CA1273575A - Pharmaceutical compositions consisting of acylated phospholipids - Google Patents

Pharmaceutical compositions consisting of acylated phospholipids

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Publication number
CA1273575A
CA1273575A CA000516015A CA516015A CA1273575A CA 1273575 A CA1273575 A CA 1273575A CA 000516015 A CA000516015 A CA 000516015A CA 516015 A CA516015 A CA 516015A CA 1273575 A CA1273575 A CA 1273575A
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cis
glycero
phospholipid
octadecenoyl
optionally
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French (fr)
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Alan J. Schroit
Rajiv Nayar
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University of Texas System
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University of Texas System
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • 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
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Dispersion Chemistry (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Pharmaceutical Compositions Consisting of Acylated Phospholipids Abstract of the Disclosure Disclosed are pharmaceutical compositions consisting of a) a synthetic phospholipid of the formula (I), wherein m represents two or three, R1 and R2 preferably represent acyl having from 10 to 20 carbon atoms, X represents the direct bond, C1-C4-alkylene, C2-C4-alkenylene, or C1-C4-alkylene or C2-C4-alkenylene substituted by hydroxy or a pharmaceutically acceptable salt thereof, b) a synthetic phospholipid of the formula (II), wherein R1 and R2 repreaent the acyl group of a saturated or an unsaeurated carboxylic acid having from 10 to 20 carbon atoms and 1 - 2 double bonds, c) a compound or a mixture of compounds having pharmacological acitivity, and, optionally, d) a phospholipid from natural sources selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phoaphatidylinositol, phosphatidylglyceride, cardiolipin and cholesterol and its derivatives, and, optionally, a pharmaceutically acceptable carrier solution buffered to pH 7,0 - 7,8, and, optionally pharmaceutically acceptable carriers in solid form.

Also disclosed are mixtures of the novel, synthetic, acylated phospholipids (I) and synthetic phosphatidylethanolamine deri-vatives, a process for the preparation of the pharmaceutical compositions, the novel, synthetic acylated phospholipids (I) themselves, a process for the preparation of the novel, synthetic, acylated phospholipids (I), and a method of use for the pharma-ceutical compositions.

Description

12735~

4-15454/~

Pharmace~tical Compositions Consistin~ of Acvlated Phospholipids Subject matter of the present invention are pharmaceutical composi-tions containing acylated phosphatidylethanolamine derivatives, phosphatidylethanolamine and compounds having pharmacological properties. The present invention also relates to mixtures of the acylated phosphatidylethanolamine derivatives and phosphatidyl-ethanolamine, a process for the preparation of the pharmaceutical compositions, and a method of use for the pharmaceutical compo-sitions.

The pharmaceutical compositions according to the present invention are bsing administered in the form of liposomes.

Pharmaceutical administration systems based on liposomes have been described in the general review issued by G. Gregoriadis, Liposome Technology, Vol. II, Incorporation of Drugs, Proteins and Genetic Material, CRC Press 1984. Such systems have the advantage that biologically active material can be introduced into tissues by phagocytosis~ especially into tissues of the reticulo-endothelial system. Por example, a transport mechanigm is known how antibiotics are being introduced into infected tissues by phagocytosis thu~
causing the improved removal or destruction of the infecting microorgani~m. Endocytosis also is a helpul mechanism in the combat of centres of inflammation. Antirheumatic pharmaceuticals encap-su1ated in liposomes are preferably lntroduced into infected tissues as compared to "healthy" tissues. Moreover, cyto~tatic agent3, commonly known as "anticancer drugs", when encapsulated in liposomes, can be introduced into specific organs of the reticulo-y ~L273~75 endothellal system (liver, spleen or marrow). Additionally, due tofiltration in the capillaries of the lung and subsequent transport by migrating monooytes, biologically active material, for example compounds having immunomodulatory properties, can be concentrated in alveolar macrophages. This results in an improved action on meta-static lung tumours and in a simultaneous reduction of toxicity.

It has now surprisingly been found that the uptake of liposomes and their endocytosis by macrophages, especially alveolar macrophages, i8 increased whenever acylated phosphatidylethanolamine derivatives are incorporated in the shell structure of the liposomes.

The present invention relates to pharmaceutical compositions consisting of a) a phospholipid of the formula sn I 1 H2-0-Rl __ 2 R20- H Q Q Q ~I), 3 Hz-0-~-0-(CH2)m-NH-C-X-C-OH

wherein m represents two or three, Rl and R2 independently of each other represent alkyl, alkenyl, or scyl each having from 10 to 20 carbon atoms, X represents the direct bond, C1-C4-alkylene, C2-C4-alkenylene, or C1-C4-alkylene or C2-C4-alkenylene substituted by hydroxy or a pharmaceutically acceptable salt thereof, b) a phospholipid of the formula ~n 1 H2-0-R3 _ 2 R40- H ~ (II), 3 H2-o- ¦~0-CH2-CH2-NH3 wherein R3 and R4 represent the acyl group of a saturated or an unsaturated carboxylic acid having from 10 to 20 carbon atoms and 1 - 2 double bonds, ~273~7S

c~ a compound or a mixture of compounds having pharmacological acitivity, and, optionally, d) a lipid selected from the group consisting of phosphatidyl-choline, phosphatidylserine, phosphatidylinositol, pho~phatidyl-glycerol, cardiolipin and cholesterol and its derivatives, and, optionally, a pharmaceutically acceptable carr~er solution buffered to pH 7,0 - ~,8, and, optionally, pharmaceutically acceptable additives.

ID the context of the description of the present invention, the general terms employed hereinbefore and hereinafter preferably haYe the following meanings:

The terms "lower" used in connection with definitions of organic radicals, for example lower alkyl, lower alkylene, lower alkoxy, lower alkanoyl, etc., mear.s that such organic radicals, unless expressly defined otherwise, contain up to 7, preferably up to 4, carbon atoms.

The nomenclature of the phospholipids of the formulae I and II is in agreement with the recommendations of the IUPAC and IUB Commission on Biochemical Nomenclature (CBN3 according to the Eur. J. of Biochem. 79, 11-21 (1977) "Nomenclature of Lipids" (sn-nomenclature, stereospecific numbering~.

Unless indicated otherwise, generic names proposed by the World Health Organisation (WHO) (Recommended International Non-properietary NameQ) are used to define the active ingredient~, whlch names have been ta~en ~rom the ~tandard text book "Pharmazeutische Chemie" (E. Schroder, C. Rufer and R. Schmiechen, Thieme Verlag Stuttgart, 1982) and the Merck Index (Tenth Edition).

In the synthetic phospholipid of the formula I (component a) ~ is preferably two.

~27357~ii Alkyl Rl and/or R2 is preferably straight-chained with an even number fro~ 10 to 20 carbon atoms, for example n-decyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl or n-icosyl.

Alkenyl Rl andlor R2 is prefe{ably straight-chained with an even number from 12 to 20 carbon atoms and a double bond, for example 9-cis-dodecenyl, 9-cis-tetradecenyl, 9-cis-hexadecenyl, 6-cis-octa-decenyl, 6-trans-octadecenyl, 9-cis-octadecenyl, 9-trans-octadecenyl or 9-cis-icosenyl.

Acyl Rl and~or R2 is preferably straight-chained with an even number from 10-20 carbon atoms, for example Clo-C20~alkanoyl or Clo alkenoyl.

Alkanoyl Rl and/or R2 is preferably n-decanoyl, n-dodecanoyl, n-tetradecanoyl, n-hexadecanoyl, n-octadecanoyl and n-icosanoyl.

Alkenoyl Rl and/or R2 is p~eferably 9-cis-dodecenoyl, 9-cis-tetra-decenoyl, 9-cis-hexadecenoyl, 6-cis-octadecenoyl, 6-trans-octa-decenoyl, 9-cis-octadecenoyl, 9-trans-octadecenoyl, ll-cis-octa-decenoyl and 9-cis-icosenoyl.

X defined as Cl-C4-alkylene is straight chained or branched C1-C4-alkylene, for example methylene, l,l-ethylene, 1,1-, 1,2- or 1,3-propylene or, preferably, 1,2-ethylene.

X defined as C2-C4-alkenylene is preferably 6traight chained alkenylene, for example vinylene, propylene, or 1,2- or 2,3-butylene.

