Indian Patents. 250478:PHARMACEUTICAL COMBINATION COMPRISING AN ANTIFUNGAL AGENT AND AN ACTIVE SUBSTANCE SELECTED FROM CARVEOL, EUGENOL, THYMOL, BORNEOL, CARVACROL, AND ALPHA- AND BETA-IONONES

Title of Invention	PHARMACEUTICAL COMBINATION COMPRISING AN ANTIFUNGAL AGENT AND AN ACTIVE SUBSTANCE SELECTED FROM CARVEOL, EUGENOL, THYMOL, BORNEOL, CARVACROL, AND ALPHA- AND BETA-IONONES
Abstract	The invention relates to a pharmaceutical composition. The inventive pharmaceutical composition comprises: at leas a firm therapeuticall-active substance which is selected from carveol, thymol, eugenol, borneol, carvacrol, alpha-jonone, betaionone and the isomers, derivatives and mixtures thereof; and at least a second therapeutically-active substance which is an antifungal agent.

Full Text	The invention relates to a pharmaceutical composition comprising two therapeutically active substances one of which exerts a potentiating action on the other, and to the use of said composition. It is known that the efficacy of therapeutic agents depends on the doses used which, in the case of partial resistance, necessitates increasing the doses of the therapeutic agents in order to attain the desired efficacy. This dose increase leads to problems with adverse effects and acute or chronic toxicity, which may considerably complicate the condition of the treated patients. Said partial resistance may turn into complete resistance. In this case, increasing the dose no longer has any beneficial therapeutic effect; only the toxic effects are observed. The treatment in such a case consists in changing the therapeutic agent. This chain of events can repeat itself and lead to the most serious situation: complete resistance to multiple therapeutic agents (multidrug resistance). For instance, in particular, immunosuppressed patients become increasingly difficult to treat and their life expectancy is correspondingly shortened. Moreover, their quality of life is substantially affected by the administration of high doses of therapeutic agents. The invention is directed at alleviating these problems by proposing to combine at least two therapeutically active substances, one of which potentiates the activity of the other, which not only makes it possible to lower the doses of each therapeutically active substance but also to treat patients afflicted with infections caused by resistant microorganisms. In this regard, the invention provides a pharmaceutical composition characterized in that it comprises: at least one first therapeutically active substance selected from the group consisting of carveol, thymol, eugenol, borneol, carvacrol, alpha-ionone, beta-ionone, and the isomers, derivatives and mixtures thereof, and, at least one second therapeutically active substance which is an antifungal agent. The first therapeutic substance can be obtained by chemical synthesis or from a plant source. Preferably, the antifungal agent in the composition of the invention is selected from the group consisting of the polyenes, the imidazoles, the triazoles, the nucleoside analogues, the allylamines, the echinocandins, the sordarins, the morpholines, griseofulvin, cyclopiroxolamine, selenium sulfide, and the mixtures thereof. More preferably, the antifungal agent is selected from the group consisting of nystatin, amphotericin B, ketoconazole, econazole, miconazole, clotrimazole, fluconazole, itraconazole, voriconazole, posaconazole, 5-fluorocytosine, naftafin, terbinafin, caspofungin, amorolfin, and 2 the derivatives and mixtures thereof. A more particularly preferred antifungal composition is a composition in which the first therapeutically active substance is carvacrol or eugenol, and the antifungal agent is fluconazole. Another more particularly preferred antifungal composition is a composition in which the first therapeutically active substance is carvacrol or eugenol, and the antifungal agent is selected from voriconazole, caspofungin, itraconazole, 5-fluorocytosine, and the mixtures thereof. The invention also proposes a kit characterized in that it comprises at least one first container containing a first therapeutically active substance selected from the group consisting of carveol, thymol, eugenol, borneol, carvacrol, alpha-ionone, beta-ionone, and the isomers and derivatives and mixtures thereof, and at least one second container containing a second therapeutically active substance which is an antifungal agent. Lastly the invention proposes a method for treating an infection due to a fungus characterized in that one administers simultaneously or sequentially to a patient having a fungal infection, at least one first therapeutically active substance selected from the group consisting of carveol, thymol, eugenol, borneol, carvacrol, alpha-ionone, beta-ionone, and the isomers and derivatives and mixtures thereof, and at least one second therapeutically active substance which is an antifungal agent. Preferably, in said method, one simultaneously or sequentially administers to a patient having a fungal infection between 10 and 200 mg/kg of body weight/day of said first therapeutically active substance, and between 2 and 100 mg/kg of body weight/day of said second therapeutically active substance which is an antifungal agent. Preferably, in this method, said first therapeutically active substance is selected from the group consisting of carvacrol, eugenol and thymol and said second therapeutically active substance is selected from the group consisting of fluconazole, voriconazole, itraconazole, 5- fluorocytosine and caspofungin, and the mixtures thereof. The invention will be better understood and other aims and advantages thereof will appear more clearly in the explanatory description which follows and which refers to the figures wherein: Figure 1 presents kinetic test results on a C. albicans culture of the fungicidal action of fluconazole alone and carvacrol alone as compared with a pharmaceutical composition according to the invention comprising fluconazole and carvacrol, and Figure 2 presents the results of in vivo tests in a systemic candidiasis model in experimentally infected mice either untreated, or treated with fluconazole alone, treated with carvacrol alone, or treated with a pharmaceutical composition according to the invention comprising fluconazole and carvacrol. 3 The pharmaceutical composition according to the invention comprises as first therapeutically active substance thymol, eugenol, carvacrol, borneol, carveol, alpha-ionone, beta-ionone, and the derivatives and isomers as well as any mixtures thereof. Said compounds have well-known antifungal properties and must be pure. Thymol, eugenol, carvacrol, borneol, carveol, alpha-ionone, beta-ionone are found in various proportions in different aromatic plant extracts, that is to say, they can be purified from such plants. However, they can quite simply be obtained by chemical synthesis. As a matter of fact, the inventors have now discovered that said compounds have a potentiating effect on many therapeutically active substances including known antifungal agents which are already used as medicaments specific in this field. The second therapeutically active substance comprised in the pharmaceutical composition of the invention is therefore an antifungal agent, which is already known as such and already used as medicament specific in this field and whose activity is potentiated. Examples of known antifungal agents already used as medicaments specific in this field which can be used in the pharmaceutical composition of the invention, and whose effect will be potentiated by the first pure therapeutically active substance, belong to three families: the pyrimidine family represented by 5-fluorocytosine, the azole family represented by fluconazole, voriconazole, itraconazole and the echinocandin family represented by caspofungin. Said compounds can be used alone, or in combination with each other. The derivatives thereof, if they have antifungal activity, can also be used. Particularly preferred are fluconazole, voriconazole, capsofungin and 5-fluorocytosine used in combination more particularly with carvacrol, eugenol and/or thymol. Of course, the pharmaceutical composition according to the invention is not restricted to the use of only those antifungal agents mentioned above. In fact, considering the potentiating effect exerted by the first therapeutically active substance defined in the invention, other known or future antifungal agents can also be successfully used. The pharmaceutical composition according to the invention can be formulated so as to be suitable for a simultaneous or sequential administration of said at least first and second therapeutically active substances. The pharmaceutical form of the pharmaceutical composition of the invention shall be adapted to its use. For example, it can be used in the form of a solution, suspension, tablet or others. The compositions for parenteral administration are generally pharmaceutically acceptable sterile solutions or suspensions which can optionally be prepared immediately before use. For the preparation of nonaqueous solutions or suspensions, it is possible to use natural vegetable oils like olive oil, sesame oil or paraffin oil or the injectable organic esters such as ethyl oleate. The sterile aqueous solutions can be composed of a solution of therapeutically 4 active substances in water. The aqueous solutions are suitable for intravenous administration in so far as the pH is properly adjusted and they are made isotonic, for example by adding a sufficient amount of sodium chloride or glucose. In fact, considering the chemical structure of antifungal agents, and secondly, considering the chemical structure of carveol, carvacrol, thymol, eugenol, borneol, alpha-ionone and beta-ionone, it is thought, without intending to be bound by this theory, that carveol, carvacrol, thymol, eugenol, borneol, alpha-ionone and beta-ionone and the isomers, derivatives and mixtures thereof, interact with the antifungal agents to form complexes having a structure which diffuses more easily into the body's physiological fluids and which diffuses more easily into the cytoplasm of target infected cells. However, it has been shown that when the different components of the pharmaceutical composition of the invention are mixed in the presence of detergents such as Tween or Triton or solvents such as ethanol or DMSO (dimethyl sulfoxide), the active molecules of the first and second therapeutically active substance associate with the molecules of the detergents and solvents and do not form potentiating complexes. Now it has been discovered that the potentiating complex forms when an aqueous agar suspension is used, as means of dispersion by viscosity. Thus, the pharmaceutical composition of the invention will preferably be prepared without detergent and without solvent. For example, it will be prepared as an aqueous suspension made viscous by the addition of agar at a non-solidifying concentration, for example from 1 to 5 grams of agar per liter of suspension. The pharmaceutical composition of the invention enables the treatment of local or systemic infections caused by resistant microorganisms using doses of each of said first and second therapeutically active substance which are lower than the doses required for treating the same infections due to susceptible microorganisms with one or the other of these same said first and second therapeutically active substances alone. In fact, the composition of the invention enables the use of doses of said first therapeutically active substance, when it is combined with said second therapeutically active substance, which are approximately three to ten times lower than the doses required when said first therapeutically active substance is used alone, and of doses of said second therapeutically