Therapeutic Efficacy of a Mixed Formulation of Conventional and PEGylated Liposomes Containing Meglumine Antimoniate, Combined with Allopurinol, in Dogs Naturally Infected with Leishmania infantum.

The treatment of dogs naturally infected with Leishmania infantum using meglumine antimoniate (MA) encapsulated in conventional liposomes (LC) in association with allopurinol has been previously reported to promote a marked reduction in the parasite burden in the main infection sites. Here, a new assay in naturally infected dogs was performed using a novel liposome formulation of MA consisting of a mixture of conventional and long-circulating (PEGylated) liposomes (LCP), with expected broader distribution among affected tissues of the mononuclear phagocyte system. Experimental groups of naturally infected dogs were as follows: LCP plus Allop, receiving LCP intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg of body weight/dose) at 4-day intervals plus allopurinol at 30 mg/kg/12 h per os (p.o.) during 130 days (LCP+Allop); LC plus Allop, receiving LC intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg/dose) plus allopurinol during 130 days (LC+Allop); Allop, treated with allopurinol only; and a nontreated control. Parasite loads were evaluated by quantitative PCR in liver, spleen, and bone marrow tissue and by immunohistochemistry in the ear skin, before treatment, just after treatment, and 4 months later. The LCP+Allop and LC+Allop groups, but not the Allop group, showed significant suppression of the parasites in the liver, spleen, and bone marrow 4 months after treatment compared to the pretreatment period or the control group. Only LCP+Allop group showed significantly lower parasite burden in the skin in comparison to the control group. On the basis of clinical staging and parasitological evaluations, the LCP formulation exhibited a more favorable therapeutic profile than the LC one, being therefore promising for the treatment of canine visceral leishmaniasis.

Mixed formulation of conventional and pegylated liposomes as a novel drug delivery strategy for improved treatment of visceral leishmaniasis.

OBJECTIVE: Test the hypothesis that pegylated meglumine antimoniate-containing liposomes (LMA) and their mixture with non-pegylated (conventional) LMA may be more effective than conventional LMA against visceral leishmaniasis (VL), because of wider drug distribution among different mononuclear phagocyte system (MPS) tissues. METHODS: Sb was determined in the blood and MPS tissues after administration of pegylated or conventional LMA intravenously to mongrel dogs naturally infected with Leishmania infantum and Swiss mice. Pegylated and conventional LMA as well as their mixture were evaluated for their antileishmanial efficacy in BALB/c infected with L. infantum through determination of parasite load in liver, spleen and bone marrow. RESULTS: An improved targeting of Sb to the bone marrow of dogs was clearly evidenced, as an important impact of pegylation. In accordance with this data, pegylated LMA significantly reduced parasite load in bone marrow of infected mice, in contrast to conventional LMA. The mixed formulation of conventional and pegylated LMA promoted parasite suppression to a higher extent in both spleen and bone marrow, compared to pegylated or conventional LMA. CONCLUSIONS: The present work establishes for the first time the potential of mixed formulations of conventional and pegylated liposomes as a drug delivery strategy for improved treatment of VL.

Mucoadhesive chitosan films as a potential ocular delivery system for ofloxacin: preliminary in vitro studies.

OBJECTIVE: The objective of the study is to evaluate the physical properties, in vitro release profile, and antibacterial efficiency of chitosan films prepared with ofloxacin. PROCEDURE: Mucoadhesive films were prepared by means of a casting and solvent evaporation technique performed in a 2 wt% acetic acid solution and distilled water. Physical properties were characterized by release and swelling studies, differential scanning calorimetry (DSC) analysis, and attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR) analysis. The in vitro evaluation of the films was performed by inhibiting Staphylococcus aureus and Pseudomonas aeruginosa through activity studies. RESULTS: Circular ofloxacin-loaded chitosan-developed films with 0.3 mg of drug weighed 7 mg were 110 µm thick and 5 mm in diameter. The DSC curve of ofloxacin-loaded chitosan films suggests an amorphous dispersion of ofloxacin within these films. ATR-FTIR analysis showed that ofloxacin is indeed present in the matrix film. The drug was released in vitro over a 1-h period. No statistical difference could be observed between the ofloxacin-loaded chitosan films and sterile disk soaked for 1 min in 0.3% commercial ofloxacin ophthalmic solution for S. aureus and P. aeruginosa (P = 0.1686, P = 0.1172, respectively).The films presented a significantly larger mean bacterial inhibition zone of S. aureus than did the commercial ciprofloxacin control disk (P = 0.0002) and a significantly larger mean bacterial kill zone of P. aeruginosa than did the commercial enrofloxacin control disk (P < 0.0001). CONCLUSIONS: Ofloxacin was successively incorporated onto chitosan films and was not inactivated during the process of manufacturing, thus preserving antibacterial proprieties.

