vials of 2 ml
capacity are filled with 1 ml of this solution, frozen in
a suitable lyophilisation apparatus and lyophllised.
An injection suspension can be reconstituted from the dry
lyophilisate in the vials by adding an aqueous solvent,
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.
34~4
for example 1 ml of distilled water. Injection suspensions
; with a particle size of <10 lu and loose agglomerates of
25-50 ~ are obtained. They have the compositîon:
Calcitonin M (base) (100%) 1.0 mg
L-methionine 1.0 mg
mannitol, free of pyrogens 25.0 mg
'~
