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Patent US6143321 - Liposomes containing active agents - Google PatentsSearch Images Maps Play YouTube News Gmail Drive More »Sign in<nobr>Advanced Patent Search</nobr>PatentsLiposomes formulations are designed to maximize the amount of poorly water-soluble active agent that can be carried by the liposome. Liposomes containing active agent in the lipid bilayer of the liposome, and/or entrapped within the liposome interior space in a micellar preparation are described. Methods...http://www.google.com/patents/US6143321?utm_source=gb-gplus-sharePatent US6143321 - Liposomes containing active agentsAdvanced Patent SearchPublication numberUS6143321 APublication typeGrantApplication numberUS 09/174,775Publication dateNov 7, 2000Filing dateOct 19, 1998Priority dateFeb 6, 1997Fee statusPaidAlso published asUS5827533, US5882679, US6296870, WO1998034597A1Publication number09174775, 174775, US 6143321 A, US 6143321A, US-A-6143321, US6143321 A, US6143321AInventorsDavid Needham, Ranjit S. SarpalOriginal AssigneeDuke UniversityExport CitationBiBTeX, EndNote, RefManPatent Citations (6), Non-Patent Citations (5), Referenced by (27), Classifications (12), Legal Events (3) External Links: USPTO, USPTO Assignment, EspacenetLiposomes containing active agentsUS 6143321 AAbstract Liposomes formulations are designed to maximize the amount of poorly water-soluble active agent that can be carried by the liposome. Liposomes containing active agent in the lipid bilayer of the liposome, and/or entrapped within the liposome interior space in a micellar preparation are described. Methods of making such liposomes are presented.
That which is claimed is: 1. A liposome containing an active agent, said liposome comprising:(a) a lipid unilaminar bilayer comprising phospholipid, lipid surfactant with a Critical Micelle Concentration (CMC) of about 1 millimolar or less, and from 3 mole percent to 60 mole percent of cholesterol; and (b) a micellar preparation comprising active agent aggregated with the lipid surfactant to form micelles, said micelles entrapped within the interior space of the liposome;and said cholesterol is contained in the lipid bilayer in an amount sufficient to increase the stability of the micelle-containing liposome compared to that which would occur in the absence of cholesterol. 2. A liposome according to claim 1, wherein said lipid bilayer further comprises active agent associated with the lipid bilayer.
12. A liposome containing paclitaxel, said liposome comprising:(a) a lipid unilaminar bilayer comprising phospholipid, lysophosphatidylcholine, and cholesterol in a mole ratio of about 8:4:3, and (b) a micellar preparation comprising paclitaxel aggregated with lysophosphatidylcholine micelles, said micelles entrapped within the interior space of the liposome. 13. A liposome according to claim 12, further comprising paclitaxel associated with the lipid bilayer.
RELATED APPLICATIONS This application is a divisional of Ser. No. 09/017,984, filed Feb. 3, 1998 which is a Continuation-in-Part of application Ser. No. 08/795,100 filed Feb. 6, 1997, now U.S. Pat. No. 5,827,533, the contents of which are hereby incorporated herein in their entirety.
FIELD OF THE INVENTION The present invention relates to liposomes containing active agents such as therapeutic drugs, where the liposome membrane is formulated to provide a liposome with increased carrying capacity for poorly water-soluble active agents. Alternatively or in addition, the active agent may be contained within, or adsorbed onto, the liposome bilayer. The active agent may be aggregated with a lipid surfactant and carried within the liposome's internal space; the liposome membrane is formulated to resist the disruptive effects of the active agent-surfactant aggregate
BACKGROUND OF THE INVENTION Liposomes consist of at least one lipid bilayer membrane enclosing an aqueous internal compartment. Liposomes may be characterized by membrane type and by size. Small unilamellar vesicles (SUVs) have a single membrane and typically range between 0.02 and 0.05 μm in diameter; large unilamellar vesicles (LUVs) are typically larger than 0.05 μm. Oligolamellar large vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 0.1 μm. Liposomes with several nonconcentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are termed multivesicular vesicles.
SUMMARY OF THE INVENTION It is therefore a general object of the present invention to provide a liposome composition and method for administering a therapeutically effective amount of a compound for an extended period in the bloodstream.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a videomicrograph of a holding pipet holding a test vesicle, and a flow pipet delivering monooleyolphosphatidylcholine (MOPC)-free bathing solution at a controlled flow rate to the exterior of the test vesicle. The vesicle projection length (Lp) inside the holding pipet can be measured.
FIG. 2 graphs the exposure of single SOPC vesicles to a flow of MOPC solution. Filled circles=vesicles with 20 mol % PEG-lipid exposed to 3 μm MOPC; open circles=vesicles with 20 mol % PEG-lipid exposed to 100 μm MOPC; open diamonds=SOPC (vesicles without any PEG-liquid) exposed to 100 μm MOPC; and a filled asterisk indicates vesicle rupture. Without PEG-liquid in the bilayer, as shown by the open diamonds, 100 μM MOPC causes rapid expansion and rupture of the vesicle. With 20 mol % PEG-lipid in the vesicle bilayer, uptake in 100 μM MOPC (open circles) is essentially the same as that observed at the CMC.
FIG. 3 graphs the exposure of SOPC vesicles containing different concentrations (mol %) of PEG(750)-lipid in the vesicle bilayer to a flow of 100 μM MOPC solution; where open diamonds=0.5% PEG; X=1.0% PEG; crosses=2.5% PEG; open boxes=4.0% PEG; inverted triangles (ν)=5.0% PEG; triangles (Δ)=10% PEG; and open circles=20% PEG. Standard deviation shown on the last data point for each concentration are averages of 10 test vesicles.
FIG. 4 graphs the uptake and desorption of lysolecithin (100 μM MOPC) by SOPC vesicles containing varied concentrations (mol %) of PEG(750)-lipid, after more than five minutes' exposure (X=0% PEG; open diamond=1.0% PEG; open triangle=2.5% PEG; open circle =10% PEG; and open square=20% PEG. At 0 mol % PEG(750)-lipid, test vesicles broke after only 50 seconds, and so desorption experiments could not be carried out. Solid lines are theoretical fits to the data.
FIG. 5B graphs the dependence of MOPC uptake into 4 mol % PEG-lipid vesicles as a function of the bathing MOPC concentration, for the concentration range of 0 μMOPC to 14 μM MOPC in the bathing solution. As indicated by an arrow, the cross-over of concentration regimes occurs close to 3 μM (the CMC of MOPC measured by DPH fluorescence).
FIG. 9 is a graph indicating stationary equilibrium uptake of MOPC into SOPC vesicles, for different concentrations of PEG-lipid in the vesicle membrane; the concentration of MOPC in the bulk solution is 100 μM. Standard Deviations are averages of 10 vesicles at each concentration. Open circles=vesicles stable in MOPC solutions and reached equilibrium uptake.
FIG. 10 is a scale drawing of a MOPC micelle at the surface of a PEG-grafted bilayer (PEG-lipid density of approximately 5 mol %), with micelle dimensions (66Å�86Å) calculated as described herein and size of each PEG "mushroom" (RF =19Å) calculated from the Flory radius as described herein.
FIG. 21 shows the calibration cure of TAXOL� in methanol which was used for the quantification of TAXOL�. TAXOL� was dissolved in methanol at a concentration ranging from 5 μM to 30 μM and the absorbance of each solution was measured at 230 nm on a UV/VIS spectrophotometer. Unknown samples containing TAXOL� were quantified against this calibration curve. y=0.002946+0.033378� R2 =0.099865.
FIG. 24 shows the maximum loading capacity of TAXOL� in micelles as MOPC concentration is increased from 0 mM to 20 mM. y=0.051984+0.19657� R2 =0.99928.
FIG. 35 depicts TAXOL� associated with MOPC micelles encapsulated in SOPC/CHOL/MOPC (16/8/6) liposomes, compared to micelle-free liposomes of the same composition. Lipids were presaturated with 1:1 mM TAXOL� and were hydrated with two different micellar-TAXOL� concentrations: 1.5 mM MOPC micelles/0.25 mM TAXOL� and 5 mM MOPC/0.7 mM TAXOL� in PBS.
Liposome preparations have been devised which avoid rapid RES uptake and which thus have an increased half-life in the bloodstream. STEALTH� liposomes (Liposome Technology Inc., Menlo Park, Calif.) include polyethyleneglycol (PEG)--grafted lipids at about 5 mol % in the lipid bilayer. See, e.g., Allen, UCLA Symposium on Molecular and Cellular Biology, 89:405 (1989); Allen et al., Biochim, Biophys. Acta 1066:29 (1991); Klibanov et al., FEBS Letters 268:235 (1990); Needham et al., Biochim. Biophys. Acta 1108:40 (1992); Papahadjopoulos et al., Proc. Natl. Acad. Sci. USA 88:11460 (1991); Wu et al., Cancer Research 53:3765 (1993); Klibanov and Huang, J. Liposome Research 2:321 (1992); Lasic and Martin, Stealth Liposomes, In: Pharmacology and Toxicology, CRC Press, Boca Raton, Fla., (1995). See also U.S. Pat. No. 5,225,212 to Martin et al.; U.S. Pat. No. 5,395,619 to Zalipsky et al; regarding liposomes containing polymer grafted lipids in the vesicle membrane. The presence of polymers on the exterior liposome surface decreases the uptake of liposomes by the organs of the RES.
In liposomes according to the present invention, the lipid bilayer contains a percentage of polymer-grafted lipids sufficient to inhibit micelle/membrane fusion. The percentage of polymer-grafted lipid required to inhibit micelle/membrane fusion will vary depending on the specific polymer used and its molecular weight, and the lipid composition of the micelles contained within the liposome. It will further be apparent to those skilled in the art that the percentage of polymer can be varied to affect the stability of liposomes in vivo, with increasing amounts of polymer used to provide increasing liposome half-life. Thus liposomes according to the present invention, may contain a minimum amount of polymer-grafted lipids sufficient to reduce (but not prevent) fusion of the membrane with micelles contained within the liposome. (e.g., 1 mole % of PEG(750) where micelles are MOPC or other low-CMC surfactants), to an amount sufficient to essentially completely inhibit such micelle/membrane fusion (e.g., ≦20 mol % PEG(750)). The specific proportions of polymer will vary depending on the polymer utilized; the composition contained within the liposome, and the desired stability (or half-life) of the liposome. One skilled in the art will be able to determine desirable proportions using techniques described herein and available in the art.
In the outer hydrocarbon chains, the last three or four carbon atoms are more nearly all-trains, and so are less liquid-like. This region is still fairly hydrophobic but will contain some water. It therefore represents a transitionary region between purely hydrophobic and purely water soluble regions. Molecules that enter this region must be fairly compatible with both the lipid chains and the solvated headgroup region and therefore are likely to be themselves amphipathic; amphiphilic active agents are likely to partition into this region of the micelle. Also, it is envisioned that by attaching a hydrophobic acyl chain to a given active agent molecule, the active agent could be anchored in the micelle as shown in FIG. 17b. The present inventors' studies with TAXOL� indicates that this is the region to which the TAXOL� molecules apparently bind. TAXOL� has a solubility of approximately 25 micromolar and so, although not completely water soluble, it is relatively water insoluble. The association of TAXOL� with a simple MOPC micelle increased the solubility of TAXOL� in direct proportion to the amount of lipid in suspension at a ratio of 1 mole TAXOL� per mole MOPC (data not shown). From spectroscopic absorption studies (data not shown) it appears that TAXOL� is in a similar water-soluble environment to that when it is alone in water solution, as opposed to some fairly hydrophobic internal micelle site. The molecule is in fact not soluble at all in alkanes such as hexane or tetradecane. A slight shift in the frequency of the absorption points to a micelle surface site for solubilization. Other non-polar agents such as Pt-Rhodoamine complexes (known to be cytotoxic to tumors) may also be solubilized at this hydrophobic/hydrophilic boundary for use in chemotherapy.