Complement activation-related pseudoallergy in dogs following intravenous administration of a liposomal formulation of meglumine antimoniate / Pseudoalergia relacionada à ativação do complemento em cães após administração intravenosa de uma formulação lipossomal de antimoniato de meglumina

The increasing use of nanotechnologies in advanced therapies has allowed the observation of specific adverse reactions related to nanostructures. The toxicity of a novel liposome formulation of meglumine antimoniate in dogs with visceral leishmaniasis after single dose has been investigated. Groups of 12 animals received by the intravenous route a single dose of liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg), empty liposomes (GII) or isotonic saline (GIII). Evaluation of hematological and biochemical parameters showed no significant changes 4 days after administration. No undesired effects were registered in the GIII. However, adverse reactions were observed in 67.7% of dogs from both groups that received liposomal formulations. The side effects began moments after bolus administration and disappeared during the first 15 minutes after treatment. Prostation, sialorrhea and defecation were the most frequent clinical signs, registered in 33.3% and 41.6 % of animals from the groups GI and GII, respectively. Tachypnea, mydriasis, miosis, vomiting and cyanosis were also registered in both groups. The adverse reactions observed in this study were attributed to the activation of the complement system by lipid vesicles in a phenomenon known as Complement Activation-Related Pseudoallergy (CARPA). The influence of the physical-chemical characteristics of liposomal formulation in the triggering of CARPA is discussed.

O crescente uso das nanotecnologias nas terapias avançadas tem permitido a observação de reações adversas específicas relacionadas às nanoestruturas. A toxicidade de uma nova formulação lipossomal de antimoniato de meglumina após dose única foi avaliada em cães com leishmaniose visceral. Grupos de 12 animais receberam por via intravenosa uma dose única de antimoniato de meglumina lipossomal (grupo I [GI], 6,5 mg Sb/kg), lipossomas vazios (GII) ou solução salina isotônica (GIII). A avaliação de parâmetros hematológicos e bioquímicos não revelou alterações significativas quatro dias após a administração. Nenhum efeito indesejável foi registrado no GIII. No entanto, reações adversas foram observadas em 67,7% dos cães de ambos os grupos que receberam formulações lipossomais. Os efeitos colaterais iniciaram momentos após a administração em "bolus" e desapareceram no decurso dos primeiros 15 minutos após o tratamento. Prostração, sialorréia e defecação foram os sinais clínicos mais frequentes, registrados em 33,3% e 41,6% dos animais dos grupos GI e GII, respectivamente. Taquipnéia, midríase, miose, vômitos e cianose também foram registrados em ambos os grupos. As reações adversas observadas neste trabalho foram atribuídas à ativação do sistema complemento pelas vesículas lipídicas em fenômeno conhecido como Pseudoalergia Relacionada à Ativação do Complemento (PARAC). A influência das características físico-químicas da formulação lipossomal no desencadeamento de PARAC é abordada.

Efficacy of combined therapy with liposome-encapsulated meglumine antimoniate and allopurinol in treatment of canine visceral leishmaniasis.

