US 2004OO37875A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0037875A1 MetSelaar et al. (43) Pub. Date: Feb. 26, 2004

(54) COMPOSITION FOR TREATMENT OF (30) Foreign Application Priority Data NFLAMMATORY DISORDERS

Jun. 13, 2002 (WO)...... WO O2/45688 A2 Jul. 12, 2000 (EP)...... OO2O4372.7 (76) Inventors: Josbert Maarten Metselaar, Utrecht (NL); Marca Henriette M. Wauben, Publication Classification Utrecht (NL); Gerrit Storm, Utrecht (NL) 51)1) Int. Cl.Cl." ...... A61K 9/12 7; A61K 31/5735 (52) U.S. Cl...... 424/450; 514/179 Correspondence Address: TRASK BRITT (57) ABSTRACT P.O. BOX 2550 A pharmaceutical composition for parenteral administration, SALT LAKE CITY, UT 84110 (US) comprising liposomes composed of non-charged vesicle forming lipids, including up to 20 mole percent of an (21) Appl. No.: 10/455,257 amphipathic vesicle-forming lipid derivatized with polyeth (22) Filed: Jun. 5, 2003 yleneglycol, and optionally including not more than 10 mole percent of negatively charged vesicle-forming lipids, the Related U.S. Application Data liposomes having a Selected mean particle diameter in the Size range between about 40-200 nm and containing a water (63) Continuation of application No. PCT/EP01/14633, Soluble corticosteroid for the site-specific treatment of filed on Dec. 7, 2001. inflammatory disorders, is provided. US 2004/0037875 A1 Feb. 26, 2004

COMPOSITION FOR TREATMENT OF liposomes are the Steroidal anti-inflammatory compounds, NFLAMMATORY DISORDERS Such as prednisone, methylprednisolone, paramethaZone, 11-fludrocortisol, triamcinolone, betamethasone and dexam CROSS-REFERENCE TO RELATED ethasone. The Steroids listed belong to the group of Steroids APPLICATION which are systemically administered. Example No. 12 is the only example of a glucocorticoid-containing PEG-liposome 0001. This application is a continuation of PCT Interna in this patent and relates to the preparation of beclometha tional Patent Application No. PCT/EP01/14633 filed on Sone dipropionate-containing PEG-liposomes. However, no Dec. 7, 2001, designating the United States of America, and in Vivo data were provided. On preparing dexamethasone published, in English, as PCT International Publication No. containing PEG-liposomes according to the disclosure in WO 02/45688 A2 on Jun. 13, 2002, the contents of the EP-0662820 and on intravenous administration of the same entirety of which is incorporated by this reference. in an in Vivo experimental arthritis model, the present inventors noted that the beneficial effects, as taught in TECHNICAL FIELD EP-0662820, could not be observed at all. There was no 0002 The invention relates generally to pharmaceutical difference in pharmacokinetic profile between a Suspension compositions, and more particularly to a pharmaceutical containing the glucocorticoid and the PEG-liposomes in composition for parenteral and intravenous administration, which the same glucocorticoid had been encapsulated. comprising liposomes composed of non-charged vesicle 0005 Since glucocorticoids often are the most effective forming lipids, including up to about 20 mole percent of an drugs in the treatment of inflammatory disorders, there is a amphipathic vesicle-forming lipid derivatized with polyeth need to provide liposomal compositions which after yleneglycol (PEG) and optionally including not more than parenteral administration can more efficiently deliver effec 10 mole percent of negatively charged vesicle-forming tive amounts of glucocorticoid at the inflamed region or lipids, the liposomes having a Selected mean particle diam tissue for enhanced and prolonged local activity. eter in the size range between about 40-200 nm and con taining a corticosteroid for the Site-specific treatment of BRIEF SUMMARY OF THE INVENTION inflammatory disorders. 