TITLE OF THE INVENTION Synthesis and Use of Cationic Steroids For Anti-inflammatory Drug Therapy

BACKGROUND OF THE INVENTION

The immunological responses to unmethylated CpG motifs on plasmid DNA (Tan et al., 1999, Hum. Gene Ther. 10:2153-2161), cationic lipid formulations (Tousignant et al., 2000, Hum. Gene Ther. 11 :2493-2513), and lipid/DNA lipoplexes (Tousignant et al., 2000, Hum. Gene Ther. 11 :2493-2513; Scheule et al., 1997, Hum. Gene Ther. 8:689-7071; Li et al., 1999, Am. J. Physiol. 276:L796-L804) present major obstacles to nonviral gene delivery. Although transgene expression occurs within hours after plasmid delivery (Li et al. 1999, Am. J. Physiol. 276:L796-L804) the immunostimulatory cytokines Tumor Necrosis Factor-α (TNF-α) and IL-6 are also detected by one hour after systemic (Tousignant et al. 2000. Hum. Gene Ther. 11 :2493- 2513) or intranasal (Scheule et al. 1997, Hum. Gene Ther. 8:689-7071) administration. Other immunomodulatory cytokines, such as IFN-γ and IL- 12, remain elevated for several days thereafter (Lasic, 1997, Liposomes in Gene Delivery, CRC Press). This rapid immune response to lipoplexes resembles the acute phase response to infection, which consists of local tissue reactions and systemic reactions by the liver, mediated chiefly by the inflammatory cytokine IL-6 (Streetz et al, 2001, Cell. MoI. Biol. 47:661- 673). The inflammatory cytokines lead to an activation of endothelial cells and an influx of neutrophils, lymphocytes, and macrophages at the site of gene delivery (Tousignant et al. 2000, Hum. Gene Ther. 1 1:2493-2513; Scheule et al. 1997, Hum. Gene Ther. 8:689- 7071). As a result, immunological responses to nonviral DNA delivery decrease the magnitude and duration of expression of the transgene and decrease the effectiveness of frequent dosing (Li et al. 1999, Am. J. Physiol. 276:L796-L804). Pharmacological doses of anti-inflammatory glucocorticoids prior or concurrent to administration of lipoplexes have a positive impact on inhibiting an immune response to a plasmid, resulting in an increased amount and duration of transgene expression, increase lifetime of plasmid, and shortened windows of time between dosing (Tan et al. 1999. Hum. Gene Ther. 10:2153- 2161; Braun et al., 1999, FEBS Letters 454:277-282; Wiseman et al., 2001, Gene Ther. 8:1562-1571).

Glucocorticoids bind the glucocorticoid receptor (GR), inducing exposure of a classical nuclear localization sequence that allows importin α/βi binding and subsequent trafficking into the nucleus (Galigniana et al., 1999, J. Biol. Chem.

274:16222-16227; Savory et al., 1999, MoI. Cell. Biol. 1025-1037). Glucocorticoids are also potent anti-inflammatory molecules that regulate the immune system by: (1) inhibiting the production or release of major cytokines such as IL-I, IL-6, TNF-α and IFN-γ; (2) decreasing the stability of mRNA encoding IL-I, IL-2, IL-6, IL-8, TNF-α, and GM-CSF; and (3) inhibiting cytokine-induced transcription by API and NF-κB via the GR (Ashwell et al., 2000, Ann. Rev. Immunol. 18:209-345; McEwan et al., 1997, Bioessays 19:153-160; Schimmer et al., 1996, in The Pharmacological Basis of Therapeutics 1459-1485, McGraw-Hill). Liganded GR forms dimers that bind 15 base- pair glucocorticoid response elements (GREs) to induce or repress transcription (Schimmer et al., 1996, in The Pharmacological Basis of Therapeutics 1459-1485, McGraw-Hill; McNally et al., 2000, Science 287:1262-1265).

Viral and non-viral vectors are used as the basis for gene delivery in current nucleic acid and gene therapy methods. However, there are concerns about the production, reproducibility, cost, and safety of viral vectors for gene therapy. As a result, work has focused on the development of nonviral vectors where the gene construct of interest is packaged by synthetic nonviral materials. Examples of such materials include polycations, dendrimers, and polysaccharides, as well as small molecule cationic lipids such as dioleylphosphatidylethanolamine (DOPE), 3-beta-[N',N'dimethylaminoethane)- carbamoyljcholesterol (DC-chol), and spermine cholesterol. For example, cationic lipids for lipofection condense plasmids, facilitate endosome escape, neutralize charge of DNA, and/or shield DNA from nucleases (Lasic, 1997, Liposomes in Gene Delivery, CRC Press).

There is a long felt need in the art for the development of new compositions and methods for anti-inflammatory drug therapy. The present invention satisfies these needs. SUMMARY OF THE INVENTION

The invention relates generally to cationic steroid pharmaceutical compositions, methods of making cationic steroid pharmaceutical compositions, and methods of using cationic steroid pharmaceutical compositions for treating diseases and disorders, including inflammation.

In one embodiment, the cationic steroid pharmaceutical composition of the invention comprises a steroid-polyamine molecule, wherein the polyamine constituent of the steroid-polyamine molecule is attached through the C-21 position of the steroid constituent of the steroid-polyamine molecule. In another embodiment, the cationic steroid pharmaceutical composition of the invention comprises a steroid-polyamine molecule, wherein the polyamine constituent of the steroid-polyamine molecule is attached through the C-21 position of two steroid constituents of the steroid-polyamine molecule, and wherein the steroid-polyamine molecule comprises an amphiphilic dimer, disubstituted polyamine, resulting from conjugation of two steroid constituents to both primary amines on the polyamine. In some embodiments, the pharmaceutical composition of the invention further comprises a lipid. In these and other embodiments, the steroid constituent of the steroid-polyamine molecule can be dexamethasone.

In another embodiment, the composition of the invention is a cationic steroid and a pharmaceutically acceptable carrier, wherein the cationic steroid is produced by a method comprising, mixing together a steroid, a conjugating reagent, and a polyamine, wherein the conjugating reagent conjugates the polyamine through the C-21 position of the steroid via the displacement of a leaving group, purifying the conjugated steroid-polyamine molecule, thereby producing a cationic steroid pharmaceutical composition. In a further embodiment, the composition of the invention has a cationic steroid and a pharmaceutically acceptable carrier, wherein the cationic steroid is produced by a method comprising, mixing together a steroid, a conjugating reagent, and a polyamine, wherein the conjugating reagent conjugates the polyamine through the C-21 position of two steroid constituents via the displacement of a leaving group, purifying the conjugated steroid-polyamine molecule, thereby producing a cationic steroid pharmaceutical composition, and wherein the steroid-polyamine molecule is an amphophilic dimer, disubstituted polyamine, resulting from conjugation of two steroid constituents to both primary amines on the polyamine. In some embodiments, the pharmaceutical composition of the invention further comprises a lipid. In these and other embodiments, the steroid constituent of the steroid-polyamine molecule can be dexamethasone.

In a further embodiment, the composition of the invention is a cationic steroid pharmaceutical composition made by a method comprising, mixing together a steroid, a conjugating reagent, and a polyamine, wherein the conjugating reagent conjugates the polyamine through the C-21 position of the steroid via the displacement of a leaving group, purifying the conjugated steroid-polyamine molecule, thereby producing a cationic steroid pharmaceutical composition. In another embodiment, the composition of the invention is a cationic steroid pharmaceutical composition made by a method comprising, mixing together a steroid, a conjugating reagent, and a polyamine, wherein the conjugating reagent conjugates the polyamine through the C-21 position of two steroid constituents via the displacement of a leaving group, purifying the conjugated steroid-polyamine molecule, thereby producing a cationic steroid pharmaceutical composition, wherein the steroid-polyamine molecule is an amphiphilic dimer, disubstituted polyamine, resulting from conjugation of two steroid constituents to both primary amines on the polyamine. In some embodiments, the pharmaceutical composition of the invention further comprises a lipid and its method of making further comprises mixing the steroid-polyamine molecule with a lipid. In these and other embodiments, the steroid constituent of the steroid-polyamine molecule can be dexamethasone.

In one embodiment, the invention is a kit for administering a cationic steroid pharmaceutical composition, wherein the cationic steroid pharmaceutical composition is made by mixing together a steroid, a conjugating reagent, and a polyamine, wherein the conjugating reagent conjugates the polyamine through the C-21 position of the steroid via the displacement of a leaving group, purifying the conjugated steroid-polyamine molecule, the kit comprising a cationic steroid pharmaceutical composition, an applicator, and an instructional material for the use thereof. In another embodiment, the invention is a kit for administering a cationic steroid pharmaceutical composition, wherein the cationic steroid pharmaceutical composition is made by mixing together a steroid, a conjugating reagent, and a polyamine, wherein the conjugating reagent conjugates the polyamine through the C-21 position of two steroids via the displacement of a leaving group, purifying the conjugated steroid-polyamine molecule, the kit comprising a cationic steroid pharmaceutical composition, an applicator, and an instructional material for the use thereof, wherein the steroid-polyamine molecule is an amphiphilic dimer, disubstituted polyamine, resulting from conjugation of two steroid constituents to both primary amines on the polyamine. In some embodiments the kit comprises a cationic steroid pharmaceutical composition that is made by mixing together a steroid, a conjugating reagent, and a polyamine with dimethylsulfoxide. In some embodiments the kit comprises a cationic steroid pharmaceutical composition that further comprises a lipid.

In another embodiment, the invention is a method of making a cationic steroid pharmaceutical composition comprising mixing together a steroid, a conjugating reagent, and a polyamine, wherein the conjugating reagent conjugates the polyamine through the C-21 position of the steroid via the displacement of a leaving group, and purifying the conjugated steroid-polyamine molecule, thereby producing a cationic steroid pharmaceutical composition. In a further embodiment, the invention is a method of making a cationic steroid pharmaceutical composition comprising mixing together a steroid, a conjugating reagent, and a polyamine, wherein the conjugating reagent conjugates the polyamine through the C-21 position of two steroids via the displacement of a leaving group, and purifying the conjugated steroid-polyamine molecule, thereby producing a cationic steroid pharmaceutical composition, wherein the steroid-polyamine molecule is an amphiphilic dimer, disubstituted polyamine, resulting from conjugation of two steroid constituents to both primary amines on the polyamine. In some embodiments, the method of making further comprises mixing together a steroid, a conjugating reagent, and a polyamine with dimethylsulfoxide. In some embodiments the method of making further comprises mixing the steroid-polyamine molecule with a lipid. In a further embodiment, the invention is a method of treating a disease or disorder in a mammal, the method comprising administering to the mammal a composition comprising a cationic steroid pharmaceutical composition made by a method comprising, mixing together a steroid, a conjugating reagent, and a polyamine, wherein the conjugating reagent conjugates the polyamine through the C-21 position of the steroid via the displacement of a leaving group, purifying the conjugated steroid-polyamine molecule, thereby producing a cationic steroid pharmaceutical composition; and wherein the cationic steroid pharmaceutical composition treats the disease or disorder, thereby treating a disease or disorder in a mammal. In some embodiments, the composition used can be made by a method comprising, mixing together a steroid, a conjugating reagent, and a polyamine with dimethylsulfoxide. In other embodiments, the composition used can be mixed together with a lipid. In some embodiments the mammal is a human. In various embodiments, the disease or disorder is selected from the group consisting of inflammation, asthma, arthritis, pain, inflammation of a joint, allergy, an autoimmune disorder, rhinitis, and chronic idiopathic urticaria.

In yet another embodiment, the invention is a method of treating a disease or disorder in a mammal, the method comprising administering to the mammal a composition comprising a cationic steroid pharmaceutical composition made by a method comprising, mixing together a steroid, a conjugating reagent, and a polyamine, wherein the conjugating reagent conjugates the polyamine through the C-21 position of two steroids via the displacement of a leaving group, purifying the conjugated steroid- polyamine molecule, thereby producing a cationic steroid pharmaceutical composition; wherein the steroid-polyamine molecule is an amphiphilic dimer, disubstituted polyamine, resulting from conjugation of two steroid constituents to both primary amines on the polyamine, and wherein the cationic steroid pharmaceutical composition treats the disease or disorder, thereby treating a disease or disorder in a mammal. In some embodiments, the composition used can be made by a method comprising, mixing together a steroid, a conjugating reagent, and a polyamine with dimethylsulfoxide. In other embodiments, the composition used can be mixed together with a lipid. In some embodiments the mammal is a human. In various embodiments, the disease or disorder is selected from the group consisting of inflammation, asthma, arthritis, pain, inflammation of a joint, allergy, an autoimmune disorder, rhinitis, and chronic idiopathic urticaria.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of preferred embodiments of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. In the drawings:

Figure 1, comprising Figures IA and IB, schematically illustrates synthesis of a cationic steroid for nucleic acid or drug delivery and anti-inflammatory activity. The 21 -hydroxy position of dexamethasone was chosen for conjugation of a cationic group since it can be substituted without loss of glucocorticoid function (Figure IA). In Figure IB it can be seen that the use of 2-iminothiolane (Traut's reagent) as a coupling reagent does not consume a cation on spermine during the synthesis of the cationic steroid, dexamethasone-spermine (DS; Product 1). Under basic conditions, the prodrug DS undergoes hydrolysis, releasing spermine and a dexamethasone-amide (DA; Product 2). Figure 2, comprising Figures 2A-2E, illustrates the optimization of cationic steroid lipoplex formulation for transfection of confluent bovine aortic endothelial cells. In Figure 2A, lipofections of 1 μg of plasmid per well (2-cm ² /well at~4 x 10 ⁵ cells/well) for expression of enhanced green fluorescent protein (EGFP) were carried out at increasing amounts of total lipid (0 to 40 μg) per μg DNA and various DS:DOPE mass ratios of 1 :0 μg/μg DSrDOPE (light gray); 2: 1 μg/μg DS:DOPE (dark gray); 1:1 μg/μg DS:DOPE (gray); 1 :2 μg/μg DS:DOPE (off-white); 0:1 μg/μg DS:DOPE (white); as well as for Lipofectamine at 6 μg lipid: 1 μg DNA when neither DS nor DOPE was present (black). Figure 2B demonstrates optimization of the lipid:DNA ratio at 1 :2 μg/μg DS:DOPE by optimizing the expression of EGFP as a function of charge ratio to DNA, and led to 10-fold enhancement of EGFP production relative to Lipofectamine. EGFP expression was normalized to the expression obtained with Lipofectamine. GFP positive BAEC imaged by epifluorescence microscopy were lipofected with Lipofectamine (Figure 2C), unconjugated dexamethasone/spermine/DOPE (Figure 2D), or DS/DOPE (E). The results indicate that dexamethasone must be conjugated to spermine to have transfer or delivery activity. Figure 3, comprising Figures 3A-3D, illustrates a FACS analysis and images of a microscopic analysis of lipofections using Lipofectamine or DS/DOPE. Confluent BAEC cells were lipofected with 6:1 Lipofectamine:DNA (A, B) or 6:12:1 DS:DOPE:DNA. (C, D) and subjected to flow cytometry (A, C) or epifluorescence microscopy (B, D). FACS analysis showed a 4.3-fold increase in percent transfection using DS/DOPE versus Lipofectamine, consistent with the increased transfection observed by direct visualization of EGFP in attached living cells.

Figure 4, comprising Figures 4A through 4M, illustrates that cationic steroids are pharmacologically active. Figures 4A through 4L are images of photomicrographs showing that a 30 minute treatment of 3T3 cells expressing GFP-GR protein with dexamethasone, DS/DOPE, or the hydrolysis product DA caused nuclear localization of GFP-GR at doses of 1000 nM (first column), 100 nM (second column), and 10 nM (third column). Untreated cells (fourth column- Figures 4D, 4H, and 4L) displayed predominantly cytosolic localization of the fluorescent glucocorticoid receptor protein (0 nM). Dexamethasone (lower panel) and DA (upper panel) induced nuclear localization at 10 nM concentrations, while DS/DOPE (middle panel) had slightly less activity at this dose. Figure 4M demonstrates that dexamethasone, the cationic steroid DS, and its hydrolysis product DA, all caused dose-dependent induction of SEAP transcription from a GRE-SEAP reporter plasmid as indicated by a fluorogenic assay for secreted alkaline phosphatase activity. Promoter construct experiments were made in triplicate, **p O.001 ; *p O.005, +p <0.05.

