CA2055990A1 - Phospholipid-vitamin derivatives and methods for preparation thereof - Google Patents

Phospholipid-vitamin derivatives and methods for preparation thereof

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Publication number
CA2055990A1
CA2055990A1 CA 2055990 CA2055990A CA2055990A1 CA 2055990 A1 CA2055990 A1 CA 2055990A1 CA 2055990 CA2055990 CA 2055990 CA 2055990 A CA2055990 A CA 2055990A CA 2055990 A1 CA2055990 A1 CA 2055990A1
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Canada
Prior art keywords
phospholipid
vitamin
ascorbate
group
phospholipase
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CA 2055990
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French (fr)
Inventor
David G. Schena
Jeffrey T. Davis
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Genzyme Corp
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David G. Schena
Jeffrey T. Davis
Genzyme Corporation
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Publication of CA2055990A1 publication Critical patent/CA2055990A1/en
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/127Liposomes
    • A61K9/1271Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers

Abstract

PHOSPHOLIPID-VITAMIN DERIVATIVES AND METHODS
FOR PREPARATION THEREOF

ABSTRACT
Novel phospholipid-vitamin derivatives are described. The phospholipid-vitamin derivatives of this invention are advantageously prepared by reacting a phospholipid with a vitamin or vitamin derivative containing a primary hydroxyl group, in the presence of a microbial phospholipase-D. The resulting phospholipid-vitamin derivatives are useful as free-radical scavengers, anti-oxidants and as fat-soluble derivatives of vitamins.

Description

!,c ~

PHOSPHOLIPID-VITAMIN DERIVA11VES AND METHODS FOR PREPA~A~ON THEREOF
. .

BACKGROUND OF Tl iE INVENTION

This Inventlon relates to phosphollpld-vltamln derivatives and to a process for their productlon by an enzymatlc transphosph~ldylatlon rooctlon. The process described In this invention leads to the re~;lospecific synth0sis of phosphollpid-vHamln 10 derivatlves whlch are novel compounds possesslng valuable chemlcal and physlcal properties. This Invention specifically pertalns to an enzymatlc process for produclng a phospholipld-vHarnin derivative, wh~ch comprises reactin~ a phospholipid wHh awater-soluble v~ltamln or vitamin derlvative In the presence of a microblal phospholipase-D, and to the resuHin~ phospholipid-vitamin derivative produced 15 thereby. By regiospecific~ is meant, In the context of this Invention, the site-speciflc reaction of the phospholipid and vitamin derivative.

It has been previously shown that the phospholipase-D enzyme (EC 3.1.4.4) from cabbage is capable of catalyzing the hydrolysis of the choline head ~roup from 20 either phosphatidylcholine or eg~ lecHhin, yieldin~ a hydrolyzate contalnin~
phosphatidic acid and the choline free base. It Is also known that the cabbage phospholipase-D whlch catalyzes the hydroiysis of e~ lecHhin, also mediates a transphosphatidylation reaction wherein the phosphatldyi moiety of phosphatid~choline is transferred to one of the followin~ primary aicohols: ~Iycerol, 25 methanol, ethanol, ethanolarnine or serine.

The patent IHerature describes the use of cabba~e phospholipase-D as a transphosphatidylation catalyst (BrHish Pat. No. 1,581,810). llle British patent relates to the transfer reaction between a phospholipid and o primary alcohol having a 30 linear or branched alkyl ~roup of up to 5 carbon atoms, optionally substituted by a hydroxyl, halogen or amino group. The patent states that when the transphosphatidylation reaction is carried out with primary alcohols of more than 5 GZ- 1 .0 7 ~

the phosphatldyi molety from a phosphollpld donor to the primary 5'-hydroxyl ~roup of varlous nucleosldes (S. Shuto et al,: Chern. Pharm. Bull., Va-i. 35, 447 (19873 ~nd S.
Shuto et al.: Chem, Pharm. i3u!1., Vol. 3~ 9 (1 98fl)). Som~ ot the resultln~ 5'~3-s~
phosphatldyi) nucleosldes prepared from anti-tumorlcldic nucleoskics (I,e. ~Fu, Ara 5 C) have been shown to have enhanced antlleukemlc activities, arK~i are o~ potentlal therapeutlc Importance.

