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WO1996005164A1 - Preparation anti-inflammatoire - Google Patents

Preparation anti-inflammatoire Download PDF

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Publication number
WO1996005164A1
WO1996005164A1 PCT/AU1995/000485 AU9500485W WO9605164A1 WO 1996005164 A1 WO1996005164 A1 WO 1996005164A1 AU 9500485 W AU9500485 W AU 9500485W WO 9605164 A1 WO9605164 A1 WO 9605164A1
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WO
WIPO (PCT)
Prior art keywords
preparation
inflammatory
fraction
compound
active
Prior art date
Application number
PCT/AU1995/000485
Other languages
English (en)
Inventor
Theodore Macrides
Nicolette Kalafatis
Original Assignee
J.W. Broadbent Nominees Pty. Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by J.W. Broadbent Nominees Pty. Ltd. filed Critical J.W. Broadbent Nominees Pty. Ltd.
Priority to EP95927574A priority Critical patent/EP0777641A1/fr
Priority to JP8506852A priority patent/JP2000505777A/ja
Priority to AU31565/95A priority patent/AU3156595A/en
Publication of WO1996005164A1 publication Critical patent/WO1996005164A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/62—Leeches; Worms, e.g. cestodes, tapeworms, nematodes, roundworms, earth worms, ascarids, filarias, hookworms, trichinella or taenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/02—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals
    • C07C233/09—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having nitrogen atoms of carboxamide groups bound to hydrogen atoms or to carbon atoms of unsubstituted hydrocarbon radicals with carbon atoms of carboxamide groups bound to carbon atoms of an acyclic unsaturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/02—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms with only carbon-to-carbon double bonds as unsaturation
    • C07C57/03—Monocarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/52—Esters of acyclic unsaturated carboxylic acids having the esterified carboxyl group bound to an acyclic carbon atom
    • C07C69/587—Monocarboxylic acid esters having at least two carbon-to-carbon double bonds