Figure 5 graphically illustrates that dexamethasone spermine (DS) binds the glycosaminoglycan hyaluronic acid (HA). In the presence of physiological salt buffer, the release of dexamethasone from hyaluronic acid contained in a dialysis membrane was extremely rapid, while DS remained tightly bound to HA for over 24 hours, and DA had intermediate release kinetics. Figure 6, comprising Figures 6A, 6B, and 6C, graphically illustrates structure and activity of a series of steroid-spermine conjugates and lipofectamine (LA) for DNA packaging and transfection in bovine aortic endothelial cells (BAEC). Dynamic light scattering (Figure 6A) is given for steroid-spermines of varying Log Psteroid (Log Psteroid = 1.6 to 2.9) at DOPE:steroid ratios of 0.5, 1, and 2 and amine:phosphate ratios ranging from 2 to 24. EGFP expression at 24 hours (Figure 6B) and 48 hours (Figure 6C) is given for each formulation tested in Figure 6 A. Lo gP is defined as the log of the octano I/water partitioning ratio.

Figure 7 depicts the structure of disubstituted spermine (D ₂ S), another product of the synthesis reaction. D2S is an amphiphilic dimer, disubstituted spermine, resulting from conjugation of dexamethasone to both primary amines on spermine. D ₂ S exhibits the characteristic cationic charge of the primary reaction product at physiological pH due to the secondary amines retained from spermine and also has additional hydrophobicity relative to DS resulting from the additional dexamethasone moiety. Figure 8 depicts the results of Nuclear Magnetic Resonance analysis of

DS.

Figure 9 depicts the results of an example experiment demonstrating the anti-inflammatory activity of DS. Dexamethasone or DS was injected intraperitoneally (i.p.) into mice, prior to thioglycollate. At a dose of 5.85 mg/kg, DS significantly inhibited the accumulation of neutrophils in the i.p. space versus vehicle (1% ethanol in PBS), resulting in a 60% reduction (P=0.0058, n=6) in neutrophil immigration.

Figure 10 depicts the results of an example experiment analyzing IFN-γ antigen, TNF-a antigen, and the transgene alkaline phosphatase in a mouse lung tissue after nasal instillation of 100 mg plasmid at Days 1 and 7 postinstillation. Figure 11 depicts the results of an example experiment staining lung tissues. Cryosections of lung at day 1 post-instillation of the AdV vector (2.5 X 10 ^u GC) stained for alkaline phosphatase (A, B, E, F, I) and adjacent sections counterstained with H&E (C, D, G, H, J). DS/DOPE targeted expression to the airways (A, B), whereas AdV vector alone resulted in uniform transgene expression (E, F). PBS controls showed no alkaline phosphatase expression (I). H&E staining demonstrated that AdV formulated with DS/DOPE (C, D) resulted in low cellular infiltration, similar to that in the PBS control (J), while AdV vector delivery alone resulted in a high cellular infiltration (G, H).

Figure 12 depicts the results of an example experiment staining lung tissue. Cryosections of lung at day 7 post-instillation of the AdV vector (2.5 X lO ¹¹ GC) stained for alkaline phosphatase (A, B) and adjacent sections counterstained with H&E (E, F). DS/DOPE targeted expression to the airways (A, B), whereas AdV vector alone resulted in uniform transgene expression (C, D). H&E staining demonstrated that AdV formulated with DS/DOPE (E, F) resulted in low cellular infiltration, while AdV vector delivery alone resulted in a high cellular infiltration (G, H). Figure 13 depicts the results of an example experiment assessing LacZ gene expression (A, B) by histochemical staining of lung tissue and adjacent sections counterstained with H&E (C, D) at day 1 post-instillation of liposome-formulated AdV- LacZ vector (10 ¹¹ GC). Both DS/DOPE and DC-Chol/DOPE formulations resulted in airway epithelial cell targeting, but the DS/DOPE formulation (A, C) exhibited less cellular infiltration compared to the DC-Chol/DOPE formulation (B, D). The presence of CD8+ T cells was assessed at days 1 (E, G, I) and 7 (F, H, J) post-instillation of 10 ¹¹ GC of AdV vector. CD8+ T cells are present when AdV was delivered alone (E, F) but not when AdV was formulated with DS/DOPE (G, H) or in PBS controls (I, J).

Figure 14 depicts the results of an example experiment evaluating the effect of adenoviral vector formulation on re-administration of vector, (a) Dosing regimen for AdV-LacZ vector (with or without formulation) or phosphate-buffered saline (PBS)-sham dosing at day 0 followed by homologous re-administration at day 21 with AdV-AIkP. (b) At day 24, AIkP gene expression was observed in the day 0 phosphate- buffered saline (PBS)-sham treatment group but was reduced by about 95% in the day 0 AdV-LacZ treatment group. The use of dexamethasone- spermine/dioleoylphosphatidylethanolamine (DS/DOPE) and two other formulations at day 0 resulted in significantly higher levels of AIkP compared to AdV vector delivered alone at day 0 (PO.05).

Figure 15 depicts the results of an example experiment evaluating neutralizing antibodies (NABs) against AdV vector in sera at day 28 (A) post-single vector administration at day 0 or (B) double administration at days 0 and 14. The titer of serum-circulating NAB for each sample was reported as the highest dilution producing >50 % cells being LacZ-positive after exposure to wild-type AdV-LacZ (multiplicity of infection = 10 ⁴ ) that had been pre-incubated in heat-inactivated diluted serum (1 hour , 37 ⁰ C) (n=3 mice per cohort). All lipid formulations (dexamethasone- spermine/dioleoylphosphatidylethanolamine (DS/DOPE), DC-cholesterol (DC-

Chol)/DOPE and dexamethasone) resulted in suppression of NAB production at day 28 to near-background levels seen with phosphate-buffered saline (PBS)-sham control (A). Suppression of circulating levels of NAB by DS/DOPE formulation was observed after homologous vector re-administration at day 14 (B). In the vector re-administration study, one mouse of the AdV-LacZ (day O)/AdV-human a 1 -antitrypsin (hAAT) (day 14) cohort displayed unusually high NAB titer, but removal of this value (n*=2 bar) still indicated that formulation of AdV-LacZ with DS/DOPE resulted in strong suppression of NAB production (*P=0.05).

Figure 16 depicts the results of an example experiment evaluating localized immune responses following vector delivery to lung. Immunostaining of CD4+ and CD8+ T-cell infiltration in lung sections at day 28 following re-administration of AdV vectors (A and B) without dexamethasone- spermine/dioleoylphosphatidylethanolamine (DS/DOPE) formulation, (C and D) with DS/DOPE formulation only at the first instillation at day 0 and (E and F) with DS/DOPE formulation used for both instillations at days 0 and 14. CD4+ T-cell staining (A, C and E) and CD8 staining (B, D and F) indicated that the use of the DS/DOPE formulation reduced CD4+ and to a lesser extent CD8+ T-cell infiltration.

Figure 17 depicts bioluminescent images showing luciferase expression at 28 days post instillation in mice treated with AAV2/9 vector preparations co-formulated DS (A) or 60/40 mol% DSZD ₂ S (B).

Figure 18 depicts bioluminescent images showing luciferase expression at 14 and 20 days post instillation in mice treated with AAV2/9 vector preparations co- formulated DS/D ₂ S.

Figure 19 depicts a graph of relative light units (RLU) of luciferase expression at 14 and 20 days post instillation in mice treated with AAV2/9 vector preparations co-formulated DSZD ₂ S. Figure 20 depicts a graph of relative light units (RLU) of luciferase expression at 14 and 20 days post instillation in mice treated with AAV2/9 vector preparations co-formulated with two ratios of DS/D ₂ S.

Figure 21 depicts cryosections of lung tissue of mice treated with AAV2/9 or AAV2/6.2 vector preparations co-formulated with DS, DS/D ₂ S, and control at day 28 post instillation stained for β-galactosidase and counter stained with nuclear fast red.

Figure 22 depicts the results of an example experiment evaluating β- galactosidase expression in lung homogenates treated with AAV2/6.2 vector preparations co-formulated with DS, DS/D ₂ S, and control at 28 days post instillation. Error bars represent one standard deviation for each group.

Figure 23 depicts the results of an example experiment evaluating β- galactosidase expression at day 56 in lung homogenates treated with AAV2/9 vector preparations co-formulated with DS, DS/D ₂ S, and control readministered at 28 days post first instillation. Figure 24 depicts the results of an example experiment assessing the level of GAPDH mRNA after treatment with TNF-α, DS, D ₂ S and dexamethasone.

Figure 25 depicts the results of an example experiment assessing the level of IL-6 mRNA, normalized to the level of GAPDH mRNA, after treatment with TNF-α, DS, D ₂ S and dexamethasone. Figure 26 depicts the results of the same example experiment depicted in

Figure 28, but depicted without TNF-α arm of the experiment.

Figure 27 depicts the results of an example experiment assessing the level of osteoprotegerin (OPG) mRNA, normalized to the level of GAPDH mRNA, after treatment with TNF-α, DS, D ₂ S and dexamethasone. Figure 28 depicts the results of an example experiment assessing the level of RANKL mRNA, normalized to the level of GAPDH mRNA, after treatment with TNF-α, DS, D ₂ S and dexamethasone.

Figure 29 depicts the results of an example experiment evaluating tartrate resistant acid phosphatase (TRAP) solution activity in BMM culture at day 5 in response to experimental treatments. Figure 30 depicts the results of an example experiment demonstrating that treatment with DS and DS/D ₂ S (10OnM) resulted in a >30-fold increase in mature osteoclasts (TRAP positive multinuclear cells) compared to dexamethasone in BVIM.

Figure 31 depicts the results of an example experiment evaluating TRAP solution activity in calvarial osteoblast cells/BMM co-culture at day 7 in response to experimental treatments.

Figure 32 depicts the results of an example experiment demonstrating that treatment with DS and DS/D ₂ S (10OnM) resulted in a similar amount or an increase (at 10OnM) in mature osteoclasts (TRAP positive multinuclear cells) compared with treatment with dexamethasone.

DETAILED DESCRIPTION General Description The invention relates generally to compositions and methods for making and using cationic steroids for preventing and diminishing inflammation in a mammal, preferably a human. The invention also relates to compositions and methods for making and using a nonviral delivery vehicle for delivering molecules such as nucleic acids or drugs to cells, both in vivo and in vitro. The invention relates specifically to a nonviral delivery vehicle comprising a cationic lipid nonviral delivery vehicle, the vehicle comprising a steroid or other hydrophobic pharmaceutical agent or drug conjugated to a polyamine and used in conjunction with a lipid. The invention also relates to modifying existing drugs to create new drug entities with new pharmacokinetic/pharmacodynamic properties. The methods of the invention include making the delivery vehicle and use of the delivery vehicle. The invention also relates to use of the delivery vehicle to transfect nucleic acids into cells. The invention further relates to a platform chemistry or delivery vehicle with inherent pharmacological or biological activity, such as anti-inflammatory activity that offers new pharmacokinetic or pharmacodynamic properties distinct from the parent pharmaceutical agent or compound, and that can be used alone or with other agents. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.

As used herein, each of the following terms has the meaning associated with it in this section. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, "alleviating a disease or disorder symptom," means reducing the severity of the symptom. A "cationic lipid" or "cationic steroid" or "cationic drug" is a lipid or steroid or drug or hydrophobic moiety which has a positive charge, or is part of a complex which has a positive charge, such as a steroid coupled with a polyamine.

A "compound," as used herein, refers to any type of substance or agent that is commonly considered a drug, or a candidate for use as a drug, as well as combinations and mixtures of the above.

The term "delivery vehicle," as used herein, refers to a molecule or composition useful for binding or carrying another molecule, such as a nucleic acid or drug, and delivering it to a target site, such as a cell. "Delivery vehicle" is used interchangeably with terms such as "drug delivery vehicle" "nucleic acid delivery vehicle," or "delivery vector."

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, are reduced.

As used herein, the term "domain" refers to a part of a molecule or structure that shares common physicochemical features, such as, but not limited to, hydrophobic, polar, globular and helical domains or properties such as ligand binding, signal transduction, cell penetration and the like. Specific examples of binding domains include, but are not limited to, DNA binding domains and ATP binding domains. An "effective amount" or "therapeutically effective amount" of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. An "effective amount" of a delivery vehicle is that amount sufficient to effectively bind or deliver a compound. "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

As used herein, the term "fragment," as applied to a protein or peptide, can ordinarily be at least about 3-15 amino acids in length, at least about 15-25 amino acids, at least about 25-50 amino acids in length, at least about 50-75 amino acids in length, at least about 75-100 amino acids in length, and greater than 100 amino acids in length. As used herein, the term "fragment," as applied to a nucleic acid, can ordinarily be at least about 20 nucleotides in length, typically, at least about 50 nucleotides, more typically, from about 50 to about 100 nucleotides, preferably, at least about 100 to about 200 nucleotides, even more preferably, at least about 200 nucleotides to about 300 nucleotides, yet even more preferably, at least about 300 to about 350, even more preferably, at least about 350 nucleotides to about 500 nucleotides, yet even more preferably, at least about 500 to about 600, even more preferably, at least about 600 nucleotides to about 620 nucleotides, yet even more preferably, at least about 620 to about 650, and most preferably, the nucleic acid fragment will be greater than about 650 nucleotides in length.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of a compound, composition, vector, or delivery system of the invention in the kit for effecting alleviation of the various diseases or disorders recited herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating the diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit of the invention can, for example, be affixed to a container which contains the identified compound, composition, vector, or delivery system of the invention or be shipped together with a container which contains the identified compound, composition, vector, or delivery system. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

An "isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g, as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

The term "nucleic acid" typically refers to large polynucleotides.

The term "oligonucleotide" typically refers to short polynucleotides, generally, no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i. e. , A, U, G, C) in which "U" replaces "T."

The term "peptide" typically refers to short polypeptides.

As used herein, the term "pharmaceutically-acceptable carrier" means a chemical composition with which an appropriate delivery vehicle and nucleic acid, drug, or compound can be combined and which, following the combination, can be used to administer the appropriate delivery vehicle and nucleic acid, drug, or compound to a subject.

As used herein, the term "physiologically acceptable" ester or salt means an ester or salt form of the active ingredient which is compatible with any other ingredients of the pharmaceutical composition, which is not deleterious to the subject to which the composition is to be administered.

"Polyamine" as used herein refers to polymers of amines as well as to other types of molecules containing amines, such as amine rich polymers or other amine containing polymers, a lysine containing peptide, and an arginine containing peptide.

"Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof.

A "polynucleotide" means a single strand or parallel and anti-parallel strands of a nucleic acid. Thus, a polynucleotide may be either a single-stranded or a double-stranded nucleic acid. "Primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single-stranded, but can be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties.

A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. The term "protein" typically refers to large polypeptides. "Slow release," as used herein, refers to delivery of a nucleic acid, drug, or molecule to a cell, tissue, or organ, wherein the nucleic acid, drug, or molecule is not all readily available because some remains bound to the delivery vehicle or to an anionic molecule and is slowly released for availability over a period of time. The period of time should be at least 10% longer than availability that is not slow release, preferably at least 25% longer, more preferably at least 35% longer, and even more preferably at least 50% longer. Such a drug or molecule can include a prodrug or steroid prodrug. "Synthetic peptides or polypeptides" mean a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Those of skill in the art know of various solid phase peptide synthesis methods.

A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs. "Tissue," as used herein, refers to the general definition of tissue which includes a collection of similar cells and the intercellular substances and spaces surrounding them, and the term is also used herein to include collections of similar cells in tissues and organs. The term "topical application," as used herein, refers to administration to a surface, such as the skin. This term is used interchangeably with "cutaneous application."

By "transdermal" delivery is intended both transdermal (or

"percutaneous") and transmucosal administration, i.e., delivery by passage of a drug through the skin or mucosal tissue and into the bloodstream. Transdermal also refers to the skin as a portal for the administration of drugs or compounds by topical application of the drug or compound thereto.

The term to "treat," as used herein, means reducing the frequency with which symptoms are experienced by a patient or subject or administering an agent or compound to reduce the frequency with which symptoms are experienced. As used herein, "treating a disease or disorder" means reducing the frequency with which a symptom of the disease or disorder is experienced by a patient. Disease and disorder are used interchangeably herein.

A "vector," as used herein, refers to either a delivery vehicle as described herein or to a vector such as an expression vector.

