NZ217431A - Synergistically antimalarial combination preparations comprising an iron(iii) chelating agent and a schizontocide - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £17431 Prior;Tv Da: ' v 5'C CoiUfj'.cie Spec:r:cjticn F.'ed: Clss'r r. T. /' ' " '" ° 217431 Patents Forn No. 5 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION ffV^ "COMBINATION PREPARATIONS FOR THE TREATMENT OF MALARIA" X/We, CIBA-GEIGY AG, a Swiss Corporation of Klybeckstrasse 141, 4002 Basle, Switzerland, hereby declare the invention, for which £/we pray that a patent may be granted to jcjs/us, and the method by which it is to be performed, to be particularly described in and by the following statement: (followed by page ! A.) c - iA- 2 1743 4-15488/- Combination preparations for the treatnent of malaria Malaria is widely known as a dangereous infectious disease that is transmitted by the Anopheles mosquito and takes the form of periodic and repeated attacks of high fever (40°-41°C) accompanied by chill. Malaria is prevalent worldwide in various forms in warm climates of high humidity, especially in subtropical and tropical zones. At the present time, it is estimated that malaria afflicts some 200-400 million people. In Africa alone, malaria kills about 10 X of those afflicted and debilitates the rest partially or completely. The disease is held responsible for about 50 % of all cases of mortality in young children. For decades, and indeed for centuries, malaria has resisted all attempts to eliminate it and it is still one of the major world health problems. Aside from comprehensive and concerted efforts to achieve a solution by ecological means (e.g. mosquito control by insecticides or, more radically still, by swamp drainage), interest centres mainly on the possibilities of the medical treatment of man as host of the disease, including especially the search for therapeutic agents for combating the parasites direct.

Malaria is in fact a generic term covering a number of diseases having a similar clinical pattern but whose parasites — parasitic protozoa belonging to the class of the sporozoa — are different species of plasmodium. These diseases attack not only humans but also mammmals. They are species-specific, i.e. they cannot be transmitted from one species of animal to another or to humans.

Human malaria takes three different forms in accordance with the clinical aspects of the disease: tertian fever whose parasites are Plasmodium vivax and P. vivale, quartan fever whose parasite is P. malariae, and the particularly dangerous subtertian or malignant •21743 fever Is.c. Malaria tropica] transmitted by P. innnaculatum or P. falciparum. The treatment and drugs administered are, however, the same for all three types.

Characteristic of all Plasmodium species is their complex life cycle, in one phase of which the parasites undergo sexual development in the intestines of the female mosquito, from where the asexual sporozoites so formed then take up residence in the mosquito's salivary gland. In a second phase, the exceedingly rapid asexual development of the sporozoites takes place in the liver and in the red blood cells of the human host. The infection is transmitted to humans by the mosquito bite, whereupon the minute unicellular sporozoites of the parasite enter the bloodstream and find their way to the liver. Once the liver cell has been infected, the sporozoite multiplies asexually to form a multicellular stage, known as the schizont, which subsequently fissions to release about 5000 to 10,000 unicellular merozoites into the bloodstream. These merozoites can, in turn, attack the liver cells, with repetition of the entire asexual reproductive cycle, or they attack the erythrocytes (red blood cells). Each merozoite invades a red blood cell, where it multiplies until the cell collapses. The components thereby set free, mainly amino acids, are utilised by the merozoite for its own metabolic processes and energy production to form 10-20 new merozoites within 48 or 72 hours (depending on the type of infection) by an extremely rapid asexual reproductive cycle (schizogony), which results in complete destruction of the host cell (Plasmodia are even superior to rapidly growing tumours in their rate of cell reproduction). The new merozoites formed attack other erythrocytes and a further reproductive cycle commences. This cyclic development is responsible for the well-known malarial symptoms. The toxic waste products of cell destruction in particular cause the high fever and chill, and the typical intermissions of 48 hours (tertian fever and falciparum malaria) to 72 hours (quartan fever) correspond to the infective cycle of the parasite in the blood. The recurring attacks gradually decrease in intensity (though not in frequency) until 2 174 they finally disappear. It is probable that, on the one hand, the vitality of the parasitic cells dimishes naturally and, on the other, that the victim develops a kind of immunity.

