WO2004014289A2 - Use of beta-cyclodextrin (cas rn: 7585-39-9) in the preparation of medicaments for the treatment of human and animal diseases caused by cryptosporidium parasites - Google Patents

Use of beta-cyclodextrin (cas rn: 7585-39-9) in the preparation of medicaments for the treatment of human and animal diseases caused by cryptosporidium parasites Download PDF

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WO2004014289A2
WO2004014289A2 PCT/ES2003/000410 ES0300410W WO2004014289A2 WO 2004014289 A2 WO2004014289 A2 WO 2004014289A2 ES 0300410 W ES0300410 W ES 0300410W WO 2004014289 A2 WO2004014289 A2 WO 2004014289A2
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cyclodextrin
treatment
preparation
human
medicaments
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WO2004014289A3 (en
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María Elvira ARES MAZÁS
José Antonio CASTRO HERMIDA
José BLANCO MÉNDEZ
Francisco Javier Otero Espinar
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Universidade De Santiago De Compostela
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Beta-CYCLODEXTRINE (CAS RN: 7585-39-9) IN THE
  • Neonatal diarrhea in domestic ruminants is one of the main causes of economic losses directly related to mortality and morbidity, with treatment costs and animal growth delays. It is a multifactorial etiology process, and there are several enteropathogenic agents that may be involved in epidemic outbreaks. In addition, animal management, nutritional status and the environment are factors that have an impact on the occurrence of the disease. The prevalence of the responsible agents differs according to the geographical area and the age of the animal.
  • the parasite has a great capacity for multiplication in the host, the minimum infectious dose being very low and offering a remarkable resistance to specific therapy (Kosek, M .; Alc ntara, C; Lima, AMA and Guerrant, LR Lancet Infec ⁇ D ⁇ s. (2001) 1: 262-269.
  • the object of the invention is the use of ⁇ -cyclodextrin in the preparation of medicaments and any kind of pharmaceutical form for the prophylactic and / or therapeutic treatment of cryptosporidiosis present in humans and animals.
  • SUBSTITUTE SHEET (RULE 26) Although certain drugs offered clinical and / or parasitological improvements, the current absence of a specific therapy against Cryptosporidium infection remains a pending issue for human and animal health.
  • Halofuginone lactate has been marketed under the name of Halocur®, formulated as an oral solution (0.5 mg / mL) and indicated for the prevention and reduction of diarrhea caused by C. parvum in fresh calves born, and must be administered once a day and at the same time for 7 consecutive days.
  • paromomycin is the most widely used and has some activity in experimental models and clinical trials (Fichtenbaum, CJ. Et al. Clin. Infec. Dis. (1993) 16: 298-300; Flanigan, TP etal. Am.
  • Nitazoxanide is one of the last drugs evaluated against human cryptosporidiosis. It is a nitrotiazole benzamide with extensive activity against protozoa, helminths and bacteria. In a study conducted in Mexico, in 15 AIDS patients with cryptosporidiosis, the resolution of the infection was observed in all patients after a 1-2 g / day regimen administered for 10-30 days (Feregrino, GM et al XI Internetional Conference on AIDS. (1996) Vancover, Canada Similar results were obtained in a study conducted in Africa (Doumbo, O. et al. Am. J. Trop. Med. Hyg. (1997) 56: 637-639.
  • inclusion complexes with cyclodextrins makes it possible to improve the solubility of the active ingredients and therefore their passage through membranes when the solubility is a limiting factor thereof.
  • the diffusion fraction increases, which can reduce the administration dose and achieve the same therapeutic effect, which represents an additional advantage of the reduction of side effects as a consequence of the high doses administered.
  • Some active ingredients with relative efficacy in the treatment of human and / or animal cryptosporidiosis have poor solubility (spiramycin, diloxanide furoate, furazolidone, halofuginone, among others) or stability problems (such as paromomycin) and their effectiveness may be enhanced by formulating as inclusion complexes with cyclodextrins.
  • G-l halof ⁇ ginone lactate and paromomycin among others, were selected for their potential anti-cryptosporidial activity.
  • some formed inclusion complexes for example diloxanide furoate and G-1 while others were unable, due to their molecular structure, to enter the cavity of ⁇ -cyclodextrin.
  • the characterization of the different complexes using suitable instrumental techniques demonstrated the formation of stable complexes in solid state and in solution, improving properties such as the solubility and dissolution rate of the drugs, and even the stability in products such as Gl, which is degraded in Aqueous solutions or suffer sublimation problems.
  • the oocystic viability was determined by release techniques and inclusion / exclusion of fluorogenic vital dyes.
  • the results obtained with both active ingredients showed that as the exposure time increases, the percentage of unlocked oocysts (empty oocysts detected by phase contrast) decreases and the number of unviable oocysts (oocysts incorporating the vital dye propidium iodide) increases. ). It was found that its formulation as inclusion complexes improves its activity.
  • Trial 4 dose every 12 hours; trial 5: two hours before causing infection and dose every
  • trial 6 dose every 12 hours
  • trial 7 dose every 8 hours.
  • ⁇ -cyclodextrin against cryptosporidiosis in domestic ruminants was evaluated.
  • newborn lambs were experimentally infected on the first day of life with 10 6 oocysts of C. parvum of sheep origin.
  • the administration of ⁇ -cyclodextrin was performed orally and in the form of an aqueous suspension, the dose being 1 g / kg body weight.
  • the lambs were treated during the first three days of life, and in the therapeutic trial the ⁇ -cyclodextrin was administered, with the same pattern, from the start of the diarrheal process and confirmation of the presence of oocysts in the stool
  • the efficacy was determined taking into account oocystic elimination, the presence of diarrhea and body weight gain on days 15 and 30 of animal life.
  • the results (Graph 1) showed that ⁇ -cyclodextrin was highly effective as a prophylactic by preventing the onset of diarrheal processes in infected lambs and preventing infection in one of them, as well as significantly decreasing the intensity and duration of the patent period .
  • Therapeutically administered it reduced the severity of the diarrheal process and shortened the period of oocystic excretion.
  • the treated animals significantly increased their body weight and no apparent toxicity or reinfections were observed.
  • cyclodextrins cause lysis of erythrocytes when dissolved, and therefore eliminate, components of their membrane such as cholesterol and certain phospholipids, phosphatidylcholine and sphingomyelin (Ohtani, Y. etal. J. Europ J. Biochem. (1989) 186: 17-22; Arima, H. et al. V International Symposium on Cyclodextrin. (1990). Paris, France); lipid compounds detected on the wall of the oocysts of C.
