ES2258415A1 - Human and animal cryptosporidiosis medicine ingredient comprises an anti-parasite agent inhibiting L selection - Google Patents

Abstract

The human and animal cryptosporidiosis medicine ingredient has the capacity to inhibit the L-selection of the Cryptosporidium sp. parasite. It is Bobel-24 (2,4,6-triiodophenol), and is incorporated in treatment and prevention medicines.

Description

Bobel-24 (2,4,6-triiodophenol) for treatment or prevention of human and animal cryptosporidiosis.

The object of the invention is the use of a  L-selectin inhibitor, lectin that binds specifically to sugary residues compatible with Gal / GalNAc, the Bobel-24, to obtain preparations Pharmacists for both preventive therapy and limiting or even healing of cryptosporidiosis, both in humans as in animals is referred to a new coating.

State of the art

Cryptosporidium sp. It is a protozoan cosmopolitan parasite, responsible for gastrointestinal processes both in man and in various animals of veterinary interest. The infection is normally self-limited, but in immunocompromised hosts it results in uncontrollable diarrhea that is characterized by high fluid loss, even
20 liters / day, and that are accompanied by abdominal pain, nausea, vomiting, malaise and fever. As the parasite is intracellular, the destruction of the intestinal epithelium leads to the release of proinflammatory molecules that favor the appearance of an intestinal inflammation that accompanies this process. The magnitude of the problem is due to the fact that in states of congenital, iatrogenic or acquired immunosuppression, the parasite is able to spread through macrophages to pulmonary or bile ducts, while the intestinal multiplication increases progressively, leading to uncontrolled diarrhea that They can lead to death since there is no specific therapy. It can also cause serious pathologies in newborns and the elderly in which and for opposite reasons the immune system is not fully functional. It is a ubiquitous protozoan that affects man and various mammals and can be acquired through drinking water, since the oocyst resists the chlorination of water. In fact, water epidemics began to be detected in 1984, highlighting that produced in Milwaukee in 1993 with approximately 30% of the affected population, some 403,000 inhabitants. The process of infection of epithelial cells by Cryptosporidium , as in other coccidia, consists of an initial step, in which adhesion of the parasite sporozoites to the plasma membrane of the epithelial cell occurs. This initial step in the parasite-host relationship is an indispensable prerequisite for subsequent invasion. On the other hand, this initial interaction of the parasite is carried out through lectins present on the surface of the sporozoite and that specifically recognize hydrocarbon epitopes with the galactose-N-acetylgalactosamine (Gal / GalNAc) sequence (X.-M. Cheng & NF LaRusso, Gastroenterology 2000; 118: 368-379).

