CA1302266C - Formulations containing unsaturated fatty acids for the synthesis of prostaglandins and hydroxy-fatty acids in biological systems - Google Patents
Formulations containing unsaturated fatty acids for the synthesis of prostaglandins and hydroxy-fatty acids in biological systemsInfo
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- CA1302266C CA1302266C CA000536688A CA536688A CA1302266C CA 1302266 C CA1302266 C CA 1302266C CA 000536688 A CA000536688 A CA 000536688A CA 536688 A CA536688 A CA 536688A CA 1302266 C CA1302266 C CA 1302266C
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
Abstract of the disclosure:
The present invention relates to formulations containing unsaturated fatty acids for the synthesis of prostaglan-dins and hydroxy-fatty acids in biological systems and to the use of such formulations for the preparation of medica-ments which are suitable for curing prostaglandin defic-iencies in humans or animal, for example healing or pre-venting diseases of the gastro intestinal tract.
The present invention relates to formulations containing unsaturated fatty acids for the synthesis of prostaglan-dins and hydroxy-fatty acids in biological systems and to the use of such formulations for the preparation of medica-ments which are suitable for curing prostaglandin defic-iencies in humans or animal, for example healing or pre-venting diseases of the gastro intestinal tract.
Description
~3~ 66 HOECHST AKTIENGESELLSCHAFT HOE 86/F 107 Dr.LA/je Formulat;ons containing unsaturated fatty acids for the synthesis of prostaglandins and hydroxy-fatty acids in 5 biolog;cal systems __ Long-chain polyunsaturated fatty acids or their esters and other derivatives are of importance as a starting material for the enzyme-catalyzed preparation of prostaglandins and hydroxy-fatty acids. Thus, for example by means of cells or of celL homogenates or cell fractions, the products, which can otherwise be prepared only by involved chemical processes, can be obtained preparatively by a b;ochemicaL
method especially from dihomo-gamma-linolen;c ac;d, arach-;don;c ac;d or e;cosapentaeno;c ac;d as the substrates.In such preparatiYe problems, the aim will be a stimulation of the formation of the desired prostaglandin. This can be achieved by addition of suitable enzyme co-factors.
However, the said fatty ac;ds can also be used for medical purposes, for example in cl;nical invest;gations of the ability of body cells, or cell homogenates or cell frac-t;ons (for example kerat;nocytes; blood vessel cells ob-tained by biopsy; cells or homogenates of the medulla of k;dney3, to metabolize the part;cular fatty acid or to convert it to prostagland;ns having a certa;n b;olog;cal act;on. In such med;cal problems, it is not imPortant whether the prostagland;n formed ;s homogeneous, but there ~;ll be an a;m of st;mulat;ng the format;on of the pros-~aglandin serv;ng as the med;cal parameter to be measured.
The present ;nvention describes novel, stable formulations which contain long-chain polyunsaturated fatty acids, by means of which preparations prostaglandins and hydroxy-fatty acids which can be used for the said purposes can advantageously be prepared.
It is already known that the conversion of polyunsaturated fatty ac;ds, such as arach;don;c ac;d, can be stimulated by prostaglandin-synthesizing enzymes w;th the aid of ~ ~3~2Z6~;
certain co-factors and, thus, the synthesis of very par-ticular prostaglandins can also be promoted. For example, in biochemical pharmacology 31 (1982), 3S91, an ;n v;tro system ;s described wh;ch converts arachidon;c ac;d by means of the enzyme obtained from seminal vesicles of sheep, in each case w;~h or without acldition of the co-factors glutathione and hydroquinone. In loc. c;t.~ page 3595, it is explained that the conversion is accelerated by the said co-factors. A similar system of arach;donic acid, homogenate of rabbit k;dney medulla, glutathione and hydroquinone is described in Life Sciences 26 (1980) 765.
For the purposes mentioned above, aqueous reagent formul-ations have now been prepared, ~hich conta;n the polyun-saturated fatty ac; d s prefe~ly in combination wqth stimulat~
enzyme co-factors. Such formulations are not only novel but also show unexpected properties. As explained in detail belo~, these stimulators without exception modify the stability of the fatty acids in a surprising manner.
Thus, within a short time, a markedly greater quantity of undesired decomposition and convers;on products of arach-idonic acid can form in arach;donic acid formulations con-taining glutath;one or hydroquinone, than is the case in formulations which do not contain these addit;ons. These byproducts are highly reactive and can trigger undes;red, even biolog;cal s;de reactions. Our own investigat;ons descr;bed in more detail below, however, sho~ that the prostagland;n synthesis can also be stimulated by mixtures which contain polyunsaturated fat~y acids, such as arachi-donic acid, together with other sulfur-containing compounds such as the disulfide lipoic acid or the reduced thiols homocysteine and cysteine or their derivat;ves acylated on the n;trogen atom, much smaller quant;ties of undes;red byproducts being formed accord;ng to the invention. This is surprising, since it was not possible to achieve these advantageous effects with other sulfùr compounds occurring in nature or derived from natural compounds, such as co-enzyme A or its scission or hydrolysis products cysteamine - ~.3~2266 and pantethe;ne, or dihydrolipoic acid, cystine and mono-or poly-thiol sugar compounds such as dithiothreitol or dithioerythritol. In the experimental part, it is stated that the undesired side reactions can be fully suppressed by an addition of lipoic acid, but can be largely, but not completely suppressed by an addit;on of the other sulfur-containing compounds accord;ng to the invent;on.
In fact, i~ is already known that the conversion of arac-h;don;c ac;d or the synthesis of certain prostaglandins can also be stimulated by an addition of substances with-out a sulfur content, for example, as cited above, by means of hydroquinone and also phenol, adrenalin, norad-renalin, tryptophan, 5-hydroxytryptophan and ascorbic acid. Surprisingly~ it has now been found that the un-desired decomposition of polyunsaturated fatty acids, for example of arachidonic acid, in an aqueous medium is con-siderably accelerated by these stimulators. The use of, for example, 2-butanol or pyridine instead of water has an additional destabilizing effect, whereas the undesired byproducts surprisingly do not arise when di~ethyl sul-foxide or ethanol or polyols such as glycerol or poly-ethylene glycol are used. Organic solvents have, however, the disadvantage that they denature enzy~es; they must therefore be removed before the enzyme reaction or diluted ~ith water. The residues ~hich remain after the removal of the solvent and which consist of an intimate mixture of unsaturated fatty acid and stimulator are, surPr;singly, aga;n unstable. It has not yet been described that sur-pr;s;ngly advantageous aqueous formuLations for the stim-ulation of the prostaglandin synthesis can be obtained with tyrosine. As explained in the experi~ental part, formulations which contain unsatura~ed fatty acids and tyrosine or tyrosine-l;ke compounds are, surprisingly, completely stable.
However, even aqueous mixtures or solutions of unsaturatecl fatty ac;d and the other abovementioned stimulators can be fully stab;lized, namely by an addition of lipids which 226~
are soluble or emuls;fiable in water, or of proteins which can be prepared, for example, from human or mammal blood, such as serum albumin or globulins, such as gamma-globulin, or porphyr;n-containing proteins such as hemo-glob;n, eethemoglobin or cytochromes. Vitam;ns of the Eseries and flavonoids, in particular those with a sugar moiety, for example rutin or troxerutin (for the nomen-clature, see "The Merck Index", 10th edition, 1983, Rahway, N.J., USA) are also suitable according to the invention, whereas sugars withou~ a flavone moiety, such as rhamnoglucose, glucose and sorbitol, are not stabiliz-ing. Surprisingly, however, the formulations according to the invention can be prepared with the aid of cyclodextrins.
With respect to stabilization with serum albumins, it is indeed known per se that albumins can bond long-chain fatty acids at an energetically low level, but it is the more surprising that the fatty acids bonded in this way behave like freely dissolved fatty acids in the reagent formulations according to the invention, that is to say they are enzymatically converted without hindrance. It has also already been described tcf., for example, Journal of Biological Chemistry 253 ~1978) 5061) that the enzymes o~ the prostagland;n synthesis conta;n heme or other por-phyrins bonded as prosthet;c groups. However, th;s req-u;res only extremely small quantit;es of porphyr;n-con-ta;n;ng enzymes, which do not have any stabilizing effect on the added quantities of unsaturated fatty acid. There is no ind7cation to be found in the literature to the effect that a stabil;zing reagent for st;~ulating the enzyme-catalyzed prostaglandin synthesis can be prepared w;th a higher proportion of porphyrin-containing proteins.
Stable formulat;ons wh;ch contain fatty ac;ds hav;ng 20 to 22 carbon atoms and 3 or more double bonds in each case and, as stabil;zers, a proportion of up to 30 percent by weight of phospholip;ds, for example lec;thin, have been d;scLosed ;n DE 34,197,996 A1. Such stable formul-at;ons can l;kewise be used according to the invent;on in ~3022~6 combination with the stimulators already mentioneda As explained ;n the experimental part, however, it is more appropriate to add more than 30 percent by weight of phospholipid, for example lecithin, to the formulations of the unsaturated fatty acids having 18 to 22 carbon atoms, because more stable mixtures are then obta;ned.
A formulation which contains up to 9U% by weight of phos-pholipid, preferably 50 to 80 percent by weight o~ phos-pholipid, is advantageous. ~ith a view to medical activ-ities, a formulation is preferred which contains fatty acids, according to the invention having 18 carbon atoms.
A formulation containing unsaturated fatty acids and a proportion of phospholipids greater than 30 percent by weight has already been proposed as a dietary product (EP 0,148,303 A1)~ Stabilizing effects of the phospho-lipids on the unsaturated fatty acids are not disclosed in EP 0,148,303 A1. Neither is there any indication of the formulat;ons, claimed in the present application and composed of unsaturated fatty acids, phospholipids and stimulators, being advantageously suitable for the uses claimed in the present application, especially for the cla;med medical applications.
The invention therefore relates to solid or l;quid mix-tures of substances or solutions, ~hich comprise A~ one or more unsaturated fatty acid(s), defined by three to five isolated double bonds and 18 to 22 car-bon atoms which are arranged in a straight chain andcan be methylated or ethylated at one or two carbon atoms in positions 2, 3, 4, 16, 17, 18, 19 or 20, as the free carboxylic acid wi~h terminal-C02H or as the ; carboxamide or as -C02X, X being a protective group which can be eliminated under acidic conditions, such as an alkyl radical, for example an ethyl or propyl radical, or a 1- or 2-lysophospholipid, for example 2-lysolecithin, or a metal or amine cation or the cationic form of an ion exchanger, L3 [)223~6 B) as stimulators ~ith, in some cases, a simultaneous stabilizing action, one or more compounds from the following groups:
B13 a phenolic compound of the formula I
R~ ~ R2 OH
in wh;ch the radicals R2 and R3 are hydroxyl groups or hydrogen and R1 is a radical -OH, -Co2H, -CH2-C02-H, -CH=CH-C02H, -CH2-CHR4R5 or ~CHOH-CH2-NH-R6 with R4 = -H or -C02H and R5 = -H or -NH2 and R6 = -H~
-CH3 or -C2H5~
B2) an indole derivative of the formula II
~3--CH2-CH-R7 II
N
in which R7 is hydrogen or -COOH, R8 ;5 hydrogen or -NH2 and R9 is nitrogen or -OH, 83) is cysteine or homocysteine, or lipo;c acid, the acylic alkyl rad;cal of wh;ch can be shortened by up to four methylene groups~
B4) a peptide comprising a max;mum of ten amino acids, wherein one or more of the amino acids are replaced by : in each case one of the compounds according to 8~) ~o B 3 ) ~
Bs) a compound according B1) to B4) which can carry a C1-C4-alkanoyl group on one N atom, B6) a flavone derivative wh;ch ;s subst;tuted by at least one hydroxyl group which can carry a sugar radical, .
~3~ 2266 B7) a salt of ~he ion;c forms of the compounds accord-;ng to B1)-B6) and, ;f appropr;ate, Bg) a carboxyl;c acid compound according to 91) to 87) which can be esterif;ed w;th an alkoxy radicaL or be ;n the form of a carboxam;de ~hich can also be mono~
or di-alkylated, and, if appropriate C) as stabilizers, one or more compounds from the follow-ing groups:
C1) d;methyl sulfoxide, ethyl alcohol, glycerol, ethylene glycol, polyethylene glycol or glycerol triacetate and C2) phospholipids~ sugar lipids, cyclodextrins, pro-teins, for example those which can be prepared from human and mammal blood, or vitamins of the E series, as a stable formulation for the synthesis of prostagland;ns and hydroxy-fatty ac;ds in biological systems.
Examples of metal cations X which can be used are ~hose of the alkal; metals, such as lithium, sodium and potas-sium, and of the alkaLine earth netals such as magnes;um and calcium, and also cationic forms of other metaLs such as alum;num, zinc and ;ron, ;f appropriate ;n a ~orm chelated with citric acid or ethylenediaminetetraacetic acid and the like. The amine cations can be those of primary, secondary or tert;ary am;nes such as the alkyl-am;nes, for example mono-, di-, and tri-methylamine or -ethylamine, -propylamine, -;sopropylamine, -butylamine, isobueylamine and -t-butylamine, and N-methyl-hexylamine, benzylamine, ~-phenyl-ethylam;ne, ethylenediamine, di-ethylenetriam;ne, pyrrol;dine, piper;dine, morpholine, piperaz;ne, mono-, di- and tri-e~hanolamine, ethyldieth-anolamine~ N-butylethanolamine, tris-~hydroxymethyl~-am;no-methane and the like. Examples of suitable amine salts are those of tryptam;ne and cysteine and the basic am;ne salts of lys;nè and arg;nine. Examples of suitable 3~226~
quaternary ammonium cat;ons are tetramethyl ammon;um, tetraethyl a~mon;um and benzyltr;methyl ammonium. These cations can aLso be used for forming salts of the an;oni~
forms of the compounds according to B1) to ~6)~ whereas S chloride and fluoride are preferred for forming salts of the cationic forms.
