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Liposomal compositions containing green porphyrins as photosensitizers are improved by enhancing the ratio of phospholipid to photosensitizer and by conducting the hydration an sizing of the liposomes in the composition at low temperature.
1. A method for making a pharmaceutical composition containing liposomes, said liposomes comprising a therapeutically acceptable amount of a hydro-monobenzoporphyrin photosensitizer and a mixture of phospholipids comprising egg phosphatidyl glycerol ("EPG") and dimyristoyl phosphatidyl choline ("DMPC"), wherein said method comprises the steps of:
a. combining the photosensitizer and the phospholipids in a molar ratio of 1:7.0 or more phospholipid in the presence of an organic solvent;
b. removing said organic solvent to form a photosensitizer:phospholipid complex;
c. hydrating said photosensitizer:phospholipid complex with an aqueous solution at a temperature below the glass transition temperature of the photosensitizer:phospholipid complex to form coarse liposomes containing said photosensitizer-phospholipid complex; and
d. homogenizing or reducing the particle size of said coarse liposomes to a particle size range of below about 300 nm at a temperature below the glass transition temperature of the photosensitizer:phospholipid complex.
2. The method of claim 1, wherein said molar ratio of hydro-monobenzoporphyrin photosensitizer to phospholipids is about 1:8.0 or more phospholipid.
3. The method of claim 1, wherein said organic solvent is methylene chloride.
4. The method of claim 1, wherein said organic solvent is removed in step "b." by evaporation under reduced pressure.
5. The method of claim 1, wherein said aqueous solution comprises a disaccharide or polysaccharide.
6. The method of claim 4, wherein said disaccharide or polysaccharide is selected from lactose or trehalose.
7. The method of claim 4, wherein the concentration ratio of disaccharide or polysaccharide to said mixture of phospholipids is about 10-20 to 0.5-6.0.
8. The method of claim 1, wherein said hydro-monobenzoporphyrin (Gp) photosensitizer has one of the formulae 1-6 set forth in FIG. 1 and has one or more light absorption maxima between 670-780 nm, or is a metalated or labeled form thereof,
each R1 and R2 is independently selected from the group consisting of carbalkoxy (2-6C), alkyl (1-6C) sulfonyl, aryl (6-10C) sulfonyl, aryl (6-10C), cyano, and --CONR5 CO-- wherein R5 is aryl (6-10C) or alkyl (1-6C);
each R3 is independently carboxyalkyl (2-6C) or a salt, amide, ester or acylhydrazone thereof, or is alkyl (1-6C); and
R4 is --CHCH2 ; --CHOR4' wherein R4' is H or alkyl (1-6C), optionally substituted with a hydrophilic substituent; --CHO; --COOR4' ; --CH(OR4')CH3 ; --CH(OR4')CH2 OR4' ; --CH(SR4')CH3 ; --CH(NR4' 2)CH3 ; --CH(CN)CH3 ; --CH(COOR4')CH3 ; --CH(OOCR4')CH3 ; --CH(halo)CH3 ; --CH(halo)CH2 (halo); an organic group of less than 12C resulting from direct or indirect derivatization of a vinyl group; or R4 is a 1-3 tetrapyrrole-type nucleus of the formula --L--P, wherein --L-- is selected from the group consisting of ##STR3## and P is a second Gp, which is one of the formulae 1-6 but lacks R4 and is conjugated to L through the position shown as occupied by R4, or another porphyrin group.
9. The method of claim 8 wherein, when P is said another porphyrin group, P has the formula: ##STR4## wherein: each R is independently H or lower alkyl (1-4C);
two of the four bonds shown as unoccupied on adjacent rings are joined to R3 ;
one of the remaining bonds shown as unoccupied is joined to R4 ; and
the other is joined to L;
with the proviso that, if R4 is CHCH2, said R3 groups cannot both be carbalkoxyethyl.
10. The method of claim 8, wherein each R3 is --CH2 CH2 COOH or salt, amide, ester or acylhydrazone thereof.
11. The method of claim 8, wherein each of R1 and R2 is carbalkoxy (2-6C).
12. The method of claim 1, wherein the photosensitizer is BPD-MA having the formula of FIG. 2.
13. The method of claim 1, wherein said hydrating step "c." is accomplished at a temperature at or below room temperature.
14. The method of claim 1, wherein said homogenizing or reducing step "d." is accomplished at a temperature at or below room temperature.
15. The method of claim 1, wherein said particle size is reduced to a range of below about 250 nm.
16. The method of claim 15 wherein the particle size is reduced to below about 220 nm.
17. A pharmaceutical composition containing liposomes in the particle size range of about 150 to 300 nm, wherein said liposomes comprise:
a. a therapeutically acceptable amount of a photosensitizer and
b. a mixture of phospholipids comprising:
(1) egg phosphatidyl glycerol ("EPG") and
(2) dimyristoyl phosphatidyl choline ("DMPC"),
wherein the molar ratio of said photosensitizer and said mixture of phospholipids is about 1:7.0 or more phospholipid.
18. The composition of claim 17, wherein said molar ratio of photosensitizer to said mixture of phospholipids is about 1:8.0 or more phospholipid.
19. The composition of claim 17, wherein said hydro-monobenzoporphyrin (Gp) has one of the formulae 1-6 set forth in FIG. 1 and has one or more light absorption maxima between 670-780 nm, or is a metalated or labeled form thereof, wherein:
each R1 and R2 is independently selected from the group consisting of carbalkoxy (2-6C), alkyl (1-6C) sulfonyl, aryl (6-10C) sulfonyl, aryl (6-10C), cyano, and --CONR5 CO-- wherein R5 is aryl (6-10C) or alkyl (1-6C);
each R3 is independently carboxyalkyl (2-6C) or a salt, amide, ester or acylhydrazone thereof, or is alkyl (1-6C); and
R4 is --CHCH2 ; --CHOR4' wherein R4' is H or alkyl (1-6C), optionally substituted with a hydrophilic substituent; --CHO; --COOR4' ; --CH(OR4' 2)CH3 ; --CH(OR4')CH2 OR4' ; --CH(SR4')CH3 ; --CH(NR4')CH3 ; --CH(CN)CH3 ; --CH(COOR4')CH3 ; --CH(OOCR4')CH3 ; --CH(halo)CH3 ; --CH(halo)CH2 (halo); an organic group of less than 12C resulting from direct or indirect derivatization of a vinyl group; or R4 is a 1-3 tetrapyrrole-type nucleus of the formula --L--P, wherein --L-- is selected from the group consisting of ##STR5## and P is a second Gp, which is one of the formulae 1-6 but lacks R4 and is conjugated to L through the position shown as occupied by R4, or another porphyrin group.
20. The composition of claim 19 wherein, when P is said another porphyrin group, it has the formula: ##STR6## wherein each R is independently H or lower alkyl (1-4C);
two of the four bonds shown as unoccupied on adjacent rings are joined to R3 ;
one of the remaining bonds shown as unoccupied is joined to R4 ; and
the other is joined to L;
with the proviso that, if R4 is CHCH2, said R3 groups cannot both be carbalkoxyethyl.
21. The composition of claim 20, wherein each R3 is --CH2 CH2 COOH or a salt, amide, ester or acylhydrazone thereof.
22. The composition of claim 19, wherein each of R1 and R2 is carbalkoxy (2-6C).
23. The composition of claim 19, wherein the hydro-monobenzoporphyrin photosensitizer is BPD-MA having the formula of FIG. 2.
24. The composition of claim 19, wherein said particle size is below about 220 nm.
The invention relates to improved pharmaceutical compositions comprising liposomes incorporating porphyrin photosensitizers and methods for making these liposomes. Specifically, the invention is directed to pharmaceutical liposome compositions comprising a hydro-monobenzo-porphrin photosensitizer and a mixture of phospholipids comprising egg phosphatidyl glycerol ("EPG") and dimyristoyl phosphatidyl choline ("DMPC") in a photosensitizer:phospholipid molar ratio of about 1:7.0 or more phospholipid. The liposomes are made in such a way that the particle size range is about 150 to 300 nm.
The photosensitizing liposome compositions are useful to mediate the destruction of unwanted cells or tissues or other undesirable materials by irradiation or to detect their presence through fluorescence. Particularly preferred hydro-monobenzoporphyrin photosensitizers used in the practice of this invention include those having one or more light absorption maxima in the range of 670-780 nm.
The use of porphyrin compounds and, in particular hematoporphyrin and hematoporphyrin derivatives (HPD), has been known for some time to be useful systemically, when combined with light, for the treatment and diagnosis of malignant cells. The porphyrins have a natural tendency to "localize" in malignant tissue, where they absorb light at certain wavelengths when irradiated, thus providing a means to detect the tumor by the location of the fluorescence. Accordingly, preparations containing the porphyrins are useful in the diagnosis and the detection of tumor tissue. (See, e.g. "Porphyrin Photosensitization", Plenum Press (Kessel et al. eds. 1983)). In addition, When exposed to light at an appropriate wavelength, the porphyrins have the ability to exhibit a cytotoxic effect on the cells or other tissues in which they are localized. (See, e.g., Diamond et al., Lancet, 2:1175-77 (1972); Dougherty et al., "The Science of Photo Medicine", 625-38 (Regan et al., eds. 1982); and Dougherty et al., "Cancer: Principles and Practice of Oncology", 1836-44 (DeVita Jr. et al. eds. 1982)). It has been postulated that the cytotoxic effect of porphyrins is due to the formation of singlet oxygen when exposed to light (Weishaupt et al., Cancer Research, 36:2326-29 (1976)). The successful treatment of AIDS-related oral Kaposi's Sarcoma with a purified form of HPD, Photofrin�, porfimer sodium, was described by Schwietzer et al., Otolaryngology--Head and Neck Surgery, 102:639-49 (1990).
In addition to systemic use for the diagnosis and treatment of tumors, the porphyrins can be used in a variety of other therapeutic applications. For example, photosensitizers are useful in the detection and treatment of atherosclerotic plaques, as disclosed in U.S. Pat. Nos. 4,517,762 and 4,577,636. U.S. Pat. Nos. 4,500,507 and 4,485,806 describe the use of radio-labeled porphyrin compounds for tumor imaging. Porphyrin compounds have also been used topically to treat various skin diseases, as disclosed in U.S. Pat. No. 4,753,958.
