A pharmaceutical composition for the treatment of a fungal skin disorder and a method for the preparation thereof

The present invention relates to a pharmaceutical composition for photodynamic treatment of a fungal skin disorder.

Several pharmaceutical compositions to treat fungal skin disorders, such as tinea and in particular nail infections are known in the art, e.g. Lamisil™ (terbinafine) . They prove to be only moderately effective, and recurrence of the infection is common. This is believed to be caused by spores (conidiae) of the dermatophyte (fungus), the spores apparently not being killed in the treatment. To overcome this problem, efforts have been made to use photodynamic treatment, which should allow a more effective approach as activated oxygen species (such as singlet oxygen) should be less discriminate when it comes to inactivation of the various stages of the dermatophyte. However, while PDT has been shown to be effective against dermatophytes such as Trichophyton rubrum, the spores of these dermatophytes appear to survive as they do with topical drugs such as Lamisil™. The object of the present invention is to provide a pharmaceutical composition capable of inactivating spores (conidiae) and hy- phae of dermatophytes.

The pharmaceutical composition according to the present invention is characterized in that said pharmaceutical composition com- prises a photosensitizer and an enhancer, wherein the photosensitizer is a cationic photosensitizer and the enhancer is chosen from the group of i) an acid, and ii) a metal chelating agent.

Surprisingly, the effectiveness of a cationic photosensitizer to inactivate dermatophytes can be extended to spores by incorpora- tion of such an enhancer in the pharmaceutical composition. The pharmaceutical composition may, in addition, comprise a pharmaceutically acceptable carrier or excipient. The cationic photosensitizer in the sense of the present invention is a photosensitizer having quater- nized nitrogen atoms, and is preferably a photosensitizer comprising at least 2, and preferably 3 or more quaternized nitrogen atoms. The presence of side chains bearing one or more negative charges is not excluded, as long as the overall charge of the photosensitizer is positive (with one or more negative counter ions being present) . The

acid may be any acid, organic or inorganic and including carbonic acid. The pharmaceutical composition is a composition that meets the requirements of a pharmaceutical composition with respect to purity, and is composed of ingredients of pharmaceutical grade. More than one enhancer may be present, preferably of both classes i) and ii) . The metal chelating agent is preferably a metal chelating compound capable of chelating Ca ²⁺ and Mg ²⁺ . A suitable example is EDTA or NaEDTA, which may act both as an acid and as a chelating agent. The term "metal chelating compound" encompasses any compound capable of bind- ing metal ions, including for example crown ethers, zeolites, ion- exchangers, proteins (calmoduline) and precipitants (such as negatively charged counter ions) . Binding of Ca ²⁺ is considered to be of particular advantage. In general, if the enhancer is an acid, the pH of the pharmaceutical composition will be 3 or higher, but below 7, in particular below 6. If the pharmaceutical composition is to be introduced in an aqueous medium before use, for example by dissolving it, said pH is the pH when the resulting pharmaceutical composition is ready for use. The skin to be treated may be subjected to a treatment for removing calcium-ions immediately before treatment by pho- todynamical therapy (PDT), e.g. using a concentrated EDTA-solution or even ample amounts of demineralised or distilled water. In case of the use of a chelating agent, any excess of the agent may subsequently washed away, again using for example demineralised or distilled water. The invention also comprises the treatment of a fungal skin condition using photodynamic treatment with a cationic photosen- sitizer, and according to a preferred embodiment the treatment involves a step of sequestering calcium, for example as described above or elsewhere in the application. The step of sequestering calcium will be performed before and/or during PDT. According to a preferred embodiment, the pharmaceutical composition has a pH of x where 3,5 < x < 6, preferably 4.5 < x < 5.7.

To maintain the pH in this range, especially after contact with the skin, the acid is preferably a weak acid, capable of buffering the pharmaceutical composition in the above ranges. The weak acid has preferably a pKa in the ranges defined above ^" for x.

According to a preferred embodiment, the pharmaceutical composition has an ionic strength of 10 mM or less, more preferably 6 mM or less, most preferably 2 mM or less.

Low ionic strength has been found to have a positive effect on the effectiveness of the photosensitizer in pharmaceutical compositions according to the present invention in the treatment of hyphae .

According to a preferred embodiment, the cationic photosensi- tizer is a photosensitizer comprising a porphyrin macrocycle, the porphyrin macrocycle comprising at least one quaternized nitrogen atom. More preferably, it has 3 quaternized nitrogen atoms. Currently most preferred is 5, 10, 15-tris (4-methylpyridinium) -20-phenyl-

[21H,23H]-porphine trichloride (Sylsens B). Important fields of application of the pharmaceutical composition according to the present invention are those wherein the fungal skin disorder is chosen from the group consisting of tinea and onychomycosis .

According to an important embodiment, the pharmaceutical compo- sition is a topical pharmaceutical composition.