The fatty acids preferred for the enzymatic reaction are those designated as 18:2 ~-6, 20:4 ~-6, 22:5 ~-6, 18:3 ~-3, 20:5 ~-3, 22:6 ~-3, 18:3 ~-6, 20-3 ~-6, 22:4 ~-6 and 22:4 ~-3, the first figure in the conventional nomenclature denoting the number of carbon atoms, the figure after the colon denoting the number of double bonds and the figure after the ~ denoting the position of the first double bond counted from the methyl end of the molecule. The products from certain fatty acids are already known, and it is also known that the rate of reaction of the unsatu-rated fatty ac;ds and the formation of defined products can be promoted by the use of certa;n stimulators. As is explained ;n more detail in the experimental part, the present invent;on makes it possible to st;mulate the for-mation of defined fatty ac;d products ;n an advantageous manner. The select;on of the composition of ~he formula-t;on accord;ng to the ;nvention with respect to the type of unsaturated fatty acid, type of stimulator and also type of stabiLizer w;ll therefore depend ent;rely on the part;cular problem.
Where the stinulators are amino acids or peptides, both the L- form and the D- form and also mixtures of the D-and L- forms can be used.
St1mu~ators of the fsrmula I ~ith R = R = -H and R
-CHz-CH(NH2)-C02H, and peptides which contain these, or lipoic acid, the side chain of which can be shortened, or flavonoids have ths particular advantage that ~hey enhance the stability of the fat~y acids even without an additional stabili~er.
~31DZ2Ei6 _ 9 _ The quantities of the unsaturated fatty acids to be used in the formulations according to the invent;on are aLso dependent on the desired application~ For the prepara-t;ve production of prostaglandins, any desired quantity of S the formulation can be apportioned~ For the preparat;on of reagent formulations according to ~he invention, wh;ch serve for use in clinical-chemical tasks, i.e~ for example for investigating the ability of cells or cell constituents, such as blood cells or biopsy material from the kidney, lung, stomach and the like, to convert unsatura~ed fatty acids or to synthesize prostaglandins with a defined bio-logical activ;ty, reagent m;xtures accord;ng to the inven-tion suffice which contain S mg to 1 pg, preferably 100 ~9 to 1 ng, of unsaturated fatty acid. The reagents can be in the form of a suspension or solution, for example ;n water, ethanol, ethylene glycol, polyethylene glycols or glycerol or m;xtures of these solvents. After the solvent has been removed, the reagents are in the form of mixtures or blends. Removal of the organic solvents is necessary in particular when the reagent mixtures are used for enzyme-catalyzed reactions in vitro.
Enzyme systems, obtainable from biological oaterial, for the preparation of prostaglandins are known per se. It is also known that, depending on the origin and nature of the biological material, different prostagland;ns can be synthesized~ The reagents according to the invention can then be used for the preparation of these prostaglandins.
For this purpose, a solution or suspension or homogenate of the biological material is mixed with the formulations accord;ng to the invention and incubated in the known manner. The synthesis of defined pros~aglandins can be controlled to a certain extent by the selection of the st;mulator. Undesired fatty acid products, such as throm-boxan or leucotr;enes, san be suppressed, according tothe state of the art, by an addition of thro~boxan inhib-;tors or lipoxygenase inhib;tors. If the unsaturated fatty acid in the reagent is in a bonded form, for example as the ester, care must be taken to convert it into the 30:~2~i6 free form before or during the enzymatic reaction, if appropriate by means of the lipase enzymes present anyway in biological material, or by an addition of suitabLe lipase enzymes or, even before the enzymatic reaction, by the known processes of chemical hydrolysis. After the extraction of the prostaglandin formed~ or of the prost-aglandins formed, by means of suitable extractants, for example ethyl acetate, the desired prostaglandin can be produced in the pure form, for example by means of known chromatographic methods, such as h;gh-pressure liquid chromatography. The quantitative proportions of the con-st;tuents according to the invention in the formulations can vary within wide limits. They depend on the nature of the unsaturated fatty acid~ on the stimulator used or on the stabilizer and also on the intended use. The ~eight ratio between unsaturated fatty acid Srelative to the free acid) and stimulator is in general between S0 and 0.1, preferably between 20 and 0.5. The quantity of ~he added stabilizer is chosen such that its proport;on by weight, relative to the free unsaturated fatty acid, is between about 0.5 and 100, preferably bet~een 1 and 15 and espec;ally between 2 and about 8 to 10.
. .
Biological systems can also be treated in vivo with the medically suitable formulations according to the invent;on, and this is ;n fact more advantageous than is possible with the individual components of the formulations accord-ing to the invention. However, a systemic ~for example intravenous) administration of the formulations accord;ng to the invention is inappropriate, because the unsatur-a~ed fatty acids can be metabolized before they reach the site of action. By contrast, for example the cells of the skin, mouth, oesophagus, nose, eyes, intestines and stomach and of the bronchial and lung tract are directly accessible.
Prostagland;n defiencies in humans or animals, for example in the gastro-intestinal tract, which lead to, for example, disturbances in gastric juice secretion and to gastro-~` 13~
intestinal lesions tulcers), can be treated, for example,with formulations according to the invention in the form of oral (peroral) medicament forms. The intestines can also be treated with medicament forms which have been coated enterically (insoluble in gastric acid). If it is desired to induce diarrhea, for example in the case of const;pation, medicament forms for rectal therapy can also be used, whereas intravaginal therapy can be used for in-duc;ng labor, for example for s~arting the birth process.
Malfunct;ons of the skin and of ~he connective tissue re-g;on below the skin can be treated by dermal formulations according to the invention. Oral formulations and sup-pos;tories for human therapy are of such a composition that they contain, per dosage unit, between 0.5 and 2,000 mg, ;n general between 2 and 1,000 mg and prefer-ably between 5 and 250 mg, of unsaturated fatty acid. In the case of illnesses res;stant to therapy, for example obstinate constipat;on, ;t can also be appropriate to apply a dosage un;t of up to 6,000 mg or more of unsatu-rated fatty acid. Surpr;sin~ly, the stra;ght-cha;n fatty acids accord;ng to the ;nvention having 18 carbon atoms and also those having 22 carbon atoms are equally effec-t;ve as therapeut;cs for gastro-;ntest;nal ulcers as the fatty acids conta;n;ng 20 carbon atoms. This is of med;-cal ;mportance, because they cause fewer side effectssuch as diarrhea. A suitable therapy comprises, for example, the administration of one, two or more, prefer-ably 3 to 8, individual dosages per day of the formula-tion accord;ng to the invention, the required quantity depending on the number of individual doses and also on the disease to be treated, and one dose can also be made up, for example, from several singLe doses admin;stered simultaneously.
It ;s known that gastro-intestinal ;ntolerances, wh;ch lead to gastro-intest;nal ulcers, can arise as undes;red side effects of certa;n med;caments. The ~ormulations accord;ng to the invent;on are also suitable for the pre-vent;on or therapy of such damage to the gastro-intest;nal ~L3~:266 mucosa. The treatment with the formulations can take place - either before, or simultaneously with, or after the admin-istration of the medicaments causing damage to the mucosa.
It is particularly advantageous to combine the reagents according to the invention with the medicaments which are causing damage to the gastric mucosa, in a suitable pharma-ceutical formulation. In this case, the components can either be compartmentalized or can be in the form of a mix-ture. Numerous medicaments which cause stomach intoler-1~ ances or gastro-intestinal lesions are known. These in-clude anti-rheumatics (steroid and non-steriod anti-inflammatory agents) such as corticosteroids (for example cortisone), sali~yl;c acid der;vat;ves (for example ace-tylsal;cylic ac;d, diflunisal) or phenylalkanoic acids (for example piroxicam, acemetacin, p;metacin, nambumetone, carprofen, priprofen, fenclofenac, sulindac, indometac;n, fenoprofen, t;aprofen;c ac;d, tolmet;n, flurbiprofen, suprofen, indoprofen, ibuprofen, naproxen, alclofenac, ketoprofen, diclofenac) or pyrazolones tphenylbutazone, metami20le), and also hypotens;ve agents (for example reserp;ne), vasodilators based on xanthine (for example pento~yfylline), tuberculostatic agents (for example rifampicin) or cytostatics (for example methohexate)~
The formuLations according to the invent;on, used medic-ally as pharmaceutical products, can conta;n the conven-~ional pharmaceutical aux;liar;es and e~c;p;ents, which are suitable f~r the preparation of orally and dermally (and also~ for example, nasally, subl;ngually, rectally and ;ntravaginalLy) active medicaments. These include, for example, starch, sucrose, calcium sulfate, gelatine, starch paste, syrup~ wheat starch and corn starch, stearic acid~ stearate salts, sugars, saccharin, food const;tuents for animals, such as fats, carbohydrates, proteins and mineral solids; preservatives, sweeteners, dyestuffs, sp;ces or suspending agents, mineral and vegetable oils such as peanut o;l, olive o;l, even;ng primrose o;l, soya oil, sunflower o;l, cotton seed oil, f;sh oil and sesame oil or fractions of these oils, both ionic and non-ionic ~.3~2~
detergents ~for example as described below), preservatives - and fungicides such as parabens, chlorobutanol, ben~yl alcohol, phenol and thimerosal, and isotonic agents such as sugar or sodium chloride. The polyunsaturated fatty acids can also be adsorbed on or mixed w;th polydextran or gels of poLyacrylamide, alkylsulfate or aLkylphosphate, or on aluminum hydroxide or silica.
The preparations for dermal administration are, for ex-ample, aqueous, alcoholic or polyol-containing solutions or suspensions, oily solutions c,r suspens;c,ns, or powders for subsequent ;ncorPorat;on into a dermally appl;cable form by add;tion of the requ;site carrier. The solutions or suspens;ons are formulated w;th the requisite pharma-ceutical auxil;aries and excipients contain;ng, for ex-ample, fats, waxes~ petroleum jelly or paraffin, and pres-ervat;ves, susPend;ng and dispers;ng agents and ;sotonic agents, such as, for example, methylparaben and propyl-paraben, sodium chloride, polyethylene glycols, especially polyethylene glycol 4000, sod;um carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidon, sorbimacrogol oleate, a condensation product of ethylene ox;de ~;th fatty acids, for example polyoxyethylene stearate, or ~ith fatty alco-hols such as, for example, heptadecaethylenoxycetanol, or with part;al esters such as, for example, polyoxyethylene-sorbitol monooleate, or hexitans originating from sorbitol, such as, for example, polyoxyethylene-sorbitan monooleate.
Suspensions in oily med;a are prepared by d;spersing the active compound in the abovementioned oils. These sus-pensions can contain agents which delay absorption, suchas, for example, alu~inum monostearate. A dry product, for example a free2e-dr;ed product, wh;ch can be m;xed ~;th a suitable pharmaceut;cal carr;er at the des;red t;me, represents a further embod;ment. Preparations from the active sompound, such as, for example, tablets, cap-sules, suppos;tories, syrups, solut;ons, suspens;ons and elix;rs, conta;n pharmaceut;cal aux;l;ar;es which render the preparation su;table for these adm;nistration forms ;n order to achieve the medical effect. The mixtures ~L3~)~2~6 accord;ng to the invention are mixed with pharmaceutical aux;liaries such as lactose, starch, acacia gum, gelatin, talc and the like and processed to g;ve capsules, tablets and the l;ke or to give conventional suppos;tory ~ompos-itions, for example those based on triglycerides, suchas, for example witepsoL suppository compositions (H.P. Fiedler, Lexikon der H;lfsstoffe fUr Pharmazie, Kosmetik und angrenzende Gebiete ~Dictionary of Auxiliar-ies for Pharmacy~ Cosmetics and Related Fields~, 1971, volume 9, pages 548-50 and 632-634, or for intravaginal suppositories US Patents 263,026 and 663,145~; if approp-riate, fatty alcohols; solid hydrocarbons, such as petrol-eum jelly or paraffin solidum; saturated fatty acids, such as lauric acid, myr;stic acid, palmitic acid and stearic acid; emulsifiers such as ethoxylated triglycer-ides and polyethoxylated vegetable oils; fatty acid sugar esters; silicones; geLatin; methyl cellulose; hydroxy-propoxy cellulnse and hydroxypropyl cellulose; polyethylene glycols; polyvinylpyrrolidone; polyvinyl alcohol; poly-acrylic acid and salts thereof, or are mixed with water,suspending agents, sucrose, preservatives or hydro-alcoholic exc;p;ents and processed to g;ve suspens;ons solut;ons or el;x;rs. lt may be desirable for the unsaturated fatty acid to be released slowly or ~ith delay from the pharm-aceutical formulation. Such a delayed release can beaccomplished in accordance with the state of the art. It depends on the pH of the b;olog;cal med;um whether the delayed release ;s accompl;shed, for example, by means of polystyrene or polyacrylic derivat;ves ~Eudragit~R)) or, for example, by bind;ng to a su;table ;on exchanger~
Enter;cally coated products, ~or example tablets, cap-sules, p;lls, beads or microbeads, can also be prepared.
They are in the form of dosage un;ts, for example as a s;ngle tablet or capsule or in the form of a collect;on of p;Lls, microbeads or microspheres which have enteric properties and a prolonged action. Such entericalLy coated products and the coating materials used for this purpose are described ;n the literature, for example in US Patents 2~093,462 and 2,196,768 (cellulose acetate -" ~30~2~6 phthalate), ~,897,121 (copolymers of styrene and maLe;c - acid), 3,081,233 and others.
