CA2228944C - Liposomal phosphodiester, phosphorothioate, and p-ethoxy oligonucleotides - Google Patents
Liposomal phosphodiester, phosphorothioate, and p-ethoxy oligonucleotides Download PDFInfo
- Publication number
- CA2228944C CA2228944C CA2228944A CA2228944A CA2228944C CA 2228944 C CA2228944 C CA 2228944C CA 2228944 A CA2228944 A CA 2228944A CA 2228944 A CA2228944 A CA 2228944A CA 2228944 C CA2228944 C CA 2228944C
- Authority
- CA
- Canada
- Prior art keywords
- oligonucleotide
- oligos
- composition
- liposomal
- ethoxy
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/312—Phosphonates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
Abstract
An improved delivery system for antisense oligonucleotides involves a liposomal composition, comprising a liposome which consists essentially of neutral phospholipids and an antisense oligonucleotide that is entrapped in the liposome and is selected from the group consisting of phosphodiester oligonucleotides, phosphorothioate oligonucleotides, and p-ethoxy oligonucleotides.
Description
LIPOSOMAL PHOSPHODIESTER, PHOSPHOROTHIOATE, AND
p-ETHOXY OLIGONUCLEOTIDES
The present invention relates to liposomal formulations of certain antisense oligonucleotides, specifically liposomal phosphodiester, phosphorothioate, and p-ethoxy oligonucleotides. The invention also relates to methods of making such formulations and methods of using such formulations in medical therapy.
Antisense oligonucleotides (oligos), complementary to specific regions of the target mRNA, have been used to inhibit the expression of endogenous genes.
When the antisense oligonucleotides bind to the target mRNA, a DNA-RNA hybrid is formed. This hybrid formation inhibits the translation of the mRNA and, thus, the gene's expression of the protein. If the protein is essential for the survival of the cell, the inhibition of its expression may lead to cell death. Therefore, antisense oligonucleotides can be useful tools in anticancer and antiviral therapies.
The main obstacles in using antisense oligonucleotides to inhibit gene expression are cellular instability, low cellular uptake, and poor intracellular delivery.
= Natural phosphodiesters are not resistant to nuclease hydrolysis; thus high concentrations of antisense oligonucleotides are needed before any inhibitory effect is observed. Modified phosphodiester analogs, such as phosphorothioates, have been made to overcome this nuclease hydrolysis problem, but they have not provided a completely satisfactory solution to the problem.
The cellular uptake of antisense oligonucleotides is low. To solve this problem, physical techniques such as calcium-phosphate precipitation, DEAE-dextran mediation, or electroporation have been used to increase the cellular uptake of oligonucleotides. These techniques are difficult to reproduce and are inapplicable in vivo. Cationic lipids, such as Lipofectin, have also been used to deliver phosphodiester or phosphorothioate oligonucleotides. An electrostatic interaction is formed between the cationic lipids and the negatively charged phosphodiester or phosphorothioate oligonucleotides, which results in a complex that is then taken up by the target cells. Since these cationic lipids do not protect the oligonucleotides from nuclease digestion, they are only useful in delivering the nuclease-resistant phosphorothioates, but not the nuclease-cleavable phosphodiesters.
Another modified phosphodiester (PD) analog that has been prepared is p-ethoxy (pE) oligos. The modifications of pE oligos are made in the phosphate backbone so that the modification will not interfere with the binding of these oligos to the target mRNA. pE oligos are made by adding an ethyl group to the nonbridging oxygen atom of the phosphate backbone, thus rendering these oligos uncharged compounds. In spite of their resistance to nucleases, the cellular uptake and intracellular delivery of pE oligos are still poor because upon internalization, these oligos remain sequestered inside the endosomal/lysosomal vacuoles, impeding their access to the target mRNA.
There is a need for improved antisense compositions for use in treatment of disease, and also a need for processes for making such improved compositions.
One aspect of the present invention is a liposomal composition of antisense oligonucleotides, comprising (a) a liposome consisting essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is a p-ethoxy oligonucleotide. The phospholipids are preferably phosphatidylcholines. An especially preferred phospholipid is dioleoylphosphatidyl choline. When the antisense oligonucleotide is a phosphodiester oligonucleotide, the preferred molar ratio of phospholipid to oligo is less than about 3,000:1. When the antisense oligonucleotide is a phosphorothioate oligonucleotide, the preferred molar ratio of phospholipid to oligo is between about 10:1 and about 50:1. When the antisense oligonucleotide is a p-ethoxy oligonucleotide, the preferred molar ratio of phospholipid to oligo is between about 5:1 and about 100:1.
According to a preferred embodiment of the invention, there is provided a liposomal composition of antisense oligonucleotides, comprising (a) a liposome which consists essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is a p-ethoxy oligonucleotide, wherein the phospholipid and p-ethoxy oligonucleotide are in a molar ratio of between about 5:1 and about 100:1.
Another aspect of the present invention is a method of inhibiting the growth of tumor cells in mammals.
The method comprises the step of administering to a mammalian subject having a tumor an amount of the above-described composition that is effective to inhibit the growth of tumor cells.
Another aspect is a use for inhibiting the growth of tumor cells in a mammalian subject having a tumor, of an - - 3a -amount effective to inhibit the growth of tumor cells of a composition comprising (a) a liposome which consists essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is selected from the group consisting of phosphodiester oligonucleotides, phosphorothioate oligonucleotides, and p-ethoxy oligonucleotides.
According to a preferred embodiment of the invention, there is provided a use for inhibiting the growth of tumor cells in a mammalian subject having a tumor, of an amount effective to inhibit the growth of tumor cells of a composition comprising (a) a liposome that consists essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is a p-ethoxy oligonucleotide.
Another aspect is a use of a composition in the manufacture of a medicament for inhibiting the growth of tumor cells in a mammalian subject having a tumor, said composition in an amount effective to inhibit the growth of tumor cells and comprising: (a) a liposome that consists essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is selected from the group consisting of phosphodiester oligonucleotides, phosphorothioate oligonucleotides, and p-ethoxy oligonucleotides.
According to a preferred embodiment of the invention, there is provided a use of a composition in the manufacture of a medicament for inhibiting the growth of tumor cells in a mammalian subject having a tumor, said composition in an amount effective to inhibit the growth of tumor cells and comprising: (a) a liposome that consists essentially of neutral phospholipids, and (b) an antisense - 3b -oligonucleotide that is entrapped in the liposome and is a p-ethoxy oligonucleotide.
Another aspect of the present invention is a method of preparing the liposomal composition of antisense oligonucleotides, comprising the steps of (a) hydrating a lyophilized composition that consists essentially of neutral phospholipids and an antisense oligonucleotide that is selected from the group consisting of phosphodiester oligonucleotides, phosphorothioate oligonucleotides, and p-ethoxy oligonucleotides, thereby forming an aqueous suspension which includes free oligonucleotide and liposomes entrapping oligonucleotide; and (b) separating the free oligonucleotide from the liposomes by dialysis. In a preferred embodiment, the aqueous suspension is sonicated before dialysis.
The compositions of the present invention constitute an improved delivery system for antisense oligos, such as those used in anti-cancer therapy. In addition to minimizing nuclease hydrolysis of the oligos, the liposomal compositions of the present invention result in increased cellular uptake and intracellular delivery of the antisense oligos, as compared to prior art compositions. Therefore, when such compositions are used to deliver oligos that inhibit the expression of a gene found in cancerous cells but not in normal cells, the therapeutic results are enhanced.
Cancers which may be treated in accordance with the present invention would be numerous, with leukemias being one prominent example.
The formulations of the present invention also enhance the incorporation of oligos in the liposomes, as compared to prior art liposomal formulations.
Figures IA and I B are photographs showing the uptake of (A) free and (B) liposomal pE oligos by ALL-1 cells.
Figure 2A shows the extent of growth inhibition that resulted when ALL-1 and HL60 cells were incubated with liposomal-pE antisense oligos targeted against the B1/A2 breakpoint junction of Bcr-Abl mRNA.
Figure 2B shows the extent of growth inhibition that resulted when ALL-1 cells were incubated with B 1 /A2 liposomal-pE antisense oligos and B2/A2 liposomal-pE control oligos.
Figure 3A shows the extent of growth inhibition that resulted when BV173 and HL60 cells were incubated with liposomal-pE antisense oligos targeting against the B2/A2 breakpoint junction of Bcr-Abl mRNA.
Figure 3B shows the extent of growth inhibition that resulted when BV173 cells were incubated with liposomal-pE antisense oligos targeting against the breakpoint junction and with control oligos targeting against the B1/A2 breakpoint junction.
Figure 4A shows the extent of growth inhibition that resulted when K562 and HL60 cells were incubated with liposomal-pE antisense oligos targeting against the B3/A2 breakpoint junction of Bcr-Abl mRNA.
Figure 4B shows the extent of growth inhibition that resulted when K562 cells were incubated with liposomal-pE antisense oligos targeting against the B3/A2 breakpoint junction and with control oligos targeting against the B1/A2 breakpoint junction.
Figure 5 shows the extent of growth inhibition that resulted when Tween 20-containing liposomal-pE oligos were incubated with BV 173 cells.
"Liposomes" is used in this patent to mean lipid-containing vesicles having a lipid bilayer, as well as other lipid carrier particles which can entrap antisense oligonucleotides. The liposomes can be made of one or more phospholipids, as long as the lipid material is substantially uncharged. It is important that the composition be substantially free of anionic and cationic phospholipids and cholesterol.
Suitable phospholipids include phosphatidyl cholines and others that are well known to persons that are skilled in this field. The liposomes can be, for example, unilamellar, multilamellar, or have an undefined lamellar structure. A pharmaceutical composition comprising the liposomes will usually include a sterile, pharmaceutically acceptable carrier or diluent, such as water or saline solution.
"Entrap," "encapsulate," and "incorporate" are used in this patent to mean that the oligo is enclosed within at least some portion of the inner aqueous space (including the interlamellar regions of the bilayer) of the liposome.
A composition of the present invention is preferably administered to a patient parenterally, for example by intravenous, intraarterial, intramuscular, intralymphatic, intraperitoneal, subcutaneous, intrapleural, or intrathecal injection, or may be used in ex vivo bone marrow purging. Preferred dosages are between 5-25 mg/kg. The administration is preferably repeated on a timed schedule until the cancer disappears or regresses, and may be in conjunction with other forms of therapy.
The making and use of the present invention is further illustrated by the following examples.
Materials Phosphodiester and phosphorothioate oligonucleotides were provided by Genta Incorporated. Phospholipids were purchased from Avanti Polar Lipids.
Oligonucleotide Labeling Phosphodiesters were labeled at 37 C for 8 h with [32Py]ATP at the 5' end by T4 kinase. The labeled oligonucleotide was precipitated with ethanol at -20 C
overnight. After washing with 70% ethanol three times, phosphodiester oligonucleotides were twice filtered with a Microcon-3 filter to separate the labeled oligonucleotides from free [32Py]ATP.
Phosphorothioates, labeled with 35S, were provided by Genta Incorporated.
