EP0794776A2

EP0794776A2 - Use of rifabutin and a combination of rifabutin and atovaquone in the treatment of pneumocystis carinii infections

Info

Publication number: EP0794776A2
Application number: EP95938501A
Authority: EP
Inventors: John Charles William Comley
Original assignee: Wellcome Foundation Ltd
Current assignee: Wellcome Foundation Ltd
Priority date: 1994-11-29
Filing date: 1995-11-29
Publication date: 1997-09-17
Also published as: GB9424013D0; WO1996016652A2; AU3987195A; WO1996016652A3; JPH10509722A

Abstract

The invention relates to combinations of atovaquone and rifabutin, their use in the treatment and prophylaxis of infections caused by P.carinii and their use in the manufacture of medicaments for the treatment and/or prophylaxis of such infections. The combinations can conveniently be administered in a single pharmaceutical formulation. Preferably, atovaquone and rifabutin are administered in a potentiating ratio so that they act in synergy.

Description

USE OF RIFABUTIN AND A COMBINATION OF RIFABUTIN AND ATOVAQUONE IN THE TREATMENT OF PNEUMOCYSTIS CARINII INFECTIONS

The present invention relates to synergistic combinations of atovaquone and rifabutin which have activity against. Pneumocystis carinii. More particularly, the invention is concerned with pharmaceutical compositions and their use in the treatment of infections caused by Pneumocystis carinii.

Atovaquone 2-[4-{4-chlorophenyl)cyclohexyl]-3-hydroxy-1 ,4-naphthoquinone is described in US Patent No. 5053432 and European Patent No. 0123238. It has been described as being useful in the treatment of Pneumocystis carinii, malaria and a number of other protozoal infections.

Rifabutin is a semisynthetic ansamycin antibiotic derived from rifamycin S. It has the chemical name 1',4-didehydro-1-deoxy-1,4-dihydro-5^,-(2-methylpropyl)- 1-oxorifamycin XIV (Chemical Abstracts Service, 9th Collective Index) or (9S, 12E, 14S, 15R, 16S, MR, 18R, 19R, 20S, 21 S, 22E, 24Z) - 6,16,18,20 - tetrahydroxy-1 '-isobutyl-14-methoxy-7,9,15,17, 19,21 ,25-heptamethyl- spiro [9,4- (epoxypentadeca[1,11 ,13]trienimino)-2H-furo[2\3\:7,8]naphth [1 ,2-c midazole- 2,4^,-piperidine]-5_I10,26-(3H,9H)-trione-16-acetate. Its structure is represented by Formula (I).

Rifabutin has activity against My∞bacterium aviυm complex (MAC) and is commonly used for treatment and prophylaxis of MAC, particularly in patients with AIDS (D.L. Cohn and C.R. Horsburgh, Opportunistic Complication of HIV 2 1, p.6-12 and S. Nightingale et al, New England Journal of Medicine 329 p828- 833). However, it does not have activity against P. carinii infections.

It has now surprisingly been found that by combining, either concomitantly or sequentially, atovaquone and rifabutin, potentiation of atovaquone's activity against P.carinii is achieved. A potentiating combination of atovaquone and rifabutin can be simply presented in a single pharmaceutical formulation.

Combination therapy is advantageous for the treatment of infections since it reduces the likelihood of drug-resistant strains emerging. Where drug combinations are synergistic, it may also be possible to reduce dosages and thus to improve drug tolerance and patient compliance. This is particularly important in immunocompromised patients such as patients with AIDS.

In a first aspect, the present invention provides a method for the treatment and/or prophylaxis of an infection caused by P. carinii in mammals, including humans, which comprises administering an effective amount of atovaquone or a physiologically acceptable salt thereof and concomitantly or sequentially administering an effective amount of rifabutin.

In a second aspect, the present invention provides atovaquone for use in the manufacture of a medicament, for administration either concomitantly or sequentially with rifabutin, for treatment and/or prophylaxis of an infection caused by P. carinii, in mammals, including humans.

