Abstract:
The present invention describes the action of a new family of drugs against a number of  Leishmania  species and  Trypanosona cruzi , for the treatment of cutaneous, mucocutaneous and visceral leishmaniosis as well as tripanosomosis. In vitro assays and studies conducted in animal models and in human patients demonstrated that the compounds had a higher activity compared to drugs in clinical use against  Leishmania mexicana amazonensis, L. donovani infantum, L. braziliensis braziliensis  and  Trypanosoma cruzi.    
     The pharmaceutical activity of compositions based on such family of compounds was evidenced and supports its human and veterinary application for the treatment of the above-mentioned diseases by using different administration routes.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention regards to human and animal health and, in general has to do with a family of chemical compounds biologically active against the etiological agents of leishmaniosis and trypanosomosis. 
       PREVIOUS ART 
       [0002]    Leishmaniosis is a disease caused by protozoan parasites of the genus  Leishmania . This genus comprises about 20 pathogenic species for human beings and animals. The disease has been described in 88 countries and 1.5-2 million new cases are reported yearly. It is estimated that 12 million people are affected by the disease worldwide and 350 million are at risk every year (CDC. http://www.dpd.cdc.gov/dpdx., Parasitic Disease information. Fact Sheet 1. April 2004). 
         [0000]    There are three clinical forms:
 
Cutaneous leishmaniosis: The cutaneous form is the commonest one, accounting for 90% of the cases. Cutaneous ulcers that take from moths to years to heal are typical. Secondary bacterial or fungal infections frequently worsen the primary lesion.
 
Visceral leishmaniosis: The liver and spleen are enlarged, the bone marrow is damaged, and the patient suffers from anemia and progressive weight loss. Most untreated cases and 15-25% of those who receive treatment in a late stage die.
 
Mucocutaneous leishmaniosis: Months to years after the primary cutaneous lesion healing has occurred, or even when the skin ulcer is still present, the parasites spread to the oral, nasal and pharyngeal mucosa. Soft tissues, mainly cartilages, are damaged, causing monstrous deformities of the face and eventually death due to breathing impairment once the laryngeal cartilages are destroyed.
 
         [0003]    The acquired immunodeficiency syndrome (AIDS) has aggravated the epidemiological situation, as patients require longer treatments and the frequency of relapses is higher (Alvar J, et. al. Clin Microbiol Rev 1997(10):298-319.) Pentavalent antimony derivatives have been the first line drugs for over 50 years. Nowadays, Pentostam® (Sodium stibogluconate) and Glucantime® (meglumine antimoniate) are the pharmaceutical forms in medical use. They are not effective against all species; being  L aethiopica  and  L. major  being the least sensible. Moreover, an increasing number of strains from traditionally sensitive species develop resistance. (Aparicio P. et. al. Terapéutica antiparasitaria, Enferm Infecc Microbiol Clin 2003; 21(10):579-94). Side effects related to pentavalent antimony derivatives have been a frequent cause of treatment discontinuation. Subclinical pancreatitis (31%), cardiotoxicity (15%) and nephrotoxicity (5%) are the major side effects. AIDS patients co-infected with  Leishmania  are notably sensitive to pancreatitis (Alvar J, et al. Clin Microbiol Rev 1997 (10): 298-319.) Amphotericin B and pentamidine are the second line drugs for the treatment of leishmaniosis. They are reserved for pentavalent-antimony-resistant cases. Two less toxic, and effective Amphotericin B lipid formulations (Alphocil® and AmBisone®); have been developed, but the majority of the affected population cannot afford to pay for the treatment. (Aparicio P, et. at. Terapéutica antiparasitaria, Enferm Infecc Microbiol C in 2003; 21 (10):579-94). 
         [0004]    The American trypanosomosis, also known as Chagas&#39; disease, is an endemic health problem in Latin America caused by  Trypanosoma cruzi  infection. Around 50 000 people die every year as a consequence. Twenty million are estimated to suffer from this disease and other 100 millions are in danger of getting sick yearly. The contaminated feces of hematophagous insects are responsible for transmitting the parasite to people. (Anonymous, http://www unl.edu.ar/eje.php?ID=1834, Sep. 15, 2005). 
         [0005]    Hundreds of structurally different compounds have been tested against  T. cruzi  during the last six decades; however, just a few of them have passed the preclinical stage with relative success. Only two trypanocidal drugs have been licensed in Latin America: nifurtimox (1972-1992) which has been eventually withdrawn from the market and benznidazole, clinically available since 1975 (Paulino A, et al. Mini Rev Med Chem 2005; 5(5):499-519, Lockman J W, Hamilton A D Curr Med Chem 2005; 12(8):945-59). 
       DESCRIPTION OF THE INVENTION 
       [0006]    The present invention describes the action of pharmaceutical compositions containing any of the six following representatives of the β-nitrovinylfuran chemical family:
   2-(2-nitrovinyl)-furan   2-bromo-5-(2-nitrovinyl)-furan   2-(2-bromo-2-nitrovinyl-furan   2-bromo-5-(2-bromo-2-nitrovinyl)-furan,   2-(2-methyl-2-nitrovinyl)-furan   2-bromo-5-(2-methyl-2-nitrovinyl)-furan   
 
