NZ207045A - Iron complexes of pyrone derivatives and pharmaceutical compositions - Google Patents

Description

207045 Priority Date(s): . ...tTTTTT Complete Specification Filed: Class: CX2f.l5(P.i>...<k?lJl?£% w.m/.ss.r • »f t <«*•«• I •• t •• t»« • ■ • » • J* f • 1LM • • p ... . _ . 3 1 MAR 19^ Publication Date: P.O. Journal, No: .... A*.

No.: D«te: NEW ZEALAND PATENTS ACT, 1953 COMPLETE SPECIFICATION PHARMACEUTICAL COMPOSITIONS k/Wt, NATIONAL RESEARCH DEVELOPMENT CORPORATION, a British Corporation established by Statute, of 101 Newington Causeway, London SEl 6BU, England, hereby declare the invention for which $ / we pray that a patent may be granted to me/us, and the method by which it is to be performed, to be particularly described in and by the following statement:- 207045 122156 PHARM&CEUTICALLY ACTIVE IRON COtfLEXBS OF 3-HYPRO XY-4-PYRONES This invention relates to iron compounds for use in pharmar-ceutical compositions for the treatment of iron deficiency anaemia.

An adequate suppLy of iron to the body is an essential requirement for tissue growth in both man and animals. Although there is, normally an ample amount of iron in the^diet, the level of absorption of iron from food is generally law so that Che supply of iron to the body can easily become critical under a variety of conditions.

Iron deficiency anaemia is commonly encountered in pregnancy and may also present a problem in the newly born, particularly in certain animal species such as the pig. Msreover, in certain pathological conditions there is a maldistribution of body iron leading to a state of chronic anaemia. This is seen in chronic diseases such as rheumatoid arthritis, certain haemolytic diseases and cancer.

Although a wide range of iron compounds is already marketed for the treatment of iron deficiency anaemia, the level of iron uptake by the body from these compounds is often quite low therd)y necessitating the administration of relatively high dosage levels of the compound. The administration of high dose, poorly absorbed, iron complexes may cause siderosis of the gut wall and a variety of side effects such as nausea, vomiting, constipation and heavy malodorous stools.

The present invention relates to a group of iron complexes which we have identified as being of particular value for use at relatively lew dosage levels in the treatment of iron deficiency anaemia. The hitherto unrecognised value of these complexes in such a context, as shown by in vivo experiments, is unexpected in view of the well known need for improved iron compounds for the treatment of iron deficiency anaemia. This is particularly so as among the compounds whose iron complexes are of the most interest for use in pharmaceutical compositions according to the present invention is a significant nurtber of compounds which are naturally occurring materials, or are readily derivable from such materials, and which have been knewn for some time to be capable of forming iron complexes. Furthermore, several of these compounds have previously been used in foodstuffs thereby indicating their notr-toxic nature and che consequent suitability for pharmaceutical use of their iron complexes.

Although certain of the iron complexes themselves have also previously be^n proposed for use in foodstuffs, as colouring , agents, it had never previously been appreciated that they have any therapeutic use and the conditions proposed for the use of such complexes as colouring agents would not generally be such as to lead to any significant physiological effect.

According to the present invention a pharmaceutical composition comprises an iron complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-A-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, together with a physiologically acceptable diluent or carrier,sa1d composition having a form as defined hereinafter.

The iron complexes of use in the present invention preferably contain iron in the ferric state. Although the use of complexes containing iron in the ferrous state may be considered, such complexes tend to be less stable and are thus of less interest.

The iron complexes- are preferably neutral and this is conveniently achieved by cooplexing with a ferric cation the appropriate number of anions derived from the hydroxypyrone (through the conversion OH —•» 0 ) necessary to produce neutrality. Preferred iron complexes of use:.in the present invention are thus the neutral 3:1 complexes containing three hydroxypyrone anions complexed with a ferric cation.

The substituted 3-hydroxy-4-pyrones may carry more than one type of aliphatic hydrocarbon group but this is not usual and, indeed, substitution by one rather than two or three aliphatic hydrocarbon groups is preferred. The term aliphatic hydrocarbon group is used herein to include both acyclic and cyclic groups which may be unsaturated or saturated, the acyclic groups having a branched chain or especially a straight chain. Groups of from 1 to 4 carbon atoms and particularly of J to 3 carbon atoms are of 207045 most interest. Saturated aliphatic hydrocarbon groups are preferred, these being either cyclic groups such as the cycloalkyl groups cyclopropyl and especially cyclohexyl or, more particularly, acyclic groups such as the alkyl groups n-propyl and isopropyl, 05 and especially ethyl and methyl. Substitution at the 2- or 6-position is of especial interest although, when the ring is substituted by the larger aliphatic hydrocarbon groups, there may be an advantage in avoiding substitution on a carbon atom alpha to the -C-C^ system. This system is involved in the complexing 0 OH with iron and the close proximity of one of the larger aliphatic hydrocarbon groups may lead to steric effects which inhibit complex formation.

Examples of specific compounds whose iron complexes are of use in the present invention are shown by the following formulae (I), 15 (II) and (III):- HO cm) in which R is a cycloalkyl or alkyl group, for example irethyl, \ ethyl, ir-propyl or isopropyl. Among these compounds 3-hydroxy~2-methyl— 4-pyrone (maltol; II, R « CH^) is of most interest, whilst 3-hydroxy-4-pyrone (pyromeconic acid; I), 3-hydroxy-6-methyl-4-pyrone (III, R » CH^) and particularly 2-ethy1-3-hydroxy-4-pyrone (ethylpyromeconic acid; II, R «* are also of especial interest.

For convenience the compound 3—hydroxy-2-methy1-4-pyrone is referred to in the following discussion under the name maltol.

^ J07045 -3tKTt986l - In Che case of certain of Che hydroxypyrones referred Co above, i.e. malcol, 3-hydroxy 6-me thy 1-4-pyrone and 2-ethyl-3-hydroxy-4-pyrone, the formation of an iron complex of the compound has been referred Co in Che literature, although it should be noted chat the procedures described in Che liceracure for Che produccion of such complexes ofcen would not provide.complexes of , a form which is preferred for use ih the present invention and Che only 3:1 complex reporced is thac of malcol. In che case of Che other hydroxypyrones, all forms of the iron complexes are novel and are included, per se, by the present invention.

The iron complexes are conveniently prepared by the reaction . of the hydroxypyrone and iron ions, the latter conveniently being derived from an iron salt, particularly a ferric halide and especially ferric chloride. The reaction is conveniently effected in a suitable mutual solvent and water may often be used for this purpose. If desired, however, an aqueous/organic solvent mixture may be used or an organic solvenC, for example echanol, methanol or chloroform and mixCures of chese solvents cogecher and/or with wacer where appropriace. In. parcicular, methanol or especially echanol may be used where ic is desired co effect the separation of at least a major part of a byproduct such as sodium chloride by precipitation whilst the iron complex is retained in solution.