A number of porphyrin photosensitizer preparations have been disclosed for therapeutic applications. A photosensitizer preparation widely used during the early stages of photodynamic therapy both for detection and treatment was a crude derivative of hematoporphyrin, also called hematoporphyrin derivative ("HPD") or Lipson derivative, prepared as described by Lipson et al., J, Natl. Cancer Inst., 26:1-8 (1961). A purified form of the active component(s) of HPD was prepared by Dougherty and co-workers by adjustment of the pH to cause aggregation, followed by recovery of the aggregate, as disclosed in U.S. Pat. Nos. 4,649,151; 4,866,168; 4,889,129; and 4,932,934. A purified form of this product is being used clinically under the trademark Photofrin�, porfimer sodium.
Of particular interest in the context of the present invention is a group of modified porphyrins, known as "green porphyrins" (Gp), having one or more light absorption maxima between about 670-780 nm. These Gp compounds have been shown to confer cytotoxicity against target cells at concentrations lower than those required for hematoporphyrin or HPD. Gp compounds can be obtained using Dieis-Alder reactions of protoporphyrin with various acetylene derivatives under the appropriate conditions. Preferred forms of Gp are the hydro-monobenzoporphyrin derivatives ("BPD's"). The preparation and use of the Gp and BPD compounds are disclosed in U.S. Pat. Nos. 4,920,143 and 4,883,790, hereby incorporated by reference into the disclosure of the present application.
While the porphyrin compounds have a natural ability to localize in neoplastic tissue, while being cleared from the normal surrounding tissue, the selectivity of the porphyrin photosensitizers is still somewhat limited. Because tumor tissue generally includes a number of different components, such as malignant cells, a vascular system, macrophages, fibroblasts, etc., the distribution of the photosensitizer within tumor tissue may be highly heterogeneous. This is especially true for those photosensitizers that are not homogeneous and that contain a mixture of components having different degrees of hydro- or liposolubility. Zhou et al., Photochemistry and Photobiology, 48:487-92 (1988). The low selectivity of some of these agents as tumor localizers may lead to side-effects, such as an undesirably systemic hypersensitivity to light. Therefore, an active area of research has been to increase the tumor selectivity of known porphyrin photosensitizers and to identify those porphyrin photosensitizers that may exhibit greater tumor-selectivity. In general, photosensitizers that are more lipophilic tend to exhibit greater tumor-targeting capability. Spikes et al., Lasers in Medical Science, 2:3, 3-15 (1986).
It has recently been shown that the encapsulation of certain drugs in liposomes, prior to administration, has a marked effect on the pharmacokinetics, tissue distribution, metabolism and efficacy of the therapeutic agent. In an effort to increase the tumor selectivity of porphyrin photosensitizers, porphyrin compounds have been incorporated into unilamellar liposomes, resulting in a larger accumulation and a more prolonged retention of the photosensitizer by both cultured malignant cells and in experimental tumors in vivo. Jori et al., Br. J. Cancer, 48:307-309 (1983); Cozzani et al., In Porphyrins in Tumor Phototherapy, 177-183, Plenum Press (Andreoni et al. eds. 1984). This more efficient targeting of tumor tissues by liposome-associated porphyrins may be due in part to the specific delivery of phospholipid vesicles to serum lipoproteins, which have been shown to interact preferentially with hyperproliferative tissue, such as tumors, through receptor-mediated endocytosis. In this manner, the selectivity of porphyrin uptake by tumors has been increased, as compared with photosensitizers dissolved in aqueous solution. See Zhou et al., supra.
Accordingly, hematoporphyrin and hematoporphyrin dimethyl esters have been formulated in unilamellar vesicles of dipalmitoyl phosphatidyl choline (DPPC) and liposomes of dimyristoyl (DMPC) and distearoyl phosphatidyl choline (DSPC). Zhou et al., supra; Ricchelli, New Directions in Photodynamic Therapy, 847:101-106 (1987); Milanesi, Int. J. Radiat. Biol., 55:59-69 (1989). Similarly, HP, Photofrin�, porfimer sodium, and tetrabenzoporphyrins have been formulated in liposomes composed of egg phosphatidyl choline (EPC). Johnson et al., Proc. Photodynamic Therapy: Mechanisms II, Proc. SPIE-Int. Soc. Opt. Eng., 1203:266-80 (1990). Further, freeze-dried pharmaceutical formulations comprising a porphyrin photosensitizer, a disaccharide or polysaccharide, and one or more phospholipids (such as EPG and DMPC) have been made. These formulations form liposomes containing an effective amount of porphyrin photosensitizer upon reconstitution with a suitable aqueous vehicle and are described in Desai et al., copending U.S. application Ser. No. 08/489,850 filed 13 Jun. 1995.
When the process of Desai et al. is applied to large batches, e.g., 5 to 10 liters or greater, up to about 100-200 liters, some decrease in potency has been observed due to loss of product during the sterile filtration step. The loss is related to the particle size reduction step. Thus, due to the importance of photodynamic therapy in the treatment of cancer, there is a continuing need to identify new photosensitizer formulations that are not only stable and capable of selective delivery of a photosensitizer to target tissues, but also that are easily manufactured on a large scale.
Farmer et al., U.S. Pat. No. 4,776,991 discloses the large-scale encapsulation of hemoglobin in liposomes having a narrow liposome size distribution comprising, as phospholipids,
(1) hydrogenated soy phosphatidyl choline ("HSPC"; approximate composition 85% distearoyl phosphatidyl choline and 15% dipalmitoyl phosphatidyl choline);
(2) cholesterol;
(3) negatively charged DMPC; and
(4) alpha-tocopherol.
(Farmer et al., column 3, lines 52-69.) These lipids are mixed in chloroform to form a solution; the chloroform is evaporated away to form a lipid film; and sterile hemoglobin is added to the film with gentle agitation at 35� C. (30�-37� C.) for 45 minutes to form multilamellar liposomes. Rotary agitation of the liposomes is continued at 4� C. (2�-6� C.) for 10-16 hours, and the liposomes are forced through a Microfluidizer� to break multilamellar liposomes and produce large unilamellar liposomes. The interaction cheer of the Microfluidizer� is maintained at 5�-7� C. (Column 4, lines 1-14; column 7, lines 3-9 and 19-21.) The lipids chosen to make the liposomes, however, must be temporarily shrunk by hyperosmotic shock with added saline prior to sterilization by pressure filtration through a standard 0.22 μm sterilizing filter. (Column 4, line 18-22; column 9, lines 55-58.)
Kappas et al., U.S. Pat. No. 5,010,073 issued 23 Apr. 1991, discloses the preparation of liposomes containing a metalloporphyrin with egg phosphatidyl choline ("EPC") being used as the lipid. The EPC is dissolved in chloroform, the metalloporphyrin is added, and the solution is evaporated to dryness. Phosphate buffered saline at room temperature is used to hydrate the lipid film, and the mixture is "vortexed vigorously." Solids are collected by centrifuging at 4� C. The weight ratio of EPC to metalloporphyrin may be greater than 10. (Kappas et al., column 6, lines 46-65.) However, Kappas et al. recommends that the resulting metalloporphyrin liposomes be sonicated prior to injection to prevent the production of large aggregates. (Column 6, line 66 through column 7, line 1.)
Schneider et al., U.S. Pat. No. 5,270,053 issued 14 Dec. 1993, discloses liposome formulations said to be free of solid particles and larger lipid aggregates. (Schneider et al., column 3, lines 52-54.) However, the presence of a specific synthetic lipid is required. (Column 2, lines 51-66.) For example, in Example 21, at columns 13 and 14, 50 grams of a mixture of two such synthetic lipids are dissolved in tertiary butanol, which is then mixed with a solution of 0.5 g zinc-phthalocyanine. Using a dynamic mixer, the solution is mixed with 10 liters of lactose medium cooled to 4� C. to produce a blue, slightly opalescent dispersion. The separation and isolation of large liposomes from the small liposomes is performed, when necessary, by conventional separation methods such as gel filtration or sedimentation. (Column 7, lines 11-17.)
Thus, there remains a need for a large-scale method to produce DMPC/EPG liposomes containing a photosensitizer in small enough particle sizes that large quantities of pharmaceutical compositions containing the liposomes are easily aseptically filtered through standard 0.22 μm sterilizing filters in an efficient manner and without the need for preparing the synthetic lipids of Kappas et al.
The present invention involves a method for making a pharmaceutical composition containing liposomes. The liposomes comprise a therapeutically acceptable amount of a hydro-monobenzoporphyrin photosensitizer and a mixture of phospholipids comprising egg phosphatidyl glycerol ("EPG") and dimyristoyl phosphatidyl choline ("DMPC"). The method of making such liposomes comprises the steps of:
a. combining the photosensitizer and the phospholipids in a molar ratio of about 1:7.0 or more phospholipid in the presence of an organic solvent;
b. removing the organic solvent to form a lipid film;
c. hydrating the lipid film with an aqueous solution at a temperature below 30� C., to form coarse liposomes containing a photosensitizer-phospholipid complex; and
d. homogenizing or reducing the particle size of the coarse liposomes to a particle size range of about 150 to 300 nm at a temperature below about 30� C.
The hydrating and homogenizing/reducing steps "c." and "d." of the invention are preferably performed at a temperature below the glass transition temperature of the photosensitizer:DMPC/EPG phospholipid mixture complex.
Maintaining the hydration temperature and the homogenizing/reducing step at a temperature below 30� C. would not have been expected to produce smaller particle sizes. In fact, the invention is contrary to the conventional wisdom that small particle sizes are achieved by increasing rather than decreasing these temperatures. See, e.g., M. Lee et al., "Size Distribution of Liposomes by Flow Field-Flow Fractionation", J. Pharm. & Biomed. Analysis, 11:10, 911-20 (1993), equation (6) showing particle diameter "d" as inversely related to temperature "T", and FIG. 6b showing liposome preparation I (prepared at about 70� C.) having smaller particle sizes than preparation II (prepared at about 23� C.).
