USE OF PARTRICIN DERIVATIVES FOR TREATING FUNGAL AND PROTOZOAL INFECTIONS * * * * * * * * The present invention realtes to the use of partricin derivatives for treating fungal and protozoal infections; in particular, the present invention concerns the use of a pharmaceutical formulation comprising a therapeutically effective quantity of a derivative of partricin A and/or B or a pharmaceutically acceptable salt thereof for the manufacture of a medicament useful in the oral treatment of fungal and/or protozoal, oral and/or intestinal infections.

Partricin is a polyene macrolide belonging to the heptane class, whose chemical structure, independently from cis/trans isomers and enantiomer forms, steric configuration, etc. is represented in The Merck Index, 13th Edition n. 7121. The two components A and B differ from each other for the presence of a methylamino or amino group, respectively, in the para position of the aromatic ring at the C-37 of the macrolide ring.

They can be obtained by isolation from the fermentation broths of Streptomyces aureofaciens NRRL3878 or from the compound mixture in the solid state, or even through specific selective fermentations.

Partricin is endowed of potent anti-fungal and anti-protozoal activity, but because of its noticeable toxicity, it has not found therapeutic use. Its very low water solubility constituted a further obstacle to its pharmaceutical use.

Pharmaceutically acceptable derivatives of partricin A and/or B are disclosed, for example, in EP-B-0434943 (corresponding to US 5296597) and in EP-B-0489308 (corresponding to US 5298495).

Generally, such derivatives of partricin A and/or B are amide or ester derivatives at the carboxy group at C-18, optionally aminoacylated at the amino group of the mycosamine moiety. Such derivatives result often more active as anti-fungal/anti-protozoal agents, in comparison to the starting partricin, and even more often result to be endowed with lower systemic toxicity and improved tolerance.

Although still scarcely soluble in a water medium"per se", the presence of basic groups in the radicals characterizing the derivatives,

permits an easy salt preparation and to reach-consequently-a high, unusual water solubility. The above mentioned patents disclose the topical use of these derivatives, f. i. as creams and ointments for dermatological use, tablets or suppositories for vaginal use, etc. , and let forecast similar uses as in collyria, lotions, topical powders, etc.; the improved bio-availability thereof after oral administration and particularly the achieved possibility of an injectable use by parenteral route, especially by intravenous route, is therein also emphasized.

We have now found that the improved water solubility, which is particularly excellent with the salt, while permitting-indeed-an optimal use of such derivatives by parenteral route and particularly by intravenous route, is however still insufficient to guarantee an acceptable absorption and a satisfactory systemic bio-availability of the partricin derivatives after oral ingestion.

In a test carried out on rats (Test 1 herebelow), it has been shown, in fact, that although the preparation of a salt of one of the above mentioned partricin derivatives led evidently to a higher absorption by oral route in comparison with the corresponding free base-which is very slightly soluble in water-, it did not yet bring to blood levels comparable-not even roughly-to those obtained by the intravenous administration.

Test 1 Mean plasma concentrations were detected in groups of male rats (N=5) following the administration of N-dimethylaminoacetyl partricin A 2-dimethylaminoethylamide base and of the diascorbate salt thereof (code: SPK-843) by oral route (100 mg/kg) and by intravenous route (1 mg/kg of only the diascorbate salt). HPLC analysis according to Kim H. and Lin C. , Antimicr. Agents Chemother. , 25,45 1984. Time Base Diascorbate Diascorbate 100 mg/kg, p. o. 100 mg/kg, p. o. 1 mg/kg, i. v. ml ml rnl 15 mit---1. 630 1 h - 0.025 1. 025 2 h 0. 016 0. 088 0.648 8 h 0. 038 0. 236 0.415 24 h 0. 014 0. 163 0. 320 48 h-0. 027 0. 160 p. o.: per os (oral administration)

It is noteworthy that the water soluble diascorbate is able to give, after oral administration, plasma concentrations much higher than the free base, and yet still extremely lower than those obtained by i. v. administration of a 100 times lower dose, thus showing that the oral bioavailability of the above derivatives is still unsatisfactory.

