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REVISTA CON-CIENCIA N°2/VOL. 4 (NOVIEMBRE 2016) Pag. 11-19 ISSN: 2310-0265

Estudios de susceptibilidad de cepas de Leishmania aethiopica frente a alcaloides de Galipea longiflora (Evanta)

Susceptibility studies on Leishmania aethiopica strains against total alkaloids from Galipea longiflora (Evanta)

GADISA ENDALAMAW1 SALAMANCA EFRAIN2 ASEFFA ABRAHAM1 TICONA JUAN CARLOS2

CORRESPONDENCIA: AGIMENEZ@MEGALINK.COM

UDAETA ENRIQUE2 FLORES NINOSKA CHUQUI ROGELIO3

GIMÉNEZ ALBERTO2*

FECHA DE RECEPCIÓN: 23/09/2016 FECHA DE ACEPTACIÓN: 3/11/2016

Resumen

El Instituto de Investigaciones Fármaco Bioquímicas (IIFB), de la Facultad de Ciencias Farmacéuticas y Bioquímicas, de la UMSA, desarrolla trabajos sobre la actividad leish- manicida, de los alcaloides totales (CAT) ob- tenidos de la corteza de la especie medicinal amazónica conocida como Evanta (Galipea longiflora) por los Pueblos Tacana, Tsimane y Mosetene.

Como parte de las actividades del Pro- yecto UMSA-ASDI “Biomoleculas de interés medicinal e industrial (antiparasitarios)” he- mos podido contar con la estadía, en el IIFB, de un investigador del Armauer Hansen Re- search Institute (AHRI) de Etiopia, lo que nos ha permitido desarrollar evaluaciones de CAT, Miltefocine y Amfotericina B, frente a cepas de Leishmania aethiopica, agente causante de las diversa formas de Leishmaniais cutá- nea en Etiopía.

Abstract

The Instituto de Investigaciones Fár- maco Bioquímicas (IIFB), at the Faculty of Pharmaceutical and Biochemical Sciences, from UMSA, carry out work related to the leishmanicidal activity of the total alkaloids (CAT) obtained from the bark of the Ama- zonian medicinal species known as Evanta (Galipea longiflora) by the Tacana, Tsimane y Mosetene people.

As part of the activities develop by the UMSA-ASDI Project “Biomolecules of me- dicinal and industrial Interest (antiparasitic)” we had a visit, in our laboratories at IIFB, of a researcher from The Armauer Hansen Re- search Institute (AHRI) from Ethiopia, dur- ing his stay we were able to carry out evalu- ations of CAT, Miltefocine and Anphotericin B, against strains of L. aethiopica, causative agent of the different manifestations of cu- taneous leishmaniasis in Ethiopia.

1. Armaeur Hansen Research Institute, Ethiopia

2. Area de Química Farmacéutica, Instituto de Investigaciones Fármaco Bioquímicas

3. Comunidad Tacana de Santa Rosa de Maravilla

* Autor correspondencia. Instituto de Investigaciones Fármaco Bioquímicas, Facultad de Ciencias Farmacéuticas y Bioquímicas, Universi- dad Mayor de San Andrés. Av. Saavedra No 2224, Miraflores La Paz, Bolivia

Un total de seis cepas, de L. aethiopi- ca, fueron adaptadas a condiciones in vitro y mostraron un comportamiento homogé- neo frente a CAT, cinco de estas cepas mos- traron un valor promedio de IC50 = 8,68 ± 1,56 mg/mL, valor algo inferior a los calcula- dos para nuestras cepas de referencia, L. ama- zonensis y L. braziliensis con IC50 = 11,73 ± 4,32 mg/mL y IC50 = 12,28 ±- 2,95 mg/mL,

respectivamente. Excepto por una cepa de

L. aethiopica que mostro valores consisten- temente más elevados que el resto con IC50

= 14,37 ± 3,58 mg/mL.

Como consecuencia de esta interacción científica, la Universidad Mayor de San Andrés (UMSA) ha firmado un Memorandum de Enten- dimiento para el desarrollo de investigaciones conjuntas, con el Armauer Hansen Research Ins- titute (AHRI), dependiente del Ministerio de Sa- lud de Etiopia y explorar la posibilidad de que nuestra experiencia de validación clínica con Evanta en el tratamiento de leishmaiasis cutá- nea, en Bolivia, podría ser replicada en Etiopía, donde se reportan entre 20,000 a 30,000 nue- vos casos de Leismaniasis por año.

A total of six strains of L. aethiopica, were adapted to in vitro a conditions, at IIFB; and did show homogenous behavior against CAT. Five of the strains, showed an average calculated value for IC50 = 8.68 ±

1.56 mg/mL, a value somewhat lower to the

calculated for the reference strains L. ama- zonensis and L. braziliensis with IC50 = 11.73

± 4.32 mg/mL and IC50 = 12.28 +/- 2.95 mg/

mL, respectively. Except for one strain that showed values somewhat higher, to the oth- er strains, consistently through our studies, with IC50 = 14.37 ± 3.58 mg/mL.