The invention also contemplates the pharmaceutical compositions themselves, which comprise liposomes including a hydro-monobenzoporphyrin photosensitizer and a mixture of DMPC and EPG phospholipids in a photosensitizer-phospholipid molar ratio of about 1:7.0 or more phospholipid, and which have a particle size of about 150 to 300 nm.
These liposome compositions provide nearly 100% encapsulation of the hydro-monobenzoporphyrin photosensitizer, which can be expensive and usually requires a complicated synthetic procedure to produce. Thus, there is no reworking necessary and very little waste of the photosensitizer. In addition, due to their small particle size, the present liposomes exhibit the improved filterability important in producing large-scale batches of a 500 ml to liter or more, as well as improved retention of photosensitizer potency.
Other objects, features, and advantages will occur from the following descriptions of the various embodiments and the accompanying drawings, in which:
FIG. 1 shows the structure of green porphyrin (Gp) compounds used in the liposomal formulations of the invention.
FIGS. 2(a-b) shows the structure of four preferred forms of the hydro-monobenzoporphyrin derivatives (BPD's) of formulas 3 and 4 in FIG. 1.
FIGS. 3(a-b) is a graphic representation of the relationship of filterability to lipid concentration.
FIG. 4 shows a proposed mechanism for the effect of temperature on the aggregation of a hydro-monobenzoporphyrin photosensitizer.
The present invention relates to a pharmaceutical liposome formulation of a hydro-monobenzoporphyrin photosensitizer for use in the photodynamic therapy or diagnosis of tumors, or for a variety of other therapeutic applications. Liposomes are completely closed, lipid bilayer membranes that contain an entrapped aqueous volume. Typically, liposomes are formed spontaneously upon the addition of an aqueous solution to a dry lipid film.
The liposomes of the invention may be unilamellar vesicles having a single membrane bilayer or multilamellar vesicles having multiple membrane bilayers, each bilayer being separated from the next by an aqueous layer. A liposome bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. The structure of the membrane bilayer is such that the hydrophobic (nonpolar) "tails" of the lipid monolayers orient themselves towards the center of the bilayer, while the hydrophilic "heads" orient themselves toward the aqueous phase. Either unilamellar or multilamellar or other types of liposomes may be used in the practice of the present invention.
In a liposome-drug delivery system, a hydrophilic therapeutic agent can be entrapped in the aqueous phase of the liposome and then administered to the patient. Alternatively, if the therapeutic agent is lipophilic, it may associate with the lipid bilayer. Liposomes may be used to help "target" a drug to an active site or to solubilize hydrophobic drugs for parenteral administration. Typically, the hydro-monobenzoporphyrin photosensitizer of the invention is relatively hydrophobic and forms a stable photosensitizer-lipid complex.
The liposomes of the present invention possess certain attributes that make them well-suited for delivering a hydro-monobenzoporphyrin photosensitizer. The liposomes formed in the present invention are "fast breaking" in that the photosensitizer-liposome combination is stable in vitro but, when administered in vivo, the photosensitizer is rapidly released into the bloodstream where it associates with serum lipoproteins. It is believed that this inhibits the photosensitizer from being accumulated in non-target tissues such as the liver.
The photosensitizers useful in the practice of this invention include the hydro-monobenzoporphyrins (the so-called "green porphyrins" or "Gp" compounds) disclosed in U.S. Pat. Nos. 4,920,143 and 4,883,790. Typically, these compounds have one or more light absorption maxima between about 670-780 nm and are poorly water-soluble (less than 1 mg/ml) or water-insoluble. Gp is preferably selected from the group consisting of those compounds having one of the formulae 1-6 set forth in FIG. 1, mixtures thereof, and the metalated and labeled forms thereof.
In FIG. 1, R1 and R2 can be independently selected from the group consisting of carbalkoxy (2-6C), alkyl (1-6C) sulfonyl, aryl (6-10C) sulfonyl, aryl (6-10C), cyano, and --CONR5 CO-- wherein R5 is aryl (6-10C) or alkyl (1-6C). Preferably, however, each of R1 and R2 is carbalkoxy (2-6C).
R3 in FIG. 1 can be independently carboxyalkyl (2-6C) or a salt, amide, ester or acylhydrazone thereof, or is alkyl (1-6C). Preferably R3 is --CH2 CH2 COOH or a salt, amide, ester or acylhydrazone thereof.
R4 is --CHCH2 ; --CHOR4' wherein R4' is H or alkyl (1-6C), optionally substituted with a hydrophilic substituent; --CHO; --COOR4' ; CH(OR4')CH3 ; CH(OR4')CH2 OR4' ; --CH(SR4')CH3 ; --CH(NR4' 2)CH3 ; --CH(CN)CH3 ; --CH(COOR4')CH3 ; --CH(OOCR4')CH3 ; --CH(halo)CH3 ; --CH(halo)CH2 (halo); an organic group of <12C resulting from direct or indirect derivatization of a vinyl group; or R4 consists of 1-3 tetrapyrrole-type nuclei of the formula --L--P, wherein --L-- is selected from the group consisting of ##STR1## and P is a second Gp, which is one of the formulae 1-6 but lacks R4 and is conjugated to L through the position shown as occupied by R4, or another porphyrin group. When P is another porphyrin group, P preferably has the formula: ##STR2## wherein each R is independently H or lower alkyl (1-4C);
two of the four bonds shown as unoccupied on adjacent rings are joined to R3 ;
one of the remaining bonds shown as unoccupied is joined to R4 ; and
the other is joined to L;
with the proviso that, if R4 is --CHCH2, both R3 groups cannot be carbalkoxyethyl. The preparation and use of such compounds is disclosed in U.S. Pat. Nos. 4,920,143 and 4,883,790, which are hereby incorporated by reference.
Even more preferred are hydro-monobenzoporphyrin compounds that are designated as benzoporphyrin derivatives ("BPD's"). BPD's are hydrolyzed forms, or partially hydrolyzed forms, of the rearranged products of formula 1-3 or formula 1-4, where one or both of the protected carboxyl groups of R3 are hydrolyzed. Particularly preferred is the compound referred to as BPD-MA in FIG. 2, which has two equally active regioisomers.
Many desirable hydro-monobenzoporphyrin photosensitizers, such as BPD-MA, are not only insoluble in water at physiological pH's, but are also insoluble in (1) pharmaceutically acceptable aqueous-organic co-solvents, (2) aqueous polymeric solutions, and (3) surfactant/micellar solutions. However, such photosensitizers can still be "solubilized" in a form suitable for parenteral administration by using a liposome composition. For example, BPD-MA can be "solubilized" at a concentration of about 2.0 mg/ml in aqueous solution using an appropriate mixture of phospholipids to form encapsulating liposomes.
The liposomes of the inventions comprise a mixture of the commonly encountered lipids dimyristoyl phosphatidyl choline ("DMPC") and egg phosphatidyl glycerol ("EPG"). The presence of DMPC is important because DMPC is the major component in the composition to form liposomes which can solubilize and encapsulate insoluble hydro-monobenzoporphyrin photosensitizers into a lipid bilayer. The presence of EPG is important because the negatively charged, polar head group of this lipid can prevent aggregation of the liposomes.
Other phospholipids, in addition to DMPC and EPG, may also be present. Examples of suitable additional phospholipids that may also be incorporated into the liposomes of the present invention include phosphatidyl cholines (PCs), including mixtures of dipalmitoyl phosphatidyl choline (DPPC) and distearoyl phosphatidyl choline (DSPC). Examples of suitable phosphatidyl glycerols (PGs) include dimyristoyl phosphatidyl glycerol (DMPG), DLPG and the like.
Other types of suitable lipids that may be included are phosphatidyl ethanolamines (PEs), phosphatidic acids (PAs), phosphatidyl serines, and phosphatidyl inositols.
The molar ratio of the hydro-monobenzoporphyrin photosensitizer to the DMPC/EPG phospholipid mixture can be as low as 1:7.0 or may contain a higher proportion of phospholipid, such as 1:7.5. Preferably, this molar ratio is 1:8 or more phospholipid, such as 1:10, 1:15, or 1:20. This molar ratio depends upon the exact photosensitizer being used, but will assure the presence of a sufficient number of DMPC and EPG lipid molecules to form a stable complex with most hydro-monobenzoporphyrin photosensitizer molecules. When the number of lipid molecules is not sufficient to form a stable complex, the lipophilic phase of the lipid bilayer becomes saturated with photosensitizer molecules. Then, any slight change in the process conditions can force some of the previously encapsulated photosensitizer to leak out of the vesicle, onto the surface of the lipid bilayer, or even out into the aqueous phase.
If the concentration of hydro-monobenzoporphyrin photosensitizer is high enough, it can actually precipitate out from the aqueous layer and promote aggregation of the liposomes. The more unencapsulated photosensitizer that is present, the higher the degree of aggregation. The more aggregation, the larger the mean particle size will be, and the more difficult aseptic or sterile filtration will be. Thus, as demonstrated in Example 1 below, even small changes in the molar ratio can be important in achieving the improved filterability sought in the invention.
Accordingly, slight increases in the lipid content can increase significantly the filterability of the liposome composition by increasing the ability to form and maintain small particles. This is particularly advantageous when working with significant volumes of 500 ml, a liter, five liters, 40 liters, or more, as opposed to smaller batches of about 100-500 ml or less. This volume effect is thought to occur because larger homogenizing devices tend to provide less efficient agitation than can be accomplished easily on a small scale. For example, a large size Microfluidizer� has a less efficient interaction chamber than that one of a smaller size.
A molar ratio of 1.05:3:5 BPD-MA:EPG:DMPC (i.e., slightly less phospholipid than 1:8.0 photosensitizer:phospholipid) may provide marginally acceptable filterability in small batches of up to 500 ml. However, when larger volumes of the composition are being made, a higher molar ratio of phospholipid provides more assurance of reliable aseptic filterability. Moreover, the substantial potency losses that are common in scale-up batches, due at least in part to filterability problems, can thus be avoided.
