Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • 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
  • A61K47/51—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 non-active ingredient being a modifying agent
  • A61K47/54—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 non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• • 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
  • A61K47/51—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 non-active ingredient being a modifying agent
  • A61K47/56—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/59—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
  • A61K47/60—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 non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• • 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
  • A61K47/6911—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 liposome
• • 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—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators

Definitions

• Enhancing intracellular delivery of liposomes and/or their contents represents one of the major remaining problems in the development of the next generation of drug delivery systems.
• general methods for increasing the interactions of liposomes with cells need to be developed.
• attempts include the use of specific targeting information on the liposome surface, such as an antibody (see, Meyer, O. et al., Journal of Biological Chemistry 273:15621-15627 (1998); Kao, G.Y. et ai, Cancer Gene Therapy 3:250-256 (1996); Hansen, C.B.
• the compounds of Formula I contain groups that give rise to compounds having the general structure of Formula II:
• "A” is a lipid, such as a hydrophobic lipid.
• "X” is a single bond or a functional group that covalently attaches the lipid to at least one ethylene oxide unit, i.e., (-CH - CH 2 -O-).
• "Z” is a single bond or a functional group that covalently attaches the at least one ethylene oxide unit to a cationic group.
• "Y” is a polycationic moiety.
• the index "n” is an integer ranging in value from about 6 to about 160.
• the present invention relates to a lipid-based drug formulation comprising:
• amphipathic lipid refers, in part, to any suitable material wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase.
• Amphipathic lipids are usually the major component of a lipid vesicle. Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxy and other like groups.
• amphipathic lipids Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols and ⁇ -acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipid described above can be mixed with other lipids including triglycerides and sterols.
• dialkylglycerolyl denotes two C ⁇ -C o alkyl chains bonded to the 1- and 2-position of glycerol by ether linkages.
• Dialkylglycerol groups have the following general formula:
• acyl groups can be saturated or have varying degrees of unsaturation.
• the 3-position of the propane molecule has a -NH- group attached.
• cationic lipid refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH.
• lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC”); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTMA”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(2,3- dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); 3 -(N-(N',N'- dimethylaminoethane)-carbamoyl)cholesterol (“DC-Choi”) and N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl am
• heterocyclyl aryl, heteroaryl, carboxyl, carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl, amido, and the like to form alkyl groups such as trifluoromethyl, 3- hydroxyhexyl, 2- carboxypropyl, 2-fluoroethyl, carboxymethyl, cyanobutyl and the like.
• alkynyl refers to branched or unbranched hydrocarbon chains containing one or more carbon-carbon triple bonds.
• aryl denotes a chain of carbon atoms which form at least one aromatic ring having preferably between about 6-14 carbon atoms, such as phenyl, naphthyl, indenyl, and the like, and which may be substituted with one or more functional groups which are attached commonly to such chains, such as hydroxyl, bromo, fluoro, chloro, iodo, mercapto or thio, cyano, cyanoamido, alkylthio, heterocycle, aryl, heteroaryl, carboxyl, carbalkoyl, alkyl, alkenyl, nitro, amino, alkoxyl, amido, and the like to form aryl groups such as biphenyl, iodobiphenyl, methoxybiphenyl, anthryl, bromophenyl, iodophenyl, chlorophenyl, hydroxyphenyl, methoxyphenyl, formylphenyl, acet
• amido denotes an amide linkage: -C(O)NR- (wherein R is hydrogen or alkyl).
• carboxyl denotes the group -C(O)O-
• carbonyl denotes the group -C(O)-
• carbonate indicates the group -OC(O)O-.
• HBS Hepes-buffered saline
• Rho-PE rhodamine-phosphatidylethanolamine
• LUNs refers to "large unilamellar vesicles.”
• the present invention provides cationic-polymer-lipid conjugates (CPLs), such as distal cationic-poly(ethylene glycol)-lipid conjugates that can be incorporated into conventional and stealth liposomes for enhancing, inter alia, cellular uptake.
• CPLs cationic-polymer-lipid conjugates
• the CPLs of the present invention have the following architectural features: (1) a lipid anchor, such as a hydrophobic lipid, for incorporating the CPLs into the lipid bilayer; (2) a hydrophilic spacer, such as a polyethylene glycol, for linking the lipid anchor to a cationic head group; and (3) a polycationic moiety, such as a naturally occurring amino acid, to produce a protonizable cationic head group.
• the present invention provides a compound of Formula I:
• post-insertion involves forming vesicles (by any method), and incubating the pre-formed vesicles in the presence of CPL under appropriate conditions (usually 2-3 hours at 60°C). Between 60-80% of the CPL can be inserted into the external leaflet of the recipient vesicle, giving final concentrations up to 7 mol % (relative to total lipid).
• the method is especially useful for vesicles made from phospholipids (which can contain cholesterol) and also for vesicles containing PEG-lipids (such as PEG-Ceramide).
• the CPL-LUNs of the present invention can be formed by extrusion.
• the film may be redissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogeneous lipid mixture which is in a more easily hydrated powder-like form.
• a suitable solvent such as tertiary butanol
• This film is covered with an aqueous buffered solution and allowed to hydrate, typically over a 15-60 minute period with agitation.
• the size distribution of the resulting multilamellar vesicles can be shifted toward smaller sizes by hydrating the lipids under more vigorous agitation conditions or by adding solubilizing detergents, such as deoxycholate.