active substance, when it is combined with said first therapeutically active substance, which are approximately two to ten times lower than the doses required when said second therapeutically active substance is used alone. The result is to offer a treatment which has the following advantages: - effective at very low doses against susceptible microorganisms, - effective against microorganisms resistant to a therapeutic agent, - effective against microorganisms resistant to several therapeutic agents, 5 - control of recurrence phenomena, - control of phenomena of resistant microorganisms selection. In all these cases, there is a notable reduction in the risks of toxicity and/or adverse effects well known to the person of the art, thanks to the potentiation which enables the administration of very low doses. In addition, the costs of producing the treatment are reduced due to the use of small quantities of active substances. The pharmaceutical compositions according to the invention can be in the form of liposomes or associated with supports such as cyclodextrins or polyethylene glycols. The pharmaceutical compositions of the invention are a simple and efficient means to combat the problems related to microbial agents in general which comprise mainly resistance to therapeutic agents and toxicity of the latter resulting from the use of high doses. In fact, carveol, thymol, eugenol, borneol, carvacrol and the derivatives, mixtures and isomers thereof, are simple molecules which have never been described as having any toxicity whatsoever and their addition with its potentiating effect on the second therapeutically active substance enables the use of much lower doses of said second therapeutically active substance. In a first variant, then, the method for treating patients having a fungal infection consists in administering to said patients the dose, determined by the physician, of the pharmaceutical composition of the invention comprising suitable doses of at least one said first therapeutically active substance, combined with suitable doses of at least one said second therapeutically substance, that is, the suitable antifungal agent. In a second variant, the method for treating patients having a fungal infection consists in sequentially administering to said patients the dose determined by the physician of at least one said first therapeutically active substance, followed by the suitable dose of at least one said second therapeutically active substance, that is, the suitable antifungal agent, or vice versa. In this regard, the invention proposes a kit comprising at least one first container containing one of said first therapeutically active substances, and at least one second container containing one of said second therapeutically active substances. Said kit enables health care personnel to prepare on demand either a mixture of suitable doses of the desired first therapeutic substance(s) and of the desired antifungal agent(s) for a simultaneous administration, or to sequentially and separately administer the suitable dose of at least one said first therapeutically active substance, followed by the suitable dose of at least one said second therapeutically active substance, that is, the suitable antifungal agent, or vice versa. However, a mixture for simultaneous use shall be preferred in order to allow the potentiation complex to form and to act immediately after administration to the patient. The invention shall become clearer in the following examples describing different 6 embodiments, which are given for purposes of illustration and not by way of limitation. EXAMPLE 1: Treatment of different Candida albicans strains with fluconazole potentiated by carvacrol (Flue P) In vitro tests: Determination of Minimal Fungicidal Concentration (MFC) on different strains of Candida albicans The experiment was carried out with several strains of Candida albicans having different susceptibilities isolated in the hospital environment from the mouth, vagina and digestive tract. The antifungal agent used was fluconazole, an azole derivative which is one of the most effective and most widely used antifungals on the market. An antifungal pharmaceutical composition was prepared by mixing fluconazole at different concentrations with carvacrol at a sub-inhibitory concentration of 0.3 g per liter of solution or excipient. This pharmaceutical composition according to the invention was named FLUC-P for potentiated fluconazole. In each case, antifungal activity was determined either with fluconazole alone, or with carvacrol alone, or with the composition of the invention. Table 1 gives the results of static tests to determine the minimal fungicidal concentration (MFC) in µg/ml. Table 1 Candida albicans in exponential growth phase Fluconazole alone Composition of the invention Carvacrol alone MFC (µg/ml) MFC (µg/ml) MFC (µg/ml) Susceptible strain 0.5 / 1000 Moderately resistant strain 150 10 1000 Highly resistant strain >300 10 1000 Table 1 shows that the composition of the invention had notable fungicidal activity on these strains with different susceptibilities, as compared with fluconazole alone or with carvacrol alone. Indeed, from Table 1, it is noted that the composition of the invention has a remarkable fungicidal activity on those strains with different susceptibilities as compared with fluconazole alone or with carvacrol alone. In fact, it can be seen that by using a carvacrol concentration of 0.3 mg/ml, which is approximately three times lower than the MFC of carvacrol alone, the fluconazole concentration which produced fungicidal activity was 15 to 30 times lower than the MFC of fluconazole alone. 7 Kinetic tests Kinetic tests were also carried out on a C. albicans strain highly resistant in order to compare the fungicidal activities of fluconazole alone at a concentration of 300 µg/ml, carvacrol alone at 300 µg/ml and the composition of the invention comprising fluconazole 150 ug/ml and carvacrol 300 µg/ml. The number of colony-forming units (CFU) was measured over time. The results, shown in Figure 1, clearly indicate that only the composition of the invention had demonstrable activity against this highly resistant strain. In vivo tests Groups of 15 mice were experimentally infected by intravenous injection of 10,000,000 cells (colony-forming units) of a Candida albicans strain with moderate resistance to fluconazole. The first group was composed of control mice which were infected and untreated. The second group was composed of infected mice treated by gavage, 24 h post- infection, with fluconazole alone at a dose of 4 mg/kg of body weight/day. The third group was composed of infected mice treated by gavage, 24 h post- infection, with carvacrol alone at a dose of 30 mg/kg of body weight/day. The fourth group was composed of infected mice treated by gavage, 24 h post- infection, with fluconazole 2 mg/kg of body weight and with carvacrol 30 mg/kg of body weight. The duration of treatment was seven days for surviving animals. The survival rate was measured over time. The results are given in Figure 2, which shows that only the mice treated with the pharmaceutical composition were still alive ten days after infection. All the other mice died between the second and seventh day post-infection. Examination of the organs of mice which died during the experiment (untreated mice and those treated with fluconazole alone or carvacrol alone) revealed high loads of Candida albicans in kidney, lung and bone marrow. In contrast, in mice treated with the composition of the invention and sacrificed one to ten days after stopping treatment, no C. albicans was found in lung and bone marrow. In kidney, only two animals still had a very low C. albicans load corresponding to 5% of that seen in the control group. The other animals treated with the composition of the invention had no fungal load in the kidneys. Here again, it clearly appears that potentiation of fluconazole by carvacrol gives surprising results regarding the reduction in the minimal fungicidal concentration and the rapidity of action in vitro. This potentiation was also found in vivo in a model of systemic infection. 8 Now, systemic infection is one of the most serious and life-threatening forms of infection and the most difficult to treat, especially in case of relapse with increasingly resistant microbes. Other data showed that the composition of the invention comprising fluconazole and carvacrol produced surprising therapeutic effects at doses which were at least two-fold lower than the doses required to treat localized experimental infections (vaginal and oral) in rats and mice. EXAMPLE 2: Treatment of different Candida albicans strains with voriconazole potentiated by carvacrol (Vorico-P) The experiment was carried out with several strains of Candida albicans having different susceptibilities isolated in the hospital environment. The antifungal agent used was voriconazole, an azole derivative and one of the most recent antifungals on the market. An antifungal pharmaceutical composition according to the invention was prepared by mixing voriconazole at different concentrations with carvacrol at a sub-inhibitory concentration of 0.3 g per liter of solution or excipient. This pharmaceutical composition according to the invention was named Vorico-P for potentiated voriconazole. In each case, antifungal activity was determined either with voriconazole alone, or with carvacrol alone, or with the composition of the invention. Table 2 gives the results of static tests to determine the minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) in µg/ml. Table 2 Candida albicans in exponential growth phase Voriconazole alone Vorico-P Carvacrol alone MIC (µg/ml) MFC (µg/ml) MFC (µg/ml) Susceptible strain Resistant strain 10 Table 2 shows that the composition of the invention had notable fungicidal activity on the voriconazole-resistant strain, as compared with voriconazole alone or with carvacrol alone. In fact, it can be seen in Table 2 that by using a carvacrol concentration of 0.3 mg/ml, which is 3.3 times lower than the MFC of carvacrol alone, the voriconazole concentration producing fungicidal activity against resistant strains was at least ten times lower than the concentration of voriconazole alone capable of exhibiting fungistatic activity. Thus, it can be seen that the potentiation of voriconazole by carvacrol not only 9 allowed a large reduction in the voriconazole dose but also transformed its fungistatic activity into a fungicidal action. EXAMPLE 3: Treatment of different Candida albicans strains with capsofungin potentiated by carvacrol (Caspo-P) The experiment was carried out with several strains of Candida albicans having different susceptibilities isolated in the hospital environment. The antifungal agent used was caspofungin, from the echinocandin family, which is one of the newest antifungals on the market. An antifungal pharmaceutical composition according to the invention was prepared by mixing caspofungin at different concentrations with carvacrol at a sub-inhibitory concentration of 0.3 g per liter of solution or excipient. This pharmaceutical composition according to the invention was named Caspo-P for potentiated caspofungin. In each case, antifungal activity was determined either with caspofungin alone, or with carvacrol alone, or with the composition of the invention. Table 3 gives the results of static tests to determine the minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) in µg/ml. Table 3 Candida albicans in exponential growth phase Caspofungin alone Caspo-P Carvacrol alone MIC (µg/ml) MFC (µg/ml) MFC (µg/ml) Susceptible strain Resistant strain 3 Table 3 shows that the composition of the invention had notable fungicidal activity on the resistant strain, as compared with caspofungin alone or with carvacrol