EP1313490A1

EP1313490A1 - Pharmaceutical composition comprising wikstromol and/or matairesinol, its use as hepatoprotectant and process for their isolation from cedrus deodara

Info

Publication number: EP1313490A1
Application number: EP00991822A
Authority: EP
Inventors: Janaswamy M. Indian Inst. Chemical Tech. RAO; Pullela V. Indian Inst. Chemical Tech. SRINIVAS; Jhillu S. Indian Inst. Chemical Tech. YADAV; K.V. Indian Inst. Chemical Tech. RAGHAVAN
Original assignee: Council of Scientific and Industrial Research CSIR
Current assignee: Council of Scientific and Industrial Research CSIR
Priority date: 2000-10-23
Filing date: 2000-10-23
Publication date: 2003-05-28
Also published as: BR0017334A; CN1484528A; JP2004512308A

Abstract

This invention relates to the isolation of two compounds namely (-)-matairesinol and (-)-wikstromol together with or associated with a therapeutically acceptable additives and useful as an antioxidants and hepatoprotective agents.

Description

PHARMACEUΗCAL COMPOSITION COMPRISING WIKSTROMOL AND/OR MATAIRES INAL, ITS USE AS HEPATOPROTECTANT AND PROCESS FOR THEIR ISOLATION FROM CEDURS DEODARA

Technical Field

This invention relates to the isolation of two compounds namely (-)- Matairesinol (4,4¹- dihydroxy-3, 3¹- dimethoxylignan-9, 9¹-olide) and (-)-wikstromol (4,4¹,8-trihydroxy-3, 3¹- dimethoxyliganan-9, 9¹-olide) from the plant Cedrus deodara in significant yields. This invention also identifies the use of the said compounds as a hepatoprotective agent, and as an antioxidant or a free radical scavenger.

Background art

The compounds (-)-Matairesinol and (-)-wikstromol as such do not find much commercial value. The structural formulae of (-)-Matairesinol and (-)-wikstromol are as shown in Figure 1. Lignans are widely distributed in angiosperms and gymnosperms. The range of their structures and biological activities is broad. Literature indicates that (-)-Matairesinol exhibits CAMP phosphodiesterase inhibition activity [W.Donald Macrae and G.H.Neil Towers, Biological activities of Lignans, Phytochemistry 23 (6), 1207-20(1984)]. (-)- Wikstromol is found active against p-388 lymphocyte leukemia and anti-HIN activity (M.K.Khamlach, R.Dhal and E.Brown; Premieres syntheses totales du (+)-wikstromol, de la (-)-Trachelogenine, de la (-)-Νortrachelogenine et des lignoides apparentes - Tetrahedran 48, 10115 - 10126 (1992)].

The wood of Cedrus deodara possesses diaphoretic, diuretic and carminative properties, and it is useful in treatment of fevers, piles and pulmonary and urinary disorders. The extract of the bark is astringent and useful for fevers, diarrhoea and dysentery. The oleoresin of deodar and the dark-colored oil obtained from the wood are valued for their application for ulcers and skin diseases. [Ref: Wealth of India, Nol.II, P.108-10 (1950 published by CSIR)].

A close look at literature suggests that the lignan, seco-isolariciresinalldiglycoside (SDG) which has been reported to possess multitude of activities is isolated from flaxseed (US Patent 5,846,944). The natural lignans (-)-matairesinol reduce the binding of H-labeled 5α- dihydrotestosterone (DHT) to human sex hormone-binding globulin (SHBG). [Matthias Schottnerand Gerhard Spiteller (J.Νat. Prod. 1998, 61, 119-121)].

P.K.Agarwal and P.Rastogi (Phytochemistry vol 21, No 6, pp 1459 - 1461, 1982) reported isolation of two lignans meso-secoisolariciresinol and cedrusinin from Cedrus deodara. The isolation of (-)-matariesional in 0.10 % yield and (+) -wikstromol in 0.124 % yield is reported from Wikstroemia viridiflora (Wikstromol, a new lignan from Wϊkstroemia viridiβora - Sheela Tandon and R.P.Rastogi; Phytochemistry 1976, vol 15, pp 1789-1791).

The extraction of the lignans and other constituents namely, (-)-nortrachelogenin, carinol and carissanol from Carissa edulis has been reported by Hans Achenbach, Reiner Waibel and Ivan Addae-Mensah, Ref. Phytochemistry, Vol.22, No.3, ρp.749-753, 1983.

