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  Pharmaceutical Patents  

 

Title:  Targeted iron chelator delivery system
United States Patent: 
8,029,795
Issued: 
October 4, 2011

Inventors:
 Gwathmey; Judith K. (Cambridge, MA)
Assignee:
  Gwathmey, Inc. (Cambridge, MA)
Appl. No.:
 11/011,750
Filed:
 December 14, 2004


 

Pharm Bus Intell & Healthcare Studies


Abstract

A targeted iron chelator delivery system that comprises an iron chelator, a targeting agent and a lipid carrier, e.g., a liposome, is provided. The iron chelator delivery system may be used to remove excess iron from specific organs such as, for example, heart and liver tissue. Methods for preparing and administering the targeted iron chelator delivery system are also provided. The iron chelator delivery system may be administered during a blood transfusion to prevent iron overload.

Description of the Invention

FIELD OF THE INVENTION

Certain examples are directed to targeted iron chelator delivery systems for removal of excess iron from targeted cells, tissues or organs, such as, for example, the liver or heart.

BACKGROUND

Iron-overload due to transfusion currently occurs with any patient who receives more than 30 or 40 transfusions over the course of his or her life. The excess iron can injure any organ in the body. The heart and liver are particularly susceptible to damage, and failure of one of these organs is often the cause of death in patients with transfusional iron-overload. Transfusion related iron-overload is a major cause of morbidity and mortality in patients with a variety of transfusion-dependent anemias, hereditary hemochromatosis, including thalassemia major (Cooley's anemia).

Transfusion associated iron-overload develops in conditions characterized by severe, life-threatening anemia where transfusions substantially prolong life expectancy (Cairo, M. (1990) Introduction. Am. J. Pediatr. Hematol. Onco. 12:1-3). The most notable causes of transfusional iron-overload are the thalassemias, mild aplastic anemia, and congenital anemia (McLaren G. M. W. et al. (1983) CRC Crit. Rev. Clin. Lab. Sci. 126:896-899). Children with sickle cell disease and children who are stroke prone with sickle cell disease who receive chronic transfusions for complications may also suffer from iron-overload (Pegelow, C. et al. (1997) J. Pediatr. 126:896-899; Adams, R., et al. (1988) NEJM 339:5-11).

Transfused red cells are engulfed and destroyed at the end of their life span by stationary reticuloendothelial cells in the liver (Kupfer cells) and the spleen. The iron from the hemoglobin is removed and stored as hemosiderin. The reticuloendothelial cells return some of this iron to the circulation coupled to transferrin, and the iron is redistributed to the cells of the body. No known physiological mechanism of iron excretion exists. Therefore, after a number of transfusions, the level of iron in the body reaches a toxic level. At that point, chelation therapy is required. Iron that is stored in hemosiderin is innocuous. This iron is in equilibrium, however, with a very small pool of so-called "free iron" in the cell. This pool of iron is so small that its size has never been satisfactorily determined. Better termed "loosely-bound iron," this material catalyzes the formation of reactive oxygen species through Fenton chemistry. These reactive oxygen species are the agents of cell injury.

Iron-overload, whether due to chronic transfusions or hereditary hemochromotosis, has a plethora of side-effects (Bonkovsky, H. (1991) American Journal of Medical Science 301:32-43), (Koren, A. et al. (1987) Am. J. Dis. Child. 141:93-96). Liver damage and heart failure are the two most common causes of death. Liver iron deposition initiates hepatic fibrosis, cirrhosis and death (Bonkovsky, H. (1991) American Journal of Medical Science 301:32-43). Congestive heart failure or death from cardiac arrhythmias is common (Koren, A. et al. (1987) Am. J. Dis. Child. 141:93-96). For disorders such as thalassemia major (by definition, transfusion-dependent thalassemia), iron-overload now is the limiting factor in survival. The advent of chronic transfusion therapy in the 1960's increased life span to the early to mid-twenties, however, the pernicious consequences of iron-overload were invariably fatal (Cooley, T. (1945) Am. J. Med. 209:561-572), (Piomelli, S. (1991) Hematol. Oncol. Clin. North. Am. 5:557-569).

Iron is one of the leading causes of pediatric poisoning deaths in the United States (Litovitz, T. L. et al. (1992) Am. J. Emerg. Med. 10:452-505). Numerous reports of serious or fatal poisonings have been cited in the medical literature (Litovitz, T. L. et al. (1992); Westlin, W. F. (1966) Clin. Pediatr. 5:531-535; Henriksson, P. et al. (1979) Scand. J. Haematol. 22:235-240) including five toddler deaths in Los Angeles county during a seven month period in 1992 (Weiss, B. et al. (1993) Morb. Mortal. Wkly. Rep. 42:111-113). It is clear that iron can cause serious morbidity and mortality, yet many clinicians and families remain unaware of the dangers of iron (Anderson, B. D. (Apr. 18, 2000) Medscape Pharmacists). Although uncommon, iron solutions may be absorbed through damaged or burned skin. Following ingestion of large amounts of iron, peak serum levels generally occur within 2 to 6 hours. After ingestion, iron in the +2 state is oxidized to the +3 state and attached to the transport protein, ferritin. The iron is then released from the ferritin to transferrin in the plasma, transported to the blood forming storage sites, and incorporated into enzymes in the body. Iron is eliminated slowly from the body. Even in states of iron overload, children may lose up to 2 mg per day. Ingestion of less than 20 mg/kg elemental iron is likely to produce GI symptoms. For patients who ingest greater than 60 mg/kg elemental iron, potentially life threatening symptoms may occur.

