WO1996034598A1 - Tissue entrapment - Google Patents
Tissue entrapment Download PDFInfo
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- WO1996034598A1 WO1996034598A1 PCT/GB1996/001072 GB9601072W WO9634598A1 WO 1996034598 A1 WO1996034598 A1 WO 1996034598A1 GB 9601072 W GB9601072 W GB 9601072W WO 9634598 A1 WO9634598 A1 WO 9634598A1
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- tumour
- concentration
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- lipid
- liposomes
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- 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
Definitions
- the present invention relates to lipid-based
- compositions for delivering diagnostic and therapeutic materials to tumours and skin and to the use of such compositions in medicine are provided.
- the present inventors have arrived at an alternative mechanism for enhancing the delivery of a payload in a lipid-based composition to a solid tumour; the mechanism will be referred to hereafter as the "trap" mechanism.
- the "trap” mechanism operates by specifically reducing loss from the tumour of the lipid-based
- composition and thus retaining greater quantities of the delivered payload within the tumour.
- the liposomes of the invention are effective to localise specifically in a solid tumour region by virtue of the extended lifetime of the liposomes in the blood stream and a liposome size which allows both extravasation into tumours, a relatively high drug carrying capacity and minimal leakage of the entrapped drug during the time required for the liposomes to distribute to and enter the tumour (the first 24 to 48 hours following
- tumour localisation takes place appears justified in the light of the data presented in the same patent application. Where there is an extended lifetime of a material in the blood stream and that material is capable of extravasation into a tumour site, there will be
- the blood clearance rate is a composite of the tissue
- tumour to blood ratios are 0.1:1 at 2h, 0.3:1 at 24h and 0.6:1 at 48h, i.e. at the later two time points the ratios have fallen with respect to the un-PEGylated control.
- This reduction in tumour to blood ratios with respect to control at late time points is what is expected in most situations when entry into one or more of the body' s liposome eliminating organs is reduced by polymer derivatisation, leading to an enhanced circulation time (see discussion on tumour to blood ratios below).
- liposomes have the maximum retention time in the blood, and that they are small enough to traverse the blood/tumour barrier. It is implicit in this view that the longer the blood circulation time, the greater the amount of liposomes delivered to the tumour. The optimum formulation of these polymer coated liposomes is therefore to be achieved by maximising the retention time in the circulation. In order to do this, a combination of cholesterol-related and other lipid composition-related improvements in half life were further improved by PEGylation. Claim 9 of the same patent application emphasises the degree of increase in plasma half life to be achieved ("several times greater" than that of liposomes in the absence of derivitisation).
- tumour to blood ratios are lower for the polymer-derivatised liposomes than the control non- derivatised liposomes and are less than 1 during some or all of the period between 24 and 48h.
- concentration ratios are very desirable in many settings. Examples include tumour imaging of vascular organs, drug and radionuclide delivery. However, the present inventors have appreciated that achieving increased tumour localisation at the expense of reducing the tumour to blood ratios is undesirable.
- the basis of this invention is an examination of the factors influencing the tumour to blood ratio. Without wishing to be bound by their theory, the present inventors believe that enhancement of tumour uptake of diagnostic and therapeutic agents, delivered as the payloads in lipid- based structures bearing hydrophilic moieties, is achieved by influencing the rates of destruction by or loss of the lipid-based structures from the tumour, such that the payload material becomes trapped within the tumour. The inventors have shown that the skin is an organ which behaves in a similar fashion to solid tumours.
- hydrophilic polymer moieties other than PEG moieties are hydrophilic polymer moieties other than PEG moieties.
- tumour to blood ratio of a liposomally encapsulated compound changes with respect to unmodified (control) liposomes.
- the tumour to blood concentration ratio will be reduced with respect to the control, particularly at late time points (e.g. 24 to 144h) and such reductions in tumour to blood ratios of formulations of liposomes with enhanced tumour uptake due to enhanced circulation lifetime, are evident in several reports as discussed above.
- tumour to blood ratios do not necessarily fall when uptake by an eliminating organ or organs is reduced.
- K4,1 elimination organ(s)
- a fourcompartment model was constructed and simulations were performed using SCOMP (aPascal program for IBMPC's written by M.S. Leaning and M.A. Boroujerdi (1991))[1].
- the model has options for the kinetics offlux between compartments. Setting fluxes to havelinearkinetics in all compartments except the elimination organ(s) and aLangmuir flux in the latter (to simulate a saturable clearance mechanism), gave realistic simulations of our own andother's data.
- Therates shown in the table define the transferrates between compartments.
- the hypothetical bolus input at time 0 was 100 in each case. Tumour:blood ratios were calculated using the model's output for the concentrations in the four compartments over time.
- tumour to blood ratio tends to fall and when it is low ( ⁇ 1) the tumour to blood ratio conversely tends to rise, with a complex change being observed at intermediate values.
- ratio of the elimination rates for the rest of the tissues and the elimination organ (s) (KO,3/KO,4) is low or high, tumour to blood ratios tend to fall and rise respectively with reduced K4,1.
- compartment 4 is not the predominating elimination organ, thus this scenario is not relevant to liposomal modifications which exclude liposomes from the RES.
- any modification reducing the destruction rate of liposomes by the tumour tissue, or reducing the egress rate from the tumour back to the blood, will increase tumour to blood ratios.
- tumour to blood ratios always rise (at the same time as the concentration of liposomes in the tumour increases) following liposome modification are: 1) where the destruction rate of liposomes by the tumour is reduced by the modification.
- tumour to blood ratio to fall, when there is reduced entry into elimination organ(s), occurs when there is reduced destruction within or egress from the tumour and is an important discriminant between the "push" and "trap” principles.
- optimisation in accordance with the invention abandons the improved circulation time and the exclusion from the reticuloendothelial system (liver and spleen) as the arbiters of modified liposome function.
- liposomes or other lipid-based structures
- tumour or skin
- trap mechanism it is necessary to consider and optimise a variety of interdependent aspects of the liposome (or other lipid-based structure) material.
- decisions are required in relation to at least the following features:
- composition of the lipid components both in terms of the individual species of lipids to be used and the relative proportions thereof.