Cryoprotective Agents and Isotonic Agents
Any cryoprotective agent known to be useful in the art of preparing freeze-dried formulations, such as di- or polysaccharides or other bulking agents such as lysine, may be used in the claimed invention. Further, isotonic agents typically added to maintain isomolarity with body fluids may be used. In a preferred embodiment, a di-saccharide or polysaccharide is used and functions both as a cryoprotective agent and as an isotonic agent.
In an especially preferred embodiment, the particular combination of the phospholipids, DMPC and EPG, and a disaccharide or polysaccharide form a liposomal composition having liposomes of a particularly narrow particle size distribution. When the process of hydrating a lipid film is prolonged, larger liposomes tend to be formed, or the photosensitizer can even begin to precipitate. The addition of a disaccharide or polysaccharide provides instantaneous hydration and the largest surface area for depositing a thin film of the drug-phospholipid complex. This thin film provides for faster hydration so that, when the liposome is initially formed by adding the aqueous phase, the liposomes formed are of a smaller and more uniform particle size. This provides significant advantages in terms of manufacturing ease.
However, it is also possible that, when a saccharide is present in the composition of the invention, it is added after dry lipid film formation, as a part of the aqueous solution used in hydration. In a particularly preferred embodiment, a saccharide is added to the dry lipid film of the invention during hydration.
Disaccharides or polysaccharides are preferred to monosaccharides for this purpose. To keep the osmotic pressure of the liposome composition of the invention similar to that of blood, no more than 4-5% monosaccharides could be added. In contrast, about 9-10% of a disaccharide can be used without generating an unacceptable osmotic pressure. The higher amount of disaccharide provides for a larger surface area, which results in smaller particle sizes being formed during hydration of the lipid film.
Accordingly, the preferred liposomal composition of the present invention comprises a disaccharide or polysaccharide, in addition to the photosensitizer and the mixture of DMPC and EPG phospholipids. When present, the disaccharide or polysaccharide is preferably chosen from among the group consisting of lactose, trehalose, maltose, maltotriose, palatinose, lactulose or sucrose, with lactose or trehalose being preferred. Even more preferably, the liposomes comprise lactose or trehalose.
Also, when present, the disaccharide or polysaccharide is formulated in a preferred ratio of about 10-20 saccharide to 0.5-6.0 DMPC/EPG phospholipid mixture, respectively, even more preferably at a ratio from about 10 to 1.5-4.0. In one embodiment, a preferred but not limiting formulation is lactose or trehalose and a mixture of DMPC and EPG in a concentration ratio of about 10 to 0.94-1.88 to about 0.65-1.30, respectively.
The presence of the disaccharide or polysaccharide in the composition not only tends to yield liposomes having extremely small and narrow particle size ranges, but also provides a liposome composition in which hydro-monobenzoporphyrin photosensitizers, in particular, may be stably incorporated in an efficient manner, i.e., with an encapsulation efficiency approaching 80-100%. Moreover, liposomes made with a saccharide typically exhibit improved physical and chemical stability, such that they can retain an incorporated hydro-monobenzoporphyrin photosensitizer compound without leakage upon prolonged storage, either as a reconstituted liposomal suspension or as a cryodesiccated powder.
Other optional ingredients include minor amounts of nontoxic, auxiliary substances in the liposomal composition, such as antioxidants, e.g., butylated hydroxytoluene, alpha-tocopherol and ascorbyl palmitate; pH buffering agents, e.g., phosphates, glycine, and the like.
Liposomes containing a selected hydro-monobenzoporphyrin photosensitizer as described herein may be prepared by combining the photosensitizer and the DMPC and EPG phospholipids (and any other optional phospholipids or excipients, such as antioxidants) in the presence of an organic solvent. Suitable organic solvents include any volatile organic solvent, such as diethyl ether, acetone, methylene chloride, chloroform, piperidine, piperidine-water mixtures, methanol, tert-butanol, dimethyl sulfoxide, N-methyl-2-pyrrolidone, and mixtures thereof. Preferably, the organic solvent is water-immiscible, such as methylene chloride, but water immiscibility is not required. In any event, the solvent chosen should not only be able to dissolve all of the components of the lipid film, but should also not react with, or otherwise deleteriously affect, these components to any significant degree.
The organic solvent is then removed from the resulting solution to form a dry lipid film by any known laboratory technique that is not deleterious to the dry lipid film and the photosensitizer. Preferably, the solvent is removed by placing the solution under a vacuum until the organic solvent is evaporated. The solid residue is the dry lipid film of the invention. The thickness of the lipid film is not critical, but usually varies from about 30 to about 45 mg/cm2, depending upon the amount of solid residual and the total area of the glass wall of the flask. Once formed, the film may be stored for an extended period of time, preferably not more than 4 to 21 days, prior to hydration. While the temperature during a lipid film storage period is also not an important factor, it is preferably below room temperature, most preferably in the range from about -20 to about 4� C.
The dry lipid film is then dispersed in an aqueous solution, preferably containing a disaccharide or polysaccharide, and homogenized to form the desired particle size. Examples of useful aqueous solutions used during the hydration step include sterile water; a calcium- and magnesium-free, phosphate-buffered (pH 7.2-7.4) sodium chloride solution; a 9.75% w/v lactose solution; a lactose-saline solution; 5% dextrose solution; or any other physiologically acceptable aqueous solution of one or more electrolytes. Preferably, however, the aqueous solution is sterile. The volume of aqueous solution used during hydration can vary greatly, but should not be so great as about 98% nor so small as about 30-40%. A typical range of useful volumes would be from about 75% to about 95%, preferably about 85% to about 90%.
Upon hydration, coarse liposomes are formed that incorporate a therapeutically effective amount of the hydro-monobenzoporphyrin photosensitizer-phospholipid complex. The "therapeutically effective amount" can vary widely, depending on the tissue to be treated and whether it is coupled to a target-specific ligand, such as an antibody or an immunologically active fragment. It should be noted that the various parameters used for selective photodynamic therapy are interrelated. Therefore, the therapeutically effective amount should also be adjusted with respect to other parameters, for example, fluence, irradiance, duration of the light used in photodynamic therapy, and the time interval between administration of the photosensiting agent and the therapeutic irradiation. Generally, all of these parameters are adjusted to produce significant damage to tissue deemed undesirable, such as neovascular or tumor tissue, without significant damage to the surrounding tissue, or to enable the observation of such undesirable tissue without significant damage to the surrounding tissue.
Typically, the therapeutically effective amount is such to produce a dose of hydro-monobenzoporphyrin photosensitizer within a range of from about 0.1 to about 20 mg/kg, preferably from about 0.15-2.0 mg/kg and, even more preferably, from about 0.25 to about 0.75 mg/kg. Preferably, the w/v concentration of the photosensitizer in the composition ranges from about 0.1 to about 8.0-10.0 g/L, when the mixture becomes such a thick gel that it is not possible to handle or administer to a subject by the usual means. Most preferably, the concentration is about 2.0 to 2.5 g/L.
The hydration step should take place at a temperature that does not exceed about 30� C., preferably below the glass transition temperature of the photosensitizer-phospholipid complex formed, even more preferably at room temperature or lower, e.g., 15�-20� C. The glass transition temperature of the photosensitizer-lipid complex can be measured by using a differential scanning microcalorimeter.
In accordance with the usual expectation that the aqueous solubility of a substance should increase as higher temperatures are used, at a temperature around the transition temperature of the complex, the lipid membrane tends to undergo phase transition from a "solid" gel state to a pre-transition state and, finally, to a more "fluid" liquid crystal state. At these higher temperatures, however, not only does fluidity increase, but the degree of phase separation and the proportion of membrane defects also increases. This results in an increasing degree of leakage of the photosensitizer from inside the membrane to the interface and even out into the aqueous phase. Once a significant amount of liposome leakage has occurred, even slight changes in the conditions such as a small drop in temperature, can shift the equilibrium away from aqueous "solubility" in favor of precipitation of the photosensitizer. Moreover, once the typically water-insoluble photosensitizer begins to precipitate, it is not possible to re-encapsulate it within the lipid bilayer. The precipitate is thought to contribute significantly to filterability problems.
In addition, the usual thickness of a lipid bilayer in the "solid" gel state (about 47 Å) decreases in the transition to the "liquid" crystalline state to about 37 Å, thus shrinking the entrapped volume available for the hydro-monobenzoporphyrin photosensitizer to occupy. The smaller "room" is not capable of containing as great a volume of photosensitizer, which can then be squeezed out of the saturated lipid bilayer interstices. Any two or more vesicles in the process of exuding photosensitizer may aggregate together, introducing further difficulties with respect to particle size reduction and ease of sterile filtration. See FIG. 4 for an illustration of one proposed mechanism to explain this surprising effect of temperature on photosensitizer liposome aggregation. Moreover, the use of higher hydration temperatures, such as, for example, about 35� to 45� C., can also result in losses of photosensitizer potency as the photosensitizer either precipitates or aggregates during aseptic filtration.
The particle sizes of the coarse liposomes first formed in hydration are then homogenized to a more uniform size, reduced to a smaller size range, or both, to about 150 to 300 nm, preferably also at a temperature that does not exceed about 30� C., preferably below the glass transition temperature of the photosensitizer-phospholipid complex formed in the hydration step, and even more preferably below room temperature of about 25� C. Various high-speed agitation devices may be used during the homogenization step, such as a Microfluidizer�, for example a Microfluidics� Model 110F; a sonicator; a high-shear mixer; a homogenizer; or a standard laboratory shaker.
It has been found that the homogenization temperature should be at room temperature or lower, e.g., 15�-20� C. At higher homogenization temperatures, such as about 32�-42� C., the relative filterability of the liposome composition may improve initially due to increased fluidity as expected, but then, unexpectedly, tends to decrease with continuing agitation due to increasing particle size.