Description

Synthesis and Hydrolysis of Cationic Steroids or Drugs for Use as Nucleic Acid and Drug Delivery Vehicles It has been discovered in the present invention that using novel methods steroids or drugs can be conjugated with a polyamine, and that the resultant molecule can bind nucleic acids, drugs, and other molecules (See Examples 1-10). The invention further encompasses methods for making steroids, drugs, or other compounds or molecules cationic. Furthermore, it has been discovered in the present invention that when coupled with a lipid, this nonviral molecule is capable of delivering nucleic acids and drugs to cells (Examples 1 and 2). It has also been discovered in the present invention that the steroid can maintain its inherent biological activity once coupled with the polyamine. It has been further discovered in the invention that various steroids, polyamines, and lipids are useful for the methods of the invention. In addition, it is disclosed herein that the delivery vehicle of the present invention has the ability to target molecules of interstitial spaces as potential delivery sites to allow slow release of a compound at a local interstitial site. Interstitial space or tissue includes extracellular space. The invention also includes methods of using the invention to treat a disease and kits to administer the nonviral delivery vehicle.

The invention also relates to methods for modifying existing drugs to create new drug entities with new pharmacokinetic/pharmacodynamic properties using the platform chemistry of the invention. The new drug entity may or may not be used with a lipid. The new drug entity may function on its own or as a pro-drug that releases an active drug.

The present invention discloses novel methods for delivering nucleic acids, including DNA, to cells and causing transfection of the DNA into a cell and expression of the DNA.

The invention should be construed to include various steroids or hydrophobic drugs as described herein, including glucocorticoids, and should not be construed to include only the steroids described herein. Such steroids included, but are not limited to, a mineral corticoid, an androgen, an estrogen, a progestagen, an analog with steroidal agonist activity, an analog with steroidal antagonist activity, an inactive structural analog, and modifications or derivatives thereof. For example, additional steroids which are useful in the invention include, cortexolone mesylate, cortisone mesylate, prednisone 21-mesylate, 3β-iodocholesterol, 16β-bromo-4-androsten-3,17- dione, 2α-bromo-5α-cholestan-3-one, 16α-bromoestradiol, 16α-bromoestrone, 16β- bromoestrone, 17α-bromopregnenolone, 17-bromoprogesterone, androsterone tosylate, cholestanol tosylate, cholesteryl tosylate, dehydroepiandrosterone tosylate, dihydrotestosterone tosylate, epiandrosterone tosylate, 1 lα-hydroxyprogesterone tosylate, 19-nortestosterone tosylate, pregnenolone tosylate, and testosterone tosylate. The invention should be construed to include active and inactive analogs, modifications, and derivatives of the steroids and drugs, as well as steroid prodrugs and prodrugs. In addition, the invention should be construed to include various polyamines, including spermine. In addition to the polyamine spermine, the invention should be construed to include any other polyamine, including, but not limited to, spermidine, polylysine, proteins, peptides, oligonucleotides, other biopolymers, synthetic polyamines, such as polyamines (for example, polyethyleneimine), a lysine containing peptide, an arginine containing peptide, a cationic polymer, and an amine rich polymer, and amino dendrimers.

In one aspect of the invention the lipid is a cationic lipid and in another aspect the lipid is a neutral lipid. Neutral lipids of the invention include, but are not limited to, DOPE, phosphatidylcholine (PC), and cholesterol. Cationic lipids of the invention include, but are not limited to, 3-beta-[N',N'dimethylaminoethane)- carbamoyl]cholesterol) (DC-Choi), N[l-(2,3-dioleyloxy)propyl [N,N,N-triethyl- ammonium (DOTMA), 2'-(l",2"-dioleoyloxypropyldimethyl-ammonium bromide)-N- ethyl-6-aminospermine tetra trifluoroacetic acid (DOSPA), l,3-bis(oleoyloxy)-3- (trimethylammonio)propane (DOTAP), and GL-67. In one aspect of the invention, the lipid is a helper lipid to aid in delivery of a nucleic acid, drug, or other compound. Furthermore, the invention should be construed to include lipids other than those described herein.

In one embodiment of the invention a steroid or drug and a polyamine such as spermine are coupled using a coupling reagent. In one aspect of the invention the reagent is 2-iminothiolane. In another aspect of the invention, a steroid or drug and spermine are also mixed with dimethylsulfoxide. In yet another aspect of the invention, a purified steroid-polyamine complex or purified drug-polyamine complex are mixed with a lipid to form a delivery vehicle. In one embodiment, the product of the synthesis reaction is dexamethasone-spermine (DS) molecule, resulting from conjugation of dexamethasone to a primary amine on spermine (see Figure IB). DS exhibits a cationic charge at physiological pH due to the secondary amines retained from spermine. In another embodiment, the product of the synthesis reaction is an amphiphilic dimer, disubstituted spermine (D ₂ S), resulting from conjugation of dexamethasone to both primary amines on spermine (see Figure 7). D ₂ S exhibits the characteristic cationic charge of the DS product at physiological pH due to the secondary amines retained from spermine, but has additional hydrophobicity relative to DS resulting from the additional dexamethasone moiety.

In Vitro Molecule Delivery with a Cationic Lipid Nonviral Delivery Vehicle

Various methods and assays have been described herein to synthesize a steroid-polyamine complex or a drug-polyamine complex of the invention and methods to add lipids to the complex to arrive at a cationic lipid nonviral delivery vehicle of the present invention. Various assays have also been disclosed herein to demonstrate the ability of the delivery vehicle to bind a compound and to deliver a compound to a cell. Preferably the cell is a mammalian cell, and more preferably the cell is a human cell. The invention includes methods of treating a disease or disorder in a mammal, using a nonviral delivery vehicle of the invention. In one aspect of the invention the vehicle delivers a nucleic acid and in another aspect the vehicle delivers a drug or another compound. In one aspect the mammal is a human. Diseases or disorders of the invention which may be treated using a delivery vehicle of the invention include, but are not limited to, inflammation, asthma, arthritis, pain, inflammation of joints, cancer, allergies, hypertension, neoplasia, hyperplasia, metastasis, claudication, intimal hyperplasia, hemophilia, anemia, coagulopathies, autoimmune disorders, duodenal ulcers, gastric ulcers, erosive esophagitis, pathological hypersecretory conditions, rhinitis, chronic idiopathic urticaria, hypersecretory conditions, heartburn, candidiasis, Helicobacter pylori infection, osteoarthritis, rheumatoid arthritis, familial adenomatous polyposis, depression, obsessive-compulsive disorder, bulimia nervosa, premenstrual dysphoric disorder, psychotic disorders, bipolar disorder, obsessive-compulsive disorder, posttraumatic stress disorder, panic disorder, panic disorder, social anxiety disorder, schizophrenia, psychotic disorders, generalized anxiety disorder, dysmenorrhea, menopausal symptoms, osteoporosis, prostate cancer, breast cancer, hypoestrogenism, Kraurosis vulvae, hypercholesterolemia, congestive heart failure, cardiac ischemic complications, myocardial infarction, hypertension, left ventricular dysfunction, type 2 diabetes, ovarian cancer, nonsmall cell lung cancer, Kaposi sarcoma, hairy cell leukemia, warts, malignant melanoma, hepatitis C, hepatitis B, non-Hodgkin lymphoma, erectile dysfunction, epilepsy, Paget disease, neutropenia, progenitor cell mobilization, heart transplant rejection, kidney transplant rejection, liver transplant rejection, psoriasis, pain, cluster headache, migraine, angina, gastritis, endometriosis, central precocious puberty, bronchospasm, gastro-esophageal reflux disease, mastocytosis, and proliferative disorders.

Some examples of diseases which may be treated according to the methods of the invention are described above. The invention should not be construed as being limited solely to these examples, as other diseases or disorders which are at present unknown, once known, may also be treatable using the methods of the invention In one embodiment of the invention, the delivery vehicle delivers a nucleic acid, steroid, drug, or compound to a cell. In one aspect of the invention the cell is selected from the group consisting of an endothelial cell, a mesenchymal cell, a neural cell, a fibroblast, neuron, a smooth muscle cell, a kidney cell, a liver cell, a myoblast, a stem cell, an embryonic stem cell, a hematopoietic stem cell, an osteoblast, a chondrocyte, a chondroblast, a monocyte, a neutrophil, a macrophage, a retinal nerve cell, and an epithelial cell. Preferably the cell is a mammalian cell. More preferably it is a human cell.

In one embodiment of the invention, the delivery vehicle delivers a nucleic acid, steroid, drug, or compound to a tissue. In one aspect of the invention, the tissue comprises muscle, mucosa, epithelial, nerve, connective, blood, stromal, heart, liver, kidney, skin, brain, intestinal, interstitial space, bone, bone marrow, joint, cartilage, tendon, esophagus, gonad, cerebrospinal fluid, pancreas, spleen, ocular, nasal cavity, and hair tissue.

The invention should not be construed as being limited solely to these examples, as other diseases and disorders treatable by the nonviral delivery vehicle of the invention which are at present unknown, once known, may also be treatable using the methods of the invention.

In one embodiment of the invention, a disease or disorder is treated by administering the delivery vehicle and a nucleic acid or drug via an oral route. Other routes of administration, include, but are not limited to intranasal, rectal, vaginal, intramuscular, topical, subdermal, sublingual, intraperitoneal, and intravenous. The invention relates to the administration of a nonviral delivery vector and an identified nucleic acid, drug, or other molecule to be delivered, in a pharmaceutical composition to practice the methods of the invention. The composition comprises the nonviral delivery vector and an identified nucleic acid, drug, or other molecule to be delivered and a pharmaceutically-acceptable carrier. For example, a nonviral delivery vehicle with which an appropriate identified nucleic acid, drug, or other molecule to be delivered, is combined, is used to administer an identified nucleic acid, drug, or other molecule to be delivered an animal. One of skill in the art would recognize that when more than one nucleic acid or drug or other compound is being administered, each additional one may be delivered with a delivery vehicle of the invention and/or additional nucleic acids, drugs, or other molecules may be delivered independent of a delivery vehicle of the invention.

The invention should not be construed to being limited solely to the isolated nucleic acids, drugs, or other molecules described herein as those which may be delivered by a delivery vehicle of the invention. The invention should be construed to include other nucleic acids, drugs or other molecules not described herein, which can also be delivered by the delivery vehicle of the invention. These other nucleic acids, drugs, or other molecules with which the delivery vehicle of the invention can bind and/or deliver include, but are not limited to, hydrophobic chemical entities, fat soluble drugs, anionic chemical entities, anionic radionucleotides, anionic or hydrophobic radioisotopes for chemotherapy, oligonucleotides, single or double stranded RNA or DNA oligonucleotides or nucleotides or fragments thereof, PCR products, RNA-DNA chimeric molecules, RNAi (RNA interference), peptide-nucleic acid (PNA), peptides or proteins containing anionic acid groups such as glutamic or aspartic acid, heparin, low molecular weight heparin, anionic glycosaminoglycans, anionic or hydrophobic fluorescent molecules, and viral particles or subtractions of virus particles. Other molecules and drugs which are included among the compounds which can be delivered by the delivery vehicle of the invention include, but are not limited to, recombinant proteins, erythropoietin, tissue plasminogen activator (tPA), tumor necrosis factor-alpha receptor, Omeprazole, Simvastatin, Atorvastatin calcium, Amlodipine besylate, Loratadine,

Lansoprazole, Epoetin alfa, Celecoxib, Fluoxetine hydrochloride, Olanzapine, Paroxetine hydrochloride, Rofecoxib, Sertraline hydrochloride, Epoetin alfa, a conjugated estrogens, Amoxicillin and clavulanate Potassium, Pravastatin sodium, Enalapril maleate, Metformin hydrochloride, Pravastatin, Losartan potassium, Ciprofloxacin hydrochloride, Risperidone, Paclitaxel, Azithromycin, interferon alpha-2b, rebavirin, Sildenafil citrate, Gabapentin, Fluticasone propionate, Alendronate sodium, Clarithromycin, Filgrastim, cyclosporine, Lisinopril dihydrate, venlafaxine HCl, human insulin, Levofloxacin, Fexofenadine, Hydrochloride, Lisinopril/lisinopril, Sumatriptan succinate, Nifedipine, Fluconazole, Ceftriaxone sodium, Famotidine, Enoxaparin sodium, Leuprolide acetate, Salmeterol xinafoate, Clopidogrel bisulfate, Lansoprazole, and Ranitidine. The delivery vehicle of the invention may also bind with anionic domains in polymers such as an anionic glycosaminoglycan, a collagen, a fibrin, a cellular glycocalyx, a red blood cell glycocalyx, a sialic acid, a sulfated glycocalyx, a deoxyribonucleic acid and a ribonucleic acid.

The invention further relates to the used of cationic steroid prodrugs and cationic prodrugs and slow release therapies. In one embodiment of the invention, the delivery vehicle or the nucleic acid, drug, compound, or molecule being delivered by the delivery vehicle may target molecules of interstitial and extracellular spaces by binding to said molecules. Such binding then allows slow release of the nucleic acid, drug, compound, or molecule at a local site. In one embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In another embodiment, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 g/kg/day. Pharmaceutically acceptable carriers which are useful include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey). The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable, aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3 -butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.

Pharmaceutical compositions that are useful in the methods of the invention may be administered, prepared, packaged, and/or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

The compositions of the invention may be administered via numerous routes, including, but not limited to, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, or ophthalmic administration routes. The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like. Pharmaceutical compositions that are useful in the methods of the invention may be administered systemically in oral solid formulations, ophthalmic, suppository, aerosol, topical or other similar formulations. In addition to the compound such as heparan sulfate, or a biological equivalent thereof, such pharmaceutical compositions may contain pharmaceutically-acceptable carriers and other ingredients known to enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, resealed erythrocytes, and immunologically based systems may also be used to administer compounds according to the methods of the invention.

Compounds which are identified using any of the methods described herein may be formulated and administered to a mammal for treatment of various diseases, disorders, or conditions described herein. The invention encompasses the preparation and use of pharmaceutical compositions comprising a compound useful for treatment of various diseases, disorders, or conditions described herein. Such a pharmaceutical composition may consist of the active ingredient alone, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of these. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

An obstacle for topical administration of pharmaceuticals is the stratum corneum layer of the epidermis. The stratum corneum is a highly resistant layer comprised of protein, cholesterol, sphingolipids, free fatty acids and various other lipids, and includes cornified and living cells. One of the factors that limits the penetration rate (flux) of a compound through the stratum corneum is the amount of the active substance which can be loaded or applied onto the skin surface. The greater the amount of active substance which is applied per unit of area of the skin, the greater the concentration gradient between the skin surface and the lower layers of the skin, and in turn the greater the diffusion force of the active substance through the skin. Therefore, a formulation containing a greater concentration of the active substance is more likely to result in penetration of the active substance through the skin, and more of it, and at a more consistent rate, than a formulation having a lesser concentration, all other things being equal.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.

Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, intrathecal or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient. In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents. Particularly contemplated additional agents include anti-emetics and scavengers such as cyanide and cyanate scavengers.

Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology. Formulations suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions or suspensions. Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.

Enhancers of permeation may be used. These materials increase the rate of penetration of drugs across the skin. Typical enhancers in the art include ethanol, glycerol monolaurate, PGML (polyethylene glycol monolaurate), dimethylsulfoxide, and the like. Other enhancers include oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone.

The source of active compound to be formulated will generally depend upon the particular form of the compound. Small organic molecules and peptidyl or oligo fragments can be chemically synthesized and provided in a pure form suitable for pharmaceutical/cosmetic usage. Products of natural extracts can be purified according to techniques known in the art. Recombinant sources of compounds are also available to those of ordinary skill in the art.

In alternative embodiments, the topically active pharmaceutical or cosmetic composition may be optionally combined with other ingredients such as moisturizers, cosmetic adjuvants, anti -oxidants, chelating agents, bleaching agents, tyrosinase inhibitors and other known depigmentation agents, surfactants, foaming agents, conditioners, humectants, wetting agents, emulsifying agents, fragrances, viscosifϊers, buffering agents, preservatives, sunscreens and the like. In another embodiment, a permeation or penetration enhancer is included in the composition and is effective in improving the percutaneous penetration of the active ingredient into and through the stratum corneum with respect to a composition lacking the permeation enhancer. Various permeation enhancers, including oleic acid, oleyl alcohol, ethoxydiglycol, laurocapram, alkanecarboxylic acids, dimethylsulfoxide, polar lipids, or N-methyl-2-pyrrolidone, are known to those of skill in the art. In another aspect, the composition may further comprise a hydrotropic agent, which functions to increase disorder in the structure of the stratum corneum, and thus allows increased transport across the stratum corneum. Various hydrotropic agents such as isopropyl alcohol, propylene glycol, or sodium xylene sulfonate, are known to those of skill in the art. The compositions of this invention may also contain active amounts of retinoids (i.e., compounds that bind to any members of the family of retinoid receptors), including, for example, tretinoin, retinol, esters of tretinoin and/or retinol and the like.

