WO2002096443A1

WO2002096443A1 - Use of a product comprising catuama extract as an antithromboembolic agent

Info

Publication number: WO2002096443A1
Application number: PCT/BR2001/000093
Authority: WO
Inventors: Osvaldo Silva Filho; Tadafissa Fujii; João BATISTA CALIXTO; Daniela De Almeida Cabrini; Alessandra Beirith; Elisabeth Soares Fernandes
Original assignee: Laboratorio Catarinense S/A.
Priority date: 2001-05-30
Filing date: 2001-07-31
Publication date: 2002-12-05
Also published as: BR0102186A

Abstract

This invention relates to the use of a product comprising Catuama extract, comprising the species Trichilia sp. (preferably of the species catigua), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae), wherein said product is an antithromboembolic agent. A product particularly encompassed by the scope of the invention is Catuama extract commercially available as CatuamaR.

Description

Title: "USE OF A PRODUCT COMPRISING CATUAMA EXTRACT AS AN ANTITHROMBOEMBOLIC AGENT, PHARMACEUTICAL COMPOSITION COMPRISING SUCH PRODUCT FOR TREATING OR PREVENTING THROMBOEMBOLIC DISORDERS, METHOD FOR TREATING THROMBOEMBOLIC DISORDERS USING SAID PRODUCT AND USE OF SAID PRODUCT FOR MANUFACTURING A PHARMACEUTICAL COMPOSITION FOR TREATING OR PREVENTING THROMBOEMBOLIC DISORDERS".

Field of the Invention The present invention relates to the use of a product comprising

Catuama extract, comprising species of Trichilia sp., particularly Trichilia ca- tigua (Meliaceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae). Background of the Invention Medicinal plants known as catuaba (Trichilia sp.) have recognized uses, due to their aphrodisiac activities, as a tonic and in the treatment of physical and mental fatigue.

Already known are, e.g., phytotherapeutic formulations prepared from extracts of catuaba plants, which can be used alone or in combination with other medicinal plant extracts, such as guarana. A number of alternative formulations containing extracts of other species of catuaba are already well-known from the state-of-the-art, all of them being related to the tonic and stimulating effect of this group of plants.

There also exists in the art phytotherapeutic products comprising a combination of extracts of plants from the Trichilia sp. species, particularly Trichilia catigua (Meliaceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae).

A commercially available product comprising extracts of the above-mentioned plants in combination with suitable carriers is Catuama®. More particularly, the product Catuama® is a phytotherapeutic widely used in Brazil. Its composition consists of 4 extracts from medicinal plants including: catuaba (Trichilia catigua, A. juss, Meliaceae - (husk)), guarana (Paullinia cupana, K., Sapinadaceae - (seed)), muirapuama (Ptychopetalum olacoides, B., Olacaceae - (root)) and ginger (Zingiber officinale, L., Zingiberaceae - (rhizome). Summary of the Invention The present invention refers to the use of a product of the extract of Catuama comprising Trichilia sp., particularly Trichilia catigua (Meliaceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae), as an antithromboembolic agent.

In another aspect, this invention refers to pharmaceutical com- positions comprising said extracts having antithromboembolic activities.

In yet another aspect, this invention refers to a method for treating and/or preventing thromboembolic disorders using said extract of Catuama.

In still another embodiment, the invention refers to the use of a product comprising extract of Trichilia sp., particularly Trichilia catigua (Meliaceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae), for preparing a pharmaceutical composition for the treatment and/or prevention of thromboembolic disorders. Detailed Description of the Invention

After extensive studies, the inventors have found that the extract of Catuama, comprising Trichilia sp., particularly Trichilia catigua (Meliaceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae), has extraordinary antithrom- boembolic activities.

