WO2000053198A1

WO2000053198A1 - Inhibitor of lipoxygenase pathways

Info

Publication number: WO2000053198A1
Application number: PCT/AU2000/000179
Authority: WO
Inventors: Theodore Macrides; Nicolette Kalafatis; Henry W. Betts
Original assignee: Pharmalink International Limited
Priority date: 1999-03-10
Filing date: 2000-03-10
Publication date: 2000-09-14
Also published as: AUPP910699A0

Abstract

A method of inhibition of a lipoxygenase pathway, particularly for the treatment of a disease or condition associated with a lipoxygenase pathway, in a human or animal patient which comprises administration to the patient of an effective amount of a lipid extract of Perna canaliculus or Mytulis edulis.

Description

INHIBITOR OF LIPOXYGENASE PATHWAYS

FIELD OF THE INVENTION

This invention relates in general to a preparation having effective activity as an inhibitor of the lipoxygenase pathways, the preparation being a lipid extract of mussels, including the New Zealand green-lipped mussel, Perna canaliculus, and the blue mussel, Mytilus edulis. In particular, the invention relates to the use of this preparation as a prophylactic or therapeutic agent in inhibition of lipoxygenase pathways, particularly the 5- and/or 12-lipoxygenase pathways, for example in the treatment of cancer by inhibiting tumour cell proliferation and tumour metastasis, as well as in the treatment of asthma, atherosclerosis and other diseases or conditions associated with a lipoxygenase pathway.

BACKGROUND OF THE INVENTION

The role of the metabolites of arachidonic acid in inflammation has been well established, and many of the current therapeutic agents for the treatment of inflammation involve the inhibition of arachidonic acid metabolism. For example, many of the non-steroidal anti-inflammatory drugs inhibit the formation of prostaglandins and thromboxane via the cyclo-oxygenase pathway.

The metabolism of arachidonic acid via the 5- lipoxygenase pathway of leukocytes leads to the formation of leukotriene B₄ (LTB₄) and the leukotrienes C₄, D₄ and E₄ (LTC₄, LTD₄ and LTE₄) as shown in Figure 1. LTB₄ is a potent chemotactic agent and is responsible for the increased number of leukocytes at sites of inflammation. LTC₄, LTD₄ and LTE₄ are very potent broncho-constricting agents produced by eosinophils in the lung, and whose production is massively increased during an asthma attack. Zileuton (Zyflo™, Abbott) is a selective, orally active inhibitor of 5- lipoxygenase, and this product has been shown to exert anti-inflammatory and antiallergic effects in animal models and humans. It is used for the prevention and chronic treatment of asthma in patients of at least 12 years of age.

As shown in Figure 1 , another major metabolite of the 5-lipoxygenase pathway is 5-hydroxyeicosatetraenoic acid (5-HETE). Until recently there were no known major physiological roles for 5-HETE. Recent research has, however, demonstrated that 5-HETE is involved in the proliferative response of cancer cells. In addition, it has also been demonstrated that the product of the 12-lipoxygenase pathway, 12-HETE, is involved in tumour metastasis.

The role of lipoxygenase metabolites such as 5-HETE and 12-HETE in cancer is demonstrated by the following observations:

(i) Increased production of 5-HETE and 12-HETE in tumour cells

In the human prostate cancer cells, 12 -lipoxygenase mRNA levels are elevated compared to normal cells, such expression correlated with poor differentiation and invasiveness of the tumour (Gao et al, 1995).

In patients with breast cancer, the levels of 12-lipoxygenase mRNA were higher compared to normal breast tissue, and similar findings were observed in cultured breast cancer cells compared to normal epithelial cells

(Natarajan et al. 1997).

Cultured human prostate cancer cells (PC-3) fed with arachidonic acid showed an increased production of 5-HETE, which was blocked by the 5- lipoxygenase inhibitor MK886 (Ghosh and Myers, 1997). Mice treated with urethane to induce lung tumours had higher levels of PGE₂ and HETEs in the lung tissue compared to control mice (Ichikawa et al., 1997).

(ii) 5-HETE stimulates tumour cell proliferation.

In the human prostate cancer cell line (PC-3) cultured with arachidonic acid the increased cell growth correlated with the amount of 5-HETE synthesised (Ghosh and Myers, 1997). The 5-lipoxygenase inhibitor MK886, but not 12-lipoxygenase inhibitors, inhibited this effect, which was reversed by the addition of exogenous 5-HETE (Ghosh and Myers, 1997).

