US20010039295A1

US20010039295A1 - N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid in iron chelating therapy

Info

Publication number: US20010039295A1
Application number: US09/748,300
Authority: US
Inventors: Raymond Bergeron
Original assignee: Individual
Current assignee: University of Florida Research Foundation Inc
Priority date: 1999-12-21
Filing date: 2000-12-21
Publication date: 2001-11-08
Also published as: EP1246618A1; US20010047108A1; WO2001045696A1; CA2394524A1; US6646157B2; AU2293101A; AU2456201A; WO2001046114A1

Abstract

The use of a mono-cationic salt of N,N′-bis(2-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid (HBED) in iron chelating therapy is disclosed. In particular, the invention relates to the intravenous use of a mono-cationic salt of HBED for treating a primate with a condition treatable by an iron chelator such as iron overload, especially transfusional iron overload.

Description

ACKNOWLEDGEMENTS

[0001] This invention was supported in part by a grant from the National Institutes of Health, No. DK49108. The U.S. Government may have rights in this invention.

INTRODUCTION

1. Technical Field

This invention relates to the use of N,N′-bis(2-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid (HBED) in iron chelating therapy. In particular, the invention relates to the intravenous use of HBED for treating a primate, in particular a human, that is afflicted with a condition treatable with an iron chelating agent, i.e., iron overload, especially transfusional iron overload.

2. Background

In humans and other primates iron is stored in the body in the form of ferritin and hemosiderin, which are protein complexes, and is transported in the plasma via another protein complex, namely transferrin. The manner in which iron is utilized is very efficient, but there is no specific mechanism that exists for the elimination of excess iron. Under conditions of iron overload the protein complexes become saturated with iron, resulting in excess iron being deposited in tissues which induces iron toxicity, and ultimately leads to peroxidative tissue damage. An iron overload condition may occur for example through a genetically determined error that results in increased absorption of iron from a normal diet or may occur through repeated blood transfusions to treat a disease state, e.g., sickle-cell anemia.

One way of treating iron overload is by administering an iron chelator. In principle, the chelator transforms the deposited iron back into a soluble form that is then capable of excretion. However, none of the available iron chelators employed for this purpose is ideal; the chelators have poor gastrointestinal absorption, or have low efficacy and/or undesirable side effects.

One iron chelator that overcomes some of these disadvantages, and is widely used in therapy, is deferoxamine B (DFO) as the methanesulfonate salt, also known as desferrioxamine B mesylate (DFOM). This is available commercially as Desferal from Novartis (previously Ciba-Giegy). In general DFOM is capable of controlling excess body iron and can prolong survival and prevent or ameliorate organ dysfunction in a person suffering from iron overload. However, DFOM is far from an ideal treatment for iron overload. It is cumbersome, inefficient, expensive, and unpleasant to the patient. Because DFOM is poorly absorbed from the gastrointestinal tract and rapidly eliminated from circulation, prolonged parenteral infusion is required for treatment; it is generally administered by a portable infusion pump for 9-12 hours daily. Not surprisingly, very few patients are capable of complying with such a demanding regimen. Additionally, DFOM is very inefficient as an iron chelator, in that typically 5% or less of the compound administered binds iron.

Yet another disadvantage is the cost of DFO, which is commercially produced by large scale fermentation of a strain of Streptomyces pilosus, a method of manufacture that is very expensive. In addition, almost all patients experience an allergic reaction to DFOM, which is uncomfortable and sometimes very painful. The allergic reaction is thought to be caused by cytokines and/or other fermentation products formed during the fermentation process that are not completely removed during purification of the crude product mixture.

It would therefore be desirable to provide a method for treating iron overload that employs a safe and inexpensive alternative to DFOM. Ideally, the iron chelator would not be associated with an allergic reaction in patients, and would be a more efficient chelator of iron than DFOM.

HBED is a compound known to be an iron chelator. U.S. Pat. No. 3,758,540 discloses that iron chelates of HBED and other compounds are useful as a source of iron in plant nutrition. See also U.S. 4,116,991. U.S. 4,528,196 discloses, on the basis of testing in rat and mouse screens, that HBED and its alkyl esters are orally active in the treatment of iron overload, and more active given orally than DFO given intraperitoneally. However, the rodent findings were not substantiated in higher animals. Testing in the Cebus apella monkey screen, which is known to be an excellent predictor of the behavior of chelators in humans, showed that DFOM administered subcutaneously has significantly higher activity than HBED or its dimethyl ester given orally (Bergeron et al., Blood, 81:2166 (1993), Peter et al., “A Comparative Evaluation of Iron Chelators in a Primate Model”, pp 373, Development of Iron Chelators for Clinical Use, Boca Raton, CRC Press (1994)). It has also been demonstrated that HBED, when given orally to human patients, provides iron excretion at a level that is ineffective for the treatment of iron overload (Grady et al., “Preliminary Results from a Phase I Clinical Trial of HBED”, in the Development of Iron Chelators for Clinical Use, Boca Raton, CRC Press (1994), pp 395).

The inventor previously found that, surprisingly, HBED as a mono-cationic salt is useful for treating iron overload when administered subcutaneously. The HBED salt upon subcutaneous administration is not associated with an allergic reaction in patients, and is a more efficient chelator of iron than DFO. See PCT Application W099/39706.

Objects of the Invention

An object of this invention is to provide an improved method for treating a condition in primates, particularly humans, which condition is treatable by an iron chelator.

It is a further object of this invention to provide a composition for the intravenous treatment of a condition that is treatable by an iron chelator, particularly where the condition is iron overload.

It is a further object of this invention to provide a process for making an intravenously administratable composition useful for treating a condition treatable with an iron chelator, particularly where the condition is iron overload.

Other objects of the invention may be apparent to one of skill in the art upon reading the following specification and claims.

SUMMARY OF THE INVENTION

The present invention relates to a method of treatment of a primate having a condition that is treatable by an iron chelator. The method comprises intravenous administration of a therapeutically effective amount of a compound of the formula (I)

namely N,N′-bis(2-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid (HBED), as a mono-cationic salt (M is a cation). The method is particularly suitable for treating iron overload in a primate, particularly a human.

Another aspect of this invention is a pharmaceutical composition comprising a pharmaceutically-acceptable excipient suitable for intravenous injection in combination with a mono-cationic salt of HBED.

Still another aspect of this invention is an article of manufacture that comprises a container containing a pharmaceutical composition comprising a therapeutically effective amount of the mono-cationic salt of HBED, and is associated with printed labeling instructions for intravenous administration of the composition to treat a condition that is treatable by an iron chelator.

Still another aspect of this invention is a process for preparing a pharmaceutical composition, which process comprises combining a pharmaceutically-acceptable excipient suitable for intravenous injection with the mono-cationic salt of HBED.