The topically active pharmaceutical or cosmetic composition should be applied in an amount effective to affect desired changes. As used herein "amount effective" shall mean an amount sufficient to cover the region of skin surface where a change is desired. An active compound should be present in the amount of from about 0.0001% to about 15% by weight volume of the composition. More preferable, it should be present in an amount from about 0.0005% to about 5% of the composition; most preferably, it should be present in an amount of from about 0.001% to about 1% of the composition. Such compounds may be synthetically- or naturally-derived. Liquid derivatives and natural extracts made directly from biological sources may be employed in the compositions of this invention in a concentration (w/v) from about 1 to about 99%. Fractions of natural extracts and protease inhibitors may have a different preferred rage, from about 0.01% to about 20% and, more preferably, from about 1% to about 10% of the composition. Of course, mixtures of the active agents, of this invention may be combined and used together in the same formulation, or in serial applications of different formulations.

The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of an aqueous gel because of repeated patient use when it is exposed to contaminants in the environment from, for example, exposure to air or the patient's skin, including contact with the fingers used for applying a composition of the invention such as a therapeutic gel or cream. Examples of preservatives useful in accordance with the invention included but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and combinations thereof. A particularly preferred preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05% to 0.5% sorbic acid.

The composition preferably includes an antioxidant and a chelating agent which inhibit the degradation of the compound for use in the invention in the aqueous gel formulation. Preferred antioxidants for some compounds are BHT, BHA, alphatocopherol and ascorbic acid in the preferred range of about 0.01% to 0.3% and more preferably BHT in the range of 0.03% to 0.1 % by weight by total weight of the composition.

Preferably, the chelating agent is present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Particularly preferred chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20% and more preferably in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition which may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are the particularly preferred antioxidant and chelating agent respectively for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefor as would be known to those skilled in the art. Controlled-release preparations may also be used and the methods for the use of such preparations are known to those of skill in the art.

In some cases, the dosage forms to be used can be provided as slow or controlled-release of one or more active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, or microspheres or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the invention. Thus, single unit dosage forms suitable for oral administration, such as tablets, capsules, gelcaps, and caplets, that are adapted for controlled-release are encompassed by the present invention. All controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood level of the drug, and thus can affect the occurrence of side effects. Most controlled-release formulations are designed to initially release an amount of drug that promptly produces the desired therapeutic effect, and gradually and continually release of other amounts of drug to maintain this level of therapeutic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body.

Controlled-release of an active ingredient can be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. The term "controlled-release component" in the context of the present invention is defined herein as a compound or compounds, including, but not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, or microspheres or a combination thereof that facilitates the controlled-release of the active ingredient.

Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent.

Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, and acacia. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl- para- hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin. Known thickening agents for oily suspensions include, for example, beeswax, hard paraffin, and cetyl alcohol. Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Powdered and granular formulations of a pharmaceutical preparation of the invention may be prepared using known methods. Such formulations may be administered directly to a subject, used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by addition of an aqueous or oily vehicle thereto. Each of these formulations may further comprise one or more of dispersing or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweetening, flavoring, or coloring agents, may also be included in these formulations.

A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in- water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

As used herein, an "oily" liquid is one which comprises a carbon- containing liquid molecule and which exhibits a less polar character than water. A formulation of a pharmaceutical composition of the invention suitable for oral administration may be prepared, packaged, or sold in the form of a discrete solid dose unit including, but not limited to, a tablet, a hard or soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, a powdered or granular formulation, an aqueous or oily suspension, an aqueous or oily solution, a paste, a gel, a toothpaste, a mouthwash, a coating, an oral rinse, or an emulsion. The terms oral rinse and mouthwash are used interchangeably herein.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for oral or buccal administration. Such a formulation may comprise, but is not limited to, a gel, a liquid, a suspension, a paste, a toothpaste, a mouthwash or oral rinse, and a coating. For example, an oral rinse of the invention may comprise a compound of the invention at about 1.4 %, chlorhexidine gluconate (0.12%), ethanol (11.2%), sodium saccharin (0.15%), FD&C Blue No. 1 (0.001%), peppermint oil (0.5%), glycerine (10.0%), Tween 60 (0.3%), and water to 100%. In another embodiment, a toothpaste of the invention may comprise a compound of the invention at about 5.5%, sorbitol, 70% in water (25.0%), sodium saccharin (0.15%), sodium lauryl sulfate (1.75%), carbopol 934, 6% dispersion in (15%), oil of spearmint (1.0%), sodium hydroxide, 50% in water (0.76%), dibasic calcium phosphate dihydrate (45%), and water to 100%. The examples of formulations described herein are not exhaustive and it is understood that the invention includes additional modifications of these and other formulations not described herein, but which are known to those of skill in the art.

A tablet comprising the active ingredient may, for example, be made by compressing or molding the active ingredient, optionally with one or more additional ingredients. Compressed tablets may be prepared by compressing, in a suitable device, the active ingredient in a free-flowing form such as a powder or granular preparation, optionally mixed with one or more of a binder, a lubricant, an excipient, a surface active agent, and a dispersing agent. Molded tablets may be made by molding, in a suitable device, a mixture of the active ingredient, a pharmaceutically acceptable carrier, and at least sufficient liquid to moisten the mixture. Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycollate. Known surface-active agents include, but are not limited to, sodium lauryl sulphate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binding agents include, but are not limited to, gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricating agents include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Tablets may be non-coated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the active ingredient. By way of example, a material such as glyceryl monostearate or glyceryl distearate may be used to coat tablets. Further by way of example, tablets may be coated using methods described in U.S. Patents numbers 4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlled release tablets. Tablets may further comprise a sweetening agent, a flavoring agent, a coloring agent, a preservative, or some combination of these in order to provide for pharmaceutically elegant and palatable preparation.

Hard capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such hard capsules comprise the active ingredient, and may further comprise additional ingredients including, for example, an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin. Soft gelatin capsules comprising the active ingredient may be made using a physiologically degradable composition, such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the invention which are suitable for oral administration may be prepared, packaged, and sold either in liquid form or in the form of a dry product intended for reconstitution with water or another suitable vehicle prior to use.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for rectal administration. Such a composition may be in the form of, for example, a suppository, a retention enema preparation, and a solution for rectal or colonic irrigation. Suppository formulations may be made by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e., about 20°C) and which is liquid at the rectal temperature of the subject (i.e., about 37°C in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols, and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.

Retention enema preparations or solutions for rectal or colonic irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, enema preparations may be administered using, and may be packaged within, a delivery device adapted to the rectal anatomy of the subject. Enema preparations may further comprise various additional ingredients including, but not limited to, antioxidants, and preservatives.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for vaginal administration. Such a composition may be in the form of, for example, a suppository, an impregnated or coated vaginally-insertable material such as a tampon, a douche preparation, or gel or cream or a solution for vaginal irrigation.

Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Douche preparations or solutions for vaginal irrigation may be made by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, douche preparations may be administered using, and may be packaged within, a delivery device adapted to the vaginal anatomy of the subject.

Douche preparations may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives. As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialytic infusion techniques. Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system.

Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets or lozenges made using conventional methods, and may, for example, 0.1 to 20% (w/w) active ingredient, the balance comprising an orally dissolvable or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder or an aerosolized or atomized solution or suspension comprising the active ingredient. Such powdered, aerosolized, or aerosolized formulations, when dispersed, preferably have an average particle or droplet size in the range from about 0.1 to about 200 nanometers, and may further comprise one or more of the additional ingredients described herein.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Genaro, ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

Typically, dosages of the compound of the invention which may be administered to an animal, preferably a human, will vary depending upon any number of factors, including but not limited to, the type of animal and type of disease state being treated, the age of the animal and the route of administration. The compound can be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even lees frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the, animal, etc. It will be recognized by one of skill in the art that the various embodiments of the invention as described above relating to methods of treating diseases, disorders or conditions, includes other diseases, disorders and conditions not described herein. The invention further includes kits for treating a disease or disorder in an animal.

In accordance with the present invention, as described above or as discussed in the Examples below, there can be employed conventional chemical, cellular, histochemical, biochemical, molecular biology, microbiology and recombinant DNA techniques which are known to those of skill in the art. Such techniques are explained fully in the literature. See for example, Sambrook et al., 1989 Molecular Cloning - a Laboratory Manual, Cold Spring Harbor Press; Glover, (1985) DNA Cloning: a Practical Approach; Gait, (1984) Oligonucleotide Synthesis; Harlow et al., 1988 Antibodies - a Laboratory Manual, Cold Spring Harbor Press; Roe et al., 1996 DNA Isolation and Sequencing: Essential Techniques, John Wiley; and Ausubel et al., 1995 Current Protocols in Molecular Biology, Greene Publishing.

Adeno-Associated Viral Vectors and Physicochemical Characteristics

In one embodiment, viral-mediated transduction can be used to deliver nucleic acid. In some embodiments, replication incompetent recombinant viral vectors are able to insert a therapeutic transgene into the host cell genome, resulting in long-term expression.

In one embodiment, the viral vector is an adeno-associated virus (AAV). AAVs are icosahedral non-enveloped single-stranded DNA viruses belonging to the parvovirus family, which require a helper virus for efficient reproduction. Adeno- associated virus (AAV) infection involves cell surface binding, viral uptake, intracellular trafficking, translocation to the nucleus, capsid uncoating, synthesis of double stranded DNA, and viral gene expression. AAV2 can infect a broad range of cell types. The ubiquitous AAV receptor is heparan sulfate proteoglycan (HSPG); however, co-receptors (such as, for example, fibroblast growth factor 1 and α _v β ₅ integrin for AAV2) have also been shown to play an important role. Different AAV serotypes exhibit different cell-type and tissue tropisms such as, for example, lung alveolar epithelium for AAV9 and heart for AAV8. Some cell types and tissue types are more difficult than others to target. For example, the respiratory epithelium presents number of natural barriers, including a well-defined mucus layer, lack of apical receptors, and a preexisting or induced immune response to AAV vectors that can cause issues with re-administration. In one embodiment, the anti-inflammatory activity of the compounds of the invention can be utilized to prevent, inhibit, attenuate or ameliorate the immune response to AAV vectors.

Inflammatory model of arthritis

Glucocorticoids have been used to treat inflammation for a diverse set of disorders including asthma, rheumatoid arthritis, and dermatological conditions. Glucocorticoids are believed to produce their effects by both genomic and non-genomic mechanisms. Glucocorticoids are known influence the regulation of numerous genes. Two genomic mechanisms of glucocorticoid action include transactivation and transrepression of gene transcription which generate the pharmacological effects of the compounds. While not wishing to be bound by any particular theory, it is believed that while transrepression of inflammatory transcription factors comprises the majority of the anti-inflammatory activity, transactivation is believed to play a larger role in the undesired side effects. Because transactivation and transrepression mechanisms of glucocorticoid genomic regulation are believed to rely on different conformations of glucocorticoid-glucocorticoid receptor (GR) complexes, differences in structure of the glucocorticoid compound can be exploited to dissociate the transrepression and transactivation activity of these compounds and thereby dissociate at least some of the anti-inflammatory effects, and undesired side affects, of glucocorticoid compounds

As disclosed herein, a comparison of DS and D ₂ S with some common glucocorticoids demonstrates that the modified structure of the glucocorticoid compounds of the invention effects the activity of the glucocorticoid compounds of the invention, presumably due, at least in part, to the presence of the cationic regions not typically present in this class of compounds. While not wishing to be bound by any particular theory, these differences in structure may affect the ability of the GR bound complexes to dimerize, leading to potential dissociation of the transrepression/transactivation activity of the compounds. It is an aspect of the invention that the glucocorticoid compounds of the invention ameliorate inflammation. It is further an aspect of the invention that the glucocorticoid compounds of the invention ameliorate inflammation and dissociate the anti-inflammatory effects from the undesired side effects of glucocorticoid compounds.

An in vitro model system that has been used to analyze the dissociated character of glucocorticoids is an induced arthritis phenotype in the bone microenvironment. It is generally accepted that prolonged glucocorticoid treatment can lead to deleterious side effects in vivo including a profound impact on skeletal remodeling and predisposition to osteoporosis. Following widespread glucocorticoid use therapeutically in the treatment of rheumatoid arthritis, a number of adverse side effects have been discovered including glucocorticoid-induced osteoporosis (GIO). Some reports have linked the GIO side effect of glucocorticoids to impairment of osteoclasts which indirectly suppresses bone formation. It's been suggested that glucocorticoids can exhibit pleiotropic effects on bone metabolism including suppression of osteoblast activity, increased osteoblast apoptosis, as well as effects on the formation, function, activity, and signaling of osteoclasts. The dissociation of transactivation/transrepression activities of glucocorticoid compound may improve the treatment of rheumatoid arthritis by reducing inflammation without at the same time inducing GIO. It is an aspect of the invention that the dissociated transactivation/transrepression activities of the glucocorticoid compounds of the invention can reduce inflammation without at the same time inducing unwanted side effects, such as, for example, GIO.

One particular signaling pathway which may be used to evaluated the dissociated character of glucocorticoid steroids is the receptor activator of NF-κB ligand (RANKL) - receptor activator of NF-κB (RANK) - osteoprotegerin (OPG) axis. Osteoblasts induce osteoclastogenesis from precursor bone-marrow macrophages (BMMs) in a contact dependent manner. RANKL binds to RANK on the surface of BMMs which initiates the activation of mature osteoclast formation demonstrated by the emergence of multinuclear tartrate-resistant acid phosphatase (TRAP) positive osteoclasts. OPG is a decoy receptor of RANKL which osteoblasts secrete in order to control the signaling of RANKL. It's been shown that inflammatory stimuli cause an imbalance in this signaling pathway via upregulation of RANKL, downregulation of OPG, as well as other possible effects. Despite reducing inflammatory cytokines, such as IL-I and IL-6, some studies have shown that dexamethasone induces similar changes in the RANKL-RANK-OPG pathway leading to increased osteoclastogenesis and bone resorption, which has been proposed as the mechanism leading to GIO. It is an aspect of the invention that the dissociated character of the glucocorticoid compounds of the invention can reduce inflammation without at the same time inducing unwanted side effects, such as, for example, increased osteoclastogenesis and bone resorption.

Experimental Examples

The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1- Synthesis and Hydrolysis of Cationic Steroids DS and D?S

There is a need in the art to be able to easily synthesize vectors or vehicles useful for nonviral delivery of molecules to and into cells. A method to fulfill this need is disclosed in the present invention.

The Materials and Methods used in the present example are now described.

Synthesis of Cationic Steroids Dexamethasone mesylate or dexamethasone (Steraloids, NH), 2- iminothiolane (Traut's reagent) (Pierce), spermine (Sigma), and DMSO (Aldrich) were used as received. A total of 105 mg (223 μmol) of dexamethasone-mesylate, 800 μl of DMSO, and 28.4 mg (206 μmol) of Traut's reagent were dissolved together prior to addition of 31.9 μl (145 μmol of spermine at room temperature. After 45 minutes, the reaction was complete by TLC. HPLC purification (60/40 0.1 %TFA/acetonitrile, Hamilton PRP-I column) and freeze-drying yielded DS as a white powder. Yield: 15.3 mg (14.0%).

Large Scale, High Yield, Multi-gram Synthesis of Steroid Mesylate Precursor for Conjugation

A 200 mL Erlenmeyer flask equipped with a magnetic stir bar and a rubber septum was charged with 5 grams of dexamethasone (12.7 mmol, 1 equivalent) and 16 mL of anhydrous pyridine. The flask was cooled to 0°C with an ice bath, and 1.2 mL of methanesulfonyl chloride was added. After 1 hour, 0.8 mL of additional methanesulfonyl chloride was added. At two hours, the solution was poured into 100 mL of cold IM HCl, forming a precipitate. The precipitate was filtered and redissolved in 250 mL of ethanol, and precipitated into 250 mL of IM HCl. The precipitate was filtered and recrystallized in ethanol, filtered, and dried under vacuum yielding 4.95 g (82.6%) of dexamethasone mesylate.