Like other intracellular parasites, Plasmodium poses a special problem as regards therapy. Aside from the different development forms, the micro-organism is almost never open to direct attack by an exogenic or endogenic inhibitor (i.e. one produced by the host), as it usually finds refuge in host cells and is thus protected by two functionally very different cell membranes of two physiologically widely different cell types (its own and that of the host cell). The functional properties of the erythrocyte membrane block access to the parasite by a number of inhibitors (such as the body's own innnunofactors) which, if the parasite had an extracellular existence, would certainly be effective. It is probably for this reason that, so far, all attempts to control the disease by immunological methods of treatment, including preventive vaccination, have failed. And so it is that, among antimalarial agents, chemo-therapeutic agents are still the most successful, e.g the classical quinine, which was for centuries the sole effective drug and, despite certain shortcomings, continues to occupy a leading position in the treatment of malaria. Over the past 50 years, numerous chemotherapeutic agents of widely varying structural classes have been developed as schizontocides, i.e. drugs that interrupt the life cycle of the Plasmodia in one of their asexual reproductive stages and inhibit cell destruction either in the liver tissue or in erythrocytes or even kill the merozoites. Exemplary of drugs belonging to the first group (tissue schizontocides) is for example primaquine [6-methoxy-8-(4'—amino-1'-methylbutylamino)q\iinoline]. In addition to quinine, the group of blood schizontocides is represented by the most successful antimalerial agent so far developed, chloroquine [7-chloro-4-(4'-diethylamino-lmethylbutylamino)-quinoline]. A survey of important known chemotherapeutic drugs will be found in the literature, q.v. Advances in Malaria Chemotherapy, WHO Technical Report Series 711; World Health Organisation, Geneva, 1S84; pages 12, 13 and 163-175. u The general effectiveness of these drugs, hovever, is diminishing rapidly with time as a result of the marked ability of Plasmodia to develop resistance to chemotherapeutic agents. Resistance to quinine was observed even at the beginning of this century. At the present time, resistance to chloroquine is increasing and intensifying the seasrch for new solutions.

The current improved knowledge of the metabolic processes in the development cycle of Plasmodium species has directed attention to the fact that the enormously rapid multiplication of the cell population and it survival must be directly dependent on an adequate supply of iron. This is especially true of the commensurably high DNA synthesis in which the iron-containing enzyme, ribonucleotide reductase, plays a key role. To ensure the multiplication of Plasmodia, iron has to enter the parasite cell by means of a suitable trasnport system. To do so it must penetrate not only the cell membrane of the parasite, but also - if the parasite is unable to take iron from the erythrocyte as host cell - the phylogenet-ically different cell membranes of the host. If it were possible to cut off the supply of iron to the enzyme, e.g. by binding it to a chelating agent, then the multiplication mechanism could be stopped or the parasite cell even killed.

It is apparent that the antibacterial activity of certain iron(III) chelating agents isolated from natural sources, in particular from micro-organisms, must be ascribed to such a mechanism.

It has been found that e.g. the desferriferrithiocine [2-(3'-hydroxy-2-pvridyl)-4-methyl-2-thiazoline-4-carboxylic acid and its semisynthetic derivatives disclosed in British patent specification 2 085 873 have certain general antibacterial activity in vitro, as has also desferriox-amine B [6,17,28-trihydroxy-7,10,18,21,29-pentaoxo-6,11,17,22,28—penta- Helv. Chim. Acta 66, 1385-9 [1963]. In the bacteria, however, merely the azatriacontanylamine], q.v. H. Bickel, H. Keberle and E. Vischer: simple case is encountered 2 17431 (aside from the fact that only the in vitro activity was established) of the iron(III) chelating agent only having to penetrate a single, viz. the bacterial, membrane.

However, in the case of the parasitic cells of a Plasmodium, success hinges on whether the chelating agent is capable of being transported not only through the cell membrane of the parasite, but also through the phvlogenetically quite different cell membrane of Che erythrocyte as host cell. Fortunately, this condition is met by the above-mentioned iron(III) chelating agents of natural origin and the semisynthetic analogs referred to above, and experimentation has shown that the growth of different Plasmodium species, including also cultures of Plasmodium falciparum in human blood cells, is not only inhibited, but that they are also killed by desferrioxamine B in vitro. In in vivo assays with rodent-specific species (P. vinckei), it has been found that 0.2 g/kg of desferrioxamine B markedly inhibits the ghrowth of the parasites and kills it completely at 1.0 g/kg, q.v. A. Jung et al., Abstracts of the Iron Club Meeting, Rennes (France), July 10-13, 1984. As the activity of desferrioxamine in animal tests and in tests on humans has been correlated in another connection, and as similar correlations are also known between Plasmodium vinckei in mice and P. falciparum in humans in the use of classical antimalarial drugs, the theoretical basis has thus been established for assessing the possibility of, and conditions for, the therepautic treatment of humans. It may be that such an analysis led to the conclusion that the necessary dose of the drug (although it is toxicologically harmless even when administered in very high doses) would not be practical and of interest for broad use. At all events, it is evident that a prejudice prevailed against the therapeutic use of desferrioxamine B or analogous iron chelating agents for the treatment of human malaria and that these were never put on trial.