- test species the performance of the lipid-based structures in question
- test species two control products which are identical in all respects to the test species save as follows: (a) first control product: this differs from the test
- test species is a liposome which comprises a therapeutic agent entrapped in unilamellar vesicles of a given size formed of a mixture of two lipid species (A and B) which cannot be PEGylated and a lipid species (C) which has the capacity to be PEGylated and, in the test
- the first control product will thus also be a unilamellar liposome, of the same size and content of therapeutic agent and formed of the same mixture of lipid species A, B and C as the test liposome (but species C is not PEGylated).
- the second control product will thus also be a unilamellar liposome of the same size and content of therapeutic agent and will be formed of a mixture of lipid species A and B only in the same relative proportions as for A and B in the test liposomes.
- the liposome is composed of a single lipid specie which is susceptible to PEGylation or of two or more lipid species, each of which is susceptible to PEGylation. Only a portion of the lipids in the test liposomes is PEGylated.
- the first control product is formed of the same lipid specie or combination of lipid species as the test liposomes but now there is no PEGylation.
- the second control product is, in these special types of case, replaced in the comparison tests by the first control product.
- test is conducted by intravenous injection of a standard dose of a diagnostic or therapeutic agent
- agent entrapped in the various liposome products into test animals with an appropriate model solid tumour.
- the blood and tumour concentrations of the agent are measured at 24 and 48 hours after the injection.
- the ratio of tumour concentration of the agent to the blood concentration of the agent achieved at either or both the 24 and 48 hour points will be greater than unity.
- invention will not be significantly lower at either 24 or 48 hours than the tumour to blood concentration ratio achieved by the first control product.
- tumour concentration at each of the 24 and 48 hour points achieved by liposomes of the invention will be greater than the tumour concentration at the same time points achieved by the first control product and also, where it is appropriate to compare with a second control product, greater than the tumour concentration at the same time points achieved by the second control product.
- optimisation of delivery of diagnostically and therapeutically effective agents to tumours or the skin by other lipid-based structures bearing hydrophilic moieties may be achieved by application of these principles in the same way as described above in relation to the use of PEGylated liposomes for delivery of agents to tumours.
- the present invention therefore provides a composition of a diagnostically or therapeutically effective agent for administration via the bloodstream to a solid tumour or the skin, the composition comprising a lipid-containing multi- molecular structure, the agent being present predominantly in the lipid-containing multi-molecular structure, wherein the lipid-containing multi-molecular structure comprises one or more hydrophobic entities bearing covalently bound hydrophilic polymer moieties, and wherein the physical form of the lipid-containing multi-molecular structure, the nature of the hydrophobic entities, the nature of the hydrophilic polymer moieties, the ratio of the polymer- bearing hydrophobic entities to non-derivatised hydrophobic entities exposed to the bloodstream and, when there are two or more hydrophobic entities, the relative proportions of the hydrophobic entities, are all selected such that: (i) on intravenous injection of the composition to an animal, where appropriate bearing a model solid tumour, the ratio of tumour concentration to blood concentration or the ratio of skin concentration to blood concentration of the agent achieved at either or both of 24 and
- the composition consists essentially of an agent associated with a lipid-containing multi-molecular structure consisting of one or more species of hydrophobic entity, each specie being susceptible to derivatisation with hydrophilic polymer moieties and where at least a portion of each of the species of hydrophobic entities is derivatised with hydrophilic moieties, the tumour concentration or skin concentration of the agent achieved by intravenous injection of the composition to an animal, where appropriate bearing a model solid tumour, is greater at 24 and 48 hours following injection than is the tumour concentration or skin concentration of the agent achieved by intravenous injection to an animal of a second control product, which is identical to the composition except that the second control product lacks any
- hydrophilic polymer modification and lacks any hydrophobic entities which are derivatised by polymer modification in the composition, or, in the case where the composition consists essentially of an agent associated with a lipid- containing multi-molecular structure which consists of one or more species of hydrophobic entity, each specie being susceptible to derivatisation with hydrophilic polymer moieties and where at least a portion of each of the species of hydrophobic entities is derivatised with hydrophilic moieties, the tumour concentration or skin concentration of the agent achieved by intravenous
- appropriate bearing a model solid tumour is greater at 24 and 48 hours following injection than is the tumour concentration or skin concentration of the agent achieved by intravenous injection to an animal of the first control product as defined above.
- agents which may be delivered by the compositions of the invention except in the sense that the agent will, of course, be one intended to be effective either in treating or diagnosing solid tumours, where the composition is optimised for delivery of the agent to a tumour, or else for treating or diagnosing skin diseases or disorders of the skin when the compositions have been optimised for delivery to the skin.
- agents which may be administered in compositions of the present invention include drugs, for instance cytotoxic and cytostatic drugs, and nucleic acids, especially DNA.
- compositions will generally provide the same dose at the target site as a conventional treatment or diagnostic composition of that agent, or possibly less than the conventional dose when the
- Doses greater than the conventional dose may be delivered when this is clinically desirable and where conventional doses are limited by toxic effects not experienced with the compositions of the invention or by the inability of conventional administration forms to deliver desired doses of the agent to the target tissue.
- multi-molecular structure is intended to encompass any structure comprising an
- the multi-molecular structures must contain at least one lipid specie and this requirement is reflected in the use of the term "lipid-containing multi-molecular structure". It should be noted that the minimum requirement for one lipid specie to be present in these structures may be satisfied by the presence of a lipid specie as the
- hydrophobic entity bearing hydrophilic polymer moieties also required as a part of the structures of the invention.
- the nature of the multi-molecular structures used in the compositions of the invention, such as liposomes will be discussed below, as will the nature of the hydrophobic entities, such as lipids and particularly phospholipids and the nature of the hydrophilic moieties, such as
- lipid-based structures polyethylene glycol residues.
- the nature of the target tissue for treatment or by the nature of the hydrophobic entities selected for use in the compositions often be dictated by the nature of the target tissue for treatment or by the nature of the hydrophobic entities selected for use in the compositions.
- the physical form of the structures will be dictated by intereactions between the therapeutic or diagnostic agent and the hydrophobic entities.
- lipids tend to form drug-lipid complexes in the form or ribbons or discoids.
- the agent may therefore be present, for instance, entrapped within the lipid-based structures or otherwise bound to the lipid- based structures.