Experimental part:
~ . .
Method 1: Investigation of the stability of unsaturated fatty acids in the formulations The stabilizing action of substances on polyunsaturated fatty acids was inuestigated as follows Test A for water-soluble substances:
0.125 mg of unsaturated fat~y acid or Na salt (see tables) (sodium arach;donate, from Sigma, Munich, Germany) is dissolved in 0.05 ml of ~ater. 0.2 ml of aqueous solution which contains 2 mg of stab;l;zer and can add;tionally contain 2 mg of st;mulator is mixed in (cf. Table 1), 0.1 ml is then taken out immediately, extracted and ana-lysed (see below). Further processing according tomodification 1 or 2.
Modification 1 for_aqueous solutions After 24 hours incubation at 37C, 0.1 ml is again taken, extracted and analysed (see below).
Mod;fication_2 for solid mixtures (lyophilisates) ._ The test solution is lyophilized, the lyophilisate is incubated for 24 hours at 37C and then dissolved again in the previous quantity of water, and 0.1 ml is takenr extracted and analysed tsee below).
Test ~ for water-insoluble substances:
0.125 mg of unsaturated fat~y acid (arachidonic acid, 5,8,11,14,17-eicosapentaenoic acid or 8,11,14-eicosatrienoic acid, from Sigma, Munich) is dissolved ` - -in 0.05 mL of ethanol or dimethyl sulfoxide. 0.2 ml of ethanol (or d;methyl sulfoxide) which contains 2 mg of stabilizer and can additionally conta;n 2 mg of She stim-ulator (cf. TabLe 1) is added thereto, and 0.1 ml ;s taken immediately and, as below, extracted and analysed. Further treatment according to modification 1 or 2.
Modification 1 for solutions After 24 hours' incubation at 37C~ 0.1 ml is again taken, mixed with 0.1 ml of water, extracted and analysed.
Modification 2 for solid mixtures The remaining solution is freed of solvent, incubated for 24 hours at 37C and then dissolved again in 0.15 ml of solvent, and 001 ml ;s taken, mixed with 0.1 ml of water, extracted and analysed.
~traction and analysis 0.05 ml of 1.2 M aqueous citric acid solut;on ;s added to the samples obta;ned, and the samples are extracted with twice 0.5 ml of ethyl acetate, the ethyl acetate is removed from the combined extracts and the residue is dissolved in 0.8 ml of methanol and analysed by means of high-pressure liquid chromatography (HPLC) (Rad Pak C-18 column, 100 x 8 mm, from ~aters, K~nigstein, Germany, 1.5 ml/minute, solvent 800 ml of methanol, 200 ml of ~ater and 0.1 ml of glacial acetic acid)~ The separa~;on is monitored uith a commercially available UV detector at 206 nm.
Method 2: Prostagland;n synthesis 0.3 ml of 0.03 M potassium phosphate buffer of pH = 8.0 contains 0.5 mg of enzyme (from seminal ves;cLes of sheep) tBiochemistry 10 (1971) 2372) or homogenate of the inner kidney medulla (L;fe Sciences 26 (1980) 765), 2.75 ~9 of 14C-arachidonic acid (0.5 ~Ci~ and 0.5 mg of stimulator, and, if appropriate, 0.5 mg of stabilizer substance (see Table 1). After 10 minutes' incubation at 37C~ the re-action is stopped with 0.05 ml of 1.2 M citric acid and the mixture is extracted with twice 0.5 ml of ethyl acetate, the combined extracts are freed of solvent, and the resi-due is dissolved in 0.2 ml of methanol, diluted 1:1 with water and separated by HPLC (nucleosil C-18 column, 125 x 4.6 mm, from ~ischoff, Leonberg, Germany, solvent 300 ml of acetonitrile, 700 ml of water and 1 ml of glacial ace-tic acid and after 30 minutes pure methanol)~ The sepa-ration of the radioactive fractions is followed by a monitor (L~ 504 from ~erthold, Wildbad, Germany). The products formed~ such as 6-keto-prostaglandin F2alpha~
prostaglandin E2, prostaglandin F2alpha and pros~aglandin D2, are separated under these conditions.
Table 1 Stimulation of the prostaglandin formation in stabilized fatty acid formulat;ons The prostaglandins PGE2, PGF2alpha and PGD2 as products from arachidonic acid where quantified by means of method 2, and the sum was related as "total prostaglandin" to 100X. The quantity of "total prostaglandin" formed ~ith-out the add;t;on of a stimulator was set at f=1; the quan-tity formed after s~imulation was related thereto. f=2 or f=3 therefore means twice or three times the stimulation under the conditions of method 2. The stability of the sample was investigated by means of method 1 (right-hand column). W;thout additions (right-hand column: A), 25-28%
of the arachidonic acid remain in aqueous solution under these conditions. Values greater than 25-28~ therefore represent a stabilizat;on, and values smaller than 25-28 repre ent a destabil;za~;on. Example No. 1 W3S carr;ed out ~ithout a stimulator. The effects according to the ;nvention can be obta;ned w;th the stabilized formulations from the correspondingly designated examples. The sub-stances test w;th respect to stabilization (see Method 1 for quantit;es) were: A) aqueous solution without o~
stab;lizer addition, 0) ethanolic solution without further - addition, C) pyridine solution, D) with rutin, predis-solved in dimethyl sulfoxide, dimethyl sulfoxide then stripped off, E) like B) ~ith lecithin, ethanol stripped off, F) like A) ~ith cytochrome c from horse heart (from Boehringer, Mannheim, Germany), G) like A) with methemo-globin (human) (from Sigman, Munich, Germany), H) like A) with bovine serum albumin (from Calbiochem, Frankfurt, Germany), J) like A) or B), but solvent s~ripped off, K) like J) with cytochrome c, L) like J) with hemoglobin (human) (from ~ehring~erke, Marburg, Germany), and M) like J) with human serum albumin (from Behringwerke, Marburg, Germany).
`" ~3~ 6 c, _ Arachidonic acid product Stability (Method 2) (% of remaining arachidonic acid Example Stimulator relative PGE2 PGF2 ~ h PGD2 by Method 1) prosta-No~ glandin ________________._ .___...___._.________~__..__.___________________________._____ f t Comparative investigation l)without stimulator l 83 2 l5 B lO0 H lO0 ~ 13 J 0 D 97 ~ lO0 F 98 M lO0 Examples of substances not according to the ;nvention , .
2) m-cresol 0.61 98 0 2 3) phenol 0.95 77 5 18 .
method especially from dihomo-gamma-linolen;c ac;d, arach-;don;c ac;d or e;cosapentaeno;c ac;d as the substrates.In such preparatiYe problems, the aim will be a stimulation of the formation of the desired prostaglandin. This can be achieved by addition of suitable enzyme co-factors.
However, the said fatty ac;ds can also be used for medical purposes, for example in cl;nical invest;gations of the ability of body cells, or cell homogenates or cell frac-t;ons (for example kerat;nocytes; blood vessel cells ob-tained by biopsy; cells or homogenates of the medulla of k;dney3, to metabolize the part;cular fatty acid or to convert it to prostagland;ns having a certa;n b;olog;cal act;on. In such med;cal problems, it is not imPortant whether the prostagland;n formed ;s homogeneous, but there ~;ll be an a;m of st;mulat;ng the format;on of the pros-~aglandin serv;ng as the med;cal parameter to be measured.
The present ;nvention describes novel, stable formulations which contain long-chain polyunsaturated fatty acids, by means of which preparations prostaglandins and hydroxy-fatty acids which can be used for the said purposes can advantageously be prepared.
It is already known that the conversion of polyunsaturated fatty ac;ds, such as arach;don;c ac;d, can be stimulated by prostaglandin-synthesizing enzymes w;th the aid of ~ ~3~2Z6~;
certain co-factors and, thus, the synthesis of very par-ticular prostaglandins can also be promoted. For example, in biochemical pharmacology 31 (1982), 3S91, an ;n v;tro system ;s described wh;ch converts arachidon;c ac;d by means of the enzyme obtained from seminal vesicles of sheep, in each case w;~h or without acldition of the co-factors glutathione and hydroquinone. In loc. c;t.~ page 3595, it is explained that the conversion is accelerated by the said co-factors. A similar system of arach;donic acid, homogenate of rabbit k;dney medulla, glutathione and hydroquinone is described in Life Sciences 26 (1980) 765.
For the purposes mentioned above, aqueous reagent formul-ations have now been prepared, ~hich conta;n the polyun-saturated fatty ac; d s prefe~ly in combination wqth stimulat~
enzyme co-factors. Such formulations are not only novel but also show unexpected properties. As explained in detail belo~, these stimulators without exception modify the stability of the fatty acids in a surprising manner.
Thus, within a short time, a markedly greater quantity of undesired decomposition and convers;on products of arach-idonic acid can form in arach;donic acid formulations con-taining glutath;one or hydroquinone, than is the case in formulations which do not contain these addit;ons. These byproducts are highly reactive and can trigger undes;red, even biolog;cal s;de reactions. Our own investigat;ons descr;bed in more detail below, however, sho~ that the prostagland;n synthesis can also be stimulated by mixtures which contain polyunsaturated fat~y acids, such as arachi-donic acid, together with other sulfur-containing compounds such as the disulfide lipoic acid or the reduced thiols homocysteine and cysteine or their derivat;ves acylated on the n;trogen atom, much smaller quant;ties of undes;red byproducts being formed accord;ng to the invention. This is surprising, since it was not possible to achieve these advantageous effects with other sulfùr compounds occurring in nature or derived from natural compounds, such as co-enzyme A or its scission or hydrolysis products cysteamine - ~.3~2266 and pantethe;ne, or dihydrolipoic acid, cystine and mono-or poly-thiol sugar compounds such as dithiothreitol or dithioerythritol. In the experimental part, it is stated that the undesired side reactions can be fully suppressed by an addition of lipoic acid, but can be largely, but not completely suppressed by an addit;on of the other sulfur-containing compounds accord;ng to the invent;on.
In fact, i~ is already known that the conversion of arac-h;don;c ac;d or the synthesis of certain prostaglandins can also be stimulated by an addition of substances with-out a sulfur content, for example, as cited above, by means of hydroquinone and also phenol, adrenalin, norad-renalin, tryptophan, 5-hydroxytryptophan and ascorbic acid. Surprisingly~ it has now been found that the un-desired decomposition of polyunsaturated fatty acids, for example of arachidonic acid, in an aqueous medium is con-siderably accelerated by these stimulators. The use of, for example, 2-butanol or pyridine instead of water has an additional destabilizing effect, whereas the undesired byproducts surprisingly do not arise when di~ethyl sul-foxide or ethanol or polyols such as glycerol or poly-ethylene glycol are used. Organic solvents have, however, the disadvantage that they denature enzy~es; they must therefore be removed before the enzyme reaction or diluted ~ith water. The residues ~hich remain after the removal of the solvent and which consist of an intimate mixture of unsaturated fatty acid and stimulator are, surPr;singly, aga;n unstable. It has not yet been described that sur-pr;s;ngly advantageous aqueous formuLations for the stim-ulation of the prostaglandin synthesis can be obtained with tyrosine. As explained in the experi~ental part, formulations which contain unsatura~ed fatty acids and tyrosine or tyrosine-l;ke compounds are, surprisingly, completely stable.
However, even aqueous mixtures or solutions of unsaturatecl fatty ac;d and the other abovementioned stimulators can be fully stab;lized, namely by an addition of lipids which 226~
are soluble or emuls;fiable in water, or of proteins which can be prepared, for example, from human or mammal blood, such as serum albumin or globulins, such as gamma-globulin, or porphyr;n-containing proteins such as hemo-glob;n, eethemoglobin or cytochromes. Vitam;ns of the Eseries and flavonoids, in particular those with a sugar moiety, for example rutin or troxerutin (for the nomen-clature, see "The Merck Index", 10th edition, 1983, Rahway, N.J., USA) are also suitable according to the invention, whereas sugars withou~ a flavone moiety, such as rhamnoglucose, glucose and sorbitol, are not stabiliz-ing. Surprisingly, however, the formulations according to the invention can be prepared with the aid of cyclodextrins.
With respect to stabilization with serum albumins, it is indeed known per se that albumins can bond long-chain fatty acids at an energetically low level, but it is the more surprising that the fatty acids bonded in this way behave like freely dissolved fatty acids in the reagent formulations according to the invention, that is to say they are enzymatically converted without hindrance. It has also already been described tcf., for example, Journal of Biological Chemistry 253 ~1978) 5061) that the enzymes o~ the prostagland;n synthesis conta;n heme or other por-phyrins bonded as prosthet;c groups. However, th;s req-u;res only extremely small quantit;es of porphyr;n-con-ta;n;ng enzymes, which do not have any stabilizing effect on the added quantities of unsaturated fatty acid. There is no ind7cation to be found in the literature to the effect that a stabil;zing reagent for st;~ulating the enzyme-catalyzed prostaglandin synthesis can be prepared w;th a higher proportion of porphyrin-containing proteins.
Stable formulat;ons wh;ch contain fatty ac;ds hav;ng 20 to 22 carbon atoms and 3 or more double bonds in each case and, as stabil;zers, a proportion of up to 30 percent by weight of phospholip;ds, for example lec;thin, have been d;scLosed ;n DE 34,197,996 A1. Such stable formul-at;ons can l;kewise be used according to the invent;on in ~3022~6 combination with the stimulators already mentioneda As explained ;n the experimental part, however, it is more appropriate to add more than 30 percent by weight of phospholipid, for example lecithin, to the formulations of the unsaturated fatty acids having 18 to 22 carbon atoms, because more stable mixtures are then obta;ned.
