WO1987007893A1

WO1987007893A1 - Pharmaceutical compositions

Info

Publication number: WO1987007893A1
Application number: PCT/GB1987/000400
Authority: WO
Inventors: Jack Silver
Original assignee: National Research Development Corporation
Priority date: 1986-06-20
Filing date: 1987-06-10
Publication date: 1987-12-30
Also published as: GB8713566D0; EP0271529A1; GB2192130A; GB8615041D0; JPS63503458A

Abstract

Neutral 3:1 hydroxypyrone:chromium(III) complexes in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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 are of value for use in the treatment of conditions such as diabetes responding to an increase in the level of chromium in the bloodstream.

Description

PHARMACEUTICAL COMPOSITIONS This invention relates to chromium compounds and to their use both as a therapeutic agent and as a dietary supplement in the treatment of chromium depletion.

An adequate supply of chromium to the body is an essential requirement for health in both animals and man. Dietary chromium deficiency may lead to the ageing-related condition of chromium depletion observed in man. Such depletion, when it occurs with excessive consumption of sugar and other carbohydrates, may result in glucose intolerance, glycosuria, hyperinsulinemia and hyperlipidemia. Consequently it has been proposed that chromium(III) supplementation might prove helpful in treating conditions such as atherosclerosis and diabetes mellitus and clinical trials have been carried out using chromium chloride to provide a chromium supplement to impaired glucose tolerance. Moreover, in European Applications Nos. 80200219.6 and 81200316.8, published as 0016496 and 0037144 respectively, various chromium(III) complexes, for example of acetyl acetone, malonaldehyde and 3-oxobutanal , are proposed for use in the treatment of chromium depletion. I have now identified a group of hydroxypyrone chromium complexes which is of particular value for use at relatively low dosage levels in the treatment of chromium deficiency. Although these hydroxypyrones are known to form various metal complexes and, indeed, a chromium complex of one of the compounds, 3-hydroxy-2- methyl-4-pyrone (commonly known as maltol), has previously been prepared for the purposes of determining its magnetic moment and ultraviolet and infrared spectra (Morita et a ., Chemistry Letters, 1975, 339), the particular value in the treatment of chromium deficiency of their chromium complexes, as shown by ijn vivo experiments, is quite unexpected. Accordingly the present invention comprises a neutral 3:1 hydroxypyrone:chromium(III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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 use in therapy.

The chromium complexes of the present invention are also of particular interest in that several of the hydroxypyrone compounds from which the complexes are derived have previously been used either as the metal-free compound or as its iron complex in foodstuffs, thereby indicating their non-toxic nature and the consequent suitability for pharmaceutical use of the chromium complexes of these compounds.

The 3:1 chromium complexes used in the present invention contain chromium in the trivalent state and are neutral, there being an internal balance of charges between the metal cation and the three monobasic, bidentate ligands bound covalently thereto which are provided by the loss of a proton from the hydroxy group of the hydroxypyrone (OH —^ 0 ). The inclusion in a complex of two or even three different ligands can produce an added dimension to the design of complexes having optimised properties for uptake inn vivo and can also present advantages in certain methods of formulation. However, complexes containing three identical hydroxypyrone ligands are more generally preferred by virtue of their greater simplicity of preparation and use.

As regards the alternative forms of hydroxypyrone ligands present in the complexes of use in the present invention, 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 1 to 4 carbon atoms and particularly of 1 to 3 carbon atoms are of 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 ethyl, n-propyl , isopropyl, and especially 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.

0 OH This system is involved in the complexing with chromium and the close proximity of one of the larger aliphatic hydrocarbon groups may lead to steric effects which inhibit complex formation.

Examples of hydroxypyrones providing ligands present in the complexes^'used in the present invention have the formula (I), specific hydroxypyrones of particular interest having the formulae (II) and (III):-