Definitions

  • This invention relates in general to an anti-inflammatory preparation, and in particular it relates to an anti-inflammatory preparation isolated and identified from lipid extracts of mussels, including the New Zealand green lipped mussel, Pema canaliculus, and the blue mussel Mytilus edulis.
  • aqueous extract showed a dose-dependent anti- inflammatory activity when administered intraperitoneally and could not be detected upon oral administration of the mussel powder. It was suggested that the water-soluble fraction therefore contained an irritant component possessing apparent anti-inflammatory activity.
  • lipid extract of Pema canaliculus and Mytilus edulis has become available through the procedure of supercritical fluid extraction (SFE).
  • SFE supercritical fluid extraction
  • the lipid rich extract is obtained as a dark yellow-brown viscous oil exhibiting strong ultraviolet absorbing character which is consistent in physical data to oil extracts obtained from earlier large scale solvent extraction procedures.
  • An anti-inflammatory preparation which exhibits significant in vivo and in vitro activity and which is substantially free of toxic side effects has now been isolated from this lipid extract.
  • the present invention provides an anti- inflammatory preparation which comprises a purified, active fraction isolated from a lipid extract of Pema canaliculus or Mytilus edulis or an active component thereof.
  • fraction means an isolated portion of the initial lipid extract which is identified as a separate region on a chromatogram and may be separated from the remainder of the lipid extract by chromatographic techniques.
  • active fraction means a fraction which exhibits in vitro and/or in vivo anti-inflammatory activity, which may be demonstrated for example by using the biological assays described herein.
  • active component means one or more compounds in an active fraction which individually or together exhibit in vitro and/or in vivo anti- inflammatory activity.
  • this purified fraction is isolated by high performance liquid chromatography (HPLC) of the lipid extract, using for example reverse phase- HPLC (RP-HPLC) as described in detail hereunder.
  • the fraction is further purified using silver ion-HPLC (Ag-HPLC).
  • HPLC high performance liquid chromatography
  • Ag-HPLC silver ion-HPLC
  • the anti-inflammatory preparation of the present invention may comprise a number of separate active constituent compounds identified by separate peaks on a chromatogram, and accordingly it is to be understood that the term "purified fraction" as used herein extends not only to the fraction including one or more of these active constituent compounds, but also to one or more of the active constituent compounds represented by these separate peaks. These peaks are believed to represent free fatty acids, but may also include fatty acid derivatives within the fraction.
  • the free fatty acid active component(s) in the anti-inflammatory preparation of this invention may also be converted into a pharmaceutically acceptable ester derivative, for example a methyl or other lower alkyl ester thereof, a pharmaceutically acceptable amide derivative, for example, an amide of the formula -NHR wherein R represents hydrogen or lower alkyl, or a pharmaceutically acceptable salt for example an alkaline or alkaline earth metal salt such as a sodium, potassium or calcium salt thereof.
  • a pharmaceutically acceptable ester derivative for example a methyl or other lower alkyl ester thereof
  • a pharmaceutically acceptable amide derivative for example, an amide of the formula -NHR wherein R represents hydrogen or lower alkyl
  • a pharmaceutically acceptable salt for example an alkaline or alkaline earth metal salt such as a sodium, potassium or calcium salt thereof.
  • the preparation of the invention comprises a purified, active fraction isolated from a lipid extract of Pema canaliculus; more preferably, it comprises a fraction having the characteristics of one of the fractions identified herein as VAR-1, RPFA2, RPFA3, RPFA4, AG5 or AG6, or an active constituent compound thereof. Chromatograms of these fractions, and in some cases molecular ion spectra of constituent compounds therof, are included herein. A particularly preferred fraction is AG5, or an active component thereof.
  • purified means that the fraction has been isolated or separated from other components of the lipid extract which do not exhibit any or any substantial anti-inflammatory activity.
  • the fraction has been purified to the extent that it may be identified by a separate peak or a number of adjacent separate peaks on a chromatogram.
  • a further aspect of this invention provides the compound, 5,11,14,17-eicosatetraenoic acid, as well as its pharmaceutically acceptable esters, amides and salts.
  • This compound may be prepared in substantially pure form, that is substantially free from other compounds with which it is usually associated in the mussel lipid extract, by chromatographic methods disclosed in detail herein.
  • the present invention also provides a pharmaceutical composition comprising the anti-inflammatory preparation broadly described above, together with one or more pharmaceutically acceptable carriers and/or diluents.
  • the invention includes a pharmaceutical composition comprising the compound 5,11 ,14,17-eicosatetraenoic acid or a pharmaceutically acceptable ester, amide or salt thereof, together with one or more pharmaceutically acceptable carriers and/or diluents.
  • Such a pharmaceutical composition may, if desired, also include one or more other pharmaceutically active components, for example, one or more known non-steroidal anti-inflammatory agents such as aspirin and indomethacin.
  • one or more known non-steroidal anti-inflammatory agents such as aspirin and indomethacin.
  • the present invention provides a method of treatment of inflammation on an inflammatory condition in an animal, including a human, which comprises administering to the animal an effective amount of an anti- inflammatory preparation as broadly described above.
  • the invention also extends to administration of an effective amount of the compound 5,11,14,17- eicosatetraenoic acid or a pharmaceutically acceptable ester, amide or salt thereof.
  • the present invention provides the use of the anti- inflammatory preparation as broadly described above, and in particular the compound 5,11,14,17-eicosatetraenoic acid or a pharmaceutically acceptable ester, amide or salt thereof, in the preparation of a medicament for treatment of inflammation or an inflammatory condition in an animal, including a human.
  • the animal treated is a human.
  • the invention provides anti-inflammatory treatment in general, and this includes by way of example, treatment of rheumatoid and osteoarthritis as well as allied inflammatory disorders.
  • the anti-inflammatory preparation of this invention has been shown to have significant anti-inflammatory activity both in vitro in inhibition of leukotriene synthesis and in vivo in the adjuvant-induced polyarthritis model in the rat. Since increased leukotriene levels have been associated with the occurrence of asthma and particularly with inflammation of the bronchi! associated with accumulation of neutrophils and the like in the bronchi. 5 , the anti-inflammatory treatment i accordance with the present invention extends to treatment of asthma as well a to treatment of arthritis and other inflammatory disorders in general.
  • the magnitude of the effective amount of the anti-inflammatory preparatio of this invention will, of course, vary with the group of patients (age, sex, etc.), th nature or the severity of the condition to be treated and the dosage form an route of administration.
  • the daily dose range for use will usually li within the range of from about 0.1 to about 100 mg per kg body weight of th patient, more preferably from about 0.5 to about 50 mg/kg/day, and even mor preferably from about 1 to about 30 mg kg/day.