It is belleved that mlcroblal phosphollpase-D enzymes hove a number of advanta~es compared to traditlonal cabba~e enzyme. For one, the relatlve 10 amount o~ phosphatldic acld produced by competitlve hydroiysis durin~ a transphosphatlciylatlon reactlon Is greatly dlminlshed In the presence of some microbial phosphollpase-D enzymes as opposed to the conventional cabba~e enzyme, whlch may oflen aive phosphatidic aclci as the maJor product. Also, the microbial enzymes, as described above, may have a wider substrate specificity for 15 the transphosphatidylation alcohol acceptor than does the cabi~e phospholipase-D. This property may allow for the synthesis of struc~urally complicated phospholipids, such as the aforementioned phosphaticiylsacchan'des and phosphatidylnucleosides.

Accordin~ly, it is one aspect of this invention to provicie novel phospholipid-vitamin derivatives which are lipophilic derivatives of a vHamin whl'ch has a primary hydroxyi group.

It Is another aspect of the present invention to provide a method for the synthesis of structurally novel phospholipid-vitamin derivatives.

It is still another ospect of this invention to provide vHamin derivatives w'lthenhanced lipophiiicity and therefore enhanced soiubiiity in lipids and enhanced transport across cell membranes.
SUMMARY OF THE lNvENrloiN

The present invention relates to novel phospholij~id-vitamin derivatives and GZ- 1 .0 `i~ ~ 2 ~

methods for thelr preparatlon. The metho~ of thls Inventlon Is an enzymatic tronsphosphatidylallon reactlon wher~ln a new phosphollpl~vitamln derlvative Is produced through lhe actlon of a mlcroblo! phosphollpas~-D which catcliyzes In are~lospeciflc manner the transfer o~ the phosphatl~lc acld m~ety from the startln~
s phosphollpld to the prirnary alcohol of the wc~ter-soluble v~tamln or vltamln derlvotive used In the reactlon. The prociucts of th~s Inventlon may also ~e obtalne~ usln~techniqueS whlch Involve coupling a vHamin havlnç~ a pr~mary hydroxyl ~roup to aphosphc~tidlc acid.

Preferred embodiments of the present ~nventlon relote to phosphollpl~
vitamin derlvatives, represented by the following forrnula:
o Il A-O-P-O-B
~R6 wherein A is a moiety represented by the following formula:

~2Rl ~H2R2 2 5 ~H2 --in which Rl and R2 both represent -O-COR3 or-O-COR4, or Rl represents-O-COR3 and R2 represents -OH, or Rt represents O-R5 and R2 represents {~COR4. in which R3, R4 and R5 are identical or different and~each represents a saturated or 30 unsaturated aliphatic hydrocarbon group having from 1 to 21 carbon utoms, ~OHrepresents a vHamin having a primary hydroxy group, and R6 is a posrt~vely charged counterion which forms a soluble salt of the phosphatidic acid. Representative GZ2- 1 .0 '`s ~
~3~
counterions may be elther or~anlc or Inor~anlc specles or~ Incl~e H, Na, K, U, ivi~, cyclohexylammonlum, ammonlum and ciimethyiamlnopyriciinlum, The present Invention also relates to a proc~ss ~or prc~iucin~ phospholipi~
5 vitamln derlvatlves by reactln~ a phospholiplci represented by the formula:

Il .
A-O-P-O-D

15 wherein A and i?~ ,are as defined above, and ~ represents the ~roup -(CH2)2N(CH3)3, -CH2CH(NH2)COOi i -CH2CH2NH2 -CH2CHOHa~20H~
-(CH2)nCH3, In which n represents a number from 1 to 5, with a vHamin or vitaminderivative having at least one primary hydroxyl group in the presence of a microbial phospholipase-D.

More specifically, the vitamin component of the vitamin derivative may be selected from, but is not limHeci to, the following: thiamine (Vrtamin Bl), riboflavin (VItamin B2), pyridoxine (Vitamin 80, pyridoxal (Vitamin i~6 aldehyde), pyridoxamine (vitamin i~ amine), ascorbic acid (Vitamin C), biotinol ~riamin H alcohol), ~
2s hydroxy-2,5,7,8- tetramethylchroman-2-carbinol (Vitamin E analo~ue), pantothenic acid, pantothenol, or 3-pyridylmethanol (niacin alcohoi). The structures of these particular vHamins or vitamin derivatives is well known in the art.

Additional aspects and understanding of the present invention may b 30 obtained by reference to the accompanying drawings.

GZ2- 1 .0 2 ~

BRIEF DESCi?lPrlON OF THi- Di?AWlNGS

Fl~ure 1 is a structural representatlon of a phosphollpld-thiamine derivatlve prepared accordin~ to the process of thls Inventlon.

Flgure 2 Is a structural representatlon of a phospholipld-pyricioxine derivatlveprepared accordln0 to the process of th!s Invention.