In a third aspect, the present invention provides rifabutin for use in the manufacture of a medicament for treatment and/or prophylaxis of an infection caused by P. carinii in mammals including humans. This medicament may be administered either concomitantly or sequentially with atovaquone.

Preferably atovaquone and rifabutin are administered concomitantly for treatment and/or prophylaxis of infections caused by P. carinii. Most preferably atovaquone and rifabutin are administered in a potentiating ratio.

Thus, according to a further aspect of the present invention, there is provided a combination of atovaquone, or a physiologically acceptable salt thereof, and rifabutin wherein atovaquone, or its salt, and rifabutin are present in a potentiating ratio.

The term 'potentiating ratio' is used herein to indicate that atovaquone and rifabutin are present in a ratio such that the antiparasitic activity of the combination is greater than that of atovaquone alone or of the additive activity that would be predicted for the combination based on the activities of the individual components. Thus the individual components act synergistically in combination provided they are present in a potentiating ratio.

A potentiating ratio, which may be successfully used to treat Pneumocystis carinii infections is in the range 1:0.1 to 1:100 of atovaquone: rifabutin. Suitably, the potentiating ratio is in the range 1:0.2 to 1:10.

A particularly suitable potentiating ratio is in the range 1 :1 to 1:3.

The present invention also provides in another aspect a method for the treatment and/or prophylaxis of Pneumocystis carinii infections in mammals, including humans, which comprises administering an effective amount of a combination of atovaquone, or a physiologically acceptable salt thereof, and rifabutin.

The hydroxyl group of atovaquone may form salts with appropriate bases, and physiologically acceptable salts of atovaquone include inorganic base salts such as alkali metal (e.g. sodium and potassium) salts and alkaline earth metal

(e.g. calcium salts); organic base salts (e.g. phenylethylbenzylamine, dibenzylethylenediamine, ethanolamine and diethanolamine salts); and amino acid salts (e.g. lysine and arginine).

The amount of a combination of atovaquone and rifabutin required to be effective against Pneumocystis carinii will, of course, vary and is ultimately at the discretion of the medical practitioner. The factors to be considered include the route of administration and nature of the formulation, the mammal's bodyweight, age and general condition and the nature and severity of the disease to be treated. In general, a suitable effective dose for administration to man for treatment of Pneumocystis carinii infections is in the range of 5mg to 1000mg of atovaquone per kilogram bodyweight per day and 5mg to 1000mg of rifabutin per kilogram bodyweight per day, for example from 10 to 500mg/kg/day of atovaquone and 20 to 500mg/kg/day of rifabutin, particularly 20 to 100mg/kg/day of atovaquone and 20 to 400mg/kg/day of rifabutin.

A suitable effective dose for administration to man for prophylaxis of Pneumocystis carinii infections is in the range of from 100 to lOOOOmg per kilogram bodyweight per week of each of atovaquone and rifabutin for example from 200 to 3000mg/kg/week of each of atovaquone and rifabutin.

It should be understood that the dosages referred to above are calculated in terms of the drugs βer se, not the salt forms.

For use according to the present invention the combination of atovaquone and rifabutin is preferably presented as a pharmaceutical formulation.

Pharmaceutical formulations comprise the active ingredients (that is, the combination of atovaquone and rifabutin) together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic and/or prophylactic ingredients.- The carriers) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

Accordingly, the present invention provides a pharmaceutical formulation comprising a combination of atovaquone and rifabutin in association with one or more pharmaceutically acceptable carriers therefor.

The present invention further provides a process for the preparation of a pharmaceutical formulation which process comprises bringing into association a combination of atovaquone and rifabutin with one or more pharmaceutically acceptable carriers therefor.

