Definitions

• the present invention relates to methods and compositions for the treatment of cancer using an oligonucleotide and an hydroxy radical up-regulator.
• the oligonucleotide is characterized by its ability to down- regulate the path by which the cell repairs oxidative damage to its DNA.
• the oligonucleotide renders the tumor cells more susceptible to eradication upon exposure to the hydroxy radical up-regulator, preferably administered substantially concomitantly with or subsequent to administration of the oligonucleotide.
• This novel treatment preferentially inhibits the proliferation or kills malignant cells but not normal cells.
• the oligonucleotide is antisense to the gene which encodes protein p53, although other antisense oligonucleotides can also be used.
• the invention also includes novel conjugates of the oligonucleotide and the hydroxy up-regulator, as well as new oligonucleotides.
• the present invention provides a method of treating cancer by preferentially inducing p53 independent apoptosis in cancer cells.
• a one-electron reduction of O2 yields superoxide ion, O2-; an additional electron yields hydrogen peroxide, H2O2; and a third electron yields a hydroxy radical, 0H ⁇ , and a hydroxide ion.
• reactive oxygen radicals are far more reactive, and hence potentially much more toxic, than is O2 itself.
• Hydroxy radicals are extremely reactive and represent the most active mutagen derived from ionizing radiation.
• the radical is highly electrophilic and reactive, with a capacity to bind the DNA to produce modified bases, such as 8-hydroxyguanine, and thymine glycol.
• the former has been detected in DNA isolated from tissues exposed to ionizing radiation or to hydrogen peroxide (H 2 0 2 ) (Kasai et al., Carcinoqenesis 7: 1849, 1986).
• H 2 0 2 hydrogen peroxide
• the latter which is an oxidation product of thymine residues in DNA, is often found in the urine of individuals who have suffered DNA damage (B. Ames, Science 221: 1256-1264, 1983).
• SOD superoxide dismutase
• This metalloenzyme is found in the cytoplasm of eukaryotic cells and contains copper and zinc; a different form is found both in mitochondria and in bacterial cells, and contains manganese; and another related iron-containing form is found in some bacteria, cyanobacteria, and some plants (see, for example, CK Matthews and KE van Holde, Biochemistry, The Benjamin/Cummings Publishing Company, Inc., Redwood City, CA, 1990).
• the wide occurrence of SOD enzymes is confirmation of the biological necessity of rapid inactivation of reactive oxygen intermediates.
• Hydrogen peroxide (H2O2), another highly reactive oxygen species, is inactivated by at least two different enzyme systems.
• the most commonly utilized is the enzyme catalase, which is a heme protein widely distributed in cells.
• the reaction involves oxidation of one molecule of H2O2 and reduction of another.
• the extremely high turnover rate of this enzyme (more than 40,000 molecules utilized per second) confirms the importance of removing excess hydrogen peroxide from the cellular micro-environment.
• erythrocytes contain a selenium-containing enzyme, glutathione peroxidase (GSH), which reduces H2O2 to water while simultaneously oxidizing the glutathione:
• GSH glutathione peroxidase
• Glutathione peroxidase contains one residue per mole of an unusual amino acid, selenocysteine, an analog of cysteine that contains selenium in place of sulfur. Glutathione synthetase and glutathione reductase are further examples of this family of scavengers.
• H2O2 is chemically inactivated by mannitol, with which it forms a stable, equimolar compound.
• hydroxy, H2O2 , and superoxide ion are considered to be interchangeable .
• vitamin A Retinol and retinoic acid
• vitamin E alpha-tocopherol
• vitamin C ascorbic acid
• trace mineral element selenium all of which serve in a variety of tissues and bodily processes as general reducing agents.
• Cancer cells particularly those which are malignant, exhibit elevated levels of free hydroxy radicals. These and other types of diseased cells do not exhibit the same degree of anti-oxidant protection as do normal cells. In particular, such cells are notably deficient in the scavenger protection systems discussed above.