There is a considerable amount of epidemiological evidence indicating an association between diet rich in fruits and vegetables and a decreased risk of cardiovascular disease and certain forms of cancer. It is generally assumed that the active principles contributing to these protective effects are nothing but antioxidant phytochemicals.

Recent research is directed to find out phytochemicals from plant sources in highlighting the role of polyphenolic compounds of plant materials as antioxidants, antimutagenic, anti inflammatory, antiatherosclerotic, antidiabetic, antihepatotoxic and antimicrobial agents. [Overview of flax lignans by Neil D. Westcott and Alister D. Muir, Crop Utilization Section, Saskatoon Research Centre, Agriculture and Agri-Food Canada, 107, Science Place, Saskatoon, SK S7N 0X2, Canada in Nolumn II - January 2000 - inform]. Alcoholic extract of stem C.deodara was found to have anti-cancer activity. [Ref. Medicinal Plants of India (ICMR) Nol.I, 1976, pp.214 and 215].

Medicinal importance of antioxidant active principles are acquiring importance because of the involvement of free radical mediated oxidative stress in age related diseases.

Free radicals are highly energized molecules that contain an unpaired electron. These are produced through normal biological and environmental processes involving oxygen and can trigger chain reactions that product more free radicals. Normally, there is a balance between the amount of free radicals generated in the body and antioxidants to protect against them. However, natural antioxidants present in the body can cope up only with the optimal generation of free radicals and any additional burden of free radicals or lack of antioxidant protection can tip this balance and lead to oxidative stress. There is a need for free radical scavengers or antioxidant principles isolated from natural resources for preparation of formulations useful in alleviating diseases. Hence, it becomes necessary to look for other lignans possessing related and important biological properties. Accordingly, the applicants conducted a detailed study on principles from Cedrus deodara and this investigation led to the isolation of two active principles namely, Matairesinol and wikstromol. These compounds, although hitherto isolated from Wi stromia spp. wherein low yields. Cedrus deodara, hence, is a new source for these lignans and their presence in this taxon in significantly high yields makes this invention more important.

Disclosure of the invention

The main object of the invention relates to use of (-)-wikstromol and/or (-)-matairesinol as free radical scavengers or antioxidants. Another object of the invention relates to use of (-)-wikstromol and useful as an hepatoprotective agent.

Yet another object of the invention is to provide methods for the isolation of (-)-wikstromol and/or (-)-matairesinol from Cedrus deodara.

Still another object of the invention is to provide methods for the treatment of artherosclerosis.

Summary of the invention

Accordingly, the invention provides novel compositions containing (-)-wikstromol and/or (-)-matairesinol and useful as free radical scavengers and antioxidants. The invention also provides novel compositions containing (-)-wikstromol and useful as hepatoprotective agent. The invention further provides methods for the isolation of (-)-wikstromol and/or (-)-matairesinol from Cedrus deodara as well as use of the compounds for the treatment of artherosclerosis.

Detailed description

Accordingly, the invention provides a composition comprising an effective amount of (-)- wikstromol together with or associated with an additive and useful as an antioxidant. In an embodiment the composition contains an effective amount of (-)-matairesinol together with additive and useful as an antioxidant. In another embodiment the composition contains an effective amount of (-)-wikstromol together with or associated with an additive and useful as hepatoprotective agent. In an embodiment the additive is selected in such a manner that they do no affect or interfere with the efficacy of active principles of the composition. The additive is such that they enhance and do not retard the activity of the active ingredients i.e. (-)-wikstromol and/or (-)-matairesinol. In another embodiment the ratio of (-)-wikstromol and/or (-)-matairesinol with the additive is in the range between 0.1 : 10 to 2: 10.

In yet another embodiment (-)-wikstromol and/or (-)-Matairesinol present in an amount of

250-300 mg per dose. Further the invention provides a process for the isolation of (-)-wikstromol and/or (-)- matairesinol from the Cedrus deodara, said process comprising the steps of: a) extraction of the pulverized plant parts oϊ Cedrus deodara with solvents to remove the essential oils; b) concentrating the extract under vacuum to obtain a residue; c) adding ethyl acetate to the residue obtained in step (b); d) separating the solvents by conventional methods; e) subjecting the residue to a first elution with about 3% methanol in chloroform to obtain (-)-matairesinol; and f) subjecting the residue of step (e) to a second elution with about 5% methanol to obtain (-)-wikstromol.