  • any suitable route of administration may be employed for providing a animal, especially a human, with an effective dosage of the anti-inflammator preparation of this invention.
  • oral, rectal, vaginal, topical, parenteral, ocular, nasal, sublingual, buccal, intravenous and the lik administration may be employed.
  • Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, suppositories, aerosols and the like.
  • Said dosage forms also include implanted slow releasing devices specifically designed for this purpose or other forms of implants modified to additionally act in this fashion.
  • compositions of this invention may also be included in the pharmaceutical compositions of this invention.
  • pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and is described by way of example in Remington's Pharmaceutica Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, U.S.A.
  • the anti-inflammatory preparation of this invention can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier and/or diluent according to conventional pharmaceutical compounding techniques.
  • a pharmaceutical carrier and/or diluent may take a wide variety of forms depending on the form of preparation desired for administration, e.g. oral or parenteral (including intravenous and intra-arterial).
  • any of the usual pharmaceutical media may be employed, for example, carriers and diluents such as water, glycols, oils, alcohols, flavouring agents, preservatives, colouring agents and the like in the case of oral liquid preparations such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric- coated by standard techniques.
  • the anti- inflammatory preparation of this invention may also be administered by controlled release means and/or delivery devices.
  • compositions of the present invention suitable for oral or parenteral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.
  • Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients.
  • the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient 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 ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the particular treatment.
  • Figure 1 shows isocratic reverse phase HPLC of fraction VAR-1.
  • FIG. 2 shows separation of mussel fatty acids (20 mg) by RP-HPLC. Chromatographic conditions: Beckmann UltrasphereTM C18 column; Flow rate, 1.5 ml/min; Mass detector Evaporator set value (ESV) +60; gas pressure, 14 psi; The relative time of elution of the constituent fatty acids in the mixture are indicated.
  • Chromatographic conditions Beckmann UltrasphereTM C18 column; Flow rate, 1.5 ml/min; Mass detector Evaporator set value (ESV) +60; gas pressure, 14 psi; The relative time of elution of the constituent fatty acids in the mixture are indicated.
  • ESV Mass detector Evaporator set value
  • FIGS 3, 4 and 5 show GCchromatog rams of the fractions RPFA-
  • Figure 6 shows separation of mussel fatty acid methyl esters, FAME
  • Figures 7 and 8 show GC chromatograms of the fractions AG5 and AG6, respectively.
  • Figures 9, 10, 11, 12, 13 and 14 show molecular ion spectra of fractions AG5 (C18:4), AG5 (C19:4), AG5 (C20:4), AG5 (C20:4), RPFA3 (C20:5) and RPFA3 (C21:5), respectively.
  • the green lipped mussel (Pema 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 powder of pulverised form.
  • SFE supercritical fluid extraction
  • 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.
  • Mycobacterium tuberculosis suspended in an oily vehicle (squalane) at a final concentration of 10 mg/ml was injected intradermally (0.05ml) into the tail of male Dark Agouti or Long Evans rats weighing between 200 to 25 Og.
  • the injected tail became inflamed (increase in volume) and reached maximal size within three to five days (primary lesion).
  • Assessment of the inflammation was made 2 weeks after tail injection. Secondary lesions of adjuvant arthritis occurred after a delay of approximately 10 days and were characterised by inflammation of non-injected sites and a decrease in body weight gain.
  • Vehicle treated controls and adjuvant controls served as reference groups to determine body weight and paw thickness changes in experimental groups.
  • Reverse phase HPLC assay is now established as a reliable measure of leukotriene production in human polymo ⁇ honuclear leukocytes (PMNLs) and the lowering of measured levels of 6-LTB 4 , 12epi, 6t-LTB 4 are formed from the non-enzymic hydrolysis of LTA 4 while LTB 4 is produced from LTA unpleasant.
  • the anti-inflammatory preparation has been examined for its effect on leukotriene production after incubation in PMNL preparation.
  • the levels of LTB 4 , 6t-LTB 4 and 12epi, 6t-LTB 4 produced were measured by the method of reverse phase HPLC according to Cleland et al. ⁇ .
  • the first preparative isolation of the crude oil was achieved by normal phase HPLC with UV detection at 280nm. This method afforded the fractionation of five fractions which were coded as 1A to 5A.
  • the polar fractions (3A, 4A, 5A) were derivatised to the fatty acid methyl esters (3ES,
  • the oil was dissolved in dichloromethane and spotted onto precoated silica gel TLC plate.
  • the eluting solvent mixture was hexane, ether, acetic acid. Visualisation of the components was achieved by spraying the plate with a 10% copper sulphate in an 8% phosphoric acid solution. The components appear as dark spots on a white background when heated to 110°C.
  • a preparative thin layer method was used to isolate the polar material (code 3PLC, R f band 0.1-0.5) from the lipophilic extract.
  • 3PLC sample was again screened for activity against the neutrophil, leukotriene synthesis inhibition in vitro model and the chronic polyarthritis in vivo animal model.
  • the results for the in vitro assay are summarised in Table 2a and support the in vivo results presented in Table 2b.
  • Polar band 3PLC has shown a significant inhibitory activity in both assays.
  • the 3PLC fraction shows UV activity with band intensity at 320, 295, 285, 275, 265 and 235nm, indicating unsaturation with possible conjugation. Further investigations will enable use of disposable extraction cartridges packed with silica to isolate the active lipids (3PLC). On the basis of the initial screening, the polar component (5% ether fraction) compared favourably to 3PLC.
  • Narp-HPLC has been utilised to isolate the lipids by carbon number in a multi-step gradient method.
  • the HPLC operating parameters are listed below:
  • the lipid fraction with chemical characteristics similar to 3PLC was injected onto the column and four fractions were collected based on their elution time and coded VAR-1 , VAR-2, VAR-3 and VAR-4, respectively.
  • Narp-HPLC The separation by Narp-HPLC is a time-windows technique and the following summarises collection character.
  • VAR-1 11-20 VAR-2 20-27 VAR-3 33-47 VAR-4 47-59 The VAR fractions have been tested by the leukotriene assay and the results are summarised in Table 3a.
  • the results using the chronic inflammation assay are shown in Table 3b. Both tests have been consistent in showing that VAR-1 has retained significant biological activity at a reduced dosage level of 10 mg/kg body weight in comparison to the earlier assays of 50 and 30 mg/kg body weight. This indicates that purification of the bioactive material has taken place with significant increase in specific activity.
  • the parameters which have been measured in Table 3b indicate that fraction VAR-1 has retained anti-inflammatory activity and significantly outperforms all other extracts so far examined.
  • a TLC analysis of VAR-1 using hexane/ether/acetic acid of proportions (80:20:2) has an R, 0.38 by visualisation with the copper sulphate acid spray.
  • the active fraction VAR-1 (identified by the time-window 11-20 minutes) has been further chromatographed using isocratic aqueous reverse phase liquid chromatography.
  • the chromatogram ( Figure 1) indicates the separation of four main compounds of similar polarity associated with eight minor constituents within a time window of 2-15 minutes.