• irradiation kills cancer cells preferentially to normal tissue and conventional radiation therapy attempts to exploit this mechanism of action, as do certain conventional chemotherapeutic agents, such as the nitrosoureas, e.g., BCNU (bis-chloroethylnitrosourea) , and the anthracycline cytotoxic antibiotics, doxorubicin and daunorubicin.
• BCNU bis-chloroethylnitrosourea
• anthracycline cytotoxic antibiotics doxorubicin and daunorubicin.
• the sensitivity of a cell and the resultant cellular response to ionizing radiation depends primarily on the presence or absence of oxygen within the cell and upon the stage of division which the cell is in at the time of irradiation.
• production of oxidative damage is initiated by a dose of radiation.
• Cells which are oxygen-rich and sensitive to radiation will be killed more effectively and efficiently than cells which are oxygen- deficient.
• the molecular pathways responsible for inherent radiation sensitivity involve the initiation and production of oxidative damage, cellular sensing of the damage, and cellular response to and repair of the damage. Repair of radiation damage is most likely an enzymatic process and radiobiologists have examined the role of certain enzymes which are responsible for the detoxification of cytotoxic oxygen-related free radicals.
• the cellular repair mechanism is located between the G ⁇ and S phase of the cell cycle.
• the gene p53 has been postulated to play a role in the repair of damaged DNA and is in fact considered to function as a cell cycle checkpoint after irradiation.
• Apoptosis is part of normal development and also can be triggered by DNA damage, such as that delivered by radiation and some chemicals, including those used in chemotherapy. It has been shown that levels of p53 protein rise dramatically after DNA damage.
• p53 is currently believed to be crucial to the apoptotic pathway induced by DNA damage.
• oligonucleotides which are antisense to p53 (and hence inhibit its activity) produce apoptosis ex vivo and in vivo in treated cells via an additional apoptosis pathway, which is p53 independent.
• oligonucleotide which acts as an oxidative repair path down- regulator.
• oligonucleotides are those which are antisense to p53.
• oligonucleotides antisense to: p21 see PCT International Publication WO 93/12251
• CDC-2 see PCT International Publication WO 93/12251
• CDK cyclin-dependent kinases
• DNA polymerase ⁇ are discussed more fully hereinafter.
• the cancer cell In addition to exposure to the oligonucleotide of this invention, the cancer cell must also be exposed to conditions which increase the net reactive oxygen content in a cell. Such conditions are produced, for example, by exposure of the cancer cell to radiation, or an agent capable of radical oxygen induced cytotoxicity, such as for example, anthracycline cytotoxic antibiotics (e.g., doxorubicin) , BCNU, BSO (buthionine sulfoximine) , hydrogen peroxide, or antisense oligonucleotide inhibitors of SOD (superoxide dismutase), catalase, GSH synthetase, GSH reductase, or GSH peroxidase.
• anthracycline cytotoxic antibiotics e.g., doxorubicin
• BCNU doxorubicin
• BSO buthionine sulfoximine
• SOD superoxide dismutase
• catalase GSH synthe
• the present invention provides a method for selectively killing cells characterized by p53 expression in cells in vivo or ex vivo.
• the method comprises administering to a mammalian or human host, or to cells harvested from such host, an oligonucleotide with a sequence complementary to p53 mRNA and an agent capable of increasing radical oxygen induced cytotoxicity.
• the oligonucleotide and agent can be administered substantially concomitantly or sequentially with either therapeutic agent being given first. However, best results are achieved when the oligonucleotide is administered sufficiently in advance to permit therapeutic blood levels to be achieved.
• the antisense p53 eliminates the p53 mediated GI checkpoint of the cell cycle, which results in an increase in the sensitivity if the targeted cells have oxidative DNA damage. Consequently, upon exposure of the cells by an agent which acts to increase radical oxygen in the cell, the cell death mediated by the anti-p53 oligonucleotide is increased dramatically, leading to delayed p53 independent apoptosis of the targeted cells with little or no adverse effect on normal tissue (as normal tissue has greater oxygen scavenging activity) .