It can be therefore observed that a very high percentage of the orally administered derivative, even in the more water-soluble form of a salt, is irremediably lost to the purpose of a systemic absorption and is then excreted as such (f. i. , mean fecal excretion of 90-99%), usually being minimally in an inactive form because of metabolic processes or of degradation induced by the strong gastric acidity, if the compound was not administered in gastro-resistant form.

Nevertheless, it has been unexpectedly found that these water soluble derivatives of partricin A and/or B are particularly useful also for the oral route, to display locally their powerful wide spectrum anti- mycotic and anti-protozoal activity.

In another test carried out on mice (Test 2 here below) infected with cultures of Candida albicans, it has been found that the above mentioned water soluble derivatives are effective against oral and intestinal infections, strongly decreasing the number of viable fungal cells and also bringing to the complete sterilization of the infected organ. The derivatives activity has been found to be significantly higher than that of the same derivative in the form of slightly soluble free base, the increase of the activity being evidently induced by the enhanced diffusion power of the compound, due to the reached water solubility.

Test 2 Four groups of five male mice each, immuno-suppressed by pre- treatment with triamcinolone acetonide (1 mg/mouse, by subcutaneous route), were treated after 2 days with 4. 2 106 cells/mouse of Candida albicans in the oral cavity, thereafter two groups were treated from the third to the fifth day with 0.5 and 1.0 mg/mouse/day of N- dimethylaminoacetyl partricin A 2-dimethylaminoethylamide diascorbate (SPK-843) by oral route whereas one group was treated with 1.0 m g/mouse/day of the corresponding free base. After 24 hours from the last

administration, the mice were subjected to an oral drawing (oral tampon) for the counting of the viable Candida cells.

According to a similar scheme, four groups of mice were treated with 5 and 10 mg/mouse/day of SPK-843 by oral route and 1 group with 10 mg/mouse/day of free base. After 24 hours from the last administration, stools were collected for the counting of the viable Candida cells. Group CFU Negative Group CFU Negative (mean/tampon) culture (mean/10 mg culture stools) Control 3. 2-105 0/5 Control 8. 4-104 0/5 (oral (intestinal infection) infection) 0. 5 mg/kg 2. 510 3/5 5 mg/kg 0. 710 4/5 Diascorbate diascorbate 1. 0 mg/kg 0. 310 4/5 10 mg/kg-5/5 diascorbate diascorbate 1. 0 mg/kg 3. 8-103 0/5 10 mg/kg 8. 5-102 1/5 free base free base CFU : Colony Forming Units The high antifungal activity of the above mentioned partricin derivative against oral and intestinal infections by Candida albicans was therefore confirmed. The activity of such derivative was anyway much more evident when the derivative is a water soluble one such as, f. i., under the form of a diascorbate salt.

It is therefore possible to observe, to the purpose of a clearly localized therapy, that the mentioned lack of systemic absorption permits the evident advantage to minimize the side effects of systemic origin.

Accordingly, the use of a pharmaceutical formulation comprising a therapeutically effective quantity of a derivative of partricin A and/or B or a pharmaceutically acceptable salt thereof for the manufacture of a medicament useful in the oral treatment of fungal and/or protozoal, oral and/or intestinal infections, can be mentioned among the aspects of the invention; preferably, the fungal infection is due to Candida albicans.

In particular, the derivative of partricin A and/or B is selected from the group consisting of an amide or an ester, unsubstituted or substituted by an aminoalkyl group, the mycosamine moiety thereof being unsubstituted or substituted by an N-aminoacyl group or a pharmaceutically acceptable salt thereof; preferably, the derivative of

partricin A and/or B is N-dimethylaminoacetyl-partricin A 2- dimethylaminoethylamide diascorbate.

The partricin derivative comprised in the pharmaceutical formulation suitable for the invention appears under the form of a yellow solid with high and poorly defined melting point, because of the gradual decomposition thereof. EP-B-0434943 and EP-B-0489308 are herein incorporated by way of reference, as far as the structure and preparation method thereof are concerned.

