CA2117749A1 - Composition for therapeutic or diagnostic use, process for its preparation and its use - Google Patents
Composition for therapeutic or diagnostic use, process for its preparation and its useInfo
- Publication number
- CA2117749A1 CA2117749A1 CA002117749A CA2117749A CA2117749A1 CA 2117749 A1 CA2117749 A1 CA 2117749A1 CA 002117749 A CA002117749 A CA 002117749A CA 2117749 A CA2117749 A CA 2117749A CA 2117749 A1 CA2117749 A1 CA 2117749A1
- Authority
- CA
- Canada
- Prior art keywords
- ldl
- agent
- active agent
- composition
- lysosomotropic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- 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
-
- 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
- A61K9/1275—Lipoproteins; Chylomicrons; Artificial HDL, LDL, VLDL, protein-free species thereof; Precursors thereof
-
- 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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6917—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a lipoprotein vesicle, e.g. HDL or LDL proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention concerns a composition comprising a carrier selected from the group consisting of lipoproteins, in particular reconstituted LDL (Low Density Lipoprotein), other microemulsion particles, liposomes and micelles, containing a therapeutically or diagnostically active, lipo- or amphiphilic agent, and associated with a suitable ligand recognizable by its specific (complementary) cell receptor, together with a lysosomotropic agent, for targeting of the active agent to a site of interest, such as cancerous tissue.
Description
W093~0~0 21 17 7 ~ 9 PCT/F193/00149 Composition for therapeutic or diagnostic use, process for its preparation and its use FIELD OF I ~ ENTION
The present invention is based on the use of a carrier selected from the qroup consistîng of lipoproteins, in particular reconsti~uted LDL (~ow Density Lipoprotein), other types of microemulcion particles, liposomes and micelles, and containi~g a therapeutically or diagnostical-ly active~ l~po- or amphiphilic ~gent, and a~sociated:with a suitable ligand recognizable by its specific (complemen-tary~ cell recep~or, together with a lysosomotropic agent, for targeting of the active agent to the site of interest/
such as cancerous tissue~ and to optimize the effect thereof a ~he said site.
BACKGROUND OF THE INVENTION
~igand speci~ic receptors on the cell surf~ce medi~te both formation on the environme~t as well as nutrients into : the cell. Such ligands to be recognized include a range of : mole~u:~ar structures, e.g~ growth factors (for exam~le platelet derived growt~ factor, en~othelial ~cell growth ~actor, tumor ~r~wth ~actor, nerve ~row~h factor~, hormones ~ , (e.g~ insulin, vasopres~in etc.), extracellular matrix protein (a.g. ooll~agen) and lipoproteins ~e.g. low densit~
:lipoprotein). In maDy cases t he~ binding of the ligand to the cell surface receptor is fo~lowe~ by a clust~ring o~
~he lig~nd-receptor cumplex and subsequent internalization i~` via cbated pits, ultimakely locating the ligand~ ln ly-so~omes. This pathway is utilized for exampIe by~viruses in their entry into cellsO
: : :
It ha be~n known ~or a long time that people suf~ering from cancer exhibit a cholesterol level in the ~erum whi~h ~ ~ is lower than normal. Cholesterol circulates in the blood :
' :
W~93/20~0() 2 1 17 7 ~ 9 PCT/Fl93/00149 in the form of a lipid-protein complex, primarily as the so-called ~ow Density Lipoprotein, LDL. LDL is a spherical macromolscule having a diameter of about ~80 ~. LDL is surrounded by a surface film consisting of phospholipids, choles~erol and sphingomyelin, and of protein/ i.e.
~polipoprotein B ~apo B). The interior of LDL is formed by chol~sterol in esterified form ~compounds of fatty acids and cholesterol), triacylglycerol, retinol, as well as other hydrocarbon soluble compounds.
Cells contain on their surfaces specific LDI,-receptors which bind to the LDL-partic:le by specifically recognizing the protein component of ~he LDL-surface, i.e. apolipopro-tein B. The density of LDL-re~eptors is much higher on cancerous cells than on normal cells and cancer cells bind LDL more effectiYelyO I~ is evident that the lower serum cholestero~ level of cancer patienks: is due to~ enhanced uptake of LDL by the cancer cells. This is easy to ra-tionaliz~ in terms of the increased :requiremen~ of com-ponents such as cholesterol for the gr ~ h~of the ~umor : cells.
The use o~reconstit~ted:LDL containing aytotoxic compou~ds ~:whlch :replac~e~the natural ap~la~;~:lipids ln~the particle 25: ~core h~s ~been describe~ The::lipids are removed from::the core of LD~ by ~irst lyophili:~ing the lipoprotein in~the presence of ~starch (Gustaf~on, A (~1965)~:J. Lipid:Res.:~:20 : : 254-264)~. A~slightly modified~techni~ue~has~been~dessribed~ :~
by~ Xrieger (Krieger,;M~ ) M~ethods~Enz~mol.~ 12~,~608~
~ 30 ~ 613). LDL is reconstituted by lyophilizlng;in the pr~sen~e ! of potato s~arch~and extracting the niu~ral lipids;with an org~nic 9alvent, such as heptàne~ The agent~to be introdu-~
ced is dissolved in an~organic solvent~and~incuba~ed With the ext~acted ~DL. The;or~anic ~sol~ent is~;~evaporated~and ~
35 ~ the ~econstituted LDL is dissolved in a buffered ~a:queous~ ;
~ solution.~
:
' :
WO~/2~)l) 2 ~ 17 7 ~1 ~ PCT/Fl93/0014(~
~ccording to an improvement to this method, described in the WO-publication 86/07540, the starch is replaced by a saccharide or a sugar alcohol, and according to this publication, a reconstituted LDL is obtained having similar properties as natural LDL, in vitro and in vivo.
However, when using e.g. reconstituted LDL as a carrier for e.g. drugs, in the form of an injectable aqueous solution, retro-endocytosis has been observed. Thus a~t~r an ini~ial decrease of the drug level in the blood, indicating upt~ke of LDL by the cell~, an increase in the level of the drug is obserYed, indic~ting expulsion of the drug from the cells. Thus it is difficult to achieve a significant accumulation of the drug in the target tissue, and conse-quently the action of the active agent cannot be fullytaken advantage of.