Furthermore, the emergence of drug resistant parasites, e.g., malaria, has intensified the search for new therapeutic approaches (e.g. drug combinations). One new approach under investigation is the administration of iron chelating agents (Cabantchik, Z. I. et al. (1996) Acta Haematol. 95:70-77; Van Zyl, R. L. et al. (1992) J. Antimicrob. Chemother. 30:273-278).

Patients with transfusion iron-overload, iron poisoning, and drug resistant parasitic diseases (e.g., malaria) are commonly treated with low molecular weight iron chelators. These compounds remove the excess, toxic iron from the patient's blood. The most commonly used drug worldwide is desferrioxamine (Desferal.RTM., Novartis). Therapy with desferrioxamine is effective, but under-utilized because of drug delivery problems. Oral absorption of desferrioxamine is very low. In some cases, desferrioxamine infusion has proven not to be adequate (Westlin, W. (1996) Clin. Ped. 5:531-535; Tenenbein, M. et al. (1992) Lancet 339:699-701; Adamson, I. Y. et al. (1993) Toxicol. Appl. Pharmacol. 120:13-19). In addition, the low molecular weight of this hydrophilic molecule (657 Da) leads to renal clearance in about 15 to 20 minutes. Consequently, the drug is given by continuous infusion over 12 to 16 hours. This is done either by subcutaneous infusion or by infusion into a permanent catheter. Such a long infusion duration is inconvenient and prone to infections and thrombosis. Desferrioxamine also has severe drawbacks in the treatment of parasitic diseases; (1) it is hydrophilic and poorly absorbed after oral administration; and (2) it is cleared rapidly after intravenous administration and iron chelators like desferrioxamine do not readily penetrate into advanced growth stages of parasitized cells (Loyevsky, M. et al. (1993) J. Clin. Invest. 91:218-224). As a consequence, continuous infusion of iron chelators like desferrioxamine over a three day period is required to obtain enhanced parasite clearance in human malaria (Mabeza, G. F. et al. (1996) Acta Haematol. 95:78-86). Nonetheless, many patients use desferrioxamine suboptimally or not at all.

No other chelator has proven clinical efficacy. Searches for clinically effective alternatives to desferrioxamine for transfusional iron-overload have thus far been futile. Some chelating agents, such as diethyltriamine pentaacetic acid (DPTA) are effective, but too toxic for clinical use. Other chelators (e.g., EDTA) bind other cations in addition to iron, making them unacceptable as treatment of transfusional iron-overload or iron poisoning.

One approach to the problem has been to immobilize desferrioxamine to a large molecular matrix, thereby extending its biological half life. Immobilized desferrioxamine depends on a shift in "pseudoequilibrium" conditions to produce a net outflux of iron from cells. The vast amount of storage iron exists inside cells, however, effectively out of the reach of immobilized desferrioxamine. The problem is that the storage iron inside the cells remains a dangerous source of free radicals until it is chelated and inactivated by the Desferrioxamine in the matrix.

U.S. Pat. No. 5,534,241 ('241 patent) discloses chelation of iron using a linked molecule having a polymeric moiety covalently bonded to a lipid soluble anchor and a plurality of chelating agents covalently bonded to the polymeric moiety. There are several drawbacks to the compound disclosed in the '241 patent. In particular, complicated chemical synthesis and purification are required to covalently link the various groups. Also, to prevent rapid degradation of the '241 patent compound, surface protection is required. In addition, the polychelating agent is not free but is instead bound to the polymeric moiety, which can limit its ability to chelate iron from cellular stores.

The only chelator currently in extensive clinical trial is deferiprone (L1). Deferiprone removes excess iron reasonably well although it falls short of desferrioxamine in this regard (Collins, A. et al. (1994) Blood 83:2329-33). The great appeal of deferiprone over desferrioxamine is its oral absorption. For many patients, the convenience of an orally active chelator might more than compensate for lesser efficacy.

A number of clinical problems cloud deferiprone's future. Severe agranulocytosis occurs in about 2% of patients (al-Refaie, F. et al. (1992) Blood 80:593-9). Other significant side-effects of deferiprone include arthralgias and severe nausea (al-Refaie, F. et al. (1995) Br. J. Haematol. 91:224-229). Because of these and other problems, deferiprone's clinical future is far from assured. Therefore, there exists a need for an improved iron chelator delivery system to remove iron-overload in a cell, tissue, or organ.

SUMMARY

In accordance with a first aspect, a targeted iron chelator delivery system for treating iron-overload in mammalian tissues is provided. The delivery system includes free iron chelator, a targeting agent and a lipid carrier. In certain examples, the iron chelator is "free" in that it is not covalently bound to either the targeting agent or the lipid carrier, and, thus may dissociate from the lipid carrier and chelate excess iron in the target tissue. In certain examples, the targeting agent and/or the lipid carrier includes one or more cationic or anionic groups.

