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Abstract
Compound alpha (Ca) is a benzimidazolic derivate which has shown a high fasciolicidal efficacy when it is given by oral via.
Solubilization of Ca would be a suitable alternative to obtain an injectable formulation. The objectives of the present study were
to evaluate the solubility of Ca in order to determine the in vitro fasciolicidal efficacy and experimentally on infected ovines.
The assays of solubility quantified by high-performance liquid chromatography (HPLC), showed that beta-cyclodextrin (-CD)
and hydroxi-propylbeta-cyclodextrin (HP--CD) solubilized in 0.015% and 1.018% respectively. The solubilization for HP--CD
was calculated as 3.95% for 5.8 mg; 10.76% for HP--CD with methanol, 39.048% for proylene-glycol (PG), 100% for glycerol
formal (GF), and 100% for combined PG and GF and water in proportions of 3:2:5 and 3:2:4. In vitro evaluation using immature F.
hepatica showed that Ca induced 100% efficacy 24 hours after solubilization with either GF or GF combined with PG and water.
The efficacy in sheep was 56.4 and 68.4% at 1 and 2 mg/kg respectively; whereas 1 mg/kg of sulphoxide metabolite induced an
efficacy of 73.9%. The HPLC assays performed showed no evidence of the sulphoxide and sulphone metabolites. It is concluded
that besides solubility and high in vitro fasciolicidal efficacy, the in vivo results in sheep showed only a moderate efficacy.
Resumen
El compuesto alfa es un derivado bencimidazlico de gran eficacia fasciolicida, que se ingiere va oral, su solubilizacin constituye
una alternativa para obtener una formulacin inyectable. Los objetivos de este estudio fueron: evaluar la solubilidad del com-
puesto alfa, determinar la eficacia fasciolicida in vitro y en ovinos experimentalmente infectados. Los ensayos de solubilidad que
fueron cuantificados mediante cromatografa de lquidos de alta resolucin, mostraron que la betaciclodextrina e hidroxipropil-
betaciclodextrina solubilizaron en 0.051% y 1.018%. Esta ltima se evalu con cinco concentraciones, mostr que a 5.8 mg la
solubilidad fue 3.95%; hidroxipropil-betaciclodextrina y metanol, 10.76%; propiln-glicol, 39.048%; glicerol formal, 100%; y gli-
cerol formal con propiln- glicol y agua en proporciones 3:2:5 y 3:2:4, 100%. La evaluacin in vitro con fasciolas inmaduras mostr
que el compuesto con glicerol formal y la combinacin de vehculos a las 24 horas fueron eficaces en 100%. La eficacia in vivo en
ovinos mostr que el compuesto alfa a dosis de 1 mg/kg fue de 56.46%, a 2 mg/kg de 68.4% y con sulfxido del compuesto alfa
a 1 mg/kg de 73.9%. No se encontr el compuesto alfa ni sus metabolitos sulfxido y sulfona en el suero determinado por medio
del cromatgrafo de lquidos de alta resolucin. Se concluye que a pesar de obtener solubilidad del compuesto alfa y alta eficacia
fasciolicida in vitro, los resultados in vivo en ovinos revelaron eficacia moderada.
2
Solubility hidroxipropil-alfaciclodextrina (HP--CD), betaci-
clodextrina (-CD), hidroxipropil-betaciclodextrina
The formation of Ca aducts with the CD was carried (HP--CD) y los cosolventes propiln-glicol* (PG)
out using the following procedure: the Ca (2 mg) was y glicerol formal** (GF), que provinieron de fuente
added to an aqueous solution (2 mL/H2O) with each comercial.***
cyclodextrins (3 mg) for triplicated,14 it was shaken
during seven days under environmental temperature Solubilidad
conditions. The CD with the best hydrosolubility
properties was evaluated in the concentrations 5.8, 2.9, Se realiz la formacin de aductos del Ca con las CD
1.45, 0.725, 0.0362 mg with 2 mg of Ca. Other assays con el siguiente procedimiento: el Ca (2 mg) se
were done using HP--CD and methanol (MeOH), it suspendi en una solucin acuosa (2 mL/H2O) con
was evaporated at room temperature and resuspended cada ciclodextrina (3 mg) por triplicado,14 se agit
in water, Ca with PG, CP with GF and the combination durante siete das a temperatura ambiente. La CD que
of cosolvents GF with PG and H2O at a proportion of proporcion mayor hidrosolubilidad se evalu en las
3:2:5 and 3:2:4. A 3.0 mg concentration of HP--CD concentraciones 5.8, 2.9,1.45, 0.725, 0.0362 mg con 2
and 2.0 mg of Ca was used in each assay by triplicated; mg de Ca. Se realizaron otros ensayos utilizando HP--
each sample was filtered through an acetate of cellulose CD y metanol (MeOH), se evapor al medio ambiente
membrane, to be injected in the high-performance y se resuspendi en agua, Ca con PG, CP con GF y
liquid chromatography (HPLC) system. The solubility la combinacin de cosolventes GF con PG y H2O en
was determined in the physical form by the vehicles proporcin 3:2:5 y 3:2:4. Se utiliz una concentracin
transparency with the compound and by the use of de HP--CD 3.0 mg y de Ca 2.0 mg en cada ensayo
HPLC.15-17 por triplicado; cada muestra se filtr a travs de una
membrana de acetato de celulosa, para ser inyectado
Chromatographic conditions en el cromatgrafo de lquidos de alta resolucin
(CLAR). Se determin la solubilidad en forma fsica
A chromatographic system with binary pump, mediante transparencia de los vehculos con el
autosampler and detector of diodes arrangement compuesto y con el uso del CLAR.15-17
(304 nm of wavelength) was used. The movable phase
consisted in methanol (40), water (40) and acetonitrile Condiciones cromatogrficas
(20) in HPLC degree, degasificator in line with helium.