in which R is a cycloalkyl or especially an alkyl group, for example methyl, ethyl, n-propyl, isopropyl or butyl, and n is 0, 1, 2 or 3 (the ring being unsubstituted by any alkyl group when n is 0). Among these compounds 2-ethyl-3-hydroxy-4-pyrone (II, R = C2H5), 3-hydroxy-2-propyl-4-pyrone (II, R = CH2CH2CH3), 3-hydroxy-2- (T-methylethyl)-4-pyrone (II, R = CH(CH3)2) and especially 3-hydroxy-2-methyl-4-pyrone (II, R = CH3; commonly known as maltol), are of most interest, although 3-hydroxy-4-pyrone (I, n = 0) and 3-hydroxy-6-methyl-4- pyrone (III, R = CH3) are also of particular interest. Among the hydroxypyrone ligands described above certain ligands or combinations of ligands are of particular value and some indication of these has already been given. One measure of the value of the different ligands is provided by the partition coefficient for the ligand-providing compound ( _part) between n-octanol and Tris hydrochloride (20 mM, pH 7.4; Tris representing 2-amino-2 hydroxymethylpropane 1,3-diol) at 20°C, this being expressed as the ratio (concentration in organic phase)/ (concentration in aqueous phase). Preferred ligands have a value of Kpart °^{r tne} ϋgand-providing compound of above 0.02 or 0.05 but less than 3.0, especially of above 0.2 but less than 1.0.

Examples of specific chromium(III) complexes of use in the present invention are those complexes containing three identical ligands drawn from the hydroxypyrones named hereinbefore as being of particular interest, especially chromium(III) (3-hydroxy-2- methyl-4-pyrone)3, chromium(III) (2-ethyl-3-hydroxy-4-pyrone)3, chromium(III) (3-hydroxy-2-(l '-methylethyl)-4-pyrone)3 and chromium(III) (3-hydroxy-2-propyl-4-pyrone) . Among complexes containing two or three different ligands, specific examples are those containing each of the possible combinations of mixed ligands selected from the group consisting of 3-hydroxy-2-methyl-4-pyrone, 2-ethyl-3-hydroxy-4-pyrone, 3-hydroxy-2-(l '-methylethyl )-4-pyrone and 3-hydroxy-2-propyl-4-pyrone, particularly those containing the first mentioned ligand as one of the three present in the complex and especially those containing only two different ligands such as chromium(III) (3-hydroxy-2-methyl-4-pyrone) (2-ethyl-3-hydroxy-4- pyrone)2 and chr^'omium(III) (3-hydroxy-2-methyl-4-pyrone)2 (2-ethyl-3-hydroxy-4-pyrone) . It will be appreciated that the names of the hydroxypyrones are used in the names of these complexes to represent the ligands derived therefrom, this usage being employed throughout the specification.

Apart from the compound chromium(III) (3-hydroxy-2-methyl-4- pyrone)3 which has been described by Morita et al_, all of the other complexes of use in the present invention are believed to be novel per se. The present invention thus further comprises a neutral 3:1 hydroxypyrone:chromium(III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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 with the exclusion of that complex in which each of the three ligands is provided by 3-hydroxy-2-methyl-4-pyrone. The chromium complexes are conveniently prepared by the reaction of the compound or compounds which provide the ligands and Cr⁺⁺⁺ ions, the latter conveniently being derived from a chromium salt, for example chromic nitrate. The reaction is conveniently effected in a suitable mutual solvent and water may be used for this purpose. If desired, however, an aqueous/organic solvent mixture may be used or an organic solvent, for example ethanol , methanol or chloroform and mixtures of these solvents together and/or with water where appropriate. In particular, an aqueous methanolic or aqueous ethanolic medium may be used for the reaction.

To prepare the chromium complexes containing a 3:1 molar proportion of ligand:chromium(III) a similar molar proportion of ligand-providing compound(s) :chromium(III) salt may conveniently be employed in the reaction mixture. Where the complex contains more than one form of hydroxypyrone ligand, the individual ligand providing compounds may conveniently be used in the reaction mixture in the same molar proportion as that of the ligands in the complex, i.e. a proportion of 1:1:1 when three different ligands are present and of 2:1 when two different ligands are present. It will be appreciated, however, that the use of such proportions will not lead exclusively to the complex containing three different ligands or to the selected complex containing two ligands of one type and one of the other since, although these forms of complex will predominate providing the ligand-providing compounds are of similar reactivity, they will be obtained in admixture with other forms of complex.