It should be appreciated that the nature of the iron complex obtained by the reaction of a hydroxypyrone and iron ions will depend both on the proportion of these two reactants and upon the pH of Che reaccion medium. Thus, for che preparacion of the 3:1 ferric complex, for example, Che hydroxypyrone and Che ferric sale are convenienCly mixed in solucion in a 3:1 molar proportion and the pH adjusted to a value in the range of 6 to 9, for example 7 or 8. If a similar excess of hydroxypyrone:iron is employed but no adjustment is made of the acidic pH which results on the admixture of the hydroxypyrone and an iron salt such as ferric chloride, then a mixture of the 2:1 and 1:1 complex will instead be obtained. 6 - l'-i v. 7045 usually have proceeded substantially to completion after 5 minutes necessary. Following separation of any precipitated by-product, 05 such as sodium chloride in the case of certain solvent systems, the reaction mixture may conveniently be evaporated on a rotary . evaporator or freeze dried to yield' the^solid iron complex. This may, if desired, be crystallised from a suitable solvent, for example water, an alcohol such as ethanol, or a solvent mixture, 10 including mixtures containing an ether. The present invention thus further includes a process for the preparation of a neutral 3.: 1 hydroxypyrone: iron (III) complex of-3-hydroxy-4-pyrone or of a 3-hydroxy-4—pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon 15 group of 1 to 6 carbon atoms, but excluding 3-hydroxy-2-methy 1-4-pyrone, which comprises reacting said hydroxypyrone with ferric ions at a pH in the range of 6 to 9 to form the 3:1 complex and isolating said complex in the solid form. iron complex in substantially pure form, i.e. substantially free from by-products of manufacture, in other cases, for example with a solid oral formulation as described hereinafter, the presence of by-products such as sodium chloride may be quite acceptable. In general, however, the neutral 3:1 hydroxypyrone:iron (III) complex 25 is of particular interest in a form which' is substantially free at least from those by-products which are complexes containing different proportions of hydroxypyrone and iron, in particular the 2:1 and 1:1 complexes. Accordingly the present invention includes an iron complex, for example the 3:1 hydroxypyrone: iron (III) complex, of 3-hydroxy-4-pyrone or of a 3-hydroxy-4- pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, but excluding 3-hydroxy-2-methy1-4—pyrone, when in a form substantially free from iron complexes of the hydroxypyrone 35 containing other proportions of iron. As indicated hereinafter, at about 20°C, although a longer reaction time may be used if Whils-t for some uses it may be appropriate to prepare the ic nay be advantageous under some circumscances for Che iron complex Co be used in admixCure with che free pyrone or a sale Chereof concaining a physiologically acceptable cacion. Ic is possible Co produce such a mixCure by mixing che two components eicher in che solid form or in soluCion, followed by isolacion of a solid mixcure in Che laccer case when a solid composicion is required. However, ic maybe more convenienc Co ob Cain such a mixcure by reaccing a molar proporcion of che hydroxypyrone and iron ions of greacer than 3:1. Ic should be scressed, however, that Che condicions as veil as Che proporcion of reacCanCs used in. Che reaccion are of importance if a mixture of Che free pyrone and Che preferred neuCral 3:1 complex is Co be obcained. In parcicular, as indicaced previously, the pH of Che reaccion mixture is parcicularly important and, because of this face, cercain prior arc procedures concerned wieh Che use of iron hydroxypyrone complexes in food colouring, for example as described in US patent 4,018,907, substantially fail Co yield che 3:1 complex even chough an excess of the hydroxypyrone is presenc, owing Co Che lack of pH conCrol.

CerCain hydroxypyrones, such as malcol, are available commercially. Wich ochers, a convenienc scarcing macerial in many instances consists of 3^hydroxy—4-pyrone which is readily obtainable by the decarboxylacion of Zje-dicarboxyS—hydroxy-4-pyrone (meconic acid). Thus, for example, 3-hydroxy~4-pyrone may be reacted wich an aldehyde Co inserc a I—hydroxyalkyl group ac Che 2-posicion, which group may Chen be reduced Co produce a 2-alkyl-3-hydroxy-4-pyrone. The preparacion of 2—eChyl-3-hydroxy-4-pyrone, ecc, by Chis route is described in the published US application serial number 310,141 (series of I960).* It will be appreciated that these are not the only routes available to these compounds and their iron complexes and that various alternatives may be used as will be apparent to Chose skilled in Che art.

The iron complexes may be formulaCed wich a physiologically accepCable diluent or carrier for use as pharmaceucicals for veCerinary or human use in a varieCy of ways. HowefBrV composicions * available on request in which the diluent or carrier is other tfrsn a noifc-e-cerile solution in water and/or an organic solvent are generally preferred. Thus, the iron complexes nay be applied as an aqueous, oily or emulsified composition incorporating a liquid diluent, which will, however, 05 most usually be employed for parenteral administration and therefore may conveniently be sterile and pyrogen free. One form of composition of particular interest thus has the "foru^.pf a sterile, injectable solution, suspension or emulsion. Oral administration is, however, more generally to be preferred for the treatment of iron deficiency anaemia in humans and the complexes of the present invention may * be given by such a route. Although compositions incorporating a 'liquid diluent may be used for oral administration, it is preferred to ase compositions incorporating a solid carrier, for example a conventional solid carrier material such as starch, lactose, 15 dextrin or magnesium stearate. The iron complex will of course be present in such a preferred composition in solid form, which form is accordingly a preferred one for the complex, and such a solid composition may conveniently be presented as some type of formed composition, for example, as tablets, capsules (including spansules), 20 etc.

Although solid compositions are preferred in many applications, liquid composicions are of inceresc in cercain particular inscances, for example human and veterinary inCramuscular administration and veterinary oral administration as discussed hereinafter. It is 25 often desirable Co produce liquid compositions containing a higher concentration Chan is readily obtainable wich a purely aqueous composition and ic has been found ChaC Chis may be done by Che use of glycols or glycol ethers, either in admixture with water or, for betcer solubilisacion, alone. The glycol eChers of particular 30 interest are the mono-ethers containing as an etherifying group an aliphatic hydrocarbon group of J to 6 carbon atoms as described above, for example a methyl group, such a glycol mono-ether being methyl ethylene glycol. In general, however, the glycols themselves are preferred. Examples of such glycols are the simple dihydroxy 35 alkanes such as ethylene glycol as well as those more complex 207045 compounds comprising two hydroxy groups attached to a chain containing both carbon and oxygen atoms, such as triethylene glycol, tetraethylene glycol and polyethylene glycol, for example of 4,000 daltons molecular weight. Triethylene glycol and especially 05 tetraethylene glycol are of particular interest in view of their very low toxicity. By using such glycols and glycol, ethers it is , possible to increase solubility for many.^complexes to 10 to 20 rag/ml.

In the case of animals, compositions for parenteral administration arc of greater interest than with humans. The problems of 10 iron deficiency anaemia in newly born pigs arise primarily during the first three weeks or so of their life when a very rapid weight gain takes place. The usual routes for administration of the iron complexes of the present invention to young piglets are parenteral, for example intramuscular, or oral, for example as a liquid 15 preparation "injected" into the mouth. However, an alternative approach is to enhance the iron content of the milk on which the piglets are feeding by treating the mother pig using oral or parenteral administration, for example with an injectable slow release preparation (such an approach may also be an interest in a 20 human context). When it is applicable to feed piglets on foodstuffs other than the milk of the mother pig, it may also be possible to effect the pharmaceutical administration of the iron complex in this other foodstuff.

Other forms of administration than by injection or through 25 the oral route may also.be considered in both human and veterinary contexts, for example the use of suppositories for human admini— stration.