X defined 8S Cl-C4-alkylene or Cz-C4-alkenylene substituted by hydroxy i8 preferably straight chained Cl-C4-alkylene substituted by 1 or, depending on the number of carbon atoms, up to 4 hydroxy ~2'73~7S

groups, for example l-hydroxy-1,2-ethylene, 1,2-dihydroxy-1,2-ethy]ene, 1- or 2-hydroxy-1,3-propylene or 1,2-dihydroxy-1,3-propylene.

A pharmaceutically acceptable salt of the phospholipid (I) i~
preferably formed by reaction with one or two equivalents of dllute aqueous alkalimetal hydroxide, for example sodium or potassium hydroxide and is, preferably, the mono- or di~odium salt.

Other pharmaceutically acceptable salts are formed by reaction with amines such as trimethyl-, ethyl-, diethyl-, or triethylamine, piperidine, piperazine, 2-hydroxyethylpiperazine, cyclohexylamine, pyrrolidine, or choline.

In the synthetic phospholipid (I) R1 and R2 preferably are straight chained alkenoyl with an even number from 10 to 20 carbon atoms, for example 9-cis~dodecenoyl, 9-cis-tetradecenoyl, 9-cis-hexadecenoyl, 6-cis-, 6-trans-, 9-cis-, 9-trans-, or 11-cis-octadecenoyl, or 9-ci~-icosenoyl, X is C2-C4-alkylene, for example 1,2-ethylene or 1,3-propylene, or C2-C4-alkenylene, for example vinylene.

Most preferred are the sodium or disodium salts of N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol3-N-hydroxysuccinyl-amine and N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phospho-ethanol-N-hydroxyglutarylamine.

In the phospholipid of the formula II (component b)) the acyl groups R3 and R4 are preferably straight chained with an even number rom 10 - 20 carbon atoms, for example clo-c2o-alkanoyl or C~o-Czo-alkenoyl, especially 9-cis-dodecenoyl, 9-ci~-tetradecenoyl, 9-cis-hexadecenoyl, 6-cis-, 6-trans-, 9-cis-, 9-trans- or ll-cis-octadecenoyl.

Most preferred is 1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phospho-ethanolamine.

i273S7~

Compounds or mixtures of compounds having pharmacological activity (component c)) are preferably drugs selected from the group con-sisting of antiphlogistics and/or antiinflammatory agents, anti-biotics, antileishmaniasis agents, antimycotics, antineoplastic agents, and compounds having immunomodulatory action.

Antiphlogistics andlor antiinflammatory agents are preferably glucocorticoids, for example cortisone, hydrocortisone, prednisone, prednisolone, fluocortolone, triamcinolone, methylprednisolone, prednylidene, paramethasone, dexamethasone, betamethasone, beclo-mPthasone, fluprednylidene, desoximethasone, fluocinolone, flumethasone, diflucortolone, clocortolone, clobetazol, or fluor-cortinebutylester, salts of substituted phenylacetic acids or
2-phenylpropionic acids, for example alclofenac, ibufenac, ibu-profen, clindanac, fenclorac, ketoprofen, fenoprofen, indoprofen, fenclofenac, diclofenac, flurbiprofen, pirprofen, naproxen, benoxa-profen, carprofen or cicloprofen; analgesically active anthranilic acid derivatives, for example of the formula ~-\ /COOH

H (III) R2\ ~-\ /R1 i!

~ 3 in which Rl, R2 and R3, independently of each other, represent hydrogen, methyl, chlorine or trifluoromethyl, for example mefenamic acid, flufenamic acid, tolfenamic acid or meclofenamic acid;
aDalgesically active anilino-substituted nicotinic acid derivatlves, for example miflumic acid, chlonixin or flunixin; analge~ically active heteroarylacetic acid~ or 2-heteroarylpropionic acids having a 2-indol-3-yl or pyrrol-2-yl radical, for example indomethacin, oxmetacin, intrazol, acemetazin, cinmetacin, zomepirac, tolmetin, ~2'73S7S
~ 7 -colpirac or tiaprofenlc acid, analgesically active indenylacetic acids, for example sulindac, aDalgesically active heteroaryloxy-acet~c acids, for example benzadac.

Antibiotics are preferably tetracycline derivatives of the formula:
Rs~ R4 ~ 3~Z ~(CH3)2 .\ /OH
I i1 i i i1 (I~), ~ / \CONHR

in which Rl represents hydrogen or pyrrolidin-l-ylmethyl, R2 represents hydrogen or hydroxy, R3 represents hydrogen, hydroxy or methyl, R4 represents hydrogen or methyl, and Rs represents hydro-gen, chlorine or dimethylamino, for example chlorotetracycline, oxytetracycllne, tetracycline, demethylchlortetracycline, meta-cycline, doxycycline, minocycline or rolitetracycline, amino-glycosides, for example kanamycin, amikacln, gentamicin Cl, C1a, C2 or C2b, sisomicin, netilmicin, spectinomycin, streptomycin, tobramycin, neomycin B, dibecacin, or kanendomycin, makrolides, for example maridomycin or erythromycin, lincomycins, for example clindamycin or lincomycin, penicillanic acid and cephalosporanic acid derivatives having antibiotic activity with 6~-or 7~-acylamino groups, which are present in fermentatively, semi-synthetically or synthetically obtainable 6~-acylaminopenicillanic acid or 7~-acyl-aminocephalosporanic acid derivatives or in 7~-acylaminocephalo-sporanic acid derivatives modified in the 3-position, for example penicillanic acid derivatives that have become known under the names penicillin G or V, phencthicillln, prop:lcillin, nafcillln, oxacillin, cloxacillin, dicloxaclllin, flucloxacillin, cyclacillin, epicillin, mecillinam, methicillin, azlocillin, sulbenicillin, ticarcillln, mezlocillin, piperacillin, carindacillin, azidocillin or ciclazillin, or cephalosporin derivatives that have become known under the names cefaclor, cefuroxime, cefazlur, cephacetrile, cefazolin, cephalexin, cefadroxil, cephaloglycin, cefoxitin, ~L2~73~7S

cephaloridine, cephsulodin, cefotiam, ce~tazidine, cefonicid, ce~otaxime, cefmenoxime~ ceftizoxi~e, cephalothin, cephradine, cefamandol, cephsnone, cephapirin, cefroxadin, cefatrizine, cefazedone, ceftrixon or ceforanid, other ~-lactam antibodies of the clavam; penem or carbapenem type, for example moxalactam, clavulanic acid, nocardicine A, sulbactam, aztreonam or thienamycine, or other antibiotics of the b1comycin, novobiocin, chlor- or thiamphenicol, rifampicin-, fosfomycin-, colistin- or vancomycin type.

Antileishmaniasi~ agents preferably are antimony compounds, for example potassium antimonyl tartrate, stibophen, sodium stibocaptate, or sodium stibogluconate.

Antimycotics are, for.example, thiocarbonic acid derivatives, for example dibenzthione, tolnaftate, or tolcidate, imidazole deri-vatives~ for example clotrimazole, miconazole, econazole, iconazole, or ketoconazole, or polyene derivatives such as nystatine, natamycine, or amphotericine B.

Antineoplastic agents preferably are alkylating agents having the bls-(2-chloroethyl)-amine group such as chlormethine, chlorambucile, melphalan, uramustine, mannomustine, extramustinephosphat, mechlor-ethaminoxide, cyclophosphamide, ifosfamide, or trifosfamide, alkylating agents having a substituted aziridine group, for example tretamine, thiotepa, triaziquone, or mitomycine, alkylating agents of the methanesulfonic ester type such as busulfane, alkylating N-alkyl-N-nitrosourea derivatives, for example carmustine, lomustine, semustine, or streptozotocine, slkylating agents of the mitobronitole, dacsrbazine, or procarbazine type, complexing sgents such as cis-platin, antimetabolites of the folic acid type, for example methotrexate, purlne derivatlves such as mercaptopurine, thioguanine, azathioprine, tiamiprine, vidarabine, or puromycine, pyrimidine derivatives, for example fluorouracll, floxuridine, tegafur, cytarabine, idoxuridine, flucytosine, antibiotics such as ~2~3~7~;
g dactinomycin, dsunorubictn, doxorubicin, mithramycin, bleomycin A2 or B2 or etoposide, or vinca alcaloids, optionally in combination with chlormethamine, prednisolone~ prednisone, or procarba~ine.