0006 The invention provides a pharmaceutical compo BACKGROUND OF THE INVENTION Sition for parenteral administration, comprising liposomes composed of non-charged vesicle-forming lipids, including 0.003 Intravenous administration of compositions based up to 20 mole percent of an amphipathic vesicle-forming on PEG-containing liposomes for the site-specific treatment lipid derivatized with polyethyleneglycol and optionally of inflamed tissues and regions is already disclosed in including not more than 10 mole percent of negatively EP-0662820. It is well known that long-circulating small charged vesicle-forming lipids, the liposomes having a sized liposomes which contain non-charged or Slightly nega Selected mean particle diameter in the size range between tively charged vesicle-forming lipids, Such as PEG-lipo about 40-200 nm and containing a water soluble corticos Somes, after intravenous administration can circulate for teroid for the Site-specific treatment of inflammatory disor many hours in a volume not larger than the general circu derS. lation and therefore, in theory, are able to deliver relatively high portions of anti-inflammatory agents via extravasation 0007. The invention also provides a use of a pharmaceu at Sites of enhanced vascular permeability common to tical composition, comprising liposomes composed of non inflamed regions. Such liposomes are of particular interest in charged vesicle-forming lipids, including up to 20 mole the treatment of inflammatory diseases, e.g., rheumatoid percent of an amphipathic vesicle-forming lipid derivatized arthritis, which is a chronic autoimmune disorder, causing with polyethyleneglycol and optionally including not more joint inflammation and progressive cartilage destruction. than 10 mole percent of negatively charged vesicle-forming Although Several types of anti-rheumatic drugs are available lipids, the liposomes having a Selected mean particle diam for use, the treatment of Severe, persistent Synovitis and eter in the size range between about 40-200 nm and con acute exacerbations may require the use of Several intrave taining a corticosteroid, for the preparation of a medicament nous injections containing high doses of glucocorticoids. effective in the Site-specific treatment of inflamed tissueS or Although Systemic corticosteroids can Suppress the Symp regions after parenteral administration, characterized in that toms of the disease, adverse effects limit their use. In the corticosteroid is used in the medicament in a water addition to this, glucocorticoids Suffer from unfavorable Soluble form. pharmacokinetic behavior: Short plasma half-life values and a large distribution volume require high and repeated admin DETAILED DESCRIPTION OF THE istration in order to reach a therapeutically effective con INVENTION centration of the drug at the desired site of action. Intra 0008. It has now been found that by incorporating a water articular injection of Steroids into the affected joints is often Soluble form of a corticosteroid in long-circulating lipo used to increase the local efficacy of the glucocorticoids and Somes, composed of non-charged vesicle-forming lipids, diminish the systemic adverse effects, but this way of including up to 20 mole percent of amphipathic vesicle administration is less comfortable for the patients and not forming lipids derivatized with polyethylene glycol (PEG) feasible when multiple Small joints are affected. and optionally including not more than 10 mole percent of 0004. Also, a significant incidence of painless destruction negatively charged vesicle-forming lipids, the liposomes of the joint may be associated with repeated intra-articular having a Selected mean particle diameter in the size range injections of glucocorticoids. According to EP-0662820-B, between about 40-200 nm, an increased localization and preferred compounds for entrapment in PEG-containing improved retention of the corticosteroid at inflamed tissue US 2004/0037875 A1 Feb. 26, 2004 after one single intravenous injection of a pharmaceutical pivalate, fluorimetholone acetate, fluprednidene acetate, hal composition comprising the liposomes, can be reached as cinonide, halometasone, hydrocortisone acetate, medrySone, compared with one single intravenous injection of an aque methylprednisolone acetate, mometaSone furoate, parameta ous Solution containing the same corticosteroid compound. Sone acetate, prednicarbate, prednisolone acetate, pred nylidene, rimeXolone, tiXocortol pivalate and triamcinolone 0009. The long-circulation liposomes according to the hexacetonide. Topical corticosteroids, which undergo fast, present invention have a circulation half life of at least 6 efficient clearance as Soon as these drugs become available hours, the circulation half life being defined as the time at in the general circulation, are of Special interest. Examples which the Second linear phase of the logarithmic liposomal thereof are budesonide, flunisolide and fluticaSone propi clearance profile reaches 50% of its initial concentration, onate. By preparing a water Soluble