Flgure 3 is a structural representation of a phosphollpid-3-pyridYlCarCinol derivative prepared accordin~ to the process of this Invention.

DETAILED DESCRli'TlON OF THE INVEiYTiON

It has now been discovered, as described in the present invention, that another group of compounds, nameiy vitamins with primary hydroxyl ~roups and their derivatives, are effective acceptors In the transphosphatidyiation reaction catalyzed by microbial phospholipcse-D enzymes. Vrtamins such as ascorbic acld (vitamin C), riboflavin ~Vitamin i32), thiamine ~itamln Bl), pyridox~ne (Vitamin B6), biotinol ~riamin i-i alcohoi), and pantothenic acid are all distin~uished as containin~
primary hydroxyi groups. It is the presence of this primary hydroxyi ~roup whichallows for the regiospecrfic enzymc~ic prociuction of the phospholipid-vHamins in the phospholipase-Dmediatedtransphosphatidylationreaction, i~nospholipid-vitamin derivatives of vriamins containing primary hydroxyi groups may clternatively be obtained by using chemical coupling of the vitamin to a phosphatidic acid.

The phospholipid-vitamin derivatives prociuced by this invention are novel compounds. In the case of vitamlns possessing a primary hydroxyi group, the intermediacy of a microbial phospholipase-D enzyme is a key factor in bypassin~ the GZ2- 1 .0 2 ~
chemlcal synthesis of such derlvatlves whlch would, In neariy every case, be expenslve and forrnldable duo to the numerous hydroxyi protect~on and deprotectlon steps requlred (e.~. for Vit~mlns C, B2 and B6~. it is the newiy discovered abllity of the mlcroblal phosphollpase-D enzYme to speclflcally cutalyze 5 ~he transfer of the phosphatidyl molety of the phospholipld to the primary alcohol of She vHamin or vitamin derivatlve whlch characterizes the present invention.

The preferred process of the present inveotion comprises reactin~ a phospholipld represented by the following formula:

o Il A-O-P-O-D
I

wherein A is a moiety represented by the following forrnula:

-CH2Rl 2s in which R1 and R2 both represent -O-COR3 or -O-COR4, or R1 represents -O-COR3 ~nd R2 represents -OH, or R1 represents O-R5 and R2 represents ~COR4, in whlch R3, R4 and R5 are identical or different and each represents a saturated or unsaturated aliphatic hydrocarbon aroup having from 1 to 21 cG~bon atoms, R~S Is a positively charged counterion, preferably selected from the ~jroup consisting of H, 30 Na, K, Ll, Mg, cyclohexylammonium, ammonium, dimethyiaminopyridinium and D
represents the group -(CH2)2N(CH3)3, -CH2CH(NH2)COOH, -CH2CH2NH2, ~H2CHOHCH20H, or -(CH2)nCH3, in which n represents a number from 1 to 5, with a vitamin or vitamin derivative having crt least one primary alcohoi ~roup in the presence of a microbial phospholipase-D.

G22- 1 .0 f',. ~
2 ~ V

rhe phospholipld-vltamln derivatives descrii~d In thls Invention cannot be easl1y prepared by uslng the conventlonal cabba~e phosphollpase-D. Thus, incubatlon of phosphatidylchollne with a vHamin or vitamln derivativs in the s presence of cabba~e enzyme showed oniy the productlon of phosphatidlc acid and chollne. Chan~ln~ tha concentration of reactants or enzyme, or chan~in~ the pH or temperature had no effect on the reaction product composition. The phospholipase-D used for the production of the aforementloned phospholipid-vitamin derivatives is ideaily derived from a mlcrobial source. The microbial source 10 can include a variety of selected mlcroor~anisms or other celis whlch have been recombinantly en~ineered to contaln a gene encodin~ for the desired phospholipase-D enzyme. The microbial phospholipase-D enzymes rnay be distinguished from the cabbage enzyme in that they are caPable of catalyzin~ theformation of a phospholipid-vitamin derivative. Examples of suitable phospholipase-15 D producin~ microorganisms are those beion~inç~ to the S~enus reptoverticillium sp.Tobie 1 shows the various strains of Streptoverticiiiiurn which produce useful phospholipase-D enzymes:

Table 1 Streptoverticillium Species which prociuce Phosphoiipase-D Enzymes CaPable of Phospholipid-Vitamin Forrnation Stv. flavopersicum Stv. hachqoensis Stv. mediocidicus Stv. roseoverticiilium Stv. albireticull Stv. Ieuteovert cl!ilum Stv. hiroshimense Stv. cinnamoneum Stv. nseoca!num GZ- 1 .0 The Streptoverticl! lum mlcroorganlsms are not the oniy mlcroblal sources of phosphollpase-D capable of catalyzlng the productlon ot phospholipld-vitaminS.
Followin~ the method of thls Invention, numerous other mlcrobes have been examlned 5 for thelr ability to produce the requlsite enzymes. Bacterlà frorn the ~enus Streptomyces and from ~he ç3enus CorYnebocterium have a~so been found to producea phospholipase-D capable of maklng the novel phospholipici-vitamlns.

