4 Doctora en Química. Laboratorio de Espectroscopía Molecular. Facultad de Ciencias. La Hechicera. ULA. 

⁵ Magister en Protozoología. Centro de Investigaciones Parasitológicas ¨José W. Torrealba¨ NURR, ULA. 

⁶ Medico Microbiologo. Laboratorio de Microbiología. Facultad de Medicina Escuela de Medicina. ULA. Valera. Venezuela. 

⁷ Licenciado en Farmacia. Laboratorio de Toxicología Facultad de Farmacia. ULA.

Corresponding autor: Laura Vásquez de Ricciardi Facultad de Medicina Escuela de Medicina Valera. Universidad de Los Andes, final calle 6 detrás del Hospital Central de Valera. Valera. Venezuela. Tele-Fax: 0271-2215382; 0271-2313317.

e-mail: lavasquez60@hotmail.com

Financial support: This work was supported in part by the CDCHT- ULA project NURR-C-271-00-B-7, CVI-NURR-0395 and FONACIT Project Frontera 98000576.

Resumen

Se estudiaron los parámetros farmacocinéticos del antimoniato de meglumina Glucantime^® después de su administración solo y en combinación con gentamicina, a objeto de determinar el efecto del aminoglucósido sobre las concentraciones plasmáticas y los parámetros farmacocinéticos del metal. Se inyectaron cuatro perros con una dosis única/día de Glucantime^® (25.65 mgkg^-1) durante 5 días y Glucantime^® + gentamicine (25.65 mg kg^-1 y 5 mg kg^-1, respectivamente). Se colectaron muestras de sangre 0.25, 0.75, 1.0, 2.0, 4.0, 8.0, 12.0 y 24.0 horas post-tratamiento. Las determinaciones de antimonio se realizaron usando espectroscopia de absorción atómica. Los resultados demuestran que la combinación modificó la depuración del metal y disminuyó sus concentraciones plasmáticas. Solo Cl_B mostró una diferencia significativa (0.353 ± 0.110 a 0.733 ± 0.33 mLh-1kg^-1), sugiriendo menor persistencia tisular con la administración conjunta. En el futuro debe aclararse si la gentamicina interfiere en el análisis de las concentraciones de antimonio o viceversa.

Palabras claves: farmacocinética antimonio, antimoniato de meglumina, Glucantime^®, gentamicina.

Abstract

The pharmacokinetic parameters of Glucantime^® was studied after alone administration and in combination with gentamicine, to determine the effect of the aminoglucoside on the plasmatic concentrations and the pharmacokinetic parameters the metal. Four injected dogs were used with unique daily dose of alone Glucantime^® (25.65 mg kg^-1) and Glucantime^® + gentamicine (25.65 mg kg^-1 and 5 mg kg^-1, respectively). The blood samples were collected 0.25, 0.75, 1.0, 2.0, 4.0, 8.0, 12.0 and 24.0 hours post-treatment. Determination of antimony was carried out using atomic absorption spectrometer. The results demonstrated that the combination modifies the depuration of the metal and their sanguine decline resulted in plasmatic concentration minors. The pharmacokinetics parameters evaluated only Cl_B showed a statistically significant difference (0.353 ± 0.110 to 0.733 ± 0.33 mL h-1kg^-1) suggesting minor tisular persistence of antimony when administered together with gentamicine. It is important to recognize in future if the gentamicine interferes with the analysis of the antimony concentration o viceversa.

Key words: Pharmacokinetic antimony, N-methylglucamine antimoniate, Glucantime, gentamicine.

Recibido: 01/09/2006 Aceptado: 20/10/2006

Chemotherapeutic treatment of cutaneous and visceral leishmaniasis has not seen progress since the time antimony was first used some fifty years ago. Its cost and levels of toxicity, the prolonged duration of the therapy, and individual variations in response to the therapy have created the need of evaluating the use of other pharmacological options such as: aminoglucoside antibiotics. The first antibiotic evaluated both clinically and experimentally was monomycine (Neal 1968). More recently, the use of aminosidine against different species of Leishmania has been experimentally evaluated Scorza et al. 1988. Different researchers have shown positive results when pentavalent antimony was used in combination with aminosidine (Chunge et al. 1990, Scott et al. 1992, Belloli et al. 1994, Tecklemariam et al. 1994, Belloli et al. 1999).