Thus, even when only two hydroxypyrones (A and B) are reacted with Cr^H"+ ions a range of different complexes may be present in the reaction mixture: CrA3, CrE$3, CrAB2 and CrB A. If it is desired to enhance the degree of admixture of different forms of complex which is obtained, the proportions of reactants may be varied to this end. Thus, for example, a 1.5:1.5 molar proportion of two different ligands may be used to encourage the formation of a mixture of the two possible types of 2:1 complex differing in the ligand which predominates. it has been found that the reaction of the ligand-providing compound(s) and the chromium ions is conveniently carried out in the presence of a controlled quantity of sodium hydroxide sufficient to raise the pH above 7 but not above 8.5, a value in the range of 7.5 to 8.5, for example about 8, being suitable. Refluxing of a solution in water at the appropriate pH of the compound(s) and the chromium salt for a period of about 24 hours has been found to lead to a good yield of the complex. The complex may conveniently be isolated from an aqueous reaction mixture by evaporation thereof and extraction of the residue with a suitable organic solvent, particularly chloroform, evaporation of the dried solvent extract usually yielding the complex as a solid when all three ligands are identical although complexes containing more than one form of ligand will usually be obtained as an oil or a glass. Such a procedure enables the complex to be obtained in a form substantially free from by-products of manufacture, apart from other chromium complexes, and in the case of the complexes containing three identical ligands also substantially free from other such complexes. The term "substantially free from" is used herein to indicate th<e presence of 10% by weight or less of the material referred to. The present invention thus further includes a process for the preparation of a chromium(III) complex as described hereinbefore, which comprises reacting one, two or three hydroxypyrones, as described hereinbefore, with chromic ions but specifically excluding the preparation of chromium(III) (3-hydroxy-2-methyl-4-pyrone)3. It will be appreciated that since each of the three hydroxypyrone anions can be bound to the chromium cation in one of two orientations the complexes may exist in different stereochemical forms (all of which may be used in the present invention), even when the three anions are identical. When the complex contains anions derived from two or three different hydroxypyrones the number of isomers will be increased further. The reaction mixture obtained from the reaction of chromic ions with the hydroxypyrone or hydroxypyrones will contain a mixture of isomers of the complex or complexes which are formed. It is possible, however, to effect some separation of these isomers by chromatography, for example on aluminium hydroxide, and certain isomers may be of particular interest either by virtue of their biological activity or their physical properties.

Certain of the ligand-providing hydroxypyrone compounds, such as maltol, are available commercially. With others, a convenient starting material in many instances consists of pyro econic acid (3-hydroxy-4-pyrone) , which is readily obtainable by the decarboxylation of meconic acid (2,6-dicarboxy-3-hydroxy-4-pyrone) , and may be reacted with an aldehyde to insert a 1-hydroxyalkyl group at the 2-position, which group may then be reduced to produce a

2-alkyl-3-hydroxy-4-pyrone. The preparation of 2-ethyl-3-hydroxy-4- pyrone, etc., by this route is described in the published US Application Serial No. 310,141 (series of 1960).

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 those skilled in the art.

The chromium complexes according to the present. invention may be formulated for use as pharmaceuticals for both veterinary, for example in an avian or particularly a mammalian context, and particularly for human use by a variety of methods. For instance, they may be applied as an aqueous, oily or emulsified composition incorporating a liquid diluent which may often be employed in injectable form for parenteral administration, therefore conveniently being sterile and pyrogen free. For certain other uses a diluent which is sterile but not necessarily pyrogen free may be appropriate. As regards liquid diluents or carriers, therefore, there is often particular interest in those which are sterile. Oral administration is, however, more usually preferred for the routine treatment of chromium depletion 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 more usual, at least in humans, to use compositions incorporating a solid carrier, for example a conventional solid carrier material such as starch, lactose, dextrin or magnesium stearate. Such solid compositions may conveniently be of a formed type, for example as tablets, capsules (including spansules), etc.