The stationary phase consisted of a C18 column of 80 mm Se utiliz un sistema cromatogrfico con bomba
in length. The suspension for evaluation was filtered with binaria, automuestreador y detector de arreglo de
membrane 0.45-m, 25 L of sample and the detection diodos, con 304 nm de longitud de onda. La fase mvil
limit of the equipment was 50 ng/mL. consisti en metanol (40), agua (40) y acetonitrilo (20)
For the calculation of the compound concentration, en grado CLAR, degasificador en lnea con helio. La
an external standard was used; with the addition method, fase estacionaria consisti en una columna C18 de 80
the percentage of recovery of the analytical technique mm de longitud. La suspensin a evaluar se filtr con
was determined in order to establish the percentage of membrana 0.45 m, se inyectaron 25 L de muestra
recovery of the compound during the extraction.18 y el lmite de deteccin del equipo fue de 50 ng/mL.
Para el clculo de la concentracin del compuesto
In vitro evaluation se emple un estndar externo, con el mtodo de
adicin se determin el porcentaje de recuperacin
Preparation of compounds for de la tcnica analtica para establecer el porcentaje de
chemotherapeutic evaluation recuperacin del compuesto durante la extraccin.18
The technique of artificial excystment was carried out Las metacercarias de Fasciola hepatica fueron producidas
as it was described previously.19 en caracoles Lymnaea humilis a partir de la infeccin
con miracidios de origen bovino obtenidos de un rastro
Operation of the screening assay local.
Statistic analysis Las Fasciolas fueron examinadas los das cero, uno
y cuatro con un microscopio invertido a 40X. La
The obtained data was submitted to an analysis of actividad de los compuestos se midi por comparacin
variance (ANOVA) to determine the differences in de la sobrevivencia de las fasciolas bajo tratamiento
efficacy between concentrations and groups of the respecto de las fasciolas testigo no tratadas. Los
evaluated compounds. The data was analyzed by the procedimientos se realizaron en condiciones aspticas
Nemenyi test using the SAS statistical package.22 con una campana de flujo laminar.
Infection Animales
On day zero, the animals were infected with 250 Se utilizaron 24 ovinos Pelibuey machos de diez meses
4
metacercariae of Fasciola hepatica. The 15 day cysts de edad, libres de fasciolosis, alojados en corrales de
were kept at 5C prior to infection. encierro convencional, alimento balanceado y agua a
libre acceso en la Universidad Autnoma de Quertaro.
Fecal analysis
Infeccin
On days 8, 70 and 90, fecal samples were collected
and they were analyzed in the search of the trematode Previa semana de adaptacin, el da cero los animales
eggs using the sedimentation test. se infectaron con 250 metacercarias de Fasciola hepatica,
que tenan 15 das de enquistadas y conservadas en
Blood samples refrigeracin a 5C.