It is also prior known to use, for drug delivery, li-posomes wherein the active agent is incorpora~ed a) in a vre surrounded by a bilayer- of a Iipid substance, typical-ly a pho~pholipid (together with e.~ cholesterol and sphing~myelin) o~ bj if the desired ~herap~utic compoun~ is àmphiphilic (e.g. an anti-sancer: phospholipid), it is incorporated in~the bilayer itself. For the said purpose ~:~25 al~o microemulsions, f:or example an emulsion of active agent, such as a lipophi~ic drug surrounded by a monolayer of phosphatidyl choline, are used.
~:~SUMMARY QF THE INV NTION
: The present invention relates to an improvement to the : known methods by means of which it is possible to achieve an enri hment of~the active agent at the target site~ This is~ made poss~ble by using, in combination, (a~ a carrier selec~ed ~rom the 7roup consisting of lipoproteins, other type~ of microemulsion particles, liposomes and micelles, and containing a therapeutically or a diagnostically 2 1 17 7 4 9 PCr/F193/01)149 effective amount of a lipo- or amphiphilic active agent, associated with at least one ligand which is complementary to and recognizable by specif ic receptor in the living cells, and (b) a lysosomotropic agent~
The invention concerns, on the one hànd t a composition f or diagnostic or ther~peutic u~;e which contains the said com}~ination, and on the other hancl a method f or the preparation of the said con~position. Especially the 10 lnventi~n concerns a composition and a method wherein the carrier is specifically reconstituted LDL, as well as its use on humans and non-human animals.
DETAILED I)ESCRIPTION OF TH[E INVENTION
The. c:omposi~ion according to the invenltic:n thus contairls at least two components, i . e . a carrier selected from the group of lipopro~eins, other types of microemulsiorl parti-cles, liposomes and micelles, and which is associated to 2 0 a suitable ligand . The c:arrier compoments are known as suc:h, as is their manner of manuf acture . cca~mon f or these carriers is that they can c:on~ain a therapeutically or ~iagnostically active agent.
The carrier is associated with a ligand which is chara¢te-: rized in ~hat it is~complementary to and oan be recognized by a specific cell receptor. The term 5':associated with" i~s intended to include also the possibility that the carrier incorporates the said liga~d, such as in the c::ase of LDL, 30 where the ligand can be the ap o B moiety included in the surf ace f ilm oî the LDL ~ The ligand is thus typically one o: the substanGes mentioned earlier~ ~ su~h as a protein~
e O g . growth f actor, a hormone, apolipoprotein, viral prvteir~, synthetic peptide etc. As stated, when the carrier 35 is~ a reconstituted LDL, thP ligand i~; the apo B-moiety, ox :a corresponding receptor rec:ognizable synthetic peptide pre~;ent on its surface. The ligand may also in all ca~;es WO 93/2û~00 ~ . rt 7 ll9 P~/FI93/00149 be a synthetic ligand made lipophilic or amphiphilic: to allow for its incvrpora~ion with the carrier, into the lipid surface of the carrier particle, provided that the liyand carries the elements recognizable by the c:ell 5 receptors . Su ::h substarlces are also well known in the arlt .
The second centrial component in the compositi OXl is the lysosomotropic agent which acs:ording to the invention is a lysosomotroplc amphiphile, and is characterized as having 10 both lysosomotropic and detergent activity~ The ly:~;osomot-ropic agents exert lysosomal blocking activity and are well know~ in the art. They may in addition be amphiphilic: such as Triton WR-1339 (American Roland Corporation, USA, 5~tyloxapol~'), weak amine~ (e.g. pK 5 to 9) such as imidazo-15 les or morpholinss containing longer hydroc:arbon alkyl oracyl chains, e . g~, N-dodecyl imidazole, or chlo oquine, ee e.g. Miller ~t al., J. Cell Biol. g7 ~ (1983) 1841-185~;
Wilson et al., Can~er R~searc:h 49, (19iB9), 507-51 0; and:
deDuve et al., -Biochem. Phia~acol. 23 (1974) 2495~ whi::h 20 are all included here for rePerence~ When used together with the c:i~rrier and~ a sociat~d: ligand, the lysosomotropic amphiphile due to its ~hydrophobiG nature readily accomoda :~ tes onto the ~:arrier/par~ le surface.: I~sosomotropio amines may:in themse1ves have~cytotoxic~properties. ~ ;
According:to the~inventioD it hias been: observed that: the presence of the lysosom~tropic amphiphile ~oes not:interfe-~: : : re~with the recognition of the ligand by the receptor,:but : ~ the carrier with iaative agent i5 taken up~from~the~:e~tra- ~:
celluIar space into the cells according tD their :conten~
0~ receptors and the activity of the endoaytic ~pathway.
However, once the carrier particles carrying the lysosomot-ropic agent ;enters the ~lysosomes, the proce~sing~ of the : :particles is blo~ked. In other w~rds, the ~ycling of ~he a~i~e agent contiainin~ particle back to the cell surface : : (rQtro-endocyt~sis) does n~t take place, and an accumu-~lation o~ the aative agent in the target site is obtained.
:~ :
W~93/20X00 2117 ~ 4 ~ PC~/FI93/00149 The composition according to the invention is preferably an aqueous solution for parenteral use, such as for injection purposes ~i~), containing a therapeutically or diagnosti~
cally effective amount of active agent containing carrier particles together with the lysosomotropic agent. The ratio (weight~ of carrier particles to the lysosomotropic agent is suitably ~uch that in each particle approximately 2 to 40 mole-% of its surface active compounds is constituted by the said lysosomotropic agents.
The composition according to the inven~ion is made by combining the active agent containing carrier with the lysosomotropic agent in a pharmaceutically suitable vehicle~ together with possible pharmaceutically accep~able adjuvants. The vehicle is normally st~rile water. The adjuvants may be any substances known per se and suitable for the purpose, the ch~ice thereof being within the knowledge of a person skilled in the art.