• Liposomes containing the CPLs of the present invention can also be used as a vector to deliver immunosuppressive or immunostimulatory agents selectively to macrophages.
• glucocorticoids useful to suppress macrophage activity and lymphokines that activate macrophages can be delivered using the liposomes of the present invention.
• the diagnostic targeting of the liposome can subsequently be used to treat the targeted cell or tissue.
• the toxin when a toxin is coupled to a targeted liposome, the toxin can then be effective in destroying the targeted cell, such as a neoplasmic cell.
• SUBST ⁇ UTE SHEET RULE 26 based drug formulation having about 0.1 to 20 mole percent of a compound of Formulae I or II.
• a method for increasing the transfection of a cell with a lipid-based drug formulation comprising: contacting the cell with a lipid-based drug formulation having about 0.1 to 20 mole percent of a compound of Formulae I or II, whereby the transfection efficiency of the lipid-based drug formulation is increased compared to the transfection efficiency of a lipid-based drug formulation without the compound of Formulae I or II.
• Transmembrane potential loading has been described in detail in U.S. Patent No. 4,885,172, U.S. Patent No. 5,059,421, and U.S. Patent No. 5,171,578, the contents of which are incorporated herein by reference.
• the transmembrane potential loading method can be used with essentially any conventional drug which can exist in a charged state when dissolved in an appropriate aqueous medium.
• the drug will be relatively lipophilic so that it will partition into the liposome membranes.
• a transmembrane potential is created across the bilayers of the liposomes or protein-liposome complexes and the drug is loaded into the liposome by means of the transmembrane potential.
• the transmembrane potential is generated by creating a concentration gradient for one or more charged species (e.g., Na + , K + and/or H + ) across the membranes.
• This concentration gradient is generated by producing liposomes having different internal and external media and has an associated proton gradient. Drug accumulation can than occur in a manner predicted by the Henderson-Hasselbach equation.
• the liposome compositions of the present invention can by administered to a subject according to standard techniques.
• pharmaceutical compositions of the liposome compositions are administered parenterally, i.e., intraperitoneally, intravenously, subcutaneously or intramuscularly. More preferably, the pharmaceutical compositions are administered intravenously by steady infusion.
• suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985).
• the pharmaceutical compositions can be used, for example, to diagnose a variety of conditions, or treat a diseased state.
• compositions for intravenous administration which comprise a solution of the liposomes suspended in an acceptable carrier, preferably an aqueous carrier.
• an acceptable carrier preferably an aqueous carrier.
• aqueous carriers can be used, e.g., water, buffered water, 0.9% isotonic saline, and the like.
• These compositions can be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
• the resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
• compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
• auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
• Distal cationic-poly(ethylene glycol)-lipid conjugates were designed, synthesized and incorporated into conventional and stealth liposomes for enhancing cellular uptake.
• the present approach uses either inert, nontoxic or naturally occurred compounds as components for the CPL synthesis.
• CPLs were synthesized with the following architectural features: 1) a hydrophobic lipid anchor of DSPE for incorporating CPLs into liposomal bilayer; 2) a hydrophilic spacer of polyethylene glycol for linking the lipid anchor to the cationic head group; and 3) a naturally occurring amino acid (L-lysine) was used to produce a protonizable cationic head group. The number of charged amino groups can be controlled during the CPL synthesis.
• DSPE-CPLs were almost quantitatively inco ⁇ orated into liposomal bilayer by a hydration-extrusion method. Quite su ⁇ risingly, in an in vitro model, it was confirmed for the first time that liposomes possessing distal positively charged polymer conjugates with preferably four or more charges efficiently bind to host cell surfaces and enhance cellular uptake in mammalian cells.
• CPL distal positively charged cationic polymer lipid conjugates
• the present approach uses inert, nontoxic and naturally occurring compounds, e.g., amino acids, as components for the CPL synthesis.
• Several CPLs were designed with the following architectural features: 1 ) a hydrophobic lipid anchor for inco ⁇ orating the CPLs into the liposomal bilayer; 2) a hydrophilic spacer for linking the lipid anchor to the cationic head group; and 3) a cationic head group.
• the amount and nature of the cationic group can be changed according to the final application.
• a naturally occurring amino acid, L-lysine was used to produce a protonizable amino group. The number of amino group can be controlled during the CPL synthesis.
• LUVs containing CPL can be formed by a detergent dialysis method.
• the LUVs contain DOPE, DODAC, PEG-Cer-C20, and CPL 4 [3.4K] (or CPL [1K]). Two preparations were made with the CPL comprising 4 mol % of the original lipids: TABLE 4
• the lipids indicated above were co-dissolved in chloroform, which was then removed under nitrogen followed by 2 hours under high vacuum.
• the dry lipid mixture (10 ⁇ mol total) was then hydrated in 83 ⁇ L of 1 M OGP and 1 mL Hepes- buffered saline (20 mM Hepes 150 mM NaCI pH 7.5) at 60°C with vortexing until all the lipid was dissolved in the detergent solution.
• the lipid-detergent mixture was transferred to Slide-A-Lyzer dialysis cassettes, and dialysed against at least 2 L HBS for 48 hours, with a least two changes of buffer in that time. Removal of detergent by dialysis results in formation of LUVs.