An innovative liposomal formulation of meglumine antimoniate (LMA) was recently reported to promote both long-term parasite suppression and reduction of infectivity to sand flies in dogs with visceral leishmaniasis. However, 5 months after treatment, parasites were still found in the bone marrow of all treated dogs. In order to improve treatment with LMA, the present study aimed to evaluate its efficacy in combination with allopurinol. Mongrel dogs naturally infected with Leishmania infantum were treated with six doses of LMA (6.5 mg Sb/kg of body weight/dose) given at 4-day intervals, plus allopurinol (20 mg/kg/24 h per os) for 140 days. Comparison was made with groups treated with LMA, allopurinol, empty liposomes plus allopurinol, empty liposomes, and saline. Dogs remained without treatment from day 140 to 200 after the start of treatment. The drug combination promoted both clinical improvement of dogs and significant reduction in the parasitic load in bone marrow and spleen on days 140 and 200 compared to these parameters in the pretreatment period. This is in contrast with the other protocols, which did not result in significant reduction of the bone marrow parasite load on day 200. Strikingly, the combined treatment, in contrast to the other regimens, induced negative quantitative PCR (qPCR) results in the liver of 100% of the dogs. Both xenodiagnosis and skin parasite determination by qPCR indicated that the drug combination was effective in blocking the transmission of skin parasites to sand flies. Based on all of the parasitological tests performed on day 200, 50% of the animals that received the combined treatment were considered cured.

Occurrence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies in dogs with visceral leishmaniasis / Ocorrência de anticorpos anti-Neospora caninum e anti-Toxoplasma gondii em cães com leishmaniose visceral

Uninfected dogs and those naturally infected with Leishmania chagasi exhibiting different clinical forms of disease were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies. Blood samples were collected from 110 mongrel dogs. Sera were tested using the indirect fluorescent antibody test (IFAT), and the animals with visceral leishmaniasis (VL) (n=60) were classified clinically. Out of the 110 sera investigated, 5 (4.5 percent) were positive for N. caninum (IFAT>50) and 36 (32.7 percent) for T. gondii (IFAT>16). Anti-L. chagasi antibody titers in asymptomatic dogs (n=10) were found to be significantly lower (P<0.05) than those in oligosymptomatic ones (n=22), which were in turn significantly lower (P<0.05) than those in symptomatic ones (n=28). No association between Leishmania and N. caninum infections was observed. Among dogs infected with L. chagasi, a tendency (P=0.053) towards an association between the infection with T. gondii and the appearance of VL symptoms was observed, suggesting that the clinical manifestation of VL in dogs may enhance their susceptibility to T. gondii. The possible influence of the immunosuppressive status of canine leishmaniasis in the different clinical forms of the disease is discussed.

A presença de anticorpos anti-Neospora caninum e anti-Toxoplasma gondii foi avaliada em cães não infectados e naturalmente infectados com Leishmania chagasi manifestando diferentes formas clínicas da enfermidade. Amostras de sangue foram coletadas de 110 cães sem raça definida. Os soros foram avaliados por meio da reação de imunofluorescência indireta (RIFI) e os animais com leishmaniose visceral (LV) (n=60) foram classificados clinicamente. Dos 110 soros analisados, 5 (4,5 por cento) foram reativos para N. caninum (RIFI>50) e 36 (32,7 por cento) para T. gondii (RIFI>16). Os títulos de anticorpos anti-L. chagasi em cães assintomáticos (n=10) foram significativamente (P<0,05) mais baixos que aqueles verificados em oligossintomáticos (n=22), que por sua vez foram significativamente menores (P<0,05) que em cães sintomáticos (n=28). Não foi observada associação entre infecções por Leishmania e N. caninum. Entre os cães infectados com L. chagasi, verificou-se uma tendência de associação (P=0.053) entre infecção com T. gondii e aparecimento de sinais clínicos da LV, o que sugere que a manifestação clínica da LV em cães pode aumentar sua susceptibilidade ao T. gondii. A provável influência do quadro de imunossupressão em diferentes formas clínicas da leishmaniose canina é abordada.

Prolonged absorption of antimony(V) by the oral route from non-inclusion meglumine antimoniate-beta-cyclodextrin conjugates.

