Use of 1,4-dihydropyridine derivatives and combinations thereof with calcitonins

Use of calcium antagonists of formula I ##STR1## wherein the substituents have various significances in the treatment of conditions related directly or indirectly to ionized calcium levels in the blood and combinations of calcium antagonists, e.g. of formula I, with calcitonins.

This invention relates to calcium antagonists. Such compounds are also 
called calcium entry blockers. Calcium antagonists represent a group of 
active substances which modulate entry of Ca (2+) ions through specific Ca 
(2+) channels in smooth muscle and are useful for the treatment of inter 
alia Angina pectoris, hypertension and for some products also migraine. 
One group of calcium antagonists are characterised by a 1,4-dihydropyridine 
structure having an aryl or heterocyclic group attached to the 4 position. 
In one aspect the invention relates to a new use of a compound of formula I 
##STR2## 
wherein R.sub.1 is hydrogen, alkyl of 1 to 6 carbon atoms, alkenyl or 
alkinyl of 3 to 6 carbon atoms, cycloalkyl of 3 to 7 carbon atoms, 
cycloalkylalkyl of 4 to 8 carbon atoms, phenylalkyl of 7 to 9 carbon atoms 
or phenylalkenyl of 9 to 12 carbon atoms, the phenyl ring being 
unsubstituted or mono-, di- or trisubstituted independently by halogen, 
hydroxy or alkyl or alkoxy of 1 to 4 carbon atoms, 
R.sub.2 and R.sub.5, independently, are hydrogen or alkyl of 1 to 6 carbon 
atoms, 
R.sub.3 and R.sub.4, independently, are alkyl of 1 to 6 carbon atoms, 
alkenyl or alkinyl of 3 to 6 carbon atoms, cycloalkyl of 3 to 7 carbon 
atoms, cycloalkylalkyl of 4 to 8 carbon atoms, alkoxy of 1 to 6 carbon 
atoms, hydroxyalkoxy of 2 to 6 carbon atoms, alkoxyalkoxy of 3 to 6 carbon 
atoms, hydroxyalkoxyalkoxy of 4 to 8 carbon atoms, alkenyloxy or 
alkinyloxy of 3 to 6 carbon atoms, cycloalkyloxy of 3 to 7 carbon atoms or 
cycloalkylalkoxy of 4 to 8 carbon atoms, 
R.sub.6 is hydrogen, halogen, alkyl or alkoxy or alkylthio or 
alkylsulfonyl, each of 1 to 4 carbon atoms, trifluoromethyl, nitro or 
hydroxy, and 
X is oxygen or sulphur. 
Certain pharmacological activities of the compounds of formula I have been 
published in e.g. European Patent specification No. 150. 
Particularly interesting compounds of this group of compounds include PY 
108-068, i.e. darodipine, i.e. 
4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethylpyridine-3,5-dicarbox 
ylic acid diethyl ester, herein-after referred to as PY; PN 200-110 i.e. 
4-(2,1,3-benzoxadiazol-4yl)-1,4-dihydro-2,6-dimethyl-3-methoxycarbonyl-pyr 
idine-5-carboxylic acid isopropyl ester, hereinafter referred to as PN; and 
PK 107-959, i.e. 
4-(2,1,3-benzothiadiazol-4-yl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarbo 
cylic acid dimethylester, hereinafter referred to as PK. 
Calcium antagonists, e.g. a compound of formula I, modulate calcium ion 
transport systems at the cellular level and this has been extensively 
published in the literature. Movements at the cellular level are, however, 
very low and changes in the total levels of calcium ions in the blood 
would not be expected. 
We have now found that ionized calcium levels in the blood are lowered to a 
significant extent by a compound of formula I over a long period of time. 
These compounds are therefore useful in the treatment of conditions 
related directly or indirectly to ionized calcium levels in the blood, 
particularly abnormal or elevated calcium levels in the blood, especially 
hypercalcaemic disorders 
Morbus Paget 
osteoporosis 
arteriosclerosis and in particular atherosclerosis (prevention or 
inhibition of plaque formation) 
algoneurodystrophy and 
acute pancreatitis. 