Such a topical composition can easily applied on that part of the skin (including nails) that is to be treated. The topical composition is preferably a gel, ointment, cream or any other viscous composition, in particular the aqueous forms thereof. The term topical composition also includes sprays, especially those with volatile solvent such as ethanol or water which allow application to the skin. Non-viscous compositions without a volatile solvent are generally not preferred because they may be more difficult to apply reliably. Water-based topical pharmaceutical compositions, that can be sprayed or rubbed onto the place to be treated, are most preferred.

The present invention also relates to the use of an enhancer for the preparation of a pharmaceutical composition for the treatment of a fungal infection, wherein the pharmaceutical composition comprises cationic photosensitizer and the enhancer is chosen from the group of i) an acid, and ii) a metal chelating agent.

Preferred embodiments are detailed in the dependent claims, with the advantages as discussed above for the pharmaceutical composition.

The present invention will now be illustrated with reference to the following experiments and the drawing where

Fig. 1 shows the fungicidal effect of Sylsens B at pH 3.5 and pH 7.4;

Fig. 2 similarly shows the fungicidal effect of Sylsens B;

Fig. 3 shows the effect of ionic strength; and

Fig. 4 shows the zeta potential of T. rubrum microconidia

(filled diamonds) and fungal hyphae (filled squares) , measured as a function of the pH . The values given are the mean values for measure- ments on 3 different microconidia and hyphae isolates including the standard deviation (SD) .

Determination of the Minimal Inhibitory Concentration (MIC)

A polycarbonate membrane filter, 25 mm in diameter and a pore size of 2 μm (Whatman, Breda, The Netherlands) was placed in the central part of a 3 cm culture dish (Greiner, Alphen aan den Rijn, The Netherlands) filled with 5 ml of Malt Extract Agar (MEA) . A circular piece of human stratum corneum (SC) with a diameter of 1 cm was subsequently placed on the membrane filter and both were gently brushed with a paintbrush dipped in 70% ethanol. A microconidia suspension isolated from Trichphyton rubrum (CBS nr . 304.60 obtained from Cen- traal Bureau voor Schimmelcultures, Utrecht, The Netherlands, and prepared as described by Smijs G.M. T. et al (2004) in "Photodynamic treatment of the dermatophyte Trichophyton Rubrum and its microcon- idia with porphyrin sensitizers. Photochemistry and PhotoBiology, 80, 197 - 202") was diluted to 1000 CFU/ml and 15 μl was inoculated on the circular sheet of human SC. The dish was placed in an incubator at 28 °C and at 17 hours after spore inoculation photodynamic treatment (PDT) was applied. At 17 hours after spore inoculation, the membrane filter with SC containing spore inoculates was transported from the MEA dish to a 3 cm culture dish filled with 1035 μl incubation medium. The incubation medium contained 1 ml of distilled water with an adjusted pH (3.5, 4.5, 5.2, or 7.4) and 35 μl of a Sylsens B stock solution (Bu- chem, Apeldoorn, the Nederland) . To optimize the contact of the inoculates with the incubation medium, the membrane filter with the SC was turned upside down during the incubation period of 2 hours. The incubation was performed under continuous shaking conditions. Shortly before the illumination period the membrane filter containing the SC was turned back (surface SC now faces the lamp) . In all cases, the illumination time was 1 hour using a red light irradiance of 30 mW/cm ² . This corresponds to a light dose of 108 J/cm ² . After the illumination the membrane filter with the SC were transported to a

fresh MEA dish, placed in a 28 °C incubator and fungal growth was followed for several weeks. Dark controls were included, i.e. the same procedure was carried out except that the inoculated microconidia on human skin were treated with solvent and/or photosensitizer in the dark. In the light controls, the microconidia were treated with solvent (only) in the presence of light. The efficacy of the treatment was expressed as a relative frequency of complete inactivation (killing) of fungal growth detected at day 8 after spore inoculation. This was assessed microscopically with the use of a Zeiss Axiovert 25 (Germany) . After day eight no re-growth could be observed by visual inspection in case a complete inactivation of spores and hyphae was obtained after PDT. The treatment effect was followed for a period of several months. Every experiment contained 4 to 6 duplicates of all conditions and the experiments were repeated 3 times at least. The results are shown in Table 1.

Fig. 1 displays the results of this experiment, for pH 3.5 and 7.4 (fungicidal effect (FE) 8 days after spore inoculation) . At an acidic pH, the cationic photosensitizer was significantly more active . A similar experiment was performed with meso-tetra (N-methyl-4- pyridyl) porphyrin tetrachloride, displaying improved activity at acidic pH, albeit to a lesser extent than Sylsens B (results not shown) .

