WO1994003169A1

WO1994003169A1 - Mixture of a free radical scavenger and an iron chelator for the treatment of sickle cell disease

Info

Publication number: WO1994003169A1
Application number: PCT/GB1993/001626
Authority: WO
Inventors: Catherine Anne Rice-Evans
Original assignee: British Technology Group Ltd
Current assignee: BTG International Ltd
Priority date: 1992-08-04
Filing date: 1993-08-02
Publication date: 1994-02-17
Anticipated expiration: 1995-02-04
Also published as: AU4719793A; GB9216506D0; ZA935647B; GB2269748A; GB9316004D0

Abstract

Free radical scavengers containing a thiol group are of value for the treatment of sickle cell anaemia and other blood disorders characterised by a change in normal red blood cell morphology and/or oxidative damage of red blood cells, the compounds being used in combination with a membrane-permeable iron chelator.

Description

MIXTURE OF A FREE RADICAL SCAVENGER AND AN IRON CHELATOR FOR THE TREATMENT OF SICKLE CELL DISEASE

Background of the invention

Field of the invention

This invention is in the field of treatment of sickle cell disease and other conditions resulting from the modification of the normal red blood cell morphology. Description of the prior art

Sickle cell disease comprises a group of disorders resulting from a hereditary defect which causes a modification of the normal AA haemoglobin to haemoglobin of the SS, SC, SD or Sβthal form and leads to a polymerisation of the haemoglobin when in the deoxy state to form a linear polymer of a sickle shape. The sickle cells are less readily able to pass througji the capillaries resulting in repeated painful crises for the patients, leading to greater deoxygenation of red cells in neighbouring blood vessels and further sickling. Peroxidation of the membrane lipids and oxidative modification of the membrane proteins have also been reported for the sickle erythrocyte. The detection of non-haem iron-containing compounds in membrane preparations from sickle erythrocytes poses the possibility that such compounds may play a catalytic role in the processes leading to membrane oxidation. The non-haem iron retained by sickle erythrocyte membranes has been suggested to comprise both iron contained within ferritin and an unidentified component able to catalyse the breakdown of lipid hydroperoxides. Studies indicate that the non-haem iron-containing compounds are associated with the membrane cytoskeleton and possibly also with polar lipid headgroups on the cytoplasmic face of the membrane. Moore et al_. (1992), Blood 7£, 1334-1341, investigated the effects of ascorbate and desferal on the membranes of sickle cells and suggest that a mixture of these two compounds may protect against oxidative stress in sickle patients. However, despite the work carried out in this area, the relationship between membrane oxidation and the altered morphological and mechanical properties of the sickle cell remains unclear. Various treatments have been developed for sickle cell disease involving the oral administration to the patient of one of several antisickling drugs having an influence on the behaviour of the haemoglobin molecules, these drugs including cyanates, urea and zinc salts. Although each of the drugs can have some beneficial effect, none of them is fully satisfactory, characteristically being highly toxic, and there is still a need for an effective treatment for alleviation of the recurrent pain crises in sickle cell disease. Summary of the invention N-acetylcysteine is a compound reported to be a scavenger of hydroxyl and ferrylmyoglobin radical species. It has been found that this compound is able to penetrate the membrane of erythrocytes and is highly effective as an antioxidant in sickle erythrocytes, modulating oxidative stress and attenuating oxidative damage. Surprisingly, it has been found that a mixture of a thiol-containing free radical scavenger and a membrane-permeable iron chelator has an antisickling effect on the morphology of the red blood cells. This mixture of compounds therefore fulfils the requirements for an antisickling agent, i.e. one which is able to pass through the cell membrane of the erythrocytes and prevent or reverse sickling. Accordingly the present invention comprises a mixture of a free radical scavenger containing a thiol group and a membrane-permeable iron chelator. Description of the preferred embodiments The preferred thiol-containing free radical scavengers of the current invention have the general formula (I) below