In accordance with another aspect, a targeted iron chelator delivery system for treating iron overload in the heart is disclosed. The delivery system includes an iron chelator and a lipid carrier. In certain examples, the lipid carrier includes an antibody that may bind to one or more cardiac proteins.

In accordance with an additional aspect, a targeted iron chelator delivery system for treating iron overload in the liver is provided. The delivery system comprises an iron chelator and a lipid carrier. The lipid carrier comprises a liver cell targeting agent for targeting at least one liver cell receptor. In certain examples, the liver cell targeting agent may be free in that it is not covalently bound to the iron chelator or the lipid carrier, but may be incorporated into the lipid carrier.

In accordance with yet another aspect, a targeted iron chelator delivery system is disclosed. The targeted iron chelator delivery system includes an iron chelator, a targeting agent, and one or more vesicles. The targeted iron chelator delivery system is designed for use in a method of treating iron-overload in a mammal by administering the delivery system to the mammal so that treatment occurs. In certain examples, the concentration of the iron chelator is about 1 .mu.M to about 100 mM. In certain examples, the cross-sectional diameter of the vesicle(s) is about 10 nm to about 10 .mu.m. In certain other examples, the vesicle(s) may be dissolved in a pharmaceutically acceptable excipient prior to administration, and the vesicle may be administered for a suitable period, e.g. about 20-30 minutes to about 3 hours. It will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure, that the administration rate may vary depending on the composition of the liposome, the nature and identity of the selected iron chelator or other species to be included in the liposome, etc.

In accordance with a method aspect, a method of preparing a targeted iron chelator delivery system is disclosed. The method includes combining a lipid carrier, an iron chelator and a targeting agent selected for targeting the heart or liver to form targeted iron chelator-encapsulated vesicles, and extracting the targeted iron chelator-encapsulated vesicles to form a targeted iron chelator delivery system.

In accordance with another method aspect, a method of preparing a targeted iron chelator delivery system is provided. The method comprises dissolving one or more phospholipids in a suitable solvent, or mixture of solvents, to form a solution comprising an aqueous phase and an organic phase. An iron chelator and a targeting agent may then be added to the solution. The solution may then be vortexed to mix the aqueous and organic phases. The organic phase may then be removed by one or more suitable techniques, such as extraction, evaporation, etc. to form iron chelator encapsulated vesicles. The vesicles may be extruded through membrane filters, and non-encapsulated iron chelator may be removed using suitable separation techniques, e.g. centrifugation. The iron chelator-encapsulated vesicles may be removed or extracted to provide a targeted iron chelator delivery system.

In accordance with yet another method aspect, a method of preparing a targeted iron chelator delivery system is disclosed. The method includes drying a mixture of one or more phospholipids in a suitable solvent, or mixture of solvents, to form vesicles. The vesicles may then be hydrated by adding a solution including iron chelator. The resulting mixture may be vortexed to form iron chelator-encapsulated vesicles. The vesicles may be extruded through a membrane filter, and then dialyzed to purify the iron chelator-encapsulated vesicles. The purified iron-chelator encapsulated vesicles provide a targeted iron chelator delivery system.

In accordance with an additional method aspect, a method for treatment of iron overload is provided. In certain examples, the iron overload results from one or more blood transfusions. The method comprises administering to a mammal in need of treatment of iron overload from a blood transfusion a therapeutic amount of the targeted iron chelator delivery system. The targeted iron chelator delivery system may be any of the exemplary targeted iron chelator delivery systems discussed herein, and other targeted iron chelator delivery systems that will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure. In certain examples, the targeted iron chelator delivery system may be dissolved in a suitable pharmaceutically acceptable carrier prior to administration to a mammal. In certain other examples, the targeted iron chelator delivery system may be administered prior to a blood transfusion, co-administered during a blood transfusion or administered after a blood transfusion. In other examples, the targeted iron chelator delivery system may be co-administered with one or more additional iron chelators, e.g. deferiprone.

In accordance with another method aspect, a method of preventing iron overload from a blood transfusion is disclosed. The method includes administering to a mammal receiving a blood transfusion an iron chelator delivery system. In certain examples, the iron chelator delivery system is co-administered with the blood transfusion.

The novel targeted iron chelator delivery systems disclosed here, and methods for their use, provide robust systems for removal of excess iron that may result from iron overload due to blood transfusion or that may result from one or more medical or genetic disorders. These and other illustrative aspects, examples and embodiments are discussed in detail below.