form of the above which is the extrapolated plasma concentration at t=0. mentioned topical Steroids and encapsulating this into PEG liposomes in accordance with the present invention, it is now 0.010 The particle size of the liposomes is an important possible to Systemically administer Such corticosteroids in feature, which was demonstrated by the fact that adminis order to reach site-specific drug delivery, thereby avoiding tration of a water Soluble corticosteroid, Such as predniso adverse effects associated with Systemic treatment and over lone disodium phosphate, in PEG-liposomes, having a mean coming problems, which are inherent to the corticosteroid, particle diameter >500 nm, did not result in a significant Such as a fast clearance. In this respect, budesonide phos decrease of paw inflammation in the rat adjuvant arthritis phate has appeared to be a Salt of great interest. model. 0014. The liposomes in accordance with the present invention may be prepared according to methods used in the 0.011) A water soluble corticosteroid in accordance with preparation of conventional liposomes and the PEG-lipo the present invention is a compound which is Soluble 1 in somes, as disclosed in e.g. EP-0662820. Passive loading of is 10 (w/v), as assessed in water or water buffered at physi the active ingredients into the liposomes by dissolving the ologic values, e.g. at pH>6.0, at a temperature between 15 corticosteroid in the aqueous phase is Sufficient in order to and 25 C. reach an encapsulation as high as possible, but other meth ods can also be used. The lipid components used in forming 0012 Water soluble corticosteroids which can be advan the liposomes may be Selected from a variety of vesicle tageously used in accordance with the present invention are forming lipids, Such as phospholipids, Sphingolipids and alkali metal and ammonium Salts prepared from corticOS Sterols. Substitution (complete or partial) of these basic teroids, having a free hydroxyl group, and organic acids, components by e.g. Sphingomyelines and ergosterol Such as (C-C) aliphatic Saturated and unsaturated dicar appeared to be possible. For effective encapsulation of the bonic acids, and inorganic acids, Such as phosphoric acid water Soluble corticosteroids in the liposomes, thereby and Sulfuric acid. Also, acid addition Salts of corticosteroids avoiding leakage of the drug from the liposomes, especially can advantageously be encapsulated in the long-circulating phospholipid components having Saturated, rigidifying acyl PEG-liposomes. If more than one group in the corticosteroid chains, have appeared to be useful. molecule is available for Salt formation, mono- as well as di-Salts may be useful. AS alkaline metal Salts, the potassium 0015 The beneficial effects observed after one single and Sodium Salts are preferred. Also, other positively or injection of the water soluble corticosteroid containing PEG liposomes according to the invention are very favorable negatively charged derivatives of corticosteroids can be when compared with the results obtained after Single, but used. Specific examples of water Soluble corticosteroids are also repeated injections of the non-encapsulated water betamethasone Sodium phosphate, desonide Sodium phoS Soluble corticosteroid in different concentrations. Adminis phate, dexamethasone Sodium phosphate, hydrocortisone tration of the non-encapsulated water Soluble corticosteroid Sodium phosphate, hydrocortisone Sodium Succinate, meth was much less effective than the encapsulated corticosteroid, ylprednisolone disodium phosphate, methylprednisolone which can be easily understood if the total amount of free Sodium Succinate, prednisolone Sodium phosphate, pred Versus encapsulated corticosteroid is considered: the free nisolone Sodium Succinate, prednisolanate hydrochloride, corticosteroid was always by a factor of at least 5-10 less prednisone phosphate, triamcinolone acetonide disodium effective, even when injected on 7 consecutive days. These phosphate and triamcinolone acetonide dipotassium phoS effects have been observed in two different animal models, phate. Viz. a rat adjuvant arthritis model and a mouse model of collagen induced arthritis. The favorable effects may be a 0013 The above-mentioned corticosteroids normally are complete and long-lasting remission of all arthritis-associ used in Systemic treatment of anti-inflammatory diseases ated Symptoms, dependent of the