- the relative proportions of hydrophobic entities which are not derivatised and of those which are derivatised with hydrophilic polymer moieties affects the surface properties of the multi-molecular structures as seen by the patient's tissues. Accordingly it is the ratio of these two types of components exposed to the bloodstream that affects the performance of the compositions of the invention. There is no particular requirement imposed by the present invention on the proportions of these different types of component in parts of the multi-molecular structures not exposed to the bloodstream. Thus, for instance, in the case of liposomes of the invention it is permissable to have assymetry between the composition of the blood-contacting external surface lipid layer of the liposomes and the composition of the internal surface lipid layer of the liposomes.
- the relative proportions of the various species of hydrophobic entities can be adjusted and optimised to provide the desired "trapping" of the agent in tumours or skin.
- model tumours since experiments for optimisation of compositions cannot normally be conducted on humans, it will be necessary to select for use in the optimisation of compositions of tumour therapeutic or diagnostic agents in accordance with the principles of the present invention, experimental animals which bear solid tumours which are representative of the human tumours which are to be treated by the optimised compositions of the invention.
- compositions for treatment or diagnosis of dermatological diseases and defects it will be necessaz ⁇ to select experimental animals which have skin which is a good model of human skin. All experimental animals should also be good models of humans as regards the pathways used for delivery of the compositions to the target tissues and as regards
- the animals referred to above are suitable species and strains of animal, preferably conventional laboratory animals such as rodents or primates, selected as models for the human therapy or diagnosis for which the agent and the composition of the invention are intended.
- animals used for administration of the composition of the invention and for the administration of the first control product and, where appropriate, the second control product will be substantially identical and will certainly be matched in accordance with normal laboratory practice.
- compositions of the invention will be greater at either or both of 24 and 48 hours than that achieved with the first product: this is preferred. However it is also possible that compositions according to the invention will give at one or both of the 24 and 48 hour time points a measured concentration ratio which is numerically less than that achieved using the first control product. This is also acceptable within the present invention (although it is less preferred) provided that the difference between the measured concentration ratios achieved with the composition of the invention and the first control product is not statistically significant. Appropriate statistical tests of significance are readily available to those skilled in the art but should be selected having regard to the
- composition of the invention remains greater than the blood concentration achieved by administration of the composition throughout the period from 24 to 48 hours after
- compositions for delivery of a particular agent to a particular type of solid tumour or to skin may be optimised by iterative steps of testing the candidate composition as described above, then modifying one or more features of the composition, retesting and further modification directed by the results of the retesting.
- the objective of optimisation will depend to some extent on the intended use of the agent in question. In the case of a therapeutic agent the most important parameter is the tumour or skin concentration of the therapeutic agent since maximising this will result in more effective delivery to the tumour or skin.
- administered may be less significant, and the duration for which the agent remains in the tumour or skin may also be almost irrelevant. What is most important usually is to ensure a high contrast between the tumour and the non- tumour tissues surrounding the tumour tissues or between the skin and other tissues and this will usually be
- tumour to blood ratio or skin to blood ratio as appropriate
- tumour concentration or the % injected dose per gram of tumour at an appropriate selection of time points (e.g. 3h,24h,48h,72h,144h), or equivalent measurements for skin as appropriate.
- liposomes as examples of lipid-based structures that may be used in the present invention and to PEG moieties as examples of the hydrophilic moieties which may be used in accordance with the present invention.
- PEG moieties as examples of the hydrophilic moieties which may be used in accordance with the present invention. The discussion below refers especially to the delivery of diagnostic and
- theraeutic agents to tumours but is also applicable to delivery of diagnostic and therapeutic agents to skin, for instance in order to detect or treat dermatological
- compositions of the present invention comprise a lipid-based structure, which may be in the form of
- lipoidal DNA This structure is thought to be due to fusion of liposomal bilayers round DNA such that a strand of duplex DNA becomes coated with a lipid bilayer.
- Other forms of DNA/lipid complexes have been observed, and are thought to be similar to hexagonal II phase lipids, but with DNA present in the 5 nm lumen of the hexagonal tubes of lipid. These tubes are packed together with their hydrophobic surfaces in contact and each bundle of tubes has an exterior lipid coat orientated with its hydrophilic surface facing the aqueous environment (such structures may lie within portions of the lipid bilayer of a liposome).
- Non-liposomal drug complexes have also been described.
- the antifungal agent Amphotericin-B has been shown to form ribbon-like structures (ABLCTM, The Liposome Company) with DMPC and DMPG (7:3) and discoidal structures with
- hydrophobic entities envisaged for use in the present invention are generally lipids but there other types of hydrophobic entities which may be used, for instance hydrophobic peptides, polypeptides and proteins.
- the main requirement is that the entities are sufficiently hydrophobic to be retained in the lipid-based structure of the compositions of the invention whilst the therapeutic or diagnostic agent is delivered to and entrapped within the target tissue.
- Some of the hydrophobic molecules in the compositions of the invention are also required to act as anchors for the hydrophilic polymer moieties which are presented on the blood-contacting surfaces of the
- compositions are discussed below in connection with the hydrophilic polymer moieties.
- the polymer moieties may be bound to any hydrophobic molecule which can be integrated into the lipid-based structure so as to anchor the polymer in the lipid-based structure, provided that the lipid-based structure is not thereby disrupted, that the anchor molecule bears a
- the polymer used to anchor the polymer moiety is a phospholipid.
- the polymer may be bound to the anchoring molecule by any known
- covalent bonding technique preferably involving binding the polymer to an amino group of the anchor molecule.
- the bond should, in addition to being covalent, be non- biodegradable in normal blood or serum for the intended duration of residence in the bloodstream (usually at least 24h and preferably 48h), non-toxic and non-immunogenic.
- the hydrophilic moieties are bonded to phospholipids as anchor molecules, the phospholipids and the bond to the hydrophilic moieties should preferably be phospholipase resistant.
- TMPEG see WO-A-90/04384, WO- 90/04606, WO-A-90/04650 and WO-A-95/06058 is preferred for coupling PEG moieties to the phospholipid.
- liposomes bearing polymer moieties may be added before liposome formation but this tends to reduce the internal space available for carrying the payload and increases the amount of polymer required to achieve the desired coverage of the external surface. It is, of course, the amount of polymer exposed to the blood stream or tissues at the external surface of the liposomes or other lipid-based structure forming the dispersed phase, which primarily influences the tumour localisation of the payload.
