CN1867328B

CN1867328B - Use of lipid glyceride in preparing medicine for treatment of neurodegenerative conditions

Info

Publication number: CN1867328B
Application number: CN200480030644XA
Authority: CN
Inventors: L·S·哈比格; M·J·利奇; M·夏里夫; P·巴拉克罗夫
Original assignee: British Technology Group Ltd
Current assignee: BTG International Ltd; British Technology Group Inter Corporate Licensing Ltd
Priority date: 2003-08-18
Filing date: 2004-08-13
Publication date: 2010-12-29
Anticipated expiration: 2024-08-13
Also published as: CN1867328A; GB0319358D0

Abstract

A method is provided for treating a patient in need of therapy for a neurodegenerative disease comprising administering to that patient a therapeutically effective dose of a lipid glyceride comprising a glycerol moiety and a fatty acid moiety, the fatty acid moiety being selected from the group consisting of gamma-linolenic acid, dihomo-gamma-linolenic acid and arachidonic acid characterised in that the selected fatty acid moiety is attached to the glycerol moiety at its sn-2 position. Preferably the method is that wherein the lipid is administered for a duration and at a dose sufficient to maintain or elevate TGF-beta1 levels in the patient to therapeutic levels.

Description

Lipid glyceride is used to prepare the application of the medicine for the treatment of neurodegenerative conditions

The present invention relates to treat neurodegenerative conditions, especially wherein transforming growth factor (TGF-β) especially the increase of TGF-β 1 be the method for useful situation.More specifically, the invention provides the treatment of neurodegenerative conditions, especially such as demyelinating disease, as the treatment of multiple sclerosis, Alzheimer and parkinson disease and the degeneration sequela relevant, for example can improve or recover neuronal function from impaired situation whereby by Remyelination with head trauma, apoplexy and intracranial hemorrhage.

The new purposes of the known and noval chemical compound that comprises the unsaturated fatty acids acid moieties also is provided, has been used to produce the medicine that effectively to treat this type of situation, the medicine of the successful level that does not reach before more specifically recovering to realize about nervous function.

Co-pending unpub patent application PCT/GB04/002089 (this paper quotes as a reference) of the inventor relates to the purposes that plant and fungal oil are used for the treatment of neurodegenerative disease.These oil have the essential fatty acid gamma-Linolenic acid (GLA) of high percentage ratio, normally You more than 40% of total sn-2 fatty acid in the sn-2 position of their lipid.

The essential fatty acid (EFA) of reporting n-3 and the unsaturated pattern of n-6 in the document in detail is useful (WO 02/02105) in multiple human physiological decease (comprising autoimmune disease).Harbige (1998) Proc.Nut.Soc.57,555-562 have summarized in the autoimmune diseased state meals and have added n-3 and n-6 acid, especially mention the evidence of the benefit of the oil that is rich in gamma-Linolenic acid (GLA) and/or linoleic acid (LA).

People such as Bates notice may be more effective in treatment inflammation and autoimmune disease with regard to having mentioned the oils and fats that comprises linoleic acid and gamma-Linolenic acid residue as far back as nineteen fifty-seven, and (7: 1 LA of Naudicelle Radix Oenotherae erythrosepalae oil: recurrence patient GLA) in test state of an illness comparison is shone more serious but find to use 3g oil/sky.

Although the cause of disease of MS is still unknown, research has shown that MS patient has higher nerve-antigen autoreactive T cell level than normal individual.These T cells are especially compared the state of activation that is in increase with myelin basic protein (MBP) with myelin oligodendrocyte glycoprotein (MOG) reaction and with normal healthy controls.Axonal injury among the MS, for example, the real process of chronic inflammatory disease, demyelination and glial cells hyperplasia (astrogliosis) is complicated, but think white matter inflammation and demyelination decision disease seriousness, and studies show that axonal injury just beginning in early days and cause deformity (people such as De Stefano, 2001) among the MS recently in this disease.

Experimental autoimmune encephalomyelitis (EAE) is the most frequently used animal model that is used for the immune-mediated effect of MS.Research in Cavia porcellus has shown that linoleic acid partly suppresses sickness rate and the seriousness (people (1978) such as Meade) of EAE.(people (1995) such as Harbige 1997b) has illustrated linoleic acid and the gamma-Linolenic acid disease modification effect to the clinical and histopathology performance of EAE.Depend on dosage, gamma-Linolenic acid is protected in acute rat EAE fully, and linoleic acid has dose-dependent effects to clinical severity, but does not eliminate this effect.

Although exist these experiments to find, recognize human diseases, multiple sclerosis, be high complexity and can increase the weight of by the activity of T cell and other immune response factors and alleviate.Based on the result who only obtains, think that the n-6 fatty acid promotes autoimmune and inflammatory diseases with linoleic acid.Verified in the mice of gamma-Linolenic acid feed of exsomatizing TGF-β 1 and PGE2 produce non-special increase.Reported TGF-the β 1 ((people (1993) such as Racke that in the EAE of acute and recurrence, shields; People such as Santambrogio (1993)), and PG inhibitor such as indomethacin increase, thereby and make described disease progression (Ovadia ﹠amp; Paterson (1982)).

Cytokine relates to the pathogeny of MS, manyly studies show that the increase of myelinotoxic inflammatory cytokine (TNF-α, IL-1 β and IFN-γ) is consistent with the recurrence phase of this disease.On the contrary, as if the level of antiinflammatory and inhibitive ability of immunity cytokine transforming growth factor-beta 1 (TGF-β 1) reduces and along with patient disease alleviates and increases in the recurrence phase.As if thereby the balance between biological activity TGF-β 1 and short inflammatory TNF-α, IL-1 β and the IFN-γ is lacked of proper care between the recurrence-catabasis at MS.

During the natural convalescent period of EAE, TGF-β 1 secreted T cell suppresses EAE effector lymphocyte, and TGF-β 1 expresses in CNS and expresses in mouth-toleration-inductive protection in EAE, TGF-β and PGE ₂In brain, express (Karpus; Swanborg (1991); People such as Khoury (1992)).Harbige (1998) infer the diet gamma-Linolenic acid to the effect of EAE by comprising the class Th of TGF-β 1 ₃Mechanism mediates and may be by the mediation of superoxide dismutase antioxidant activity.

It is effective at the EAE animal model that is used for identification of M S candidate that borage oil (common 20-23% gamma-Linolenic acid of per 100% content of fatty acid and 34-40% linoleic acid) and mucor javanicus (Mucor javanicus) fungal oil (see figure 1) have been proved to be, and never be proved to be in human diseases significantly effectively.(EPO: linoleic acid: gamma-Linolenic acid 7: 1) part suppresses sickness rate and the seriousness (Mertin﹠amp of EAE in the rat to contain the high-level linoleic oil of being rich in of low-level gamma-Linolenic acid; Stackpoole, 1978), and the Naudicelle that relates to above of Bates research causes the patient to be worsened.Although patients with multiple sclerosis uses borage oil and other to contain the oil of GLA/LA in about in the past 30 years, as Radix Oenotherae erythrosepalae oil, most of patients can not be from this disease recovery, and performance does not significantly improve, and potential disease sustainable development is up to death.

Proposed to use and particularly be rich in gamma-Linolenic acid and linoleic borage oil as the immunosuppressant method in the multiple sclerosis (US 4,058,594) is provided.Crucial is that the dosage of being advised is 2.4g oil every day, and the actual evidence of effect is not provided.This dosage is more much lower than the invalid low 5g/ days dosage of finding in PCT/GB04/002089 research in human body.

Also show other more noticeable immunosuppressant treatments, comprise that T cell depletor and regulator are also effective in the EAE model as the ring phosphonic amide, when these were used for people's multiple sclerosis, though symptom improves, potential disease continued to worsen.In fact the T cell produces useful cytokine at human body, as TGF-β 1, and deleterious cytokine.Institute of Neurology, the David Baker of UK is the difference between the article that in UK MS on the 10th Frontiers meeting May in 2004 with title is " Everything stops EAE, nothing stops MS " has been summed up in EAE and MS effectively in Britain multiple sclerosis association (UK MS Society).

Clear only immunosuppressant can not be cured MS.This is what almost to determine because except autoimmune disease, among the MS patient basic Metabolic disorder cause that film is unusual, cytokine imbalance and immune attack subsequently and pathological changes.Although the patient enters the remission stage in recurrence-alleviation disease, potential demyelination but continues to carry out.

MS " goldstandard (gold standard) " treatment remains interferon, as with β-

With other interferon formulations.The treatment of this goldstandard only solves, for example, 30% patient's needs and even in these patients, doing well,improving also is limited to the recurrence seriousness that reduces.Although symptom can alleviate in a certain proportion of patient, because potential degeneration, this disease is tended to further develop disabled and dead.

In their also unpub PCT/GB04/002089 research, the inventor has determined " high dose " treatment with the triglyceride oil of following content of fatty acid that contains high-level sn-2 gamma-Linolenic acid (is gamma-Linolenic acid at sn-2＞40% residue) and suit astoundingly, can realize the improvement of nearly all symptom significant level of MS, this method is better than the improvement that current goldstandard treatment provides.Contained the preparation of gamma-Linolenic acid and do not achieve success with other before considering, as Naudicelle research, this success is especially wondrous.

In PCT/GB04/002089 studies have shown that during 18 months, the patient who takes the selected high sn-2 gamma-Linolenic acid borage oil of high dose (15g/ days) demonstrate important (p＜0.001) of EDSS score and significantly improve, relapse rate reduces, the objective measurement of the improvement of the sx of muscular spasticity and pain perception symptom and cognitive function.The 5g/ of this borage oil days low dosage does not have effect.

The patient who takes the maximum dose level of this borage oil keeps peripheral blood lymphocytes (PBMC) the generation level of TGF-β 1 in experimental period, their proinflammatory cytokine TNF-α and IL-1 β are significantly and obvious (＜70%) reduces and they have PBMC film long-chain omega-6 fatty acid dihomo-gamma-linolenic acid (DHLA) and the arachidonic acid (AA) that keeps or increase, and compare patient's these fatty acid losses during experimental period of taking placebo.

Estimate that this immunosuppressant will reduce active pathological changes and neurodegenerative increase, the maintenance and/or the increase of the fixed crucial film lipid component of the high obvious target of sn-2 GLA oil treatment, described composition is special loss in MS, this correction with metabolic deficiency is consistent, and described metabolic deficiency can not effectively be treated by current therapy.Low dosage (5g/ days) does not have this true this deduction of support of effect.

Known gamma-Linolenic acid (18:3n-6, perhaps GLA) is changed into long-chain omega 6 polyunsaturated fatty acid dihomo-gamma-linolenic acid and arachidonic acid (people 1994 such as Phylactos, people such as Harbige 1995,2000) in vivo fast.Therefore, in order to determine how to increase among the MS level of film long-chain omega-6 fatty acid, the inventor has summarized the result that they obtain with the oil of several GLA of containing: fungus (from mucor javanicus) and plant (Borago officianalis, Radix Oenotherae erythrosepalae Oenothera spp. or black-currant Ribes spp) and synthetic three-GLA oil is as the GLA delivery system result that experimental animal model obtained in (being called chronic recurrence experimental autoimmune encephalomyelitis (CREAE)) in the body of MS.

Unlike MS, in the rat EAE induce the histologic characteristics that do not produce demyelination people 1988 such as () Brosnan, but to induce acute single-phase disease pattern, the feature of MS be the CNS demyelination and occupy the majority in the case of clinical recurrence-alleviation.Yet the feature of chronic recurrence and demyelination EAE model (CREAE) is demyelination and recurrence phase.In that to illustrate myelin oligodendrocyte glycoprotein (MOG) be neural antigen target (people 1999 such as Genain) important among the MS and illustrate with MBP and compare, peripheral blood autoreactivity lymphocyte is to the bigger replying (people 1993 such as Kerlero de Rosbo of this neural antigen in MS, 1997) under the situation, the inductive CREAE of MOG has become selected animal model, its have with MS in the closely similar feature (people 1997 such as Fazakerely of observed feature, people such as Genain 1999, people such as Amor 1994).