However, although solid compositions may be preferred for the treatment of chromium depletion in certain contexts, liquid compositions are of interest in other contexts, for example for human and veterinary intramuscular administration and for veterinary oral administration as discussed hereinafter. It is in the area of liquid compositions that the complexes of the present invention containing more than one form of ligand can have a particular advantage. Thus, when using such complexes it is usually possible to produce liquid compositions containing a higher concentration of chromium in complex form than is readily obtainable with a complex in which the three ligands are identical. It is believed that the reason for this lies in the diversity of different stereoisomers of one complex which can arise when more than one form of ligand is present and which can be augmented by the presence of .several different complexes in a reaction mixture obtained from the reaction with chromic ions of more than one ligand producing hydroxypyrone compound as mentioned hereinbefore. Thus, a 3:1 chromium(III) complex containing three identical asymmetric ligands can exist in four stereoisomeric forms but when a complex contains two different ligands, it can exist in eight stereoisomeric forms and even more forms can exist if three different ligands are present. Although the four stereoisomers of a CrA3 form of complex will generally co-crystallise with ease, for a CrAB2 form of complex the increased number of stereoisomers, and usually also the presence of other 3:1 ligand:iron(III) complexes, prevents such co-crystallisation and ensures that the product is a liquid with enhanced solubility as compared with the usually solid and possibly crystalline CrA3 complexes. Other forms of administration than by injection or through the oral route may also be considered in both human and veterinary contexts, for example the use of suppositories or pessaries for human administration. Another form of pharmaceutical composition of some particular interest is one for buccal or nasal administration which may take the form of an aerosol preparation or of lozenges or pastilles.

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 of a unit dose. Whilst the dosage of the chromium complex given will depend on various factors, including the particular complex or complexes which are employed in the composition, it may be stated by way of guidance that maintenance of the amount of chromium present in the human body at satisfactory level will often be achieved using a dosage, in terms of the chromium content of the compound, which lies in a range from about 0.15 to 100 ug/kg or even up to 500 ug/kg of body weight and often in a range of from 1 to 100 yg/kg, for example of 1 to 10 μg/kg, veterinary doses being on a similar yg/kg body weight ratio. Where appropriate more than one such dosage may be administered per day but usually within a daily total of the highest level quoted above, although it will be appreciated that it may be appropriate under certain circumstances_,,- to give dosages either below or above the levels quoted. In general, the aim should be to provide the amount of chromium required by the patient without administering any undue excess so that higher dosage levels will be employed in the case of severe chromium deficiency and in diabetes thereapy, and the lower dosage levels will be employed for maintaining adequate chromium dietary levels. The properties of the pharmaceutical compositions according to the present invention are particularly suited to the achievement of such an aim.

It will be appreciated from the foregoing discussion that more than one chromium complex according to the present invention may be contained in the pharmaceutical composition and, indeed, other active compounds may also be included in the composition, for example other compounds having the ability to facilitate the treatment of chromium depletion and/or compounds of use in the treatment of other metal deficiencies, such as of iron and zinc, as well as various vitamins, minerals, etc. The present invention thus includes a pharmaceutical composition comprising a neutral 3:1 hydroxypyrone:chromium(III) complex in which each ligand is separately provided by ^• 3-hydroxy-4-pyrone or 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. Moreover, the invention also includes the use of a neutral 3:1 hydroxypyrone: chromium(III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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 chromium in a patient's body.

The chromium complexes described herein are particularly suited to the treatment of chromium depletion, including prophylactic use, such treatment being of especial interest in humans but also in a veterinary context. Thus, the complexes are readily soluble in the pH range of 6 to 10 and have been shown by i_n vitro tests to be stable to both oxidation and reduction. Moreover, the complexes show a considerable degree of stability i_n vivo so that they are not substantially affected, for example, by the acidic conditions of the stomach, thereby considerably simplifying their formulation in pharmaceutical compositions. In particular, the complexes are able to permeate biological membranes and have been shown by i_n vivo tests in rats to exert a significant level of control over glucose levels both in normals and in rats in which diabetes has been induced using streptozotocin. The present invention thus includes a method for the treatment of a patient to effect an increase in the level of chromium in the patient's bloodstream which comprises administering to said patient an amount effective to achieve such an increase of a neutral 3:1 hydroxypyrone:chromium(III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or a 3-hydroxy-4-pyrone in which one or more of the hydogen atoms attached to ring carbon atoms are replaced by an aliphatic hydrocarbon group of 1 to 6 carbon atoms. In addition to use in the form of a purely pharmaceutical composition, the chromium complexes described herein are of interest for use as an active ingredient of foodstuffs. Such a use is of particular interest in relation to animal foodstuffs, the complex being mixed in with the nutritional components thereof such as roughage-providing and protein-containing materials.

The present invention thus further extends to a foodstuff which comprises a neutral 3:1 hydroxypyrone:chromium(III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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 nutritional material.

