Clinical Adverse Reactions Induced By a Liposome Formulation in Naturally Infected Dogs with Leishmania (Leishmania) chagasi
R.R. Ribeiro; V.A. Freitas; S.M. Silva; W.M. Sampaio; M.S.M.Michalick; F. Frézard
Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil
The leishmaniasis are a group of complex syndromes caused by 24 species of Leishmania, which are naturally transmitted between animals, including the man, by sand flies vectors of the genus Phlebotomus and Lutzomyia. Among the different clinical forms of the disease, the visceral leishmaniasis (VL) is the most severe. The dog has been implicated as an important reservoir of the parasites since the discovery of canine leishmaniasis (CL) and in the New World, Leishmania (Leishmania) chagasi is the specie often isolated in patients with VL. The pentavalent antimony compounds, e.g., meglumine antimoniate (MA), are the drugs of choice for the treatment of leishmaniasis in human and dogs. However, conventional antimony treatment produces unsatisfactory results in dogs. The use of drug delivers, e.g., liposomes, is an alternative extremely promising to provide satisfactory therapeutic effect in CL (Valladares et al. 2001). When compared with conventional therapy, the liposome formulations of antimony were hundreds of times more effective in the treatment of VL in mice, hamsters and dogs. Despite the increase of leishmanicidal activity of MA, there is no commercial formulation lipid based Sb available. Furthermore, the development of some experimental formulations can produce acute toxicity in dogs (Nieto et al. 2003). In the field of nanotechnology, an important phenomenon of acute immune toxicity was reported after administration of liposomal drugs (Ambisome®, Doxil® and Dauno Xome®), radiocontrast media and micellar solvents containing amphophilic lipids (Szebeni 2005). There is substantial evidence suggesting that these acute allergic reactions are not mediated by preexisting IgE antibodies (Hypersensitivity Type I), but for complement activation (Szebeni 2005). As part of continuous efforts made by the Brazilian Group for Research and Leishmanicidal Drugs Development, Federal University of Minas Gerais, to improvement of products developed (Demicheli 1999; Demicheli & Frézard 2001; INPI/2640), this study determined and discussed the profile of clinical adverse reactions in naturally infected dogs with L.(L.) chagasi, after single dose of liposomal MA.
Materials and Methods
Thirty-six mongrel dogs (weighing 815 kg) naturally infected with L.(Leishmania) chagasi, exhibiting different clinical forms of canine leishmaniasis (Mancianti et al. 1988), were identified and captured during an epidemiological survey carried out by Control Zoonosis Center in Santa Luzia, Minas Gerais state, Brazil. All animals were found to be positive by IFAT (= 1:40 dilutions) and ELISA (optical density > 0.100; = 1:400 dilutions). In addition, parasitological diagnosis was performed by observation of parasite forms in both cytological examinations and/or cultures of bone marrow aspirates. During the whole experimental period, the dogs were housed in a screened kennel to avoid reinfection and received drinking water and a balanced feed ad libitum (Pedigree Champ®, Effem). MA was synthesized from an equimolar mixture of N-methylglucamine and pentavalent antimony oxyhydrated in water. The resulting product contained approximately 29% of antimony by weight, as determined by plasma emission spectroscopy (ICP-OES) using a PerkinElmer Optima 3000 plasma emission spectrometer. MA-containing liposomes with reduced size were prepared as described previously (Frézard et al. 2000). Drug containing liposomes were separated from nonencapsulated drug by centrifugation (14,000Xg, 30 min). The concentration of encapsulated antimony and the phospholipid concentration were determined in the resulting liposome suspension, using previously described colorimetric assays (Stewart 1980). The drug encapsulation efficiency and the final Sb/lipid ratio were equal to 40±4% and 0.25:1 (w/w), respectively. The final vesicular suspension was sized by photon correlation spectroscopy at 25°C with a 90° scattering angle and using a channel correlator (ZEN3500, Malvern Instruments, UK) in conjunction with a laser of wavelength 532nm. The mean hydrodynamic diameter of the vesicles was 400nm, with a mean polydispersity factor of 0.3. Empty liposomes with a comparable mean hydrodynamic diameter were obtained using the same method as that described above, except that the MA solution was replaced by a 0.65 M N-methylglucamine aqueous solution at pH 7.2. The dogs were stratified by weight, sex and clinical forms (Mancianti et al. 1988) and randomly distributed into three groups, each group containing initially 4 asymptomatic, 4 oligosymptomatic and 4 symptomatic dogs. Group I was treated with a single dose of liposomal MA at 6.5 mg Sb/kg/dose. Group II received a single dose of antimony free liposomes given at the same lipid dose as in group I. Group III received a single dose of isotonic saline given at the same volume as in group I. Following a single intravenous bolus injection, the animals were monitored continuously for clinical and behavioral alterations during 4 days. Temperature, food and water intake were registered and heart and respiratory frequencies were measured. The present research adhered to the Principles of Laboratory Animal Care (NIH publication #8523, revised in 1985) and received approval from the Ethical Committee for the use of Experimental Animals (CETEA) of the UFMG (Brazil) protocol n° 123/05.