Other aspects may become apparent upon further reading of this patent application.

DESCRIPTION OF PRESENTLY PREFERRED EMBODIMENTS

Method of Treatment

One aspect of this invention is a method of treating a primate, particularly a human, that has a condition that is treatable by an iron chelator. The method comprises the intravenous administration of a therapeutically-effective amount of a mono-cationic salt of HBED. A condition treatable by an iron chelator may occur, e.g., through a genetically determined error that results in increased absorption of iron from a normal diet or may occur through repeated blood transfusions to treat a disease state. Such conditions in which iron chelators may be used include malaria, cancer, HIV infections, inflammatory bowel disease, host v. graft rejection, graft v. host rejection, reperfusion injury, neurological disorders, and iron overload. The condition in which this method is most useful is “iron overload.” This condition is characterized by greater than normal focal or generalized deposition of iron within body tissues.

When such focal or generalized deposition is associated with tissue injury, with total body iron greater than about 15 grams, it is known as hemochromatosis, which may be primary or secondary. Primary hemochromatosis most commonly arises from an autosomal recessive trait linked to the histocompatibility locus on chromosome 6 that results in increased absorption of iron from a normal diet. This form of primary hemochromatosis affects 3 to 8 people per thousand. Primary hemochromatosis may also occur as a result of other genetically determined errors including conditions identified as atransferrinemia, thalassemia major, and y-linked hypochromic anemia. While the Merck Manual, 15th Edition, sets forth the classification of these conditions, differential diagnosis is difficult. Diagnosis will depend on the history of iron administration, the examination of relatives of the patient, the degree of iron overload, and the presence or absence of localizing signs.

Hemochromatosis rarely occurs before middle age. Typical manifestations are cirrhosis of the liver, brown pigmentation of the skin, diabetes mellitus, and cardiomyopathy, which may be manifested by cardiomegaly, congestive failure, and arrhythmias or conduction disturbances. In the case of pituitary failure, testicle atrophy and loss of libido may be seen. Abdominal pain, arthritis and chondrocalcinosis occur less often.

Focal hemosiderosis (the accumulation of hemosiderin in tissues) chiefly occurs in the lungs and kidneys. Pulmonary hemosiderosis may be due to recurrent pulmonary hemorrhage which occurs as an idiopathic entity, e.g., as part of Goodpasture's Syndrome.

Diagnosis of the above mentioned conditions may be found in Merck's Manual. Each of these conditions should be considered as a condition that is treatable by an iron chelator.

Secondary hemochromatosis or hemosiderosis may result from increased parenteral iron intake such as through repeated transfusion (transfusional iron overload). Repeated transfusions are often required for various diseases, such as, for example, sickle-cell anemia, thalassemia, Cooley's anemia, and myelodysplasia. Secondary hemochromatosis or hemosiderosis may also be caused by iron dextran taken intramuscularly, increased iron absorption due to increased iron ingestion, or may be due to anemia with erythroid hyperplasia, or may possibly be linked to megadoses of Vitamin C. Focal hemosiderosis may be pulmonary, renal, or hepatic.

As mentioned herein before, the key to the treatment of a condition treatable by an iron chelator is the intravenous administration of an unbound form of the compound HBED as shown in Formula I hereinbefore, as a mono-cationic salt. The HBED salt is “unbound” when it is not chemically or physically attached to another moiety, such as, for example, a liposome or micelle.

One advantage of intravenous administration of HBED for treatment of secondary hemochromatosis is that it can be readily administered during the transfusion, if desired.

The mono-cationic salt of HBED is one that is “pharmaceutically-acceptable” i.e. a salt that retains the biological effectiveness and properties of HBED, and that is not biologically or otherwise undesirable.

A pharmaceutically-acceptable mono-cationic salt may be considered a base addition salt and may be prepared from an inorganic or organic base. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, and ammonium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally-occurring substituted amines, and cyclic amines. They include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, N-alkylglucamines, theobromine, purines, piperidine, N-ethylpiperidine and tetra-n-butylammonium hydroxide. Preferred are the mono-sodium salt and the mono-ammonium salt.

The term “treatment” as used herein covers any treatment of a condition treatable by an iron chelator in a primate, particularly a human, and includes:

(i) preventing the condition from occurring in a subject which may be predisposed to the condition but has not yet been diagnosed as having it;

(ii) inhibiting the condition, e.g., arresting or slowing its development; or

(iii) relieving the condition, e.g., causing regression of the condition.

The method of administration that has been found to be particularly useful is the intravenous administration of the mono-cationic salt of HBED. “Intravenous administration” means that the drug is delivered in an appropriate composition directly into the veins, e.g., by injection. An intravenous injection can be of a small volume for a short duration, typically via a syringe. Alternatively, an intravenous injection can be of a larger volume for a longer duration, sometimes referred to as an infusion and frequently involving the use of what is known colloquially as an “IV bag” or “IV bottle”. The injection or infusion may be of a solution of a formulation comprising the active entity. Generally, the intravenous administration will be done with excipients that are suitable for intravenous administration, which means that the excipients will meet USP considerations in being appropriate for injectable compositions. Thus, the composition will need to be sterile to avoid any complications due to insterility at the injection site.

The amount of the active ingredient that will be present in the composition to be administered is a therapeutically-effective amount, that is, an amount which is sufficient to result in successful treatment as defined above when administered to a mammal exhibiting a condition treatable by an iron chelator, e.g., a disease state of iron overload. The therapeutically effective amount will vary depending on the subject and disease state being treated, the severity of the affliction and the manner of administration, and may be determined routinely by one of ordinary skill in the art in light of the disclosure of this specification. The typical daily dose of HBED varies according to individual needs, the severity of the condition to be treated and the specific salt chosen. Generally, about 5 micromoles (μmoles) to about 500 μmoles of the active moiety, i.e., HBED, will be administered to a patient on a per kilogram (Kg) basis. Suitable doses are in the general range from about 1 to about 200 mg/Kg body weight of the recipient per day, preferably in the range from about 5 to about 100 mg/Kg, most preferably in the range from about 10 to about 80 mg/Kg, of the active moiety (HBED).