• the lipid samples were fractionated on a column of Sepharose CL-4B (see Figures 8A and 8B). The fractionation profiles show LUVs formed with either CPL 4 [3.4K] or CPL 4 [1K].
• vesicles were formed using a detergent dialysis method (see, Wheeler, J.J., et al. (1999) Stabilized plasmid-lipid particles: construction and characterization. Gene Therapy 6, 271-281, the teachings of which are inco ⁇ orated herein by reference).
• the lipids, as described in Example II, were co-dissolved in chloroform in the appropriate ratios, following which the chloroform was removed under a stream of nitrogen and placed under high vacuum for 2 hours.
• Vesicles of DOPC and DOPC/Chol were prepared by extrusion as previously described (Hope, M.J., et al, (1985) Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential. Biochim. Biophys. Acta 812, 55-65).
• CPLs cationic PEG-lipids
• the free CPL elutes in a broad peak centered at 16 mLs, which is separate from the vesicle peak, allowing for easy isolation of the CPL-LUV.
• the DSPE- CPL-Q5 is retained and does not exchange out of the vesicles.
• the CPL-LUN fraction from Figure 9(B) was re-eluted on the column of Sepharose CL-4B. As shown in Figure 9(C), all of the CPL remains with the LUVs. The effects of incubation temperature and time on the insertion process are shown in Figure 10.
• DSPE-CPL-Q1 was incubated in the presence of DOPE/DODAC/PEGCerC20 (84/6/10) at room temperature, 40°C, and 60°C, with aliquots withdrawn at 1, 3, and 6 hours.
• the highest insertion levels were achieved at 60 °C, which was therefore used in subsequent insertions. Although slightly higher insertion was obtained at 6 hr, we chose 3 hr to minimize sample degradation.
• CPL-LUVs were examined by fluorescence microscopy, using a rhodamine filter. While control LUVs exhibited no signs of aggregation, significant levels were observed for CPL-LUVs. However, it was found that addition of 40 mM CaCl 2 completely prevented this effect.
• oligopeptides see, Zalipsky et al, Bioconjugate Chemistry 6, 705-708 (1995); Zalipsky et al, Bioconjugate Chemistry 8, 111-118 (1997)) oligosaccharides (see, Zalipsky et al, Bioconjugate Chemistry 8, 111-118 (1997)), folate (see, Gabizon et al, Bioconjugate Chemistry 10, 289-298 (1999); Lee et al, Journal of Biological Chemistiy 269, 3198-3204 (1994); Reddy et al.
• this composition is representative of the many sterically-stabilized drug delivery systems which contain PEG-lipids. Insertion of CPLs leads to localization of positive charge above the surface PEG layer, thereby allowing electrostatic interactions between the CPLs and cell surfaces. This should lead to increased cellular interactions for both conventional- and PEG-containing liposomes.
• the CPLs are conjugates of DSPE, a dansyl-lysine moiety, the hydrophilic polymer PEG 34 oo, and a mono- or multivalent cationic headgroup.
• the PEG functions as a spacer, separating the charged headgroup from the lipid anchor and vesicle surface.
• Incubation of a wide variety of neutral and cationic LUVs with micellar CPLs resulted in the inco ⁇ oration of up to 6-7 mol % (relative to total vesicle lipid) of CPL in the outer vesicle monolayer (see tables in Figures 23 and 24).
• the insertion efficiency was quite high, with approximately 70 - 80% of added CPL inco ⁇ orating into the LUVs (see tables in Figures 23 and 24).
• the most important factors influencing the CPL insertion levels were the incubation temperature ( Figure 10) and initial CPL/lipid ratio ( Figure 11).
• the composition of the liposome was found to affect the final CPL levels to a lesser degree (see tables in Figures 23 and 24).
• the CPL-LUV could be efficiently separated from free CPL by gel exclusion chromatography. Similar insertion levels were obtained for all CPLs, with headgroup charges ranging from one to four charges per molecule . With this knowledge, vesicles could be prepared containing a desired level of CPL with reasonable accuracy.
• the protocol described for insertion of CPL into conventional and sterically-stabilized CPL is ideal for demonstrating the methodology using in vitro applications.
• the added positive charge is physically distant from the surface, and is available for interactions with cells. This is particularly important for polymer-coated vesicles that are designed for minimal interactions with serum proteins and cells such as macrophages.
• this system may not be ideal for in vivo applications, where it may be desirable to initially hide or screen the CPL charge to reduce clearance and allow accumulation of the vesicles at the tissue of choice.
• alternative embodiments employ shorter PEG spacer chains in the CPL, or longer PEG chains in the PEG-Cer molecules.
• the PEG-Cer molecules are known to exchange out of the particle during circulation see, Webb et al, Biochimica et Biophysica Acta 1372, 272- 282 (1998)], which would leave the CPL exposed for cellular interactions.
• the cationic liposomes employed in the present study are composed of a fusogenic lipid (DOPE), a cationic lipid (DODAC), and a stabilizing lipid (PEG-Cer-C20), the latter of which imparts long-circulating properties to the vesicles.
• DOPE fusogenic lipid
• DODAC cationic lipid
• PEG-Cer-C20 stabilizing lipid
• This lipid composition was modeled after a new class of lipid-based DNA carrier systems known as stabilized plasmid-lipid particles (SPLPs) see, Wheeler et al. Gene Therapy 6, 271-281 (1999)). SPLPs are small (70 nm) particles that encapsulate a single plasmid molecule.