Liposomal-phosphodiester Preparation Phosphodiester oligonucleotides dissolved in distilled water were mixed with phospholipids in the presence of excess t-butanol so that the final volume of t-butanol in the mixture was 80-90%. Trace amounts of [3H]cholestanyl ether and [32P]phosphodiester were also added to the mixture as lipid and oligonucleotide markers, respectively. The mixture was vortexed before being frozen in an acetone/dry ice bath. The frozen mixture was lyophilized and hydrated with hepes buffered saline (1 mM Hepes and 10mM NaCl) overnight. Liposomes were twice sonicated for 10 min in a bath type sonicator.
Liposomal-phosphorothioate Preparation Liposomal-phosphorothioates were prepared similarly to that described for liposomal-phosphodiesters, except phosphorothioates, instead of phosphodiesters, were used. Also, [35S]phosphorothioates, instead of [32P]phosphodiesters, were used as the oligonucleotide marker.
Separation of Free Oligonucleotides from those Incorporated in Liposomes The separation of free phosphodiester or phosphorothioate oligonucleotides from those incorporated in liposomes was done by dialyzing the mixture against an excess 2500-fold volume of RPMI medium at room temperature overnight. Aliquots of the preparation were taken before and after dialysis for liquid scintillation counting to assess the incorporation of phosphodiester or phosphorothioate oligonucleotides in liposomes.
Development of liposomal-phosphodiesters Dioleoylphosphatidylcholine (DOPC) lipids were chosen for the phosphodiester (PD) incorporation because they are neutral while PD is negatively charged. By using this lipid, electrostatic repulsion (which can lower incorporation) may be reduced. Positively charged lipids were not used because they may induce non-specific cellular toxicity. The initial attempts of incorporating PD
oligonucleotides into liposomes were done by using freeze-and-thaw, and dehydration-rehydration methods.
(A) Freeze-and-thaw (FT) Method.
[3H]labeled DOPC lipids were evaporated from their organic solvent under nitrogen gas to form a lipid film. After vacuum desiccation, the lipid film was hydrated with hepes buffered saline (1 mM Hepes, 10 mM NaCl, pH 8.0) and sonicated in a bath type sonicator. These pre-formed liposomes were then mixed with [32P]labeled PD oligonucleotides at a 100 or 1000 to 1 molar ratio. The whole mixture was frozen in an acetone/dry ice bath for 5-10 minutes and thawed at room temperature for 25-30 minutes. This process was repeated three times before the sample was loaded on a BioGel A0.5M column for separation of free PD
oligonucleotides from liposomal-PD. Aliquots were taken before and after the freeze-thaw procedure and were sent to liquid scintillation counting to determine the incorporation of phosphodiesters into liposomes.
Incorporation was determined by [32P1PD/umole f3H1DOPC after freeze-thaw X100%
[32P]PD/ mole [3H]DOPC before freeze-thaw There was no incorporation (Table 1).
(B) Dehydration-Rehydration (DR) Method [3H]labeled DOPC was evaporated from the organic solvent under nitrogen gas to form a lipid film. After vacuum desiccation, the lipid film was hydrated with hepes buffered saline and sonicated in a bath type sonicator. These pre-formed liposomes were then frozen in an acetone-dry ice bath and lyophilized. The dried lipids were then rehydrated with distilled water containing [32P]phosphodiester oligonucleotides. The molar ratio of DOPC to PD was either 100/1 or 1000/1.
The mixture was loaded on a BioGel A0.5M column for separation of free PD
oligonucleotides from liposomal-PD. Aliquots were taken before and after column loading and were sent to liquid scintillation counting. The incorporation was < 5 %
(Table 1).
Table 1 Comparison of Freeze-thaw (FT) and Dehydration-rehydration (DR) methods on the incorporation of PD into DOPC liposomes.
Molar ratio Methods % incorporation DR 2.0 DR 4.6 With 500/1 and 5000/1 molar ratios of DOPC to PD, 0 and 5.2%
incorporation were obtained, respectively.
It was then found that by adding t-butanol to the mixture before freezing and lyophilization, incorporation was raised to 11.9%. Also, by reducing the volume of distilled water added during the rehydration process from 200 to 50 l, 11.9 versus 1.8% incorporation was obtained with 1000/1 molar ratio. Effect of molar ratio on the incorporation was again measured with these improved conditions (Table 2).
Table 2 Effect of lipid to oligonucleotides molar ratio on the incorporation of PD
into liposomes.
Molar ratio % incorporation 1000/1 11.9 2000/1 17.0 3000/1 13.4 4000/1 5.5 500011 9.2 PD dissolved in distilled water was mixed with DOPC in the presence of excess t-butanol so that the final volume of t-butanol in the mixture was 80-90%
before being frozen in an acetone/dry ice bath and lyophilized. This was to avoid the step of preparing pre-formed liposomes. With this procedure, similar level of incorporation (16.2%) was obtained at the 2000/1 molar ratio.
It was decided to change the method of separation of free PD from liposomal-PD because the recovery of lipids and PD were < 50%. Two other separation methods were used: Microcon-10 filters and dialysis (Table 3).
Table 3 Comparison of different methods of separating free PD from PD incorporated in liposomes.a Method % Incorporation % Lipid and PD recovery Column 11.9 40-50 Filter 44.7 60-70 Dialysis 76.7 >90 a DOPC was used to incorporate PD oligonucleotides at a 1000/1 molar ratio.
When the liposomal mixture was sonicated for 10 min before the dialysis separation process, similar level of incorporation was obtained. Another lipid, dimyristoyl phosphatidylcholine, was also used for PD incorporation. There was > 85 % incorporation.
Development of liposomal-phosphorothioates Similar incorporation protocol was used with phosphorothioates (PT) since PT and PD are structural analogs. Various molar ratios of DOPC to PT were used (Table 4). The effect of sonication of the liposomal mixture (before dialysis) was also studied.
Table 4 Effect of lipid to oligonucleotide molar ratios on the incorporation of PT
into liposomes.
% incorporation Molar ratio without sonication with sonication 10/1 >90 >90 5011 >90 >90 100/1 45.8 55.5 200/1 44.1 49.1 50011 27.8 47.0 1000/1 25.1 42.1 Incorporation of p-ethoxy oligos into liposomes pE oligos were purchased from Oligos Therapeutics (Wilisonville, OR).
Phospholipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL).
WO 97(407794 PCT/US96/14146 Oligo labeling pE oligos were labeled at 37 C for 24 h with [32Py]ATP at the 5' end by T4 polynucleotide kinase, and then precipitated with ethanol at -20 C overnight.
They were then washed with 70% ethanol three times to separate the labeled oligo from free [32Py]ATP.
(b Liposome preparation pE oligos dissolved in distilled H2O were mixed with phospholipids at various molar ratios in the presence of excess t-butanol so that the final volume of t-butanol in the mixture was at least 95%. Trace amounts of [3H]cholestanyl ether and [32P]pEs were also added to the mixture as lipid and oligo markers, respectively. The mixture was vortexed, frozen in an acetone/dry ice bath and then lyophilized. The lyophilized preparation was hydrated with Hepes buffered saline (1 mM Hepes and 10 mM NaC1) at a final oligo concentration of 10-100 M. The liposomal-p-ethoxy oligos were sonicated for 10-20 min in a bath type sonicator.
Separation of free pE oligos from those incorporated in liposomes The separation of free pE oligos from those incorporated in liposomes was done by dialyzing (MW cutoff = 12-14,000) against 1000-fold excess of Hepes buffered saline at room temperature overnight. Aliquots of liposomal-pE oligos were taken before and after dialysis for liquid scintillation counting to access the incorporation of pE oligos in liposomes.
(d) Incorporation efficiency The lipid phosphatidylcholine (PC) was chosen for the incorporation of pE
oligos because both PC and pE oligos are neutral molecules so they should be compatible. Among all the different PCS, dioleoyl PC (DOPC) was chosen because it has a chain-melting phase transition temperature at -15 to -20 C. Thus, at room temperature, DOPC is in the liquid crystalline phase which is the ideal phase to prepare liposomes.
To incorporate pE oligos into liposomes, different molar ratios of pE oligos were mixed with DOPC together in the presence of excess of t-butanol. Trace amounts of radio labeled pE oligos and DOPC were included in the mixture. The DOPC/pE oligos mixtures were frozen in a dry ice/acetone bath before being lyophilized. The lyophilized DOPC/pE oligo powder was then hydrated with Hepes buffered saline so that the final oligo concentration was 10 .tM. pE oligos were successfully incorporated into DOPC liposomes, ranging between 28 to 83 %
efficiency (Table 5). The incorporation efficiency was dependent on the molar ratios of DOPC to pE oligos: 10 > 100 > 5 > 1000:1.
Table 5 Effect of molar ratio of DOPC to pE oligos on the incorporation of pE oligos Molar ratio of Incorporation DOPC:pE oligos efficiency (%)a 5:1 45 10:1 83 100:1 71 1000:1 28 aThe incorporation efficiency values were obtained from three separate experiments.
Delivery of pE oligos to leukemic cells After achieving a high incorporation efficiency (> 80%) of pE oligos into liposomes, we then proceeded to test whether these liposomal-pE oligos can reach the cytoplasm in which the mRNA is located. We had purchased a 16-mer pE oligo labeled with rhodamine at the 5' end so that we could visualize the localization of the pE oligos by fluorescent microscopy.
Incubation of pE oligos with leukemic cells ALL-1 cells, which are human acute lymphocytic leukemic cells, were used.
Fifty thousand ALL-i cells/well were plated in a 24-well plate in 0.3 mL of medium. After 2 h of plating, final concentrations of 16 M of liposomal or free pE oligos conjugated with rhodamine were added to ALL-1 cells. After 24 h of incubation, the cells were thrice washed with phosphate buffered saline before being viewed under a confocal laser scanning microscope. See Figure 1 (Uptake of (A) free or (B) liposomal-pE oligos by ALL-1 cells).
Our data indicates that when incorporated into liposomes, higher amounts of pE oligos were taken up by the ALL-1 cells. The liposomes were able to deliver the pE oligos to the cytoplasm.
Growth inhibition of liposomal-pE oligos on leukemic cells We then proceeded to test whether liposomal-pE oligos can specifically inhibit the growth of leukemic cells. We have used three different kinds of human leukemic cell lines: ALL-1 (acute lymphocytic leukemia), BV173 and K562 (both are chronic myelogenous leukemia). All three cell lines contain the rearranged Philadelphia (Ph) chromosome which arises from a reciprocal translocation of chromosomes 9 and 22. This translocation results in the relocation of the c-Abl protooncogene from chromosome 9 onto the 3' end of the breakpoint cluster region (Bcr) of chromosome 22, thus producing a hybrid Bcr-Abl gene. The breakpoint junctions where the Bcr and the Abl genes fuse are different in the three cell lines.
