Nanostructured gadolinium (III) micelles: Synthesis, characterization, cytotoxic activities, and MRI applications in vivo.

Gadolinium-based contrast agents (GBCAs) are used in around 40 % of MRI procedures. Despite initial perceptions of minimal risk, their long-term use has emphasized the need to reduce toxicity and develop more efficient GBCAs with extended blood retention. Advancements in nanomaterials have led to improved GBCAs, enhancing MRI diagnostics. This study synthesizes and characterizes nanostructured gadolinium(III) micelles as superior MRI contrast agents. The complexes, [Gd(L)2], where L is a ligand of the N-alkyl-N-methylglucamine surfactant series (L8, L10 or L12, L10), form nanostructured micelles in aqueous solution. Gd(L8)2 and Gd(L10)2 relaxivities remained stable across concentrations. Compared to Gd-DTPA, Gd(III) micelles showed enhanced T1-weighted MRI contrast. Gd(L12)2 micelles exhibited cytotoxicity against B16F10 melanoma cells (IC50 42.5 ±â€¯2.2 µM) and L292L929 fibroblasts (IC50 52.0 ±â€¯2.5 µM), with a selectivity index of 1.2. In vivo application in mice brain T2-weighted images suggests nanostructured Gd(III) micelles are promising MRI contrast agents for targeting healthy organs or tumors.

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.

Reactive oxygen species generating photosynthesized ferromagnetic iron oxide nanorods as promising antileishmanial agent.

Aim: To investigate the photodynamic therapeutic potential of ferromagnetic iron oxide nanorods (FIONs), using Trigonella foenum-graecum as a reducing agent, against Leishmania tropica. Materials & methods: FIONs were characterized using ultraviolet visible spectroscopy, x-ray diffraction and scanning electron microscopy. Results: FIONs showed excellent activity against L. tropica promastigotes and amastigotes (IC50 0.036 ± 0.003 and 0.072 ± 0.001 µg/ml, respectively) upon 15 min pre-incubation light-emitting diode light (84 lm/W) exposure, resulting in reactive oxygen species generation and induction of cell death via apoptosis. FIONs were found to be highly biocompatible with human erythrocytes (LD50 779 ± 21 µg/ml) and significantly selective (selectivity index >1000) against murine peritoneal macrophages (CC50 102.7 ± 2.9 µg/ml). Conclusion: Due to their noteworthy in vitro antileishmanial properties, FIONs should be further investigated in an in vivo model of the disease.

Cytotoxicity and apoptotic activity of novel organobismuth(V) and organoantimony(V) complexes in different cancer cell lines.

Novel organobismuth(V) and organoantimony(V) complexes of Ph3ML2 type were synthesized, in which L = deprotonated 2-acetylbenzoic acid (2AcBH), 4-acetylbenzoic acid (4AcBH) or 5-acetylsalicylic acid (5AcSH) and M = bismuth(V) or antimony(V). Complexes [Ph3Bi(2AcB)2] (1) [Ph3Sb(4AcB)2] (2), [Ph3Bi(4AcB)2] (3) and [Ph3Sb(5AcS)2(.)CHCl3] (4) were characterized by elemental analysis, IR, and NMR. Crystal structures of 2 and 4 were determined by single crystal X-ray diffraction. In vitro cytotoxic activities against cancerous (human chronic myelogenous leukemia, K562 and murine metastatic melanoma, B16F10) and healthy non-cancerous (murine fibroblasts, L929 and murine melanocytes, Melan-A) cells showed that, compared to free ligands, both of the metal complexes are more active as anticancer agents at low concentration in cancerous cell lines, but also possessed toxic effect at comparatively higher concentration towards the non-cancerous cells. The organobismuth(V) complex Ph3Bi(2AcB)2 was found to be more active than the Ph3BiCl2 metal precursor against the tumor cell lines and exhibited the highest selectivity index. Moreover, evaluation of the pro-apoptotic activity of Ph3Bi(2AcB)2 in B16F10 cells, by quantifying the cellular DNA using flow cytometry, indicates that cell cycle arrest and cell apoptosis contribute to the drug cytotoxicity. This work supports the great potential of organobismuth(V) dicarboxylate complexes as anticancer agents.

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.

Novel triphenylantimony(V) and triphenylbismuth(V) complexes with benzoic acid derivatives: structural characterization, in vitro antileishmanial and antibacterial activities and cytotoxicity against macrophages.