DETAILED DESCRIPTION OF CERTAIN EXAMPLES

It will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure, that examples of the targeted iron chelator delivery system disclosed here represent a significant technological advance. Efficacious targeted iron chelator delivery systems may be designed to provide rapid iron removal in a specific cell, tissue, organ or organs with minimal toxicity and minimal side-effects. The target iron chelator delivery system disclosed here provides numerous advantages over existing therapies designed to remove excess iron. For example, the kidneys rapidly excrete small, hydrophilic iron chelator molecules, such as desferrioxamine. Without wishing to be bound by any particular scientific theory, examples of the iron chelator delivery system disclosed here, e.g., iron chelators associated with lipid carriers, liposomes, and one or more targeting agents, may be designed to be too large to be filtered by the renal glomeruli. For example, small liposomes that are about 10 nm in diameter are of sufficient size to be above the filtration limit of the glomeruli of the kidneys. The biological half-life of the iron chelator may be extended using the targeted iron chelator delivery system disclosed here and certain specific cells, tissues or organs may be targeted to reduce the amount of iron present in the targeted cells, tissues or organs.

The more user-friendly terms "include" and "includes," as used throughout this disclosure, should be understood to be interchangeable with the open ended terms "comprises" and "comprising."

Conventional iron chelation therapy, e.g. desferrioxamine therapy, may cost between about $12,000 and $15,000 per year. Much of the expense involves the portable home infusion pumps, associated equipment (e.g., sterile infusion tubing and needles), and home nursing visits. These costs may be reduced by using the targeted iron chelator delivery system disclosed here. In addition, examples of the targeted iron chelator delivery system provided here may be made available to underdeveloped countries that lack the financial resources needed for conventional iron chelation therapies. Also, the targeted iron chelator delivery system disclosed here reduces and/or eliminates the problems of local reactions associated with the subcutaneous administration of some iron chelators. For example, the chelator diethyltriamine pentaacetic acid (DTPA) has been used to treat iron-overload, but this compound can cause severe local reactions that has led to discontinuation of its use. Intravenous administration, although more efficacious, is associated with the risk of infection, atrial thrombosis, and subclavical thrombosis. In addition, an indwelling catheter may curl into the atrium causing cardiac irritation and thrombosis, and right atrial thrombosis can lead to life threatening pulmonary embolism. Examples of the targeted iron chelator delivery system disclosed here may overcome or avoid these and other problems associated with iron chelation therapy. In at least certain examples, the targeted iron chelator delivery system provided here delivers iron chelator more efficiently than conventional methods. Lower amounts of iron chelator may be used and combined with lipid carriers, e.g., vesicles, to deliver the iron chelator to a specific cell, tissue or organ to increase the local concentration of the iron chelator. Other advantages of certain examples of the targeted iron chelator delivery system include, but are not limited to, reduced toxicity based on the administration of a lower dose for a shorter period of time, targeted delivery of the iron chelator without high renal clearance, an increase in the half life of the iron chelator via targeted delivery of the drug to the heart and the liver, thus, reducing the amount of drug needed, and entrapment of the drug in the liver up to 5 days, thus, allowing a longer period of time for iron chelation. Some iron chelator from the liver parenchyma can also redistribute to other tissues and bind free iron.

In accordance with certain examples, a targeted iron chelator delivery system for treating iron-overload in mammalian tissues is provided. In certain examples, the targeted iron chelator delivery system comprises an iron chelator, a targeting agent, and a lipid carrier. As used here "targeted" refers to specificity or selectivity of the iron chelator delivery system for a specific cell type, tissue or organ. For example, in iron chelator delivery systems targeted at reducing iron-overload in the heart, the system may include a recognition site, e.g., a site or binding portion of an enzyme, protein, antibody, etc., for one or more cardiac markers, such as polysaccharides, lipids, proteins, etc. In iron chelator delivery systems targeted at reducing iron-overload in the liver, the system may include one or more recognition sites for a liver receptor or marker, e.g., a protein, polysaccharide, lipid, etc. located primarily in the liver. Similarly, the iron chelator delivery system may be configured to target any specific tissue or organ by including a molecule or group that may recognize or bind to a marker on the target tissue or organ. Suitable markers include, for example, those markers discussed above, e.g. proteins, lipids, polysaccharides, etc., and other markers that will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure.

In accordance with certain examples, the iron chelator of the targeted iron-chelator delivery system is selected for its ability to remove iron, e.g. free iron and/or bound iron, in one or more specific tissues or organs. The term "iron chelator" refers to a compound that can remove excess iron from the patient's blood, tissues and/or organs. In certain examples, the iron chelator may be selected such that it binds tightly to iron. In other examples, the iron chelator is selected so that it binds to iron with a lower binding constant than normal hemoglobin. In certain examples, two or more iron chelators are used to provide enhanced removal of iron from a specific cell-type, tissue or organ. For example, a first iron chelator may be selected that has a high iron-binding constant but has low bioavailability, and a second iron chelator may be selected that has high bioavailability but binds iron with a lower binding constant than the first iron chelator. Other examples of combining two or more iron chelators will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure. Exemplary iron chelators for use in the targeted iron delivery system disclosed here, include but are not limited to, hydrophilic molecules having charged groups that may bind to or associate with iron. In certain examples, the iron chelator includes one or more amino or carboxy groups that can bind to iron. Depending on the pH and on the nature of the group, the charged groups may be positively charged or negatively charged. Exemplary iron chelators include, but are not limited to, desferrioxamine, deferiprone, PIH (pyridoxal isonicotinoyl hydrazone), rhodotorulic acid, HBED (N,N'-Bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid), HBPD (N,N'-Bis(2-hydroxybenzyl)propylene-1,3-diamine-N,N-diacetic acid), 2,3-dihydroxybenzoic acid, DTPA (diethyltriamine pentaacetic acid), and iron chelators produced by bacterial siderophores. The concentration of the iron chelator may vary depending on numerous factors including, for example, the binding constant of the iron/iron chelator complex, bioavailability, clearance rate, etc. In certain examples the iron chelator is present in a concentration from about 1 nM to about 500 mM, more particularly from about 500 nM to about 250 mM, e.g. about 1 .mu.M to about 100 mM. In other examples, the iron chelator is present in an effective or therapeutic amount. As used here "therapeutic amount" refers to an amount sufficient to reduce, alleviate or ameliorate pathological symptoms associated with a disease or disorder. It should be understood that a therapeutic amount does not necessarily remove all excess iron, but instead removes sufficient amounts of iron such that the symptoms are reduced, alleviated or ameliorated. It will be within the ability of the person of ordinary skill in the art, given the benefit of this disclosure, to select suitable concentrations of iron chelator for use in the targeted iron chelator delivery system disclosed here.