According to a preferred embodiment the above mentioned partricin derivative has a water solubility >0.1% which permits dissolving locally the complete dose of the medicament.

Because of the partricin derivative powerful anti-mycotic activity and for the requested local activity thereof, the therapeutically effective quantity of such derivative is very low and preferably ranges between 0.1 mg and 200 mg, in particular between 1 and 50 mg.

According to another preferred embodiment, the formulation suitable for the invention is under the form of granules, powders or mouth-soluble tablets ; in particular, the granules or powders are dissolved, suspended or diluted in a watery, hydroalcoholic, hydroglycolic, polyglycolic or oily vehicle.

A pharmaceutical formulation comprising a thickening and/or surfactant agent is further preferred; the formulation, according to another preferred embodiment, is under the form of a gastro-resistant tablet, in particular protected by an overcoating or undercoating protective layer.

In general and for all the therapeutic uses, the above mentioned partricin derivative can be diluted with suitable quantities of pharmaceutically acceptable, solid or liquid, excipients, as well as with disintegrating, preservative, edulcorating, thickening, anti-oxidant agents and the like, as well known to the average man skilled in the art.

The pharmaceutical formulation which can be considered to be suitable for the invention permits to vehiculate properly the medicament to the oral and/or intestinal infection site, and include f. i. solutions and suspensions in watery, hydroalcoholic, hydroglycolic, polyglycolic or oily

vehicle, etc. , also added with thickening and surfactant agents, etc. , and in particular mouth-soluble tablets, and also powders and granules to optionally dissolve/suspend/dilute before using in any liquid vehicle, as known to the average man skilled in the art, for the oral cavity disinfection; all of mentioned formulations, as well as capsules, tablets and in particular gastro-resistant tablets for the intestinal disinfection.

In any case, storing the formulation in an inert environment (under nitrogen) and far from light, and the presence of a protective layer against atmospheric agents, such as humidity, oxygen, etc. , are generally preferable.

Such protective layer, particularly useful for the preservation of tablets and gastro-resistant tablets, is constituted by substances suitable to create a protective and compact film, not permeable to environmental agents, and it is particularly conceived as an overcoating or more often as an undercoating film.

Another aspect of the present invention is a pharmaceutical formulation comprising N-dimethylaminoacetyl-partricin A 2- dimethylaminoethylamide diascorbate and showing the characteristics, forms, excipients, protective layers, etc. above mentioned.

The following examples illustrate the invention without limiting it.

Example 1 1 g of granules for oral use contains: Active ingredient N-dimethylaminoacetyl partricin A 2-dimethylaminoethylamide diascorbate 10 mg Excipients Sodium carboxymethylated-starch 100 mg Sodium citrate 30 mg Lean cocoa powder 10 mg Cocoa fluid extract 10 mg Silicon dioxide precipitate 10 mg Alkylparabens (Na salt, mix) 10 mg Saccharose 820 mg Procedure (for 1000 g of granules)

Sieve the active ingredient (code SPK-843), Na carboxymethylated- starch, sodium citrate, cocoa powder, silicon dioxide and icing sugar, then put the resulting mixture into the mixer and moisten with 100 ml of purified water. Mix for 20 min. , put onto a rolling granulator and dry in an air-cabinet at 45°C, under ventilation for one night.

Put into the mixer, moisten with cocoa fluid extract, dry again at 45°C and regularize in a rolling granulator and sieve at 50 mesh/cm2.

Then put it into powder mixer, add the parabens and mix for 2-3 minutes.

Granule use: before using it, 1 g of granules is poured into 5 ml of water and homogenized. After every oral application (1 ml), the remaining must be preserved in the refrigerator for 3-10 days.