Compounds or mixture~s of compounds having immunomodulatory action are, for example, mursmylpeptides of the formula R3~

(V), ~ HCOC~3 R2-C' \
CORl wherein Rl represents the L-Ala-D-isoGln-L-Ala-2-(1,~-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamide, L-Ala-D-Glu-(cgam~a-L-Ala-2-(l~2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamide, L-Ala-D-isoGln-OH, L-Ala-D-Gln-NH2-~-n-butylester, L-Ala-D-isoGln-L-(stearoyl)-Lys, L-Val-D-Gln-NH2-~-n-methylester, L-Ala-D-isoGln-L-Ala-1,2-dipalmitoyl-sn-glycerineester or the L-Ala-D-isoGln-L-Ala-cholesterineester group, R2 represents hydrogen, methyl or n-propyl, R3 represents hydrogen, n-stearoyl, 10-(2,3-dimethoxy-1,4-dioxo-5-methyl)-2,5-cyclohexadienoyl, 2-behenoyloxy-2-methyl-propanoyl or n-octanoyl, and R4 represents hydrogen or n-octanoyl, as well as the 2-palmitoylthio derivative thereof, lipopeptide~ such as n-lauroyl-L-Ala-D-isoGln-(m-DAP-Gly)-NHz, n-lauroyl-L-Ala-D-isoGln-(L-DAP-Gly)-NH2, n-lauroyl-L-Ala-D-isoGln-(L-Lys-D-Ala)-NH2, n-octanoyl-L-Ala-D-isoGln-(L-Lys-D-Ala)-NH2 or palmitoyl-Cys-((2R)-2,3-dilauroyloxy-propyl)-Ala-D-Glu-(Gly-taurine-Na)-NH2, or are lymphokine~ which are produced by lymphocytes, monocytes or macropha~es after ~timulation by antigenes or mitogenes.

Lymphokines are especially gamma interferon, especially natural or recombinant human gamma interferon, especially human gamma-inter-feron obtainable according to the European Patent Appli-~2~3S~ 21~9~6~;6 cations 63,482 (10-27-82); 77,670 (4-27-83); 83,777 (7-20-83);
88,540 (9-1~-83~; 89,676 (9-28-83); 95,350 ~11-30-83); 99,084 (1-25-84); 110,044 (6-13-84); 112,967 (7-11-84); and the Interna-tional (PCT) Applications (WO) 83/04,053 (11-24-83) or WO 84/02,129 (6-7-84).
Preferred is human gamma-interferon of the following amino acid sequences:

H2N-Cys-Tyr-Cys-Gln-Asp-Pro-Tyr-Val-Gln-Glu-Ala-Glu-Asn-Leu-Lys-Lys-Tyr-Phe-Asn-Ala-Gly-His-Ser-Asp-Val-Ala-Asp-Asn-Gly-Thr-Leu-Phe-Leu-Gly-Ile-Leu-Lys-Asn-Trp-Lys-Glu-Glu-Ser-Asp-Arg-Lys-Ile-Net-Gln-Ser-Gln-Ile-Val-Ser-Phe-Tyr-Phe-Lys-Leu-Phe-Lys-Asn-Phe-Lys-Asp-Asp-Gln-Ser-Ile-Gln-Lys-Ser-Val-Glu-Thr-Ile-Lys-Glu-Asp-Met-Asn-Val-Lys-Phe-Phe-Asn-Ser-Asn-Lys-Lys-Lys-Arg-Asp-Asp-Phe-Glu-Lys-Leu-Thr-Asn-Tyr-Ser-Val-Thr-Asp-Leu-Asn-Val-Gln-Arg-Lys-Ala-Ile-His-Glu-Leu-Ile-Gln-Val-Met-Ala-Glu-Leu-Ser-Pro-Ala-Ala-Lys-Thr-Glu-Lys-Arg-Lys-Arg-Ser-Gln-Met-Leu-Phe-Gln-Gly-Arg-Arg-Ala-Ser-Gln-OH, according to the European Patent Application 121,157 (10-10-87) and H2N-Cys-Tyr-Cys-Gln-Asp-Pro-Tyr-Val-1.ys-Glu-Ala-Glu-Asn-Leu-Lys-Lys-Tyr-Phe-Asn-Ala-Gly-His-Ser-Asp-Val-Ala-Asp-Asn-Gly-Thr-Leu-Phe-Leu-Gly-Ile-Leu-Lys-Asn-Trp-Lys-Glu-Glu-Ser-Asp-Arg-Lys-Ile-Met-Gln-Ser-Gln-Ile-Val-Ser-Phe-Tyr-Phe-Lys-Leu-Phe-Lys-Asn-Phe-Lys-Asp-Asp-Gln-Ser-Ile-Gln-Lys-Ser-Val-Glu-Thr-Ile-Lys-Glu-Asp-Met-Asn-Val-Lys-Phe-Phe-Asn-Ser-Asn-Lys-Lys-Lys-Arg-Asp-Asp-Phe-Glu-Lys-Leu-Thr-Asn-Tyr-Ser-Val-Thr-Asp-I.eu-Asn-Val-Gln-Arg-L.ys-Ala-Ile-llis-Glu-Leu-Ile-G:Lrl-Val-Met-Ala-Glu-Leu-Ser-Pro-Ala-Ala-Lys-Thr-Gly-Lys-Arg-Lys-Arg-Ser-Gln-Met-Leu-Phe-Arg-Gly-Arg-Arg-Ala-Ser-Gln-OH, according to the British Patent Specification 2,107,718, human interleukine 2 obtainable, for example, from the culture filtrates of cell cultures of leukaemia or lymphoma cells after activation or ~2~357S

stimulation with human T-cell mitogenes and by purification with reverse phase HPLC, culture filtrates that contain mixtures of compounds known as migration inhibitlon factor ~MIF~, leukocytes migration inhibition factor, macrophages activating factor (MAF), colony stimulating factor, as well as interleukine 1 and 2 and gamma interferon and which are obtained from cultures of human T-lympho-cytes from the spleen or from peripheral blood after stimulatlon by antigenes or mitogenes, or example human T-cell leukaemia-lymphoma virus (HTLY I or II), phytohaemagglutinine, or concanavaline, especially those culture filtrates or isolates that contain a high percentage of macrophage activating factor (~A~).

Preferred are N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine--2-(1,2-dipalmitoyl-sn-glycero-3~hydroxyphosphoryloxy)-ethylamide, sodium-N-acetyl-~-muramyl-L-alanyl-D-isoglutamine or sodium-~-acetyldesmethylmuramyl-L-alanyl-D-isoglutamine of the formula V, optionally in combination ~ith purified, natural or recombinant human gamma interferon.

Lipids (component d)) selected from the group consisting of phos-phatidylcholine, phosphatidylserine, phosphatidylinositol, phos-phatidylglycerol and cardiolipin, are synthetic phospholipids or are mixtures of phospholipids having various acyl groups of different molecular weight and structure, for example soy bean or chicken egg phosphatidylcholine or phosphatidylchinoline from bovine brain, bovine liver or porcine liver, phosphatidylserine from bovine brain, phosphatidylinositol from soybean or from yeast~ phosphatidyl-glycerol from egg yolk, or cardiolipin from bovine heart.

Derivatives of cholesterol are for example, chol0stane, coprostane, ergosterol or stigmasterol.