dose. In addition thereto, and disorders. Since it has been proven by the present in the mouse model, a reduced cartilage erosion was inventors that by using a water Soluble form of a corticOS observed one week after treatment at the time the inflam teroid in PEG liposomes, having a specified Small mean mation had returned again. particle diameter, effective targeting of the drug to arthritic Sites, by Systemic administration, occurs, the present inven 0016 Since in Experimental Autoimmune Encephalomy tion can advantageously be applied to corticosteroids, elitis ultra high doses of methylprednisolone have been which, for a variety of reasons, normally are used for topical shown to be more efficient in induction of T cell apoptosis use. Such corticosteroids include, for example, alclometha than the “standard” dose of 10 mg/kg used in multiple Sone dipropionate, amcinonide, beclomethasone monopro Sclerosis therapy, it has been investigated whether a long pionate, betamethasone 17-Valerate, ciclomethasone, clobe circulating lipoSome formulation containing prednisolone is tasol propionate, clobetaSone butyrate, deprodone Superior to methylprednisolone pulse therapy in induction of propionate, desonide, desoxymethasone, dexamethasone T cell apoptosis in Situ. It has been observed that liposomal acetate, diflucortolone Valerate, diflurasone diacetate, diflu prednisolone phosphate given at 10 mg/kg augments T cell cortolone, difluprednate, flumetasone pivalate, flunisolide, apoptosis in Situ rapidly and the reduced infiltration of T fluocinolone acetonide acetate, fluocinonide, fluocortolone cells and macrophages leads to an ameliorated disease US 2004/0037875 A1 Feb. 26, 2004 activity of Adoptive Transfer Experimental Autoimmune (Sigma-Aldrich) or budesonide 21-phosphate (prepared by Encephalomyelitis (AT-EAE). As the liposomes can extrava Syncom, Groningen, the Netherlands) respectively was Sate and accumulate in inflamed tissue with a disrupted used. blood-brain-barrier, liposomal prednisolone phosphate could be a therapeutic alternative to methylprednisolone, which needs a higher dosage and would therefore cause Example III more Systemic side-effects. These findings may have impli 0026 Assessment of Therapeutic Efficacy in Rat Adju cations for the treatment of inflammatory autoimmune dis eases of the Central Nervous System (CNS) such as multiple vant Arthritis Model Sclerosis. 0027 Lewis rats were immunized subcutaneously at the 0017 Surprisingly, although small, rigid PEG-containing tail base with heat-inactivated Mycobacterium tuberculosis liposomes are known to hardly release their contents, the in incomplete Freund's adjuvant. Paw inflammation started PEG-liposomes according to the present invention were able between 9 and 12 days after immunization, reached maxi to effectively deliver the water soluble corticosteroid at the mum Severity approximately after 20 days, and then gradu desired Site of action resulting in a complete remission of the ally resolved. inflammation. 0028 Assessment of the disease was performed by visu 0.018. The following examples further illustrate the ally Scoring paw inflammation Severity, maximum Score 4 invention. per paw, and measuring disease-induced body weight loss. The therapeutic efficacy of liposomal prednisolone phos EXAMPLES phate, prepared according to example 1, on these variables was compared with equal doses unencapsulated drug. Rats Example I were treated when the average score >6 (at day 14 or 15 after 0.019 Preparation of Prednisolone Phosphate-Containing disease induction). PEG-Liposomes 0029. A complete remission of the inflammation process 0020 750 mg of dipalmitoyl phosphatidylcholine in 4 out of 5 rats was observed within 3 days after treatment (DPPC) (Lipoid Ludwigshafen), 250.8 mg of cholesterol with a single dose 10 mg/kg liposomal prednisolone phos (Sigma Aldrich) and 267.6 mg of PEG-distearoylphosphati phate (average score 0.1+0.1, compared to 11.8+1.9 of the dylethanolamine (PEG-DSPE) (Avanti Polar Lipids) were PBS-treated rats). Unencapsulated prednisolone phosphate weighed and mixed in a 100 ml round-bottom flask. The did not significantly alter the course of the disease (p>0.05, lipids were dissolved in about 30 ml of ethanol. Thereafter, Kruskall-Wallis test, non-parametric). In contrast, also 1 evaporating to dryneSS in a Rotavapor during 1 hour under mg/kg liposomal prednisolone phosphate was effective vacuum at 