Example 2 Each gastro-resistant tablet contains: Active ingredient N-dimethylaminoacetyl partricin A 2-dimethylaminoethylamide diascorbate 10 mg Excipients Starch 40 mg Sodium citrate 30 mg Talc 10 mg Magnesium stearate 5 mg Lactose 185 mg Enteric coating (25 mg): Shellac 1. 5 mg Cellulose acetophtalate 9 mg Diethyl phtalate 5. 4mg Talc 9 mg Procedure (for 10, 000 tablets) The active ingredient (code SPK-843), together with starch, sodium citrate, lactose and half of talc and magnesium stearate, were put into the mixer, stirred for 15 minutes and pre-compressed in big tablets. After grinding in the granulator with a UNI No. 16 sieve and adding the remaining quantity of talc and magnesium stearate, mix again for 15

minutes and compress the granules in 280 mg tablets. Tablets were put into a coating pan and coated with 20% of Shellac@ dissolved in isopropanol. After drying in an air cabinet at 35-40°C under air ventilation for one night, the coated tablets were coated up to a mean weight of 305 mg/tablet, with a suspension composed of cellulose acetophtalate (5% by weight), diethyl phtalate (3%), talc (5%), isopropanol (43.5%), ethyl acetate (43.5%).

In the end the coated tablets were dried in a cupboard at 35-40°C under air ventilation for one night.

Example 3 Each gastro-resistant tablet contains: Active ingredient N-dimethylaminoacetyl partricin A 2-dimethylaminoethylamide diascorbate 25 mg Excipients Starch 40 mg Sodium chloride 30 mg Sodium citrate 30 mg Magnesium stearate 5 mg Microcrystalline cellulose 180 mg Enteric coating (40 mg) Shellac 1. 5mg Cellulose acetophtalate 14. 8mg Diethyl phtalate 8. 9 mg Talc 14. 8 mg Procedure (for 20,000 tablets) The active ingredient (code SPK-843), together with starch, sodium citrate and microcrystalline cellulose and half of the quantity of sodium chloride and magnesium stearate were mixed and pre-compressed. After disintegration of tablets, sieve and add the remaining quantity of sodium chloride and magnesium stearate and mix again, then finally compress into 310 mg tablets. Then coat in a coating pan with a solution of Shellac@, dry and coat again with a solution of cellulose acetophtalate and diethyl phtalate, up to a total dry weight of 350 mg, according to Example 1.

Example 4 Each gastro-resistant tablet contains: Active ingredient N-dimethylaminoacetyl partricin A 2-dimethylaminoethylamide diascorbate 50 mg Excipients Lactose 200 mg Pre-gelatinized starch 40 mg Talc 10 mg Magnesium stearate 2. 5 mg Enteric coating methacrylic acid copolymer 14 mg Triethyl citrate 3 mg Polysorbate 0. 4mg Sodium lauryl sulphate 0.1 mg Dyes (E171/E172) 0.2/0. 1 mg Talc 4. 2mg Overcoating Polyvinyl alcohol 13. 5 mg Pullulant 3. 5 mg Polyethylene glycol 6000 1. 8 mg Talc 1. 2mg Procedure (for 10,000 tablets) The active ingredient (SPK-843) and excipients were mixed and sieved and then directly compressed. The resulting tablets were put into a coating pan and coated 1: 3 with a solution/suspension of the enteric coating mixture in water at 40°C up to an increase of 22 mg in weight.

After drying the tablets at 40°C under ventilation for one night, they were further coated with the overcoating protective mixture obtained by dissolving the polyvinyl alcohol in water 1: 10 at 50°C, by cooling, adding the other components and returning to 40°C.

The overcoating was carried out up to a weight increase of 20 mg, then finally drying the resulting tablets in an air cabinet at 40°C under ventilation for one night.

Example 5 Each gastro-resistant tablet has the same quantities of the active ingredient, excipients, gastro-resistant coating and layer of protective coating, as per Example 4.

Adversely from Example 4, the tablets were first coated with a protective coating mixture (polyvinyl alcohol, Pullulant, polyethyleneglycol 6000 and talc in water), therefore as an undercoating, and afterwards with the mixture for enteric coating.

Example 6 Each gastro-resistant tablet comprised 25 mg of the partricin A derivative of the Example 1 as an active ingredient and excipients and enteric coating as per Example 3, and overcoating or undercoating protective layer as per Examples 4 or 5 respectively.