In ALL-1 cells, the breakpoint junction is Bcr exon 1/ Abl exon 2. In BV173 cells, the breakpoint junction is Bcr exon 2/ Abl exon 2. In K562 cells, the breakpoint junction is Bcr exon 3/ Abl exon 2. All these hybrid genes produce a novel Bcr-Abl fusion protein, which has enhanced tyrosine kinase activity that has been linked to the pathogenesis of the leukemias. Thus, inhibition of the production of the Bcr-Abl protein may lead to leukemic cell growth inhibition and even cell death.
To specifically inhibit the production of the Bcr-Abl protein, we have decided to target the antisense sequences against the breakpoint junctions of the Bcr-Abl mRNA
which is only found in Ph chromosome-positive leukemic cells, but not normal cells. This way, we hope to induce minimal non-specific toxic side effects as only leukemic, not normal, cell growth will be affected.
Sequences of the pE antisense oligos (written from 5' to 3' end) antisense against Bcr exon 1/ Abl exon 2 (B1/A2) found in ALL-1 cells GAAGGGCTTCTGCGTC
antisense against Bcr exon 2/ Abl exon 2 (B2/A2) found in BV173 cells CTGAAGGGCTTCTTCC
antisense against Bcr exon 3/ Abl exon 2 (B3/A2) found in K562 cells GGGCTTTTGAACTCTGCT
(b Delivery of liposomal-pE oligos to leukemic cells Ten thousand ALL-1 or BV 173 cells or five thousand K562 cells were plated per well in a 96-well plate in 100 L of RPMI medium containing 10% fetal calf serum. After 2 h of plating, final concentrations of 0 - 10 M of liposomal-pE
oligos were added to leukemic cells. The cells were incubated with liposomal-pE
oligos for 5 days. HL60 cells, a human promyelocytic cell line which does not have the Philadelphia chromosome, were used as control cells. They were plated under the same conditions at ten thousand cells/well.
(c) Determination of the viability of the leukemic cells At the end of the incubation, 100 gL of medium were added to each well which makes the final volume of each well to be 200 L. Then 50 L of cells were aliquoted and added to 96-well plates containing 130 L. of medium and 20 L
of alamarBlue dye. The cells will be incubated for 4-8 more hours at 37 C before being read directly on a microplate reader (Molecular Devices, Menlo Park, CA) at 570 and 595 nm. The alamarBlue dye incorporates an oxidation-reduction indicator that changes color in response to chemical reduction of growth medium resulting from cell growth. The difference in absorbance between 570 and 595 nm will be taken as the overall absorbance value of the leukemic cells. The viabilities of leukemic cells treated with liposomal-pE oligos will be compared with those of the control untreated cells.
When ALL-1 and HL60 cells were incubated with liposomal-pE antisense oligos targeted against the B1/A2 breakpoint junction of Bcr-Abl mRNA, a dose-dependent growth inhibition of ALL-1, but not HL60, cells was observed (Fig.
2A). Similarly growth inhibition was observed with BV173 and K562 cells when they were incubated with liposomal-pE antisense oligos targeting against the and B3/A2 breakpoint junctions of Bcr-Abl mRNA, respectively (Fig. 3A, 4A).
Under identical conditions, HL60 cells were not growth-inhibited.
To ensure that the growth inhibitory effects were sequence-dependent, the Ph chromosome-positive, cell lines were incubated with antisense and control liposomal-pE oligos. When ALL-1 cells were incubated with the B1/A2 liposomal-pE antisense oligos and the B2/A2 liposomal-pE control oligos, growth inhibition was induced (Fig. 2B). However, the B1/A2 antisense oligos induced a much greater inhibitory effect. Similarly, higher inhibitory effects on BV173 and cells were found with the corresponding liposomal-pE antisense oligos than with the control oligos (Fig. 3B, 4B).
We also found that by including the detergent Tween 20 in the liposomal-pE
oligo mixture, the potency of the inhibitory effects of the liposomal-pE
oligos was increased. We added Tween 20 at 5 % (wt. of pE oligos) in the liposomal-pE
oligo mixture. Then the mixture was vortexed, frozen in an acetone/dry ice bath before being lyophilized. The dried mixture was then hydrated and sonicated as stated previously. When Tween 20-containing liposomal-pE oligos were added to BV173 cells, 100% growth inhibition was observed at 5 M (Fig. 5) whereas under the same conditions, 100% growth inhibition with normal liposomal-pE oligos (no Tween 20) was observed at 10 M instead.
* * *
The preceding description of specific embodiments of the present invention is not intended to be a complete list of every possible embodiment of the invention.
Persons skilled in this field will recognize that modifications can be made to the specific embodiments described here that would be within the scope of the present invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Board of Regents, The University of Texas System (ii) TITLE OF INVENTION: LIPOSOMAL PHOSPHODIESTER, PHOSPHOROTHIOATE, AND p-ETHOXY OLIGONUCLEOTIDES
(iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Arnold, White & Durkee (B) STREET: 750 Bering Dr., Ste. 400 (C) CITY: Houston (D) STATE: TX
(E) COUNTRY: USA
(F) ZIP: 77057-2198 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy Disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version 41.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/520,385 (B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Goodman, Kenneth D.
(B) REGISTRATION NUMBER: 30,460 (C) REFERENCE/DOCKET NUMBER: UTFC433P--(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 713/787-1460 (B) TELEFAX: 713/789-2679 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GAAGGGCTTC TGCGTC
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CTGAAGGGCT TCTTCC
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: õ
GGGCTTTTGA ACTCTGCT
p-ETHOXY OLIGONUCLEOTIDES
The present invention relates to liposomal formulations of certain antisense oligonucleotides, specifically liposomal phosphodiester, phosphorothioate, and p-ethoxy oligonucleotides. The invention also relates to methods of making such formulations and methods of using such formulations in medical therapy.
Antisense oligonucleotides (oligos), complementary to specific regions of the target mRNA, have been used to inhibit the expression of endogenous genes.
When the antisense oligonucleotides bind to the target mRNA, a DNA-RNA hybrid is formed. This hybrid formation inhibits the translation of the mRNA and, thus, the gene's expression of the protein. If the protein is essential for the survival of the cell, the inhibition of its expression may lead to cell death. Therefore, antisense oligonucleotides can be useful tools in anticancer and antiviral therapies.
The main obstacles in using antisense oligonucleotides to inhibit gene expression are cellular instability, low cellular uptake, and poor intracellular delivery.
= Natural phosphodiesters are not resistant to nuclease hydrolysis; thus high concentrations of antisense oligonucleotides are needed before any inhibitory effect is observed. Modified phosphodiester analogs, such as phosphorothioates, have been made to overcome this nuclease hydrolysis problem, but they have not provided a completely satisfactory solution to the problem.
The cellular uptake of antisense oligonucleotides is low. To solve this problem, physical techniques such as calcium-phosphate precipitation, DEAE-dextran mediation, or electroporation have been used to increase the cellular uptake of oligonucleotides. These techniques are difficult to reproduce and are inapplicable in vivo. Cationic lipids, such as Lipofectin, have also been used to deliver phosphodiester or phosphorothioate oligonucleotides. An electrostatic interaction is formed between the cationic lipids and the negatively charged phosphodiester or phosphorothioate oligonucleotides, which results in a complex that is then taken up by the target cells. Since these cationic lipids do not protect the oligonucleotides from nuclease digestion, they are only useful in delivering the nuclease-resistant phosphorothioates, but not the nuclease-cleavable phosphodiesters.
Another modified phosphodiester (PD) analog that has been prepared is p-ethoxy (pE) oligos. The modifications of pE oligos are made in the phosphate backbone so that the modification will not interfere with the binding of these oligos to the target mRNA. pE oligos are made by adding an ethyl group to the nonbridging oxygen atom of the phosphate backbone, thus rendering these oligos uncharged compounds. In spite of their resistance to nucleases, the cellular uptake and intracellular delivery of pE oligos are still poor because upon internalization, these oligos remain sequestered inside the endosomal/lysosomal vacuoles, impeding their access to the target mRNA.
There is a need for improved antisense compositions for use in treatment of disease, and also a need for processes for making such improved compositions.
One aspect of the present invention is a liposomal composition of antisense oligonucleotides, comprising (a) a liposome consisting essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is a p-ethoxy oligonucleotide. The phospholipids are preferably phosphatidylcholines. An especially preferred phospholipid is dioleoylphosphatidyl choline. When the antisense oligonucleotide is a phosphodiester oligonucleotide, the preferred molar ratio of phospholipid to oligo is less than about 3,000:1. When the antisense oligonucleotide is a phosphorothioate oligonucleotide, the preferred molar ratio of phospholipid to oligo is between about 10:1 and about 50:1. When the antisense oligonucleotide is a p-ethoxy oligonucleotide, the preferred molar ratio of phospholipid to oligo is between about 5:1 and about 100:1.
According to a preferred embodiment of the invention, there is provided a liposomal composition of antisense oligonucleotides, comprising (a) a liposome which consists essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is a p-ethoxy oligonucleotide, wherein the phospholipid and p-ethoxy oligonucleotide are in a molar ratio of between about 5:1 and about 100:1.
Another aspect of the present invention is a method of inhibiting the growth of tumor cells in mammals.
The method comprises the step of administering to a mammalian subject having a tumor an amount of the above-described composition that is effective to inhibit the growth of tumor cells.
Another aspect is a use for inhibiting the growth of tumor cells in a mammalian subject having a tumor, of an - - 3a -amount effective to inhibit the growth of tumor cells of a composition comprising (a) a liposome which consists essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is selected from the group consisting of phosphodiester oligonucleotides, phosphorothioate oligonucleotides, and p-ethoxy oligonucleotides.
According to a preferred embodiment of the invention, there is provided a use for inhibiting the growth of tumor cells in a mammalian subject having a tumor, of an amount effective to inhibit the growth of tumor cells of a composition comprising (a) a liposome that consists essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is a p-ethoxy oligonucleotide.
Another aspect is a use of a composition in the manufacture of a medicament for inhibiting the growth of tumor cells in a mammalian subject having a tumor, said composition in an amount effective to inhibit the growth of tumor cells and comprising: (a) a liposome that consists essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is selected from the group consisting of phosphodiester oligonucleotides, phosphorothioate oligonucleotides, and p-ethoxy oligonucleotides.
According to a preferred embodiment of the invention, there is provided a use of a composition in the manufacture of a medicament for inhibiting the growth of tumor cells in a mammalian subject having a tumor, said composition in an amount effective to inhibit the growth of tumor cells and comprising: (a) a liposome that consists essentially of neutral phospholipids, and (b) an antisense - 3b -oligonucleotide that is entrapped in the liposome and is a p-ethoxy oligonucleotide.
Another aspect of the present invention is a method of preparing the liposomal composition of antisense oligonucleotides, comprising the steps of (a) hydrating a lyophilized composition that consists essentially of neutral phospholipids and an antisense oligonucleotide that is selected from the group consisting of phosphodiester oligonucleotides, phosphorothioate oligonucleotides, and p-ethoxy oligonucleotides, thereby forming an aqueous suspension which includes free oligonucleotide and liposomes entrapping oligonucleotide; and (b) separating the free oligonucleotide from the liposomes by dialysis. In a preferred embodiment, the aqueous suspension is sonicated before dialysis.