• the 4 mol % CPL shows the greatest increase in transfection: approximately 4500 times higher, followed by the 3% and then the 2% CPL samples. Therefore, the presence of the CPL, DSPE-Quad5 in the SPLP increased in both uptake and transfection to levels comparable to or above those achieved with the complexes.
• the final samples were prepared to contain 2, 3, or 4 mol % of the CPL.
• the dansyl assay involved preparing a standard curve of 0.5 to 2.5 mol % of dansylated CPL in BBS and determining the concentration of the CPL in the sample.
• the phospholipid was extracted from the SPLP by extracting the lipid using the Bligh-Dyer extraction technique (Bligh & Dyer, 1952) and then performing a Fiske- Subarrow assay on the organic phase of the extraction.
• the PicoGreen assay was performed by comparing the sample in the presence of PicoGreen and Triton X-100 using a DNA standard curve. The final % insertion of the CPL was determined by dividing the CPL concentration by the lipid concentration.
• the optimal time for insertion of the CPL into the SPLP was determined using SPLP prepared with 0.5 mol % Rh-DSPE. 15 nmol of the dansylated CPL (DSPE- Quad5) was mixed with 200 nmol of the labeled SPLP and the sample was incubated at 60°C for various time points (0.5, 1, 2, 3, and 4 hours). At these time points the sample was removed from the water bath and was passed down a Sepharose CL-4B column. The major fraction was collected from the column and the dansyl to rhodamine fluorescence ratios were measured.
• Freeze-Fracture EM Freeze-fracture EM was performed on the 2%, 3%, and 4% CPL samples by methods which will be described by K. Wong 5. Serum Stability of Particles: The stability of the DNA within these
• the rhodamine fluorescence of the lysate was then measured on a fluorometer using a ⁇ ex of 560 nm and a ⁇ em of 600 nm using slit widths of 10 and 20 nm, respectively. An emission filter of 430 nm was also used. A 1.0 L microcuvette was used. The lipid uptake was determined by comparison of the fluorescence to that of a lipid standard (5 nmol). This value was then normalized to the amount of cells present by measuring the protein in 50 ⁇ L of the lysate using the BCA assay.
• BHK cells were plated in 24-well plates in complete media. These were incubated overnight at 37°C in 5% CO 2 .
• SPLP, SPLP + 75mM CaCl 2 , DOPE:DODAC (1 :1)/DNA complexes, and CPL-SPLP systems (2, 3, and 4 mol % CPL) containing 2.5 ⁇ g of DNA were made up to lOO ⁇ L using HBS or HBS + 75mM CaCl 2 and were placed on the cells. Then 400 ⁇ L of complete media was added to this. At 4 and 9 hours, the transfection media was removed and replaced with complete media containing penicillin and streptomycin for a complete 24 hour transfection.
• the cells were lysed with lysis buffer containing Triton X-100. Following this lysis, 10- 20 ⁇ L of the lysated was transferred to a 96-well luminescence plate. The luminescence of the samples on the plate were measured using a Luciferase reaction kit and a plate luminometer. The luciferase activity was determined by using a luciferase standard curve and was normalized for the number of cells by measuring the protein with the BCA assay on 10-20 ⁇ L of the lysated.
• the CPL, DSPE-Quad5 will be used in the following studies. Its structure is shown in Figure 16A. This molecule possesses four positive charges at the end of a PEG oo molecule, which has been covalently attached to the lipid DSPE. The inco ⁇ oration of this CPL into empty liposomes of the same composition as the SPLP has been described previously in the above examples.
• the DSPE-Quad5 was inco ⁇ orated into SPLPs containing DOPE:PEG- CerC20:DODAC (84: 10:6) at various concentrations of the CPL (from 2-4 mol %).
• the inco ⁇ oration efficiencies for the various CPL percentages were between 70 and 80% of the initial.
• gel filtration chromatography was employed in order to separate the SPLPs possessing the CPL from the uninco ⁇ orated CPL.
• a typical column profile for the 3% DSPE-Quad5 is shown in Figure 19A.
• the CPL, lipid, and DNA all eluted from the column at the same time in a single peak. There was however a small amount of uninco ⁇ orated CPL that eluted at a later stage.
• the diameter of these particles containing the CPL was determined by QELS to be 125 mn compared to the SPLP, which had a diameter of 109 nm. To observe the structure of these particles compared to the SPLP in the absence of the CPL, freeze- fracture EM was performed
• the serum stability of the SPLP in the presence and absence of various amounts of the CPL was assayed (data not shown). Incubating free DNA with 50% mouse serum for only 1 hour results in its complete degradation. The serum stability of the CPL-SPLPs was similar to that for the SPLP system. This indicates that the DNA in the CPL-SPLP is as protected as that in the SPLP system without CPL.
• Figure 21 shows the time course for the uptake of rhodamine labeled SPLP in the presence (2, 3, or 4 mol %) and absence of the DSPE-Quad5 (0%).
• the uptake of the 4% system is higher than the 3% system, which is higher than the 2% system, and all three are much higher than the system without CPL.
• Fig. 22 shows 4 h and 9 h time points of the same formulations.
• Distearoylphosphatidylcholine was purchased from Northern Lipids (Vancouver, Canada).