The present invention thus further includes a pharmaceutical composition comprising a physiologically effective amount of a 30 neutral 3:1 hydroxypyrone:iron(III) complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of I to 6 carbon atoms, together with a physiologically acceptabe diluent or carrier, but exghfcgifig any liquid 35 which is noir-sterile and non-pyrogen f/4 N 207045 - Compositions may be formulated in unit dosage form, i.e. in the form of discrete portions containing a unit dose, or a multiple or sub—unit dose. Whilst the dosage of hydroxypyrone iron complex given will depend on various factors, including the particular 05 compound which is employed in the composition, it may be stated by way of guidance that maintenance at a satisfactory level of the , amount of iron present in the human'bod^ will often be achieved using a daily dosage, in terms of the iron content of the compound, which lies in a range from about 0.1 to 100 mg and often in a range from 0.5 to 10 mg, for example 1 or 2 mg, veterinary doses / being on a similar g/kg body weight ratio. However, it will be "apppreciated that it may be appropriate under certain circumstances to give daily dosages either below or above these levels. In general, the aim should be to provide the amount of iron required 15 by the patient without administering any undue excess and the properties of the pharmaceutical compositions according to the present invention are particularly suited to the achievement of this aim. Similarly, the concentration of iron in the pharmaceutical composition in the form of the hydroxypyrone complex may 20 vary quite widely, for example over a range from 0.01 to 20% w/w. However, it is more usual for the concentration to exceed 0.01Z w/w and it may often exceed 0.05 or 0.1% w/w, whilst a more usual limit for the upper end of the range is 13Z w/w. A common range of concentration is 0.05 to 5% w/w, for example 0.2 to 0.5, 1 or 25 2Z w/w.

Where desired, more than one hydroxypyrone iron complex as described above may be present in the pharmaceutical composition or indeed other active compounds may be included in the composition, for example compounds having the ability to facilitate the treatment 30 of anaemia, such as folic acid. Another additional component which may be included in the composition, if desired, is a source of zinc. Iron compounds used in the treatment of iron deficiency % anaemia can inhibit the mechanism of zinc uptake in the body and this can cause serious side effects^£B=feh£L foetus when treating 207045 - I I - anaemia is in a pregnant female. Ic is believed, however, that the iron complexes of the presenC invention have a further advantage in that they either do not have this effect or exhibit the effect at a lever level than the compounds at present used in the treatment 05 of anaemia. Accordingly, it may often be the case that the level of zinc providing compound added to the composition may not require to be high or, with preferred formulatiotrs of the iron complexes, ^ may be dispensed with altogether.

The present invention thus further includes the use of an 10 , iron complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, for the manufacture of a medicament for use in effecting an increase in the level of iron in a patient's blood-15 stream.

We have found that the iron complexes described herein are particularly suited to the treatment of iron deficiency anaemia, both in humans and also in a veterinary context and particularly for the treatment of various -mammalian species, especially pigs.

Thus, the chelating agents which they contain, and particularly maltol, have a high affinity for iron (log^S^ = 30 for maltol) but a lower affinity for copper (II), zinc (II), calcium and magnesium.

Both the high affinity of maltol for iron and its low affinity for calcium are reflected in its.Ksoj_ value £ log is defined as being equal to log^^^'^ + 21 - [ PKSp + n 1°S a^(H+) + m log a^(Ca++)j where lo8>3Fe(L)n the emulative affinity constant of the ligand in question for iron (III), pK is the negative logarithm 40? SP of the solubility product for Fe(OH)^ and has a value of 39, n and m are the nuo&er of hydrogen and calcium ions, respectively, 30 which are bound to the ligand, and a^(H+) and a^(Ca++) are the affinities of the ligand for hydrogen ions and calcium ions, respectivelyJ. In order to solubilise iron (III) hydroxide, log Kgoj must be greater than 0. The value of Kgo^ for maltol is 8.0 and this is also sufficiently large to pjj^tent appreciable -so 207045 competition from phytate, phosphate, thiols and other potential ligands likely to occur in the intestinal lumen. In order to exchange iron efficiently with transferrin, the log K ^ value should be close to that of apotransferrin, which is 6.0, so that 05 maltol is also suitable in this respect. Moreover, although the neutral 3:1 maltol:iron CHI) complex is thermodynamically stable ' (thermodynamic stability constant « 30) -it is also extremely labile and is therefore afcle to donate iron to high affinity sites, such as- those found in apotransferrin. The half, life for 10 ' the exchange of iron (III) between the maltol complex and apotransferrin is 1 minute whereas, by contrast, the corresponding figure ' for the complex of EDTA with iron (III) is 4 days.

It will be appreciated, however, that in addition to possessing properties such as thosre described above for iron maltol, a compound 15 which is to act as a source of iron through oral administration is required to to show a high level of membrane permeability. An indication of the properties of a compound in this respect is provided by the value of the partition coefficient (K ) obtained pore on partition between n—octanol and Tris hydrochloride (20 mM, pH 7.4; Tris representing 2—amino-2-hydroxymethylpropane 1,3- diol) at 20°C and expressed as the ratio (concentration of compound in organic phase)/(concentration of compound in aqueous phase).

The value of for the neutral 3:1 maltol:iron (III) complex is 0.5, which is well placed in the preferred range of 0.2 to 1.0 and compares favoura&liy with the figures of 0.001 and 0.0015 for the EDTA:iron (III) complex and iron (III) ascorbate, respectively. ^ The value of the iron complexes of the present invention is confirmed by various in vitro and ui vivo tests. Thus, their ability to permeate biological menfcranes is confirmed in practice 59 by tests of the ability of the Fe labelled iron complexes to permeate erythrocytes. Moreover, iron complexes of the present invention have been found to exhibit a high level of efficiency in promoting iron uptake, as measured in the rat small intestine, as 207045 13 - compared with a range of other iron complexes currently* marketed for the treatment of iron deficiency anaemia. In vivo experiments in the cat and rat have confirmed the value of iron mal Col compounds as a source of iron, the iron uptake obtained either on intravenous 05 administration or on direct administration into the snail intestine being markedly superior to that obtained with commercially available iron compounds such as iron sulphate, iron EDTA and iron gluconate! It was found from these experiments that 'the iron was not excreted to any significant extent in the urine but became generally 10 distributed throughout the body, the complexes donating iron to , transferrin to an equilibrium level once they are present in the bloodstream.

Certain aspects of their formulation may enhance the activity of the complexes in particular contexts. Thus, although the neutral 3:1 ferric complexes are st&le over a wide pH range from about 4 or 5 up to JO, they will dissociate at the pH values of less than 4 prevailing in the stomach to form a mixture of the 2:1 and I:) complex together with the free hydroxypyrone, and it has 59 been found that the blood levels of Fe achieved on administration 20 of the 3:1 complex into the small intestine are much higiher than when administration is made into the stomach. However, when the stomach contents is flushed to the small intestine in in. vivo cat experiments an increase of iron uptake occurs almost immediately. The undesirable effects of this dissociation on iron uptake may be 25 countered by using one or more of the following procedures in the formulation of the iron complex. Firstly, one of several variations may be employed which avoid or reduce exposure of the iron complex' to the acidic conditions of the stomach. Such approaches may range from a controlled release system, for example one based upon a 30 polymer, which simply provides a delayed release of the complex with time, through a system which is resistant to dissociation under acidic conditions, for example by the use of buffering, to a system which and is biased towards release under conditions such as prevail in the small intestine, for example a pH sensitive system yliich is stabilised 35 towards a pH of i to 3 such as prevails in the stomach birt not one of 7 to 9 such as prevails in the small intestine. Since the pH of the stomach is higher after a meal, it may be advantageous, whatever method of formulation is used, to administer the iron complexes at such a time.