- the present invention places no particular limit on the quantity of hydrophilic polymer moieties to be exposed on the blood-contacting surface of the lipid-based structures other than the requirements imposed by the optimisation of the composition for its intended use.
- the hydrophobic entities exposed at the blood-contacting surface of the lipid-based structures may be so derivatised if desired or necessary to achieve the therapuetic or diagnostic goal.
- the content of the derivatisable hydrophobic entities and the degree of derivatisation are such that at least 2% of the total hydrophobic entities exposed at the blood-contacting surface are derivatised with hydrophilic moieties. More preferably from 20 to 100% of the derivatisable hydrophobic entities are actually derivatised.
- hydrophilic moieties for hydrophobic entities not exposed to the blood- contacting surfaces of the lipid-based structures is less critical. It may be convenient that the proportion of the hydrophobic entities which are derivatised with hydrophilic polymer moieties will be substantially constant throughout the lipid-based structures of the invention and in the case where all the derivatisation of the hydrophobic entities is conducted before assembly of the lipid-based structures, the production process makes it almost inevitable that the composition of the lipid-based structures will be
- the polymer moieties may be of any suitable polymer
- hydrophilic polymer though preferably polyethylene glycol (PEG) is used, especially PEG'S of molecular weight from 250 to 12000 and more preferably PEG 5000.
- PEG polyethylene glycol
- the diagnostic or therapeutic agent is at least partially associated with the lipid-based structures.
- Preferably at least 50% by weight of the diagnostic or therapeutic agent is associated with the lipid-based structures, for instance by entrapment within or between the lipid bilayers or in the enclosed aqueous environment of liposomes or incorporated into the lipid bilayer thereof.
- tumour to blood ratios in conjunction with tumour liposome concentrations, or direct measurements of flux rates from tumours and elimination rates within tumours, and thus allow the construction of a liposomally entrapped compound with optimum retention within tumours and acceptable tumour to blood ratios.
- tumours vary qualitatively and the blood/tumour barrier is not always identical, it is not possible to disclose a single formulation with optimum properties.
- Example 9 there is up to 21 & 40 mol% of PEG-PE on the exterior surface (depending on the
- the preferred linkage is more stable to enzymatic degradations than ester or amide linkages which are subject to cleavage by esterases and amidases respectively.
- Succinyl ester linkages may additionally undergo hydrolysis (Carter and Meyerhoff, J. Immunol. Methods, 1985, 81 , 245- 257). With biodegradable linkages, degradation in the tumour milieu might ensue. A variety of linkages have been assessed (as micelles) for stability in serum [Parr et al, Biochim. Biophys. Acta, 1195: 21-30,(1994)].
- Succinate- linked PEG-lipid which contains an ester bond, was most susceptible to loss of PEG.
- the preferred linkage should not generate a net negative charge when the amino group is substituted, in contrast to other linkages, such as the carbamate linkage which has been shown by Woodle et al, (Biophysical Journal, 61: 902-910, (1992)) to consume the positive charge of the NH 2 group, leaving phosphatidyl ethanolaxnine with a net negative charge.
- the generation of a net negative charge on the lipid by PEGylation may also be disadvantageous because it might render the liposome susceptible to removal by macrophages (which have a
- Factors influencing tumour-retention of liposomes differ from those slowing blood clearance rates:- 1) A slowing of blood clearance rate can be achieved by altering the lipid composition of the liposome but this does not in itself necessarily produce significant
- Reticuloendothelial system uptake is one of the major routes of elimination and is a saturable process (as indicated by the capacity for RES blockade).
- monocyte and receptor mediated endocytosis (lymphocytes).
- lipid exchange in the tumour environment may not have the same consequences as in the blood.
- longevity in the blood were to be partially achieved by lipid composition, that might be substantially altered (e.g. by cholesterol loss) in the tumour milieu.
- lipid composition that might be substantially altered (e.g. by cholesterol loss) in the tumour milieu.
- the impact of PEGylation is also likely to be different if the major influences on clearance rates for the two sites (blood and tumour) have different mechanisms. Uptake by large organs, such as the skin, has the major impact on tissue distribution rate, hence on blood levels at early time points, whereas uptake by elimination organs such as liver and spleen influences the overall elimination rate and hence blood levels.
- pinocytic vesicle shuttle 50 nm particles
- Fenestrated capillaries offer four routes across the endothelium:
- pinocytic vesicle shuttle (5 - 30 nm particles);
- diaphragm fenestrae (porosity unknown);
- Discontinuous capillaries offer two routes:
- modified liposomes are spared from the removal system for particulates.
- compositions of the present invention are:
- compositions for administration in any conventional pharmaceutically acceptable manner.
- the compositions may be presented as dry powders, such as lyophilised liposomes, for reconstitution with sterile water or water for injection.
- the compositions may be presented as dry powders, such as lyophilised liposomes, for reconstitution with sterile water or water for injection.
- the compositions may be presented as dry powders, such as lyophilised liposomes, for reconstitution with sterile water or water for injection.
- the compositions may be presented as dry powders, such as lyophilised liposomes, for reconstitution with sterile water or water for injection.
- compositions may be presented as aqueous dispersions or suspensions ready for injection or as concentrates suitable for dilution, for instance with sterile water or water for injection, so as to form injectable products.
- compositions of the invention when formulated for
- compositions of the invention which are presented as dry powders or
- injectable products may also contain conventional additives to aid reconstitution or dilution thereof.
- compositions of the invention where necessary after reconstitution or dilution, are administered to patients in need thereof, for instance patients having or suspected to have solid tumours or dermatological diseases or disorders, in suitable amounts to achieve the necessary therapeutic or diagnostic dose at the target site for the desired duration, without causing clinically unacceptable side effects.
- the compositions are administered by injection by any conventional route which will afford access to the bloodstream for the multi-molecular
- compositions will be administered by the intravenous, intramuscular or parenteral route.
- the compositions may be injected in a single dose, as divided doses or by infusion over a period of from several minutes to several hours or even days as appropriate.
- Fig 1. shows the percent of dose injected per gram of tumour tissue plotted as a bar chart against DSPE content of administered PEGylated liposomes (see Example 4) .
