Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
w o 93/06823 2 0 9 7 611 PCT/EP92/02269
VFRBENONE, HAVING ANTIELASTASE ACTIVITY,AGAINST PUUMONARY EMPHYSEMA
The present invention relates to the use of a
terpene derivative, namely (lS)(-)-4,6,6-
trimethylbicyclo-(3,1,1)-hept-3-ene-2-one (hereinafter
named verbenone) of formula
S O
~ 3
C~
~3
for the preparation of a medicament useful in the
treatment of pulmonary emphysema. The invention also
relates to a verbenone-cyclodextrin complex and the
pharmaceutical compositions containing i~.
Pulmonary emphysema is a progressive respiratory
disorder characterized by an abnormal and permanent
increase in the volume of the air spaces located
distally to the terminal bronchiole, with destruction
of the alveolar septum.
A number of experimental and clinical data
generally ascribe the emphysema pathogenesis to an
unbalance of the protease-antiprotease ratio at the
alveolus level. Under normal conditions, alveolar
proteases exert an essential activity, which is the
lysis of cell fragments, thrombi and particles. Said
activity is counterbalanced be endogenous antiproteases
~ 1 antitrypsin [a-lAT], ~-2 macroglob~lin [~-2M],
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W093/06823 2 0 9 7 611 PCT/EP92/02269
Bronchial Mucus Inhibitor [B.M.I.]), which yrotect the
Dulmonary tissue from the protease action. The loss of
the normal balance between proteases and antlproteases
can be attributed to three factors: 1) congenital lack
in antiproteases (particularly ~-lAT); 2) excessive
protease release at the alveolus level; 3) partial
inactivation of antiproteases (~-lAT1 in the presence
of a normal or enhanced protease production. Said
factors are generally worsened by tobacco smoke.
Among alveolar proteases, leukocytal elastase
proves to have the most marked digestive activity on
all the pulmonary connective components, particularly
on elastin, the fibres of which constitute the alveolar
septa, thus giving lung the functional elasticity.
Besides the traditional treatments, which cannot
remedy the elastin destruction, therapies have been
studied to restore elastin synthesis and to protect
elastin from elastases action. Satisfactory
experimental results were obtained both administering
human a-lAT intravenously and stimulating the hepatic
synthesis thereof by administration of danatrol, which
is a testosterone semi-synthetic derivative. However,
both methods suffers from drawbacks, the first method
being particularly complex and expensive, whereas the
~econd has antigonadotropinic effects.
Therefore there i5 the need for a medicament
exerting an antielastase activity, directly or indi-
rectly, and also having poor side-effects.
Now it has been found that verbenone exerts an
unpredictable antielestase activity at the lung level,
and, dl~e to said activity, it is use~ul in the
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W093/06823 2 0 9 7 6 11 PCT/EP92/02269
treatment of emphysema.
Verbenone is a monoterpene derivative havins
antiinflammatory and mucolytic activities and it is
used as a mucolytic and fluidifying agent in the acute
and chronic disorders of the respiratory tract.
As a consequence of further researches, verbenone
showed a surprising inhibiting activity on the elastase
action, particularly on the neutrophylic one. Said
activity was unforeseeable from the pharmacological and
clinical studies which evidenced the mucolytic and
antiinflammatory activities of verbenone and the use
thereof in therapy.
The use of verbenone in the treatment of pulmonary
emphysema is extremely advantageous. In fact, pulmonary
emphysema can be favoured, or worsened, by the
simultaneous presence of other pulmonary disorders,
such as bronchitis and/or pneumonia, other infective
forms, asthma, which involve obstructions of the
respiratory tract; therefore a medicament which can at
the same time have antiinflammatory and mucolytic
activities and protect elastin of the alveolar septa is
obviously useful.
Tests were carried out in vitro to evaluate
verbenone activity on the release of neutrophilic
elastin from polymorphonuclear granules. Said tests
evidenced the marked antielastase activity of
verbenone, which activity is not dose-related nor
cyto~oxic.
The results from said tests are reported
hereinbelow.
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W093/06823 PCT/EP92/02269
Separation of neutro~hilic polymorphonuclears
Polymorphonuclear phagocytes were separated
starting from heparinized peripheral blood (60 cc) from
healthy volunteers or from buffy coats (leukocytes and
platelets enrichments).
Neutrophilic polymorphonuclears were separated by
means of a 6~ colloidal dextran gradient, then the
erythrocytes were lysated with an ammonium chloride
solution.
Neutro hilic elastase release
p
100 ,ul of cells at a concentration of lxlO7/ml,
pretreated with cytochalasin B (5 ~g/ml) for 10', were
incubated or 20' at 37C in a microtiter plate in the
presence or in the absence of 50 yl of a challenge
consisting ~f fMLP (formyl-methionyl-leuc~l-peptide) at
a 10-6 molar inal concentration. After said
incubation, the plate was centrifuged and 100 ~1 of
supernatant were collected and incubated for 60-120' at
37C with a specific substrate for the neutrophilic
elastase (methoxysuccinyl-ala-ala-pro-val-p-Na) to a 1
mM final concentration. The reaction was stopped by
adding 50 ~1 of 1 N acetic acid and reading was carried
out by means of a multiscan photometer at a 405 nm wave
length. The values of the absorbance of samples
challenged with f.~LP were compared to those from
unchallenged cells, to obtain a ~ value corresponding
to the amount of neutrophilic elastase released
following to challenge. Any interferences of verbenone
on the neutrophilic elastase release were tested by
addition of different concentrations of the medicament
directly to the cell suspension during the test.