  • the analytical separation when compared to known standards (palmitic and myristic acid) indicate that the sample VAR-1 is a mixture of fatty acids.
  • the operating conditions of this analysis are summarised as follows:
  • VAR-1 1:100 85 100 100
  • VAR-2 1:100 91 100 100
  • VAR-4 1:100 51 51 44 (5.2 mg/ml) 1:1000 6 0 0
  • 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 C0 2 cylinders connected in parallel and placed on a weighing scale for recharging when appropriate.
  • the extraction unit can be supplied with liquid SC-C0 2 and SC-C0 2 .
  • SC-C0 2 liquid SC-C0 2
  • 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 C0 2 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 C0 2 .
  • the tailing unit traps the gaseous C0 2 from the evaporator unit and returns the volatile components to the evaporator.
  • Mussel powder (300 g) was charged to the extraction unit (leaching column). SC-C0 2 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.
  • the FA fraction was collected and were subsequently reduced in volume to an oily residue (24 %, w/w) by rotary evaporation. The residues were transferred to vials and further dried under a stream of nitrogen until constant in weight. Purity of the free fatty acid fraction was verified by TLC on aluminium-backed silica plates.
  • TLC Thin Layer Chromatography
  • HPTLC aluminium sheets coated with silica gel 60 F 254 were cut to an appropriate size (10 cm x 20 cm) and used witho prewashing. Mussel fractions and the lipid standards were dissolved i dichloromethane.
  • the direct sample application method was performed using micropipette (Becton-Dickinson and Co., Parsippany, NJ, USA) and compared t the standard.
  • the chromatographic separation was carried out in hexane-dieth ether-acetic acid (80+20+1, v/v/v). Bands were identified after spraying with 10 copper sulphate in 8% phosphoric acid reagent and charring at 110°C for 20 min (Bitman and Wood, 1982).
  • Model 150A An Applied Biosystems chromatograph (Model 150A) consisted of a Mode 1400A dual pump solvent delivery system (Applied Biosystems, Foster City, Ca USA) and a Model 1783A absorbance controller unit.
  • the inclusion of a splitte (consisting of a stainless steel block with 3 apertures) permitted preparativ collection of the mussel fatty acids.
  • the column was diverted to a sampl collector (Gilson Instruments, Model 201, V lliers, LE, BEL, IR).
  • the mussel fatty acids were diluted to 500 g/ml in 1mL AcCN and THF (1:1, v/v) and an aliquot of 45 ⁇ L was injected on column.
  • the samples were filtered before column injection through a 0.45 ⁇ m nylon syringe filter (Activon Scientific Products, Thornleigh, Australia).
  • the above preparations were stored at -10°C during the course of this work.
  • an initial solvent composition consisting of 13% water - 87% acetonitrile (AcCN) was held for 35 mins to isolate the individual species according to their carbon number and degree of unsaturation. At 40 mins., the final solvent composition was 100% AcCN (segment 2) by linear solvent strength gradient elution of AcCN into 13% water-AcCN. Segment 3 permitted elution of long chained fatty acids. The column was regenerated (Segment 4-5) as preparation for the next sample. The flow rate was maintained at 1.5 mL min. 1.4.2 Silver ion chromatography Chromatographic system
  • a ChromspherTM column 10 ⁇ m, 5 //m, 25 cm x 10mm (ID) (Chrompack, Netherlands).
  • a binary gradient was prepared with solvent reservoir A; dichloroethane-dichloromethane 1:1 (v/v) and solvent reservoir B; dichloroethane- dichloromethane-methanol-acetonitrile 4/4/1/1, (v/v/v/v).
  • the ACS mass detector parameters were set to +40 and the gas pressure 14 psi. The flow rate was maintained at 2.0 mL/min.
  • Mussel fatty acids were isolated by RP-HPLC and methylated by dissolving in 12.5% BF 3 methanol 9 . After the mixture was refluxed for 1 h. at 70°C, heptane solvent (20m L) was added and refuxed for a further 15 min. The organic layer is washed first with saturated sodium chloride solution (15mL). Methyl terf-butyl ether (2x50 mL) was added and the extract was washed with water (2x20 mL). The FAME mixture was dried over anhydrous sodium sulphate powder, filtered, concentrated and chromatographed on a silica gel G60 column previously conditioned with hexane.
  • Injector temperature was set at 260°C and the linear velocity of the helium gas was 20 cm/min.
  • GC analysis was carried out on a Hewlett-Packard 5890 GC equipped with an HP1 column (12 mm x 0.2 mm x 0.33 ⁇ m) with an inlet pressure of 5 psi.
  • the oven temperature was held at 75°C for 2 min. then heated at 30°C/min. to 300°C with a final holding time of 9 min. Injections were spitless.
  • An injector temperature of 200°C and a detector temperature of 300°C were used.
  • Electron impact gas chromatography mass spectroscopy (El GC/MS) was performed on a Hewlett Packard 5890 GC (Finnigan MAT INCOS 50 MS). Chromatographic conditions were identical to those described above. The interface temperature was 280°C, the injector 250°C and the ion source temperature was 180°C. A scan range of 50 to 500 Da at 0.6 sec/scan was used.
  • PEG Positive ion chemical ionisation
  • the separation of the mussel extract into lipid class was facilitated by open column flash chromatography. Preliminary separation of lipids according t polarity was achieved on silica gel. Cholesterol esters and triglycerides were eluted in dichloromethane solvent. The free fatty acids were eluted in hexane MTBE (80 + 10, v v).
  • Ascending one dimensional TLC was used to screen the fractions from the preparative column chromatography of the crude mussel extract. Separation on silica gel plates resolved the lipid classes. Rapid analysis occurred following development in 80+20+1 (v/v/v) hexane-diethyl ether-acetic acid. The components are resolved into black spots on a white background by spraying with a copper sulphate reagent upon drying in a 110°C oven for 20 minutes. The free fatty acid components was verified at R, equivalent to 0.34.
  • RPFA-2 The RP fractions were concentrated by rotary evaporation at 40°C and reconstituted in methanol. The fractions were screened by an in vitro model of inflammation by leukotriene synthesis 10 .
  • RPFA samples have been further analysed by high resolution GC.
  • parent compound i.e. total free fatty acids isolated by open column chromatography (see materails and methods 1-2).
  • the RP fractions were profiled by high resolution GC by the conditions described in Section 1.4.3. Known fatty acids were identified by direct comparisons of their retention times to those of a commercial standard (Nu-Chek- Prep Inc. (Elysian, MO, USA).
  • the GC chromatograms of RPFA-2, RPFA-3 and RPFA-4 as their methyl esters are shown in Figures 3, 4 and 5, respectively.
  • the peak corresponding to 26.93 mins. is common to RPFA-3 and RPFA-4 and was deemed to be contain the bio-active constituent. Focus was given to fraction RPFA-3 which is rich in component at 26.93 mins. and has also exhibited in vitro activity.
  • the active isolate, RPFA3 was further purified by AG-HPLC using a binary gradient program (Section 1.4.2).
  • the fractions from silver ion chromatography (see Figure 6) were separated into eight constituents; the major component at 46.9 min. corresponding to docosahexaenoic acid (DHA).
  • Interbands AG2 and AG4 were not included in the in vitro assay due to scarcity of material within these time windows.
  • AG8 (1:100 dilution). At the low concentrations of 1:1000 and 1:10000, AG5 exhibited low inhibitory activity due to experimental insolubility in the test medium.
  • the time elution of AG5 and AG6 is comparable to the retention of authentic standards containing tetraene and pentaene components.
  • Peak at 26.001 mins. is known and has been identified as arachidonic acid methy ester. Peak at 27.542 mins is known and has been identified as eicosapentaenoic aci methyl ester (EPA).