FIG. 19 shows a schematic of a PEG-lipid micelle (drawn approximately to scale). The hydrodynamic radius measured by dynamic light scattering, for micelles made from lipid-PEG1900 is approximately 85 Å to 100 Å (Needham, (1992)). If the lipid acyl chains and phospholipid headgroup that comprise the core of the micelle take up 25 Å of the total radius, then the polymer extends approximately 60-75 Å out from this core, which is in close agreement with the polymer extension obtained for the same molecule in bilayers from x-ray diffraction experiments. (Needham (1992)). These results indicate an extended, "brush-like" conformation for this non-absorbing, but grafted, polymer.
The micelles utilized in the present invention can be spherical or non-spherical (ellipsoid or even tubular). The association of active agents with the micelle forming surfactants may in some cases transform the shape of the original solubilizing micelle (as discussed below). That these different geometries exist van be appreciated by considering the geometric requirements that aggregation number places on shape. Micelles formed by simple amphiphiles (e.g., C12) are often small with an aggregation number of about 100 (molecules per micelle). Even these micelle are too large to be accommodated within a spherical geometry, given that the radius is limited by the extension length of the hydrocarbon chains. To increase volume with one dimension fixed, the micelles must take up an ellipsoidal shape. This aspect of micellar and other structures is discussed below.
In this treatment, a balance between attractive (hydrophobic--tending to reduce area per molecule) and repulsive (inter-headgroup--tending to increase area per molecule) interactions determines an optimum area per headgroup (ao) at which the total interfacial free energy per molecule (μo N) in an aggregate is a minimum. The energy varies parabolically (and elastically) about a minimum at a certain headgroup area. The most favored structure (micelle, bilayer or inverted micelle) depends on this minimum area, the incompressible volume of the hydrocarbon chains and the maximum effective length (the critical chain length lc) that the hydrocarbon chains can assume.
For lipids of the optimal area ao, hydrocarbon volume v and critical chain length lc, the value of the dimensionless parameter, v/ao lc, will determine whether the lipids assemble into spherical micelles (v/ao lc <1/3); non-spherical micelles (1/3<v/ao lc <1/2); vesicles or bilayers (1/2<v/ao lc <1); or inverted micelles (v/ao lc <1). Each of these structures corresponds to the minimum sized aggregate in which the lipids have minimum free energy, i.e., a-ao.
R=3v/ao so that, only if R≦1c, i.e., v/ao lc ≦1/3 will the amphiphiles pack into a spherical micelle. For example, the parameters for SDS in water are: lc =17 Å, ao =62 Å2, v=350 Å3 and SDS forms a relatively spherical micelle, v/ao lc ≈1/3.
Similarly, non-spherical, micelles exist if the v/ao lc ratio is between 0.33 and 0.5. For MOPC, the parameters are lc =33 Å, ao =44 Å2 ; MOPC forms an elliptical micelles as shown in FIG. 10. These slight changes in molecular shape (projected area of acyl chain and chain length) might also come about if there is a strong association between the MOPC molecule and a relatively rigid hydrophobic, but still amphipathic, active agent. Thus active agent incorporation into a spherical micelle might change it into an ellipse or even a cylinder or disc. An example of mixed micelle is the combination of a small surfactant (high cmc) such as glycocholate, and a larger two chain surfactant such as a phospholipid (very low cmc). At certain ratios these two molecules will combine to form the mixed micelle, in which the high cmc surfactant that prefers to pack as a spherical surface occupies the edges of a low cmc lipid that prefers to be flat.
Even for the "bare" lipid bilayer surface, we expect that there will be some activation energy barrier for the formation of the transition state complex, where the bilayer lipids and micelle lipids mix. Clearly this activation energy barrier must not be too large compared to the available thermal energy (k3 T, 2.5 kJ/mol), since micelles readily fuse with the membrane. FIG. 15 shows a sketch of the transition state complex for a micelle approaching a bare membrane. The micelle is like a small liquid drop, and therefore curvature of the lipid interfaces involved in the transition state complex is expected, and suggest a complex involving the formation of some transitionary bridge or "stalk". The curvature that is required to make such a fusion "stalk" is in the wrong direction for lysolipids to accomodate--their large headgroup/acyl chain area ratio makes them prefer to opposite convex curvature (as in the micelle surface). Thus, for the fusion transition state complex to form, lipids from the bilayer must make the "stalk". Now, as shown herein, the exposure of a bilayer vesicle to a lysolipid solution leads to the rapid uptake of monomer, i.e., even though micelles can be excluded by some mechanism, by the time the micelle reaches the bare membrane, the bilayer will not be simply composed of just the original phospholipids but will also contain approximately 5 mol % MOPC. Before we can discuss how cholesterol may reduce the tendency of a micelle to fuse with the bilayer (i.e., make the formation of a transition state complex more unlikely), the kinds of interactions that cholesterol has with phospholipids (and with their acyl chains in particular) must be considered.
The present invention provides compositions and methods of assembly of lipid and surfactant materials such that normally water-insoluble drugs, such as TAXOL�, can be suspended in aqueous solution at concentrations far in excess of their aqueous solubility limit. In one embodiment, liposomes contain the drug in the liposome membrane facilitated by co-inclusion of lysolipid. The drug and lipid components are co-dissolved in a suitable solvent; after evaporation and hydration in aqueous buffer, liposomes are formed that carry the drug in the liposome membrane. The lipid component solubilizes the drug, which is then contained within (or adsorbed onto) the liposome membrane. An exemplary liposome containing TAXOL� as the active agent carried by the liposome membrane is described herein. Amounts of TAXOL� in such liposomes prepared according to the present invention can reach levels of at least about 10 mole %, 11 mol %, 12 mol %, 13 mol %, 14 mol %, 15 mol %, 16 mol %, 17 mol % or more, relative to SOPC in the membrane.
A further embodiment of the present invention provides liposomes that contain the drug within the liposome interior; the drug is solubilized in a micelle suspension of a suitable surfactant. This surfactant-drug suspension is then encapsulated in a lipid formulation by hydrating a pre-equilibrated lipid film (equilibrated with both drug and surfactant) with the micelle suspension and extruding the mixture to form large unilamellar vesicles (LUVETS) by known extrusion techniques. The present invention utilized a micelle surfactant with sufficiently low critical micelle concentration, and includes cholesterol in the bilayer membranes of the liposomes at sufficient concentration that the micelle suspension does not dissolve the lipid bilayers. The system is stabilized by first equilibrating all phases with surfactant and drug, and by using a particular lipid/cholesterol/surfactant composition that optimizes loading of the target drug by the lipid bilayer membranes. An exemplary liposome loaded with TAXOL� is described in detail herein. Amounts of TAXOL� in such liposomes prepared according to the present invention can reach levels of at least about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, 10 mol % or more, relative to SOPC in the liposome.
The components of liposomes that contain micelles are: (i) membrane bilayer-forming material, usually, but not exclusively, from the class of common membrane phospholipids and cholesterol; (ii) low CMC surfactant, usually but not exclusively a lysolipid, such as monooleoylphosphatidylcholine; (iii) the active agent or drug (an exemplary drug discussed herein is TAXOL�) and (iv) aqueous buffer.
Mutual solubilities exist between the various components of the present drug delivery system. The amount to which one component can partition into the phase formed by another component determines the capacity of the micelle-liposome system to carry drug, and determines the stability of the encapsulated micelle system. This amount depends on the solubility limit that a particular component has in the particular phase in question. The solubility limit is commonly known to those skilled in the art as the maximum solute addition into a solvent phase without super saturation. For example, cholesterol will dissolve into a bilayer phase composed of phospholipid up to a solubility limit of about 60 mol % (Needham and Nunn, Biophys. J. 58:997 (1990)). Thus the bilayer phase is the solvent for the cholesterol solute, up to the point at which cholesterol precipitates in the aqueous phase as a cholesterol rich crystallite. Similarly, lysolipid can dissolve into phospholipid bilayers up to the point at which the phase change from bilayer to micelle occurs. With regard to drug, a given drug might have limited solubility in the aqueous phase, but when associated with lysolipid micelles or phospholipid bilayers, can be co-dissolved into an aqueous phase to much higher concentration, increasing the solubility limit of the drug in aqueous media by orders of magnitude. This is the case for TAXOL�, where its solubility limit in aqueous media is in the micromolar range but, as demonstrated in the following examples, in the presence of 20 mM LPC the solubility limit can be increased to 4 mM (an increase of about 4000 times).
EXAMPLE 1 Materials and Methods Vesicle and Lysolipid Solution Preparation
Lipid vesicles were made from 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC) and 1,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine-N-PEG-750 (DMPE-PEG(750)-lipid) (Avanti Polar Lipids Inc., Alabaster, Ala., USA) using the procedure described by Needham and Zhelev In Vesicles Rosoff (Ed.), Marcel Dekker, New York and Basel (1996). Briefly, chloroform solutions of the particular lipids were mixed to obtain the desired molar ratios. Thirty (30) microliters of a lipid solution were spread onto a roughened TEFLON� (E. I. du Pont de Nemours and Co.) disc and the chloroform evaporated under nitrogen. Final traces of solvent were removed by placing the disc in an evacuated desiccator for 2 hours (Needham and Nunn, Biophys. J. 58:997 (1997)). The so-formed dried lipid layers were prehydrated with water vapor in a nitrogen carrier gas and then fully hydrated overnight in a sucrose solution at 40� C. The osmolarity of the solution was 200 mOsm. Both the pure SOPC and SOPC/PEG(750)-lipid vesicles were re-suspended in a glucose solution of 205 mOsm for use in micromanipulation experiments as described below.
An experimental chamber 3 mm thick and open at both sides was utilized for micromanipulation of vesicles. Experiments were performed at room temperature (23� C.) using an inverted NIKON� microscope with 60� oil immersion objective. The microscope images were recorded using a COHU CCD camera. The micropipets were made of 0.75 capillary glass tubing pulled to a fine point with a vertical pipette puller and cut to the desired diameter with a microforge. The pipettes were connected to a manometer system that allowed the applied pressures to be changed and measured to an accuracy of 2 microatmospheres. The micropipet suction pressures were measured by differential transducer (Validyne DP15-24). The measured pressures together with real time were multiplexed on the recorded images with a multiplexer (Model 401, Vista Electronics, La Mesa, Calif., USA). The recorded images were used to measure the change of the vesicle projection length inside the holding pipet during uptake and desorption of lysolecithin, and to calculate the membrane tension. This was done using calibrated video calipers (Model 305, Vista Electronics).