In accordance with other examples, the lipid carrier of the targeted iron chelator delivery system may take numerous forms including, for example, micelles, vesicles, liposomes, etc. In certain examples, the amount of lipid used is above the critical micelle concentration such that micelles or vesicles are the predominant form in solution. In some examples, lipid bilayers associate to form unilamellar vesicles, paucilamellar vesicles or multilamellar vesicles, e.g. small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs), targeted chelate nanoparticles, polymeric carriers, etc. The term "liposome" is used in the broad sense and should be understood to include unilamellar vesicles, paucilamellar vesicles, multilamellar vesicles and other forms and configurations that vesicles can take. In certain examples, the lipid carrier may be formed and the iron chelator can be disposed within a cavity of the lipid carrier, e.g. is encapsulated within the lipid carrier. In other examples, the iron chelator may be intercalated in a membrane of the lipid carrier or associated with the outer surface of the lipid carrier. For example, a hydrophilic iron chelator, such as desferrioxamine, may be combined with a lipid carrier. In yet other examples, the iron chelator may be coated with the lipids of the lipid carrier, e.g., iron chelators associated, intercalated, or attached to the surface of lipid molecules or to the lamellae of a lipid carrier. Other examples of lipid carrier formulations for the present iron chelator delivery system include, for example, the following: systems used with amphotericin B, which involve complexing the active ingredient with phospholipids as used in the formulation for ABELCET.RTM. (The Liposome Company, Inc., Princeton N.J.); cholesteryl sulfate complexes for injection similar to the formulation used for AMPHOTEC.RTM. (Sequus Pharmaceuticals, Menlo Park, Calif.) which comprises a sterile, pyrogen-free, lyophilized powder for reconstitution and intravenous administration, e.g., a formulation comprising a complex of desferrioxamine and cholesteryl sulfate (upon reconstitution a colloidal dispersion of microscopic disc-shaped particles result); and a single bilayer liposomal drug delivery system such as used with AmBisome.RTM. (Nexstar Pharmaceuticals, Boulder Colo. (taken over by Gilead Sciences Foster City, Calif.)), wherein single bilayer liposomes are used. In certain examples, the drug becomes active when the lipid carrier, e.g., the liposome, fuses with cells to release its content. In some examples, chelates may be attached to targeted carriers by bonds that are broken when the carriers are internalized into iron overloaded cells. Without wishing to be bound by any particular scientific theory, the iron chelator can interact with the liposome structure through numerous forces, e.g. hydrophobic interactions, hydrogen bonding, van der Waals interactions, and the like, such that the iron chelator is associated with the liposome for a sufficient time to allow for delivery of the iron chelator delivery system to a specific target cell, tissue, organ or system.

In accordance with other examples, the lipid carrier of the targeted iron chelator delivery system may comprise numerous different lipids, e.g., polar, non-polar, charged, uncharged, amphipathic lipids, or may comprise substantially a single type of lipid or a single lipid. Exemplary lipids for use in the lipid carrier disclosed here include phospholipids, glycerophospholipids, ether glycerophospholipids, sphingolipids, waxes and suitable molecules having a polar head group and a non-polar tail. Exemplary phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylinositol. The lipid carrier may further include additional compounds, such as, for example, terpenes, steroids, and the like that can change the properties of the lipid carrier, e.g. can alter the membrane fluidity of the lipid carrier. In certain examples, the lipid carrier includes cholesterol within the membrane. The lipid carriers used in the targeted iron chelator delivery system disclosed here have a number of advantages over currently used options, such as red cell ghosts, as encapsulation agents. For example, vesicles may be manufactured, while red cells ghosts are harvested. Use of red cell ghosts also entails some exposure, even slight, to biological pathogens. Finally, lipid carriers, e.g., liposomes, avoid alloimmunization issues because they lack red cell antigens (Rose, W. et al. (1990) Blood 76:1431-7).