This unsatisfactory situation has changed radically in the light of the instant invention, for it has now been found that, surprisingly, the iron chelating agents referred to above are effective against r~~ 217431 chloroquine-resistant strains of Plasmodium falciparum in substantially lower dosage than against normal strains, i.e. against the parasites which, on account of their resistance to conventional ■" - chemotherapeutic agents, are regarded as one of the most difficult problems in the fight against malaria.

In addition, the further surprisng fact has been discovered that iror.(III) chelating agents of the desferrioxamine type substantially ' potentiate the effectiveness of the cited schizontocides (e.g. chloroquine) not only when used against Plasmodium strains which are resistant to chloroquine, but also against those which are generally sensitive to chloroquine.

An experimental comparison of the effectiveness of desferrioxamine B against cultures of P. falciparum in human erythrocytes has shown that the same growth inhibition of a chloroquine-resistant strain compared with a strain cultured under the same conditions and having general sensitivity to chloroquine is achieved in the absence of chloroquine in an approximately three times lower concentration of desferrioxamine. This concentration is further halved in the presence of an intrinsically ineffective concentration of chloroquine. A similarly surprising result is obtained by treating a strain of P. falciparum which is generally sensitive to chloroquine ^ with a combination of chloroquine and desferrioxamine B. Both components are employed in concentrations which, individually, would effect a 20 % growth inhibition. Combined administration effects a growth inhibition of 70-100 % and clearly demonstrates the surprising synergistic effect of both components.

Hence the present invention relates to a combination preparation comprising two components in a synergistically effective ratio, namely an iron(III) chelating agent selected from the class consisting of desferrioxamine and desferriferrithiocine and the pharmacologically effective derivatives thereof as component A, and a conventional antimalerial agent selected from the category of the schizontocides as component B, each component being contained in a / - 217431 separate pharmaceutical composition or both together in a combined pharmaceutical composition, optionally in admixture with at least one pharmaceutical excipient. A preferred pharmaceutical composition contains a combination of both components defined above, together with optional conventional pharmaceutical excipients- The invention further relates to a process for the preparation of such a combination preparation or pharmaceutical composition as herein defined comprising their components, together with optional conventional pharmaceutical excipients. The invention relates further to a process for the preparation of such a combination preparation or composition as herein defined comprising their components by conventional pharmaceutical methods, and to the use of said combination preparation or pharmaceutical composition for the treatment of human malaria. In addition, the invention relates to a suitable therapeutic method of treating human malaria by simultaneous or alternate administration of an iron(III) chelating agent selected from the class consisting of desferrioxamine B and des— ferriferrithiocine and the pharmacologically effective derivatives thereof, and a conventional antimalarial agent selected from the category of the schizontocides, in a synergistically effective ratio and in amounts which, together, are effective against the parasites of malaria, which method comprises administering each component in an individual dosage form or administering both components in the form of a combination preparation or together in the form of a single pharmaceutical compostion, especially one as defined above.

The malarial diseases to be treated will be understood as those comprising infections transmitted by Plasmodium species, e.g. by strains of P. vivax or P. ovale (the parasites of tertian fever), P. malariae (the parasite of quartan fever) and, in particular, P. falciparum or P. immaculatum (the parasites of malignant malaria), as well as mixed infections of two or more strains of the same or of another Plasmodium species or, also reinfections with the same strain, but in a postponed phase. In particular, all above aspects of the invention relate to the treatment of malignant malaria transmitted by Plasmodium falciparum. The above aspects also 217431 relate especially to the treatment of malarial diseases caused by Plasmodium strains which are resistant to conventional chemotherapeutic agents, in particular to quinine and, most particularly chloroquine, as well as to other analogous antimalarial egents, and, first and foremost, to the treatment of the disease in its acute phase, i.e. during the multiplication of Plasmodia in human host erythrocytes.