The pharmaceutical compositions according to the present invention, when applied in the form of liposomes, are characterized by their excellent phagocytosis. For example, phsgocytosis of multilamellar liposomes consisting of a 3:7 molar mixture of sodium-N-[1,2-di-1;;~73~;

~9-cis-octadecenoyl)-sn-glycero-3-phospoethanol]-N-hydroxysuccinyl-amine (I) and dioleoylphosphatidylethanolamine (II) or sodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxyglutarylamine (I) and dioleoylphosphatidylethanola~ie (II) by mouse peritoneal macrophages is higher than phagocytosis of multi-lamellar liposomes consisting exclusively of phosphatidylethanol-amine or of a 3:7 molar mixture of phosphatidylserine and phos-phatidylcholine. This can be demonstrated in-~itro by incubation of mouse psritoneal macrophagas with multilamellar liposomes containing trace amounts of l2sI as a liposome marker. At regular intervals the cultures are washed and the amount of cell-associated radiation is determined. Moreover, liposomes consisting of phospholipids of formula I and II in a molar ration of 3:7 containing immunomodu-lators such as MDP and gamma interferon show higher activation of macrophages to the tumoricidal state at low doses than liposomes consisting of phophatidyl choline and phosphatidyl serine containing the same amount of MDP and gamma-interferon. This can also be shown in vitro by plating peritoneal mouse macrophages in culture wells and activating the macrophages with multilamellar liposomes con-sistlng of sodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phos-phoethanoll-N-hydroxysuccinylamine (I) and dioleoylphosphatidyl-ethanolamine (II) in a molar 3:7 ratio containing MDP and gamma interferon and with multilamellar liposomes consisting Df phos-phatidylcholine and phosphatidylserine in the same molar ratio containing MDP and gamma interferon. The liposome preparations are used at a concentration of 100 nmol of total phospholipid per well and contains 6 units recombinant immune gamma interferon and 0,2 g MDP. After washing the wells 104[12sI]iododeoxyuridine - labeled BL6 melanoma cells are added. Cytoxity is determined after 72 hourH of cocultivation by meflsUring the radioactivity associated with the adherent viable target cells after washing the cultures three times with Hank's balanced salt solution. Percent cytotoxity can be calculated with respect to the counts per minute in control cultures containing unactivated macrophages and target cells.

~2~73~i7~;

The pharmaceutical composltions according to the present invention, when applied in the form of llposomes, are also characterized by their excellent release properties at low pH-values. The release properties of liposomes consisting of disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol~-~-hydroxysuccinylamine and 1,2-di-(9-cls-octadecenoyl)-sn-glycero-3-phosphoethanolamine in a molar ratio of 3 : 7 are analyzed according to the fluorescence method to measure the lea~age from liposomes at low pH-values as described by Ellens et al. Biochemistry 1984, 23, 1532-1538. To investigate the pH dependence of leakage, liposomes are injected into buffer solutions ranging from pH 4.0 to 7.4 and the percentage of the entrapped 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and p-xylylene-bis-pyridinium dibromide (DPX) which replace the encapsuled pharmaceutica in this assay, is determined. Complete encapsulation of the water soluble fluorophore ANTS complexed with the "quencher" DPX extinguishes most of the ANTS fluorescence.
Leakage of ANTS from the liposomes could be followed by the increase in fluorescence due to the relief of DPX "quenching".

It has now been found that there is essentially no leakage above pH 6Ø However, when the pH i5 decreased, there is a concomitant enhancement in the amount of ANTS/DPX release from the liposomes with half maximal release occurring at approximately pH 4.5 and complete release at about 4Ø

It is known that pathologic tissues have an ambient pH that is considerably lower than that of normal tissues. For example of primary tumors, metastasis, inflammation, and infection have reduced local pH-environments. The llposomes of the prosent invention, therefore, would deliver their content, for example antlinflammatory drugs or immunomodulators spQcifically to the site of inflammation, the primary tumor or metastasis and release these drugs in the acidic environment of these pathologic tissuas. For example, liposomes consisting of a 3:7 molar mixture of disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol~-N-hydroxy-succinylamine (I) and dioleoylphosphatidylethanolamlne release more ~2t7~i7~

of their content, for example an effective dose of recombinant human gamma interferon, at low pH values, e.g. pH 49 than at higher or neutral pH values. This can be shown in-vitro in buffer solutions of pH 7.4 and pH 4 by determing at regular time intervals the radiation of marked 125 I-Interferon released from the lipids. For example, at a pH-value of about 4 more than 25 % of enclosed gamma~interferon are released from liposomes after 180 minute~.

ThPrefore, the pharmaceutical compositions according to the present invention, when applied in the form of liposomes, are excellent administration systems for drug delivery to the reduced local pH
environment of pathologic tissues. Therefore, they are especially useful in the cancer chemotherapy for combating metastatic tumor cells.

Aqueous liposome dispersions wherein the phospholipids of the formulae I and II are the encapsulating material and compounds or a mlxture of compounds having pharmacologic activity are encapsulated, optionally after concentration or isolation of the liposomes, for example in the (ultra)centrifuge, are suitable for therapeutic purposes for parenteral (bukkal, lingual, sub-lingual, i.v., i.c., topical, s.c., i.m. or nasal) administration.

For parenteral administration (topical) the liposome-containing aqueous di6persion can be mixed with customary thickeners, for example hydroxypropylmethylcellulose, suitable preservatives, antioxidants and perfumes, and can be used in the form of a lotion or a gel for application to the skin or mucous membranes, For parenteral administratlon, the aqueou~ disper~ion of the enriched liposomes can be ~uspended in a suitable carrler liquid, for example sterlle, calcium free, isotonic sodium chloride or glucose solution, optionally buffered to pH 7.2 - 7.4.

~ 735~75 The dosage of the active ingredient to be administered is generally the highest and lowest dose amount as prescribed, for example in the Deutsches Arzneimittelbuch (DAB) [German Pharmacopoieia~ for the specific active ingredient for the particular form of admini-stration, the age of the patient and the health of the patient.
Aqueous liposome dispersions also have the advantage, however, that actlve ingredients administered in smaller doses may, neverth~less~
pass to the receptors and produce a therapeutic effect, or, on administration of higher doses, undesirable side effects may be avoided.

The preferred dosage amount for the liposome encapsulated immuno-modulators of the muramylpeptide or lipopeptide type is about 0.001 up to 10 mg/kg body weight per dose. For human gamma interferon or mixtures containing MAF the preferred dosage amount is about 0.01 ml liposome dispersion per kilo body weight containing 100 - 1000 units of gamma interferon or MAF. If muramylpeptides are administered in combination with gamma interferon, it is estimated that the highest dose to be applied to a human of about 70 kg weight is about 10 mg of liposomes per kilo body weight containing 3 microgramm of the muramylpeptlde and 1500 units of gamma interferon. The highest and tbe lowest dose of the encapsulated material, the concentration of the phospholipids in the aqueous phase as well as the concentration of the encapsulated compounds can be varied according to results to be e~tablished experimentally in clinical trials.

The present invention preferably relates to pharmaceutical composi-tions consisting of a~ a phospholipid of the for~ula I, wherein m represents two, R1 and are defined as above~ X represents C1-CI~-alkylene, C2-C4-alkenyl-ene or C1-C~-alkylene sub~tituted by hydroxy or a pharmaceutically acceptable salt thereof, b) a phospholipid of the formula II, wherein R~ and R4 independently of each other represent straight chained C1o-C20-alkanoyl or C1o-C20-alkenoyl~

~3~7S

c) a compound or a combination of compounds having pharmacological activity, and, optionally, a pharmaceutically acceptable carrier solution buffered to pH 7.2-7.4.

More preferably, the present invention relates to pharmaceutical compositions consisting of a) a phospholipid of the formula I, wherein m represents two, R1 and R2 are defined as above, X represents C2-C4-alkylene or C2-C4-alkenylene, or a pharmaceutically acceptable salt thereof, b) a phospholipid of the formula II, wherein R3 and R4 independently of each other represent straight chained C1o-C2~-alkanoyl or C 1 o-C 2 o-alkenoyl, c) a compound or a combination of compounds selected from the group consisting of antiphlogistics and/or antiinflammatory agents, antibiotics, antileishmaniasis agents, antineoplastic agents and immunomodulators and, optionally, a pharmaceueically acceptable carrier solution buffered to pH 7.2 - 7.4.

The present inventi~n specifically relates to pharmaceutical compositions consisting of a) a phospholipid of the formula I, wherein m represents two, R1 and R2 independently of each other represent straight chained alkanoyl or alkenoyl with an even number from 10 to 20 carbon atoms, X
represents C2-C4-alkylene, for example 1,2 ethylene or 1,3-propylene, or C2-C4-alkenylene, for example vinylene, or a pharma~
ceutically acceptable salt thereof, b) a phospholipid of the formula II, wherein R3 and R4 represent straight chained C1o-C2D-alkenoyl with an even number from 10 to 20 carbon atoms, ~2~3S7~;

c) a compound or a combination of compounds selected from the group consisting ~f antiphlogistics and/or antiinflammatory agents, antibiotics, antlneoplastic agents and immunomodulators and, optionally, a pharmaceuticallY acceptable carrier solution buffered to pH 7.2-7.4.