The production of the aforementloned phosphoiipid-vitamins may be carried o out uslng either purified mlcrobial phosphollpase-D enzymes or by usin~ the crude broth following fermentatlon of the phosphollpase-D produclng or~anlsm. The phospholipase-D enzymes may be used In solutlon or may be Immobilized onto solidsupports, For example, the following solici supports may be used to immobilize phospholipase-D; octyl-sepharose CL-48, phenyl-sepharose CL-4B, butyl-toyopeari 15 ~550C, controlled glass beads, celite, Amberiite lon-exchcYnge resins, or glutaraldehyde linked polvinylchloride.

The transphosphatidylation reactlon to produce a phospholipic~vHamin can be advantageously conducted by contacting the phosphoiipid wHh the vitamln or 20 vitamin derivative in the presence of the microbial phospholipase D in a solvent rnixture.
The solvent mlxture may be either a monophasic aqueous buffer system or a biphasic mlxture of an aqueous solvent and an organic solvent. The aqueous solvents may contain additlves which act to promote the enzymatic reactivHy or to stabilize the enzyme. For example, the aqueous solvents may contain buffers sucn as acetic ocid, 25 citric acid or phosphoric acid or neutral salts such as calclum acetate, calcium chloride or sodium acetate. Examples of the organic solvents which may be used include aliphatlc hydrocarbons such as hexane, pentane or octane, aromatic hydrocarbons such as benzene or toluene, ethers such as tert-butyldimethyl ether, diethyl ether or tetrahydrofuran, esters such as ethyl acetate and halogenated hydrocarbons such as 30 methylene chloride or chloroform. The preferred solvent system is a mixture of an aqueous sodium acetate buffer and an ether. The mixing ratio of aqueous to or~anlc solventscanbeintherangeof 100:1 to 1:100(v/v),witharat~of 1:1 (v/v)being prefened.

GZ2- 1 .0 ... . . . . .... ..

~ ~ rs~

rhe reactlon may be carrlcd out from about 0~C to abs~ut 80'C, preferabiy between 20'C and 30~C for the StrePtov~rtlc Inum and Strepto~yces i~hosphollpase-D
catalyzed reactlons. The reactlon tlme may be frorn 10 mlnutes to 10 days, but is 5 preferably between 1 and 24 hours. If necessa~y, the course of the reactlon may i~
monitored by a technlque such as 11 C (thln iayer chromato~raphY) or HPLC thl0h pressure llquld chromato~raphy). By monHoring the ~ormatlon of product and disappearance of substrate, the proper reactlon tlme rnay be attained, 10The phospholipid-vitamin derivatlves formed may be isolated by a variety of known technlques. These technlques Include column chromato~raphY on sllica or alumina gel or on an lon-exchan~e resin, hi~hperformance llquici chromatography, or by precipitation as various saits from or0anic solvents. These saH forms include sodlum, potassium, barium, trialkylammonium or calclum saHs of the phospholipid-vHamin lS derivative. The phosphoiipid-vHamin derivatives may be further purified by recrystailization from organic soivents.

The phospholipid-vitamin derivatives of this inventlon are believed to be usefuias liposome-forming substances or as therapeutic açlents thernselves, particular~ In the 2 o treatment of diseases where there is evidence of a deflciency In the ability to transport vitamins across the cellular membrane. By appending either the vitamin C (ascorbate) or a vitamin E derivative to a phospholipid, novel materials with potent anti-oxidant properties may be prepared. rhese anti-oxidant phospholipic~vitamins are usefui for various applications in the scaven~in~ of oxy~en free-radicais which may cataiyze lipid 25 peroxidation. Thus, incorporation of the anti-oxidant phospholipid-vitamin derivatives described in this invention into liposomal forrnulations contcinin~ other unsaturated phospholipids wiil protect the liposomal components from the deleterious effect of oxidative d~mage.