In cultured human breast cancer cells (HS578T), the lipoxygenase inhibitors nordihydroguairetic acid and esculetin, but not the cyclo-oxygenase inhibitor piroxicam, suppressed cell growth (Hofmanova et al, 1996).

In patients with breast cancer, the 5-lipoxygenase inhibitor, tamoxifen, has been clinically effective in prolonging life (Tavares et al, 1987).

Both in in vivo and in vitro studies of colon cancer in mice using

MAC26 and MAC13 tumour cells, low concentrations of linoleic acid and arachidonic acid stimulated cell growth. This growth was inhibited by cyclo- oxygenase and lipoxygenase inhibitors indomethacin and BWA4C (Hussey and Tisdale, 1996). In the human pancreatic cell line (Panc-1), the 5-lipoxygenase inhibitor MK886 induced cell death (Anderson et al , 1998).

In mice with Lewis lung cancers, cancer cell growth and metastasis was inhibited following administration i.p. of minocycline and phenidone- cyclo-oxygenase and lipoxygenase inhibitors respectively (Teicher et al. , 1994).

(Hi) 12-HETE promotes tumour metastasis

Tumour metastasis is characterised by a variety of quite distinct physiological processes including detachment of tumour cells from the primary tumour, intravasation from the primary tumour site into the blood stream, adherence to blood vessel endothelium at a remote sight, induction of endothelial cell retraction and extravasation and migration to a new tissue site. Interactions between tumour cells and platelets which produce 12- HETE are very important in the process of retraction and extravasation (Honn et al, 1994a)

Cultured amelanotic melanoma cells (B16a) are found in two forms.

The high metastatic cells (HM340) generated high levels of 12-HETE and low levels of 5-HETE, whereas the low metastatic line (HL180) generated only low amounts of both HETEs. The lipoxygenase inhibitor N-benzyl-N- hydroxy-5-phenylpentanamide inhibited 12-HETE production and the ability to adhere to endothelial cells and the formation of new tumours in the lung

(tiu et al, 1994).

The exogenous addition of 12-HETE induces time- and dose- dependent endothelial cell retraction in both large and micro-vessels (Honn et al , 1994a, 1994b).

12-HETE regulates the expression of receptor-mediated adhesion of tumour cells to endothelial cells, sub-endothelial matrix and fibronectin (Honn et al, 1988). Pretreatment of murine melanoma tumour cells with exogenous 12- HETE enhances αllbβ-integrin mediated adhesion to and spreading on fibronectin (Timar, et al, 1992).

In rat prostate adenocarcinoma, 12 HETE increases the motility and invasion of tumour cells (Lui et al , 1997).

Recent studies (Steinber, 1999; Cyrus et al, 1999) have demonstrated the role of lipoxygenases, particularly 12/15-lipoxygenases, in the.pathogenesis of atherosclerosis, and suggest that inhibition of these lipoxygenases may decrease disease progression. Atherosclerosis is regarded as the underlying cause of myocardial infarction, stroke and vascular occlusive disease of the extremities, and is the leading cause of mortality in countries such as the United States of America. Accordingly, inhibitors of lipoxygenases have a role in the prevention and/or treatment of atherosclerosis.

SUMMARY OF THE INVENTION

International Patent Application No. PCT/AU96/00564 discloses a preparation having anti-inflammatory activity, particularly anti-arthritic activity, which comprises a lipid extract of Perna canaliculus or Mytilus edulis rich in non-polar lipids, which is prepared by supercritical fluid extraction from crude mussel powder.

In work leading to the present invention, it has been demonstrated that the lipid extract disclosed in International Patent Application No. PCT/AU96/00564 is an effective inhibitor of LTB₄ and 5-HETE synthesis in isolated human polymorphonuclear neutrophils and of 12-HETE production by human platelets.

Accordingly, in one aspect the present invention provides a method of inhibition of a lipoxygenase pathway, particularly the 5-lipoxygenase pathway and/or the 12-lipoxygenase pathway, which comprises administration of an effective amount of a lipid extract of Perna canaliculus or Mytulis edulis.

In another aspect, the present invention provides a method for inhibition of leukotriene synthesis, particularly inhibition of LTB₄, LTC₄, LTD₄ and LTE₄ synthesis, which comprises administration of an effective amount of a lipid extract of Perna canaliculus or Mytulis edulis.

In a further aspect, the present invention provides a method for the treatment of a disease or condition associated with a lipoxygenase pathway, particularly the 5-lipoxygenase pathway and/or the 12-lipoxygenase pathway, in a human or animal patient which comprises administration to the patient of an effective amount of a lipid extract of Perna canaliculus or Mytulis edulis.