Large scale, multi-gram, high yield synthesis of N-\3-( i 4-\(3- aminopropyl)amino]butyl ) amino)propy 1 ] -4- [( ^" 9-fluoro- 11,17-dihydroxy- 16-methyl-3 ,20- dioxopregna-l,4-dien-21-yDsulfanyl1 butanimidamide-TFA salt (1, Dexamethasone- Spermine, DS)

A l-L round-bottom flask equipped with a magnetic stir bar and rubber septum was purged with nitrogen and charged with 600 mL of USP-grade dry ethanol, 60 mL of anhydrous THF, 4.95 grams (10.5 mmol, 1.05 equivalents) of dexamethasone- mesylate and 10 grams (19.5 mmol, 4.95 equivalents) of spermine. 2-Iminothiolane, 1.38 grams (10 mmol, 1.0 equivalents) in 3 mL of water, was added dropwise to the solution over 5 minutes with vigorous mixing. The solution changed color from clear to clear light yellow. The reaction was monitored by TLC and by analytical HPLC. After three hours at room temperature, the TLC spot at R _t = 0 (the charged DS) (R, =0.7 minutes DS by analytical HPLC) was maximized, and dexamethasone-mesylate (R _t 0.47, 2.8 minutes HPLC) spot minimized. The crude reaction mixture was diluted with 15.25 mL (198 mmol, 19.8 equivalents) of trifluoroacetic acid, forming a white precipitate (spermine tetra TFA salt). Solvent was removed with a rotary evaporator and 50 mL of ethanol was added, forming a white precipitate (spermine-4TFA). The pH 3 solution was filtered twice to remove spermine tetratrifluoroacetic acid salt, and ethanol was removed with a rotary evaporator. 50 mL of water was added to the yellow viscous oil, yielding a precipitate (dexamethasone and dexamethasone mesylate), and the solution was filtered through 0.2 μM filter to yield a clear yellow liquid. Water was removed with a lyophilizer over several days to yield dexamethasone-spermine tetratrifluoroacetic acid salt. Yield: 8.228 grams (7.17 mmol, 72%).

TLC R _t =0, 50/50 Hexane/THF, analytical HPLC R ₄ =0.7 minutes. ¹ H NMR (500MHz, DMSO) δ= 6.23 (d, J = 0.023, 1 H, C2 ), 6.01 (s, 1 H, C4), 5.33 (d, J = 0.01, 1 H, C17-OH), 5.04 (s, 1 H, CI l-OH), 4.14 (d, J = 0.02, 1 H, CI l-OH), 3.63 (dd, J = 0.39, 0.03,2 H ₅ C21), 3.37 (s, 20 H, Spermine), 3.27 (s, 2 H, Spermine), 3.93 (s, 4 H, Spermine), 2.61 (m, 1 H, C6a), 2.37 (m, 1 H, C6b), 2.33 (m, 1 H, C8), 2.17 (d, J = 0.02, 1 H, C12), 2.1 (q, J = 0.02, 1 H,C14), 1.87 (m, 4 H, Spermine), 1.78 (m, J = 0.01, 1 H, CJa), 1.63 (m, 2 H, Spermine), 1.58 (m, 1 H, C 15a), 1.48 (s, 3 H, Cl 9), 1.46 (s, 1 H, CT), 1.07 (m, 1 H, C15b), 0.87 (s, 3 H, Cl 8), 0.78 (d, 3 H, C22); HRMS (FAB ⁺ ) C ₃₈ H ₆₃ FN ₅ O4S: [M+H] ⁺ calcd 678.4428, found 678.4429.

Instrumentation/Semi-preparative Purification The LC-MS system consisted of a Shimadzu (Columbia, MD) LC-20AB solvent delivery system and Shimadzu SIL-20A autosampler coupled to Shimadzu SPD-

2OA dual wavelength UV- Vis detector and Shimadzu LCMS 201 OEV single quadrupole . mass spectrometer. Purification was adapted from the method described in Gruneich et al.

(2004, Gene Ther. 11:668-674). The semi-preparative separation system consisted of the Shimadzu instrument coupled to a Hamilton (Reno, NV) PRP-I column (150mm x 10mm i.d., 10μm particle size). The mobile phase flow rate was 4mL/min with a starting ratio of 90% mobile phase A (water) and 10% mobile phase B (acetonitrile). The elution profile consisted of: (i) an isocratic step to 16% B for 30 minutes and (ii) 30% B for 30 minutes to separate the reaction products. Fractions were collected as either TFA or formate salts followed by complete solvent removal by lyophilization. Final products were dissolved in either nuclease free water or methanol/chloroform (50/50 vol%) at 5-6 mg/ml.

Analytical Characterization

Analytical characterization was performed with the Shimadzu instrument coupled to a Hamilton PRP-I column (150 mm x 2.1 mm i.d., 5μm particle size). The mobile phase flow rate was 0.25mL/min with a starting ratio of 90% mobile phase A

(water) and 10% mobile phase B (acetonitrile). The elution profile consisted of: (i) an isocratic step to 16% B for 60 minutes and (ii) 30% B for 60 minutes to quantify purity.

Mass spectrometry was performed on the eluent using electrospray ionization (ESI) in positive ion mode with a scanned m/z range from 160-2000. ¹ H and ¹³ C NMR analysis was performed with a Bruker (location) AVANCE III 500MHz instrument using a dual

5mm cryoprobe or a Bruker DMX 600 using a 5mm TXI 3 axis grad probe.

Other methods which were used but not described herein are well known and within the competence of one of ordinary skill in the art of chemistry, immunology, and cellular and molecular biology.

The Results of the experiments described in this example are now presented.

Synthesis of cationic dexamethasone prodrug

The 21 -hydroxy group of dexamethasone is not required for antiinflammatory activity, and therefore is an ideal choice for conjugation to a polycation (Figure IA) (Schimmer et al., 1996, in The Pharmacological Basis of Therapeutics, 1459- 1485, eds. Hardman and Limbird, McGraw-Hill, New York). A one-pot reaction between spermine, 2-iminothiolane (Traut's reagent) and dexamethasone mesylate yielded the dexamethasone-^permine conjugate, (DS), as the major product (Figure IB). Traut's reagent is selectively ring-opened by the primary amines (Hermanson, 1996, Bioconjugate Techniques, Academic Press) on spermine, forming a hydrolytically sensitive amidimide bond (Hermanson, 1996, Bioconjugate Techniques, Academic Press) between spermine and iminothiolane and a reactive thiolate anion that reacts with the α- keto mesylate on the 21 position of dexamethasone mesylate (Simmons et al., 1980, J. Org. Chem. 45:084-3088), yielding an α-keto thioether linkage between the dexamethasone and iminothiolane. The conjugation reaction was complete in 45 minutes by TLC. ¹ H NMR of DS confirmed the presence of the 3-bis-enone and 11- and 17- hydroxy groups required for glucocorticoid activity (Figure IA) as well as ¹ H signals from the conjugated spermine and the 21-α-keto thioether group. Hydrolysis of DS in IM NaOH for 20 minutes resulted in the cleavage of the amidimide linkage between spermine and iminothiolane, forming a dexamethasone-amide (DA) (Figure IB), which has a 21 -substituted butyl thioether amide side-chain on dexamethasone. For DA, Figure IB: CaIc: C: 63.26; H: 7.35; N: 2.84; Found: C: 63.44; H: 7.27; N: 2.83. Water solubility: DS> 100,000 mg/1; DA 60 mg/1; (dexamethasone 100 mg/1).

There is a need in the art for a nonviral cationic lipid delivery vehicle which can deliver such molecules as nucleic acids or drugs to cells, interstitial sites, organs, or tissues. Further, there is a need in the art for a cationic steroid compositions which can prevent and/or ameliorate inflammation. This invention satisfies this need.

Example 2- Molecule Delivery In Vitro with a Nonviral Cationic Steroid Delivery Vehicle

The Materials and Methods used in the present example are now described.

Cell Culture and Lipofection

Bovine aortic endothelial cells (BAEC, passage 4-13) were passaged at a 1 :3 split to 24-well culture plates, and then grown to dense confluence before lipofection. Growth medium was Dulbecco's modified Eagle's medium (DMEM) containing 10% heat inactivated charcoal-filtered (to remove steroid hormones) fetal calf serum

(Hyclone), 0.30 mg/ml glutamine, 150 U/ml penicillin, and 0.15 mg/ml streptomycin (Gibco). 293 cells were grown under identical conditions. The plasmids pEGFP-N3 and pGRE-SEAP (Clontech) were used for expression of EGFP or secreted alkaline phosphatase under the regulation of CMV or GRE promoters, respectively. In some experiments, Lipofectamine reagent (Invitrogen) containing 2: 1 (μg:μg) mixture of polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl ]-N, JV-dimethyl- 1 - propanaminium trifluoroacetate (DOSPA). -DOPE was used according to manufacturer's instructions. In other experiments, Lipofectamine 2000 (Invitrogen), in a 1 : 1 (wt) ratio with nucleic acid, was used according to the manufacturer's instructions.

For fluorescence microscopy and flow cytometry, pEGFP-N3 plasmid (Clontech, Palo Alto, CA) was used to generate GFP as the fluorescent reporter transgene protein. Cells were transfected in 6-well plates with each condition in duplicate. One day after GFP transfection, cells were imaged and then harvested in 500ul PBS and kept on ice until analysis. A BD Biosciences (Franklin Lakes, NJ) FACSCalibur flow cytometer was used to obtain fluorescence data with 50,000 counts recorded per condition. For the luminescence assays, pGL4.75 plasmid (Promega, Madison, WI) was used to generate renilla luciferase as the reporter transgene protein. Cells were transfected in 96-well plates with 8 replicates of each condition. To measure transgene expression, EnduRen Live Cell Substrate (Promega) was added 24 hours after transfection and luminescence was measured 90 minutes following addition of the reagent. Cell viability was determined by adding an equal volume of Cell Titer GIo

(Promega) and measuring luminescence 30 minutes after addition. Luminescence in both assays was measured with an EnVision Multilabel Plate Reader (Perkin Elmer, Wellesley, MA).

Preparation and characterization of lipid assemblies and lipoplexes

To form the DS:DOPE lipid solution, 67 μl of a 10 mg/ml solution of DS in ethanol and 27 μl of a 50 mg/ml solution of DOPE in ethanol were vortexed together.

After solvent evaporation with a N ₂ stream, 1 mL of sterile Millipore water was added to the lipid film and the solution was sonicated for 10 minutes. The solution (2 μg of lipid per μl of water) was stored at 4 ⁰ C and retained lipofection activity for over 6 months

(data not shown). The hydrodynamic diameter of DS/DOPE (1 μg/2 μg) obtained by dynamic light scattering (DynaPro 99 instrument) was 70 to 150 nm without DNA, while lipoplexes with 1 μg DNA and 0 to 20 equivalents of DS and 0 to 20 equivalents of DOPE had sizes ranging from 200 to 500 nm.

Fluorescence GFP measurement

For lipofection, a solution of DS/DOPE in 125 μl OptiMEM I (Gibco) was mixed with a solution of DNA in 125 μl OptiMEM I, incubated for 30 to 60 minutes, and then overlaid on BAEC cells for 2 hours, followed by PBS rinse and addition of growth media. Fluoresence was measured after 48 hours. At lipid concentrations exceeding charge ratios of 6: 1 DS:DNA, the formulations began to display cytotoxicity in BAEC as indicated by altered morphology. EGFP expression of transfections was monitored in duplicate (Figure 2A) or triplicate (Figure 2B) with a fluorescent plate reader (Labsystems Fluoroskan Ascent; 485 nm/538 nm filter pair) with background subtraction using the auto fluorescence of non-transfected BAEC. EGFP expression was calibrated using 0 to 200 ng recombinant GFP (Clontech) in 750 μl PBS. In assays of EGFP in lysates, lipofected cells were trypsinized, pelleted (200 xg, 8 minutes), resuspended in 150 μl PBS, and subjected to 3X freeze/thaw at -78 ⁰ C. The lysate was centrifuged at 13,500 RPM (5 minutes) in an Eppendorf Minifuge. Supernatant was collected, and pooled with supernatant from PBS washed and pelleted cells (total volume 750 μl), and EGFP fluorescence was measured (Ex 485, Em 515; SLM fluorometer). In some experiments, flow cytometry of lipofected and trypsinized cells expressing EGFP was performed at the University of Pennsylvania Flow Cytometry Core Facility using a FACSCalibur instrument.

The Results of the experiments described in this example are now presented.

In vitro gene delivery with cationic steroids

Using the neutral lipid dioleylphosphatidylethanolamine (DOPE) and 1 μg plasmid/well, we measured gene expression obtained with varying amounts of DS (0 to 20 μg) and varying amounts of DOPE (0 to 20 μg (Figure 2A). In the case where neither DS nor DOPE was present, Lipofectamine was used (6 μg/μg-DNA) as a benchmark. The mass ratio of 1 :2 of DS:DOPE (light bars, Figure 2A) provided high EGFP expression while minimizing the use of the DS conjugate and minimizing the total lipid load below 10 μg total lipid/μg DNA. In a separate set of experiments maintaining the DSrDOPE mass ratio constant at 1 :2, the amount of DS relative to DNA was systematically varied from 1 to 10 charge equivalents (0.9 to 9 μg DS per μg DNA), assuming an average charge of the spermine of DS to be 3.8 per molecule (Geall et al., 2000, Bioconjug. Chem. 11 :314-326) and no net charge contribution from DOPE. The optimal lipofection plateaued at a charge ratio of 6 DS: 1 DNA, giving more than 10-fold increase in the amount of transgene expression relative to Lipofectamine reagent. Increases beyond a charge ratio of 6:1 DS:DNA provided no increase in expression (Figure 2B). To test if gene transfer activity of DS was merely associated with the DNA binding ability of spermine and the hydrophobic character of dexamethasone, DNA was combined with spermine, dexamethasone, and DOPE (all unconjugated) using the same molar ratios and concentrations as in the conjugated DS/DOPE transfection (Figure 2D). Only when the dexamethasone was conjugated with spermine was EGFP expression detected (Figure 2E). Spermine alone or dexamethasone alone had no detectable gene transfer activity. Using a flow cytometry cutoff of 100 F.I. to define percent transfection (Subramanian et al., 1999, Nat. Biotech. 17:873-877), a 4.3-fold increase was observed in percent transfection over Lipofectamine from 5.9 % to 25.5 % (Figure 3). Lipofection of subconfluent (proliferating) BAEC with DS/DOPE yielded a 4.6-fold increase in percent transfection over Lipofectamine from 16.0 % with Lipofectamine to 73.8 % lipofection with DS/DOPE (data not shown). Although hydrolysis of DS to DA is accelerated in IM NaOH, the amidimide bond in DS appeared relatively stable in neutral pH when formulated with DOPE, since DS/DOPE formulations stored for six months at 4°C in water retained their lipofection activity.

Synthesis and Use of Other Cationic Steroids in Delivery Vehicles

The conjugation of spermine to steroids was carried out to create molecules useful for DNA transfer to mammalian cells. 21 -chloro-

17hydroxyprogesterone, cholesterol tosylate, hydrocortisone mesylate, or 17 α-mesylate- estradiol-3 -acetate was mixed with DMSO and Traut's reagent followed by addition of spermine. The resulting cationic steroids displayed gene transfer activity when used with the neutral lipid, DOPE, on 293 cells.

Other compounds which were coupled with spermine and tested on bovine aortic endothelial cells include 11-deoxycortisone, 11-deoxycortisol, Cortisol, and corticosterone. Twenty-one formulations were tested and had varying effects on EGFP expression (see Figure 6).

Use of Other Cationic and Hydrophobic Compounds Based on the structural resemblance of tamoxifen (estrogen antagonist) and 4-hydroxytamoxifen to other cationic steroids (e.g., dexamethasone-spermine) or cationic cholesterol derivatives, the gene transfer activity of tamoxifen and 4-hydroxy tamoxifen was measured. To examine whether tamoxifen or 4-hydroxytamoxifen (a more active metabolite) can function as nucleic acid transfer reagents, drug was formulated with DOPE at various ratios and then mixed with DNA. GFP expression was measured in 293 cells and the results indicate that these additional steroids can be used as well in a delivery vehicle of the invention.