         [0013]    Besides the β-nitrovinylfuran derivatives, these pharmaceutical compositions contain vehicles, excipients, solvents and adjuvants of pharmaceutical use. Solvents are preferably apolar ones, such as sunflower oil, petrolatum or Mygliol 810. These pharmaceutical compositions contain β-nitrovinylfuran derivatives at concentrations ranging from of 0.01 to 10%. The exact composition depends on the particular compound and the route of administration. The choice of a specific administration route, either topical or systemic. is determined by the clinical form of the disease. Trypanosomosis as well as mucocutaneous and visceral leishmaniosis are treated by routes that ensure a systemic distribution of the drug. On the contrary, cutaneous leishmaniosis and those cutaneous manifestations of the above-mentioned clinical forms of leishmaniosis are treated by both topical and systemic routes. 
         [0014]    The activity of compounds was tested against three  Leishmania species , which were representative of species causing the cutaneous mucocutaneous and visceral forms of leishmaniosis. They were also tested against  Trypanosoma cruzi , the etiological agent of Chagas&#39; disease. 
     
    
     
       DESCRIPTION OF THE FIGURES 
         [0015]      FIG. 1 : In vivo activity of 2-bromo-5-(2-bromo-2-nitrovinyl)-furan and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan against experimental cutaneous leishmaniosis. Early stage lesions treated every 24 h. 
           [0016]      FIG. 2 : In vivo activity of 2-bromo-5-(2-bromo-2-nitrovinyl)-furan and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan against experimental cutaneous leishmaniosis. Early stage lesions treated every 12 h. 
           [0017]      FIG. 3 : In vivo activity of 2-bromo-5-(2-bromo-2-nitrovinyl)-furan and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan against chronic experimental cutaneous leishmaniosis. Effect during the first week of treatment. 
           [0018]      FIG. 4 : In vivo activity of 2-bromo-5-(2-bromo-2-nitrovinyl)-furan and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan against chronic experimental cutaneous leishmaniosis as measured by changes in lesion size from the beginning to the end of therapy. 
       
    
    
     EXAMPLES FOR REALIZATION 
     Example 1 
     In Vitro Activity of β-nitrovinylfuran Derivatives Against  Leishmania  Promastigotes 
       [0019]    Mean inhibitory concentrations (Cl50%) against  L. mexicana amazonensis, L. donovani infantum  and  L. braziliensis braziliensis  promastigotes were determined according to the method described by Bodley A L et al (J Infect Dis 1995; 172(4):1157-9.). Minimal parasiticidal concentrations (CP100, minimal concentrations capable of abolishing the motility of all parasites in the culture) were also determined. Amphotericin B and Glucantime® were tested as reference drugs. 
         [0020]    All the β-nitrovinylfuran derivatives inhibited parasite growth at considerably low concentrations. Similarly, the compounds produced parasite death at low concentrations during the first 2-3 h following their addition to the culture media. Amphotericin B was more active than the β-nitrovinylfuran derivatives, but Glucantime® was significantly less active. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 In vitro activity of β-nitrovinylfuran derivatives against 
               
               
                   L. m. amazonensis ,  L. d. infantum  and  L. b. braziliensis  promastigotes 
               
             
          
           
               
                   
                 
                   amazonensis 
                 
                 
                   braziliensis 
                 
                 
                   Infantum 
                 
               
             
          
           
               
                 Compound 
                 CI50 
                 CP100 
                 CI50 
                 CP100 
                 CI50 
                 CP100 
               
               
                   
               
             
          
           
               
                 A 
                 0.66 
                 1.12 
                 0.20 
                 1.29 
                 0.35 
                 1.75 
               
               
                 B 
                 0.45 
                 2.08 
                 0.18 
                 0.61 
                 0.23 
                 0.51 
               