The solvents used in step (a) are hexane and chloroform.

In another embodiment, the plant parts of Cedrus deodara such as bark and leaves are used for extraction. In yet another embodiment, the wasted plant parts of Cedrus deodara are employed for isolation of the said compound. Preferably, the waste left after extraction of essential oil from the plant parts is used in the process.

There is enough literature, for example the article in Inform Vol.11, pg.118-121, that suggests that oxygen free radicals are responsible for development of arteriosclerosis. And, that, antioxidants are useful to retard hypercholestrolemic arteriosclerosis.

Accordingly, it is a further feature of the invention to treat arteriosclerosis, wherein a composition containing an effective amount of (-)-wikstromol or (-)-matairesinol is administered to a subject in need thereof.

(-)-Wikstromol or (-)-matairesinol may be administered together with or in combination with therapeutically acceptable additives. The effective amount of (-)-wikstromol or (-)- matairesinol that may be administered to a subject can be readily determined by a person skilled in the art. However, it is recommended that the dosage of (-)-wikstromol or (-)- matairesinol administered may be in the range of 250 to 300 mg per dose, twice a day.

While it is possible to administer the composition oral as well as systemic routes, the oral route achieves desired best results. Compositions employing (-)-wikstromol or (-)-matairesinol may be prepared by conventional methods as known in the art. The compositions may be in the form of tablets, capsules or syrups, etc. Suitable additives as known in the art may be selected for the preparation of these compositions.

In essence, the focus of the invention is to provide methods for using (-)-wikstromol and/or (-)-matairesinol compositions useful as a free radical scavenger and antioxidant and methods for using (-)-wikstromol as a hepatoprotective agent. The heartwood of Cedrus deodara finds extensive use in essential oil industry. The oil by name 'cedar wood' oil finds application in flavor and fragrances. The heartwood powder after extraction of essential oil is a by-product and waste. This invention relates to isolation and purification of the compounds (-)-Matairesinol and (-)-wikstromol from Cedrus deodara and their use as antioxidants and hepatoprotective agent. This present invention relates to the isolation of two compounds namely (-)- Matairesinol and (4,4¹-dihydroxy-3, 3¹- dimethoxylignan-9, θ^olide) and (-)- wikstromol (4,4^X,8- trihydroxy-3, 3^x-dimethoxyliganan-9, 9¹-olide) from a new source Cedrus deodara. This invention also relates to new use of the compounds as an antioxidant, as as hepatoprotective agent and for the treatment of atheresclorosis.

The present invention embodies isolation of (-)-Matairesinol and (-)-wikstromol, two antioxidant principles from an entirely new source and their free radical scavenging property compared with known biologically proved anti free radical agents, anti dyslipidemic and anti hepatotoxic efficacies. Brief description of accompanying drawings

The invention is illustrated by the accompanying drawings wherein:

Fig.l: represents the structural formulae of (-)-Matairesinol and (-)-Wikstromol.

Fig. 2(a)&(b): are graphical representations depicting free-radical activity of (-)- Matairesinol and (-)- Wikstromol.

Fig. 3: is a graphical representation of the hepatoprotective activity of (-)-wikstromol.

Some of the embodiments of the present invention are represented by the following examples, which should not be construed as limitations on the inventive scope of the invention. Examples 1 Experimental protocol a process for the isolation of Matairesinol and Wikstromol.

200 g of dried wood powder of Cedrus deodara was loaded in a soxhlet apparatus. The powder was first extracted with hexane to remove the essential oil composition. The residue from the extraction of hexane was further extracted with chloroform. The chloroform extract was concentrated under vacuum. The thick syrupy residue was dissolved in ethyl acetate for about 50g of residue around 60ml of ethyl acetate. The isolation of residue in ethyl acetate was added drop wise to hexane around 5Lt. The solid separated 35-g was filtered off.

The solid was loaded on silica gel column 60 120 mesh, 3.5 -cm dia. Column loaded to a height of 60cm. Initially, the column was eluted with chloroform followed by 3% methanol in chloroform of get Matairesinol.

Further elution of the column with 5% methanol in chloroform yielded (-)- Wikstromol.