~20 The dosage used naturally depends on ~he drug or diagnostic agent usedt: as well as the condition to be treated or diagnozed~ The amount to be administered for any specific purpose can~readily be determined by a person skilled in 2:5 the axt. : ~ :
,, ;
As stated above, a cording to a pr~ferred embodiment, the carrier is comprised of reconstituted LDL. Such reconstitu-ted LDL is ma~e by a method comprising the steps of lyvphilizing LD~ in the presence of a protec~ing substan-ce, extracting the lyophili2ed LDL with an organic solven~, incubating a therapeutically or diagnostica:lly effective lipo- or amphiphilic agent in an or~anlc solvent with ~he extracted LDL, evaporatin~ the solvent and dissolving the product in a~ agueous buf~ered s~lution to remove any non-incIuded active agent and separating the L~L e~g. ~y ul~racentrifugatio~.
W0~3/2~00 2 1 1 ~ 7 4 9 PCT/Fl93/00149 The LDL to be reconstitutecl is usually is~lated from human serum by differential ultracentrifugational flotation in the density range of l.Olg < ~ < ~.063 g/ml. Alternative techniques include, for instance, rate zonal ultracentrifu-gation.
The protecting substance may be any of thP ~ubstances known for the purpo~e, such as starch, in particular potat.o starc~, but also a sugar derivative as is disclosed in the WO publicati~n 86/07540.
The sol~renk used for the extraction of 1:he neutral lipids from the lyophilized LDL is suitably a non-polar sol~ent, such as heptane, hexane, pentane, petroleum ether, octane or their mixtures.
The solvent used for dissolving the active agent and for incub~ting with the lyophilized and extracted LDL, is al~o preferably a non-polar solvent such as ~the ones li~ted ; above.
According to another mode of the invention, the carrier p~rticle~ can be a liposome. The ~echni~ue~ for making ~25 lipo~omes and for including active agent~, such as a;drug th~reinO are very well known in ~he art. : :
In ~his: ca~e the li~and can comprise a suit~ble subs~ance :
recognizable by a specifi~cell surface receptor, such a~
~hose listed earlier, and which i5 incorporated in the liposome shell during the manufa~ture th~r~of.
According to the invention also a microemulsion can: be used, the prepara~ion of which is known in the art (e.~.
35 Wal h, M. T. e~ al, Methods in:Enzymology, vol. 128 582 (1986). The same ligands as th9se m~ntioned above may be used~
WO ~/20~0(~ 2 1 1 rJ rl 4 9 PCl`/F193/0014') The active agent is an agent for diagnostic or thPrapeutic purposes. It can be lipophilic or amphiphilic, although the border line between these two types is diffuse. As a diagnostic agent e.g. light sensitizers, such as hemato-porphyrins, radiosensitizers, such as boronated fatty acid esters, or x~ray contrast agents can be used. As a thera peutic agent, preferably anti cancer drugs are used, such as doxorybicin, dauno~ycin, l~hexadecyl 2-methyl 3-phospho-ch~line etc.
The following examples illu trate the invention withoutlimiting the same.
Example LDL was isolated by diff~-rential ultracentrif~agational f lotation in the density range 1.1019 ~ d ~ 1. OÇ3 g/ml ~
Subsequently, KBr used for the ad justment of appropriate densities was removed~by ext:ensive dialysis against 0.3 mM
EDTA, pH 7.0 at 4 C~ Thereafter the lipopxotein was concen~rat2d by ultrafiltration ~sing: an ~micon XM-300 membrane and the concentration of chole5ter~1 was mea5ured.
Pure potato starch (60 m~ from Sigma) was dissloved in ~he LDL-svlution contai~in~ 16 ~mol~s of cholest~rol in a total volume of 1.5 ml. The resulting 501ution was then fro2en ~n liquid nitrogen and lyophilized. The core lipi~s of ~he lyophiliæed LPL~were then extracted with 4 x ~0 ml of~ice-cold heptane. The core lipids were replaced with a cytoto 30~ xic compound, namely:cholestaryl ester of chlorambucil (15 mg di~solved in 1.$ ml of heptane~ which con~ained a 3H-cholesteryl moiety as a marker for the quantitation of tissue distrlbution o~ the cytotoxic compound. The so~tion was fir~t incu~ated at -10 ~C ~or 90 minutes, wherQafter the solvent was removed under a gentle 6tream of nitrogen while: keeping the 5ample on an ice water bath. The dry residue was then dis501ved in 3.5 ml of 10 mM Tricine 21 1 77~-.9 WO ~3/~0~ PCr/F193/00149 buf f er, pH 8 . ~, and incubated at ~ 4 ~ C f or 4 8 hours . In order to separate the choleslteryl chlorambucil loaded LDL, the solutic7n was centrifuged a~ ~Ooo rpm and the superna~
tant collectec~. To the superna~ant solution contair~ g the 5 cyto~tat-LDL pa~ticles, was then added 50 ,uL of Triton ~
1339, and the mixture was incubated at + 4 C overnight. In this manner an injectable preparation ~as obtained. Tht s can be used as such, or if necessary, it can be irradial:ed by bath type sonication to speed up the dispersion into lû small particles.
EXPERIMENTAL
~ he preparation obtained in tha Example ab,c>ve was used as 15 such and injec~ed i . v. into rats bearing P~ous sarcoma inoculated in their periton~um,. The average weight OI ~he developed tumor~ was 0. 9 g~ The animals were killed after O . 5, 1. 0 and 3 hrs ~Erom th injection and the radioac~ivi ties in different ~i~3sues we!re determined. In the followirlg 20 table, t~st animals de~ignated A ~s:) E are compared to the values from a control rat (killed at 1 hr) without the implanted sarcoma.
Control A B C D E
2~ hrs: 1 : 0 . 5 1 ï ~ 3 3 Gpm / q " __ blood 131 3 50 152 140 20 32 heart 19 83 ~6 8~ 5a~ ~9 ~ :
kidney 22 ~ 44 56 72 : 56 ~ ~5 3û pancreas44 23 lOg 161 8g 94 spleen 47 33 120 153 213 15î
liver 85 7g 189 195 299 1~7:
tumor - 23 459 530 296 47~
~ From the results it can be ~een ~hat the LDL-chlorambucil ester acGumulatss in the tumor, and that the degree of expulsion is low, whereas its level in the blood decre~es WO ~3~20~00 2 117 7 4 ~ pcr/Fl93/ool49 rapidly to a low level.