The invention more specifically relates to pharmaceutical compo-sitions consisting of a) a phospholipid of the formula I, wherein m represents two, R1 and R2 independently of each other represent 9-cis-dodecenoyl, 9-cis-tetradecenoyl, 9-cis-hexadecenoyl, 6-cis-, 6-trans-, 9-Ci9-, 9-trans-, or ll-cis-octadecenoyl, or 9-cis-icosenoyl, X represents C2-C4-alkylene, for example 1,2-ethylene or 1,3-propylene, or C2-C4-alkenylene, for example vinylene, or a pharmaceutically acceptable ~alt thereof, b) a phospholipid of the formula II, wherein R3 and R4 independently of each other represent 9-cis-dodecenoyl, 9-cis-tetradecenoyl, 9-cis-hexadecenoyl, 6-cis-, 6~trans-, 9-cis-, 9-trans-, or ll-cis-octadecenoyl, or 9-cis-icosenoyl, c) a compound or a combination of compounds selected from the group consisting of antiphlogistics and/or antiinflammatory agent~, antibiotlcs, antineoplastic agents and immunomodulator~, and, optionally, a pharmaceutically acceptable carrier ~olution buffered to pH 7.2-7.4.

Especially, the present invention relates to pharmaccutical campo-sitions consisting of a) sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxysuccinylamine or sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol~-N-hydroxyglutarylamine, b) 1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanolamine, ~273~7~

c~ a compound or a combination of compounds selected from the group consisting of antiphlogistic and/or antiinflammatory agents, antibiotics, antineoplastic agents, and immunomodulators, and, optionally, a pharmaceutically acceptable carrier solution buffered to pH 7.2 - 7.4.

Of high preference are pharmaceutical compositions consisting of a) sodium or disodium-N-[1,2-di-(9-cis-octadecenGyl)-sn-glycero-3-phosphoethanol]-N-hydroxysuccinylamine or sodium or disodium-N-l1,2-di-(9-cis-octadecenoyl)-sn-~lycero-3-phsophoethanol]-N-hydroxyglutarylamine, b) l,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanolamine, c) a compound or a combination of compounds selected from the group consisting of diclofenac, pirprofen, mitomycin, cytarabine, dactino-mycin, daunorubicin, doxorubicine, etoposide, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamide, N-acetylmuramyl-L-alanyl-D-glut-amic acid-(Cr-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-hydroxy-phosphoryloxy)-ethylamide-disodium salt, N-acetyl-D-muramyl-L-alanyl-D-isoglutamine-sodium salt, N-acetyldesmethyl-muramyl-L-alanyl-D-isoglutamine-sodium salt, N-acetylmuramyl-L-alanyl-D-glut-amine-~-n-butylester, N -(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N -stearoyl-L-lysine, 6-0-stearoyl-N-acetylmuramyl-L-alanine-D-isoglutamine and lymphokines, and, optionally, a pharmaceutically acceptable carrier solutlon buffered to p~ 7.2 - 7.4.

Especially preferred are pharmaccutical compoDition~ consisting oE

a) sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxysuccinyla~ine or sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxyglutarylamine, ~273~

b) 1,2-di-(9-cis-octadecenoyl)-sn~glycero-3-phosphoethanolamine, c) a compound or a combination of compounds o~ the group consisting of diclofenac, pirprofen, mitomycin, cytarabine, dactinomycin, daunorubicin, doxorubicine, etoposide, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-hydroxy-phosphoryloxy)-ethylamide, sodium-~-acetyl-D-mùramyl-L-alanyl-D-isoglutamine, sodium-N-acetyldesmethyl-muramyl-L-alanyl D-iso-glutamine, purified, natural or recombinant human gamma interferon, interleukine 2, and compounds obtained from cultures of human I-lymphocytes from the spleen or from peripheral blood after stimulation by antigenes or mitogenes and which are characterized by a high percentage of macrophage activating factor (MAF), and, optionally, a pharmaceutically acceptable carrier solution buffered to pH 7.2 - 7.4.

Most preferred are pharmaceutical compositions consisting of a) sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phospho-ethanol]-N-hydroxysuccinylamine or sodium or disodium-N-¦1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxyglutarylamine, b) 1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanolamine, c) a compound or a combination of compounds consisting of N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipal~itoyl-sn-hydroxyphosphoryloxy)-ethylamide, sodium-N-acetyl-D-muramyl-L-alanyl-D-isoglutamine, sodium-N-acetyldesmethylmuramyl-L-alanyl-D-glutamine, and purified, natural or recomblnant human gamma interferon, and, optionally7 n pharmaceutically acccptable carrier solution buffered to pH 7.2-7.4.

The invention also relates to mixtures of synthetic phospholipids of the formulae I and II and, optionally, a lipid selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, and, ~73~

optionally, cholesterol and its derivatives, especially mixtures wherein the ratio of the phospholipld (I~ to the phospholipid (II) is from about 10 to 90 to about 50 to 50 mole per cent. The ratio of 30 to 70 mole per cent is especially preferred. The mixtures are useful for preparing liposomes in an aqueous phase containing the component c) - compounds or a combination of compounds having pharmacological activity -.

The invention also relates to a process for the preparation of the pharmaceutical compositions mentioned above or of the mixture mentioned above, characterized in that a~ a homogeneous mixture consisting of phospholipids of the for-mulae I and II, A ]ipophilic compound or mixture of compounds having pharmacological activity and, optionally, a llpid mentioned above from natural sources is prepared and, optionally, the homogeneous mixture thus obtained is dispersed in an aqueous phase or, b) a homogeneous mixture consisting of phospholiplds of the for-mulae I and II and, optionally, a lipid mentioned above from natural sources is prepared and, optionally, the homogeneous mixture thus obtained is dispersed in an aqueous phase containing a hydrophilic compound or mixture of compounds having pharmacological activity and, if necessary, the aqueous dispersion thus obtained is buffered to pH 7,0 - 7,8 and, if desired, non-encapsulated lipids and~or compounds having pharmacological activlty are separated from the aqueous phase and/ or the liposomes thus obtained are concentrated or separated off from the aqueous phase.

The homogenou~ mixture is prepared by formation of a film or of a lyophilisate.

The film is prepared according to method a) by dissolving the phospholipids (I) and (II) ~md the lipophilic compound or mixture of compounds and, optionally, a lipid mentioned above from natural ~273~;75 sources or according to method b) by dissolving the phospho-lipids (I) and (II) and, optionally~ a lipid mentioned above ~rom natural sources in an organic solvent and stripplng the solvent.

Suitable solvent~ are, for example, unsubstituted or substituted, for example halogenated, aliphatic or cycloaliphatic hydrocarbons, for example n-hexane, cyclohexane, methylenechloride, or chloroform, alcohols, for example methanol or ethanol, lower alkanecarboxylic acid esters or amides, for example acetlc acid ethylester or dimethylformamide, or ethers, for example diethylether, tetra-hydrofurane or dioxane, or mixtures of these solvents. The organic solvent i8 subsequently stripped by applying a vacuum, preferably a high vacuum, or by blowing off with an inert gaæ, for example nitrogen.

The lyophilisate is formed according to method a) by dissolving the phospholipids (I) and (II) and the lipophilic compounds or mixture of compounds or according to method b) by dissolving the phospho-lipids (I) and (II) in an organic solvent according to the method as described in the U.S. Patent Specification No. 4,311,712. Suitable solvents are in the solid form together with the phospholipids (I) and (II) at the temperature of the lyophilisation process and are having a melting point of more than 0C, for example glacial acetic acid, benzene or dioxane, especially tert-butanol.

A homogeneous mixture may also be prepared by spray-drying a solution of the phospholipids (I) and (II) and of the encapsulating material in an organic solvent having a low boiling point such as chloroform. A pow~er is obtalned by this method.

The ratio of the phospholipld component (I) to the phosphollpid component (II) in the homogenous mixture is approximately 10 v. 90 up to 50 v. 50 mole per cent. Preferred is the ratio 30 v. 70 mole per cent. The approximate ratio of the molar amounts of the en-~2~

capsulated material (gamma-interferon~ divided by the total amount of the phospholipids ~I) and (II) is about O.OOOl to O.l v. l.0, preferably 0.005 to O.Ol v. 0.1.