A number of pharmacological drugs have lipophilic and cationic groups. These compounds are in a class of molecules that can bind DNA and have gene transfer activity in the presence or absence of neutral lipids such as DOPE or cholesterol.

Representative examples of other drugs that reside in the class of molecules which are cationic and lipophilic include, but is not limited to: rantidine HC, propoxyphese- N/ AP AP, tamoxifen, verapamil SR, triamterene w/HCTZ, trimox, acyclovir, cyclobenzaprine, methylphenidate, amitriptyline, trimethoprim/sulfa, ipratropium bromide, methotrexate, diltazem CD, norvasc, prozac and sarafem, Vasotec, zestril, effexor, prinivil, imitrex, serevent, zoloft, and paxil.

These results describe a new approach to nonviral nucleic acid and drug delivery by using a novel cationic prodrug or drug vehicle. Dexamethasone, a potent glucocorticoid recognized to enhance gene delivery in vivo, was conjugated with spermine via an iminothiolane linkage. This molecule was designed to combine the gene delivery properties of the clinically relevant cationic lipids such as DC-Choi, DMRIE, DOTMA, and GL-67, with the added functionality of hydrolyzing to release pharmacologically active drug. This procedure yielded a pharmacologically active prodrug that facilitates nucleic acid and drug packaging and delivery. The use of pharmacologically active cationic steroids also allows the exploitation of anionic biopolymers such as glycosaminoglycans in the body to serve as natural occurring depots for local drug delivery.

Example 3- Pharmacological Activity of Cationic Steroids

The Materials and Methods used in the present example are now described.

Glucocorticoid receptor localization

The 3T3 cell line 3676 expressing green fluorescent protein (GFP)- glucocorticoid receptor chimeric protein from a tetracycline regulated promoter (Walker et al., 1999, Methods 19:386-393) was used to measure GR receptor localization into the nucleus. Cells were maintained in growth media supplemented with 100 μg/ml geneticin. The cells were incubated for 30 minutes with dexamethasone, DS, or DA ranging in concentration from 10 nM to 1000 nM, and nuclear translocation of GFP-GR was visualized at 4OX using a Hamamatsu CCD camera and Leica DM IRBE fluorescent microscope.

Induction of transcription from GRE

The levels of GRE induction using DS and its hydrolysis product DA were compared with dexamethasone using a GRE-SEAP promoter construct assay (Clontech). 293 cells were lipofected with GRE-SEAP plasmid in triplicate using 1 μg plasmid and 6 μg Lipofectamine per well in 24-well plates. After 120 minutes, the lipofection media was replaced with 500 μl of growth media, and the cells were treated with dexamethasone, DS/DOPE, or DA over a range from 1 nM to 10,000 nM. After 24 hr of induction, 50 μl aliquots of growth media were analyzed for alkaline phosphatase activity using the SEAP fluorogenic assay following the manufacturer's instructions. The Results of the experiments described in this example are now presented.

Pharmacological activity of catiom ^' c steroid Delivery of dexamethasone, DS/DOPE, or DA at 10 to 1000 nM of steroid pharmacophore caused rapid nuclear localization of a glucocorticoid competent GFP-GR chimeric protein (Figure 4A) stably expressed in 3T3 cells. The majority of the GFP-GR fluorescence localized within the nucleus in less than 1 hr similar to the localization induced by dexamethasone. Partial nuclear import of GFP-GR by 10 nM DS/DOPE and complete nuclear import at 100 nM, suggested that the apparent K _D for this compound was approximately 10-fold higher than that of dexamethasone (K _D ~ 1 nM; Ashwell et al., 2000, Ann. Rev. Immunol. 18:209-345), potentially due to reduced access to the cytosolic GR caused by endosome sequestration or association of DS with anionic elements in the cytosol. DA appeared to have similar apparent K _D to that of dexamethasone since full nuclear localization was observed at 10 nM. While nuclear localization of GR is one test of glucocorticoid activity, a second test of pharmacological activity was also employed, the ability to induce gene expression from a glucocorticoid responsive promoter. Both DS and its hydrolysis product DA induced dose-dependent transcription from a GRE promoter construct (pGRE-SEAP), displaying an EC50 of ~ 10 to 100 nM relative to dexamethasone in 293 cells. Using Student's T Test (one tailed and two-sample unequal variance), at 100 nM concentrations, all forms of the steroid (dexamethasone, DS, or DA) induced statistically significant levels of SEAP transcription from a GRE promoter.

Example 4- Binding of a Cationic Lipid Delivery Vehicle to a Glycosaminoglycan Glycosaminoglycans are anionic interstitial molecules which can be targeted as potential delivery sites to allow slow release of a drug, compound, or nucleic acid at a local interstitial site. There is a need in the art for a nonviral delivery vehicle which can deliver such compounds as nucleic acids or drugs to such an interstitial site, allowing them to be released in a slow release fashion. The present invention fills this need. The Materials and Methods used in the present example are now described.

Hyaluronic acid release assay Hyaluronic acid sodium salt (Rooster Comb HA; Sigma) was diluted in

PBS at 1 mg/ml to mimic an anionic glycosaminoglycan at a nominal interstitial concentration. Equal molar amounts (1 μmol of the steroid moiety of DS, DA, or dexamethasone were added to separate HA solutions (in duplicate). The resulting mixtures were injected into 1 mL Slide-a-lyzer dialysis slides (Pierce; MWCO = 10,000 Da), and dialyzed against 50 mL PBS at room temperature. The time course of steroid release from the dialysis membrane was followed by measuring the increase of absorbance of aliquots from the dialysis buffer at 240 nm (Abs _max for dexamethasone).

The Results of the experiments described in this example are now presented.

Binding to hyaluronic acid in vitro

Polymer-based implants are well developed for steroid delivery (as in the Norplant technology) yet the exploitation of naturally occurring anionic biopolymers as a depot is an attractive mechanism for long term, localized anti-inflammatory therapy. The results demonstrated that dexamethasone rapidly eluted from a prototypical extracellular matrix constituent hyaluronic acid (HA) in less than 5 hours. In contrast, only a small fraction of the cationic steroid DS eluted from HA in a 24 hour period, while over half of the DA eluted (Figure 5). The half-life for dissociation from HA in PBS was calculated to be 2, 14, and 56 hr for dexamethasone, DA, and DS, respectively. New nonviral drug delivery vehicles for nucleic acid and gene delivery and slow release prodrug applications have now been made and described herein that are easily synthesized, result in high levels of transfection or delivery when used to deliver nucleic acids, genes, drugs, or other compounds, and have their own inherent properties that add to the therapeutic potential of the delivered product.

Example 5- In vivo activity of DS in thioglycollate (TG) model of inflammation The Materials and Methods used in the present example are now described.

Mouse treatments Male, 6- to 8-week-old (20 g) BALB/c mice, six per group, were used.

Mice were injected i.p. with either vehicle (200 mL of 1% ethanol in PBS), dexamethasone (Dex, a dose of 2 mg/kg in 200 mL of 1% ethanol in PBS), or an equimolar amount (102 nmol of drug/animal) of DS (5.85 mg/kg in 200 mL of 1% ethanol in PBS). After 30 minutes, mice were injected i.p. with 1.5 mL of 3% TG in PBS containing 1% ethanol. At 4 hour post-TG, the i.p. fluid was collected and neutrophils were counted.

The Results of the experiments described in this example are now presented.

Reduction of neutrophil migration

At 4 hour postchallenge, DS significantly (P=0.0058, n=6) inhibited the accumulation of neutrophils in the i.p. space versus vehicle (1% ethanol in phosphate- buffered saline (PBS)), resulting in a 60% reduction in neutrophil immigration (Figure 9). Similar results were obtained with groups pretreated with dexamethasone or DS 48 hours prior to TG challenge (not shown). All animals remained healthy in all groups, indicating that DS at the tested dose was not lethal.

Example 6- Mouse Pulmonary DNA Transfer The Materials and Methods used in the present example are now described.

DNA delivery by nasal instillation and measurement of cytokines

For studies of in vivo gene delivery, mice (C57/B6) were anesthetized with ketamine/zylazine (i.p.). A total of eight mice per group received 50 ml of lipid/plasmid (100 mg pCB-AP (6075 bp) in 10% sucrose) by nasal instillation during inspiration following the approach of Lee et al. (1996, Hum Gene Ther 1996 7:1701— 1717). Either DS or DC-Choi (Avanti Polar) was used at a molar ratio of one sterol per base and one cationic lipid per DOPE, resulting in a cationic lipid:DOPE:phosphate molar ratio of 1 : 1 : 1. Mice were weighed and euthanized at 1 or 7 days postinstillation. The lungs from each mouse was harvested and weighed. One half of each lung was homogenized in 2 ml of IX Reporter Gene Assay Lysis Buffer (Roche Diagnostics) and clarified by centrifugation (10 minutes, 3000 r.p.m.). Supernatants were stored at -8O ⁰ C until assayed in triplicate using the SEAP Chemiluminescent kit (Roche Diagnostics). Levels of TNF-α and IFNγ were quantified against mouse standards using ELISA (Pierce Endogen) at Day 1 , based on peak inflammatory responses expected at this time.

The Results of the experiments described in this example are now presented.

Reduction of TNF-α and IFNY

Having demonstrated the anti-inflammatory activity of DS at a relevant pharmacological dose in the TG challenge model (see Example 5), a set of experiments was conducted with lipoplexes to evaluate both the gene transfer activity and the anti- inflammatory activity of DS. DC-Choi was chosen as a commercially available reference lipid since it is sterol-based and has been used in animal and human clinical trials and is commonly formulated with the neutral lipid DOPE. This avoids referencing DS:DOPE against other nonsterol-cationic lipids used with cholesterol, for example, as the neutral lipid. At a molar ratio of cationic sterol lipid:DOPE:base of 1 : 1 : 1 used to deliver 100 mg of plasmid (50 mL intranasal instillation), DS:DOPE resulted in a statistically significant 64% reduction (P=O.08, n=4) of IFNγ/gram of homogenized lung at Day 1 compared to DCChol:DOPE (Figure 10). DS lipoplexes resulted in less TNF-α expression on average compared to DC-Choi lipoplexes at Day 1 , but did not reach statistical significance in this small cohort study due, at least in part, to the high variability of TNF-α expression in the DC-Choi group. At Day 1, DS:DOPE resulted in a statistically significant increase of 3.22-fold (P=0.029) over DC-Chol:DOPE in the expression of the alkaline phosphatase transgene. At Day 7, one animal of the DS:DOPE cohort expressed 415 times more alkaline phosphatase than the other three members of the cohort. By eliminating this overexpressor, the DS:DOPE cohort resulted in a statistically significant 6.78-fold increase (P=0.0015, n=3) in transgene expression compared to the DC-Choi cohort (Figure 10). No member of the DCChol:DOPE group expressed more transgene than the lowest expressor of the DS:DOPE group at Day 1 or Day 7 postinstillation. Transgene expression actually increased from Days 1 to 7 in the DS:DOPE cohort. As expected for lipoplexes, the transgene expression level obtained with DS:DOPE was about 150-fold less than that achieved with intranasal instillation of 2.5X10 ¹¹ adenoviral (Ad5.CB.AP) particles expressing alkaline phosphatase from a matched promoter (169 ± 152 ng alkaline phosphatase/gram at Day 1 postinstillation, n=3).

Example 7- Lipoplex Formulation of adenovirus vectors (AdV) for Targeting to Mouse Airway Epithelium

The Materials and Methods used in the present example are now described.

Virus preparation and formulation

E 1/E3 -deleted replication-deficient, recombinant adenovirus vectors (2- 4.5 X 10 ¹² GC/ml stock) expressing either a LacZ or a AIkP reporter were created as previously described in Gao et al. (1996, J. Virol. 70:8934-8943) by homologous recombination between the shuttle plasmid that carries the transgene and the 5' end of the adenovirus genome with El deleted and 3' end of H5.sub360 in which the E3 gene was inactivated by a small deletion. The recombinant viruses were propagated in 293 cells and purified by the standard CsCl gradient sedimentation method. The replication- deficient AAV2/5CBntLacZ contains the LacZ gene with a nucleus localization sequence at its N-terminus, under the transcriptional control of the chicken h-actin promoter. The AAV (2/5) vector (~2 X lO ¹² GC/ml stock) was produced by transcapsidation in 293 cells using a triple transfection method and purified by CsCl gradient sedimentation as described in Gao et al. (2002, Proc. Natl. Acad. Sci. USA 99:11854-11859). For in vivo studies, DS/DOPE (51 Ag total lipid/1011 GC), DC-Chol/DOPE (40 μg total lipid/10 ¹¹ GC), or dexamethasone (29.4 μg/1011 GC) was added to an equal volume of AdV-LacZ, AdV-AIkP, or AAV-LacZ vector and incubated at room temperature for 15 to 30 minutes prior to use. The in vitro studies used varying lipid concentrations as indicated elsewhere herein and lipid/vector complexes were prepared as described elsewhere herein.

Cell culture and in vitro gene transfer studies

A549 (ATCC CCL-185) and MDCK (ATCC CCL-34) cells were grown in DMEM (GIBCO, Carlsbad, CA, USA) supplemented with 10% newborn calf serum (GIBCO), 2% penicillin/streptomycin (Cambrex, Baltimore, MD, USA), and 1% 1- glutamine (GIBCO) and maintained at 37 ⁰ C and 5% CO2. For transfections/transductions, MDCK and A549 cells were seeded at 2 X 10 ⁴ cells/well in 24-well plates and grown to confluency. Prior to transfection/transduction, wells were examined under the light microscope for general uniformity and health and then cells from at least one well were then harvested and a cell count was performed using a hemacytometer. All vector and liposome formulations were prepared in Opti-MEM and then 300 μl of the transfection/transduction mixture was added to PBS-rinsed cells. The mixture was incubated with the cells for 2 hours at 37 ⁰ C, then removed, and fresh growth medium added. At 24 hours after transduction, cells from each well were harvested and lysed using h-galactosidase reporter gene assay lysis buffer (Roche, Indianapolis, IN, USA) according to the manufacturer's instructions. Samples were collected and centrifuged at maximum speed for 2 minutes and analyzed immediately or stored at - 8O ⁰ C. LacZ gene expression was assayed by using 50 μl of cell extract per well in a 96- well plate using a chemiluminescence h-galactosidase reporter gene assay kit (Roche). Signal was detected by integrating light production from the chemiluminescent substrate for 5 seconds on a fluorescence/luminescence plate reader (Fluoroskan Ascent FL, Labsystems).

In vivo delivery to mouse lungs

C57B1/6 mice (6 to 8 weeks of age) were anesthetized by an i.p. injection of a 3:2 mixture of xylazil (20 mg/ml):ketamine (100 mg/ml). For dosing, mice were suspended from their dorsal incisors (hind quarters supported) and a dose of 10 ⁿ GC of AdV or AAV vector was delivered as a 50-μl bolus into the nostrils using a gel-loading tip (Finnpippette). Lungs were harvested at 1, 7, or 21 days and inflated with a 1 :1 mixture of PBS and OCT embedding compound. One lobe was then submerged in OCT, frozen in isopentane cooled with liquid nitrogen, and cryosectioned (10 Am). Sections were fixed in 0.5% glutaraldehyde in PBS (Electron Microscopy Sciences, Hatfield, PA, USA) for 10 minutes (48 ⁰ C) and then washed twice in PBS/1 mM MgC12 (48 ⁰ C) and stained with X-gal substrate (5-bromo-4-chloro-3-indoyl-h-d-galactopyranoside) for 16 hours at 37 ⁰ C to reveal LacZ-positive cells, NBT/BCIP substrate (Roche) for 16 hours at room temperature to reveal AlkP-positive cells, or H&E. After being washed in PBS, sections were counterstained with nuclear fast red, dehydrated through a graded ethanol series and xylene, coverslipped, and imaged. Slides were labeled with only a single number and were ordered randomly to eliminate bias. Scores from 0 (normal, no inflammation) to +++ (extensive inflammation) were given to each numbered slide, and a short description was also provided. The other lobe was placed in 2 ml of Lysis Buffer (Roche) and placed on ice. Directly after all samples were collected, each sample was homogenized for approximately 10 seconds. The instrument was washed between each sample using 70% ethanol and PBS. Samples were then immediately centrifuged at 3000 rpm (Sorvall RT7; Dupont) for 10 minutes. The supernatant was removed and stored at - 8O ⁰ C prior to analysis of transgene and cytokine expression.