• DODAP or AL-1 l,2-dioleoyloxy-3-dimethylammoniumpropane
• Cholesterol was purchased from Sigma Chemical Company (St. Louis, Missouri, USA).
• PEG-ceramides were synthesized by Dr. Zhao Wang at Inex Pharmaceuticals Co ⁇ . using procedures described in PCT WO 96/40964, inco ⁇ orated herein by reference. [ 3 H] or [ 14 C]-CHE was purchased from NEN (Boston, Massachusetts, USA).
• lipids were > 99% pure. Ethanol (95%), methanol, chloroform, citric acid, HEPES and NaCI were all purchased from commercial suppliers. Lipid stock solutions were prepared in 95% ethanol at 20 mg/mL (PEG- Ceramides were prepared at 50 mg/mL).
• SALPs are first prepared according to the methods set out in PCT Patent Application No. WO 98/51278, published 19 November 1998, and inco ⁇ orated herein by reference. See also, J.J. Wheeler et al, (1999), Gene Therapy, 6, 271-281. Briefly, a l ⁇ mer of [3H]-phosphorofhioate oligodeoxynucleotide Inx-6295 (human c-myc) having sequence 5' T AAC GTT GAG GGG CAT 3' (SEQ ID.
• the antisense-lipid mixture was subjected to 5 cycles of freezing (liquid nitrogen) and thawing (65°C), and subsequently was passed 10X through three stacked 100 nm filters (Poretics) using a pressurized extruder apparatus with a thermobarrel attachment (Lipex Biomembranes). The temperature and pressure during extrusion were 65°C and 300-400 psi (nitrogen), respectively.
• the extruded preparation was diluted with 1.0 mL of 300 mM citric acid, pH 3.8, reducing the ethanol content to 20%.
• the extruded sample was dialyzed (12 000- 14 000 MW cutoff; SpectraPor) against several liters of 300 mM citrate buffer, pH 3.8 for 3-4 hours to remove the excess ethanol.
• the sample was subsequently dialyzed against HEPES-buffered saline (HBS), pH 7.5, for 12-18 hours to neutralize the DODAP and release any antisense that was associated with the surface of the vesicles.
• Encapsulation was assessed either by analyzing the pre-column and post-column ratios of [ ⁇ ]-antisense and [ 14 C]-lipid or by determining the total pre-column and post-column [ ⁇ ]-antisense and [ l4 C]-lipid radioactivity.
• distal positively charged cationic poly(ethylene glycol) lipid conjugates were synthesized and assessed for their efficacy at enhancing the cellular uptake of CPL-inco ⁇ orated liposomes. It was confirmed that distal charged polymer conjugates bound to a liposome surface enhanced liposome uptake in mammalian cells in vitro.
• CMC critical micelle concentration
• CPLi Compared to a control, reduced cell uptake was observed for CPLi, a moderate increase for CPL 2 (2 fold), and a large increase for both CPL 4 and CPL 8 .
• the similar degree of increase resulting from CPL and CPL 8 indicates a charge density of four in the CPLs satisfies the requirement for maximum enhanced cellular uptake.
• DSPC l,2-Distearoyl-sn-glycero-3- phosphocholine
• DSPE l,2-distearoyl-sn-glycero-3-phosphoethanolamine
• the phosphate concentration of the CPL was determined using the Fiske- Subarrow phosphorus assay (see, Fiske, C.H., and Subbarow, Y. (1995) The colorimetric determination of phosphorous. J. Biol. Chem. 66, 375-400.).
• the primary amine concentration in the CPL was determined using the fluorophore, fluorescamine.
• a fluorescamine solution (0.6mg/mL) in acetone was prepared.
• An aliquot of CPL solution in FIBS (2-4 ⁇ L) was made up to 250 ⁇ L with 200 mM sodium borate, pH 8.0. To this mixture, 50 L of the fluorescamine solution was added dropwise with vortexing, followed by 1700 ⁇ L of water.
• tBoc-NH-PEG 340 o-CO 2 -(N ⁇ - dansyl)iysine-NHS (2) was prepared as follows. A solution of tBoc-NH-PEG 34 oo-CO 2 - (N ⁇ -dansyl)lysine (1) (500 mg, 132 ⁇ mol) and NHS (31.5 mg, 274 ⁇ mol) in 2 mL of dry chloroform was added to DCC (42.8 mg, 207 ⁇ mol) dissolved in 1 mL of dry chloroform. The reaction mixture was stirred for 2 h at room temperature.
• DSPE 120.6 mg, 161 ⁇ mol
• Dansylated CPLi (4) Trifluoroacetic acid (TFA) (2 mL) was added to a solution of dansylated CPLi-tBoc (3) (550 mg, 121 ⁇ mol) in 2 mL of chloroform and stirred for 4 h at room temperature. The solution was concentrated to a thick paste and chloroform/ether washed three times. After the removal of ether, the solid was dissolved in 6 mL of chloroform/methanol (2:1) and washed with 1.2 mL of 5% sodium bicarbonate. The chloroform phase was extracted, dried and redissolved in 6 mL chloroform/methanol (2:1) and washed with 1.2 mL distilled water.