As a consequence of our scientific inter-

action, the Universidad Mayor de San An- drés (UMSA) has signed a Memorandum of Understanding for the development of joint research with the Armauer Hansen Research Institute (AHRI) that belongs to the Ministry of Health in Ethiopia, and explore the possi- bilities to replicate the Bolivian clinical val- idation experience of Evanta in the treat- ment of cutaneous leishmaniasis, in Ethiopia where the annual incidence is estimated to be between 20, 000 to 30, 0000.

PALABRAS CLAVE

Bolivia, Etiopia, L. aetiopica, L.amazonensis, L.braziliensis, susceptibilidad Alcaloides, Galipea longiflora

KEY WORDS

Bolivia, Ethiopia, L. aetiopica, L.amazonensis, L.braziliensis, suceptibility Alkaloids Galipea longiflora

INTRODUCTION

Leishmaniasis is part of the group of forgotten or neglected tropical di- seases and one of the most important vector-borne diseases in humans. This disease could be caused by various species of Leishmania parasites, most of which are zoonotic. Different parasite species are associated with diffe- rent clinical forms of the disease. Many species of Leishmania cause skin ul- cers and nodules, leading to cutaneous leishmaniasis (CL) which may be local (LCL) or diffuse (DCL). Some species of these organisms can also affect mu- cous membranes and may cause injury and disfigure the nose, causing mu- cocutaneous leishmaniasis (MCL). Other species damage internal organs and cause visceral leishmaniasis (VL), the latter is fatal if not treated in time (Blac- kwell JM et al. 2009). Leishmaniasis predominantly affectspoor people in Afri- ca, Latin Amerca and Asia. Globally estimated It is estimated to be endemic in 98 countries with annual incidence of 0.9 to 1.6 million cases per year

There are about 20 species of the Leishmania cause leishmaniasis in human (Table 1). These species belong to the Trypanosomatidae family that includes at least eight different Genera. The different clinical manifestations associated with

different Leishmania species. . Currently, the genus Leishmania is divided into two groups: the sub-Genus Leishmania and sub-Genus Viannia (Ardaya 2015).

In the Old World, leishmaniasis could be grouped into two classes, cuta- neous (CL), associated with the L. major, L. tropica and L. aethiopica complexes, and visceral (VL), involves up to 4 different species all associated with L. dono- vani complex (Ayele & Ali 1984). VL is endemic in many tropical and subtropical areas of the world, is the most severe form of leishmaniasis, almost always fa- tal if left untreated (Lyons et al. 2003). It is estimated that between 200,000 and 400,000 new cases of VL occur in the world each year (WHO, 2013 ) and of the- se, East Africa has the second highest number of cases, after the Indian subcon- tinent (Alvar et al. 2007 and Alvar et al 2012). In the New World, CL infections (LCL and DCL) are associated with L. mexicana and L. braziliensis complexes. MCL, is most often associated with L. braziliensis and L. panamensis, although other species may be involved. The DCL occurs with some species of the pa- rasite and due to host immune factors, while VL is associated with L. chagasi.

In Bolivia, an estimated 0.8 millones of individuals are at high risk of be- coming infected with leishmaniasis, the disease is spread in 7 of the 9 de- partments (except Oruro and Potosi). It is estimated that there are approxi- mately 2,500 new cases per year based on published data, L braziliensis species (CL and MCL) and L. amazonensis (CL) have a clear dominance in na- tional epidemiological indices. L. lainsoni (CL and MCL) and L. chagasi (VL) are presented in rare and few reports (Rojas et al. 2009; Martinez et al. 2002 and Mollinedo et al. 2000). In Bolivia, cases of leishmaniasis tend to increase every year, as well as other vector transmitted diseases like Dengue, Malaria, Cha- gas Disease (Garcia et al. 2009 and INLASA 2012).

Table 1. Taxonomic Classification of Pathogenic Parasites of Leishmania

Adapted from Bañuls et al 2007

Although no official data is available, it is estimated that the population at high risk of contracting some form of leishmaniasis in Ethiopia, is about 30mi- llion inhabitants and the total number of cases diagnosed as positive, each year, is estimated at around 20,000 to 30,000, with 25% of cases of VL (L. donovani) and 75% of cases of CL (L. aethiopica), the latter comes in three different clinical forms (LCL, MCL and DCL). LCL injuries by L .aethiopica, do not form open so- res, as with strains in the New World, (L. mexicana complex). The injuries caused by L. aethiopica are closed (nodules) and is often at the site of vector inocula- tion. Although, occasionally, severe and persistent LCL, MCL and DCL cause dis- figuring and often require prolonged treatment schemes with very low success rate. In the case of DCL, definitive cure is almost never achieved, and relapse is common (Hailu et al. 2006; Lemma et al. 1969; Bryceson 1969 and Sarojini 1984).