• the anti-p53 oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human p53 gene and is hybridizable to the mRNA transcript. In a preferred embodiment, the oligonucleotide comprises between about a 10-mer and about a 30-mer oligodeoxyribonucleotide containing a sequence selected from the group consisting of:
• the agent capable of radical oxygen induced cytotoxicity can be, for example, a radiosensitizer, a chemotherapeutic agent which generates radical oxygen, or an oligonucleotide capable of interaction with cells of the host causing the formation of hydroxy radicals (e.g., see Patrick L. Iversen United States Patent Application Serial Number 07/735,067, filed July 25, 1991 entitled INHIBITION OF MUTAGENICITY
• cytotoxic agents that have a lethal effect on target cells with the patient, wherein the lethal effect may be enhanced by one or more sensitizing agents, may be used in the method of the invention. Included are radiation (whether the radiation therapy is delivered internally or administered by external means) and cytotoxic drugs. Examples of some of the many such drugs are nitrogen mustards such as L-phenylalanine nitrogen mustard (melphalan), anthracycline chemotherapeutics such as daunorubicin and doxorubicin, platinum compounds such as cis-diamino dichloro platinum (cisplatin) .
• nitrogen mustards such as L-phenylalanine nitrogen mustard (melphalan)
• anthracycline chemotherapeutics such as daunorubicin and doxorubicin
• platinum compounds such as cis-diamino dichloro platinum (cisplatin) .
• Radioisotopes include, but are not limited to, the radionuclide metals 186 Re, 188 Re, 64 Cu, 67 Cu, 109 Pd, 212 Bi, 203 Pb, 212 Pb, 211 At, 97 Ru, 105 Rh, 198 Au, 199 Ag, and 131 I. These radioisotopes generally will be bound to carrier molecules (e.g., are in the form of a chelate-antibody conjugate) when administered to a patient. Examples of suitable internally delivered radiotherapeutic agents are the metal radionuclide chelates which are conjugated to antibodies as described in European Patent Application Publication No. 188,256. Radiation administered by external means includes external beam radiation such as cobalt therapy.
• sensitizing agent depends on such factors as the particular type of tumor to be treated and the desired cytotoxic agent to be administered. For example, certain drugs have been reported to sensitize cells to therapeutic radiation, as discussed above.
• U.S. Pat. No. 4,628,047 reports the use of diltiazem (chemical name: d-3-acetoxy-cis- 2 , 3-dihydro-5- [ 2- (dimethylamino ) ethyl ] -2- (p- methoxyphenyl ) l,5-benzo-thiazepin-4 (5H) -one) to enhance the sensitivity of a variety of types of cancer cells toward cytotoxic agents such as doxorubicin.
• a preferred sensitizer is BSO, a synthetic amino acid that inhibits gamma-glutamylcysteine synthetase and leads to a marked decrease of glutathione (GSH) in cells.
• GSH glutathione
• BSO is available from Chemical Dynamics Corporation, South Plainfield, N.J. Additional combinations of sensitizers and cytotoxic agents may be identified through such methods as in vitro assays using cultured cells which correspond to the desired target cells (e.g., a specific cancer cell line). Assays for determining whether BSO is effective in lowering glutathione synthetase levels in a particular type of cell line have been developed (Cancer Treatment Reports, Vol. 69, No. 11, pp. 1293-1296 [1985]).
• Another embodiment of this invention provides novel conjugates comprising the oligonucleotide bound through a suitable linking moiety to a suitable sensitizing agent, such as buthionine sulfoximine or superoxide dismutase and the like, such that upon exposure to the target cells it is able to increase the concentration of hydroxy radicals to which the cellular DNA of the host is exposed.