The present invention in one aspect provides a method of treating a subject 
suffering from a condition related directly or indirectly to ionized 
calcium levels in the blood, in particular from a hypercalcemic disorder, 
Morbus Paget, osteoporosis, arteriosclerosis, algoneurodystrophy or acute 
pancreatitis, which comprises administering a compound of formula I to the 
subject. In another aspect the present invention provides a method of 
reducing ionized calcium levels in a subject which comprises administering 
a compound of formula I to a subject in need of such treatment. 
The compound of formula I preferably is PY, PK or especially PN. 
The effect of the compounds of the invention in lowering calcium levels is 
indicated in any standard animal test for hypercalcemic activity. In one 
test the compound is administered to rabbits (e.g. ca. 1 kg in weight) 
either as a suspension e.g. 0.1 to 5 mg (e.g. 0, 1 mg) per ml of solution 
(i.e. blood plasma with ca. 1-5% alcohol) and 0.1-5 mg/kg animal body 
weight by intravenous infusion (0.5 ml per hour) or orally e.g. 1 to 5 
mg/kg animal body weight as a suspension in distilled water. A significant 
lowering of calcium ions is observed using standard measuring techniques 
for calcium ions in the blood, e.g. using a calcium ion selective 
electrode, or by photometric methods. 
One method for measuring calcium ion concentrations is as follows: 
The test involves continuous measurement of the ionized calcium 
concentration in the blood of young rabbits by means of a flow-through 
system containing Ca.sup.2+ -selective liquid membrane electrodes, based 
on the neutral carrier ETH 1001, inserted into an extracorporeal shunt 
established by means of indwelling catheters between the femoral artery 
and the femoral vein. 
The blood is pumped from the artery to the vein, through the system 
containing the Ca.sup.2+ -selective electrodes, at a flow rate of 18 ml/h. 
A second peristaltic pump transports a reference electrolyte solution at a 
rate of 0.9 ml/h past a first, reference electrode to a second electrode, 
known as the common electrode, where it meets and mixes with the 
circulating blood. The mixed fluids are then returned to the animal's 
circulatory system. 
The concentration of ionized calcium in the blood is measured 
potentiometrically as follows: The difference in potential between the 
reference electrode and the Ca.sup.2+ -selective electrode is measured 
differentially relative to the common electrode. The difference is 
amplified and the analogue signal produced by the amplifier is processed 
by a digital voltmeter and continuously recorded as a function of time, 
the difference in voltage being proportional to the Ca.sup.2+ 
concentration. 
The ionized calcium concentration in blood (1.1-1.2 mmol/liter) is about 
10.sup.5 higher than the detection limit of the electrodes in aqueous 
solutions of calcium (10.sup.-5 -10.sup.-6 mmol/liter). The method is 
capable of detecting a change of 0.003 mmol/liter or 0.3% in the Ca.sup.++ 
concentration. 
The anti-atherogenic activity has been confirmed also by measurement of the 
inhibition of neointimal lesion development in the rat carotid artery 
(balloon catheterization test, essentially as published in A. W. Clowes et 
al., Lab. Invest. 49 [1983]208-215). 
In a first experiment a group of 12 rats was given PN 250 .mu.g/kg/day in 
10% ethanol-water s.c. A control group of 9 animals was non-injected and a 
further control group of 7 animals was vehicle-injected. Animals were 
randomized into 3 groups for catheterization and were weighed every other 
day throughout the study. Rats were anesthetized with ether inhalation. 
The thoracic region was shaved and the external branch of the left common 
carotid artery was isolated by an incision from the lower mandible to the 
clavicle, followed by retracting the submaxillary gland (lateral) and 
several muscle bundles including the sternohyoideus (medial), 
sternomastoideus (lateral) and digastricus posterior belly (lateral). The 
external carotid was carefully separated from surrounding tissue and guy 
sutures were placed distal to the superior thyroid artery (cephalad end 
ligated) and proximal (end open) to the heart for retraction. Lidocaine 
was applied to vasodilate during catheter introduction. Following an 
incision with iris scissors, a Fogarty arterial embolectomy catheter (No. 