alone. In fact, it can be seen in Table 3 that by using a carvacrol concentration of 0.3 mg/ml, which is 3.3 times lower than the MFC of carvacrol alone, the caspofungin concentration producing fungicidal activity was at least six times lower than the concentration of caspofungin alone capable of exhibiting fungistatic activity. Thus, it can be seen that the potentiation of caspofungin by carvacrol not only allowed a large reduction in the caspofungin dose but also transformed its fungistatic activity into a fungicidal action. EXAMPLE 4: Treatment of different Candida albicans strains with 5- 10 fluorocytosine potentiated by eugenol (Fluoro-P) The experiment was carried out with several strains of Candida albicans having different susceptibilities isolated in the hospital environment. The antifungal agent used was 5- fluorocytosine, from the pyrimidine group, which is one of the oldest antifungal agents. An antifungal pharmaceutical composition according to the invention was prepared by mixing 5- fluorocytosine at different concentrations with eugenol at a sub-inhibitory concentration of 0.5 g per liter of solution or excipient. This pharmaceutical composition according to the invention was named Fluoro-P for potentiated 5-fluorocytosine. In each case, antifungal activity was determined either with 5-fluorocytosine alone, or with eugenol alone, or with the composition of the invention. Table 4 gives the results of static tests to determine the minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) in µg/ml. Table 4 Candida albicans in exponential growth phase 5-fluorocytosine alone Fluoro-P Eugenol alone MIC (µg/ml) MFC (µg/ml) MFC (µg/ml) Susceptible strain 2.5 / 2000 Resistant strain 25 5 2000 Table 4 shows that the composition of the invention had notable fungicidal activity on the resistant strain, as compared with 5-fluorocytosine alone or with eugenol alone. In fact, it can be seen in Table 4 that by using a eugenol concentration of 0.5 mg/ml, which is four times lower than the MFC of eugenol alone, the 5-fluorocytosine concentration producing fungicidal activity on resistant strains was five times lower than the concentration of 5-fluorocytosine alone capable of exhibiting fungistatic activity. Thus, it can be seen that the potentiation of 5-fluorocytosine by eugenol not only allowed a large reduction in the 5-fluorocytosine dose but also transformed its fungistatic activity into a fungicidal action. EXAMPLE 5: Enlargement of the spectrum of action of fluconazole potentiated by carvacrol to different Aspergillus niger strains The experiment was carried out with several strains of Aspergillus niger which were not susceptible to fluconazole. The antifungal agent used was fluconazole, an azole derivative 11 and one of the most widely used antifungals. The biggest drawback of fluconazole is that it does not act on infections due to filamentous fungi. Aspergillosis, caused by microorganisms from the family Aspergillus sp, represents the most common and most difficult-to-treat infection. An antifungal pharmaceutical composition according to the invention was prepared by mixing fluconazole at different concentrations with carvacrol at a sub-inhibitory concentration of 0.25 g per liter of solution or excipient. This pharmaceutical composition according to the invention was named Fluc-P for potentiated fluconazole. In each case, antifungal activity was determined either with fluconazole alone, or with carvacrol alone, or with the composition of the invention. Table 5 gives the results of static tests to determine the minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) in µg/ml. Table 5 Candida albicans in exponential growth phase Fluconazole alone Fluc-P Carvacrol alone MIC (µg/ml) MFC (µg/ml) MFC (µg/ml) 12 different strains of Aspergillus niger >1000 150 500 Table 5 shows that the composition of the invention had notable fungicidal activity on Aspergillus niger, as compared with fluconazole alone or with carvacrol alone. In fact, it can be seen in Table 5 that the use of a carvacrol concentration of 0.25 mg/ml, which is two times lower than the MFC of carvacrol alone, combined with fluconazole at a concentration of 150 µg/ml, led to an outstanding fungicidal activity that fluconazole alone was unable to provide even at a concentration of 1000 µg/ml. Thus, it can be seen that the potentiation of fluconazole by carvacrol not only allowed a large reduction in the fluconazole dose with species normally treated with fluconazole, but also enlarged the spectrum thereof to filamentous fungi from the Aspergillus genus which are normally not susceptible to fluconazole. The method for treating a fungal infection consists in administering simultaneously or sequentially to a patient having a fungal infection, the dose determined by the physician of at least one first therapeutically active substance selected from the group consisting of carveol, thymol, eugenol, borneol, carvacrol, and the isomers and derivatives and mixtures thereof, and the determined dose of at least one second therapeutically active substance which is an antifungal agent. Generally, one simultaneously or sequentially administers to a patient having a fungal infection between 1 and 3000 mg/kg of body weight/day of at least one first 12 therapeutically active substance selected from the group consisting of carveol, thymol, eugenol, borneol, carvacrol, alpha-ionone, beta-ionone and the isomers and derivatives and mixtures thereof, and between 1 and 20 mg/kg of body weight/day of at least one second therapeutically active substance which is an antifungal agent. In fact, the use of an amount less than 1 mg/kg of body weight/day of said first therapeutically active substance, in particular carvacrol, does not produce the desired potentiation effect. On the other hand, the use of an amount greater than 3000 mg/kg of body weight/day of said first therapeutically active substance, in particular carvacrol, does not further increase the potentiation effect and poses a greater risk of toxicity. Likewise, the use of an amount less than 1 mg/kg of body weight/day of said second therapeutically active substance which is an antifungal agent, in particular fluconazole, does not produce the desired therapeutic effect and the use of an amount greater than 20 mg/kg of body weight/day, in particular of fluconazole, does not improve the therapeutic effect and increases the risk of toxicity. Thus, in a preferred manner, one simultaneously or sequentially administers to a patient having a fungal infection 30 mg/kg of body weight/day of at least one first therapeutically active substance selected from the group consisting of carveol, thymol, eugenol, borneol, carvacrol, alpha-ionone, beta-ionone and the isomers and derivatives and mixtures thereof, and 2 mg/kg of body weight/day of at least one second therapeutically active substance which is an antifungal agent. More particularly, in the case of Candida albicans infection, one simultaneously or sequentially administers to the patient: - 30 mg/kg of body weight/day of carvacrol, and - 2 mg/kg of body weight/day of fluconazole. Of course, the invention is in no way restricted to the embodiments described and illustrated herein which are given solely by way of example. On the contrary, the invention comprises all the technical equivalents of the methods described herein as well as the combinations thereof where such are carried out in the spirit of the invention. 13 WE CLAIM: 1. A pharmaceutical composition characterized in that it comprises: - at least one first therapeutically active substance selected from the group consisting of carveol, thymol, eugenol, borneol, carvacrol, alpha-ionone, beta-ionone, and the isomers and derivatives and mixtures thereof, and, - at least one second therapeutically active substance which is an antifungal agent. 2. Composition as claimed in claim 1, characterized in that the antifungal agent is selected from the group consisting of the pyrimidines, the azole family, the echinocandin family, and the mixtures thereof. 3. Composition as claimed in claim 1 or 2, characterized in that the antifungal agent is selected from 5-fluorocytosine, fluconazole, voriconazole, itraconazole and caspofungin, and derivatives and mixtures thereof. 4. Composition as claimed in any one of claims 1 to 3, characterized in that said first therapeutically active substance is eugenol or carvacrol. 5. Composition as claimed in any one of claims 1 to 4, characterized in that said first therapeutically active substance is carvacrol and the antifungal agent is selected from fluconazole, voriconazole and caspofungin, and derivatives and mixtures thereof. 6. Composition as claimed in claim 5, characterized in that the antifungal agent is fluconazole. 7. Composition as claimed in any one of claims 1 to 4, characterized in that said first therapeutically active substance is eugenol and the antifungal agent is 5- fluorocytosine. 8. Composition as claimed in any one of claims 1 to 7, characterized in that said first and second therapeutically active substances are suspended in an aqueous agar solution. 9. Composition as claimed in any one of claims 1 to 8, characterized in that said 14 composition does not include any detergent or solvent. 10. Composition as claimed in any one of claims 1 to 9, characterized in that it contains between 10 and 200 mg/kg of body weight/day of said first therapeutically active substance, and between 2 and 100 mg/kg of body weight/day of said second therapeutically active substance. 11. Kit characterized in that it comprises: at least one first container containing a first therapeutically active substance selected from the group consisting of carveol, thymol, eugenol, borneol, carvacrol, alpha-ionone, beta-ionone and the isomers and derivatives and mixtures thereof, and - at least one second container containing a second therapeutically active substance which is an antifungal agent. 12 Kit as claimed in claim 11, characterized in that the antifungal agent is selected from the group consisting of the pyrimidines, the azole family, the echinocandin family, and the mixtures thereof. 13. Kit as claimed in claim 11 or 12, characterized in that the antifungal agent is selected from 5-fluorocytosine, fluconazole, voriconazole, itraconazole and caspofungin, and derivatives and mixtures thereof. 14. Kit as claimed in any one of claims 11 to 13, characterized in that said first therapeutically active substance is eugenol or carvacrol. 15. Kit as claimed in any one of claims 11 to 14, characterized in that said first therapeutically active substance is carvacrol and the antifungal agent is selected from fluconazole, voriconazole and caspofungin, and derivatives and mixtures thereof. 16. Kit as claimed in claim 15, characterized in that the antifungal agent is fluconazole. 17. Kit as claimed in any one of claims 11 to 14, characterized in that said first therapeutically active substance is eugenol and the antifungal agent is 5- fluorocytosine. 18. Kit as claimed in any one of claims 11 to 17, characterized in that it contains between 15 10 and 200 mg/kg of body weight/day of said first therapeutically active substance, and between 2 and 100 mg/kg of body weight/day of said second therapeutically active substance. 19. A pharmaceutical composition and a kit substantially such as herein described with reference to the accompanying drawings and as illustrated in the foregoing examples The invention relates to a pharmaceutical composition. The inventive pharmaceutical composition comprises: at leas a firm therapeuticall-active substance which is selected from carveol, thymol, eugenol, borneol, carvacrol, alpha-jonone, betaionone and the isomers, derivatives and mixtures thereof; and at least a second therapeutically-active substance which is an antifungal agent.