40 C., followed by flushing with nitrogen gas (p<0.05, Kruskall-Wallis, non-parametric). With respect to during 1 hour took place. weight loSS, only 10 mg/kg liposomal prednisolone phos 0021 1200 mg of prednisolone disodium phosphate phate had a significant effect (p<0.05, one-way ANOVA). (OPG Nieuwegein) were weighed and dissolved in 12 ml of Within 8 days, these rats regained initial weight. Other sterilized PBS. The solution was added to the dry lipid film treatment groups continued Suffering from progressive and Shaken for one hour in the presence of glass beads in weight loSS. order to enable complete hydration of the lipid film. 0030 No effect of a single injection unencapsulated 0022. The liposomal suspension was transferred to an prednisolone phosphate was observed. Therefore, it was extruder (Avestin, maximum volume 15 ml) and extruded decided to inject 10 mg/kg and 20 mg/kg daily for 7 dayS. under pressure, using nitrogen gas, 6 times through 2 pore Both treatment regimens reduced inflammation Scores from filters one placed on top of the other, having a pore size of an average of 6.5-0.56 (day 14) to average values around 200 and 100 nm respectively, 100 and 50 nm respectively, 5.0 from day 15 until day 21 (control treatment with daily and 50 and 50 nm respectively. Subsequently the liposomal saline reached a maximum of 10.6+1.3 on day 20). How Suspension was dialyzed in a dialyzing compartment (Slide ever, Single injections of 10 mg/kg and 20 mg/kg liposomal A-Lyzer, 10.000 MWCO) 2 times during 24 hours against 1 prednisolone phosphate at day 14 resulted in disappearance liter of Sterilized PBS. of adjuvant arthritis (AA) symptoms until day 20. Control 0023 The mean particle size of the liposomes was deter treatment with empty liposomes did not result in an altered mined by means of light Scattering (Malvern Zeta-sizer) and progression of the disease (p>0.05). was found to be 93.1+1.2 nm, the polydispersity index being 0.095+0.024. The encapsulation efficiency of the predniso 0031. The liposomal compositions of example 2 were lone phosphate was determined by means of a HPLC also tested in the adjuvant arthritis model. Liposomal dex method and was found to be between 3 and 4%. The amethasone phosphate proved to be more effective than Suspension of liposomes was Stored in a nitrogen atmo either liposomal prednisolone phosphate or liposomal sphere at 4 C. and found to be stable for about 2 months. betamethasone phosphate. A dose of 2 mg/kg was equally effective as 10 mg/kg liposomal prednisolone phosphate. 2 Example II mg/kg liposomal betamethasone phosphate proved to be more effective than 2 mg/kg liposomal prednisolone phos 0024 Preparation of Other Water Soluble Corticosteroid phate, however, in contrast to 2 mg/kg liposomal dexam Containing PEG-Liposomes ethasone phosphate, no complete reversal was obtained. 0.025 Example I was repeated but instead of prednisolone Liposomal budesonide 21-phosphate (1 mg/kg) produced at disodium phosphate dexamethasone disodium phosphate least equally effective disease Suppression as compared to (OPG Nieuwegein), betamethasone disodium phosphate liposomal dexamethasone phosphate. US 2004/0037875 A1 Feb. 26, 2004

Example IV 5. The pharmaceutical composition of claim 2, wherein the corticosteroid comprises a topically applied corticoster 0.032 Assessment of Therapeutic Efficacy in AT-EAE oid. 0.033 Adoptive Transfer Experimental Autoimmune 6. The pharmaceutical composition of claim 5, wherein Encephalomyelitis (AT-EAE) was induced in female Lewis the topically applied corticosteroid is Selected from the rats by intravenous injection of 107 MBP-specific T cells. group consisting of budeSonide, flunisolide, fluticasone pro 0034 10 mg/kg prednisolone phosphate containing lipo pionate, and mixtures thereof. Somes, as prepared according to Example I, were applied 7. A method for site-specific treatment of inflamed tissues intravenously at 42 hours and 18 hours prior to Sacrifice. or regions in a Subject, the method comprising: Another group received 50 mg/kg methylprednisolone intra venously at 18 hours and 6 hours before perfusion. Control parenterally administering to the Subject a pharmaceutical rats received empty liposomes and/or Saline at equivalent composition comprising: time points. a corticosteroid in water-Soluble form, and 0.035 T cells or macrophages in spinal cord were detected immunohistochemically in paraffin embedded