The orally active composition comprising meglumine antimoniate (MA) and beta-cyclodextrin (beta-CD) differs markedly from conventional drug-CD complexes, since it combines a water-soluble drug and a hydrophilic CD. In order to obtain insights into the mechanism(s) responsible for the improved oral delivery of the drug, physicochemical and pharmacokinetic studies were carried out. The composition investigated here was prepared at a 7:1 antimony(Sb)/beta-CD molar ratio, a condition that improves its solubility in water and allows the oral administration of a high dose of Sb in large animals. It was characterized by circular dichroism, (1)H-NMR, ESI-MS and photon correlation spectroscopy. Pharmacokinetic data were obtained in Beagle dogs after oral administration of the composition at 100 mg Sb/kg. (1)H-NMR and ESI-MS data supported the formation of non-inclusion complexes between MA and beta-CD. Sub-micron assemblies were also evidenced that slowly dissociate and presumably release the MA drug, upon reconstitution of the composition in water. Pharmacokinetic studies of MA and MA/beta-CD in dogs showed a prolongation of the serum mean residence time of Sb from 4.1 to 6.8 h, upon complexation of MA with beta-CD. Evidence was also obtained that Sb remains essentially under the form of pentavalent Sb-meglumine complex, following gastro-intestinal absorption from the MA/beta-CD composition. In conclusion, the present data support the model that the sustained drug release property of 7:1 MA/beta-CD composition resulted in the prolongation of MA absorption by the oral route and, consequently, in the increase of the drug mean residence time in serum.

Pentavalent antimonials: new perspectives for old drugs.

Pentavalent antimonials, including meglumine antimoniate and sodium stibogluconate, have been used for more than half a century in the therapy of the parasitic disease leishmaniasis. Even though antimonials are still the first-line drugs, they exhibit several limitations, including severe side effects, the need for daily parenteral administration and drug resistance. The molecular structure of antimonials, their metabolism and mechanism of action are still being investigated. Some recent studies suggest that pentavalent antimony acts as a prodrug that is converted to active and more toxic trivalent antimony. Other works support the direct involvement of pentavalent antimony. Recent data suggest that the biomolecules, thiols and ribonucleosides, may mediate the actions of these drugs. This review will summarize the progress to date on the chemistry and biochemistry of pentavalent antimony. It will also present the most recent works being done to improve antimonial chemotherapy. These works include the development of simple synthetic methods for pentavalent antimonials, liposome-based formulations for targeting the Leishmania parasites responsible for visceral leishmaniasis and cyclodextrin-based formulations to promote the oral delivery of antimony.

Expression of IFN-gamma, TNF-alpha, IL-10 and TGF-beta in lymph nodes associates with parasite load and clinical form of disease in dogs naturally infected with Leishmania (Leishmania) chagasi.

American visceral leishmaniasis is a zoonosis of the New World. Dogs are the main reservoir of the disease and there is much interest in the understanding of mechanisms implicated in protection against canine infection. Nevertheless, most studies in dogs have not been carried out in organs that are targets of infection. This work is first to report the profile of cytokines and parasite burdens, as determined by real-time PCR, in the lymph nodes of dogs naturally infected with Leishmania chagasi. With this purpose, 18 mongrel dogs were divided in three groups: control non-infected dogs (n=6) and naturally infected animals with L. chagasi, asymptomatic (n=6) and symptomatic (n=6). Parasite burden in lymph nodes was 73-fold greater in symptomatic than asymptomatic animals. Prescapular lymph nodes of asymptomatic dogs had the highest expression of IFN-gamma and TNF-alpha and low parasite burden, indicating that these cytokines play a role in protection against infection. Highest expression of IL-10 and TGF-beta and high parasite burden were observed in symptomatic dogs, suggesting a role for these cytokines in the progression of disease. Hence, the balance of expression of IFN-gamma and TNF-alpha (protective) and IL-10 and TGF-beta (disease progression) in lymph nodes determine parasite burden and clinical expression in naturally infected dogs.

Reduced tissue parasitic load and infectivity to sand flies in dogs naturally infected by Leishmania (Leishmania) chagasi following treatment with a liposome formulation of meglumine antimoniate.