Show that from the evidence of inventor's CREAE and the research of rat EAE feed (72%w/w 18:3n-6 GLA) does not provide protection (seeing Table 3) at EAE for black-currant (blackcurrant) seed oil of enrichment.Importantly, the black-currant seed oil has low sn-2GLA, and most GLA are at sn-1 and sn-3 position (Lawson and Hughes 1988).In addition, contain three GLA structuring triacylgcerol (TG-GLA) partly the protection effect (table 2) that is similar to the borage oil that is used for CREAE is provided.This will be also is important consistent with sn-2 GLA, that is, remove and may experience oxidation, only remaining sn-2 GLA through enzymatic in vivo in the face of sn-1 and sn-3 GLA outward.Known specific lipase can be removed sn-1 and sn-3 fatty acid but obviously protection sn-2 position produces this selective hydrolysis (Lawson and Hughes 1988, Kyle 1990) from the triacylgycerol molecule in vivo.

This summary makes that the inventor supposes to have the sn-2-acid and gamma-linolenic, the glyceride of dihomo-gamma linolenic acid or arachidonic acid residue is in proofreading and correct the MS metabolism even be better than high sn-2-acid and gamma-linolenic borage oil in their the previous test.This will allow to take in the lipid of low dosage more and/or may reduce the treatment time that causes beneficial effect.

The table 3 of EP 0520624 (Efamol Holdings) has compared the content of triglyceride of Radix Oenotherae erythrosepalae and borage oil, has instructed for multiple GLA and has replied imbalance, and the former is more effective in treatment than the latter.This document points out that borage oil has 27 kinds of different triglyceride compositions, and only 20% composition has sn-2 GLA.Page 3 40-42 is capable, and the biological test of pointing out has shown that in fact equivalent GLA may have very different effects when providing GLA with different oil source.Crucially, it instructs the reader then, a kind of specific fraction that exists in Radix Oenotherae erythrosepalae oil (EPO) rather than the borage oil cause the former at rising PGE1 (seeing EP 0520624 chart page 4 and table 2) thus cause outstanding effect in the antiinflammatory effect: this component is accredited as two-linoeoy1-list-γ-Caulis et Folium Lini base-glycerol (DLMG), states that it is the 18-19% of total triglyceride among the EPO.Crucially, the 6th page clearly to instruct GLA be not important in sn-1,2 or 3 position for this effect.

People such as Dines (1994) are at Proceedings of the PhysiologicalSociety, Aberdeen Meeting 14-16 September 1994 reports once more point out also that with the research of the oil treatment diabetic neuropathy neuronal damage that contains gamma-Linolenic acid of type that EP0520624 advocates borage oil is not very effective in this neural degeneration of treatment, and Radix Oenotherae erythrosepalae oil is effective.This article concludes that borage oil contains active other compositions of interference GLA.

The inventor is in view of their result about high sn-2-gamma-Linolenic acid borage oil, begin now to illustrate in fact at glyceride, especially the existence of sn-2-gamma-Linolenic acid, dihomo-gamma-linolenic acid or arachidonic acid residue causes treating effect among EAE, CREAE and the human diseases MS in the triglyceride.

In first aspect, the invention provides treatment needs the patient's of neurodegenerative disease treatment method, this method comprises described patient's administering therapeutic effective dose lipid glyceride of fixed structure really, described glyceride comprises the glycerol part of carrying out esterification with one or more fatty acid parts, and the feature of described method is that described lipid has the fatty acid part that is selected from gamma-Linolenic acid, dihomo-gamma-linolenic acid and arachidonic residue in the sn-2 position.

Especially advantageously Zhi Liao neurodegenerative disease relates to the disease of demyelination.The potential neural degeneration of the special prevention of the present invention is also recovered neuronal function.Particularly, described method normalization neuron film is formed, and the ratio of the cytokine of the TGF-β 1/TNF α ratio of the spontaneous release of PBMC of getting well and TGF-β 1 and other PBMC release.The most advantageously, this method stops all types of multiple sclerosiss, especially recurrence neural degeneration that alleviate, among former carrying out property and the chronic progressive external MS and partially or completely recover neuronal function, as by for example MRI or cat scan or by the EDSS score measured.These class methods can also be used for the treatment of the cerebral lesion behind apoplexy, head trauma and the intracranial hemorrhage, wherein have demyelination or neuronal damage in described apoplexy, head trauma and intracranial hemorrhage.In addition, provide in other chronic demyelinations of treatment, as the application in Alzheimer and the parkinson disease.

Preferably, use described lipid with the persistent period and the dosage that TGF-β level among the patient are kept or be elevated to treatment level.Treatment level refers to consistent with the health volunteer at least level.Preferably, described dosage for for example after using 18 months every day the TGF-β 1/TNF-α ratio from the spontaneous release of the isolating peripheral blood lymphocytes of blood samples of patients (PBMC) be 0.4 to 3.0, at least 0.5, more preferably at least 0.75, most preferably at least 1.Preferably, described dosage produces TGF-β 1/IL-1 β ratio after 18 months and is at least 0.5 for for example using every day in blood samples of patients, and more preferably at least 0.75, most preferably at least 1.Preferably, after 12 months, more preferably produce described level after 6 months.

Usually, during dosage forms for oral administration, the amount of the lipid of using every day is 0.5 to 30g, more preferably 1 arrives 20g, most preferably 1 arrives 18g, common 3 to 5g.

When sn-2 partly was the sn-2 part of gamma-Linolenic acid residue, dosage can be in the higher end portion of these scopes, when sn-1 and sn-3 partly are relative inertness, for example, was the acid that metabolism is used, during as satisfied fatty acid especially.When sn-2 partly is the sn-2 part of dihomo-gamma-linolenic acid residue, dosage can be littler, and when sn-2 partly was the sn-2 part of arachidonic acid residue, effect was higher, but administration should be more careful, because may have unwanted side effect in higher level.

More preferably, described method is characterised in that described lipid is the monoglyceride, diglyceride or the triglyceride that contain at least one sn-2 gamma-Linolenic acid, dihomo-gamma-linolenic acid or arachidonic acid moieties of following general formula I:

Formula I

R wherein ¹And R ³Be independently selected from hydrogen and acyl group,

R ²Be selected from the group that gamma-Linolenic acid, dihomo-gamma-linolenic acid and arachidonic acid residue constitute.

For the present invention, the end that acyl group is defined as at the hydrocarbyl chain of the optional replacement that is selected from alkyl and alkenyl chain comprises at least one carbonyl, and this carbonyl is directly connected on the oxygen of glycerol residue shown in the formula 1 by its carbon.

Preferably, acyl group R ¹And R ³Be formula-CO-(CH ₂) _n-CH ₃Saturated fatty acid part, wherein n is for being selected from 1 to 22, more preferably 4 to 16, even more preferably 5 to 12,6 to 10 integer most preferably.Especially preferably, acyl group is sad and acyl group capric acid, especially has 1 of gamma-Linolenic acid, dihomo-gamma-linolenic acid or arachidonic acid moieties, 3-two caprylins or 1,3-two caprins in the sn-2 position.

Preferably, being used for glyceride of the present invention is triglyceride.

US 4701469 has described some potential triglyceride that are used for the nutraceutical purposes; the inventor has determined that they can be used for the inventive method; although US 4701469 only describes 1 especially; 3-dioctyl triglyceride; wherein sn-2 acid is EFA; 1 of preparation has only been described, 3-two caprylyl eicosapentaenoic acid glycerols.It is said that these triglyceride can be used in particular for immunomodulating etc.,, be not listed in the purposes in the immunosuppressant among neural degeneration and the MS although pointed out numerous disease.

Although most preferably, be included in radicals R in the formula I chemical compound ¹To R ³Be simple satisfied fatty acid or naturally occurring fatty acid,, but have other possibilities as medium chain or long-chain fatty acid with structure or metabolic function.Especially preferably, fatty acid is to be mainly used in energy-producing those fatty acids of metabolism.When fatty acid is structural fatty acid, when promptly being used for film, they are such as gamma-Linolenic acid, linoleic acid, dihomo-gamma-linolenic acid and arachidonic acid residue expediently.Residue refers to that the fatty acid carboxyl esterification goes up the back rest parts to one of hydroxyl of glycerol molecule.

Other preferred acid that are used for sn-1 and sn-3 are selected from opposite to the fatty acid that mainly is directed to the structural membrane storehouse the energy-producing fatty acid of human body metabolism: this type of preferred acid comprises oleic acid and Palmic acid.

As sn-1 and sn-3 fatty acid chain (R ¹And R ³) when unsaturated, it also can be the fatty acid chain of other essential fatty acid, as n-3 acid, as parinaric acid, eicosapentaenoic acid and docosahexenoic acid (docosahyexanoicacid).When fatty acid is optionally substituted, they will preferably be replaced by hydroxyl, oxo, carboxyl, alkyl, alkenyl and alkoxyl.Hydrocarbyl chain is preferably long 1 to 30 carbon atom, more preferably 4 to 28 carbon atoms, the more preferably hydrocarbyl chain of 4 to 24 carbon atoms.Most preferably, hydrocarbyl chain is the hydrocarbyl chain of fatty acid, more specifically the hydrocarbyl chain of list or polyunsaturated fatty acid.

Many preferred lipids that are used for the inventive method are known and can be by chemical method preparations known in the art.For example, many is obtainable by commercial sources, and as three γ-linolenin, it is called TLG, but is called GGG at this paper, has reflected radicals R ¹R ²R ³Be identical, wherein G represents the gamma-Linolenic acid residue.

GGG can obtain from Nu-Check-Prep Inc by commercial sources.EP 0300844 has described the synthetic of it, uses the ester exchange based on base catalysis of glyceryl triacetate and gamma-Linolenic acid methyl ester to obtain mixture, and this mixture contains 80%GGG, unreacted gamma-Linolenic acid methyl ester and 10% mono-and diglycerides.

Triarachidin is known and can for example obtains on a small quantity from Sigma by commercial sources.Synthesized AAA owing to have the immobilized-lipase (US 4888324) that the angiogenesis enhanced activity applies for a patent from arachidonic acid by using.

Yet; although can use three and two-gamma-Linolenic acid, dihomo-gamma-linolenic acid or arachidonic acid triglyceride or triglyceride; but the inventor preferably uses list-gamma-Linolenic acid, dihomo-gamma-linolenic acid or arachidonic acid sn-2 ester triglyceride, the enhanced activity that keeps sn-2 gamma-Linolenic acid, dihomo-gamma-linolenic acid or arachidonic acid moieties to provide about desirable film normalization and disease modification effect because they use less immunomodulating and short scorching fatty acid gamma-Linolenic acid, dihomo-gamma-linolenic acid or arachidonic acid.

Preferred new lipid can obtain by process and the method that proposes among this paper embodiment.Most preferred lipid is such lipid, wherein only gamma-Linolenic acid, dihomo-gamma-linolenic acid or an arachidonic acid moieties in the sn-2 esterification to glycerol, flank sn-1 and sn-3 acid are undersaturated medium chain or long chain acid.

Thereby another aspect of the present invention provides new lipid disclosed herein, comprises the chemical compound of formula II

R wherein ¹And R ³Identical and be-C (O) (CH ₂) _nCH ₃, wherein n is selected from 4 to 14, more preferably is selected from 6 to 10, most preferably is selected from 7,8 or 9, R ²Be selected from γ-Caulis et Folium Lini acyl group, two height-γ-Caulis et Folium Lini acyl group and arachidonic acyl group.