Table 1 μM Sylsens B pH 3.5 pH 5.2 pH 7.4

0 0 (4) 0 (4) 0 (12) 0 (dark control) 0 (4) 0 (4) 0 (6)

0 5 0 (6) 0 (3) 0 (6)

1 0 (6) 0 (3) 0 (6) 1 (dark control) 0 (2) 0 (2) 0 (2)

5 0 2 (10) 0 43 (7) 0 4 (12) 5 (dark control) 0 (2) 0 (4) 0 (6) 6 0 5 (2) 1 (4) 02 (12) 6 (dark control) 0 (6)

7 1 (4) 8 0 75 (4) 1 (4) 0 6 (12) 8 (dark control) 0 (6)

9 1 (4)

10 0 8 (10) 1 (9) 0 6 (12) 10 (dark control) 0 (2) 0 (4) 0 (6)

15 1 (4) 1 (4) 0 7 (12) 15 (dark control) 05 (6)

20 1 (4) 0 6 (12) 20 (dark control) 0 (2) 0 (2) 0 3 (6)

30 1 (4) 1 (5) 0 6 (12) 30 (dark control) O ₅ (2) 1 (2) 0 3 (6)

40 1 (4) 1 (12) 40 (dark control) 0 (6)

50 1 (10) 1 (3) 0 9 (12) 50 (dark control) 0 5 (2) 1 (2) 0 5 (6)

65 1 (4) 1 (12) 65 (dark control) 1 (2) 1 (6)

80 1 (8) 1 (3) 1 (12) 80 (dark control) 0 5 (4) 1 (2) 1 (6)

MIC ² > 10 5 - 6 ≥ 50

< 15

Incubation : 2 hours in water medium with adj usted pH

Illumination: 1 hour, 30 mW/cm ² (red light) .

¹ The fungicidal effect was determined at 8 days after spore inoculation .

² Minimum Inhibitory Concentration in μM (under the given test circumstances)

³ The dark toxicity is in the same range.

⁴ The fungal growth itself or the germination process (low number of CFU in the light and dark controls) is slow at this pH.

PDT of T. rubrum 17 hours after spore inoculation using the ex- vivo model, different concentrations of Sylsens B and a different pH (water medium) . The numbers indicate the relative frequency of the fungicidal effect ¹ . Numbers between brackets give the number of tests performed. It is obvious from these results that the lowest MIC is found at pH 5.2 indicating that at this pH the sensitizer, upon illumination, is optimal effective as a fungicidal agent.

The experiment with Sylsens B was repeated more extensively, for a longer period of time and with more samples. More specifically, the photodynamic efficacy of Sylsens B towards T. rubrum was tested at 17 (fig. 2a), 48 (fig. 2b) and 72 hours (fig. 2c) after spore inoculation on human SC, at the following incubation pH values: Filled diamonds: pH 3.5, filled squares: pH 5.2 and filled triangles: pH 7.4. Application of PDT was performed using the ex-v±vo model with a 2 hours incubation period in distilled water of adjusted indicated pH, followed by 1 hour illumination (30 mW/cm ² , red light) . Values given are the mean values for 5 different experiments and their standard error of mean (SEM) . For the 17-hour stage, dark controls dis- played 8 to 12 cfu. For the 48-hour and 72 hour stage, dark controls displayed 8 to 12 cfu up to 80 μM Sylsens B and 5 to 8 cfu for higher concentrations. The light controls displayed for all stages 8 to 12 cfu.

Ionic strength

Reference is made to fig. 3. The photodynamic efficacy of Sylsens B towards T. rubrum was tested at 17 (filled circles) , 48 (filled triangles) and 72 hours (filled squares) using different molarities of a citric acid/sodium citrate buffer of pH 5.2. Applica- tion of PDT was performed using the ex-vivo model with a 2 hours incubation period in a citric acid/sodium citrate buffer of pH 5.2 of different molarity, followed by 1-hour illumination (30 mW/cm ² , red light) . The Sylsens B concentration was fixed at 10 μM for PDT application at 17 hours after spore inoculation, 80 μM for application at 48 hours after spore inoculation and 160 μM for application at 72 hours after spore inoculation. Values given are the mean values for 3 different experiments and their SEM.

Both light and dark controls displayed 8 to 12 cfu for all the growth stages.

In the above experiments, deuteroporphyrin monomethylether (DP me) , an anionic porphyrin, was used as a control. Being negatively charged, this porphyrin failed to display the observed pH effect. In addition, experiments have been conducted to show that the observed pH effect is not due to different efficacies of Sylsens B at those different pH values. In particular the production of ¹ O ₂ was substan- tially constant over the pH range used. In addition, experiments conducted to determine the Zeta potential (using a Zetasizer 2000/3000, Malvern Instruments Ltd, Worcs, UK) of microconidia showed that these are negatively charged at a pH above 3.5 (fig. 4), and hence more likely to bind a cationic photosensitizer . Because skin has an iso- electric range centered at a pH of about 5.5, skin will generally be less negatively charged below this pH and hence less likely to bind a cationic photosensitizer. The effect of calcium-ions was investigated by determining the photodynamic effect in the presence of 5 mM CaCl ₂ at pH 5.2. This eliminated binding of Sylsens B to the fungus culti- vated in suspension. The same effect could be achieved using a positively charged dye, Alcian Blue, which at 160 μM competed with Sylsens B (160 μM) for binding to the fungus, reducing the effectiveness of Sylsens B to inactivate the fungus. So, PDT may even be used to inactivate (kill) the growing fungus if measures are taken to prevent naturally present calcium from interfering with the treatment, in particular by using a chelating compound such as EDTA.