The present study was designed to investigate the impact of gentamicine on antimony levels and its possible influence on pharmacokinetic of metal.

Animal Four two-year old healthy female hybrid dogs were used, averaging 15 kilograms of weight. The criteria of exclusion were anemia, lengthering of abnormal prothrombine time, and deterioration in renal, pancreatic or hepatic functioning. Three days prior to testing, blood and urine values were measured for platelet count and concentration levels of glycaemia, urea, creatinine, fractionate and total bilirrubine, amylases, proteins and transaminases.

The Ethical Committee of the Center of Parasitological Research "José Witremundo Torrealba" of Los Andes University, approved all procedures carried out in this study

Drugs, doses, and routes of administration. A daily dose of Glucantime^®, Specie Rhone-Poulnec Rorer in ampoules of 5 ml with 1.5 of N-methylglucamine NMG-Sb (427.5 mg SbV) Lote 456 Man 0698-Exp 0603, was administered for five days via a subcutaneous injection (25.65 mg.Kg^-1 of antimony).

A daily dose of gentamicine, Servipharm in ampoules of 2 ml with 80 mg of gentamicine sulfate Lote 801 Exp 01.2001, was administered for five days via an intramuscular injection in the right posterior leg, at a rate of 5 mg.Kg^-1.

Methods

A controlled cross-experiment was carried out, the animals were distributed into two groups (two dogs each), and the study was done in two steps. In a first step, a controlled experiment was carried out. Two animals (Group 1) were injected with NMG-Sb to evaluate the pharmacokinetic parameters of the pentavalent antimony; whereas the other two animals for (Group 2) were injected with both NMG-Sb plus gentamicine, to assay for changes in the pharmacokinetic parameters resulting from the combined presence of the drugs. In a second step carried out a month after, the two dogs from Group 1 were injected with the combination, whereas the two dogs from Group 2 were injected with NMG-Sb alone.

Blood sampling. A blood sample was obtained previous to drug administration, followed by time sampling 0.25 - 0.75 - 1 - 2 - 4 - 8 - 12 - 24 hours after the first day of drug administration. A last sample was taken on the fifth day prior to administering the drugs in order to determine drug concentration at steady state. From each sample, 2 ml of blood were used to determine total antimony content. Another 2 ml of blood were centrifuged at 1500 g to separate its serum, which was then frozen in vials and tested for gentamicine content.

Quantification of antimony and gentamicine content.  Lyophilized blood samples were processed using an absorption spectrophotometer (Varian Techron model AA-1475). The resulting limits of detection (set at 3 times standard deviation of target value) were 2.8 ng-L for antimony III (SbIII) and 3.2 ng-L for antimony V (SbV). Total antimony content was calculated as the sum of the concentrations of both species (Petit de Peña et al. 2001).

Sample analyses of gentamicine were carried out using an analyzer TDx and immunofluorescent assay (Abbott Laboratories). The limit of detection was set at 0.01 µg/ml.

Pharmacokinetic analysis. Standard methods of pharmacokinetic analysis were use (Rowland & Tozer 1980, Wagner 1975). Concentration curves of plasma vs. time (Cp vs. t) at logarithmic scale were plotted for the results obtained, which included Cpmax, Tmax, Ka, half-life (t_½), elimination constant (K_el), distribution volume Vd, clearance Cl_B, and the area under the curve (AUC) was calculated using the method of residuals.

Statistical analysis. Pharmacokinetic parameters were analyzed through a paired t-test using the statistical program SPSS version 10.0. A statistical significant value of p<0.05 was established.