At present there is no specific treatment against this parasite, so cryptosporidiosis in immunocompetent individuals, being a self-limited process, is resolved by symptomatic treatments. However, because the process is uncontrollable in immunodeficiency states of very different origin, and may sometimes be the cause of a fatal outcome, the development of effective treatments is urgent. It must be taken into account that this pathology affects not only humans, but also causes large losses in the livestock sector, since, for example, and due to its form of transmission and development, it affects remarkably newborns. Due to the situation, the need for prophylaxis in individuals and animals at risk is generally accepted, since the infective dose is extremely low. To this end, numerous compounds of different groups have been used, such as macrolides, among which spiramycin or azithomycin, benzeneacetonitrile derivatives such as diclazuril or Letrazuril, or others with different chemical structures; among all of them, the most used has been Paromomycin, toxic at therapeutic doses and ineffective in many cases; The research is therefore still very active, promising trials are published with different compounds such as Maduramycin, Alborixin, Indinavir, etc. (CL Chapel & PC Okhuysen 2003. Curr Opin Infect Dis 15 (5): 523-7; R Mele, MA Morales, F Tosini & E Pozio (2003). Int J Parasitol 33 (7): 757-764; R. Fayer 1997. CRC Press.). The compounds used in the fight against this parasite, have very different mechanisms of action. One of the most explored forms of action in recent years has been the prevention of infection, that is, preventing the penetration of the parasite into the cells. For example, different proteases that play a fundamental role in the penetration of parasitic protozoa into their host cells have been studied (US 5750496; ES 2131467); deepening especially in the field of serine proteases. Patenting the use of different inhibitors thereof for the treatment of Cryptosporidiosis (US 5,750,496; ES 2131467). Great attention has also been given to the recipient by means of which the first contact between the host cell and the parasite is made. In the latter field, and in the case of Cryptosporidium it has been observed that glycoprotein epitopes with the Gal / GalNac sequence found in the apical membrane of epithelial cells and that sporozoite-specific lectins and specific for Gal / GalNac are involved and they play a fundamental role in the adhesion of Cryptosporidium parvum to the cells of the intestinal and biliary epithelium, as a previous and indispensable step for the subsequent invasion of the cells by C. parvum (X.-M. Cheng & NF LaRusso. Gastroenterology 2000; 118 : 368-379). In this field, the international patent WO 96/24368 stands out, which protects the use of different lectins for the treatment of oral and food diseases, including Cryptosporidiosis. On the other hand, it is known that L-selectin, a fundamental lectin in the process of initial adhesion and subsequent bearing of leukocytes on the membranes of the vascular endothelium, has several receptors among which is GlyCAM-1, which has a structure sugared, sulfated and branched with abundance of O-glycans containing Gal / GalNAc sequences (S Hemmerich, H Lefflers & SD Rosen 1995. Jour Biol Chem 270 (20): 12035-12047).

The product Bobel-24, is a trisubstituted 2,4,6 phenolic derivative, namely 2,4,6 triiodophenol, which is used as indicated in patents ES 2171532 and EP 707849 as a non-steroidal anti-inflammatory, for the treatment of the different diseases caused in humans and animals by their inhibitory activity of 5-lipoxygenase; including inflammatory diseases of the gastrointestinal tract including ulcerative colitis, Crohn's disease, chronic intestinal pathology, rectitis and lymphoplasmocytic enteritis. Not mentioning in any case Cryptosporidiosis as a possible target of the product Bobel-24, although this is a parasitosis that occurs with inflammation of the intestinal tract. This may be due to the fact that, although intestinal inflammation is present in Cryptosporidiosis, it cannot be considered as a notable sign of it, and in any case, the application of anti-inflammatory agents with 5-lipoxygenase inhibitor activity will not lead to themselves to the resolution of this parasitic infection. Along these same lines, it should be finally pointed out that Cryptosporidiosis cannot simply be considered a leukotriene-mediated disease, which is the fundamental claim of patent ES 2171532; since its pathology is due to the multiplication of the parasite inside the cells of the intestinal epithelium and the consequent destruction of it. However, if claimed in said patent, its usefulness for other infectious diseases such as Herpes simplex or Herpes Zoster, leishmaniosis or septic shock. On the other hand, its inhibitory capacity of L-selectin and the enzyme inducible nitric oxide synthase (iNOS) is also known (García Capdevilla et al . 1997. J. Chromatogr. B, 708: 169-175; Lopez-Belmonte J , Norton JA 1997. Inflamation Res. 46 (suppl.3), S253; Lopez-Belmonte J & Norton JA 1999. Mediators of Inflam. 8 (suppl. 1) S 154), the object of the present invention being the prevention of infection and elimination of the parasite based on the inhibitory capacity of the L-selectin that the product Bobel-24 possesses.

The invention

Accordingly, the present invention relates to the use of the product Bobel-24 (2,4,6-triiodophenol), alone or in combination with other agents, in the preparation of medicaments for the treatment or prevention of human or animal Cryptosporidiosis, based on its inhibitory character of L-selectin, which is a lectin present on the surface of the sporozoites of Cryptosporidium sp., protozoan parasite responsible for said disease , and which specifically recognizes hydrocarbon epitopes with the galactose-N-acetylgalactosamine sequence (Gal / Ga1NAc) present in the apical membrane of epithelial cells.