• TFA Trifluoroacetic acid
• Dansylated CPL 2 -tBoc (5) A solution of N ⁇ ,N ⁇ -di-tBoc-L-lysine-N- hydroxysuccinimide ester (105 mg, 236 ⁇ mol) in 2 mL dry chloroform was gradually added to a solution of dansylated CPLi (4) (510 mg, 112 ⁇ mol) in 2 mL chloroform containing 200 ⁇ L triethylamine and stirred at room temperature for 3 h. The completion of the reaction was indicated by the disappearance of primary amine as visualized by ninhydrin assay on TLC.
• the reaction mixture was concentrated to a thick paste and chloroform/ether washed ( ⁇ 3 times) until the disappearance of excess N ⁇ ,N ⁇ -di-tBoc-L- lysine-N-hydroxysuccinimide ester as checked by TLC.
• the product was dissolved in 6 mL chloroform/methanol (2:1) and washed with 1.2 mL 0.1 M HCl.
• the chloroform phase was extracted, dried, redissolved in 6 mL chloroform/methanol (2: 1) and washed with 1.2 mL distilled water.
• the chloroform phase was concentrated to a thick paste and the purified compound was obtained through a chloroform/ether wash and vacuum dried. Yield: 510 mg (96%).
• CPL -tBoc (7) was the same as that of CPL 2 -tBoc (5) by reacting N ⁇ ,N ⁇ -di-tBoc-L-lysine-N-hydroxysuccinimide ester (170 mg, 383 ⁇ mol) with dansylated CPL 2 (6) (455 mg, 95 ⁇ mol). Yield: 475 mg (96%).
• TLC sica gel
• chloroform methanol 85: 15
• CPL 8 -tBoc (9) was the same as that of CPL 2 -tBoc (5) by reacting N ⁇ ,N ⁇ -di-tBoc-L-lysine-N-hydroxysuccinimide ester (70 mg, 158 ⁇ mol) with dansylated CPL 4 (8) (100 mg, 19 ⁇ mol). Yield: 112 mg (96%).
• TLC sica gel chloro form/methanol (85:15) R/0.58. ⁇ NMR (CDCI 3 ).
• the CPL were synthesized by repeated coupling reaction steps involving amines and NHS-activated carbonate groups as outlined in Figure 29. This consists of (a) inco ⁇ orating the dansyl fluorescent label to the hydrophilic PEG spacer, (b) coupling of the DSPE anchor, and (c) attachment of the cationic headgroup to the lipid.
• the heterobifunctional PEG polymer tBoc-NH-PEG 340 o-CO 2 -NHS (MW 3400) was chosen for two reasons. Firstly, it was commercially available. Secondly, it is insoluble in ether that provided a very convenient means of purifying its derivatives, 1 - 10. Other reagents were used in excess to ensure the complete conversion of the PEG polymer to its derivatives. The excess reagents were soluble in ether and therefore could be removed by washing in ether during purification.
• Inco ⁇ oration of the fluorescent label, N ⁇ -dansyl lysine, to the PEG polymer by coupling the ⁇ -amino group of dansyl lysine with the NHS activated carbonate of PEG gave the lysine derivative 1.
• the DSPE anchor was coupled via intermediate 2 that was formed by the esterification of 1 using NHS and DCC.
• the resulting PEG lipid, 3, was deprotected by removing the tBoc to form CPLi, 4, with one positive charge.
• the positive charges in the other CPL are carried by the amino groups of lysine.
• the 1H NMR spectra showed well-resolved resonances for the PEG, tBoc and acyl chains of DSPE at approximately 3.61, 1.41 and 1.21 ppm, respectively, and for the resonances of the dansyl moiety (aromatic protons at 7.1-8.5 ppm; methyl protons at 2.8-3.0 ppm). From the integrated signal intensities of the former three peaks, it was found that the ratio of tBoc/PEG or tBoc/DSPE was 1.0, 2.1, 4.0, and 8.1 for CPLi-tBoc, CPL 2 -tBoc, CPL 4 -tBoc, and CPL 8 - tBoc, respectively.
• the CPL described here possess several attributes which may increase their usefulness relative to other cationic lipids. Firstly, the phospholipid anchor will readily allow efficient inco ⁇ oration of CPL into liposomal systems. Secondly, the dansyl label will permit accurate and convenient quantification of the CPL in the bilayer using fluorescence techniques. Finally, the valency of the cationic headgroup in the CPL can easily be modified using lysine residues.
• tBoc tert-butyloxycarbonyl
• tBoc-NH-PEG ⁇ ooo-CO 2 -NHS tBoc protected and NHS activated PEG 1 000
• CPL cationic poly(ethylene glycol) lipid conjugate
• CPLi CPL with one positive charge
• CPL 2 CPL with two positive charges
• CPL 4 CPL with four positive charges
• DCC N,N'-dicyclohexyl-carbodiimide
• DCU dicyclohexyl urea
• NHS N-hydroxysuccinimide
• DSPE l,2-distearoyl-sn-glycero-3-phosphoethanolamine
• PEGiooo poly( ethylene glycol) with an average
• Fluorescamine was obtained from Molecular Probes (Eugene,OR). Trifluoroacetic acid, diethyl ether, methanol, triethylamine, and chloroform were obtained from Fisher Scientific (Vancouver, BC). All other reagents were used without further purification.