- Fig. 2 shows the percent of dose injected per gram of liver, spleen and tumour tissue plotted as a bar chart against DSPE content of administered PEGylated liposomes (see Example 4).
- Fig. 3 correlates the percent of dose injected pergram of tumour with the percent of dose injected per gram of liver (a) or spleen (b), (see Example 6).
- Fig. 4 shows the percent of 111 Indium retained plotted as bar charts at 13 (a), 19 (b), 22 (c) and 49 (d) days of storage against DSPE content of liposomes (see Example 6).
- Fig. 5 shows the percent of 111 Indium retained plotted as bar charts at lh and 24h after exposure to citrated fresh frozen plasma against PEGylated DSPE content of liposomes administered (see Example 6) .
- Fig. 6 shows, (a) a plot of the percent dose injected per gram of tumour tissue versus latency (percent
- Fig. 7 and 8 each show plots of percent 111 Indium retained versus percent DSPE in the PEGylated liposomes exposed for either lh (A) or 24 - 25h (B) to various types of plasma (see Example 6)
- Fig. 9 plots the percent 1T1 Indium retained against time (minutes) of exposure to fresh frozen citrated plasma (see Example 6).
- Fig. 10 plots the percent 111 Indium retained after incubation in plasma for lh (A) and 24h (B) versus DSPE content in unPEGylated and PEGylated liposomes (see Example 6).
- Fig. 11 plots the percent of injected dose per g of kidney tissue at 1 h (upper panel) and 25 h (centre panel) against 111 Indium released in vitro by exposure to mouse plasma (percent of total) and, as a bar chart, against DSPE content (mol%) of liposomes (bottom panel), (see Example 6).
- Fig. 12 plots the percent of injected dose of
- Fig. 13 plots the percent of dose injected per gram of tissue for various organs against DSPE content (mol%) of administered PEGylated liposomes (left hand series of graphs) and the organ:blood ratios of those doses against DSPE content (mol%) (right hand series of graphs) (see Example 7).
- Fig. 14 plots percent of llx Indium loaded in liposomes against DSPE content (mol%) of the liposomes (see Example 8).
- Fig. 15 plots 111 In counts against elution volume (ml) for various DSPE contents of liposomes (see Example 8).
- Fig. 16 plots percent of llx Indium entrapped in
- Fig 17. plots percent of injected dose of 125 I per gram of blood at various times (h) post injection (see Example 9).
- Fig. 18 plots the percent of 125 I injected per gram of organ for PEGylated (closed circles) and unPEGylated (open circles) liposomes at various times (h) post injection of the liposomes (left hand series of graphs) and the
- Fig. 19 shows the area under the curve (AUC's) over the period 1 to 144 h taken from the graphs in Fig. 18 for PEGylated (hatched) and unPEGylated (opened) liposomes (upper panel) and the corresponding tumour to organ ratios (lower panel) for the various tissues tested (see Example 9).
- Fig. 20 plots the percent dose of 111 In per gram of organ against the time (h) post injection of liposomes containing 5 % DSPE and 33 % cholesterol (triangles) and 40 % DSPE without cholesterol (squares) (left hand series of graphs) and corresponding organ:blood ratios (right hand series of graphs) (see Example 9).
- Fig. 21 shows the percent of injected dose per gram of tissue for blood (upper panel) and tumour (lower panel) for various liposome compositions and for Free 111 In-NTA
- Figure 22 shows a 19 F-nmr trace of TMPEG-5000 in DMSO (see Example 11).
- Figure 23 is a graph of the strength at various times of the 19 F signal at 62.5 ppm (TMPEG-5000) expressed
- Figure 24 shows a 19 F-nmr trace of TMPEG-5000 in 50 mM borate pH 9.3 containing 250 mM sucrose after 80 min incubation when all the intact TMPEG had disappeared (see Example 11).
- Figure 25 is a graph of relative signal strength versus time for various species detected by 19 F-nmr when TMPEG-5000 was exposed to a) 50 mM borate pH 9.3 containing 250 mM sucrose and b) 20 mM HEP ⁇ S pH 7.4 containing 290 mM sucrose (see Example 11).
- Figure 26 shows the percent of injected dose per gram of tissue for blood (upper panel) and tumour (lower panel) plotted against time post-injection (h) for 111 In (circles) and 125 I-TI (squares) administered in liposomes.
- the liposomes were typically prepared by extrusion of a 10 mg/ml liposomal suspension.
- lipid films with a total content of 50 mg phospholipid (PL) were prepared by mixing quantities (shown in Table 1, ⁇ l) of DSPC (at a
- lonophore A23817 was incorporated into the lipid bilayer at a molar concentration of 0.1 ⁇ mol per 50 mg total
- Cholesterol Hexadecylether 25 ⁇ Ci
- Cholesterol Hexadecylether 25 ⁇ Ci
- the solvent was evaporated carefully. This can be done in many different ways, for example evaporation by blowing nitrogen gas as follows: the outlet was placed 5 cm above the surface of the solvent and the nitrogen flow was adjusted to avoid bubbling at the solvent/air interface. The pressure of nitrogen has to be adjusted according to the number of outlets.
- the liposomal suspension was then subjected to 5 cycles of freezing and thawing by immerising the tube in liquid N 2 for 1-2 min (or the time required for the
- liposomal preparation to be frozen
- immersion in water at 65°C for 1-2 min (or the time required to have a liquid liposomal suspension).
- the liposomal suspension was then extruded at 65oc (temperature provided by a thermobarrel connected to a recirculating water bath) through polycarbonate filters (double stack filters) as follows: through 0.4 micron filters 5 times; through 0.2 micron 5 times and through 0.1 micron 10 times. This produces liposomes of average size circa 100 nm.
- the liposomes with entrapped contents were then separated from the unentrapped water soluble components by exchanging the buffer, e.g. by gel permeation
- liposomes were collected with the void volume and NTA (or other water soluble contents) with the total volume of the column.
- the PD-10 column was equilibrated with the appropriate buffer and loaded with 2 ml (maximum) of the extruded liposomal suspension. After collecting fraction 1, fractions 2 to 30 were eluted with 300 ⁇ l buffer. The location of the liposomes was established by quantifying the lipid label (i.e. 3 H) by scintillation counting. Fractions containing liposomes were then pooled and lipid content established by the 3 H content and also by estimation of phosphorus.