As used herein "antithromboembolic activities" includes activities related to disorders such as: unstable angina or acute myocardial infarction; ischemic coronary syndromes in patients having undergone coronary angio- plasty or arteriectomy; ischemic heart complications related to the abrupt obstruction of the treated coronary artery; thromboembolic disorders of any etiology or localization, including those from surgical procedures and delivery complications and thromboangiitis obliterans, Raynaud's disease, diabetes, acrocyanosis etc., such as: intermittent claudication; trophic disorders, pre- gangrene, varicose ulcers, paresthesia, nocturnal cramps, cold extremities; disorders associated to consumption coagulopathy, thrombosis coagulopa- thy in patients suffering from nephrotic syndrome, as well as digestive dis- eases coagulopathy, and also those with inadequate or no response to heparin; in pre-eclampsia; thromboembolic diseases (specially in general and orthopedic surgery), peripheral vasculopathies and cerebrovascular alterations, prevention of postsurgical venous thrombosis, of myocardial rein- farction, of atherosclerotic injuries, and vascular diseases in patients with platelet hyperactivity; postthromboembolic disorders treated with streptoqui- nase such as, e.g., in disseminated intravascular coagulation, myocardial infarction, inhibition of coagulation upon using extracorporeal circulation (surgeries) or hemodialysis; already existing deep venous thrombosis, prophylaxis of deep venous thrombosis and recurrences, prophylaxis of pulmo- nary thromboembolism; prophylaxis and therapeutics of hyperlipidemia; treatment and/or prophylaxis of atherosclerotic events in patients with atherosclerosis caused by recent stroke or acute myocardial infarction (AMI); existing peripheral arterial disease; treatment and/or prophylaxis of pulmonary embolia, acute and subacute peripheral arterial thrombosis and chronic occlusive arterial disease, retinal artery or central vein occlusion; treatment and/or prevention in phlebology: varices and varicosities; venous failure (edema, weight sensation in the legs, etc.), consequences of thrombophlebitis, pre-ulcerous conditions and varicose ulcers, and posttraumatic edemas. A number of products comprising varying concentrations of ex- tract of the above plants are commercially available. The use thereof, so far recommended in the art, is related to the treatment of physical and mental fatigues, neuromuscular asthenia and weariness.

Studies and research now carried out by the present inventors show new antithromboembolic activities related to products based on the above mentioned extracts as confirmed by the data and tests disclosed herein.

The concentration of the extract of each plant of Trichilia sp., particularly Trichilia catigua, Paullinia cupana, Ptychopetalum olacoides and Zingiber officinale, in the product or pharmaceutical composition of the present invention ("Product comprising extract of Catuama") is as follows:

Solid formulation:

Component % (m/m) Generic Preferred

Extract of Trichilia sp (specially Trichilia 5 to 50 30 to 50 catigua)

Extract of Paullinia cupana 2 to 30 10 to 21

Extract of Ptychopetalum olacoides 0.2 to 15.0 5.0 to 12

Extract of Zingiber officinale 0.50 to 3.0 0.5 to 1.50

Suitable excipient 2.0 to 92.30 15.5 to 54.5

In its dry and excipient-free form, extract of Catuama comprises:

The product may comprise usual excipients for formulation such as preservatives, colorants, carriers, etc. Adequate excipients are well known by those skilled in the art and do not constitute limiting aspects of the invention.

For the purposes of the present invention, all plants of the genus Trichilia were found to be useful, such as, e.g., T. catigua A. Juss., T claus- seni C. DC, T casaretti C. DC, T pallida Swartz. and T. elegans A. Juss. According to a preferred embodiment of the present invention, it was found that, among the genera comprised of species Trichilia sp., Trichilia catigua is particularly suitable for the intended purposes. Additionally, the materials extracted from Trichilia sp. are preferably fragments of the whole plant, more preferably stalk, which are advantageously used as extract, more preferably they are formulated with pharmaceutically acceptable inert carriers. Formulations of Trichilia sp. useful for the present invention can be administered, e.g., orally in the form of tablets, coated tablets, hard and soft gelatin cap- sules, solutions, emulsions and suspensions; or rectally, in the form of suppositories. Suitable carriers include, but are not limited to, lactose, starch or derivatives thereof, talc, stearic acid or salts thereof in the case of solid formulations for oral administration. Suitable carriers for soft gelatin capsules include vegetable oils, waxes, fats, semi-solid and liquid polyols. Solutions may be prepared comprising selected carriers such as water, polyols and carbohydrates. In the case of suppositories, suitable carriers comprise natural or hardened oils, waxes, fats and polyols.