In addition to such in vivo uses of the complexes as described hereinbefore the chromium complexes described herein are also of interest in other contexts, for example as a source of chromium in tanning, dyeing and the preservation of wood and paper.

The invention is illustrated by the following Examples and it will be noted that Examples 3 and 4 illustrate the ability of the chromium complexes of the present invention to enhance oral glucose tolerance and produce a beneficial reduction in blood glucose levels. EXAMPLES Example 1 Preparation of chromium(III) (maltol)3

A solution of maltol (7.56 g) and Cr( θ3)3-9H2θ (8.0 g) in water (250 ml) is adjusted to pH 8 with aqueous sodium hydroxide, refluxed for about 24 hours and then evaporated to dryness under reduced pressure. The resultant green powder is stirred in cold chloroform (200 ml) until the bulk has dissolved (ca. 30 minutes) and the mixture is then filtered. The chloroform solution is dried and evaporated under reduced pressure to give chromium(III) (maltol )3 as dark green crystals (7.05 g, ~ 80%), υ_maχ (nujol) 1601, 1567, 1552, 1502, 632, 558, 540 shoulder, 483, 408, 390 cm^-1, λmaχ(H2θ) 429 nm. Example 2 Preparation of chromium(III) (2-ethyl_3-hydroxy-4-pyrone)3 A solution of 2-ethyl-3-hydroxy-4-pyrone (8.28 g) and

Cr(Nθ3)3.9H2θ (8.0 g) in water (250 ml) is adjusted to pH 8 with aqueous sodium hydroxide, refluxed for about 24 hours and then evaporated to dryness under reduced pressure. The- resultant green powder is stirred in cold chloroform (200 ml) until the bulk has dissolved (ca_.. 30 minutes) and the mixture is then filtered. The chloroform solution is dried and evaporated under reduced pressure to give chromium(III) (2-ethyl-3-hydroxy-4-pyrone)3 as dark green crystals in 50-60% yield, υ_max (nujol) 1590, 1560, 1330, 1270, 1194, 986, 940, 840, 720, 540, 480 cm^-1. Example 3

In vivo tests of the activity of chromium(lll) (maltol)3 in rats

(1) A group of 16 adult rats was injected intravenously with citrate buffer and then maintained for a period of 14±1 days on drinking water containing chromium(III) (maltol)3 in an amount providing a dosage level of 2 yg of the complex per 100 g of the rat's weight each day. At the end of this period the rats were divided into two groups, one group of 8 rats being given an oral dose of glucose in water at a level of 2 yg/kg and the second group of 7 rats being given an oral dose of the same volume (1 ml/10 g of the rat's body weight) of the 0.9% w/v saline. The rats of each group were then anaesthetised and their blood glucose levels measured over a period of 180 minutes from the administration of the glucose solution or saline (time = 0). A similar experiment was carried out with a control group of 15 rats which were maintained on plain drinking water, these rats then being subdivided into two groups, one group of 9 rats receiving glucose solution and the other group of 7 rats receiving saline. The results obtained are shown as an average for each group with indication of the S.E. (Standard Error) in Figure 1 (chromium treated rats) and Figure 2 (control rats), there being a significant difference (P < 0.01) between the level of glucose measured at 30 minutes in the glucose treated rats maintained on the chromium complex and in the control group. (In these and all other Figures the saline load is shown by an open circle and the glucose load by a solid circle.)

(2) The experiments described in (1) were repeated using rats (both chromium treated and control groups) which had been rendered diabetic by the administration of streptozotocin in citrate buffer given intravenously at a 60 mg/kg dosage level at the initiation of the period of 14±1 days. The chromium treated group contained 10 rats of which 4 were subsequently treated with glucose solution and 6 with saline, all 10 being given drinking water providing twice the dosage level used in (1), i.e. 4 yg of the complex per 100 g of the rat's weight each day. The control group contained 11 rats of which 5 were subsequently treated with glucose solution and 6 with saline. The results obtained are shown as an average for each group with an indication of the S.E. in Figure 3 (chromium treated rats) and Figure 4 (control rats), there being a significant difference (P < 0.05 or < 0.01, shown as * and ** respectively) at all points except zero time in the glucose treated rats maintained on the chromium complex and in the control group.