• a suitable sensitizing agent such as buthionine sulfoximine or superoxide dismutase and the like
• an embodiment of this invention is a conjugate represented by the following formula: R-L-X wherein R represents an antisense oligonucleotide having a sequence essentially complementary to a sequence of RNA transcribed from a target gene selected from the group consisting of p53, p21, glutathione synthetase, and a DNA polymerase involved in repair of oxidative DNA damage; L represents a linking moiety, preferably selected from those disclosed in U.S. 5,112,954 (see also Iversen et al, J Gen Virol. 1989 Oct; 70 (Pt 10): 2673-82), and X represents an agent capable of radical oxygen induced cytotoxicity.
• the oligonucleotide Upon administration of the conjugate, the oligonucleotide will bind to the cellular membrane and release the biologically active sensitizing agent. The result is an increase in the number of hydroxy radicals within the cell and a decrease in the ability of the cell to scavenge oxygen radicals within the cell.
• the oligonucleotide increases the cell's sensitivity to the increased amount of hydroxy radicals by inhibiting the function of the p53 gene. Inhibition of the p53 gene results in a failure of the cell to arrest its cycle at the GI checkpoint and prevents DNA repair. The cell will continue its cycle, inducing cell death via apoptosis, due to the excessive amount of unrepaired DNA damage.
• the target cells may be cancer cells such as leukemia cells or any cancer which produces elevated levels of p53 are susceptible to this therapeutic regimen.
• Figure 1 shows the results of a flow cytometry study to examine the effects of an oligonucleotide on cell death.
• the addition of OL(l)p53 to primary AML cell culture increases the rate of cell death via apoptosis in the culture.
• a 0 percent of apoptotic cells;
• D u percent of intact, proliferating cells.
• Panels A, C, E represent patient cells prior to incubation with 0L(l)p53 (SEQ ID N0:1) ex-vivo.
• Panel B, D, F represent patient cells following 10 day incubation with i ⁇ M OL(l)p53.
• the insert box in the lower left indicates the A 0 region.
• the present invention provides a method for selectively inducing apoptosis by enhancing the action of hydroxy radicals against the DNA of a particular type of target tissue compared to non-target tissue.
• the method comprises administering to a mammalian or human host a sensitizing agent and an oligonucleotide, either separately or concomitantly, wherein the compound is preferential for a certain target site in vivo , such as a cancer cell.
• Oligonucleotides complementary to and hybridizable with any portion of the mRNA transcript of p53, p21, CDC-2, CDK, or DNA polymerase ⁇ are, in principle, effective for inhibiting translation of the transcript, and capable of inducing the effects herein described. However, it is known that there are preferred sites to which the oligonucleotide is most preferably directed. For example, with respect to p53, translation is most effectively inhibited by blocking the mRNA at a site within a region defined by exon 10 or exon 11 (i.e., the oligonucleotide is complementary to a portion of exon 10 or exon 11 of p53 mRNA) .
• Oligonucleotides hybridizable to the mRNA transcript finding utility include not only native polymers of the biologically significant nucleotides, but also oligonucleotide species which have been modified for improved stability and/or lipid solubility. For example it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting a methyl group or sulfur group in the internucleotide phosphodiester linkage The phosphorothioates, in particular, are stable to nuclease cleavage and lipid-soluble. Consequently second and later generation molecular modifications of the native oligomer are included within the scope of this invention.
• the sensitizing agent is an agent that increases the sensitivity of a cell to the effects of a cytotoxic, oxygen- generating agent.
• the oxygen-generating agent may be an oligonucleotide antisense to any of the mRNA of any of the oxygen radical scavenger proteins, a chemotherapeutic agent, or radiosensitizer, such as BSO, or any compound which will increase the amount of hydroxy radicals in the cell.
• chemotherapeutic agent such as BSO
• radiosensitizer such as BSO
• antisense oligonucleotides designed to the mRNA of the tumor suppressor gene p53 were shown in vivo to inhibit or kill tumor cell targets in a nucleic acid sequence specific manner.