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  • Epidemiology (AREA)
  • Rheumatology (AREA)
  • Engineering & Computer Science (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Pain & Pain Management (AREA)
  • Immunology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La préparation anti-inflammatoire comprend une fraction active, purifiée qui a été isolée d'un extrait lipidique de Perna canaliculus ou Mytilus edulis, ou un composant actif de ceux-ci. Le composé d'acide 5,11,14,17-eicosatétraénoïque peut être un constituant principal de la fraction active. Une forme sensiblement pure de l'acide 5,11,14,17-eicosatétraénoïque est isolée et des esters, amides et sels pharmaceutiquement acceptables de cet acide ont été identifiés. Ces composés sont utilisés dans des traitements et préparations anti-inflammatoires.
PCT/AU1995/000485 1994-08-11 1995-08-11 Preparation anti-inflammatoire WO1996005164A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP95927574A EP0777641A1 (fr) 1994-08-11 1995-08-11 Preparation anti-inflammatoire
JP8506852A JP2000505777A (ja) 1994-08-11 1995-08-11 抗炎症製剤
AU31565/95A AU3156595A (en) 1994-08-11 1995-08-11 Anti-inflammatory preparation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPM7405 1994-08-11
AUPM7405A AUPM740594A0 (en) 1994-08-11 1994-08-11 Anti-inflammatory preparation