2F) was inserted into the external branch of the carotid, passed into the 
aortic arch and slightly withdrawn to assure inflation of the catheter 
within the common carotid artery. The catheter was inflated using 
approximately 900 mmHg air pressure, drawn distally and deflated. This was 
repeated three times to assure complete de-endothelialization of the 
common carotid. The catheter was removed, the artery ligated and the 
incision closed. Under these conditions the carotid is completely 
de-endothelialized. 
Animals were sacrificed 14 days after ballooning in the same sequence as 
the catheterization. Thirty minutes before fixation the animals were 
injected with a 2.25% Evan's Blue dye solution (1.5 mg/kg iv) to 
differentiate between the neointimal and re-endothelialized areas. Areas 
of the carotid that have endothelial regrowth exclude the dye and appear 
white. The remaining areas that lack endothelium are stained blue. Whole 
body (beating heart) perfusion fixations (90-110 mmHg) were then performed 
on anesthetized (sodium pentobarbital, 50.0 mg/kg ip) animals using 200 ml 
of 1% glutaraldehyde in 0.15M sodium cacodylate buffer (pH 7.4, 37.degree. 
C., 410 mOsm) followed by 400 ml of 3% glutaraldehyde in 0.15M sodium 
cacodylate. The entry site of the fixative was a puncture in the left 
ventricle, with efflux collected by vacuum suction from the right atrium. 
The thoracic aorta flow was restricted to optimize carotid perfusion. 
Successful fixation was judged by upper body rigidity and an absence of 
blood from the large arteries. 
In a second experiment the procedure as described above was followed, with 
groups consisting of non-injected controls (n=8), animals treated with PY 
(n=5) or PN (n=8). Animals received drug for 2 days before catheterization 
and to the conclusion of the study (14 days). PY and PN were given at 1.0 
mg/kg in 10% EtOH-water s.c. All animals (controls and drug-treated) were 
randomized into 3 groups for catheterization. 
After each experiment the fixed carotid was removed and cut into 3 segments 
(distal, central, proximal). Tissue processing of only the central segment 
included 18 hours in 3% buffered glutaraldehyde, buffer rinsing, 
dehydration in an ascending ethanol series and infiltration with Spurr's 
resin. The samples were embedded with cross-sectional orientation so that 
sectioning (0.5 .mu.m) would include blue areas which have not 
re-endothelialized and show continued proliferative responses. A two-step 
polychromatic stain utilizing toluidine blue and 1.0% basic fuchsin 
rendered histological differentiation to nuclear, cytoplasmic and 
extracellular connective matrix components. 
All light microscopic histology slides were randomized, encoded and 
evaluated utilizing a Zeiss standard microscope and the Videoplan 
computerized image analyzer. Vessel measurements included maximum lesion 
height. Image calibration and magnification checks were performed for 
every group of slides analyzed. Analysis of groups was done using 
parametric and non-parametric statistical methods. 
PN and PY were tolerated at all doses used, with control and treated 
animals showing similar weight gains. Maximal lesion height (.mu.m) are 
tabulated in Table 1. Non-injected and s.c. vehicle controls (Expt. 1) 
gave equivalent values and have been combined for statistical purposes. 
TABLE 1 
______________________________________ 
MAXIMAL INTIMAL HEIGHT (.mu.m) FROM CONTROLS 
AND TREATED ANIMALS 
Dose 
(mg/kg) Intima p-Value 
______________________________________ 
Expt. 1 
Controls -- 84.9 .+-. 21.3 
-- 
n = 16 
PN 0.25 63.2 .+-. 19.9 
p = 0.006 
n = 11 
Expt. 2 
Controls -- 72.4 .+-. 17.4 
-- 
n = 8 
PY 1.0 58.6 .+-. 18.1 
p = 0.097 
n = 5 
PN 1.0 38.9 .+-. 22.0 
p = 0.002 
n = 8 
______________________________________ 
The p values refer to comparisons between control and treated groups. 
