PHARMACEUTICAL COMPOSITION COMPRISING AN IRON CHELATOR

Description

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions comprising iron chelating agents. More in particular, it relates to compositions which are suitable for parenteral administration and capable of providing slow and sustained release of an incorporated active compound. In a further aspect, the invention relates to drug carriers which may be incorporated in pharmaceutical compositions to effect sustained release, and which are particularly suitable for the delivery of iron chelating agents. The invention further addresses methods for preparing such compositions, powders and kits from which the composition may be reconstituted, and the uses of the compositions.

BACKGROUND

Iron chelators have been used in diagnosis and therapy for many years. They can be classified using a number of criteria such as their origin (synthetic versus biologically produced molecules), their interaction with solvents such as water (hydrophobic versus hydrophilic) or their stoichiometric interaction. An example of an iron chelator used in therapy is desferrioxamine. For example, the drug product Desferal ^® comprises desferrioxamine in the form of its mesylate salt.

Desferrioxamine is used in the treatment of chronic iron overload, such as in transfusional haemosiderosis, thalassaemia major, sideroblastic anaemia, autoimmune haemolytic anaemia, and other chronic anaemias, idiopathic (primary) haemochromatosis in patients in whom concomitant disorders preclude phlebotomy, iron overload associated with porphyria cutanea tarda in patients unable to tolerate phlebotomy. Moreover, desferrioxamine is indicated for treating acute iron poisoning, chronic aluminium overload in patients with end-stage renal failure with aluminium-related bone disease, dialysis encephalopathy or aluminium-related anaemia.

More recently, it was discovered that iron chelators may be useful for preventing neuronal degeneration, treating neuronal injuries and supporting neuronal regeneration.

Ben-Shachar D. et al. (J. Neurochem., vol. 56, 1991, 1441-1444) discloses retardation of 6-hydroxydopamine-induced degeneration of nigrostriatal dopamine neurons by administration of the iron chelator desferrioxamine (Desferal).

To avoid the need of direct injection into the brain for the treatment of neurodegenerative diseases and in particular Parkinson's disease, novel iron chelating agents were developed and disclosed e.g. in WO 00/74664, which were believed to have a substantially increased lipophilicity. Based on the modified physicochemical properties of the new compounds, it was suggested that these may be able to cross the blood-brain barrier, so that they penetrate into the brain tissues after intravenous administration, and thus enable a more feasible and convenient therapeutic regimen. On the other hand, it is well known that the development of new therapeutic entities is always challenging, time- and cost consuming and, most significantly, associated with substantial clinical risks. In effect, very few of the novel drug candidates with a promising

pharmacological profile in vitro or even in preclinical studies actually prove to be safe and effective in subsequent clinical studies. With respect to the compounds disclosed in WO 00/74664, it is not known whether they are indeed clinically effective in the treatment of Parkinson's disease or any other condition associated with either neuronal degeneration or with the need of neuronal regeneration.

It is further postulated that the beneficial effect of iron chelators in neuronal regeneration may be related to their ability to inhibit or delay the formation of scars. These clinical applications of iron chelator are disclosed, for example, in WO 98/51708 and in EP 0 878 480. Administration via any oral or injectable route including direct local injection into the affected tissue is suggested.

In order to use iron chelators for new therapeutic applications, it is desirable that they are provided in formulations which can be directly administered into the affected tissues and regions, and which have a prolonged effectiveness, preferably much longer than that of the known and marketed aqueous solutions, in order to avoid frequent administration into sensitive tissues or organs.

A few efforts to develop sustained release formulations of iron chelators are known. For example, Lowther N. et al. (Drug Dev. Ind. Pharm., vol. 25, iss. 11, 1999, 1157-1166) describe oil-based, injectable depot formulations of desferrioxamine-B n-decanesulfonate. The oily carrier liquid, in which the active ingredient is incorporated in suspended form, comprises sesame oil, ethyl oleate, and typically at least one surfactant. The release profiles of the compositions demonstrate that all of the desferrioxamine-B n-decanesulfonate is in sustained release form. On the other hand, the surfactant in the compositions, such as Tween ^® 80 and Span ^® 85, may cause tissue irritation. Furthermore, it appears that the in vitro release of the active compound from

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the suspensions extended over a period of about 12 hours, which is probably not long enough for avoiding repeated parenteral administration.

Schlicher, E.J. et al. (Int. J. Pharm. vol. 153, 1997, 235-245), describe microparticle compositions of desferrioxamine. The microparticles were made from polylactide-co-glycolide, a hydrophobic biodegradable polymer. However, the drug load capacity of the described microparticles is too low to accomodate a dose of the active compound which would be effective over a period of at least several days. Moreover, polylactide-co-glycolide is known to degrade into acidic reaction products that may render the microclimate within the microparticles substantially acidic, which may lead to the degradation of the acid-labile desferrioxamine.

US 2005/175684 Al discloses targeted liposomal compositions of iron chelators such as desferrioxamine. The compositions are capable of providing sustained release and prolonged activity. They are prepared by combining the iron chelator, one or more vesicle-forming lipids, and at least one targeting agent in such a way that the iron chelator is liposomally encapsulated. Subsequently, the liposomal delivery system is purified from any non- liposomally encapsulated iron chelator by a separation method such as centrifugation or dialysis, so that the composition comprises the drug substance in liposomal form only. A drawback of these liposomes is that the industrial manufacture of sterile lipid vesicles carrying targeting ligands on their surface is not well-established and expected to be very costly. Furthermore, the preferred targeting ligands, especially peptides and proteins, are often immunogenic and thus capable of inducing adverse reactions in patients.

Similarly, Postma N. S. et al. (J. Control. ReI., vol. 58, 1999, 51-60) have prepared and tested liposomally encapulated desferrioxamine. Two types of liposomes are compared, of which the first type represents "fluid" liposomes at

body temperature, whereas the second type of liposomes is described as "rigid". In both cases, the liposomes comprising desferrioxamine are first prepared by a reversed-phase evaporation method and then separated from non-liposomal drug by repeated ultracentrifugation and washing. While some sustained release effect has been demonstrated through biodistribution studies in mice, the suitability and therapeutic effectiveness of the compositions has not been established.