A formulation which contains up to 9U% by weight of phos-pholipid, preferably 50 to 80 percent by weight o~ phos-pholipid, is advantageous. ~ith a view to medical activ-ities, a formulation is preferred which contains fatty acids, according to the invention having 18 carbon atoms.
A formulation containing unsaturated fatty acids and a proportion of phospholipids greater than 30 percent by weight has already been proposed as a dietary product (EP 0,148,303 A1)~ Stabilizing effects of the phospho-lipids on the unsaturated fatty acids are not disclosed in EP 0,148,303 A1. Neither is there any indication of the formulat;ons, claimed in the present application and composed of unsaturated fatty acids, phospholipids and stimulators, being advantageously suitable for the uses claimed in the present application, especially for the cla;med medical applications.
The invention therefore relates to solid or l;quid mix-tures of substances or solutions, ~hich comprise A~ one or more unsaturated fatty acid(s), defined by three to five isolated double bonds and 18 to 22 car-bon atoms which are arranged in a straight chain andcan be methylated or ethylated at one or two carbon atoms in positions 2, 3, 4, 16, 17, 18, 19 or 20, as the free carboxylic acid wi~h terminal-C02H or as the ; carboxamide or as -C02X, X being a protective group which can be eliminated under acidic conditions, such as an alkyl radical, for example an ethyl or propyl radical, or a 1- or 2-lysophospholipid, for example 2-lysolecithin, or a metal or amine cation or the cationic form of an ion exchanger, L3 [)223~6 B) as stimulators ~ith, in some cases, a simultaneous stabilizing action, one or more compounds from the following groups:
B13 a phenolic compound of the formula I
R~ ~ R2 OH
in wh;ch the radicals R2 and R3 are hydroxyl groups or hydrogen and R1 is a radical -OH, -Co2H, -CH2-C02-H, -CH=CH-C02H, -CH2-CHR4R5 or ~CHOH-CH2-NH-R6 with R4 = -H or -C02H and R5 = -H or -NH2 and R6 = -H~
-CH3 or -C2H5~
B2) an indole derivative of the formula II
~3--CH2-CH-R7 II
N
in which R7 is hydrogen or -COOH, R8 ;5 hydrogen or -NH2 and R9 is nitrogen or -OH, 83) is cysteine or homocysteine, or lipo;c acid, the acylic alkyl rad;cal of wh;ch can be shortened by up to four methylene groups~
B4) a peptide comprising a max;mum of ten amino acids, wherein one or more of the amino acids are replaced by : in each case one of the compounds according to 8~) ~o B 3 ) ~
Bs) a compound according B1) to B4) which can carry a C1-C4-alkanoyl group on one N atom, B6) a flavone derivative wh;ch ;s subst;tuted by at least one hydroxyl group which can carry a sugar radical, .
~3~ 2266 B7) a salt of ~he ion;c forms of the compounds accord-;ng to B1)-B6) and, ;f appropr;ate, Bg) a carboxyl;c acid compound according to 91) to 87) which can be esterif;ed w;th an alkoxy radicaL or be ;n the form of a carboxam;de ~hich can also be mono~
or di-alkylated, and, if appropriate C) as stabilizers, one or more compounds from the follow-ing groups:
C1) d;methyl sulfoxide, ethyl alcohol, glycerol, ethylene glycol, polyethylene glycol or glycerol triacetate and C2) phospholipids~ sugar lipids, cyclodextrins, pro-teins, for example those which can be prepared from human and mammal blood, or vitamins of the E series, as a stable formulation for the synthesis of prostagland;ns and hydroxy-fatty ac;ds in biological systems.
Examples of metal cations X which can be used are ~hose of the alkal; metals, such as lithium, sodium and potas-sium, and of the alkaLine earth netals such as magnes;um and calcium, and also cationic forms of other metaLs such as alum;num, zinc and ;ron, ;f appropriate ;n a ~orm chelated with citric acid or ethylenediaminetetraacetic acid and the like. The amine cations can be those of primary, secondary or tert;ary am;nes such as the alkyl-am;nes, for example mono-, di-, and tri-methylamine or -ethylamine, -propylamine, -;sopropylamine, -butylamine, isobueylamine and -t-butylamine, and N-methyl-hexylamine, benzylamine, ~-phenyl-ethylam;ne, ethylenediamine, di-ethylenetriam;ne, pyrrol;dine, piper;dine, morpholine, piperaz;ne, mono-, di- and tri-e~hanolamine, ethyldieth-anolamine~ N-butylethanolamine, tris-~hydroxymethyl~-am;no-methane and the like. Examples of suitable amine salts are those of tryptam;ne and cysteine and the basic am;ne salts of lys;nè and arg;nine. Examples of suitable 3~226~
quaternary ammonium cat;ons are tetramethyl ammon;um, tetraethyl a~mon;um and benzyltr;methyl ammonium. These cations can aLso be used for forming salts of the an;oni~
forms of the compounds according to B1) to ~6)~ whereas S chloride and fluoride are preferred for forming salts of the cationic forms.
The fatty acids preferred for the enzymatic reaction are those designated as 18:2 ~-6, 20:4 ~-6, 22:5 ~-6, 18:3 ~-3, 20:5 ~-3, 22:6 ~-3, 18:3 ~-6, 20-3 ~-6, 22:4 ~-6 and 22:4 ~-3, the first figure in the conventional nomenclature denoting the number of carbon atoms, the figure after the colon denoting the number of double bonds and the figure after the ~ denoting the position of the first double bond counted from the methyl end of the molecule. The products from certain fatty acids are already known, and it is also known that the rate of reaction of the unsatu-rated fatty ac;ds and the formation of defined products can be promoted by the use of certa;n stimulators. As is explained ;n more detail in the experimental part, the present invent;on makes it possible to st;mulate the for-mation of defined fatty ac;d products ;n an advantageous manner. The select;on of the composition of ~he formula-t;on accord;ng to the ;nvention with respect to the type of unsaturated fatty acid, type of stimulator and also type of stabiLizer w;ll therefore depend ent;rely on the part;cular problem.
Where the stinulators are amino acids or peptides, both the L- form and the D- form and also mixtures of the D-and L- forms can be used.
St1mu~ators of the fsrmula I ~ith R = R = -H and R
-CHz-CH(NH2)-C02H, and peptides which contain these, or lipoic acid, the side chain of which can be shortened, or flavonoids have ths particular advantage that ~hey enhance the stability of the fat~y acids even without an additional stabili~er.
~31DZ2Ei6 _ 9 _ The quantities of the unsaturated fatty acids to be used in the formulations according to the invent;on are aLso dependent on the desired application~ For the prepara-t;ve production of prostaglandins, any desired quantity of S the formulation can be apportioned~ For the preparat;on of reagent formulations according to ~he invention, wh;ch serve for use in clinical-chemical tasks, i.e~ for example for investigating the ability of cells or cell constituents, such as blood cells or biopsy material from the kidney, lung, stomach and the like, to convert unsatura~ed fatty acids or to synthesize prostaglandins with a defined bio-logical activ;ty, reagent m;xtures accord;ng to the inven-tion suffice which contain S mg to 1 pg, preferably 100 ~9 to 1 ng, of unsaturated fatty acid. The reagents can be in the form of a suspension or solution, for example ;n water, ethanol, ethylene glycol, polyethylene glycols or glycerol or m;xtures of these solvents. After the solvent has been removed, the reagents are in the form of mixtures or blends. Removal of the organic solvents is necessary in particular when the reagent mixtures are used for enzyme-catalyzed reactions in vitro.
Enzyme systems, obtainable from biological oaterial, for the preparation of prostaglandins are known per se. It is also known that, depending on the origin and nature of the biological material, different prostagland;ns can be synthesized~ The reagents according to the invention can then be used for the preparation of these prostaglandins.
For this purpose, a solution or suspension or homogenate of the biological material is mixed with the formulations accord;ng to the invention and incubated in the known manner. The synthesis of defined pros~aglandins can be controlled to a certain extent by the selection of the st;mulator. Undesired fatty acid products, such as throm-boxan or leucotr;enes, san be suppressed, according tothe state of the art, by an addition of thro~boxan inhib-;tors or lipoxygenase inhib;tors. If the unsaturated fatty acid in the reagent is in a bonded form, for example as the ester, care must be taken to convert it into the 30:~2~i6 free form before or during the enzymatic reaction, if appropriate by means of the lipase enzymes present anyway in biological material, or by an addition of suitabLe lipase enzymes or, even before the enzymatic reaction, by the known processes of chemical hydrolysis. After the extraction of the prostaglandin formed~ or of the prost-aglandins formed, by means of suitable extractants, for example ethyl acetate, the desired prostaglandin can be produced in the pure form, for example by means of known chromatographic methods, such as h;gh-pressure liquid chromatography. The quantitative proportions of the con-st;tuents according to the invention in the formulations can vary within wide limits. They depend on the nature of the unsaturated fatty acid~ on the stimulator used or on the stabilizer and also on the intended use. The ~eight ratio between unsaturated fatty acid Srelative to the free acid) and stimulator is in general between S0 and 0.1, preferably between 20 and 0.5. The quantity of ~he added stabilizer is chosen such that its proport;on by weight, relative to the free unsaturated fatty acid, is between about 0.5 and 100, preferably bet~een 1 and 15 and espec;ally between 2 and about 8 to 10.
. .
Biological systems can also be treated in vivo with the medically suitable formulations according to the invent;on, and this is ;n fact more advantageous than is possible with the individual components of the formulations accord-ing to the invention. However, a systemic ~for example intravenous) administration of the formulations accord;ng to the invention is inappropriate, because the unsatur-a~ed fatty acids can be metabolized before they reach the site of action. By contrast, for example the cells of the skin, mouth, oesophagus, nose, eyes, intestines and stomach and of the bronchial and lung tract are directly accessible.
Prostagland;n defiencies in humans or animals, for example in the gastro-intestinal tract, which lead to, for example, disturbances in gastric juice secretion and to gastro-~` 13~
intestinal lesions tulcers), can be treated, for example,with formulations according to the invention in the form of oral (peroral) medicament forms. The intestines can also be treated with medicament forms which have been coated enterically (insoluble in gastric acid). If it is desired to induce diarrhea, for example in the case of const;pation, medicament forms for rectal therapy can also be used, whereas intravaginal therapy can be used for in-duc;ng labor, for example for s~arting the birth process.
Malfunct;ons of the skin and of ~he connective tissue re-g;on below the skin can be treated by dermal formulations according to the invention. Oral formulations and sup-pos;tories for human therapy are of such a composition that they contain, per dosage unit, between 0.5 and 2,000 mg, ;n general between 2 and 1,000 mg and prefer-ably between 5 and 250 mg, of unsaturated fatty acid. In the case of illnesses res;stant to therapy, for example obstinate constipat;on, ;t can also be appropriate to apply a dosage un;t of up to 6,000 mg or more of unsatu-rated fatty acid. Surpr;sin~ly, the stra;ght-cha;n fatty acids accord;ng to the ;nvention having 18 carbon atoms and also those having 22 carbon atoms are equally effec-t;ve as therapeut;cs for gastro-;ntest;nal ulcers as the fatty acids conta;n;ng 20 carbon atoms. This is of med;-cal ;mportance, because they cause fewer side effectssuch as diarrhea. A suitable therapy comprises, for example, the administration of one, two or more, prefer-ably 3 to 8, individual dosages per day of the formula-tion accord;ng to the invention, the required quantity depending on the number of individual doses and also on the disease to be treated, and one dose can also be made up, for example, from several singLe doses admin;stered simultaneously.
It ;s known that gastro-intestinal ;ntolerances, wh;ch lead to gastro-intest;nal ulcers, can arise as undes;red side effects of certa;n med;caments. The ~ormulations accord;ng to the invent;on are also suitable for the pre-vent;on or therapy of such damage to the gastro-intest;nal ~L3~:266 mucosa. The treatment with the formulations can take place - either before, or simultaneously with, or after the admin-istration of the medicaments causing damage to the mucosa.
It is particularly advantageous to combine the reagents according to the invention with the medicaments which are causing damage to the gastric mucosa, in a suitable pharma-ceutical formulation. In this case, the components can either be compartmentalized or can be in the form of a mix-ture. Numerous medicaments which cause stomach intoler-1~ ances or gastro-intestinal lesions are known. These in-clude anti-rheumatics (steroid and non-steriod anti-inflammatory agents) such as corticosteroids (for example cortisone), sali~yl;c acid der;vat;ves (for example ace-tylsal;cylic ac;d, diflunisal) or phenylalkanoic acids (for example piroxicam, acemetacin, p;metacin, nambumetone, carprofen, priprofen, fenclofenac, sulindac, indometac;n, fenoprofen, t;aprofen;c ac;d, tolmet;n, flurbiprofen, suprofen, indoprofen, ibuprofen, naproxen, alclofenac, ketoprofen, diclofenac) or pyrazolones tphenylbutazone, metami20le), and also hypotens;ve agents (for example reserp;ne), vasodilators based on xanthine (for example pento~yfylline), tuberculostatic agents (for example rifampicin) or cytostatics (for example methohexate)~
The formuLations according to the invent;on, used medic-ally as pharmaceutical products, can conta;n the conven-~ional pharmaceutical aux;liar;es and e~c;p;ents, which are suitable f~r the preparation of orally and dermally (and also~ for example, nasally, subl;ngually, rectally and ;ntravaginalLy) active medicaments. These include, for example, starch, sucrose, calcium sulfate, gelatine, starch paste, syrup~ wheat starch and corn starch, stearic acid~ stearate salts, sugars, saccharin, food const;tuents for animals, such as fats, carbohydrates, proteins and mineral solids; preservatives, sweeteners, dyestuffs, sp;ces or suspending agents, mineral and vegetable oils such as peanut o;l, olive o;l, even;ng primrose o;l, soya oil, sunflower o;l, cotton seed oil, f;sh oil and sesame oil or fractions of these oils, both ionic and non-ionic ~.3~2~
detergents ~for example as described below), preservatives - and fungicides such as parabens, chlorobutanol, ben~yl alcohol, phenol and thimerosal, and isotonic agents such as sugar or sodium chloride. The polyunsaturated fatty acids can also be adsorbed on or mixed w;th polydextran or gels of poLyacrylamide, alkylsulfate or aLkylphosphate, or on aluminum hydroxide or silica.