Another aspect of the present invention provides the method for the chemical compound of synthetic general formula III

R wherein ¹And R ³Identical and be-C (O) (CH ₂) _nCH ₃, wherein n is selected from 4 to 14, more preferably is selected from 6 to 10, most preferably is selected from 7,8 or 9, R ²Be γ-Caulis et Folium Lini acyl residue, two height-γ-Caulis et Folium Lini acyl residue and arachidonic acyl residue, described method comprises

With 1,3-dihydroxy acetone and formula X-C (O) (CH ₂) _nCH ₃, wherein X is selected from the chemical compound reaction of Cl, Br and I,

Obtain corresponding 1,3-two-(C (O) (CH ₂) _nCH ₃) ₂-ketonic compound,

The reduction ketone group obtains corresponding 1,3-two-(C (O) (CH ₂) _nCH ₃) ₂-alcohol,

And with itself and γ-Caulis et Folium Lini acyl chlorides or two height-γ-Caulis et Folium Lini acyl chlorides or arachidonic acyl chloride reaction.

The method that synthetic general formula I V chemical compound is provided on the one hand again of the present invention

R wherein ¹To R ³Identical and be selected from γ-Caulis et Folium Lini acyl residue, two height-γ-Caulis et Folium Lini acyl residue or arachidonic acyl residue, described method comprises corresponding γ-Caulis et Folium Lini acyl chlorides, two height-γ-Caulis et Folium Lini acyl chlorides or arachidonic acyl chlorides and glycerine reaction.

Be described below among the synthetic and figure below of some chemical compounds in these chemical compounds and shown synthetic schemes.

For example, can implement a step esterification of glycerol, it uses GLA and coupling agent, as DCCI/DMAP (1,1-dicyclohexylcarbodiimide/4-dimethylaminopyridine) coupling reagent.But this method obtains good yield produces impurity, unless it is removed, otherwise makes that final oil is muddy.Can use coupling agent prevent as EDCI (1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride) as described in problem, described coupling agent produces the water solublity by-product, these by-products are more easily removed.Jpn.Kokai Tokkyo Koho JP 05310638 A2 22Nov 1993Heisei, but 6pp. has described use DCCI and similar different prepared in reaction three-α-linolenin (LnLnLn, wherein Ln is a linoleic acid).

A kind of alternative approach provides two step sequences, and it utilizes GLA-C1 (from gamma-Linolenic acid and oxalyl chloride preparation) and the reaction of glycerol dichloromethane/pyridine, and this method has good yield, reaches 250g by the column chromatography productive rate during amplification.Jpn.Kokai Tokkyo Koho JP04328199 A2 17 Nov 1992 Heisei, 5pp. (Japan) Concentration ofa-linolenic triglyceride by flash chromatography.Ando, Yukiki, Watanebe, Yoichi, Takagi, (Nisshin Oil MillsLtd Japan) describes the relevant still different technology that is used for purification three-α-linolenin (LnLnLn) to Yoshiaki.

Comparative example tricaprin (decanoin) is can be from the known compound of Sigma acquisition by commercial sources.By with methyl caprate and sodiumglyceroxide reaction, obtain described chemical compound (seeing E.S.Lutton and A.J.Fehl, Lipids, 5,90-99 (1970)) by the column chromatography purification crude product subsequently.

A kind of alternative approach relates to the acid catalyzed reaction of glycerol and capric acid, four crystallizations subsequently (seeing L.H.Jenson and A.J.Mabis, Acta Cryst., 21,770 (1966)).

The applicant also provides and has improved one's methods, and it allows the decanoyl chloride reaction more than glycerol and 3 equivalents and passes through recrystallization purifying decanoin product.

Other aspects of the present invention provide the purposes as above-mentioned triglyceride oil, are used for the medicine of the neurodegenerative disease that production for treating the inventive method proposes.Especially preferred medicine is used for stoping and reversing the neural degeneration of all types of multiple sclerosiss, especially recurrence partially or completely recovery that alleviate, former carrying out property and chronic progressive external neural degeneration and neuron integrity function is as measuring by for example MRI or cat scan or by the EDSS score.Can treat other TGF-β 1 as described above and reply disease.

Can be used for lipid of the present invention by known any conventional vector administration in the pharmacy.Most convenient ground, with them with pure oil or with the mixture of food, to contain this type of oily capsule form or to use with the enteric coating form.Those skilled in the art will expect other forms, see Remington Pharmaceutical Sciences, the 19th edition.

Those skilled in the art will recognize that other useful reagent can be united with described lipid is used for the present invention or forms the part of therapeutic scheme with described lipid.These reagent can be the ion channel blocking agent, for example, and sodium channel blockers, interferon (α, β or γ), T cell depletor, steroid or other palliatives.To also recognize when immune and inflammatory response are subjected to regulating, consider the complicated character of these systems, will need carefully to carry out this type of associating.Yet, consider delayed response to oil of the present invention, before TGF-β 1 horizontal normalization, may be useful, as long as extra treatment does not hinder this normalization process at the shorter effect reagent in first middle of the month for the treatment of.

Being used for the synthetic of struetural lipid of the present invention (structured lipids) describes hereinafter with the synthetic of comparative example.Some of these lipids are new, and other are known, but also are not used in treatment of the present invention.

Only with reference to following non-limiting table, embodiment and accompanying drawing the present invention is described by way of example now.Those skilled in the art will expect falling into other embodiments of the scope of the invention according to these.

Table

Table 1: shown multiple triglyceride oil composition % total fatty acid content and the protection effect in EAE.

Table 2: the parameter that has shown three treatment groups in the high sn-2 GLA borage oil test of describing among the PCT/GB04/002089.

Table 3: the GLA that has shown various ways is to the EAE incidence rate in the SJL mice and the influence of clinical score: low score shows the therapeutic effect of improvement.

Table 4: black-currant (Blackcurrent) oil that shows enrichment---vegetable oil of the still low sn-2 GLA of a kind of high GLA---can not mate fungus and borage oil among the EAE.

Accompanying drawing:

Fig. 1: show placebo and high sn-2 gamma-Linolenic acid, the PCT/GB04/002089 test oil, the human MS patient of processing is spontaneous peripheral blood lymphocytes cytokine production in the time of 18th month.

Fig. 2: show and compare that the high sn-2 GLA borage oil of placebo and low dosage (5g/ days) represents with rectangular histogram that to the influence of human MS patient EDSS score the x axle is treatment month number with high dose (15g/ days).

Fig. 3: shown the influence of the high sn-2 GLA of placebo, low dosage and high dose borage oil to the average relapse rate of human MS patient (%) with rectangular histogram, the x axle is a month number.

Fig. 4: shown the synthetic reaction scheme that is used for the single fat acid triglyceride of the inventive method and purposes.

Fig. 5: the reaction scheme that has shown synthetic control compound tricaprin.

Fig. 6: the reaction scheme that has shown synthetic fatty acid mixed triglyceride CGC of the present invention.

Fig. 7: the reaction scheme that has shown synthetic fatty acid mixed triglyceride C-DHGLA-C of the present invention.

Fig. 8: shown synthetic control compound GCG, 1,3-two caprylyls, the reaction scheme of 2-gamma-Linolenic acid.

Fig. 9: the reaction scheme that has shown synthetic fatty acid mixed triglyceride C-AA-C of the present invention.

Figure 10-19 has shown the result as the research of EAE in SJL and C57BL mouse that provides among the following embodiment.(DHLA＝DHGLA：A＝AA)

Embodiment

High sn-2 borage oil (PCT/GB04/002089) test

28 active recurrence-alleviations (recurring secondary at 18 months before this) patients with multiple sclerosis (age 18 was by 65 years old) enters in the double blinding placebo-controlled trial to study in 18 months encapsulated borage oil to the influence of clinical activity and experiment parameter.This oil is high sn-2 gamma-linoleic acid (GLA) content (＞40% sn-2 residue is a gamma-linoleic acid), has low monene (for example, erusicacid) content and do not add vitamin E---a kind of known immunomodulator.

Recruit the patient in two tame inner city hospitals from neurological outpatient clinic; When (baseline) visit for the first time, obtain hospital's informed consent.Exclusion standard is included in preceding 3 months any type of steroid or immunosuppressive drug treatment, gestation, hyperlipemia, uses aspirin or related drugs and vitimin supplement or fatty acid regularly.

Only satisfy that the patient of standard comprises in test below all: (a) can understand fully and can cancel agreement at any time harmlessly in the treatment prerequisite for informed consent; (b) age 18 is to the sex out-patient of 60 years old (comprise 18 and 60 years old); (c) the verified clinical definite recurrent MS that is diagnosed as; (d) had 3 times clinical recurrence on the books in the past in 2 years at least; (e) have 0.0-5.5 (comprising 0.0 and 5.5) disabled must the graduation of baseline expansion (Expanded Disability Scoring Scale, EDSS) score, condition are the deteriorations that they are documented; (f) testing confirmation by medical history, health check-up and clinical chemistry, urine and hematology is healthy except the MS related symptoms.

By Pharmacy Department the patient is divided into three groups at random, every group comprises 12 patients:

● first clinical group (n=12) accepts placebo (5g PEG400)

● second clinical group (n=12) accepts low dosage (5g) refine Borage officinalis

● the 3rd clinical group (n=12) accepts high dose (15g) refine Borageofficinalis.

Be added to the form (for low dosage 5/ day, 15/ day high dose) of gram oil capsule every day 1, continue 18 months.Borage officinalis oil and omega 6 polyunsaturated fatty acid are to It is generally accepted composition of food into to human consumption safety (GRAS).Not classification or labelling requirement under the EC regulation.Clinical assessment comprises: expand disabled grade score (EDSS) and clinical recurrence record.Take venous blood (50ml) to be used for laboratory research at the 1st, the 3rd, the 6th, the 12nd, the 15th and 18th month that adds.

Biochemistry below each visit research and amynologic parameter with and treatment before data relatively and between the group data relatively:

● the external peripheral blood lymphocytes cytokine production that stimulates and do not stimulate: relate to the change among pathogenetic TGF-β 1, IFN-γ, TNF-α, IL-1 β, IL-6 and the IFN-β of MS.Cytokine and related gene expression.

● solubility adhesion molecule in the serum, especially ICAM-1 and VCAM-1

● peripheral blood lymphocytes film fatty acid and blood plasma lipoid fatty acid component.

The result table 1 and 2 and Fig. 1 to 5 in show.

Elementary result parameter is that baseline (the 0th month) and treatment finish clinical recurrence number between (18th month).The secondary result parameter comprises: to the time of the clinical recurrence first time; Recurrence seriousness (as by EDSS score and the assessment of use steroid therapy); With compare with baseline the 3rd, 6,9,12 and the change of 18 months EDSS and its be defined as EDSS and increase by at least 1.0 points, continue 3 months, perhaps EDSS increases by at least 1.5 points from baseline EDSS, continues 3 months.

11 patients are in placebo group, and 7 patients take the low dosage borage oil, and 10 patients take the high dose borage oil.The drug resistance of being studied is good, and does not have serious adverse events at 18 months duration of test.

The separation of PBMC and cultivation

With the heparinization whole blood with equal-volume Hanks balanced salt solution (Sigma, UK) dilution and with the blood of gained dilution at Lymphoprep (Nycomed, Oslo, Norway) higher slice., remove PBMC from the interface and use the Hanks solution dilution after 30 minutes with the 800g density centrifugation.Then by with centrifugal 10 minutes of 250g with cell washing 2 times.Final of gained is suspended in the culture medium again, and (Sigma, (Sigma UK) forms with 10% autologous plasma this culture medium UK) to add 2mML-glutamine, 100U penicillin and 100 μ g streptomycins by the RPMI-1640 culture medium.(Bibby Sterilin Ltd, Stone UK) add 2 * 10 to tissue culture tube ⁶Individual/ml PBMC (＞95% survival is judged by trypan blue exclusion) also uses 5%CO at 37 ℃ ₂Cultivated 24 hours.Determine in the dynamic experiment in front that antigen concentration, cell density and incubation time produce (data not shown) to determine the maximum cell factor.Also prepare conventional cytospin prepared product to be used for differential count subsequently.After the cultivation,, remove the gained supernatant then, aliquot and-70 ℃ of preservations by removing cell from culture in centrifugal 10 minutes with 250g.