In the area of Pharmaceutical Chemistry at the IIFB (AQF-IIFB), at the Faculty of Pharmaceutical and Biochemical Sciences, UMSA, within the "Project UMSA-SIDA In- fections Diseases: Evanta in the Treatment of cutaneous leishmaniasis", we have pro- duced Evanta based treatments (creams and syrups) (Tacana 1999 and Beatriz 2006), and the effectiveness assessed in a clinical trial (Phase II, treatment of CL), in a com- parative treatment versus Glucantime. The study has been carried out in the Hospi- tal of Palos Blancos, from June 2007 to December 2012, with patients diagnosed with CL caused by L. amazonensis and L. braziliensis. A total of 60 individuals (patients with primary infection, over 18 and under 50 years old, as inclusion criteria), were treated, the results of our studies of comparative efficacy for Evanta (29 patients, creams and syrups) are bordering the 68% cure of patients and over 90% cure of patients treated with Glucantime (31 patients, intramuscular) (Magariños 2013).

Based on the information gathered in the last 10 years, from field and cli- nic experiences developed in Bolivia, and adding research results from our la- boratories, at IIFB, using Evanta (CAT), we can suggest that our experiences of validation of traditional medicines, for the treatment of cutaneous leishma- niasis, could be replicated in Ethiopia (OMS, 2013), where there are reported up to ten times more new cases of leishmaniasis per year, compared to the es- timated 2,500 cases per year, in Bolivia.

MATERIALS Y METHODS

Leishmania aethiopica strains from AHRI

The Leishmania aethiopica, strains were donated by AHRI, these were ob- tained from patients ulcers, from a total of ten strains received, six were able to adapt to in vitro cultures at IIFB and were used in the present study.

Reference Leishmania strains at IIFB

Leishmania braziliensis (M2904 C192 RJA) and Leishmania amazonensis (Clon 1 NHOM-BR-76-LTB-012). Leishmania lainsoni (INL 125-11)

Drugs preparation.

The drugs used in the study were Amphotericin-B (ANFOTERICIN-CRI- TÁLIA), Miltefosine (IMPA VIDO) and Total Alkaloids from Galipea longiflora (CAT-IIFB). Amphotericin-B was diluted in Schneider medium (1mg/mL) and dilutions with concentrations ranging from 0.62 to 0.01μg/mL were prepared. Miltefosine and CAT were diluted with DMSO (10mg/mL), and dilutions with concentrations ranging from 100 to 1.5μg/mL were prepared. Final concen- tration of DMSO was kept below 1%.

Leishmanicidal test

The activity was measured on in vitro cultures of the Leishmania parasi- te in promastigote forms, (modified from Salamanca et al 2008), cultivated at 26°C in Schneider medium (pH 6.8) supplemented with inactivated (56°C x 30min) calf bovine serum (10%). Parasites in logarithmic phase of growth, at a concentration of 1x106parasites/mL, were distributed on a 96 micro well pla- tes and the different concentration of the drugs were added. The micro well plates were incubated for 72hrs at 26°C. After incubation, a solution of XTT (1mg/mL) in PBS (pH 7.0 at 37°C) with PMS (Sigma-Aldrich, 0.06mg/mL), was added (50μL/well), and incubated again for 4hrs at 26°C. DMSO (1%) and Anphotericine B (0.5 mg/mL) were used as reference drugs during the evalua- tions, were done by sextuplicate. Optical density of each well was obtained with a Synergy HT microplate reader with I 200-450nm. The IC50 values were calculated using The Gen5 program (Biotek).

RESULTS

Because Leishmaniasis is a neglected disease, lack of attention also hap- pens with research and development of new diagnostic tools, drugs or vac- cines, because there is no market interested in social groups suffering from the disease (WHO). The misuse and mismanagement of drugs can lead to the emergence of resistance forms (Gil et al. 2007) such is the case with Glucanti- me, which is well documented in countries like India (Osorio et al 2005), Bra- zil (Nascimento-Zauli et al. 2010) and Bolivia (10% of treatment failure, Ber- mudez et al. 2006) and such behavior has been observed within patients with visceral L. coinfected with HIV (Koert et al 2011). While in a clinical trial using Miltefosine, in Palos Blancos, Bolivia, only 50% of patients with MCL were cu- red (Soto et al. 2007).