• the anti-p53 oligonucleotides can be synthesized and purified according to the published methods for oligonucleotide synthesis. These methods are generally described, for example, in Stec and Zon, J. Chromatoqr. 326: 263, 1985; Tyer et al. (Nucl. Acids Res. 18: 2855, 1990; U.S.
• Patent 5,264,423, European Patent Application EP 0 288 163 A2 or Winnacker, From Genes to Clones : Introduction to Gene Technology, VCH Verlagsgesellschaft mbH (H. Ibelgaufts trans. 1987) .
• oligonucleotide synthesis can be used to prepare the anti-p53 oligonucleotides, they are most conveniently prepared using any of the commercially available, automated nucleic acid synthesizers.
• the phosphorothioate antisense oligonucleotides of SEQ ID NOS: 1-2 were prepared using an Applied Biosystems, Inc. DNA synthesizer (Model 380B) according to the manufacturer's protocols using phosphoramidite chemistry.
• the phosphorothioate oligonucleotides used were from the group consisting of: OL(l)p53, (SEQ ID N0:1) directed against a region in exon 10 of p53; p53t, (SEQ ID NO: 2) directed against a region in exon 11 of p53; ISIS- 1082 ( 5 ' -TCCTAGGTCC ATGTCGTACGC-3 • ; SEQ ID N0:3), a control sequence directed against a region in Herpes Virus; and FRED ( 5 ' -CCTCGGTCCC CCCTCGTCCC-3 ' ; SEQ ID N0:4), a control sequence with the reverse orientation of SEQ ID N0:1 (OL(l)p53).
• Oligonucleotides were synthesized on an Applied Biosystems (Foster City, CA) Model 380A DNA synthesizer. Phosphorothioate oligonucleotides were synthesized and purified according to the methods described in Stec and Zon, J. Chromatoq . » 326: 263-280, and in Applied Biosystems, DNA Synthesizer, User Bulletin, Model 380A/380B/381A/391-EP, December 19891
• EXAMPLE 1 Oligonucleotide-Induced Lipid Peroxidation Methods for Human Patients.
• Three human patients with acute myelogenous leukemia or myelodisplastic syndrome were administered the oligonucleotide 0L(l)p53 at a dose of 0.2 mg/kg/hr over a ten (10) day period.
• Urine samples were collected. 1 ml of 30 mM thiobarbituric acid (TBAR) and 500 ⁇ L of 1% trichloroacetic acid was added to 500 ⁇ L of urine.
• the samples were acidified to pH 1.5 with concentrated hydrochloric acid (HC1) and heated to 100°C for 45 minutes and read at 532 nm using a spectrophotometer (Gilford) to determine the amount of TBAR products in the urine.
• HC1 concentrated hydrochloric acid
• MTT Assay 5 mg/ml of MTT was added to each of the wells on the 96-well plate. The plate was incubated for 2 hours. The MTT was removed from the plate and 100 ⁇ L of dimethylsulfoxide (DMSO) was added to each well. The optical density (OD) of each well was determined at 540 nm on a Molecular Devices Plate Reader.
• DMSO dimethylsulfoxide
• the following example demonstrates the ability of antisense oligonucleotides, directed toward the p53 gene, to increase the rate of cell death through the mechanism of apoptosis.
• Blast cells from peripheral blood samples of two patients with acute myelogenous leukemia (AML) were used.
• Primary suspension cultures were performed according to the methods described by Bayever et al (Leukemia and Lymphoma) .
• Leukemic blasts were isolated from heparinizedperipheral blood by Ficoll-Hypaque (Pharmacia LKB Biotechnology, Piscataway, NJ) separation and depleted from T-cells adhered to AET-treated Sheep-erythrocyte (Sigma, St. Louis, Mo.).