Preferably, the lipid extract is an extract rich in non-polar lipids as described in International Patent Application No. PCT/AU96/00564, particularly a lipid extract prepared by supercritical fluid extraction from crude mussel powder.

The present invention extends to the use of a lipid extract of Perna canaliculus or Mytulis edulis in the preparation of a composition for use in inhibition of a lipoxygenase pathway, particularly in inhibition of the 5-lipoxygenase pathway and/or the 12-lipoxygenase pathway.

The invention also extends to the use of a lipid extract of Perna canaliculus or Mytulis edulis in the preparation of a composition for use in inhibition of leukotriene synthesis, particularly inhibition of LTB₄, LTC₄, LTD₄ and LTE₄ synthesis.

In yet another aspect, this invention extends to the use of a lipid extract of Perna canaliculus or Mytilus edulis in the preparation of a composition for use in treatment of a disease or condition associated with a lipoxygenase pathway, particularly the 5-lipoxygenase pathway and/or the 12-lipoxygenase pathway, in a human or animal patient.

The present invention also extends to a composition for inhibition of a lipoxygenase pathway, particularly the 5-lipoxygenase pathway and/or the 12- lipoxygenase pathway, which comprises a lipid extract of Perna canaliculus or Mytilus edulis, together with one or more pharmaceutically acceptable carriers and/or diluents.

As used herein, the term "treatment" extends to both prophylactic and therapeutic treatment of the particular disease or condition in the patient.

As used herein, the term "disease or condition associated with a lipoxygenase pathway" is used to encompass all diseases or conditions in which metabolites of a lipoxygenase pathway (particularly the 5-lipoxygenase pathway and/or the 12-lipoxygenase pathway) play a role, and in which at least partial inhibition of the lipoxygenase pathway can provide an effective treatment. These diseases or conditions include, by way of example: • respiratory diseases or conditions such as asthma, bronchial disease and chronic obstructive pulmonary disease (COPD);

• vascular diseases or conditions such as atherosclerosis, coronary artery diseases, hypertension and sickle cell disease-associated vaso-occlusion; skin diseases or conditions such as various dermatitis, psoriasis and atopic eczema;

• gastrointestinal diseases or conditions such as inflammatory bowel disease, ulcerative colitis, Crohn's disease, pancreatitis and periodontal disease;

• cancers such as bowel cancer and prostate cancer; sarcoidosis; • septic shock; musculo-skeletal diseases or conditions such as arthritis, including polyarthritis and rheumatoid arthritis; leukemia; diabetes; allergy including otitis media and ocular allergy; uveitis; dysmenorrhoea; kidney diseases or conditions such as glomerulonephritis and nephrotic syndrome; and • prostate diseases or conditions such as benign prostate hyperplasia.

Throughout this specification, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

DETAILED DESCRIPTION OF THE INVENTION

As described above, work leading to the present invention has demonstrated that the lipid extract described herein is an effective inhibitor of the 5-lipoxygenase and 12-lipoxygenase pathways, inhibiting production of metabolites of these pathways including LTB₄, LTC₄, LTD₄ and LTE₄, 5-HETE and 12-HETE. Since LTB₄, LTC₄₎ LTD₄ and LTE₄ production is massively increased during an asthma attack, inhibition of production of these leukotrienes is a mechanism of action whereby the lipid extract may be effective in the treatment of asthma. Similarly, since it has been demonstrated that 5-HETE is involved in the proliferative response of cancer cells, and that 12-HETE is involved in tumour metastasis, inhibition of 5-HETE and/or 12-HETE synthesis is a mechanism of action whereby the lipid extract may be effective in the treatment of cancer. Preferably, the lipid extract which is used in the methods of the present invention is an extract prepared by supercritical fluid extraction (SFE) of freeze- dried powdered mussel using a cryogenic fluid (such as cryogenic fluid CO₂) as the extracting medium. This method of preparation is fully described in International Patent Application No. PCT/AU96/00564, the contents of which are incorporated herein by reference. In comparison to solvent extraction techniques, supercritical fluid extraction using cryogenic fluid C0₂ produces a lipid extract rich in non-polar lipids, particularly in free fatty acids. While the exact composition of the lipid extract has not yet been established, it is known to contain not only free fatty acids (including unsaturated fatty acids), but also triglycerides and cholesterol esters.