30One currentiy avaiiable commercial lipophilic vHamin derivative, aliithiamine, is a lipid soluble form of Vitamin Bl which is administered to individuais suffering from GZ2- 1 .0 ~c~
3 ~ t7' ~
thiamlne deflclencles. AllHhiamlne is able to cross the cellu~r membrane bllayer with moderate efficlency, and once In the cytoplasm allithlomlne is metabol!zed to thiamlne, The phosphatldyi-thlamlne deriv~tive whlch Is readiiy ovallable accordln~ to thls Inventlon may prove to be superlor to allHhlamlne In its ai~ility to cross the cellular 5 bilayer membrane, thus provldin~ an aHernate strate~y for the de ively of thlamine to vitamln B1 deficlent cells.

Covalent lipophllic vitamln C derivatlv0s, wHh 6~palmitoyi ascorbate beln~
the most wldely describeci (Swern et ai., Soap, Vol 20, p.224, 1943), have a number of o valuable commerclal applicatlons, for exam;~le as anti-oxldant aciciitlves In fooci and oll preparations. Ronoxan A Is a commerclal formulatlon of ~O-paimHoyi ascorbate, a-tc~copherol (Vitamin i~, and e~j~ lecithin which Is useci as an antl-oxidant to protect fats and oils containin~j unsaturated fatty acids a~ainst air oxidation (G. Pon0racz, Int. J.
Vitamin Nutr, Res. Vol. 43 (4) p. 517, (1973)). The ~palmitoyi ascorbate when 15 combined wrth tocopherol and lecithin has a syner~istic effect in protectin~ fats frorn clir oxidation (see Table 2 below, from Pon~racz 1. J. V. N. R., 43(4) p. 517 (1973)).

Table 2 -StabilHy of Butterfat to Oxidation Anti- x'l~m~/a fat) Peroxide Values coPm) 3 4 5 6 (days) Control 265 - - -200T 9.9 56.3 11û
500 AP+200 T 1.3 3.7 4.4 11 500AP+lOOT+500Lec 0.5 1.0 1.2 1.5 G22- 1 .0 .. . . .. .

! l ~ f~
Table 2 AP = ~PamltoylAscorbate r = a-Tocopherol Lec = E~ Lecithln Althou~h Hs antloxldant propertles are Impressive, ~paimitoyl ascorbate has poor solubility and a very slow dlssolution rate In fats and oiis ~i, Klaul p. 16 In VHamin C, Edited by G. G. Birch and K. J. Parker John Wiley i~N, i~. 1974). These 10 drawbacks represent a serlous problern for many practlcal appllcotions of i~onoxan A. One advanta~e of the phosphatldyl-ascorbate derivatlves described by the present Inventlon Is that they display enhanced solubllity and Increased dlssolutlon rates in fats and oiis.

The Interactlon of ~O-palm'rtoyl ascori~ate wHh the phospholipid bilayers of DPPC, DPPE, and DMPE liposomes has been investi~cfed throu~h tt~e use of infrared spectroscopic studies ~Kohler, Mantch, and Casal, J. Can. Chem. 6~ p. 983 (1988)). It was found that incorporation of ~GpalmHoyl ascorbate into ~he pi~ospholipid bilayer which makes up a liposome induced a lar~e increase (15-25C) n the phase 20 ~ransHion temperature for these phospholipids. The physical effect of this increase in phase transition temperature is manifested by a correspondin~ increase in stability of the liposome.

The ai~ove~escribed phospholipid-vrtamins producec accordin~ to this 25 invention may also be useful In research applications for the stuciy of vitamin - transport across cellular membranes.

Further understanding of the invention will be had by a detailed study of the followin~ examples. These examples are ~jiven by way of illustration and are not30 intended to limrt the inventlon.

Example 1 Preparation of 1 ~-Dimyristoyi-sn-Glycer~3-Phosphoryiffliamine (DMP-T) by Transphosphatidylation of Dimyristoylphosphatidylcholine with Thicmine (VHamin B1), in the Presence of Microbial Phospholipase-D.

GZ2- 1 .0 .. . . .

.