The preparations for dermal administration are, for ex-ample, aqueous, alcoholic or polyol-containing solutions or suspensions, oily solutions c,r suspens;c,ns, or powders for subsequent ;ncorPorat;on into a dermally appl;cable form by add;tion of the requ;site carrier. The solutions or suspens;ons are formulated w;th the requisite pharma-ceutical auxil;aries and excipients contain;ng, for ex-ample, fats, waxes~ petroleum jelly or paraffin, and pres-ervat;ves, susPend;ng and dispers;ng agents and ;sotonic agents, such as, for example, methylparaben and propyl-paraben, sodium chloride, polyethylene glycols, especially polyethylene glycol 4000, sod;um carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidon, sorbimacrogol oleate, a condensation product of ethylene ox;de ~;th fatty acids, for example polyoxyethylene stearate, or ~ith fatty alco-hols such as, for example, heptadecaethylenoxycetanol, or with part;al esters such as, for example, polyoxyethylene-sorbitol monooleate, or hexitans originating from sorbitol, such as, for example, polyoxyethylene-sorbitan monooleate.
Suspensions in oily med;a are prepared by d;spersing the active compound in the abovementioned oils. These sus-pensions can contain agents which delay absorption, suchas, for example, alu~inum monostearate. A dry product, for example a free2e-dr;ed product, wh;ch can be m;xed ~;th a suitable pharmaceut;cal carr;er at the des;red t;me, represents a further embod;ment. Preparations from the active sompound, such as, for example, tablets, cap-sules, suppos;tories, syrups, solut;ons, suspens;ons and elix;rs, conta;n pharmaceut;cal aux;l;ar;es which render the preparation su;table for these adm;nistration forms ;n order to achieve the medical effect. The mixtures ~L3~)~2~6 accord;ng to the invention are mixed with pharmaceutical aux;liaries such as lactose, starch, acacia gum, gelatin, talc and the like and processed to g;ve capsules, tablets and the l;ke or to give conventional suppos;tory ~ompos-itions, for example those based on triglycerides, suchas, for example witepsoL suppository compositions (H.P. Fiedler, Lexikon der H;lfsstoffe fUr Pharmazie, Kosmetik und angrenzende Gebiete ~Dictionary of Auxiliar-ies for Pharmacy~ Cosmetics and Related Fields~, 1971, volume 9, pages 548-50 and 632-634, or for intravaginal suppositories US Patents 263,026 and 663,145~; if approp-riate, fatty alcohols; solid hydrocarbons, such as petrol-eum jelly or paraffin solidum; saturated fatty acids, such as lauric acid, myr;stic acid, palmitic acid and stearic acid; emulsifiers such as ethoxylated triglycer-ides and polyethoxylated vegetable oils; fatty acid sugar esters; silicones; geLatin; methyl cellulose; hydroxy-propoxy cellulnse and hydroxypropyl cellulose; polyethylene glycols; polyvinylpyrrolidone; polyvinyl alcohol; poly-acrylic acid and salts thereof, or are mixed with water,suspending agents, sucrose, preservatives or hydro-alcoholic exc;p;ents and processed to g;ve suspens;ons solut;ons or el;x;rs. lt may be desirable for the unsaturated fatty acid to be released slowly or ~ith delay from the pharm-aceutical formulation. Such a delayed release can beaccomplished in accordance with the state of the art. It depends on the pH of the b;olog;cal med;um whether the delayed release ;s accompl;shed, for example, by means of polystyrene or polyacrylic derivat;ves ~Eudragit~R)) or, for example, by bind;ng to a su;table ;on exchanger~
Enter;cally coated products, ~or example tablets, cap-sules, p;lls, beads or microbeads, can also be prepared.
They are in the form of dosage un;ts, for example as a s;ngle tablet or capsule or in the form of a collect;on of p;Lls, microbeads or microspheres which have enteric properties and a prolonged action. Such entericalLy coated products and the coating materials used for this purpose are described ;n the literature, for example in US Patents 2~093,462 and 2,196,768 (cellulose acetate -" ~30~2~6 phthalate), ~,897,121 (copolymers of styrene and maLe;c - acid), 3,081,233 and others.
Experimental part:
~ . .
Method 1: Investigation of the stability of unsaturated fatty acids in the formulations The stabilizing action of substances on polyunsaturated fatty acids was inuestigated as follows Test A for water-soluble substances:
0.125 mg of unsaturated fat~y acid or Na salt (see tables) (sodium arach;donate, from Sigma, Munich, Germany) is dissolved in 0.05 ml of ~ater. 0.2 ml of aqueous solution which contains 2 mg of stab;l;zer and can add;tionally contain 2 mg of st;mulator is mixed in (cf. Table 1), 0.1 ml is then taken out immediately, extracted and ana-lysed (see below). Further processing according tomodification 1 or 2.
Modification 1 for_aqueous solutions After 24 hours incubation at 37C, 0.1 ml is again taken, extracted and analysed (see below).
Mod;fication_2 for solid mixtures (lyophilisates) ._ The test solution is lyophilized, the lyophilisate is incubated for 24 hours at 37C and then dissolved again in the previous quantity of water, and 0.1 ml is takenr extracted and analysed tsee below).
Test ~ for water-insoluble substances:
0.125 mg of unsaturated fat~y acid (arachidonic acid, 5,8,11,14,17-eicosapentaenoic acid or 8,11,14-eicosatrienoic acid, from Sigma, Munich) is dissolved ` - -in 0.05 mL of ethanol or dimethyl sulfoxide. 0.2 ml of ethanol (or d;methyl sulfoxide) which contains 2 mg of stabilizer and can additionally conta;n 2 mg of She stim-ulator (cf. TabLe 1) is added thereto, and 0.1 ml ;s taken immediately and, as below, extracted and analysed. Further treatment according to modification 1 or 2.
Modification 1 for solutions After 24 hours' incubation at 37C~ 0.1 ml is again taken, mixed with 0.1 ml of water, extracted and analysed.
Modification 2 for solid mixtures The remaining solution is freed of solvent, incubated for 24 hours at 37C and then dissolved again in 0.15 ml of solvent, and 001 ml ;s taken, mixed with 0.1 ml of water, extracted and analysed.
~traction and analysis 0.05 ml of 1.2 M aqueous citric acid solut;on ;s added to the samples obta;ned, and the samples are extracted with twice 0.5 ml of ethyl acetate, the ethyl acetate is removed from the combined extracts and the residue is dissolved in 0.8 ml of methanol and analysed by means of high-pressure liquid chromatography (HPLC) (Rad Pak C-18 column, 100 x 8 mm, from ~aters, K~nigstein, Germany, 1.5 ml/minute, solvent 800 ml of methanol, 200 ml of ~ater and 0.1 ml of glacial acetic acid)~ The separa~;on is monitored uith a commercially available UV detector at 206 nm.
Method 2: Prostagland;n synthesis 0.3 ml of 0.03 M potassium phosphate buffer of pH = 8.0 contains 0.5 mg of enzyme (from seminal ves;cLes of sheep) tBiochemistry 10 (1971) 2372) or homogenate of the inner kidney medulla (L;fe Sciences 26 (1980) 765), 2.75 ~9 of 14C-arachidonic acid (0.5 ~Ci~ and 0.5 mg of stimulator, and, if appropriate, 0.5 mg of stabilizer substance (see Table 1). After 10 minutes' incubation at 37C~ the re-action is stopped with 0.05 ml of 1.2 M citric acid and the mixture is extracted with twice 0.5 ml of ethyl acetate, the combined extracts are freed of solvent, and the resi-due is dissolved in 0.2 ml of methanol, diluted 1:1 with water and separated by HPLC (nucleosil C-18 column, 125 x 4.6 mm, from ~ischoff, Leonberg, Germany, solvent 300 ml of acetonitrile, 700 ml of water and 1 ml of glacial ace-tic acid and after 30 minutes pure methanol)~ The sepa-ration of the radioactive fractions is followed by a monitor (L~ 504 from ~erthold, Wildbad, Germany). The products formed~ such as 6-keto-prostaglandin F2alpha~
prostaglandin E2, prostaglandin F2alpha and pros~aglandin D2, are separated under these conditions.
Table 1 Stimulation of the prostaglandin formation in stabilized fatty acid formulat;ons The prostaglandins PGE2, PGF2alpha and PGD2 as products from arachidonic acid where quantified by means of method 2, and the sum was related as "total prostaglandin" to 100X. The quantity of "total prostaglandin" formed ~ith-out the add;t;on of a stimulator was set at f=1; the quan-tity formed after s~imulation was related thereto. f=2 or f=3 therefore means twice or three times the stimulation under the conditions of method 2. The stability of the sample was investigated by means of method 1 (right-hand column). W;thout additions (right-hand column: A), 25-28%
of the arachidonic acid remain in aqueous solution under these conditions. Values greater than 25-28~ therefore represent a stabilizat;on, and values smaller than 25-28 repre ent a destabil;za~;on. Example No. 1 W3S carr;ed out ~ithout a stimulator. The effects according to the ;nvention can be obta;ned w;th the stabilized formulations from the correspondingly designated examples. The sub-stances test w;th respect to stabilization (see Method 1 for quantit;es) were: A) aqueous solution without o~
stab;lizer addition, 0) ethanolic solution without further - addition, C) pyridine solution, D) with rutin, predis-solved in dimethyl sulfoxide, dimethyl sulfoxide then stripped off, E) like B) ~ith lecithin, ethanol stripped off, F) like A) ~ith cytochrome c from horse heart (from Boehringer, Mannheim, Germany), G) like A) with methemo-globin (human) (from Sigman, Munich, Germany), H) like A) with bovine serum albumin (from Calbiochem, Frankfurt, Germany), J) like A) or B), but solvent s~ripped off, K) like J) with cytochrome c, L) like J) with hemoglobin (human) (from ~ehring~erke, Marburg, Germany), and M) like J) with human serum albumin (from Behringwerke, Marburg, Germany).
`" ~3~ 6 c, _ Arachidonic acid product Stability (Method 2) (% of remaining arachidonic acid Example Stimulator relative PGE2 PGF2 ~ h PGD2 by Method 1) prosta-No~ glandin ________________._ .___...___._.________~__..__.___________________________._____ f t Comparative investigation l)without stimulator l 83 2 l5 B lO0 H lO0 ~ 13 J 0 D 97 ~ lO0 F 98 M lO0 Examples of substances not according to the ;nvention , .
2) m-cresol 0.61 98 0 2 3) phenol 0.95 77 5 18 .
4) L-phenylalanine 0.9 80 2 18 ) trans-cinnamic acidO 9 Examples of compounds according to the invention of the formula I
6) Rl --cM2-~NH2 5~5 81 B lO0 X lO90 R2 ~ R~ ~-H C 67 M lO0 7) Rl -CH2;CH2~H2 4.0 80 3 17 A 87 8) Rl ~-CH2-CH2-CD2H 4,; 83 1 16 A 89 R2 ~ R3 ~-H
~ ~3~;~Z~6 9) Rl Y-ai2~ NH2 9.4 7014 16 ~is F, 97 O2H ~ 97 G96 Rz ~-OH
R3 ~-H
10) ~ CH2~ NH2 6.0 5235 13 A 17 H100 R2 a-OH B 95 X98 R3 ~-H C 9 M100 11) ~1 '-CH2- ~ N~ GH3 (Stimulation: posit;ve) A (Stabilization: positive) 12) Rl ~-C~OH-CH2-NH2 8.3 66 17 17 A 11 F 99 R2 ~-OH ' B 93 G 95 R3 ~-~ D 93 M lOO
13) Rl ~-CHOH-CM2-NHCH3 13 70 12 18 A 9 H 97 R2 ~-OH B 92 J 0 R3 ~-H E 94 M 100 14) Rl W-OH 3,2 69 12 19 A 7 Ç g9 R2 ~ R3 -H D 97 ~ 97 15) Rl ~-CO2H 5,1 71 12 17 A 22 ~ 98 R2 ~-OCH3 B lOO G 96 R3 .-H D 98 J 17 16) ~1 '-c~2H 4.5 69 13 18 A 21 F 98 _ R~ ~ R~ --H B 98 G 96 17) R3 ~-H 13 D 99 HM 919968 : Examples of compounds of the formula II, according to the invention 18) Rl -COzH 9.B 76 3 21 A 18 19) Rl '-C2~ lO,l ~9 ~ 18 ~ 17 E 9S
20~ R2 ~ R~ S~l 82 lC0 F 98 ~L3(~22iEiil~
..