The yield (-)-Matairesinol is around 0.06g.

The yield (-) wikstromol is around 8.0g.

The compounds (-)-Matairesinol and (-) wikstromol were obtained in 90% purity. The spectrochemical and physical properties of (-) matairesinol and wikstromol are as under:

(-)-Matairesinol:

1. Molecular formula C₂oH₂ Oe

2. 1H-NMR: δ 2.53(4H, m), 2.95(2H, br s), 3.86 (6H, s), 4.20-4.40(2H, m), 5.50(2H, - OH), 6.40-6.80(6H,m). ,

3. ¹³C-NMR: δ 34.48(C-8), 38.10(0-8^'), 40.90(C-7), 46.60(C-7^'), 46.60(07), 55.70 (2 X -OMe). 71.3(O-CH₂-O), 111.01, 111.53, 114.11, 114.40, 121.21, 121.95, 128.32, 129.45, 129.70, 144.30, 146.38(12 X Ar-C), 178,64(-C=O)

5. IR: Cm^-1 3560(-OH), 1765 ( γ -lactone)

6. [α]_D : - 37.09 (28°C)

(-)-Wikstromol

1. Molecular formula : C₂oH₂₂O₇ 2. 1H-NMR : 2.40-2.55(2H, m), 2.65-2.80 (2H, m), 3.10-3.20(1H, d), 3.85(6H, d), 3.95(2H,br d) 5.60(2H, d), 6.50-6.80(6H, m)

3. ¹³C-NMR: δ 31.50(07), 41.90(08), 43.74(07^'), 55.94(2 X -OMe), 70.26(O-CH₂-O), 76.33(-C-OH) 111.55, 112.81, 114.35, 114.56, 116.82, 121.42, 123.12, 126.20,

130.35, 144.27, 144.95, 146.59(12 X Ar-C)

4. MS : 3740 ")

5. [α]_D : - 30.90 (28°C)

Example - 2

In vitro evaluation of free radical scavenging antioxidant potency:

Antioxidant activity of the compounds ((-)-Matairesinol and (-)-wikstromol) were tested for their capacity/potency to scavenger most widely used free radical 2,2-diphenyl-l-picryl hydroxyl radical (DPPH). The well-accepted and tested probucol and α-tocopherol were taken as reference compounds, lmg/ml DMSO concentration of the compounds [(-)- Matairesinol and (-)-wikstromol] were prepared and subsequently, serially diluted to lower concentrations with DMSO 100 ml of test compounds were reconstituted to 1ml in Tris- HC1 buffer (pH 7.4) equal volume of 500 um of DPPH radical dissolved in ethanol was reacted with this. After incubation for 45 minutes in dark, the absorbency at 517nm was recorded. Radical with no compound was taken as control for calculation of % radical scavenging activity. All the readings were recorded in triplicate. Results (Figs. 2(a)&(b) & 3) show that compounds under consideration possess more potent antioxidant/radical scavenging potency than reference standards. Figures 2(a)&(b) shows the data wherein the antioxidant activity of (-)-Matairesinol and (-)- Wikstromol in comparison to commonly used Producol and Vitamin-E. It is well observed that the test compounds consistently show higher antioxidant activity than reference compounds in much lower concentrations.

Example 3 Carbon tetrachloride is a classical method to induce free radical mediated hepatotoxicity. In order to evaluate the free radical mediated hepatotoxicity and hepatoprotective properties in the compound (-)-wikstromoi, this method was employed. Male wistar rats were selected for study. The compound was dissolved in a small amount of DMSO and reconstituted in gum acacia/water. The compound was given one hour before CC1 administration to the over-night fasted rats in the dose of 250mg/kg-body weight orally. After one hour mixture of CC1₄ and liquid paraffin (1 : 1, v/v) in the dose of 2.5ml/kg was given orally. Fasting was continued. However, water was provided ad-libitum. After 5hrs of CC1 administration, blood was taken out from retroorbital plexus. Serum aspartase transaminase and serum alanine transaminase was measured as a marker of hepatotoxicity. Table 1 and fig.3 show significant protection offered by the compound, (-)-wikstromol. Figure 3 shows that (-)- wikstromol significantly prevented CC1₄ induced toxicity in rats.

Table 1

Table 1. Serum Aspartate transaminase (AST) and Alanine transaminase (ALT) status before oral administration of compound and CC1 and 5 hours after administration. (Values are mean ± SD, no in parenthesis are no. of animals in each group).