Full Text

The invention relates to a pharmaceutical composition comprising two therapeutically
active substances one of which exerts a potentiating action on the other, and to the use of
said composition.
It is known that the efficacy of therapeutic agents depends on the doses used which,
in the case of partial resistance, necessitates increasing the doses of the therapeutic agents
in order to attain the desired efficacy. This dose increase leads to problems with adverse
effects and acute or chronic toxicity, which may considerably complicate the condition of the
treated patients.
Said partial resistance may turn into complete resistance. In this case, increasing the
dose no longer has any beneficial therapeutic effect; only the toxic effects are observed.
The treatment in such a case consists in changing the therapeutic agent.
This chain of events can repeat itself and lead to the most serious situation: complete
resistance to multiple therapeutic agents (multidrug resistance).
For instance, in particular, immunosuppressed patients become increasingly difficult
to treat and their life expectancy is correspondingly shortened. Moreover, their quality of life
is substantially affected by the administration of high doses of therapeutic agents.
The invention is directed at alleviating these problems by proposing to combine at
least two therapeutically active substances, one of which potentiates the activity of the other,
which not only makes it possible to lower the doses of each therapeutically active substance
but also to treat patients afflicted with infections caused by resistant microorganisms.
In this regard, the invention provides a pharmaceutical composition characterized in
that it comprises:
at least one first therapeutically active substance selected from the group
consisting of carveol, thymol, eugenol, borneol, carvacrol, alpha-ionone, beta-ionone, and
the isomers, derivatives and mixtures thereof,
and,
at least one second therapeutically active substance which is an antifungal
agent.
The first therapeutic substance can be obtained by chemical synthesis or from a plant
source.
Preferably, the antifungal agent in the composition of the invention is selected from the
group consisting of the polyenes, the imidazoles, the triazoles, the nucleoside analogues, the
allylamines, the echinocandins, the sordarins, the morpholines, griseofulvin, cyclopiroxolamine,
selenium sulfide, and the mixtures thereof.
More preferably, the antifungal agent is selected from the group consisting of nystatin,
amphotericin B, ketoconazole, econazole, miconazole, clotrimazole, fluconazole, itraconazole,
voriconazole, posaconazole, 5-fluorocytosine, naftafin, terbinafin, caspofungin, amorolfin, and
2