Preferably, a high pressure device such as a Microfluidizer� is used for agitation. In microfluidization, a great amount of heat is generated during the short period of time during which the fluid passes through a high pressure interaction chamber. In the interaction chamber, two streams of fluid at a high speed collide with each other at a 90� angle. As the microfluidization temperature increases, the fluidity of the membrane also increases, which initially makes particle size reduction easier, as expected. For example, filterability can increase by as much as four times with the initial few passes through a Microfluidizer� device. The increase in the fluidity of the bilayer membrane promotes particle size reduction, which makes filtration of the final composition easier. In the initial several passes, this increased fluidity mechanism advantageously dominates the process.
However, as the number of passes and the temperature both increase, more of the hydrophobic hydro-monobenzoporphyrin photosensitizer molecules are squeezed out of the liposomes, increasing the tendency of the liposomes to aggregate into larger particles. At the point at which the aggregation of vesicles begins to dominate the process, the sizes cannot be reduced any further. Surprisingly, particle sizes actually then tend to grow through aggregation.
For this reason, in the method of the invention, the homogenization temperature is cooled down to and maintained at a temperature no greater than room temperature after the composition passes through the zone of maximum agitation, e.g., the interaction chamber of a Microfluidizer� device. An appropriate cooling system can easily be provided for any standard agitation device in which homogenization is to take place, e.g., a Microfluidizer�, such as by circulating cold water into an appropriate cooling jacket around the mixing chamber or other zone of maximum turbulence. While the pressure used in such high pressure devices is not critical, pressures from about 10,000 to about 16,000 psi are not uncommon.
As a last step, the compositions of the inventions are preferably aseptically filtered through a filter having an extremely small pore size, i.e., 0.22 μm. Filter pressures used during sterile filtration can vary widely, depending on the volume of the composition, the density, the temperature, the type of filter, the filter pore size, and the particle size of the liposomes. However, as a guide, a typical set of filtration conditions would be as follows: filtration pressure of 15-25 psi; filtration load of 0.8 to 1.5 ml/cm2 ; and filtration temperature of about 25� C.
A typical general procedure is described below with additional exemplary detail:
(1) Sterile filtration of organic solvent through a hydrophobic, 0.22 μm filter.
(2) Addition of EPG, DMPC, hydro-monobenzoporphyrin photosensitizer, and excipients to the filtered organic solvent, dissolving both the excipients and the photosensitizer.
(3) Filtration of the resulting solution through a 0.22 μm hydrophobic filter.
(4) Transfer of the filtrate to a rotary evaporator apparatus, such as that commercially available under the name Rotoevaporator�.
(5) Removal of the organic solvent to form a dry lipid film.
(6) Analysis of the lipid film to determine the level of organic solvent concentration.
(7) Preparation of a 10% lactose solution.
(8) Filtration of the lactose solution through a 0.22 μm hydrophilic filter.
(9) Hydration of the lipid film with a 10% lactose solution to form coarse liposomes.
(10) Reduction of the particle sizes of the coarse liposomes by passing them through a Microfluidizer� three times.
(11) Determination of the reduced particle size distribution of liposomes.
(12) Aseptic filtration of the liposome composition through a 0.22 μm hydrophilic filter. (Optionally, the solution may first be pre-filtered with a 5.0 μm pre-filter.)
(13) Analysis of photosensitizer potency.
(14) Filling of vials with the liposome composition.
(15) Freeze-drying.
Once formulated, the liposome composition of the invention may be freeze-dried for long-term storage if desired. For example, BPD-MA, a preferred hydro-monobenzoporphyrin photosensitizer, has maintained its potency in a cryodesiccated liposome composition for a period of at least nine months at room temperature, and a shelf life of at least two years has been projected. If the composition is freeze-dried, it may be packed in vials for subsequent reconstitution with a suitable aqueous solution, such as sterile water or sterile water containing a saccharide and/or other suitable excipients, prior to administration, for example, by injection.
Preferably, liposomes that are to be freeze-dried are formed upon the addition of an aqueous vehicle contain a disaccharide or polysaccharide during hydration. The composition is then collected, placed into vials, freeze-dried, and stored, ideally under refrigeration. The freeze-dried composition can then be reconstituted by simply adding water for injection just prior to administration.
Particle Size
The liposomal composition of the present invention provides liposomes of a sufficiently small and narrow particle size that the aseptic filtration of the composition through a 0.22 μm hydrophilic filter can be accomplished efficiently and with large volumes of 500 ml to a liter or more without significant clogging of the filter. A particularly preferred particle size range is below about 300 nm, more preferably below from about 250 nm. Most preferably, the particle size is below about 220 nm.
As seen above, the invention controls three major parameters that can affect the ease of particle size reduction to an unexpected degree. As a result, the filterability, particularly with standard aseptic filtration, is significantly improved in the liposome composition of the invention. These parameters are (1) suitable molar ratio of hydro-monobenzoporphyrin photosensitizer to DMPC-EPG lipid mixture; (2) temperature during the hydration step; and (3) temperature during the homogenization or size reduction step. Of these three factors, the photosensitizer/lipid molar ratio appears to have the greatest effect.
Filterability can be tested by passing a liposome composition through a Microfluidizer� three times and withdrawing a sample with a syringe. The syringe is connected to a 0.22 μm hydrophilic filter and then placed in a syringe pump. The constant rate of piston movement is set at 10 ml/min, and filtrate is collected until the filter becomes blocked by large aggregates of liposome. The volume of the filtrate is then measured and recorded in terms of ml/cm2 or g/cm2, with a square centimeter being the effective filtration area. Thus, filterability for the purposes of the invention is defined as the maximum volume or weight of liposomal composition that can be filtered through a 0.22 μm filter.
Administration and Use
The use of the hydro-monobenzoporphyrin photosensitizers incorporated in the liposomes of the invention is typically for the diagnosis or treatment of cancer. The liposomal compositions are useful in sensitizing neoplastic cells or other abnormal tissue, including infectious agents, to destruction by exposure with light, preferably, visible light. Upon photoactivation, the photosensitizer thought to promote the formation of singlet oxygen, which is responsible for the cytotoxic effect. By incorporating the photosensitizer in the liposomes of the present invention, more efficient sensitization of tumor tissues can be obtained.
In addition, when the photosensitizers of the invention are photoactivated by appropriate excitation wavelengths, they are typically able to fluoresce visibly. This fluorescence can then be used to localize the tumor or other target tissue.
Generally speaking, the concentration of the hydro-monobenzoporphyrin photosensitizer in the liposome depends upon the nature of the photosensitizer used. When BPD-MA is used, for example, the photosensitizer is generally incorporated in the liposomes at a concentration of about 0.10% up to 0.5% w/v. If freeze-dried and reconstituted, this would typically yield a reconstituted solution of up to about 5.0 mg/ml photosensitizer.
The liposome compositions of the invention are typically administered parenterally. Injection may be intravenous, subcutaneous, intramuscular, intrathecal, or even intraperitoneal. However, the liposomes could also be administered by aerosol intranasally or intrapulmonarally.
The quantity of hydro-monobenzoporphyrin photosensitizer liposome formulations to be administered depends on the choice of active ingredients, the conditions to be treated, the mode of administration, the individual subject, and the judgement of the practitioner. Generally speaking, however, dosages in the range of 0.05-10 mg/kg may be needed. The foregoing range is, of course, merely suggestive, as the number of variables in regard to an individual treatment regime is large. Therefore, considerable excursions from these recommended values are expected.
For use as a diagnostic in localizing tumor tissue or in localizing atherosclerotic plaques, the pharmaceutical compositions of the invention are administered systemically in the same general manner as is known with respect to photodynamic therapy. The waiting period to allow the drugs to clear from tissues to which they do not accumulate is approximately the same, for example, from about 30 minutes to about 10 hours. After the compositions of the invention have been permitted to localize, the location of the target tissue is determined by detecting the presence of the photosensitizer.
For diagnosis, the hydro-monobenzoporphyrin photosensitizer compounds incorporated into liposomes may be used along with, or may be labeled with, a radioisotope or other detecting means. If this is the case, the detection means depends on the nature of the label. Scintigraphic labels such as technetium or indium can be detected using ex vivo scanners. Specific fluorescent labels can also be used but, like detection based on fluorescence of the photosensitizers themselves, these labels can require prior irradiation.
For activation of the photosensitizer hydro-monobenzoporphyrin of the invention, any suitable absorption wavelength is used. This can be supplied using the various methods known to the art for mediating cytotoxicity or fluorescence emission, such as visible radiation, including incandescent or fluorescent light sources or photodiodes such as light emitting diodes. Laser light can also be used for in situ delivery of light to a localized photosensitizer. In a typical protocol, for example, several hours prior to irradiation, approximately 0.5-1.5 mg/kg of a green porphyrin photosensitizer is injected intravenously and then excited by an appropriate wavelength.
The methods of preparing the liposomal compositions of the present invention, the compositions themselves, and the method of using them in photodynamic treatment are described in greater detail in the examples below. These examples are readily adapted to the production and use of analogously described liposomes by simple substitutions of appropriate hydro-monobenzoporphyrin photosensitizers, additional phospholipids or alternative methods. The following examples are being presented to describe the preferred embodiments, utilities and attributes of the present invention, but they not meant to limit the invention. For example, although BPD-MA is used as the hydro-monobenzoporphyrin photosensitizer to form liposomes, the invention is not intended to be limited to this particular photosensitizer.
Effect of Molar Ratio on Filterability
Three 100-ml batches of BPD-MA liposomes were prepared at room temperature (about 20� C.), using the following general procedure. BPD-MA, butylated hydroxytoluene ("BHT"), ascorbyl palmirate, and the phospholipids DMPC and EPG were dissolved in methylene chloride, and the resulting solution was filtered through a 0.22 μm filter. The solution was then dried under vacuum using a rotary evaporator until the amount of methylene chloride in the solid residue was not detectable by gas chromatography.
A 10% lactose/water-for-injection solution was then prepared and filtered through a 0.22 μm filter. The lactose/water solution was added to the flask containing the solid residue of the photosensitizer/phospholipid. The solid residue was dispersed in the 10% lactose/water solution and stirred for about one hour, cooled, and passed through a homogenizer. The solution was then filtered through a 0.22 μm Durapore, hydrophilic filter.