RNHCHC0 H t (I) R' in which R is hydrogen, a group CH3(CH )_n ^c0 in which n is 0 or 1, or a group HS(CH2)m^C0 iⁿ which m is 1 or 2 and R¹ is hydrogen or a mercaptomethyl , methyl or ethyl group with the proviso that the compound of formula (I) contains a single SH group, the compound optionally being in the form of a physiologically acceptable salt. Preferred compounds are those in which R is other than hydrogen and/or R' is hydrogen or mercaptomethyl. Examples of specific compounds, in increasing order of interest, are cysteine (R is H, R' is a mercaptomethyl group), N-mercaptoacetylglycine (R is HSCH2CO, R¹ is H), N-mercaptopropionylglycine (R is HSCH₂CH2C0, R' is H), and N-acetylcysteine (R is CH3CO, R¹ is a mercaptomethyl group), including the physiologically acceptable salts of these compounds.

Compounds containing a thiol group (SH) such as N-acetylcysteine (NAC) and 2-mercaptopropionylglycine (MPG) are known for their efficacy in restoring the activity of protein bound sulphydryl groups. The ability of NAC to protect cells and tissues against oxidative damage has been attributed to radical scavenging and to the enhancement of the availability of cysteine, a substrate for glutathione synthesis. Markers of free radical-mediated damage in sickle erythrocytes have been recognised for a number of years, for example, reduced glutathione levels are diminished on average by 20%, and increased levels of lipid peroxides and their secondary metabolites have been identified in sickle erythrocyte membranes. However, it could not have been predicted that a mixture of such thiol-containing free radical scavengers and a membrane-permeable iron chelator as well as being an antioxidant would also be an antisickling agent, since oxidative stress, as relieved by antioxidant compounds is not the trigger for sickling.

The detection of non-haem iron-containing compounds in membrane preparations from sickle erythrocytes poses the possibility that such compounds may play a catalytic role in the processes leading to membrane oxidation. The non-haem iron retained by sickle erythrocyte membranes has been suggested to comprise both iron contained within ferritin and an unidentified component able to catalyse the breakdown of lipid hydroperoxides. Studies indicate that the non-haem iron-containing compounds are associated with the membrane cytoskeleton and possibly also with polar lipid headgroups on the cytoplas ic face of the membrane.

Suitable iron chelators for incorporation into the mixture of the invention are hydroxypyridone compounds. Hydroxypyridone chelators may take various forms as described, for example in UK Patents 2118176B, 2136807B, 2146990B and 2146989B, and in their equivalents, for example US Patents 4840958, 4585780, 4587240, 4666927 and 4912118, the disclosure of all of which is incorporated herein.

Preferred hydroxypyridone chelators are:

(1) a 3-hydroxypyrid-2-one or 3-hydroxypyrid-4-one in which the hydrogen atom attached to the nitrogen atom is replaced by an aliphatic acyl group, by an aliphatic hydrocarbon group, or by an aliphatic hydrocarbon group substituted by one or more substituents selected from aliphatic acyl, alkoxy, cycloalkoxy, aliphatic amide, aliphatic ester, halogen and hydroxy groups and, optionally, in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic acyl, alkoxy, cycloalkoxy, aliphatic amide, aliphatic ester, halogen or hydroxy group, by an aliphatic hydrocarbon group, or by an aliphatic hydrocarbon group substituted by an alkoxy, cycloalkoxy, aliphatic ester, halogen or hydroxy group, or a physiologically acceptable salt thereof;