In a human being treated for transfusional iron overload, a mono-cationic salt of HBED is preferably administered during the transfusion. For example, in treating sickle cell anemia patients, transfusions should be given only when the anemia is more severe than usual (during aplastic crises accompanying severe infection). Treatment with the mono-cationic salt of HBED accompanies the transfusion. Accepted indications for transfusions include cardiopulmonary symptoms (especially when the hemoglobin count is <5 gm/dL), cardiopulmonary signs (such as high output cardiac failure or hypoxemia) or for other life-threatening conditions which could be alleviated by an increase of oxygen in the blood (such as sepsis, severe infection, cerebrovascular accident, organ failure). Transfusions also reduce surgical morbidity for sickle cell anemia patients. Chronic transfusion therapy appears to limit recurrences of cerebrovascular bleeding and is particularly useful for young patients, under 18 years old, who have had strokes. Chronic transfusion therapy is usually continued for more than three years. Chronic transfusion therapy is also indicated where sickle cell anemia patients have recalcitrant leg ulcers and may be helpful during pregnancy. Similarly, thalassemia major patients are treated by blood transfusions. Chronic transfusion therapy is valuable in the suppression of abnormal hematopoiesis.

The use of a mono-cationic salt to prevent iron overload in chronic transfusion therapy is especially important in the treatment of young children with sickle cell anemia.

Transfusions are performed most often using red blood cells instead of whole blood. The red blood cell solution is more viscous than whole blood and is diluted with 0.9% sodium chloride prior to administration. The rate of transfusion should be one unit of red blood cells administered in not more than two hours. Prolonged transfusion increases the hazard of bacterial contamination. An 18-gauge needle, or larger, is desirable. Smaller needles, within limits, may be used for young people.

For a more complete reference to transfusion methods see The Merck Manual, 6^thEdition, pp. 1180.

Pharmaceutical Compositions of the Invention

Broadly, a composition of the invention is the combination of the mono-cationic salt of HBED with a pharmaceutical excipient that is suitable for I.V. administration. Generally, the compositions of the invention may fall into one of three (3) categories:

1. a solution that is ready for administration,

2. a dry soluble composition that is ready to be combined with a diluent just prior to use (ie., reconstitutable), and

3. a liquid concentrate ready for dilution prior to administration.

A reconstitutable composition is a sterile solid packaged in a dry form. A reconstitutable composition is preferred because it is more stable when stored as a dry solid rather than in a solution ready for immediate administration. The reconstitutable mono-cationic salts of HBED dissolve rapidly, e.g., in one minute or less. The dry solid is usually packaged in a sterile container with a butyl rubber closure to ensure the solid is kept at an optimal moisture range. A reconstitutable dry solid is formed by dry fill, spray drying, or freeze drying methods. Descriptions of these methods may be found in Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 1, pps. 215-227.

Generally a solution ready for intravenous injection will be a volume greater than 1 ml, preferably more than 10 ml but generally less than 100 ml, although the rate of administration must be carefully controlled by the physician administering the solution. Subcutaneous injection is generally limited to a volume of 2 ml, and intramuscular injection is normally limited to 3 ml. Intravenous injection preferably employs dilute aqueous solutions. The I.V. injection should be as close to isotonic as possible to minimize tissue damage and irritation, reduce hemolysis of blood cells, and prevent electrolytic imbalance that may occur during administration. However, it is not always essential that the I.V. injection be isotonic. Intravenous injection allows administration of a solution (along with a blood transfusion, for example) that is not isotonic as long as the administration is slow enough to allow dilution of the solution by the blood. Hypertonic solutions are often used in subcutaneous and intramuscular injections to facilitate absorption of drug due to the resulting local effusion of tissue fluids. However, the injection of hypertonic solutions may cause immediate or delayed pain.

The liquid intravenous compositions are generally dilute and the component present in the highest proportion is the vehicle. The vehicle normally has no therapeutic activity and is nontoxic, but presents the active constituent to the body tissues in a form appropriate for absorption. Absorption normally will occur most rapidly and completely when the mono-cationic salt of HBED is presented as an aqueous solution. Limits for particulate matter occurring in intravenous fluids, or large-volume injections used for single-dose infusions, are defined in the USP. Limits also apply to multiple-dose injections, small-volume injections, or injections prepared by reconstitution from sterile solids. The USP defines particulate matter as extraneous, mobile, undissolved substances, other than gas bubbles, unintentionally present in parenteral solutions. The total numbers of particles having effective linear dimensions equal to or larger than 10 μm and larger than 25 μm are counted. The intravenous fluid meets the requirements of the test if it contains not more than 50 particles per mL which are equal to or larger than 10 μm, and not more than 5 particles per mL which are equal to or larger than 25 μm in linear dimension. Preferably, the vehicle of greatest value for this intravenous composition is an aqueous vehicle comprising water that meets the USP specification for water for injection. Generally, water of suitable quality will either be prepared by distillation or reverse osmosis to meet these USP specifications. The appropriate specifications are spelled out in Remington: The Science and Practice of Pharmacy” 19th Ed. at pps. 1526-1528.

Additional substances may be included in the intravenous compositions of this invention to improve or safeguard the quality of the composition. Thus, an added substance may affect solubility, provide for patient comfort, enhance the chemical stability, or protect the preparation against the growth of microorganisms. Thus, the composition may include an appropriate solubilizer, substances to act as antioxidants and substances that act as a preservative to prevent the growth of microorganisms. These substances will be present in an amount that is appropriate for their function, but will not adversely affect the action of the composition as a treatment for iron overload. Examples of appropriate solubilizers include detergents such as Tween. Examples of appropriate antimicrobial agents include thimerosal, benzethonium chloride, benzalkonium chloride, phenol, methyl p-hydroxybenzoate and propyl p-hydroxybenzoate. Appropriate antioxidants may be found in Remington at p. 1529.

Because the mono-cationic salts are soluble and form a solution in the pH range of 7.0-8.0, no buffer is generally needed. If needed, the addition of a buffer to the intravenous composition prevents changes in pH of the composition that may occur during storage due to breakdown of components or interaction with the container or the atmosphere. The ideal buffer system should have a buffer capacity adequate to maintain the pH of the preparation at a stable storage value while permitting the preparation to adjust to the pH of the blood as the preparation is introduced into the body. Ideally, the pH of the buffer will be about pH 7.4, the pH of the blood. pH values that vary significantly from pH 7.4 may cause complications. Fluids that have a pH of above 9 can cause tissue necrosis when introduced into the body. Fluids with a pH of below 3 can cause extreme pain and phlebitis after administration. For intravenous preparations, a wider range of pH's are acceptable particularly if the solution is to be added to transfusion fluid. The pH of solutions to be administered by IV should fall within the range of 3.0-10.5. This increase in range is allowed because of the buffering capacity of the blood. A suitable buffer system can be designed for specific drugs from knowledge of the pH profile of the drug in solution. Other methods of administration, such as subcutaneous and intramuscular injections, require that the solution fall within a narrower pH range of 4.0 and 9.0.

Generally the sterile composition of this invention will comprise about 0.01% by wt. to about 50% by wt. of the mono-cationic salt of HBED, with the remainder being the appropriate excipient or excipients.