               
                 C 
                 0.63 
                 1.77 
                 0.25 
                 1.25 
                 0.36 
                 2.01 
               
               
                 D 
                 0.25 
                 1.53 
                 0.26 
                 0.49 
                 0.35 
                 0.86 
               
               
                 E 
                 0.34 
                 3.49 
                 0.35 
                 4.84 
                 0.31 
                 1.27 
               
               
                 F 
                 0.20 
                 1.42 
                 0.24 
                 1.32 
                 0.20 
                 0.62 
               
               
                 Amphotericin B 
                 0.027 
                 0.051 
                 0.013 
                 0.028 
                 0.013 
                 0.016 
               
               
                 Glucantime ® 
                 1916 
                 &gt;4250 
                 1893 
                 &gt;4250 
                 1925 
                 &gt;4250 
               
               
                   
               
               
                 Legend: 
               
               
                 A: 2-(2-nitrovinyl)-furan 
               
               
                 B: 2-bromo-5-(2-nitrovinyl)-furan 
               
               
                 C: 2-(2-bromo-2-nitrovinyl)-furan 
               
               
                 D: 2-bromo-5-(2-bromo-2-nitrovinyl)-furan 
               
               
                 E: 2-(2-methyl-2-nitrovinyl)-furan 
               
               
                 F: 2-bromo-5-(2-methyl-2-nitrovinyl)-furan 
               
             
          
         
       
     
       Example 2 
     In Vitro Activity Against  Trypanosoma cruzi    
       [0021]    The in vitro activity of the β-nitrovinylfuran derivatives was tested against trypomastigotes of  T. cruzi  grown in a rat myoblast cell line. Minimal parasiticidal concentrations (minimal concentration that immobilizes all the parasites in the culture) and mean inhibitory concentrations (concentration that reduces the number of parasites to 50% of those found in the un-treated control cultures) were determined according to Buckner&#39;s method (Antimicrob Agents Chemother 1996; 40(11):2592-7). The experiment evidenced that the test compounds had an in vitro parasiticidal activity superior to that of benznidazole. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 In vitro effect β-nitrovinylfuran derivatives against  T. cruzi   
               
             
          
           
               
                   
                 CI50 
                   
                 CP100 
                   
               
             
          
           
               
                   
                 Compound 
                 μg/mL 
                 μM 
                 μg/mL 
                 μM 
               
               
                   
                   
               
             
          
           
               
                   
                 A 
                 1.8 
                 12.6 
                 10.0 
                 71.9 
               
               
                   
                 B 
                 1.2 
                 5.7 
                 3.3 
                 15.3 
               
               
                   
                 C 
                 3.7 
                 16.9 
                 10.0 
                 45.9 
               
               
                   
                 D 
                 4.1 
                 13.9 
                 10.0 
                 33.7 
               
               
                   
                 E 
                 4.7 
                 31.0 
                 30.0 
                 196.1 
               
               
                   
                 F 
                 5.3 
                 23.0 
                 30.0 
                 129.3 
               
               
                   
                 Benznidazole 
                 0.9 
                 3.3 
                 &gt;40 
                 &gt;154 
               
               
                   
                   
               
               
                   
                 Legend: 
               
               
                   
                 A: 2-(2-nitrovinyl)-furan 
               
               
                   
                 B: 2-bromo-5-(2-nitrovinyl)-furan 
               
               
                   
                 C: 2-(2-bromo-2-nitrovinyl)-furan 
               
               
                   
                 D: 2-bromo-5-(2-bromo-2-nitrovinyl)-furan 
               
               
                   
                 E: 2-(2-methyl-2-nitrovinyl)-furan 
               
               
                   
                 F: 2-bromo-5-(2-methyl-2-nitrovinyl)-furan 
               
             
          
         
       
     
       Example 3 
     Toxicological Studies 
       [0022]    The same animal model used for in vivo anti-leishmanial tests was used to perform comparative toxicological studies of the β-nitrovinylfuran derivatives. The main excipient of the compositions used with this purpose was the sunflower oil. The mean lethal doses (LD50) of each β-nitrovinylfuran derivative and Amphotericin B were estimated in female Balb/c mice (18-20g body weight), treated by a single intraperitoneal injection. The maximal non-lethal doses (LD0) were calculated from the dose-mortality curves. The maximal tolerated doses (MTD, maximal dose causing neither mortality nor body weight losses over 10% respect to the initial weight) by repeated intraperitoneal administration every 12 or 24 h during 14 days were determined as well. Results indicated that the β-nitrovinylfuran derivatives had a relatively wide range of LD50&#39;s, which were all superior to that of Amphotericin B. The MTD determined during the course of the present series of experiments allowed choosing a proper dose for in vivo anti-leishmanial activity studies. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Mean lethal doses (LD50), Maximal non-lethal doses 
               
               
                 (LD0) and Maximal tolerated doses (MTD) of β-nitrovinylfuran 
               
               
                 derivatives and Amphotericin B. 
               