A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practised using any mode of administration that is medically acceptable, meaning any mode that produces therapeutic levels of the active component of the invention without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, transdermal or parenteral (e.g. subcutaneous, intramuscular and intravenous) routes.

Compositions comprising the lipid extract may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing the active component into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active component into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the active component, in liposomes or as a suspension in an aqueous liquid or non-liquid such as a syrup, an elixir, or an emulsion.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active component which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. A sterile injectable preparation may be formulated as a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in polyethylene glycol and lactic acid. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Other delivery systems can include sustained release delivery systems. Preferred sustained release delivery systems are those which can provide for release of the active component of the invention in sustained release pellets or capsules. Many types of sustained release delivery systems are available. These include, but are not limited to: (a) erosional systems in which the active component is contain within a matrix, and (b) diffusional systems in which the active component permeates at a controlled rate through a polymer.

The formulation of such therapeutic compositions is well known to persons skilled in this field. Suitable pharmaceutically acceptable carriers and/or diluents include any and all conventional solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active component, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

Oral administration will be preferred for many conditions because of the convenience to the patient, although parenteral administration or localised sustained delivery may be more desirable for certain treatment regimens.

The active component is administered in therapeutically effective amounts. A therapeutically effective amount means that amount necessary at least partly to attain the desired effect, or to delay the onset of, inhibit the progression of, or halt altogether, the onset or progression of the particular condition being treated. Such amounts will depend, of course, on the particular condition being treated, the severity of the conditions and individual patient parameters including age, physical condition, size, weight and concurrent treatment. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a lower dose or tolerable dose may be administered for medical reasons, psychological reasons or for virtually any other reasons.

It is especially advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the human or animal patients to be treated; each unit containing a predetermined quantity of active component calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent. The specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active component and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active component for the particular treatment.

Generally, daily doses of active component will be from about 0.01 mg/kg per day to 1000 mg/kg per day. Small doses (0.01-1 mg) may be administered initially, followed by increasing doses up to about 1000 mg/kg per day. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localised delivery route) may be employed to the extent patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of the active component.

In the accompanying figures:

Figure 1 shows the metabolism of arachidonic acid via the lipoxygenase pathways.

Figure 2 shows the effect of lipid extract on platelet 12-HETE and neutrophil 5-HETE synthesis.

Further features of the present invention are more fully described in the following Example(s). It is to be understood, however, that this detailed description is included solely for the purposes of exemplifying the present invention, and should not be understood in any way as a restriction on the broad description of the invention as set out above. EXAMPLE 1

A PREPARATION OF LIPID EXTRACT A.1 Raw Material The green lipped mussel (Perna canaliculus) is harvested on the south coast of New Zealand at which time the total mussel is stabilised with tartaric acid. Freeze drying results in a dry power of pulverised form.

A.2 Extraction of Lipids The technique of supercritical fluid extraction (SFE) is utilised to extract the biologically active lipids from the crude mussel powder. Cryogenic fluid C0₂ is used as the extracting medium. The CO₂ is expanded to atmospheric pressure and the extract is presented as a concentrated oil. The powder yields 3-3.5% of oil.

A.3 Profile of the crude oil

The extractable oil is orange amber in colour and is a viscous liquid at ambient temperature. The extract is stored below 4°C and is handled in a nitrogen atmosphere. The crude oil shows strong UV activity and is protected from light to minimise the polymerisation of double bond components.

B PILOT SCALE SUPERCRITICAL FLUID EXTRACTION

Extraction of total lipids in freeze-dried mussel powder, Perna canaliculus was performed on a pilot scale SFE unit undertaken at the Food Research Institute (Department of Agriculture, Werribee, Vic, Australia). B.1 Instrumentation

Extractions were performed on a pilot scale extraction unit consisting of five basic sub-units (Distillers MG Limited., England, UK). The five basic units comprise: Carbon dioxide supply, Solids extraction, Primary separation, Evaporation and Tailing units.

The carbon dioxide supply unit consists of two CO₂ cylinders connected in parallel and placed on a weighing scale for recharging when appropriate. The extraction unit can be supplied with liquid SC-CO₂ and SC- CO₂. For this work the SFE unit was operated using SC-CO₂. Solid material was placed in the leaching column and the primary separator facilitates separation of extracted material by reduction of pressure (which allows extract to settle), adsorption or liquid extraction. The fluid extract was passed into the evaporation unit to evaporate the CO₂ by the use of internal heating tubes. The vapour may contain volatiles and thus it is subsequently passed to the tailing column to be scrubbed by pure liquid CO₂. The tailing unit traps the gaseous CO₂ from the evaporator unit and returns the volatile components to the evaporator.