The combination of atovaquone and rifabutin may conveniently be presented as a pharmaceutical formulation in unit dosage form. A convenient unit dose formulation contains the active ingredients in amounts of from 10mg to 3g each, e.g. 50mg to 3g each. Typical unit doses may contain for example 100mg of atovaquone and 300mg of rifabutin or 50mg of atovaquone and 150mg of rifabutin.

Pharmaceutical formulations include those suitable for oral, rectal and parenteral (including subcutaneous, intradermal, intramuscular and intravenous), administration as well as administration by naso-gastric tube. The formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of the active ingredients. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compounds in a free- flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent. Moulded tablets may be made by moulding an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored.

Capsules may be prepared by filling the active ingredients, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein the active ingredients together with any accessory ingredient(s) are sealed in a rice paper envelope. The combination of atovaquone and rifabutin may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged e.g. in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water liquid emulsion.

Formulations for oral administration include controlled release dosage forms, e.g. tablets, wherein the active ingredients are formulated in an appropriate release - controlling matrix, or are coated with a suitable release - controlling film. Such formulations may be particularly convenient for prophylactic use.

The active ingredients may also be formulated as a solution or suspension suitable for administration via a naso-gastric tube.

Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of the active combination with the softened or melted carrier(s) followed by chilling and shaping in moulds.

Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of the active combination in aqueous or oleaginous vehicles. Injectible preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers which are sealed after introduction of the formulation until required for use. Alternatively, the active ingredients may be in powder form which are constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.

The combination of atovaquone and rifabutin may also be formulated as a long- acting depot preparation, which may be administered by intramuscular injection or by implantation e.g. subcutaneously or intramuscularly. Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion- exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.

It should be understood that in addition to the aforementioned carrier ingredients the pharmaceutical formulations for the various routes of administration described above may include, as appropriate one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.

Methods for preparing atovaquone are described in EP 123,238, and one specific method is illustrated in Example 1.

Example 1

2-ftrans-4-(4-Chlorophenyl.cvclohexyn-3-hvdroxy-1.4-naphthoαuinone a) 4-(4-Chlorophenyl.cvclohexane-1-carboxylic Acid

Acetyl chloride (30g) and finely powdered aluminium chloride (60g) were stirred together in carbon disulphide (120 ml) and then cooled to -50°C, in a C0₂/oxitol bath. Cyclohexene (30 g), previously cooled to -50°C, was added dropwise during 10 minutes while maintaining the temperature of the reaction mixture at below -20°C. The mixture was stirred at -50°C for a further 60 minutes and the solvent then decanted to leave a gummy orange complex. A little chlorobenzene was added as the material warmed to ambient temperature; the remainder of the chlorobenzene (total 300 ml) was then added, the so-obtained solution heated at 40°C for 3 hours with stirring, poured onto a mixture of ice and concentrated hydrochloric acid and the organic layer separated, washed with 2M hydrochloric acid, 2M sodium hydroxide and water, dried over anhydrous sodium sulphate and evaporated to dryness. The product was distilled in vacuo. the fraction boiling at 140-154°C (0.1 mm Hg) collected, diluted with an equal volume of petroleum ether (40-60), cooled to -6°C and a continuous stream of nitrogen gas bubbled through, and the separated colourless solid recovered.

Bromine (2.8ml) was added to a solution of sodium hydroxide (6.2g) in water (42 ml) at 0°C. The above-obtained substituted hexahydroacetophenone (3.1g) was dissolved in dioxan (15 ml) and the cold hypobromite solution then added, keeping the reaction mixture at below 20°C. The reaction mixture was stirred at ambient temperature for 6 hours^'* then allowed to stand overnight. Sodium metabisulphite was added to destroy excess hypobromite, the mixture cooled and then acidified to give a colourless solid. The solid was filtered off, washed with water, dried and recrystallised from ethanol to give 4-(4-chlorophenyl) cyclohexane-1-carboxylic acid, m.p. 254-256°C.