To 268 m~j of dlmyristoyiphosPhG~ldylCholine (DMPC) dissoved In 10 ml of ether was added 20 ml of 0.11 M thlamlne-HCL In 50 mM socilum acetate (pH 5.0) buffer. TQ the resultln~ blphaslc mlxture was added 10 ml ot pure StreptomVceS s~.
phosphollpase-D (60 U/ml, obtalned from Si~ma) In 50 mM socilum acetate buffer (pH 6.2). Thc resuHln~;~ emulslon was vl~orously stirrod at room temperature and the course of the transphosphatidylation reaction was monitored by thin-l~yer chromato~raphy (rLC) by assayin~ for the disappearance of DMPC and the simultaneous appearance of DMP-T. After stlrrin~ for 4 h, the rea~tion had proceeded to completion. The reaction mixture was diluted with 60 ml of CHCI3, the organic layer was separated and collected, dried over sodium suifate, and concentrated in vacuo to ~ive 321 m~ of a whlte solid (67% molar yleld). This crude solid was purified by silica ~el chromato~raphy usin~ 25:4 CHC13:MeOH:H20 as eluent. Fractions containin0 UV active material which stained positive ~t phosphorus (usin0 molybedenum stain) were pooied and concentrated in vacuo to ~jive 280 rn~(58~ molar yield) of pure DMP-T as a whHe solid. Characterization of the DMP-T ~ave the following values: R~ = 0.26. silica ~el 60 F254 (Merck), CHC13-MeOH-H20 (65:25:4):
UV max (chloroform) 268 nm (E 3.9 mM): 400 MHZ ~H-NMi~ (CDC13)~10.0(s) 1 H, 8.10(s) 1 H, 5.62(s) 1 H, 5.18(s) 1 H,4.32(dd) 1 H, 4.05(m) 3H, 3.~m) 2H, 3.~s) 2H,3.10(m~ 2H, 2 0 2.5~(s) 3H, 2.45~s) 3H,2.25(dc~) 4H,1.40 1.18(m) 52i~i, 0.~8(t) 6H. The structure of this product is shown In i~i~ure 1.

Example 2 Prepara~ion of 1 2-Dimyristoyi-sn-Glycero-3-Phophorylpyridoxine (DMP-P) by Transphosphatidylation of Dimyristoyiphosphatidylcholine (DMPC) wHh Pyridoxine (V'ltamin B~), in the Presence of Microbial Phospholipas~D.

To 500 m~ of dimyristoylphosphatidylcholine (DMPC) dissoived in 12 ml of methylene chlofide was added 50 ml of 0.7 M sodium pyridoxine in 50 mM calcium acetate (pH ~.0) buffer. To the resuHin~ biphasic mixture was ad~ed 1 ml of crude Stre~toverticillium flavoPersicum phospholipase-D (10 U/ml, where U represents aDPPC hydrolysis activity unit) in a 50 mM sodium acetate (pH 6.2~ buffer.

GZ~- 1.0 !j',.,.; ~
J ~

The resultlng blphaslc solutlon was vlgorously stlrred at room temperatur and the course of the transphosphatldyiatlon reactlon was monitored by thln iayer chromatography (TLC) by assayln~ for the disappear<: nce of DMPC anci tha S slrnultaneous appearance of DMP-P. After stlrrlng tor 18 hr., ti~? reactlon was Jud~ed complete by rLC. The methylene chlorlde layer was separated and dried over sodlum sulfate and concentrated In vacuo to ~Ive 500 mç~ of c~ white solid.
Characterization of the resultln~ DMP-P gave the followlr~: m.p. = 139-143C, Rf =
0.56, silica gel ~0 F254 (Merck), CHC13, MEOH, H20 (~5.25:4); HPLC 0a e min. The structure of this product is shown In Fl~ure 2.

Example 3 Preparatlon of 1,2-Dimyristoy~sn-Glycer~3-Phosphory~i'yridylcarbinol (DMP-i~ by Transphosphatidyiation of Dimyristoylphosphatldylcholine (DMi'C) wHh 3-pyrldylcarbinol (niacinol) in the presence of Microblal Phospholipas~D.

To 500 m~ of dimyristoyl phosphatidylcholine (DMPC) d-~so~ed in 5 ml of methylene chloride was added 10 ml of 0.9 M 3-pyridylcarbinol buffered to pH 6. To the resuHln~ biphasic m~lxture was added 2 ml of crude StrePtoverticillium flavoperslcum phospholipase D (10 Ulml, where U represents a DPPC hydrolysis activity unit) in a 50 mM sodium acetate (pH ~.2) buffer.

The resultin~ biphasic solutlon was vl0orous~ stirred at room temperaturs and the course of the transphosphatidylation reaction was monitored by thin layer chromatography ~TLC) by assayin~ ~or the disappearance of DMPC and the simultaneous appearance of D~P-N. A~ter stirrin0 for 18 hr. the reaction wasJudged complete by ~C. The methylene chloride layer was sep~rated and the aqueous layer washed wHh 10 rnl methylene chloride. The combined or0anic layers were dried over sodium sulfate and concentrated in vacuo to ~Ive 258 mg of white solid (510. Charactereation of the resulting DMP-N ~ave the followin~: mp 129-135C, 3 o RP: 0.4 ~UV active), silica ç~el 60 F254 (Merck), CHC13, MEOH, H20 (65:25:4). ll~e structure of this product is shown in Figure 3.