B.2 Pilot plant extraction procedure

Mussel power (300 g) was charged to the extraction unit (leaching column). SC-C0₂ was delivered at a flow rate of 3.0 kg/h for two hours per extraction. Extractor temperature was set at 40 °C and the extractor pressure at 310 bar (4,500 psi). The evaporator temperature was held constant at 40 °C. The mussel lipid extracts were stored under nitrogen at -

10°C in amber glass sealed containers. EXAMPLE 2

Recent research (see above) has demonstrated that 5-HETE is involved in the proliferative response of cancer cells. In addition, it has also been demonstrated that the product of the 12-lipoxygenase pathway, 12-HETE, is involved in tumour metastasis (see above). As a result of this overwhelming evidence, studies were undertaken to examine the inhibitory effect of the lipid extract prepared by SFE as described in Example 1 on the production of 12-HETE by platelets, and to compare it with its inhibitory effects on 5-HETE synthesis by neutrophils.

To do this, isolated human platelets or neutrophils were pre-incubated with different doses of the lipid extract for 10 minutes, before 12-HETE synthesis was initiated by the addition of 10 μM arachidonic acid and 5 μM A23187 (calcium ionophore). Synthesis was allowed to proceed for 5 minutes before it was terminated by the addition of citric acid. Figure 2 shows the effect of increasing concentrations of the lipid extract on both platelet 12-HETE and neutrophil 5-HETE synthesis. 50% inhibition of 12-HETE and 5-HETE was achieved with approximately 10 μg/ml and 30 μg/ml lipid extract respectively.

EXAMPLE 3

In a further series of experiments, the effect of the lipid extract of Example 1 was tested on the production of 5-HETE, LTB₄ and all-trans isomers of LTB₄ in human neutrophils. As can be seen in Table 1 , 50 /yg/ml lipid extract inhibited LTB₄ synthesis by 62%, the all-trans isomers by 77 and 87% respectively, and 5-HETE synthesis by 88%.

TABLE 1

EXAMPLE 4

The aim of this study was to assess efficacy and safety of a lipid extract prepared by SFE as described in Example 2 in treatment of patients with bronchial asthma. The lipid extract was encapsulated with olive oil as carrier.

Forty patients (14 males and 26 females, aged 18-62 years, median age 40 years) with atopic steroid-naive asthma were enrolled in double-blind randomized placebo control study at the Hospital Therapeutic Clinic of Pavlov's Medical University, St Petersburg, Russia. Thirty patients were treated with the lipid extract (2 capsules, twice daily) for 8 weeks and 10 patients were treated with placebo (olive oil capsules). Inhalations of β₂-antagonists (salbutamol, fenoterol) were used by each group on demand. Patients were diagnosed according to the American Thoracic Society's definition of asthma. Diagnosis was based upon clinical history, reversibility of FEV, more than 15%. The patients' mean of duration asthma was 5,8+0,9 years (mean±sem) and their mean FEV₁ at the time of the study was 86,3 ±3,3% predicted (mean+sem). The study was approved by the Local Ethics Committee. The informed consent of the participants was obtained in writing.

Pulmonary function tests included airway resistance, specific airway conductance ("Respiratory system 3000", Ohio Medical Products, Madison, USA), forced vital capacity, FEV^ mid-expiratory flow at 25, 50 and 75% of vital capacity ("Pneumoscreen II", Jaeger, Hoechberg, Germany). For assessment of peak flow rate, individual peak-flow meters were used (Vitalograph for Allersearch, Ireland). The concentrations of eosinophil cationic protein (ECP) were determined using radioimmunoassay (Pharmacia & Upjohn, Uppsala, Sweden). The concentration of hydrogen peroxide in exhaled air condensates was measured using horse radish peroxidase-catalysed oxidation of tetramethylbenzidine. Students paired two-tailed t-test was used for statistical methods (Microsoft Excel 5, Statistica for Windows 5). P value less than 0.05 was considered significant.

The results of the study are shown in Tables 2 and 3. The lipid extract had a positive effect on clinical symptoms, peak expiratory flow (PEF) rate and concentration of hydrogen peroxide in exhaled air condensate. There was no improvement in the placebo treated group. No side effects were observed in either group of asthmatic patients during the treatment with the lipid extract or placebo. Table 4 provides a summary analysis of these results.