In addition to the carriers, the formulations of the Catuama extract according to the present invention may contain preservative agents, solubilizing agents, stabilizers, wetting agents, emulsifiers, sweeteners, coloring agents, flavoring agents, tonicity adjustment substances, buffers, coating agents or antioxidants.

However, an effective dosage for administration to humans was found to be in the range from 10 mg to 0.5 g of Catuama extract. In the case of pharmaceutical formulations containing Catuama extract, the intended effects can be effectively obtained using from 0.2 to 50% by weight of said extract, based on the total formulation. The invention will now be described specifically referring to the applicant's product having the name Catuama®, that is already commercialized in Brazil for the treatment of several chronic diseases such as physical and mental fatigue, neuromuscular asthenia and weariness. The pharma- ceutical formulations available allow the product to be administered orally. Another advantage of the product is associated with the lack of any reported undesirable or side effects, even when the product is used for long periods of time.

Together, the results discussed herein show that the Catuama extract referred to in this invention (specially Catauma®) exhibits antithromboembolic effects, specially when used regularly and for extended periods of time.

The present invention relates to the Catauma extract effects on pathologies involving vascular alterations essentially in combating and/or preventing thromboembolic disorders. Cardiovascular dysfunctions, specially those related to atherosclerosis and vascular integrity of small arteries are, without doubt, relevant and are frequently associated with the occurrence of high mortality rates. Several substances released by endothelial cells, including NO (nitric oxide) and prostacyclins, inter alia, are responsible for vascular smooth muscle relaxation and inhibition of platelet aggregation. More and more such substances have their activity associated with diverse pathologies. The decrease in the NO content is usually associated with a reduction in the activity of the enzyme NO synthase, changes in calcium influx and the increase of apoptosis of endothelial cells. The deficiency in en- dothelium-dependent vascular contractility may also cause important alterations in the contractile response by various neurotransmitters and local factors, thus worsening the vascular "rigidity" state. A change in the vascular endothelium may cause hyperplasia and an increase in platelet adhesion, which in turn, releases potent growing factors. Vascular and extravascular endothelial intervention can thus be useful for the treatment and prevention of cardiovascular and/or atherosclerotic pathologies in young and old patients. Thus, the inhibiting effect on platelet aggregation observed for the Catuama extract is of importance, principally in cases where there is a need for chronic administration. The product does not cause alterations in blood coagulation, thus having reduced side effects. Brief Description of the Drawings

Figure 1 shows the plasma platelet aggregation profile in healthy human volunteers (A). Platelet aggregation was induced by the addition of PAF (100 nM) in the presence or absence of Catuama extract. The effect of Catuama extract (10-100 μg/sample) on the plasma PAF (100 nM)-induced platelet aggregation of healthy human volunteers (B) is also shown. Each group represents the average of 6 volunteers and the vertical bars represent the E.P.M. It significantly differs from the control group, **P < 0.01.

Figure 2 shows the plasma platelet aggregation profile in healthy human volunteers (A). Platelet aggregation was induced by the addition of ADP (10 μM) in the presence or absence of Catuama extract. The effect of Catuama extract (10-100 μg/sample) on the plasma ADP (10 μM)-induced platelet aggregation of healthy human volunteers (B) is also shown. Each group represents the average of 6 volunteers and the vertical bars represent the E.P.M. It significantly differs from the control group, **P < 0.01. Figure 3 shows the effect of Catuama extract (10-400 μg/ sample) on ADP (20 μM)-induced platelet aggregation in plasma from guinea-pig blood. Each group represents the average of 6 animals and the vertical bars represent the E.P.M. It significantly differs from the control group, **P < 0.01. Figure 4 shows the effect of the chronic treatment (seven days) in rats with Catuama extract (20 mg/kg) orally or a carrier on ADP (1-10 μM)- induced platelet aggregation in rat blood. Each group represents the average of 4 to 5 animals and the vertical bars represent the E.P.M.