Analysis was performed using a one-tail Student t-test. Similar values 
were obtained using non-parametric statistical analyses. All other values 
are mean .+-.SD for number of animals indicated. 
It can be seen from Table 1 that when given at 0.25 mg/kg/d PN inhibited 
lesion development by 26% (Expt. 1). Increasing the dose to 1.0 mg/kg/d 
resulted in 46% inhibition, whereas PY inhibited 19% at a similar dose 
(Expt. 2). Vessel diameters (determined from cross-sections of perfusion 
fixed vessels) were comparable in all three experiments. 
This effect appears to be independent of blood pressure lowering effects or 
platelet involvement. 
The compounds of the invention are therefore useful in the treatment of 
conditions related directly or indirectly to ionized calcium levels in the 
blood, in particular in the treatment of 
hypercalcaemic disorders 
Morbus Paget 
osteoporosis 
arteriosclerosis and in particular atherosclerosis (prevention or 
inhibition of plaque formation) 
algoneurodystrophy and 
acute pancreatitis. 
Preferred is the use in the treatment of arteriosclerosis and in particular 
atherosclerosis. 
An indicated daily dosage is in the range of from about 0.2 to about 350 
mg, preferably 1 to 70 mg, especially 1 to 10 mg i.v., and from about 2 to 
2000 mg per os or from about 1 to about 200 mg sublingually for interval 
therapy. Preferred for PY, PN and PK is a daily dosage of from about 0.2 
mg to about 10 mg i.v., from about 2 to about 50 mg per os or from about 1 
to about 50 mg sublingually. Especially for interval therapy the unit 
dosage may be administered in divided dosages e.g. 3 times a day 
containing from about 0.1 to about 150 mg i.v. or about 0.7 to about 700 
mg per os or about 0.3 to about 70 mg sublingually of the compound admixed 
with a solid pharmaceutical carrier or diluent. 
Additionally we have found that combinations of calcium antagonists, 
hereinafter referred to as "the compounds of the invention", e.g. calcium 
antagonists having a 1,4-dihydropyridine structure having an aryl or 
heterocyclic group attached to the 4 position, especially compounds of 
formula I, with calcitonins, have particularly advantageous properties. 
We have found that coadministration of calcium antagonists with calcitonins 
produces especially advantageous results. Such calcitonins are of any 
natural or synthetic origin and include human calcitonin, fish calcitonin 
and their derivatives, e.g. Salmon calcitonin, eel calcitonin, its 
derivative 1,7-Asu-eel calcitonin, hereinafter referred to as Elcitonin, 
and porcine calcitonin. Salmon calcitonin, hereinafter SMC, is preferred. 
In standard hypocalcaemic tests it is found that the compounds of the 
invention affect beneficially the hypocalcemic effect of calcitonins, in 
particular provide a prolongation thereof. For example in the rabbit test 
mentioned above initially calcitonin is injected at a dose of 0.1 to 5 
international units per animal kg weight about 30 minutes before the test 
starts. The compound of the invention is administered in accordance with 
the details given above. The hypocalcemic effect of the calcitonin is 
measured with regard to time. It is observed that the compounds of the 
invention prolong the effects of the calcitonin. 
An indicated daily dose of calcitonin is in the range 50 to 100% that 
normally used for known uses, e.g. 5 to 100 micrograms. This may be 
conveniently administered in unit dosage forms containing 10 to 100 
international units. The administration route is preferably parenteral, 
e.g. intramuscular, subcutaneous or intravenous. An alternative mode of 
administration is nasal administration. The compounds of the invention, 
and calcitonin, may be administered in any conventional manner used for 
standards in the same indications as mentioned above. 
Results obtained in the rabbit test mentioned above with the preferred 
compounds PN and SMC are as follows: 
______________________________________ 
PN alone: 
Lowering of ionized 
Posology Dose calcium.sup.1) 
______________________________________ 
Intravenous infusion 
0.5 mg/0.5/over 1 
10-20% 
hour 
Oral 1 mg/kg 5-10% 
______________________________________ 
.sup.1) By calcium ion selective electrode 
The intravenous experiment is repeated measuring total calcium 
photometrically. Drop in calcium levels over 11/2 hours=23%, over 21/2 
hours=30%. 