A particularly convenient approach to a controlled release composition involves encapsulating the iron complex by a material which is resistant to dissociation in the stomach but which is adapted towards dissociation in the small" intestine (or possibly, if the dissociation is slow, in the large intestine). Such encapsulation may be achieved with liposomes, phospholipids ' generally being resistant to dissociation under acidic conditions. .The liposomally entrapped 3:1 iron(III) complexes can therefore survive the acid environment of the stomach without dissociating to the 2:1 and 1:1 complexes, and the free hydroxypyrone. On entry into the small intestine, the pancreatic enzymes rapidly destroy the phospholipid-dependent structure of the liposomes thereby releasing the 3:1 complex. Liposome disruption is further facilitated by the presence of bile salts. However, it is usually it is more convenient to effect the encapsulation, including micro-encapsulation, by the use of a solid composition of a pH sensitive nature.

The preparation of solid compositions adapted to resist dissociation under acidic conditions but adapted towards dissociation under non—acidic conditions is well known in the art and most often involves the use of enteric coating, whereby.tab lets, capsules, etc, or the inidividual particles or granules contained therein, are coated with a suitable material. Such procedures are described, for example, in the article entitled "Production of enteric coated capsules" by Jones in Manufacturing Chemist and Aerosol News, May 1970, and in such standard reference books as "Pharmaceutical Dosage Forms, Volume III by Liebermann and Lackmann (published by Marcel Decker). One particular method of encapsulation involves the use of gelatine capsules coated with a cellulose acetate phthalate/ diethylphthalate layer. This coating protects the gelatin capsule from the action of water under the acid conditions of the stomach where the coating is protonated and therefore stable. The coating is ho/ever destabilised under the neutral/ alkaline conditions of the intestine where it is not protonated, thereby allowing water to act on the gelatin. Once released in the intestine the rate of permeation of the intestine wall by the , water soluble 3:1 ironClII) complex is relatively constant irrespective of the position within the intestine, i.e. whether in the jejunum, ileum or large intestine. Other examples of methods of formulation which may be used include the use of polymeric hydrogel * formulations which do not actually encapsulate the iron complex 'but which are resistant to dissociation under acidic conditions.

A s-econd approach to countering the effect of the acidic conditions prevailing in the stomach involves formulation of the complex in the pharmaceutical composition together with the metal—free hydroxypyrone from which it is derived or a salt thereof containing a physiologically acceptable cation. The dissociation of the neutral 3:1 ferric complex, for example, involves various equilibria between this complex, the 2:1 and 1:1 complexes, and the metal-free compound, so that the presence of the latter will inhibit this dissociation. Any proportion of the free compound can be advantageous in this context but little further advantage accrues from increasing the proportion beyond a certain level. A preferred range for the molar proportion of the free compound present in compositions .according to the'present invention is thus from 0 to 100 moles of free hydroxypyrone: 1 mole of iron complex, particularly of the neutral 3:1 iron (III) complex. Conveniently, a proportion of up to no more than 20, 30 or 50 moles:! mole is used with a lcwer level of 1 or 2 moles: 1 mole, although to obtain a marked effect upon dissociation of the iron complex a proportion of at least 5 or 10 moles:1 mole is usually employed. Thus a range of particular interest for the molar proportion of uncomplexed hydroxypyrone: iron complex is from 1:1 to 100:1, for example from 5:1 to 50:1 or from 10; 1 to 20:1. It will be appreciated that a solid mixture of the hydroxypyrone and the corresponding 207045 3:1 iron(III) complex in a proportion of from 1:1 to J00:1 is novel and that such novel mixtures are included by the present invention. The use of such a mixture is an important feature of the present invention since in principle it can enable one to 05 obtain almost quantitative uptake of iron from the complex.

The use of an uncomplexed hydroxypyrone or a salt thereof in ' admixture with its iron complex may alsa-have another advantage in addition to the prevention of dissociation of the iron complex under acidic conditions. Thus, in certain pathological conditions 10 y there may be an excess of iron deposited at certain sites even though the patient exhibits an overall anaemia. In these patients' the use of such a mixture has the advantage that the iron complex will remedy the overall anaemia whilst the free hydroxypyrone will act to remove iron from pathological to physiological sites. 15 However, although it is preferable for the hydroxypyrone present in an iron donor to be rapidly metabolized in order that iron may be efficiently transferred to the binding proteins and eventually to the iron requiring mechanisms within the body, it is preferable for a hydroxypyrone being used as an iron remover not to be rapidly 20 metabolized so that it remains in the system, taking up iron, for an extended period. Thus, for example, maltol is rapidly metabolized and is therefore particularly suited for use as an iron complex, but for this same reason it is not appropriate for use in the free form. It is also the case that different compounds may 25 function more efficiently either in the free form as an iron remover or in complex form as an iron donor for quite other reasons. Accordingly the present invention includes a mixture of an iron complex of 3-hydroxy- 4- pyrone or of a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon 30 atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, together with a different such 3-hydroxy— 4-pyrone or a salt thereof containing a physiologically acceptable cation.

Alternatively, the different 3-hydroxy-4-pyrone may be „ replaced by a quite different form of iron chelatjj Examples of such other iron chelating agents whipft may b^^^ed * tnfl .joctW* 207045 - 16a - include the substituted 3-hydroxypyrid-2-ones and -4-ones, and 1-. hydroxypyrid-2-one and siistituted I-hyiroxypyrid-2-ones (and salts of these various pyridones containing a physiologically acceptable cation) described in UK Patent 2118176 (Application 05 8308056) and UK Patent Applications 2 136 807 (8308054) and 2 146 990 (8325496).

When a free hydroxy-4-pyrone, hydroxypyrid-2-one, hydroxypyrid— 4-one or salt thereof, or other iron chelating agent is present in admixture with the iron complex of a hydroxy-4-pyrone for the 10 . purpose of acting as an iron remover, then the amount of this ' agent used may be different than when a. free hydroxypyrone -necessarily corresponding to that present in the iron complex is present primarily to prevent dissociation. Thus the daily dosage of the iron complex may be as above and the daily dosage of the 15 free iron chelating agent, particularly vhen this is a hydroxypyrid-2- or -4—one or a 1—hydroxypyrid-2-one, may be that quoted in the co-pending applications referred to above, i.e. about 0.1 g to 5 g for human use, particularly 0.5 g to 2 g, from which it will be seen that the proportion of iron complex and free iron chelating 20 agent used in such a context may extend across a wide range but preferred amounts of the free iron chelating agent tend to be higher than when this is necessarily a hydroxypyrone.

In addition to the pharmaceutical uses of the iron complexes discussed above they are also of potential interest as a source of 25 iron in various other ..contexts including cell and bacterial growth, plant growth, and the control of iron transport across menbranes. This invention is illustrated by the following Examples:- E XA.~MPI.ES Example 1 The preparation of iron maltol A chloroform solution of maltol is mixed with a 1M solution of ferric chloride in ethanol to provide a 3:1 molar ratio of maltol:iron in the mixture. After 5 minutes at 20°C, a 10 molar excess of solid sodium caxbonate is added and the mixture is 207045 - I6b — stirred for 10 minutes. The mixture is then filtered and the solvent evaporated to give the neutral complex containing maltol and the ferric cation in 3:1 proportion. Recrystallisation of the 3:1 complex from ethanol gives wine red needle crystals in an 05 essentially quantitative yield, m.p. 275°,V (nujol) 1600cm"'. max The use of an excess of maltol above the 3:1 molar ratio ' leads to an essentially quantitative yield of a solid mixture of the excess maltol and the 3:1 iron maltol complex on rotary evaporation, this mixture not being deliquescent.