             
          
           
               
                   
                 LD50 
                 LD0 
                 MTD 
                 MTD 
               
               
                 Compound 
                 (mg/Kg) 
                 (mg/Kg) 
                 (every 24 h) 
                 (every 12 h) 
               
               
                   
               
             
          
           
               
                 A 
                 68 
                 50 
                 55 
                 — 
               
               
                 B 
                 73 
                 45 
                 20 
                 — 
               
               
                 C 
                 51 
                 35 
                 — 
                 — 
               
               
                 D 
                 24 
                 15 
                 5 
                 2-3 
               
               
                 E 
                 80 
                 65 
                 60 
                 — 
               
               
                 F 
                 246 
                 200 
                 100 
                 100 
               
               
                 Ampho- 
                 22 
                 15 
                 — 
                 — 
               
               
                 tericin B 
               
               
                   
               
               
                 Legend: 
               
               
                 A: 2-(2-nitrovinyl)-furan 
               
               
                 B: 2-bromo-5-(2-nitrovinyl)-furan 
               
               
                 C: 2-(2-bromo-2-nitrovinyl)-furan 
               
               
                 D: 2-bromo-5-(2-bromo-2-nitrovinyl)-furan 
               
               
                 E: 2-(2-methyl-2-nitrovinyl)-furan 
               
               
                 F: 2-bromo-5-(2-methyl-2-nitrovinyl)-furan 
               
             
          
         
       