Using the foregoing procedure, three different preparations of the BPD-MA liposomal composition, each having a different molar ratio of photosensitizer:EPG:DMPC, was prepared as follows:
TABLE 1______________________________________ Formulations Molar Molar Molar Ratio Ratio RatioComponents 1.05:3:5 1.0:3:5 1.0:3:7______________________________________Photosensitizer 0.21 g 0.21 g 0.21 gEPG 0.65 g 0.68 g 0.68 gDMPC 0.94 g 0.99 g 1.38 gButylated hydroxy 0.0002 g 0.0002 g 0.0002 gtoluene ("BHT")Ascorbic acid 0.002 g 0.002 g 0.002 g6-palmitateLactose NF 10 g 10 g 10 gcrystallineinjectableWater for 100 ml 100 ml 100 mlinjection qs______________________________________
The filterability of these three batches was tested according to the following method: After the liposome composition had been passed three times through a Microfluidize�, a sample was withdrawn with a syringe. The volume of the sample withdrawn depended on a visual estimation of the filterability of the composition. The syringe was connected to a 0.22 μm hydrophilic filter and was then placed in a syringe pump. The rate of piston movement was set at 10 ml/min, and filtrate was collected until the filter became blocked by large liposome aggregates. The volume of the filtrate was measured and recorded as ml/cm2. The results are summarized below in Table 2.
TABLE 2______________________________________Formulations Total Lipid(BPD-MA: Concentration FilterabilityEPG:DMPC) (% w/v) (g/cm2)______________________________________Molar Ratio 1.59 1.571.05:3:5Molar Ratio 1.67 3.581.0:3:5Molar Ratio 2.06 12.61.0.:3:7______________________________________
Table 1 indicates that the filterability of the first formulation (slightly less phospholipid than a 1:8.0 photosensitizer/DMPC-EPG lipid mixture) was about 2.3 times less than the second formulation (which had a molar ratio of 1:8.0) and about eight times less than the 1:10.0 molar ratio of the third formulation. FIG. 3 shows a plot of the filterability in g/cm2 versus total lipid concentration (%, w/v). The relationship was surprisingly linear, with an r-square of 0.9985. The following empirical equation was obtained from this plot:
______________________________________Filterability = 23.36 Total Lipid Concentration (%, w/v)! - 35.51!______________________________________
According to the slope of this equation, for every one-gram increase in DMPC or EPG lipid present, a 23-fold improvement in the filterability of the liposome formulation was achieved. The plot further indicates that, if the total lipid concentration became less than 1.52% with a BPD-MA photosensitizer concentration of 2.1 mg/ml, the liposome composition would not be capable of filtration through a 0.22 μm filter at all.
A larger batch of BPD-MA liposomes (1.2 liters, i.e., 12 times the 100-ml batches of Example 1) was prepared using the molar ratio 1:3:5 (photosensitizer:EPG:DMPC) at room temperature (about 20� C.). The contents of composition is described below in Table 3:
TABLE 3______________________________________ Mole-Compo- Molar No. of cularnents Amount Ratio Moles Weight______________________________________BPD-MA 2.60 g 1 0.00362 718EPG 8.38 g 3 0.01086 772DMPC 12.26 g 5 0.01811 677BHT 0.0024 g -- -- --Ascor- 0.024 g -- -- --bicAcid6-Pal-mitateLactose 120 g -- -- --NFCrys-tallineInjec-tableWater 1.2 -- -- --for litersInjec-tion qs______________________________________
A double thickness of lipid film was used in this larger batch to provide a more strenuous test of the molar ratio of 1:8.0. The results showed a significant improvement in filterability, as compared with a batch 1/12th the size with only slightly less of the DMDC and EPG lipids, as seen below in Table 4:
TABLE 4______________________________________Formu-lations Potency(BPD- Batch Filtera Pre- Post-MA:PG: Size bility filtra- filtra-DMPC) (ml) (g/cm2) tion tion______________________________________Molar 100 1.57 2.11 2.10ratio1.05:3:5Molar 100 3.58 2.05 2.02ratio1:3:5Molar 100 12.60 2.11 2.23ratio1:3:7Molar 1,200 5.00 2.09 2.08ratio1:3:5______________________________________
The slight increase in lipid content greatly increased the filterability of the liposome composition. Moreover, the yield was nearly 100%, with a 99.5% (by HPLC analysis) photosensitizer potency being maintained after sterile filtration.
Four batches of BPD-MA liposome compositions, each having the same photosensitizer/DMPC-EPG lipid molar ratio (1.05:3:5), were prepared using different film hydration temperatures. Data for these four batches, presented below in Table 5, were compared to demonstrate the relationship of potency loss and film hydration temperature.
TABLE 5______________________________________ Hydration Temperature Potency LossBatch (�C.) (%)______________________________________1 30 9.52 Room 4.8 temperature (about 25� C.)3 30 16.74 40 85.0______________________________________
The results showed that an increase in hydration temperature above room temperature was associated with an undesirable, significant loss in the potency of the photosensitizer.
A pair of 0.53-liter batches (photosensitizer:EPG:DMPC=1.05:3:5) was used to study the relationship between filterability and homogenization temperature. The compositions were prepared as described above in Example 1 using a hydration temperature of 45� C. and a Microfluidizer� with the outlet temperature set at 35� C. The data showed that, at high hydration and homogenization temperatures, the relative filterability of BPD-MA liposome compositions increased after the initial passes through a Microfluidizer� (#1 to #2), as expected, but then decreased for additional passes (#2 to #4), as seen below:
TABLE 6______________________________________Pass # Filterability (ml/cm2)______________________________________1 0.102 0.273 0.224 0.19______________________________________
The second 0.53-liter batch (photosensitizer:EPG:DMPC=1.05:3:5) was used to study the relationship between the number of passes through a Microfluidizer�, filterability, and particle size distribution. In this batch, both a high hydration temperature (40� C.) and a high homogenization temperature (Microfluidizer� outlet temperature set at 42� C.) were used. The data is listed below in Table 7.
TABLE 7______________________________________ Particle Size Filterability DistributionPass # (ml/cm2) (+50 nm)______________________________________1 N/A 7102 N/A 7103 0.25 2654 0.25 2505 0.50 2506 0.50 2477 0.30 3028 0.25 3029 0.25 295______________________________________
The data show that the filterability increased from passes #3 to #5, as expected, but then dropped back to the original value in passes #6 to #9. In contrast, the average particle size of the liposomes in the composition was reduced from 710 nm at pass #1 to as small as 247 nm at pass #6. However, as additional passes were made, the particle size unexpectedly increased to about 300 nm.
A 100-ml batch of BPD-MA liposomes is prepared at room temperature (about 20� C.) using the following general procedure. BPD-MA, butylated hydroxytoluene ("BHT"), ascorbyl palmirate, and the phospholipids DMPC and EPG are dissolved in methylene chloride. The molar ratio of photosensitizer:EPG:DMPC is 1.0:3:7 and has the following composition:
______________________________________Photosensitizer 0.21 gEPG 0.68 gDMPC 1.38 gBHT 0.0002 gAscorbic acid 6- 0.002 gpalmitateLactose NF 10 gcrystallineinjectableWater for injection 100 mlqs______________________________________
Using the above formulation, the total lipid concentration (% w/v) is about 2.06. The resulting solution is filtered through a 0.22 μm filter and then dried under vacuum using a rotary evaporator. Drying is continued until the amount of methylene chloride in the solid residue is no longer detectable by gas chromatography.
A 10% lactose/water-for-injection solution is then prepared and filtered through a 0.22 μm filter. Instead of being warmed to a temperature of about 35� C., the lactose/water solution is allowed to remain at room temperature (about 25� C.) for addition to the flask containing the solid residue of the photosensitizer/phospholipid. The solid residue is dispersed in the 10% lactose/water solution at room temperature, stirred for about one hour, and passed through a Microfluidizer� homogenizer three to four times with the outlet temperature controlled to about 20�-25� C. The solution is then filtered through a 0.22 μm Durapore, hydrophilic filter.
The filterability of the batch is tested by the following procedure: After the liposome composition has been passed three times through a Microfluidizer�, a sample is withdrawn with a syringe. The volume of the sample withdrawn is about 20 ml, depending on a visual estimation of the filterability of the composition and assessment of the quality of the liposomes. The syringe is connected to a 0.22 μm hydrophilic filter and is then placed in a syringe pump. The rate of piston movement is set at 10 ml/min, and filtrate is collected until the filter becomes blocked by large liposome aggregates. The filtrate is measured and recorded as ml/cm2 or g/cm2.
The filterability of the composition in g/cm2 is typically greater than about 10. Moreover, the yield is about 100% by HPLC analysis, with photosensitizer potency typically being maintained even after sterile filtration. Average particle sizes vary from about 150 to about 300 nm (�50 nm).
It will be clear to those skilled in the art that modifications and/or variations of the disclosed subject matter can be made without departing from the scope of the invention claimed below.
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Donald Miller
Author of New York Times Bestsellers "Blue Like Jazz" and "A Million Miles in a Thousand Years"
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Donald Miller is a best-selling American author and public speaker based in Portland, Oregon, who focuses on Christian spirituality as "an explanation for beauty, meaning, and the human struggle."
Miller's first book, "Prayer and the Art of Volkswagen Maintenance," chronicled a cross-country road trip he took at age 21 after leaving home. Republished in 2005 as "Through Painted Deserts," it was commercially successful. Miller wrote "Blue Like Jazz" in 2003, which would slowly become a New York Times Bestseller. The movie "Blue Like Jazz" was released in 2008. Miller had also written the screenplay along with Steve Taylor and Ben Pearson.
In 2004, Don released "Searching for God Knows What," a book about how the Gospel of Jesus explains the human personality. "Searching" has become required reading at numerous colleges across the country. Don's 2006 recent project was a book about growing up without a father called "To Own a Dragon." Don is the founder of The Belmont Foundation, a not-for-profit foundation which partners with local churches to create mentoring programs for young men growing up without fathers.
Don's next book, "A Million Miles in a Thousand Years," was released in late 2009.
Miller serves on President Barack Obama's Task Force on Fatherhood and Healthy Families.