  • ⁇ -cyclodextrin is not toxic since it is not absorbed through the gastrointestinal tract, thus preventing its accumulation in organs (e, T. and Uekama, KJ Pharmac. Science. (1997) 86: 147-162) .
  • Toxicity studies showed that, orally, LD 50 values exceed 12.5; 18.5 and 5 g / kg live weight for mice, rats and dogs respectively (Szejtli, J. Cyclodextrins in Technology. (1988). J. Szejli (Ed.) Kluwer Academic Publishers, Dordrecht, The Netherlands).
  • ⁇ -cyclodextrin In addition to being a biodegradable product, it is used in aquaculture for the preparation of pharmaceutical formulations for the treatment of farmed fish.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to the use of β-cyclodextrin (CAS RN: 7585-39-9) in the preparation of medicaments for the treatment of human or animal diseases caused by Cryptosporidium parasites. According to the invention, the β-cyclodextrin is administered orally alone or as inclusion complexes by way of a prophylactic and/or therapeutic agent in the treatment of animal cryptosporidiosis. The aforementioned β-cyclodextrin and the complexes thereof can be administered as such or inserted in drinking water and mixed with feed, with various different formulations being used to facilitate the administration thereof. Moreover, the β-cyclodextrin and the complexes thereof can be used to promote the elimination of infecting forms of Cryptosporidium (oocysts) in waste water and purines.

Description

UTILIZACIÓN DE LA beta-CICLODEXTRINA (CAS RN: 7585-39-9) EN LAUSE OF Beta-CYCLODEXTRINE (CAS RN: 7585-39-9) IN THE
PREPARACIÓN DE MEDICAMENTOS PARA EL TRATAMIENTO DE LASPREPARATION OF MEDICINES FOR THE TREATMENT OF
ENFERMEDADES HUMANAS O ANIMALES CAUSADAS POR PARÁSITOSHUMAN OR ANIMAL DISEASES CAUSED BY PARASITES
PERTENECIENTES AL GÉNERO CRYPTOSPORIDIUM. La diarrea neonatal en rumiantes domésticos es una de las principales causas de pérdidas económicas directamente relacionada con la mortalidad y morbilidad, con los costes de tratamiento y los retrasos en el crecimiento de los animales. Es un proceso de etiología multifactorial, y son varios los agentes enteropatógenos que pueden estar implicados en brotes epidémicos. Además, el manejo de los animales, estado nutricional y el entorno son factores que repercuten en la aparición de la enfermedad. La prevalencia de los agentes responsables difiere según el área geográfica y la edad del animal. Sin embargo, diversos estudios señalan a Cryptosporidium parvum como el enteropatógeno más comúnmente diagnosticado en las ganaderías bovina, ovina y caprina (de Graaf, D.C.; Vanopdenbosch, E.; Ortega-Mora, L.M.; Abbassi, H and Peeters, J.E. Int. J. Parasitol. (1999) 29:1269-1287). Las formas infectantes, los ooquistes, se eliminan en grandes cantidades en las heces de los individuos parasitados durante la fase aguda de la infección, tienen un pequeño tamaño (4,5-5 μm) y son resistentes a las condiciones ambientales y a los desinfectantes habitualmente empleados. Además, el parásito presenta una gran capacidad de multiplicación en el hospedador, siendo la dosis mínima infectante muy baja y ofreciendo una resistencia notable a la terapia específica (Kosek,M.; Alc ntara, C; Lima, A.M.A. and Guerrant, L.R. Lancet Infecí. D¿s.(2001) 1: 262- 269.BELONGING TO THE GENDER CRYPTOSPORIDIUM. Neonatal diarrhea in domestic ruminants is one of the main causes of economic losses directly related to mortality and morbidity, with treatment costs and animal growth delays. It is a multifactorial etiology process, and there are several enteropathogenic agents that may be involved in epidemic outbreaks. In addition, animal management, nutritional status and the environment are factors that have an impact on the occurrence of the disease. The prevalence of the responsible agents differs according to the geographical area and the age of the animal. However, several studies point to Cryptosporidium parvum as the most commonly diagnosed enteropathogen in cattle, sheep and goats (from Graaf, DC; Vanopdenbosch, E .; Ortega-Mora, LM; Abbassi, H and Peeters, JE Int. J Parasitol. (1999) 29: 1269-1287). The infectious forms, the oocysts, are eliminated in large quantities in the feces of the parasitized individuals during the acute phase of the infection, have a small size (4.5-5 μm) and are resistant to environmental conditions and disinfectants usually employees. In addition, the parasite has a great capacity for multiplication in the host, the minimum infectious dose being very low and offering a remarkable resistance to specific therapy (Kosek, M .; Alc ntara, C; Lima, AMA and Guerrant, LR Lancet Infecí D¿s. (2001) 1: 262-269.
Paralelamente, la frecuente presencia de C. parvum como parásito oportunista en enfermos inmunocomprometidos, así como su asociación con el incremento en la morbilidad y malnutrición infantil en países en vías de desarrollo, son hechos que denotan la importancia de la cryptosporidiosis (Fayer, R. Cryptosporidium and Cryptosporidiosis (R. Fayer (Ed.). CRC Press, Boca ratón, Florida. 1997). Por otra parte, Cryptosporidium ha emergido como un nuevo agente contaminante de medios hídricos, aguas residuales y purines entre otros, al estar implicado en brotes epidémicos desencadenados por el consumo de agua de bebida contaminada que alertaron a la opinión pública, ya que es el mecanismo de transmisión más importante por sus potenciales repercusiones numéricas (Smith, H.V. and Rose, J.B . Parasitol. Today. (1998) 14:14-22).At the same time, the frequent presence of C. parvum as an opportunistic parasite in immunocompromised patients, as well as its association with the increase in infant morbidity and malnutrition in developing countries, are facts that denote the importance of cryptosporidiosis (Fayer, R. Cryptosporidium and Cryptosporidiosis (R. Fayer (Ed.). CRC Press, Boca Raton, Florida. 1997) On the other hand, Cryptosporidium has emerged as a new pollutant for water, wastewater and slurry among others, being involved in epidemic outbreaks triggered by the consumption of contaminated drinking water that alerted public opinion, since it is the most important transmission mechanism due to its potential numerical repercussions (Smith, HV and Rose, JB. Parasitol. Today. (1998) 14: 14-22).