, The partition coefficient Kpart (concentration in n-octanol/ concentration in aqueous phase) between n-octanol and Tris hydro- ' chloride (20 mM, pH 7.4) of maltol and of its 3:1 iron complex is -4 measured at 10 Mby spectrophotometry. Acid washed glassware is used throughout and, following mixing for 1 minute, the aqueous/ 15 n-octanol mixture is centrifuged at 1000 g for 30 seconds. The two resulting phases are separated for a concentration determination by spectrophotometry on each. For maltol, the range 220-340 nm is used for the concentration determination whilst for the complex the range 340-640 nm is used. Typically, a value of 0.66 is 20 obtained for maltol and of 0.50 for its iron complex, whilst comparative experiments on iron(III) EDTA and iron(III) ascorbate give much smaller values of 0.001 and 0.0015, respectively.

The ability of the iron complex of maltol to bind to haemo- '59 glob in is investigated by studying the elution profile of a Fe 59 label when a mixture of haemoglobin and the Fe-labelled complex ' (at 1 mM concentration) in NaCl (130 m*0 buffered to pH 7.4 by Tris hydrochloride is applied to a PD-10 column (Sephadex G-10 gel permeation column - Pharmacia). Typically, no evidence is found 30 for binding of the complex to haemoglobin which is an advantageous finding since such binding reduces availability of the iron.

The ability of the iron complex of maltol to bind to bovine serum albumen (BSA) is investigated through a similar procedure in which the complex is applied to a column with BSA ra^t^rf^s^Jian 35 haemoglobin. The iron complex also shows littlerability tfd^ind to BSA. 'c eJ 207045 Example 2 In vitro tests on permeation of iron complexes into human erythrocytes The accumulation of iron by human erythrocytes which are 05 associated with the iron complex of maltol described in Example 1, and various other iron compounds by vay of comparison, was studied by incubating the erythrocytes for 1 hour at 37°C in a medium 59 consisting of the Fe labelled iron compound in aqueous sodium chloride (130 mM) buffered to a pH of 7.4 by Tris hydrochloride.

Following this period of incubation an aliquot of the erythrocyte/ medium mixture was placed above a layer of silicone oil (p ■ 1.06) and the erythrocytes separated by ceatrlfugation through the oil. 59 The Fe levels associated with the erythrocytes and the incubation medium were then counted and presented as a distribution ratio 15 (concentration in erythrocytes/ concentration in medium). The ratios obtained for the various Iron compounds after incubation for 1 hour are shown in Table 1 where it will be seen that the uptake of Iron is clearly much greater with the iron maltol complex than with the other compounds. Values of less than 0.1 are probably 20 associated with binding to the external surface and do not represent transmembrane movement of iron. Moreover, although a period of 1 hour was employed in order to facilitate monitoring of the more slowly permeating iron compounds, the uptake of the iron maltol complex reached equilibrium at the level shown after about 15 25 minutes.

Table 1 Concentration Distribution Compound (mM) ratio Fe111 (maltol)j 3 1.60 Fe** gluconate 1 0.08 Fe1*1 ascorbate 1 0.12 Fe*** citrate 1 0.05 Fe*** EDTA 1 0.05 When the above described procedure was applied using ratios of maltol to Iron of less than 3:1 larger apparent distribution ratios were observed than 1.60. However, this is explained by the non-specific binding of the positively charged 2:1 and 1:1 maltol: 05 iron complexes to the surface of the erythrocytes which possesses a net negative charge, being rich in both phosphate and sulphate 59 moieties. Experiments to determine the percentage of Fe associated with erthrocyte ghosts after lysis confirm this hypothesis. In one experiment, lysis was initiated by a small 10 volume of 102 v/v Triton X100 and in a second experiment by a 10 fold excess of water. In each case the resulting ghosts were centrifuged through silicone oil (p ■ 1.02) and, as will be seen from Table 2, very little of the 3:1 maltol:iron complex was found to be bound to the membranes, in contrast with the situation with 15 the 2:1 and 1:1 complexes. Such binding is of course undesirable as the complex is likely to remain tightly bound to the membrane by electrostatic Interactions and not be transmitted across it. 207045 Table 2 Ratio of maltol:iron Iron associatled with with ghosts (£) Triton lysis Hypotonic lysis 0:1 100 — 1:1 55 63 2:1 22 39 3:1 <1 <5 Example 3 In vitro tests on permeation of rat jejunal sac by iron complexes The iron uptake into the serosal space of the rat jejunal sac was compared for the iron complex of maltol described in Example 1 05 and various other iron compounds by way of comparison. Rats (male Sprague Dawley, 60 g) were killed and the jejunum removed, everted and cut into three segments (4 cm length). The segments were tied at both ends, filled with Krebs Ringer buffer (0.2 ml) and incubated 59 in Krebs Ringer buffer containing the appropriate Fe compound at 37°C for periods up to 90 minutes. The contents of the sac 59 were counted for Fe and measured spectrophotometrically.

The results obtained for the iron maltol complex and for 6 other iron compounds which are each contained in preparations marketed for the treatment of iron deficiency anaemia are shown in 15 Table 3, the iron uptake for each at 15 and 60 minutes after the initiation of the experiment being shown relative to that for ferric chloride as 1. It will be seen that the Iron maltol complex provides a level of iron uptake which is significantly higher than the levels observed for any of the 6 compounds in current use for 20 the treatment of iron deficiency anaemia. The uptake of the iron maltol complex was linear for a period of 90 minutes. Moreover, the uptake increased linearly as the concentration of the complex was Increased over a range from 0.5 to 10 mM, so It does not show saturation kinetics and the process is thus non-facilitated and therefore should occur in all natural membranes.

Table 3 Compound Relative iron uptake minutes 60 minutes FeCl3 1 1 Fe111 (maltol)3 AO .8 Fe** sulphate 2.A 1.4 Fe** fumarate 4.0 1.8 Fe** gluconate 1.6 0.8 Fe** succinate 2.0 1.0 » HI Fe ascorbate 0.4 0.8 Fe*** citrate 2.0 1.8 The procedure described above was used to compare the uptake of iron from buffer containing differing molar proportions of maltolriron. The results obtained are presented in Table 4 which shows the amount of iron transferred via the maltol complex into the serosal contents of the sac, the basal uptake of iron measured in a control experiment being subtracted in each case. It will be seen that the amount of iron transferred in the case of a 3:1 molar proportion of maltol:iron(III) is much higher than in the other two cases and, moreover the low, but significant level of iron uptake observed in the case of a 2:1 ratio is attributed to the proportion of the 3:1 complex (containing 13% of the total iron) present under these conditions. 207045 Table 4 Maltol/iron Iron uptake (molar ratio) (n mole) 1:1 1.6 2:1 4.0 3:1 .0 Example 4 In vivo test of action of Iron compounds In the rat The action of the Iron complex of maltol described in Example 1 was compared with that of iron(II) sulphate, iron(III) EDTA (1:1 O5 molar ratio) and iron(II) gluconate.

Groups of rats (300-350 g) were anaesthetised with nembutal (0.25 ml) and then with ether. A mid-line incision 59 was made and the Fe labelled sample (100 wg Fe, 10 yCi) was passed into the lumen of the duodenum via a small Incision. The abdominal well was then closed with a suture. The animals were sacrificed 1, 2, 4 and 6 hours after the administration of the 59 compound and the various organs were monitored for their Fe content. The data is presented as histograms in Figures 1 to 4 which relate to iron maltol, iron sulphate, iron EDTA and iron 15 gluconate, respectively, and show the levels of ^Fe in cpm after various time intervals for the different organs, the data in each case representing a mean of the results for three individual animals. In the case of the data for blood and sternum (bone marrow) the counts given are cpm/ml and cpm/g respectively, whilst 20 in all other cases they are the total cpm counts. The various histograms have been normalised and consequently are directly comparable.