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Dallas, TX (SportsNetwork.com) - Erik Cole scored twice as the Dallas Stars took a 4-1 victory over the Nashville Predators on Friday.
Jamie Benn and Ryan Garbutt each posted a goal for the Stars, who have won four of their last five games. Kari Lehtonen made 33 saves in the win.
"We put the puck in the right spot a lot of the times and were able to create some pressure, and we have numbers back when teams are coming out," said Dallas head coach Lindy Ruff. "We have to give our forwards a lot of credit for coming back and doing the work their doing and getting shot leads and blocking more shots."
Kevin Klein scored the only goal of the game for the Predators, who matched a season-high with their fifth straight loss. Carter Hutton allowed all four goals on 34 shots.
"We've been giving up a lot of early goals and tonight I can't blame the goaltender, Carter (Hutton) was pretty decent tonight and our goaltending was adequate tonight," said Phoenix head coach Barry Trotz. "We needed some guys to snipe, we had Lehtonen down a couple of times and they didn't snipe."
Dallas took the initial lead in the game as Benn took the puck down the left wing and skated behind the net before wrapping the puck in at the right side just 42 seconds in.
Nashville tied the game at 7:14 of the second period as Colin Wilson skated from behind the net to the low left circle and held up. He then sent the puck to the slot where a flying Klein one-timed it home for his first of the season.
Dallas, though, made it a 2-1 game just 24 seconds later as Cole scored his first of the game on a wrister from the right circle with traffic in front.
The Stars then made it a 3-1 contest at 14:04 of the second as Cole skated down the right wing to the front and slammed on the brakes before switching to the backhand and stuffing it in past the left pad of Hutton.
It was a 4-1 game at 4:09 of the third as Benn sent a shot on net from the right point that was tipped by Cody Eakin then tipped by Garbutt into the net.
Dallas hosts St. Louis on Sunday ... Nashville hosts Los Angeles on Saturday
... Nashville took a 3-1 win against Dallas at home on Dec. 12 ... Both teams
were 0-for-3 on the power play.
Oklahoma running back Keith Ford has been suspended indefinitely for violating academic and team rules, Sooners coach Bob Stoops confirmed Monday.
Qualifying for NASCAR national touring series races at Daytona International Speedway and Talladega Superspeedway will no longer feature a pack of cars running together.
If Teddy Bridgewater taught us anything, it's trust the tape, at least over a pro day.
The Boston Red Sox have released pitcher Felipe Paulino.
Minnesota Timberwolves coach Flip Saunders announced that center Nikola Pekovic would miss the remainder of the season after sustaining an ankle injury in March.
The Minnesota Wild agreed to terms on entry-level contracts with goaltender Brody Hoffman and defenseman Zach Palmquist on Monday.


text:
After numerous heart surgeries, local woman to run in Fargo half-marathon
When 30-year Katie Sandberg of Detroit Lakes was a freshman in college, she remembers walking up only one flight of stairs to the MSUM library and becoming so embarrassed she hid behind the bookcases.
"I was so out of breath, and there were sharp pains in my lungs, so I hid behind those bookshelves from my friends until I could catch my breath enough to talk — one flight of stairs," said Sandberg.
Are you a newspaper subscriber but you don't have a Digital Access account yet? https://secure.forumcomm.com/?publisher_ID=40&event=subscriber/lookup.
You will need your subscription account number and phone number. Not sure if you have an account? Email us at firstname.lastname@example.com and we can help you.


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More Videos...
08 Charger SRT8 vs Regal GS Drag Race
6-30-10 - Lebanon Valley Drag Way - test and tune - GS was seriously Boosted
Buick Regal GS vs Cobalt SS S/C
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car and the guy with the cobalt tell me that he was just stage 1 but I
think it was a Stage 2 because I race some cobalt in the past and a stage 1
is not fast has he is I smoke him but he was catching a little bit before I
let off throttle at the end
Drag St-Eustache ete 2010
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mille ;)
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Be carefull out there things happen fast on the track.. subaru sti hits
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The Buick Avenir concept is a flagship sedan exploring progressive design,
withall-new levels of passenger well-being and technology integration. 2016
Buick Avenir
The interior of the Avenir was designed specifically with the driver
andpassenger's state of well-being in mind. It was designed to rejuvenate
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1987 turbo-t (13.23 @ 109) vs supercharged 3800 buick regal gs (14.01@103)
1987 Buick turbo-T racing a fwd
supercharged 3800 Buick Regal at Earlville, IA.
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**I made an editing error at the beginning. Thanks a bunch to Streetside
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Hello and welcome to Saabkyle04! YouTube's largest collection of automotive
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identical except for badging and interior trim. It wasn't until you
stepped it on up to the GNX did you get the truly different package (which
I will explain in the video). The GN is arguably the most menacing looking
buick of all time with it's black monochrome paint scheme and turbo charged 3.8 V6 pumping out amazing
performance numbers for the time period, even today! A unique experience
to be able to check out one of these beasts. During this presentation, we
will take a 1st person look at what the vehicle is all about beginning with
the start up, performance data, fuel economy, the occasional track data,
and build quality. Also, I will teach you how to use most of the interior
and exterior features in a detailed fashion, that before, you could only
get from going to a dealership yourself! Throughout the video, I will
highlight key styling and unique differences about the vehicle, any
available options, and of course it would not be an enthusiast car video
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FOR MORE INFORMATION ABOUT THE CHANNEL SEE BELOW: In this channel you will
find in depth reviews/tour of automobiles from all over the world,
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gives the viewer the chance to view the most detailed 1st person look you
can get without going to an actual dealer yourself! Every video is
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presentations. I treat every vehicle with the same respect whether a 1960
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vehicles from past, present, and future while broadening the knowledge of
the automotive enthusiast. You will see everything from vintage, brand new,
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power-plant thanks to its 264 cubic inch V8, and style that was, well...
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the old Buick a bit differently though. He knew that with a few little
touches (like a 502 cubic inch big-block, modern suspension, and a new set
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( http://www.TFLcar.com ) The 2014 Buick Regal GS is the sportiest car in
Buick's new 2014 line-up. The big news for 2014 is a new refreshed
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improved and that the GS button in the car makes for a fun and exciting
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Buick Regal. Is this newest Buick Regal GS the real deal. You'll have to
watch Roman's review to find out.
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Twitter: ( https://www.twitter.com/tflcar )
and now even Truck Videos on YouTube at:
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Similar 1/4 mile timeslips to browse:
1987 Buick Regal Turbo-T: 8.690 @ 158.460
Dan Strezo, Engine: 274 c.i., Turbos: T88-T4 Tires: MT 29.5x10.5 Non-W
1986 Buick Regal Grand National T-type: 8.900 @ 154.000
Jack Cotton, Engine: 4.1l, V6, Turbos: single Turbonetics T76 Tires: MT 29-10.5-15
1984 Buick Regal : 9.610 @ 146.000
1981 Buick Regal GS: 9.980 @ 139.071
RONALD BEY, Engine: 494 ci, Tires: FUTURA
1987 Buick Regal : 10.200 @ 132.120
Rich Lenz, Engine: 454, Supercharger: none Turbos: none
1986 Buick Regal : 10.270 @ 132.970
Bob Heinz,
1986 Buick Regal T-Type WH1 Designers Series: 10.750 @ 122.000
Banning Cohen, Engine: .040 over stock block 3.8v6, Turbos: PT67 Tires: 28x11.50x15 Hooser QT Pro
1987 Buick Regal Turbo T: 11.251 @ 121.000
John, Engine: 231, Turbos: 1 Tires: Mickey Thompson ET Radials
1986 Buick Regal limited: 11.290 @ 120.320
timmy, Engine: 464 buick, Tires: 205x15 ft 28x10.5x15 micky thom rear
1987 Buick Regal T-Type: 11.500 @ 120.000
Bob V, Engine: 3.8 L, Turbos: CPT-65 Tires: BFG DR
1987 Buick Regal Limited T: 11.670 @ 117.000
Timothy Dirks, Engine: 3.8, Turbos: Stage V Ball Bearing Tires: Hoosier QTP 26x9
1987 Buick Regal Turbo: 12.060 @ 114.280
Wes Ingram, Engine: 3.8 V6 stock, Turbos: 46 Tires: G60 slicks
1987 Buick Regal Turbo T: 12.090 @ 111.980
Bill Heckman, Engine: 231 CI /Stock, Supercharger: no Turbos: PE 51 Tires: MT ET Drag Slicks
1987 Buick Regal : 12.160 @ 113.010
Chris Walker, Engine: 231, Turbos: TA-49 Tires: Radial TA's
1987 Buick Regal Turbo T: 12.314 @ 110.480
JOHN DICARLO, Engine: 231 V6, Turbos: TE-44 Tires: Sumitomo street tires
1981 Buick Regal : 12.535 @ 113.460
George Pantazelos, Engine: 455 Buick, Supercharger: none Turbos: none Tires: Goodyear
1982 Buick Regal : 12.660 @ 107.130
Bob Holifield, Engine: 355 Chevy, Tires: Goodyear (F) M/T ET Drag (R)
2001 Buick Regal GS: 12.664 @ 111.780
Brian Atwood, Engine: 3800 Series III, Supercharger: Gen V Eaton M90 (stock) Tires: BALD Nitto 555r's
2000 Buick Regal GS: 12.693 @ 109.090
Bob Carboni, Engine: 3.8L supercharged L67, Supercharger: Eaton M90 Turbos: T72 Tires: MT ET Street
2001 Buick Regal GS: 12.882 @ 107.970
Brian Atwood, Engine: 3.8 Series III, Supercharger: Eaton M90 Gen V Tires: Nitto 555R
©2015 DragTimes - Disclaimer - Contact Us


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West Nusa Tenggara Province Sports & Outdoors
West Nusa Tenggara Province Hotels
Qunci Villas
I stayed in Qunci Villas for 5 nights in January 2011 - it was a beautiful stay for a number of...more
Tambora Hotel
Hotel Tambora is located in the west of Sumbawa Besar city about one and a half kilometres from the...more
The Lombok Lodge
Jl. Oberoi, Mataram, 83001, Indonesia
Satisfaction: Excellent
Good for: Business
Tao' Kombo Travel Lodge
Gili Meno, Indonesia
Satisfaction: Very Good
Good for: Couples
Sheraton Senggigi Beach Resort
Its located at west part of Lombok island. 1,5 hour from airport and half hour from Bangsal harbour....more
Pool Villa Club
Jalan Pantai Senggigi, Mataram, 83010, Indonesia
Satisfaction: Excellent
Good for: Business
Jeeva Klui Resort
Best experience ever, staff were always helpful and courteous Breakfast are all al carte, no...more
Lombok Garden Hotel
Jl. Bung Karno No. 7, (formerly Hotel Granada), Mataram, 83231, Indonesia
Satisfaction: Very Good
Good for: Solo
Novotel Lombok
E' successo per caso, non era previsto, ma come tante le cose che accadono per caso si è rivelato un...more
Top West Nusa Tenggara Province Hotels
- Sumbawa Besar Hotels
- 45 Reviews - 205 Photos
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Belief Quotes and Sayings - Page 4
Never believe anything on the basis of belief alone.