In conclusion, this study has revealed beneficial effects of the lipid extract in mild asthmatic patients. TABLE 2

Values are present as mean ± sem. *p < 0.05 versus baseline

TABLE 3

Values are present as mean ± sem. TABLE 4

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Claims

1. A method of inhibition of a lipoxygenase pathway, particularly the 5- lipoxygenase pathway and/or the 12-lipoxygenase pathway, which comprises administration of an effective amount of a lipid extract of Perna canaliculus or Mytulis edulis.

2. A method for inhibition of leukotriene synthesis, particularly inhibition of LTB₄, LTC₄, LTD₄ and LTE₄ synthesis, which comprises administration of an effective amount of a lipid extract of Perna canaliculus or Mytulis edulis.

3. A method for the treatment of a disease or condition associated with a lipoxygenase pathway, particularly the 5-lipoxygenase pathway and/or the 12-lipoxygenase pathway, in a human or animal patient which comprises administration to the patient of an effective amount of a lipid extract of Perna canaliculus or Mytulis edulis.

4. A method according to any one of claims 1 to 3, wherein the lipid extract is an extract rich in non-polar lipids.

5. A method according to claim 4, wherein the lipid extract is prepared by supercritical fluid extraction from crude mussel powder.

6. A method according to claim 3, wherein the treatment is treatment of cancer, particularly inhibition of tumour cell proliferation or inhibition of tumour metastasis.

7. A method according to claim 3, wherein the treatment is treatment of asthma.

8. A method according to claim 3, wherein the treatment is treatment of atherosclerosis.

9. Use of a lipid extract of Perna canaliculus or Mytulis edulis, in the preparation of a composition for use in inhibition of a lipoxygenase pathway, particularly in inhibition of the 5-lipoxygenase pathway and/or the 12- lipoxygenase pathway.

10. Use of a lipid extract of Perna canaliculus or Mytulis edulis, in the preparation of a composition for use in inhibition of leukotriene synthesis, particularly inhibition of LTB₄, LTC₄, LTD₄ and LTE₄ synthesis.

11. Use of a lipid extract of Perna canaliculus or Mytulis edulis, in the preparation of a composition for use in treatment of a disease or condition associated with a lipoxygenase pathway, particularly the 5-lipoxygenase pathway and/or the 12-lipoxygenase pathway, in a human or animal patient.

12. A composition for inhibition of a lipoxygenase pathway, particularly the 5- lipoxygenase pathway and/or the 12-lipoxygenase pathway, which comprises a lipid extract of Perna canaliculus or Mytulis edulis, together with one or more pharmaceutically acceptable carriers and/or diluents.

- -

AMENDED CLAIMS

[received by the International Bureau on 3 July 2000 (03.07.00); original claims 1-12 replaced by new claims 1-13 (2 pages)]

1 A method of inhibition of the synthesis of 5-HETE and/or 12-HETE, which comprises administration of an effective amount of a lipid extract of Perna canaliculus or Mytulis edulis.

2 A method for the treatment of cancer in a human or animal patient, which comprises administration to the patient of an effective amount of a lipid extract of Perna canaliculus or Mytulis edulis.

3. A method according to claim 1 or claim 2, wherein the lipid extract is an extract rich in non-polar lipids.

4. A method according to claim 3, wherein the lipid extract is an extract prepared by supercritical fluid extraction

5. A method according to claim 4, wherein the lipid extract is an extract prepared by supercritical fluid extraction from crude mussel powder.

6. A method according to claim 2, wherein the treatment is inhibition of tumour cell proliferation or inhibition of tumour metastasis.

3 Use of a lipid extract of Perna canaliculus or Mytulis edulis, in the preparation of a composition for use in inhibition of the synthesis of 5-HETE and/or 12-HETE.

8. Use of a lipid extract of Perna canaliculus or Mytulis edulis, in the preparation of a composition for use in treatment of cancer in a human or animal patient.

. Use according to claim 7 or claim 8, wherein the lipid extract is an extract rich in non-polar lipids.

10. Use according to claim 9, wherein the lipid extract is an extract prepared by supercritical fluid extraction.

11. Use according to claim 10, wherein the lipid extract is an extract prepared by supercritical fluid extraction from crude mussel powder.

12. A composition for use in inhibition of the synthesis of 5-HETE and/or 12- HETE, which comprises a lipid extract of Perna canaliculus or Mytulis edulis, together with one or more pharmaceutically acceptable carriers and/or diluents.

13. A composition for use in the treatment of cancer in a human or animal patient, which comprises a lipid extract of Perna canaliculus or Mytulis edulis, together with one or more pharmaceutically acceptable carriers and/or diluents.