The toxicity and antileishmanial effectiveness of a novel liposome formulation of meglumine antimoniate in mongrel dogs with visceral leishmaniasis (VL) obtained from a region where VL is endemic in Brazil have been investigated. Groups of 12 animals received by the intravenous route four doses (with 4-day intervals) of either liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg of body weight/dose), empty liposomes (GII), or isotonic saline (GIII). Evaluation of markers of hematopoietic, hepatic, and renal functions before and just after treatment showed no significant change. On the other hand, transitory adverse reactions, including prostration, defecation, tachypnea, and sialorrhea, were observed during the first 15 min after injections in GI and GII. Parasitological evaluation of sternal bone marrow 4 days after the last dose showed a significant reduction of parasite burden in GI, compared to the other groups. Immunocytochemical evaluations of the skin, bone marrow, cervical lymph nodes, livers, and spleens of dogs for parasites, 150 days after treatment, indicated significant parasite suppression (higher than 95.7%) in the lymph nodes, livers, and spleens of GI, compared to control groups. Feeding of Lutzomyia longipalpis phlebotomines on dogs from GI, 150 days after treatment, resulted in a significant reduction of sand fly infection efficiency, compared to feeding on animals from GII and GIII. This is the first report of both long-term parasite suppression and reduction of infectivity to sand flies in naturally infected dogs following treatment with a liposome-encapsulated drug. Importantly, this was achieved using a 20-fold-lower cumulative dose of Sb than is used for conventional antimonial treatment.

In vitro binding and survival assays of Leishmania parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi.

BACKGROUND: There are a few works considering the characterization of canine monocyte-derived macrophages as well as a standardized procedure for isolation, culture, and infection of these cells with Leishmania. We have performed several modifications in order to improve the canine monocyte-derived macrophage cultures. In addition, we have done a comparative study between monocytes and monocyte-derived macrophages from dogs naturally and experimentally infected with L. chagasi. RESULTS: In the presence of exogenous serum, opsonized Leishmania promastigotes binds better to monocytes/macrophages than without serum. Otherwise, this binding occurs due to the strict correlation between the opsonized biologic particles with the third receptor of the complement (CR3-CD11b/CD18). In fact, our assays with CD11b confirmed the importance of this receptor for canine cells and the L. chagasi experimental system. Moreover, monocytes obtained from naturally infected dogs have shown a higher number of monocytes bounded to promastigotes. The experimental results regarding survival have shown that promastigote forms of opsonized L. chagasi were more infective, because we found higher numbers of promastigotes bound to the different cells. As a consequence, after forty-eight hours of binding, higher numbers of amastigotes appeared inside monocyte-macrophages. CONCLUSION: These studies have given support to continue comparative studies involving canine monocytes, monocyte-derived macrophages and peritoneal macrophages. Since we have standardized the canine cell culture, we are looking forward to determining the phenotypic properties of these cells before and after L. chagasi infection using flow cytometry.

Improved targeting of antimony to the bone marrow of dogs using liposomes of reduced size.

A novel liposomal formulation of meglumine antimoniate (MA), consisting of vesicles of reduced size, has been evaluated in dogs with visceral leishmaniasis to determine its pharmacokinetics as well as the impact of vesicle size on the targeting of antimony to the bone marrow. Encapsulation of MA in liposomes was achieved through freeze-drying of empty liposomes in the presence of sucrose and rehydration with a solution of MA. The resulting formulation, with a mean vesicle diameter of about 400 nm, was given to mongrel dogs with visceral leishmaniasis as an i.v. bolus injection at 4.2 mgSb/kg of body weight. The pharmacokinetics of antimony were assessed in the blood and in organs of the mononuclear phagocyte system and compared to those achieved with the free drug and the drug encapsulated in large sized liposomes (mean diameter of 1200 nm). The targeting of antimony to the bone marrow was improved (approximately three-fold) with the novel liposomal formulation, when compared to the formulation of MA in large sized liposomes. This study provides the first direct experimental evidence that passive targeting of liposomes to the bone marrow of dogs is improved by the reduction of vesicle size from the micron to the nanometer scale.