In accordance with the practice of this invention, it has been found that (-)-matairesinol and (-)-wikstromol are isolated from a new source namely the plant Cedrus deodara in significant yields. Also, it has been found that (-)-matairesinol and (-)-wikstromol show antioxidant properties and hepatoprotective properties.

ADVANTAGES

Antioxidant compounds recently have attracted the attention due to their broad spectrum activities in disorders of multiple origin viz., coronary heart disease, cancer, diabetes, rheumatic disorders and inflammatory condition where free radicals in causing/fostering the disease play important role. Much attention is being directed now to harness and harvest the antioxidant compounds from natural resources.

The compounds (-)-matairesinol and (-)-wikstromol are used in pure form.

Hence, the usage is more advantageous than a mixture of compounds having similar properties, which are in current use. It is also important to note that the process of isolation of (-)-Matairesinol and (-)- wikstromol is highly economical when compared to the isolation of tocopherol which is a widely used antioxidant. (-)-Matairesinol and (-)-wikstromol are used at a high degree of purity of over 90%. They are found to be highly effective when administered at a dosage of 250mg/kg of body weight in an animal.

Claims

1. A pharmaceutical composition useful as an antioxidant or as a free radical scavenger, said composition comprising an effective amount of (-)-wikstromol and/or (-)-matairesinol together with or associated with an additive.

2. A composition as claimed in claim 1 wherein, the additive is selected in such a manner such that it does not affect or interfere with efficacy of (-)-wikstromol and/or (-)-matairesinol.

3. A composition as claimed in claim 1 wherein, the additive is selected from nutrients such as carbohydrates, proteins, sugar and pharmaceutically acceptable carriers.

4. A composition as claimed in claim 1 wherein, the ratio of (-)-wikstτomol and/or (-)-matairesinol with the additives is in the range between 0.4:10 to 2:10.

5. A composition as claimed in claims 1 wherein, the amount of (-)-wikstromol or (-)-matairesinol is 250 to 300mg per dose.

6. A pharmaceutical composition useful as a hepatoprotective agent, said composition comprising an effective amount of (-)-wikstromol together with or associated with an additive.

7. A composition as claimed in claims 2 wherein, the additives is selected in such a manner such that it does not affect or interfere with efficacy of (-)-wikstromol.

8. A composition as claimed in claim 2 wherein, the additive is selected from nutrients such as carbohydrates, proteins, sugar and pharmaceutically acceptable carriers.

9. A composition as claimed in claim 2 wherein, the ratio of (-)-wikstromol with the additives is in the range between 0.4: 10 to 2: 10.

10. A composition as claimed in claims 2 wherein, the amount of (-)-wikstromol is 250 to 300mg per dose.

11. A process for the isolation of (-)-wikstromol or (-)-matairesinol from the plant Cedrus deodara, said process comprising the steps of : a) extracting the pulverized plant parts of Cedrus deodara with solvents to remove the essential oils; b) concentrating the extract under vacuum to obtain a residue; c) adding ethyl acetate to the residue obtained in step (b); d) separating the solvents by conventional methods; e) subjecting the residue to a first elution with about 3% methanol in chloroform to obtain (-)-matairesinol; and f) subjecting the residue of step (e) to a second elution with about 5% methanol to obtain (-)-wikstromol.

12. A process as claimed in claim 11 wherein the solvents are hexane and chloroform.

13. A process as claimed in claim 11 wherein, the plant parts of Cedrus deodara are selected from the waste material of Cedrus deodara left after the extraction of essential oils.

14. A process as claimed in claim 11 wherein, the yield of (-)-wikstromol is about 4% of the plant dried material.

15. A process as claimed in claim 11 wherein, the yield of (-)-matairesinol is about 0.03% of the plant dried material.

16. A process as claimed in claim 11 wherein, the compound (-)-matairesinol has the following spectrochemical and physical properties, a) Molecular formula C oH₂₂O6 b) 1H-NMR: δ 2.53(4H, m), 2.95(2H, br s), 3.86 (6H, s), 4.20-4.40(2H, m),