In the appended drawing, Fig. 1 illustrates simila~c tests and it shows the level of chlorambucil ester-LDL in the 5 blood and in the tumor, respe~tively, when injected as described above but in the absence of a ly50somo~ropic agent (Triton WR 1339). In Fig.. 1 the absci~sa indie:~ate~;
the time and the ordinate the amount [ in promille) in blood and tulslor respectively, calculated from that injected.
10 ~ig. 2 ~;hows in yraphîc: form the mean value~; for the levels obtained above, when the same I,DL has been injected in the presence of khe ly~;osomotropic agentL From the Figures it is eviden1: that in the absence of a lysoRomotropic agent, after an initial transient decrease, the level in the blood 15 increases rapidly, whereas the opposite is true f or the level in the tumour.
On the other hand, when using a preparation according to the invention, ~thè level ~f reconstltu~ed~ LDL in the blood 2 0 decreases, and the l~vel thereof in the tumour inc:reases, ndicating: that retrc~endocy~osis is ~prevented by the incorporation of the lysosomotropic agent according to the in~entior~
:
~: :
:
The present invention is based on the use of a carrier selected from the qroup consistîng of lipoproteins, in particular reconsti~uted LDL (~ow Density Lipoprotein), other types of microemulcion particles, liposomes and micelles, and containi~g a therapeutically or diagnostical-ly active~ l~po- or amphiphilic ~gent, and a~sociated:with a suitable ligand recognizable by its specific (complemen-tary~ cell recep~or, together with a lysosomotropic agent, for targeting of the active agent to the site of interest/
such as cancerous tissue~ and to optimize the effect thereof a ~he said site.
BACKGROUND OF THE INVENTION
~igand speci~ic receptors on the cell surf~ce medi~te both formation on the environme~t as well as nutrients into : the cell. Such ligands to be recognized include a range of : mole~u:~ar structures, e.g~ growth factors (for exam~le platelet derived growt~ factor, en~othelial ~cell growth ~actor, tumor ~r~wth ~actor, nerve ~row~h factor~, hormones ~ , (e.g~ insulin, vasopres~in etc.), extracellular matrix protein (a.g. ooll~agen) and lipoproteins ~e.g. low densit~
:lipoprotein). In maDy cases t he~ binding of the ligand to the cell surface receptor is fo~lowe~ by a clust~ring o~
~he lig~nd-receptor cumplex and subsequent internalization i~` via cbated pits, ultimakely locating the ligand~ ln ly-so~omes. This pathway is utilized for exampIe by~viruses in their entry into cellsO
: : :
It ha be~n known ~or a long time that people suf~ering from cancer exhibit a cholesterol level in the ~erum whi~h ~ ~ is lower than normal. Cholesterol circulates in the blood :
' :
W~93/20~0() 2 1 17 7 ~ 9 PCT/Fl93/00149 in the form of a lipid-protein complex, primarily as the so-called ~ow Density Lipoprotein, LDL. LDL is a spherical macromolscule having a diameter of about ~80 ~. LDL is surrounded by a surface film consisting of phospholipids, choles~erol and sphingomyelin, and of protein/ i.e.
~polipoprotein B ~apo B). The interior of LDL is formed by chol~sterol in esterified form ~compounds of fatty acids and cholesterol), triacylglycerol, retinol, as well as other hydrocarbon soluble compounds.
Cells contain on their surfaces specific LDI,-receptors which bind to the LDL-partic:le by specifically recognizing the protein component of ~he LDL-surface, i.e. apolipopro-tein B. The density of LDL-re~eptors is much higher on cancerous cells than on normal cells and cancer cells bind LDL more effectiYelyO I~ is evident that the lower serum cholestero~ level of cancer patienks: is due to~ enhanced uptake of LDL by the cancer cells. This is easy to ra-tionaliz~ in terms of the increased :requiremen~ of com-ponents such as cholesterol for the gr ~ h~of the ~umor : cells.
The use o~reconstit~ted:LDL containing aytotoxic compou~ds ~:whlch :replac~e~the natural ap~la~;~:lipids ln~the particle 25: ~core h~s ~been describe~ The::lipids are removed from::the core of LD~ by ~irst lyophili:~ing the lipoprotein in~the presence of ~starch (Gustaf~on, A (~1965)~:J. Lipid:Res.:~:20 : : 254-264)~. A~slightly modified~techni~ue~has~been~dessribed~ :~
by~ Xrieger (Krieger,;M~ ) M~ethods~Enz~mol.~ 12~,~608~
~ 30 ~ 613). LDL is reconstituted by lyophilizlng;in the pr~sen~e ! of potato s~arch~and extracting the niu~ral lipids;with an org~nic 9alvent, such as heptàne~ The agent~to be introdu-~
ced is dissolved in an~organic solvent~and~incuba~ed With the ext~acted ~DL. The;or~anic ~sol~ent is~;~evaporated~and ~
35 ~ the ~econstituted LDL is dissolved in a buffered ~a:queous~ ;
~ solution.~
:
' :
WO~/2~)l) 2 ~ 17 7 ~1 ~ PCT/Fl93/0014(~
~ccording to an improvement to this method, described in the WO-publication 86/07540, the starch is replaced by a saccharide or a sugar alcohol, and according to this publication, a reconstituted LDL is obtained having similar properties as natural LDL, in vitro and in vivo.
However, when using e.g. reconstituted LDL as a carrier for e.g. drugs, in the form of an injectable aqueous solution, retro-endocytosis has been observed. Thus a~t~r an ini~ial decrease of the drug level in the blood, indicating upt~ke of LDL by the cell~, an increase in the level of the drug is obserYed, indic~ting expulsion of the drug from the cells. Thus it is difficult to achieve a significant accumulation of the drug in the target tissue, and conse-quently the action of the active agent cannot be fullytaken advantage of.