The dispersion is carried out by mechanical agitation (shaking, stirring, Vortex mixer) thé aqueous phase to which according to method a) the homogenous mixture of the phospholipids (I) and (II) and the lipophilic compounds or the lipophilic mixture of compounds having pharmacological activity have been added. According to method b) the aqueous phase containlng the hydrophilic compounds or mixture of compounds having pharmacological properties is added to the homogeneous mixture of the phospholipids (I) and (II).

A mixture of small, large, unilamellar or multilamellar ~iposomes is formed spontaneously at a high rate without supplying external energy. Approximately O.l to 40 per cent per weight, preferably 2 to 20 per cent per weight, of the homogeneous mixture relative to the total weight of the aqueous dispersion can be dispersed in the aqueous pha~e. Such dispersions can further be diluted to sbout 1 micromole lipid per ml. Such liposome dispersions have entrapped approximately 2.5 microliters oE the aqueous phase per micromole of the lipid.

Acidic or basic aqueous dispersions are buffered to approximately pH 7.0 - 7.8, preferably 7.2 - 7.4. Preferably, the dispersion is carried out in an aqueous phase having a pH fsom 7.2 to 7.4.

Method a) is preferred in the event that lipophilic, water in-soluble compounds are encapsulated in liposomes, for example lipophilic muramyltripeptides.

Method b) is preferred in the event that hydrophilic water insoluble compounds are encapsulated in liposomes, for example cytarabine or cytostatic compounds such as trifosfamide.

- ~3 -The preparation of the pharmaceutical compositions according to the present invention in the form of lipo~omes can also be carried out by other methods known in the art for preparlng liposomes, for example by sonication with ultrasonic waves, by inEusion methods or reversed phase evaporation.

The dispersion step is performed at temperatures below 60, pre-ferably at room temperature. In case of a potential thermal sen-sitivity of the encapsulated material, the dispersion is carried out under cooling and, optionally, under inert gas atmosphere, for example nitrogen or argon atmosphere.

The liposomes obtained ran be made storage stable in the aqueous phase up to several weeks or months after addition of stabilizers, for example mannite or lactose.

The size o~ the liposo~es formed depends, inter alia, on the structure of the active ingredient and the lipid component, the mixing ratio of the components and the concentration of these components in the aqueous dispersion. Thus, for example, by in-creasing or reducing the concentration of the lipid components aqueous phases having a high content of small or large liposomes are produced.

The separation of small liposomes from large liposomes i8 effected by means of conventional separation methods, for example sedi-mentation of the large liposomes in an ultracentrifuge, by gel filtration or extrusion through straight-pored filters. For example, on centrifuging, for example from 5 to 60 minute~ in n rotational field giving rise to an inertial force equivalent to a gravitational field of 5000-40 000 x g, large liposomes are deposited at the bottom of the vessel, whilst small liposomes remain dispersed and can be decanted off. After repeated centrifugation, complete separation of large liposomes from small liposomes is achieved.

214c,9--6986 Preferably, liposomes are separated from the aqueous phase in the event that according to method b) the aqueous phase contains non-encapsulated water insoluble compounds or pharmaceuticals.
Especially, water soluble antineoplastic agents, for example alkylating agents such as cyclofosfamide, should be separated off by filtration, ultrafiltration, dialysis or by centrifugation in order to prevent eventual side effects caused by non-encapsulated com-pounds. The liposome fraction can be admixed with a carrier solution buffered to pH 7.2 - 7.4, for example isotonic, sterile sodium chloride solution buffered to p~ 7.2 - 7.4.

Liposomes in the aqueous phase having a diameter greater than 6.0 x 10 ~ m, for example large multilamellar liposomes, can be separated off by gel filtration, for example with Sepharose or Sephacryl as carriers.

By extrusion through straight-pored filters, for example membrane filters of the Acrodisc~, Nucleopore~ or polycarbonate type having a pore diameter of approximately 1.0 x 10 6 _ 1.0 x 10 8 m at a pressure of approximately from 0.1 to 1.5 bar and a filtration rate of approximately 20 ml/h, a particularly uniform size distribution of the liposomes is obtained.

The formation of liposomes and their content in the aqueous phase can be detected in a manner known per se by using various physical analytical methods, for example by microscopy of freeze-fracture samples and thin sections in an electroll microscope, by X-ray re~raction, by clynamlc light scal:ter:Lng, by mass determination of the filtrate in an analytical ultracentrifuge and, especially, by spectroscopy, for example in the nuclear magnetic resonance spectrum (lH l3C and 3~P)-Synthetic phospholipids of the formula I are known. Their prepara-tion as intermediates has been described in the European Patent Application No. 56992 (7-23-82).

- 25 - ~148~-69~6 The phospholi.pids of the formula II are all known. Some of them are commercially available (Avanti,Fluka, Serva, Sigma).

The pharmaceuticals mentioned above, especiaily the antiphlogistic, antirheumatic, antileishmaniasis agents, antimycotics, antibiotics or antineoplastic agents are all known, see, for example, MERCK Index, Ienth Edition.

The preparation of muramylpeptides of the formula V has been des-cribed in the British Patent Specification 1,570,625, and in the European Patent Applications 25,495 (3-25-81) and 21,367 (1-7-81).
Immunomodulators of the lipopeptide type are also known, see Euro-pean Patent Application 114,787 and European Patent Specifica-tion 330.

The preparation of purified, natural or recombinant, gamma inter-feron has been described in the European Patent Applications 63,482 (10-27-82); 77,670 (4-27-83); 83,540 (7-13-83); 89,676 (9-28-83~;
95,350 (11-30-83); 99,084 (1-25-84); 110,044 (6-13-84); 112,976 (7-11-84); 121,157 (10-10-84) in the British Patent Specification 2,107,718 as well as in the International (PCT) Applications (W0) 83/04053 (11-24-83) or WO 84/02129 (6-7-84).

The preparation of purified interleukine 2 is described in the ~uropean Pa~ent Application 106,179 (4-25-84) and in the US Patent Spec1fication 4,448,879.

The buffer solutions of pH 7,0 to 7,8 preferably are sterile phosphate bufEer solutions based on the dihydrogenphospllate/
hydrogenphosphate equilibrium (KH2P0l,/Na2HP0l,). The prepa~ation of these buffer solutions is descrlbed in standard manuals, for example "Hager's Handbuch der Pharmazeutischen Praxis", Springer Verlag, Vol. 1, pg. 357-359. Especially sterile, isotonic calcium-free buffer solution of pH 7.2 (Dulbecco) or Hank's Balanced Salt Solution (M.A. Bioproducts, Walkersville MD USA) is used.

.

~2'73~

The following examples are illustrating the invention without limiting the scope thereof. Temperatures are given in degrees Celsius.

Example 1: a) In a round flask 84.70 mg ~Q.098 mmol) disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxysuccinylamine and 16~.10 mg (0.226 mmol) 1,2-di-(9-cis-octa-decenoyl)-sn-glycero-3-phosphoethanolamine are dissolved in a sufficient amount of tert-butanol until both lipids are dissolved.
The solution is filtered under sterile conditions over Acrodisc~
filter (2.0 x 10 7 m) and is bottled in a sterile vial. This vial is frozen at -45. A vacuum is applied to the frozen vial and the solvent is removed until room temperature has been reached. The vial is sealed under inert gas atmosphere, for example argon atmosphere.