The antimalarial agents belonging to the category of the schizontocides eligible for use in the practice of this invention are generally known conventional chemotherapeutic agents which are administered for inhibiting the growth of and killing merozoites (the asexual growth forms of Plasmodia), especially in human erythrocytes. To this category belong principally chloroquine [7-chloro-4-(4'-diethylamino-1'-methylbutylamino)quinolineJ in the form of the free base or of a pharmaceutically acceptable acid addition salt thereof, especially the diphosphate or sulfate, and analogous 4-3minoquinolines, e.g. amodiaquine [7-chloro-4-(3'-dimethylaminomethyl)-4'-hydroxyphenylamino)quinoline] and the acid addition salts thereof such as the hydrochloride, as well as in particular quinine and its analogs, e.g. mefloquine ((2-piperidyl)-(2,8-bistrifluoromethyl—4-quinolyl)carbinol] and halofantrine f(2-dibutylaminoethyl)-(1,3-dichloro-6-trifluoromethyl-9-phenanthrolyl)carbinol], each in the form of the free base or of a pharmaceutically acceptable acid addition salt. Examples of further blood schizontocides are certain natural antibiotics such as tetracycline, sulfonamides of the sulfadioxine type [N'-(5,6-di-ethoxy-4-pyrimidinyl)sulfanilimide] and sulfalene type [N'-3-methoxy-2-pyrazinyl)sulfanilimide, sulfones of the dapsone type [4,4'-diaminodiphenylsulfone], sesquiterpenoids of the artemisinine type (qinghaosu), dihydroartemisinine methyl ether (artemether) and the sodium salt of dihydroartemisinine hemi— succinate (artesumatesodium), as well as inhibitors of dihydrofolate reductase such as proguanil [N1-(4-chlorophenyl)—N5-isopropyl-diguanide] and pyrimethamines [2,4-diamino-5-(4-chlorophenyl)-6-ethylpyrimidine]. The normal therapeutic dose of all these compounds 2 t 74 is generally known. In the therapeutic method of this invention, it is possible to administer the minimum doses of conventional dosage which are ordinarily employed for the particular case, such as treatment of the acute phase or for aftertreatment, or it is even possible to administer doses below the normal dosage limit. In some circumstances, especially for combating resistant Plasmodium strains, it may be expedient to maintain the conventional dosage of the chemotherapeutic component in the combination of this invention in order to achieve a substantially better and/or shorter curative therapy compared with conventional therapy. As the dosage of all known blood schizontocides is in approximately the same order of magnitude, the following particulars may serve as general guidelines for the entire category. Chloroquine, for example, will be administered orally to adults suffering from a medium-severe attack of falciparum (malignant) malaria at the start of the acute phase (on the first day) in a daily dose of about 900 mg, which dose will then be reduced to about 300 tag on the two following days. The equivalent daily dose of quinine will be about 1.8 g administered orally during the entire treatment or, if administration is made parenterally (i.v. or by infusion), up to about 1.8 g on the first day and about 1.2 g on the following days. The daily dose will be administered in about 2-4, usually 3, individual doses, and treatment will be continued until the symptoms disappear. For aftertreat— ment and/or preventive administration, a single daily dose of 300 mg of chloroquine administered orally once per week will be regarded as adequate.

The antimalarial agents of this category are preferably admninis— tered in conventional pharmaceutical dosage forms such as capsules, tablets and syrups on the one hand, and injection solutions on the other.

The iron(III) chelating agent eligible for use in the practice of this invention is in particular desferrioxamine B in the form of the free base or of a pharmaceutically acceptable acid addition salt such as the hydrochloride and, preferably, the methanesulfonate 17431 (mesylate), q.v The Merck Index, No. 2839, page 412, 10th Edition, Merck & Co. Inc., Rahway, N.J., USA, 1983. As medicament for the treatment of malaria in the practice of this invention, the iron chelating agent is made available in particular in a form suitable for parenteral administration, e.g. as an aqueous solution. The iron chelating agent, in particular a pharmaceutically acceptable acid addition salt of desferrioxamine B, e.g. the mesylate, is preferably administered parenterally in a daily dose of 0.2 to 5 g, in particular of about 0.2 to 1.5 g and, most preferably, of about 0.5 to 1.0 g. The dosage depends on the weight and individual condition of the patient and, in particular, on the sensitivity of the parasite. It is especially preferred to administer 0.5 g twice daily and to continue with this dose over 1-2 weeks until all symptoms have disappeared. In the initial stages and, in particular, during attacks, this daily dose can be increased up to a four-fold one.