Four groups of six animal each were selected, each one Muestras de sangre
based on the Fasciola egg counts from samples taken
on day 8. 23 Las muestras de sangre se recolectaron, conservaron y
se procesaron para la extraccin del suero y posterior
Treatments inyeccin en el CLAR.18,22
6
Cuadro 1
PORCENTAJE DE SOLUBILIDAD DEL COMPUESTO ALFA UTILIZANDO
DIFERENTES VEHCULOS
SOLUBILITY PERCENTAGE OF THE COMPOUND ALPHA USING
DIFFERENT VEHICLES
2 Water LDD
2 Alpha cyclodextrin with water 0
2 Hydroxy propyl alpha cyclodextrin with water 0
2 Beta cyclodextrin with water 0.051
2 Hydroxy propyl alpha cyclodextrin with water 1.018
2 Hydroxy propyl alpha cyclodextrin with water 3.95
2 Hydroxy propyl alpha cyclodextrin with methanol plus water 10.76
2 propylene glycol with water 39.048
2 Glycerol formal with water 100
2 Glycerol formal with propylene glycol and water/3:2:5 ml 100
2 Glycerol formal with propylene glycol and water/3:2:4 ml 100
*Mean determinated by high-performance liquid chromatography
LOD = Limit of detection. A concentration below 50 ng/mL is not detected
Cuadro 2
EFICACIA FASCIOLICIDA in vitro DEL COMPUESTO ALFA SOLUBILIZADO
CON DIFERENTES COSOLVENTES
In vitro FASCIOLICIDAL EFFICACY OF THE COMPOUND ALPHA SOLUBILIZED
WITH DIFFERENTS COSOLVENTS
Dose mg/L
10 Efficacy % 50 Efficacy %
Compound/vehicle 24 hours 96 hours 24 hours 96 hours
Ca with hydroxypropyl-betacyclodextrin / 0.5 mg/L 60 60 60 60
Ca with PG 50 60 50 60
Ca with PG 30 40 30 40
Ca with GF 100 100 100 100
Ca with GF 100 100 100 100
Ca with GF combined PG and H2O /3:2:5 100 100 100 100
Ca with GF combined PG and H2O /3:2:5 100 100 100 100
Ca with GF combined PG and H2O /3:2:4 100 100 100 100
Ca with GF combined PG and H2O /3:2:4 100 100 100 100
Triclabendazole with GF 100 100 100 100
GF with H2O / 2 ml 0 0 0 0
PG with H2O / 2 ml 0 0 0 0
Hydroxypropyl-beta-cyclodextrin/ 5.6 mg/L with
0 0 0 0
H2O/ 2 ml
RPMI medium / 1.6 ml 0 0 0 0
Ca = Compound alpha PG= Propylen glycol RPMI= Culture medium
GF = Glycerol formal H2O = Water
Fasciolas per ovine between treatments but it was with del tratamiento no fue estadsticamente significativa
respect to the control group (P < 0.05). entre tratamientos (P > 0.05).
Even when the mean size (length) of the Fasciolas En la reduccin de Fasciolas adultas se hall que el
from the groups varied, the difference was only numeric. nmero promedio de Fasciolas por grupo fue: Grupo
The efficay percentage was: Group 1, 51.48%; Group 1, 36.29; Grupo 2, 37.79; Grupo 3, 33.96; Grupo 4,
2, 49.58%; Group 3, 54.6%. The analysis of the mean 74.96. El anlisis del nmero de fasciolas por ovino
length of the Fasciolas shows no significant difference, muestra que no hay diferencia significativa, por lo cual
so there are differences between the treatments and no hay diferencias entre los tratamientos pero s entre
the control group (P < 0.05). stos y el testigo (P < 0.05).
The Ca kinetic determinated by HPLC showed En el tamao promedio (longitud) de las Fasciolas de
that in all serum samples no Ca was detected or its los grupos, aunque vari, slo hubo diferencia numrica.
sulphoxide and sulphone metabolites, the limit of El porcentaje de eficacia fue: Grupo 1, 51.48%; Grupo
detection of the equipment was 2ng/mL. 2, 49.58%; Grupo 3, 54.6%. El anlisis de la longitud
total promedio de las Fasciolas muestra que no hay
Discussion diferencia significativa, por lo cual hay diferencias entre
los tratamientos y con el testigo (P < 0.05).
Solubility of Ca La cintica del Ca determinado por el CLAR mostr
que en todas las muestras de suero no se encontr el
The technique to determine the solubility of Ca was Ca ni sus metabolitos sulfxido y sulfona, el lmite de
validated in the HPLC, demonstrating that it is a deteccin del equipo fue de 2 ng/mL.
suitable method to separate components in a mixture
and is very specific for the recognition of one or several Discusin
analites of interest; in addition, it allows an excellent
separation of the individual components to recognize Solubilidad del compuesto alfa
them easily.14,26,27
The hidrosolubility of HP--CD detected by the La tcnica para determinar la solubilidad del compuesto
HPLC is due, in general, to the stechiometry of the alfa fue validada en el cromatgrafo de lquidos de alta
majority of the complexes formed with CD is in resolucin, por lo cual se demuestra que se trata de un
relation 1:1,28 the increase of solubility joined to the mtodo adecuado para separar componentes en una
increase of HP--CD concentration is the result of a mezcla y es muy especfico para el reconocimiento de
greater amount of HP--CD molecules than that of Ca, uno o varios analitos de inters; adems, permite una
which favors the formation of aducts between both excelente separacin de los componentes individuales
compounds. para reconocerlos fcilmente.14,26,27
8
The HP--CD with methanol improved the solubility La hidrosolubilidad de la HP--CD detectada por
of the compound alpha in 10.95%, the obtained el CLAR se debe a que, en general, la estequiometra
solubility was low due to the size of the cyclodextrin de la mayora de los complejos formados con CD es
cavity, 29 its low solubility in water of 18.5 g/L at 25C17 en relacin 1:1,28 el aumento de solubilidad aunado a
for pharmaceutical compounds with molecular weights incremento de la concentracin HP--CD es resultado
between 200 and 800 g/mol30 and aqueous solubility de mayor cantidad de molculas de HP--CD que de
of 60% (w/v), 27 joined to the molecular weight of Ca Ca, lo que favorece la formacin de aductos entre los
and the stechiometry of the majority of the complexes dos compuestos.