In the above-described experimental set-up, over time the test vesicle area either reached a maximum or the vesicle broke down. To desorb MOPC from an intact test vesicle, the vesicle was once more exposed to a MOPC-free bathing solution (not shown). Exposure of a MOPC-containing vesicle to lysolipid-free solution resulted in a rapid decrease of membrane area until a minimum area is attained. This minimum final area is similar to, if not the same as, the vesicle area before MOPC uptake, demonstrating (a) essentially complete desorption of lysolipid and (b) reversible lysolipid exchange. The measured projection lengths (Lp), together with the measured pipet radius and the outside vesicle radius, can be used to calculate the original vesicle area (Ao) and the area change due to MOPC uptake (ΔA). As initially the MOPC would only be expected to enter the outer monolayer, the relative fractional area change ΔA/Ao was converted to the total number of lipids in the outer monolayer of the bilayer by using the approximation developed by Zhelev:
ΔA ASOPC AMOPC                                                 (Equation 1)
where ΔA is the change in vesicle area due to MOPC uptake relative to the starting area, ASOPC is the area per lipid molecule of 67 Å2, AMOPC is the area per MOPC molecule of 35 Å2 (Zhelev, Biophys J. 71:257 (1996)).
The critical micelle concentration (CMC) of the lysolipid was measured independently by DPH fluorescence following the method in Chattopadhyay and London, Analytical Biochem. 139:408 (1984). Briefly, a series of solutions containing increasing concentrations of MOPC in the range 10-7 M to 10-4 M were prepared. Diphenyl hexatriene (DPH), a lipid soluble fluorescent dye, was added to these samples and the intensity of fluorescence was measured by spectrofluorimeter at and emission wavelengths of 358 nm and 430 nm, respectively. The CMC was determined by plotting the fluorescence intensity (in arbitrary units) versus concentration of MOPC solutions, made up by diluting a 100 mM stock solution of MOPC (solubilized initially by Tetrahydrofuran) in water (Chattopadhyay and London, Analytical Biochem. 139:408 (1984)).
EXAMPLE 3 Geometric Characteristics of MOPC Micelle The geometric characteristics of MOPC micelles were calculated using simple molecular shape considerations; these considerations were checked against surfactants for which micelle size and shape have been measured. Israelachvili et al., Quar. Rev. Biophys. 13:121 (1980), have shown that the size and shape of lipid aggregates depend on the area of the surfactant headgroup, the length of its hydrocarbon chain, and the chain volume. These molecular characteristics are used to `construct` an MOPC micelle with minimum exposed hydrophobic area. It is assumed that the micelle hydophobic region is incompressible, does not have void space at the center, and one of the dimensions of the micelle hydrophobic core does not exceed twice the length of a lysolipid hydrocarbon chain. Because the lysolipid making up MOPC micelles is a single chain molecule, it is expected that its micelle will be axisymmetric (Bendedouch et al., J. Phys. Chem. 87:153-159 (1983)). As the cross-sectional area of a single hydrocarbon chain (approximately 34 Å2 ; McIntosh et al., Biochemistry 34:8520 (1995)) is smaller than the area per headgroup (approximately 44 Å2 ; Mattai and Shipley, Biochim. Biophys. Acta 859:257 (1986)), the relevant molecular area for the packing of single chain phosphatidylcholines in micelles is the headgroup area.
lmax =(1.5+1.265 (Nh -1)) Å
Vh =(27.4+26.9 (Nh -1)) Å3 Using the above characteristics for dicaproylphosphatidylcholine and diheptanoylphosphatidylcholine, the minimum exposed hydrophobic area for their micelles is found when the micelles are made of 19 monomers and 26 monomers, respectively. The number of monomers per micelle (or the micelle aggregation number) for these two short chain lipids, determined from small angle neutron scattering experiments, is 19 and 27, respectively, (Lin et al., J. Am. Chem. Soc. 109:2321 (1987)). Thus, the aggregation numbers calculated from the geometric approach correspond to the ones measured experimentally, and validate the present approach to calculating geometric characteristics of MOPC micelles.
FIG. 3 graphs the exposure of the SOPC vesicles containing different concentrations (mol %) of PEG(750)-lipid to a flow of 100 μM MOPC solution; open diamonds=0.5% PEG; X=1.0% PEG; crosses=2.5% PEG; open boxes=4.0% PEG; inverted triangles (v)=5.0% PEG; triangles (.increment.)=10% PEG; and open circles=20% PEG. The standard deviation shown on the last data point for each concentration are averages of 10 test vesicles. The data are fitted with an exponential that represents uptake into the outer monolayer of the bilayer Zhelev, Biophys J. 71:257 (1996).
The simplest thermodynamic approach is to assume that there is only one chemical potential in the system, that of the MOPC monomer, i.e., the phospholipid is virtually insoluble and so is not exchanged, at least not initially, although phospholipid exchange with micelles appears to be involved in the ultimate dissolution of the membrane (Evans et al., In Bile Acids in Gasteroenterology Basic and Clinical Advances, Hoffman et al. (Eds.) Kluwer Academic Publishers, Doderecht, Boston and London, pp. 59-68 (1994)). Evans et al. (1994) found that the uptake of bile salts into lipid bilayers followed an ideal mixing relation at thermodynamic equilibrium. The balance of chemical potentials for the monomer in the bulk solution and the membrane (Evans et al., 1994) can be written in a simplified form: ##EQU3## where, Xm is the mole fraction of MOPC in the membrane, Xs is the mole fraction of MOPC in the bulk solution, and the "constants" term contains the reference chemical potentials, and excess energy due to applied membrane tension. The prefactor in this relationship: ##EQU4## is the ratio of monomer aggregate number in the bilayer to the number in aqueous solution so that any value different than 1:1 indicates non-ideality of the system.
EXAMPLE 6 Stationary Equilibrium Model for MOPC Uptake Inhibition by PEG-Lipid At stationary (kinetic) equilibrium, the amount of MOPC partitioning into a lipid membrane is determined by the balance between MOPC taken up as both monomer and micelle, and the desorption of MOPC monomers (Needham and Zhelev, Ann. Biomed. Egr. 23:287 (1995); Zhelev, Biophys J. 71:257 (1996)). The data presented herein establish that the presence of only a few mol % of PEG(750)-lipid in the vesicle membrane inhibits the partitioning of MOPC micelles (up to 500 μM solution concentration) into the membrane while, even up to 20 mol % PEG-lipid does not affect the exchange of MOPC monomers both into and out of the membrane. The PEG-graft acts to selectively sample the aggregate distribution of the surfactant solution itself and thus the data, even below and at the CMC, cannot be fit with a single "on" rate constant as the PEG concentration in the membrane is changed.
While not wishing to be held to a single theory, the present inventors note that the simplest interpretation of this reduced uptake as a function of PEG-lipid density is to treat the uptake as an activation step, i.e., the polymer layer presents an activation energy barrier to close approach of the micelle to the bilayer surface. The presence of grafted PEG(750) creates an apparent "surface pressure" in the region next to the membrane surface. This apparent "surface pressure" restricts the access of MOPC micelles to the membrane surface. As mentioned herein, for MOPC exchange, micelle-membrane fusion is coupled with monomer uptake and desorption. This coupling results in a stationary MOPC concentration in the membrane which is set by the magnitude of the rates of the transport processes. Therefore, for MOPC concentrations below the CMC, or above the CMC, the equilibrium amount of MOPC in the membrane is calculated from the adsorption and desorption rates and appropriate bulk concentrations of the monomer and micelle species (Zhelev, Biophys J 71:257 (1996)): ##EQU6## where Cm is the membrane concentration of MOPC (calculated as a number of molecules per unit membrane area); C'b∞ is the bulk concentration of MOPC for concentrations up to the CMC, i.e., the concentration of monomer; C"b∞ is the bulk MOPC concentration above the CMC, i.e., corresponding to the amount of MOPC in micelles; k'bm is the rate of MOPC monomer uptake, k"bm is the rate MOPC micelle-membrane fusion; and kmb is the rate of MOPC monomer desorption from the bilayer.
The process of micelle-membrane fusion is not well understood, even for unmodified bilayers. In the model used herein, micelle-membrane fusion is considered to be a first order reaction (Glasstone et al., The Theory of Rate Processes McGraw-Hill Co., New York (1941)). It is assumed that MOPC molecules have two quasistationary states: micelle state and bilayer state. The transition from the micelle state into the bilayer state occurs through an apparent "activation state", which is characterized by an apparent barrier energy. When there is grafted PEG(750) on the membrane surface, the barrier energy increases because MOPC micelles must cross the region occupied by the PEG(750) mushrooms. The increase of the barrier energy due to the presence of PEG therefore represents the additional work required to transport the micelles through the mushroom region. This work is calculated by multiplying the area in the mushroom region occupied by the micelles, and the apparent "surface pressure" of the mushrooms for a given surface density. The increase of the barrier energy, due to the work required for micelle transport through the mushroom region gives the correction factor for the apparent decrease of the rate of micelle membrane fusion as compared to membranes without PEG(750). In this case, the rate of micelle-membrane fusion in the presence of PEG(750) k"bm(PEG) is equal to the product of the same rate for membranes without PEG(750) k"bm0 and the exponential correction factor: ##EQU7## where nPEG is the PEG(750) molar concentration in the membrane; al is the area per molecule of the bilayer lipid; and am is the cross sectional area of the micelle projected at the surface that corresponds to the point of micelle-membrane fusion (see FIG. 11A).
Fixed Mol % PEG-Lipid (4 Mol %); Variable MOPC Bulk Concentration (0.025 μM-500 μM)
EXAMPLE 8 Encapsulation of Fluorescent Micelles in Lipid Vesicles The following experiment demonstrated that concentrations of micelles at 100 microMolar or more could be stably encapsulated inside lipid vesicles, and that the vesicles are in fact stable until acted upon by a force that breaks the vesicle and releases the micellar contents.
Two different dyes were used: 2-(4,4-difluoro-5-(4-phenyl-1.3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (abbreviated to β BODIPY-lipid); and 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine) (abbreviated to (NBD-PC(16)).
This trial used SOPC+20 mol % PEG(2000)-lipid (0.2 mg/ml, 2�10-4 M total lipid) with 100 micromolar LPC. This combination did not form giant lipid vesicles but formed small 1 micrometer sized vesicles; the vesicles were stabilized against LPC disruption (results not shown) but the micropipet disruption test could not be carried out because the vesicles were too small. Although a fluorescence test was not carried out on this preparation, one would not expect to have been able to distinguish fluorescence in the membrane (edge fluorescence) versus the internal bulk volume (homogeneous bulk fluorescence) because the size of the liposomes approached the wavelength of light. Nevertheless, liposomes were prepared with this formulation, and the natural tendency of this formulation to favor small vesicles may be useful where vesicles of small diameter and containing micellar preparations are desired, for example for intravenous drug delivery. Such formulations can be used to provide extruded unilamellar vesicles of 200 nm diameter containing micelles.
EXAMPLE 9 Quantification and Preparation of Drug-Associated Micelles When formulating a drug into a micelle-liposome preparation according to the present invention, the solubility limit of the drug in the micelle phase must be determined. The present example utilized TAXOL�, a drug with poor water solubility (about 1 micromolar).
The concentration of TAXOL� in aqueous solution or aqueous micelle suspension was detected and measured using spectrophotometric absorbance at a wavelength of 230 nm (FIG. 21). The concentration of TAXOL� in a solution or suspension could be measured in the range of 5-50 micromolar. For concentrations higher than 50 micromolar, samples were diluted in methanol and the original concentrations were back-calculated.
Samples were prepared containing the required aliquot of TAXOL� in methanol and MOPC (a micelle forming surfactant) in chloroform. These components were mixed together in glass culture tubes, and the mixture was dried and kept under vacuum for approximately 15 hours to evaporate the solvent. MOPC was chosen because it has an acyl chain composition that matches the di-chain phospholipid, stearoyloleoylphosphatidylcholine, that was used to form the lipid bilayers of encapsulating liposomes, as discussed below. Additionally, MOPC has a Critical Micelle Concentration (CMC) in the micromolar range; we have found that for bilayers to be stable in the presence of micelles, the micelle-forming surfactant must have a low CMC. Surfactants with high CMCs (e.g., in the mM range) readily dissolve lipid membranes, even when the membranes contain cholesterol.