Publications (1)

Publication Number Publication Date
WO1996005164A1 true WO1996005164A1 (fr) 1996-02-22

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1995/000485 WO1996005164A1 (fr) 1994-08-11 1995-08-11 Preparation anti-inflammatoire

Country Status (6)

Country Link
EP (1) EP0777641A1 (fr)
JP (1) JP2000505777A (fr)
AU (1) AUPM740594A0 (fr)
CA (1) CA2196422A1 (fr)
WO (1) WO1996005164A1 (fr)
ZA (1) ZA956661B (fr)

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0982034A1 (fr) * 1998-08-26 2000-03-01 aristavet Veterinärspezialitäten GmbH & Co. KG Composition contenant de la glucosamine et un extrait de Perna canaliculus
GB2347349A (en) * 1997-05-21 2000-09-06 Macfarlane Lab New Zealand Lim A synergistic composition comprising mussel protein extract and glycosaminoglycan suitable for treatment of arthritis
WO2000053198A1 (fr) * 1999-03-10 2000-09-14 Pharmalink International Limited Inhibiteur des mecanismes d'action de la lipo-oxygenase
WO2000056164A1 (fr) 1999-03-22 2000-09-28 Kal Kan Foods, Inc. Aliment destine au maintien d'articulations saines et a alleger des symptomes d'arthrite chez des animaux de compagnie
WO2000071140A3 (fr) * 1999-05-21 2001-05-31 Foodscience Corp Procedes et compositions de modulation de la reponse immunitaire et de traitement des maladies inflammatoires
US6262119B1 (en) 1996-04-12 2001-07-17 Peptide Technology Limited Methods of treating immunopathologies using polyunsaturated fatty acids
US6346278B1 (en) 1995-09-11 2002-02-12 Pharmalink International Limited Lipid extract having anti-inflammatory activity
WO2002024211A1 (fr) * 2000-09-22 2002-03-28 Pharmalink International Limited Preparation immunomodulatrice
WO2005073354A1 (fr) * 2004-01-30 2005-08-11 Bionovate Limited Extraction d'un solvant de lipides, par exemple des acides gras essentiels
WO2005112960A1 (fr) * 2004-05-19 2005-12-01 Bionovate Limited Traitement de l'asthme et de l'arthrite et d'autres maladies inflammatoires
WO2005112910A1 (fr) * 2004-05-19 2005-12-01 Bionovate Limited Combinaisons d'acide hyaluronique et d'acides gras polyinsatures
WO2005115076A3 (fr) * 2004-05-31 2006-05-11 Al Tayeb Fayez Hussien Nouveau produit presentant une efficacite elevee et des effets indesirables reduits au minimum, et procede associe
WO2006052150A1 (fr) * 2004-11-15 2006-05-18 University Of Otago Composition d'extrait de moule a activite anti-inflammatoire renforcee
WO2006128244A1 (fr) * 2005-06-03 2006-12-07 Mc Farlane Marketing (Aust.) Pty. Ltd. Extrait lipidique de moule et procede de preparation
US20100297254A1 (en) * 2007-12-12 2010-11-25 Dan Bright Nutraceutical composition and methods of use
US20110045099A1 (en) * 2007-12-12 2011-02-24 Dan Bright Nutraceutical composition and methods of use
WO2014143614A1 (fr) 2013-03-11 2014-09-18 Jan Remmereit Compositions lipidiques contenant des acides gras bioactifs
WO2018117867A1 (fr) * 2016-12-20 2018-06-28 Sanford Limited Compositions et/ou extraits bioactifs de moules
US10376550B2 (en) 2007-12-12 2019-08-13 Dacy Tech Pty Ltd. Nutraceutical composition and methods of use
US10993925B2 (en) 2015-09-18 2021-05-04 Sciadonics, Inc. Lipid formulations containing bioactive fatty acids
US11058656B2 (en) 2015-11-25 2021-07-13 Sciadonics, Inc. Lipid formulations containing bioactive fatty acids

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0010061A1 (fr) * 1978-09-25 1980-04-16 Stuart John Mc Farlane Préparations pharmaceutiques contenant de l'extrait de mollusque
WO1985005033A1 (fr) * 1984-05-01 1985-11-21 Broadbent, James, Meredyth Extrait de moules stabilise