(3) The experiments described in (2) with streptozotocin treated rats were repeated with 6 week old rats but on this occasion the chromium treated group of rats received a 4 yg/100 g/day dose of chromiu (III) (maltol )3 for 14 days in their drinking water prior to the administration of the streptozotocin as described in (2) and were then maintained on plain drinking water for a period of 12±1 days prior to the administration of the glucose solution or saline. Once again the control group were treated similarly apart from being maintained on plain drinking water for the periods both before and after the administration of streptozotocin. In this case a group of 10 chromium treated rats was used, of which 8 were subsequently treated with glucose solution and 2 with saline, and a group of 23 control rats, of which 13 were subsequently treated with glucose solution and 10 with saline.

The results obtained are shown as an average for each group with indication of the S.E. in Figure 5 (chromium treated rats) and Figure 6 (control rats), there being a significant difference (P ( 0.05, i 0.01 or (0.001, shown as *, ** and *** respectively) between the level of glucose measured at all points except zero time and 30 minutes in the glucose treated rats maintained on the chromium complex and in the control group. Example 4 In vivo tests of the activity of chromium(III) (maltol)3 in neonatal rats

(1) A group of 16 neonatal rats was injected intraperitoneally with citrate buffer at the age of 2 days and subjected to cardiac puncture under ether anaesthesia at the age of 4 days (this is required to check the hyperglycaemic status of the rats used in (2) which are treated with streptozotocin and is carried out on the rats of all groups for uniformity). At the age of 6 weeks drinking water containing chromium(III) (maltol )3 was supplied, the amount of the complex contained in the water providing a dosage level of 2 yg/100 g/day. At the age of 8 weeks the rats were divided into two groups, one group of 10 rats being given an oral dose of glucose in water at a level of 2 yg/kg and the second group of 6 rats being given an oral dose of the same volume of saline. The rats of each group were then anaesthetised and their blood glucose levels measured over a period of 180 minutes from the administration of the glucose solution or saline (time = 0).

An exactly similar experiment was carried out with a control group of 13 rats which were maintained on plain drinking water from age 6 weeks to age 8 weeks, the rats then being subdivided into two groups, one group of 6 rats receiving glucose solution and the other group of 7 rats receiving saline.

The results obtained are shown as an average for each group with an indication of the S.E. in Figure 7 (chromium treated rats) and Figure 8 (control rats), there being a significant difference (P < 0.01) between the level of glucose measured at 30 minutes in the glucose treated rats maintained on the chromium complex and in the control group. (2) The experiments described in (1) above were repeated using rats (both chromium treated and control groups) which had been rendered diabetic by the administration of streptozotocin in citrate buffer given intraperitoneally at a 90 mg/kg dosage level at the age of 2 days. No statistically significant difference was observed between the treated and control groups. The experiments described in (1) were therefore repeated again using a group of 20 rats maintained on plain drinking water, of which 13 are subsequently treated with glucose solution and 7 with saline, but on this occasion using neonates from mothers who were maintained on drinking water containing chromium(III) (maltol )3 at 2 yg/100 g/day for 6 to 9 days prior to delivery. This group of neonatal rats was compared with the control group of rats of the first repetition of the experiments described in (1) which received streptozotocin in citrate buffer but no chromium complex (total group of 16 of which 8 subsequently received glucose solution and 8 subsequently received saline).

The results obtained as an average for each group with an indication of the S.E. are shown in Figure 9 (rats from chromium treated mothers) and Figure 10 (control rats), there being a significant difference (P < 0.05) between the level of glucose measured at 60, 90 and 120 minutes in the glucose treated rats from chromium treated mothers and in the control group.

Claims

1. A neutral 3:1 hydroxypyrone:chromium(III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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 use in therapy.

2. A neutral 3:1 hydroxypyrone:chromium(III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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 with the exclusion of that complex in which each of the three ligands is provided by 3-hydroxy-2-methyl-4-pyrone.

3. A complex according to Claim 1 or 2, in which the or each aliphatic hydrocarbon group is an acyclic group of 1 to 4 carbon atoms.

4. A complex according to Claim 1 or 2, in which each ligand is separately provided by 3-hydroxy-4-pyrone or a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by the same or different substituents selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.

5. A complex according to any of Claims 1 to 4, in which the substituted 3-hydroxy-4-pyrone has a single substituent at the 2- or 6-position.