To this vial containing a lyophilisate of the ltpid components mentioned above, 2.5 ml of a sterile~ phosphate buffered (pH 7.2 - 7.4), calcium free sodium chloride solution (Dulbecco) containing doxorubicin in a concentration of 4 gJl are added with a sterile syringe. The vial is then shaken for ten minute~ on a standardized laboratory shaker (Vortex, speed 6) and is placed in a centrifuge. After centrifugation in a gravitational field of about 40 t X g for about 60 minutes, the supernatant is decanted. The liposome dispersion is resuspended in 2,5 ml 0.85 % sterile, phosphate buffered (pH 7.2 - 7.4) sodium chloride solution (Dulbecco). The centrifugation and resuspension are repeated until the supernatant is free of doxorubiein. The liposome dispersion obtained is suitable for parenteral administration.

b) PreParation of sodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-~lycero-3phosphoethanolL-N-hvdroxvsuccinylamine 200.6 mg (270 micromol) 1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanolamine are dried under nitrogen and placed under high vacuum for two hours. The dry lipid is dissolved in 10 ml of freshly distilled pyridine containing 54 mg (540 micromol) succinic acid anhydride. The reaction mixture is stirred for two hours under ~3~5 21489-65~6 nitrogen atmosphere at 55. The excess pyridine is removed by evaporization under reduced pressure and the excess succinic acid anhydride is hydrolyzed by suspending the residue in a mixture of chloroform-methanol-0.58 % aqueous sodium chloride solution (1:2:0.8 vlv). The product is extracted by partitioning after addition of one volume chloroform and one volume 0.58 % aqueous sodium chloride solution. The lower phase is washed three times with a mixture of chloroform-methanol-0.58 % aqueous sodium chloride solution (3:48:47 v/v). The solvent is removed by rotary evaporation and the residue is resuspended in chloroform. Rf (Merck silicagel 60 plates): 0,375 (chloroform, methanol, water-65125/4); UV:
25~.6 nm (broad); m.p. 154C.

Example 2: In a manner analogous to Example 1 aqueous liposome dispersions are prepared containing 84.70 mg (0.098 mmol) disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxy-succinylamine and 168.10 mg (0.226 mmol) 1,2-di-(9-cis-octa-decenoyl)-sn-glycero-3-phosphoethanolamine and 0.1 mg up to 10 mg N-acetyl-L-muramyl-L-alanyl-D-isoglutamine-sodium salt or 0.1 mg up to 10 mg N-acetyldesmethylmuramyl-L-alanyl-D-isoglutamine-sodium salt or 1000 to 100.000 units of recombinant human immune gamma-interferon obtainable according to EP-A-121,157 (10-10-87-Kyowa Hakko Kogyo Co.) or a combination of 1000 to 1000,000 units of this recombinant human immune gamma-interferon with 50-200 microgramm sodium -N-acetyl-D-muramyl-L-alanyl-D-isoglutamine or sodium-N-acetyldesmethylmuramyl-L-alanyl-D-isoglutamine.

Example 3: a) In a round flask 84.70 mg (0.098 molo:l) disod~um-N-[1,2-di-(9-cis-octadecenoyl)-sll-glycero-3~phosphoethanol]-N-hydroxysuccinylamine and 168.10 mg (0.226 mmol) 1,2-di-(9-cis-octa-decenoyl)-sn-glycero-3-phosphoethanolamine are dissolved in a sufficient amount of tert-butanol until both lipids are dissolved.
The solution is filtered under sterile conditions over Acrodisc~
filter (2.0 x 10 m) and is bottled in a sterile vial. This vial is rotated at 1750 rpm (rotations per minute) and the solvent is , ' ~q7357S

removed in a stream of purified, filtered tat the pressure of 1 bar~
dry nitrogen. The vial is evacuated in a high vacuum of 6.0 x 10 2 mbar and is stable under argon inert gas atmosphere.

To this vial containing a thin film of the lipid components men-tioned above, 2.5 ml of a sterile, phosphate buffered (pH 7.2 - 7.4), calcium free sodlum chloride solution (Dulbecco) containing diclofenac in a concentration of 2 g/l are added with a sterile syringe. The vial is then shaken for ten minutes on a standardized laboratory shaker ~Vortex, speed 6) and is placed in a centrifuge. After centrlfugatlon in a gravitational field of about 40,000 x g for about oO minutes the supernatant ls decanted. The liposome dlspersion ls resuspended in 2,5 ml 0.85 % sterile, phosphate buffered (pH 7.2 - 7.4) sodlum chloride solution (Dulbecco) and is suitable for parenteral administration.

Example 4: In a round flask 0.1 mg N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmltoyl-sn-glycero-3-hydroxy-phosphoryloxy)-ethylamlde (preparation accordlng to European Patent Speclfication 25,495), 84,70 mg (0.098 mmol) disodium-N-11,2-di-(9-cis-octadecenoyl~-sn-glycero-3-phosphoethanol3-N-hydroxysuc-cinylamine and 168.10 mg (0.226 mMol) 1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanolamlne are dlssolved ln a sufficient amount of sterlle tert-butanol until all components are dissolved.
The solution i8 filtered under sterile conditlons over Acrodisc~
fllter (2.0 x 10 7m~ and is bottled in a sterlle vial. The vial is rotated at 1750 rpm and the solvent is blown off ln a stream of purifled, filtered (at the pressure of 1 bar) dry nitrogen. Tha vial ls evacuated in a high vacuum of 6.0 x 10 2 mbar nnd 1~ sealed under argon inert gas atmosphare.

To this vial containing a thin film of the components mentioned above 10 ml of a sterile, phosphate buffered (pH 7.2 - 7.4), calcium free sodium chloride solution (Dulbecco) are added with a sterile syringe. The vial is then shaken for 10 minutes on a standardized ~73~7~

laboratory shaker (Vortex, speed 6~. The liposome dispersion obtalned is storage stable at 4 and i9 suitable for parenteral administration.

Example 5: In a round flask 0.1 mg N-acetylmuramyl-~-alanyl-D-iso-glutaminyl-L-alanine-2-(1,2 dipalmitoyl-sn-glycero-3-hydroxy-phosphoryloxy)-ethylamide (preparation according to European Patent Specification 25,495), 84.70 mg (0.098 mmol) disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxysuc-cinylamine and 168.10 mg (0.226 mmol) 1,2-di-(9-cis-octadecenoyl)~
sn-glycero3-phosphoethanolamine are dissolved in a sufficient amount of sterile tert-butanol until all components are dissolved.
The solution is filtered under sterile conditions over Acrodisc~
filter (2.0 x lO 7m) and is bottled in a sterile vial. This vial is frozen at -45. A vacuum is applied to the frozen vial and the solvent is removed until room temperature has been reached. The vial is sealed under argon inert gas atmosphere.

To this vial containing a lyophilisate of the components mentioned above 10 ml of a sterile, phosphate buffered (pH 7.2 - 7.4), calcium free sodium chloride solution (Iulbecco) are added with a sterile syringe. The vial i~ then shaken for lO minutes on a standardized laboratory shaker (Vortex, speed 6). The liposome dispersion obtained is storage stable at 4 and is suitable for parenteral administration.

Example 6: In a manner analogous to Examples 4 or 5 liposome dispersions are prepared containing 0,1 mg to lO mg N-acetylmuramyl-L-alanyl-D-lsoglutaminyl-L-alanine-2-(1,2-dlpalmitoyl-sn-Klycero-3-hydroxyphosphoryloxy)-ethylamide, 84.70 mg (0.098 mmol) disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxy-succinylamine and 168.10 mg (0.226 mmol) 1,2-di-(9-cis-octade-cenoyl)-sn-glycero-3-phosphoethanolamine.

7~

Exam~ 7: In a manner analogous to Example 1 liposome dispersions are prepared containing 86,3 mg (0,098 mmol~ disodium-N-[1,2-di-(9-cis-octadecenoyl~-sn-glycero-3-phosphoethanol]-N-hydroxyglutaryl-amine and 168,1 mg (0,026 mmol) 1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanolamine and 0,1 mg up to 10 mg N-acetyl-L-muramyl-L-alanyl-D-isoglutamine-sodium salt or 0,1 mg up to 10 mg N-acetyl-desmethylmuramyl-L-alanyl-D-isoglutamine sodium salt.