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0010061A1 (fr) * 1978-09-25 1980-04-16 Stuart John Mc Farlane Préparations pharmaceutiques contenant de l'extrait de mollusque
WO1985005033A1 (fr) * 1984-05-01 1985-11-21 Broadbent, James, Meredyth Extrait de moules stabilise

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
ARZNEIMITTEL FORSCHUNG/DRUG RESEARCH, Volume 30, No. (ii), 1980, EDITIO CANTOR, AULENDORF, GERMANY, K.D. RAMSFORD et al., "Gastroprotective and Anti-Inflammatory Properties of Green Lipped Mussel (Perna Canaliculus) Preparation", pages 2128-2132. *
BIOCHEMICAL JOURNAL, Volume 291, No. 3, 1993, BIOCHEMICAL SOCIETY, LONDON, ENGLAND, JAVIER NAVAL et al., "Alternative Route for the Biosynthesis of Polyunsaturated Fatty Acids in K562 Cells", pages 841-845. *
BIOCHIMICA ET BIOPHYSICA ACTA, Volume 1167, No. 2, 1993, ELSEVIER, NETHERLANDS, SAEREE JAREONKITMONGKOL et al., "Occurrence of Two Nonmethylene-Interrupted 5 Polyunsaturated Fatty Acids in a 6-Desaturase-Defective Instant of the Fungus Mortierella Alpina 1S-4", pages 137-141. *
COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY, Volume 107B, No. 2, 1994, PERGAMON PRESS, LONDON, VALERY M. DEMBITSKY et al., "Comparative Study of the Endemic Freshwater Fauna of Lake Baikal-III. Phospholipid and Fatty Acid Compositions of the Amphiphod Crustacean of the Genus Enlimnogammarus", pages 317-323. *
DERWENT ABSTRACT, Accession No. 89-333329/46, Class B05; & DE,C,3 814 047 (FRATZER) 16 November 1989. *
JAOCS: JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY, Volume 61, No. 3, 1984, AMERICAN OIL CHEMISTS SOCIETY, CHAMPAIGN, ILLINOIS, USA, J.R. VICKERY et al., "The Fatty Acid Composition of Gymnospermae Seed Leaf Oils", pages 573-575. *
JAOCS: JOURNAL OF THE AMERICAN OIL CHEMISTS SOCIETY, Volume 65, No. 4, 1988, AMERICAN OIL CHEMISTS SOCIETY, CHAMPAIGN, ILLINOIS, USA, M.S.F. LIE KEN JIE et al., "Lipids in Chinese Medicine. Characterisation of All Cis-5, 11, 14, 17-Eicosatetraenoic Acid in Biota Orientalis Seed Oil and a Study of Oxo-Furanoid Esters Derived From Biota *
LIPIDS, Volume 21, No. 9, 1986, AMERICAN OIL CHEMISTS SOCIETY, CHAMPAIGN, ILLINOIS, USA, TORU TAKAGI et al., "Fatty Acids in Echinoidea: Unusual Cis-5-Olefinic Acids as Distinctive Lipid Components in Sea Urchins", pages 558-565. *
LIPIDS, Volume 27, No. 7, 1992, AMERICAN OIL CHEMISTS SOCIETY, CHAMPAIGN, ILLINOIS, USA, IKUO IKEDA et al., "5c, 11c, 14c - Eicosatrienoic Acid and 5c, 11c, 14c, 17c - Eicosatetraenoic Acid of Biota Orientalis Seed Oil Affect Lipid Metabolism in the Rat", pages 500-504. *
PATENT ABSTRACTS OF JAPAN, C-1159, page 63; & JP,A,05 276 964 (SUNTORY LTD) 26 October 1993. *
YUKAGAKU/JOURNAL OF THE JAPAN OIL CHEMISTS SOCIETY, Volume 31, No. 9, 1982, TOKYO, JAPAN, YUTAKA ITABASHI et al., "Cis-5-Olefinic Nonmethylene-Interrupted Fatty Acids in Lipids of Seeds, Arils and Leaves of Japanese Yew", pages 574-579. *
YUKAGAKU/JOURNAL OF THE JAPAN OIL CHEMISTS SOCIETY, Volume 35, No. 7, 1986, TOKYO, JAPAN, TORU TAKAGI et al., "Fatty Acid Composition of Bivalves From Japanese Waters", pages 517-521. *

Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6346278B1 (en) 1995-09-11 2002-02-12 Pharmalink International Limited Lipid extract having anti-inflammatory activity
US6262119B1 (en) 1996-04-12 2001-07-17 Peptide Technology Limited Methods of treating immunopathologies using polyunsaturated fatty acids
GB2347349A (en) * 1997-05-21 2000-09-06 Macfarlane Lab New Zealand Lim A synergistic composition comprising mussel protein extract and glycosaminoglycan suitable for treatment of arthritis
EP0982034A1 (fr) * 1998-08-26 2000-03-01 aristavet Veterinärspezialitäten GmbH & Co. KG Composition contenant de la glucosamine et un extrait de Perna canaliculus
WO2000053198A1 (fr) * 1999-03-10 2000-09-14 Pharmalink International Limited Inhibiteur des mecanismes d'action de la lipo-oxygenase
RU2230458C2 (ru) * 1999-03-22 2004-06-20 Кэл Кэн Фудз, Инк. Корм для домашних животных, способ его приготовления и способ терапии для поддержания здоровья суставов и ослабления артритных симптомов у домашних животных
WO2000056164A1 (fr) 1999-03-22 2000-09-28 Kal Kan Foods, Inc. Aliment destine au maintien d'articulations saines et a alleger des symptomes d'arthrite chez des animaux de compagnie
US6977084B2 (en) 1999-03-22 2005-12-20 Bui Linh M Pet food containing Perna canaliculus for maintenance of joint health and alleviation of arthritic symptoms
US6596303B1 (en) 1999-03-22 2003-07-22 Mars Incorporated Pet food for maintenance of joint health and alleviation of arthritic symptoms in companion animals
WO2000071140A3 (fr) * 1999-05-21 2001-05-31 Foodscience Corp Procedes et compositions de modulation de la reponse immunitaire et de traitement des maladies inflammatoires
US7229646B2 (en) 1999-05-21 2007-06-12 Foodscience Corporation Methods and compositions for modulating immune response and for the treatment of inflammatory disease
US6921543B2 (en) 2000-09-22 2005-07-26 Pharmalink International Limited Immunomodulatory preparation
WO2002024211A1 (fr) * 2000-09-22 2002-03-28 Pharmalink International Limited Preparation immunomodulatrice
WO2005073354A1 (fr) * 2004-01-30 2005-08-11 Bionovate Limited Extraction d'un solvant de lipides, par exemple des acides gras essentiels
US7572464B2 (en) 2004-01-30 2009-08-11 Bionovate Limited Solvent extraction of lipids such as essential fatty acids
WO2005112960A1 (fr) * 2004-05-19 2005-12-01 Bionovate Limited Traitement de l'asthme et de l'arthrite et d'autres maladies inflammatoires
WO2005112910A1 (fr) * 2004-05-19 2005-12-01 Bionovate Limited Combinaisons d'acide hyaluronique et d'acides gras polyinsatures
WO2005115076A3 (fr) * 2004-05-31 2006-05-11 Al Tayeb Fayez Hussien Nouveau produit presentant une efficacite elevee et des effets indesirables reduits au minimum, et procede associe
WO2006052150A1 (fr) * 2004-11-15 2006-05-18 University Of Otago Composition d'extrait de moule a activite anti-inflammatoire renforcee
WO2006128244A1 (fr) * 2005-06-03 2006-12-07 Mc Farlane Marketing (Aust.) Pty. Ltd. Extrait lipidique de moule et procede de preparation
US20100297254A1 (en) * 2007-12-12 2010-11-25 Dan Bright Nutraceutical composition and methods of use
US20110045099A1 (en) * 2007-12-12 2011-02-24 Dan Bright Nutraceutical composition and methods of use
US8431169B2 (en) * 2007-12-12 2013-04-30 Dacy Tech Pty Ltd Nutraceutical composition and methods of use
US10376550B2 (en) 2007-12-12 2019-08-13 Dacy Tech Pty Ltd. Nutraceutical composition and methods of use
US10154979B2 (en) 2013-03-11 2018-12-18 Sciadonics, Inc. Lipid compositions containing bioactive fatty acids
WO2014143614A1 (fr) 2013-03-11 2014-09-18 Jan Remmereit Compositions lipidiques contenant des acides gras bioactifs
US10537542B2 (en) 2013-03-11 2020-01-21 Sciadonics, Inc. Lipid compositions containing bioactive fatty acids
US10980763B2 (en) 2013-03-11 2021-04-20 Sciadonics, Inc. Lipid compositions containing bioactive fatty acids
US11628152B2 (en) 2013-03-11 2023-04-18 Sciadonics, Inc. Lipid compositions containing bioactive fatty acids
US10993925B2 (en) 2015-09-18 2021-05-04 Sciadonics, Inc. Lipid formulations containing bioactive fatty acids
US11058656B2 (en) 2015-11-25 2021-07-13 Sciadonics, Inc. Lipid formulations containing bioactive fatty acids
WO2018117867A1 (fr) * 2016-12-20 2018-06-28 Sanford Limited Compositions et/ou extraits bioactifs de moules

Also Published As

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CA2196422A1 (fr) 1996-02-22
ZA956661B (en) 1996-09-11
JP2000505777A (ja) 2000-05-16
EP0777641A1 (fr) 1997-06-11
AUPM740594A0 (en) 1994-09-01

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