Method of Preparation

Another aspect of this invention flows from the discovery of the useful results obtained by intravenous administration of the drug. This aspect is a method of preparing a pharmaceutical composition suitable for intravenous administration which process comprises combining the mono-cationic salt of HBED with a pharmaceutical excipient suitable for intravenous administration under conditions that are effective in providing a sterile composition suitable for intravenous administration. Thus, in preparing the compositions of this invention, care must be taken to ensure the final composition is sterile and suitable for intravenous injection. The process will generally follow currently approved good manufacturing procedures (GMP) in order to result in the desired intravenous product. When employing aqueous vehicles, the USP water must be used for preparing the composition itself and preferably is used in preparing the equipment for preparing the composition. The general considerations for preparing intravenous preparations may be found in Remington: The Science and Practice of Pharmacy, 19th Ed. Chap. 87.

The following reaction sequence sets forth a representative method for making a mono-cationic salt useful in the invention. In the sequence, “M” represents a monovalent cation.

In reaction sequence I and throughout the specification, certain abbreviations are used as follows:



C	=	centigrade
DCM	=	dichioromethane (methylene chloride)
DMSO	=	dimethyl sulfoxide
EtOH	=	ethanol
gal	=	gallon
IPA	=	isopropanol
Kg	=	kilogram
L	=	liter
MeOH	=	methanol
mg	=	milligram
ml	=	milliliters
MOH	=	akaline hydroxide (M⁺= monocation)
NaBH₄	=	sodium borohydride
THF	=	tetrahydrofuran
TLC	=	thin layer chromatography

In the first Step A, ethylenediamine (available from Sigma, P.O. Box 14508, St. Louis, Mo. 63178) is reacted with 2 molar equivalents of o-hydroxybenzaldehyde (also available from Sigma as salicylaldehyde) in a suitable solvent such as an alkanol, particularly ethanol or EtOH/5% IPA, to form the compound of Formula (1). Generally this is carried out at a temperature of about 0° C. to about 20° C. with cooling, as the reaction is exothermic. Generally the pressure will be about atmospheric with the reaction taking place under an atmosphere of a non-reactive gas such as nitrogen. The reaction time will generally be about 5 to about 24 hours, at least about 16 hours.

In the second Step B, the compound of Formula (1) is reacted with a reducing agent to hydrogenate the double bonds to form the compound of Formula (2). Generally, this is done with sodium borohydride in a suitable solvent or solvent mixture. A suitable solvent is, e.g., a mixture of ethanol (e.g. denatured), methanol, and tetrahydrofuran (THF). Alternatively, a mixture of THF and EtOH/5% IPA may be used. The solvent is present in a quantity sufficient to dissolve compound (1) and the sodium borohydride. The ratio of THF to the other component will be about 2:1 to about 5:1. This step is carried out at a temperature of about 0° C. to about 50° C., preferably less than 30° C. Generally the pressure will be about atmospheric with the reaction taking place under an atmosphere of a non-reactive gas such as nitrogen. The reaction time will generally be about 10 to about 20 hours.

In Step C, once the compound of Formula (2) is prepared, it is reacted with at least 2 molar equivalents, e.g. 2.1, of tertiary butyl ester of a haloacetic acid, e.g. bromoacetic acid. This is done in the presence of a weak base such as potassium carbonate and an organic solvent such as DMSO. It may be useful to include t-butyl ammonium bromide. This results in the tertiary butyl ester of HBED that is shown as Formula (3) in reaction sequence I. This step is carried out at a temperature of about 10° C. to about 50° C. and atmospheric pressure. Other useful solvents include, e.g., chloroform. The reaction time will generally be about 10 to about 100 hours, preferably about 40 to 48 hours.

In Step D, the resulting ester is then hydrolyzed with a weak acid, such as formic acid, in a non-aqueous environment to give the compound identified as Formula (4), which is HBED itself. This step is carried out at a temperature of about 20° C. to about 65° C. and atmospheric pressure. The reaction time will generally be about 10 to about 100 hours, generally less than 50.

In Step E, HBED is then reacted with one molar equivalent of an organic or inorganic base to give the cationic salt, e.g. the monocationic salt shown as Formula (5) in Step E. The pharmaceutically acceptable monocationic salts prepared in accordance with the process of this invention are preparable from an inorganic or organic base. The salt derived from inorganic bases include, but are not limited to, the cations sodium, potassium, lithium, and ammonium. These are readily obtained from the corresponding hydroxides. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines; substituted amines including naturally-occurring substituted amines and cyclic amines. These include methylamine, ethylamine, isopropylamine, trimethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, N-alkylglucamines, theobromine, purine and the like. The mono-sodium salt and the monoammonium salt are the two compounds that are particularly preferred. This step is carried out at a temperature of about 2° C. to about 50° C. (preferably about 40-45° C.) and atmospheric pressure. The reaction time will generally be about 1 to about 5 hours, e.g. about 1.5 to 2.5 hours.

Article of Manufacture

Another aspect of this invention is an article of manufacture that comprises a container holding a composition of this invention, which is suitable for intravenous administration, in combination with printed labeling instructions providing instructions for how to administer the composition intravenously. A therapeutically-effective amount of the composition will be contained in any suitable container that will not significantly interact with the composition and will be labeled with the appropriate labeling that indicates it will be for intravenous use only. Associated with the container will be the labeling instructions for the use of the mono-cationic salt of HBED in treating the condition. The labeling will be consistent with the method of treatment as described hereinbefore. The container which holds the composition of this invention may be a container having a liquid composition suitable for injection which has an appropriate needle for injection and/or a syringe so that the patient, doctor, nurse, or other practitioner can administer the drug. The composition can be delivered by small volume injection or by infusion, with coinfusion during transfusion being particularly preferred. Alternatively, the composition in the container may be a dry composition containing the mono-cationic salt of HBED, to be combined with, e.g., an aqueous vehicle to dissolve the composition. Representative examples of a suitable container are described in Remington's Pharmaceutical Sciences, Eighteenth Edition, 1990, Mack Publishing Company, p. 1572-1576. Whatever the particular container is, the labeling is important to provide guidance for the method of treatment.

EXAMPLES

The following examples are given as nonlimiting representations of various aspects of the invention to enable one of ordinary skill in the art to make and use the invention. In the examples, deferoxamine B (DFO) in the form of the methanesulfonate salt (DFOM-trade name: DESFERAL) was manufactured by Novartis (Basel, Switzerland). HBED hydrochloride dihydrate was obtained from Strem Chemical Co. (Newburyport, Mass.). Cremophor RH-40 was obtained from BASF (Parsippany, N.J.). Sprague-Dawley rats were purchased from Charles River (Wilmington, Mass.). [0066] Cebus apella monkeys were obtained from World Wide Primates (Miami, Fla.). All reagents and standard iron solutions were obtained from Aldrich Chemical Co. (Milwaukee, Wis.).