______________________________________ 
SMC + PN (given at the same dosage as 
above 30 minutes after SMC): 
SMC dose: 1 international unit/kg intravenous 
______________________________________ 
PN posology Prolongation of SMC 
hypercalcemic effect.sup.1) 
intravenous infusions 
2-5 hours 
oral 1-3 hours 
______________________________________ 
.sup.1) By calcium ion selective electrode 
PN may thus be administered to larger mammals at an oral dose of from 2 to 
20 mg. SMC may be co-administered at about 70% of the normal dosage of the 
same duration for the same mode of administration. 
The compounds of the invention and calcitonin may be administered in the 
same way as for other uses and in free form or in salt form as 
appropriate. The same formulations may be used. 
In another aspect the present invention provides a pharmaceutical 
composition comprising a compound of the invention and a calcitonin. In a 
yet further aspect the invention provides a compound of the invention and 
a calcitonin in association therewith for use in the treatment of 
conditions related directly or indirectly to ionized calcium levels in the 
blood, in particular in the treatment of 
hypercalcaemic disorders 
Morbus Paget 
osteoporosis 
arteriosclerosis and in particular atherosclerosis (prevention or 
inhibition of plaque formation) 
algoneurodystrophy and 
acute pancreatitis. 
In a further aspect the invention provides a pack or dispenser device 
containing a pharmaceutical composition comprising a compound of the 
invention and additionally a pharmaceutical composition containing a 
calcitonin together with instructions for use in the indications mentioned 
above. 
In a further aspect the invention provides the use of a compound of formula 
I in the manufacture of a pharmaceutical composition for the treatment of 
conditions related directly or indirectly to ionized calcium levels in the 
blood, in particular of conditions as mentioned above. 
The compounds of the invention may be administered on their own or in the 
form of pharmaceutical compositions. Such compositions conveniently 
contain more than 1% by weight of the compound of the invention and may be 
prepared by conventional techniques to be in conventional forms, for 
example capsules, tablets, suppositories, dispersible powders, 
suspensions, for enteral or parenteral administration. Suitable 
pharmaceutical diluents or carriers include, for example, alcohols, e.g. 
polyethyleneglycol, polyvinylpyrrolidone, mannitol and lactose as well as 
suitable preserving agents, such as ethyl-p-hydroxybenzoate, suspending 
agents such as methyl cellulose, tragacanth and sodium alginate, wetting 
agents such lecithin, polyoxyethylene stearate and polyoxyethylene 
sorbitan mono-oleate, granulating and disintegrating agents such as starch 
and alginic acid, binding agents such as starch, gelating and acacia, and 
lubricating agents such as magnesium stearate, stearic acid and talc, in 
order to provide an elegant and palatable pharmaceutical preparation. 
In view of the low solubility of many of the compounds of the invention it 
is preferred to employ solid forms. It is preferred to use pharmaceutical 
compositions formulated to facilitate rapid absorption of a compound of 
the invention. For example oral pharmaceutical compositions may be 
employed and formulated to dissolve rapidly in the mouth, e.g. sub-lingual 
tablets and capsules. Alternatively the pharmaceutical compositions may be 
in powder or liquid form for administration as a spray or mist into the 
oral cavity. 
It is contemplated that a spray applicator, e.g. an atomizer, may be used 
for administering such a spray or mist. Such spray applicators are known, 
for example atomizers for administering a liquid spray, and powder blowers 
which may be constructed to receive a cartridge containing a unit dosage 
of a liquid or powder pharmaceutical composition, break the cartridge, and 
expel the contents in the form of a spray or mist. Alternatively a 
pressurized container may incorporate a pharmaceutical composition in the 
form of a powder or liquid and compressed gas for expelling the 
compositions. 
Naturally metering devices may be incorporated to facilitate administration 
of a predetermined amount of the pharmaceutical composition. 