In general, a single dose of liposomal formulations, both of liposome in the absence of Sb (n = 12), as of liposome containing MA (n = 12), resulted in similar profile and intensity of adverse reactions. The clinical reactions began moments after bolus administration and disappeared during the first 15 minutes, involving 67.7% of dogs from both groups that received liposomal formulations. There were not registered adverse reactions of any kind in saline group. The clinical condition apparently not influenced the toxicity of liposomal formulations. Prostation, sialorrhea, and defecation were the most frequent signs, affecting between 33.3 and 41.6% of animals from the groups GI and GII. Tachypnea, mydriasis and miosis were observed in 3 (16.6%, GI) and 4 (25%, GII) animals. Tremor muscle, tachycardia, urination and cyanosis were observed one time (8,3%) only in group that received empty liposomes. On the other hand, two out of 12 dogs just in the group I manifesting each xerostomia (8.3%) and hypotension (8.3%). Vomiting was registered in one animal for each group, resulting in 8.3% of frequency.
The Brazilian Group for Research and Leishmanicidal Drugs Development has been dedicated to design of liposomal formulations of MA. Since the first assay involving liposomal formulations in canine model that our group record clinical acute adverse reactions. However, determining the profile of reactions in dogs with different clinical forms of leishmaniasis and discussion of the trigger mechanism and your relation with characteristics of the liposomal formulation had not been made. Like us, the increasingly use of advanced therapies based on nanotechnology have allowed the observation ever more of signs the acute immune toxicity with distinct characteristics of hypersensitivity reactions groups. In these cases, the allergen active the complement system and the phenomenon have been tentatively named Complement Activation Related Pseudoallergy (CARPA). The liposomes are able to activate the complement system. Natural antibodies against phospholipids and cholesterol are present in all animal species (Alving & Wassef 1992) and the connection of these proteins to lipids of liposomes would allow the activation of complement through the classic route (Alving & Wassef 1992). There also the possibility of activating the complement cascade activate by the alternative route (Alving & Wassef 1992). It is very likely that the acute clinical adverse reactions observed in this study were consequence of the action of anaphylatoxins (C3a and C5a) released after activation of the complement cascade by liposomal vesicles. Most likely, the increase of the pulmonary arterial pressure, the reduction of the cardiac debit (Szebini et al. 2000) and the increase of pulmonary and peripheral vascular resistance, generated by the products of the complement, provided a transitional circulatory collapse that resulted in tissue hypoxia. Because of this, the sympathetic nervous system was stimulated and resulted in some the clinical signs observed: tachycardia, tachypnea, mydriasis and xerostomia. In order compensatory, the parasympathetic nervous system was also stimulated providing the reactions of miosis, sialorrhea, vomiting, urination and defecation. The occurrence of clinical effects just after the contact with allergen is a common symptom between CARPA and classical hypersensitivity type I (IgE mediated), but the high reaction rate observed (66.6% of animals) and spontaneous resolution of adverse reactions are features exclusive of the activation of complement. In CARPA, the toxicity can reach the peak between one and five minutes after the administration of the formulation and in this study the side effects have always been included in the first fifteen minutes after application of the formulations. The clinical forms of leishmaniasis apparently not modified the profile and intensity of side effects. The equal participation of different clinical conditions in the groups was important not only because it involves the diverse realities that are encountered in clinical veterinary practice, but also because the development of the disease can enhance the acute toxicity of formulations and may influence response to treatment (Amusategui 1998). The presence of dicetylphosphate in the composition of the formulation ensures anionic character of the vesicles and this is other evidence that contributing to our theory for the reason that the binding of antibodies to liposomes is accelerated by the presence of negative charge in the membrane (Szebini et al. 2000). The side effects could not be attributed to antimony, because there was no difference between the group treated with antimony liposomal (GI) and the free drug (GII). Furthermore, the toxic effects of the metal is chronic, does not disappear spontaneously and involve arthralgia, myalgia, diarrhea, anorexia and inflammation at the site of inoculation (Alvar et al. 2004). Costa Val (2004) described occurrence of like adverse reactions in dogs (n = 11) treated with empty liposome. No effects were observed in the groups that received saline, AM free or encapsulated in liposome. Although the lipid composition used was the same, the mean hydrodynamic diameter of the vesicles (1200 nm) was greater than that used in this study (400 nm). In theory, the binding of antibodies to liposomes and subsequent activation of the complement system should be proportional to the total vesicular surface area exposed to plasma (Szebeni et al. 2000). Comparing the results of Costa Val (2004) and ours, it is likely that smaller surface area of contact in micrometric vesicles and lowest dose of Sb utilized (3.8 mg Sb/kg) providing less activation of complement, however Costa Val registered higher reaction rate (81.8%, n = 11) in dogs. This fact could be explained by the individual variation of the manifesting of reactions in CARPA, which is a characteristic of the phenomenon (Devine et al. 1994; Nieto et al. 2003). No hematologic, hepatic and renal laboratory toxicity were observed in dogs 96 hours after administration of formulations (data no showed). Although the activation of complement appears to be an intrinsic property of lipid bilayers formed by cholesterol and phospholipids electrically charged (Devine et al. 1994; Szebini 1998), laboratory changes associated of systems carriers of drugs can be observed in the absence of clinical effects. Administration the antimony liposomal formulation based on phosphatidylcholine (10 nm) in dosage of Sb 9.8 mg/kg through intravenous and subcutaneous routes did not resulted in adverse reactions, but the authors alerted to the possible risk of induction of thrombocytopenia and bleeding related to the use of liposomes consisting of cholesterol, phosphatidylcholine and dicetylphosphate (Valladares et al. 2001). Since the speed of entry of liposomes in the vascular system is considered critical element for activation of the complement (Szebeni et al. 2000), the intravenous administration of this formulation by continuous infusion in fluid therapy can reduce or eliminate some or all of the side effects observed, without changing the characteristics of the product. In future, testing for complement activation in vitro and in vivo may be useful to determining the mechanism of immunotoxicity of the formulation, according with recommended by US Food and Drug Administration (FDA).
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