CA2097611A1 - Verbenone, having antielastase actibity, against pulmonary emphysema - Google Patents

Abstract

The use of a terpene derivative for the preparation of a medicament useful in the treatment of pulmonary emphysema.

Description

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

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~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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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 - ;- .. .. .. .

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.

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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: . ~ ' . : : ' ' The results are shown in 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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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.

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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Claims (5)

1. The use of [(15)(-)-4,6,6-trimethylbicyclo-(3,1,1)-hept-3-ene-2-one] of formula for the preparation of a medicament having antielastase activity.

2. The use of [(lS)(-)-4,6,6-trimethylbicyclo-(3,1,1)-hept-3-en-2-one] for the preparation of a medicament useful for the treatment of pulmonary emphysema.

3. The [(lS)(-4,6,6-trimethylbicyclo-(3,1,1)-hept-3-en-2-one]-cyclodextrin complex.

4. Pharmaceutical compositions containing the complex of claim 3.

5. Compositions according to claim 4, in form of capsules, pills, tablets, sugar-coated pills, granulates, syrups, drinkable solutions, nasal drops, solutions for aerosol or inhalation, suppositories, injectable solutions or suspensions for both the intravenous and intramuscular routes.