• the filtrate containing tBoc-NH-PEG ⁇ ooo-CO 2 -(N ⁇ -dansyl)lysine-NHS (2), was slowly added to a solution of DSPE (365 mg, 488 ⁇ mol) in 3 mL of dry chloroform and 300 ⁇ L of triethylamine.
• DSPE 365 mg, 488 ⁇ mol
• the dissolution of DSPE in dry chloro form, and triethylamine required warming to 65 °C.
• the reaction mixture was stirred overnight at room temperature, it was filtered to remove some precipitate (unreacted DSPE) and dried to a viscous paste.
• the paste was dissolved in chloroform methanol (2: 1), washed with dilute HCl and water as before.
• Dansylated CPL 2 -tBoc (5) A solution of N ⁇ ,N ⁇ -di-tBoc-L-lysine-N- hydroxysuccinimide ester (350 mg, 789 ⁇ mol) in 3 mL dry chloroform was gradually added to a solution of dansylated CPLi (4) (750 mg, 400 ⁇ mol) in 3 mL chloroform containing 300 ⁇ L triethylamine and stirred at room temperature for 3 h. The completion of the reaction was indicated by the disappearance of primary amine as visualized by ninhydrin assay on TLC.
• CPL -tBoc (7) was the same as that of CPL 2 -tBoc (5) by reacting N ⁇ ,N ⁇ -di-tBoc-L-lysine-N-hydroxysuccinimide ester (500 mg, 1127 ⁇ mol) with dansylated CPL 2 (6) (650 mg, 292 ⁇ mol). Besides washing with dilute HCL and water no further attempts were made to purify CPL -tBoc before deblocking to generate CPL 4 . Yield: 800 mg (Crude). TLC (silica gel) chloroform /methanol (85:15) ⁇ .58 (dansyl peak only).
• CPL (8) Dansylated CPL (8).
• the synthesis of CPL (8) was the same as that of CPLi (4) by deprotecting dansylated CPL -tBoc (7) (800 mg).
• the final product was purified by column chromatography using silica gel 60, 70 - 230 mesh, and chloroform methanol ammonia solution (65:25:4 v/v). Yield: 300 mg (38%).
• TLC sica
• Phospholipid Assay Phospholipid was determined by first extracting the lipids from SPLP using the Bligh-Dyer technique, and then measuring phosphate in the organic phase according to the Fiske-Subbarow method (see, Bligh EG, Dyer WJ A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959; 37: 91 1-917; and Fiske CH, Subbarow Y. The colorimetric determination of phosphorous. J Biol Chem 1925; 66: 375-400.).
• DNA Assay DNA content was measured using the PicoGreen Assay kit
• the serum stability of the SPLP-CPL containing various % of CPL were determined by mixing the particles with mouse serum to a final serum concentration of 50% v . These mixtures were then incubated for 0, 1, 2, or 4 hours at 37°C. At these time points, a volume of the mixture containing about 1 ⁇ g of plasmid DNA was removed and the DNA was extracted from the lipid and protein using a phenohchloroform extraction. The resulting DNA solutions were then run on a 1% agarose gel following which the DNA was transferred to nitrocellulose and a Southern blot was performed.
• Spectrophotometer using a ⁇ ex of 560 nm and a ⁇ em of 600 nm with slit widths of 10 and 20 nm, respectively. An emission filter of 430 nm was also used. Lipid uptake was determined by comparison of the fluorescence in the lysate to that of a lipid standard and normalized to the amount of cells as determined by the BCA protein assay (Pierce, Rockford, IL).
• SPLP-CPL4 exhibit enhanced uptake into BHK cells and dramatically enhanced transfection potency.
• the next set of experiments was aimed at determining the influence of inco ⁇ orated CPL on the uptake of SPLP into BHK cells and the resulting transfection potency of the SPLP-CPL system.
• SPLP containing up to 4 mol % CPL were prepared in the presence of 40 mM CaCl 2 and were added to BHK cells (final CaCl 2 concentration 8 mM) and incubated for varying times. The cells were then assayed for associated SPLP-CPL as indicated in Methods.
• the transfection properties of SPLP, SPLP-CPL 4 and plasmid DNA- cationic lipid complexes were examined using the incubation protocol usually employed for complexes. This consisted of incubation of 10 4 BHK cells with SPLP, SPLP-CPL 4 and complexes containing 0.5 ⁇ g pCMVLuc for 4 h, followed by removal of SPLP, SPLP-CPL 4 or complexes that are not associated with the cells, replacement of the media, incubation for a further 20 h and then assaying for luciferase activity.
• the SPLP-CPL 4 preparations contained 7 mM CaCl 2 in the incubation medium. As shown in Figure 33, the presence of the CPL 4 resulted in dramatic increases in the transfection potencies of the SPLP system. SPLP-CPL 4 containing 4 mol % CPL 4 exhibited luciferase expression levels some 3xl0 3 higher than achieved with SPLP. (.see.Mok et al, Biochim Biophys Acta, 1419:137-150 (1999)).
• SPLP-CPL4 Uptake of SPLP-CPL into BHK cells was monitored following a 4 h incubation in the presence of 0-14 mM MgCl 2 and CaCl 2 . As shown in Figure 35 the amount of SPLP-CPL 4 taken up by BHK cells is the same for both Mg 2+ and Ca 2+ - containing media. The uptake of the SPLP-CPL 4 decreases as the concentration of divalent cations increases, which likely arises due to shielding of the negatively charged binding sites for the CPL 4 on the surface of the BHK cells. 5. SPLP-CPL4 exhibit transfection potencies in vitro that are comparable to or greater than achieved using complexes.