US 5,043,166 describes liposomes loaded with a cytostatic agents from the class of anthracycline glycoside antibiotics such as doxorubicin, which is known to be a highly cardiotoxic compound. While liposomal encapsulation by itself already reduces the cardiotoxicity of the drug substance, the document suggests that a further reduction may be achieved by the co-encapsulation of a lipophilic free radical quencher and a water soluble iron chelator such as (des)ferrioxamine. It is hoped that the auxiliary compounds will reduce peroxidative lipid damage through the drug substance in vivo. The document further teaches the preparation of the respective liposomes, which is conducted using the traditional film method. After the preparation, the liposomes are sized by extrusion through filter membranes, and the non- encapsulated active ingredient is removed by centrifugation, ultrafiltration, gel filtration, or treatment with an ion exchange resin with the effect that the final composition comprises only encapsulated active ingredients.

Similarly, US 5,023,087 discloses liposomes providing sustained release for various types of active compounds. Again, the additional incorporation of an iron chelator such as desferrioxamine as a protective agent is suggested in order to increase the tolerability of the composition by reducing some of its toxic effects. Moreover, the liposomes are adapted for intramuscular and/or subcutaneous administration. The preparation of the liposomes is similar to that taught in US 5,043,166. The preparation method is designed to provide drug- and protective agent-loaded liposomes which are purified from non-

encapsulated active agents and which release the incorporated compounds slowly after injection.

Thus, there remains a need for improved compositions for the parenteral administration of iron chelating agents.

It is therefore an object of the invention to provide pharmaceutical compositions suitable for the delivery of iron chelators which do not have the disadvantages and limitations of the known compositions. Another object is to provide compositions incorporating iron chelating agents which are suitable for parenteral administration, and which are useful for the treatment of injured nervous tissue, without requiring repeated administration. Other objects are directed to the preparation of such compositions and to powders and kits which may be reconstituted to obtain the compositions prior to their use. Further objects will become apparent on the basis of the following description and claims.

SUMMARY OF THE INVENTION

According to the invention, a pharmaceutical composition for parenteral administration is provided which comprises an active compound selected from the group of physiologically acceptable iron chelating agents, wherein at least a fraction of the active compound is incorporated in sustained release form. The composition is further characterised in that is comprises a second fraction of the active compound which is incorporated in immediate release form. In another embodiment, the composition is further characterised by high viscosity and its viscoelastic behaviour at room temperature

One of the preferred iron chelators is desferrioxamine (DFO), which is known to be an effective iron chelator in human therapy, and which has been used to

treat iron poisoning. Another preferred iron chelator is desferasirox. Desferrioxamine and desferasirox may be incorporated in form of a pharmaceutically acceptable salt, such as the methane sulfonate in the case of desferrioxamine.

At least a fraction of the iron chelator comprised in the composition is incorporated in sustained release form. According to one of the preferred embodiments, another fraction of the iron chelator is present in immediate release form. The sustained release characteristics may be achieved by incorporating the respective drug fraction within sustained release particles, such as polymeric microparticles or lipid particles.

A feature of the composition in one of the preferred embodiments is its relatively high viscosity, which is substantially above that of typical aqueous compositions for parenteral administration. The high viscosity may be useful to increase the residence time of the composition at the site of administration, and thus enhance its effectiveness.

The composition is particularly useful for administration directly into nervous tissue, such as intralesional, intraneural, intraspinal, intrathecal, or intramedullary administration. One of the preferred uses is the administration into injured nerves to promote tissue regeneration or delay scar formation.

For stability reasons, it may be preferred to provide and store the composition in dry form, such as a lyophilised powder. From such powder, it may be reconstituted with a suitable liquid carrier prior to administration. The powder and, optionally, the liquid carrier may be part of a pharmaceutical kit.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a spinal cord lesion of a rat after 7 days of treatment with placebo (a), a composition having low viscosity and no visco-elastic behavior (b) and composition of the invention (c) containing a fraction of the active compound, incorporated in sustained release form, and a second fraction of the active compound incorporated in immediate release form and having high viscosity and a viscoelastic behaviour.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a pharmaceutical composition for parenteral administration which comprises an active compound selected from the group of physiologically acceptable iron chelating agents. At least a fraction of the active compound is incorporated in sustained release form. The composition further comprises a second fraction of the active compound which is incorporated in immediate release form. In another embodiment, the composition is further characterised on its high viscosity and its viscoelasticity at room temperature. Dynamic viscosity is the commonly used form of viscosity, often abbreviated to just viscosity. The units are either the SI units of pascal seconds (Pa s) or the poise (P).

Thixotropic liquids exhibit a time-dependent response to shear strain rate over a longer period than that associated with changes in the shear strain rate. They may liquefy on being shaken and then solidify (or not) when this has stopped.

The methods to measure and determine viscosity, viscoelasticity and thixotropic behaviour by rotation rheometers like ARES - AR Rheometers are

described in detail e.g. in Physical Pharmacy by Alfred N. Martin, James Swarbrick, and Arthur Cammerata. S. R. van Tomme et al. describe a method of measuring rheological properties viscosities, which can be used in this invention (Biomaterials 26 (2005), 2129-2135). The respective disclosure is incorporated by reference.

In a further aspect, a pharmaceutical composition is provided which is obtainable by a method comprising the formation of a liquid or gel-type aqueous suspension of sustained release particles loaded with an active compound selected from the group of physiologically acceptable iron chelating agents. The method is further characterised in that a step of removing dissolved active compound which is not incorporated in, or associated with the sustained release particles from the loaded sustained release particles is absent.

As used herein, an active compound is a chemical or biological substance or mixture of substances which is useful for the diagnosis, prevention or treatment of diseases, symptoms, and other conditions of the body, or for influencing a body function. The composition of the invention comprises at least one, and optionally two or more of such active compounds. Other expressions which may be used as synonyms for active compounds are drugs, drug substances, active ingredients, active agents and the like.