- the liposomes can then be loaded with 111 In (if they had been produced in the presence of for instance NTA) by incubation with 111 ln ( 111 Indium hydrochloride formulated in 0.04 M HCl) at a ratio of for example 0.8 mCi per 10 mg of phospholipid.
- the liposomal suspension was adjusted to a final
- phopholipid concentration of 2 mg/ml in a reaction mixture containing TMPEG 166 mg/ml (alternative strategies are discussed below).
- the PEGylated liposomes were collected free of
- fraction 1 fractions 2 to 30 were eluted with 500 ⁇ l buffer and fractions 31 to 40 with 2 ml of buffer. PEGylated liposomes were collected with the void volume.
- the liposomes were constructed using 50 mM phosphate pH 7.4 containing 250 mM sucrose and NTA;
- the buffer used for the chromatographic separation of NTA was 50 mM phosphate pH 7.4 containing 250 mM sucrose; 4) Before Indium loading liposomes were filtered through 0.2 ⁇ filter (Acrodisc 13) to remove any aggregates;
- the 111 Indium loaded was 0.5 mCi per 10 mg phospholipid
- the final concentration of phospholipid in the PEGylation reaction was 3.5 mg/ml containing TMPEG at 150 mg/ml.
- an alternative and possibly preferable PEGylation scheme is to add TMPEG in a step-wise fashion so that it is at sub-aggregation, sub- fusogenic doses during the early stages of the reaction. If necessary, excess TMPEG can be removed between addition of aliquots of TMPEG.
- the liposomes were typically prepared by extrusion of a 10 mg/ml liposomal suspension.
- lipid films with a total content of 40 mg phospholipid (PL) were prepared by mixing DOPC (at a concentration of 20 mg/ml in chloroform) and DOPE (at a concentration of 10 mg/ml in
- the lipid film was produced as in Example 1.
- 4 ml of the buffer containing the water soluble component (s) to be entrapped e.g 125 I- tyraminylinulin, TI
- the mixtures were then vortexed vigorously.
- the mixtures were subjected to several cycles of warming up to 65°c (2 min) and vortexing (1 min) until complete dispersion of the lipid.
- the liposomal suspension was then subjected to 5 cycles of freezing and thawing by immersing the tube in liquid N 2 for 1-2 min (or the time required for the liposomal preparation to be frozen) followed by
- the liposomal suspension was then extruded at circa 65oC through polycarbonate filters (double stack filters) as follows: through 0.4 micron filters 5 times; through 0.2 micron 5 times and through 0.1 micron 10 times. This produces liposomes of average size circa 100 nm.
- the liposomes with entrapped contents were then separated from the unentrapped water soluble components using 10 ml syringe barrel Sepharose CL-4B column using 20 mM HEPES 145 mM NaCl buffer pH 7.4 Liposomes were collected with the void volume and TI (or other water soluble
- the extruded liposomes loaded with the appropriate contents were then PEGylated by reaction with TMPEG for 2h at room temperature in 20 mM HEPES 145 mM NaCl pH 7.4.
- the liposomal suspension was adjusted to a final phospholipid concentration of 3.4 mg/ml in a reaction mixture containing TMPEG 70 mg/ml.
- PEG-liposomes should have an essentially identical biological distribution, irrespective of whether they are prepared by incorporation of PEG-lipid or
- PEGylated on the exterior even though PEG-DSPE in the latter case constitutes only 5 mol% PEG-DSPE with respect to total lipid.
- One difference will be that the carrying capacity will be reduced by PEG-DSPE to an extent
- PEG is known to exclude proteins from surfaces. Heavy PEGylation will lose significant proportions of the liposomal cavity. This should not however affect the biological behaviour.
- Distearoylphosphatidylethanolamine (20 mg, 27 ⁇ mol) was dissolved in 3 ml of dry chloroform/dry methanol (5/2 vol/vol) with slight warming.
- Tresyl-monomethoxy PEG-5000 150 mg, 27 ⁇ mol dissolved in 1 ml of dry chloroform/dry methanol (5/2) was added.
- the mixture was stirred at 50°c in the presence of sodium carbonate (290 mg) until the ninhydrin positive DSPE spot detectable on thin layer chromatography disappeared. After removing the sodium carbonate by centrifugation, the MPEG derivative was precipitated from dry diethyl ether and dried under reduced pressure.
- Tumour to blood ratios were substantially above 1 and either increased or were maintained with respect to unPEGylated controls.
- the amount of PEG optimal for exclusion from the liver and spleen will not necessarily be identical to that giving the best localisation in the tumour.
- Figure 2 compares the effect of different mole percent of PEGylated DSPE in DSPE-DSPC liposomes (prepared by
- the figures 3a and 3b show results for individual mice and confirm the results of Figure 2.
- Most of the improvement in tumour concentration e.g. between 3 to 8.5 % injected dose per gram tumour
- occurs without any reciprocal change in liver or spleen concentration these are both circa 15 % injected dose per gram for the majority of tumour values over the above range.
- some factor other than exclusion from the RES of the liver and spleen must account for the
- Figure 4a-c shows the latency of liposomes containing 111 indium chelated to NTA. Irrespective of the level of
- DSPE:DSPC liposomes with 0 to 100 mol% PEGylated DSPE showed negligible loss of contents when stored for at least 19 days in 50 mM phosphate 250 mM sucrose pH 7.4 at 4oc.
- Free l ⁇ : ⁇ Indium (and excess TMPEG) was removed from loaded liposomes before storage using a Sepharose CL-4B 20 ml syringe barrel column. After storage for the times indicated, leakage of liposomally entrapped 111 Indium was assessed using paper chromatography of Whatman 4 filter paper and appropriate buffer (depending on liposome pH), run for approximately 8 cm. The upper and lower sections were counted for 111 In. The latency was corrected for free counts at time zero. Similar results were obtained with unPEGylated liposomes after 22 days storage (Figure 4c). Storage of an 111 Indium loaded
- PEGylated liposome preparation for 49d without removal of the TMPEG and buffer 50 mM borate 250 mM sucrose pH 9.3 also resulted in a similar moderate loss of contents (Figure 4d) to that seen with PEGylated liposomes from which TMPEG was removed prior to storage.