Particularly preferred formulations for the treatment of malaria are about 2.5 to 20 %, preferably about 5 to 10 %, aqueous solutions of pharmaceutically acceptable acid addition salts of desferrioxamine B, which solutions are prepared directly or shortly before administration from dry-filled ampoules or from other stable and sterile stored formulations of the salt and from distilled or demineralised water. For example, it is possible to prepare 10 % or less concentrated solutions of desferrioxamine mesylate using commercially available 500 mg dry-filled ampoules of DESFERAL® (registered trademark of Ciba-Geigy AG) and, for example, distilled water or physiological sodium chloride solutions. Such solutions can be administered parenterally e.g. by intramuscular injection, e.g. into the glutaeus maximus, or into the anterior thigh muscle or deltoid muscle, as well as by intravenous or subcutaneous injection or by infusion. Particularly suitable for self-medication or for administration by untrained personnel, e.g. health workers in developing countries, is slow subcutaneous injection, e.g. into the abdominal skin, using a portable infusion pump and a flexibly 217431 - n - mounted injection syringe, over the course of several hours once or twice daily or continuously during the day or night, as is done for treating diseases such as thalassemia.

The preparation of desferrioxamine B and of pharmaceutically acceptable salts, in particular the hydrochloride, is described in British patent specification 999,583 in combination with US-patent 3,153,621, the latter describing the manufacture of the necessary starting material. Said r British specification also describes the preparation of pharmaceutical formulations such as dry-filled ampoules, suppositories and capsules. Further suitable salts are for example those with methanesulfonic, sulfuric, phosphoric, acetic, glycolic, lactic, succinic, maleic, fumaric, malic, tartaric, citric, ethanesulfonic and hydroxyethane-sulfonic acid.

The eligible iron(III) chelating agent is likewise the above-mentioned desferriferrithiocine and its derivatives, in particular pharmaceutically acceptable salts, preferably alkali metal salts and alkaline earth metal salts, and pharmacologically effective analogs, in particular the lower alkane esters described in British patent specification 2 085 873.

Suitable formulations of iron chelating agents of this type are those for enteral such as oral administration, as well as those for parenteral such as subcutaneous administration, said formulations containing the chelating agent alone or in admixture with a pharmaceutically acceptable excipient. The dosage is about the same as that indicated above and ^ likewise depends on the age, weight and indiividual condition of the t patient, on the sensitivity of the parasite and on the mode of administration. The pharmaceutical compositions contain from about 10 to 95 %, preferably from about 20 to 90 % (weight/weight) of the iron chelating agent in solid formulations, from about 5 to 20 %, preferably about 10 %, in liquid 2 1743 formulations, and may be in the form of dosage units such as dragees, tablets, capsules, suppositories or also ampoules and dry-filled ampoules.

The combination preparation of this invention may preferably consist of two individual pharmaceutical compositions, each containing one of the two components in a synergistically effective ratio. This alternative is especially appropriate if each of the two components achieves maximum effectiveness in another dosage form. Thus, for example, desferrioxamine B as the preferred component A, or an acid addition salt thereof, is fully effective only when administered parenterally, whereas the preferred component B, e.g. chloroquine, is not suitable for parenteral administration and exhibits far fewer side-effects when administered orally. Such a combination preparation then preferably consists of two matching sets of dosage units in an obligatory scheme, each containing one component. Administration itself need not always be made simultaneously. Thus by maintaining a fixed overall dosage it is possible to divide the daily dose of one component into a number of individual doses differing from the number of individual doses into which the daily dose of the other component is divided. For example, if necessary, an injection solution will be administered 3 times daily in combination with ,<'■ 2 tablets 3 times daily, corresponding to the pharmokinetics of both V components. Wherever possible, however, it is preferred to combine both components in a single pharmaceutical composition such as a conventional injection solution or, preferably, in a conventional oral dosage form. Compositions in the form of dosage units are especially preferred. All such pharmaceutical compositions are formulated and processed in accordance with the known general rules and formulation methods of standard pharmaceutical practice.

A synergistically effective ratio is one in which the combination of both components produces an effect which is more potent than the mere additive effect of the individual components. Such a synergistic ratio may vary within wide limits, its maximum effectiveness being dependent, inter alia, on the specific choice of the com— 2 1743 ponents and, in particular, on the respective sensitivity (or resistance) of a Plasmodium strain to each component. This optimum ratio can be determined for each individual case and combination by simple, generally known test methods. As, in general, parasite strains of the same or at least similar resistance occur in the endemic centres of malaria, such a determination usually has general validity for the whole region. Preferably the combination preparation contain both components in a ratio from about 4:1 to 1:4, in particular from about 2:1 to 1:2, said ratio being based on amounts by weight of the components or, preferably, on a comparable activity basis (such as the IC50 for a specific Plasmodium).