Example 1

This example explains how to prepare and handle [0067] Cebus apella monkeys for testing to determine the effectiveness of the process and composition of this invention.
A. Iron Loading of [0068] Cebus apella Monkeys
After intramuscular anesthesia with ketamine, an intravenous infusion is started in a leg vein. Iron dextran is added to approximately 90 mL of sterile normal saline and administered to the animals by slow infusion at a dose of 200 to 300 mg iron per Kg body weight over 45 to 60 minutes. Two to three infusions, separated by between 10 and 14 days, may be necessary to provide about 500 mg iron per Kg body weight. After administration of iron dextran, the serum transferrin iron saturation rises to 70-80%. The serum half-life of iron dextran in humans is 2.5 to 3.0 days. At least twenty half-lives, sixty days, passes before using any of the animals in experiments evaluating iron-chelating agents. [0069]
B. Iron-balance Studies in [0070] Cebus apella Monkeys
Seven days before the administration of the drug, the animals are placed in metabolic cages and started on a low-iron liquid diet. The monkeys are maintained on the low-iron liquid diet for the duration of the experiment. They are given food according to their body weight, and intake is very carefully monitored. [0071]
Three days prior to drug administration, day −2 to day 0, baseline iron intake and output values are measured. This same measurement is made for day +1 to day +3. The total amount of iron intake is compared with the total amount of iron excreted. [0072]
C. Primate Fecal and Urine Samples [0073]
Fecal and urine samples are collected at 24 hour intervals. The collections begin four days prior to the administration of the test drug and continue for an additional five days after the drug is given. Fecal samples are assayed for the presence of occult blood, weighed, and mixed with distilled deionized water prior to autoclaving for 30 minutes. The mixture is then freeze-dried, and a known portion of the powder is mixed with low-iron nitric acid and refluxed for 24 hours. Once any particulate matter in the digested samples is removed by centrifugation, iron concentrations are determined by flame atomic absorption. Monkey urine samples are acidified and reconstituted to initial volume after sterilization, if necessary. [0074]

Example 2

This example explains how to make a mono-cationic salt of HBED. The formula numbers refer to the compounds in the reaction sequence set forth in the specification under the “Method of Preparation” section. [0075]
A. Mono-sodium HBED [0076]
(1) Ethylenediamine (MW=60.1, d=.899) (475 g, 528 ml, 7.91 moles) was added at a rapid dropwise rate from a constant pressure addition funnel to a solution of salicylaldehyde (MW=122.12, d=1.146) (1.93 Kg, 1.68 liter (L), 15.8 moles, 2.0 equiv.) over 1.15 hr. The exothermic reaction was kept below reflux using a water bath periodically. During the addition, another 300 ml of EtOH was added to facilitate stirring, which was continued overnight. The reaction mixture was cooled for 1 h in an ice bath and filtered to give yellow iridescent flakes and a brown solution. The solid product was washed with EtOH (1.2 L) and air dried to produce 2314 g of a compound of formula 1 (quantitive yield) (MW-268.32). [0077]
(2) To a stirred solution of the compound of formula (1) (2122 g, 7.91 moles) in 7 L of EtOH (SD 3) and 17 L of THF was added NaBH[0078] ₄(628 g, 16.6 moles, 2.1 equiv in 50 g portions). The addition required 6 hr. The reaction was allowed to continue overnight and then concentrated in vacuo to remove the solvents, the residue was dissolved in dichloromethane (DCM:17 L), washed with water (3 L), dried over NIC₂SO₄, and then filtered. Concentration in vacuo to approximately 4 L gave a heterogeneous mixture which was filtered and the resulting solid was washed with 1:1 DCM:hexane. It was washed with another 2 L of hot DCM. Hexane (2 L) was added to the DCM, and a precipitate formed that was filtered off. Hexane (6 L) was added to the mixture and a precipitate formed. The DCM/hexane mixtures were filtered, and all the resulting filtered solids dried to give 876 g (41% , 3.22 moles) of a white solid identified as formula 2, TL (9:1:0.3 DCM:MeOH:NEt₃) one spot, mp 122-125° C.
(3) Potassium carbonate (691 g, 5.0 moles, 3.0 equiv.) was added to a stirred solution of the compound of formula (2) (450 g, 1.64 moles) in DMSO (1.9 L). The mixture was cooled to 12°-14° C. using a water bath, and tert-butyl bromoacetate (683 g, 3.50 moles, 2.1 equiv.) was added over 30 min. After 2 days at room temperature, the reaction was poured into DCM (1.5 L). This was filtered and washed with water (7×1.5 L), dried (Na[0079] ₂SO₄), and filtered. The DCM was removed in vacuo to give a white foam. This was dissolved in warm (45° C.) benzene (375 mL). The solution was added to hexane (2.5 L) with efficient stirring. After 15-20 min., a precipitate formed. The solution was cooled in an ice bath for 2 hrs. The product was filtered, washed with hexane, air dried, and then dried in vacuo under high vacuum at 30-35° C. to give 450 g of a white solid identified as formula 3 (0.9 moles, 54%) mp=80-81° C.
(4) Cold formic acid (1 L) was added to the compound of formula 3 (450 g, 0.9 moles). After stirring at room temperature for 5 days, the reaction mixture was poured into diethyl ether (4 L) while swirling to give 2 layers. The top layer was decanted and discarded. The lower layer was poured into stirred diethyl ether (3 L), giving a white solid. The liquid phase was decanted, and the remaining solid was dissolved in absolute EtOH (1.5 L, no heat). This was poured into stirred diethyl ether (2.5 L) to again give a white solid and clear solution. After decanting the liquid, the solid was dissolved in absolute EtOH (1.6 L, no heat) and stirred overnight. The solution was poured into stirred diethyl ether (3.5 L). The resulting solid was filtered, washed with ether, then quickly transferred to a flask, and toluene (0.5 L) was added. This was removed in vacuo at 25° C. The white solid was dried overnight in a vacuum oven at 25° C. to give 145 g of a white solid. This solid was treated with acetonitrile (1.4 L) while being stirred, then water (140 mL) was added. The resulting heterogeneous mixture was stirred for 1 hr. and then filtered. The solid was washed with acetonitrile then DCM, giving 105 g of HBED after drying at 25° C. under high vacuum overnight. A portion of this material (94 g) this was treated with absolute EtOH (450 mL) and stirred for 4 h. It was filtered, washed with more EtOH (150 mL) and then dried overnight under high vacuum at 25° C., giving 89.5 g (0.23 moles, 26% yield) of HBED, shown as formula 4. [0080]
(5) HBED (89 g, 0.23 moles) was placed into a 1 L Erlenmeyer flask with a magnetic stir bar, and water (JT Baker, cat #4218-03,100 ml) was added. This heterogeneous solution was stirred and cooled in an ice bath for 10 min., then a solution of NaOH in water (13.29 g, 0.23 moles (prepared from Alfa Aesar cat #41281 and JT Baker water, 340 mL) was added dropwise over 30 min. After the addition was finished, pH=7.2. A precipitate began to form, which redissolved upon addition of more water (100 mL). Filtration through 0.45 micron filters (Whatman, PTFE), concentration of the filtrate in vacuo at 25-30° C., and further drying under high vacuum at room temperature for 2.5 days gave (91 g) white solid, confirmed to be HBED, monosodium salt, the compound of formula 5 where M is Na. [0081]
B. Other Mono-cationic Salts of HBED. [0082]
By following the procedure of part A of this example but substituting other bases for NaOH in step (5), other salts are obtained, such as: [0083]
mono-ammonium HBED, [0084]
mono-potassium HBED, [0085]
mono-lithium HBED, and the like. [0086]