formed with CD, that is in relation 1:1, which decreases La HP--CD con metanol mejor la solubilidad del
the formation of aducts. This result contrasts with the compuesto alfa en 10.95%, la solubilidad obtenida fue
previous observations where HP--CD increased the baja debido al tamao de la cavidad de la ciclodextrina,29
water solubility of albendazole. 14,26 su solubilidad baja en agua de 18.5 g/L a 25C17 para
The PG did not show a suitable solubility due to the compuestos farmacuticos con pesos moleculares
vehicle density, but its use with HP--CD increases the entre 200 y 800 g/mol30 y una solubilidad acuosa del
solubility of compounds. 31 60% (w/v),27 aunado al peso molecular del Ca y la
With GF and the combinations of vehicles a 100% estequiometra de la mayora de los complejos formados
of concentration was obtained; because it is a binary con CD, que es en relacin 1:1, lo cual disminuye la
solvent that solubilizes a great variety of hydrophobic formacin de aductos. Este resultado contrasta con las
and hydrophilic compounds, uses cosolvency to observaciones previas en donde la HP--CD aument la
increase the compounds solubility to an acceptable solubilidad en agua del albendazol.14,26
concentration for intramuscular formulations and El PG no mostr una adecuada solubilidad debido
improves the bioavailability of the little polar active quiz a la densidad del vehculo, pero su uso con HP-
principles and also can be used in high doses of more -CD aumenta la solubilidad de compuestos.31
than 4 000 mg/kg in rats. 31-33 Con el GF y las combinaciones de vehculos se obtuvo
The suitable solubility obtained by the vehicles 100% de concentracin; pues es un solvente binario que
cosolvence has been used successfully in the solubiliza gran variedad de compuestos hidrofbicos
formulation of ivermectins and rifampicin.34,35 e hidroflicos, utiliza cosolvencia para incrementar la
solubilidad de compuestos en concentracin aceptable
In vitro evaluation para formulaciones intramusculares y mejora la
biodisponibilidad de los principios activos poco polares,
Previous studies show that the compound alpha has puede ser usado a altas dosis de ms de 4 000 mg/kg en
high efficacy in vitro against F. hepatica,6-8 thus solubility ratas.31-33
with the used vehicles does not affect the efficacy of the La adecuada solubilidad obtenida por la
compound when obtaining 100% of efficacy against cosolvencia de los vehculos ha sido utilizada con xito
the trematode. en la formulacin de ivermectinas y rimfampicina.34,35
The low efficacy obtained with the solubilized Ca is Estudios previos muestran que el compuesto alfa tiene
because by oral route there is a ruminal preactivation alta eficacia in vitro contra F. hepatica,6-8 por lo cual la
of the sulphoxide and sulphone metabolites. The Ca solubilidad con los vehculos utilizados no afecta la
passes to the plasma in suitable concentration to exert eficacia del compuesto al obtenerse 100% de eficacia
certain efficacy in sheep and goats. 6,7,11 Is probable contra el trematodo.
that Ca remains in the tissue compartment when is
injected intramuscularly, little compound passes to the Evaluacin in vivo en ovinos
sanguineous torrent and arrives in low concentration
to the liver to be metabolized in sulphoxide and La baja eficacia del Ca solubilizado se debe a que
sulphona; this behavior is associated with its physico- por va oral tiene una preactivacin en rumen en
chemical characteristics that favors a low volume of sus metabolitos sulfxido y sulfona, el primero, pasa
distribution, thus at greater dose the concentration a plasma en concentracin adecuada para tener
and the fasciolicidal efficacy increases. alta eficacia en ovinos y caprinos.6,7,11 Es probable
It should be considered that in the site of injection que el Ca al ser inyectado va intramuscular quede
the disposition of the deposited compound depends en el compartimiento tisular, poco compuesto pasa
on the balance between variables, such as: compound al torrente sanguneo y llega baja concentracin al
affinity with the formulated vehicle, injected volume, hgado para ser metabolizado en sulfxido y sulfona;
10
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