In accordance with certain other examples, the targeting agent of the targeted iron chelator delivery system is selected for its ability to recognize a specific marker. The targeting agent may take numerous forms depending on the type of marker selected and depending on the dosage requirements, bioavailability, clearance rate, etc. The targeting agent typically is a protein that can recognize one or more sites on the target cell, tissue or organ to deliver the lipid carrier containing the iron chelator to that cell, tissue or organ or within suitable proximity to allow the iron chelator to bind to excess iron. Suitable targeting agents will be selected by the person of ordinary skill in the art, given the benefit of this disclosure. Exemplary targeting agents include enzymes, antibodies, e.g. monoclonal and polyclonal antibodies, and other biomolecules that can bind to a protein, polysaccharide, lipid, etc. with high specificity. Such targeting agents may be reconstituted in the lipid carrier using numerous methods including, for example, those methods described in U.S. Pat. No. 4,483,929, the entire disclosure of which is incorporated herein by reference for all purposes. Other suitable methods for incorporating targeting agents will be recognized by the person of ordinary skill in the art, given the benefit of this disclosure.

In certain examples, the targeting agent is an antibody that is designed to bind to one or more cardiac markers, e.g., one or more up-regulated modulatory proteins present in pathogenic cardiocytes. Cardiac failure as a result of iron-overload is of great clinical importance. The heart can be targeted for delivery of suitable amounts of the iron chelator delivery system, disclosed here. In certain examples, desferrioxamine may be combined with a lipid carrier by placing a positive or negative charge on the lipid carrier and/or by attaching antibodies specific to cardiac, vascular, endothelial, and matrix proteins to the lipid carrier. For example, the targeting agent may be designed to target myosin, troponin, or myosin light chain proteins, vasculature proteins, endothelial cells, or matrix proteins. The lipid carrier may also be tagged with cardiac imaging labels. In addition, selected delivery routes and an increase in the half life of the lipid carrier can enhance delivery to the heart.

In certain other examples, the targeting agent is designed to target one or more liver markers. For example, the targeting agent can be designed to target one or more liver receptors that bind to asialoglycoprotein, galactose and mannose. In addition and without wishing to be bound by any particular scientific theory, most of the lipid carrier molecules are trapped by the liver and engulfed by the Kupfer cells and hepatocytes in the liver. The targeted iron-chelator delivery system disclosed here takes advantage of this seemingly unfavorable side-effect. Reticuloendothelial (RE) cells can engulf the iron chelator delivery system, which places the iron chelator in a position to intercept iron as it is released from erythrocytes degraded by the liver. Other suitable cardiac and liver markers for targeting will be selected by the person of ordinary skill in the art, given the benefit of this disclosure.

In accordance with certain other examples, one or more tags may be added to the lipid carrier. The tags can provide information about the local environment of the delivery system, e.g. whether or not the delivery system has been taken-up by a cell, tissue or organ. Suitable tags include but are not limited to fluorescent tags, such as fluorescein isothiocyanate (FITC), didansyl chloride, and other suitable probes that can be reacted with the lipid carrier or the targeting agent of the targeted iron chelator delivery system disclosed here. In certain examples, one or more magnetically active tags such as nitroxide spin labels, magnetically active nuclei, etc. may be used. In other examples, one or more radioactive tags, such as .sup.35S, .sup.32P, .sup.99Tm, .sup.111In, etc. can be added to the iron chelator delivery system. In certain other examples, one or more colorimetric labels, e.g. colored dyes, enzymes that can react with a substrate to produce a colored product, etc., may be added to the iron chelator delivery system. Other suitable tags will be readily selected by the person of ordinary skill in the art, given the benefit of this disclosure.

In accordance with certain other examples, a targeted iron chelator delivery system for treating iron overload in the heart is disclosed. The targeted iron chelator delivery system includes an iron chelator, such as, for example, any one or more of the iron chelators discussed herein. In certain examples, the iron chelator typically is selected from one or more of the following: desferrioxamine, deferiprone, PIH, rhodotorulic acid, HBED, HBPD, 2,3-dihydroxybenzoic acid, DTPA, and iron chelators produced by bacterial siderophores. In certain examples, the iron chelator is present in a therapeutic amount. In other examples, the concentration of iron chelator is about 1 .mu.M to about 100 mM, more particularly from about 100 .mu.M to about 10 mM, e.g. from about 1 mM to about 5 mM. The delivery system further includes a lipid carrier, such as those discussed herein. In certain examples, the lipid carrier comprises an antibody for targeting at least one cardiac protein, e.g. cardiac myocyte proteins, vasculature proteins, endothelial cells, matrix proteins, myosin, troponin, and myosin light chain. In certain other examples, the lipid carrier is a liposome. In examples where the lipid carrier is a liposome, the lipid carrier may have a cross-sectional diameter of about 10 nm to about 10 .mu.m. In examples using liposomes, the iron chelator may be encapsulated within the central cavity of the liposome, intercalated into the liposome bilayer, or associated with the outer surface of a membrane of the liposome. In examples using multilamellar liposomes, the iron chelator may be encapsulated between lamellae of the liposomes, intercalated into one or more of the bilayers of the multilamellar liposomes, or associated with the outer surface of the multilamellar liposomes.