Figure 5 shows the effect of the sub-chronic treatment (seven days) in rats with Catuama extract (200 mg/kg) or a carrier on the inhibition of ADP (10-20 μM)-induced platelet aggregation caused by acetylsalicylic acid in rat blood. Each group represents the average of 4 to 5 animals and the vertical bars represent the E.P.M.

Figure 6 shows the effect of Catuama extract (40-80 μg/ml) on in vitro clotting time by the prothrombin method. The results are expressed as the average ± E.P.M. The asterisks indicate the significance when com- pared to the control group, ^*p < 0.05 (n = 6).

Figure 7 shows the effect of Catuama extract (40-80 μg/ml) on in vitro clotting time by the activated partial thromboplastin method. The results are expressed as the average ± E.P.M. The asterisks indicate the significance when compared to the control group, ^*p < 0.05 (n = 6). The illustrative test examples below are given for a better description of the present invention. However, the data and procedures illustrated therein refer to certain embodiments of the present invention and are not to be construed as limiting the scope thereof.

The following tests were carried out using a composition of ex- tracts in the solid and dry form (herein referred to as Catuama extract) as follows:

A - Effect on platelet aggregation factor (ADP)- or adenosine diphosphate- induced platelet aggregation (PAF) in platelet-rich plasma from rabbit, rat and human blood. Platelet-rich plasma (PRP) was obtained by centrifuging whole blood at 1500 rpm for 10 minutes at room temperature. After separation of PRP, the remaining blood was subjected to a new centrifugation at 3500 rpm for 10 minutes in order to obtain the respective platelet-poor plasma (PPP). Immediately after obtaining the PRP, it was transferred, by means of an automatic pipette, into plastic tubes, which were sealed with parafilm and used within a period of, at most, 3 hours after the collection of the material. Platelet counting was carried out with an 1% ammonium oxalate solution using an optical microscope.

The aggregometer consists of a photometer kept at 37°C and equipped with a device which maintains the sample under continued stirring and to which a recorder can be coupled allowing the accompaniment of light transmission variation , while platelet response occurs (Zucker, 1989). Born and Cross's turbidimetric method (1983) is based on the principle of trans- mittance increase through a platelet solution when aggregation is induced.

The aggregation tests were carried out in a final volume of 470 μl, as follows: aliquots of PRP (450 μl) were transferred into silicone glass cuvettes, to which 10 μl of the platelet aggregation-inducing drugs or compounds to be tested were added.

PAF (100 nM)- or ADP (10 μM)-induced platelet aggregation was verified in blood of healthy male human volunteers in the presence or ab- sence of Catuama extract (10-100 μg/sample) or a carrier.

PAF (10 nM)- and ADP (20 μM)-induced platelet aggregation were verified in blood of male and female guinea-pigs in the presence or absence of Catuama extract (10-400 μg/sample) or a carrier.

The results from the platelet aggregation assays are shown be- low:

The incubation of PRP with Catuama extract at varying concentrations (10-100 μg/sample) resulted in concentration-dependent inhibition on PAF (100 nM)-induced platelet aggregation (figure 1) or ADP (100 μM)- induced platelet aggregation (figure 2) in human blood. Maximum Inhibi- tions and 50% Inhibitory Concentrations (Cl₅₀s) are shown in Table 1.

In plasma from guinea pigs, incubation of PRP with Catuama extract (10-1000 μg/sample) inhibited ADP (20 μM)-induced platelet aggregation (figure 3). The Cl₅₀ and Maximum Inhibition of Catuama extract are shown in Table 1. Table 1 - 50% Inhibitory Concentrations (Cl₅₀) and Maximum Inhibitions (l_max) obtained for the Catuama extract in PAF (10-100 nM)- or ADP (10-20 μM)- platelet aggregation in human and guinea pig plasma.

The 50% Inhibitory Concentrations (Cl₅₀) are given under μg/sample and Maximum Inhibitions (l_max) are given under % maximum effect. Each group represents the average of 6 animals.