• DOPE:DODAC (1:1) complexes 1.5:1, c.r.
• DOPE/DOTMA [1 : 1] complexes 1.5:1 c.r.
• the transfection potency of the SPLP-CPL4 increases markedly with increased incubation times, suggesting that a limiting factor for transfection achieved at a 4 h incubation time was the rate of uptake of the SPLP-CPL system.
• transfection levels are achieved that are comparable to those achieved by Lipofectin or DOPE/DODAC complexes.
• SPLP-CPL4 are non-toxic and efficient transfection agents.
• SPLP-CPL contains low levels of cationic lipid and are potentially less toxic than complexes.
• the toxicity of SPLP-CPL4 and complexes was assayed by determining cell viability following a 48 h exposure to levels of SPLP-CPL and complexes corresponding to 0.5 ⁇ g plasmid and -30 nmol total lipid. As shown in Figure 37B, SPLP-CPL 4 exhibit little if any toxicity. Cell survival was only 30% after a 48 h incubation with Lipofectin complexes whereas -95% of the cells were viable following a 48 hour incubation with SPLP-CPL 4 .
• the efficiency of transfection is also an important parameter.
• the proportion of cells transfected were estimated using plasmid carrying the green fluorescent protein (GFP) gene. Transfection was detected by expression of the fluorescent protein inside a cell employing fluorescence microscopy. As shown in Figure 37A and 38B, approximately 35% of the cells at 24 h and 50% at 48 h were transfected by SPLP-CPL 4 , with no apparent cell death. In contrast, Lipofectin complexes exhibit maximum transfection efficiencies of less than 35% and only -50% cell survival after the 24 h transfection period. Similar low transfection efficiencies and high toxicities were also seen with DOPE:DODAC complexes.
• CPL 4 increases the transfection potency of the SPLP system.
• the presence of the CPL increases the uptake of SPLP into the BHK cells, however the increase in transfection potency is almost entirely dependent on the additional presence of Ca 2+ . It may be noted that, following an 8 h incubation, the presence of 4 mol % CPL increases the uptake of SPLP into BHK cells by approximately 50-fold, whereas the transfection potency (in the presence of Ca 2+ ) is increased by a factor of -10 4 .
• SPLP-CPL 4 are markedly less toxic to BHK cells in tissue culture. This is presumably related to the low proportions of cationic lipid contained in SPLP as compared to complexes.
• the transfection potency and efficiency of SPLP-CPL 4 is clearly comparable to the levels that can be achieved with complexes. It should be noted that this finding suggests that models of transfection by complexes that involve.
• the superiority of SPLP-CPL 4 compared to commercially available complex systems e.g. Lipofectin
• the size and serum stability of the SPLP-CPL 4 compared to complexes are important parameters for effective gene delivery systems, especially if one wishes to approach the capabilities of viral systems.
• the SPLP-CPL 4 have been shown here to be of relative small size (-100 nm) compared to complexes, which are frequently on the order of microns in diameter. The small size should allow for accumulation at sites with larger fenestration (e.g. tumors, and inflammation sites), (see, Kohn et al. Lab Invest; 67:596-607 (1992)).
• DNA in the SPLP-CPL 4 was shown to be protected from the external environment (t.e. inaccessible to degradation by DNase within serum), whereas DNA in complexes is susceptible to DNase. (see, Wheeler et al, Gene Therapy; 6:271-281 (1999)).
• This example shows transfection rates of BHK cells by long- versus short- chained CPLs.
• CPL (PEG 3.4k) and CPL(PEG Ik) were generated and each inserted into a separate SPLP system containing PEG- 2 ooo-Cer C20 as described above.
• Figure 38 illustrates transfection rates of the CPLs having a PEG 3.4k versus a CPL having a PEG Ik.
• the short-chained PEG in the CPL results in a decrease by a factor of about 4 compared to the transfection by the long chained CPL.
• the long chain CPL (PEG 4 oo) sticks out above the surface, whereas the short chain CPL (PEGiooo) is buried (masked) in the surface of the SPLP.
• the reduced in vitro transfection of the short chain CPL clearly suggests that it has improved in vivo circulation.
• This in vivo example discloses pharmacokinetics and biodistribution of CPL 4 -l-k LUNs (SPLPs containing short chain CPLs) in C57/bl6 mice.
• SPLPs containing short chain CPLs Different SPLP formulations containing increasing amounts of CPL-4-lk are assayed in vivo to determine optimal clearance characteristics.
• mice were treated with 3 [H]CHE-LUV administered by tail vein IN. in a total volume of 200 ⁇ l . Mice receive one treatment only. At the indicated time-points mice are weighed, sacrificed, and blood will be collected by cardiac puncture then evaluated for 3 [H]CHE. Formulations are expected to be well tolerated. Mice are treated according to certified animal care protocols. Any mice exhibiting signs of distress associated with the treatment are terminated at the discretion of vivarium staff. All mice are terminated by CO 2 inhalation followed by cervical dislocation. Measurement of 3 [H]CHE from blood is determined according to standard protocols. In vivo pharmacokinetics of SPLP containing short chain CPL 4 are illustrated in Figure 40.

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