El objeto de la invención es la utilización de la β-ciclodextrina en la preparación de medicamentos y cualquier clase de forma farmacéutica para el tratamiento profiláctico y/o terapéutico de la cryptosporidiosis presente en humanos y animales. HOJA DE SUSTITUCIÓN (REGLA 26) Aunque ciertos fármacos ofrecieron mejorías clínicas y/o parasitológicas, la ausencia actual de una terapia específica frente a la infección por Cryptosporidium sigue representando una cuestión pendiente para la sanidad humana y animal.The object of the invention is the use of β-cyclodextrin in the preparation of medicaments and any kind of pharmaceutical form for the prophylactic and / or therapeutic treatment of cryptosporidiosis present in humans and animals. SUBSTITUTE SHEET (RULE 26) Although certain drugs offered clinical and / or parasitological improvements, the current absence of a specific therapy against Cryptosporidium infection remains a pending issue for human and animal health.
En rumiantes recién nacidos y de hasta tres meses de edad, en la actualidad, tan sólo el lactato de halofuginona, la paromomicina y el decoquinato han demostrado ser parcialmente efectivos en la prevención y en el tratamiento de la cryptosporidiosis, al disminuir el período de excreción ooquística y la gravedad de la diarrea, cuando se administran durante períodos que oscilan entre 3 y 21 días e incluso hasta 8 semanas. Sin embargo, en algunos casos, ciertos autores comprobaron la aparición de reinfecciones asintomáticas una vez suspendido el tratamiento (Nillacorta, I.; Peeters, J.E.; Nanopdenbosch, E.; Ares-Mazás, E. and Heys, H.. Antimicrob. Agents Chemother. (1991) 35: 283-287; Fayer; R. and Ellis, W. J. Parasitol. (1993) 79:771-774; Νaciri, M.; Mancassola, R.; Yvoré, P. and Peeters, J.E. Vet. Parasitol. (1993) 45: 199-207; Mancassola, R.; Richard, A. andΝaciri, M. Vet. Parasitol. (1997) 69:31- 37; Causapé, A.C.; Sánchez- Acedo, C; Quílez, J.; del Cacho, E. and Viu, M. Res. Rev. Parasitol. (1999) 59: 41-46; Viu, M.; Quílez, J.; Sánchez-Acedo, C; del Cacho, E. andLópez- Bernad, F. Vet. Parasitol. (2000) 90:283-287). De ellos y parauso exclusivamente veterinario, el lactato de halofuginona ha sido comercializado bajo el nombre de Halocur®, formulado como solución oral (0,5 mg/mL) e indicado para la prevención y reducción de diarreas ocasionadas por C. parvum en terneros recién nacidos, debiendo ser administrado una vez al día y a la misma hora durante 7 días consecutivos.In newborn ruminants and up to three months of age, at present, only halofuginone lactate, paromomycin and decokinate have been shown to be partially effective in the prevention and treatment of cryptosporidiosis, by decreasing the period of excretion oocystic and the severity of diarrhea, when administered during periods ranging from 3 to 21 days and even up to 8 weeks. However, in some cases, certain authors verified the appearance of asymptomatic reinfections once treatment was discontinued (Nillacorta, I .; Peeters, JE; Nanopdenbosch, E .; Ares-Mazás, E. and Heys, H .. Antimicrob. Agents Chemother. (1991) 35: 283-287; Fayer; R. and Ellis, WJ Parasitol. (1993) 79: 771-774; Νaciri, M .; Mancassola, R .; Yvoré, P. and Peeters, JE Vet. Parasitol. (1993) 45: 199-207; Mancassola, R .; Richard, A. andΝaciri, M. Vet. Parasitol. (1997) 69: 31-37; Causapé, AC; Sánchez-Acedo, C; Quílez, J .; del Cacho, E. and Viu, M. Res. Rev. Parasitol. (1999) 59: 41-46; Viu, M .; Quílez, J .; Sánchez-Acedo, C; del Cacho, E. and López- Bernad, F. Vet. Parasitol. (2000) 90: 283-287). Of these and exclusively veterinary parauso, halofuginone lactate has been marketed under the name of Halocur®, formulated as an oral solution (0.5 mg / mL) and indicated for the prevention and reduction of diarrhea caused by C. parvum in fresh calves born, and must be administered once a day and at the same time for 7 consecutive days.
En el hombre, y debido a que el curso clínico de la cryptosporidiosis depende fundamentalmente del estado inmunitario, las opciones terapéuticas varían considerablemente. En adultos inmunocompetentes y en niños, la infección suele ser autolimitada por lo que no está indicado tratamiento. Sin embargo, al tratarse de una enfermedad diarreica se aconseja la hidratación. En enfermos de SIDA, en los que la infección puede cronificarse, el tratamiento ideal implica la restauración parcial de la función inmune con terapia antirretroviral. Palella, FJ. etal. NewEng. J. Med. (1998) 338:853-860, comprobaron que la morbilidad y mortalidad por infecciones oportunistas han decrecido enormemente con el empleo de esta terapia. Incluso varios estudios demostraron un descenso en la prevalencia de la cryptosporidiosis subsecuente a la aplicación de este tratamiento (Kim, L.S. et al. Clin. Infec. Dis. (1998) 27:655-656; Lemoing, V. et al. AIDS (1998) 12: 1395-1397). Además, varios trabajos han demostrado la resolución de la diarrea por Cryptosporidium coincidiendo con un aumento en el número de linfocitos T CD4 paralelo a la administración de la terapia antirretroviral triple (Carr, A. et al. Lancet (1998) 351:256-261; Foudraine, Ν.A. eí al. AIDS (1998) 12:35-41). Si este tratamiento antirretroviral no es posible, pueden ser considerados varios antibióticos que demostraron una cierta eficacia frente a Cryptosporidium, como la paromomicina, la nitazoxanida y la azitromicina. De ellos, la paromomicina es la más ampliamente empleada y presenta cierta actividad en modelos experimentales y ensayos clínicos (Fichtenbaum, CJ. et al. Clin. Infec. Dis. (1993)16:298-300; Flanigan, T.P. etal. Am.In man, and because the clinical course of cryptosporidiosis depends fundamentally on the immune status, the therapeutic options vary considerably. In immunocompetent adults and children, the infection is usually self-limited, so no treatment is indicated. However, being a diarrheal disease, hydration is advised. In AIDS patients, in whom the infection may become chronic, the ideal treatment involves partial restoration of immune function with antiretroviral therapy. Palella, FJ. etal. NewEng J. Med. (1998) 338: 853-860, found that morbidity and mortality from opportunistic infections have decreased greatly with the use of this therapy. Even several studies demonstrated a decrease in the prevalence of cryptosporidiosis subsequent to the application of this treatment (Kim, LS et al. Clin. Infec. Dis. (1998) 27: 655-656; Lemoing, V. et al. AIDS ( 1998) 12: 1395-1397). In addition, several studies have demonstrated the resolution of Cryptosporidium diarrhea coinciding with an increase in the number of CD4 T lymphocytes parallel to the administration of triple antiretroviral therapy (Carr, A. et al. Lancet (1998) 351: 256-261 ; Foudraine, Ν.A. and al. AIDS (1998) 12: 35-41). If this antiretroviral treatment is not possible, several antibiotics that demonstrated a certain efficacy against Cryptosporidium, such as paromomycin, nitazoxanide and azithromycin, can be considered. Of these, paromomycin is the most widely used and has some activity in experimental models and clinical trials (Fichtenbaum, CJ. Et al. Clin. Infec. Dis. (1993) 16: 298-300; Flanigan, TP etal. Am.