Example 3

The monosodium salt of HBED in distilled H[0087] ₂O was given to the monkeys intravenously (IV) at a single dose of 75 μmol/kg (34.6 mg/kg n=4). In the iron loaded Cebus apella monkey, the IV push injection of HBED monosodium salt produced a net iron excretion (efficiency 12.2±1.4%, 70% of the iron was in the stool, 30% in the urine) that was nearly three times that observed after similar doses of subcutaneous DFOM. The drug has also been given as an IV bolus injection to conscious rats at a dose of 200-300 mg/kg with no apparent side-effects.

Example 4

Owing to the profound impact that DFO administered IV can have on blood pressure and cardiac function (Whitten, D. F.; Gibson, G. W.; Good, M. H.; Goodwin, J. F.; Brough, A. J. Studies in Acute Iron Poisoning. I. Desferrioxamine in the Treatment of Acute Iron Poisoning: Clinical Observations, Experimental Studies, and Theoretical Considerations. [0088] Pediatrics 1965, 36, 322-335), the manufacter recommends that the drug be given by this route at doses not to exceed 15 mg/kg/h (22 μmol/kg/h). In addition, patients suffering from acute iron toxicity should be given DFO IV only under circumstances of cardiovascular collapse, and even then at doses not to exceed 15 mg/kg/h for the first 1000 mg. Subject IV dosing, if needed, must be at a slower rate not to exceed 125 mg/h. Upon consideration of this, this example compares the impact of DFO and NaHBED administered IV on blood pressure and heart rate in normotensive rodents. The chelators were administered at a dose of 300 μmol/kg in a 0.5-ml volume either by IV bolus (n=5 for both drugs) or by IV infusion over a 20-min period (n=5 for NaHBED, n=4 for DFO) in accordance with the following procedure.
The basic procedure has been described in an earlier publication (Bergeron, R. J.; Wiegand, J.; Sninsky, C. A.; Katovich, M. J. The Impact of Polyamine Analogues on the Blood Pressure of Normotensive and Hypertensive Rats. [0089] Clin. Exp. Hypertension 1995, 17, 1197-1217). Briefly, normotensive adult male Sprague Dawley rats (400 g) were anesthetized with sodium pentobarbital (55 mg/kg IP). After the neck and shoulder areas were prepared for surgery, an incision was made into the neck area. The proximal end of the carotid artery was tied off with 3-0 silk; the other end was clamped. Once a piece of PE-50 tubing was inserted approximately 3 cm into the artery and tied into place; the tubing was skin tunneled to the shoulder area. The jugular vein was cannulated using a similar procedure. Both of the tubings were flushed with heparin (100 U/ml); a stylet was inserted.
Approximately 24 h after the surgery, the animals were placed into individual cages and were connected to a blood pressure transducer (ADInstrurnents, Inc., Milford, MA) via the carotid catheter. The blood pressure transducer and the MacLab Bio Amplifier were in turn connected to a Mac Bridge 4 (ADInstruments, Inc., Milford, Mass.), a computer-controlled tnansducer interface. The data were then transmitted to the MacLab 4e data acquisition system powered by a Macintosh Quadra 650. The MacLab Chart program was used to display and analyze the data. [0090]
After a ½-h stabilization period, 0.5 ml of saline was administered as an IV bolus via the jugular vein catheter, and the blood pressure and heart rate were monitored for 5 min. Then, the compound of interest was administered IV at a dose of 300 μmol/kg in a 0.5-ml volume of dH[0091] ₂O as a bolus (n=5 for both drugs). Once the drug administration was completed, additional saline was given to make sure that all of the drug had been delivered. The blood pressure and heart rate readings were taken for 1 h post-drug, during which the animals were unrestrained.
Rats receiving the drug as a 20-min IV infusion (n=5 for HBED, n=4 for DFO) were allowed to acclimate as above. Due to the dead space of the tubing, the infusion pump was allowed to run for approximately four minutes before starting the timer. The drug was then given for an additional 20 min and the pump was stopped; no additional saline was given as a flush. Blood pressure and heart rate were monitored during the infusion and for one hour thereafter. [0092]
When rodents were given 300 μmol/kg DFO as an IV bolus, there was a 25% decrease in blood pressure that did not return to baseline levels until 35 min post-drug (P<0.001 for t=5.5 to 15 min and P<0.005 for t=25 min). The heart rate in these animals also increased by 16% and likewise did not return to predrug levels until >35 min post-dosing (p<0.001 for t=5.5 to 35 min). When DFO was administered at 300 μmol/kg as a 20-min IV infusion, no effect on either blood pressure or heart rate was recorded. In contrast there was no effect on either blood pressure or heart rate in rats given the same dose of NaHBED as an IV bolus or as a 20-min IV infusion. The lack of an effect of NaHBED administered IV on either blood pressure or heart rate maims NaHBED an attractive therapeutic for the treatment of acute iron poisoning. [0093]