In accordance with certain other examples, a targeted iron chelator delivery system for targeting the heart is disclosed. The targeted iron chelator delivery system includes an iron chelator and a carrier, such as, for example, a lipid carrier. The lipid carrier includes a targeting agent for targeting the heart. The targeting agent and/or lipid carrier may include one or more cationic and/or anionic groups. In certain examples, the cationic or anionic groups are carboxy groups, amino groups, hydroxy groups and other suitable positively and/or negatively charged groups.

In accordance with certain other examples, a targeted iron chelator delivery system for treating iron overload in the liver is provided. The delivery system includes an iron chelator, and a lipid carrier. The lipid carrier includes a liver cell targeting agent for targeting at least one liver cell receptor or liver cell protein. Exemplary liver receptors and proteins include, but are not limited to, receptors that bind to asialoglycoprotein (e.g. hepatocyte asialoglycoprotein receptor), galactose, and mannose (e.g. a Kupffer cell mannose receptor), and receptors on liver endothelial cells. The iron chelator may be selected from any of those iron chelators discussed herein, e.g. desferrioxamine, deferiprone, PIH, rhodotorulic acid, HBED, HBPD, 2,3-dihydroxybenzoic acid, DTPA, and iron chelators produced by bacterial siderophores, and other suitable iron chelators selected by the person of ordinary skill in the art, given the benefit of this disclosure. The concentration of the iron chelator can vary, for example, from about 1 .mu.M to about 100 mM. In certain examples, the iron chelator is present in a therapeutic amount. The lipid carrier may take the form of liposomes, multilamellar vesicles and other ordered structures that may be formed with lipids and/or phospholipids.

In accordance with certain examples, a method of preparing a targeted iron chelator delivery system is disclosed. The method includes combining a lipid carrier, an iron chelator and a targeting agent selected for targeting the heart or liver to form targeted iron chelator-encapsulated vesicles. The targeted iron-chelator encapsulated vesicles may then be extracted or removed to provide a targeted iron chelator delivery system. The iron chelator, lipid carrier and targeting agent each may be selected from any of the iron chelators, lipid carriers and targeting agents discussed herein and other suitable iron chelators, lipids carriers and targeting agents that will be selected by the person of ordinary skill in the art, given the benefit of this disclosure.

In accordance with certain other examples, a method of preparing a targeted iron chelator delivery system is provided. The method includes dissolving suitable phospholipids, e.g. phosphatidylcholine and optionally a steroid, e.g. cholesterol, in a suitable solvent, e.g. dichloromethane, ether, chloroform, methanol, etc., or solvent mixture, to form a solution comprising an aqueous phase and an organic phase. To this solution is added an iron chelator and a targeting agent. The solution with added iron chelator and targeting agent is vortexed to mix the aqueous and organic phases. The solution is then subjected to an extraction step and/or an evaporative step to remove the organic phase. Such extraction or evaporative step may be performed under vacuum, e.g. using a rotovap or similar device, to form iron chelator-encapsulated vesicles, e.g. iron chelator encapsulated liposomes. The vesicles may then be extruded through suitable membrane filters. Non-encapsulated iron chelator may then be removed by filtration, centrifugation or other suitable separation techniques. The iron chelator encapsulated-liposomes may then be removed to provide a targeted iron chelator delivery system, which can be stored until use.

In accordance with yet other examples, a method of preparing a targeted iron chelator delivery system is disclosed. The method includes drying a mixture of a lipid carrier, e.g. phosphatidylcholine and optionally a steroid, e.g. cholesterol, in a suitable solvent, e.g. dichloromethane, ether, chloroform, methanol, etc., or mixture of solvents, to form vesicles. The mixture may be dried under vacuum, under nitrogen, under argon, or under inert environments. The dried mixture can be hydrated by adding an aqueous solution of iron chelator followed by vortexing to form iron chelator-encapsulated vesicles. The iron-chelator encapsulated vesicles may be extruded through membrane filters. The vesicles may be centrifuged or dialyzed to purify the iron chelator-encapsulated vesicles.

In accordance with certain other examples, the targeted iron-chelator delivery system disclosed here can be pre-administered, co-administered, or post-administered to subjects receiving transfusion. For example, mammals, e.g., humans or animals, often receive blood on a three to six week schedule, depending on the severity of their anemia. The targeted iron chelator delivery system disclosed here may be used to prevent or reduce transfusional iron-overload. For example, desferrioxamine may be combined with a lipid carrier, e.g., a liposome, and the resulting product can be administered pre-transfusion, during transfusion, or at the end of a transfusion session. The targeted iron chelator delivery system can be administered continuously, intermittently, periodically, etc., and may be administered using suitable routes, such as oral administration, rectal administration, subcutaneous administration, iv or arterial infusion or injection, administered in a suppository, nasally, and other suitable methods for administration that will be selected by the person of ordinary skill in the art, given the benefit of this disclosure. Such treatment using the targeted iron chelator system disclosed here would allow significant improvement over the current daily use of an infusion pump for 12-16 hours intervals. For example, thirty grams of desferrioxamine encapsulated by vesicles may be sufficient to supply enough chelator for a month. This approach would be simpler than any available to date, including oral administration of deferiprone. The person of ordinary skill in the art will recognize, given the benefit of this disclosure, that the exact dosage and dose schedule may vary depending on the subject's characteristics, e.g. age, weight, health, human mammal, non-human mammal, etc. In some examples, the targeted iron chelator delivery system may be mixed or added to the blood transfused into the mammal, e.g. the blood and iron chelator delivery system are mixed and then the combination is transfused into a human or non-human mammal. It is a significant advantage that examples of the targeted iron chelator delivery system disclosed here can be administered prophylactically to prevent iron overload in individuals prior to, or during, blood transfusion.