Man prefers to believe what he prefers to be true.
– Francis Bacon
When others in doubt, your strong belief steers.
– Toba Beta
Believing is easier than thinking. Hence so many more believers than thinkers.
~Bruce Calvert
It is ok to doubt what you have been taught to believe.
You can fly without wings if you believe.
There are two different types of people in the world, those who want to know, and those who want to believe.
– Friedrich Nietzsche
It takes courage to act, takes faith to continue acting and it takes belief to act right.
The really important thing about religion is that your faith and your belief help you to live a better life, sustain you in sorrow, and make you face the end of life with serenity.
Belief is the death of intelligence.
– Robert Anton Wilson
Superstitions are habits rather than beliefs.
– Marlene Dietrich
Thinking ends where belief begins.
A belief which leaves no place for doubt is not a belief; it is superstition.
– Jose Bergamin
Don't be changed by the world and evil temptations. But change the world by staying true to what you believe in.
We all are born non-believers, but we are taught to believe in what our parents believe in.
Killing is not so easy as the innocent believe.
– J.K. Rowling
Belief is when someone else does the thinking.
~Buckminster Fuller
Belief creates the actual fact.
Believe in everything because everything's reachable. (B. I. E. B. E. R)


text:
Find commute times to your work, school, gym and more.
Ellipse at Fairfax Corner
Contact this property
Floor Plans
Prices, specials, features and availability subject to change.
Contact for details (571) 388-2992
Business Center, Clubhouse, Fitness Center, Hot Tub, Media Center, On Site Maintenance, On Site Management, Playground, Swimming Pool, Tennis Court(s)
Barbecue Grill, Bocce Ball Court, Car Wash Area, Close to Vienna Metro (Orange Line), Dog Park, Media Lounge with Fireplace, Pet Friendly Community, Picnic Area, Short Term Available, Trail for: Biking, Hiking, or Jogging, Walking Trail
Property Details
Brand new designer finishes and a fantastic location near Wegman's - all at an unbeatable price! Make the move today!
Discover apartment living that offers the ideal combination of comfort, convenience and luxury at Ellipse at Fairfax Corner. Our apartments in Fairfax, Virginia feature beautifully renovated interiors and access to resort-style amenities that make apartment living a true pleasure. Enjoy the finer things in life in a location that promises the best Fairfax has to offer. We have eight unique floor plans from which you can choose ranging from 700 to more than 1,100 square feet. No matter which layout you choose, you can look forward to enjoying a private patio or balcony, a cozy fireplace and a gourmet kitchen with updated cabinetry and countertops. You can start your day off right wi
Leasing Terms
Our lease terms are: 3 months, 6 months, 9 months, 12 months, We offer multiple lease terms (Please note that lease terms may vary, are subject to change without notice, and are based on availability. Inquire with property staff for complete details)
Dog Policy: Maximum 2 dogs per apartment. Restrictions: Pit Bull, Staffordshire Terrier, Rottweiler, Doberman Pinscher, Chow, Spitz, Akita, and any other combination of the restricted breeds.
Cat Policy: Maximum 2 cats per apartment.
Pet Policy
Dogs Allowed. Cats Allowed. Pets Accepted. Call for service animal policy.
Community Information
404 units
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Scores are compared to the national average. The higher the bar, the higher the crime risk. The lower the bar, the lower the crime risk.
near Fairfax, 22030Transit
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Office Hours
Monday-Wednesday: 9:00am-6:00pm
Thursday: 9:00am-7:00pm
Friday: 9:00am-6:00pm
Saturday: 10:00am-5:00pm
Sunday: 12:00pm-5:00pm
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(340 ratings)
September 28, 2010
By: YouPorn.com
This video is part of the following collections:
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June 26, 2013, opinion of the U.S. Supreme Court in United States vs. Windsor, the case challenging the federal Defense of Marriage Act. Click here for full coverage of the same-sex marriage case.
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( Patrick Tehan )
Bigfoot is my BFF
A visit to the Bigfoot Discovery Museum in Felton will bolster your belief or seal your skepticism.
(Photo by Kevin Winter/Getty Images)
Angelina Jolie inspires Nickelodeon crowd
'Maleficent' actress tells kids being different is a good thing, and it's OK to cause a little trouble.


text:
"Irene Samuel" Chairs Israeli Brunch
Active co-chairmen of the annual Israeli Brunch of National Council of Jewish Women, front row, l-r, Mrs. G. Diamond (Leslie) and Mrs. C. Diamond (Isabelle), are shown here with some of the Brunch committee heads, standing from left to right, Mesdames J. Bass, D. Tessler (Francis), L. Pearlman (Marg) and I. Lerner (Celia). Not shown in the picture are: Mesdames I. Bernstein, R. Toban, R. Loomer and A. Nemetz. In keeping with the accepted practice of National Council of Jewish Women for branches to assume responsibility of major Section projects, Irene Samuels Branch in Vancouver Section, have for many years successfully chaired and convened the Annual Israeli Brunch. Original article in the Jewish Western Bulletin, December 4, 1964:


text:
Roll up your sleeves for a two-day, introductory workshop on how to prepare an effective electronic Common Technical Document (eCTD) submission. This in-person workshop includes skill-building, interactive exercises, case studies and small group discussions.
Rockville, MD4/1/2015
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Local or global—RAPS has you covered with offices in Washington, DC, Shanghai and Singapore. There are RAPS chapters in Canada, Israel, Switzerland, Taiwan and throughout the United States, and local networks in Australia, Belgium, Brazil, Germany, India, Mexico, The Netherlands, Poland and Singapore.
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overview of expedited pathways for US medical devices that are alternatives to Traditional
510(k)s and Premarket Approval Applications (PMAs). These include Special
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Regulatory Focus is pleased to team with Tarius, a regulatory information services provider, to routinely offer insight about upcoming FDA Scientific Advisory Committee (SAC) meetings for CDER, CBER, and the Office of the Commissioner.
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#228 of 289
Charlotte, NC
Comment, Like, Favorite
Expert Printing and Assembly
Shipping Within 2 - 3 Business Days
Millions of Satisfied Customers
Stepping Out Into The Spotlight
Sue Melvin
Digital Art - Digital Photograph
I used a digital art technique called "out of frame," "out of bounds" or "oob" to create a 3-dimensional illusion of this cute little eastern bluebird stepping out into the spotlight.
*This image has been featured in three FAA groups*
June 10th, 2011
Viewed 545 Times - Last Visitor from Borger, TX on 03/27/2015 at 2:44 AM
Sales Sheet
eastern bluebird canvas prints
eastern bluebird framed prints
eastern bluebird throw pillows
More from Sue Melvin
Temple City, CA - United States
Very captivating, 3D art - love it.
Gallatin, TN - United States
I'm a fan too Sue. This piece came out wonderful! I love doing the OOB pictures. Very nice work indeed!
Chicago, IL - United States
Such a cute little guy! I also like this OOF work Sue! ...He definitely looks like he would enjoy being in a bit of the spotlight :) f/v
Charlotte, NC - United States
Wow... Randy! Are you sure you really like this OOF creation??? LOL You just made my day, my friend! How kind of you to promote my work in this major way, too! All very much appreciated! :-))
Iola, WI - United States
Totally awesome imagery, Sue, as this is one your best of the best! Love the OOF idea and a great form of artistic endeavor, and thanks much for sharing! Got my fave, vote, Tweet, FB, and g+1 as it is out of this world!
Bentonville, AR - United States
Love little Bluebirds--voted
Beloit, WI - United States
How cool is that?!?! Great picture, Sue! Voted.
Orange Park, FL - United States
Sue, Congratulations on your 2nd place win in the OOF - Out Of Frame - Birds contest! Outstanding image! v/f
Charlotte, NC - United States
Brian- thanks! Always enjoy your OOF contests and was honored to be among the winners! I appreciate the feature of this photo in your OOF - Out of Frame group! :-)
Pasadena, MD - United States
Gallatin, TN - United States
Congrats on your win! Great work!
Sydney, Ne - Australia
Another gorgeous birdie capture, Sue... and your out of frame work is stunning. V&F
Charlotte, NC - United States
Debra- thanks so much for featuring this photo in your Designs With Photography group! :-)
Oklahoma City, OK - United States
Wow! I love this, Sue. Very special work.
Lakeland, FL - United States
Sue this is brilliant, you've brought him right off the canvas.
Livonia, Mi - United States
Really stunning, Sue! In the spotlight, indeed!
Pueblo, CO - United States
Sue~Love this image. Instant vote & fave.
Charlotte, NC - United States
John- thanks so much for featuring this photo in your Creativity group! :-)
Lake Country, BC - Canada
I have no idea how you do these, Sue, but they are terrific and must be very popular!! SU
Sandy, UT - United States
I've not learned this beauiful digital technique. This is so very gorgeous. Sue! Looks 3-D.
Cebu, B7 - Philippines
Sue, this is beautiful! The bluebird seemed to have arrived at another world.
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Beverly Hills, CA (United States)
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