Example 5

This example provides modified method for making the mono-sodium salt of HBED. The compounds represented by formulae numbers (1)-(5) may be found in Reaction Sequence I of this specification. [0094]
A. One hundred twenty L of a mixture of EtOH with 5% IPA is charged to a 50 gal reactor. The reactor is purged with nitrogen and a nitrogen bleed is established for the rest of step A. The reactor is stirred at medium speed and 30 Kg of salicylaldehyde is charged to the reactor. The reaction mixture is then cooled to less than 10° C. The temperature is kept at less than about 20° C. while 7.38 Kg of ethylenediamine is charged to the reactor. The mixture in the reactor is stirred for at least 16 hours at room temperature. The reactor contents are centrifuged then washed with 20 L of EtOH/5% IPA. The material is collected and dried in a Stokes oven at 70° C.±5° C. with at least 26″ vacuum for at least 16 hours. This provides the product indicated in Reaction Sequence I as formula (1). [0095]
B. 274 L of THF is charged into a clean 100 gal reactor. The reactor stirring mechanism is started at a medium speed. 113 L of a mixture of EtOH with 5% IPA, along with 34 Kg of the product of Part A of this example is charged to the reactor. The reactor is then purged with nitrogen and nitrogen bleed is used throughout the following process. The reaction mixture is cooled to less than 10° C. and 10.110 Kg of sodium borohydride is charged to the reactor while keeping the temperature less than 30° C. The mixture in the reactor is stirred for at least 12 hours at room temperature and a sample is removed to determine if there is any starting material left in the reactor. Once the reaction is complete, the reactor contents are heated to reflux under vacuum and the solvent is stripped until 30 gal remain in the reactor. Then the vacuum is released and the reaction mixture is cooled to less than 30° C. 274 L of DCM are then added to the stirring contents of the reactor and half the reaction mixture is transferred to an adjacent reactor. 100 L of deionized water is added to each reactor and the resulting mixture in each reactor is stirred for at least 15 minutes. The stirring is stopped and the layers are allowed to settle for at least 15 minutes. The bottom organic layers are drained into a stainless steel tank and the aqueous layer is drained and discarded. 10 Kg of sodium sulfate is added to the organic layers in the stainless steel tank, the resulting mixture is stirred for at least 15 minutes and then allowed to settle for at least 15 minutes. The solid material is filtered on a ceramic funnel and the filtrate is returned to a reactor and washed with 20 L of DCM. The reactor contents are refluxed at atmospheric pressure and the solvent is stripped to one-half the original volume. The mixture is then cooled to less than 25° C. and the material is filtered on a ceramic funnel sending the filtrate to an adjacent reactor A. The solid obtained in the previous step is suspended in 70 L of DCM. The DCM mixture in the reactor is stirred at medium speed for at least 12 hours and the resulting material is filtered on a ceramic funnel and collected for the next step. The filtrate is set aside in a vessel B. The resulting material is shown as Formula (2) in Reaction Sequence I. The filtrate collected previously in reactor A is then mixed with 120 ml of hexane and the resulting mixture is stirred for at least 3 hours. The resulting solid material is filtered on a ceramic funnel and collected for the next step, also as Formula (2). To the filtrate obtained in vessel B 70 L of hexane is added the mixture in the reactor is stirred for at least 2 hours, filtered on a ceramic tunnel collected and packaged. This is further material shown as Formula (2) in Reaction Sequence I. [0096]
C. 128 L of chloroform is charged into a clean 100 gal reactor. The stirring speed is kept at 60-90 rpm for this step. 16.3 Kg of the product identified as Formula (2) is added to the reactor. The reactor is heated to about 45° C. Keeping the temperature between 45° C. and 50° C., 25.5 Kg of potassium carbonate is added to the reactor. The resulting reaction mixture is cooled to 40-45° C. and 2.65 Kg tetrabutyl ammonium bromide is added. The reaction mixture is cooled to 35-40° C. and stirring is continued. 25.5 Kg of t-butyl bromoacetate is then added to the reactor and the mixture in the reactor is allowed to stir for at least 48 hours at 35-40° C. Samples are removed from the reactor to determine if any starting material remains. Stirring is continued until no more starting material is detected. The reaction contents are centrifuged and washed with 20 L of chloroform. The filtrate is returned along with the wash to a 100 gal reactor. The reactor is then stirred and charged with 60 L of deionized water and stirred for at least 15 minutes. The stirring is stopped, the layers are allowed to separate for at least 15 minutes, and the bottom organic layer is drained into a tank. The aqueous layer is drained into tanks and discarded. Sodium sulfate (3.2 Kg) is then charged to the tank containing the organic layer and stirred for at least 15 minutes and then filtered on a ceramic funnel. The filtrate is send to a reactor which is then heated under vacuum of at least 20″ and the solvent is stripped to an oily material keeping the temperature less than about 40° C. The vacuum is released and the reaction mixture is cooled to 20-30° C. 86 L of hexane are added to the stirring contents of the reactor to form solid material that settles out. The reactor contents are centrifuged and washed with about 10 L of hexane. The solid product is collected and dried in a Stokes oven at 60±5° C. with at least a 26″ vacuum for at least 16 hours. This provides the di-t-butyl ester shown as Formula (3) in Reaction Sequence I. [0097]
D. Into each of 2 clean 50 L flasks, 25.75 L of formic acid is charged. Moderate stirring is started in the flask and continued while 12.9 Kg of the t-butyl ester of HBED from the previous is added. Each flask is heated to 40±5° C. and the temperature is kept between 40-45° C. for at least 48 hours with continued stirring. A sample is removed from the reactors to determine if there are any starting materials available. Once there are none, the reaction mixture is cooled to less than 25° C. 245 L of di-isopropyl ether is then charged into a 100 gal tank. The di-isopropyl ether is stirred and the contents of each 50 L flask is added to the 100 gal tank. The mixture in the tank is stirred for at least 20 minutes then allowed to settle for at least 30 minutes. The solvent layer (the upper layer) is removed and discarded. Next, 86 L of a mixture of EtOH/5% IPA is added to the tank. The mixture is stirred until fully dissolved, then 135 L of di-isopropyl ether is added and stirred for at least 30 minutes. After settling, the material is filtered on a ceramic funnel and washed with 20 L of di-isopropyl ether. 49 L of a mixture of EtOH and IPA is charged into a 65 gal tank. The material in the 65 gal tank is stirred and the solid from the ceramic funnel is charged into the tank the mixture is stirred until dissolved. 74 L of di-isopropyl ether is added to the stirring contents of the 65 gal tank and the resulting solid material is filtered on a ceramic funnel and washed with 10 L of di-isopropyl ether. The sample, if more than 94% pure, is collected and dried in a Stokes oven at 60°±5° C. with at least a 26″ vacuum for at least 16 hours. This results in HBED shown as Formula (4) in the Reaction Sequence. [0098]
E. 10 L of pyrogen-free water (Water For Injection—WFI) is added into a clean pre-rinsed 50 L flask. The contents are stirred at about 80-120 rpm and 3.00 Kg HBED is added to the stirred contents of the flask. A mixture of 309 g of sodium hydroxide and 8.00 L of WFI water is prepared to form a sodium hydroxide solution. Over the course of 1.5 to 2.5 hours the sodium hydroxide solution is charged to the flask keeping the temperature below 25° C. Once a pH range of 7.1 to 7.3 is reached, the mixture in the flask is allowed to stir for at least 15 minutes and water is added until all material goes into solution. The solution is sent through a Whatman PTFE 0.45 micron disposable cartridge filter into a 5 g glass carboy. The solution is lyophilized using 1.5 L solution per tray. The material is frozen at −20° C., lyophilized at 0° C., and dried at 20° C. Each batch is dried to less than 5% water, as determined by the Karl Fisher technique. This provides HBED as the mono-sodium salt shown in as Formula (5) is the reaction sequence. This material is shown to be a hydrate. [0099]
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [0100]
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. [0101]

Claims

The subject matter claimed is:

1. An aqueous pharmaceutical composition suitable for intravenous injection in a human comprising an aqueous solution of a therapeutically effective amount of a mono-cationic salt of N,N′-bis(2-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid.