It is also prior known to use, for drug delivery, li-posomes wherein the active agent is incorpora~ed a) in a vre surrounded by a bilayer- of a Iipid substance, typical-ly a pho~pholipid (together with e.~ cholesterol and sphing~myelin) o~ bj if the desired ~herap~utic compoun~ is àmphiphilic (e.g. an anti-sancer: phospholipid), it is incorporated in~the bilayer itself. For the said purpose ~:~25 al~o microemulsions, f:or example an emulsion of active agent, such as a lipophi~ic drug surrounded by a monolayer of phosphatidyl choline, are used.
~:~SUMMARY QF THE INV NTION
: The present invention relates to an improvement to the : known methods by means of which it is possible to achieve an enri hment of~the active agent at the target site~ This is~ made poss~ble by using, in combination, (a~ a carrier selec~ed ~rom the 7roup consisting of lipoproteins, other type~ of microemulsion particles, liposomes and micelles, and containing a therapeutically or a diagnostically 2 1 17 7 4 9 PCr/F193/01)149 effective amount of a lipo- or amphiphilic active agent, associated with at least one ligand which is complementary to and recognizable by specif ic receptor in the living cells, and (b) a lysosomotropic agent~
The invention concerns, on the one hànd t a composition f or diagnostic or ther~peutic u~;e which contains the said com}~ination, and on the other hancl a method f or the preparation of the said con~position. Especially the 10 lnventi~n concerns a composition and a method wherein the carrier is specifically reconstituted LDL, as well as its use on humans and non-human animals.
DETAILED I)ESCRIPTION OF TH[E INVENTION
The. c:omposi~ion according to the invenltic:n thus contairls at least two components, i . e . a carrier selected from the group of lipopro~eins, other types of microemulsiorl parti-cles, liposomes and micelles, and which is associated to 2 0 a suitable ligand . The c:arrier compoments are known as suc:h, as is their manner of manuf acture . cca~mon f or these carriers is that they can c:on~ain a therapeutically or ~iagnostically active agent.
The carrier is associated with a ligand which is chara¢te-: rized in ~hat it is~complementary to and oan be recognized by a specific cell receptor. The term 5':associated with" i~s intended to include also the possibility that the carrier incorporates the said liga~d, such as in the c::ase of LDL, 30 where the ligand can be the ap o B moiety included in the surf ace f ilm oî the LDL ~ The ligand is thus typically one o: the substanGes mentioned earlier~ ~ su~h as a protein~
e O g . growth f actor, a hormone, apolipoprotein, viral prvteir~, synthetic peptide etc. As stated, when the carrier 35 is~ a reconstituted LDL, thP ligand i~; the apo B-moiety, ox :a corresponding receptor rec:ognizable synthetic peptide pre~;ent on its surface. The ligand may also in all ca~;es WO 93/2û~00 ~ . rt 7 ll9 P~/FI93/00149 be a synthetic ligand made lipophilic or amphiphilic: to allow for its incvrpora~ion with the carrier, into the lipid surface of the carrier particle, provided that the liyand carries the elements recognizable by the c:ell 5 receptors . Su ::h substarlces are also well known in the arlt .
The second centrial component in the compositi OXl is the lysosomotropic agent which acs:ording to the invention is a lysosomotroplc amphiphile, and is characterized as having 10 both lysosomotropic and detergent activity~ The ly:~;osomot-ropic agents exert lysosomal blocking activity and are well know~ in the art. They may in addition be amphiphilic: such as Triton WR-1339 (American Roland Corporation, USA, 5~tyloxapol~'), weak amine~ (e.g. pK 5 to 9) such as imidazo-15 les or morpholinss containing longer hydroc:arbon alkyl oracyl chains, e . g~, N-dodecyl imidazole, or chlo oquine, ee e.g. Miller ~t al., J. Cell Biol. g7 ~ (1983) 1841-185~;
Wilson et al., Can~er R~searc:h 49, (19iB9), 507-51 0; and:
deDuve et al., -Biochem. Phia~acol. 23 (1974) 2495~ whi::h 20 are all included here for rePerence~ When used together with the c:i~rrier and~ a sociat~d: ligand, the lysosomotropic amphiphile due to its ~hydrophobiG nature readily accomoda :~ tes onto the ~:arrier/par~ le surface.: I~sosomotropio amines may:in themse1ves have~cytotoxic~properties. ~ ;
According:to the~inventioD it hias been: observed that: the presence of the lysosom~tropic amphiphile ~oes not:interfe-~: : : re~with the recognition of the ligand by the receptor,:but : ~ the carrier with iaative agent i5 taken up~from~the~:e~tra- ~:
celluIar space into the cells according tD their :conten~
0~ receptors and the activity of the endoaytic ~pathway.
However, once the carrier particles carrying the lysosomot-ropic agent ;enters the ~lysosomes, the proce~sing~ of the : :particles is blo~ked. In other w~rds, the ~ycling of ~he a~i~e agent contiainin~ particle back to the cell surface : : (rQtro-endocyt~sis) does n~t take place, and an accumu-~lation o~ the aative agent in the target site is obtained.
:~ :
W~93/20X00 2117 ~ 4 ~ PC~/FI93/00149 The composition according to the invention is preferably an aqueous solution for parenteral use, such as for injection purposes ~i~), containing a therapeutically or diagnosti~
cally effective amount of active agent containing carrier particles together with the lysosomotropic agent. The ratio (weight~ of carrier particles to the lysosomotropic agent is suitably ~uch that in each particle approximately 2 to 40 mole-% of its surface active compounds is constituted by the said lysosomotropic agents.
The composition according to the inven~ion is made by combining the active agent containing carrier with the lysosomotropic agent in a pharmaceutically suitable vehicle~ together with possible pharmaceutically accep~able adjuvants. The vehicle is normally st~rile water. The adjuvants may be any substances known per se and suitable for the purpose, the ch~ice thereof being within the knowledge of a person skilled in the art.