b, 2-f4-(4-chlorophenyl .cvclohexyl ^*-3-chloro-1.4-naphthoαuinone

A mixture of 2-chloro-1 ,4-naphthoquinone (3.95g, 0.02 mol), 4-(4- chlorophenyl)cyclohexane-1-carboxylic acid (4.9g, 0.02 mol) and powdered silver nitrate (1.05g, 0.0062 mol) was heated to reflux with vigorous stirring in 40 ml of acetonitrile. A solution of ammonium persulphate (12.0g, 0.0525 mol) in 50 ml of water was added dropwise over 1 hour. The mixture was refluxed for 3 hours then cooled in ice for 30 mins, after which it was filtered, and the residual sticky solid extracted twice with boiling chloroform to remove inorganic material. The chloroform was removed by evaporation to leave a yellow-brown solid (ca 2.7g). This was dissolved in 40 ml of boiling acetonitrile; a little insoluble material was removed by filtration. On cooling, the title compound separated as yellow crystals, (550 mg) m.p. 172-175°C.

NMR, dH(δβ-DMSO) 8.05 (2H, mult., β-naphth), 7.85(2H, mult., a-naphth), 7.30 (4H, s., PhH), 3.30 (1H, br.t., CH), 2.67 (1H, br.t., CH), 1.2-2.4 (8H, mult., 4xCH₂).

c) 2-f4-f4-chlorophenyl.cvclohexyn-3-hvdroxy-1.4-naphthoαuinone

The product of stage (b) was suspended in 10 ml of boiling methanol and 0.55g of potassium hydroxide in 5.5 ml of water was added dropwise over 15 mins. The mixture was refluxed until a dark red solution formed, (after ca. 6 hrs) when 2 ml of concentrated hydrochloric acid was cautiously added dropwise. The mixture was cooled and filtered, and the solid residue washed thoroughly with water. The water washings were re-acidified and filtered. The combined solid residues (500 mg) mp 200-209°, were recrystallised from acetonitrile to give the title product as the trans-isomer (300 mg) m.p. 216-219°C.

BIOLOGICAL TEST RESULTS

Example 2

Comparison of atovaquone alone with atovaαuone/rifabutin combination for prophylaxis of P. carinii.

Prophylactic antipneumocystis activity was determined in SCID (C.B.-17lcr cruscid) mice artificially infected with P. carinii. This SCID mouse model has been described by J.C.W. Comley and A.M. Sterling in J.Eukaryotic

Microbiology. Briefly the protocol involved starting Pneumocystis-free female

SCID mice (20-25g) on dexamethasone dissolved in the drinking water (at

2mg/l) 7 days prior to intratracheal (IT) inoculation with a single dose of cryopreserved mouse P. carinii (-20,000 cysts). Each drug or control group consisted of at least 10 mice. Drugs were evaluated for prophylaxis by administration once a day from day 1 post infection (p.i.) until day 42 p.i. (i.e. 42 doses). All drugs were administered orally, by gavage. In combination studies mice were always dosed with atovaquone first, with rifabutin administered immediately afterwards. All drugs trials were terminated on day 43 p.i. 24 hours after the last drug dose. Mice were anaesthetized with halothane, killed by cervical dislocation, the lungs removed en bloc and impression smears prepared. The presence of P. carinii in lung impression smears was rapidly and unambiguously identified by immunofluorescence. The intensity of the PCP was graded by scanning the impression smears and assigning, on a semi-logarithmic basis, one of the following infection scores: 0 = no infection evident; + 1 = very weak infection; + 2 = mild infection; + 3 = moderate infection; + 4 = heavy infection. Scores were an agreed assessment reached following two independent blind examinations.

The results are presented in Table 1 as follows:

Table 1

Effect of atovaquone (ATQ) in combination with rifabutin (RIF) on the prophylaxis of PCP in SCID mice.