In accordance with certain examples, the targeted iron chelator delivery system may be dissolved in a pharmaceutically acceptable carrier prior to administration. As used herein "pharmaceutically acceptable carrier" includes any and all excipients, solvents, dispersion media, coatings, antibacterial and antifungal agents, toxicity agents, buffering agents, absorption delaying or enhancing agents, surfactants, and micelle forming agents, lipids, liposomes, and liquid complex forming agents, stabilizing agents, and the like. Suitable media and agents for pharmaceutically active substances will be readily selected by the person of ordinary skill in the art, given the benefit of this disclosure. Supplementary active compounds may also be incorporated into the compositions.

In accordance with additional examples, a targeted iron chelator delivery system is provided. The targeted iron chelator delivery system includes an iron chelator, a targeting agent, and one or more vesicles. The targeted ion chelator delivery system is configured for use in a method of treating or preventing iron-overload in a mammal. The delivery system is administered to the mammal so that treatment occurs, e.g. so that a suitable amount of excess iron is removed from the target cell, tissue or organ. In certain examples, the concentration of the iron chelator is about 1 .mu.M to about 100 mM, and the size of the vesicle is about 10 nm to about 10 .mu.m. The vesicle may be dissolved in a pharmaceutically acceptable excipient prior to administration, and can be administered for a suitable period, e.g. intravenous administration for about 20-30 minutes to about 3 hours at a rate of about 50 cc/hour in normal saline (0.9% NaCl).

In certain examples the iron chelator delivery system takes the form of a gel, a lozenge, a spray, a tablet, an oral suspension, etc. The delivery system may be administered locally by injection, by catheterization, etc., may be administered systemically by intravenous infusion or arterial infusion, may be administered topically, subcutaneously, may be administered rectally, may be administered by inhalation and through other suitable methods. In certain examples, the targeted iron chelator delivery system is delivered through a catheter that is inserted proximate to the target organ. For example, a catheter may be inserted into the hepatic artery to provide delivery of the iron chelator delivery system to the liver, may be inserted into the inferior vena cava or a coronary artery to provide delivery of the iron chelator delivery system to the heart, etc. The exact dosage may vary depending on the desired level of iron to be removed, the nature of the iron chelator, etc. In certain examples, the dosage is selected such that from about 1 to about 30 mg of iron chelator/kg of body weight is administered at each dose, more particularly about 5 to about 25 mg of iron chelator/kg of body weight is administered at each dose, e.g. about 10, 15 or 20 mg iron chelator/kg of body weight is administered at each dose. The iron chelator delivery system may be administered continuously, hourly, daily, weekly, monthly, semi-monthly, or other suitable schedule. In certain examples the delivery system may be administered in bolus or may be administered intermittently. In examples where the delivery system takes the form of a liquid, suspension or solution, the delivery system may be administered at a rate of about 50 cc/hour to about 250 cc/hour, more particularly from about 100 cc/hour to about 200 cc/hour, e.g., about 150 cc/hour.

In accordance with certain examples, the targeted iron chelator delivery system may take the form of a dried powder, e.g. a lyophilized powder, such that storage and transport of the delivery system is simplified. Prior to administration, the dried powder can be rehydrated in a suitable solvent or excipient and administered to a human or non-human mammal as prophylaxis to prevent iron overload or to treat iron overload in the human or the non-human mammal in need of such treatment.

In accordance with certain examples, methods and compositions comprising liposomes and antibiotics or antifungals are provided. For example, a commercially available liposome that carries an antifungal is AMBISOME.RTM. available from Fujisawa Healthcare, Inc. (Deerfield, Ill.). The antibiotic or antifungal liposomes can be administered alone or co-administered with one or more of the targeted iron chelator delivery systems disclosed herein, e.g., to prevent or treat secondary infection. The exact administration rate of antibiotic and antifungal containing liposomes may vary, and in certain examples, the liposomes are administered at a rate of about 50-250 cc/hour in normal saline (0.9% NaCl), more particularly about 150-250 cc/hour in normal saline, e.g., about 200-250 cc/hour in normal saline.
 

Claim 1 of 17 Claims

1. A targeted iron chelator delivery system for targeting the heart or liver, the targeted iron chelator delivery system comprising: an iron chelator; and a lipid carrier comprising: a targeting agent for targeting the heart or liver, a phosphatidylcholine, a cholesterol, a phosphatidylethanolamine, and one or more cationic lipids, wherein the one or more cationic lipids are present in an amount effective to selectively increase uptake of the targeted iron chelator delivery system by the heart or liver.
 

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