J. Med. (1996) 100:370-372). Una combmaciónparomomicina-azitromicina ha sido propuesta por Smith, N.H. et al. Infec. Dis. (1998) 178:900-903) pudiendo ser consideradas terapias experimentales (Crabb, J.H. Adv. Parásito! (1998) 40:121-149.J. Med. (1996) 100: 370-372). A combination paromomycin-azithromycin has been proposed by Smith, N.H. et al. Infec Dis. (1998) 178: 900-903) and can be considered experimental therapies (Crabb, J.H. Adv. Parasite! (1998) 40: 121-149.
La nitazoxanida es uno de los últimos fármacos evaluados frente a la cryptosporidiosis humana. Es un nitrotiazol benzamida con amplia actividad frente a protozoos, helmintos y bacterias. En un estudio realizado en México, en 15 enfermos de SIDA con cryptosporidiosis, se observó la resolución de la infección en la totalidad de los enfermos tras una pauta de 1-2 g/día administrada durante 10-30 días (Feregrino, G.M. et al. XI Internetional Conference on AIDS. (1996). Vancover, Canadá. Resultados similares fueron obtenidos en un estudio llevado a cabo en África (Doumbo, O. et al. Am. J. Trop. Med. Hyg. (1997) 56: 637-639.Nitazoxanide is one of the last drugs evaluated against human cryptosporidiosis. It is a nitrotiazole benzamide with extensive activity against protozoa, helminths and bacteria. In a study conducted in Mexico, in 15 AIDS patients with cryptosporidiosis, the resolution of the infection was observed in all patients after a 1-2 g / day regimen administered for 10-30 days (Feregrino, GM et al XI Internetional Conference on AIDS. (1996) Vancover, Canada Similar results were obtained in a study conducted in Africa (Doumbo, O. et al. Am. J. Trop. Med. Hyg. (1997) 56: 637-639.
En el caso de algunos fármacos ensayados en el tratamiento de la cryptosporidiosis humana y animal, hemos podido detectar ciertas características físico-químicas que nos inducen a plantear que una mejora en sus propiedades biofarmacéuticas repercutiría favorablemente en su eficacia. Algunos de los problemas que frecuentemente presentan los fármacos son su baja solubilidad y estabilidad. El descubrimiento de las ciclodextrinas supuso un avance notable en la solución de estos problemas.In the case of some drugs tested in the treatment of human and animal cryptosporidiosis, we have been able to detect certain physicochemical characteristics that lead us to suggest that an improvement in their biopharmaceutical properties would have a favorable impact on their efficacy. Some of the problems that drugs frequently present are their low solubility and stability. The discovery of cyclodextrins was a remarkable advance in the solution of these problems.
La formación de complejos de inclusión con ciclodextrinas permite mejorar la solubilidad de los principios activos y por consiguiente su paso a través de membranas cuando la solubilidad es un factor limitante de la misma. Por otra parte, al mejorar la solubilidad del principio activo, aumenta la fracción que difunde, con lo cual se puede disminuir la dosis de administración y lograr un mismo efecto terapéutico, lo que supone una ventaja adicional de la disminución de los efectos secundarios como consecuencia de las altas dosis administradas.The formation of inclusion complexes with cyclodextrins makes it possible to improve the solubility of the active ingredients and therefore their passage through membranes when the solubility is a limiting factor thereof. On the other hand, by improving the solubility of the active substance, the diffusion fraction increases, which can reduce the administration dose and achieve the same therapeutic effect, which represents an additional advantage of the reduction of side effects as a consequence of the high doses administered.
Algunos principios activos con relativa eficacia en el tratamiento de la cryptosporidiosis humana y/o animal presentan escasa solubilidad (espiramicina, furoato de diloxanida, furazolidona, halofuginona, entre otros) o problemas de estabilidad (como por ejemplo la paromomicina) y su eficacia puede ser mejorada formulándose como complejos de inclusión con ciclodextrinas.Some active ingredients with relative efficacy in the treatment of human and / or animal cryptosporidiosis have poor solubility (spiramycin, diloxanide furoate, furazolidone, halofuginone, among others) or stability problems (such as paromomycin) and their effectiveness may be enhanced by formulating as inclusion complexes with cyclodextrins.