The dried MOPC/TAXOL� film was then hydrated with buffer (10 mM phosphate buffer in 0.8% saline at pH 7.4), centrifuged at 2000 rpm for ten minutes to spin down any undissolved TAXOL� (depending on the ratio of MOPC to TAXOL�, the drug can precipitate out as small crystals detectable with an optical light microscope). The remaining supernatant is then dissolved in methanol and assayed for TAXOL� content. TAXOL� was quantified against the calibration curve of TAXOL� in methanol (FIG. 21). The graph of FIG. 22 represents the maximum amount of TAXOL� in aqueous micelle suspension as a function of time after preparation.
Data for uptake of TAXOL� into MOPC micelle suspensions is shown in FIG. 22. It was found that TAXOL� can be solubilized up to 1 mM in 5 mM MOPC, compared to its natural solubility in water of about 1 micromolar. For 0.2 mM TAXOL�, the TAXOL� is stable in the micelle suspension for at least 24 hours. With increasing amounts of TAXOL� in the suspension, the TAXOL� has a tendency to precipitate from the micelles and form crystals in the aqueous phase. This precipitation, however, does not occur significantly for the first four hours, but then proceeds rapidly as higher amounts of TAXOL� are solubilized. At 1 mM in micelle suspension, the TAXOL� is 1000 times its natural solubility limit, and thus is extremely oversaturated. When TAXOL� crystals are separated from the micelle suspension, our analysis shows that they do not contain any MOPC, suggesting that TAXOL� precipitates alone and partitions from the micelle suspension into its own crystallites. TAXOL� thus has a stronger interaction with itself than with the solubilizing micelle. Therefore, in preparing TAXOL� formulations (and possibly other drug formulations) it is necessary to monitor the amount of drug associated with the micelles as a function of time after rehydration. In the present case, TAXOL� should be formulated into its final encapsulated form within four hours. Once encapsulated, any precipitation of drug into the aqueous phase would remain trapped inside the lipid capsule, and thus retained in the drug delivery system.
TAXOL� was found to be largely insoluble in hexane. The absorbance spectrum of TAXOL� in water, ethanol, and the LPC micelle suspension were similar to each other and all were different than that in hexane. From these experiments, we determined that TAXOL� is not dissolved in the largely hydrophobic (oily) micelle interior, but rather is associated with the surface of the micelle, probably intercalating into the headgroup and outer chain regions. Those skilled in the art will appreciate that each drug will have its own specific interaction with micelles and may be solubilized by the micelle in any one, or combinations, of the four regions of the micelle as depicted in FIG. 23.
To evaluate how much TAXOL� can be solubilized by MOPC micelles as a function of MOPC concentration, we measured the amount of TAXOL� solubilized by MOPC micelles as the concentration of MOPC was increased from 0 to 20 mM. FIG. 24 shows that the amount of TAXOL� solubilized increases linearly with MOPC concentration at a rate of 1 mole of TAXOL� per 5 moles MOPC. It is expected that TAXOL� will keep pace with MOPC concentration, possibly up to the solubility limit of MOPC which is in the molar range.
Sample preparations of TAXOL� solubilized by MOPC micelles can be summarized as follows: place required aliquots from stocks of TAXOL� and MOPC in a culture tube; evaporate solvents by sparging nitrogen; keep culture tubes overnight under vacuum in a desiccator; hydrate TAXOL�/MOPC films with PBS and vortex for five minutes; centrifuge at 2000 rpm for 10 minutes; measure TAXOL� in the supernatant against calibration curve.
EXAMPLE 10 Quantification of Drug Loading into Bilayer Drug may also partition into or be adsorbed at the interface of a bilayer membrane. The present Example quantified the amount of drug partitioning into an otherwise empty liposome (i.e., that partitions into the liposome membrane and internal aqueous space). Experiments were carried out where MOPC and cholesterol were systematically varied in 16 mM SOPC with one component variation at a time.
The desired components were dissolved in chloroform and the solvent was dried off in a round bottom flask using rotavapor by heating the flask at about 50 degree C, under vacuum. Flasks were then evacuated for 15 hours to remove the last traces of solvent. The lipid/drug mixture was then hydrated with PBS buffer by gently etching the dried lipid films with a TEFLON� bead, and a liposome suspension was made and processed within two hours according to the method developed by Hope et al., 812:55 Biochem Biophys Acta (1985). This preparation method produces Large Unilamellar Vesicles by Extrusion Technique (LUVETs) of a desired size, in this case 200 nm.
The liposomes were then separated from the unencapsulated TAXOL� by size exclusion chromatography (SEC). SEPHAROSE� CL-4B gel was used to make 5 cm long and 1 cm wide SEC mini columns. The gel was added to the columns and the columns were centrifuged at 2800 rpm for ten minutes. These minicolumns were pre-equilibrated with empty liposomes of the same concentration to avoid the loss of any lipids on to the columns when samples having TAXOL� are passed through the columns. An aliquot of liposome sample is added carefully and gently in the center of the column. The sample containing columns were further centrifuged at 2900 rpm for ten minutes. The cleaned liposome fraction eluted out of the column was assayed for TAXOL� content in all cases by dissolving an aliquot of lipid/TAXOL� mixture in methanol and using the spectrophotometric assay (as described above; at 230 nm). In some cases, the cleansed liposomes were also evaluated for lipid content using Bartlett's phosphate assay. There was very little loss of lipid onto the SEC column when the columns were pre-conditioned with empty liposomes.
Using a fixed SOPC/MOPC mole ratio of 16/4 (as mM aqueous concentration), we measured the amount of TAXOL� associated with liposomes as made, and during processing, and compared these numbers to the initial amount of TAXOL� mixed with the lipid. Samples were formed by mixing all the components together in chloroform and evaporating the solvent. The lipid films were hydrated with PBS and liposomes produced. At this lipid concentration (16 mM) the liposomes held about 1.7 mM of TAXOL� after being processed through extrusion and cleaned through SEC columns.
FIG. 25 shows that the initial measured amount of TAXOL� solubilized in the liposome decreases due to losses during processing. It was found that TAXOL� could be solubilized in the liposome preparation before extrusion up to 3.5 mM. Beyond this point the suspension was not homogeneous and insoluble material existed. The maximum amount of TAXOL� taken up in this preparation (which must be largely taken up by the membrane, since water solubility of TAXOL� is about 1 micromolar) was about 1.7 mM. This was observed after cleaning liposomes using SEC columns, and for a phospholipid concentration of 16 mM, i.e., a mole ratio of about 0.11 TAXOL�/phospholipid, which represents one TAXOL� molecule per eleven lipid molecules.
This experiment demonstrates that the liposome bilayer is a significant sink for, and therefor potential transporter of, TAXOL�. This recognition that there is a tendency for TAXOL� to partition into the bilayer phase allows the preparation of a liposome system that carries additional amounts of TAXOL�, by pre-loading the bilayers with TAXOL� at the maximum mole ratio prior to the encapsulation of a drug/micelle phase. The experiment also demonstrates that TAXOL� can be lost from the liposome preparation onto filters and columns. However, clinical formulations of such drug delivery liposomes may not require cleaning, and thus may retain more TAXOL� than cleaned liposomes.
This same experiment was carried out for several compositions: 16/0, 16/2, 16/4, and 16/6 SOPC/MOPC. The maximum uptake values after processing (extrusion and cleaning via SEC column) are summarized in FIG. 26. The data show that for pure phospholipid (that is, in the absence of MOPC), only 0.5 mM TAXOL� is retained in the liposome (3 mol % TAXOL� in the phospholipid bilayers). However, by adding MOPC in the bilayer, much more TAXOL� is solubilized in the bilayer phase. At a composition of 16/4 (SOPC/MOPC), 1.7 mM TAXOL� (i.e., 11 mol %) can be solubilized. This amount exceeds previous reported values in the literature that used pure phospholipid liposomes. While not wishing to be held to a particular theory of the present invention, we suspect that MOPC softens the bilayer interface and allows greater partitioning of TAXOL� into the headgroup region of the bilayer.
The effect of varying the cholesterol content of the liposome was studied. As shown in FIG. 27, the inclusion of 33 mol % cholesterol into SOPC membranes reduced the maximum amount of TAXOL� that could be solubilized by the liposome preparation to 15 micromolar. With 50 mol % cholesterol in the bilayers, only 1 micromolar TAXOL� could be solubilized after processing and cleaning by SEC column.
The experiment indicates that a liposome system composed of cholesterol-rich membranes, by itself, will not be an effective delivery system for a poorly water soluble drug such as TAXOL�. While not wishing to be held to a single theory, the mechanism seems to be associated with the state of condensation of the bilayer interface, i.e., cholesterol-rich membranes become increasingly less compressible with increased cholesterol content in the membranes. The highly condensed and stiff cholesterol-rich membrane does not appear to allow the TAXOL� molecules to penetrate and be solubilized by the membrane.
These experiments measured TAXOL� solubilized by liposome membranes as cholesterol content was varied in liposomes made from fixed SOPC/MOPC 16/6. Data are presented in FIG. 28, and show that for 16 mM of SOPC with 6 mM MOPC, 1.7 mM TAXOL� can be solubilized. But with the addition of 33 mol % cholesterol and 50 mol % cholesterol, the amount of solubilized TAXOL� decreased to 0.6 mM and 0.015 mM, respectively. SOPC alone loads about 0.5 mM TAXOL� (as discussed before in relation to FIG. 27). Thus, interestingly, even though the addition of cholesterol to SOPC membranes dramatically reduced their ability to solubilize TAXOL� (apparently due to the condensation of the membrane surface), the addition of MOPC restored this capacity from 15 micromolar to 0.6 mM for the 33 mol % cholesterol membranes.
In many clinically approved treatments using liposomes, the administered dose is about 100 mM to about 200 mM lipid. The formulation (SOPC/CHOL/MOPC 16/8/16) can be scaled up, e.g., to 100 mM SOPC, which allows 6.4 mM of TAXOL� to be solubilized. The lipid formulation is used in the micelle-liposome construct described below, but it is interesting to note that even in liposomes that do not contain micelles, the present liposome formulation has the capacity to solubilize even greater amounts of TAXOL� if the cholesterol is left out and only SOPC/MOPC composition is used. A similar scale up from the 16/6 formulation, which solubilized 1.7 mM TAXOL�, is expected to therefore solubilize 11 mM TAXOL� (i.e., 11 mol % relative to SOPC).
These present experiments demonstrate that a poorly water-soluble drug such as TAXOL� can be lost from the liposome preparation onto filters and in columns during cleaning of the liposomes. If such cleaning is not required for clinical formulations, it will be apparent that such formulations may carry even greater amounts of drug, e.g., greater amounts of TAXOL� than described herein. Based on the data presented herein, the amount of TAXOL� solubilized could reach 37.5 mM for a 200 mM lipid sample, if the sample is not cleaned through columns as described above.