- Figure 7 compares the latency result with citrated human plasma (from Figure 5a and b) to the latency for liposomes exposed to heparinised mouse plasma for 1 and 24/25h.
- mouse plasma led to less loss of latency at 24/25h, particularly evident at 40 - 100 % PEGylated DSPE, the mouse plasma results, like the human plasma results are still showing an inverse relationship with tumour 111 Indium concentration and are thus indicating that plasma induced loss of latency is not a reliable predictor of PEG-liposome behaviour with respect to tumour drug
- Figure 8 compares the latency result with citrated human plasma (from Figure 5a and b) to the latency for liposomes exposed to citrated human plasma which had been heat treated at 56oC for 45 min to remove complement and other heat labile lipid transfer factors (previously reported). As with mouse plasma, there was some difference after 24h incubation, evident with 40 - 100 % PEGylated DSPE liposomes, but there is still an inverse relationship with tumour 111 Indium
- FIG. 13 shows the tissue concentrations (left panels) and tissue:blood ratios. The tissue concentrations were not corrected for the blood content of the organs. The concentrations of 111 Indium at 24h rose in all organs with PEGylated liposomes containing increasing amounts of DSPE (left hand panels). In all organs there was a trend of decreasing tissue:blood ratios (right hand panels) up to 20 mol% DSPE, implying that the increase in blood levels was not accompanied by a parallel increase in tissue levels, hence that the PEG-lipid was slightly reducing entry into these tissues as well as its effect on liver and spleen (or that the "push" principle is predominating in these normal tissues).
- PEGylated liposomes with 40 or more mol% either maintained this reduced tissue:blood ratio (e.g. lung) or showed a slight increase, but never in excess of the unPEGylated 0 % DSPE control.
- PEG-DSPE would be unable to form Hexagonal II (the 5 nm core would have difficulty accommodating the bulky PEG headgroup).
- PEG-lipid estimates based on thin layer chromatography may be underestimates of the PEGylation status of the contents-bearing liposomes, whose behaviour is
- Example 1 The selection of pH 9.3 in Example 1 was based on
- PEGylation period can be used to overcome the delay in
- DSPE:DSPC liposomes also showed a similar relationship between the increments in tumour to blood ratios and skin to blood ratios and showed that the effect was related to the concentration of PEGylated DSPE in the liposomes.
- PEG concentration of PEGylated DSPE in the liposomes.
- Table 4 shows the blood and skin results and skin to blood ratios for a range of different liposomal formulations.
- the liposomal preparation should have utility in delivery of compounds selectively to skin.
- Figure 21 shows the effect of different lipid
- compositions E and F had markedly reduced latency on exposure to plasma (see Example 6).
- preparations and others, A, C and D in Figure 21) contained 111 Indium chelated to NTA.
- NTA is a
- FIG. 22 shows the 19 F-nmr of TMPEG (the preparation used in Examples 1 and 2 in dimethyl ⁇ ulphoxide (d6). The two triplets were observed, centred at -60.7752 ppm and
- TFESA trifluorethane sulphonic acid
- the most likely explanation for the -120ppm specie is that it is free fluoride ions released by a side reaction of the TMPEG.
- reaction mixtures may need to have serial aliquots of fresh TMPEG added to obtain high levels of PEGylation.
- reaction mixtures may need to have serial aliquots of fresh TMPEG added to obtain high levels of PEGylation.
- tyraminylinulin TI
- 111 Indium chelated to NTA As outlined above the former is small and rapidly removed via renal excretion once leaked from the liposome. In contrast, 111 lndium is transferred from NTA to plasma or extravascular proteins after liposome disruption. In order to assess how this affects our selection criteria, these two types of
- liposomal contents were assessed in the same liposomes.
- DOPC:DOPE (79:21 mol%) containing ionophore A23817 (1.08 mg per mg of lipid) were prepared by extrusion of the lipid suspension (10 mg/ml) in Hepes 20 mM pH 7.4, sodium chloride 145 mM and NTA 1 mM (the lipid suspension was obtained by vortexing followed by several cycles of warming up to 65oc for 2 min and vortexing for 1 min and then subjected to 5 cycles of freezing and thawing and subsequent extrusion as in Example 2).
- the buffer was subsequently exchanged to Hepes 20 mM pH 7.4, sodium chloride 145 mM using a PD-10 column.
- 111 In loading 1.2 ml of liposomes at 3 mg/ml were incubated with 0.3 mCi of 111 In for 30 min at 65oc.
- 111 ln loaded liposomes were isolated by GPC in a PD-10 column.
- the incorporation of 111 Indium to the liposomes was circa 100 % as shown by paper chromatography as described in Example 6.
- the extruded liposomes loaded with 111 In were then PEGylated by reaction with TMPEG for 2h at room temperature in Hepes 20 mM pH 7.4 containing sodium chloride 145 mM as described in Example 2.
- Figure 26 shows the blood pharmacokinetics and tumour biodistribution for 111 ln and 125 I-labeleld TI entrapped in DOPC:DOPE ((79:21) mol%) liposomes.
- Blood levels for 111 ln were slightly greater than blood levels for 125 I-labelled TI at all time points.
- the tumour biodistribution was very different for the two contents: while levels of 111 ln were raising with time post-injection until they reached a plateau, levels of 125 I-labelled TI decreased with time post-injection. This behaviour is consistent with leakage of at least one of the contents from the liposomal vehicle.