the derivatives and mixtures thereof.
A more particularly preferred antifungal composition is a composition in which the first
therapeutically active substance is carvacrol or eugenol, and the antifungal agent is
fluconazole.
Another more particularly preferred antifungal composition is a composition in which the
first therapeutically active substance is carvacrol or eugenol, and the antifungal agent is
selected from voriconazole, caspofungin, itraconazole, 5-fluorocytosine, and the mixtures
thereof.
The invention also proposes a kit characterized in that it comprises at least one first
container containing a first therapeutically active substance selected from the group consisting
of carveol, thymol, eugenol, borneol, carvacrol, alpha-ionone, beta-ionone, and the isomers and
derivatives and mixtures thereof, and at least one second container containing a second
therapeutically active substance which is an antifungal agent.
Lastly the invention proposes a method for treating an infection due to a fungus
characterized in that one administers simultaneously or sequentially to a patient having a fungal
infection, at least one first therapeutically active substance selected from the group consisting
of carveol, thymol, eugenol, borneol, carvacrol, alpha-ionone, beta-ionone, and the isomers and
derivatives and mixtures thereof, and at least one second therapeutically active substance
which is an antifungal agent.
Preferably, in said method, one simultaneously or sequentially administers to a patient
having a fungal infection between 10 and 200 mg/kg of body weight/day of said first
therapeutically active substance, and between 2 and 100 mg/kg of body weight/day of said
second therapeutically active substance which is an antifungal agent.
Preferably, in this method, said first therapeutically active substance is selected from
the group consisting of carvacrol, eugenol and thymol and said second therapeutically active
substance is selected from the group consisting of fluconazole, voriconazole, itraconazole, 5-
fluorocytosine and caspofungin, and the mixtures thereof.
The invention will be better understood and other aims and advantages thereof will
appear more clearly in the explanatory description which follows and which refers to the figures
wherein:
Figure 1 presents kinetic test results on a C. albicans culture of the fungicidal
action of fluconazole alone and carvacrol alone as compared with a pharmaceutical
composition according to the invention comprising fluconazole and carvacrol, and
Figure 2 presents the results of in vivo tests in a systemic candidiasis model in
experimentally infected mice either untreated, or treated with fluconazole alone, treated with
carvacrol alone, or treated with a pharmaceutical composition according to the invention
comprising fluconazole and carvacrol.
3

The pharmaceutical composition according to the invention comprises as first
therapeutically active substance thymol, eugenol, carvacrol, borneol, carveol, alpha-ionone,
beta-ionone, and the derivatives and isomers as well as any mixtures thereof.
Said compounds have well-known antifungal properties and must be pure.
Thymol, eugenol, carvacrol, borneol, carveol, alpha-ionone, beta-ionone are found in
various proportions in different aromatic plant extracts, that is to say, they can be purified from
such plants. However, they can quite simply be obtained by chemical synthesis.
As a matter of fact, the inventors have now discovered that said compounds have a
potentiating effect on many therapeutically active substances including known antifungal agents
which are already used as medicaments specific in this field.
The second therapeutically active substance comprised in the pharmaceutical
composition of the invention is therefore an antifungal agent, which is already known as such
and already used as medicament specific in this field and whose activity is potentiated.
Examples of known antifungal agents already used as medicaments specific in this field
which can be used in the pharmaceutical composition of the invention, and whose effect will be
potentiated by the first pure therapeutically active substance, belong to three families: the
pyrimidine family represented by 5-fluorocytosine, the azole family represented by fluconazole,
voriconazole, itraconazole and the echinocandin family represented by caspofungin.
Said compounds can be used alone, or in combination with each other. The derivatives
thereof, if they have antifungal activity, can also be used.
Particularly preferred are fluconazole, voriconazole, capsofungin and 5-fluorocytosine
used in combination more particularly with carvacrol, eugenol and/or thymol.
Of course, the pharmaceutical composition according to the invention is not restricted to
the use of only those antifungal agents mentioned above. In fact, considering the potentiating
effect exerted by the first therapeutically active substance defined in the invention, other known
or future antifungal agents can also be successfully used.
The pharmaceutical composition according to the invention can be formulated so as to
be suitable for a simultaneous or sequential administration of said at least first and second
therapeutically active substances.
The pharmaceutical form of the pharmaceutical composition of the invention shall be
adapted to its use. For example, it can be used in the form of a solution, suspension, tablet or
others. The compositions for parenteral administration are generally pharmaceutically
acceptable sterile solutions or suspensions which can optionally be prepared immediately
before use.
For the preparation of nonaqueous solutions or suspensions, it is possible to use natural
vegetable oils like olive oil, sesame oil or paraffin oil or the injectable organic esters such as
ethyl oleate. The sterile aqueous solutions can be composed of a solution of therapeutically
4

active substances in water. The aqueous solutions are suitable for intravenous administration in
so far as the pH is properly adjusted and they are made isotonic, for example by adding a
sufficient amount of sodium chloride or glucose.
In fact, considering the chemical structure of antifungal agents, and secondly,
considering the chemical structure of carveol, carvacrol, thymol, eugenol, borneol, alpha-ionone
and beta-ionone, it is thought, without intending to be bound by this theory, that carveol,
carvacrol, thymol, eugenol, borneol, alpha-ionone and beta-ionone and the isomers, derivatives
and mixtures thereof, interact with the antifungal agents to form complexes having a structure
which diffuses more easily into the body's physiological fluids and which diffuses more easily
into the cytoplasm of target infected cells.
However, it has been shown that when the different components of the pharmaceutical
composition of the invention are mixed in the presence of detergents such as Tween or Triton
or solvents such as ethanol or DMSO (dimethyl sulfoxide), the active molecules of the first and
second therapeutically active substance associate with the molecules of the detergents and
solvents and do not form potentiating complexes.
Now it has been discovered that the potentiating complex forms when an aqueous agar
suspension is used, as means of dispersion by viscosity.
Thus, the pharmaceutical composition of the invention will preferably be prepared
without detergent and without solvent. For example, it will be prepared as an aqueous
suspension made viscous by the addition of agar at a non-solidifying concentration, for example
from 1 to 5 grams of agar per liter of suspension.
The pharmaceutical composition of the invention enables the treatment of local or
systemic infections caused by resistant microorganisms using doses of each of said first and
second therapeutically active substance which are lower than the doses required for treating
the same infections due to susceptible microorganisms with one or the other of these same
said first and second therapeutically active substances alone. In fact, the composition of the
invention enables the use of doses of said first therapeutically active substance, when it is
combined with said second therapeutically active substance, which are approximately three to
ten times lower than the doses required when said first therapeutically active substance is used
alone, and of doses of said second therapeutically active substance, when it is combined with
said first therapeutically active substance, which are approximately two to ten times lower than
the doses required when said second therapeutically active substance is used alone.
The result is to offer a treatment which has the following advantages:
- effective at very low doses against susceptible microorganisms,
- effective against microorganisms resistant to a therapeutic agent,
- effective against microorganisms resistant to several therapeutic
agents,
5