(2) a l-hydroxypyrid-2-one in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by a substituent selected from aliphatic acyl, aliphatic amide, cyano, aliphatic ester, halogen and hydroxy groups, alkoxy and cycloalkoxy groups and alkoxy and cycloalkoxy groups substituted by an alkoxy, cycloalkoxy, aliphatic amide, aliphatic ester, halogen or hydroxy group, and aliphatic hydrocarbon groups and aliphatic hydrocarbon groups substituted by an alkoxy, cycloalkoxy, aliphatic ester, halogen or hydroxy group, but excluding compounds in which said replacement of hydrogen atoms is effected only by substituents selected from aliphatic hydrocarbon groups, halogen groups and aliphatic hydrocarbon groups substituted by a halogen group, or a physiologically acceptable salt thereof; or (3) a compound in which two or more rings, being selected from 3-hydroxypyrid-2-ones, 3-hydroxypyrid-4-ones and l-hydroxypyrid-2- ones, are covalently linked with the rings retaining their adjacent hydroxy and oxo groups [so-called poly(hydroxypyridone) compounds]. Hydroxypyridone compounds of types (1), (2) and (3) are described in detail in UK patents GB 2118176B and 2136807B; GB 2146990B; and GB 2146989B, respectively, which relate to the use of such compounds for the removal of toxic amounts of metal from the body, and the various preferred and specific types of compound described in these patents may be used in the present invention. Certain compounds of types (2) and (3) are also described in U.S. Patent 4,698,431, the disclosure of which is incorporated herein.

Among the compounds of these earlier patents which may be mentioned particularly are those in which the rings are unsubstituted apart from linking groups in the case of those compounds containing two or more rings, and, for both these compounds and the single ring compounds, those in which the rings are substituted by aliphatic hydrocarbon groups (in the case of the 3-hydroxypyridones and particularly on the nitrogen atom of the ring), by aliphatic hydrocarbon groups substituted by a hydroxy or cycloalkoxy or particularly an alkoxy group or by more than one, for example two of such groups (in the case of the 1- and 3-hydroxypyridones and particularly on the nitrogen atom of the ring in the latter case), or by an aliphatic amide group or an alkoxy or cycloalkoxy group substituted by an alkoxy, cycloalkoxy, aliphatic amide or hydroxy group (in the case of the 1-hydroxy- pyridones). The size of the aliphatic hydrocarbon and alkyl or cycloalkyl moieties in such groups (including those in the aliphatic amide groups) is conveniently in a range of C-j_8, particularly Cι_6, for example Cη—4, such as methyl and ethyl. The hydroxypyridones of use in the present invention are discussed in detail in the earlier patents but compounds of particular interest in the context of the present invention are water soluble, neutral compounds of a hydrophilic character. As explained -in these earlier patents it is possible to balance the presence of a hydrophilic substituent by the presence of one or more hydrophobic substituents but in order to achieve the appropriate hydrophilic character in the compound it is preferred, for example, that the number of carbon atoms in the aliphatic hydrocarbon, alkoxy and cycloalkoxy moieties and such substituted moieties is limited. Thus the aliphatic hydrocarbon groups are conveniently of 1 to 6 carbon atoms, especially 1 to 4 and more especially 1 to 3 carbon atoms, when these groups are not themselves substituted. Acyclic rather than cyclic groups are preferred, as are saturated rather than unsaturated groups, the preferred unsubstituted aliphatic hydrocarbon groups and those in the aliphatic amide groups being the alkyl groups ethyl and particularly methyl. The substituted aliphatic hydrocarbon groups are conveniently of 1 to 6 carbon atoms, preferably of 1 to 4 carbon atoms, especially of 1 , 2 or 3 carbon atoms. Once again acyclic rather than cyclic, and saturated rather than unsaturated groups are preferred, the preferred substituted aliphatic hydrocarbon groups being substituted methyl, ethyl, isopropyl and n-propyl groups, the three latter alkyl groups conveniently being substituted terminally. Alkoxy groups are preferred to cycloalkoxy groups and the former are conveniently of 1 to 6 carbon atoms, preferably of 1 to 4 carbon atoms and especially of 1 or 2 carbon atoms. Aliphatic amide groups conveniently contain no more than one aliphatic hydrocarbon group which may, for example, be ethyl or especially methyl .