In another experimental group, groups of rats were treated orally (v.°), once a day, for seven days, with Catuama extract (200 mg/kg), acetyl- salicylic acid (100 mg/kg) or with a carrier only (control group) . On the seventh day, 6 hours after the last treatment, blood was collected from these animals and the PRP was subjected to ADP (0,5-20 μM)-induced platelet aggregation.

A possible interference of the treatment on the inhibition of platelet aggregation caused by acetylsalicylic acid (50-800 μg/sample) was also checked in the plasma of the groups treated subchronically (seven days) with the Catuama extract (200 mg/kg).

The results from the platelet aggregation assays, after sub- chronic treatment, are shown hereinbelow.

Subchronic treatment (seven days) in rats with Catuama extracts (200 mg/kg) orally did not significantly change ADP (0.5-20 μM)-induced platelet aggregation (figure 4). The CE₅₀s for ADP (0.5-20 μM) in the control group or the group treated with Catuama extract were: 2.50 (0.86-7.24) and 2.66 (1.28-5.54) μM, respectively. The CE₅₀s for ADP were calculated from the concentration-response curve with five points (distinct for each group) of concentrations of ADP.

The inhibition of platelet aggregation caused by acetylsalicylic acid (50-800 μg/sample) was not significantly changed when the rats were sub- chronically (seven days) treated with Catuama extract (200 mg/kg) (figure 5). B - Measurement of the prothrombin time (PT) and the activated partial thromboplastin time (PTT_A) Reconstituted Simplastin^® Excel S, heated at 37°C, was the re- actant used for determining PT and the in vitro assay was carried out according to the methodology described (Pelzer et al., 1968; Tanabe et al., 1999). Samples of plasma (0.1 ml) containing Catuama extract (40-80 μg/ml), heparin or water from milli-Q (control group) were placed into plastic test tubes (disposable, 8 ml) and kept in a water bath at 37°C for 3 to 10 minutes. Then 0.2 ml of "Simplastim^® Excel S" (previously heated) was rapidly added to the tubes containing the different plasmas and, at the same time, the quantification of the time required to form the clot was initiated. The clotting time for the different samples was measured in seconds and carried out in duplicate.

To determine PTT_A, reactant Platelin^® LS was used and an in vitro assay was carried out according to the methodology previously described (Tanabe et al., 1999). Samples of plasma (0.1 ml) containing Ca- tuama extract (40-80 μg/ml), heparin or water from milli-Q (control group) were placed into plastic test tubes (disposable, 8 ml), 0.1 ml of reactant was added and then they were kept in a water bath at 37°C for 5 minutes. Following this, 0.1 ml of the calcium chloride solution (25 mM) was added and, at the same time, the quantification of the time required to form the clot was initiated. The clotting time for the different samples was measured in seconds, with the experiments being carried out in duplicate.

The results obtained from the assays of measurement of the prothrombin time (PT) and the activated partial thromboplastin time (PTT_A) are shown hereinbelow. Incubation of Catuama extract (at concentrations varying from 40 to 80 μg/ml) was not able to cause any changes in the clotting time, both when analyzed by the prothrombin method (figure 6) and the activated partial thromboplastin (figure 7). On the other hand, incubation of heparin (100 μg/ml) used as the reference drug caused a significant increase in the clot- ting time by the two methods used.

Claims

1. Use of a product comprising Catuama extract, comprising the species Trichilia sp., Paullinia cupana (Sapindaceae), Ptychopetalum olacoi^¬ des (Olacaceae) and Zingiber officinale (Zingiberaceae), wherein said product is an antithromboembolic agent.

2. The use of claim 1 , wherein the composition of said product is as follows

Solid formulation:

Component % (m/m) Generic Preferred

Extract of Trichilia sp (specially Trichilia 5 to 50 30 to 50 catigua)

Extract of Paullinia cupana 2 to 30 10 to 21

Extract of Ptychopetalum olacoides 0.2 to 15.0 5.0 to 12

Extract of Zingiber officinale 0.50 to 3.0 0.5 to 1.50

Suitable excipient 2.0 to 92.30 15.5 to 54.5 or in its dry and excipient-free form, comprises

3. The use of any of the preceding claims, wherein said product is Catuama^®.

4. The use of any of the preceding claims, for the treatment and/or prevention of unstable angina or acute myocardial infarction, or is- chemic coronary syndromes in patients having undergone coronary angio- plasty or arteriectomy.