- Table 5 shows the blood and tumour concentrations and the tumour to blood concentration ratios at 24 h post- injection for 111 In and 125 I-labelled TI entrapped in control and PEGylated D0PC:D0PE lipsomes. For both contents, the tumour to blood concentration ratios were greater with the PEGylated liposomes than with the control liposomes
Abstract
Description
Claims
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JP8533133A JPH11504915A (en) | 1995-05-03 | 1996-05-03 | Tissue entrapment |
EP96912158A EP0825850A1 (en) | 1995-05-03 | 1996-05-03 | Tissue entrapment |
AU55097/96A AU5509796A (en) | 1995-05-03 | 1996-05-03 | Tissue entrapment |
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GB9509016.3 | 1995-05-03 | ||
GBGB9509016.3A GB9509016D0 (en) | 1995-05-03 | 1995-05-03 | Tissue entrapment |
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EP (1) | EP0825850A1 (en) |
JP (1) | JPH11504915A (en) |
AR (1) | AR001858A1 (en) |
AU (1) | AU5509796A (en) |
CA (1) | CA2218932A1 (en) |
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WO (1) | WO1996034598A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5820873A (en) * | 1994-09-30 | 1998-10-13 | The University Of British Columbia | Polyethylene glycol modified ceramide lipids and liposome uses thereof |
WO1998046275A2 (en) * | 1997-04-11 | 1998-10-22 | The Board Of Regents Of The University Of Michigan | Blood-pool carrier for lipophilic imaging agents |
US5885613A (en) * | 1994-09-30 | 1999-03-23 | The University Of British Columbia | Bilayer stabilizing components and their use in forming programmable fusogenic liposomes |
US6645463B1 (en) | 1994-05-16 | 2003-11-11 | The Board Of Regents Of The University Of Michigan | Blood-pool selective carrier for lipophilic imaging agents |
US6673364B1 (en) | 1995-06-07 | 2004-01-06 | The University Of British Columbia | Liposome having an exchangeable component |
US6734171B1 (en) | 1997-10-10 | 2004-05-11 | Inex Pharmaceuticals Corp. | Methods for encapsulating nucleic acids in lipid bilayers |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0354855A2 (en) * | 1988-08-11 | 1990-02-14 | Terumo Kabushiki Kaisha | Liposomes on which adsorption of proteins is inhibited |
WO1990004384A1 (en) * | 1988-10-20 | 1990-05-03 | Royal Free Hospital School Of Medicine | Liposomes |
WO1991005546A1 (en) * | 1989-10-20 | 1991-05-02 | Liposome Technology, Inc. | Solid tumor treatment method and composition |
WO1991016040A1 (en) * | 1990-04-18 | 1991-10-31 | Takeda Chemical Industries, Ltd. | Liposome composition |
US5264221A (en) * | 1991-05-23 | 1993-11-23 | Mitsubishi Kasei Corporation | Drug-containing protein-bonded liposome |
WO1994007466A1 (en) * | 1992-10-07 | 1994-04-14 | Liposome Technology, Inc. | Compositions for treatmewnt of inflamed tissues |
WO1994021235A1 (en) * | 1993-03-23 | 1994-09-29 | Liposome Technology, Inc. | Enhanced circulation effector composition and method |
WO1994022429A1 (en) * | 1993-03-31 | 1994-10-13 | Liposome Technology, Inc. | Solid-tumor treatment method |
-
1995
- 1995-05-03 GB GBGB9509016.3A patent/GB9509016D0/en active Pending
-
1996
- 1996-05-03 EP EP96912158A patent/EP0825850A1/en not_active Withdrawn
- 1996-05-03 AU AU55097/96A patent/AU5509796A/en not_active Abandoned
- 1996-05-03 CA CA 2218932 patent/CA2218932A1/en not_active Abandoned
- 1996-05-03 JP JP8533133A patent/JPH11504915A/en active Pending
- 1996-05-03 AR AR33640796A patent/AR001858A1/en unknown
- 1996-05-03 WO PCT/GB1996/001072 patent/WO1996034598A1/en not_active Application Discontinuation
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0354855A2 (en) * | 1988-08-11 | 1990-02-14 | Terumo Kabushiki Kaisha | Liposomes on which adsorption of proteins is inhibited |
WO1990004384A1 (en) * | 1988-10-20 | 1990-05-03 | Royal Free Hospital School Of Medicine | Liposomes |
WO1991005546A1 (en) * | 1989-10-20 | 1991-05-02 | Liposome Technology, Inc. | Solid tumor treatment method and composition |
WO1991016040A1 (en) * | 1990-04-18 | 1991-10-31 | Takeda Chemical Industries, Ltd. | Liposome composition |
US5264221A (en) * | 1991-05-23 | 1993-11-23 | Mitsubishi Kasei Corporation | Drug-containing protein-bonded liposome |
WO1994007466A1 (en) * | 1992-10-07 | 1994-04-14 | Liposome Technology, Inc. | Compositions for treatmewnt of inflamed tissues |
WO1994021235A1 (en) * | 1993-03-23 | 1994-09-29 | Liposome Technology, Inc. | Enhanced circulation effector composition and method |
WO1994022429A1 (en) * | 1993-03-31 | 1994-10-13 | Liposome Technology, Inc. | Solid-tumor treatment method |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6645463B1 (en) | 1994-05-16 | 2003-11-11 | The Board Of Regents Of The University Of Michigan | Blood-pool selective carrier for lipophilic imaging agents |
US7582279B2 (en) | 1994-05-16 | 2009-09-01 | The Board Of Regents Of The University Of Michigan | Blood-pool carrier for lipophilic imaging agents |
US5820873A (en) * | 1994-09-30 | 1998-10-13 | The University Of British Columbia | Polyethylene glycol modified ceramide lipids and liposome uses thereof |
US5885613A (en) * | 1994-09-30 | 1999-03-23 | The University Of British Columbia | Bilayer stabilizing components and their use in forming programmable fusogenic liposomes |
US6673364B1 (en) | 1995-06-07 | 2004-01-06 | The University Of British Columbia | Liposome having an exchangeable component |
WO1998046275A2 (en) * | 1997-04-11 | 1998-10-22 | The Board Of Regents Of The University Of Michigan | Blood-pool carrier for lipophilic imaging agents |
WO1998046275A3 (en) * | 1997-04-11 | 1999-01-28 | Univ Michigan | Blood-pool carrier for lipophilic imaging agents |
US6734171B1 (en) | 1997-10-10 | 2004-05-11 | Inex Pharmaceuticals Corp. | Methods for encapsulating nucleic acids in lipid bilayers |
Also Published As
Publication number | Publication date |
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EP0825850A1 (en) | 1998-03-04 |
AU5509796A (en) | 1996-11-21 |
AR001858A1 (en) | 1997-12-10 |
JPH11504915A (en) | 1999-05-11 |
CA2218932A1 (en) | 1996-11-07 |
GB9509016D0 (en) | 1995-06-21 |
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