The active compound or, if more than one active compounds are present, at least one of the active compounds, is a physiologically acceptable iron chelating agent. Physiologically acceptable primarily means that the iron chelator is safe and tolerable in view of the intended use. It does not mean that the compound is free of side effects, but that the side effects appear acceptable in view of the benefits a patient may receive from the compound.

A chelating agent, or chelator, is a substance whose molecules are capable of forming complexes such as chelates with partner molecules. Iron chelators are particularly capable of chelating iron ions - often also of other metal ions - and thereby reducing the activity of the iron. For example, iron chelators such as desferrioxamine can be used to reduce the toxicity of iron in the event of iron poisoning.

Several iron chelators are known and have been suggested or used for therapeutic purposes. For example, the iron chelator may be selected from the group of inhibitors of the enzyme prolyl-4-hydroxylase; N-oxaloglycine; pyridine derivatives, such as 5-arylcarbonyamino- or 5-arylcarbamoyl- derivatives, 2-carboxylate, 2,5 dicarboxylate, their ethyl esters or ethyl amides or -5-acyl sulfonamides, 2,4 dicarboxylate, their ethyl esters or ethylamides, or dimethoxyethylamides; 3,4 bipyridine, such as 5 amino-6- (lH)-one, l,6-dihydro-2-methyl-6-oxo-5-carbonitril; 2,2'-bipyridine, such as 5,5'-dicarboxylic acid or its pharmaceutically acceptable salts, 4,4'-dicarboxylic acid ethyl ester or ethyl amide; 3,4'-dihydroxybenzoate, such as the diethyl ester; proline and its structural and functional analogues; beta - aminopropionitrile; desferrioxamine; desferasirox; anthracyclines; 2,7,8- trihydroxy anthraquinones, fibrostatin-C; coumalic acid or its pharmaceutically acceptable salts; and 5-oxaproline.

Recently, it was discovered that iron chelators may be useful for treating neuronal injuries and supporting neuronal regeneration. It is believed that their beneficial effect in this therapeutic indication is related to their ability to inhibit or delay the formation of scars. It is further assumed at present that the inhibitory effect of iron chelators on scar formation may be mediated by the inhibition of iron-dependent enzymes involved in the formation of collagen, in particular of prolyl-4-hydroxylase. The use of iron chelators for treating neuronal injuries is disclosed, for example, in WO 98/51708, the teachings of which are fully incorporated herein by reference.

In one of the preferred embodiments, the iron chelator is desferrioxamine, or a salt, derivative, isomer, or solvate thereof. In another preferred embodiment, the iron chelator is desferasirox, or a salt, derivative, isomer, or solvate thereof. Desferasirox is 4-[3,5-Bis (2-hydroxyphenyl)-lH-l,2,4- triazole-1-yl] benzoic acid. Desferrioxamine (N-[5-(3-[(5-aminopentyl)- hydroxycarbamoyl]-propionamido)pentyl]-3-([5-(N-hydroxyaceta mido)- pentyl]-carbamoyl)-propionohydroxamic acid) has multiple carbonyl and hydroxyl groups that provide electrons to coordinate with those in Fe ³⁺ . The compound chelates iron in a one-to-one ratio. The affinity of desferrioxamine to divalent ions such as Fe ²⁺ is substantially lower.

Desferrioxamine (mono)methane sulphonate, also referred to as desferrioxamine mesylate, is available in form of the product Desferal ^® for the treatment of chronic iron overload, such as in transfusional haemosiderosis, thalassaemia major, sideroblastic anaemia, autoimmune haemolytic anaemia, and other chronic anaemias, idiopathic (primary) haemochromatosis in patients in whom concomitant disorders preclude phlebotomy, iron overload associated with porphyria cutanea tarda in patients unable to tolerate phlebotomy. Moreover, desferrioxamine is indicated for treating acute iron poisoning, chronic aluminium overload in patients with end-stage renal failure with aluminium-related bone disease, dialysis encephalopathy or aluminium- related anaemia.

Preferably, desferrioxamine methane sulphonate is selected as iron chelating agent according to the invention. Also preferred are other salts of desferrioxamine, such as desferrioxamine chloride, sulfonate, sulfate, mesylate, n-decanesulfonate, and embonate.

Desferrioxamine methane sulphonate is very hydrophilic, highly water-soluble and poorly lipid-soluble. It is rather unstable in acidic aqueous solutions. In

humans, its apparent half life in the bloodstream after intramuscular injection is initially about 2.4 hours, with a terminal half-life of about 6 hours. It is poorly absorbed after oral administration, as characterised by an oral bioavailability of only about 2%.

If desferrioxamine desferrioxamine methane sulphonate is selected as iron chelator, its content in the composition of the invention is preferably at least about 0.1% (w/w), such as from about 0.1% (w/w) to about 50% (w/w). More preferably, it is at least about 1% (w/w), such as from about 1% (w/w) to about 30% (w/w). Another presently preferred content is at least 5% (w/w), such as from about 5 to about 25% (w/w), and such as about 20% (w/w).

Optionally, the composition may comprise one or more further active ingredients. If another active compound is present, it is preferred that such further compound is selected from the group of inhibitors of collagen synthesis, antibodies with affinity for collagen IV, laminin, and/or entactine; compounds stimulating the growth of nervous tissue, chemokines from the SDF-I family, in particular SDF- lγ. Such compounds may have a potential for producing similar, complementary or synergistic effects to those of the iron chelator, and the respective drug combinations could be useful e.g. in the treatment of nervous tissue injuries, as is one of the preferred uses of the composition of the invention.

The composition is adapted for parenteral administration. As used herein, parenteral administration includes any invasive route of administration, such as subdermal, intradermal, subcutaneous, intramuscular, locoregional, intratumoral, intraperitoneal, interstitial, intralesional, intravenous, intraarterial, intraneural, intraspinal, intrathecal, or intramedullary administration. Moreover, administration may be by injection, infusion, insertion, or implantation. A preferred mode of administering the composition

of the invention is by injection into nervous tissue, such as intralesional, intraneural, intraspinal, intrathecal, or intramedullary injection.