G7~- 1 .0

Claims (20)

1. A phospholipid-vitamin derivative represented by the formula:

wherein A is represented by the formula:

in which R1 and R2 both represent -O-COR3 or -O-COR4, or R1 represents -O-COR3 and R2 represents -OH, or R1 represents O-R5 and R2 represents -O-COR4, in whichR3, R4 and R5 are identical or different and each represents a saturated or unsaturated aliphatlc hydrocarbon group having from 1 to 21 carbon atoms, B-OH
represents a vitamin having a primary hydroxyl group, and R6 is a positively charged counterion.
2. The phospholipid-vitamin derivative of Claim 1 wherein R6 is selected from the group consisting of H, Na, K, Li, Mg, cyclohexylammonium, ammonium and dimethylaminopyridinium.
3. The phospholipid-vitamin derivative of Claim 1 wherein the vitamin is selected from the group consisting of L-ascorbic acid, L-sodium ascorbate, L-potassium ascorbate, L-lithium ascorbate, L-calcium ascorbate, 2-sulfoascorbate, GZ2-1.0 2-0-alkyl ascorbate, 2,3,0-dialkylascorbate, mono(dihydrogen phosphate) ascoribate, thiamine, riboflavin, pyridoxal, pyridoxine, biotinol, pantothenic acid, pantothenol, 6-hydroxy-2-5,7,8-tetramethylchroman-2-carbinol, pyridoxamine and 3-pyridylcarbinol.
4. rhe phospholipid-vitamin derivative of Claim 1 which is selected from the group consisting of 1,2-dimyristoyl-3-glycero-3-phosphorylthiamine, 1,2-dimyristoyl-sn-glycero-3-phosphorylpyridoxine,1,2-dimyristoyi-sn-glycero-3-phosphoryl-3-pyridylcarbinol.
5. A method for making phospholipid-vitamin derivatives which comprises reacting a phospholipid represented by the formula:

wherein A is represented by the formula:

in which R1 and R2 both represent -O-COR3 or -O-COR4, or R1 represents -O-COR3 and R2 represents -OH, or R1 represents O-R5 and R2 represents -O-COR4, in whichR3, R4 and R5 are identical or different and each represents a saturated or unsaturated aliphatic hydrocarbon group having from 1 to 21 carbon atoms, R6 is a positively charged counterion and D represents the group -(CH2)2N(CH3)3, -CH2CH(NH2)COOH, -CH2CH2NH2, -CH2CHOHCH2OH, or -(CH2)nCH3, in which n GZ2-1.0 represents a number from 1 to 5, with a vitamin or vitamin derivative selected from the group consisting of L-ascorbic acid, L-sodium ascorbate, L-potassium ascorbate, L-lithium ascorbate, L-calcium ascorbate, 2-O-sulfoascorbate, 2-O-alkyl ascorbate, 3-O-alky1 ascorbate, 2,3-O-dialkylascorbate, mono(dihdyrogen phosphate) ascorbate,thiamine, riboflavin, pyridoxal, pyridoxine, biotinol, pantothenic acid, pantothenol, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carbinol, pyridoxamine or 3-pyridylcarbinol, in the presence of phospholipase-D.
6. The method of Claim 5 wherein R6 is selected from the group consisting of H, Na, K, Ll, Mg, cyclohexylammonium, ammonium, and dimethylaminopyridine.
7. The method as claimed in Claim 5 in which the phospholipid is at least one member selected from the group consisting of egg lecithin, soy lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and an alkyl ether of phosphatidic acid.
8. The method as claimed in Claim 7 in which the vitamin derivative is L-ascorbic acid or a salt thereof.
9. The method as claimed in Claim 7 in which the vitamin derivative is 6-hydroxy-2,5,7,8-tetramethylchroman-2-carbinol.
10. The method as described in Claim 7 in which said phospholipase-D is derived from a microbial source selected from the group consisting of the bacterial genus Streptoverticillium, Streptomyces and Corynebacterium.
11. The method as described in Claim 10 in which said phospholipase-D is derived from Streptomyces sp.
12. The method as described in Claim 5 wherein the reaction is carried out by contacting the phospholipid with the ascorbate in an aqueous solvent or in a GZ2-1.0 biphasic mixture consisting of an aqueous solvent and an organic solvent.
13. The method as claimed in Claim 12 wherein said organic solvent is at least one member selected from the group consisting of benzene, chloroform, diethyl ether, ethyl acetate, hexane, methylene chloride, petroleum ether, t-butyldimethyl ether and toluene.
14. The method as claimed in Claim 5 wherein the phospholipase-D enzyme is immobilized on a solid support.
15. The method as claimed in Claim 5 wherein the reaction is carried out at a reaction temperature of from about 10°C to about 90°C.
16. The method of Claim 5 in which the pH of the aqueous solvent is from about pH 3 to about pH 8.
17. A phospholipid-vitamin derivative which is prepared according to the method of Claim 5.
18. A liposomal formulation comprising at least one lipid and the phospholipid-vitamin derivative of Claim 1.
19. The formulation of Claim 18 wherein the lipid is a phospholipid.
20. The formulation of Claim 18 wherein the lipid is a fat or oil.