A comparison between Figures 1 and 2 shows that the neutral 3:1 maltol:iroa(III) complex is markedly superior to 25 iron(II) sulphate for the introduction of iron via the rat intestine. The gut washings (which contain non-absorbed iron) show a much lower level of counts for the maltol complex, and the 2070. counts associated with the gut wall, liver, blood, bone max^row and spleen are correspondingly greater. It is clear from Figuije 1 chat ^Fe associated with maltol enters the intestine wall ^ery rapidly and from there it is efficiently removed by the blojbd 05 supply. Iron is deposited in the bone marrow continuously "if throughout the 6 hour period at an apparently constant rat?£ I The maltol complex is also more efficient than iron(II$0 EDTA as shown by Figure 3. With the later complex, tha guej washings remain high for 4 hours and may be presumed tcc decrease only due to the effect of natural bowel movements translocating ' material from the portion under investigation to lower -portions of •the intestine. The levels in the intestine wall and blood ^re extremely lew. Although iron is transferred to both btfne narrow and spleen, this is at reduced rates as compared to those tained vith the maltol complex. As shown by Figure 4, iron(IB) gluconate proved more effective than the sulphate or the EDTA conrplekt although deposition in the gut wall was less than that xfc served with the maltol complex. The decrease was reflected ir* thef lower 59 levels of Fe in both bone-marrow and the spleen, the Idiff^rence 20 being particularly marked after 6 hours. In view of th'e tmi^h higher levels of Fe trapped in the intestine wall in .'the t£ase of the maltol complex, it may be predicted that this compound facilitates a more prolonged supply of iron than iron(II) gluconate.

This test illustrates the superiority of the neutral ^):1 25 maltol:iron(III) complex as compared with three commonly us^d "soluble iron" preparations for the movement of iron acros^ the rat jejunal wall into the blood circulation, the iron maltci^. being very rapidly removed from the lumen of the intestine.

Example 5 In vivo test of action of iron complexes in the cat ^ The action of the iron complex of maltol described in Example I was compared with that of iron(III) EDTA (1:1 molar ratio) ^rhich is one of the iron compounds currently marketed for the treatment of.iron deficiency anaemia. Cats were anaesthetised with dhlora-35 lase (60 rag/kg) and pentobarbitone sodium (60 mg/kg) (i.p.)i, having been kept free of food for 18 hours. In (ea&fcTlnMiWil the 207045 - 23 - ■ trachea was cannulated to maintain a clear airway and to allow positive pressure artificial respiration if necessary. The left femoral vien was cannulated for the intravenous administration of drugs and physiological saline solution. Arterial blood pressure was monitored by a Washington pressure transducer through a cannula inserted into the femoral artery of the right hind leg. Arterial blood samples were taken at appropriate Intervals from a short cannula Inserted into an external carotid artery. Body temperature was monitored with a rectal thermometer. Each animal was given heparin (1000 iu/kg) as anticoagulant and additional small amounts of pentabarbltone sodium if needed to maintain a satisfactory level of anaesthesia.

In those animals where the iron compounds were to be administered into the duodenum, a mid-line incision was made in the abdomen to reveal the intestines. A cannula was then inserted through a small cut such that its tip rested approximately 5 cm below the opening of the bile duct. The cannula was then sutured In place and the abdominal wall closed with stitches.

The iron maltol complex (100 yg Fe) alone (3:1 molar ratio of maltol:iron) and together with a large excess of maltol (40:1 molar ratio of maltol:iron) was Injected intravenously in separate experiments and 0.25 ml samples of blood were taken at intervals. The apparent volume of distribution of the compound was calculated by extrapolation of the log-linear blood concentration curve to zero. (The volume corresponds to a value between that of the total extracellular space of the animal and the blood volume.) 59 Elimination of Fe from the blood followed first order kinetics with a rate constant of -0.022/raimite in the presence and absence 59 of excess maltol, as illustrated in Figure 5 which shows the Fe level in the blood in cpm/0.25 ml plotted against time in the case of one typical experiment of each type in an individual cat. 59 The distribution of Fe in the tissues of the animal after the same intravenous experiment to which Figure 5 relates (the lower end of the ordinate in this Figure represents the background level) was Investigated and the typical results are shown in 59 Table 5. The amount of Fe administered in this experiment 207045 05 r) y was 4/aC1 or 2.2 x 10 cpm. It will be seen that approximately 10% of the dose was located in the combined tissue of the heart, liver and spleen. As less than 0.2% of the dose was located in the 59 urine, the bulk (approximately 902) of the Fe was almost certainly directed to the bone marrow and extremely high levels were found to be located In the sternum. , As indicated previously, the "maltol complex is able to donate iron rapidly to transferrin and It Is hypothesised that such an exchange occurs as soon as the complex is delivered to the plasma; and that the Initial plateau (represented in Figure 5 by a dotted line) represents saturation of the plasma transferrin pool with 59 When there is net donation of Iron from the plasma Into the organs of the animal, the blood levels of radioactivity begin to fall, the major route of transfer of iron bound to transferrin 59, 59 Fe. being to the bone marrow, liver and spleen. Binding of transferrin prevents Its excretion in the urine.

Table 5 (Iron maltol, i.v) Fe to Tissue Total tissue weight (g) Sample weight (8) 59 Net Fe content (cpm/g) Met 59 total Fe content (cpm) Heart . 14.4 ' 0.91 •. 490 • 7,056 Liver 105 1.3 510 53,550 Spleen 8.4 0.86 14,890 125,076 Kidney 12.2 1.05 546 6,661 Skeletal muscle - 1.85 0 0 Sternum - 1.2 3,200 - (bone marrow) Urine — 1 152 <3,000 59, Identical experiments carried out with '"Fe labelled iron(III) EDTA gave a entirely different picture a^^l-g^e for the results of a typical experiment illustrated in F^gi 207045 (In which the lower end of Che ordinate represents the background 59 level) and Table 6 (the amount of Fe administered in this experiment was 2/u.Cl but the figures given in the table have been adjusted to correspond to a dosage of 2.2 x 10^ cpm in order to facilitate comparison with Table 5). In this experiment the radioactivity in the blood showed no initial plateau. Instead, loss of radioactivity followed at least a two-component"process such that a large amount found its way to the urine rather than to the tissues. The rate constant of the elimination from the blood of the Linear phase of the regression was 0.023/minute. The concentration of radioactivity in the kidney and urine, and not in the bone marrov or spleen, would indicate that iron in this form does not appear to be able to attach to transferrin in the plasma and protect, itself from urinary excretion. The combined tissue of heart, Liver and spleen contained only 1Z of the original dose at the end of the experiment, whereas the urine contained over 50%. This is in accord with the fact that EDTA does not exchange iron with transferrin rapidly.

Table 6 (Iron EDTA, i.v.) Tissue Total tissue weight (g) Sample weight (g) 5D Net J Fe content (cpm/g) Net 59 total Fe content (cpm) Heart .5 1.01 209 3,248 Liver 75 1.21 261 19,600 Sternum - 0.28 1,164 - (bone marrow) Spleen 11.2 0.89 162 1,814 Kidney 19.2 1.47 1,134 21,770 Skeletal muscle - 2.59 95 - ' Urine 19 ml 2 ml 62,156 1,180,900 The iron maltol complex (100jug Fe) was also administered to the duodenum of the cat in the presence of a 40 f^M lepras of '•rs < * cl 207045 maltol followed by 5 ml of 150 ml Tris hydrochloride buffer (pH 7.4). 59 In this case the Fe content of the blood, as shown in Figure 7, reaches a maximum level 2 hours after the initial administration (the readings start at about 300 cpm/0.5 ml which represents the 59 05 background reading). The distribution of Fe in the tissues of the animal after the same duodenal experiment to which Figure 7 relates were investigated and the'* typical results are shown in 59 Table 7. The amount of Fe administered in this experiment was 10/xCt or 5.327 x 10^ cpm into a 2.9 kg cat. It will be seen 59 that the distribution of the Fe after 4 hours was similar to that after Intravenous infusion, with low levels in the kidney and urine and high levels in both the spleen and bone marrow.