6. A complex according to Claim 1, in which each ligand is separately provided by 3-hydroxy-4-pyrone, 3-hydroxy-2-methyl-4- pyrone, 3-hydroxy-6-methyl-4-pyrone, 2-ethyl-3-hydroxy-4-pyrone, 3-hydroxy-2-(l '-methylethyl)-4-pyrone or 3-hydroxy-2-propyl-4- pyrone.

7. A complex according to any of Claims 1 to 5, in which each ligand is provided by the same hydroxypyrone.

8. A complex according to Claim 1, in which each ligand is provided by the same hydroxypyrone, the hydroxypyrone being 3-hydroxy-2-methyl-4-pyrone, 2-ethyl-3-hydroxy-4-pyrone, 3-hydroxy-2-(l '-methylethyl)-4-pyrone or 3-hydroxy-2-propyl-4- pyrone.

9. Chromium(III) (3-hydroxy-2-methyl-4-pyrone)3, for use in therapy.

10. A complex according to Claim 2, in which each ligand is provided by the same hydroxypyrone, the hydroxypyrone being 2-ethyl-3-hydroxy-4-pyrone, 3-hydroxy-2-(l '-methylethyl)-4-pyrone or 3-hydroxy-2-propyl-4-pyrone.

11. Chromium(III) (2-ethyl-3-hydroxy-4-pyrone)3.

12. A pharmaceutical composition comprising a complex according to Claim 2 together with a physiologically acceptable diluent or carrier.

13. A pharmaceutical composition comprising a neutral 3:1 hydroxypyrone:chromium(III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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, but excluding any liquid which is non-sterile.

14. A pharmaceutical composition comprising a neutral 3:1. hydroxypyrone:chromium(III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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.

15. A pharmaceutical composition according to Claim 14, which is adapted for oral^' administration.

16. A pharmaceutical composition comprising a neutral 3:1 hydroxypyrone:chromium(III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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, said composition being of injectable form.

17. A pharmaceutical composition according to any of Claims 12 to 16, in which the complex is as defined in any of Claims 3 to 7.

18. A pharmaceutical composition according to Claim 12, in which the complex is as defined in Claim 10 or 11. ⁵

19. A pharmaceutical composition according to any of Claims 13 to 16, in which the complex is as defined in Claim 8 or 9.

20. A pharmaceutical composition according to any of Claims 12 to 19 in unit dosage form.

21. A foodstuff comprising a neutral 3:1 hydroxypyrone: ° chromiu (III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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, together with a 5 nutritional material.

22. A method for the treatment of a patient to effect an increase in the level of chromium in the patient's bloodstream which comprises administering to said patient an amount effective to achieve such an increase of a neutral 3:1 hydroxypyrone: chromium(III) complex in which each ligand is separately provided by 3-hydroxy-4-pyrone or 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.

23. A method according to Claim 22, in which the or each aliphatic hydrocarbon group is an acyclic group of 1 to 4 carbon atoms.

24. A method according to Claim 22, in which each ligand is separately provided by 3-hydroxy-4-pyrone or a 3-hydroxy-4-pyrone in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by the same or different substituents selected from the group consisting of methyl, ethyl, n-propyl and isopropyl.

25. A method according to Claim 24, in which the substituted 3-hydroxy-4-pyrone has a single substituent at the 2- or 6-position.

26. A method according to Claim 22, in which each ligand is separately provided by 3-hydroxy-4-pyrone, 3-hydroxy-2-methyl-4- pyrone, 3-hydroxy-6-methy1-4-pyrone, 2-ethyl-3-hydroxy-4-pyrone, 3-hydroxy-2-(l '-methylethyl)-4-pyrone or 3-hydroxy-2-propyl-4- pyrone.

27. A method according to Claim 22, in which each ligand is provided by the same hydroxypyrone.

28. A method according to Claim 22, in which each ligand is provided by the same hydroxypyrone, the hydroxypyrone being 3-hydroxy-2-methyl-4-pyrone, 2-ethyl-3-hydroxy-4-pyrone,

3-hydroxy-2-(l '-methylethyl)-4-pyrone or 3-hydroxy-2-propyl-4- pyrone.

29. A method according to Claim 22, in which the complex is chromium(111) (3-hydroxy-2-methy1-4-pyrone)3.

30. A method according to Claim 22, in which the patient is suffering from diabetes.