• the cells were cultured at 5 x 10 5 /ml in 24- well plates in Iscove's medium (Sigma), 12.5% FCS (Hyclone Laboratories, Logan, UT), 12.5% horse serum (GIBCO, Grand Island, NY), 1 ⁇ g/ml Gentamicin (Sigma) and 1% hydrocortisone (Abbott Laboratories, Chicago, IL) in humid air 5% C0 2 at 37° C and oligonucleotides (OL(l)p53, SEQ ID N0:1; and FRED) were added 24 hours later at l ⁇ M. Viable cell counts were determined by Trypan blue exclusion from aliquots removed every 2 to 3 days.
• thymo ⁇ ytes were obtained from young (4-6 week old) BDF1 mice supplied by Jackson Laboratories, Bar Harbor, Maine. The mice were humanely euthanized, and thymic tissue removed and minced. A cell suspension was prepared by repeated gently aspiration of the cells into a 1 ml syringe without a needle. The cells were washed 3 times and gently resuspended in RPMI 1640 with 20% fetal calf serum at 2 x 10 6 cells/ml. The thymocytes were then treated dexamethasone (1 ⁇ M/4 hours) or hydrocortisone acetate at lO ⁇ M (overnight).
• the cells were incubated overnight at 4°C and then allowed to warm to room temperature for 30 minutes in the dark prior to analyzing. Cells were then analyzed on an EPICS Elite flow cytometer using Coulter software. The percentage of apoptotic cells was calculated from the forward angle light scatter (FS) versus linear red fluorescence histogram by gating on the A 0 population. Apoptotic cell numbers were expressed as a percentage of total cells incorporating dye after exclusion of non-staining, non-viable cells and debris. For each sample 10 4 cell events were recorded on the FS versus linear red fluorescence histogram.
• FS forward angle light scatter
• Apoptotic cell numbers were expressed as a percentage of total cells incorporating dye after exclusion of non-staining, non-viable cells and debris.
• Ifosfamide (IFEX, Bristol-Meyers Oncology): 1.33 g/M 2 IV on each of days 4 through 7.
• Mitoxantrone hydrochloride (NOVANTRONE, Lederle Laboratories) 10 mg/M 2 IV on day 4 only.
• Etoposide (VEPESID, Bristol-Meyers Oncology) 80 mg/M 2 IV on each of days 4 through 7.
• Patients are regularly evaluated for toxicity and response of their disease. Patients receive weekly CBC (complete blood count) and platelet counts. Cycles are repeated every 4 weeks for a maximum of 6 cycles. Patients are assessed with respect to response every cycle for disease measurable by physical examination. Patients are classified with respect to response (complete response, partial response, stable response, or progressive disease). Patients are expected to show enhanced clinical response with this therapy as opposed to traditional MINE therapy, which does not include use of the antisense oligonucleotide SEQ ID NO:l (0L(l)p53). OL(l)p53 is expected to sensitize the cancer cells to the effects of MINE by inhibiting the cell cycle repair mechanism, thereby preferentially killing cancer cells via p53-independent apoptosis.
• the oligonucleotide which acts as an oxidative repair path down- regulator is administered to a patient prior to, substantially concomitantly with, or simultaneously with administration of the agent capable of radical oxygen induced cytotoxicity.
• the two active agents can be in conjugated or unconjugated form.
• the amount of each agent administered is such that the combination of the two types of agents is therapeutically effective.
• the dosages will vary in accordance with such factors as the condition of the patient, the type of cancer being treated, the type of agents being administered.
• the cytotoxic reactive oxygen increasing agents generally preferred are those well known in the art. Recommended dosages and dosage forms for most of these agents have been established and can be obtained from conventional sources, such as the Physicians Desk Reference, published by Medical Economics Company, Inc., Oradell, NJ 07649 or the
• a method for treating cancer characterized by p53 expression is provided.
• Such cancers include those of the bladder, brain, breast, cervix, colon, esophagus, larynx, liver, lung, ovary, pancreas, prostate, skin, stomach and thyroid.
• An oligonucleotide effective against the type of cancer with which a patient is afflicted is administered to the patient prior to or substantially concomitantly with administration of an agent capable of radical oxygen induced cytotoxicity (and which effectively increases the net reactive oxygen content of the cells upon exposure thereto) .