The compositions of the present invention constitute an improved delivery system for antisense oligos, such as those used in anti-cancer therapy. In addition to minimizing nuclease hydrolysis of the oligos, the liposomal compositions of the present invention result in increased cellular uptake and intracellular delivery of the antisense oligos, as compared to prior art compositions. Therefore, when such compositions are used to deliver oligos that inhibit the expression of a gene found in cancerous cells but not in normal cells, the therapeutic results are enhanced.
Cancers which may be treated in accordance with the present invention would be numerous, with leukemias being one prominent example.
The formulations of the present invention also enhance the incorporation of oligos in the liposomes, as compared to prior art liposomal formulations.
Figures IA and I B are photographs showing the uptake of (A) free and (B) liposomal pE oligos by ALL-1 cells.
Figure 2A shows the extent of growth inhibition that resulted when ALL-1 and HL60 cells were incubated with liposomal-pE antisense oligos targeted against the B1/A2 breakpoint junction of Bcr-Abl mRNA.
Figure 2B shows the extent of growth inhibition that resulted when ALL-1 cells were incubated with B 1 /A2 liposomal-pE antisense oligos and B2/A2 liposomal-pE control oligos.
Figure 3A shows the extent of growth inhibition that resulted when BV173 and HL60 cells were incubated with liposomal-pE antisense oligos targeting against the B2/A2 breakpoint junction of Bcr-Abl mRNA.
Figure 3B shows the extent of growth inhibition that resulted when BV173 cells were incubated with liposomal-pE antisense oligos targeting against the breakpoint junction and with control oligos targeting against the B1/A2 breakpoint junction.
Figure 4A shows the extent of growth inhibition that resulted when K562 and HL60 cells were incubated with liposomal-pE antisense oligos targeting against the B3/A2 breakpoint junction of Bcr-Abl mRNA.
Figure 4B shows the extent of growth inhibition that resulted when K562 cells were incubated with liposomal-pE antisense oligos targeting against the B3/A2 breakpoint junction and with control oligos targeting against the B1/A2 breakpoint junction.
Figure 5 shows the extent of growth inhibition that resulted when Tween 20-containing liposomal-pE oligos were incubated with BV 173 cells.
"Liposomes" is used in this patent to mean lipid-containing vesicles having a lipid bilayer, as well as other lipid carrier particles which can entrap antisense oligonucleotides. The liposomes can be made of one or more phospholipids, as long as the lipid material is substantially uncharged. It is important that the composition be substantially free of anionic and cationic phospholipids and cholesterol.
Suitable phospholipids include phosphatidyl cholines and others that are well known to persons that are skilled in this field. The liposomes can be, for example, unilamellar, multilamellar, or have an undefined lamellar structure. A pharmaceutical composition comprising the liposomes will usually include a sterile, pharmaceutically acceptable carrier or diluent, such as water or saline solution.
"Entrap," "encapsulate," and "incorporate" are used in this patent to mean that the oligo is enclosed within at least some portion of the inner aqueous space (including the interlamellar regions of the bilayer) of the liposome.
A composition of the present invention is preferably administered to a patient parenterally, for example by intravenous, intraarterial, intramuscular, intralymphatic, intraperitoneal, subcutaneous, intrapleural, or intrathecal injection, or may be used in ex vivo bone marrow purging. Preferred dosages are between 5-25 mg/kg. The administration is preferably repeated on a timed schedule until the cancer disappears or regresses, and may be in conjunction with other forms of therapy.
The making and use of the present invention is further illustrated by the following examples.
Materials Phosphodiester and phosphorothioate oligonucleotides were provided by Genta Incorporated. Phospholipids were purchased from Avanti Polar Lipids.
Oligonucleotide Labeling Phosphodiesters were labeled at 37 C for 8 h with [32Py]ATP at the 5' end by T4 kinase. The labeled oligonucleotide was precipitated with ethanol at -20 C
overnight. After washing with 70% ethanol three times, phosphodiester oligonucleotides were twice filtered with a Microcon-3 filter to separate the labeled oligonucleotides from free [32Py]ATP.
Phosphorothioates, labeled with 35S, were provided by Genta Incorporated.
Liposomal-phosphodiester Preparation Phosphodiester oligonucleotides dissolved in distilled water were mixed with phospholipids in the presence of excess t-butanol so that the final volume of t-butanol in the mixture was 80-90%. Trace amounts of [3H]cholestanyl ether and [32P]phosphodiester were also added to the mixture as lipid and oligonucleotide markers, respectively. The mixture was vortexed before being frozen in an acetone/dry ice bath. The frozen mixture was lyophilized and hydrated with hepes buffered saline (1 mM Hepes and 10mM NaCl) overnight. Liposomes were twice sonicated for 10 min in a bath type sonicator.
Liposomal-phosphorothioate Preparation Liposomal-phosphorothioates were prepared similarly to that described for liposomal-phosphodiesters, except phosphorothioates, instead of phosphodiesters, were used. Also, [35S]phosphorothioates, instead of [32P]phosphodiesters, were used as the oligonucleotide marker.
Separation of Free Oligonucleotides from those Incorporated in Liposomes The separation of free phosphodiester or phosphorothioate oligonucleotides from those incorporated in liposomes was done by dialyzing the mixture against an excess 2500-fold volume of RPMI medium at room temperature overnight. Aliquots of the preparation were taken before and after dialysis for liquid scintillation counting to assess the incorporation of phosphodiester or phosphorothioate oligonucleotides in liposomes.
Development of liposomal-phosphodiesters Dioleoylphosphatidylcholine (DOPC) lipids were chosen for the phosphodiester (PD) incorporation because they are neutral while PD is negatively charged. By using this lipid, electrostatic repulsion (which can lower incorporation) may be reduced. Positively charged lipids were not used because they may induce non-specific cellular toxicity. The initial attempts of incorporating PD
oligonucleotides into liposomes were done by using freeze-and-thaw, and dehydration-rehydration methods.
(A) Freeze-and-thaw (FT) Method.
[3H]labeled DOPC lipids were evaporated from their organic solvent under nitrogen gas to form a lipid film. After vacuum desiccation, the lipid film was hydrated with hepes buffered saline (1 mM Hepes, 10 mM NaCl, pH 8.0) and sonicated in a bath type sonicator. These pre-formed liposomes were then mixed with [32P]labeled PD oligonucleotides at a 100 or 1000 to 1 molar ratio. The whole mixture was frozen in an acetone/dry ice bath for 5-10 minutes and thawed at room temperature for 25-30 minutes. This process was repeated three times before the sample was loaded on a BioGel A0.5M column for separation of free PD
oligonucleotides from liposomal-PD. Aliquots were taken before and after the freeze-thaw procedure and were sent to liquid scintillation counting to determine the incorporation of phosphodiesters into liposomes.
Incorporation was determined by [32P1PD/umole f3H1DOPC after freeze-thaw X100%
[32P]PD/ mole [3H]DOPC before freeze-thaw There was no incorporation (Table 1).
(B) Dehydration-Rehydration (DR) Method [3H]labeled DOPC was evaporated from the organic solvent under nitrogen gas to form a lipid film. After vacuum desiccation, the lipid film was hydrated with hepes buffered saline and sonicated in a bath type sonicator. These pre-formed liposomes were then frozen in an acetone-dry ice bath and lyophilized. The dried lipids were then rehydrated with distilled water containing [32P]phosphodiester oligonucleotides. The molar ratio of DOPC to PD was either 100/1 or 1000/1.
The mixture was loaded on a BioGel A0.5M column for separation of free PD
oligonucleotides from liposomal-PD. Aliquots were taken before and after column loading and were sent to liquid scintillation counting. The incorporation was < 5 %
(Table 1).
Table 1 Comparison of Freeze-thaw (FT) and Dehydration-rehydration (DR) methods on the incorporation of PD into DOPC liposomes.
Molar ratio Methods % incorporation DR 2.0 DR 4.6 With 500/1 and 5000/1 molar ratios of DOPC to PD, 0 and 5.2%
incorporation were obtained, respectively.
It was then found that by adding t-butanol to the mixture before freezing and lyophilization, incorporation was raised to 11.9%. Also, by reducing the volume of distilled water added during the rehydration process from 200 to 50 l, 11.9 versus 1.8% incorporation was obtained with 1000/1 molar ratio. Effect of molar ratio on the incorporation was again measured with these improved conditions (Table 2).
Table 2 Effect of lipid to oligonucleotides molar ratio on the incorporation of PD
into liposomes.
Molar ratio % incorporation 1000/1 11.9 2000/1 17.0 3000/1 13.4 4000/1 5.5 500011 9.2 PD dissolved in distilled water was mixed with DOPC in the presence of excess t-butanol so that the final volume of t-butanol in the mixture was 80-90%
before being frozen in an acetone/dry ice bath and lyophilized. This was to avoid the step of preparing pre-formed liposomes. With this procedure, similar level of incorporation (16.2%) was obtained at the 2000/1 molar ratio.
It was decided to change the method of separation of free PD from liposomal-PD because the recovery of lipids and PD were < 50%. Two other separation methods were used: Microcon-10 filters and dialysis (Table 3).
Table 3 Comparison of different methods of separating free PD from PD incorporated in liposomes.a Method % Incorporation % Lipid and PD recovery Column 11.9 40-50 Filter 44.7 60-70 Dialysis 76.7 >90 a DOPC was used to incorporate PD oligonucleotides at a 1000/1 molar ratio.
When the liposomal mixture was sonicated for 10 min before the dialysis separation process, similar level of incorporation was obtained. Another lipid, dimyristoyl phosphatidylcholine, was also used for PD incorporation. There was > 85 % incorporation.
Development of liposomal-phosphorothioates Similar incorporation protocol was used with phosphorothioates (PT) since PT and PD are structural analogs. Various molar ratios of DOPC to PT were used (Table 4). The effect of sonication of the liposomal mixture (before dialysis) was also studied.
Table 4 Effect of lipid to oligonucleotide molar ratios on the incorporation of PT
into liposomes.
% incorporation Molar ratio without sonication with sonication 10/1 >90 >90 5011 >90 >90 100/1 45.8 55.5 200/1 44.1 49.1 50011 27.8 47.0 1000/1 25.1 42.1 Incorporation of p-ethoxy oligos into liposomes pE oligos were purchased from Oligos Therapeutics (Wilisonville, OR).
Phospholipids were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL).
WO 97(407794 PCT/US96/14146 Oligo labeling pE oligos were labeled at 37 C for 24 h with [32Py]ATP at the 5' end by T4 polynucleotide kinase, and then precipitated with ethanol at -20 C overnight.
They were then washed with 70% ethanol three times to separate the labeled oligo from free [32Py]ATP.