     
       Example 4 
     Treatment of Experimental Cutaneous Leishmaniosis with 2-5 bromo-5-(2-bromo-2-nitrovinyl)-furan and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan. Early Stage Lesions Treated Every 24 h. 
       [0023]    Balb/c mice were experimentally infected with  L. m. amazonensis  promastigotes by intradermal injection in the footpads and six weeks later they were treated with the test compounds by intraperitoneal route. The product 2-bromo-5-(2-bromo-2-nitrovinyl)-furan was administered at a dose of 5 mg/Kg; the 2-bromo-5-(2-methyl-2-nitrovinyl)-furan, at 50 mg/Kg and Amphotericin B, at 1 mg/Kg. Animals were daily dosed for 14 days. Miglyol 810, the vehicle used for the β-nitrovinylfuran derivatives, was similarly administered to a group of mice. A group of infected mice that did not receive any treatment at all was also included as control. 
         [0024]    The lesion growth curves ( FIG. 1 ) evidenced that mice treated with either 2-bromo-5-(2-bromo-2-nitrovinyl)-furan or 2-bromo-5-(2-methyl-2-nitrovinyl)-furan had a minimal lesion growth during the first week of treatment. Those animals treated with 2-bromo-5-(2-methyl-2-nitrovinyl)-furan showed a small decrease in lesion size. Lesion growth during that week was statistically lower on the test group compared to the rest of the groups. During the second week of treatment the growth rate was comparable in all the experimental groups. However, the differences established during the first week of treatment with 2-bromo-5-(2-bromo-2-nitrovinyl)-furan remained for two other weeks after treatment had finished. The group of mice treated with Amphotericin B evolved similarly to controls; however, during the first week after discontinuation of treatment they showed a remarkable decrease of lesion growth rate. 
       Example 5 
     Treatment of Experimental Cutaneous Leishmaniosis with 2-bromo-5-(2-bromo-2-nitrovinyl)-furan and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan. Early Stage Lesions Treated Every 12 h. 
       [0025]    The experiment was essentially carried out in a similar way as the one described above. However, 2-bromo-5-(2-bromo-2-nitrovinyl,-furan was administered at a dose of 2 mg/Kg and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan at 100 mg/Kg. Treatments were administered by intraperitoneal route, but every 12 h instead of 24 h. 
         [0026]    The lesions of mice treated with 5-(2-bromo-2-nitrovinyl)-furan decreased during the first week of treatment ( FIG. 2 ), been statistically smaller (P&lt;0.001) than those of control mice. The following week, lesions grew at a similar rate as control and the next week, a decline in lesion growth rate was then noticed. Seven days after finishing treatment, mice treated with 5-(2-bromo-2-nitrovinyl)-furan had lesions statistically (P&lt;0.05) smaller than those of controls. 
         [0027]    Lesion growth rate of mice treated with either 2-bromo-5-(2-methyl-2-nitrovinyl)-furan or 2-bromo-5-(2-bromo-2-nitrovinyl)-furan was significantly lower compared to controls. Similarly, both products showed higher activity than Amphotericin B. 
       Example 6 
     Treatment of Experimental Cutaneous Leishmaniosis with 2-bromo-5-(2-bromo-2-nitrovinyl)-furan and 2-bromo-5-(2-methyl-2-nitrovinyl)-furan. Chronic Lesions Treated every 12 h. 
       [0028]    Mice developed chronic leishmanial lesions at eighteen weeks post-infection. Lesions were ulcerated and covered with a thick crust. The average dorso-plantar diameter of the infected foot was 1.23±0.24 cm and the lateral diameter was 1.24±0.19 cm. At that stage the treatment started twice a day with either 2-bromo-5-(2-bromo-2-nitrovinyl)-furan at 2 mg/Kg; 2-bromo-5-(2-methyl-2-nitrovinyl)-furan at 50 mg/Kg or Amphotericin B at 5 mg/Kg every other day. A control group formed by infected non-treated mice was also included in the experiment. All drugs were administered by intraperitoneal route for 14 days. During the first week of treatment with 2-bromo-5-(2-bromo-2-nitrovinyl)-furan mice showed a decrease of the dorso-plantar diameter ( FIG. 3 ) of the infected foot that differed from the control group (P&lt;0.05). Although lateral diameter of the lesions of these mice also revealed a decrease, it was comparable to the diameter of control mice (P&gt;0.05). The 2-bromo-5-(2-methyl-2-nitrovinyl)-furan did not prove to be effective for the treatment of chronic lesions. Amphotericin B was not effective, since neither the dorso-plantar nor the lateral diameters differed statistically from the lesions of the control group (P&gt;0.05). 
         [0029]    By the end of treatment ( FIG. 4 ), mice treated with 2-bromo-5-(2-bromo-2-nitrovinyl)-furan evidenced a decrease of lesion size. Lesions of these mice were significantly different to the ones of control mice in both the dorso-plantar (P&lt;0.05) and lateral (P&lt;0.01) diameters. These mice also differed from those treated with Amphotericin B (P&lt;0.05). On the contrary, treatment with 2-bromo-5-(2-methyl-2-nitrovinyl)-furan was ineffective. 
         [0030]    Animals treated with Amphotericin B incremented their lesion dorso-plantar diameters as controls (P&gt;0.05). Although the lateral diameter diminished in the lesions of Amphotericin B-treated mice, the change was not different (P&gt;0.05) to that found in controls. 
         [0031]    The graphic illustrates that control mice incremented their lesions mainly by the dorso-plantar diameter. Interestingly, the therapeutic effect of the test compounds is more evident in relation to the lateral diameter. 
         [0032]    The present study showed that 2-bromo-5-(2-bromo-2-nitrovinyl)-furan has i vivo activity against  Leishmania , as demonstrated in the biomodel of experimental cutaneous leishmaniosis developed in Balb/c mice inoculated with  L. m. amazonensis . On the other hand, although higher doses of the compound 2-bromo-5-(2-methyl-2-nitrovinyl)-furan are needed, an in vivo effect against the experimental disease was also showed. 
       Example 7 
     Treatment of Human Cutaneous Leishmaniosis with 2-bromo-5-(2-bromo-2-nitrovinyl)-furan 
       [0033]    One hundred patients with parasitological diagnose of cutaneous leishmaniosis were treated. Patients were topically treated, once a day, for 21 days with an oily ointment containing 0.15% 2-bromo-5-(2-bromo-2-nitrovinyl)-furan as active ingredient and petrolatum as the main excipient. 
         [0034]    Ninety percent of patients showed total cure of the cutaneous lesions. The time of recovery depended on the severity of lesions and varied from 10 to 21 days. At the end of the treatment period, the 90% of patients that had recovered evidenced not only the cure of the ulcers, but the local and systemic symptoms due to the primary effect of the parasite infection and the secondary bacterial or fungal contaminants had disappeared as well. 
         [0035]    Patients did not evidence any side effect that impeded the continuation of therapy as conceived in the protocol.