21) Rl D-C02H 5,7 80 3 17 A 18 G 962) R2 ~-H B 99 H 97 R3 ~-OH F 9~ J 12 22) Rl ~H 10.6 75 5 20A 16 F 98 R2 W-NHz B 99 ~ 98 R3 ~-OH E 96 L 100 23) R1 --H 6.1 81 3 16A 20 F 98 Rz ~-NHz D lOO G 97 R3 ~-H E 971) H 500 Examples of pept;des according to the invent;on, containing tyrosine (Tyr) or tryptophan (Trp) 2~) Srp-Srp ll 7 75 2 23A 17 D 97 25) Gly-T2p-Gly 13,0 79 1 20A lB H 99 G 97 ~ 99 26) Trp-Ala 12.6 77 1 22~ 19 E 92 27) Leu-T~p 17.0 77 1 22A 20 F 98 28) Trp-Met-Asp-Phe11~8 73 7 20A 17 D 971) 29~ lou~ 5et 10.8 69 9 22A 18 H 99 ~ S9 J
D 971~ 109 30) Tyr~r-l)~r-OCH3 5.~ 75 ~ 11 A 94 J 98 ~1) ~r~Trp 6.~ 73 3 14. A 80 D 98 32) Cyclo-Trp-Tyr tStimulation: positive) (Stabilization: positive) Examples of sulfur-containing compounds according to the invention 33) L-cysteine 4.9 56 17 27 A 9 D 95 -- D 943)M 96 34) D-cysteine ~,5 63 14 23 ~ 1~ H 97 D 96 J 52) D 95 ~ 98 . . :
3~ 6 -: - 22 -~5) Glutathione 13.5 lO0 0 0 A 0 H 9l B ~0 J 0 56) Lipoic acid 5,5 ~ ~9 8 ~ 92 G 100 F lO0 L lO0 ~7) N-Acetylcysteine 4.0 61 14 25 A 9 E 93 ~ 93 F 98 D 95 t~ 98 38) TyT-Cys-5lu-Hls--ph~-Ar~-Trp-Gly tStimulation positive) M 100 Sulfur-containing compounds not according to the inven~ion ~9) ~ithïothreitol 0~8 40) Dithioerythritol 0,85 4l) Cystine l.0 85 0 l5 ~2) Dihydrolipoic acid l.0 4~) Cysteamine 0 9 ~ Mercaptoethanol 0 95 : ~) Coenzyme A l.0 -~) Pantetheine o gs l) _ 3) instead or arachidonic acid, 5~8~ 4~17~-eicosapentaenoic acid was used in 1), 8,11,14-e;cosatrienoic acid was used in 2), and 9,12,15-linolenic acid was used-in 3)~-4): Hare gamma-globulin tfrom Calbiochem, Frankfurt) ~3~ 6 Table 2 Investigations of the properties, accorcling to the invention, of vitamins, flavonoids, cyclodextrins and (sugar )lipid compounds. The fatty acids used in the stabilization experiment are indicated in brackets.
(A = arachidonic acid, E = 5,8,11,14,17-eicosapentaenoic acid~
Arachidonic acid product Stabilization (Method 2) (% remaining unsaturated Example Stimulator relatlive PGE2 2alpha 2 fatty acid by prosta-No. glandin ~_~_____~___~_~_v__~_~o_~_~o_______.~_.~.__.__...~_._._.._._._._._..
~_, , _ , , , . ,, . _ _. . .
Vitamins not accordjng to the invention A7) Vjta~in Qlo 0. 8 8 78 14 8~ (A) 48) Vitamin Bl~ 6~ ~A3 49~ Yi~in C 0% (A) Vitamins according to the invention ~0~ DL-alpha-Tocopher~l 78~ (A) Sl) D-~lphaeTocoph Iy 0,~ tO ~ lS 98% (A) Sugar-containing flavonoids according to the invention 52) Ru~in 6 76 6 18 97~ (A~
53) Phlorizin ~ gO 2 18 97~ (E) ~ Naringin 2 U 2 lS 96~ (A) Flavonoids according to the invention without a sugar moiety 5~ Naringenin l~S 80 0 20 96% ~A~
~6) Kaempferol 0~5 76 ll 13 9e% tE) ~:~[)2;~
57) Quercetin ~3 72 18 10 92~(E) 58~ Catechin 0.2 70 18 12 ~(A) S9) Epicatechin 0.15 71 17 12 ~9~(A) 60) Phloretin 0.15 76 11 13 97~ (A) 61) Morin 0.08 54 27 19 100%(E~
All the flavonoids investigated stabilize the polyunsaturated fatty acids. The sugar-containing flavonoids are particularly suitable according to the invention, because they stimulate the formation of the prostaglandins. Particularly for the use according to the invention in vitro, the flavonoids without a sugar moiety are less suitable, because they inhibit the prostaglandin synthesis.
C~clodextrins accordin~
to the invention 62 ) lpha~Dextrin (Cyclohe~taaloylose) 60~ (A) 63~ beta-De~trin ~Cycloheptaa~Dylose) 90: (A) 64) gar~a-Dextrir, (Cyclooctaamylose) 75~ (A) - Lip;ds according to the invention (from Serva, Heidelberg) 65) ~croscpa1oitate 91~ (A) secaraee7 9~(E) 66) ~c~se ~lmitate stcarate15 92S (A) SP~-li 67) ~a dioctYlsu~fosuccinate 94~ (A) ~xamples of stable formulations accord;ng to the invention, ~ ;ncluding pharmaceutical formulations Examples 68) to 70) _ _ _ _ _ .
50 mg of sodium arachidonate, 500 mg of bov;ne serum al-bumin and 500 mg of L-tyrosine are dissolved under sterile conditions in 10 ml of twice distilled water and, likewise under sterile conditions, stored in a brown bottle. 93.5%
of the arachidonic acid originally present are recovered after 6 weeks' storage at room temperature, whereas the same sodium arachidonate solution without additions vir-tually no longer contain any arachidonic acid after this time (content < 0.8%). The stabilized solution prepared in this way Example 68) filled into a drinking ampoule for medical purposes, ExampLe 6~) treated ~ith 15 mg of seminal vesicles of sheep and incubated for 60 minutes at 37C, and the prostaglandins formed are then extracted and preparatively isolated - as described in Method 2, or or Example 70) used for the clinical-biochemical deter-mination of the thrombophilia of test subjects (patients), by adding 1 ml of lyzed platelet-rich blood plasma from these test subjects to 0.01 ml of this stabilized sodium arachidonate solution and extracting after 30 minutes' incubation at 25C. The prostaglandin D2 formed (Method 2) is used as a measure of the thrombophilia of the patient.
Example 71) 100 mg of 5,8~11,14,17-eicosapentaenoic acid and 500 mg of rutin are taken up together in 15 ml of dime$hyl sulfoxide, the dimethyl sulfoxide is completely stripped off ar,d the residue is transferred, together with ~l311~2~
acetyl salicylic acid crys~als (S00 mg of R 95 D, from RUhm Pharma, Darmstadt~ Federal Republic of Germany) into size 0 gelat;n capsules (from Kapsugel, Basel, SwitzerLand~.
Example 72) 1 9 of L-alpha-lecithin, beta, gamma-dipal-mitoyl (from Calbiochem GmbH, Frankfurt/Main, Germany), 100 mg of arachidonic acid, 100 mg of lipoic acid and 100 mg of DL-alpha-tocopherol acetate (from Aldrich, Steinheim, Germany) are stirred into 10 ml of twice dis-tilled water, and the mixture is homogenized by ultra-sonic treatment.
Example 73) 170 mg of wheat starch, 450 mg of lactose, 20 mg of magnesium stearate, 5 mg of sodium arachidonate, 10 mg of tryptophan and 20 mg of troxerutin (from Aldrich, Steinheim, Germany) are ground, intimately mixed and pressed to give a tablet.
Example 74? 150 mg of wheat starch, 435 mg of lactose, 15 mg of magnesium stearate~ 5 mg of sodium arachidonate and 15 mg of lipoic acid are intimately mixed by grinding and pressed to give a tablet.
-Example 75) 10 mg of sodium arachidonate are taken up with 20 mg of serotonine and 100 mg of hemoglob;n (reduced,from Calbiochem GmbH, Frankfurt) in 3O5 ml of water (twice distilled) and lyophili~ed. The residue can be processed to give dosage units ;n capsules (size 0 gelatin capsules from Kapsugel, ~asel, Sw;tzerland).
Example 7$) 15 mg of 8,11,14-eicosatrieno;c acid are taken up with 50 mg of N-acetylcysteine and 100 mg of cytochrome c from horse heart ~from Aldrich, Steinheim, Germany) in 2 ml of water ttwice distilled).
ExampLe 77) 40 mg of arachidonic acid are taken up under sterile conditions with 180 mg of beta-cyclodextrin and 60 mg of 5-hydroxytryptamin in 50 ml of water (twice distilled).
` ~30Z;2~6 Example 78 a-f) 6 ~atches a, b, c, d, e and f are prepared . . . .. __ ~ ~hich each contain~ in 1 ml of ethanol, 0.7 mg of arach-idonic acid and, respectively~ a) 1, b) 5, c) 10, d) 25, e) 50 and f) lO0 mg of lipoic acid. After the ethanol has been stripped off, these batches can be kept for at least 12 days at 37C. The determination according to Method 2 gave still 99 2% of arachidonic acid in the batches after 12 days.
F xamples 79) - 82~
0.125 mg of arachidonic acid are taken up in 0.25 ml of water, which contains 5 or 25 or 50 or 75 percent by ~eight of dimethyl sulfoxide, and the mixtures are incub-ated at 37C. Extraction and analysis are carried out after 24 hours. A typical analysis gave ~ of dimethyl sulfox;de % of arach;donic acid .
Example 79) 5 54 Example 80) 25 75 Example 81) 50 83 Example 82) 75 94 -Example 03) 2 g of arachidonic acid and 1 9 of tyrosamine are st;rred into 5 ml of glycerole triacetate and uni-formly distributed by ultrason;c treatment at 30C.
6) Rl --cM2-~NH2 5~5 81 B lO0 X lO90 R2 ~ R~ ~-H C 67 M lO0 7) Rl -CH2;CH2~H2 4.0 80 3 17 A 87 8) Rl ~-CH2-CH2-CD2H 4,; 83 1 16 A 89 R2 ~ R3 ~-H
~ ~3~;~Z~6 9) Rl Y-ai2~ NH2 9.4 7014 16 ~is F, 97 O2H ~ 97 G96 Rz ~-OH
R3 ~-H
10) ~ CH2~ NH2 6.0 5235 13 A 17 H100 R2 a-OH B 95 X98 R3 ~-H C 9 M100 11) ~1 '-CH2- ~ N~ GH3 (Stimulation: posit;ve) A (Stabilization: positive) 12) Rl ~-C~OH-CH2-NH2 8.3 66 17 17 A 11 F 99 R2 ~-OH ' B 93 G 95 R3 ~-~ D 93 M lOO
13) Rl ~-CHOH-CM2-NHCH3 13 70 12 18 A 9 H 97 R2 ~-OH B 92 J 0 R3 ~-H E 94 M 100 14) Rl W-OH 3,2 69 12 19 A 7 Ç g9 R2 ~ R3 -H D 97 ~ 97 15) Rl ~-CO2H 5,1 71 12 17 A 22 ~ 98 R2 ~-OCH3 B lOO G 96 R3 .-H D 98 J 17 16) ~1 '-c~2H 4.5 69 13 18 A 21 F 98 _ R~ ~ R~ --H B 98 G 96 17) R3 ~-H 13 D 99 HM 919968 : Examples of compounds of the formula II, according to the invention 18) Rl -COzH 9.B 76 3 21 A 18 19) Rl '-C2~ lO,l ~9 ~ 18 ~ 17 E 9S
20~ R2 ~ R~ S~l 82 lC0 F 98 ~L3(~22iEiil~
..
21) Rl D-C02H 5,7 80 3 17 A 18 G 962) R2 ~-H B 99 H 97 R3 ~-OH F 9~ J 12 22) Rl ~H 10.6 75 5 20A 16 F 98 R2 W-NHz B 99 ~ 98 R3 ~-OH E 96 L 100 23) R1 --H 6.1 81 3 16A 20 F 98 Rz ~-NHz D lOO G 97 R3 ~-H E 971) H 500 Examples of pept;des according to the invent;on, containing tyrosine (Tyr) or tryptophan (Trp) 2~) Srp-Srp ll 7 75 2 23A 17 D 97 25) Gly-T2p-Gly 13,0 79 1 20A lB H 99 G 97 ~ 99 26) Trp-Ala 12.6 77 1 22~ 19 E 92 27) Leu-T~p 17.0 77 1 22A 20 F 98 28) Trp-Met-Asp-Phe11~8 73 7 20A 17 D 971) 29~ lou~ 5et 10.8 69 9 22A 18 H 99 ~ S9 J
D 971~ 109 30) Tyr~r-l)~r-OCH3 5.~ 75 ~ 11 A 94 J 98 ~1) ~r~Trp 6.~ 73 3 14. A 80 D 98 32) Cyclo-Trp-Tyr tStimulation: positive) (Stabilization: positive) Examples of sulfur-containing compounds according to the invention 33) L-cysteine 4.9 56 17 27 A 9 D 95 -- D 943)M 96 34) D-cysteine ~,5 63 14 23 ~ 1~ H 97 D 96 J 52) D 95 ~ 98 . . :
3~ 6 -: - 22 -~5) Glutathione 13.5 lO0 0 0 A 0 H 9l B ~0 J 0 56) Lipoic acid 5,5 ~ ~9 8 ~ 92 G 100 F lO0 L lO0 ~7) N-Acetylcysteine 4.0 61 14 25 A 9 E 93 ~ 93 F 98 D 95 t~ 98 38) TyT-Cys-5lu-Hls--ph~-Ar~-Trp-Gly tStimulation positive) M 100 Sulfur-containing compounds not according to the inven~ion ~9) ~ithïothreitol 0~8 40) Dithioerythritol 0,85 4l) Cystine l.0 85 0 l5 ~2) Dihydrolipoic acid l.0 4~) Cysteamine 0 9 ~ Mercaptoethanol 0 95 : ~) Coenzyme A l.0 -~) Pantetheine o gs l) _ 3) instead or arachidonic acid, 5~8~ 4~17~-eicosapentaenoic acid was used in 1), 8,11,14-e;cosatrienoic acid was used in 2), and 9,12,15-linolenic acid was used-in 3)~-4): Hare gamma-globulin tfrom Calbiochem, Frankfurt) ~3~ 6 Table 2 Investigations of the properties, accorcling to the invention, of vitamins, flavonoids, cyclodextrins and (sugar )lipid compounds. The fatty acids used in the stabilization experiment are indicated in brackets.