Specific examples of substituted aliphatic hydrocarbon group substituents are hydroxymethyl , 2-hydroxyethyl , 3-hydroxypropyl , methoxymethyl , 2-methoxyethyl , 3-methoxypropyl , 2-methoxy-l- methylethyl, ethoxymethyl , 2-ethoxyethyl , 3-ethoxypropyl , and

2-ethoxy-l-methylethyl , the groups in which the oxygen atom is attached to the bonding carbon atom of the group being of less interest in the case of N-substituents in view of the lesser

I I ι i stability of the grouping -N-C-0- as compared with -C-C-0-. As regards both the single ring compounds and those containing two or more rings, among the 3-hydroxypyridones it is the 3-hydroxypyrid-4-ones which are of especial interest in the present invention and there is also particular interest in the l-hydroxypyrid-2-ones. Thus, a first preferred group of compounds consists of single ring 3-hydroxypyrid-4-ones in which the carbon atoms of the ring each separately are either unsubstituted or are substituted by an aliphatic hydrocarbon group, for example as described above, conveniently at the 6 or more preferably at the 2 position. It will be appreciated that the term aliphatic hydrocarbon group as used herein includes both acyclic and cyclic groups which may be unsaturated or more preferably saturated, the acyclic groups having a branched or more preferably a straight chain. Groups of 1 to 6 carbon atoms, particularly of 1 to 4 and more particularly of 1 to 3 carbon atoms are of more interest. The nitrogen atom is preferably substituted either by an aliphatic hydrogen group, particularly a methyl or ethyl group or by a hydroxy-substituted or more particularly an alkoxy, for example methoxy or ethoxy, substituted aliphatic hydrocarbon group of 1 to 6 carbon atoms, particularly an ethyl, propyl or isopropyl group substituted at the 2- or 3-position (as appropriate).

Specific examples of such compounds are 3-hydroxy-l ,2-dimethylpyrid-4-one, 1-eth l-3-hydroxy-2-methyl-pyrid-4-one, 3-hydroxy-l-(2'-hydroxyethyl)-2-methylpyrid-4-one,

3-hydroxy-1-(2'-hydroxy-1 '-methylethyl)-2-methylpyrid-4-one, 3-hydroxy-l-(2'-hydroxypropyl)-2-methylpyrid-4-one, 3-hydroxy-1-(3'-hydroxyprop l)-2-methylpyrid-4-one, 3-hydroxy-1-(2'- ethoxyethyl)-2-methylpyrid-4-one, 3-hydroxy-1-(2'-methoxy-1 '-methylethyl)-2-methylpyrid-4-one, 3-hydroxy-1-(2'-methoxypropyl)-2-methylpyrid-4-one, 3-hydroxy-1-(3'-methoxypropyl)-2-methylpyrid-4-one, 1-(2'-ethoxyethyl)-3-hydroxy-2-methylpyrid-4-one, 1-(2'-ethoxy-1 '-methylethyl)-3-hydroxy-2-methylpyrid-4-one, l-( '-ethoxypropy!)-3-hydroxy-2-methylpyri -4-one, l-(3'-ethoxypropyl)-3-hydroxy-2-methylpyrid-4-one, and salts thereof.

Of the above list of compounds, 3-hydroxy-l-(2'-hydroxyethyl)- 2-methylpyrid-4-one and 3-hydroxy-l-(2'-hydroxy-l '-methylethyl)-2- methylpyrid-4-one and salts thereof are particularly preferred. Other specific examples include analogues of the compounds described above in which the methyl group at the 2-position is replaced by an ethyl group and salts thereof, a preferred group of compounds again being those in which the hydrogen atom attached to the nitrogen atom is replaced by a hydroxy-substituted or alkoxy-substituted aliphatic hydrocarbon group, for example 3-hydroxy-l-(2-hydroxyethyl)-2-ethylpyrid-4-one and 3-hydroxy-l-(2-hydroxy-l '-methylethyl)-2-ethylpyrid-4-one. However, the particularly preferred compounds are those hydroxypyridones of type (1) in which the hydrogen atom attached to the nitrogen atom is replaced by an aliphatic hydrocarbon group and, optionally, in which one or more of the hydrogen atoms attached to ring carbon atoms, particularly that at the 2-position, are replaced by an aliphatic hydrocarbon group, particularly 1 ,2-diethyl-3-hydroxypyrid-4-one and its salts.