CD8+ immunohistochemistrv on lung cryosections

The lung sections (10 μM) were air dried and fixed in cold (-2O ⁰ C) acetone for 5 minutes. Once dried the sections were blocked with 1% goat serum/PBS for 15 minutes. The primary antibodies (rat anti-CD8 (BD Pharmingen, 550281)) were then added at a 1 :20 dilution in PBS/1% goat serum and incubated at room temperature for 1 hour. Following three 5-min washes in PBS, the secondary antibody (FITC anti-rat) was added at a 1 :200 dilution in PBS/1% goat serum and incubated in the dark at room temperature for 30 minutes. Following three 5-min washes in PBS, the sections were mounted in Vectashield with DAPI and viewed under a fluorescence microscope.

Transgene detection AdV-AIkP vector transduction was measured using the Secreted Alkaline Phosphatase Chemiluminescent Gene Reporter Assay (Roche) according to the manufacturer's instructions. All values were normalized to total protein content in the sample using the Bio-Rad protein assay (-0.5 μg/ml per sample).

Statistical analysis

Values are reported as means ± standard deviation. Treated groups of mice were n = 4, unless otherwise indicated. All quantitative analyses were performed in duplicate or triplicate for each lung sample. In vitro studies were performed in triplicate wells and transgene expression was quantified for each well in duplicate. Statistical analysis was performed using JMP software. Analysis of variance tests were performed to determine difference between groups; P<0.05 was considered significant. Within groups, the Student t test was used to determine significance when P<0.05.

The Results of the experiments described in this example are now presented.

Reduction of inflammation by Adv formulated with DS To evaluate whether DS/DOPE was effective at both increasing AdV vector-mediated gene transduction to airway epithelial cells and reducing associated inflammation, an AdV vector expressing human placental alkaline phosphatase (AIkP) (2.5 X lO ¹¹ genome copies (GC)) was delivered to mice by intranasal instillation either alone or in a complex with DS/DOPE. At day 1 or 7, the lungs were removed and one lung was homogenized and the other frozen for cryosectioning. Analysis of lung homogenates for AIkP revealed that gene expression was reduced by 4-fold on day 1 (11.0 ± 5 pg/g vs. 46 ± 30 pg/g) and 146-fold on day 7 (1.2 ± 0.5 pg/g vs. 180 ± 148 pg/g) when the AdV vector was formulated with DS/DOPE (51 μg DS/DOPE with 10 ⁿ GC AdV). Histochemical analysis of lung sections stained for AIkP revealed that, although total AIkP staining was substantially decreased by the formulation of the AdV vector with DS/DOPE, the lipoplex resulted in the marked targeting of AIkP expression to the airway epithelial cells, whereas AdV vector alone resulted in transgene expression in both alveoli and airways at both days 1 and 7 (Figures 11 and 12). Furthermore, hematoxylin/eosin (H&E) staining showed extensive cellular infiltration at both time points when the AdV vector was delivered alone. In contrast, when we formulated the AdV vector with DS/DOPE no irregularities were seen in the lung compared to PBS controls (Figures 11 and 12). These results indicate that DS/DOPE was effective at reducing the inflammation in the lung while targeting transgene expression to the airway epithelial cells.

The targeting effects of DS/DOPE on AdV vector delivery to mouse lung epithelium shown in Figures 11 and 12 were likely due to the physical-chemical effects of liposomes. To test this possibility, we chose DC-Choi as a representative sterol-based reference cationic lipid to DS. At day 1 post-instillation of the AdV-LacZ vector (10 ¹¹ GC) with DS/DOPE or DC-Chol/DOPE (Figure 13 A-13D), both liposome formulations led to targeting of the transgene expression mainly to airway epithelial cells, though not only so. Consistent with our prior studies of plasmid delivered with DS/DOPE to mouse lung that reduced inflammation relative to DC-Chol/DOPE (see Example 6), the delivery of AdV-LacZ vector with DS/DOPE produced less cellular infiltration than that obtained with DC-Chol/DOPE (Figures 13 A-13D).

The data disclosed herein suggest that the targeting action of DS/DOPE is a property achieved with liposomes in general and does not require the pharmacological activity of DS. One possible explanation for the reduced cellular infiltrate observed with DS/DOPE formulated AdV vector (Figures 11 and 12) is the lower total gene expression obtained by this formulation. However, DC-Chol/DOPE caused a marked cellular infiltration at a similarly low total transgene expression (Figures 13B and 13D). This comparison suggests the pharmacological action of DS reduced cellular infiltration caused by the AdV vector.

To quantify our qualitative observations of inflammation in the lung, representative slides from each group were randomly numbered and ordered (AdV-, AdV + DS/DOPE-, AdV + DC-Chol/DOPE-, AdV + dexamethasone-, and PBS-treated) and then they were analyzed by a pathologist blinded to the treatment groups. Scores were defined as 0 (normal, no inflammation) to +++ (extensive inflammation). A short description, along with a score, was provided by the pathologist for each slide. The AdV- LacZ vector appeared to be less inflammatory (+/- at day 1, + at day 7) than the AdV- AIkP vector (++ at day 1, +++ at day 7). The AdV-AIkP vector-treated tissue was characterized by mononuclear infiltrates and congestion of blood vessels, and at day 7 there was evidence of alveolar hemorrhage. As expected, scores at day 7 were consistently higher than scores at day 1. At day 7, there were no signs of inflammation of the airways treated with either AdV vector formulated with DS/DOPE (0, normal) or dexamethasone (0, normal). However, for airways treated with the AdV vector formulated with DC-Chol/DOPE, at day 7 there was mild (+) monocytic infiltrate and congestion. The DC-Chol/DOPE-formulated AdV-LacZ vector-treated airways had levels of inflammation similar to those seen when the AdV-LacZ vector was delivered alone at both days 1 and 7 (+/- at day 1, + at day 7). This data indicates that the pharmacological activity of DS is responsible for the reduction in inflammation when AdV vector is formulated with DS/DOPE. Further evidence to support that the pharmacological activity of DS was related to the observed reduction in cellular infiltration came from staining cryosections for the presence of CD8+ T cells at days 1 and 7 (Figure 13). Lungs treated with DS/DOPE-formulated AdV vector showed little to no positive staining for CD8+ T cells. However, lungs that were treated with only AdV vector resulted in a significant incidence of CD8+ cells (Figures 13E and 13F). These data further support that the pharmacological activity of DS interferes with the immune response to the AdV vector when delivered to the lung by intranasal instillation.

Example 8- Transgene expression following single or repeated administration of AdV vector to lung

The Materials and Methods used in the present example are now described.

Virus preparation and formulation El/E3-deleted replication deficient, recombinant AdV vectors (2-4.5 X 10 ¹² particle/ml) expressing either β-galactosidase or hAAT were generated as described previously in Gao et al. (1996, J Virol 70:8934-8943). The recombinant viruses were propagated in 293 cells and purified by the CsCl gradient sedimentation method. For in vivo studies, DS/DOPE (51 mg total lipid), DC-Chol/DOPE (40 mg total lipid) or dexamethasone (29.4 mg) was added to an equal volume of AdV-LacZ or AdV-hAAT vector (10 ' p) and incubated at RT for 15-30 minutes prior to use.

In vivo delivery to mouse lungs C57B1/6 mice (6-8 weeks of age) purchased from Charles River

Laboratories were anesthetized by an i.p. injection of a 3:2 mixture of xylazil (20 mg/ml)/ketamine (100 mg/ml). For dosing, mice were suspended from their dorsal incisors (hind quarters supported) and a dose of 10 ¹¹ p of AdV-LacZ vector was delivered as a 50 ml bolus to the both nostrils using a gel-loading tip (Finnpippette). Serum was collected at intermediate time points via retro-orbital bleed. In some experiments, a second dose of AdV-hAAT was delivered. At necropsy, the lungs and serum were harvested for further analysis. One lobe of the lung was inflated with PBS/optimal cutting temperature (OCT) (1 :1) mixture, covered in OCT and frozen in isopentane cooled with liquid nitrogen, and cryosectioned (10 mm). Sections were fixed and processed as described previously in Bell et al. (2005, Histochem Cell Biol 124:77- 85). The other lung lobe was placed in 2 ml of Lysis Buffer (Roche, Indianapolis, IN, USA) and placed on ice. Lungs were homogenized as described previously (Price et al., 2005, MoI Therapy; 12:502-509) and the supernatant was transferred to a sterile Eppendorf tube until analysis of transgene expression. Serum was collected and stored at -2O ⁰ C.

Transgene detection

AdV vector-mediated β-galactosidase gene expression in lung homogenate was analyzed by using the β-galactosidase ELISA kit (Roche) according to the manufacturer's instructions. All values were normalized to total protein content in the sample quantified using the Bio-Rad Protein Assay. ELISAs were performed on sera samples to detect hAAT transgene expression. For AdV vector-mediated AIkP gene expression in lung homogenate, the AIkP chemiluminescent detection kit was used (Roche) according to the manufacturer's instructions.

NAB detection

'8431' cells were seeded onto flat-bottom 96-well cell culture plates at 5 X 10 ⁴ cells per well. At 16 hours postseeding, the cells were infected with 200 particles of wild-type AdV/cell for 3 hours at 37 ⁰ C. Sera samples were heat-inactivated at 56 1 C for 35 minutes, diluted 1 :10 in 50 ml of serum-free Dulbecco's modified Eagle's medium and diluted twofold in a 96-well round-bottom tissue culture dish. A total of 50 ml of serum-free Dulbecco's modified Eagle's medium containing AdV-LacZ (multiplicity of infection = 10 ⁴ ) was added to both the diluted sera and a control serum sample and incubated for 1 hours at 37 ⁰ C. The media containing the wild-type AdV was aspirated and replaced with 100 ml of the AdV/serum mix and incubated for 1 hours at 37 ⁰ C. FCS/Dulbecco's modified Eagle's medium (DMEM) 20% (100 ml) was added to each well and incubated for 48 hours at 37 ⁰ C. Transduction was evaluated by counting LacZ- positive cells in each well under high-power magnification. The titer of NAB for each sample was reported as the highest dilution with which less than 50% of the cells were LacZ positive.

CD4+ and CD8+ immunohistochemistry on lung cryosections

The lung sections (10 mm) were air-dried and fixed in -2O ⁰ C cold acetone for 5 minutes. Once dried the sections were blocked with 1% goat serum/PBS for 15 minutes. The primary antibodies (rat anti-CD8; BD Pharmingen, 550281 and rat anti- CD4; BD Pharmingen, 550280) were then added at a 1 :20 dilution in PBS/1% goat serum and incubated at RT for 1 hour. Following three 5 minutes washes in PBS, the secondary antibody (fluorescein isothiocyanate anti-rat) was added at a 1 :200 dilution in PBS/1% goat serum and incubated in the dark at RT for 30 minutes. Following three 5 minutes washes in PBS, the sections were mounted in Vectashield with 4,6-diamidino-2- phenylindole and viewed under a fluorescence microscope.

Statistical analysis Values are reported as mean ± s.d. Number of mice per group is three, unless otherwise stated. All quantitative analyses of lung samples were performed in duplicate or triplicate. Statistical analysis was performed using JMPTM software. Analysis of variance tests were performed to determine difference between groups, P<0.05 was considered significant. Within groups, the Student's t-test was used to determine significance.

The Results of the experiments described in this example are now presented.

Transgene expression

On problem with viral gene delivery is the inability to achieve significant levels of transgene expression upon homologous vector re-administration due to blocking of transduction by serum-circulating NAB and immunological clearance of vector through cytotoxic T lymphocyte activation upon the second administration. After using various formulations for the first instillation of AdV-LacZ (10 ¹¹ particle/mouse, intranasal, day 0), vector was then re-administered to mice at day 21 using the same serotype AdV vector (but without formulation) expressing a different transgene (alkaline phosphatase; AIkP) to directly evaluate the impact of the first AdV vector administration on the second AdV vector administration (see Figure 14A). This experimental design would result in the activation of significant levels of serum-circulating NAB to the AdV vector capsid and potentially activate cytotoxic T lymphocytes specific to the vector capsid and/or the transgene resulting in decreased AIkP gene expression. Three days after AdV-AIkP vector delivery (Day 24), AIkP gene expression was detectable in all treatment groups. As expected, delivery of AdV-LacZ vector without formulation at day 0 resulted in a striking 95% reduction (P=O.04) in AIkP gene expression at day 24 compared with phosphate-buffered saline (PBS)-sham delivery at day 0 (Figure Ib), confirming a strong immunological response following the initial administration of AdV-LacZ vector. Formulation of the AdV-LacZ vector with DS/DOPE at day 0 resulted in a significantly higher (P=0.05) level of AIkP gene expression at day 24 (3 days post- AdV-AIkP vector instillation) compared to that achieved when mice were preadministered with either AdV-LacZ alone or in formulation with DC-Chol/DOPE at day 0. AIkP gene expression was also numerically greater with DS/DOPE formulation compared to dexamethasone formulation; however, the difference did not reach statistical significance, β-galactosidase expression, from the first AdV vector dose, was undetectable (as expected) in all groups by day 24. Formulation of AdV-LacZ vector with DS/DOPE at day 0 allowed homologous AdV vector re- administration at day 21 , resulting in AIkP gene expression that was comparable to that achieved after a single administration of AdV-AIkP vector at day 21 in mice pretreated with PBS-sham at day 0.

Effect of formulation on the generation of neutralizing antibodies (NAB) against AdV vector

To assess the impact of lipid formulation on the production of NAB in serum following AdV vector delivery to lung, the levels of serum-circulating NAB of each treatment group were quantified. Specifically, the levels of serum-circulating NAB at day 28 following a single administration of AdV vector were dramatically reduced by formulation of AdV vector with DS/DOPE, DC-Chol/DOPE or dexamethasone (Figure 15A), indicating that either a cationic liposome formulation (DS or DC-Choi) or a glucocorticoid prevented the development of NAB. Since DS/DOPE is a liposome formulation with glucocorticoid activity that appeared to facilitate AdV vector re- administration (Figure 14), NAB production at day 28 following AdV vector re- administration was also evaluated. For the second administration at day 14, an AdV vector expressing human αl -antitrypsin (hAAT) was used to avoid a combined response to vector and transgene products (Figure 15B). The treatment group that received two doses of AdV vector with no formulation (n=3) had a high titer of serum-circulating NAB (1 :3700). However, this high titer was attributable to a large degree to a single outlier (1 : 10,240). As such, Figure 15B also displays the NAB data with this outlier omitted (n=2). As expected from the hAAT gene expression results in Figure 14, the use of DS/DOPE to formulate the AdV vector delivered at day 0 followed by a second dose of AdV vector alone (no formulation) at day 14 reduced serum-circulating NAB assayed at day 28. Formulation of the second dose of AdV vector with DS/DOPE delivered at day 14 further reduced the generation of NAB assayed at day 28.

Effect of AdV vector formulation on pulmonary T-cell-mediated response In light of the data depicted in Figures 14 and 15 , which demonstrate the inflammatory response to the AdV vector and the anti-inflammatory activity of the DS/DOPE formulation, the role of formulation on cellular responses was evaluated. The localized pulmonary response at day 28 to AdV vector (± DS/DOPE formulation for the first or second vector instillation) was assessed by immunostaining for CD4+ and CD8+ T-cell infiltration in frozen lung sections (Figures 16A- 16F). Delivery of AdV-LacZ at day 0 followed by AdV-hAAT at day 14 (both doses delivered with no formulation) in either vector instillation resulted in a striking elevation of CD4+ and CD8+ T lymphocytes surrounding the conducting airways at day 28 (Figures 16A and 16B). The magnitude of CD4+ or CD8+ T-cell infiltration at day 28 was reduced when the AdV- LacZ vector was formulated with DS/DOPE for the first delivery at day 0, even when the AdV-hAAT vector was delivered with no formulation at day 14 (Figures 16C and 16D). CD4+ and CD8+ T-cell infiltration at day 28 was further reduced when the AdV-LacZ vector was formulated with DS/DOPE for the first instillation at day 0 and the AdV- hAAT vector formulated with DS/DOPE for the second instillation at day 14 (Figures 16E and 16F). It was previously determined that the DS/DOPE formulation promotes targeting of the AdV vector to the conducting airways. (Price et al., 2005, MoI Therapy 12:502-509) This transduction pattern was also correlated with CD4+ and CD8+ T-cell infiltration, which was reduced and localized to the conducting airway epithelium when the delivered AdV vector was formulated with DS/DOPE. Similar reductions of the localized CD4+ T-cell infiltration were observed for the dexamethasone formulation, but not for the DC-Chol/DOPE formulation (data not shown). Example 9- Co-formulation of AAV vectors with DS and mixtures of DS/D2S

The Materials and Methods used in the present example are now described.