For example, a combination preparation for separate administration of each component may consist of an approximately 5-15 % (w/v), preferably 10-12 %, injection solution of desferrioxamine B mesylate and 100-300 mg, preferably about 150 mg, of chloroquine in the form of tablets or dragees, administered as normal daily dose to adults in a ratio of 5-10 ml of injection solution (corresponding to about 250-1500 mg, preferably about 500—1000 mg, of iron chelating agent) to 1-3 tablets (corresponding to about 100-900 mg, preferably about 250-500 mg, of schizontocide) divided into 2-6, preferably 2-4, portions. In severe cases, and especially at the start of the acute phase, the dose can temporarily be doubled. Lower doses, coresponding to body weight, are administered to children. Instead of the mesylate it is also possible to use other suitable acid addition salts of desferrioxamine B or desferriferrithiocine and salts thereof. Chloroquine can also be replaced by another antimalarial agent, e.g. one of those referred to above, in effective equivalent dosage. A pharmaceutical composition comprising a combination of both components is e.g. an injection solution containing about 5-10 % (w/v) of quinine (as dihydrochloride) together with about 5-10 % (w/v) of desferrioxamine B (as mesylate), which solution can be administered daily parenterally in several (2-4, preferably 3) doses, preferably as an infusion over 1—3 hours in an overall daily dose of about 5-25 ml, preferably about 10-20 ml. Pharmaceutical compositions containing a combination of 21743 t both components for oral administration in dosage units are for example capsules, dragees or tablets which contain about 100-300 mg of desferriferrithiocine (or a salt thereof) together with about 100-300 mg of chloroquine (or an analogous antimalerial agent) preferably in equal amounts by weight, and which are administered to an adult 1-6 times, preferably 2-4 times, daily (corresponding to a daily dose of 100-1600 mg, preferably 200—900 mg) of each component. • The pharmaceutical compositions of the present invention are t obtained in a manner known per se, e.g. by conventional mixing, ^ granulating, sugar-coating, dissolving or lyophilising methods. : Accordingly, tablet and sugar-coated tablet cores for oral admini— '• t stration can be obtained by combining the active ingredients with i-. solid carriers, if desired granulating the resultant mixture, and j~ processing the mixture or granulate, if desired or necessary after |" the addition of suitable adjuncts, to tablets or sugar-coated tablet • cores. j i i Suitable carriers are in particular fillers such as sugar, for ! t example lactose, saccharose, mannitol or sorbitol, cellulose • preparations and/or calcium phosphates, e.g. tricalcium phosphate or j i calcium biphosphate, and also binders such as starch pastes, e.g. { maize, corn, rice or potato starch, gelatin, tragacanth, methyl j cellulose and/or polyvinylpyrrolidone, and/or, if desired, disinte— j grators, such as the above-mentioned starches, also carboxyraethyl I starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a i salt thereof such as sodium alginate. Adjuncts are in particular ! i glidants and lubricants, for example silica, talc, stearic acid or K salts thereof such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable * coatings which can be resistant to gastric juices, using inter alia V-'- concentrated sugar solutions which may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, *1 shellac solutions in suitable organic solvents or mixtures or solvents or, for the preparation of coatings which are resistant to r gastric juices, solutions of suitable cellulose preparations such as j i 2 1743 acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments can be added to the tablets or dragee coatings, for example to identify or indicate different doses of active ingredient .

The following Examples serve to illustrate the invention.

Example I: Combination preparation for separate administration of the components.

Component A: Dry-filled ampoules containing 250 mg and 500 mg of iron(III) chelating agent. ml ampoules are each filled with 2.5 ml of a 10 % (w/v) filtered and sterilised aqueous solution of desferrioxamine B mesylate and lyophilised in conventional manner. The injection solution is prepared by addition of a corresponding amount (2.5 - 5.0 ml) of water or physiological solution (sterilised).

In similar manner, dry-filled ampoules containing 500 mg of desferrioxamine B mesylate are prepared for formulating 5 ml of a 10 % (w/v) of injection solution.

Component B: 150 mg tablets of blood schizontocide Composition of a tablet: chloroquine sulfate (micronised) 150.0 mg corn starch 50.0 mg colloidal silica 5.0 mg gelatin 5.0 mg cellulose (microcrystalline) 75.0 mg sodium carboxymethyl starch 20.0 mg magnesium stearate 1.5 mg 306.5 mg 217431 Preparation of 100,000 tablets kg of chloroquine sulfate and 5-0 kg of corn starch are raixed with 0.5 kg of colloidal silica and the mixture is moistened with a solution of 0.5 kg of gelatin in 5.0 kg of distilled water (30°C). This moist mixture is passed through a 3 mm sieve and dried at 45°C (fluidised bed drier). The dry granulate is forced through a 0.8 mo sieve, mixed with a presieved mixture of 7.5 kg of microcrystalline cellulose and 2.0 kg of sodium carboxymethyl starch and 0.15 kg of magnesium stearate, and the whole is compressed to tablets weighing 306.5 mg.