The following summarizes one protocol for preparing and assessing liposome-solubilized TAXOL�; make stocks of lipids and drug in chloroform; mix aliquots of lipids, cholesterol and/or drug in a 100 mL round bottom flask; evaporate solvents on rotavapor under vacuum by heating at 50 degrees C. and keep flasks under vacuum overnight; hydrate the lipid films with PBS solution and make MLVs by gently etching the lipid films with a TEFLON� bead and letting equilibrate for one hour; extrude using lipex extruder through a stack of two 0.2 micron polycarbonate fibers eight times to product LUVETs; make minicolumns from SEPHAROSE� CL-4B in 5 cm syringes, drain out ethanol by constantly flushing with about 200 mL of water and centrifuge at 2800 rpm to remove water in a centrifuge tube; precondition the minicolumns with blank liposomes by gently adding liposomes in the center of SEPHAROSE� column and centrifuge again; similarly add TAXOL� samples in the column and centrifuge, and collect sample at the bottom of culture tubes; take required aliquots in methanol and measure absorbance at 230 nm.
EXAMPLE 11 Partitioning of MOPC into the Bilayer Phase for Micelle Hydration As noted above, each component of a liposome system has the ability to partition into each compartment of the system.
In liposomes containing a drug/micelle suspension, partitioning of MOPC into the SOPC or SOPC/CHOL liposome bilayer can lead to a depletion of MOPC from the (equilibrated) drug/micelle suspension. In liposomes containing TAXOL�/micelle suspensions, this redistribution of micelle-forming MOPC into the bilayer phase was found to also release TAXOL� from its solubilized state at the surface of the micelle into the aqueous solution. The TAXOL� rapidly exceeded its solubility limit and readily precipitated.
SOPC lipid films were made as described above, in the concentration range of 2 mM to 24 mM, having 50% mol % cholesterol in the membranes. Hydration of cholesterol-rich films was done using 1.5 mM micellized MOPC having 0.25 mM TAXOL� in PBS. The rest of the sample processing is the same as described above in Example 10.
As shown in FIG. 30, paradoxically, an increase in the total lipid concentration (i.e., greater number of liposomes per ml and therefore greater encapsulated volume) resulted in a decreased amount of TAXOL� being associated with the liposomes. This effect was due to the partitioning of MOPC into the bilayer phase leaving the TAXOL� to precipitate. In other words, TAXOL� that was previously solubilized at 250 times its natural solubility limit, readily precipitated as the micelle population was depleted by MOPC uptake into the bilayer phase. The methods of the present invention overcome this problem by presaturating the bilayer phase with MOPC such that he micelle phase is not depleted. Based on the experiments shown in Example 5 herein, we made an initial estimate and presaturated the lipid film with 20 mol % MOPC. Note that MOPC was chosen as the lysolipid for this system because it matches the acyl chain composition of the bilayer lipid SOPC and so has a large solubility in the membrane without destroying it.
When presaturation of the bilayer phase with 20 mol % MOPC (in relation to SOPC) is carried out prior to the addition of micellized TAXOL�, the amount of TAXOL� associated with the liposome fraction increases to about 100 micromolar measured with lipid concentration over the range shown in FIG. 30, i.e. from 2 to 48 mM lipid. This trend demonstrates the need to presaturate the bilayers with MOPC to maximize the drug carrying ability of liposomes according to the present invention.
The size of liposomes made using various combinations of components was measured, as shown in FIG. 31. The combinations were: SOPC/CHOL membranes hydrated with PBS (SOPC/CHOL); SOPC/CHOL/MOPC membranes hydrated with PBS (SOPC/CHOL/MOPC); SOPC/CHOL/MOPC membranes hydrated with MOPC (SOPC/CHOL/MOPC/MOPC); and SOPC/CHOL/MOPC hydrated with MOPC micelles containing TAXOL� (SOPC/CHOL/MOPC/MOPC/TX). Lipids were 40 mM, MOPC was 20 mol % with respect to SOPC; MOPC concentration in micellar form was 2.5 mM, and TAXOL� was 0.25 mM in 2.5 mM MOPC micelles.
Liposome size was measured using quasi-elastic light scattering. The size of the liposomes did not vary depending on the liposome composition. The inclusion of MOPC in the lipid, or rehydration with MOPC micelles or micelles that contain TAXOL�, did not destabilize or dissolve the liposomes.
EXAMPLE 12 Optimization of MOPC Presaturation with Respect to TAXOL� Loading in Liposomes for Micelle Hydration The examples provided above indicated that, to develop a liposome formulation with optimized loading of a poorly water-soluble drug (such as TAXOL�), one must allow for the partitioning of micelle MOPC into the bilayer phase, in order to stabilize the micellized drug suspension that is placed within the liposome.
We investigated the amount of MOPC needed to optimize TAXOL� loading in liposomes, using SOPC/CHOL bilayers (16/16) with MOPC concentrations in the bilayers ranging from 2 mM to 8 mM. Hydration of the lipid films was done using 1.5 mM MOPC in micellized form having 0.25 mM TAXOL� in PBS.
FIG. 32 shows that, for a 1/1 SOPC/CHOL bilayer at 16 mM SOPC, the amount of TAXOL� loaded into the liposome preparation increases with increasing amounts of MOPC presaturation. A maximum of almost 100 micromolar TAXOL� loading is achieved for MOPC presaturation of from about 6 mM to about 8 mM final concentration. It is therefore desirable to presaturate the bilayers with both TAXOL� and MOPC to preserve the micellized drug suspension intact and to fully exploit the capacity of the liposome bilayer to also adsorb TAXOL�.
EXAMPLE 13 Optimization of the Amount of Cholesterol in the Bilayer Phase for Micelle Hydration As described above in Example 7, a certain amount of cholesterol is required to stabilize the liposome bilayer phase in the presence of a micellized drug preparation. Previously, for micelle hydration, we have simply used an amount of cholesterol close to the maximum saturation of approximately 50 mol % cholesterol. However, this amount may not be optimal, especially in the presence of MOPC. We therefore investigated the relationship between the amount of TAXOL� that could be loaded into a liposome (in the bilayer phase and encapsulated in a micellar preparation) as a function of the amount of cholesterol present in a bilayer having a fixed SOPC/MOPC ratio of 16/6, at a lipid concentration of 16 mM. Hydration of lipid films was done with 1.5 mM micellized MOPC having 0.25 mM TAXOL� in PBS.
FIG. 33 shows that TAXOL� loading increased with decreasing cholesterol content of the bilayers, reaching a maximum at 16/8/6 (i.e., SOPC/CHOL mole ratio of 2:1). TAXOL� loading seemed to decrease at lower cholesterol concentrations, which may be due to the instability of the bilayer to micelles and MOPC. As discussed above, bilayers without cholesterol are not stable in the presence of such high concentrations of MOPC micelles.
EXAMPLE 14 Optimization of Concentration of Micellized TAXOL� We optimized the concentration of micellized TAXOL� used to hydrate a SOPC/CHOL/MOPC/TAXOL� film. Having shown earlier (Example 9) that the limiting amount of TAXOL� that could be solubilized by MOPC micelles was at a mole ratio of 0.2 (i.e., about 5 moles of MOPC per mole TAXOL�), we kept this ratio and increased the overall concentration of each from 1.5 mM MOPC to 5 mM MOPC. FIG. 34 shows the comparison of two formulations having different compositions which were hydrated with 1.5 mM MOPC having 0.25 mM TAXOL�, as well as with 5 mM micellized MOPC loaded with 0.7 mM TAXOL� in PBS.
FIG. 34 clearly shows that for a MOPC/TAXOL� concentration of 5 mM/0.7 mM, the amount of TAXOL� that can be loaded into the liposome system is 400 micromolar when the total lipid concentration is 16 mM, i.e., a final TAXOL�-to-lipid mole ratio of 2.5 mole %.
EXAMPLE 15 Quantification of TAXOL� Associated with Micellized MOPC and with Liposome Bilayer As described in earlier examples, presaturation of a lipid phase with both MOPC and TAXOL�, prior to hydration with micelle-solubilized drug, increases the drug-carrying capacity of the resulting liposome. We found that 6 mM MOPC was needed to presaturate 16 mM SOPC bilayers, and 8 mM of cholesterol was required in these SOPC/MOPC bilayers to give maximum TAXOL� loading. Also, after liposomes are processed by extrusion and SEC columns, the maximum amount of TAXOL� that can be loaded to otherwise empty bilayers (i.e., liposomes without any micelle suspension in the interior) is about 0.6 mM, which is about half of the 1.1 mM TAXOL� in the initial lipid films.
Having determined these conditions, we next quantified the amount of TAXOL� associated with micelles that was in fact encapsulated in liposomes. Lipid films were made from 16 mM SOPC and preloaded with 6 mM MOPC, 8 mM cholesterol, and 1.1 mM TAXOL�, by preparing a thin film of all these components together in chloroform. These lipid films were hydrated with either 1.5 mM micellized MOPC loaded with 0.25 mM TAXOL� in PBS, or with 5 mM micellized MOPC loaded with 0.7 mM TAXOL� in PBS. FIG. 35 shows the comparison of these two hydration processes, along with a control (no micelle used for lipid hydration).
As shown earlier in FIG. 29 and repeated again here for the control, the 16/8/6 composition can solubilize 0.6 mM TAXOL� when hydrated with simple PBS buffer (no micelles). When this composition is hydrated with a MOPC/TAXOL� suspension of 1.5/0.25 (mM), there is an increase in the total solubilized TAXOL� to 0.8 mM.
When the concentration of the micellized TAXOL� suspension is increased to 5/0.7 (mM), the amount of TAXOL� solubilized by the entire system is 1.2 mM.
From these data, we can therefore estimate the amount of TAXOL� that is partitioned into the micelle phase and encapsulated within the liposome interior space, as distinct from TAXOL� solubilized by the bilayer. As shown in FIG. 35, any TAXOL� that is loaded, over and above 0.6 mM, must be in the micelle phase. Thus, for the 1.5/0.25 sample, about 0.2 mM of TAXOL� is contained in the micelle phase, and for the 5/0.7 sample, about 0.6 mM is entrapped with the micelles. This data indicates that TAXOL� can be solubilized in both the bilayer and micelle phases in approximately equal amount at these bilayer lipid concentrations (16/8/6 mM).
EXAMPLE 16 Optimization of Total Concentration of TAXOL� in Micelle Loaded Liposomes The present examples demonstrate that the total loading capacity of liposomes for poorly water-soluble drugs can be increased when SOPC bilayers are preloaded with MOPC. Using TAXOL�, loading capacity of liposomes is increased up to 22 mM when SOPC bilayers (200 mM) are preloaded with MOPC. As described in Example 10 and shown in FIGS. 25 and 26, SOPC/MOPC bilayers can solubilize even larger amounts of TAXOL� that, if not columned (i.e., cleaned), could approach 37.5 mM TAXOL� (i.e., 19 mol % with respect to lipid).
The present examples further demonstrate that liposomes containing micellized drug preparations require cholesterol in the lipid bilayers, to stabilize the liposome membrane against the deleterious effects of the micelles contained within. However, cholesterol reduces the amount of TAXOL� that can be solubilized by the bilayer phase of the liposome. Although TAXOL� solubilization is decreased in the lipid bilayer, the micellized TAXOL� in the liposome interior acts to replace the TAXOL�-carrying capacity lost in the bilayer. Scaling up the liposome-micelle formulation described herein from 16 mM to 200 mM SOPC, one could expect about 10-15 mM TAXOL� (i.e., 5 to 7.5 mol % TAXOL� based on SOPC concentration). As described above, if these preparations are not cleaned using SEC columns, one could expect to achieve up to 15 to 18 mM TAXOL� (7.5-9 mol %).