~20 The dosage used naturally depends on ~he drug or diagnostic agent usedt: as well as the condition to be treated or diagnozed~ The amount to be administered for any specific purpose can~readily be determined by a person skilled in 2:5 the axt. : ~ :
,, ;
As stated above, a cording to a pr~ferred embodiment, the carrier is comprised of reconstituted LDL. Such reconstitu-ted LDL is ma~e by a method comprising the steps of lyvphilizing LD~ in the presence of a protec~ing substan-ce, extracting the lyophili2ed LDL with an organic solven~, incubating a therapeutically or diagnostica:lly effective lipo- or amphiphilic agent in an or~anlc solvent with ~he extracted LDL, evaporatin~ the solvent and dissolving the product in a~ agueous buf~ered s~lution to remove any non-incIuded active agent and separating the L~L e~g. ~y ul~racentrifugatio~.
W0~3/2~00 2 1 1 ~ 7 4 9 PCT/Fl93/00149 The LDL to be reconstitutecl is usually is~lated from human serum by differential ultracentrifugational flotation in the density range of l.Olg < ~ < ~.063 g/ml. Alternative techniques include, for instance, rate zonal ultracentrifu-gation.
The protecting substance may be any of thP ~ubstances known for the purpo~e, such as starch, in particular potat.o starc~, but also a sugar derivative as is disclosed in the WO publicati~n 86/07540.
The sol~renk used for the extraction of 1:he neutral lipids from the lyophilized LDL is suitably a non-polar sol~ent, such as heptane, hexane, pentane, petroleum ether, octane or their mixtures.
The solvent used for dissolving the active agent and for incub~ting with the lyophilized and extracted LDL, is al~o preferably a non-polar solvent such as ~the ones li~ted ; above.
According to another mode of the invention, the carrier p~rticle~ can be a liposome. The ~echni~ue~ for making ~25 lipo~omes and for including active agent~, such as a;drug th~reinO are very well known in ~he art. : :
In ~his: ca~e the li~and can comprise a suit~ble subs~ance :
recognizable by a specifi~cell surface receptor, such a~
~hose listed earlier, and which i5 incorporated in the liposome shell during the manufa~ture th~r~of.
According to the invention also a microemulsion can: be used, the prepara~ion of which is known in the art (e.~.
35 Wal h, M. T. e~ al, Methods in:Enzymology, vol. 128 582 (1986). The same ligands as th9se m~ntioned above may be used~
WO ~/20~0(~ 2 1 1 rJ rl 4 9 PCl`/F193/0014') The active agent is an agent for diagnostic or thPrapeutic purposes. It can be lipophilic or amphiphilic, although the border line between these two types is diffuse. As a diagnostic agent e.g. light sensitizers, such as hemato-porphyrins, radiosensitizers, such as boronated fatty acid esters, or x~ray contrast agents can be used. As a thera peutic agent, preferably anti cancer drugs are used, such as doxorybicin, dauno~ycin, l~hexadecyl 2-methyl 3-phospho-ch~line etc.
The following examples illu trate the invention withoutlimiting the same.
Example LDL was isolated by diff~-rential ultracentrif~agational f lotation in the density range 1.1019 ~ d ~ 1. OÇ3 g/ml ~
Subsequently, KBr used for the ad justment of appropriate densities was removed~by ext:ensive dialysis against 0.3 mM
EDTA, pH 7.0 at 4 C~ Thereafter the lipopxotein was concen~rat2d by ultrafiltration ~sing: an ~micon XM-300 membrane and the concentration of chole5ter~1 was mea5ured.
Pure potato starch (60 m~ from Sigma) was dissloved in ~he LDL-svlution contai~in~ 16 ~mol~s of cholest~rol in a total volume of 1.5 ml. The resulting 501ution was then fro2en ~n liquid nitrogen and lyophilized. The core lipi~s of ~he lyophiliæed LPL~were then extracted with 4 x ~0 ml of~ice-cold heptane. The core lipids were replaced with a cytoto 30~ xic compound, namely:cholestaryl ester of chlorambucil (15 mg di~solved in 1.$ ml of heptane~ which con~ained a 3H-cholesteryl moiety as a marker for the quantitation of tissue distrlbution o~ the cytotoxic compound. The so~tion was fir~t incu~ated at -10 ~C ~or 90 minutes, wherQafter the solvent was removed under a gentle 6tream of nitrogen while: keeping the 5ample on an ice water bath. The dry residue was then dis501ved in 3.5 ml of 10 mM Tricine 21 1 77~-.9 WO ~3/~0~ PCr/F193/00149 buf f er, pH 8 . ~, and incubated at ~ 4 ~ C f or 4 8 hours . In order to separate the choleslteryl chlorambucil loaded LDL, the solutic7n was centrifuged a~ ~Ooo rpm and the superna~
tant collectec~. To the superna~ant solution contair~ g the 5 cyto~tat-LDL pa~ticles, was then added 50 ,uL of Triton ~
1339, and the mixture was incubated at + 4 C overnight. In this manner an injectable preparation ~as obtained. Tht s can be used as such, or if necessary, it can be irradial:ed by bath type sonication to speed up the dispersion into lû small particles.
EXPERIMENTAL
~ he preparation obtained in tha Example ab,c>ve was used as 15 such and injec~ed i . v. into rats bearing P~ous sarcoma inoculated in their periton~um,. The average weight OI ~he developed tumor~ was 0. 9 g~ The animals were killed after O . 5, 1. 0 and 3 hrs ~Erom th injection and the radioac~ivi ties in different ~i~3sues we!re determined. In the followirlg 20 table, t~st animals de~ignated A ~s:) E are compared to the values from a control rat (killed at 1 hr) without the implanted sarcoma.
Control A B C D E
2~ hrs: 1 : 0 . 5 1 ï ~ 3 3 Gpm / q " __ blood 131 3 50 152 140 20 32 heart 19 83 ~6 8~ 5a~ ~9 ~ :
kidney 22 ~ 44 56 72 : 56 ~ ~5 3û pancreas44 23 lOg 161 8g 94 spleen 47 33 120 153 213 15î
liver 85 7g 189 195 299 1~7:
tumor - 23 459 530 296 47~
~ From the results it can be ~een ~hat the LDL-chlorambucil ester acGumulatss in the tumor, and that the degree of expulsion is low, whereas its level in the blood decre~es WO ~3~20~00 2 117 7 4 ~ pcr/Fl93/ool49 rapidly to a low level.