Example 2: Dry-filled ampoules for combined administration of both components, each containing 300 tag of desferrioxamine B mesylate and 300 mg of quinine hydrochloride 7-10 nil ampoules are filled in succession with 3 ml of a sterilised and filtered aqueous 10 % (w/v) solution of quinine dihydrochloride and 2 ml of a sterilised and filtered aqueous 15 « (w/v) solution of desferrioxamine B mesylate and lyophilised under conventional conditions. (Alternatively, it is possible to fill the ampoules with 6 ml of an aqueous solution prepared by dissolving 100 g of each component in water and sterilised by filtration).

The injection solution is prepared by dissolving the dry solid under sterile conditions by addition of a corresponding amount (5-10 ml) of sterilised water or physiological solution.

Example 3: Dragees containing c. 100 mg of component A [iron(III) chelating agentJ and 100 mg of component B (blood schizontocide) are prepared as follows: J . ■ 217431 Composition of a drawee core: component A (micronised) component B (micronised) corn starch tricalcium phosphate polyvinylpyrrolidone K 25 magnesium stearate sodium carboxymethyl starch 100.0 mg 100.0 mg 75.0 mg 75.0 mg 15.0 mg 2.0 mg 33.0 mg 400.0 mg Preparation of 50.000 drawees A mixture of 5 kg of micronised component A, 5 kg of micronised component B, 3.75 kg of corn starch and 3.75 kg of tricalcium phosphate is granulated with a solution of 0.75 kg of polyvinylpyrrolidone K 25 in 5 kg of distilled water by fluidised bed granulation. The granulated material is passed through a 1 mm sieve and dried at 45°C and then mixed with 0.1 kg of magnesium stearate and 1.65 kg of sodium carboxymethyl starch and the mixture is compressed to 400 mg domed tablets.

Preparation of 6.6 kg of sugar-coated dragees 6 kg of the dragees cores are coated in portions to a weight of 410 mg in a coating pan of 45 cm diameter with a sugar syrup (2 parts of sugar and 1 part by weight of distilled water) in which 1.5 % of polyvinylpyrrolidone K 25 and 1 % of polyethylene glycol 6000 is dissolved and 20 % of talcum is suspended. Then sugar syrup (2 parts of sugar and 1 part of water) is applied to a final weight of 440 mg. The dragees are finally given a gloss with a 2 % solution of carnauba wax in trichloroethylene.

Desferriferrithiocine (or an equivalent amount of the sodium or potassium salt is used as component A, and chloroquine (or an equivalent mount of amodiaquine, artemisinine or a derivative thereof, sulfadioxine or pyrimethamine) is used as component B. 18 - f WslX Example 4: Gelatin capsules containing 125 mg of iron(III) chelating agent as component A and 125 mg of blood schizontocide as component B are prepared as follows: Composition of a drv-filled capsule Preparation of 10.000 dry-filled capsules 1.25 kg of micronised desferriferrithiocine is intimately mixed with 1.25 kg of micronised chloroquine diphosphate and, if necessary, triturated. To this mixture are added 0.49 kg of micronised lactose and 0.01 kg of magnesium stearate and the mixture is passed through a sieve and homogenised. The powder is sieved and filled dry, in portions, into gelatin capsules.

In a manner similar to that described in the preceding Examples 1—4 it is possible to process the other components mentioned in the description to analogous medicaments.

Test Report Test Micro-organisms a) The basic organism cell line A , which has normal sensitivity to chloroquine, is reproduced by conventional culturing of the strain Plasmodium falciparum in a 2 % (v/v) suspension of human erythrocytes . desferriferrithiocine chloroquine diphosphate lactose 125.0 mg 125.0 mg 49.0 mg magnesium stearate 1.0 mg 350.0 mg 2 I 74 3 1 b) Cell line A of the above strains cultured under similar standard conditions as under a), but with the addition of successively increasing concentrations of chloroquine until the formation of a chloroquine-resistant cell line B. which continues to exhibit normal growth (the same rate of growth as cell line A) in culture media with a 500 jiM concentration of chloroquine.