The preparation plasma sample

With the centrifugal 10ml heparinized blood of 250g 10 minutes.Remove the gained plasma layer then, aliquot and-70 ℃ of preservations.

Detect proinflammatory cytokine

With by the obtainable one-tenth antagonist (R that can detect cytokine of commercial sources with the ELISA form; D systems Ltd, Abingdon UK) detects TNF-α, IL-1 β and IFN-γ in cell culture supernatant and the blood plasma.Sensitivity to TNF-α and IFN-γ ELISAs is 15.6-1000pg/ml, is 3.9-250pg/ml for the sensitivity of IL-1 β.

The active TGF-β 1 of detection of biological

By sensitivity is the obtainable E of commercial sources of 15.6-1000pg/ml _Max(Promega, Southampton UK) detect biological activity TGF-β 1 in cell culture supernatant and the blood plasma in the ELISA system.

Statistical analysis

With the difference of Si Shi t check and the thinner intracellular cytokine generation of Man-Huai U check (Mann-Whitney U-test), and think that difference is significant less than 0.05 the time when the p value.

The result

Two patients produce diarrhoea, confirm that afterwards they have taken the borage oil of high dose.A patient suffers from diarrhoea slightly, but the second place patient is medium serious, and this patient had been interrupted this research medicine afterwards.Record does not interrupt and suffers from diarrhoea after drug withdrawal stopping, and occurs diarrhoea when still stimulating again again.Therefore, this patient withdraws from from test.Residue patient with the treatment of high dose borage oil shows fabulous clinical improvements on all primary and secondary result standards.For example, 6 months their average EDSS scores of treatment back are from baseline EDSS be improved (Fig. 1).More importantly, treat after 6 months, when comparing with recurrence number in the placebo group, the clinical recurrence average has significantly reduced (Fig. 2).On the contrary, the patient who accepts the low dosage borage oil does not show any clinical improvements when with the placebo group comparison.Except its beneficial effect to the MS disease activity, the high dose borage oil also provides the certain sx to muscle spasm state (stiff) and pain perception symptom, and the cognitive function that improves is provided.

As seeing from figure below, in high dose group, 9,12 and after 18 months relapse rate drop to 0.The increase of seeing in the time of 15th month is the patient who withdraws from owing to from this group.

Be that three brief medical histories are in order to illustrate the treatment benefit of the high sn-2 GLA of high dose borage oil below.Preceding two medical histories are from test, and the 3rd be test back patient, obtain MRI research from this patient.

Patient 1 (treatment):

First place patient is 48 years old women, and she has suffered from clinical activity, recurrence remission form MS 9 years.She works as full-time manager in local health bureau (Health Authority) at first, but she can not carry out her responsibility owing to serious MS.Therefore, she is secretarial job on the side afterwards, but still has difficulty aspect motion because muscle rigidity and sensation are disorderly.She also experiences serious clinical recurrence, and recurrence in average per 9 months once.Most these recurrences cause being admitted to hospital and carry out steroid therapy.Consider her MS that enlivens, she is raised the borage oil test.Not relevant with research adverse events was taken medicine after 4 months, and she is the good improvement of experience aspect walking and sensory symptoms.

About 9 months of treatment back, she is health and can begin full-time job enough.In addition, she kept not recurring in 18 months persistent period of clinical trial.After off-test, the treatment record shows that she takes the high dose borage oil.

Patient 2 (contrast):

The second place case is 46 years old women, and she has also suffered from the clinical activity recurrence alleviates MS 8 years.She is initially the shop-assistant, but is unemployed after being diagnosed as MS.

Her symptom comprises movable difficulty and the pain perception symptom in two legs.She has experienced clinical recurrence in clinical trial three times in preceding 2 years, and has been admitted to hospital and has carried out steroid therapy twice.Therefore, she is raised in the borage oil test, but her walking continues to worsen.Enter test after 6 months, she need use crutch and accept the baclofen treatment to reduce the extremity spasticity.About 10 months of beginning borage oil test back, she is admitted to hospital and carries out steroid therapy because of serious clinical recurrence.She suffered from the bladder imbalance afterwards and brought into use wheelchair to grow distance.Open the treatment record after off-test in 18 months, what find that she takes is placebo.Afterwards, she brings into use walking frame for the distance above 50 yards.

The patient 3: treatment (outside the test)

The 3rd case is 26 years old man, and he is diagnosed as MS in April calendar year 2001.His symptom was beginning in 1999, and he states the intractable pain of disperse at that time, and this pain influences a plurality of parts of health, especially influences the left side of breast and abdomen.Then be that hands and foot are intermittent numb, be accompanied by the undulatory property weakness.The distressful bladder symptom that also has frequent micturition and urgent micturition form.The diagnosis of calendar year 2001 MS is based on his recurrence relief of symptoms, and is confirmed by the nuclear magnetic resonance (MRI) of positive cerebrospinal fluid analysis and brain, and MRI shows in two brain hemisphere and has a plurality of abnormal findings of white matter.Symptom does not respond multiple pharmacotherapy.

In April, 2003, beginning per os supplementary copy invention high dose borage oil.The patient is reported in and begins this per os and replenish in 3 months of administration his symptom and significantly improve.His pain perception symptom complete obiteration.He does not just have numbness or weakness from May, 2003 at report, and notices the remarkable improvement of his bladder control.Per os replenishes administration and does not cause adverse events.Carry out the improvement of repetition brain MRI with Mr.'s N symptom of confirming to be reported.Repeat MRI and show that the size of abnormal findings of white matter and distribution reduce.

Embodiment: structure sn-2 lipid

Among all embodiment below, obtain more high-purity by using more highly purified initial substance gamma-Linolenic acid, dihomo-gamma-linolenic acid or arachidonic acid (as obtaining) from for example Sigma Aldrich.GLA95 is meant 95% pure gamma-Linolenic acid.

Synthesizing of 1: three γ linolenin of synthetic embodiment

1) acyl chlorides method

2.0g (7.2mmol, 3.1 equivalents) GLA95 (95% pure gamma-Linolenic acid) is dissolved among the 10mlDCM.Dropwise add 1.01g (0.71ml, 8.0mmol, the 3.4 equivalents) oxalyl chloride that is dissolved in 5ml DCM under the nitrogen in 2-3 minute.Stirred overnight at room temperature.The vacuum concentration reactant mixture is removed DCM and excessive oxalul chloride.In 2-3 minute, this acyl chlorides dropwise is added under the nitrogen in the stirred mixture of 215mg (2.3mmol, 1 equivalent) glycerol, 0.58ml (3.1 equivalent) pyridine and 10ml DCM then.Stir the mixture under the room temperature and spend the night.The pyridine hydrochloride that forms of filtering and wash then with DCM.Solution is washed with 1 * 4ml water, 0.1N HCl, 5% sodium bicarbonate and 5%NaCl.Use dried over mgso, filter, and vacuum concentration obtains yellow oil.Purification should oil on silicagel column, with containing the hexane of 10% ether as eluting solvent.Obtain clear, colorless oil, its sample ester exchangeization is also analyzed by GC subsequently.This product contains 96.3% GLA.

2) DCCI method

Under the nitrogen 2.19g GLA95 (3.15 equivalent), 230mg (1 equivalent) glycerol, 153mg DMAP (0.5 equivalent) are stirred in 10ml DCM.Add the 1.85g DCCI (3.6 equivalent) that is dissolved among the 5ml DCM.Stirred reaction mixture spends the night under the room temperature nitrogen.The DCU that filter to form also washs with DCM.With DCM with 1 * 5mlN HCl, water, 5% sodium bicarbonate and water washing.Use dried over mgso, filter, and vacuum concentration obtains oil.Purification should oil on silicagel column, with containing the hexane of 10% ether as eluting solvent.Obtain the muddy a little oil of 1.47g (67%).The sample ester exchangeization of this product is also analyzed by GC subsequently.This product contains 95.8%GLA.

Amplify

20g (0.072mol, 3.1 equivalents) GLA95 (gamma-Linolenic acid, 95%) are dissolved among the 100mlDCM.Add 13.7g (9.3ml, 0.11mol, 4.78 equivalents) oxalyl chloride under the nitrogen in 3-4 minute.Stirred reaction mixture spends the night under the nitrogen.The vacuum concentration reactant mixture is removed DCM and excessive oxalul chloride.In about 5 minutes, this oil droplet is added under the nitrogen in the stirred mixture of 2.14g (0.023mol, 1 equivalent) glycerol, 100ml DCM and 5.8ml (5.68g, 0.072mol, 3.1 equivalents) pyridine then.Stir the mixture under the room temperature and spend the night.The pyridine hydrochloride that forms of filtering and wash then with DCM.DCM solution is washed with 1 * 25ml water, 10% sodium bicarbonate, 0.1N HCl, 5%NaCl.(forming emulsion in this process, especially when beginning).With the DCM dried over mgso, filter, and vacuum concentration obtain brown oil (～21g).

Purification should oil on silicagel column, at first uses the hexane that contains 5% ether to use the hexane that contains 10% ether then.Obtain 15.6g (77% productive rate) clean oil.By tlc, this material contains small-amount free GLA.(this material had been purified afterwards).

The extensive amplification

Repeat top reaction with 10 times of scales.Thereby, use 200g GLA95,1 L DCM, 137g oxalyl chloride and 21.4g glycerol.When adding acyl chlorides, remain on below 35 ℃ with the psychrolusia reaction mixture and with temperature.Produce the 250g brown oil.With its purification on the 500g silicagel column at first.Be dissolved in oil in the 200ml hexane and be applied to pillar.At first use hexane, then with the hexane that contains 5% ether, then with the hexane eluting that contains 10% ether.Collect fraction and, finally produce two batches of oil by the tlc analysis.The first fraction A (66g) contains impurity and a small amount of GLA (moving slowly than TGL) that moves in a small amount of forward position, and the second fraction B (99g) does not have the forward position to move impurity and contains a little GLA.

Repeat to react on a large scale and obtain as above two kinds of fraction with 169g GLA.85g " A " fraction and 54g " B " fraction are arranged specifically.Merge two batches " A " and repurity on the 500g silicagel column.Handle " B " fraction (also in this batch product, adding the 15g material that reacts from a small scale) in a similar manner.

Purification also finally obtains 259g oil from some top fraction once more.This oil of distillation arrives constant weight-256g on rotary evaporator under fine vacuum.This represents 65% gross production rate.

Product analysis

GC

With the small sample ester exchange and carry out GC and analyze:

GLA content is 97.1%.Major impurity is a linoleic acid-1.91%.

Note: be used for synthetic initial GLA95 and contain 96.2%GLA and 2.42% linoleic acid.

HPLC

(Hypersil C18 4.6 * 100mm) carries out the HPLC method, with 80/20 acetonitrile/THF eluting to use reversed-phase column.Detect by 210nm UV.This shows that product is three kinds of mixture of ingredients.Main peak (93.6%) is needed product.More slowly the impurity of Yi Donging (account for product 5.0%) may be GGLI triglyceride (LI=linoleic acid).Second impurity moves and accounts for 1.4% of product slightly soon.

Note: obviously different between the triglyceride that is absorbed in different content of fatty acid at 210nm place.For example, the UV of three γ linolenins absorb be trilinolenin 5-6 doubly.

Sum up

Prepared 254g three-6 by two step acyl chlorides approach from 96.2%GLA, 9, the 12-glyceryl linolenate) (the acid and gamma-linolenic triglyceride, three γ linolenins, GGG).It is transparent, light yellow oil, and it is preserved under nitrogen in cold closet.GLA content is 97.1% and does not detect C20:1, C22:1 or C24:1 acid.HPLC purity is 93.6%.

Use GLA98 (98% gamma-Linolenic acid: Scotia) or more highly purified initial substance can realize easily that high-purity GGG's more is synthetic.