(A = arachidonic acid, E = 5,8,11,14,17-eicosapentaenoic acid~
Arachidonic acid product Stabilization (Method 2) (% remaining unsaturated Example Stimulator relatlive PGE2 2alpha 2 fatty acid by prosta-No. glandin ~_~_____~___~_~_v__~_~o_~_~o_______.~_.~.__.__...~_._._.._._._._._..
~_, , _ , , , . ,, . _ _. . .
Vitamins not accordjng to the invention A7) Vjta~in Qlo 0. 8 8 78 14 8~ (A) 48) Vitamin Bl~ 6~ ~A3 49~ Yi~in C 0% (A) Vitamins according to the invention ~0~ DL-alpha-Tocopher~l 78~ (A) Sl) D-~lphaeTocoph Iy 0,~ tO ~ lS 98% (A) Sugar-containing flavonoids according to the invention 52) Ru~in 6 76 6 18 97~ (A~
53) Phlorizin ~ gO 2 18 97~ (E) ~ Naringin 2 U 2 lS 96~ (A) Flavonoids according to the invention without a sugar moiety 5~ Naringenin l~S 80 0 20 96% ~A~
~6) Kaempferol 0~5 76 ll 13 9e% tE) ~:~[)2;~
57) Quercetin ~3 72 18 10 92~(E) 58~ Catechin 0.2 70 18 12 ~(A) S9) Epicatechin 0.15 71 17 12 ~9~(A) 60) Phloretin 0.15 76 11 13 97~ (A) 61) Morin 0.08 54 27 19 100%(E~
All the flavonoids investigated stabilize the polyunsaturated fatty acids. The sugar-containing flavonoids are particularly suitable according to the invention, because they stimulate the formation of the prostaglandins. Particularly for the use according to the invention in vitro, the flavonoids without a sugar moiety are less suitable, because they inhibit the prostaglandin synthesis.
C~clodextrins accordin~
to the invention 62 ) lpha~Dextrin (Cyclohe~taaloylose) 60~ (A) 63~ beta-De~trin ~Cycloheptaa~Dylose) 90: (A) 64) gar~a-Dextrir, (Cyclooctaamylose) 75~ (A) - Lip;ds according to the invention (from Serva, Heidelberg) 65) ~croscpa1oitate 91~ (A) secaraee7 9~(E) 66) ~c~se ~lmitate stcarate15 92S (A) SP~-li 67) ~a dioctYlsu~fosuccinate 94~ (A) ~xamples of stable formulations accord;ng to the invention, ~ ;ncluding pharmaceutical formulations Examples 68) to 70) _ _ _ _ _ .
50 mg of sodium arachidonate, 500 mg of bov;ne serum al-bumin and 500 mg of L-tyrosine are dissolved under sterile conditions in 10 ml of twice distilled water and, likewise under sterile conditions, stored in a brown bottle. 93.5%
of the arachidonic acid originally present are recovered after 6 weeks' storage at room temperature, whereas the same sodium arachidonate solution without additions vir-tually no longer contain any arachidonic acid after this time (content < 0.8%). The stabilized solution prepared in this way Example 68) filled into a drinking ampoule for medical purposes, ExampLe 6~) treated ~ith 15 mg of seminal vesicles of sheep and incubated for 60 minutes at 37C, and the prostaglandins formed are then extracted and preparatively isolated - as described in Method 2, or or Example 70) used for the clinical-biochemical deter-mination of the thrombophilia of test subjects (patients), by adding 1 ml of lyzed platelet-rich blood plasma from these test subjects to 0.01 ml of this stabilized sodium arachidonate solution and extracting after 30 minutes' incubation at 25C. The prostaglandin D2 formed (Method 2) is used as a measure of the thrombophilia of the patient.
Example 71) 100 mg of 5,8~11,14,17-eicosapentaenoic acid and 500 mg of rutin are taken up together in 15 ml of dime$hyl sulfoxide, the dimethyl sulfoxide is completely stripped off ar,d the residue is transferred, together with ~l311~2~
acetyl salicylic acid crys~als (S00 mg of R 95 D, from RUhm Pharma, Darmstadt~ Federal Republic of Germany) into size 0 gelat;n capsules (from Kapsugel, Basel, SwitzerLand~.
Example 72) 1 9 of L-alpha-lecithin, beta, gamma-dipal-mitoyl (from Calbiochem GmbH, Frankfurt/Main, Germany), 100 mg of arachidonic acid, 100 mg of lipoic acid and 100 mg of DL-alpha-tocopherol acetate (from Aldrich, Steinheim, Germany) are stirred into 10 ml of twice dis-tilled water, and the mixture is homogenized by ultra-sonic treatment.
Example 73) 170 mg of wheat starch, 450 mg of lactose, 20 mg of magnesium stearate, 5 mg of sodium arachidonate, 10 mg of tryptophan and 20 mg of troxerutin (from Aldrich, Steinheim, Germany) are ground, intimately mixed and pressed to give a tablet.
Example 74? 150 mg of wheat starch, 435 mg of lactose, 15 mg of magnesium stearate~ 5 mg of sodium arachidonate and 15 mg of lipoic acid are intimately mixed by grinding and pressed to give a tablet.
-Example 75) 10 mg of sodium arachidonate are taken up with 20 mg of serotonine and 100 mg of hemoglob;n (reduced,from Calbiochem GmbH, Frankfurt) in 3O5 ml of water (twice distilled) and lyophili~ed. The residue can be processed to give dosage units ;n capsules (size 0 gelatin capsules from Kapsugel, ~asel, Sw;tzerland).
Example 7$) 15 mg of 8,11,14-eicosatrieno;c acid are taken up with 50 mg of N-acetylcysteine and 100 mg of cytochrome c from horse heart ~from Aldrich, Steinheim, Germany) in 2 ml of water ttwice distilled).
ExampLe 77) 40 mg of arachidonic acid are taken up under sterile conditions with 180 mg of beta-cyclodextrin and 60 mg of 5-hydroxytryptamin in 50 ml of water (twice distilled).
` ~30Z;2~6 Example 78 a-f) 6 ~atches a, b, c, d, e and f are prepared . . . .. __ ~ ~hich each contain~ in 1 ml of ethanol, 0.7 mg of arach-idonic acid and, respectively~ a) 1, b) 5, c) 10, d) 25, e) 50 and f) lO0 mg of lipoic acid. After the ethanol has been stripped off, these batches can be kept for at least 12 days at 37C. The determination according to Method 2 gave still 99 2% of arachidonic acid in the batches after 12 days.
F xamples 79) - 82~
0.125 mg of arachidonic acid are taken up in 0.25 ml of water, which contains 5 or 25 or 50 or 75 percent by ~eight of dimethyl sulfoxide, and the mixtures are incub-ated at 37C. Extraction and analysis are carried out after 24 hours. A typical analysis gave ~ of dimethyl sulfox;de % of arach;donic acid .
Example 79) 5 54 Example 80) 25 75 Example 81) 50 83 Example 82) 75 94 -Example 03) 2 g of arachidonic acid and 1 9 of tyrosamine are st;rred into 5 ml of glycerole triacetate and uni-formly distributed by ultrason;c treatment at 30C.
Claims (21)
1. A formulation for the synthesis of prostaglandins and hydroxy-fatty acids in biological systems, which comprises:
A) one or more unsaturated fatty acid(s), defined by three to five isolated double bonds at 18 to 22 carbon atoms which are arranged in a straight chain and can be methylated or ethylated at one or two carbon atoms in positions 2, 3, 4, 16, 17, 18, 19 or 20, as the free carboxylic acid with terminal -CO2H, or as the carb-oxamide or as -CO2X, X being a protective group which can be eliminated under acidic conditions or a 1- or
A) one or more unsaturated fatty acid(s), defined by three to five isolated double bonds at 18 to 22 carbon atoms which are arranged in a straight chain and can be methylated or ethylated at one or two carbon atoms in positions 2, 3, 4, 16, 17, 18, 19 or 20, as the free carboxylic acid with terminal -CO2H, or as the carb-oxamide or as -CO2X, X being a protective group which can be eliminated under acidic conditions or a 1- or
2-lysophospholipid or a metal or amine cation or the cationic form of an ion exchanger, and B) as stimulators with, in some cases, a simultaneous stabilizing action, one or more compounds from the following groups:
B1) a phenolic compound of the formula I
I
in which the radicals R2 and R3 are hydroxyl groups or hydrogen and R1 is a radical -OH, -CO2H, -CH2-COOH, -CH=CH-CO2H, -CH2-CHR4R5 or -CHOH-CH2-NH-R6 with R4 = -H or -CO2H and R5 = -H or -NH2 and R6 =
-H, -CH3 or -C2H5, B2) an indole derivative of the formula II
II
in which R7 is hydrogen or -COOH, R8 is hydrogen or -NH2 and R9 is hydrogen or OH, B3) cysteine, homocysteine or lipoic acid, the acyclic alkyl radical of which can be shortened by up to four methylene groups, B4) a peptide comprising a maximum of ten amino acids, wherein one or more of the amino acids are replaced by one of the compounds according to B1) to B3) in each case, B5) an amino compound according to B1) to B4) wherein one N atom can be substituted by a C1-C4-alkanoyl group, B6) a flavone derivative which is substituted by at least one hydroxyl group which can carry a sugar radical, B7) a salt of the ionic forms of the compounds accord-ing to B1) - B6) and, B8) a carboxylic acid compound according to B1) to B7) which can be esterified with an alkoxy radical or be in the form of the carboxamide which can also be mono- or di-alkylated.
2. A formulation as claimed in claim 1, which comprises as stabilizers, one or more compounds from the following groups:
C1) dimethyl sulfoxide, ethanol, polyols or polyol esters and C2) phospholipids, sugar lipids, cyclodextrins, proteins, cytochromes of the C series or vitamins of the E series, in the solid or liquid form.
B1) a phenolic compound of the formula I
I
in which the radicals R2 and R3 are hydroxyl groups or hydrogen and R1 is a radical -OH, -CO2H, -CH2-COOH, -CH=CH-CO2H, -CH2-CHR4R5 or -CHOH-CH2-NH-R6 with R4 = -H or -CO2H and R5 = -H or -NH2 and R6 =
-H, -CH3 or -C2H5, B2) an indole derivative of the formula II
II
in which R7 is hydrogen or -COOH, R8 is hydrogen or -NH2 and R9 is hydrogen or OH, B3) cysteine, homocysteine or lipoic acid, the acyclic alkyl radical of which can be shortened by up to four methylene groups, B4) a peptide comprising a maximum of ten amino acids, wherein one or more of the amino acids are replaced by one of the compounds according to B1) to B3) in each case, B5) an amino compound according to B1) to B4) wherein one N atom can be substituted by a C1-C4-alkanoyl group, B6) a flavone derivative which is substituted by at least one hydroxyl group which can carry a sugar radical, B7) a salt of the ionic forms of the compounds accord-ing to B1) - B6) and, B8) a carboxylic acid compound according to B1) to B7) which can be esterified with an alkoxy radical or be in the form of the carboxamide which can also be mono- or di-alkylated.
2. A formulation as claimed in claim 1, which comprises as stabilizers, one or more compounds from the following groups:
C1) dimethyl sulfoxide, ethanol, polyols or polyol esters and C2) phospholipids, sugar lipids, cyclodextrins, proteins, cytochromes of the C series or vitamins of the E series, in the solid or liquid form.
3. A formulation as claimed in claim 1, wherein the polyols or polyol esters to be used as stabilizers of group C1) are glycerole, ethylene glycol, polyethylene glycol or-glycerole triacetate respectively.
4. A formulation as claimed in claim 1, which comprises, as the fatty acids of group A), 18:2 .omega.-6, 20:4 .omega.-6, 22:5 .omega.-6, 18:3 .omega.-3, 20:5 .omega.-3, 22:6 .omega.-3, 18:3 .omega.-6, 20:3 .omega.-6, 22:4 .omega.-6 or 22:4 .omega.-3 fatty acids and, as the stimulators of group B), compounds of the formula I, wherein R2 and R3 are hydrogen and R1 is a radical CH2-CH(NH2)COOH or peptides which contain such compounds, or lipoic acid, the side chain of which can be shortened by up to 4 CH2 groups, or a flavone derivative, no addition of a stabilizer from group C) being necessary in some cases.
5. A formulation as claimed in claim 1, wherein the stabilizers of group C) are betadextrin, lecithin, proteins which can be prepared from human or mammal blood, or cytochromes of the C series.
6. A formulation as claimed in claim 1 or 4, wherein ruin or troxerutin are used as the sugar-containing flavone derivatives.
7. A formulation as claimed in claim 5, which comprises, as the stabilizers of the group C), hemoglobin or methemo-globin, globulin or serum albumin.
8. A formulation as claimed in claim 1, 2 or 3, which comprises, as the fatty acids of group A), arachidonic acid, 5,8,11,14,17-eicosapentaenoic acid, 8,11,14-eicosa-trienoic acid or 9,12,15-linolenic acid.
9. A formulation as claimed in claim 1, wherein the weight ratio of unsaturated fatty acid from group A) and stimulator from group B) is between 50 and 0.1.
10. A formulation as claimed in claim 9, wherein the weight ratio is between 20 and 0.5.
11. A formulation as claimed in claim 1, wherein the weight ratio of unsaturated fatty acid from group A) and stabilizer from group C) is between 100 and 0.5.