EXAMPLE 17 Addition of Polyethylene Glycol to Liposomes The addition of polymers to the exterior surface of liposomes produced according to the present invention provides additional desirable characteristics.
Patent CitationsCited PatentFiling datePublication dateApplicantTitleUS5023086 *Dec 20, 1988Jun 11, 1991Micro-Pak, Inc.In lipid vesiclesUS5755788 *Apr 30, 1996May 26, 1998Rutgers, The State UniversityProsthesis and implants having liposomes bound thereto and methods of preparationUS5827533 *Feb 6, 1997Oct 27, 1998Duke UniversityLiposomes containing active agents aggregated with lipid surfactantsWO1992022249A1 *Mar 31, 1992Dec 23, 1992Evan C UngerMethods for providing localized therapeutic heat to biological tissues and fluids using gas filled liposomesWO1994013265A1 *Dec 10, 1993Jun 23, 1994Milton G SmithA free radical quenching liposomal compositionWO1995008986A1 *Sep 27, 1994Apr 6, 1995Panayiotis Peri ConstantinidesCamptothecin formulations* Cited by examinerNon-Patent CitationsReference1 *Devlin, B. P. et al., A Kinetic Study of the Polyelectrolyte Induced Reorganization of Lipid Bilayers, Am. Chem. Soc. Div. Polym. Chem. vol. 28, No. 2, (1987), pp. 50 51.2Devlin, B. 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Effect of Bilayer Composition On the Phase Behavior Liposomal Suspensions Containing Poly(ethylene glycol) Lipids, Macromolecules, vol. 28, No. 23 (1995) pp. 7693-7699.5 *International Search Report for corresponding International Application No. PCT/US99/12964.* Cited by examinerReferenced byCiting PatentFiling datePublication dateApplicantTitleUS6497896Feb 12, 2001Dec 24, 2002Supergen, Inc.Aqueous suspension; thermal sterilizationUS6509027Feb 12, 2001Jan 21, 2003Supergen, Inc.Anticancer agentUS6878693Sep 28, 2001Apr 12, 2005Solubest Ltd.Hydrophilic complexes of lipophilic materials and an apparatus and method for their productionUS6964778 *Sep 6, 2002Nov 15, 2005Health Research, Inc.Temperature controlled content release from liposomesUS6991805 *Sep 6, 2002Jan 31, 2006Health Research, Inc.Temperature sensitive control of liposome-cell adhesionUS7081450Sep 26, 2002Jul 25, 2006Solubest Ltd.Water insoluble or soluble active compound wrapped with an amphiphilic polymer in a non-crystalline manner toform a nano-sized molecular entity in which no valent bonds are formedUS7108863Mar 26, 2002Sep 19, 2006Alza CorporationLiposome composition for improved intracellular delivery of a therapeutic agentUS7749485May 31, 2005Jul 6, 2010Bracco Research S.A.Liposomal assembly for therapeutic and/or diagnostic useUS7901709Apr 12, 2005Mar 8, 2011Duke UniversityActive agent, a gel-phase lipid bilayer membrane of a phospholipid and a surface active agent contained in the bilayer membrane; increasing the percentage of active agent released at the phase transition temperature of the lipid bilayerUS8142814 *Jan 30, 2004Mar 27, 2012Ferro CorporationMethod and apparatus for supercritical fluid assisted particle productionUS8173621Jun 5, 2009May 8, 2012Gilead Pharmasset LlcNucleoside cyclicphosphatesUS8563530Mar 31, 2011Oct 22, 2013Gilead Pharmassel LLCPurine nucleoside phosphoramidateEP2075014A2May 27, 2003Jul 1, 2009Angiotech International AgCompositions and methods for coating medical implantsEP2609923A2Mar 31, 2011Jul 3, 2013Gilead Pharmasset LLCNucleoside PhosphoramidatesEP2610264A2May 20, 2010Jul 3, 2013Gilead Pharmasset LLCN-[(2'r)-2'-deoxy-2'-fluoro-2'-methyl-p-phenyl-5'-uridylyl]-l-alanine 1-methylethyl ester and process for its productionEP2671888A1Dec 23, 2009Dec 11, 2013Gilead Pharmasset LLC3',5'-cyclic nucleoside phosphate analoguesWO2002076428A1 *Mar 26, 2002Oct 3, 2002Alza CorpLiposome composition for improved intracellular delivery of a therapeutic agentWO2010075517A2Dec 23, 2009Jul 1, 2010Pharmasset, Inc.Nucleoside analogsWO2010075549A2Dec 23, 2009Jul 1, 2010Pharmasset, Inc.Nucleoside phosphoramidatesWO2010075554A1Dec 23, 2009Jul 1, 2010Pharmasset, Inc.Synthesis of purine nucleosidesWO2010111807A1 *Apr 3, 2009Oct 7, 2010Wuhan Docan Pharmaceutical Co., Ltd.A polysaccharide liposome, the preparation method and use of itWO2010135569A1May 20, 2010Nov 25, 2010Pharmasset, Inc.N- [ (2 ' r) -2 ' -deoxy-2 ' -fluoro-2 ' -methyl-p-phenyl-5 ' -uridylyl] -l-alanine 1-methylethyl ester and process for its productionWO2011123645A2Mar 31, 2011Oct 6, 2011Pharmasset, Inc.Nucleoside phosphoramidatesWO2011123668A2Mar 31, 2011Oct 6, 2011Pharmasset, Inc.Stereoselective synthesis of phosphorus containing activesWO2011123672A1Mar 31, 2011Oct 6, 2011Pharmasset, Inc.Purine nucleoside phosphoramidateWO2012075140A1Nov 30, 2011Jun 7, 2012Pharmasset, Inc.CompoundsWO2013026989A2Aug 22, 2012Feb 28, 2013TrophosLiposome comprising at least one cholesterol derivative* Cited by examinerClassifications U.S. Classification424/450, 424/9.51, 424/1.21, 424/94.3, 424/9.321International ClassificationA61K9/107, A61K9/133, A61K9/127Cooperative ClassificationA61K9/1075, A61K9/1271European ClassificationA61K9/107D, A61K9/127BLegal EventsDateCodeEventDescriptionApr 16, 2012FPAYFee paymentYear of fee payment: 12May 7, 2008FPAYFee paymentYear of fee payment: 8May 4, 2004FPAYFee paymentYear of fee payment: 4RotateOriginal ImageGoogle Home - Sitemap - USPTO Bulk Downloads - Privacy Policy - Terms of Service - About Google Patents - Send FeedbackData provided by IFI CLAIMS Patent Services©2012 Google

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3 bedroom detached house for sale in Littleton, Winchester, Hampshire, SO22
www.rightmove.co.uk/property/63146852
Charming detached family home packed with character and a wealth of splendid features sat on a stunning plot with ample gardens and parking. The property is approached via a double five bar gate which allows access to the shingle parking area leading up to the front door. On entry to the house you are welcomed by a spacious hallway which allows access to all the principle rooms on the ground floor. There is a splendid living room, good size family room and beautifully extended kitchen dining room with doors out to the rear garden. The kitchen is well appointed being fitted with a range of matching units and integral appliances. There is also a useful utility room. The three bedrooms can all be found on the first floor along with the family bathroom. Outside the house features ample front and rear gardens. The rear garden is enclosed and mainly laid to lawn with side access provided via a gate. The front features a gated shingle driveway providing parking with a further section of garden/vegetable plot to the front of this.
Property ref: 121_1496_3912094
To view this media, please visit the on-line version of this page at www.rightmove.co.uk/property-for-sale/property-63146852.html
Disclaimer - Property reference 3912094. The information displayed about this property comprises a property advertisement. Rightmove.co.uk makes no warranty as to the accuracy or completeness of the advertisement or any linked or associated information, and Rightmove has no control over the content. This property advertisement does not constitute property particulars. The information is provided and maintained by Charters Estate Agents Ltd, Winchester. Please contact the selling agent or developer directly to obtain any information which may be available under the terms of The Energy Performance of Buildings (Certificates and Inspections) (England and Wales) Regulations 2007 or the Home Report if in relation to a residential property in Scotland.
25 Jul 2014 33a Main Road, SO22 £795,000
06 Dec 2013 34 Main Road, SO22 £395,000

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Soldiers of Fortune (Audiobook) by Jana DeLeon | Audible.com
Series: Miss Fortune Mysteries, Book 6
4.5 out of 5 stars 4.7 (2,115 ratings)
Back to 4 stars
By green ice cream garden on 09-12-16
By green ice cream garden on 09-01-16
5 out of 5 stars 1,919
By jaenpop on 05-03-16
By Jill on 11-05-19
By Susan on 06-26-18
By Jean on 03-28-16
My go to feel good series!
By Dee Norenberg on 02-12-19
By Rusty on 02-09-16
By Andrew on 07-06-19
4.5 out of 5 stars 5,540
4 out of 5 stars 2,295
Love this series to pieces!
By K. April Holgate on 01-31-15
Who killed Helena Henry? The shorter list may be who didn’t want her dead. Psychic Raissa Bordeaux thought she’d left her former life behind, hidden away in her eclectic shop in the French Quarter. But when a child is kidnapped and Helena Henry appears, Raissa risks her true identity by offering the police help with the case. New Orleans Detective Zach Blanchard doesn’t believe in psychic hooey and is certain Raissa didn’t get her facts about the case from a crystal ball.
By K. April Holgate on 02-01-15
From New York Times and USA Today best-selling author Jana DeLeon, the sixth installment in the best-selling Miss Fortune series.
When Celia Arceneaux was elected mayor of Sinful, Louisiana, Fortune Redding expected trouble. The woman has an unmatched talent for making the worst of any situation, especially if it means getting revenge on her sworn enemies, Ida Belle and Gertie.
At the Fourth of July celebration, an explosion rocks Sinful, and it's not from fireworks. Most people assume it was a moonshine still, but when evidence points to a meth lab deep in the bayous of Sinful, Fortune realizes how dire the situation is for the tiny town.
With Carter on medical leave and no one left at the sheriff's department who can handle the investigation, Fortune, Ida Belle, and Gertie decide to take matters into their own hands. This is (another) mission for Swamp Team 3.
©2015 Jana DeLeon (P)2016 Audible, Inc.
Not blazing but okay
This isn't one of my favorite in the series, but it's still okay. I do enjoy that themes are always current.
Fun suspense mystery with great narration!
This wonderful Miss Fortune continues with Book 6. It's great humor wrapped around a light suspense mystery.
Fun story. Well paced. Well told. Well narrated. Pretty much the same as the same as the prior books. But still fun.
5 Stars and So Funny
So freaking funny!!! Soldiers of Fortune is book 6 in the Miss Fortune Mystery series. This series is set in Sinful, Louisiana, USA and follows Fortune, who is a CIA assassin in hiding, and old ladies Gertie and Ida Belle, who are lifelong Sinful residents and ex-spy war veterans, as they help solve a string of various crimes and murders.
This book and the entire series is full of humor. I stumbled across these books when I decided to check out a new-to-me genre called Cozy Mysteries and someone recommended this series. Once I listened to the first book, I couldn't stop. I have been listening to all of the books back to back.
In Soldiers of Fortune, a meth lab explodes in the bayou. The ladies jump into action to try to solve the case while Carter is supposed to still be on medical leave after someone tried to kill him in the last book. Also, the votes are being recounted for the new town mayor because Celia won the election but someone had stuffed the ballot boxes, but in the meantime, Celia is in control and as expected, she is a nightmare and causing more problems than anything else.