Compare lipid 1: synthetic Tricaprin (decanoin)

On a small scale

Room temperature nitrogen stirs glycerol (3.0g, 0.0325mol, 1 equivalent), pyridine (8.1ml, 0.10mol, 3.1 equivalents) and dichloromethane (100ml) down.Dropwise add decanoyl chloride (21ml, 19.25g, 0.10mol, 3.1 equivalents) then in 5 minutes, externally cool off to maintain the temperature at 30-35 ℃ with water-bath.When adding was finished, (DMAP (0.12g, 1mmol, 0.03 equivalent) and at room temperature stirring the mixture under the nitrogen spent the night to add 4-dimethylaminopyridine.By removing by filter sedimentary pyridine hydrochloride and using washed with dichloromethane.Use the cleaning mixture and the filtrate of aqueous solution (20ml) the washing merging of 5% sodium chloride, 5% sodium bicarbonate, 0.1N hydrochloric acid and 5% sodium chloride then.Use MgSO then ₄Dry methylene chloride layer and solvent removed in vacuo.Residual oil leaves standstill crystallization.This material obtains 15.6g (86% productive rate) wax shape white solid from isopropyl alcohol (40ml) recrystallization.

Analyze

GC-99.8% purity

HPLC

(C18 4.6 * 100mm, ACN/THF 85/15 1ml/min, λ 210nm)-94.9% purity

On a large scale

Repeat top reaction with 15 times of scales.Room temperature nitrogen stirs glycerol (45.0g, 0.49mol, 1 equivalent), pyridine (121.5ml, 1.50mol, 3.1 equivalents) and dichloromethane (1.5L) down.Dropwise add decanoyl chloride (315ml, 288.8g, 1.50mol, 3.1 equivalents) then in 15 minutes, externally cool off to maintain the temperature at 30-35 ℃ with water-bath.When adding was finished, (DMAP (1.8g, 15mmol, 0.03 equivalent) and stirring the mixture under room temperature nitrogen spent the night to add 4-dimethylaminopyridine.By removing by filter sedimentary pyridine hydrochloride and using washed with dichloromethane.Use the cleaning mixture and the filtrate of aqueous solution (300ml) the washing merging of 5% sodium chloride, 5% sodium bicarbonate, 0.1N hydrochloric acid and 5% sodium chloride then.Use MgSO then ₄Dry methylene chloride layer and solvent removed in vacuo.Residual oil leaves standstill crystallization.This material obtains 228g (86% productive rate) wax shape white solid from isopropyl alcohol (400ml) recrystallization.

Analyze

GC-99.8% purity

HPLC

(C18 4.6 * 100mm, ACN/THF 85/15 1ml/min, λ 210nm)-94.9% purity

Produce another batch product and obtain the 44g product with top small-scale batch products merging and from the isopropyl alcohol recrystallization.Batch (268g) above merging also analyzes again:

GC

99.9% purity

HPLC

97.9％

Sum up

By one step process (scheme given below) from decanoyl chloride (98%) prepared the 263g decanoin (tricaprin, CCC).It is white, low melting point solid and it is preserved under nitrogen in cold closet.C content is that 99.9% and HPLC purity of content of fatty acid is 97.9%.

Synthetic embodiment 2:2-acid and gamma-linolenic 1, and 3-two caprins (1,3-two capric acid 2-18 carbon three (6-Z, 9-Z, 12-Z) olefin(e) acid glyceride or CGC)

This triglyceride is new.Unlike CGC, its isomer C LnC (Ln=alpha-linolenic acid) (sees people Biotechnol.Lett. such as K.Long through identifying, 20,369-372 (1998) and H.Mu, people Eur.J.Lipid Sci.Technol. such as P.Kalo, 102,202-211 (2000)) be the composition of Oleum Cocois.In addition, CLxC (Lx=does not specify the linolenic acid of position of double bond) (see people J.Dairy Sci. such as J.Gresti, 76,1850-1869 (1993)) has been described.

Two kinds of intermediate that are used for synthetic CGC be known (see people Arzneim-Forsch./Drug Res. such as L.E1 Kihel, 46,1040-1044 (1996) and US4178299).The final step that describes below is novel and first two steps also are creative, because they are more suitable for large-scale production than the method for report in the past.

By with 1,3-two caprins and the prepared in reaction CGC of GLA-acyl chlorides in dichloromethane-pyridine.By 1, the sodium borohydride reduction of 3-didecyl acyloxy third-2-ketone preparation 1,3-two caprins, 1,3-didecyl acyloxy third-2-ketone is again by with 1,3-dihydroxy acetone and decanoyl chloride prepared in reaction.Must produce body 1 in the middle of the handled, 3-two caprins can experience acyl migration because it is exposed to acid, alkali and Re Shi.Preparation 1 has been described, the older method of 3-two caprins (see people Chem.Physics Lipids such as A.P.J.Mank, 16,107-114 (1976)).

General to the catalysis addition of ethylene oxidic ester (from chloropropylene oxide) by capric acid, synthetic 1 flexibly, the captivation of 3-diglyceride and triglyceride is lower, and this is because stricter reaction condition and acyl group branch problem.By column chromatography purification end-product CGC careful on silica gel, described chromatography is removed by-product.

On a small scale

1,3-didecyl acyloxy third-2-ketone

Under the room temperature nitrogen with decanoyl chloride (40.0ml, 36.8g, 0.19mol, 1.98 equivalents) and in 10-15 minute, dropwise join 1,3-dihydroxy acetone dimer (8.68g, 0.048mol, 1.0 equivalent), pyridine (15.6ml, 0.19mol), 4-dimethylaminopyridine (0.18g, 0.0014mol, 0.03 equivalent) and dichloromethane (DCM is in the suspension of stirring 150ml).Keep the temperature of reactant mixture to be lower than 30 ℃ by cooling in psychrolusia.Stirred reaction mixture spends the night under the room temperature nitrogen.Pyridine hydrochloride by removing by filter formation also washs with DCM.Use 5%NaCl, 5%NaHCO then ₃, 0.1N HCl, 5%NaCl 1 * 25ml partly wash the filtrate and the cleaning mixture of merging.With solution MgSO ₄Dry and vacuum concentration obtains light yellow semisolid.Then it is obtained white solid from methanol (150ml) crystallization.Productive rate is 28.2g (73%).

1,3-two caprins

With top ketone (28.2g, 0.071mol) be dissolved in oxolane (THF, 200ml) in.Add entry (10ml) then, cooling solution to 5 ℃, and 10 ℃ down by part add a sodium borohydride (5.38g, 0.14mol).Stirring at room reactant mixture 1h, vacuum concentration is removed THF then.Residue distributes between ethyl acetate and 5% sodium chloride solution.With ethyl acetate aqueous phase extracted and use MgSO once more ₄Dry extract and the vacuum concentration that merges becomes waxy solid.It is obtained 11.2g (40%) white solid for twice from the hexane crystallization.(by HPLC, 99%+ purity).

2-gamma-Linolenic acid 1,3-two caprins (CGC)

With gamma-Linolenic acid (GLA95,8.34g, 0.03mol) be dissolved in dichloromethane (DCM, 60ml).Gained solution stir under the room temperature nitrogen and in 5 minutes, dropwise add oxalyl chloride (3.9ml, 5.67g, 0.044mol).With mixture at room temperature stir spend the night and vacuum concentration remove DCM and excessive oxalul chloride.Then in 15 minutes with residual oily acyl chlorides (GLA-C1) 10-15 ℃ dropwise (ice/water cooling) add 1,3-two caprin (11.2g, 0.028mol), DCM (50ml), pyridine (2.42ml, 2.37g, 0.03mol) and 4-dimethylaminopyridine (0.10g, 0.0008mol, 0.03 equivalent) the solution of stirring in.Keep temperature by ice-water cooling, stirred reaction mixture spends the night under the room temperature nitrogen.By removing by filter pyridine hydrochloride and washing with DCM.Use 5%NaCl, 5%NaHCO then ₃, 0.1N HCl, 5%NaCl 1 * 20ml partly wash the cleaning mixture and the filtrate of merging.With solution MgSO ₄Dry also solvent removed in vacuo.By the residual brown oil of column chromatography purification on silica gel.Obtain 10.3g (56%) water white oil with 5% ether/hexane eluting then with hexane.By ¹³C NMR and GLC confirm structure.Measure purity by HPLC.

On a large scale

1,3-didecyl acyloxy third-2-ketone

Under the room temperature nitrogen with decanoyl chloride (272ml, 250g, 1.3mol, 2 equivalents) and in 10-15 minute, dropwise join 1,3-dihydroxy acetone dimer (59.1g, 0.65mol, 1.0 equivalent), pyridine (106ml, 103.7g 1.3mol), 4-dimethylaminopyridine (2.38g, 0.02mol, 0.03 equivalent) and dichloromethane (DCM is in the suspension of stirring 750ml).Keep the temperature of reactant mixture to be lower than 30 ℃ by cooling in psychrolusia.Stirred reaction mixture spends the night under the room temperature nitrogen.Pyridine hydrochloride by removing by filter formation also washs with DCM.Use 5%NaCl, 5%NaHCO then ₃, 0.1N HCl, 5%NaCl 1 * 150ml partly wash the filtrate and the cleaning mixture of merging.With solution MgSO ₄Dry and vacuum concentration obtains light yellow semisolid.Then it is obtained white solid from methanol (500ml) crystallization.Productive rate is 158g (60%).

1,3-two caprins

With top ketone (158g, 0.40mol) be dissolved in oxolane (THF, 2.25L) in.Add entry (50ml) then, cooling solution to 5 ℃, and below 10 ℃ by part adding a sodium borohydride (5.66g, 1.5 equivalents).By HPLC (C18 uses ACN with 1ml/min eluting λ 210nm) monitoring reactant mixture (note: in fact only add about 4.5g boron hydride) because all SM react.Stirring at room reactant mixture 1h, vacuum concentration is removed THF then.Residue distributes between ethyl acetate and 5% sodium chloride solution.With ethyl acetate aqueous phase extracted and use MgSO once more ₄Dry extract and the vacuum concentration that merges becomes waxy solid.It is obtained 96g (60%) white solid for twice from the hexane crystallization.(by HPLC, 98% purity).

2-gamma-Linolenic acid 1,3-two caprins (CGC)

With gamma-Linolenic acid (GLA95,120.2g, 0.43mol) be dissolved in dichloromethane (DCM, 750ml).Gained solution is in stirring under the room temperature nitrogen and dropwise added oxalyl chloride (55.7ml, 82.3g, 0.65mol, 1.5 equivalents) in 15 minutes.With mixture at room temperature stir spend the night and vacuum concentration remove DCM and excessive oxalul chloride.Then in 30-40 minute with residual oily acyl chlorides (GLA-C1) 10-15 ℃ dropwise (ice/water cooling) add 1,3-two caprin (164.7g, 0.41mol), DCM (650ml), pyridine (33.3ml, 32.5g, 0.41mol) and 4-dimethylaminopyridine (1.50g, 0.012mol, 0.03 equivalent) the solution of stirring in.Stirred reaction mixture spends the night under the room temperature nitrogen.By removing by filter pyridine hydrochloride and washing with DCM.With 5%NaCl, 5%NaHCO ₃, 0.1N HCl, 5%NaCl 1 * 150ml partly wash the cleaning mixture and the filtrate of merging.With solution MgSO ₄Dry and solvent removed in vacuo obtains brown oil (275g).

The scale of three kinds of top reactions is maximum-norms of the every kind of reaction of implementing.Borohydride reduction also produces a kind of by-product with variable productive rate except producing 1 outside 3-two caprins.It is isolating pure 1 that the existence of this by-product greatly influences institute, the productive rate of 3-two caprins; Can only remove described by-product by twice crystallization of crude product.Because by column chromatography purification end-product CGC, so be used for 1 of final step, 3-two caprins must be pure as far as possible.

Produce the thick CGC of about 440g from top reaction, it is a brown oil.On a series of silicagel columns, use hexane, then by this thick CGC product of 2-3% ether/hexane purification.Purification needs 7 or 8 pillars, uses 3-4 kilogram silica gel, and 25-30 rises solvent (reclaiming solvent makes this numeral reduce-in fact use more than 100 liters).