- control of recurrence phenomena,
- control of phenomena of resistant microorganisms selection.
In all these cases, there is a notable reduction in the risks of toxicity and/or adverse
effects well known to the person of the art, thanks to the potentiation which enables the
administration of very low doses.
In addition, the costs of producing the treatment are reduced due to the use of small
quantities of active substances.
The pharmaceutical compositions according to the invention can be in the form of
liposomes or associated with supports such as cyclodextrins or polyethylene glycols.
The pharmaceutical compositions of the invention are a simple and efficient means to
combat the problems related to microbial agents in general which comprise mainly resistance to
therapeutic agents and toxicity of the latter resulting from the use of high doses.
In fact, carveol, thymol, eugenol, borneol, carvacrol and the derivatives, mixtures and
isomers thereof, are simple molecules which have never been described as having any toxicity
whatsoever and their addition with its potentiating effect on the second therapeutically active
substance enables the use of much lower doses of said second therapeutically active
substance.
In a first variant, then, the method for treating patients having a fungal infection consists
in administering to said patients the dose, determined by the physician, of the pharmaceutical
composition of the invention comprising suitable doses of at least one said first therapeutically
active substance, combined with suitable doses of at least one said second therapeutically
substance, that is, the suitable antifungal agent.
In a second variant, the method for treating patients having a fungal infection consists in
sequentially administering to said patients the dose determined by the physician of at least one
said first therapeutically active substance, followed by the suitable dose of at least one said
second therapeutically active substance, that is, the suitable antifungal agent, or vice versa.
In this regard, the invention proposes a kit comprising at least one first container
containing one of said first therapeutically active substances, and at least one second container
containing one of said second therapeutically active substances.
Said kit enables health care personnel to prepare on demand either a mixture of
suitable doses of the desired first therapeutic substance(s) and of the desired antifungal
agent(s) for a simultaneous administration, or to sequentially and separately administer the
suitable dose of at least one said first therapeutically active substance, followed by the suitable
dose of at least one said second therapeutically active substance, that is, the suitable antifungal
agent, or vice versa. However, a mixture for simultaneous use shall be preferred in order to
allow the potentiation complex to form and to act immediately after administration to the patient.
The invention shall become clearer in the following examples describing different
6

embodiments, which are given for purposes of illustration and not by way of limitation.
EXAMPLE 1: Treatment of different Candida albicans strains with fluconazole
potentiated by carvacrol (Flue P)
In vitro tests: Determination of Minimal Fungicidal Concentration (MFC) on different
strains of Candida albicans
The experiment was carried out with several strains of Candida albicans having
different susceptibilities isolated in the hospital environment from the mouth, vagina and
digestive tract. The antifungal agent used was fluconazole, an azole derivative which is one of
the most effective and most widely used antifungals on the market. An antifungal
pharmaceutical composition was prepared by mixing fluconazole at different concentrations
with carvacrol at a sub-inhibitory concentration of 0.3 g per liter of solution or excipient. This
pharmaceutical composition according to the invention was named FLUC-P for potentiated
fluconazole. In each case, antifungal activity was determined either with fluconazole alone, or
with carvacrol alone, or with the composition of the invention.
Table 1 gives the results of static tests to determine the minimal fungicidal
concentration (MFC) in µg/ml.
Table 1

Candida albicans in
exponential growth
phase Fluconazole alone Composition of the
invention Carvacrol alone
MFC (µg/ml) MFC (µg/ml) MFC (µg/ml)
Susceptible strain 0.5 / 1000
Moderately resistant
strain 150 10 1000
Highly resistant strain >300 10 1000
Table 1 shows that the composition of the invention had notable fungicidal activity
on these strains with different susceptibilities, as compared with fluconazole alone or with
carvacrol alone.
Indeed, from Table 1, it is noted that the composition of the invention has a
remarkable fungicidal activity on those strains with different susceptibilities as compared with
fluconazole alone or with carvacrol alone.
In fact, it can be seen that by using a carvacrol concentration of 0.3 mg/ml, which is
approximately three times lower than the MFC of carvacrol alone, the fluconazole concentration
which produced fungicidal activity was 15 to 30 times lower than the MFC of fluconazole alone.
7

Kinetic tests
Kinetic tests were also carried out on a C. albicans strain highly resistant in order to
compare the fungicidal activities of fluconazole alone at a concentration of 300 µg/ml, carvacrol
alone at 300 µg/ml and the composition of the invention comprising fluconazole 150 ug/ml and
carvacrol 300 µg/ml. The number of colony-forming units (CFU) was measured over time.
The results, shown in Figure 1, clearly indicate that only the composition of the
invention had demonstrable activity against this highly resistant strain.
In vivo tests
Groups of 15 mice were experimentally infected by intravenous injection of
10,000,000 cells (colony-forming units) of a Candida albicans strain with moderate resistance to
fluconazole.
The first group was composed of control mice which were infected and untreated.
The second group was composed of infected mice treated by gavage, 24 h post-
infection, with fluconazole alone at a dose of 4 mg/kg of body weight/day.
The third group was composed of infected mice treated by gavage, 24 h post-
infection, with carvacrol alone at a dose of 30 mg/kg of body weight/day.
The fourth group was composed of infected mice treated by gavage, 24 h post-
infection, with fluconazole 2 mg/kg of body weight and with carvacrol 30 mg/kg of body weight.
The duration of treatment was seven days for surviving animals. The survival rate
was measured over time. The results are given in Figure 2, which shows that only the mice
treated with the pharmaceutical composition were still alive ten days after infection. All the
other mice died between the second and seventh day post-infection.
Examination of the organs of mice which died during the experiment (untreated
mice and those treated with fluconazole alone or carvacrol alone) revealed high loads of
Candida albicans in kidney, lung and bone marrow.
In contrast, in mice treated with the composition of the invention and sacrificed one
to ten days after stopping treatment, no C. albicans was found in lung and bone marrow.
In kidney, only two animals still had a very low C. albicans load corresponding to 5%
of that seen in the control group. The other animals treated with the composition of the
invention had no fungal load in the kidneys.
Here again, it clearly appears that potentiation of fluconazole by carvacrol gives
surprising results regarding the reduction in the minimal fungicidal concentration and the
rapidity of action in vitro.
This potentiation was also found in vivo in a model of systemic infection.
8

Now, systemic infection is one of the most serious and life-threatening forms of
infection and the most difficult to treat, especially in case of relapse with increasingly resistant
microbes.
Other data showed that the composition of the invention comprising fluconazole and
carvacrol produced surprising therapeutic effects at doses which were at least two-fold lower
than the doses required to treat localized experimental infections (vaginal and oral) in rats and
mice.
EXAMPLE 2: Treatment of different Candida albicans strains with
voriconazole potentiated by carvacrol (Vorico-P)
The experiment was carried out with several strains of Candida albicans having
different susceptibilities isolated in the hospital environment. The antifungal agent used was
voriconazole, an azole derivative and one of the most recent antifungals on the market. An
antifungal pharmaceutical composition according to the invention was prepared by mixing
voriconazole at different concentrations with carvacrol at a sub-inhibitory concentration of 0.3 g
per liter of solution or excipient. This pharmaceutical composition according to the invention
was named Vorico-P for potentiated voriconazole. In each case, antifungal activity was
determined either with voriconazole alone, or with carvacrol alone, or with the composition of
the invention.
Table 2 gives the results of static tests to determine the minimal inhibitory
concentration (MIC) and the minimal fungicidal concentration (MFC) in µg/ml.
Table 2