A second preferred group of compounds consists of single ring l-hydroxypyrid-2-ones in which the carbon atoms of the ring are substituted, conveniently at the 6- or 4-position by an alkoxy, alkoxyalkoxy or aliphatic amide substituent. Specific examples of such compounds are l-hydroxy-4-(2'-methoxyethoxy)-pyrid-2-one, l-hydroxy-4-(2'-ethoxyethoxy)-pyrid-2-one, and 6-carbamoyl-l- hydroxypyrid-2-one and its 6-(N-methylcarbamoyl) and 6-(N-ethylcarbamoyl) analogues, and salts thereof.

A third preferred group of compounds, of poly(hydroxypyridone) compounds, consists of compounds containing three 3-hydroxypyrid-2- or -4-one rings in which the carbon and nitrogen atoms of the rings are either unsubstituted apart from linking groups or are substituted by an aliphatic hydrocarbon group, for example as described above, particularly at the 6-, or especially at the 2-position in the case of the 4-ones. A fourth preferred group, of poly(hydroxypyridone) compounds, consists of compounds containing three l-hydroxypyrid-2-one rings in which the carbon atoms of the rings are either unsubstituted apart from linking groups or are substituted by an aliphatic hydrocarbon group or an aliphatic amide group, for example as described above, particularly at the 6- or 4-position. Linkage of the rings through the carbon atoms thereof or particularly through the nitrogen atoms thereof for the 3-hydroxypyridones is conveniently effected by linking groups which are of an aliphatic hydrocarbon nature or which additionally contain one or more groups

I ^m

-NH-, -N-, -C0NH- and -CON^. Specific examples of such compounds are described in GB 2146989B. Particularly preferred are the compounds 6B, 6C, 8, 9 and 10 listed in Table 1 of that patent, with the compound 8, N,N-di-[2-(3-hydroxy-2-oxopyrid-l-ylacetamido)- ethyl]-2-(3-hydroxy-2-oxopyrid-l-ylacetamido)ethylamine, being especially preferred for use in the current invention.

The hydroxypyridones may be prepared by procedures described in the earlier patents referred to hereinbefore. In particular, the C-carbamoyl and C-alkylated-carbamoyl 1-hydroxypyridones may be prepared as described in US Patent 4,698,431 and the N-alkoxyalkyl and N-hydroxyalkyl 3-hydroxypyrid-4-one compounds may be prepared as described in UK patent GB 2136807B. This latter route involves reaction of a corresponding 3-hydroxy-4-pyrone, or a 3-hydroxy-4- pyrone containing groups convertible to the C-substituents present in the desired hydroxypyridone, with a compound R'NI.2 in which R' represents the group present on the nitrogen atom of the desired compound or a group convertible thereto, the reaction being carried out in the presence of a base, for example an alkali metal hydroxide such as sodium hydroxide. This procedure is specifically exemplified in GB 2136807B for 3-hydroxy-l-(2'-hydroxyethyl)-2- methylpyrid-4-one and compounds containing larger N-hydroxyalkyl groups, and may be applied in an exactly analogous fashion to the preparation of the corresponding N-alkoxyalkyl compounds. Thus the use of 2-methoxyethylamine in place of 2-hydroxyethylamine in Example 11 of GB 2136807B will yield the compound 3-hydroxy-l-(2'- methoxyethyl)-2-methylpyrid-4-one. Where appropriate, the hydroxypyridones may be used in the form of a physiologically acceptable salt. Thus salts may be formed between the anion produced by the loss of the hydroxy group proton and a cation such as an alkali metal ion, for example Na⁺, quaternary ammonium ions or protonated amines such as tris

(tris represents 2-amino-2-hydroxymethyl propane 1,3-diol). As an alternative to salt formation with a base, the nitrogen atom of a 3-hydroxypyridone ring may be sufficiently basic, particularly in the case of the 3-hydroxypyrid-4-ones, for salt formation to be effected through reaction with an acid such as hydrochloric acid. The proportion of membrane-permeable iron chelator, for example a hydroxypyridone compound, to the thiol-containing free radical scavenger may vary. However, by way of guidance it may be stated that a ratio by weight of the hydroxypyridone or other chelator.thiol-containing compound is from 5:1 to 0.2:1, particularly from 3:1 to 0.5:1, for example 1:1, is often-suitable (when salts of iron chelators are used similar ratios apply but based upon the amount of the compound in the salt).