Compositions containing calcitonins are well known. They may be e.g. liquid 
for nasal or perenteral administration. If desired the calcitonins may be 
combined with a compound of the invention in a appropriate form, e.g. in 
liquid form. 
All these devices and the techniques used for formulating suitable 
pharmaceutical compositions are well known. 
The pack or dispenser device may contain a plurality of unit dosage forms 
containing a compound of the invention. These may be packed in metal or 
plastic foil, e.g. as a blister pack. The pack or dispenser may be 
provided with instructions for administration of a compound of the 
invention in the treatment of the conditions mentioned above.

The following Examples are illustrative of compositions for use in the 
invention. 
EXAMPLE 1 
Hard gelatine capsules for oral administration 
Hard gelatine capsules containing the ingredients indicated below may be 
prepared by conventional techniques, and be administered once a day for 
the treatment of conditions related directly or indirectly to ionized 
calcium levels in the blood: 
______________________________________ 
Ingredient Weight 
______________________________________ 
PN 10.0 mg 
Polyvinylpyrrolidone 30.0 mg 
Lactose 148.5 mg 
Corn starch 60.0 mg 
Magnesium stearate 1.5 mg 
250.0 mg 
______________________________________ 
EXAMPLE 2 
Tablets for sublingual administration 
______________________________________ 
Ingredient Weight 
______________________________________ 
PN 10.0 mg 
Polyethyleneglycol 6000 
33.0 mg 
Mannitol 50.0 mg 
Polyvinylpyrrolidone 4.5 mg 
Talc 2.0 mg 
Magnesium stearate 0.5 mg 
100.0 mg 
______________________________________ 
EXAMPLE 3 
Soft gelatine capsules for sublingual administration 
______________________________________ 
Ingredient Weight 
______________________________________ 
PN 10.0 mg 
Polyethyleneglycol 2000 
100.0 mg 
Polyethyleneglycol 400 140.0 mg 
250.0 mg 
______________________________________ 
Sufficient amounts of the above components are mixed in conventional manner 
and filled into gelatine capsules or compressed to tablets, which are 
administered once a day for the treatment of conditions related directly 
or indirectly to ionized calcium levels in the blood. 
EXAMPLE 4 
Salmon calcitonin ampoules 
Ampoules suitable for parenteral administration and containing the 
following sterile ingredients may be produced in conventional manner. The 
ampoules may be used with any of Examples 1 to 3, at a dose of 1 to 4 
ampoules daily. 
______________________________________ 
Constituents Weight 
______________________________________ 
Synthetic salmon calcitonin 
0.0000725 
g .sup.+) 
(polyacetate, polyhydrate) 
Glacial acetic acid 0.0020 g 
Sodium acetate 0.0020 g 
Sodium chloride 0.00750 g 
Water for injection up to 
1.0040 g 
1.0 ml 
______________________________________ 
.sup.+) corresponding to 0.000050 g of synthetic salmon calcitonin 
EXAMPLE 5 
Composition containing salmon calcitonin suitable for nasal administration 
______________________________________ 
Ingredient Quantity (per ml) 
______________________________________ 
1) Salmon calcitonin (active ingredient) 
0.1375 mg 
10% excess 0.01375 mg 
0.15125 mg 
2) NaCl 7.5 mg 
3) Benzalkonium chloride 
0.1 mg 
4) HCl (1N) added to ph 3.7 
5) Distilled water to an end volume 
of 1.0 ml 
______________________________________ 
Components 1) to 3) are combined under protection of nitrogen gas (on a 
scale to produce a final volume of 2500 ml) in conventional manner, with 
10% of salmon calcitonin being added to allow for loss at filtration. 4) 
is then added to bring the pH to 3.7 and distilled water added to an 
end-volume of 2500 ml. The obtained solution is filtered (e.g. using a 0.2 
.mu.m filter) to give a composition suitable for nasal dispensor with a 
solution volume of 2 ml. The composition comprises ca. 550 MRC-units 
active ingredient/ml, and the applicator delivers a quantity comprising 55 
units per actuation. 
Example 5 is administered concomitantly with anyone of examples 1 to 3.