Candida albicans in
exponential growth
phase Voriconazole alone Vorico-P Carvacrol alone
MIC (µg/ml) MFC (µg/ml) MFC (µg/ml)
Susceptible strain Resistant strain 10 Table 2 shows that the composition of the invention had notable fungicidal activity
on the voriconazole-resistant strain, as compared with voriconazole alone or with carvacrol
alone.
In fact, it can be seen in Table 2 that by using a carvacrol concentration of 0.3
mg/ml, which is 3.3 times lower than the MFC of carvacrol alone, the voriconazole
concentration producing fungicidal activity against resistant strains was at least ten times lower
than the concentration of voriconazole alone capable of exhibiting fungistatic activity.
Thus, it can be seen that the potentiation of voriconazole by carvacrol not only
9

allowed a large reduction in the voriconazole dose but also transformed its fungistatic activity
into a fungicidal action.
EXAMPLE 3: Treatment of different Candida albicans strains with
capsofungin potentiated by carvacrol (Caspo-P)
The experiment was carried out with several strains of Candida albicans having
different susceptibilities isolated in the hospital environment. The antifungal agent used was
caspofungin, from the echinocandin family, which is one of the newest antifungals on the
market. An antifungal pharmaceutical composition according to the invention was prepared by
mixing caspofungin at different concentrations with carvacrol at a sub-inhibitory concentration of
0.3 g per liter of solution or excipient. This pharmaceutical composition according to the
invention was named Caspo-P for potentiated caspofungin. In each case, antifungal activity
was determined either with caspofungin alone, or with carvacrol alone, or with the composition
of the invention.
Table 3 gives the results of static tests to determine the minimal inhibitory
concentration (MIC) and the minimal fungicidal concentration (MFC) in µg/ml.
Table 3

Candida albicans in
exponential growth
phase Caspofungin alone Caspo-P Carvacrol alone
MIC (µg/ml) MFC (µg/ml) MFC (µg/ml)
Susceptible strain Resistant strain 3 Table 3 shows that the composition of the invention had notable fungicidal activity
on the resistant strain, as compared with caspofungin alone or with carvacrol alone.
In fact, it can be seen in Table 3 that by using a carvacrol concentration of 0.3
mg/ml, which is 3.3 times lower than the MFC of carvacrol alone, the caspofungin concentration
producing fungicidal activity was at least six times lower than the concentration of caspofungin
alone capable of exhibiting fungistatic activity.
Thus, it can be seen that the potentiation of caspofungin by carvacrol not only
allowed a large reduction in the caspofungin dose but also transformed its fungistatic activity
into a fungicidal action.
EXAMPLE 4: Treatment of different Candida albicans strains with 5-
10

fluorocytosine potentiated by eugenol (Fluoro-P)
The experiment was carried out with several strains of Candida albicans having
different susceptibilities isolated in the hospital environment. The antifungal agent used was 5-
fluorocytosine, from the pyrimidine group, which is one of the oldest antifungal agents. An
antifungal pharmaceutical composition according to the invention was prepared by mixing 5-
fluorocytosine at different concentrations with eugenol at a sub-inhibitory concentration of 0.5 g
per liter of solution or excipient. This pharmaceutical composition according to the invention
was named Fluoro-P for potentiated 5-fluorocytosine. In each case, antifungal activity was
determined either with 5-fluorocytosine alone, or with eugenol alone, or with the composition of
the invention.
Table 4 gives the results of static tests to determine the minimal inhibitory
concentration (MIC) and the minimal fungicidal concentration (MFC) in µg/ml.
Table 4

Candida albicans in
exponential growth
phase 5-fluorocytosine alone Fluoro-P Eugenol alone
MIC (µg/ml) MFC (µg/ml) MFC (µg/ml)
Susceptible strain 2.5 / 2000
Resistant strain 25 5 2000
Table 4 shows that the composition of the invention had notable fungicidal activity
on the resistant strain, as compared with 5-fluorocytosine alone or with eugenol alone.
In fact, it can be seen in Table 4 that by using a eugenol concentration of 0.5 mg/ml,
which is four times lower than the MFC of eugenol alone, the 5-fluorocytosine concentration
producing fungicidal activity on resistant strains was five times lower than the concentration of
5-fluorocytosine alone capable of exhibiting fungistatic activity.
Thus, it can be seen that the potentiation of 5-fluorocytosine by eugenol not only
allowed a large reduction in the 5-fluorocytosine dose but also transformed its fungistatic
activity into a fungicidal action.
EXAMPLE 5: Enlargement of the spectrum of action of fluconazole
potentiated by carvacrol to different Aspergillus niger strains
The experiment was carried out with several strains of Aspergillus niger which were
not susceptible to fluconazole. The antifungal agent used was fluconazole, an azole derivative
11

and one of the most widely used antifungals. The biggest drawback of fluconazole is that it
does not act on infections due to filamentous fungi. Aspergillosis, caused by microorganisms
from the family Aspergillus sp, represents the most common and most difficult-to-treat infection.
An antifungal pharmaceutical composition according to the invention was prepared by mixing
fluconazole at different concentrations with carvacrol at a sub-inhibitory concentration of 0.25 g
per liter of solution or excipient. This pharmaceutical composition according to the invention
was named Fluc-P for potentiated fluconazole. In each case, antifungal activity was
determined either with fluconazole alone, or with carvacrol alone, or with the composition of the
invention.
Table 5 gives the results of static tests to determine the minimal inhibitory
concentration (MIC) and the minimal fungicidal concentration (MFC) in µg/ml.
Table 5

Candida albicans in
exponential growth
phase Fluconazole alone Fluc-P Carvacrol alone
MIC (µg/ml) MFC (µg/ml) MFC (µg/ml)
12 different strains of
Aspergillus niger >1000 150 500
Table 5 shows that the composition of the invention had notable fungicidal activity
on Aspergillus niger, as compared with fluconazole alone or with carvacrol alone.
In fact, it can be seen in Table 5 that the use of a carvacrol concentration of 0.25
mg/ml, which is two times lower than the MFC of carvacrol alone, combined with fluconazole at
a concentration of 150 µg/ml, led to an outstanding fungicidal activity that fluconazole alone
was unable to provide even at a concentration of 1000 µg/ml.
Thus, it can be seen that the potentiation of fluconazole by carvacrol not only
allowed a large reduction in the fluconazole dose with species normally treated with
fluconazole, but also enlarged the spectrum thereof to filamentous fungi from the Aspergillus
genus which are normally not susceptible to fluconazole.
The method for treating a fungal infection consists in administering simultaneously
or sequentially to a patient having a fungal infection, the dose determined by the physician of at
least one first therapeutically active substance selected from the group consisting of carveol,
thymol, eugenol, borneol, carvacrol, and the isomers and derivatives and mixtures thereof, and
the determined dose of at least one second therapeutically active substance which is an
antifungal agent.
Generally, one simultaneously or sequentially administers to a patient having a
fungal infection between 1 and 3000 mg/kg of body weight/day of at least one first
12