2. The composition of

claim 1

, wherein the mono-cationic salt is the mono-sodium salt.

3. The composition of

claim 1

, wherein the mono-cationic salt is the mono-ammonium salt.

4. The composition of

claim 1

, wherein the pharmaceutical composition is a unit dosage of about 1 ml to about 100 ml in total volume.

5. The composition of

claim 1

, wherein the pharmaceutical composition is unsaturated to prevent crystallization of the salt from the solution.

6. The composition of

claim 1

, wherein the salt in the pharmaceutical composition is present at a concentration of about 1% to about 30% w/v.

7. The composition of

claim 1

, which has a pH of about 7.0 to about 8.0.

8. The composition of

claim 1

, which additionally contains at least one of an antioxidant or an antimicrobial agent.

9. A dry pharmaceutical composition suitable for reconstitution with water to form an aqueous solution suitable for intravenous injection in a human, which composition comprises a mono-cationic salt of N,N′-bis(2-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid.

10. The composition of

claim 9

, wherein the mono-cationic salt is the mono-sodium salt.

11. The composition of

claim 9

, wherein the mono-cationic salt is the mono-ammonium salt.

12. The composition of

claim 9

as a unit dosage form, wherein the mono-cationic salt is present in an amount sufficient to make an unsaturated aqueous solution of the salt having a volume of about 1 milliliter to about 100 milliliters.

13. The composition of

claim 9

, which, when reconstituted with water, forms a solution having a concentration of about 1% to about 30% w/v.

14. The composition of

claim 9

15. A method of treating a human having a condition that is treatable by an iron chelator, which method comprises intravenously administrating a composition comprising a therapeutically effective amount of a mono-cationic salt of N,N′-bis(2-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid.

16. The method of

claim 15

, wherein the condition is iron overload.

17. The method of

claim 16

, wherein the condition is primary hemochromatosis.

18. The method of

claim 16

, wherein the condition is secondary hemochromatosis.

19. The method of

claim 16

, wherein the condition is focal hemosiderosis.

20. The method of

claim 16

, wherein the condition is transfusional iron overload.

21. The method of

claim 20

, wherein the human has thalassemia major.

22. The method of

claim 20

, wherein the human has sickle cell anemia.

23. The method of

claim 22

, wherein the aqueous solution is administered to a sickle cell anemia patient during the time the patient is receiving a blood transfusion.

24. The method of

claim 20

, wherein the human has myelodysplasia.

25. The method of

claim 24

, wherein the aqueous solution is administered to a myelodysplasia patent during the time the patient is receiving a blood transfusion.

26. The method of

claim 15

, wherein the composition is an aqueous solution.

27. The method of

claim 15

, wherein the composition is administered to a patient receiving a blood transfusion during the time period when the transfusion is being received by the patient.

28. The method of

claim 27

, wherein the composition is administered to the patient along with the blood transfusion.

29. An article of manufacture that comprises a container containing a pharmaceutical composition comprising a mono-cationic salt of N,N′-bis(2-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid, wherein the container holds a therapeutically-effective amount of the compound and is associated with printed labeling instructions for intravenous administration of the composition to treat a condition in a human that is treatable by an iron chelator.

30. The article of manufacture of

claim 29

, wherein the mono-cationic salt is the mono-sodium salt.

31. The article of manufacture of

claim 29

, wherein the mono-cationic salt is the mono-ammonium salt.

32. The article of manufacture of

claim 29

33. The article of manufacture of

claim 29

, wherein the composition is dry and when reconstituted with water forms an aqueous solution.

34. The article of manufacture of

claim 29

, wherein the composition is dry and when reconstituted with water forms a solution having a concentration of about 1% to about 30% w/v.

35. The article of manufacture of

claim 29

36. The article of manufacture of

claim 29

, wherein the condition is primary hemochromatosis, secondary hemochromatosis, or focal hemosiderosis.

37. The article of manufacture of

claim 29

, wherein the condition is transfusional iron overload caused by transfusion treatment of sickle cell anemia, thalassemia major or myelodysplasia.

38. The article of manufacture of

claim 29

, wherein the pharmaceutical composition is an aqueous solution in which the salt is present at a concentration of about 1% to about 30% w/v.

39. A process for preparing a pharmaceutical composition suitable for intravenous injection and treatment of a condition in a human treatable by an iron chelator which process comprises dissolving a mono-cationic salt of N,N′-bis(2-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid in water.

40. The process of

claim 39

, wherein the mono-cationic salt is the mono-sodium salt.

41. The process of

claim 39

, wherein the mono-cationic salt is the mono-ammonium salt.

42. The process of

claim 39

43. The process of

claim 39

, wherein the pharmaceutical composition has a pH of about 7.0 to about 8.0.

44. The process of

claim 39

, wherein the pharmaceutical composition is unsaturated to prevent crystallization of the salt.

45. The process of

claim 39

46. A process for preparing a dry, pharmaceutical composition that is reconstitutable with water and that forms a solution in water suitable for intravenous injection for treatment of a condition in a human treatable with an iron chelator, which process comprises forming essentially pure mono-cationic salt of N,N′-bis(2-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid and optionally adding an antioxidant or an antimicrobial agent.

47. The process of

claim 46

, wherein the mono-cationic salt is the mono-sodium salt.

48. The process of

claim 46

, wherein the mono-cationic salt is the mono-ammonium salt.

49. The process of

claim 46

, wherein the pharmaceutical composition is present in an amount sufficient to make an unsaturated aqueous solution of about 1 milliliter to about 100 milliliters.

50. The process of

claim 49

, wherein the resulting aqueous solution has a pH of about 7.0 to 8.0.

51. The process of

claim 49

, wherein the resulting aqueous solution has a concentration of about 1% to about 30% w/v.