By HPLC (C18 4.6 * 100mm, with 85/15ACN/THF with the 1ml/min eluting, ultraviolet detection λ 210nm) detect products therefrom---a kind of purity of transparent almost colourless oil (264 gram) is 96.4%.GC shows the 66.1/33.9C/G ratio.NMR the analysis showed that this product has correct CGC structure and is at least 95% purity: δ _c(500MHz, CDCl ₃) 172.65 (2-GLA carbonyls), 173.25 (1,3-capric acid carbonyl).Signal ratio: 2.04: 1.Do not have signal at 173.0 places, show not have 1.3-GLA.Small-signal 172.79 may be the oleic acid impurity in GLA or the 2-capric acid.

Sum up

Prepared 264g1 by three one step process (scheme given below) from decanoyl chloride (98%), and 3-two capric acid-2-gamma-Linolenic acid glyceride (1,3-two caprins-2-GLA, CGC).It is almost colourless oil (yellow a little) and preserves in cold closet under nitrogen.HPLC purity is 96.4%.

Synthetic embodiment 3

1,3-two capric acid-2-dihomo-gamma-linolenic acid ester (1,3-two capric acid 2-20 carbon-(8Z, 11Z, 14Z)-trienic acid glyceride or C (DHLA) C)

That this triglyceride appears as is new-also there is not to find a list of references about it.

With DHLA (3.93g, 12.8mmol, 1 equivalent) be dissolved in dichloromethane (DCM, 20ml) in and under room temperature nitrogen, stir.Dropwise add oxalyl chloride (1.69ml, 2.46g, 19.4mmol, 1.5 equivalents) in 1-2 minute, and at room temperature stir and spend the night.Vacuum concentration gained solution is removed DCM and excessive oxalul chloride.Then remaining oily acyl chlorides (DHLA-C1) was dropwise added 1 in 5 minutes under 25 ℃, 3-two caprin (4.91g, 12.2mmol, 0.95 pyridine (0.98ml equivalent),, 0.96g 12.1mmol, 0.95 equivalent) and 4-dimethylaminopyridine (DMAP, 8mg, 0.07mmol, 0.03 equivalent) stirred mixture in.Reaction temperature is elevated to 32 ℃ during adding.Monitor 30-35 ℃ of reaction stirred and by HPLC.1.5 stopped reaction after hour.The sedimentary pyridine hydrochloride of filtering also washs with DCM.Use 1 * 10ml of 5% sodium chloride, 5% sodium bicarbonate, 0.1N hydrochloric acid and 5% sodium chloride partly to wash the filtrate and the cleaning mixture of merging then.Use MgSO then ₄Drying solution and vacuum concentration obtain crude product, and it is Huang-orange oil (8.9g, it is 86% that HPLC measures purity).Should go up chromatography at silica gel (250g) by oil.Obtain the product of purification with hexane and diethyl ether-hexane (2-6%) eluting, it is a light yellow oil.Obtain C (DHLA) C with decolorizing carbon processing hexane solution and solvent removed in vacuo, it is clear, colorless oil (6.48g, HPLC record purity 98.9%).

Synthetic embodiment 4

Triarachidin (three (20 carbon) four (5-Z, 8-Z, 11-Z, 14Z) olefin(e) acid glyceride) or AAA)

Arachidonic acid (50.9g, 0.17mol, 3 equivalents) is dissolved in dichloromethane, and (DCM 175ml) and under room temperature nitrogen stirs.In 5 minutes, add oxalyl chloride (21.9ml, 31.9g, 0.25mol, 4.4 equivalents) and temperature raises 4 ℃ to the solution that stirs then.Stir the gained yellow-green mixture under the room temperature and spend the night, vacuum concentration is removed DCM and excessive oxalul chloride then.Then to glycerol (5.11g, 0.055mol, 1 equivalent), pyridine (13.5ml, 13.2g, 0.17mol, 3 equivalents) and 4-dimethylaminopyridine (DMAP, 0.20g, 0.002mol, 0.03 equivalent) (25 ℃) of preheating stir the mixture and in 15 minutes, dropwise add residual oily acyl chlorides (A-C1).The temperature of reactant mixture is elevated to 42 ℃ during adding, and observes gentle reflux.Stir the mixture and monitor at 30-40 ℃ by HPLC.After 2 hours, do not observe further formation product.The sedimentary pyridine hydrochloride of filtering also washs with DCM.Use 1 * 50ml of 5% sodium chloride, 5% sodium bicarbonate, 0.1N hydrochloric acid and 5% sodium chloride partly to wash the filtrate and the cleaning mixture of merging then.Use MgSO then ₄Drying solution and vacuum concentration obtain crude product, and it is Huang-orange oil (57g).Should go up column chromatography at silica gel (about 600g) by oil.Obtain 22.8g oily product with hexane and diethyl ether (2-4%)-hexane eluting.Produce second crowd (17.8g) from the 39.8g arachidonic acid.These two batches are merged and vacuum is removed residual solvent and obtained the mobile light yellow oil of 40.5g (43%).HPLC purity 84.8%GLC analyzes 94.3% AA (arachidonic acid).

Compare lipid 2

1,3-two (18 carbon-6Z, 9Z, 12Z-alkene acyloxy) third-2-ketone

(1,3-two (γ-Caulis et Folium Lini acyloxy) third-2-ketone, Gong) intermediate of stage 1GCG

With gamma-Linolenic acid (GLA95,197g, 0.71mol, 2.2 equivalents) be dissolved in dichloromethane contained in the 2L 3 neck flasks (DCM, 600ml) in.Room temperature nitrogen stirs gained solution down.Dropwise add oxalyl chloride (93ml, 136g, 1.07mol, 3.3 equivalents) at 15-20 ℃ in 15 minutes.Stir under the room temperature brown mixture overnight then vacuum concentration remove DCM and excessive oxalul chloride.Then under the room temperature nitrogen to 1,3-dihydroxy acetone dimer (28.99g, 0.32mol, 1.0 pyridine (52ml equivalent),, 50.9g 0.64mol, 2.0 equivalents), 4-dimethylaminopyridine (2.36g, 0.02mol, 0.06 equivalent) and dichloromethane (DCM dropwise added residual oily acyl chlorides (GLA-C1) at 25 ℃ in the stirring the mixture 600ml) in 20 minutes.Allow the temperature of reactant mixture be elevated to 40 ℃, then restir mixture 2 hours (by the HPLC monitoring) under nitrogen.The pyridine hydrochloride that filtering forms also washs with DCM.Use 1 * 150ml of 5% sodium chloride, 5% sodium bicarbonate, 0.1N hydrochloric acid and 5% sodium chloride partly to wash the filtrate and the cleaning mixture of merging then.Use MgSO then ₄Drying solution and vacuum concentration obtain about 200g yellow oil.By this material of column chromatography partial purification on silica gel (600g).Use ether-hexanes mixtures (2-15%) eluting to obtain the 42g light yellow oil then with hexane.Should go up chromatography and obtain product (95.9% purity) with 1-10% ether-hexane eluting then with hexane at silica gel (600g) once more by oil, it is a light yellow oil.Productive rate is 42g (17%).

1,3-two (18 carbon-6Z, 9Z, 12Z-alkene acyloxy) propan-2-ol

(1,3-two (γ-Caulis et Folium Lini acyloxy) propan-2-ol or 1,3-two-γ-linolenin, GolG) intermediate of stages 2 GCG

With 1,3-two (γ-Caulis et Folium Lini acyloxy) third-2-ketone (GonG, 25.5g, 0.04mol, 1 equivalent) is dissolved in oxolane, and (THF is 375ml) and in the water (12.7ml).At-20 ℃ of vigorous stirring solution, note keeping reaction temperature to be lower than-15 ℃.In 3 minutes, add a sodium borohydride (790mg, 0.02mol, 1.25 equivalents) to solution by part through stirring.-20 ℃ of stirred reaction mixtures 10 minutes, add hexane (380ml) then again.(2 * 200ml) wash MgSO to mixture water that will be still cold then ₄Drying, and vacuum concentration obtains the title mixture, it is brown oil (27.8g) (HPLC purity 82.6% is less than 1% material that shifts).Prepare another batch and obtain the 50g crude product with first merging.Go up this material of purification by column chromatography at silica gel (400g).Obtain the 36.1g product with hexane and diethyl ether-hexanes mixtures (5-20%) eluting, it is canescence oil (91.5% purity).

(as if prompting should note not allowing chemical compound spend the night on silica gel, because it experiences the transferance reaction, obtains GGol)

1,3-two-γ-linolenin 2-decanoin (1, two 18 carbon of 3--(6Z, 9Z, 12Z)-trienic acid 2-caprin or GCG)

With decanoyl chloride (13.5ml, 12.4g, 0.065mol, 1.1 equivalent) in about 10 minutes, add 1,3-two-γ-linolenin (36.1g, 0.059mol, 1 equivalent), anhydrous pyridine (5.7ml, 5.6g, 0.07mol, 1.1 equivalents), 4-dimethylaminopyridine (0.2g, 0.002mol, 0.03 equivalent) and dichloromethane (DCM is in the solution of stirring 150ml).In adition process, maintain the temperature at 17 ℃-23 ℃.Monitor 30-35 ℃ of reaction stirred and by HPLC then.Add other 1-2ml decanoyl chloride behind 1h, 1.5h and the 2h.As if as measuring by HPLC, adding in addition increases the conversion to product.Behind the 3h, filter reaction mixture is also used the DCM wash filtrate.Use 1 * 50ml of 5% sodium chloride, 5% sodium bicarbonate, 0.1N hydrochloric acid and 5% sodium chloride partly to wash the filtrate and the cleaning mixture of merging then.Use MgSO then ₄Dry DCM extract and vacuum concentration obtain crude product, and it is light yellow oil (a HPLC purity 90%).Should oil by going up column chromatography purification at silica gel (600g).Obtain product (GCG) with hexane and diethyl ether-hexane (1.5-2.5 then 3.5%) eluting, it is clean oil (35.5g, a HPLC purity 96.1%).By the further chromatography of some fraction of small amount of impurities obtains the pure lipid of other 7.5g to only containing.

Synthetic embodiment 5

1, and 3-two capric acid 2-arachidonic acid glycerides (1,3-two capric acid 2-20 carbon four-(5-Z, 8-Z, 11-Z, 14-Z) olefin(e) acid glyceride or CAC)

This triglyceride is known.Safflower oil is applied to the composition that CAC has been accredited as lymph lipid behind the rat.WO03 013,497 has described and has contained arachidonic triglyceride (producing by cultivating Mortierella alpina), and it can be used for the disease that brain hypofunction causes, and is particularly useful for cognitive the enhancing.It is known being used for the synthetic two kinds of intermediate of CAC.

From 1, the purification of synthetic CAC of 3-two caprins and CAC all is new.

Here pass through 1,3-two caprins prepare CAC with the arachidonic acyl chloride reaction that is dissolved in dichloromethane-pyridine.By sodium borohydride reduction 1,3-didecyl acyloxy third-2-ketone preparation 1,3-two caprins, 1,3-didecyl acyloxy third-2-ketone is again by decanoyl chloride and 1,3-dihydroxy acetone prepared in reaction.Necessary handled intermediate 1,3-two caprins can experience acyl migration because it is exposed to acid, alkali and Re Shi.Synthetic 1 by capric acid to the catalysis addition of ethylene oxidic ester (from chloropropylene oxide), 3-two caprins than out-of-date methods 6 because stricter reaction condition and acyl group branch problem and captivation is lower.By column chromatography purification end-product CAC careful on silica gel, described chromatography is removed by-product.