• an agent capable of radical oxygen induced cytotoxicity and which effectively increases the net reactive oxygen content of the cells upon exposure thereto
• each immunoconjugate comprises a cleavable linkage so that the oligonucleotide and the cytotoxic agent are released at the target site.
• the doses and schedules of the free oligonucleotide, the free agent capable of radical oxygen induced cytotoxicity the conjugated oligonucleotide/agent will vary depending on the age, health, sex, size and weight of the patient, the route of administration, the toxicity of the drugs and the relative susceptibilities of the cancer to the oligonucleotide and cytotoxic agent. These parameters can be determined for each system by well-established procedures and analysis e.g., in phase I, II and III clinical trials.
• the preferred dosage of the oligonucleotides of the present invention is that which is necessary to attain a concentration in blood of from about 0.01 to about 1 micromoles/l .
• This concentration can be achieved in a variety of ways. Doses of between about 0.05 and about 0.2 mg/kg/hour by continuous IV infusion has been found to be acceptable. Greater or lesser amounts of oligonucleotide may be administered as required.
• the antisense oligonucleotides, or conjugates thereof can be combined with a pharmaceutically acceptable carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
• a suitable liquid vehicle or excipient such as distilled water, physiological saline, aqueous solutions of dextrose and the like.
• the p53 mRNA antisense oligonucleotides are preferably administered intravenously.
• the pharmaceutical compositions of this invention may contain suitable excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
• Oral dosage forms encompass tablets, dragees, and capsules. Preparations which can be administered rectally include suppositories.
• Other dosage forms include suitable solutions for administration parenterally or orally, and compositions which can be administered buccally or sublingually .
• the pharmaceutical preparations of the present invention are manufactured in a manner which is itself well known in the art.
• the pharmaceutical preparations may be made by means of conventional mixing, granulating, dragee- making, dissolving, or lyophilizing processes.
• the process to be used will depend ultimately on the physical properties of the active ingredient used.
• Suitable excipients are, in particular, fillers such as sugars, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch, paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
• fillers such as sugars, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch, paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth,
• disintegrating agents may be added, such as the above-mentioned starches as well as carboxymethylstarch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
• Auxiliaries are flow-regulating agents and lubricants, for example, such as silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol .
• Dragee cores may be provided with suitable coatings which, if desired, may be resistant to gastric juices.
• concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
• suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used.
• Dyestuffs and pigments may be added to the tablets of dragee coatings, for example, for identification or in order to characterize different combinations of active compound doses .
• Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
• the push-fit capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added.
• Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of the active compounds with a suppository base.
• Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols, or higher alkanols.
• gelatin rectal capsules which consist of a combination of the active compounds with a base.
• Possible base material include, for example liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
• Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water- soluble or water-dispersible form.
• suspensions of the active compounds are appropriate oily injection suspensions may be administered.
• Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
• Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran, optionally, the suspension may also contain stabilizers.
• the active ingredients may be administered by a variety of specialized delivery techniques.
• the compounds of the present invention may also be administered encapsulated in liposomes, pharmaceutical compositions wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers.
• the active ingredient depending upon its solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension.
• the hydrophobic layer generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
• the diameters of the liposomes generally range from about 15 nm to about 5 microns .
• ADDRESSEE Zarley, McKee, Thomte, Voorhees, & Sease
• MOLECULE TYPE CDNA
• HYPOTHETICAL NO
• ANTI-SENSE YES
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTI-SENSE YES
• MOLECULE TYPE CDNA
• HYPOTHETICAL NO
• ANTI-SENSE YES
• MOLECULE TYPE cDNA
• HYPOTHETICAL NO
• ANTI-SENSE NO
• SEQUENCE DESCRIPTION SEQ ID NO: 4: CCTCGGTCCC CCCTCGTCCC 20

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  • 1996-11-21 EP EP96941450A patent/EP0941124A4/de not_active Withdrawn
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