Group Drug Dose Mean Infection SEM % No.lnf. / Statist. No. Group (mgkg/dayp.o.) Score¹ CONTROL² No.Exam³ Signif.

1 CONTROL Untreated 3J73 0.19 100 11/11

2 ATQ 50 2.30 0.14 62 10/10 a

3 ATQ 25 2.82 0.28 76 11/11 a

4 RIF 200 3.18 0.17 85 11/11

5 RIF 100 3.73 0.13 100 11/11

6 ATQ+RIF 50+200 0.40 0.15 11 4/10 a.b.c,

7 ATQ+RIF 50+100 0.18 • 0.12 5 2/11 a.b.c,

8 ATQ+RIF 25+200 0.64 0.23 17 5/11 a.b.c,

9 ATQ+RIF 25+100 0.55 0.24 15 4/11 a.b.c,

1 ) the calculated mean infection scores (± standard error of mean (SEM));

2) a percentage of the infection score in the control (% control);

3) the ratio of the number of mice infected with P. carinii over the total number of mice remaining in each group at the end of the experiment (No. Inf/No. Exam).

Statistically significant differences in the infection score between control and drugs groups in the same drug study were determined by a t-Test (two-sample assuming unequal variances) computed using commercial software (Microsoft Excel).

^statistically significant (P<0.05) from the CONTROLS (Group 1 )

^-statistically significant (P<0.05) from corresponding ATQ Group (2 or 3)

^c-statistically significant (P<0.05) from corresponding RIF Group (4 or 5).

Claims

1. A method for the treatment and/or prophylaxis of an infection caused by P. carinii in a mammal which comprises administering atovaquone or a physiologically acceptable salt thereof and administering rifabutin in amounts such that when both atovaquone or a physiologically acceptable salt thereof and rifabutin have been administered the mammal has received an effective treatment amount.

2. A method according to claim 1 wherein the atovaquone or a physiologically acceptable salt thereof and rifabutin are administered concomitantly.

3. A method according to claim 1 or claim 2 wherein the atovaquone or a physiologically acceptable salt thereof and rifabutin are administered in a potentiating ratio.

4. The use of atovaquone or a physiologically acceptable salt thereof for the manufacture of a medicament for administration either concomitantly or sequentially with rifabutin, for treatment and/or prophylaxis of an infection caused by P. carinii in mammals.

5. The use according to claim 4 wherein the medicament is adapted for administration concomitantly with rifabutin.

6. The use of rifabutin for the manufacture of a medicament for treatment and/or prophylaxis of an infection caused by P. carinii in mammals.

7. The use of rifabutin for the manufacture of a medicament for administration either concomitantly or sequentially with atovaquone or a physiologically acceptable salt thereof for treatment and/or prophylaxis of an infection caused by P. carinii in mammals.

8. A combination of atovaquone or a physiologically acceptable salt thereof and rifabutin.

9. A combination according to claim 8 wherein the atovaquone or a physiologically acceptable salt thereof and rifabutin are present in a potentiating ratio.

10. A combination according to claim 9 wherein the ratio of atovaquone:rifabutin is in the range 1:1 to 1:3.

11. A combination according to any of claims 8 to 10 for use in the treatment and/or prophylaxis of an infection caused by P. carinii in a mammal.

12. Use of a combination according to any of claims 8 to 11 for the manufacture of a medicament for the treatment and/or prophylaxis of an infection caused by P. carinii in a mammal.

13. A method for the treatment and/or prophylaxis of an infection caused by P. carinii in mammals which comprises administration of an effective amount of a combination according to any of claims 8 to 11.

14. A pharmaceutical formulation comprising a combination according to any of claims 8 to 11 in association with one or more pharmaceutically acceptable carriers therefor.

15. A product comprising atovaquone or a physiologically acceptable salt thereof and rifabutin as a combined preparation for simultaneous, separate or sequential use in therapy and/or prophylaxis of an infection caused by

P. carinii .