At least a fraction of the iron chelator incorporated in the composition, and optionally all of the iron chelator, is in sustained release form. More preferably, the fraction which is in sustained release form is less than the total incorporated dose of the iron chelator. Sustained release is understood herein as any type of slow release, often also referred to as controlled release, prolonged release, extended release, delayed release, or the like. The profile of drug release over time may be linear or non-linear, such as faster in the beginning and slower towards the end of the release period, or vice versa.

Being in sustained release form means that the respective fraction of the iron chelator is formulated and/or processed in such as way that it is released slowly after administration. The sustained release characteristics may be achieved by generally known formulation methods and techniques, some of which will be described further below.

In one of the preferred embodiments, the iron chelator which is in sustained release form is released from the composition over a period of at least about 24 hours after administration. More preferably, the sustained release dose fraction is released over at least about 48 hours, or over at least about 72 hours. If the composition is intended for the treatment of injured nerves, and if it is intended that a single administration is used to achieve the desired therapeutic effect, it is preferred that the iron chelator is released over at least about 4 days, or at least about 5 days or even one week, depending on variable factors such as the size of the lesion and the number of injection sites per lesion. In further embodiments, the release profile is adapted to provide release over at least about 2 weeks, 3 weeks, or even 4 weeks. Without wishing to be bound by theory, it is believed that the sustained release of the iron chelator may or should be adapted to achieve a sufficient delay of scar

formation that the regeneration and growth of the nervous tissue at the lesion is not inhibited.

The release of the iron chelator is not easily determined in vivo. Therefore, the preferred duration of drug release refers to the release profile as measured in vitro by an appropriate method and at 37 ⁰ C, using an appropriate medium such as buffer or water for injection. For example, the following method may be used to determine the in vitro release from a composition comprising sustained release particles:

A sample of 2 g of the composition is diluted with 18 g water for injection. The resulting dispersion is washed by diafiltration with a six-fold (120 g) quantity of water for injection, resulting in the removal of about 99.5% of the free - i.e. not particle-associated or encapsulated - active ingredient. Of the washed dispersion, 10 ml are withdrawn, filled into a vial, and incubated at 37 ⁰ C. The vials are sampled and analysed for free and particle-associated or encapsulated active ingredient.

The inventors have further discovered that, in addition to providing a fraction of the iron chelator in sustained release form, it is highly beneficial to also provide some of the active compound in immediate release form. Immediate release is, in the context of the present invention, to be understood as release within the first 12 hours of administration, and more preferably as release within the first 6 hours of administration, or even within the first 2 hours. In particular, a fraction of an active compound is in immediate release form if it is dissolved in a coherent aqueous liquid phase of the composition which is administered, and not encapsulated in or associated with sustained release particles such as liposomes. Such fraction of active compound in immediate release form is typically present if a composition comprising sustained release particles loaded with active compound is prepared without any step of removing active compound which is not encapsulated in, or associated with

the particles from the loaded particles, such as by repeated washing and centrifugation, filtration, dialysis, or chromatography. Alternatively, it is possible to obtain a composition comprising an immediate release fraction by dissolving active compound in a composition which already comprises sustained release particles loaded with active compound, or by combining sustained release particles loaded with active compound with a solution of the active compound. For the avoidance of doubt, a single centrifugation step for the purpose of concentrating the composition and/or the sustained release particles comprised therein does not represent a step of removing or separating dissolved active compound.

According to one of the embodiments, the composition comprises at least about 5% (w/w) and up to about 80% (w/w) of the incorporated active compound in sustained release form, and consequently at least about 20% (w/w) and up to about 95% (w/w) in immediate release form. In another embodiment, the weight ratio of the sustained release fraction to the immediate release fraction is up to about 1 : 1.

Another feature of a preferred embodiment is that the composition is adjusted to have a viscoelasticity at room temperature (preferably about 20 ⁰ C). Viscoelasticity is the capability of hydrocolloids to form gels of various strengths, dependent on their structure, concentration, ionic strength, pH and temperature. The combined viscosity and gel behaviour can be examined by determining the effect that an oscillating force has on the movement of the material.

Without wishing to be bound by theory, it is believed that the viscoelasticity and high viscosity of the composition of the invention can result in a prolonged residence time at the site of administration, which may enhance the sustained effectiveness of the composition. Thus, the sustained release of at least some of the active agent and the viscosity-induced residence at the injection site

may be synergistic in effecting a long-lasting therapeutic activity in the tissue region which is to be treated, such as the injured nerve or spinal cord.

It is preferred that the dynamic viscosity of the composition is at least about 5 mPa*s at room temperature, or at least about 10 mPa*s or even 20 mPa*s, respectively. In yet further embodiments, the dynamic viscosity of the composition is at least about 50 mPa*s, or at least about 100 mPa*s, respectively.

The composition is further formulated as a viscoelastic gel, which means that the viscosity is so high that the material behaves like an elastic solid if only low shear forces are exerted, and like a viscous fluid when the shear force exceeds a threshold which is defined as the yield point. A gel is a semisolid system with a finite, usually rather small, yield stress.

The beneficial effect of an increased viscosity may potentially be further enhanced by adapting the composition to have a shear-thinning behaviour. Shear-thinning means that a material becomes less viscous upon increased shear stress. Shear-thinning is also sometimes referred to as pseudoplastic behaviour. For administration purposes, a shear-thinning behaviour of a viscous liquid or of a gel above the yield point is rather useful as it means that the composition can be injected rather easily and rapidly without having to apply too much force. Shear-thinning behaviour in aqueous systems is known to be induced by certain excipients, such as (optionally cross-linked) polyacrylates, magnesium aluminum silicate, or alginates.

In another aspect, the composition is preferably shear thinning and at the same time thixotropic, which means that after applying shear stress it takes some time for the original viscosity or gel strength to recover. Such behaviour may - again without wishing to be bound by theory - be achieved, for example, by using the electrostatic interactions between oppositely charged

components, e.g. molecules, colloids, particles etc. For example, a cationic ion chelator salt such as desferrioxamine mesylate may be combined with an anionically charged particle, such as an anionically charged lipid, e.g. phosphatidylglycerol. It is believed that the electrostatic interactions between the desferrioxamine and the lipid, or lipid particle, may be at least partially disrupted upon applying shear stress, and that returning from the state of flow after removal of shear stress allows the interactions to form again, leading to an increased viscosity. Again, such behaviour may be useful for enabling the administration of a composition which is highly viscous or even gel-like at rest, having a long residence time at the site of administration, but still being well syringeable, i.e. relatively easy to inject.