There was a point in this book where I was laughing so hard that I had to keep rewinding so I wouldn't miss anything and finally had to pause it to get myself under control.
The narrator does a good job and I really like that it is the same narrator for each of the books.
Great series. Start at book one and go forward. I have been on a Fortune binge - from book one to this one and each one in one sitting. I love these characters, the author and the reader.
Maurice R. McKeon Jr
This is a very light and funny book. Sometimes as a man I like a comic mystery and this is it for me. The only problem is that the narrator does not pronounce the Cajun names LaBlanc and Hebert correctly. I say this because I am from Louisiana.
I love the added Hebert Bros and new twists that segue into the next book.
Eddy Metz
loved it, great series. Laugh out loud adventures solving mysteries. Winning combination of comedy and mystery, plus great narration
Crawspace
great, easy read. I listen to this while I drive to and from work. makes my drive even more enjoyable.
mariahthruthedark
Jana DeLeon is one of my favorite authors and this series is my favorite! For sheer, laugh-out-loud humor this can't be beat. My only aggravation is the narrator mispronounces our southern Louisiana names, and I assure you that mobsters, born and bred in these swamps, wouldn't have a New York/New Jersey accent. But that's definitely worth putting up with to be able to listen to these books!
FORUNE BOOK 6
What did you like most about Soldiers of Fortune?
THE WHOLES STORY FROM START TO END
MISS FORTUNE AND HER TWO SIDE KICKS
What does Cassandra Campbell bring to the story that you wouldn’t experience if you had only read the book?
CASSANDRA BRINGS THE PEOPLE TO LIFE AND I SPEND MOST OF MY TIME LAUGHING AT THE POSITIONS THEY FIND THEM SELVES IN
OH YEAH I WOULD BE FIRST THROUGH THE DOOR
I HAVE ALL MISS FORTUNE BOOKS SO FAR IF YOU ARE FED UP AND FEELING DOWN GET THESE BOOKS THEY WILL HAVE YOU LAUGHING AND WILL CHEER YOU UP
Light hearted and plenty of Bayou action. Great listen and well narrated love the series

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4-star hotel with 2 restaurants, near Seminyak Square
Presidential Penthouse, 1 King Bed, Pool View - Guest Room [3/42]
Suite - Guest Room [4/42]
Premium Room - Guest Room [5/42]
Suite - In-Room Amenity [7/42]
Suite - Bathroom [9/42]
Sundeck [10/42]
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Food and Drink [16/42]
Hallway [19/42]
Hotel Interior [20/42]
Street View [21/42]
Deluxe Room - Guest Room [22/42]
Premium Room - Guest Room [23/42]
Superior Room - Guest Room [24/42]
Premium Room - Guest Room [25/42]
Presidential Penthouse, 1 King Bed, Pool View - Living Room [26/42]
Presidential Penthouse, 1 King Bed, Pool View - Bathroom [27/42]
Premium Room - Bathroom [28/42]
Premium Room - Guest Room [30/42]
Premium Room - Guest Room [31/42]
Presidential Penthouse, 1 King Bed, Pool View - Room Service – Dining [32/42]
Deluxe Room - Guest Room [33/42]
Presidential Penthouse, 1 King Bed, Pool View - Bathroom Shower [35/42]
Superior Room - Bathroom [36/42]
Superior Room - Guest Room [37/42]
Presidential Penthouse, 1 King Bed, Pool View - Living Room [38/42]
Presidential Penthouse, 1 King Bed, Pool View - Minibar [39/42]
Premium Room - In-Room Amenity [40/42]
Premium Room - In-Room Amenity [41/42]
Superior Room - In-Room Amenity [42/42]
Presidential Penthouse, 1 King Bed, Pool View - Guest Room
Presidential Penthouse, 1 King Bed, Pool View - Living Room
Presidential Penthouse, 1 King Bed, Pool View - Bathroom
Presidential Penthouse, 1 King Bed, Pool View - Room Service – Dining
Presidential Penthouse, 1 King Bed, Pool View - Bathroom Shower
Presidential Penthouse, 1 King Bed, Pool View - Minibar
3 - Daun Bali Seminyak Hotel, Seminyak, Presidential Penthouse, 1 King Bed, Pool View, Guest Room
4 - Daun Bali Seminyak Hotel, Seminyak, Suite, Guest Room
6 - Daun Bali Seminyak Hotel, Seminyak, Deluxe Room, Guest Room
7 - Daun Bali Seminyak Hotel, Seminyak, Suite, In-Room Amenity
9 - Daun Bali Seminyak Hotel, Seminyak, Suite, Bathroom
11 - Daun Bali Seminyak Hotel, Seminyak, Sundeck
21 - Daun Bali Seminyak Hotel, Seminyak, Street View
22 - Daun Bali Seminyak Hotel, Seminyak, Deluxe Room, Guest Room
23 - Daun Bali Seminyak Hotel, Seminyak, Premium Room, Guest Room
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26 - Daun Bali Seminyak Hotel, Seminyak, Presidential Penthouse, 1 King Bed, Pool View, Living Room
27 - Daun Bali Seminyak Hotel, Seminyak, Presidential Penthouse, 1 King Bed, Pool View, Bathroom
29 - Daun Bali Seminyak Hotel, Seminyak, Hotel Bar
32 - Daun Bali Seminyak Hotel, Seminyak, Presidential Penthouse, 1 King Bed, Pool View, Room Service – Dining
33 - Daun Bali Seminyak Hotel, Seminyak, Deluxe Room, Guest Room
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35 - Daun Bali Seminyak Hotel, Seminyak, Presidential Penthouse, 1 King Bed, Pool View, Bathroom Shower
37 - Daun Bali Seminyak Hotel, Seminyak, Superior Room, Guest Room
38 - Daun Bali Seminyak Hotel, Seminyak, Presidential Penthouse, 1 King Bed, Pool View, Living Room
39 - Daun Bali Seminyak Hotel, Seminyak, Presidential Penthouse, 1 King Bed, Pool View, Minibar
40 - Daun Bali Seminyak Hotel, Seminyak, Premium Room, In-Room Amenity
42 - Daun Bali Seminyak Hotel, Seminyak, Superior Room, In-Room Amenity
Lowest price$61$32
Finns Recreation Club - 41 min walk
Legian Beach - 2.3 mi / 3.7 km
Presidential Penthouse, 1 King Bed, Pool View
Great Value in Seminyak Area
Great Value for this small boutique style. Great room size with modern style and clean. This is a good option if you need a good room to stay in seminyak.
this was a good hotel, close to everything, easy to find and very clean and modern. white beds are comfy
The hotel is new and clean. The only downside is that they are still doing minor construction which causes some early morning noise when you are trying to sleep in.
The hotel was construct in 2016 so all is new

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text:
Colonel Richard Henry Beddome (11 May 1830 – 23 February 1911) was a British military officer and naturalist in India who became chief conservator of the Madras Forest Department. In the mid 19th century, he extensively surveyed several remote and then-unexplored hill ranges in Sri Lanka and south India, including those in the Eastern Ghats such as Yelandur, Kollegal, Shevaroy Hills, Yelagiri, Nallamala Hills, Vishakapatnam hills and the Western Ghats such as Nilgiri hills, Anaimalai hills, Agasthyamalai Hills and Kudremukh. He described many species of plants, amphibians and reptiles from southern India and Sri Lanka, and several species from this region described by others bear his name.
Richard was the eldest son of Richard Boswell Brandon Beddome, solicitor, of Clapham Common, S.W. He was educated at Charterhouse School and trained for the legal profession, but preferred to join the East India Company at the age of 18 and joined the 42nd Madras Native Infantry as a cadet at Jabalpur.[1][2]
He entered the Army, obtaining a direct cadetship in 1848 in the East India Company's service, and sent to India was posted to the 42nd Madras Native Infantry[3] He was with that regiment at Jabalpur in 1856, serving as Quartermaster and Interpreter of the regiment, and from there he went to Secunderabad. Soon after his arrival at Madras, at the end of 1856, he was appointed to the Madras Forest Department, and never rejoined his regiment.
The following are dates of his commissions: Ensign, 20 January 1848; Lieutenant, l0 November 1853; Captain, 18 February 1861; Major, 20 January 1868; Lieutenant-Colonel, 20 January 1874; Colonel, 20 January 1879.[1]
In 1857 he was selected on account of his devotion to botany and natural history as an assistant to Dr. Hugh Cleghorn, the first conservator of the newly formed Forest Department of the Madras Presidency. He succeeded Cleghorn in 1859 and remained Chief Conservator until 1882.[1]
He became a member of the University of Madras in 1880. He made many floral studies in India and Ceylon including Trees of the Madras Presidency in 1863 and Handbook of the ferns of British India, Ceylon and Malaya Peninsula in 1892. He also studied reptiles, amphibians and molluscs.
Colonel Beddome was essentially a botanist. In the study of the flora of South India he devoted the best days of his life. The result was the publication of a series of valuable works containing figures of many species. The drawings were executed with great accuracy by the native draughtsmen he had trained to draw.
His botanical publications included The Flora Sylvatica for Southern India, 1869–73; Ferns of Southern India, 1873; Ferns of British India, 1876; Forester's Manual of Botany for Southern India, 1869–74; Icones Plantarum Indies Orientalis, 1874.[4][5]
On Reptilia and Batrachia he wrote at least fifteen papers, and described over 40 new species of reptiles[6] and amphibians.[7][8]
The following is a complete list of his papers on Mollusca: Descriptions of some new Operculated Land-shells from Southern India and Ceylon, 1S75. Descriptions of Land-shells from the Island of Koror, Pelew Group, 1889. Descriptions of some new Land-shells from the Indian Region, 1891. Notes on Indian and Ceylonese species of Glessula, 1906. Descriptions of Labyrinthus cuclausus and Neocyclotus Belli, n.spp., from Colombia, 1908 and in conjunction with H. H. Godwin-Austen: New species of Cyclophorus and a Spiraculum from the Khasi and Naga Hills, Assam.[1]
Beddome formed a fine collection of land shells from India and other parts of the world, among which the minute forms were his favourites. His collection of specimens was presented to the British Museum at difficult times, and some specimens were also left with the Indian Museum at Calcutta.
The standard author abbreviation Bedd. is used to indicate this individual as the author when citing a botanical name.[12]
Some of the species named in honor of him are listed below:
Rhinolophus beddomei, lesser woolly horseshoe bat, a species of bat in the family Rhinolophidae.
Otocryptis beddomii, Beddome's kangaroo lizard, of the family Agamidae, endemic to India.
Ristella beddomii, Beddome's cat skink, endemic to India.
Cnemaspis beddomei, Beddome's day gecko, a species of gecko found in the Western Ghats.
Kaestlea beddomei, a species of blue-tailed skink.
Ophisops beddomei, Beddome's snake-eye, a species of lizard in the family Lacertidae found in the Western Ghats of India.
Typhlops beddomei, Beddome's worm snake, a species of blind snake found in southern India.
Uropeltis beddomii, Beddome's shield-tail snake, of the family Uropeltidae, endemic to India.
Boiga beddomei, Beddome's cat snake, a species of snake found in Sri Lanka and India (Western Ghats).
Amphiesma beddomei, Beddome's keelback, a species of snake found in the Western Ghats in India,
Calliophis beddomei, Beddome's coral snake, a species of snake in the family Elapidae.
Ichthyophis beddomei, Beddome's caecilian, a species of caecilian in the family Ichthyophiidae.