Table 7 (Iron maltol, per duodenum) Tissue Total tissue weight (g) Sample weight (g) 59 Net Fe content (cpm/g) Net 59 total Fe content (cpm) Heart 14 0.633 50 1,106 Liver 81 1.45 400 32,400 Spleen 12.7 1.19 3,783 48,047 Kidney 14.4 0.835 79 1,138 Sternum 1.26 790 7,905 (bone marrow) Bile ~5 ml 1 ml 2,200 -11,000 Urine —10 ml 1 ml 22 -220 59 When Fe labelled lron(III) EDTA was administered duodenally in the same manner, the plasma levels of radioactivity hardly exceeded the background level and are therefore not illustrated in a Figure. 59 The distribution of Fe in the tissues of the animal after the same duodenal experiment were investigated and the typical results 59 are shown in Table 8. The amount of Fe administered in this 207045 - 27 -59 vilL be seen that, although some Fe entered the tissues, rather low levels were detected In the spleen and bone narrow (sternum) whereas a large proportion of the dose was located In the urine. Table 8 05 (Iron EDTA, per duodenum) O Tissue Total tissue weight (g) Sample weight (8) Net 59Fe content (cpm/g) Net 59 total Fe content (cpm) Heart .3 1.18 188 2,878 Liver 59.3 0.78 499 29,574 Kidney 11.3 0.90 1,762 19,913 Spleen 4.4 0.42 200 880 Sternum - 0.78 917 - (bone marrow) Skeletal muscle - 1.48 117 - Urine ml ml 36,306 544,596 Example 6: Polymer formulation of iron maltol A solution of ferric chloride (concentration between 1 and 5Z w/v) , together with maltol in a weight ratio of 8 parts by 10 weigtit of maltol to 1 part by weight of ferric chloride, is prepared in a 4.5:4.5:1 v/v/v mixture of chloroform:methanol:vater. Sodium carbonate is added in a 10 molar excess over the iron content in order to remove hydrochloric acid and the precipitated NaCl and Na^CO^ are filtered off. The preparation is contacted with a 15 cross-linked polyethylene glycol hydrogel material to effect take up of the solution by the polymer and provide a polymer formulation of iron maltol in the presence of excess maltol.

Example 7 Capsule formulation of iron maltol 20 A preparation of iron(III) maltol in admixture with maltol (containing 1 part by weight of Iron to 10 parts by wa£ \ r % c E V^ ■: ^ 207045 maltol) is obtained by the addition of a 1M ethanollc solution of ferric chloride to a methylene chloride solution of the appropriate amount of maltol, followed after 5 minutes at 20°C by treatment with a 10 molar excess of solid solution carbonate, stirring for 10 minutes, filtration and evaporation of the solvents.

The resulting solid iron(lll) maltol preparation is divided Into 50 mg quantities and added to standard gelatine capsules (16 x 5 mm), each capsule containing 5 mg of iron. The capsules are then coated with a cellulose acetate phthalate/dlethyl- 2 phthalate layer (6 mg coat per cm of capsule surface) in a small scale procedure analogous to the procedure described by Jones, Ibid. A proportion of the capsules are treated to add a second similar coating.

Such capsules are resistant to dissociation in the stomach but will undergo dissociation In the intestine. Thus, when treated at 37°C with dilute aqueous hydrochloric acid (pH 2.0) the singly coated capsules are typically stable for 30 minutes but in Krebs Ringer bicarbonate solution (pH 7.4) at 37°C they dissociate to release the iron complex within 1 minute. The doubly coated capsules are typically stable at pH 2.0 for 20 hours, again dissociating within 1 minute at pH 7.4.

Example 8 Liposome formulation of Iron maltol (A) A solution of egg yolk phosphatidyl chlorine^*^ (40 mg) and cholesterol (40 mg) in chloroform (1 ml) is rotary evaporated in a 50 ml round bottomed flash to form a thin lipid film. An aqueous solution of the 3:1 neutral iron(III) maltol complex (6 ml, 1 mg/ml) is added to the flask and the mixture is vibrated for 15 minutes. Centrifugation (3,000 rev/minute for 10 minutes) yields multilammelar liposomes containing iron(III) maltol.

In a modification of this procedure the phospholipid may be varied among egg yolk phosphatidyl chlorine, dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine together with a preparation of cholesterol varying from 0 to 1 moles of cholesterol per mole of phospholipid. 207Q45 (B) A chloroform solution (2 ml) of the 3:1 neutraL lron(III) maltol complex (5 mg/ml) is added to egg yolk phosphatidylcholine (100 mg) and cholesterol (50 mg). The solution is rotary evaporated to yield a deep red skin on the surface of a round 05 bottomed flask. Addition of 6 ml of a buffered solution of sodium chloride (100 mM, Trls.HCl: 20 mM, pH 7.4) followed by shaking for 15 minutes leads to a finely dispersed lipid-iron(III) maltol preparation. Centrlfugation at 3000 revs/minute for 10 minutes yields a liposome preparation which can be readily freeze dried. 10 The entrapment of iron(III) maltol using this method Is particularly efficient.

Liposomes produced by either method are resistant to dissociation in the stomach but will undergo dissociation in the intestine. 34B 207045 WHAT WE CLAIM IS:

Claims (48)

CLAIM-

1. A pharmaceutical composition comprising a physiologically effective amount of a neutral 3:1 hydroxypyrone:iron(III) complex of 3-hydroxy-A-pyrone or of a 3-hydroxy-A-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, together with a physiologically acceptable diluent or carrier, but excluding any liquid which is non-sterile and non-pyrogen free.

2. A pharmaceutical composition comprising a physiologically effective amount of a neutral 3:1 hydroxypyrone:iron(III) complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, together with a physiologically acceptable solid carrier.

3. A pharmaceutical composition according to Claim 2, which is adapted for oral administration.

4. A pharmaceutical composition according to Claim 3, which is formed.

5. A pharmaceutical composition according to Claim 4, which is formed as t& lets or capsules.

6. A pharmaceutical composition according to Claim 2 in suppository form.

7. A pharmaceutical composition comprising a physiologically effective amount of a neutral 3:1 hydroxypyrone:iron(III) complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, together with a physiologically acceptable diluent or carrier which includes a glycol or glycol ether.

8. A pharmaceutical composition according to Claim 7 which includes a polyethylene glycol.

9. A pharmaceutical composition comprising a physiologically effective amount of a neutral 3:1 hydroxypyrone:iron(III) complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon, atoms, together with a physiologically acceptable diluent, the ^cptBe^ition being of a sterile injectable form. ,i'y :/A Ni 3«

10. a pharmaceutical composition comprising a physiologically effective amount of a neutral 3:1 hydroxypyrone:iron(III) complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-A-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, together with a physiologically acceptable diluent or carrier, I the composition b eing of a delayed release form.

11. A pharmaceutical composition according to Claim 10, which is adapted for release of the Iron complex in the intestine rather than in the stomach.