(b Liposome preparation pE oligos dissolved in distilled H2O were mixed with phospholipids at various molar ratios in the presence of excess t-butanol so that the final volume of t-butanol in the mixture was at least 95%. Trace amounts of [3H]cholestanyl ether and [32P]pEs were also added to the mixture as lipid and oligo markers, respectively. The mixture was vortexed, frozen in an acetone/dry ice bath and then lyophilized. The lyophilized preparation was hydrated with Hepes buffered saline (1 mM Hepes and 10 mM NaC1) at a final oligo concentration of 10-100 M. The liposomal-p-ethoxy oligos were sonicated for 10-20 min in a bath type sonicator.
Separation of free pE oligos from those incorporated in liposomes The separation of free pE oligos from those incorporated in liposomes was done by dialyzing (MW cutoff = 12-14,000) against 1000-fold excess of Hepes buffered saline at room temperature overnight. Aliquots of liposomal-pE oligos were taken before and after dialysis for liquid scintillation counting to access the incorporation of pE oligos in liposomes.
(d) Incorporation efficiency The lipid phosphatidylcholine (PC) was chosen for the incorporation of pE
oligos because both PC and pE oligos are neutral molecules so they should be compatible. Among all the different PCS, dioleoyl PC (DOPC) was chosen because it has a chain-melting phase transition temperature at -15 to -20 C. Thus, at room temperature, DOPC is in the liquid crystalline phase which is the ideal phase to prepare liposomes.
To incorporate pE oligos into liposomes, different molar ratios of pE oligos were mixed with DOPC together in the presence of excess of t-butanol. Trace amounts of radio labeled pE oligos and DOPC were included in the mixture. The DOPC/pE oligos mixtures were frozen in a dry ice/acetone bath before being lyophilized. The lyophilized DOPC/pE oligo powder was then hydrated with Hepes buffered saline so that the final oligo concentration was 10 .tM. pE oligos were successfully incorporated into DOPC liposomes, ranging between 28 to 83 %
efficiency (Table 5). The incorporation efficiency was dependent on the molar ratios of DOPC to pE oligos: 10 > 100 > 5 > 1000:1.
Table 5 Effect of molar ratio of DOPC to pE oligos on the incorporation of pE oligos Molar ratio of Incorporation DOPC:pE oligos efficiency (%)a 5:1 45 10:1 83 100:1 71 1000:1 28 aThe incorporation efficiency values were obtained from three separate experiments.
Delivery of pE oligos to leukemic cells After achieving a high incorporation efficiency (> 80%) of pE oligos into liposomes, we then proceeded to test whether these liposomal-pE oligos can reach the cytoplasm in which the mRNA is located. We had purchased a 16-mer pE oligo labeled with rhodamine at the 5' end so that we could visualize the localization of the pE oligos by fluorescent microscopy.
Incubation of pE oligos with leukemic cells ALL-1 cells, which are human acute lymphocytic leukemic cells, were used.
Fifty thousand ALL-i cells/well were plated in a 24-well plate in 0.3 mL of medium. After 2 h of plating, final concentrations of 16 M of liposomal or free pE oligos conjugated with rhodamine were added to ALL-1 cells. After 24 h of incubation, the cells were thrice washed with phosphate buffered saline before being viewed under a confocal laser scanning microscope. See Figure 1 (Uptake of (A) free or (B) liposomal-pE oligos by ALL-1 cells).
Our data indicates that when incorporated into liposomes, higher amounts of pE oligos were taken up by the ALL-1 cells. The liposomes were able to deliver the pE oligos to the cytoplasm.
Growth inhibition of liposomal-pE oligos on leukemic cells We then proceeded to test whether liposomal-pE oligos can specifically inhibit the growth of leukemic cells. We have used three different kinds of human leukemic cell lines: ALL-1 (acute lymphocytic leukemia), BV173 and K562 (both are chronic myelogenous leukemia). All three cell lines contain the rearranged Philadelphia (Ph) chromosome which arises from a reciprocal translocation of chromosomes 9 and 22. This translocation results in the relocation of the c-Abl protooncogene from chromosome 9 onto the 3' end of the breakpoint cluster region (Bcr) of chromosome 22, thus producing a hybrid Bcr-Abl gene. The breakpoint junctions where the Bcr and the Abl genes fuse are different in the three cell lines.
In ALL-1 cells, the breakpoint junction is Bcr exon 1/ Abl exon 2. In BV173 cells, the breakpoint junction is Bcr exon 2/ Abl exon 2. In K562 cells, the breakpoint junction is Bcr exon 3/ Abl exon 2. All these hybrid genes produce a novel Bcr-Abl fusion protein, which has enhanced tyrosine kinase activity that has been linked to the pathogenesis of the leukemias. Thus, inhibition of the production of the Bcr-Abl protein may lead to leukemic cell growth inhibition and even cell death.
To specifically inhibit the production of the Bcr-Abl protein, we have decided to target the antisense sequences against the breakpoint junctions of the Bcr-Abl mRNA
which is only found in Ph chromosome-positive leukemic cells, but not normal cells. This way, we hope to induce minimal non-specific toxic side effects as only leukemic, not normal, cell growth will be affected.
Sequences of the pE antisense oligos (written from 5' to 3' end) antisense against Bcr exon 1/ Abl exon 2 (B1/A2) found in ALL-1 cells GAAGGGCTTCTGCGTC
antisense against Bcr exon 2/ Abl exon 2 (B2/A2) found in BV173 cells CTGAAGGGCTTCTTCC
antisense against Bcr exon 3/ Abl exon 2 (B3/A2) found in K562 cells GGGCTTTTGAACTCTGCT
(b Delivery of liposomal-pE oligos to leukemic cells Ten thousand ALL-1 or BV 173 cells or five thousand K562 cells were plated per well in a 96-well plate in 100 L of RPMI medium containing 10% fetal calf serum. After 2 h of plating, final concentrations of 0 - 10 M of liposomal-pE
oligos were added to leukemic cells. The cells were incubated with liposomal-pE
oligos for 5 days. HL60 cells, a human promyelocytic cell line which does not have the Philadelphia chromosome, were used as control cells. They were plated under the same conditions at ten thousand cells/well.
(c) Determination of the viability of the leukemic cells At the end of the incubation, 100 gL of medium were added to each well which makes the final volume of each well to be 200 L. Then 50 L of cells were aliquoted and added to 96-well plates containing 130 L. of medium and 20 L
of alamarBlue dye. The cells will be incubated for 4-8 more hours at 37 C before being read directly on a microplate reader (Molecular Devices, Menlo Park, CA) at 570 and 595 nm. The alamarBlue dye incorporates an oxidation-reduction indicator that changes color in response to chemical reduction of growth medium resulting from cell growth. The difference in absorbance between 570 and 595 nm will be taken as the overall absorbance value of the leukemic cells. The viabilities of leukemic cells treated with liposomal-pE oligos will be compared with those of the control untreated cells.
When ALL-1 and HL60 cells were incubated with liposomal-pE antisense oligos targeted against the B1/A2 breakpoint junction of Bcr-Abl mRNA, a dose-dependent growth inhibition of ALL-1, but not HL60, cells was observed (Fig.
2A). Similarly growth inhibition was observed with BV173 and K562 cells when they were incubated with liposomal-pE antisense oligos targeting against the and B3/A2 breakpoint junctions of Bcr-Abl mRNA, respectively (Fig. 3A, 4A).
Under identical conditions, HL60 cells were not growth-inhibited.
To ensure that the growth inhibitory effects were sequence-dependent, the Ph chromosome-positive, cell lines were incubated with antisense and control liposomal-pE oligos. When ALL-1 cells were incubated with the B1/A2 liposomal-pE antisense oligos and the B2/A2 liposomal-pE control oligos, growth inhibition was induced (Fig. 2B). However, the B1/A2 antisense oligos induced a much greater inhibitory effect. Similarly, higher inhibitory effects on BV173 and cells were found with the corresponding liposomal-pE antisense oligos than with the control oligos (Fig. 3B, 4B).
We also found that by including the detergent Tween 20 in the liposomal-pE
oligo mixture, the potency of the inhibitory effects of the liposomal-pE
oligos was increased. We added Tween 20 at 5 % (wt. of pE oligos) in the liposomal-pE
oligo mixture. Then the mixture was vortexed, frozen in an acetone/dry ice bath before being lyophilized. The dried mixture was then hydrated and sonicated as stated previously. When Tween 20-containing liposomal-pE oligos were added to BV173 cells, 100% growth inhibition was observed at 5 M (Fig. 5) whereas under the same conditions, 100% growth inhibition with normal liposomal-pE oligos (no Tween 20) was observed at 10 M instead.
* * *
The preceding description of specific embodiments of the present invention is not intended to be a complete list of every possible embodiment of the invention.
Persons skilled in this field will recognize that modifications can be made to the specific embodiments described here that would be within the scope of the present invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Board of Regents, The University of Texas System (ii) TITLE OF INVENTION: LIPOSOMAL PHOSPHODIESTER, PHOSPHOROTHIOATE, AND p-ETHOXY OLIGONUCLEOTIDES
(iii) NUMBER OF SEQUENCES: 3 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Arnold, White & Durkee (B) STREET: 750 Bering Dr., Ste. 400 (C) CITY: Houston (D) STATE: TX
(E) COUNTRY: USA
(F) ZIP: 77057-2198 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy Disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version 41.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/520,385 (B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Goodman, Kenneth D.
(B) REGISTRATION NUMBER: 30,460 (C) REFERENCE/DOCKET NUMBER: UTFC433P--(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 713/787-1460 (B) TELEFAX: 713/789-2679 (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GAAGGGCTTC TGCGTC
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CTGAAGGGCT TCTTCC
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: õ
GGGCTTTTGA ACTCTGCT
Claims (13)
1. A liposomal composition of antisense oligonucleotides, comprising (a) a liposome consisting essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is a p-ethoxy oligonucleotide.
2. The composition of claim 1, wherein the phospholipids are phosphatidylcholines.
3. The composition of claim 1, wherein the phospholipids are dioleoylphosphatidyl choline.
4. The composition of any one of claims 1 to 3, wherein the p-ethoxy oligonucleotide consists essentially of a nucleic acid molecule having the sequence GAAGGGCTTCTGCGTC.
5. The composition of any one of claims 1 to 3, wherein the p-ethoxy oligonucleotide consists essentially of a nucleic acid molecule having the sequence CTGAAGGGCTTCTTCC.
6. The composition of any one of claims 1 to 3, wherein the p-ethoxy oligonucleotide consists essentially of a nucleic acid molecule having the sequence GGGCTTTTGAACTCTGCT.
7. The composition of any one of claims 1 to 6 wherein the neutral phospholipid and p-ethoxy oligonucleotide are in a molar ratio of between about 5:1 and about 100:1.
8. A use for inhibiting the growth of tumor cells in a mammalian subject having a tumor, of an amount effective to inhibit the growth of tumor cells of a composition comprising (a) a liposome consisting essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is a p-ethoxy oligonucleotide.
9. A use of a composition in the manufacture of a medicament for inhibiting the growth of tumor cells in a mammalian subject having a tumor, said composition comprising: (a) a liposome consisting essentially of neutral phospholipids, and (b) an antisense oligonucleotide that is entrapped in the liposome and is a p-ethoxy oligonucleotide.