Said use of the Bobel 24 serves to obtain  pharmaceutical or veterinary compositions intended for both a preventive therapy, as limiting or even curative of the referred pathology. These compositions can be usable well in injectable form, in dragee capsules or tablets, or by any means of the usual ones in clinic, being able also be used in any dose per individual and day.

Embodiment

Experiences have been conducted to determine the usefulness of Bobel-24 in inhibiting the " in vitro " invasion of MDCK cells by sporozoites of C. parvum , as well as their development. For this, the previously described C. parvum culture procedure was followed (X. You, MJ Arrowood, M. Lejkowski, L Xie, RF Schinazi, JR Mead. 1996. FEMS Microbiology Letters 136: 251-256). MDCK cells were seeded in two-well Lab-Tek Chamber Slide ™ cabins, to obtain 60-80% confluence at 96 h and perform the infection. 1.2x106 oocysts were used per well and an induction of the exquisition was carried out with 0.75% NaT at 37 ° C for 45 min. Quadruplicates of each sample were used, including: a positive infection control without treatment; a control of parasite growth inhibition by treatment with Paromomycin 2 mg / ml; a control of inhibition of sporozoite binding to cells with 0.2 mg / ml mucin, maintained for 30 min. at 37 ° C prior to infection; a solvent toxicity control of the product, using 0.2% DMSO; In the experiment, Bobel-24 was used at the concentration of 90 µM, exploring three possibilities, a pre-incubation of the sporozoites for 30 min at 37 ° C, a pretreatment of the MDCK cells for 30 min. at 37 ° C, a post-treatment for 24 hours. After infection the medium was changed at 3 h, and the treatments were maintained 24 h. At 48 h, the camerites were revealed by an immunofluorescence as described previously, counting 20 fields per well studied (X. You, MJ Arrowood, M. Lejkowski, L Xie, RF Schinazi, JR Mead. 1996. FEMS Microbiology Letters 136: 251-256). The results obtained (figure 1) indicate that at the concentration studied, approximately 100% inhibition of Crypstosporidium growth in cells is achieved , when the treatment with Bobel-24 is maintained 24h. However, when only a pre-incubation of the sporozoites is performed with the Bobel-24, a significant reduction of the infection is achieved, approximately 49%. These results suggest that the Bobel-24 product, in addition to interfering with the initial penetration of sporozoite into the cells in culture, must present some additional mechanism of action by which it facilitates the elimination of the parasite from the cultures, since only with post-treatment an inhibition of almost 100% growth is reached. It should be noted that the concentration of Bobel-24 studied (90 µM) does not produce an appreciable cytotoxic effect.

The growth of Cryptosporidium parvum in the different conditions studied is shown in Figure 1, which is attached , both in terms of the number of developmental states per microscopic field studied in the different conditions (A), and in terms of percentage of infection with respect to the positive control of growth that are considered 100% (B).

Industrial application

The above results constitute the basis for the industrial preparation of pharmaceutical formulations containing Bobel-24 alone or in combination with other materials that complement or synergize its action, as an active material against cryptosporidium infection.

Claims (3)

1. Use of Bobel-24 (2,4,6-triiodophenol), alone or in combination with other agents, in the preparation of medicaments for the treatment or prevention of human or animal Cryptosporidiosis, characterized essentially by the inhibition produced by said L-selectin product present in the sporozoites of Cryptosporidium sp., parasite responsible for the disease. 2. Pharmaceutical or veterinary compositions whose active ingredients are Bobel-24 (2,4,6-triiodophenol), according to the first claim, Usable either in injectable form, in capsules, dragees or tablets, or by any means of the usual in clinic. 3. Pharmaceutical or veterinary compositions whose active ingredients are Bobel-24 (2,4,6-triiodophenol) according to claims above, usable in any dose per individual and day.