12. A pharmaceutical composition comprising a physiologically effective amount of a neutral 3:1-hydroxypyrone:iron(III) complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, together with a physiologically acceptable diluent or carrier, the composition being of a form resistant to dissociation under aqueous acidic conditions.

13. A pharmaceutical composition according to Claim 12, in which the iron complex Is encapsulated by a material resistant to dissociation under aqueous acidic conditions.

14. A pharmaceutical composition according to Claim 13,. in which the Iron complex is encapsulated by a solid material which is resistant to dissociation under aqueous acidic conditions but which is adapted for 'dissociation under aqueous non-acidic conditions.

15. A pharmaceutical composition according to any one of the •preceding claims, in which the or each aliphatic hydrocarbon group is of 1 to 4 carbon atoms.

16. A pharmaceutical composition according to any one of the preceding claims, in which the or each aliphatic hydrocarbon group is a cycloalkyl or alkyl group.

17. A pharmaceutical composition according to any one of the preceding claims, in which the or each aliphatic hydrocarbon group is an acyclic group. 3X- 20704S

18 . A pharmaceutical composition according to any one of Claims 1 to 14, in which the complex is of a 3-hydroxy-4-pyrone wherein one or more of the hydrogen atoms attached to ring carbon atoms are replaced by the same or different substituents selected from Che group consisting of methyl, ethyl, n-propyl and Isopropyl.

19. A pharmaceutical compositioh according to Claim 13, in which the or each substituent is a methyl group. »

20. A pharmaceutical composition-according to any one of the preceding claims, in which the substituted 3-hydroxy-4-pyrone has a single substituent at the 2- or 6-position.

21- !A pharmaceutical composition according to any one of Claims 1 to 14, in which the complex is of 3-hydroxy-4-pyrone, 3-hydroxy-2-■ methyl-4-pyrone, 3-hydroxy-6-methyl-4-pyrone or 2-ethyl-3-hydroxy-4-pyrone.

22. A pharmaceutical composition according to Claim 21, in which the complex is of 3-hydroxy-2-methyl-4-pyroae.

23. ! A pharmaceutical composition according to Claim 21,, in which the complex is of 2-ethyl-3-hydroxy-4-pyrone.

24.I A pharmaceutical composition according to any one of the preceding claims in which the neutral 3:1 complex is substantially free from complexes containing other proportions of the hydroxypyrone and lron(IIl) .

25. A pharmaceutical composition according to Claim 24,. in which the Iron complex is in substantially pure form.

26. A pharmaceutical composition according to any one of the preceding claims, which contains an iron chelating agent as an. additional active component thereof.

27. A pharmaceutical composition according to Claim 26, in which the iron chelating agent is uncomplexed 3-hydroxy-4-pyrone or an uncomplexed 3-hydroxy-4-pyrone in which at least one of the hydrogen atoms attached to ring carbon atoms is replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, or a salt thereof containing a physiologically acceptable cation.

28. -A pharmaceutical composition according to Claim 27, which contains a complexed hydroxypyrone together with the same hydroxy-pyrone or a salt thereof in uncomplexed form.

29. A pharmaceutical composition according to Claim 28, which comprises the neutral 3:1 iron complex of 3-hydroxy-2-methyl-4-pyrone and uncomplexed 3-hydroxy-2-methyl-4-pyrone or a salt thereof. 207045 - 33 -

30. A pharmaceutical composition comprising: (a) a physiologically effective amount of a neutral 3:1 hydroxypyrone:iron(III) complex of 3-hydroxy-4-pyrone or of a 3-hydroxy—4— pyrone in which one or more of the hydrogen atoms attached Co ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon acoms; and (b) an amount effective to inhibit dissociation of said complex' at a pH less than 4 of the same hydroxypytone, or a salt thereof containing a physiologically acceptable cation, in uncomplexed form; together with a physiologically acceptable diluent or y carrier.

31. A pharmaceutical composition according to Claim 30, in which Che complex is as defined in any of Claims 15 to 23.

32. A pharmaceutical composicion according to any one of Claims 28 to 31 , in which the molar proportion of uncomplexed hydroxypyrone:iron complex is from 3:1 Co 100:1.

33. A pharmaceutical composition according to any one of the preceding claims, which contains folic acid as an additional active component thereof.

34. A pharmaceutical composition according to any one of the preceding claims in unit dosage form.

35. A pharmaceutical composition according to any one of the preceding claims for human use.

36. A neutral 3:1 hydroxypyrone:iron(III) complex of 3-hydroxy—4-pyrone or of a 3-hydroxy-4^-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms, but excluding 3-hydroxy-2-methyl- 4-pyrone.

37. A complex according to Claim 36, being the neutral 3:1 iron complex of 3-hydroxy—4-pyrone, 3-hydroxy—6—methyl-4-pyrone or 2- e thy 1- 3—hydr oxy-lr- pyrone. 207045 - 34 -

38. A complex according co Claim 36,being che neutral 3:1 iron complex of 2-echyI-3-hydroxy-4-pyrone.

39. A complex according Co Claim 36, 37 or 38,,' being substancially free from iron complexes concaining other proportions of che hydroxypyrone and iron(III).

40. A mixture in the solid form of (a) a neucral 3:1 hydroxypyrone: iron (III) complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4—pyrone in which one or more of che hydrogen atoms attached Co ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 Co 6 carbon acoms; and (b) che same hydroxypyrone, or a sale thereof concaining a physiologically acceptable cation, in uncomplexed form; the molar proportion of uncompltexed hydroxypyrone:iron complex being from 1:1 to 100:1.

41 . .A mixture of (a) a neucral 3:1 hydroxypyrone:lron(III) complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone In which one or more of the hydrogen atoms attached to ring carbon atoms is replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms; and (b) an iron chelating agent other than the particular hydroxypyrone which is present in the canplex, in uncatplexed form.

42. A mixture of (a) a neutral 3:1 hydroxypyrone:lron(III) complex of 3-hydroxy-4-pyrone or of a 3-hydroxy—4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms Is replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms; and (b) a different hydroxypyrone being 3-hydroxy-4-pyrone or a 3-hydroxy-4-pyrone In which one or more of the hydrogen atoms attached to ring carbon atoms is replaced by an aliphatic hydrocarbon group of 1. to 6 carbon atoms, or a salt thereof containing a physiologically acceptable cation, in uncomplexed form.

43. A mixture according to Claim 40, 41 or 42. which the complex is as defined in anyone of Claims .15 to 23.1

44. A pharmaceutical composition comprising a complex according to any one of Claims 36 to 39 or a mixture according to any r of Claims 41 to 43, together with a physiologically acceptable diluent 03 -35- 207045

45. A method of increasing the level of iron in the blood of a non-human patient comprising administering an iron complex of 3-hydroxy-4-pyrone or of a 3-hydroxy-4-pyrone in which one of more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms.

46. A process for Che preparation of a neutral 3:1 hydroxypyrone: iron (III) complex of 3-hydraxy-4-pyxone or of a 3-hydraxy-4-pyrone in which one or more of the hydrogen acoms attached to ring caxbon atoms are replaced by an aliphatic hydrocarbon group of 1 Co 6 carbon acorns, but excluding 3-hydroxy-2-methy 1-4-pyrone, which comprises reacting the hydroxypyrone with ferric Ions at a pH in the range of 6 to 9 Co form Che 3:1 complex and isolating said complex in the solid form.

47. A process- according to Claim 46,' in which the complex is as defined in any- of Claims 15 to 25.

48. A pharmaceutical composition according to Claim 1 or 30 substantially as described with reference to the Examples. By Xis/Their authorised Agent A. J. PARK & SON) Per,