Investigamos la aplicación tanto in vitro como in vivo de nuevas formulaciones farmacéuticas con complejos de inclusión con β-ciclodextrina en el tratamiento de la cryptosporidiosis. Los principios activos, furoato de diloxanida, un derivado furánico sintéticoWe investigated the application both in vitro and in vivo of new pharmaceutical formulations with inclusion complexes with β-cyclodextrin in the treatment of cryptosporidiosis. The active substances, diloxanide furoate, a synthetic fury derivative
(G-l), lactato de halofαginona y paromomicina entre otros, se seleccionaron por su potencial actividad anticryptosporidial. De entre los fármacos ensayados algunos formaron complejos de inclusión (por ejemplo furoato de diloxanida y G-l) mientras que otros fueron incapaces, debido a su estructura molecular, de incorporarse a la cavidad de la β-ciclodextrina. La caracterización de los diferentes complejos empleando técnicas instrumentales adecuadas demostró la formación de complejos estables en estado sólido y en disolución, mejorando propiedades como la solubilidad y velocidad de disolución de los fármacos, e incluso la estabilidad en productos como el G-l, que se degrada en disoluciones acuosas o sufre problemas de sublimación.(G-l), halofαginone lactate and paromomycin among others, were selected for their potential anti-cryptosporidial activity. Among the drugs tested, some formed inclusion complexes (for example diloxanide furoate and G-1) while others were unable, due to their molecular structure, to enter the cavity of β-cyclodextrin. The characterization of the different complexes using suitable instrumental techniques demonstrated the formation of stable complexes in solid state and in solution, improving properties such as the solubility and dissolution rate of the drugs, and even the stability in products such as Gl, which is degraded in Aqueous solutions or suffer sublimation problems.
Para evaluar la actividad in vitro, ooquistes purificados de C. parvum se sometieron durante diferentes tiempos de exposición (30, 60, 90 y 120 minutos) a la acción, en solución acuosa, de los principios activos seleccionados: furoato de diloxanida (10 mg/kg peso vivo) y derivado furánico G-l (20 mg/kg peso' vivo) y a dos complejos de inclusión: furoato de diloxanida: β-ciclodextrina (1:1) y derivado furánico G-l: β-ciclodextrina (2,5%). Paralelamente, se realizaron controles con agua destilada y β-ciclodextrina (2,5%).To evaluate in vitro activity, purified C. parvum oocysts were subjected to different active times (30, 60, 90 and 120 minutes) to the action, in aqueous solution, of the selected active ingredients: diloxanide furoate (10 mg / kg live weight) and Flanic derivative Gl (20 mg / kg ' live weight) and two inclusion complexes: diloxanide furoate: β-cyclodextrin (1: 1) and Flanic derivative Gl: β-cyclodextrin (2.5%) . In parallel, controls were carried out with distilled water and β-cyclodextrin (2.5%).
La viabilidad ooquística se determinó mediante técnicas de desenquistamiento y de inclusión/exclusión de colorantes vitales fluorogénicos. Los resultados obtenidos con ambos principios activos demostraron que a medida que aumenta el tiempo de exposición, disminuye el porcentaje de ooquistes desenquistados (ooquistes vacíos detectados por contraste de fases) y aumenta el número de ooquistes inviables (ooquistes que incorporan el colorante vital yoduro de propidio). Se comprobó que su formulación como complejos de inclusión mejora su actividad.The oocystic viability was determined by release techniques and inclusion / exclusion of fluorogenic vital dyes. The results obtained with both active ingredients showed that as the exposure time increases, the percentage of unlocked oocysts (empty oocysts detected by phase contrast) decreases and the number of unviable oocysts (oocysts incorporating the vital dye propidium iodide) increases. ). It was found that its formulation as inclusion complexes improves its activity.
Sorprendentemente, el control de la β-ciclodextrina (2,5%) mostró un efecto importante sobre la viabilidad ooquística, observado tan sólo transcurridos los primeros 30 minutos de exposición. Esta inesperada acción también se detectó a los distintos tiempos, obteniendo unos resultados al final del ensayo, que demuestran una mayor actividad de la β- ciclodextrina que del furoato de diloxanida y de su complejo de inclusión, y ligeramente inferiores a los obtenidos con el derivado furánico. Además, la acción de la β-ciclodextrina llegó a ser superior a la causada por ambos principios activos durante los primeros 90 minutos, y solamente fue superada cuando el derivado furánico se formuló con β -ciclodextrina.Surprisingly, the control of β-cyclodextrin (2.5%) showed an important effect on oocystic viability, observed only after the first 30 minutes of exposure. This unexpected action was also detected at different times, obtaining results at the end of the test, which demonstrate a greater activity of β-cyclodextrin than of diloxanide furoate and its inclusion complex, and slightly lower than those obtained with the derivative furious In addition, the action of β-cyclodextrin became superior to that caused by both active ingredients during the first 90 minutes, and was only overcome when the fury derivative was formulated with β-cyclodextrin.
Posteriormente, mediante bioensayos en ratones lactantes Swiss CD-1, se evaluáronlas eficacias profiláctica y terapéutica del furoato de diloxanida y su correspondiente complejo de inclusión formulado en la proporción mo mol para la dosis de 10 mg/kg peso vivo, y del derivado furánico G-l y sus correspondientes complejos de inclusión formulados en la proporción mohmol y con un exceso de β-ciclodextrina (2,5%), ensayados para las dosis deSubsequently, by bioassays in Swiss CD-1 lactating mice, the prophylactic and therapeutic efficacy of diloxanide furoate and its corresponding inclusion complex formulated in the mole ratio for the dose of 10 mg / kg body weight and of the live weight were evaluated. Flanic derivative Gl and its corresponding inclusion complexes formulated in the mohmol ratio and with an excess of β-cyclodextrin (2.5%), tested for the doses of
10, 20, 25 y 30 mg/kg peso vivo de principio activo. La determinación de la eficacia se realizó estableciendo el porcentaje de animales infectados y la intensidad de infección a los 7 días postinfección mediante examen de la totalidad del contenido intestinal homogeneizado. Se comprobó, bajo ambos regímenes de tratamiento, una reducción significativa en la intensidad de parasitación tanto con los principios activos como con sus correspondientes complejos de inclusión respecto a las carnadas control. De nuevo, el control realizado con β-ciclodextrina ofreció resultados inesperados, por lo que, se evaluaron ambas eficacias profiláctica y terapéutica de una suspensión acuosa de β-ciclodextrina ( 2,5%> ) administrada tanto en una única dosis como en dosis múltiples. Se consiguió erradicar la infección cuando se administró β-ciclodextrina dos horas antes de provocar la infección y los días 1 y 2 postinfección cada 12 horas (Tabla 1).10, 20, 25 and 30 mg / kg live weight of active substance. The determination of efficacy was made by establishing the percentage of infected animals and the intensity of infection at 7 days post-infection by examining the entire homogenized intestinal content. Under both treatment regimes, a significant reduction in the intensity of parasitization was verified both with the active ingredients and with their corresponding inclusion complexes with respect to the control baits. Again, the control performed with β-cyclodextrin offered unexpected results, therefore, both prophylactic and therapeutic efficacy of an aqueous suspension of β-cyclodextrin (2.5%>) administered both in a single dose and in multiple doses were evaluated. . The infection was eradicated when β-cyclodextrin was administered two hours before causing the infection and on days 1 and 2 post-infection every 12 hours (Table 1).