5. The use of any of the preceding claims, for avoiding ischemic heart complications related to the abrupt obstruction of the treated coronary artery.

6. The use of any of the preceding claims, for the treatment of thromboembolic disorders of any etiology or localization, including those from surgical procedures and delivery complications; disorders associated to consumption coagulopathy, thrombosis coagulopathy in patients suffering from nephrotic syndrome, as well as digestive diseases coagulopathy, and also those with inadequate or no response to heparin; or in pre-eclampsia.

7. The use of any of the preceding claims, for the treatment of thromboembolic diseases (specially in general and orthopedic surgery), peripheral vasculopathies and cerebrovascular alterations, prevention of postsurgical venous thrombosis, prevention of myocardial reinfarction, of atherosclerotic injuries, and vascular diseases in patients with platelet hyper- activity

8. The use of any of the preceding claims, for postthromboembo- litic treatment with streptoquinase such as, e.g., in the disseminated intra- vascular coagulation, myocardial infarction, inhibition of coagulation upon using extracorporeal circulation (surgeries) or hemodialysis

9. The use of any of the preceding claims, for the treatment and/or prophylaxis of hyperlipidemias.

10. The use of any of the preceding claims, for the treatment and/or prophylaxis of atherosclerotic events in patients with atherosclerosis caused by recent stroke or acute myocardial infarction (AMI) or existing peripheral arterial disease.

11. The use of any of the preceding claims, for the treatment and/or prophylaxis of pulmonary embolia, acute and subacute peripheral arterial thrombosis and chronic occlusive arterial disease, retinal artery or central vein occlusion.

12. The use of any of the preceding claims, for the treatment and/or prevention of pulmonary thromboembolia.

13. The use of any of the preceding claims, for the treatment and/or prevention in phlebology: varices and varicosities; venous failure (edema, weight sensation in the legs etc.), thromboangiitis obliterans, Ray- naud's disease, diabetes, acrocyanosis, trophic disorders, pre-gangrene, paresthesia, nocturnal cramps, cold extremities; consequences of thrombophlebitis, pre-ulcerous conditions and varicose ulcers, and posttraumatic edemas.

14. A pharmaceutical composition comprising Catuama extract, comprising the species Trichilia catigua (Meliaceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae) for the treatment and/or prevention of any thromboembolic disorders.

15. The pharmaceutical composition of claim 14, wherein said product has a composition as defined in claim 2.

16. The pharmaceutical composition of any of claims 13, 14 or

15, wherein said product is Catuama^®.

17. The pharmaceutical composition of any of the preceding claims, wherein said pharmaceutical composition is used for the treatment and/or prevention of any of the disorders as described in claims 2 to 14.

18. A method for treating and/preventing thromboembolic disorders, comprising administering a product of Catuama extract comprising the species Trichilia sp. (Meliaceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae) to a patient in need thereof.

19. The method of treatment and/prevention of claim 18, wherein said product has a composition as defined in claim 2.

20. The method of treatment and/prevention of any of the pre- ceding claims, wherein said product is Catuama^®.

21. The method of treatment and/prevention of any of the preceding claims, wherein said method is used for the treatment and/or prevention of any disorders as described in claims 2 to 14.

22. Use of a product comprising Catuama extract, comprising the species Trichilia sp. (Meliaceae), Paullinia cupana (Sapindaceae), Ptychopetalum olacoides (Olacaceae) and Zingiber officinale (Zingiberaceae), wherein said product is used for preparing a pharmaceutical composition for the treatment and/or prevention of thromboembolic disorders.

23. The use of claim 22, wherein said product has the composition as defined in claim 2.

24. The use of any of claims 22 or 23, wherein said product is Catuama^®.

25. The use of any of claims 22, 23 or 24, wherein said product is used for the treatment and/or prevention of any of the disorders as described in claims 2 to 14.