5.50(2H, -OH), 6.40-6.80(6H,m). c) ¹³C-NMR: δ 34.48(08), 38.10(08^'), 40.90(07), 46.60(07^'), 46.60(07), 55.70 (2 X -OMe). 71.3(O-CH₂-O), 111.01, 111.53, 114.11, 114.40, 121.21, 121.95, 128.32, 129.45, 129.70, 144.30, 146.38(12 X Ar-C), 178,64(-C=O) e) IR: Cm^-1 3560(-OH), 1765 ( γ -lactone) f) [α]_D : - 37.09 (28°C)

17. A process as claimed in claim 11 wherein, the compound (-)-wikstromol has the following spectrochemical and physical properties. a) Molecular formula : C₂₀H₂₂O₇ b) 1H-NMR : 2.40-2.55(2H, m), 2.65-2.80 (2H, m), 3.10-3.20(1H, d), 3.85(6H, d), 3.95(2H,br d) 5.60(2H, d), 6.50-6.80(6H, m) c) ¹³C-NMR: δ 31.50(07), 41.90(08), 43.74(07^'), 55.94(2 X -OMe), 70.26(O-CH₂-

O), 76.33(-OOH) 111.55, 112.81, 114.35, 114.56, 116.82, 121.42, 123.12, 126.20, 130.35, 144.27, 144.95, 146.59(12 X Ar-C) e) [α]_D : - 30.90 (28°C) 18. Use of (-)-wikstromol and/or (-)-matairesinol to manufacture a composition for the treatment of artherosclerosis.

19. Use as claimed in claim 18 wherein, (-)-wikstromol and/or (-)-matairesinol is administered together with or in combination with therapeutically acceptable additives.

20. Use as claimed in claim 18 wherein, the additives is selected in such a manner such that it does not affect or interfere with efficacy of (-)-wikstromol and/or (-)-matairesinol.

21. Use as claimed in claim 18 wherein, the additive is selected from nutrients such as carbohydrates, proteins, sugar and pharmaceutically acceptable carriers.

22. Use as claimed in claim 18 wherein, the ratio of (-)-wikstromol and/or (-)-matairesinol with the additives is in the range between 0.4: 10 to 2: 10.

23. Use as claimed in claim 18 wherein, (-)-wikstromol or (-)-matairesinol is administered in the range of 250 to 300 mg per dose, twice a day.

24. Use as claimed in claim 18 wherein, (-)-wikstromol or (-)-matairesinol are administered orally.

25. A method of use of (-)-wikstromol and/or (-)-matairesinol as an antioxidant and as a free radical scavenger, comprising the steps of administration of an effective amount of

(-)-wikstromoI and/or (-)-matairesinol to a subject in need thereof.

26. A method as claimed in claim 25 wherein, (-)-wikstromol and/or (-)-matairesinol is administered together with or in combination with therapeutically acceptable additives.

27. A method as claimed in claim 25 wherein, the additives is selected in such a manner such that it does not affect or interfere with efficacy of (-)-wikstromol and/or (-)- matairesinol.

28. A method as claimed in claim 25 wherein, the additive is selected from nutrients such as carbohydrates, proteins, sugar and pharmaceutically acceptable carriers.

29. A method as claimed in claim 25 wherein, the ratio of (-)-wikstromol and/or (-)- matairesinol with the additives is in the range between 0.4: 10 to 2: 10.

30. A method as claimed in claim 25 wherein, (-)-wikstromol or (-)-matairesinol is administered in the range of 250 to 300 mg per dose, twice a day.

31. A method as claimed in claim 25 wherein, (-)-wikstromol or (-)-matairesinol are administered orally.

32. Use of (-)-wikstromol for the manufacture of a composition useful as a hepatoprotective agent.

33. Use as claimed in claim 32 wherein, (-)-wikstromol is administered together with or in combination with therapeutically acceptable additives.

34. Use as claimed in claim 32 wherein, the additives is selected in such a manner such that it does not affect or interfere with efficacy of (-)-wikstromol.

35. Use as claimed in claim 32 wherein, the additive is selected from nutrients such as carbohydrates, proteins, sugar and pharmaceutically acceptable carriers.

36. Use as claimed in claim 32 wherein, the ratio of (-)-wikstromol with the additives is in the range between 0.4: 10 to 2:10.

37. Use as claimed in claim 32 wherein, (-)-wikstromol is administered in the range of 250 to 300 mg per dose, twice a day.

38. Use as claimed in claim 32 wherein, (-)-wikstromol is administered orally.