Where desired more than one hydroxypyridone compound may be used, for example both a single ring bidentate compound and a three ring hexadentate compound, the proportions given then applying to the total of hydroxypyridone compounds, and more than one thiol- containing compound may be used in the mixture.

The mixture may consist only of the iron chelator, for example the hydroxypyridone(s) and the thiol-containing compound(s), but in many other instances the mixture may contain other ingredients.

According to a second aspect of the invention, there is provided the use of a mixture of a free radical scavenger containing a thiol group and a membrane-permeable iron chelator in therapy.

Preferably the use of the mixture of thiol-containing free radical scavenger and a membrane-permeable iron chelator is preventative, i.e. administered to the patient before a pain crisis occurs, but may be curative, i.e. administered during or after the occurrence of pain crises. As indicated, the thiol-containing free radical scavenger may, if desired, be used in the form of a physiologically acceptable salt, particularly one formed with a physiologically acceptable base. Examples of suitable bases are the alkali metal hydroxides, for example sodium hydroxide, quaternary ammonium hydroxides and amines such as tris (tris representing 2-amino-2-hydroxymethyl propane 1 ,3-diol) .

The mixture of thiol-containing free radical scavenger and the membrane-permeable iron chelator may be administered to the recipient by a route selected from oral, parenteral (including subcutaneous, intrader al, intramuscular and intravenous) and rectal. Particular compositions are those in an orally digestible or sterile injectable form including forms suitable for delayed release. Solid forms include powders, tablets, granules, capsules, sachets and suppositories. Liquid forms include solutions, suspensions and emulsions. The formulation may be administered in unit dosage form, or in multiple unit dosage form to provide the desired therapeutic effects. Although separate administration of the components with consequent formation of the mixture i_n vivo may be considered, formation of the mixture before administration in vivo (where applicable) is preferred.

The present invention finds particular use in the treatment of patients suffering from sickle cell anaemia or other blood disorder characterised by a change in normal red blood cell morphology and/or oxidative damage of red blood cells which comprises administering to a patient in need thereof a therapeutically effective dosage of a mixture of a free radical scavenger containing a thiol group and a membrane-permeable iron chelator. This effective amount may vary depending on a number of factors including the physical status of the patient and the type of blood disorder and the severity thereof involved.

A further formulation of the mixture of thiol-containing free radical scavenger and a membrane-permeable iron chelator according to the invention is a form suitable for the extra-corporeal treatment of blood from the patient. As one possibility such treatment may be conducted in a batch-wise manner by removing an appropriate volume of blood, admixing it with the compound and transfusing the treated blood back into the patient. As an alternative possibility the treatment may be on a continuous basis, analogous to the well-known techniques for haemodialysis, whereby for a period of time blood is continuously withdrawn, admixed with the compound and passed back into the patient. Both procedures should be conducted under sterile conditions and may be repeated as often as necessary. In either case the treatment may be monitored to maintain a therapeutically effective amount of thiol-containing free radical scavenger in the blood. An effective blood concentration of thiol-containing free radical scavenger will generally be in the range 2 to 20 mg/kg, preferably in the range 4 to 15 and most preferably in the range 5 to 10 mg/kg with an optimum concentration of 8 mg/kg.

When formulated for use the mixture will take the form of a pharmaceutical composition together with a physiologically acceptable diluent or carrier as disclosed hereinbefore. The present invention therefore further includes a pharmaceutical composition which comprises a mixture of a free radical scavenger containing a thiol group and an iron chelator, preferably a hydroxypyridone compound, for example as described hereinbefore, together with a physiologically acceptable diluent or carrier.

The invention will be illustrated by the following Examples.