Assav: I. The test organisms are cultured under the conditions conventionally employed for culturing plasmodia on a 2 % suspension of human erythrocytes, supplemented with 50 mg/Jl of hypoxanthine and with the optional addition of the tested inhibitory substance in the desired concentration. The initial parasitemia (= the percentage of erythrocytes infected at the start by plasmodia) is 1-2 %. The result (inhibitory action) is assessed as the IC50. i.e. the concentration of the active ingredient which effects a lowering of the final parasitemia by 50 %, i.e. 50 % growth inhibition.

II. An alternative assay is carried out without the addition of hypoxanthine, but maintaining all other conditions. The initial parasitemia is 0.5 %.

Preliminary assays (Sensitivity to chloroquine) Cell line A: In assay I without chloroquine, the plasmodia multiply within 48 hours by a factor of 7-9; IC50 * ~50 jtM of chloroquine over 2 cycles, corresponding to a normal sensitivity to chloroquine of other strains.

In assay II, the IC50 is c. 10-15 jiM of chloroquine over 2 cycles.

Cell line B: In assay I at a chloroquine concentration of 500 the multiplication factor is again 7-9 (i.e. it is equal to that of cell line A without chloroquine); IC50 = ~800 jiM of chloroquine over 2 cycles.

I 17431 Similar results are also obtained using sulfadioxine and pyrimethamine instead of chloroquine.

Principal assay I (Sensitivity to desferrioxamine B) j- Cell line A: Assay I: IC50 "17 : 3 |iM of desferrioxamine B over 2 cycles.

Assay II: ICso ■ 5 i 2 viM of desferrioxamine B over 2 cycles.

Cell line B: a) without the addition of chloroquine in the test tnedium: t Assay I: ICso ■ 6 i 1.5 jiM of desferrioxamine B over j v- 2 cycles. } \ - Assay II: ICso ■ 2 ! 1 |lM of desferrioxamine B over ' r 2 cycles. ! t _ b) with addition of chloroquine in a > 400 nM concentration (i.e. below the resistance N threshhold) Assay I: ICso = 3 I 1 ]iM of desferrioxamine B over 2 cycles.

Assay II: IC50 ■ 0.6 ± 0.1 pM of desferrioxamine over 2 cycles.

Principal assav II The following inhibitory concentrations are determined with Plasmodium falciparum of cell line A (chloroquine-sensitive) in assay I: chloroquine IC;o 0 10 jiM desferrioxamine B mesylate IC20 * 9 }*M Chloroquine at 10 jiM and desferrioxamine B mesylate at 9 jiM combined effect a 70-100 % growth inhibition of the above cell line.

I . 1 (■ . 1 l 217431 21

Claims (11)

WHAT WE CLAIM IS:-

1. A combination preparation comprising an iron(III) chelating agent selected from the class consisting of desferrioxamine 3, pharmaceutically acceptable acid addition salts of desferrioxamine 3, desferriferrithiocine, pharmaceutically acceptable salts of desferriferrithiocine, and lower alkyl esters of desferriferrithiocine as component A, and a conventional antimalarial agent selected from the category of the schizontocides as component B, each component being contained in a separate pharmaceutical composition or both components being contained together in a single pharmaceutical composition, optionally in admixture with at least one pharmaceutical excipient, said components being in a synergistically effective ratio to each other.

2. A combination preparation according to claim 1, wherein component A is in the form of an injection solution and component B is in the form of an oral dosage form.

3. A combination preparation according to claim 1, which contains both components in a synergistically effective ratio in the form of a single pharmaceutical composition.

6. A combination preparation according to claim 3 for parenteral administration.

5. A combination preparation according to claim 3 for oral administration.

6. A combination preparation according to claim 1, which contains, as component A, desferrioxamine B in the form of the free base or of a pharmaceutically acceptable acid addition salt.

7. A combination preparation according to claim 5, which contains desferriferrithiocine or a pharmaceutically acceptable salt thereof as component A. 2!7431 - 22 -

8. A combination preparation according to claim 6 or claim 7, which contains, as component S, chloroquine in the form of the free base or of a pharmaceutically acceptable acid addition salt.

9. A combination preparation according to claim 6, which contains, as component B, quinine in the form of the free base or of a pharmaceutically acceptable acid addition salt.

10. A combination preparation according to claim 1, wherein the synergistic ratio of component A to component 3 is 4:1 to 1:4, based on amounts by weight.

11. A combination preparation according to claim 1, wherein the synergistic ratio of component A to component 3 is 2:1 to 1:2, based on the relative effectiveness in terms of inhibiting concentrations as comparable activity basis. 2 6 OCT 1989 &