10. The use of claim 7 or 8, wherein the neutral phospholipid and p-ethoxy oligonucleotide are in a molar ratio of between about 5:1 and about 100:1.
11. A method of preparing a liposomal composition of antisense oligonucleotides, comprising the steps of:
(a) hydrating a lyophilized composition consisting essentially of neutral phospholipids, and an antisense oligonucleotide that is a p-ethoxy oligonucleotide, thereby forming an aqueous suspension which includes free p-ethoxy oligonucleotide and liposomes entrapping p-ethoxy oligonucleotide; and (b) separating the free p-ethoxy oligonucleotide from the liposomes by dialysis.
(a) hydrating a lyophilized composition consisting essentially of neutral phospholipids, and an antisense oligonucleotide that is a p-ethoxy oligonucleotide, thereby forming an aqueous suspension which includes free p-ethoxy oligonucleotide and liposomes entrapping p-ethoxy oligonucleotide; and (b) separating the free p-ethoxy oligonucleotide from the liposomes by dialysis.
12. The method of claim 11, where the aqueous suspension is sonicated before dialysis.
13. The method of claim 11 or 12, wherein the neutral phospholipid and p-ethoxy oligonucleotide are in a molar ratio of between about 5:1 and about 100:1.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/520,385 | 1995-08-29 | ||
| US08/520,385 US5855911A (en) | 1995-08-29 | 1995-08-29 | Liposomal phosphodiester, phosphorothioate, and P-ethoxy oligonucleotides |
| PCT/US1996/014146 WO1997007784A2 (en) | 1995-08-29 | 1996-08-26 | LIPOSOMAL PHOSPHODIESTER, PHOSPHOROTHIOATE, AND p-ETHOXY OLIGONUCLEOTIDES |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2228944A1 CA2228944A1 (en) | 1997-03-06 |
| CA2228944C true CA2228944C (en) | 2011-10-18 |
Family
ID=24072378
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2228944A Expired - Fee Related CA2228944C (en) | 1995-08-29 | 1996-08-26 | Liposomal phosphodiester, phosphorothioate, and p-ethoxy oligonucleotides |
Country Status (11)
| Country | Link |
|---|---|
| US (4) | US5855911A (en) |
| EP (1) | EP0847272B1 (en) |
| JP (1) | JP4291412B2 (en) |
| AT (1) | ATE253897T1 (en) |
| AU (1) | AU6912996A (en) |
| CA (1) | CA2228944C (en) |
| DE (1) | DE69630691T2 (en) |
| DK (1) | DK0847272T3 (en) |
| ES (1) | ES2210385T3 (en) |
| PT (1) | PT847272E (en) |
| WO (1) | WO1997007784A2 (en) |
Families Citing this family (59)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5855911A (en) * | 1995-08-29 | 1999-01-05 | Board Of Regents, The University Of Texas System | Liposomal phosphodiester, phosphorothioate, and P-ethoxy oligonucleotides |
| US6977244B2 (en) | 1996-10-04 | 2005-12-20 | Board Of Regents, The University Of Texas Systems | Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides |
| US6894031B1 (en) * | 1996-11-21 | 2005-05-17 | Cedars-Sinai Medical Center | Pituitary tumor transforming gene (PTTG) carboxy-terminal peptides and methods of use thereof to inhibit neoplastic cellular proliferation and/or transformation |
| US6565889B2 (en) * | 1996-12-02 | 2003-05-20 | The Regents Of The University Of California | Bilayer structure which encapsulates multiple containment units and uses thereof |
| US7262173B2 (en) | 1997-03-21 | 2007-08-28 | Georgetown University | Chemosensitizing with liposomes containing oligonucleotides |
| US6126965A (en) * | 1997-03-21 | 2000-10-03 | Georgetown University School Of Medicine | Liposomes containing oligonucleotides |
| US6835395B1 (en) | 1997-05-14 | 2004-12-28 | The University Of British Columbia | Composition containing small multilamellar oligodeoxynucleotide-containing lipid vesicles |
| JP4656675B2 (en) | 1997-05-14 | 2011-03-23 | ユニバーシティー オブ ブリティッシュ コロンビア | High rate encapsulation of charged therapeutic agents in lipid vesicles |
| DK1017365T3 (en) * | 1997-06-23 | 2004-03-29 | Alza Corp | Liposome enclosed polynucleotide composition and method |
| DE69834038D1 (en) * | 1997-07-01 | 2006-05-18 | Isis Pharmaceutical Inc | COMPOSITIONS AND METHOD FOR THE ADMINISTRATION OF OLIGONUCLEOTIDES OVER THE DISHES |
| DE19740092A1 (en) * | 1997-09-12 | 1999-03-18 | Ulrich Dr Med Hengge | New liposome comprises lipid envelope based on natural or synthetic skin |
| US7704962B1 (en) * | 1997-10-03 | 2010-04-27 | Board Of Regents, The University Of Texas System | Small oligonucleotides with anti-tumor activity |
| US7285288B1 (en) * | 1997-10-03 | 2007-10-23 | Board Of Regents, The University Of Texas System | Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides |
| CA2850318A1 (en) | 1999-02-26 | 2000-08-31 | The University Of British Columbia | Trpm-2 antisense therapy |
| US7112337B2 (en) | 1999-04-23 | 2006-09-26 | Alza Corporation | Liposome composition for delivery of nucleic acid |
| AU7868400A (en) | 1999-10-08 | 2001-04-23 | Alza Corporation | Neutral-cationic lipid for nucleic acid and drug delivery |
| FR2802422B1 (en) | 1999-12-21 | 2002-08-09 | Capsulis | MIXED STRUCTURES RESULTING FROM THE INCORPORATION OF A BIOLOGICAL MACROMOLECULE, PARTICULARLY DNA, IN A STRUCTURED PHASE OF AMPHIPHILES AND VESICLES OBTAINED FROM THESE STRUCTURES |
| US7569551B2 (en) | 2000-02-25 | 2009-08-04 | The University Of British Columbia | Chemo- and radiation-sensitization of cancer by antisense TRPM-2 oligodeoxynucleotides |
| WO2002022642A1 (en) | 2000-09-14 | 2002-03-21 | The University Of British Columbia | Antisense insulin-like growth factor binding protein (igfbp)-2-oligodeoxynucleotides for prostate and other endocrine tumor therapy |
| WO2003061386A1 (en) * | 2002-01-03 | 2003-07-31 | Board Of Regents, The University Of Texas System | Wt1 antisense oligos for the inhibition of breast cancer |
| KR101166214B1 (en) * | 2002-01-17 | 2012-07-16 | 더 유니버시티 오브 브리티쉬 콜롬비아 | Bispecific antisense oligonucleotides that inhibit igfbp-2 and igfbp-5 and methods of using same |
| EP1530636B1 (en) | 2002-08-21 | 2010-08-18 | The University Of British Columbia | Treatment of melanoma by reduction in clusterin levels |
| ES2345330T3 (en) | 2002-10-02 | 2010-09-21 | The University Of British Columbia | OLIGONUCLEOTIDOS FOR THE TREATMENT OF PROSTATE CANCER AND OTHER CANCERES. |
| MXPA05010499A (en) | 2003-03-31 | 2006-05-25 | Johnson & Johnson | Lipid particles having asymmetric lipid coating and method of preparing same. |
| WO2005018493A1 (en) | 2003-08-14 | 2005-03-03 | Boston Scientific Limited | Medical slings |
| CA2539727C (en) * | 2003-10-01 | 2016-11-01 | The University Of British Columbia | Bispecific oligonucleotide for the treatment of cns malignancies |
| CA2542886A1 (en) | 2003-11-05 | 2005-05-19 | Neelima M. Bhat | Enhanced b cell cytotoxicity of cdim binding antibody |
| WO2005051351A2 (en) | 2003-11-21 | 2005-06-09 | Alza Corporation | Gene delivery mediated by liposome-dna complex with cleavable peg surface modification |
| WO2005094899A1 (en) * | 2004-04-02 | 2005-10-13 | The University Of British Columbia | Clusterin antisense therapy for treatment of cancer |
| CA2570305C (en) | 2004-06-14 | 2015-03-10 | Boston Scientific Scimed, Inc. | Systems, methods and devices relating to implantable supportive slings |
| BRPI0517057A (en) | 2004-11-05 | 2008-09-30 | Palingen Inc | composition to induce cell membrane injury; composition for increasing cell membrane injury in a lymphoid cell; composition to permeabilize a cell; composition to induce cell membrane injury in b cells; composition for increasing cell membrane injury induced by a cell membrane injury antibody; method of treating a mammal suffering from a distinct condition due to hyperproliferation of cells; method for killing a cancer cell; method for inducing cell membrane injury in a lymphoid cell in a human patient; method for inducing cell membrane injury; method for permeabilizing a cell; method of purging the bone marrow of malignant b cells from a patient with this need; kit for determining the dose limit for a multipurpose agent that induces injury to the cell membrane in a mammal; kit for determining the dose limit for a cell membrane injury antibody in a mammal; use of a polyvalent cell membrane injury agent; and use of a cell membrane injury antibody |
| EP1814595B1 (en) * | 2004-11-23 | 2014-01-08 | The University Of British Columbia | Treatment of cancer with a combination of an agent that perturbs the egf signaling pathway and an oligonucleotide that reduces clusterin levels |
| AU2006236453B2 (en) | 2005-01-25 | 2012-02-23 | Board Of Regents, The University Of Texas System | Delivery of siRNA by neutral lipid compositions |
| WO2006108144A2 (en) * | 2005-04-06 | 2006-10-12 | Boston Scientific Scimed, Inc. | Systems, devices, and methods for sub-urethral support |
| US8043205B2 (en) | 2005-07-13 | 2011-10-25 | Boston Scientific Scimed, Inc. | Snap fit sling anchor system |
| US7878969B2 (en) | 2005-07-25 | 2011-02-01 | Boston Scientific Scimed, Inc. | Pelvic floor repair system |
| US7981023B2 (en) * | 2005-07-25 | 2011-07-19 | Boston Scientific Scimed, Inc. | Elastic sling system and related methods |
| US8273429B2 (en) * | 2006-01-19 | 2012-09-25 | Federal-Mogul World Wide, Inc. | Fabric for end fray resistance and protective sleeves formed therewith and methods of construction |
| FR2900576B1 (en) | 2006-05-03 | 2008-09-19 | Centre Nat Rech Scient | PROCESS FOR EXTEMPORANEOUS AND REVERSIBLE CONCENTRATION OF LIPOSOMES. |