Two novel organoantimony(V) and two organobismuth(V) complexes of the type ML2 were synthesized, with L = acetylsalicylic acid (HL1) or 3-acetoxybenzoic acid (HL2) and M = triphenylantimony(V) (M1) or triphenylbismuth(V) (M2). Complexes, [M1(L1)2] (1), [M1(L2)2]∙CHCl3 (2), [M2(L1)2], (3) and [M2(L2)2] (4), were characterized by elemental analysis, IR and NMR. Crystal structures of triphenylantimony(V) dicarboxylate complexes 1 and 2 were determined by single crystal X-ray diffraction. Structural analyses revealed that 1 and 2 adopt five-coordinated extremely distorted trigonal bipyramidal geometries, binding with three phenyl groups in the equatorial position and two deprotonated organic ligands (L) in the axial sites. The metal complexes, their metal salts and ligands were evaluated in vitro for their activities against Leishmania infantum and amazonensis promastigotes and Staphylococcus aureus and Pseudomonas aeruginosa bacteria. Both the metal complexes showed antileishmanial and antibacterial activities but the bismuth complexes were the most active. Intriguingly, complexation of organobismuth(V) salt reduced its activity against Leishmania, but increased it against bacteria. In vitro cytotoxic test of these complexes against murine macrophages showed that antimony(V) complexes were the least toxic. Considering the selectivity indexes, organoantimony(V) complexes emerge as the most promising antileishmanial agents and organobismuth(V) complex 3 as the best antibacterial agent.

Complexes of different nitrogen donor heterocyclic ligands with SbCl3 and PhSbCl2 as potential antileishmanial agents against Sb(III)-sensitive and -resistant parasites.

Novel trivalent antimony complexes with the nitrogen donor heterocyclic ligand 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen) or dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) have been synthesized by the reaction with SbCl3 or PhSbCl2. The crystal structures of [Sb(phen)Cl3] and [PhSb(phen)Cl2]CH3COOH were determined and shown to adopt a distorted square pyramid geometry with a five-coordinated Sb center. Surprisingly, all the complexes, the ligands and PhSbCl2 showed very high antileishmanial activities, with IC50 in the nanomolar range against Sb(III)-sensitive and -resistant Leishmania infantum (syn. Leishmania chagasi) and Leishmania amazonensis strains. These compounds were much more active against these Leishmania strains than the old trivalent drug potassium antimonyl tartrate. [PhSb(phen)Cl2]CH3COOH complex was found to be the most active compound and the lack of cross-resistance of PhSbCl2 suggests that the transport pathways of this compound across the cell membrane differ from those responsible for the resistance of Leishmania to Sb(OH)3. In the case of the complexes with PhSbCl2, our data supports the model that both ligand and metal contributed to the overall activity of the complex. Furthermore, among the complexes with SbCl3, only bipy showed an improved activity upon complexation. Cytotoxicity evaluations of these compounds against murine peritoneal macrophages showed high selective indexes in the range of 7-70 for [Sb(phen)Cl3], [Sb(bipy)Cl3] and [Sb(dpq)Cl3] complexes, being much more selective than potassium antimonyl tartrate. In conclusion, this study presents a set of new antileishmanial agents including one of the most active Sb-based compounds ever reported, which can contribute to the development of new chemotherapeutic strategies against leishmaniasis including Sb-resistant cases.

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.

Greater binding affinity of trivalent antimony to a CCCH zinc finger domain compared to a CCHC domain of kinetoplastid proteins.

It has been reported recently that Sb(III) competes with Zn(II) for its binding to the CCHC zinc finger domain of the HIV-1 NCp7 protein, suggesting that zinc finger proteins may be molecular targets for antimony-based drugs. Here, the interaction of Sb(III) with a CCCH zinc finger domain, which is considered to play a crucial role in the biology of kinetoplastid protozoa, has been characterized for the first time. The binding characteristics of Sb(III) were compared between a CCCH-type peptide derived from a kinetoplastid protein and two different CCHC-type zinc finger peptides. The formation of 1 : 1 Zn-peptide and Sb-peptide complexes from the different peptides was demonstrated using circular dichroism, UV absorption, fluorescence spectroscopies and ESI-MS. Titration of the Zn-peptide complexes with SbCl(3) was performed at pH 6 and 7, exploiting the intrinsic fluorescence of the peptides. The differential spectral characteristics of the peptides allowed for competition experiments between the different peptides for binding of Zn(II). The present study establishes that Sb(III) more effectively displaces Zn(II) from the CCCH peptide than CCHC ones, as a result of both the greater stability of the Sb-CCCH complex (compared to Sb-CCHC complexes) and the lower stability of the Zn-CCCH complex (compared to Zn-CCHC complexes). Comparison of the binding characteristics of Sb(III) or Zn(II) between the CCHC-type peptides with different amino acid sequences supports the model that not only the conserved zinc finger motif, but also the sequence of non-conserved amino acids determines the binding affinity of Sb(III) and Zn(II). These data suggest that the interaction of Sb(III) with CCCH-type zinc finger proteins may modulate, or even mediate, the pharmacological action of antimonial drugs.

Antimony(V) and bismuth(V) complexes of lapachol: synthesis, crystal structure and cytotoxic activity.