Tabla 1. Eficacias profiláctica y terapéutica de la β-ciclodextrina evaluadas en ratones lactantes infectados con 2,5 x 104 ooquistes de C. parvum. Ensayo n° Intensidad de infección* Ratones infectados (%)Table 1. Prophylactic and therapeutic efficacy of β-cyclodextrin evaluated in lactating mice infected with 2.5 x 4 4 oocysts of C. parvum. Test n ° Intensity of infection * Infected mice (%)
1 (control infección) 2,6 ± 1,6 1001 (infection control) 2.6 ± 1.6 100
2 (eficacia profiláctica: monodosis) 0,3 ± 0,1 1002 (prophylactic efficacy: single dose) 0.3 ± 0.1 100
3 (eficacia terapéutica: monodosis) 0,2 ± 0,1 1003 (therapeutic efficacy: single dose) 0.2 ± 0.1 100
4 (eficacia profiláctica: dosis múltiples) 0, 1 ± 0, 1 1004 (prophylactic efficacy: multiple doses) 0, 1 ± 0, 1 100
5 (eficacia profiláctica: dosis múltiples) 0,0 ± 0,0 0,05 (prophylactic efficacy: multiple doses) 0.0 ± 0.0 0.0
6 (eficacia terapéutica: dosis múltiples) - 0,1 ± 0,0 33,36 (therapeutic efficacy: multiple doses) - 0.1 ± 0.0 33.3
7 (eficacia terapéutica: dosis múltiples) 0,1 ± 0,0 6,27 (therapeutic efficacy: multiple doses) 0.1 ± 0.0 6.2
* media ± DE x 106 ooquistes/totalidad del intestino homogeneizado.* mean ± SD x 10 6 oocysts / total homogenized intestine.
Ensayo 4: dosis cada 12 horas; ensayo 5: dos horas antes de provocar la infección y dosis cadaTrial 4: dose every 12 hours; trial 5: two hours before causing infection and dose every
12 horas; ensayo 6: dosis cada 12 horas; ensayo 7: dosis cada 8 horas.12 hours; trial 6: dose every 12 hours; trial 7: dose every 8 hours.
Por otra parte, se evaluaron las eficacias profiláctica y terapéutica de la β-ciclodextrina frente a la cryptosporidiosis en rumiantes domésticos. Para ello, corderos recién nacidos se infectaron experimentalmente el primer día de vida con lxlO6 ooquistes de C. parvum de origen ovino. La administración de β-ciclodextrina se realizó por vía oral y en forma de suspensión acuosa siendo la dosis de 1 g/kg peso vivo. En los ensayos de eficacia profiláctica, los corderos se trataron durante los tres primeros días de vida, y en el ensayo terapéutico la β- ciclodextrina se administró, con igual pauta, a partir del inicio del proceso diarreico y confirmación de la presencia de ooquistes en las heces. La eficacia se determinó teniendo en cuenta la eliminación ooquística, la presencia de diarrea y la ganancia de peso corporal los días 15 y 30 de vida de los animales. Los resultados (Gráfico 1) demostraron que la β-ciclodextrina fue altamente efectiva como profiláctico al impedir la aparición de procesos diarreicos en los corderos infectados y evitar la infección en uno de ellos, así como al disminuir notablemente la intensidad y duración del período de patencia. Administrada terapéuticamente, disminuyó la gravedad del proceso diarreico y acortó el período de excreción ooquística. Los animales tratados aumentaron significativamente su peso corporal y no se observaron toxicidad aparente ni reinfecciones.On the other hand, the prophylactic and therapeutic efficacy of β-cyclodextrin against cryptosporidiosis in domestic ruminants was evaluated. For this, newborn lambs were experimentally infected on the first day of life with 10 6 oocysts of C. parvum of sheep origin. The administration of β-cyclodextrin was performed orally and in the form of an aqueous suspension, the dose being 1 g / kg body weight. In the prophylactic efficacy trials, the lambs were treated during the first three days of life, and in the therapeutic trial the β-cyclodextrin was administered, with the same pattern, from the start of the diarrheal process and confirmation of the presence of oocysts in the stool The efficacy was determined taking into account oocystic elimination, the presence of diarrhea and body weight gain on days 15 and 30 of animal life. The results (Graph 1) showed that β-cyclodextrin was highly effective as a prophylactic by preventing the onset of diarrheal processes in infected lambs and preventing infection in one of them, as well as significantly decreasing the intensity and duration of the patent period . Therapeutically administered, it reduced the severity of the diarrheal process and shortened the period of oocystic excretion. The treated animals significantly increased their body weight and no apparent toxicity or reinfections were observed.