2-arachidonic acid 1,3-two caprins (CAC)

With arachidonic acid (AA96,8.34g, 0.03mol) be dissolved in dichloromethane (DCM, 60ml) in.Room temperature nitrogen stir down gained solution and in 5 minutes, dropwise add oxalyl chloride (3.9ml, 5.67g, 0.044mol).The vacuum concentration that spends the night then of stirring the mixture under the room temperature is removed DCM and excessive oxalul chloride.(ice/water cooling) dropwise adds 1 with residual oily acyl chlorides (GLA-C1) down at 10-15 ℃ in 15 minutes then, 3-two caprin (11.2g, 0.028mol), DCM (50ml), pyridine (2.42ml, 2.37g, 0.03mol) and 4-dimethylaminopyridine (0.10g, 0.0008mol, 0.03 equivalent) the solution of stirring in.Keep temperature by ice-water cooling.Stirred reaction mixture spends the night under the room temperature nitrogen.By removing by filter pyridine hydrochloride and washing with DCM.Use 1 * 20ml of 5% sodium chloride, 5% sodium bicarbonate, 0.1N hydrochloric acid and 5% sodium chloride partly to wash the cleaning mixture and the filtrate of merging then.Use MgSO then ₄Drying solution and vacuum concentration remove and desolvate.By the residual brown oil of the column chromatography purification on the silica gel.Obtain 10.3g (56%) water white oil with 5% ether/hexane eluting then with hexane.By ¹³C NMR and GLC confirm structure.Measure purity by HPLC.

On a large scale

1,3-didecyl acyloxy third-2-ketone

1,3-two caprins

2-arachidonic acid 1,3-two caprins (CAC)

With arachidonic acid (AA96,78.8g, 0.26mol) be dissolved in dichloromethane (DCM, 425ml).Gained solution is in stirring under the room temperature nitrogen and dropwise added oxalyl chloride (33.9ml, 49.4g, 0.39mol, 1.5 equivalents) in 15 minutes under 15-20 ℃.With mixture at room temperature stir spend the night and vacuum concentration remove DCM and excessive oxalul chloride.Then in 30-40 minute with residual oily acyl chlorides (GLA-C1) 10-15 ℃ dropwise (ice/water cooling) add 1,3-two caprin (94.2g, 0.24mol), DCM (450ml), pyridine (19.1ml, 18.6g, 0.24mol) and 4-dimethylaminopyridine (1.721.50g, 0.014mol, 0.06 equivalent) the solution of stirring in.Stirred reaction mixture spends the night under the room temperature nitrogen.By removing by filter pyridine hydrochloride and washing with DCM.Use 5%NaCl, 5%NaHCO then ₃, 0.1N HCl, 5%NaCl 1 * 150ml partly wash the cleaning mixture and the filtrate of merging.With solution MgSO ₄Dry and solvent removed in vacuo obtains brown oil (171g).

The scale of three kinds of top reactions is maximum-norms of the every kind of reaction of implementing.Borohydride reduction also produces a kind of by-product with variable productive rate except producing 1 outside 3-two caprins.It is isolating pure 1 that the existence of this by-product greatly influences institute, the productive rate of 3-two caprins; Can only remove described by-product by twice crystallization of crude product.Because by column chromatography purification end-product CAC, so be used for 1 of final step, 3-two caprins must be pure as far as possible.

Produce the thick CAC of about 412g from top reaction, it is a brown oil.On a series of silicagel columns, use hexane 1-3% ether/this material of hexane purification then.Purification needs 7 or 8 pillars, uses 3-4 kilogram silica gel and 100 liters of solvents.

By HPLC (C184.6 * 100mm, with 85/15ACN/THF with the 1ml/min eluting, ultraviolet detection λ 210nm) detect products therefrom---a kind of purity of transparent very light yellow oil (295 gram) is 95.8%.GC shows that the C/A of 66.3/32.1 is than (bringing 1.6% impurity into by 5% impurity among the A).

Sum up

Prepared 295g 1 from decanoyl chloride (98%) and arachidonic acid (95%) by three-step approach (scheme given below), and 3-two capric acid-2-arachidonic acid glyceride (1,3-two caprins-2-AA, CAC).It is very flaxen oil and preserves under the nitrogen in cold closet.HPLC purity is 95.8%.

Synthetic embodiment 7

2-gamma-Linolenic acid 1, and the 3-glyceryl dioleate (1,3-two (18 carbon)-9Z-olefin(e) acid 2-18 carbon three (6-Z, 9-Z, 12-Z) olefin(e) acid glyceride or OGO)

This triglyceride is known: prepared the form of carbon-14 labelling and prepared normal unlabelled form by the synthetic lipase that uses of biochemistry by conventional chemical is synthetic.OGO is not but that the isomer OOG of borage main body of oil it is main component (9%).It is known being used for the synthetic two kinds of intermediate of CGC.Final step is new.

Think CGC purposes, it is from 1, the synthetic and purification of 3-glyceryl dioleate all is new.Usually, triglyceride CXC is more preferred than OXO on patent and product cost.

Here pass through 1, the GLA-acyl chloride reaction in 3-glyceryl dioleate and the dichloromethane-pyridine prepares OGO.By 1, the sodium borohydride reduction of 3-dioleoyl third-2-ketone preparation 1, the 3-glyceryl dioleate, 1,3-dioleoyl third-2-ketone is by oleoyl chloride and 1,3-dihydroxy acetone prepared in reaction.Necessary handled intermediate 1, the 3-glyceryl dioleate can experience acyl migration because it is exposed to acid, alkali and Re Shi.By list-trityl glycerol or ethylene oxidic ester preparation 1, the 3-glyceryl dioleate than out-of-

date methods

7,8 because the rapid and acyl group branch problem of multistep and captivation is lower more.By column chromatography purification end-product OGO careful on silica gel, described chromatography is removed by-product.

On a small scale

1,3-dioleoyl third-2-ketone

With 155.1g oleic acid (155.1g, 0.55mol, 1.0 equivalents, Croda 094RV05192) be dissolved in dichloromethane (DCM, 500ml).Agitating solution and dropwise add 104.4g (1.5 equivalent) 1ml oxalyl chloride (104.4g, 71.8ml, 0.82mol, 1.5 equivalents) in about 20 minutes under room temperature (RT) nitrogen at 15-20 ℃.Stirred reaction mixture spends the night under the room temperature.Vacuum is removed excessive oxalul chloride and DCM and in 15-20 minute residual oily acyl chlorides is dropwise added 1 under room temperature nitrogen, 3-dihydroxy acetone dimer (22.5g, 0.24mol monomer), (DCM is in the suspension of stirring 500ml) for pyridine (40.4ml), 4-dimethylaminopyridine (1.83g) and dichloromethane.Keep the temperature of reactant mixture to be lower than 20 ℃ by cooling in ice/water-bath.Stirred reaction mixture spends the night under the room temperature nitrogen.By removing by filter pyridine hydrochloride and washing with DCM.Use 1 * 150ml of 5% sodium chloride, 5% sodium bicarbonate, 0.1N hydrochloric acid and 5% sodium chloride partly to wash the filtrate and the cleaning mixture of merging then.Use MgSO then ₄Drying solution and vacuum concentration become orange/brown semisolid.It is ground and leaves in refrigerator overnight in methanol.Obtain 51.3g rice white solid from Di Iso Propyl Ether (DIPE) and methanol crystallization precipitated solid (HPLC purity 90%) then, its HPLC purity is 95%.Obtain the product of 41g (27%) 98% purity from the further crystallization of DIPE/ methanol.

1, the 3-glyceryl dioleate

With top ketone (32.8g, 0.053mol) be dissolved in oxolane (THF, 250ml) in.Add entry (10ml) then, cooling solution to 5 ℃, and below 10 ℃ by part adding a sodium borohydride.Follow the tracks of reaction with HPLC (C18, ACN/THF 90/10,2ml/min flow velocity, λ 210nm), after all starting ketones all react, stop to add boron hydride and (add 830mg, 0.022mol).Vacuum concentrated mixture is removed THF then.Residue distributes between ethyl acetate and water.With ethyl acetate aqueous phase extracted and use MgSO once more ₄Dry extract that merges and vacuum concentration one-tenth oil (～33g), solidify during this oil cooling.Under-20 ℃ from 100ml hexane crystallized product spend the night (HPLC purity 68%).(92% purity 21.1g) obtains 18.28g (56% productive rate) product, and its HPLC purity is 97.5% from this product of hexane (50ml) recrystallization.

2-acid and gamma-linolenic 1,3-glyceryl dioleate (O-G-O)

With gamma-Linolenic acid (GLA 95,41.2g, 0.15mol, 1.1 equivalents) be dissolved in dichloromethane (DCM, 250ml).Gained solution is in stirring under the room temperature nitrogen and dropwise added oxalyl chloride (19.1ml, 28.2g, 0.22mol, 1.65 equivalents) in 5 minutes.With mixture at room temperature stir spend the night and vacuum concentration remove DCM and excessive oxalul chloride.Then in 15 minutes with residual oily acyl chlorides (GLA-C1) 10-15 ℃ dropwise (ice/water cooling) add 1,3-glyceryl dioleate (83.5g, 0.13mol), DCM (250ml), pyridine (10.9ml, 10.6g, 0.14mol) and 4-dimethylaminopyridine (0.49g, 0.004mol, 0.15 equivalent) the solution of stirring in.Keep temperature by ice-water cooling.Stirred reaction mixture spends the night under the room temperature nitrogen.By removing by filter pyridine hydrochloride and washing with DCM.Use 5%NaCl, 5%NaHCO then ₃, 0.1N HCl, 5%NaCl 1 * 80ml partly wash the cleaning mixture and the filtrate of merging.With solution MgSO ₄Dry also solvent removed in vacuo.On silica gel, pass through the residual brown oil of column chromatography purification.Obtain 63.6g (54%) water white oil with 5% ether/hexane eluting then with hexane.Measure purity by HPLC.

Sum up

Prepared 64g 1 by three-step approach (scheme given below) from oleoyl chloride (98%), and 3-oleic acid-2-gamma-Linolenic acid glyceride (1,3-two oleic acid-2-GLA, OGO).It almost is water white oil (band yellow) slightly and it is preserved under nitrogen in cold closet.HPLC purity is 89.4%.

Struetural lipid ¹³C NMR data

GGG δ _c(125.7MHz, CDCl ₃) 172.69 (1C, C-2 carbonyls), 173.09 (2C, C-1, C-3 carbonyls)

CGC δ _c(125.7MHz, CDCl ₃) 172.76 (1C, C-2 carbonyls), 173.17 (2C, C-1, C-3 carbonyls)

CAC δ _c(125.7MHz, CDCl ₃) 172.65 (1C, C-2 carbonyls), 173.28 (2C, C-1, C-3 carbonyls)

C (DHLA) C δ _c(125.7MHz, CDCl ₃) 172.83 (1C, C-2 carbonyls), 173.30 (2C, C-1, C-3 carbonyls)

GCG δ _c(125.7MHz, CDCl ₃) 172.91 (1C, C-2 carbonyls), 173.11 (2C, C-1, C-3 carbonyls)

OGO δ _c(125.7MHz, CDCl ₃) 172.69 (1C, C-2 carbonyls), 173.25 (2C, C-1, C-3 carbonyls)

AAA δ _c(125.7MHz, CDCl ₃) 172.66 (1C, C-2 carbonyls), 173.04 (2C, C-1, C-3 carbonyls)

CCC δ _c(125.7MHz, CDCl ₃) 172.81 (1C, C-2 carbonyls), 173.21 (2C, C-1, C-3 carbonyls)

Experimental procedure

Has the proton of inhibition NOE-decoupled with 5-mm broadband probe 21 ℃ of collections on the Joel 500MHz spectrometer of 125.728MHz operation ¹³C NMR spectrum.It is the selected mode and only carry out gate during the 14.89s acquisition time of uncoupling that Waltz uncouples.Relaxation delay was set to 30 seconds and the pulse angle is 90 °.Used spectrum window is about 35ppm (from 173.5 to 172.6ppm), has the 170ppm side-play amount.Spectrum is inherent with reference to the CDCl at 77.0ppm ₃Usually, depend on the concentration and the purity of sample, for the roughly number of times that reaches the scanning that enough signal to noise ratio collects is 300-1200 time.Total acquisition time of experiment is 2-8h, for example, and 1272 scanning; 65,536 of data points.In the time that acquisition time may be reduced, use concentrated solution up to 20%w/v.The chemical shift of quoting becomes along with solution concentration.