The significant adverse effects reported, such as myalgia, arthralgia, ano- rexia, nausea and headache and the high costs, in the case of antimonials, sti- mulate that many patients do not conclude the treatment, favoring the reac- tivation of the injury, mucous damage and the emergence of drug resistance

15

(Clem A, 2010 and Bhandari et al. 2012). The disadvantages associated with the available drugs and the variability of response to those by Leishmaniasis species, have evidenced the urgent need to develop alternative treatments and monitor treatment efficacy and emerging resistance to drugs in use

The medicinal plant Galipea longiflora (Evanta) is used in the treatment of cutaneous leishmaniasis by different Amazonian ethnic groups, in Bolivia, and we evaluated the response of six Leishmania aethiopica strains, being the spe- cies responsible of the different forms of cutaneous leishmaniasis in Ethiopia, and compared with the response observed for the reference strains at IIFB, when the different strains were exposed to the total alkaloids (CAT) of Evan- ta. The results are shown on Table 2.

Table 2. Susceptibility test of Leishmania strains against Amphotericin B, Miltefosine and CAT

DISCUSSIONS

In Bolivia leishmaniasis infection are caused mainly by L braziliensis (CL and MCL) and L. amazonensis (CL) both species have a clear dominance in na- tional epidemiological indices. While L. lainsoni (CL and MCL) and L. chagasi (VL) are presented in rare and few reports (Rojas E et al. 2009).

From the six strains of L. aethiopica included in the present studies, five showed homogeneous behavior against all drugs, with an average calculated value for IC50=8.68 ± 1.56μg/mL, when exposed to CAT, a value somewhat in- ferior to the calculated for the reference strains L. amazonensis, and L. brazi- liensis with IC50=11.73 ± 4.32μg/mL and IC50=12.28 ± 2.95μg/mL, respectively, but closer to the L. lainsoni, IC50=9.7 ± 1.45 μg/mL. (except L. aethiopica 5, with values somewhat superior to the other strains consistently through our studies: IC50 = 14.37 ± 3.58μg/mL against CAT and Miltefosine IC50=3.7 ± 2.39μg/mL).

All six L. aethiopica showed homogeneous behavior against Amphoteri- cin B (average IC50=0.22±0.02μg/mL) somewhat lower values when compared with the reference strains L. braziliensis (IC50=0.39±0.06μg/mL) and L. amazo- nensis (IC50=0.33±0.04μg/mL), a similar trend is observed when compared all

L. aethiopica strains against Miltefosine (IC50=2.26±0.76μg/mL) with L. brazi-

liensis and L. amazonensis (IC50=7.5±1.48μg/mL, and IC50=6.7±0.6μg/mL, res- pectively), Interesting to notice that susceptibility values for the L. aethiopica strains are much closer to the values calculated for the less common L. lainsoni strain in Bolivia (IC50=0.22±0.02μg/mL and IC50=3.5±0.35μg/mL, respectively).

The Instituto de Investigaciones Fármaco Bioquímicas (IIFB) belonging to the Faculty of Biochemical and Pharmaceutical Sciences at UMSA, has been working in the analytical (Herrera et al. 2008 and Limachi 2013) chemical (Lla- nos et al. 2009; Ticona 2005 and 2008; Espinoza 2012) biological (Salamanca

2008 and Herrera 2008) and pharmaceutical formulations (Rodriguez 2006) assessments of the total alkaloids (CAT) produced by Evanta (Galipea longiflo- ra) since 2000 (Gimenez et al. 2005). Moreover, in collaboration with resear- chers from Brazil, through the CYTED Ibero-american research network, CAT gastro-protective et al. 2009) and anti-nociceptive (Campos-Buzzi et al 2010) effects were assessed, and results on G. longiflora shows significant inhibition of ulcer formation by decreasing gastric secretion and increase gastric mucus content. Therefore, the IIFB, has produced Evanta based treatments which were used in a clinical study on patients diagnosed with LC, caused by L. ama- zonensis and L. braziliensis, in Palos Blancos Hospital (2007-12), with efficacy bordering the 68% cure of patients.

CONCLUSIONS

Our in vitro studies developed at UMSA by researchers from IIFB and AHRI, using strains of L. aethiopica and different local leishmania spp strains circulating in Bolivia, against total alkaloids of Evanta (CAT), have shown that

L. aethiopica parasites (media IC50=6.93+/-1.89) and the Bolivian strains (me- dia IC50=11.24+/-2.89) are sensitive to Evanta alkaloids, suggesting that our experience in clinical validation, previously carried out in Bolivia, could be re- plicated in Ethiopia, where an estimated of 20,000 to 30,000 new cases of leis- hmaniasis are reported each year.

ACKNOWLEDGMENTS

The author wish to thank the UMSA-ASDI Institutional strengthening Pro- gram and the Biomolecules (antiparasitic) project, for supporting this study. The IIFB wish to recognize the CIPTA for collaborating on our studies on the Tacana’s traditional knowledge.

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