Aunque desconocemos el mecanismo de acción por el cual la β-ciclodextrina impide la implantación de la infección, la literatura consultada y ciertas observaciones realizadas, nos conducen a plantear posibles mecanismos de acción. Así, es conocido el hecho de que las ciclodextrinas provocan la lisis de los eritrocitos al disolver, y por tanto eliminar, componentes de su membrana como el colesterol y ciertos fosfolípidos, la fosfatidilcolina y la esfingomielina (Ohtani, Y. etal. J. Europ. J. Biochem. (1989) 186: 17-22; Arima, H. etal. V International Symposium on Cyclodextrin. (1990). París, Francia); compuestos lipidíeos detectados en la pared de los ooquistes de C. parvum (Mitschler, R.R. et al. J. Eukar. Microbiol. (1994) 41: 8-12). Cuando nosotros observamos microscópicamente, y utilizando una solución de verde malaquita, ooquistes de C. parvum expuestos a la acción in vitro de la β- ciclodextrina, pudimos comprobar que algunos incorporaban el colorante, lo que indica una alteración en la permeabilidad de la pared ooquística. Además, los resultados de los ensayos de viabilidad demostraron que estos ooquistes son permeables al colorante ioduro de propidio. Ciertos autores (Horan, P.K. y Kappler, J.W. J. Inmuno!. (1977) 18: 309-316) indican que sólo las células que presentan membranas alteradas o rotas, pueden ser teñidas por este colorante vital. Nosotros sospechamos que la β-ciclodextrina modifica, quizá al captar algunos de estos lípidos, la integridad de la pared ooquística que sirve de barrera protectora a los esporozoítos invasivos.Although we do not know the mechanism of action by which β-cyclodextrin prevents the implantation of the infection, the literature consulted and certain observations made, lead us to propose possible mechanisms of action. Thus, it is known that cyclodextrins cause lysis of erythrocytes when dissolved, and therefore eliminate, components of their membrane such as cholesterol and certain phospholipids, phosphatidylcholine and sphingomyelin (Ohtani, Y. etal. J. Europ J. Biochem. (1989) 186: 17-22; Arima, H. et al. V International Symposium on Cyclodextrin. (1990). Paris, France); lipid compounds detected on the wall of the oocysts of C. parvum (Mitschler, R.R. et al. J. Eukar. Microbiol. (1994) 41: 8-12). When we observed microscopically, and using a solution of malachite green, C. parvum oocysts exposed to the in vitro action of β-cyclodextrin, we could see that some incorporated the dye, which indicates an alteration in the permeability of the oocystic wall . In addition, the results of the feasibility tests demonstrated that these oocysts are permeable to the propidium iodide dye. Certain authors (Horan, P.K. and Kappler, J.W. J. Immuno !. (1977) 18: 309-316) indicate that only cells that have altered or broken membranes can be stained by this vital dye. We suspect that β-cyclodextrin modifies, perhaps by capturing some of these lipids, the integrity of the oocystic wall that serves as a protective barrier to invasive sporozoites.
Por otra parte, Comini, S.; Olivier, P.; Riottot, M. and Duhamel, D. Clin.Chim. Acta (1994) 228: 94-181, demostraron la afinidad de la β-ciclodextrina por el ácido taurocólico, empleado en nuestros ensayos in vitro, como su sal sódica en la técnica de desenquistamiento. Recientemente, Gold, D.; Stein, B. and Tzipori, S. J. Parásito!. (2001) 87: 997-1000, observaron que la presencia de esta sal biliar en cultivos celulares estimula la liberación de los esporozoítos. Tal vez, la formación de los correspondientes complejos de inclusión (ácido taurocólico: β-ciclodextrina) justifiquen el descenso en el porcentaje de ooquistes desenquistados tras su exposición in vitro a la acción de la β-ciclodextrina, respecto al control con agua destilada. Además, hemos de comentar que Harp, J.A. J. Parasitol. (1999) 85: 952-955, comprobó que ciertos azúcares, como la sacarosa y en menor medida la isomaltosa, reducen significativamente la intensidad de infección por C. parvum en ratones lactantes y que este efecto depende de la presencia y concentración de los azúcares en el intestino del hospedador. Así, el autor comprobó mediante exámenes histológicos una hipervacuolización de los enterocitos del íleon transcurridas 24 horas del tratamiento con sacarosa, sugiriendo que este azúcar interacciona con el epitelio intestinal al inducir cambios osmóticos, haciendo que las células sean menos susceptibles a la infección por el parásito. En nuestro laboratorio realizamos estudios histológicos de intestino de ratones experimentalmente infectados y tratados con β-ciclodextrina, administrada a la misma dosis y pauta de tratamiento con la que se logró erradicar la infección y no observamos cambios morfológicos en las células epiteliales con respecto a los correspondientes estudios realizados con animales que recibieron agua destilada.On the other hand, Comini, S .; Olivier, P .; Riottot, M. and Duhamel, D. Clin.Chim. Acta (1994) 228: 94-181, demonstrated the affinity of β-cyclodextrin for taurocolic acid, used in our in vitro assays, as its sodium salt in the release technique. Recently, Gold, D .; Stein, B. and Tzipori, SJ Parasite! (2001) 87: 997-1000, observed that the presence of this bile salt in cell cultures stimulates the release of sporozoites. Perhaps, the formation of the corresponding inclusion complexes (taurocolic acid: β-cyclodextrin) justify the decrease in the percentage of oocysts released after their in vitro exposure to the action of β-cyclodextrin, with respect to control with distilled water. In addition, we have to comment that Harp, JAJ Parasitol. (1999) 85: 952-955, found that certain sugars, such as sucrose and to a lesser extent isomalt, significantly reduce the intensity of C. parvum infection in lactating mice and that this effect depends on the presence and concentration of sugars in the intestine of the host. Thus, the author verified by histological examinations a hypervacuolization of the enterocytes of the ileum after 24 hours of treatment with sucrose, suggesting that this sugar interacts with the intestinal epithelium by inducing osmotic changes, making the cells less susceptible to infection by the parasite . In our laboratory we perform histological studies of the intestine of mice experimentally infected and treated with β-cyclodextrin, administered at the same dose and treatment schedule with which the infection was eradicated and we did not observe morphological changes in epithelial cells with respect to the corresponding ones studies conducted with animals that received distilled water.
Finalmente, en estudios electroforéticos llevados a cabo en nuestro laboratorio con extractos solubles de ooquistes de C. parvum sometidos, previamente y durante 24 horas a la acción de la β-ciclodextrina, revelaron unos patrones polipeptídicos en los que se detectó la ausencia de la fracción proteica comprendida entre 15,7-17,5 kDa, considerada como altamente inmunogénica. Así, Tilley, M.; Fayer, R.; Guidry, A.; Upton, SJ. and Blagburn, B.L. Infecí. Immuno!. (1990) 58: 2966-2971, demostraron la presencia de una glucoproteína de 14,5-16,5 kDa en las superficie de los esporozoítos que fue uno de los principales antígenos reconocidos por el calostro bovino hiperinmune. Estudios posteriores demostraron que en ratones lactantes experimentalmente infectados, el tratamiento con anticuerpos monoclonales frente a esta glucoproteína, incluso a dosis bajas, redujo significativamente el número de estados de desarrollo endógeno del parásito en el tracto intestinal y disminuye la producción ooquística (Tilley, M.; Upton, S.J.; Fayer, R.; Barta, J.R.; Chrisp, CE.; Freed, P.S.; Blagburn, B.L.; Anderson, B.C. and Barnard, S.M. Infecí. Immuno!. (1991) 59: 1002-1007.Finally, in electrophoretic studies carried out in our laboratory with soluble extracts of C. parvum oocysts submitted, before and for 24 hours to the action of β-cyclodextrin, revealed some polypeptide patterns in which the absence of the fraction was detected protein between 15.7-17.5 kDa, considered as highly immunogenic. Thus, Tilley, M .; Fayer, R .; Guidry, A .; Upton, SJ. and Blagburn, B.L. I infected. Immuno! (1990) 58: 2966-2971, demonstrated the presence of a 14.5-16.5 kDa glycoprotein on the surface of the sporozoites that was one of the main antigens recognized by hyperimmune bovine colostrum. Later studies showed that in experimentally infected lactating mice, treatment with monoclonal antibodies against this glycoprotein, even at low doses, significantly reduced the number of endogenous development states of the parasite in the intestinal tract and decreases oocystic production (Tilley, M. ; Upton, SJ; Fayer, R .; Barta, JR; Chrisp, CE .; Freed, PS; Blagburn, BL; Anderson, BC and Barnard, SM Infeci. Immuno !. (1991) 59: 1002-1007.