GZ2-1.0
CA 2055990 1990-11-22 1991-11-21 Phospholipid-vitamin derivatives and methods for preparation thereof Abandoned CA2055990A1 (en)

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EP0655239A1 (en) * 1993-11-25 1995-05-31 Lipotec, S.A. Liposomes encapsulating doxorubicine
EP0948341A1 (en) * 1996-09-17 1999-10-13 Amur Pharmaceuticals, Inc. Phospholipid drug derivatives
WO2000022094A2 (en) * 1998-10-09 2000-04-20 Kansas University Medical Center Methods for inhibiting oxidative modification of proteins
US6472400B1 (en) 1995-09-12 2002-10-29 University Of Kansas Medical Center Advanced gylcation end-product intermediaries and post-Amadori inhibition
US6472411B1 (en) 1995-09-12 2002-10-29 University Of Kansas Medical Center Advanced glycation end-product intermediaries and post-amadori inhibition
WO2003020941A1 (en) * 2001-08-28 2003-03-13 Degussa Food Ingredients Gmbh Method for the production of phospholipids
US6716858B1 (en) 1995-08-28 2004-04-06 Kansas University Medical Center Methods for inhibiting diabetic complications
US6730686B1 (en) 1995-09-12 2004-05-04 Kansas University Medical Center Methods for inhibiting oxidative modification of proteins
US6740668B1 (en) 1995-08-28 2004-05-25 Kansas University Medical Center Methods for inhibiting diabetic complications
US6750209B1 (en) 1995-09-12 2004-06-15 Kansas University Medical Center Advanced glycation end-product intermediaries and post-amadori inhibition
US7030146B2 (en) 1996-09-10 2006-04-18 University Of South Carolina Methods for treating diabetic neuropathy

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0655239A1 (en) * 1993-11-25 1995-05-31 Lipotec, S.A. Liposomes encapsulating doxorubicine
WO1995014459A1 (en) * 1993-11-25 1995-06-01 Lipotec, S.A. Liposomes for the encapsulation of doxorubicine
US6740668B1 (en) 1995-08-28 2004-05-25 Kansas University Medical Center Methods for inhibiting diabetic complications
US6716858B1 (en) 1995-08-28 2004-04-06 Kansas University Medical Center Methods for inhibiting diabetic complications
US6472411B1 (en) 1995-09-12 2002-10-29 University Of Kansas Medical Center Advanced glycation end-product intermediaries and post-amadori inhibition
US6472400B1 (en) 1995-09-12 2002-10-29 University Of Kansas Medical Center Advanced gylcation end-product intermediaries and post-Amadori inhibition
US6730686B1 (en) 1995-09-12 2004-05-04 Kansas University Medical Center Methods for inhibiting oxidative modification of proteins
US6750209B1 (en) 1995-09-12 2004-06-15 Kansas University Medical Center Advanced glycation end-product intermediaries and post-amadori inhibition
US7030146B2 (en) 1996-09-10 2006-04-18 University Of South Carolina Methods for treating diabetic neuropathy
EP0948341A4 (en) * 1996-09-17 2003-05-21 Supergen Inc Phospholipid drug derivatives
EP0948341A1 (en) * 1996-09-17 1999-10-13 Amur Pharmaceuticals, Inc. Phospholipid drug derivatives
WO2000022094A3 (en) * 1998-10-09 2001-02-22 Kansas University Medical Ct Methods for inhibiting oxidative modification of proteins
WO2000022094A2 (en) * 1998-10-09 2000-04-20 Kansas University Medical Center Methods for inhibiting oxidative modification of proteins
WO2003020941A1 (en) * 2001-08-28 2003-03-13 Degussa Food Ingredients Gmbh Method for the production of phospholipids
US7067292B2 (en) 2001-08-28 2006-06-27 Bioghurt Biogarde Gmbh & Co. Kg Method for the production of phospholipids

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