CXAMPLCS r.xample 1 : Formulation of Pharmaceutical Compositions

(A) Tablets of the following composition are prepared: mg/tablet N-acetylcysteine and 1 ,2-diethyl-3-hydroxypyrid-4-one mixture (1:1 w/w; micronised) 250

'Avicel' (microcrystalline cellulose) 38 polyvinylpyrrolidone 3 alginic acid 6 magnesium stearate 3 The micronised mixture is mixed with 'Avicel' and polyvinylpyrrolidone is added, dissolved in sufficient industrial methylated spirits (74° OP) to produce a mass suitable for granulating. The mass is granulated through a 20 mesh sieve and the resultant granules are dried at a temperature not exceeding 50°C. The dried granules are passed through a 20 mesh sieve and the alginic acid and magnesium stearate are then added and mixed with the granules. The product is compressed into tablets each weighing 300 mg on 3/8 inch flat bevelled edge divided punches.

(B) Tablets of the following composition are prepared: mg/tablet

N-acetylcysteine and 1 ,2-diethyl-3-hydroxypyrid-4-one mixture (1:1 w/w; micronised) 250 'Avicel' (microcrystalline cellulose) 134 polyvinylpyrrolidone 4 alginic acid 8 magnesium stearate 4

The tablets are prepared by essentially the same procedure as described in (A) and are compressed at a tablet weight of 400 mg on 7/16 inch flat bevelled edge punches.

(C) Tablets of the following composition are prepared: mg/tablet

N-acetylcysteine and 1 ,2-diethyl-3-hydroxypyrid-4-one mixture (1:1 w/w; micronised) 250 lactose (300 mesh) 19 maize starch 15 gelatine 10 magnesium stearate 6

The micronised mixture is mixed with lactose and half the total quantity of maize starch required, and a 5% solution of gelatine in water is added to the mass. The product is granulated through a 16_^mesh sieve, and the resultant granules are dried to constant weight at a temperature not exceeding 50°C. The dried granules are passed through a 20 mesh sieve and mixed with magnesium stearate and the remainder of the maize starch. The product is compressed at a 300 mg tablet weight on 3/8 inch flat bevelled edge divided punches.

Claims

C LAIMS

1. A mixture of a free radical scavenger containing a thiol group and a membrane-permeable iron chelator.

2. A mixture according to Claim 1 in which the free radical scavenger containing a thiol group is a compound of the general formula (I)

RNHCHC0₂H

I (I) R' in which R is is hydrogen, a group CH3(CI.2)_nC0 in which n is 0 or 1, or a group HS(CH2)rη 0 in which m is 1 or 2 and R' is hydrogen or a mercaptomethyl, methyl or ethyl group, with the proviso that the compound of formula (I) contains a single SH group, or a physiologically acceptable salt thereof.

3. A mixture according to Claim 2, in which the compound of formula (I) is cysteine, N-acetylcysteine, N-mercaptopropionyl- glycine, N-mercaptoacetylglycine or a physiologically acceptable salt thereof.

4. A mixture according to Claim 2, in which the compound of formula (I) is N-acetylcysteine or a physiologically acceptable salt thereof.

5. A mixture according to any one of Claims 1 to 4, in which the membrane-permeable iron chelator is a hydroxypyridone compound.

6. A mixture according to Claim 5, in which the hydroxypyridone compound is:

(1) a 3-hydroxypyrid-2-one or 3-hydroxypyrid-4-one in- which the hydrogen atom attached to the nitrogen atom is replaced by an aliphatic acyl group, by an aliphatic hydrocarbon group, or by an aliphatic hydrocarbon group substituted by one or more substituents selected from aliphatic acyl, alkoxy, cycloalkoxy, aliphatic amide, aliphatic ester, halogen and hydroxy groups and, optionally, in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an aliphatic acyl, alkoxy, cycloalkoxy, aliphatic amide, aliphatic ester, halogen or hydroxy group, by an aliphatic hydrocarbon group, or by an aliphatic hydrocarbon group substituted by an alkoxy, cycloalkoxy, aliphatic ester, halogen or hydroxy group, or a physiologically acceptable salt thereof;