Gráfico 1. Representación gráfica del número de corderos con diarrea y de la intensidad de parasitación por C. parvum en el grupo control experimental no tratado y en los grupos tratados con β-ciclodextrina Grupo control experimental no tratadoGraph 1. Graphical representation of the number of lambs with diarrhea and the intensity of parasitization by C. parvum in the untreated experimental control group and in the groups treated with β-cyclodextrin Untreated experimental control group
Figure imgf000009_0004
Figure imgf000009_0001
Figure imgf000009_0004
Figure imgf000009_0001
Días de eliminación ooquísticaDays of oocystic elimination
Grupo profilácticoProphylactic group
Figure imgf000009_0002
Figure imgf000009_0002
D2 D3 D4 D5 D6 D7 D8 D9 DIO Dll D12 D13 D15 D17 D19 D21 D23D2 D3 D4 D5 D6 D7 D8 D9 DIO Dll D12 D13 D15 D17 D19 D21 D23
Días de eliminación ooquísticaDays of oocystic elimination
Grupo terapéuticoTherapeutic group
Figure imgf000009_0003
Figure imgf000009_0003
Días de eliminación ooquísticaDays of oocystic elimination
La β-ciclodextrina administrada por vía oral no es tóxica ya que no se absorbe a través del tracto gastrointestinal, impidiéndose así su acumulación en órganos ( e, T. and Uekama, K. J. Pharmac. Science. (1997) 86: 147-162). Estudios de toxicidad demostraron que, vía oral, los valores de DL50 superan a 12,5; 18,5 y 5 g/kg peso vivo para ratones, ratas y perros respectivamente (Szejtli, J. Cyclodextrins in Technology. (1988). J. Szejli (Ed). Kluwer Academic Publishers, Dordrecht, Holanda). Tampoco se observaron efectos tóxicos en ratas cuya dieta fue suplementada en un 10% con β-ciclodextrina durante 90 días (Irie, T. and Uekama, K. J. Pharmac. Science. (1997) 86: 147-162). Por otra parte, la β-ciclodextrina, además de ser un producto biodegradable, se utiliza en acuicultura para la elaboración de formulaciones farmacéuticas destinadas al tratamiento de peces de cultivo. (Luzardo-Alvarez,The orally administered β-cyclodextrin is not toxic since it is not absorbed through the gastrointestinal tract, thus preventing its accumulation in organs (e, T. and Uekama, KJ Pharmac. Science. (1997) 86: 147-162) . Toxicity studies showed that, orally, LD 50 values exceed 12.5; 18.5 and 5 g / kg live weight for mice, rats and dogs respectively (Szejtli, J. Cyclodextrins in Technology. (1988). J. Szejli (Ed.) Kluwer Academic Publishers, Dordrecht, The Netherlands). Nor were toxic effects observed in rats whose diet was supplemented by 10% with β-cyclodextrin for 90 days (Irie, T. and Uekama, KJ Pharmac. Science. (1997) 86: 147-162). On the other hand, β-cyclodextrin, In addition to being a biodegradable product, it is used in aquaculture for the preparation of pharmaceutical formulations for the treatment of farmed fish. (Luzardo-Alvarez,
A. et al. Aquaculíure 622027 (2002) (en prensa).A. et al. Aquaculíure 622027 (2002) (in press).
Los resultados obtenidos demuestran que la β-ciclodextrina presenta una mayor eficacia que los principios activos ensayados solos o formando complejos de inclusión. The results obtained demonstrate that β-cyclodextrin has a greater efficacy than the active ingredients tested alone or forming inclusion complexes.

Claims

REIVINDICACIONES
1.- Utilización de la β-ciclodextrina en la preparación de medicamentos para el tratamiento de las enfermedades humanas o animales causadas por parásitos pertenecientes al género Crypíosporidium. 1.- Use of β-cyclodextrin in the preparation of medicines for the treatment of human or animal diseases caused by parasites belonging to the genus Crypíosporidium.
2.- Utilización de la β-ciclodextrina en la preparación de medicamentos como coadyuvante o formando complejos de inclusión, como agente profiláctico o terapéutico, para el tratamiento de las enfermedades humanas o animales causadas por parásitos pertenecientes al género Crypíosporidium.2.- Use of β-cyclodextrin in the preparation of drugs as an adjuvant or forming inclusion complexes, as a prophylactic or therapeutic agent, for the treatment of human or animal diseases caused by parasites belonging to the genus Crypíosporidium.
3.- Utilización de la β-ciclodextrina en la preparación de medicamentos, según las reivindicaciones anteriores, caracterizado porque actúa como agente reductor de la viabilidad y capacidad infectante de ooquistes de parásitos pertenecientes al género Cryptosporidium presentes en medios hídricos, como aguas residuales y purines entre otros.3. Use of β-cyclodextrin in the preparation of medicaments, according to the preceding claims, characterized in that it acts as a reducing agent of the viability and infective capacity of oocysts of parasites belonging to the genus Cryptosporidium present in water media, such as wastewater and slurry. among others.
A.- Utilización de la β-ciclodextrina en la preparación de medicamentos, según las reivindicaciones anteriores, en cualquier clase de formas de administración: disuelta o dispersada en agua, mezclada con el pienso, en formas sólidas como polvos, pellets, cápsulas, o comprimidos entre otros. A.- Use of β-cyclodextrin in the preparation of medicaments, according to the preceding claims, in any kind of administration forms: dissolved or dispersed in water, mixed with the feed, in solid forms such as powders, pellets, capsules, or tablets among others.
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