| US9381209B2 (en) * | 2007-03-05 | 2016-07-05 | The University Of British Columbia | Treatment of squamous cell carcinoma with HSP27 antisense oligonucleotides and radiotherapy |
| CN102112110A (en) * | 2008-06-06 | 2011-06-29 | 米尔纳医疗股份有限公司 | Novel compositions for the in vivo delivery of RNAi agents |
| ES2710109T3 (en) | 2010-12-17 | 2019-04-23 | Inst Nat Sante Rech Med | Nucleic acids that target TCTP for use in the treatment of chemoresistant or hormone-resistant cancers |
| CA2864394C (en) | 2011-03-02 | 2021-10-19 | Jack Roth | A method of predicting a response to a tusc2 therapy |
| US9113991B2 (en) | 2011-05-12 | 2015-08-25 | Boston Scientific Scimed, Inc. | Anchors for bodily implants and methods for anchoring bodily implants into a patient's body |
| US9636201B2 (en) | 2011-05-12 | 2017-05-02 | Boston Scientific Scimed, Inc. | Delivery members for delivering an implant into a body of a patient |
| JP6335796B2 (en) | 2012-02-08 | 2018-06-06 | アイジーエム バイオサイエンシズ インク．Ｉｇｍ Ｂｉｏｓｃｉｅｎｃｅｓ Ｉｎｃ． | CDIM binding protein and use thereof |
| CN103565745A (en) | 2012-08-10 | 2014-02-12 | 德克萨斯州大学系统董事会 | Neuroprotective liposome compositions and methods for treatment of stroke |
| WO2014195754A1 (en) | 2013-06-05 | 2014-12-11 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Hydrophobically modified antisense oligonucleotides comprising a triple alkyl chain |
| WO2014195755A1 (en) | 2013-06-05 | 2014-12-11 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Hydrophobically modified antisense oligonucleotides comprising a ketal group |
| SG10202104058XA (en) | 2015-10-14 | 2021-05-28 | Bio Path Holdings Inc | P-ethoxy nucleic acids for liposomal formulation |
| WO2017106814A1 (en) | 2015-12-18 | 2017-06-22 | The Board Of Regents Of The University Of Texas System | Therapeutics for preterm labor management |
| EP3512503A4 (en) * | 2016-09-14 | 2020-05-06 | Nanyang Technological University | Liposomal formulations |
| WO2018053232A1 (en) | 2016-09-16 | 2018-03-22 | Bio-Path Holdings, Inc. | Combination therapy with liposomal antisense oligonucleotides |
| RU2755903C2 (en) | 2016-10-12 | 2021-09-22 | Борд Оф Риджентс, Дзе Юниверсити Оф Техас Систем | Methods and compositions for tusc2-immunotherapy |
| US20210115451A1 (en) * | 2017-04-19 | 2021-04-22 | Bio-Path Holdings, Inc. | P-ethoxy nucleic acids for igf-1r inhibition |
| US20200038429A1 (en) * | 2017-04-19 | 2020-02-06 | Bio-Path Holdings, Inc. | P-ethoxy nucleic acids for igf-1r inhibition |
| CA3058018A1 (en) | 2017-04-19 | 2018-10-25 | Bio-Path Holdings, Inc. | P-ethoxy nucleic acids for stat3 inhibition |
| JP2023522103A (en) | 2020-04-20 | 2023-05-26 | ボード オブ リージェンツ，ザ ユニバーシティ オブ テキサス システム | Biologically active dry powder compositions and methods of making and using the same |
| WO2022040435A1 (en) | 2020-08-19 | 2022-02-24 | The Board Of Regents Of The University Of Texas System | Nanodrugs for targeted drug delivery and use thereof |
Family Cites Families (82)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH621479A5 (en) | 1977-08-05 | 1981-02-13 | Battelle Memorial Institute | |
| US4394448A (en) | 1978-02-24 | 1983-07-19 | Szoka Jr Francis C | Method of inserting DNA into living cells |
| US4469863A (en) | 1980-11-12 | 1984-09-04 | Ts O Paul O P | Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof |
| US4480041A (en) * | 1982-07-09 | 1984-10-30 | Collaborative Research, Inc. | Use of phosphotriester intermediates for preparation of functionalized liposomes |
| FR2540122B1 (en) | 1983-01-27 | 1985-11-29 | Centre Nat Rech Scient | NOVEL COMPOUNDS COMPRISING A SEQUENCE OF OLIGONUCLEOTIDE LINKED TO AN INTERCALATION AGENT, THEIR SYNTHESIS PROCESS AND THEIR APPLICATION |
| US4721612A (en) * | 1984-04-12 | 1988-01-26 | The Liposome Company, Inc. | Steroidal liposomes |
| US5202429A (en) | 1986-07-09 | 1993-04-13 | The Wistar Institute | DNA molecules having human BCL-2 gene sequences |
| US5049388A (en) * | 1986-11-06 | 1991-09-17 | Research Development Foundation | Small particle aerosol liposome and liposome-drug combinations for medical use |
| US5094785A (en) | 1986-12-10 | 1992-03-10 | Ciba Corning Diagnostics Corp. | Process for stabilizing liposomes |
| US4920016A (en) | 1986-12-24 | 1990-04-24 | Linear Technology, Inc. | Liposomes with enhanced circulation time |
| US4837028A (en) | 1986-12-24 | 1989-06-06 | Liposome Technology, Inc. | Liposomes with enhanced circulation time |
| US5030442A (en) * | 1987-03-30 | 1991-07-09 | Liposome Technology, Inc. | Non-crystalline minoxidil composition |
| US4904582A (en) | 1987-06-11 | 1990-02-27 | Synthetic Genetics | Novel amphiphilic nucleic acid conjugates |
| US5178875A (en) * | 1991-01-14 | 1993-01-12 | The Board Of Regents, The University Of Texas System | Liposomal-polyene preliposomal powder and method for its preparation |
| US4950432A (en) * | 1987-10-16 | 1990-08-21 | Board Of Regents, The University Of Texas System | Polyene microlide pre-liposomal powders |
| US5188897A (en) * | 1987-10-22 | 1993-02-23 | Temple University Of The Commonwealth System Of Higher Education | Encapsulated 2',5'-phosphorothioate oligoadenylates |
| US4924624A (en) * | 1987-10-22 | 1990-05-15 | Temple University-Of The Commonwealth System Of Higher Education | 2,',5'-phosphorothioate oligoadenylates and plant antiviral uses thereof |
| WO1989006977A1 (en) * | 1988-02-04 | 1989-08-10 | Board Of Regents, The University Of Texas System | Formulation and use of retinoids in treatment of cancer and other diseases |
| US5098890A (en) * | 1988-11-07 | 1992-03-24 | Temple University-Of The Commonwealth System Of Higher Education | Antisence oligonucleotides to c-myb proto-oncogene and uses thereof |
| US5831066A (en) | 1988-12-22 | 1998-11-03 | The Trustees Of The University Of Pennsylvania | Regulation of bcl-2 gene expression |
| US5734033A (en) * | 1988-12-22 | 1998-03-31 | The Trustees Of The University Of Pennsylvania | Antisense oligonucleotides inhibiting human bcl-2 gene expression |
| US5248671A (en) * | 1989-02-15 | 1993-09-28 | Board Of Regents, The University Of Texas System | Methods and compositions for treatment of cancer using oligonucleotides |
| US5087617A (en) * | 1989-02-15 | 1992-02-11 | Board Of Regents, The University Of Texas System | Methods and compositions for treatment of cancer using oligonucleotides |
| FR2644080A1 (en) * | 1989-03-13 | 1990-09-14 | Trizac Jacques | DEVICE FOR FILTRATION AND EVACUATION OF SMALL PARTICLES, WASTES AND TRENDS OF VARIOUS NATURE |
| EP0469082B1 (en) | 1989-04-18 | 1995-04-05 | Vestar, Inc. | Liposomal targeting of ischemic tissue |
| US5112962A (en) * | 1989-04-19 | 1992-05-12 | Northwestern University | Labile anchors for solid phase polynucleotide synthesis |
| US5227170A (en) * | 1989-06-22 | 1993-07-13 | Vestar, Inc. | Encapsulation process |
| US5100662A (en) * | 1989-08-23 | 1992-03-31 | The Liposome Company, Inc. | Steroidal liposomes exhibiting enhanced stability |
| US5542935A (en) | 1989-12-22 | 1996-08-06 | Imarx Pharmaceutical Corp. | Therapeutic delivery systems related applications |
| US5469854A (en) * | 1989-12-22 | 1995-11-28 | Imarx Pharmaceutical Corp. | Methods of preparing gas-filled liposomes |
| CA2033725C (en) | 1990-01-24 | 2001-05-29 | Folker Pittrof | Pharmaceutical and cosmetic compositions containing a salt of cholanic acid |
| US5279833A (en) | 1990-04-04 | 1994-01-18 | Yale University | Liposomal transfection of nucleic acids into animal cells |
| US5264618A (en) * | 1990-04-19 | 1993-11-23 | Vical, Inc. | Cationic lipids for intracellular delivery of biologically active molecules |
| AU649673B2 (en) * | 1990-04-30 | 1994-06-02 | Isis Pharmaceuticals, Inc. | Oligonucleotide modulation of arachidonic acid metabolism |
| US5665710A (en) * | 1990-04-30 | 1997-09-09 | Georgetown University | Method of making liposomal oligodeoxynucleotide compositions |
| DE59108644D1 (en) | 1990-07-02 | 1997-05-07 | Hoechst Ag | Oligonucleotide analogs with terminal 3'-3 'or. 5'-5 'internucleotide linkages |
| US5135917A (en) * | 1990-07-12 | 1992-08-04 | Nova Pharmaceutical Corporation | Interleukin receptor expression inhibiting antisense oligonucleotides |
| US5378825A (en) | 1990-07-27 | 1995-01-03 | Isis Pharmaceuticals, Inc. | Backbone modified oligonucleotide analogs |
| WO1992005272A1 (en) * | 1990-09-17 | 1992-04-02 | The Regents Of The University Of California | Method and composition for controlling proliferation of cells |
| US5271941A (en) * | 1990-11-02 | 1993-12-21 | Cho Chung Yoon S | Antisense oligonucleotides of human regulatory subunit RI.sub.α of cAMP-dependent protein kinases |
| DE4110085A1 (en) * | 1991-03-27 | 1992-10-01 | Boehringer Ingelheim Int | New 2'O-alkyl-oligo-ribonucleotide(s) with 8-35 nucleotide units - useful as anti-sense oligo-nucleotide(s), primers and probes |
| FR2677272B1 (en) * | 1991-06-05 | 1995-03-03 | Biovecteurs As | PARTICULATE VECTOR WITH SELECTIVE TROPISM, PREPARATION METHOD AND PHARMACEUTICAL COMPOSITION. |
| CA2103377A1 (en) * | 1991-06-18 | 1992-12-19 | Bruno Calabretta | Selective inhibition of leukemic cell proliferation by bcr-abl antisense oligonucleotides |
| US5525719A (en) | 1991-08-30 | 1996-06-11 | Chemgenes Corporation | N-protected-2'-O-methyl-and N-protected-3'-O-methyl-ribonucleosides and their phosphoramidite derivatives |
| DE69233046T2 (en) * | 1991-10-24 | 2004-03-04 | Isis Pharmaceuticals, Inc., Carlsfeld | DERIVATIZED OLIGONUCLEOTIDS WITH IMPROVED CAPACITY |