In the appended drawing, Fig. 1 illustrates simila~c tests and it shows the level of chlorambucil ester-LDL in the 5 blood and in the tumor, respe~tively, when injected as described above but in the absence of a ly50somo~ropic agent (Triton WR 1339). In Fig.. 1 the absci~sa indie:~ate~;
the time and the ordinate the amount [ in promille) in blood and tulslor respectively, calculated from that injected.
10 ~ig. 2 ~;hows in yraphîc: form the mean value~; for the levels obtained above, when the same I,DL has been injected in the presence of khe ly~;osomotropic agentL From the Figures it is eviden1: that in the absence of a lysoRomotropic agent, after an initial transient decrease, the level in the blood 15 increases rapidly, whereas the opposite is true f or the level in the tumour.
On the other hand, when using a preparation according to the invention, ~thè level ~f reconstltu~ed~ LDL in the blood 2 0 decreases, and the l~vel thereof in the tumour inc:reases, ndicating: that retrc~endocy~osis is ~prevented by the incorporation of the lysosomotropic agent according to the in~entior~
:
~: :
:
Claims (14)
1. Composition for therapeutical or diagnostical use comprising, in combination, (a) a carrier selected from the group consisting of lipoproteins, other types of microemul-sion particles, liposomes and micelles, and containing a therapeutically or a diagnostically effective amount of a lipo- or amphiphilic active agent, associated with at least one ligand which is complementary to and recognizable by a specific cell receptor, and (b) a lysosomotropic agent.
2. Composition according to claim 1, wherein the carrier is reconstituted Low Density Lipoprotein (LDL), the ligand being an apolipoprotein B moiety on its shell surface.
3. Composition according to claim 1 or 2, wherein the lysosomotropic agent is Triton WR 1339 ethyl oleate.
4. Composition according to claim 1 or 2, wherein the therapeutically or diagnostically active agent is selected from the group consisting of light sensitizers, radiosensi-tizers, x-ray contrast agents and anti-cancer drugs.
5. Composition according to claim 4, wherein the anti-cancer drug is chlorambucil cholesteryl ester.
6. Composition for therapeutical use comprising, in combination, a reconstituted Low Density Lipoprotein (LDL) containing chlorambucil cholesteryl ester and Triton WR
1339 ethyl oleate.
1339 ethyl oleate.
7. Process for the preparation of a composition for therapeutic or diagnostic use comprising the steps of combining a carrier selected from the group consisting of lipoproteins, other types of microemulsion particles, liposomes and micelles, containing a therapeutically or a diagnostically effective amount of a lipo- or amphiphilic active agent, and associated with at least one ligand which is complementary to and recognizable by a specific cell receptor, with a lysosomotropic agent and a pharmaceutical-ly suitable vehicle and optional further adjuvants.
8. Process for the preparation of a composition according to claim 2, comprising lyophilizing LDL in the presence of a protecting substance, extracting the lyophilized LDL with an organic solvent, incubating the therapeutically or diagnostically active agent in an organic solvent with the extracted LDL, removing the solvent, dissolving the reconstituted LDL in an aqueous buffered solution, and recovering the reconstitiuted LDL and combining a thera-peutically or diagnostically effective amount of the reconstituted LDL with a lysosomotropic agent and a suitable vehicle and optionally further adjuvants.
9. Process according to claim 8, wherein the lysosomot-ropic agent is Triton WR 1339 ethyl oleate.
10. Process according to claim 8 or 9, wherein the thera-peutically or diagnostically active agent is selected from the group consisting of light sensitizers, radiosensiti-zers, x ray contrast agents and anti-cancer drugs.
11. Process according to claim 10, wherein the anti-cancer drug is chlorambucil cholesteryl ester.
12. Process according to claim 8, wherein the therapeuti-cally active agent is chlorambucil cholesteryl ester and the lysosomotropic agent is Triton WR 1339 ethyl oleate.
13. Process according to claim 9, wherein the protecting substance is starch.
13. Process according to claim 9, wherein the protecting substance is starch.
13. Process according to claim 9 wherein the solvent used for extraction of the LDL is heptane.
14. Use of a composition according to claim 1 or a composi-tion prepared according to claim 7 for therapeutic or diagnostic treatment of humans and non-human animals.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US86525692A | 1992-04-08 | 1992-04-08 | |
| US865,256 | 1992-04-08 | ||
| PCT/FI1993/000149 WO1993020800A1 (en) | 1992-04-08 | 1993-04-07 | Composition for therapeutic or diagnostic use, process for its preparation and its use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2117749A1 true CA2117749A1 (en) | 1993-10-28 |
Family
ID=25345059
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002117749A Abandoned CA2117749A1 (en) | 1992-04-08 | 1993-04-07 | Composition for therapeutic or diagnostic use, process for its preparation and its use |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0634926A1 (en) |
| JP (1) | JPH07505408A (en) |
| KR (1) | KR950700726A (en) |
| AU (1) | AU3892593A (en) |
| CA (1) | CA2117749A1 (en) |
| WO (1) | WO1993020800A1 (en) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ275054A (en) * | 1993-11-08 | 1997-04-24 | Peptide Delivery Systems Pty L | Diagnosing atherosclerosis or coronary artery disease using labelled composition which mimics essential features of an exogenous lipoprotein |
| DE19500665A1 (en) * | 1995-01-12 | 1996-07-18 | Axel Prof Dr Haase | Process for the spatially resolving imaging of an area of a biological object with the help of electromagnetic rays using contrast media |
| GB2321455A (en) | 1997-01-24 | 1998-07-29 | Norsk Hydro As | Lipophilic derivatives of biologically active compounds |
| US5827533A (en) * | 1997-02-06 | 1998-10-27 | Duke University | Liposomes containing active agents aggregated with lipid surfactants |