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W093/06823 2 0 9 7 6 1 1 PCT/EP92/02269
The results are shown in Table 1.
TABLE 1
Modulation of the elastase release fro~ neutrophilic
polymorphonuclears by addition of verbenone to the
cells during the test, at different concentrations.
-
% inhibitioncompared to the control
Control
Verb. 1 mM 90%
Verb. 0,1 mM 86%
Verb. 10 uM 94%
Verb. 1 pM 94%
Verb. 0,1 yM 92%
Verb. 10 nM 89%
Verb. 1 nM 83%
Verb. 0,1 nM 96%
Verb. 10 pM 94%
This marked inhibiting activity is unlikely to be
ascribed to a toxic action of the medicament on the
cells. In fact the cell viability, as repeatedly
evaluated by the tripan bleu exclusion test, turned out
to be always higher than 90% after incubation of the
cells with verbenone in concentrations varying from 1
mM to lQ pM.
From the industrial point of view, verbenone is
used as the active ingredient for the preparation of a
medicament useful for the treatment of pulmonary
emphysema.
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W093/06X23 2 0 9 7 61 1 PCT/EP92/02269
For the preparation medicaments, verbenone has the
drawbacks to be insoluble in water and to have a very
unpleasant taste, therefore it cannot be used for
aqueous liquid pharmaceutical forms, such as syrups,
S oral solutions, nasal drops, aqueous solutions for
aerosol and nebulizat on.
It has been found that the verbenone-cyclodextrin
complex has high water solubility and, moreover,
verbenone unpleasant taste is no more perceptible.
Therefore, the verbenone-cyclodextrin complex is a
further object of invention. A further advantage of the
complex of the invention is provided by its increased
bioavailability.
The pharmaceutical forms comprising the verbenone-
cyclodextrin complex can be prepared, for example,according to the methods described in "Remington's
Pharmaceutical Sciences Handboo~", Hack Pub. Co., U~A.
Examples of pharmaceutical forms are capsules,
pills, tablets, sugar-coated pills, granulates, syrups,
drinkable solutions, nasal drops, solutions for aerosol
or inhalation, suppositories, injectable solutions or
sus~ension~ both for the intravenous and the
intramuscular routes.
The dosages will depend on the severity of the
disorder, the age, weight and sex of the patient and on
the clinician's advise. Examples of dosages comprise
single or repeated administrations of the medicament
containing verbenone or the verbenone-cyclodextrin
complex so as to attain a daily dosage from 10 to 500
mg of the active ingredient. The preparation of the
verbenone-cyclod_xtrin complex is illustrated in the
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W093/06823 2 0 9 7 ~11 PCT/EP92/02269
followin~ examples.
EXAMPLE 1
A ~-ciclodextrin amount equal to 0.1 mole is
weighed and placed into a mortar of suitable size.
Verbenone ~0.1 mole), previously diluted with 15 ml of
96% ethanol, is added, and the mixture is mixed with a
pestle to obtain an homogeneous mass. 20 ml of
distilled water are added, and a mixture is obtained
which hardens upon mixing. Said mixture is worked in a
~ortar for about 15 minutes, then it is dried in oven
thermostatized at 60C.
The obtained complex is in form of a flowing
powder which is completely odourless.
Verbenone titre ~ 80% on theoretical.
XA~PL~ 2
5.80 g o verbenone were dissolved in 100 ml of
95C ethanol (to obtain 50 g of a mixture), then they
were added to 44.3 g of B-cyclodextrin in a round
bottom flas~. The mixture is evaporated to dryness,
keeping temperature below 35C, then it is placed into
in a thermostatized oven at about 60C until complete
dryness. After drying, the product is sieved through a
~6 mesh sieve.
The obtained product is an equimolar mixture (1:1)
of verbenone and cyclodextrin (50 g theoretical, 49 g
found) .
~XAMPL~ 3
68 g of B-Cyclodextrin are dispersed in 500 ml of
distilled water (50 mmoles) and the solution is heated
to 65C under stirring, until complete dissolution.
7.51 g of L-verbenone (50 mmoles) are added to this
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W093/06823 2 0 9 7 fi 1 1 PCTIEP.92/02269
solution, with stirring. The mixture is left to cool to
room temperature with stirring for 60 minutes, then it
is kept at +4C for 6 hours.
The solid phase is recovered by filtration and the
solid is placed into a convection oven at 40C for 18
hours, to obtain 64 g of a white powdery product,
containing from 11.3 to 12.0% w/w of L-verbenone.
Yield _ 84.65~.
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