According to the invention, at least a fraction of the dose of the iron chelator incorporated in the composition is in sustained release form. To the formulation expert, a number of techniques are known to formulate the iron chelator in sustained release form. For example, the formulation as a gel may provide sustained release of the active compound. Various types of injectable gel compositions have been described in the past, which may be used and adapted to incorporate the iron chelator.

In one of the known concepts, polymeric gel formulations are designed which are highly shear thinning and thixotropic. By applying shear force prior to administration, the viscosity of these gels is substantially reduced, allowing for injection with a relatively small needle, whereas the gel strength is recovered slowly after administration. According to another concept, liquid compositions are formulated which, after administration, form gels in response to changes of their environment, such as pH, temperature, ionic strength. According to a third approach, liquid polymer formulations comprising a non-aqueous solvent are injected. Upon administration, the solvent diffuses away from the injection site, which leads to the precipitation of polymeric particles or to the formation of a gel. Biodegradable injectable gels have been discussed in detail by A.

Hatefi et al., Journal of Controlled Release 80 (2002), 9-28, which document is incorporated herein by reference.

Another type of injectable gel may be formulated on the basis of the excipient, sucrose acetate isobutyrate. The respective excipient and formulation technology is known as Saber™. The composition initially comprises a small amount of a pharmaceutically acceptable organic solvent, by virtue of which the composition is in liquid state for administration. After administration, the solvent is rapidly absorbed from the composition which then solidifies into a gel.

If the composition is selected to be in the form of a gel or a liquid that is converted into a gel upon administration, the incorporated iron chelator is released slowly primarily because of the slow diffusion of the active compound within the gel. Additional interactions between the active compound and the gel or any of its components may further slow the release of the iron chelator.

According to another embodiment, at least a fraction of the iron chelator is formulated within, encapsulated in, or associated with, sustained release particles. Preferably, this dose fraction is less than the total incorporated dose, such as from about 5 to about 80% (w/w) of the total dose. In other embodiments, the dose fraction incorporated within, or associated with, the sustained release particles is from about 10 to about 50% (w/w), or from about 15 to about 30% (w/w).

As used herein, particles are defined as small articles of solid, semisolid or liquid material regardless of their shape, composition, or internal morphology. For particles in the low colloidal size range, a clear distinction between the solid, liquid and semi-solid states may not even be possible or appropriate. The particles useful as sustained release particles in the composition of the invention may represent one or more of the following: polymeric, lipidic, or

lipoidal microparticles or nanoparticles, liposomes, niosomes, micelles, inverse micelles, and cross-linked micelles.

As used herein, microparticles are defined as substantially solid or semisolid particles having a weight- or volume average diameter in the region of about 0.1 μm to about 1.000 μm, but usually of about 1 μm to about 500 μm, regardless of their composition, geometrical shape, or internal structure. For example, spherical microparticles, which are often referred to as microspheres, are included in the term microparticles, just as capsular structures, such as microcapsules. Several other synonyms may exist to describe microparticles as defined above.

Nanoparticles are usually understood as any particles having a number-, weight- or volume average diameter in the region of about 1 to about 1.000 nm. Again, the term is used regardless of their composition, geometrical shape, or internal structure. Nanospheres and nanocapsules are examples of nanoparticles.

Lipid particles represent a category of particles characterised by the composition. Depending on their size, lipid particles may also be categorised as micro- or nanoparticles. Lipid particles do not require a certain morphology.

Liposomes represent one type of lipid particles. More precisely, liposomes are artificial lipid bilayer vesicles of various sizes and structures. Unilamellar vesicles are liposomes defined by a single lipid bilayer enclosing an aqueous space. In contrast, oligo- or multilamellar vesicles comprise several membranes. Typically, the membranes are roughly 3-5 nm thick and composed of amphiphilic lipids, such as phospholipids of natural or synthetic origin. Optionally, the membrane properties are modified by the incorporation of other lipids such as sterols or cholic acid derivatives. Liposomes with particularly flexible membranes based on phospholipids with a low phase

transition temperature (i.e. below body temperature) are sometimes referred to as transfersomes.

Depending on their diameter and number of bilayer membranes, liposomes may also be classified as multilamellar vesicles (MLV, two or more bilayers, typically above approx. 150-200 nm), small unilamellar vesicles (SUV, one single bilayer, typically below about 100 nm), multivesicular vesicles (MVV, several vesicular structures within a larger vesicle), and large unilamellar vesicles (LUV, one single bilayer, typically larger than about 100 nm).

In one of the preferred embodiments, the active ingredient is encapsulated within, or associated with, vesicle-forming lipids dispersed in an aqueous phase which is in the form of a viscoelastic gel. It should be noted that it is not essential that any or all of the vesicle-forming lipids are actually present in the form of liposomes in a gel-type composition. For example, the lipids may form other colloidal structures than liposomes which are also capable of exerting a sustained-release effect on those molecules of the active compound which are associated with such structures. Optionally, a gel-type composition may also simultaneously comprise lipid vesicles and other colloidal structures.

Various methods are commonly used to prepare liposomes and other lipid particles and to incorporate active ingredients, including the film method, sonication, detergent dialysis, ethanol injection, ether infusion, reverse phase evaporation, extrusion, and high pressure homogenisation. These methods, the resulting products and their properties are described in more detail in Kerby et al., Liposomes, in : Encyclopedia of controlled drug delivery, vol. 1, 461-492 (John Wiley & Sons, 1999) which is incorporated herein by reference.

Lipid nanoparticles - often called solid lipid nanoparticles (SLN) - are colloidal particles without vesicular structure. They are based on lipids or lipoidal excipients and can be loaded with various lipophilic or poorly water-soluble

active compounds. One of the common methods for making these particles is high pressure homogenisation.