Duttaphrynus beddomii, Beddome's toad endemic to the Western Ghats of India.
Raorchestes beddomii, Beddome's bush frog of the family Rhacophoridae found in the Western Ghats of India.
Nyctibatrachus beddomii, Beddome's night frog of the family Nyctibatrachidae endemic to Western Ghats of India.
Indirana beddomii, Beddome's litter frog of the family Ranixalidae found in the Western Ghats of India.
Mariaella beddomei, a species of gastropod.
Hemiplecta beddomii, a species of gastropod.
Cycas beddomei, a species of Cycad from India.
Syzygium beddomei, a species of plant in the Myrtaceae family. It is endemic to India.
Cynometra beddomei, a species of plant in the Fabaceae family. It is endemic to India .
Psychotria beddomei, a species of plant in the Rubiaceae family. It is endemic to India .
Litsea beddomei, a species of plant in the Lauraceae family. It is endemic to India.
Beddomixalus bijui, a species of tree frog in the family Rhacophoridae. It is endemic to India.
Beddome R.H. 1875. Descriptions of some new operculated land-shells from Southern India and Ceylon. Proceedings of the Zoological Society of London 1875(3): 442–453; pls. 52–53.
^ a b c d e Godwin-Austen, H.H. (1912). "Obituary notices. Colonel Richard Henry Beddome". Proceedings of the Malacological Society of London. 10: 50–53. ^ Kumar, C Satish (2011). "Richard Henry Beddome (1830–1911): A Centennial Remembrance" (PDF). Rheedea. 21 (1): 108–109. ^ Smith, M.A. 1931. The Fauna of British India, Including Ceylon and Burma. Reptilia and Amphibia. Vol. I.—Loricata, Testudines. London: Secretary of State for India in Council. (Taylor and Francis, printers). xxvii+ 185 pp. + Plates I-II. (Beddome, p. 9).
^ Smith, Malcolm (1940). "A facsimile of R. H. Beddome's articles on Indian reptiles 1862–1870". Archives of Natural History. 1 (10): 273–334. doi:10.3366/jsbnh.1940.1.10.273. ^ Turner, Ian M. "The angiosperm taxa of R.H. Beddome with notes on the dates of publication of two serially published works." (PDF). Ann. Bot. Fennici. 49: 289–304. ^ The Reptile Database. www.reptile-database.org.
^ Amphibian Species of the World 5.5, an Online Reference. research.amnh.org/vz/amphibia.
^ Ganesh, S.R. (2011). "Richard Henry Beddome and south India's herpetofauna - a tribute on his centennial death anniversary.". Cobra. 4 (2): 1–11. ^ Bedd., International Plant Names Index, 2005, retrieved 10 February 2011 ^ "Married". Southern Reporter and Cork Commercial Courier. 20 February 1862. p. 3 – via British Newspaper Archive. ^ "India, Births and Baptisms, 1786-1947". The Church of Jesus Christ of Latter-Day Saints. Children of Richard and Maria Beddome. Retrieved 8 February 2015. ^ "Author Query for 'Bedd.'". International Plant Names Index. External links[edit]
Beddome, R.H. (1864). "Contributions to the botany of Southern India". Madras Journal of Literature and Science. series III. 1: 37–59. Handbook to the ferns of British India (1892)
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Cobblestones Museum (Greytown) - 2018 All You Need to Know BEFORE You Go (with Photos) - TripAdvisor
Greytown Vacation Packages
“Excellently presented look into bygone days”
Great way to spend time in this beautiful town,after paying a modest entrance fee,to two very... read more
We purchased our entry tickets from two lovely ladies, giving freely of their time to the... read more
All reviews early settlers printing press small town local history fire station little gem new building two hours pleasant surprise fascinating visit historic village beautiful grounds entry fee new zealand great place to visit chocolate shop printing shop
Excellently presented look into bygone days
Great way to spend time in this beautiful town,after paying a modest entrance fee,to two very welcoming ladies,enjoyed the slow meandering around this well presented and thought provoking look into the past.Amazing how the building where transhipped so cleverly and precisely.Thankyou to the volunteers and...More
We purchased our entry tickets from two lovely ladies, giving freely of their time to the museum. The museum itself is well worth a visit. I'm not a New Zealander but found it most interesting to see the how, what and why of 19th century...More
Leigh36, Guest Relations Manager at Cobblestones Museum, responded to this reviewResponded October 13, 2018

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Garden experts urge holidaymakers not to bring overseas... | Daily Mail Online
Garden experts urge holidaymakers not to bring overseas plants back to UK
Published: 19:04 EST, 6 June 2018 | Updated: 19:04 EST, 6 June 2018
Almost one in 10 people planning to go abroad in the next year would consider bringing back plants with them, raising the chance of new pests and diseases arriving in the UK.
A poll for the Royal Horticultural Society (RHS) found more than half of people were planning a trip overseas in the next 12 months, and of those 9% would consider bringing back plants, seeds, bulbs, tubers or cuttings.
It could mean many holidaymakers bringing back plant material in their luggage and raising the risk of importing new pests and diseases that could damage gardens, the RHS said.
Damage to fuchsia from gall mite which was first brought into the UK on illegally imported cuttings (Andrew Halstead/RHS/PA)
While there are some restrictions on importing plants in personal baggage, Britons travelling abroad should avoid bringing back any plants, the RHS urged as it unveiled the survey findings during the Chatsworth Flower Show.
British gardens face threats from new plants and diseases from abroad, such as “game changing” bacterium Xylella fastidiosa which can hit more than 350 different types of plant, with lavender, hebe, rosemary and flowering cherry at high risk.
Fuschia gall mite, which disfigures plants and is now rife in the South East, has been attributed to an enthusiast for the plant illegally importing cuttings from South America.
While clothes, alcohol and fridge magnets are the most common things bought on foreign holidays, plants, flowers, wooden items and seeds are among the items those quizzed for the poll say they have brought back.
Plants were bought from garden centres, supermarkets, local markets, or even collected from gardens or the wild.
Nearly a third (31%) of those responding to the survey of 2,066 people by YouGov are planning trips to Italy, France or Spain in the next 12 months – all countries where Xylella has already been found, the RHS said.
Its call to holidaymakers not to bring back plants from their foreign travels is backed by the Environment Department (Defra), which is launching a Don’t Risk It campaign this summer to raise awareness of the problems of bringing back cut flowers, fruit, vegetables and plant material from holiday spots.
Sue Biggs, director general of the Royal Horticultural Society, said: “For many people, wandering the olive groves of Italy and lavender fields of France are as much a part of the holiday experience as the cities and beaches.
“But we’re asking people to leave these beautiful plants where they are for future visitors to enjoy and not to bring them back home with them.
“This is vital if we are going to win the fight to protect our gardens against the growing threat of pests and diseases.”
This summer we will be launching our ‘Don’t Risk It’ campaign to raise awareness of the risks of bringing back plant materials from holiday destinations. Find out more about our #TreeHealthResilienceStrategy https://t.co/oLAgWpmrgE pic.twitter.com/DkzQoknL7m
— Defra UK (@DefraGovUK) May 25, 2018
Defra’s chief plant health officer Nicola Spence said: “We’ve invested more than £4.5 million to strengthen our border biosecurity, recruiting new plant inspectors and enhancing training.
“Our inspectors now make more interceptions of harmful organisms than any other EU member state.
“But we can’t eliminate all risks and we all have a part to play in protecting our plants and trees.
“Through our Don’t Risk It campaign we’ll be asking everyone to enjoy the exotic plants and flowers they see on their holidays but only bring them back to the UK in their memories and pictures.”
Garden experts urge holidaymakers not to bring overseas...

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Douglas E. Faries - Semantic Scholar
Haya Ascher-Svanum64
Jeffrey A Lieberman57
Joseph P Mcevoy51
Marvin S Swartz50
Diana O Perkins48
84Haya Ascher-Svanum
74Ralf W. Dittmann
60David R. Coghill
52Tamas Treuer
OBJECTIVE Atomoxetine is an investigational, nonstimulant pharmacotherapy being studied as potential treatment for attention-deficit/hyperactivity disorder (ADHD). It is thought to act via blockade of the presynaptic norepinephrine transporter in the brain. We assessed the efficacy of 3 doses of atomoxetine compared with placebo in children and adolescents… (More)
BACKGROUND Atomoxetine is a nonstimulant drug being studied for the treatment of attention-deficit/hyperactivity disorder (ADHD). Atomoxetine is a highly specific inhibitor of the presynaptic norepinephrine transporter with minimal affinity for other noradrenergic receptors or other neurotransmitter transporters or receptors. Results of 2 proof-of-concept… (More)
Amy R Perwien, Douglas E. Faries, Christopher J . Kratochvil, Calvin R Sumner, Douglas K. Kelsey, Albert J. Allen
Despite significant functional impairments associated with attention-deficit hyperactivity disorder (ADHD) and the growing appreciation of the importance of health-related quality of life (HRQL) assessment in children with chronic disorders, relatively few studies have examined the impact of ADHD treatment on HRQL. This investigation examines the effect of… (More)
Douglas E. Faries, John M Herrera, Jyoti N Rayamajhi, David J. DeBrota, Mark A. Demitrack, William Z. Potter
In clinical studies of antidepressants, the Hamilton Depression Rating Scale (HAMD) total score has been the gold standard instrument for establishing and comparing the efficacy of new treatments. However, the HAMD is a multidimensional measure, which may reduce its ability to detect differences between treatments, in particular, changes in core symptoms of… (More)
Haya Ascher-Svanum, Douglas E. Faries, Baojin Zhu, Frank R. Ernst, Marvin S. Swartz, Jeffrey W. Swanson
BACKGROUND Relatively little is known about the relationships between medication adherence and long-term functional outcomes in the treatment of schizophrenia. To extend previous research, we prospectively examined the relationships between adherence with any antipsychotic medication and functional outcomes among schizophrenia patients treated over a 3-year… (More)
OBJECTIVE To prospectively measure the link between depressive symptoms and functional outcomes in the long-term treatment of people with schizophrenia. METHODS Data were drawn from a large, multi-site, 3-year, prospective, naturalistic, observational study, in which subjects with schizophrenia were assessed at enrollment and at 12-month intervals… (More)
Amy R Perwien, Christopher J . Kratochvil, Douglas E. Faries, Brigette S Vaughan, Thomas Spencer, Ronald T. Brown
OBJECTIVE Numerous investigations have examined the efficacy of pharmacological treatment for attention-deficit/hyperactivity disorder (ADHD) in children. However, relatively few studies have addressed the impact of treatment on long-term subjective, psychosocial outcomes, such as health-related quality of life (HRQL). This study examines the long-term… (More)
Joseph Biederman, John H. Heiligenstein, +6 authors Kory J. Schuh
OBJECTIVE The efficacy of atomoxetine was assessed in school-age girls with attention-deficit/hyperactivity disorder (ADHD). Atomoxetine is a potent inhibitor of the presynaptic norepinephrine transporter with minimal affinity for other noradrenergic receptors or for other neurotransmitter transporters or receptors. METHODS A total of 291 children who… (More)
Shuyu Zhang, Douglas E. Faries, Maureen Vowles, David Michelson
The development of rating scales for attention-deficit/hyperactivity disorder (ADHD) has traditionally focused on parent-or teacher-rated scales. However, clinician-based instruments are valuable tools for assessing ADHD symptom severity. The ADHD Rating Scale IV (ADHD RS), clinician administered and scored, has been validated as a useful instrument to… (More)

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