Results

Systematic observations brought about between 0.25 and 120 hours from the initial administration of NMG-Sb only and NMG-Sb plus gentamicine showed differences in the plasma concentration of antimony between both groups. At 0.25 hours, total average antimony concentration levels in the control group were higher than in the experimental group. At 24 hours, the experimental group showed a concentration 0.57 µg/ml half equivalent from the control group, the obtained differences were not statistically significant (Table 1).

Figure 1 compares the lowering medium values of antimony content in blood across time. Both showed decay in two phases. In the line corresponding to the NMG-Sb injected animals the first 4 hours the plasma concentration fell from 9.23 ± 4.82 µg mL^-1 to 4.82 ± 3.47 µg mL^-1, indicating a rapid distribution. In the second phase, the concentration fell more slowly and the drug was still detectable at 24 hrs (1.00µg mL^-1 ± 0.22), whereas the line corresponding to the NMG-Sb plus gentamicine showed two peak concentrations between 0 and 4 hours, similarly, the second phase concentration fell more slowly and the drug was systematically minor with combination. It should be stated that calculations of the pharmacokinetic parameters related to the apparent phase of elimination were carried out beginning the eighth hour (Tassi 1994), as it was thought best considering that drug administration was done subcutaneously. The complete curve was taken for AUC values.

Fig. 1. Blood reduction mean in antimony levels as a function of time, for Glucantime^® alone and for Glucantime^® plus gentamicine

Table 2 shows the results of the statistical analysis for the different pharmacokinetic parameters used. Peak concentrations of antimony for all four animals when injected with NMG-Sb only oscillated between 2.47 and 19.99 µg/ml with an average value of 9.51 (7.40) µg/ml. Peak concentrations of antimony for all four animals when injected with NMG-Sb plus gentamicine oscillated between 7.43 and 11.52 µg/ml. The obtained differences were not statistically significant. The time needed to reach peak concentrations of antimony was 2.25 hours (range of 1 to 4 hours) for NMG-Sb animals and 1.41 hours (range of 0.8 to 2 hours) for NMG-Sb + gentamicine animals, which was not statistically significant. From the pharmacokinetic parameters assayed, only the clearance showed significant differences between both groups (p<0.048). The half-life of animals with NMG-Sb only was 10.02 ± 3.5 hours, and for animals NMG-Sb plus gentamicine was 17.60 ± 4.87 hours. It should be noted that the constant of elimination values (K_el) diminished to 0.075 h^-1 for the first group and to 0.041 h^-1 for the second group (differences between both groups were not statistically significant).

It should be stated that of the paraclinic criteria evaluated before and after drug injection, serum creatinine levels increased temporarily in one of the experimental animals.

Discussion

Lack of research in the pharmaceutical industry to promote the synthesis of new drugs aimed to the treatment of leishmaniasis has brought about the need to evaluate combinations well-known pharmaceuticals, in an effort at increasing their potency and effectiveness.

The present work was carried-out based on the results obtained by Belloli et al. (1994), who studied the disposition of antimony and aminosidine in dogs following separated and combined administrations. The researchers reported a synergistic effect of both compounds, interpreted as a consequence of the similarities in the pharmacokinetic patterns of both. Furthermore, they reported that aminosidine slowed down the elimination of antimony, due to the significant increase in the half-life of antimony and the reduction in its clearance when both compound when administered together. This does not seem to be applicable to the case of gentamicine, as there were significant differences in the rate of body clearance (Cl_B). A possible explanation for a gentamicine-induced increase in the rate of elimination of antimony might be that the antibiotic somehow modifies the binding of the metal to tisular proteins allowing the changes in presence of the metal in tissues. Plasma levels of antimony were found lower, which might be explained by the well-known effect that metal-binding to serum proteins has over its elimination via glomerular filtration (Camner et al. 1986).

The antimony half-life obtained in our results when administered only is more prolonged than the ones previously reported for intravenous and subcutaneous administration (Chulay et al. 1988; Tassi 1992; Belloli et al. 1999). The difference might be explained by differences in diffusion mechanisms related to subcutaneous drug injection.