tis liposomes composed of non-charged vesicle-forming Sue and apoptosis was assessed by the TUNEL assay and by lipids, including up to 20 mole percent of an amphi morphological criteria. Student t test for grouped data was pathic vesicle-forming lipid derivatized with poly used for Statistical analysis. The rate of T cell apoptosis in ethyleneglycol, Spinal cord tissue was significantly augmented by liposomal prednisolone phosphate (39.4+6.8%, p<0.0001 vs. wherein the liposomes have a Selected mean particle 16.1-4.3% in the control group, all data given as meantSD). diameter in a size range between about 40-200 nm. Methylprednisolone as an internal control lead to a rate of 8. The method according to claim 7 wherein the pharma 30.8+8.0% T cell apoptosis (p<0.01 vs. Controls). As a result ceutical composition further comprises up to 10 mole per of the increase in apoptosis, T cell infiltration was clearly cent of negatively charged vesicle-forming lipids in the reduced by liposomal prednisolone phosphate (45+12 T liposome. cells/mm), which was statistically significant compared to controls (115+51 T cells/mm, p<0.05) as well as compared 9. The method according to claim 8, wherein the corti to methylprednisolone (96+19 T cells/mm, p<0.05). As costeroid comprises a Systemically effective corticosteroid. another aspect of inflammation the macrophage infiltration 10. The method according to claim 8, wherein the corti was significantly reduced by liposomal prednisolone phoS costeroid comprises a topically effective corticosteroid. phate (31st13 macrophages/mm) compared to controls 11. The method according to claim 8 for the site-specific (78+37 macrophages/mm, p<0.05) and compared to meth treatment of rheumatoid arthritis in the Subject. ylprednisolone (66+25 macrophages/mmi, p<0.05). Even 12. The method according to claim 9 for the site-specific though the Adoptive Transfer model was chosen to investi treatment of rheumatoid arthritis in the Subject. gate rapid mechanisms, a therapeutic benefit from liposomal prednisolone phosphate could be observed within 42 hours, 13. The method according to claim 10 for the site-specific achieving a clinical Score of 2.80.2 compared to controls treatment of rheumatoid arthritis in the Subject. (3.2+0.3, p<0.01), which was Superior to methylpredniso 14. The method according to claim 8 for the site-specific lone (3.2+0.3, p<0.05 vs. liposomal prednisolone phos treatment of multiple Sclerosis in the Subject. phate). 15. The method according to claim 9 for the site-specific treatment of multiple Sclerosis in the Subject. What is claimed is: 16. The method according to claim 10 for the site-specific 1. A pharmaceutical composition for the Site-specific treatment of multiple Sclerosis in the Subject. treatment of inflammatory disorders by parenteral adminis 17. A parenteral pharmaceutical composition for alleviat tration, the pharmaceutical composition comprising: ing Symptoms associated with rheumatoid arthritis or mul a corticosteroid in water-Soluble form, and tiple Sclerosis in a Subject, Said parenteral pharmaceutical composition comprising: liposomes composed of non-charged vesicle-forming lip ids, including up to 20 mole percent of an amphipathic a corticosteroid in water-Soluble form, Said corticosteroid vesicle-forming lipid derivatized with polyethyleneg Selected from the group consisting of budesonide, lycol, flunisolide, fluticaSone propionate, prednisolone, dex wherein Said liposomes have a Selected mean particle amethasone, methylprednisolone, and mixtures thereof, diameter in a size range between about 40-200 nm. liposomes composed of non-charged vesicle-forming lip 2. The pharmaceutical composition of claim 1 further ids, including up to 20 mole percent of an amphipathic comprising: Vesicle-forming lipid derivatized with polyethyleneg up to 10 mol % of negatively charged vesicle-forming lycol, and lipids in Said liposomes. up to 10 mol % of negatively charged vesicle-forming 3. The pharmaceutical composition of claim 2, wherein lipids, the corticosteroid comprises a Systemically administered corticosteroid. wherein Said liposomes have a Selected mean particle 4. The pharmaceutical composition of claim 3, wherein diameter in a size range of between about 40 and about the Systemically administered corticosteroid is Selected from 200 nm. the group consisting of prednisolone, dexamethasone, meth ylprednisolone, and mixtures thereof.