| US7001587B2 (en) | 2001-10-24 | 2006-02-21 | The Regents Of The University Of California | Measurement of protein synthesis rates in humans and experimental systems by use of isotopically labeled water |
| DE60324393D1 (en) | 2002-02-12 | 2008-12-11 | Univ California | NONINVASIVE MEASUREMENT OF BIOSYNTHESIS AND DEFROSTING SPEEDS OF BIOLOGICAL MOLECULES WHICH ARE ACCESSIBLE TO A DIRECT SAMPLING, OR NOT EASILY ACCESSIBLE, BY INSTALLING A MARKER IN METABOLIC DERIVATIVES AND KATABOLE PRODUCTS |
| WO2004011426A2 (en) | 2002-07-30 | 2004-02-05 | The Regents Of The University Of California | Method for automated, large-scale measurement of the molecular flux rates of the proteome or the organeome using mass spectrometry |
| ATE415486T1 (en) | 2002-09-13 | 2008-12-15 | Univ California | METHOD FOR MEASURING SPEEDS OF CHOLESTEROL REVERSE TRANSPORT IN VIVO AS AN INDEX OF ANTI-ARTHEROGENESIS |
| EP1558293A4 (en) | 2002-11-04 | 2006-10-25 | Univ California | Deuterated glucose or fat tolerance tests for high-throughput measurement of the metabolism of sugars or fatty acids in the body |
| US7262020B2 (en) | 2003-07-03 | 2007-08-28 | The Regents Of The University Of California | Methods for comparing relative flux rates of two or more biological molecules in vivo through a single protocol |
| US20050202406A1 (en) | 2003-11-25 | 2005-09-15 | The Regents Of The University Of California | Method for high-throughput screening of compounds and combinations of compounds for discovery and quantification of actions, particularly unanticipated therapeutic or toxic actions, in biological systems |
| TW200538738A (en) | 2004-02-20 | 2005-12-01 | Univ California | Molecular flux rates through critical pathways measured by stable isotope labeling in vivo, as biomarkers of drug action and disease activity |
| JP2014526685A (en) | 2011-09-08 | 2014-10-06 | ザ・リージェンツ・オブ・ザ・ユニバーシティ・オブ・カリフォルニア | Metabolic flow measurement, imaging, and microscopy |
| CA2858368A1 (en) | 2011-12-07 | 2013-06-13 | Glaxosmithkline Llc | Methods for determining total body skeletal muscle mass |
| US9134319B2 (en) | 2013-03-15 | 2015-09-15 | The Regents Of The University Of California | Method for replacing biomarkers of protein kinetics from tissue samples by biomarkers of protein kinetics from body fluids after isotopic labeling in vivo |
| KR102387248B1 (en) | 2021-07-06 | 2022-04-15 | (주)효림세울 | Equipment for manufacturing sirofil Composite Yarn and sirofil Composite Yarn and method for manufacturing sirofil Composite Yarn |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE456136B (en) * | 1985-06-17 | 1988-09-12 | Oncholab Ab | PROCEDURE FOR THE PREPARATION OF A LIPOPHILIC BIOLOGY ACTIVE SUBSTANCE BASED BEARING BASIS OF RECONSTITUTED LDL (Layer Density Lipoprotein) |
-
1993
- 1993-04-07 AU AU38925/93A patent/AU3892593A/en not_active Abandoned
- 1993-04-07 JP JP5518017A patent/JPH07505408A/en active Pending
- 1993-04-07 WO PCT/FI1993/000149 patent/WO1993020800A1/en not_active Application Discontinuation
- 1993-04-07 CA CA002117749A patent/CA2117749A1/en not_active Abandoned
- 1993-04-07 EP EP93907890A patent/EP0634926A1/en not_active Withdrawn
-
1994
- 1994-10-07 KR KR1019940703558A patent/KR950700726A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| JPH07505408A (en) | 1995-06-15 |
| KR950700726A (en) | 1995-02-20 |
| WO1993020800A1 (en) | 1993-10-28 |
| EP0634926A1 (en) | 1995-01-25 |
| AU3892593A (en) | 1993-11-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2117749A1 (en) | Composition for therapeutic or diagnostic use, process for its preparation and its use | |
| KR100889139B1 (en) | Irinotecan preparation | |
| Maruyama et al. | Phosphatidyl polyglycerols prolong liposome circulation in vivo | |
| JPH10501534A (en) | Sphingosomes for enhanced drug delivery | |
| JP2004525138A (en) | Liposomal compositions for improved intracellular delivery of therapeutic agents | |
| YUDA et al. | Prolongation of liposome circulation time by various derivatives of polyethyleneglycols | |
| Allen | Stealth liposomes: five years on | |
| CN105012956A (en) | Difunctional tumor targeted liposome drug-delivery system and preparation and application thereof | |
| JP4874097B2 (en) | Liposomes containing poorly water-soluble camptothecin | |
| US20040213835A1 (en) | Method to reduce liposome-induced complement activation | |
| US20040062797A1 (en) | Release of therapeutic agents in a vessel or tissue | |
| WO2000009071A2 (en) | A novel liposomal formulation useful in treatment of cancer and other proliferation diseases | |
| Samadi-Baboli et al. | Preparation of low density lipoprotein-9-methoxy-ellipticin complex and its cytotoxic effect against L1210 and P 388 leukemic cells in vitro | |
| Freund | Biodistribution and gastrointestinal drug delivery of new lipidic multilamellar vesicles | |
| Gregoriadis | Liposomes as Carriers of Drugs Observations on Vesicle Fate after Injection and Its Control | |
| KR100996975B1 (en) | Liposome coated with protein to prolong circulation time in bloodstream and preparation method thereof | |
| JPS63221837A (en) | Lipid film composition | |
| US20240094197A1 (en) | Method for removing protein corona of the surface of nanometer particle | |
| MAITANI et al. | Effects of orally administered liposomes with soybean-derived sterols and their glucosides on rat body weight | |
| WO2003022250A2 (en) | Unilamellar vesicles stabilized with short chain hydrophilic polymers | |
| ZA200507816B (en) | Liposome composition for reduction of liposome-induced complement activation | |
| Scherphof et al. | In Vivo Uptake and Processing of Liposomes by Parenchymal and Non-Parenchymal Liver Cells; Application to Immunotherapeutic Treatment of Hepatic Metastases | |
| KR100768265B1 (en) | Heparin coated liposomes to prolong circulation time in bloodstream and preparation method thereof | |
| Scherphof et al. | Lipid flow phenomena between liposomes, lipoproteins and cell membranes; Applications in drug delivery | |
| CN113116821A (en) | Medicine composition for treating hepatitis B and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 19970407 |