Micelles, inversed micelles and mixed micelles are particularly small colloidal structures with various shapes and a length or diameter of typically about 5 to about 100 nm, formed through the association of amphiphilic molecules such as detergents. In contrast to liposomes, these structures are based on monolayers which are less stable and tend to disassemble upon dilution.

Micelles may be cross-linked to form stable nanoparticles. Further details on micelles are e.g. disclosed in WO 02/085337 and EP-A 730 860, whose teachings are incorporated herein by reference.

Niosomes are colloidal structures similar to liposomes, except that the vesicle membranes are predominantly composed of nonionic amphiphilic compounds instead of ionic components.

Preferably, the mean diameter of the sustained release particles, if present in the composition of the invention, is selected from about 50 nm to about 100 μm, and more preferably from about 100 nm to about 50 μm. A mean or average particle diameter, as used herein, refers to the number average diameter of a sample as measured by laser diffraction or photon correlation spectroscopy or an equivalent method, unless stated otherwise.

If particles are selected to provide for the sustained release of at least a fraction of the iron chelator, it is also preferred that these particles are dispersed in a liquid phase which is at least partially composed of water. In addition to water, the liquid phase may comprise further liquid consituents, such as one or more pharmaceutically acceptable solvents, e.g. ethanol, acetone, glycerol, propylene glycol, polyethylene glycol, ethyl acetate, N- methylpyrrolidone, dimethylsulphoxide, or dimethylformamide. More preferably, however, the liquid is only water. On the other hand, the aqueous

phase may comprise solutes and, optionally, other particles, such as dissolved active ingredient, dissolved excipients, or suspended active ingredient.

In one of the preferred embodiments, the composition comprises lipid particles, which are optionally liposomes. These lipid particles preferably have a mean diameter from about 100 nm to about 10 μm, at least in their non- aggregated form. In another embodiment, their mean diameter is below about 5 μm.

The lipid particles are preferably composed of, or based on, amphiphilic lipids. Phospholipids are examples of suitable amphiphilic lipids. Useful phospholipids for composing the lipid particles include, for example, lecithin, hydrated lecithin, phosphatidylcholine, hydrated phosphatidylcholin, dimyristoyl phosphatidylcholine, dilauryloyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, l-myristoyl-2- palmitoyl l-palmitoyl-2-myristoyl phosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, dioleoyl phosphatidylglycerol, dipalmitpyl phosphatidic acid, and dipalmitoyl phosphatidic acid. Lecithins are mixtures of phosphatides, usually extracted from natural sources such as egg yolk or soy beans; they usually also contain substantial amounts of non-phospholipid components. Phosphatidylcholin is typically an extracted fraction of phospholipids from lecithin, predominantly comprising phosphatidylcholins, usually with a large content of l-palmitoyl-2-oleoylphosphatidylcholin if it is derived from egg yolk.

In another preferred embodiment, the lipid particles comprise at least one zwitterionic phospholipid and at least one negatively charged phospholipid. This is particularly useful if the iron chelator is desferrioxamine, or a salt of desferrioxamine, such as the mesylate. A useful zwitterionic phospholipid is, for example, a phosphatidylcholin or mixture of phosphatidylcholins. A useful

negatively charged phospholipid is, again by way of example, a phosphatidylglycerol or mixture of phosphatidylglycerols. The ratio between the zwitterionic and the negatively charged phospholipid may optionally be selected from about 1 : 9 to about 9 : 1. More preferably, it is from about 2 : 8 to about 8 : 2, or according to another embodiment, from about 4 : 6 to about 6 : 4, such as 1 : 1.

Without wishing to be bound by theory, it is believed that the negatively charged phospholipid may interact with the positively charged desferrioxamine, possibly contributing to the sustained release characteristics, and perhaps also increasing the viscosity of the composition and providing a shear-thinning and/or thixotropic rheological behaviour.

The content of the particles in the composition should be selected as adequate for binding, encapsulating, or incorporating at least a fraction of the iron chelator present in the composition to provide sustained release characteristics. Depending on the size, structure and composition of the particles, they may also have various capacities of being loaded with the active compound that should be taken into account. Typically, the particles will represent from about 0.1% to about 35% (w/w) of the composition, and preferably from about 10 % to about 30% (w/w), such as about 15%, about 20%, or about 25% (w/w). This is particularly preferred if the particles are lipid or phsopholipid particles, and/or if the iron chelator is desferrioxamine or a salt thereof.

In one of the specific embodiments which are presently preferred, the composition is an aqueous composition comprising about 5% to about 30% (w/w) of a desferrioxamine salt and about 1% to about 20% (w/w) phospholipid particles having a mean diameter from about 200 nm to about 5 μm, wherein at least a fraction of the desferrioxamine salt is associated with the phospholipid vesicles, and wherein the phospholipid vesicles comprise a

zwitterionic phospholipid such as phosphatidylcholin and a negatively charged phospholipid such as phosphatidylglycerol.

The drug load of the particles may be in the range of about 1% to about 50% (w/w), depending on the nature of the iron chelator and that of the particles. A particularly useful range is from about 5% to about 25% (w/w), or from about 10% to about 20% (w/w), in particular if the particles are lipid particles and/or if the iron chelator is desferrioxamine or a salt thereof.

In this context, the drug load refers to the amount of active compound by weight which is encapsulated or incorporated within, or associated with, or bound to the particles, relative to the weight of the particles. It can be determined by separating the particles from the liquid phase in which they are dispersed, such as by gentle centrifugation, chromatography, filtration, diafiltration, tangential flow filtration, or dialysis, and analysing the quantity of active ingredient in the particles and in the liquid phase.

As the composition of the invention is intended for parenteral administration, and in particular for the injection into nervous tissue, such as for intralesional, intraneural, intraspinal, intrathecal, or intramedullary administration, it is necessary that it is sterile and free of endotoxins. Moreover, it is preferred that the composition has a physiologically acceptable pH and osmolality. The pH may be adjusted between about 3 and about 8, depending on the selected iron chelator. Whenever possible, the pH should be adjusted within the range of about 4 to about 7.5, which is also the preferred range in the case of desferrioxamine being selected as iron chelator.