12. A formulation as claimed in claim 11, wherein the weight ratio is between 15 and 1.
13. A formulation as claimed in claim 1, which is in a form to be administered dermally, orally, perorally or as suppositories.
14. A formulation as claimed in claim 13 which contains, per dosage unit, between 0.5 and 2,000 mg of unsaturated fatty acid.
15. A formulation as claimed in claim 13 which contains, per dosage unit, between 5 and 250 mg of unsaturated fatty acid.
16. A formulation as claimed in claim 1, 2 or 3 wherein the unsaturated fatty acids are at least partially in a retarded form.
17. A medicament package, wherein the substances as claimed in claim 1, 2 or 3 are provided separately, at least one substance being separate and others being com-bined, but to be administrated together.
18. The use of a formulation as claimed in claim 1, 2 or 3 for the preparation of a medicament for curing prostaglandin deficiencies in humans or animals.
19. The use of the formulation as claimed in claim 1, 2 or 3 for the preparation of a medicament for curing or preventing diseases of the gastro-intestinal tract.
20. The formulation as claimed in claim 1, 2 or 3 for use in the preparation of a medicament for curing prostaglandin deficiencies in humans or animals.
21. The formulation as claimed in claim 1, 2 or 3 for use in the preparation of a medicament for curing or preventing diseases of the gastro-intestinal tract.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3615710.4 | 1986-05-09 | ||
| DE19863615710 DE3615710A1 (en) | 1986-05-09 | 1986-05-09 | PREPARATIONS FOR THE SYNTHESIS OF PROSTAGLANDINES AND HYDROXY FATTY ACIDS IN BIOLOGICAL SYSTEMS |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1302266C true CA1302266C (en) | 1992-06-02 |
Family
ID=6300504
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000536688A Expired - Fee Related CA1302266C (en) | 1986-05-09 | 1987-05-08 | Formulations containing unsaturated fatty acids for the synthesis of prostaglandins and hydroxy-fatty acids in biological systems |
Country Status (16)
| Country | Link |
|---|---|
| US (1) | US5043328A (en) |
| EP (1) | EP0244832B1 (en) |
| JP (1) | JPS62267222A (en) |
| KR (1) | KR950008306B1 (en) |
| AT (1) | ATE77549T1 (en) |
| AU (1) | AU603574B2 (en) |
| CA (1) | CA1302266C (en) |
| DE (2) | DE3615710A1 (en) |
| DK (1) | DK167518B1 (en) |
| ES (1) | ES2051705T3 (en) |
| GR (1) | GR3005768T3 (en) |
| HU (1) | HU201671B (en) |
| IE (1) | IE60273B1 (en) |
| IL (1) | IL82459A (en) |
| PT (1) | PT84841B (en) |
| ZA (1) | ZA873299B (en) |
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|---|---|---|---|---|
| US5334612A (en) * | 1988-11-09 | 1994-08-02 | Asta Pharma Aktiengesellschaft | Pharmaceutical compositions containing as active substance sulphur-containing carboxylic acids and their use in combating retroviruses |
| FR2639828B1 (en) * | 1988-12-01 | 1993-11-05 | Lvmh Recherche | USE OF KAEMPFEROL AND CERTAIN DERIVATIVES THEREOF FOR THE PREPARATION OF A COSMETIC OR PHARMACEUTICAL COMPOSITION |
| ES2033193B1 (en) * | 1990-10-30 | 1994-01-16 | Ganadera Union Ind Agro | FAT MIXTURE FOR CHILD AND ADULT NUTRITION. |
| US5399584A (en) * | 1992-05-05 | 1995-03-21 | The Procter & Gamble Company | Use of flavone derivatives for gastroprotection |
| US5569670A (en) * | 1992-06-05 | 1996-10-29 | Asta Medica Aktiengesellschaft | Combination medications containing alpha-lipoic acid and related |
| EP0679057B1 (en) * | 1993-01-15 | 1999-08-18 | Abbott Laboratories | Structured lipids |
| GB9715444D0 (en) * | 1997-07-22 | 1997-09-24 | Scotia Holdings Plc | Therapeutic and dietary compositions |
| JP2003514855A (en) * | 1999-10-13 | 2003-04-22 | チャコン，マルコ，エー． | Therapeutic intervention mimicking the effects of calorie restriction |
| AU781718B2 (en) * | 1999-11-02 | 2005-06-09 | Depomed, Inc. | Pharmacological inducement of the fed mode for enhanced drug administration to the stomach |
| US6228367B1 (en) * | 1999-12-22 | 2001-05-08 | Renew Life, Inc. | Food supplement formulation |
| CA2304906A1 (en) * | 2000-04-07 | 2001-10-07 | 1411198 Ontario Limited | 13-hode, a regulator of vascular biocompatibility and an inhibitor of cell hyperplasia |
| US7098352B2 (en) * | 2001-11-16 | 2006-08-29 | Virtus Nutrition Llc | Calcium salt saponification of polyunsaturated oils |
| MXPA04012740A (en) * | 2002-07-02 | 2005-03-23 | Galileo Pharmaceuticals Inc | Compositions and methods for reduction of inflammatory symptoms and/or biomarkers in female subjects. |
| US20050164908A1 (en) * | 2004-01-23 | 2005-07-28 | Ginsberg Myron D. | Neuroprotective complex for treatment of cerebral ischemia and injury |
| GB0403247D0 (en) | 2004-02-13 | 2004-03-17 | Tillotts Pharma Ag | A pharmaceutical composition |
| EP1765964A4 (en) * | 2004-04-30 | 2007-11-07 | Polyunsaturated fatty acid monovalent and divalent metal salt synthesis | |
| WO2006054316A1 (en) * | 2004-11-16 | 2006-05-26 | Magene Life Sciences Private Limited | Method(s) of preparation, stabilization, composition, and administration of gamma-linolenic acid for brain tumors |
| US20070082063A1 (en) * | 2005-10-11 | 2007-04-12 | Douglas Bibus | Risk assessment and correction of membrane damage of the upper GI tract |
| DK2349250T3 (en) * | 2008-10-31 | 2017-07-03 | Lipid Pharmaceuticals Ehf | Fatty acids for use as a medicament |
| WO2012010415A1 (en) * | 2010-07-22 | 2012-01-26 | Merz Pharma Gmbh & Co. Kgaa | Pharmaceutical composition containing a tryptophan derivative |
| WO2013087808A1 (en) | 2011-12-15 | 2013-06-20 | Merz Pharma Gmbh & Co. Kgaa | Pharmaceutical composition comprising a pyrazolopyrimidme and cyclodextrin |
| KR101991544B1 (en) | 2012-04-16 | 2019-06-20 | 로디아 오퍼레이션스 | Fluoropolymer compositions |
| GB201216385D0 (en) | 2012-09-13 | 2012-10-31 | Chrysalis Pharma Ag | A pharmaceutical composition |
| AU2014225619A1 (en) * | 2013-03-06 | 2015-10-15 | Rutgers, The State University Of New Jersey | Nutritional supplement/feed formula and methods of use thereof to reduce development of Osteochondrosis Dissecans (OCD) lesions |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE237029C (en) * | ||||
| FR2197605A1 (en) * | 1972-09-06 | 1974-03-29 | Williams John | Foodstuff or skin nutrient to combat cholesterolaemia - contains arachidonic acid or gamma linolenic acid or esters and salts thereof |
| JPS537492B2 (en) * | 1973-02-12 | 1978-03-18 | ||
| GB1476624A (en) * | 1973-05-30 | 1977-06-16 | Cepbepe | Tranquiliser medicaments |
| DE2813814A1 (en) * | 1978-03-31 | 1979-10-18 | Boehringer Mannheim Gmbh | Increasing the enteral reabsorption of pharmaceuticals - by administration together with an aminoacid or a peptide |
| IE53332B1 (en) * | 1980-03-14 | 1988-10-26 | Efamol Ltd | Pharmaceutical compositions |
| JPS5735512A (en) * | 1980-06-27 | 1982-02-26 | Nippon Oil & Fats Co Ltd | Preventive and remedy for thrombosis |
| JPS5813541A (en) * | 1981-07-16 | 1983-01-26 | Kureha Chem Ind Co Ltd | Cyclodextrin clathrate compound of eicosapentaenoic acid or docosahexaenoic acid |
| DE3366506D1 (en) * | 1982-03-01 | 1986-11-06 | Efamol Ltd | Pharmaceutical composition |
| FR2522935A1 (en) * | 1982-03-10 | 1983-09-16 | Chevalier Jacques | NOVEL PRODUCTS BASED ON MILK AND ITS DERIVATIVES RICH IN POLYUNSATURATED FATTY ACIDS AND THEIR PREPARATION PROCESS |
| JPS58164508A (en) * | 1982-03-26 | 1983-09-29 | Eisai Co Ltd | Composition for external use containing isoprenyl-carboxylic acid |
| IE54795B1 (en) * | 1982-04-29 | 1990-02-14 | Efamol Ltd | Pharmaceutical composition |
| US4668704A (en) * | 1982-08-09 | 1987-05-26 | Regents Of The University Of California | Method for protecting and healing gastro-duodenal mucosa and the liver of mammals |
| JPS61500432A (en) * | 1982-12-09 | 1986-03-13 | ビルトン，ジエラルド エル． | Easily absorbable fatty acid emulsion |
| GB8302708D0 (en) * | 1983-02-01 | 1983-03-02 | Efamol Ltd | Pharmaceutical and dietary composition |
| GB2140806B (en) * | 1983-05-28 | 1987-10-28 | Sekimoto Hiroshi | Stabilisation of unsaturated fatty acids, fish oils or fish |
| CA1239587A (en) * | 1983-10-24 | 1988-07-26 | David Rubin | Combined fatty acid composition for lowering blood cholestrol and triglyceride levels |
| ATE33333T1 (en) * | 1984-01-11 | 1988-04-15 | Von Mletzko Armin Dr | DIETETIC. |
| JPS60184011A (en) * | 1984-03-02 | 1985-09-19 | Itaru Yamamoto | Immuno-regulator |
| IT1176916B (en) * | 1984-10-10 | 1987-08-18 | Elvira Pistolesi | PHARMACEUTICAL OR DIETETIC COMPOSITION WITH HIGH ANTI-THROMBOTIC AND ANTI-ARTERIOSCLEROTIC ACTIVITY |
| JPS62187425A (en) * | 1985-10-14 | 1987-08-15 | Michio Nakanishi | Composition containing linolenic acid and its derivative |
| DE3719097C1 (en) * | 1987-06-06 | 1988-06-09 | Fratzer Uwe | Medicament containing eicosapentaenoic acid and docosahexaenoic acid as unsaturated fatty acids as well as vitamin E. |
-
1986
- 1986-05-09 DE DE19863615710 patent/DE3615710A1/en not_active Withdrawn
-
1987
- 1987-05-06 AT AT87106520T patent/ATE77549T1/en not_active IP Right Cessation
- 1987-05-06 EP EP87106520A patent/EP0244832B1/en not_active Expired - Lifetime
- 1987-05-06 ES ES87106520T patent/ES2051705T3/en not_active Expired - Lifetime
- 1987-05-06 DE DE8787106520T patent/DE3779958D1/en not_active Expired - Fee Related
- 1987-05-08 AU AU72641/87A patent/AU603574B2/en not_active Ceased
- 1987-05-08 JP JP62110953A patent/JPS62267222A/en active Pending
- 1987-05-08 ZA ZA873299A patent/ZA873299B/en unknown
- 1987-05-08 PT PT84841A patent/PT84841B/en not_active IP Right Cessation
- 1987-05-08 KR KR1019870004500A patent/KR950008306B1/en not_active IP Right Cessation
- 1987-05-08 HU HU872088A patent/HU201671B/en not_active IP Right Cessation
- 1987-05-08 DK DK235687A patent/DK167518B1/en not_active IP Right Cessation
- 1987-05-08 IL IL8245987A patent/IL82459A/en not_active IP Right Cessation
- 1987-05-08 CA CA000536688A patent/CA1302266C/en not_active Expired - Fee Related
- 1987-05-11 IE IE120387A patent/IE60273B1/en not_active IP Right Cessation
-
1989
- 1989-02-01 US US07/304,717 patent/US5043328A/en not_active Expired - Lifetime
-
1992
- 1992-09-24 GR GR920401779T patent/GR3005768T3/el unknown
Also Published As
| Publication number | Publication date |
|---|---|
| KR870010867A (en) | 1987-12-18 |
| AU603574B2 (en) | 1990-11-22 |
| HUT44433A (en) | 1988-03-28 |
| IE60273B1 (en) | 1994-06-29 |
| US5043328A (en) | 1991-08-27 |
| ZA873299B (en) | 1987-11-02 |
| JPS62267222A (en) | 1987-11-19 |
| IL82459A0 (en) | 1987-11-30 |
| PT84841A (en) | 1987-06-01 |
| DK167518B1 (en) | 1993-11-15 |
| IL82459A (en) | 1994-07-31 |
| DE3615710A1 (en) | 1987-11-26 |
| EP0244832A2 (en) | 1987-11-11 |
| GR3005768T3 (en) | 1993-06-07 |
| ATE77549T1 (en) | 1992-07-15 |
| DE3779958D1 (en) | 1992-07-30 |
| KR950008306B1 (en) | 1995-07-27 |
| DK235687D0 (en) | 1987-05-08 |
| PT84841B (en) | 1990-02-08 |
| HU201671B (en) | 1990-12-28 |
| AU7264187A (en) | 1987-11-12 |
| ES2051705T3 (en) | 1994-07-01 |
| DK235687A (en) | 1987-11-10 |
| EP0244832B1 (en) | 1992-06-24 |
| EP0244832A3 (en) | 1989-11-29 |
| IE871203L (en) | 1987-11-09 |
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