Analysis of in vivo transduction and co-formulation of cationic lipids and AAV

Cationic liposomes (DSZD ₂ S) in a 1 : 1 mole ratio with DOPE (51 μg total lipid) were mixed with AAV (10 ¹¹ GC/dose) in equal volume at room temperature for 15 minutes prior to instillation. C57B1/6 mice (6 to 8 weeks of age) were anesthetized by an i.p. injection of a 3:2 mixture of xylazil:ketamine. For dosing, mice were suspended from their dorsal incisors (hind quarters supported) and a dose of 10 ¹¹ GC of AAV vector was delivered as a 50 μl bolus into the nostrils.

To evaluate luciferase transgene expression, 250 μl of a 15mg/ml luciferin solution (Caliper Life Sciences, Hopkinton, MA) was injected per 25 gram body mass by i.p. injection. Five minutes after luciferin injection the mice were anesthetized by an i.p. injection of a 3:2 mixture of xylazil:ketamine. Animals were imaged 15 minutes after the original luciferin injection with a high sensitivity CCD Xenogen IVIS bioluminescent imaging system (Caliper Life Sciences).

For lacZ transgene expression evaluation, lungs were harvested and inflated with a 1 : 1 mixture of PBS and OCT embedding compound. One lobe was then submerged in OCT, frozen in isopentane cooled with liquid nitrogen, and cryosectioned (10 μM). Sections were fixed in 0.5% glutaraldehyde in PBS (Electron Microscopy Sciences, Hatfield, PA) for 10 minutes (4°C) and then washed twice in PBS/1 mM MgC12 (4°C) and stained with X-gal substrate (5-bromo-4-chloro-3-indoyl-β-D- galactopyranoside) for 16 hours at 37°C to reveal LacZ-positive cells. After being washed in PBS, sections were counterstained with nuclear fast red, dehydrated through a graded ethanol series and xylene, coverslipped, and imaged. The other lobe was placed in 2 ml of Lysis Buffer (Roche, Indianapolis, IN) and placed on ice. Directly after all samples were collected, each sample was homogenized for approximately 10 seconds. The instrument was washed between each sample using 70% ethanol and PBS. Samples were then immediately centrifuged at 3000 rpm (Sorvall; Thermo Scientific, Waltham, MA) for 10 minutes. The supernatant was removed and stored at -8O ⁰ C prior to analysis of transgene expression. A mouse β-galactosidase enzyme-linked immunosorbent assay (ELISA) (Roche) was used on lung homogenates according to the manufacturer's instructions to quantitatively measure LacZ gene expression. All values were normalized to total protein content in the sample using the BCA protein assay (Pierce Biotechnololgy, Inc., Rockford, IL).

The Results of the experiments described in this example are now presented.

Effect of co-formulation on transduction efficiency, tropism and re-administration

To assess whether co-formulation of AAV vectors affected transduction efficiency and tropism, two serotypes of AAV, (AAV2/6.2 and AAV2/9), each carrying a luciferase transgene reporter, were co-formulated with two cationic lipid formulations (DS and 60/40 mol% DS/D ₂ S). Bioluminescent imaging (Figure 17) was performed to assess both qualitative and semi-quantitative changes in expression compared to a control group which was exposed to AAV vector preparation without lipid formulations.

The cohort treated with AAV2/9 co-formulated with DS shown in Figure 17A showed significant transduction of the nasal epithelium and liver, but minimal luminescence in the lung. By comparison, the co-formulation with 60/40 mol% (DS/D ₂ S) mixture showed higher levels of luminescence in the lung compared to DS and control at day 28 (Figure 17B), as well as when compared with control at days 14 and 20 (Figure 18). This increased transduction efficiency in the lung could either be attributed to a lower potential for transcytosis of the epithelium in the lung for the vectors, or improved targeting resulting from the lipid mixture. A follow-up study was performed to look at gene expression for the same conditions in larger cohorts as a function of time. The results confirmed the initial study and showed an increase in transgene expression for AAV2/6.2 in the lung at Day 7 through Day 31 for both lipid formulations compared to AAV2/6.2 formulated with vehicle control (Figure 19). Similarly, AAV2/9 co-formulated with the lipids resulted in an increase in transgene expression in the lung at Day 7 through Day 31 for the two lipid formulations compared to AAV2/9 formulated with vehicle control (data not shown). Further, the co-formulation with 60/40 mol% (DS/D ₂ S) mixture showed higher average levels of luminescence in the lung compared to the co- formulation of 80/20 mol% (DS/D ₂ S), as well as with DS and control at days 14 and 28 (Figure 20). Based on the bioluminescent data, additional studies were conducted with a lacZ transgene in order to assess overall transduction efficiency, tropism within regions of the lung, as well as re-administration potential. Histological analysis of lung cross- sections from cohorts treated with AAV2/9 showed that both DS and DS/D ₂ S mixtures resulted in increased levels of conducting airway transduction (Figure 21). The natural tropism for AAV2/9 is to the alveolar epithelium with little to no expression in the conducting airway; therefore, both lipid groups appear to have altered the cellular targets for this serotype. Significant transduction of the alveolar epithelium is still evident, possibly indicating that co-formulation does not completely change the capsid binding affinity, or that the co-formulation conditions were not optimized. For AAV2/6.2, co- formulation with DS and to a lesser extent the mixture of DS/D ₂ S showed increased transduction in the conducting airway, with no expression in the alveolar epithelium. AAV2/6.2 is a variant of the surface capsid of AAV6 which exhibits natural tropism for the conducting airway of the lung; therefore, AAV2/6.2 vectors preparations co- formulated with these cationic lipids do not appear to have significantly altered the tropism.

Quantification of transduction efficiency for the lacZ transgene product was measured in the lung homogenate by ELISA normalized to total protein in each sample. The results show that both cohorts treated with AAV2/6.2 vectors co-formulated with cationic lipids (DS and 60/40 mol% DS/D ₂ S) expressed higher average transgene levels compared to control (Figure 22). These results confirmed the qualitative analysis of histological sections from several mice from each cohort and previous results observed with the luciferase transgene. A high degree of variability was noted in each experimental group leading to large standard deviations; therefore statistical significance of the data was not obtained. The high degree variability is believed to be due to inconsistent distribution of vectors among the lobes of the lung. Effect of co-formulation on re-administration

One problem with viral gene delivery is the inability to achieve significant levels of transgene expression upon homologous vector re-administration due to blocking of transduction by serum-circulating NAB and immunological clearance of vector through cytotoxic T lymphocyte activation upon the second administration. After using various formulations for the first instillation of AAV2/9-LacZ (10 ¹¹ particle/mouse, intranasal, day 0), vector was then re-administered to mice at day 28 using the same AAV2/9 vector (but without formulation) (see Figure 23). This experimental design would result in the activation of significant levels of serum-circulating NAB to the AdV vector capsid and potentially activate cytotoxic T lymphocytes specific to the vector capsid and/or the transgene. Twenty-eight days after the second administration at day 28 (i.e., Day 56), LacZ gene expression was detectable in all treatment groups. Formulation of the AAV2/9-LacZ vector with DS/D ₂ S at day 0 resulted in a 2.8-fold higher (P=O.1) level of LacZ gene expression at day 56 (28 days post-second administration) compared to that achieved when mice were preadministered with AAV2/9-LacZ alone. LacZ gene expression was 2-fold greater with the DS formulation compared to the readministered control; however, the difference did not reach statistical significance. Formulation of AAV2/9-LacZ vector with DS, or with DS/D ₂ S at day 0 allowed homologous AAV2/9- LacZ re-administration at day 28, resulting in increased LacZ gene expression.

Example 10- Effects of DS and D?S in an in vitro inflammatory model

An in vitro inflammatory model of bone remodeling was used to assess pharmacological activity of the cationic steroids of the invention because prolonged glucocorticoid treatment in the bone microenvironment has been shown to be able to reduce inflammation, but can also lead to a profound, negative impact on skeletal remodeling. The activity of cationic steroids of the invention was measured in this model to assess dissociated transrepression/transactivation character of these compounds. The results disclosed herein show that DS and D ₂ S reduce RANKL, increase osteoprotegerin OPG, and reduce inflammatory cytokine IL-6 transcripts. Culture of bone marrow- derived macrophage (BMM) and calvarial cells also demonstrated altered activity for DS and D ₂ S compared to control, suggesting that these compounds exhibit dissociated glucocorticoid character in the murine bone microenvironment.

The Materials and Methods used in the present example are now described.

Calvarial and BMM cell culture and Osteoclastogenesis

Murine osteoclasts were prepared from bone marrow cells using two standard methods. Bone marrow cells were obtained by flushing femurs and tibias from C57B1/6 mice. For co-cultures with osteoblasts, bone marrow cells (I X lO ⁵ cells/well in 96-well plates) were co-cultured with calvarial osteoblasts (I X lO ⁴ cells/well) in α- minimal essential medium containing 10% fetal bovine serum and supplemented with 20 nM lα, 25(OH) ₂ D ₃ and 1 μM prostaglandin E ₂ (PGE ₂ ) for 9 days. For stromal cell-free cultures, bone marrow cells were cultured with Macrophage Colony-Stimulating Factor (M-CSF) (30 ng/ml) for 3 days in α-minimal essential medium containing 10% fetal bovine serum, and attached cells were used as osteoclast precursors (BMMs). BMMs were subsequently differentiated into osteoclasts with M-CSF (30 ng/ml) and RANKL (i.e., TRANCE) (100 ng/ml) for 4 days. Fresh α-minimal essential medium containing M- CSF and RANKL (i.e., TRANCE) was supplied on day 3. Co-cultures treated with Vitamin D3 (VitD3) were treated at 20 nM. Cells were then fixed with 10% formalin and TRAP stained. TRAP(+) MNCs containing more than three nuclei or those that contained more than three nuclei and larger than 100 μm in diameter were counted as osteoclasts. TRAP solution activity was measured at an absorbance of 405nm.

Quantitative Real-Time CqRT-PCR)

For transcript analysis, total RNA was isolated using the Absolutely RNA microprep kit (Stratagene, La Jolla, CA). RNA was reverse transcribed using Superscript III reverse transcriptase and oligo(dT) (Invitrogen, Carlsbad, CA), and complementary DNA was purified using Qiagen's PCR purification kit (Valencia, CA). Complementary DNA from untreated samples was used for optimizing PCR conditions, with primers targeting the gene of interest. For the gene RANKL, the forward primer was 5'- CACACCTCACCATCAATGC-3' and the reverse primer was 5'- AGTCTGTAGGT ACGCTTCC-3', for the OPG gene the forward primer was 5'- CCGAGAGTGT AGAGAGG A-3' and the reverse primer was 5'- CTGCTCGCTCGATTTG-3', for the IL-6 gene the forward primer was 5'- AATGATGGATGCTACCAAACTG-S' and the reverse primer was 5'-

TTCTGTATCTCTCTGAAGGACT-3'. In order to normalize the expression levels, GAPDH was used as a housekeeping gene with forward primer 5'- CCACTCTTCCACCTTCG-3' and reverse primer 5'-GTGGTCCAGGGTTTCTTAC-S'. A standard curve was created using the untreated controls and the optimized PCR conditions for each set of primers on a Roche LightCycler 480 (Indianapolis, IN). Quantitative real-time PCR was performed using the LightCycler FastStart DNA MasterPLUS SYBR Green I kit with melting curve analysis provided by the LightCycler software.

The Results of the experiments described in this example are now presented.

In vitro bone culture inflammatory model

An in vitro bone culture signaling model was developed to characterize the dissociated character of DS and D ₂ S because active glucocorticoids exhibit measurable pleiotropic effects in the model system. For example, dexamethasone induces changes in the RANKL-RANK-OPG pathway leading to increased osteoclastogenesis and bone resorption, which were evaluated by histological analysis and qRT-PCR. TNF-α is utilized as an inflammatory cytokine to initiate the NF-κB pathway and subsequent effect on RANKL. Both cationic glucocorticoids and dexamethasone are then administered to determine the anti-inflammatory effect (for example, a reduction in IL-6) and the effect on RANKL.

An experiment was conducted to assess whether DS, D ₂ S, and dexamethasone affect osteoblasts in a dose dependent manner, prior to induction of an inflammatory state with TNF-α. The results disclosed herein show that both DS and D ₂ S exhibited anti -inflammatory activity, in the absence as well in the presence of Vitamin D3, as measured by the reduction in IL-6 transcripts (DS = 17% and D ₂ S = 28% reduction over control); however, neither decreased the transcript level to the extent of dexamethasone (72%) as shown in Figures 25 and 26. TNF-α was used as a positive control (Figure 25). Figure 26 depicts the same data but without showing the TNF-α arm. Figure 24 shows the transcript level of GAPDH as a negative control. These results indicate that both DS and D ₂ S retain glucocorticoid activity of the dexamethasone constituent, but that the modification of their structures diminished their effect in this model system.

Having established that both DS and D ₂ S exhibit anti-inflammatory activity, additional analysis was conducted to assess the level of relevant transcript expression. Specifically, OPG (Figure 27) and RANKL (Figure 28) transcripts were quantified to assess whether DS or D ₂ S altered the OPG/RANKL ratio which could indicate potential dissociated character and therapeutic value. The OPG/RANKL ratio for the negative control was 0.88. The OPG/RANKL ratio after treatment with the positive control, TNF-α, was 0.12. The OPG/RANKL ratio after treatment with dexamethasone was 0.18, after treatment with DS was 1.67, and after treatment with D ₂ S was 1.76. These results showed the expected activity of both TNF-α and dexamethasone, because both known to cause a decrease in the OPG/RANKL ratio compared to untreated controls. Interestingly, while both DS and D ₂ S retained anti-inflammatory glucocorticoid activity, they also resulted in greater OPG:RANKL ratios than control. These results demonstrate the dissociated character of the molecules of the invention, because they decreased IL-6 but did not induce the secondary side-effects compared to dexamethasone.

Osteoclastogenesis Additional analysis was conducted to visualize the effects of these compounds on osteoclastogenesis. TRAP is a marker of osteoclast differentiation. The results disclosed herein demonstrate that there is a striking contrast between dexamethasone and DS/D ₂ S treatment. In BMM cultures treated with TRANCE and M- CSF, treatment with 60/40 mol% DS/D ₂ S or 80/20 mol% DS/D ₂ S resulted in a >30 fold increase (at 100 nM) in mature osteoclasts (TRAP positive multinuclear cells) compared with treatment with dexamethasone (Figures 29 and 30) at day 5. The total TRAP solution activity differences between the DS or DSZD ₂ S groups was less than 2 fold in both cases (Figures 29 and 30). In co-cultures treated with Vitamin D3, treatment with 60/40 mol% DSZD ₂ S or 80Z20 mol% DSZD ₂ S resulted in a similar amount or an increase (at 10OnM) in mature osteoclasts (TRAP positive multinuclear cells) compared with treatment with dexamethasone (Figures 31 and 32) at day 7. The total TRAP solution activity differences between the 60/40 mol% DS/D ₂ S and 80/20 mol% DSZD ₂ S groups was less than 2 fold in both cases (Figures 31 and 32).

The data disclosed herein indicate that the cationic glucocorticoids of the invention do not significantly increase osteoclastic activity as measured by TRAP as compared with control. Furthermore, these data indicate that the TRAP activity demonstrated is primarily from phenotypically mature (multinuclear) osteoclasts. By comparison, dexamethasone induced a large increase in TRAP activity, but very low numbers of mature osteoclasts which indicates a change in the normal signaling pathway. The data disclosed herein suggest that DS and D ₂ S are active glucocorticoids and that they do not induce the same side-effects as dexamethasone.

Since osteoclast apoptosis is followed by recruitment of osteoblast progenitor cells in the signaling cascade to re-mineralize sites of resorption, the increase in TRAP activity and suppression in the number of mature multinuclear osteoclasts resulting from dexamethasone treatment may indicate a potential mechanism responsible for the glucocorticoid-induced side-effects. DS and D ₂ S did not cause the same changes in osteoclastogenesis. Therefore, this suggests different effects than dexamethasone.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.