(2) a l-hydroxypyrid-2-one in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by a substituent selected from aliphatic acyl, aliphatic amide, cyano, aliphatic ester, halogen and hydroxy groups, alkoxy and cycloalkoxy groups and alkoxy and cycloalkoxy groups substituted by an alkoxy, cycloalkoxy, aliphatic amide, aliphatic eter, halogen or hydroxy group, and aliphatic hydrocarbon groups and aliphatic hydrocarbon groups substituted by an alkoxy, cycloalkoxy, aliphatic ester, halogen or hydroxy group, but excluding compounds in which said replacement of hydrogen atoms is effected only by substituents selected from aliphatic hydrocarbon groups, halogen groups and aliphatic hydrocarbon groups substituted by a halogen group, or a physiologically acceptable salt thereof; or

(3) a compound in which two or more rings, being selected from 3-hydroxypyrid-2-ones, 3-hydroxypyrid-4-ones and l-hydroxypyrid-2- ones, are covalently linked with the rings retaining their adjacent hydroxy and oxo groups.

7. A mixture according to Claim 6, in which the hydroxypyridone is a 3-hydroxypyrid-4-one of type (1) or a physiologically acceptable salt thereof.

8. A mixture according to Claim 7, in which the hydroxypyridone is a 3-hydroxypyrid-4-one in which the hydrogen atom attached to the nitrogen atom is replaced by an aliphatic hydrocarbon group and, optionally, in which one or more of the hydrogen atoms attached to ring carbon atoms are replaced by an^* aliphatic hydrocarbon group, a hydroxy substituted aliphatic hydrocarbon group or an alkoxy substituted hydrocarbon group.

9. A mixture according to Claim 8, in which the hydrogen atom attached to the carbon atom at position 2 of the hydroxypyridone is replaced by an aliphatic hydrocarbon group.

10. A mixture according to Claim 9, in which the aliphatic hydrocarbon group is methyl or ethyl.

11. A mixture according to Claim 8, in which the hydroxypyridone compound is 3-hydroxy-l-(2'-hydroxyethyl )-2-methylpyrid-4-one, 3-hydroxy-l-(2'-hydroxy-l '-methylethyl)-2-methylpyrid-4-one,

1 ,2-diethyl-3-hydroxypyrid-4-one, 3-hydroxy-l-(2-hydroxy-l '-methyl- ethyl)-2-ethylpyrid-4-one, or a physiologically acceptable salt thereof.

12. A mixture according to Claim 11, in which the 3-hydroxypyrid-4-one is 1 ,2-diethyl-3-hydroxypyrid-4-one or a physiologically acceptable salt thereof.

13. A mixture according to Claim 6, in which the hydroxypyridone is a poly(hydroxypyridone) compound of type (3) containing three l-hydroxypyrid-2-one rings.

14. A mixture according to Claim 13, in which the compound is

N, -di-[2-(3-hydroxy-2-oxopyrid-l-ylacetamido)-ethyl]-2-(3-hydroxy- 2-oxopyrid-l-ylacetamido)-ethylamine, or a physiologically acceptable salt thereof.

15. A mixture according to any of Claims 1 to 14, in which the ratio by weight of the membrane permeable iron chelator:the free radical scavenger containing a thiol group is in the range 3:1 to 0.5:1.

16. A mixture as defined in any of Claims 1 to 15 for use in therapy.

17. The use of a mixture as defined in any of Claims 1 to 15 for the manufacture of a medicament for use in the treatment of sickle cell anaemia.

18. The use according to Claim 16, in which the treatment is preventative.

19. The use according to Claim 16, in which the treatment is curative.

20. A pharmaceutical composition comprising a mixture according to any of Claims 1 to 15 and a physiologically acceptable diluent or carrier.

21. A pharmaceutical composition according to Claim 20, which is adapted for oral administration.

22. A pharmaceutical composition according to Claim 20, which is adapted for parenteral administration.

23. A pharmaceutical composition according to Claim 20, which is adapted for extra-corporeal treatment of a patient's blood.

24. A method of treatment of a patient who has or is susceptible to develop sickle cell anaemia which comprises administering a therapeutically effective amount of a mixture according to any of Claims 1 to 16.