Claims (12)

What is claimed is:
1. A pharmaceutical composition consisting of a) a phospholipid of the formula (I), wherein m represents two or three, R1 and R2 independently of each other represent alkyl, alkenyl, or acyl each having from 10 to 20 carbon atoms, X represents the direct bond, C1-C4-alkylene, C2-C4-alkenylene, or C1-C4-alkylene or C2-C4-alkenylene substituted by hydroxy or a pharma-ceutically acceptable salt thereof, b) a phospholipid of the formula (II), wherein R3 and R4 represent the acyl group of a saturated or an unsatura-ted carboxylic acid having from 10 to 20 carbon atoms and 1 - 2 double bonds, c) a compound or a mixture of compounds having pharmacological acitivity, and, optionally, d) a lipid selected from the group consisting of phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardio-lipin and cholesterol and its derivatives, and, optionally, a pharmaceu-tically acceptable carrier solution buffered to pH 7,0 - 7,8, and, optionally, pharmaceutically acceptable additives.
2. A pharmaceutical composition according to claim 1, consisting of a) a phospholipid of the formula I, wherein m represents two, R1 and R2 are defined as in claim 1, X represents Cl-C4-alkylene 9 C2-C4-or C1-C4-alkylene substituted by hydroxy or a pharmaceutically accep-table salt thereof, b) a phospholipid of the formula II, wherein R3 and R4 independently of each other represent straight chained C10-C20-alkanoyl or C10-C20-alkenoyl, c) a compound or a combination of compounds having pharmacological activity, and, optionally, a pharmaceutically acceptable carrier solution buffered to pH 7.2-7.4.
3. A pharmaceutical composition according to claim 1, consisting of a) a phospholipid of the formula I, wherein m represents two, R1 and R2 are defined as in claim 1, X represents C2-C4-alkylene, or C2-C4-alkenylene, or a pharmaceutically acceptable salt thereof, b) a phospholipid of the formula II, wherein R3 and R4 independently of each other represent straight chained C10-C20-alkanoyl or C10 alkenoyl, c) a compound or a combination of compounds selected from the group consisting of antiphlogistics and/or antiinflammatory agents, anti-biotics, antileishmaniasis agents, antineoplastic agents and immuno-modulators and, optionally, a pharmaceutically acceptable carrier solution buffered to pH 7.2 - 7.4.
4. A pharmaceutical composition according to claim 1, consisting of a) a phospholipid of the formula I, wherein m represents two, R1 and R2 independently of each other represent straight chained alkanoyl or alkenoyl with an even number from 10 to 20 carbon atoms, X represents C2-C4-alkylene, or C2-C4-alkenylene, or a pharmaceutically acceptable salt thereof, b) a phospholipid of the formula II, wherein R3 and R4 represent straight chained C10-C20-alkenoyl with an even number from 10 to 20 carbon atoms, c) a compound or a combination of compounds selected from the group consisting of antiphlogistics and/or antiinflammatory agents and immuno-modulators and, optionally, a pharmaceutically acceptable carrier solution buffered to pH 7.2-7.4.
5. A a pharmaceutical composition according to claim 1, consisting of a) a phospholipid of the formula I, wherein m represents two, R1 and R2 independently of each other represent 9-cis-dodecenoyl, 9-cis-tetra-decenoyl, 9-cis-hexadecenoyl, 6-cis-, 6-trans-, 9-cis-, 9-trans-, or 11-cis-octadecenoyl, or 9-cis-icosenoyl, X represents 1,2-ethylene, or 1,3-propylene, vinylene, or a pharmaceutically acceptable salt thereof, b) a phospholipid of the formula II, wherein R3 and R4 independently of each other represent 9-cis-dodecenoyl, 9-cis-tetradecenoyl, 9-cis-hexa-decenoyl, 6-cis-, 6-trans-, 9-cis-, 9-trans-, or 11-cis-octadecenoyl, or 9-cis-icosenoyl, c) a compound or a combination of compounds selected from the group consisting of antiphlogistics and/or antiinflammatory agents, anti-biotics, antineoplastic agents and immunomodulators, and, optionally, a pharmaceutically acceptable carrier solution buffered to pH 7.2-7.4.
6. A pharmaceutical composition according to claim 1, consisting of a) sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phos-phoethanol]-N-hydroxysuccinylamine or sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxyglutarylamine, b) 1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanolamine, c) a compound or a combination of compounds selected from the group consisting of antiphlogistics and/or antiinflammatory agents, antibiotics, antineoplastic agents, and immunomodulators, and, optionally, a pharma-ceutically acceptable carrier solution buffered to pH 7.2 - 7.4.
7. A pharmaceutical composition according to claim 1, consisting of a) sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phos-phoethanol]-N-hydroxysuccinylamine or sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxyglutarylamine, b) 1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanolamine, c) a compound or a combination of compounds selected from the group consisting of diclofenac, pirprofen, mitomycin, cytarabine, dactino-mycin, daunorubicin, doxorubicine, etoposide, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphor-yloxy)-ethylamide, N-acetylmuramyl-L-alanyl-D-glutamic acid-(C.gamma.-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamide-disodium salt, N-acetyl-D-muramyl-L-alanyl-D-isoglutamine-sodium salt, N-acetyldesmethylmuramyl-L-alanyl-D-isoglutamine-sodium salt, N-acetyl-muramyl-L-alanyl-D-glutamine-.alpha.-n-butylester, N.alpha.-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N.gamma.-stearoyl-L-lysine, 6-O-stearoyl-N-acetyl-D-muramyl-L-alanine-D-isoglutamine and lymphokines, and, optionally, a pharmaceutically acceptable carrier solution buffered to pH 7.2 - 7.4.
8. A pharmaceutical composition according to claim 1, consisting of a) sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phos-phoethanol]-N-hydroxysuccinylamine or sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxyglutarylamine, b) 1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanolamine, c) a compound or a combination of compounds selocted from the group consisting of diclofenac, pirprofen, mitomycin, cytarabine, dactino-mycin, daunorubicin, doxorubicine, etoposide, N-acetyl-D-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-hydroxy-phosphoryloxy)-ethylamide, sodium-N-acetyl-D-muramyl-L-alanyl-D-isoglut-amine, sodium-N-acetyldesmethylmuramyl-L-alanyl-D-isoglutamine, purified, natural or recombinant human gamma interferon, interleukine 2, and compounds obtained from cultures of human T-lymphocytes from the spleen or from peripheral blood after stimulation by antigenes or mitogenes and which are characterized by a high percentage of macrophage activating factor (MAF), and, optionally, a pharmaceutically acceptable carrier solution buffered to pH 7.2 - 7.4.
9. A pharmaceutical composition according to claim 1, consisting of a) sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phos-pho-ethanol]-N-hydroxysuccinylamine or sodium or disodium-N-[1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanol]-N-hydroxyglutarylamine, b) 1,2-di-(9-cis-octadecenoyl)-sn-glycero-3-phosphoethanolamine, c) a compound or a combination of compounds consisting of N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-hydroxy-phosphoryloxy)-ethylamide, sodium-N-acetyl-D-muramyl-Lalanyl-D-isoglut-amine, sodium-N-acetyldesmethylmuramyl-L-alanylD-isoglutamine, and purified, natural or recombinant human gamma interferon and, optionally, a pharmaceutically acceptable carrier solution buffered to pH 7.2-7.4.
10. A pharmaceutical composition according to claim 1, wherein the ratio of the phospholipid component (I) to the phospholipid component (II) is approximately 10 - 90 to approximately 50 - 50 mole per cent.
11. A pharmaceutical composition according to claim 1, wherein the ratio of the phospholipid component (I) to the phospholipid component (II) is approximately 30 - 70 mole per cent.
12. A mixture suitable for the preparation of a pharmaceutical composi-tion according to claim 1 containing a) a phospholipid of the formula I, b) a phospholipid of the formula II, and, optionally, a lipid from natural sources selected from the group consisting of phosphatidyl-choline, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin and, optionally, cholesterol and its derivatives.
CA000516015A 1985-08-19 1986-08-15 Pharmaceutical compositions consisting of acylated phospholipids Expired - Lifetime CA1273575A (en)

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WO1991014423A1 (en) * 1990-03-20 1991-10-03 Otsuka Pharmaceutical Co., Ltd. Liposome preparation
JPH08510748A (en) * 1993-05-21 1996-11-12 ザ リポソーム カンパニー、インコーポレーテッド Reduction of physiological side effects induced by liposomes
ATE198419T1 (en) * 1994-10-14 2001-01-15 Liposome Co Inc ETHERLIPID LIPOSOMES AND THEIR THERAPEUTIC USE
US6589503B1 (en) 1998-06-20 2003-07-08 Washington University Membrane-permeant peptide complexes for medical imaging, diagnostics, and pharmaceutical therapy
US8038984B2 (en) 1998-06-20 2011-10-18 Washington University Membrane-permeant peptide complexes for treatment of sepsis
US7306784B2 (en) 1998-06-20 2007-12-11 Washington University Membrane-permeant peptide complexes for medical imaging, diagnostics, and pharmaceutical therapy
US7803351B2 (en) 2004-08-20 2010-09-28 Washington University Blood brain barrier permeation peptides
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