therapeutically active substance selected from the group consisting of carveol, thymol, eugenol,
borneol, carvacrol, alpha-ionone, beta-ionone and the isomers and derivatives and mixtures
thereof, and between 1 and 20 mg/kg of body weight/day of at least one second therapeutically
active substance which is an antifungal agent.
In fact, the use of an amount less than 1 mg/kg of body weight/day of said first
therapeutically active substance, in particular carvacrol, does not produce the desired
potentiation effect.
On the other hand, the use of an amount greater than 3000 mg/kg of body
weight/day of said first therapeutically active substance, in particular carvacrol, does not further
increase the potentiation effect and poses a greater risk of toxicity.
Likewise, the use of an amount less than 1 mg/kg of body weight/day of said
second therapeutically active substance which is an antifungal agent, in particular fluconazole,
does not produce the desired therapeutic effect and the use of an amount greater than 20
mg/kg of body weight/day, in particular of fluconazole, does not improve the therapeutic effect
and increases the risk of toxicity.
Thus, in a preferred manner, one simultaneously or sequentially administers to a
patient having a fungal infection 30 mg/kg of body weight/day of at least one first therapeutically
active substance selected from the group consisting of carveol, thymol, eugenol, borneol,
carvacrol, alpha-ionone, beta-ionone and the isomers and derivatives and mixtures thereof, and
2 mg/kg of body weight/day of at least one second therapeutically active substance which is an
antifungal agent.
More particularly, in the case of Candida albicans infection, one simultaneously or
sequentially administers to the patient:
- 30 mg/kg of body weight/day of carvacrol, and
- 2 mg/kg of body weight/day of fluconazole.
Of course, the invention is in no way restricted to the embodiments described and
illustrated herein which are given solely by way of example.
On the contrary, the invention comprises all the technical equivalents of the
methods described herein as well as the combinations thereof where such are carried out in the
spirit of the invention.
13

WE CLAIM:
1. A pharmaceutical composition characterized in that it comprises:
- at least one first therapeutically active substance selected from the group consisting
of carveol, thymol, eugenol, borneol, carvacrol, alpha-ionone, beta-ionone, and the
isomers and derivatives and mixtures thereof,
and,
- at least one second therapeutically active substance which is an antifungal agent.
2. Composition as claimed in claim 1, characterized in that the antifungal agent is
selected from the group consisting of the pyrimidines, the azole family, the
echinocandin family, and the mixtures thereof.
3. Composition as claimed in claim 1 or 2, characterized in that the antifungal agent is
selected from 5-fluorocytosine, fluconazole, voriconazole, itraconazole and
caspofungin, and derivatives and mixtures thereof.
4. Composition as claimed in any one of claims 1 to 3, characterized in that said first
therapeutically active substance is eugenol or carvacrol.
5. Composition as claimed in any one of claims 1 to 4, characterized in that said first
therapeutically active substance is carvacrol and the antifungal agent is selected
from fluconazole, voriconazole and caspofungin, and derivatives and mixtures
thereof.
6. Composition as claimed in claim 5, characterized in that the antifungal agent is
fluconazole.
7. Composition as claimed in any one of claims 1 to 4, characterized in that said first
therapeutically active substance is eugenol and the antifungal agent is 5-
fluorocytosine.
8. Composition as claimed in any one of claims 1 to 7, characterized in that said first
and second therapeutically active substances are suspended in an aqueous agar
solution.
9. Composition as claimed in any one of claims 1 to 8, characterized in that said
14

composition does not include any detergent or solvent.
10. Composition as claimed in any one of claims 1 to 9, characterized in that it contains
between 10 and 200 mg/kg of body weight/day of said first therapeutically active
substance, and between 2 and 100 mg/kg of body weight/day of said second
therapeutically active substance.
11. Kit characterized in that it comprises:
at least one first container containing a first therapeutically active substance
selected from the group consisting of carveol, thymol, eugenol, borneol, carvacrol,
alpha-ionone, beta-ionone and the isomers and derivatives and mixtures thereof, and
- at least one second container containing a second therapeutically active
substance which is an antifungal agent.
12 Kit as claimed in claim 11, characterized in that the antifungal agent is selected from
the group consisting of the pyrimidines, the azole family, the echinocandin family,
and the mixtures thereof.
13. Kit as claimed in claim 11 or 12, characterized in that the antifungal agent is selected
from 5-fluorocytosine, fluconazole, voriconazole, itraconazole and caspofungin, and
derivatives and mixtures thereof.
14. Kit as claimed in any one of claims 11 to 13, characterized in that said first
therapeutically active substance is eugenol or carvacrol.
15. Kit as claimed in any one of claims 11 to 14, characterized in that said first
therapeutically active substance is carvacrol and the antifungal agent is selected
from fluconazole, voriconazole and caspofungin, and derivatives and mixtures
thereof.
16. Kit as claimed in claim 15, characterized in that the antifungal agent is fluconazole.
17. Kit as claimed in any one of claims 11 to 14, characterized in that said first
therapeutically active substance is eugenol and the antifungal agent is 5-
fluorocytosine.
18. Kit as claimed in any one of claims 11 to 17, characterized in that it contains between
15

10 and 200 mg/kg of body weight/day of said first therapeutically active substance,
and between 2 and 100 mg/kg of body weight/day of said second therapeutically
active substance.
19. A pharmaceutical composition and a kit substantially such as herein described with
reference to the accompanying drawings and as illustrated in the foregoing examples

The invention relates to a pharmaceutical composition. The inventive pharmaceutical
composition comprises: at leas a firm therapeuticall-active substance which is selected from
carveol, thymol, eugenol, borneol, carvacrol, alpha-jonone, betaionone and the isomers,
derivatives and mixtures thereof; and at least a second therapeutically-active substance
which is an antifungal agent.

Documents:

04483-kolnp-2007-abstract.pdf

04483-kolnp-2007-claims.pdf

04483-kolnp-2007-correspondence others.pdf

04483-kolnp-2007-description complete.pdf

04483-kolnp-2007-drawings.pdf

04483-kolnp-2007-form 1.pdf

04483-kolnp-2007-form 2.pdf

04483-kolnp-2007-form 3.pdf

04483-kolnp-2007-form 5.pdf

04483-kolnp-2007-international publication.pdf

04483-kolnp-2007-pct priority document notification.pdf

4483-KOLNP-2007-CORRESPONDENCE OTHERS 1.1.pdf

4483-KOLNP-2007-CORRESPONDENCE-1.2.pdf

4483-KOLNP-2007-CORRESPONDENCE-1.3.pdf

4483-KOLNP-2007-FORM 1-1.1.pdf

4483-KOLNP-2007-FORM 1-1.2.pdf

4483-kolnp-2007-form 13.pdf

4483-KOLNP-2007-FORM 18.pdf

4483-KOLNP-2007-FORM 26.pdf

4483-KOLNP-2007-FORM 3-1.1.pdf

4483-KOLNP-2007-GRANTED-CLAIMS.pdf

4483-KOLNP-2007-PETITION UNDER RULE 137.pdf

4483-KOLNP-2007-REPLY TO EXAMINATION REPORT.pdf

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Patent Number

250478

Indian Patent Application Number

4483/KOLNP/2007

PG Journal Number

02/2012

Publication Date

13-Jan-2012

Grant Date

05-Jan-2012

Date of Filing

21-Nov-2007

Name of Patentee

ADVANCED SCIENTIFIC DEVELOPMENTS

Applicant Address

10 RUE LOUKSOUS, QUARTIER EI HANA, 20200 CASABLANCA

Inventors:

#	Inventor's Name	Inventor's Address
1	REMMAL, ADNANE	RÉSIDENCE RACHIDIA II, BP 6233 ADARISSA, 30000 FES

PCT International Classification Number

A61P 31/00

PCT International Application Number

PCT/IB2006/001329

PCT International Filing date

2006-05-15

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	PCT/IB2005/001317	2005-05-13	IB