The osmolality can perhaps not be adjusted to a physiological value, in particular if a high concentration of an osmotically highly active iron chelator such as desferrioxamine or a salt thereof is required. On the other hand, for tolerability reasons it is presently preferred that the osmolality is not more

than about 1000 mOsmol/kg, such as from about 400 to about 800 mOsmol/kg. On the other hand, it is not known at present which osmolalities are tolerable with regard to certain modes of administration, in particular intralesional, intraneural, intraspinal, intrathecal, and it may be possible that even higher osmolalities than 1000 mOsmol/kg will be found acceptable.

The composition may comprise one or more further excipients, which are preferably selected from pharmaceutically acceptable excipients for parenteral dosage forms. For example, it may comprise one or more pH-modifiers, osmotic agents, viscosity-increasing agents, gel-forming agents, antioxidants, radical scavengers, preservatives, surfactants, stabilisers, solvents, bulking agents, lyophilisation protectants. Examples of such excipients are generally known to a person skilled in the technical field of the invention.

Among the preferred further excipients are pH-modifiers, in particular selected from bases and alkaline buffer salts, and viscosity-icreasing agents. Also preferred among the further excipients, if any are needed, are stabilisers, lyophilisation protectants, and bulking agents.

In the case that a stabiliser is needed in order to achieve the desired product performance or shelf life of the composition, or in order that the composition can be freeze-dried for an increased storage time, such stabiliser may be selected from the group of sugars or sugar alcohols, such as lactose, dextrose, sucrose, mannitol, sorbitol, xylitol; or polysaccharides such as dextran or polydextran; or native or synthetic amino acids such as glycine; or water- soluble peptides, including oligo- and polypeptides. It should be noted that some of these stabilisers also have the function of bulking agents and lyophilisation protectants in freeze-dried compositions.

The composition of the invention may be prepared by various methods. If the composition is designed as a polymeric gel, it may, for example, be prepared

by combining the respective polymer, such as polylactide-co-glycolide, with a suitable, pharmaceutically acceptable organic solvent, such as benzyl alcohol, benzyl benzoate, diacetin, tributyrin, triethyl citrate, tributyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, triethylglycerides, triethyl phosphate, diethyl phthalate, diethyl tartrate, polybutene, glylcerin, ethylene glycol, polyethylene glycol, octanol, ethyl lactate, propylene glycol, propylene carbonate, ethylene carbonate, butyrolactone, ethylene oxide, propylene oxide, N-methyl-2-pyrrolidone, 2-pyrrolidone, glycerol formal, methyl acetate, ethyl acetate, methyl ethyl ketone, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, caprolactam, decylmethylsulfoxide, oleic acid, 1- dodecylazacycloheptan-2-one, or mixtures thereof, in the presence of the iron chelator and, optionally, further excipients.

The preparation of injectable gels is further described by A. Hatefi et al., Journal of Controlled Release 80 (2002), 9-28.

If the composition is designed to comprise polymeric sustained release microparticles, such as polylactide-co-glycolide or similarly hydrophobic biodegradable microparticles, they may, for example, be prepared by the well- described emulsion-based processes of microparticle formation, such as solvent evaporation or solvent extraction. Preferably, a double emulsion of the type w/o/w is prepared from an inner aqueous phase which comprises the iron chelator and, optionally, further excipients, an organic phase comprising the polymer(s) and, optionally, further excipients, and an aqueous continuous phase, usually comprising at least one surfactant or polymeric stabiliser. From the organic phase of this double emulsion, the organic solvent is subsequently removed by extraction and/or evaporation, leading to the solidification of the polymer into microparticles.

Alternative methods of making polymeric microparticles include spray drying, spray freeze drying, supercritical fluid processing, spray congealing,

electrostatic assembling, emulsion polymerisation, and phase separation or coacervation. The methods for making microparticles are further described in detail in E. Mathiowitz et al., Microencapsulation, in : Encyclopedia of Controlled Drug Delivery (ed. E. Mathiowitz), Vol. 2, p. 493-546, John Wiley & Sons (1999), which is incorporated herein by reference.

The microparticles can be combined with an additional amount of active ingredient, which is preferably the same as the active ingredient incorporated in the micropartciles, but optionally also a different compound, with an aqueous carrier in which the particles are suspended and administered, and optionally with further excipients. If the iron chelator is desferrioxamine, and if the microparticles are prepared from polymers like polylactide, polyglycolide, or polylactide-co-glycolide which degrade into acidic reaction products in situ, it may be preferred to incorporate at least one pH-adjusting or buffering excipient in order to avoid the degradation of the acid-labile active compound.

Various methods are also known for preparing lipid particles including liposomes, such as those describen in Kerby et al., Liposomes, in : Encyclopedia of controlled drug delivery, vol. 1, 461-492 (John Wiley & Sons, 1999) which is incorporated herein by reference.

According to the present invention, it is preferred that the composition, if designed to comprise lipid particles or liposomes as sustained release particles, is prepared by combining one or more particle-forming lipids, the iron chelator, and water, optionally in the presence of further active and/or inactive ingredients to form an aqueous dispersion. Subsequently, the dispersion may be homogenised, using a suitable homogenising tool, such as an Ultraturrax ^® or a high-pressure homogeniser. The homogenisation is preferably carried out at elevated temperature, such as at a temperature which is above the phase transition temperature range, or above the melting temperature range, of the lipid(s) selected for particle formation.

The homogenisation temperature may thus be selected in response to the selection of the lipid or lipids, ensuring that these are in a fluid or liquid state rather than solid. Typcally, the temperature will be in the range of about 0 °C to about 75 ⁰ C, which will cover most phospholipids which, at present, appear pharmaceutically acceptable for parenteral administration. More preferably, the temperature is from about 20 ⁰ C to about 70 ⁰ C.

If the lipid particles are designed to incorporate or bind only a fraction of the iron chelating agent, the remaining fraction of the active compound in the composition is either dissolved, or dissolved and suspended, in the liquid continuous phase of the particle dispersion.