Biological study

Chronic recurrent experimental autoimmune encephalomyelitis (CREAE) research

EAE induces and clinical assessment

In C57B1/6 and SJL mice, induce CREAE.(Morris-Downes as previously mentioned, MM., Deng people 2002) 100 μ g neural antigen peptide MOG 35-55 (aminoacid sequence MEVGWYRSPFSRVVHLYRNGK Genemed Synthesis in radiophosphorus acid buffer saline (PBS) is made a bet in the 0th day and the 7th day to animal skins, Inc) or 1mg mouse spinal cord homogenate (SCH) subcutaneous injection, wherein adding 480 μ g mycobacterium tuberculosis (mycobacteria tuberculosis) and 60 μ g Mycobacteriabutyricium (DIFCO, Detroit, USA) incomplete Freund's adjuvant (DIFCO, Detroit, USA) in by the emulsifying in 10 minutes of supersound process under the room temperature.Except optimizing disease, mice also is dissolved in bordetella pertussis (Bordetella pertussis) toxin 200ng (intraperitoneal) among the PBS after with the MOG antigen immune and in the 0th, 1,7 and 8 day 1h of SCH and 24h acceptance.

Since the 5th day animal weighed and check the clinical neurology symptom every day and according to the hierarchy plan classification of confirming in the past (Morris-Downes, people such as MM. 2002 and other document) by two experienced research worker: 0=is normal; 1=limping tail and foot; The righting reflex that 2=is impaired; 3=part back acroparalysis; 4=is the back acroparalysis fully; 5=is dying; 6=death.It is little by 0.5 that the animal that shows the symptom lighter than the clinical symptom of common observed grade gets the pointed grade of proportion by subtraction.

List of references

Morris-Downes, MM., wait people (2002) .Pathologicl andregulatory effects of anti-myelin antibodies in experimentalallergic encephalomyelitis in mice.J.Neuroimmunol.125.114-124.

By the nonparametric statistics analysis is each test group and the average group EAE score (Mann Whitney U check) relatively of matched group separately.

All MOG-CREAE research all comprises treatment matched group (C-C-C or be selected from the saline of studying above).At every kind of struetural lipid of three dosage level tests, all treatments are the 7th day 2 weeks of dosage forms for oral administration after inoculation all.All treatment groups all will contain 10 animals.When (the 21st day) finished in research, remove decerebrate and spinal cord and handle the symptom of half sample with mononuclear cell leukocyte-infiltration site and demyelination around the research CNS blood vessel.

Study as follows:

Research 2: EAE in spinal cord homogenate thing (SCH) the SJL mice.

EAE induces: the 0th day+the 7th day subcutaneous 1mg SCH.0th, 1,7 and 8 day intraperitoneal 200ng pertussis toxin, PT.10 mice/groups.Handled mice with CCC or CGC on the from the 7th to 21 day.

SCH EAE in the research 3:SJL mice: treat and be PSD 7 to 21, comprise these two days.

MOG EAE in the research 4:C57BL mice: treat and be PSD7 to 21, comprise these two days.

SCH EAE in the research 5:SJL mice: treat and be PSD5 to 18, comprise these two days.

MOG EAE in the research 6:C57BL mice: treatment is the 5th to 21 day (comprising these two days), and just treatment is the 5th to 15 day (comprising these two days) in the C-DHLA-C group.Eliminate animal at PSD25.[,, being used for histologic analysis in PSD 20 samplings] from 5 animals of GGG 150 μ l treatment groups with from 2 animals of GGG 350 μ l treatment groups from 3 animals of contrast CCC treatment group from 5 animals of not treatment group.

SCH EAE in the research 7:SJL mice

Be treated to the 6th to 20 day (comprising these two days).

Spinal cord homogenate (SCH) in the research 2:-SJL mice :-tried

CGC(50/150/350μl)；CCC(350μl)

GGG.(50/350μl)

[observing serious disease]

Research 3-SCH/SJL mice :-tried

CCC(50/150/350μl)

CGC(25/50/150/350μl)

GGG(50/150/350μl)

OGO(25/50/150/350μl)

[observing serious disease]

Research 4-MOG/C57BL mice :-tried

CCC(50/150/350μl)

CGC(25/50/150/350μl)

GGG(50/150/350μl)

OGO(25/50/150/350μl)

Research 6-MOG/C57BL mice :-tried

CCC(150μl)

C-DHLA-C(50μl)

CAC(50/350μl)

AAA(50/150μl)

GCG(50μl)

CGC(50μl)

GGG.(150/350μl)

[pathology: CCC; GGG]

The histological examination of brain of being submitted to and spinal cord sample shows typical case's infringement of experimental allergic encephalomyelitis.

Feature local and the disperse infringement is neurogliosis, the formation of myelin cavity, axonal degeneration and the perivascular cuffing with lymphocyte, macrophage and neutrophil cell.

Spinal cord lesion is usually located under the mantle in the white matter, and cerebral lesion appears in the alba usually.More serious in the damage ratio brain in spinal cord, have encephaloclastic all animals and all in spinal cord, have infringement, but not all animal with spinal cord lesion all has the infringement in the brain.

Use 5 hierarchy systems of sxemiquantitative to summarize the variation of seriousness between the individual mice.

Not subject mice has histology's score of 3-4, and its EAE score with 1.5-3 is relevant.A mice does not show Histological change substantially, has 0 score.In the mice of GGG treatment, great majority do not show unusually.Two mices from this group have histology's score of 2 and 3 respectively, and its EAE seriousness score with 1 and 1.5 is relevant.

Show among result Figure 11 to 20 below of four researchs.

These results show that chemical compound G-G-G, A-A-A, C-G-C, C-DHGLA-C and C-A-C can both reduce the seriousness of CREAE, and chemical compound G-C-G and C-C-C can not treat this disease.If adjusting dosage thinks that chemical compound O-G-O is also effective.

As pointing out in description, the arachidonic acid compound is effectively, but causes some animal dead.The animal that survives has the disease that reduces greatly.The dosage that can further reduce these chemical compounds is to provide the survival with satisfied treatment.

Some researchs demonstrate bell response curve for Compound C-G-C and G-G-G, show that very high dosage is not optimum, as top given.Can be by the technical staff, for example, rise in proportion and the TGF-β 1/TNF-α release ratio automatically from PBMC of monitoring changes and determines this type of dosage easily by dosage.

Consider the result of the high sn-2 gamma-Linolenic acid of PCT/GB04/002089, low sn-2black-current oil and G-C-G lack the low dosage effect of C-G-C and C-DHGLA-C among effect and Figure 20 in CREAE, can see that sn-2-gamma-Linolenic acid, dihomo-gamma-linolenic acid and arachidonic acid lipid provide new MS treatment, it is considerably beyond any current therapeutic outcome, because infringement obtains repairing and difficult symptom is resolved: also do not solve so far in other treatment and reduce EDSS in the several years.

List of references

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Many kinds of oil compositions of table 1 (the total FAs of %) feature and their protection effects in EAE

Treatment	18：2n-6	18：3n-6	18：2n-6/18：3n-6	18：1n-9	The sickness rate of EAE
						FGO?	17?	20?	0.6?	35?	0/10?
BOO?	37?	24?	1.5?	15?	3/10?
						EPO?	71?	9.4?	7.5?	9?	7/10?
SAF?	66?	-?	-?	17?	9/10?
						Contrast	-?	-?	-?	-?	9/10?

FGO, fungal oil; BOO, borage oil; EPO, Radix Oenotherae erythrosepalae oil; SAF, safflower oil.

Table 2

Treatment group-PCT/GB04/002089 borage oil-MS test

?

The women

The male

Average relapse rate (in the past in 2 years)

Average basic EDSS

Number

Group

Placebo

7?

4?

2.6?

3.9?

11?

?

Low dosage

5?

2?

2.9?

3.5?

7?

?

High dose

8?

2?

3.4?

2.8?

10?

Total

?

20?

8?

2.9?

3.4?

28?

Table 3

The molecular species of triacylglycerol ester-GLA (TG-GLA), ethyl ester-GLA (EE-GLA) and PCT/GB04/002089 Borago Officinalis oil-GLA (BOR-GLA) is relatively among the inductive CREAE of MOG in the SJL mice

Treatment	Number with EAE	Average clinical score
			Contrast
	10/11?	3.3±1.3?
			EE-GLA ^a	5/6?	3.0±0.8?
TG-GLA ^a	3/6?	1.0±1.3 ^c
			BOR-GLA ^b	3/6?	1.0±1.2 ^c

^aAnimal is given 100 μ l tried lipid; ^bGive 250 μ l BOR-GLA.With compare significant difference, ^cP＜0.05

The black-currant seed oil (73%GLA) of table 4 enrichment is to the influence of EAE sickness rate

Note: Blackcurrant oil delayed onset does not still provide protection fully.Back 7 days feed animals of sensitization (immunity).

Claims

1. the lipid glyceride that structure is clear and definite is used to prepare the purposes of demyelination neurodegenerative disease medicine, this glyceride is by partly forming with the glycerol of one or more fatty acid part esterifications, feature is that described lipid has the fatty acid part that is selected from gamma-Linolenic acid and dihomo-gamma-linolenic acid in the sn-2 position

Feature is that described lipid is monoglyceride or the triglyceride of formula II

R wherein ¹And R ³Identical and be selected from H and-C (O) (CH ₂) _nCH ₃, wherein n is selected from 4-16,

R ²Be selected from γ-Caulis et Folium Lini acyl group and two height-γ-Caulis et Folium Lini acyl group.

2. each purposes in the aforementioned claim, feature are that described medicine is used for lipid glyceride administration every day with 3-20g.

3. the purposes of claim 1, feature is that described medicine is used for lipid glyceride administration every day with 2-5g.

4. each purposes in the aforementioned claim, feature are that described neurodegenerative disease is a multiple sclerosis.

5. the purposes of claim 1, feature is that described treatment is used for the neuronal damage that neural degeneration sequela relevant with head trauma, apoplexy and intracranial hemorrhage or Alzheimer or parkinson disease cause.

6. the purposes of claim 1, wherein carry out described treatment with certain persistent period and dosage administration every day, described persistent period and dosage enough produce at least 0.75 TGF-β 1/TNF-α ratio after using 18 months every day, this TGF-β 1/TNF-α is than from by the spontaneous release of the isolating peripheral blood lymphocytes of blood samples of patients.

7. the purposes of claim 1, wherein carry out described treatment with certain persistent period and dosage administration every day, described persistent period and dosage enough produce at least 0.75 TGF-β 1/IL-1 β ratio after using 18 months every day, this TGF-β 1/IL-1 β is than from by the spontaneous release of the isolating peripheral mononuclear cells of blood samples of patients.

8. the lipid glyceride of the formula II that a structure is clear and definite

R wherein ¹And R ³For-C (O) (CH ₂) ₈CH ₃, R ²Be selected from γ-Caulis et Folium Lini acyl group and two height-γ-Caulis et Folium Lini acyl group.

9. the lipid glyceride that the structure of claim 8 is clear and definite, feature is R ²Be γ-Caulis et Folium Lini acyl residue.

10. the lipid glyceride of the formula II that a structure is clear and definite

R wherein ¹And R ³For-C (O) (CH ₂) ₈CH ₃, R ²Be selected from γ-Caulis et Folium Lini acyl group and two height-γ-Caulis et Folium Lini acyl group, described lipid glyceride is used for the treatment of.

11. the lipid glyceride that the structure that is used for the treatment of of claim 10 is clear and definite, feature is R ²Be γ-Caulis et Folium Lini acyl residue.