It seems likely that a competition between gentamicine and N-methylglucamine antimoniate (NMG-Sb) takes place during renal excretion. Our results suggest that an interaction between the antimony-containing drug and gentamicine exists, as the half-life of elimination for both was prolonged. Finally, it is important to recognize in future research if the gentamicine interferes with the analysis of the antimony concentration o vice versa.

Acknowledgment: We thank Drs. Nestor Añez, Ernesto Palacios Prü and Mireya Parilli for critical review of the manuscript.

Referencias

1. Neal R. The effect of antibiotics of the neomycin group on experimental cutaneous leishmaniasis. Am. Trop. Med Parasitol 1968; 12:54-68.        [ Links ]

2. Scorza, J; Hernández, A; Villegas, E; Marcucci, M; Araujo, P. Comprobación clínica del sinergismo entre Glucantime y la Gabbromicina en el tratamiento de la leishmaniasis tegumentaria en Trujillo, Venezuela. Bol Dir Malar & Saneam. Amb 1988; 28: 23-5.        [ Links ]

3. Chunge, C; Owate, J; Pamba, H; Donno, L. Treatment of visceral leishmaniasis in Kenya by aminosidine alone or combined with sodium stibogluconate. Trans Roy Soc Tro Med Hyg; 1990; 84: 221-225.        [ Links ]

4. Scott, J; Davidson, R; Moody, A; Grant, H; Fremingham, D; Scott, M; Olliaro, P; Bryceson, A. Aminosidine (paromomycin) in the treatment of leishmaniasis imported into the United Kingdom. Trans Roy Soc Trop Med Hyg; 1992; 86: 617-619.        [ Links ]

5a. Belloli, C; Ceci, L; Carlis, S; Tassi, P; Montesissa, C; De Natale, G; Marcotrigiano, P; Ormas, P. Disposition of antimony and aminosidine in dog after administration separately and together: implications for the therapy of leishmaniasis. Res Vet Sci; 1994; 58(2): 123-127.        [ Links ]

5b. Belloli, C; Crescenzo, G; Carli, S; Zaghini, A; Mengozzi, G; Bertini S; Ormas P. Disposition of antimony and aminosidine combination after multiple subcutaneous injection in dogs. Veterinary Journal. 1999; 157(3):315-321.        [ Links ]

6. Tecklemariam, S; Gebre, A; Frommel, D; Miko, T; Ganlov, G; Bryceson; A. Aminosidine ant its combination with sodium stibogluconate in the treatment of diffuse cutaneous leishmaniasis caused by Leishmania aethiopica. Tran Roy Soc Trop Med Hyg; 1994; 88:334-339.        [ Links ]

7. Petit de Peña, Y; Vielma, O; Burguesa, J; Burguesa, M; Rondón, C; Carrero, P. On line determination of antimony three (III) and antimony five (V) in liver tissue and whole blood by flow - injection hydride generation - atomic absortion spectrometry. Talanta 2001; 55: 743-754.        [ Links ]

8. Rowland, M; Tozer, T. Clinical Pharmacokinetics. Concepts and Applications. Lea and Febiger Philadelphia. 1980.        [ Links ]

9. Wagner, JG. Fundamentals of Clinical Pharmacokinetics Drugs Intelligence Publications ING. Hamilton. 1975.        [ Links ]

10.Tassi, P. Pharmacokinetic of N-methylglucamin antimoniate after intravenous, intramuscular and subcutaneous administration in the dog. Res. in Veter. Sci 1994; 56:144-150.        [ Links ]

11.Camner, P; Clarkson, T; Nordberg, G. Routes of exposure, dose and metabolism of metals. In Handbook on the Toxicology of Metals 2nd Ed. Ed Friberg and V.B Vouk . Amsterdam. Elsevier Science. 1986. pp. 85-127        [ Links ]

12. Chulay, L; Fleckenstein, L; Smith, D. Pharmacokinetics of antimony during treatment of visceral leishmaniasis with sodium stibogluconate and meglumine antimoniate. Trans Roy Soc Trop Med & Hyg; 1988; 82:69-72.        [ Links ]