As the composition is intended for parenteral administration, it must be supplied in sterile form. This may be achieved by aseptic processing, using pre-sterilsed equipment, ingredients, and containers. Alternatively, the composition is sterilised in the final containers, which is the preferred option if at all feasible. If heat sterilisation is not possible, sterilisation by irradiation is preferred. For example, the composition may be sterilised by gamma- irradiation, using a dose of at least about 15 kGy, and preferably not more than about 35 kGy, such as about 25 kGy.

For product stability reasons, it may not be feasible to achieve a commercially acceptable shelf life when storing the composition at room temperature or under refrigeration, such as at 2 ⁰ C to 8 ⁰ C. In this case, the composition may frozen and stored at a temperature of e.g. -20 ⁰ C.

In another preferred embodiment, the composition is designed as a powder which is reconsitituted to a liquid composition prior to administration. This option may also overcome problems associated with a limited stability of the ready-to-use composition. As used herein, a powder is a substantially dry

material composed of multiple fine particles suspended in air or another gas phase. The powder may be agglomerated to a porous solid form, as is often the case with lyophilisates. In this form, it is no longer free-flowing, but still represents a powder as defined herein.

To prepare a powder, the composition may be prepared in liquid or gel-like form by the method described above, and then dried, such as by lyophilisation. According to this embodiment, it appears useful that the composition further comprises at least one excipient which serves as a bulking agent, lyophilisation aid, and/or stabiliser. Preferably, this at least one excipient is dissolved in the continuous liquid phase of the composition. The excipient is preferably selected from the group of sugars or sugar alcohols such as lactose, dextrose, sucrose, mannitol, sorbitol, xylitol; polysaccharides such as dextran or polydextran; native or synthetic amino acids such as glycine; and water-soluble peptides, including oligo- and polypeptides, such as gelatin.

It is further preferred that the drying step is carried out in such a way that the residual water content is less than about 5% (w/w), and more preferably less than about 3% (w/w). In a further embodiment, the residual water content is less than about 2% (w/w). Such residual water content may be achieved by the selection of process parameters, such as drying temperature or duration, as generally known to the person skilled in the technical field.

The powder may be provided as part of a pharmaceutical kit. Apart from the powder, such kit may comprise a sterile liquid for reconstituting the injectable composition from the powder.

As mentioned herein-above, the composition of the invention is particularly useful for direct injection into an injured or inflamed region of nervous tissue, such as intralesional, intraneural, intraspinal, intrathecal, and/or

intramedullary injection. Such administration is highly promising for the treatment of acute or chronic diseases and conditions associated with neurotraumatic and/or neurodegenerative symptoms in humans or animals. Such diseases and conditions may be associated with traumatic injuries or inflammation of neurons, optionally resulting from axonic lesions, injuries of the spinal cord, injuries of the brain, injuries of a peripheral nerve, multiple sclerosis, stroke, and resections of tumours of the central nervous system.

In one of the preferred embodiments, the composition is used for the treatment of spinal cord or peripheral nerve injuries. It has been found that a single administration of the composition (per lesion) may be sufficient to provide the desired effect of scar inhibition for a period of time that is long enough to allow for nerve growth and/or regeneration. For large lesions, it may be useful to select more than one site of injection to better cover the whole lesion, which is still considered as only one administration according to the present invention. The long-lasting effectiveness of the composition is particularly beneficial in that, typically, no repeated administrations are needed.

It has also been found that the volume of administration may be selected to enhance the effectiveness of treatment. Preferably, the composition is injected at a volume which is in the range of about 30% to about 150% of the volume of the lesion. More preferably, the injection volume is from about 50% to about 100% of the volume of the lesion, such as about 70%. In order to practise this embodiment, the volume of the lesion must be estimated or measured. A preferred method of determining the lesion volume is by magnetic resonance imaging.

The invention is further illustrated by the following examples.

Example 1 : Preparation of a viscous gel-like composition of desferrioxamine mesylate

Desferrioxamine mesylate (70 g), egg phosphatidylcholin (17,5 g), and egg phosphatidylglycerol (17,5 g) were weighed and mixed with water for injection (245 g). The crude mixture was heated to about 60 ⁰ C in a water bath. Subsequently, the mixture was homogenised using a high-speed homogeniser (Ultraturrax ^® ). It was observed that the dispersion was milk-like, and white or off-white in appearance. No washing or separation step was performed to remove any dissolved desferrioxamine mesylate which was not encapsulated in, or associated with, the phospholipid. The homogenised dispersion was filled into 6 ml vials and capped with aluminum caps. The vials were subsequently sterilised by gamma-irradiation at a dose of 25 kGy.

A sample of the composition was analysed by laser diffraction (Malvern Mastersizer ^® ), indicating a mean particle size of about 70 μm. The pH was about 4.9, and the osmolality 1005 mθsmol/kg. The composition was highly viscous, having a viscosity of approximately 65 mPa*s.

Example 2: Preparation of a composition of desferrioxamine mesylate

A desferrioxamine mesylate composition comprising lipid particles was prepared according to example 1. but with lower content of the active and a different ratio (3: 1) of the two lipids. Again, no washing or separation step was performed to remove any dissolved desferrioxamine mesylate which was not encapsulated in, or associated with the phospholipid.

Example 3: Comparative In vivo effectiveness

Four female Wistar rats (200-250 g) received a defined mechanical spinal cord injury performed with a Scouten Wire knife (Kopf Instruments). Subsequently, a volume of 20 μl of the composition prepared according to example 1 was injected into each of the rats' lesion using a Hamilton syringe.

The rats were sacrificed after 7 days, and their lesions were examined. All treated rats showed inhibited scar formation. Upon the exposure with a reagent containing labelled collagen antibodies, no presence of the antibodies at the lesions was detected (see figure 3c). In contrast, control animal which had received an equivalent aqueous lipid particle composition without desferrioxamine mesylate had formed scars, which could be visualised with labelled collagen antibodies (see figure 3a) . Usually, scar formation is expected within about 7 days without treatment.