| CA2124673C (en) * | 1991-12-04 | 2008-08-05 | John Browse | Fatty acid desaturase genes from plants |
| US5279957A (en) * | 1992-04-30 | 1994-01-18 | Washington University | cDNA encoding human phospholipase A2 polypeptide |
| EP0646178A1 (en) * | 1992-06-04 | 1995-04-05 | The Regents Of The University Of California | expression cassette with regularoty regions functional in the mammmlian host |
| US5320962A (en) * | 1992-07-22 | 1994-06-14 | Duke University | DNA encoding the human A1 adenosine receptor |
| NL9201440A (en) * | 1992-08-11 | 1994-03-01 | Univ Leiden | Triantennary cluster glycosides, their preparation and application. |
| AU5092893A (en) * | 1992-09-02 | 1994-03-29 | Georgetown University | Method of encapsulating anthracycline glycosides in liposomes |
| US6001992A (en) | 1999-01-07 | 1999-12-14 | Isis Pharmaceuticals Inc. | Antisense modulation of novel anti-apoptotic bcl-2-related proteins |
| WO1994008625A1 (en) | 1992-10-21 | 1994-04-28 | Temple University - Of The Commonwealth System Of Higher Education | Combination of antineoplastic agent and antisense oligonucleotides for treatment of cancer |
| US6015886A (en) | 1993-05-24 | 2000-01-18 | Chemgenes Corporation | Oligonucleotide phosphate esters |
| US5417978A (en) * | 1993-07-29 | 1995-05-23 | Board Of Regents, The University Of Texas System | Liposomal antisense methyl phosphonate oligonucleotides and methods for their preparation and use |
| FR2710074B1 (en) | 1993-09-15 | 1995-12-08 | Rhone Poulenc Rorer Sa | GRB3-3 gene, its variants and their uses. |
| JPH08505882A (en) * | 1993-11-05 | 1996-06-25 | アムジエン・インコーポレーテツド | Method for preparing liposome and method for encapsulating substance |
| US5651981A (en) | 1994-03-29 | 1997-07-29 | Northwestern University | Cationic phospholipids for transfection |
| US5696248A (en) | 1994-06-15 | 1997-12-09 | Hoechst Aktiengesellschaft | 3'-modified oligonucleotide derivatives |
| US5908635A (en) * | 1994-08-05 | 1999-06-01 | The United States Of America As Represented By The Department Of Health And Human Services | Method for the liposomal delivery of nucleic acids |
| IT1275862B1 (en) * | 1995-03-03 | 1997-10-24 | Consiglio Nazionale Ricerche | ANTI-SENSE TRANSCRIPT ASSOCIATED WITH SOME TYPES OF TUMOR CELLS AND SYNTHETIC OLIGODEOXYNUCLEOTIDES USEFUL IN DIAGNOSIS AND TREATMENT |
| ATE206131T1 (en) * | 1995-03-13 | 2001-10-15 | Aventis Pharma Gmbh | PHOSPHONOMONOESTERNUCLIC ACIDS, METHOD FOR THEIR PRODUCTION AND THEIR USE |
| EP0841068B1 (en) | 1995-06-01 | 2006-07-12 | Kishimoto, Tadamitsu | Leukemic cell growth inhibitor containing antisense oligonucleotide derivative against wilms' tumor gene (wt1) |
| US6326487B1 (en) | 1995-06-05 | 2001-12-04 | Aventis Pharma Deutschland Gmbh | 3 modified oligonucleotide derivatives |
| US5705385A (en) | 1995-06-07 | 1998-01-06 | Inex Pharmaceuticals Corporation | Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer |
| EP1489184A1 (en) | 1995-06-07 | 2004-12-22 | Inex Pharmaceutical Corp. | Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer |
| US5756122A (en) * | 1995-06-07 | 1998-05-26 | Georgetown University | Liposomally encapsulated nucleic acids having high entrapment efficiencies, method of manufacturer and use thereof for transfection of targeted cells |
| US6096720A (en) * | 1995-08-01 | 2000-08-01 | Novartis Ag | Liposomal oligonucleotide compositions |
| US5855911A (en) * | 1995-08-29 | 1999-01-05 | Board Of Regents, The University Of Texas System | Liposomal phosphodiester, phosphorothioate, and P-ethoxy oligonucleotides |
| DE19532553A1 (en) * | 1995-09-04 | 1997-03-06 | Hoechst Ag | Process for the preparation of substituted N-ethyl-glycine derivatives |
| US6120794A (en) | 1995-09-26 | 2000-09-19 | University Of Pittsburgh | Emulsion and micellar formulations for the delivery of biologically active substances to cells |
| US6977244B2 (en) * | 1996-10-04 | 2005-12-20 | Board Of Regents, The University Of Texas Systems | Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides |
| US5874224A (en) | 1997-03-11 | 1999-02-23 | Incyte Pharmaceuticals, Inc. | Growth factor receptor binding protein |
| US6126965A (en) | 1997-03-21 | 2000-10-03 | Georgetown University School Of Medicine | Liposomes containing oligonucleotides |
| GB9711919D0 (en) | 1997-06-09 | 1997-08-06 | Ciba Geigy Ag | Oligonucleotide derivatives |
| DK1017365T3 (en) | 1997-06-23 | 2004-03-29 | Alza Corp | Liposome enclosed polynucleotide composition and method |
| WO1999001139A1 (en) | 1997-07-03 | 1999-01-14 | Thomas Jefferson University | An improved method for design and selection of efficacious antisense oligonucleotides |
| US7285288B1 (en) * | 1997-10-03 | 2007-10-23 | Board Of Regents, The University Of Texas System | Inhibition of Bcl-2 protein expression by liposomal antisense oligodeoxynucleotides |
| US6136965A (en) | 1998-07-02 | 2000-10-24 | The Regents Of The University Of California | Deoxynucleic alkyl and alkoxy thiourea compounds |
| US6211349B1 (en) | 1998-12-30 | 2001-04-03 | Oligos Etc., Inc. | Protonated/acidified nucleic acids and methods of use |
| US6211162B1 (en) | 1998-12-30 | 2001-04-03 | Oligos Etc. Inc. | Pulmonary delivery of protonated/acidified nucleic acids |
| US7795232B1 (en) | 2000-08-25 | 2010-09-14 | Genta Incorporated | Methods of treatment of a bcl-2 disorder using bcl-2 antisense oligomers |
-
1995
- 1995-08-29 US US08/520,385 patent/US5855911A/en not_active Expired - Lifetime
-
1996
- 1996-08-26 ES ES96929888T patent/ES2210385T3/en not_active Expired - Lifetime
- 1996-08-26 CA CA2228944A patent/CA2228944C/en not_active Expired - Fee Related
- 1996-08-26 EP EP96929888A patent/EP0847272B1/en not_active Expired - Lifetime
- 1996-08-26 DK DK96929888T patent/DK0847272T3/en active
- 1996-08-26 AT AT96929888T patent/ATE253897T1/en not_active IP Right Cessation
- 1996-08-26 JP JP51064297A patent/JP4291412B2/en not_active Expired - Fee Related
- 1996-08-26 WO PCT/US1996/014146 patent/WO1997007784A2/en active IP Right Grant
- 1996-08-26 DE DE69630691T patent/DE69630691T2/en not_active Expired - Lifetime
- 1996-08-26 PT PT96929888T patent/PT847272E/en unknown
- 1996-08-26 AU AU69129/96A patent/AU6912996A/en not_active Abandoned
-
1998
- 1998-07-09 US US09/112,869 patent/US6042846A/en not_active Expired - Lifetime
-
2003
- 2003-06-16 US US10/462,172 patent/US7176302B2/en not_active Expired - Fee Related
-
2007
- 2007-02-13 US US11/674,563 patent/US7754872B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| WO1997007784A3 (en) | 1997-04-24 |
| CA2228944A1 (en) | 1997-03-06 |
| PT847272E (en) | 2004-04-30 |
| ATE253897T1 (en) | 2003-11-15 |
| EP0847272A2 (en) | 1998-06-17 |
| WO1997007784A2 (en) | 1997-03-06 |
| EP0847272B1 (en) | 2003-11-12 |
| US7754872B2 (en) | 2010-07-13 |
| ES2210385T3 (en) | 2004-07-01 |
| US20070202157A1 (en) | 2007-08-30 |
| DE69630691T2 (en) | 2004-11-25 |
| US7176302B2 (en) | 2007-02-13 |
| US5855911A (en) | 1999-01-05 |
| JP4291412B2 (en) | 2009-07-08 |
| AU6912996A (en) | 1997-03-19 |
| US20040005353A1 (en) | 2004-01-08 |
| DK0847272T3 (en) | 2004-03-22 |
| DE69630691D1 (en) | 2003-12-18 |
| US6042846A (en) | 2000-03-28 |
| JPH11512099A (en) | 1999-10-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2228944C (en) | Liposomal phosphodiester, phosphorothioate, and p-ethoxy oligonucleotides | |
| EP0711149B1 (en) | Liposomal antisense methyl phosphonate oligonucleotides and methods for their preparation and use | |
| US6096720A (en) | Liposomal oligonucleotide compositions | |
| Akhtar et al. | Interactions of antisense DNa oliginucletide analogs with phospholid membranes (liposomes) | |
| Leonetti et al. | Encapsulation of c-myc antisense oligodeoxynucleotides in lipid particles improves antitumoral efficacy in vivo in a human melanoma line | |
| CA2163234A1 (en) | Inhibition of proliferation of vascular smooth muscle cell | |
| KR20120013336A (en) | Pharmaceutical composition containing a drug and sirna | |
| AU2009330859A1 (en) | Pharmaceutical composition containing an anionic drug, and a production method therefor | |
| JP2022506503A (en) | Modified double chain oligonucleotide | |
| Hu et al. | Programmable fusogenic vesicles for intracellular delivery of antisense oligodeoxynucleotides: enhanced cellular uptake and biological effects | |
| US7262173B2 (en) | Chemosensitizing with liposomes containing oligonucleotides | |
| Yang et al. | Targeted delivery of antisense oligodeoxynucleotides to folate receptor-overexpressing tumor cells | |
| KR101678327B1 (en) | WEAKLY ACIDIC pH-RESPONSIVE PEPTIDE AND LIPOSOME CONTAINING SAME | |
| US5935937A (en) | Compositions and methods for inducing apoptosis | |
| Tari et al. | Oligonucleotide therapy for hematological malignancies | |
| WO1996024334A9 (en) | Lipid constructs for cytoplasmic delivery of agents | |
| Wong et al. | A lipid-based delivery system for antisense oligonucleotides derived from a hydrophobic complex | |
| US7393951B2 (en) | Antisense oligonucleotide having anticancerous activity | |
| Webb et al. | 10 Lipid-Based Carriers for the Systemic | |
| Webb et al. | Lipid-based carriers for the systemic delivery of antisense drugs | |
| Louie | Stabilized antisense-lipid particles with enhanced intracellular delivery properties |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |
Effective date: 20150826 |