Antimony(V) and bismuth(V) complexes of lapachol have been synthesized by the reaction of Ph3SbCl2 or Ph3BiCl2 with lapachol (Lp) and characterized by several physicochemical techniques such as IR, and NMR spectroscopy and X-ray crystallography. The compounds contain six-coordinated antimony and bismuth atoms. The antimony(V) complex is a monomeric derivative, (Lp)(Ph3Sb)OH, and the bismuth(V) complex is a dinuclear compound bridged by an oxygen atom, (Lp)2(Ph3Bi)2O. Both compounds inhibited the growth of a chronic myelogenous leukemia cell line and the complex of Bi(V) was about five times more active than free lapachol. This work provides a rare example of an organo-Bi(V) complex showing significant cytotoxic activity.

Interaction of arsenite with a zinc finger CCHC peptide: evidence for formation of an As-Zn-peptide mixed complex.

The interaction of arsenite with a Cys(3)His (CCHC) zinc finger model (34-51) HIV-1 nucleocapsid protein p7 (NCp7) peptide in the absence and presence of Zn(II) was studied using fluorescence spectroscopy, CD (circular dichroism) and ESI-MS (Electrospray Ionization Mass Spectrometry). We found that arsenic forms different complexes with the free peptide and the zinc finger peptide. In the former case the peptide conformation differed greatly from that of the zinc finger, whereas in the second case a mixed As-Zn-peptide complex was formed with partial preservation of zinc finger conformation. An apparent stability constant was estimated for the mixed As-Zn-peptide complex (K=2083 M(-1) and 442 M(-1) at 25°C and pHs 6 and 7, respectively). Our study also shows that the interaction of arsenic with the CCHC motif is facilitated by glutathione (GSH), through formation of a GS-As-peptide conjugate.

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.

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.

Role of residual Sb(III) in meglumine antimoniate cytotoxicity and MRP1-mediated resistance.

Despite the clinical use of pentavalent antimonials for more than half a century, their metabolism in mammals and mechanisms of action and toxicity remain poorly understood. It has been proposed that the more active and toxic trivalent antimony form Sb(III) plays a critical role in their antileishmanial activity and toxicity. The aim of this work was to investigate the role of residual Sb(III) both in the antileishmanial/antitumoral activities of the pentavalent meglumine antimoniate and in the MRP1 (multidrug resistance-associated protein 1)-mediated resistance to this drug. Samples of meglumine antimoniate differing in their amount of residual Sb(III) (meglumine antimoniate synthesized either from SbCl(5) or from KSb(OH)(6) as well as commercially-available meglumine antimoniate) were evaluated in vitro and in vivo on Leishmania amazonensis infections, as well as for their cytotoxicity to normal and MRP1-overexpressing GLC4 cell lines. Although in vitro the two most effective drugs contained the highest levels of Sb(III), no correlation was found in vivo between the antileishmanial activity of meglumine antimoniate and its residual Sb(III) content, suggesting that residual Sb(III) contributes only marginally to the drug antileishmanial activity. On the other hand, the GLC4 cells growth inhibition data strongly suggests a marked contribution of residual Sb(III). Additionally, the potassium salt of antimoniate (non-complexed form of Sb(V)) was found to be more cytotoxic than meglumine antimoniate. Although MRP1-overexpressing GLC4 cells showed a marked resistance to trivalent antimonials, cross-resistance to meglumine antimoniate was observed only for the products that contained relatively high levels of Sb(III) (at least 0.03% by weight), suggesting that MRP1 mediates resistance to Sb(III) but not to Sb(V). In conclusion, our data strongly suggest that residual Sb(III) in pentavalent antimonial drugs does not contribute significantly to their antileishmanial activity, but is responsible for their cytotoxic activity against mammalian cells and the MRP1-mediated resistance to these drugs.

Thiol-induced reduction of antimony(V) into antimony(III): a comparative study with trypanothione, cysteinyl-glycine, cysteine and glutathione.

Gluthathione (GSH) has been previously shown to promote the reduction of pentavalent antimony (Sb(V)) into the more toxic trivalent antimony (Sb(II)) in the antimonial drug, meglumine antimonate. However, this reaction occurred at acidic pH (pH 5) but not at the pH of the cytosol (pH 7.2) in which GSH is encountered. The aim of the present study was to further characterize the reaction between thiols and antimonial drugs, addressing the following aspects: (i) the reducing activity of cysteine (Cys) and cysteinyl-glycine (Cys-Gly), expected to be the predominant thiols in the acidic compartments of mammalian cells; (ii) the reducing activity of trypanothione (T(SH)2), the main intracellular thiol in Leishmania parasites; (iii) the influence of the state of complexation of Sb(V) on the rate of Sb(V) reduction. We report here that Cys, Cys-Gly and T(SH)2 did promote the reduction of Sb(V) into Sb(III) at 37 degrees C. Strikingly, the initial rates of reduction of Sb(V) were much greater in the presence of Cys-Gly, Cys and T(SH)2 than in the presence of GSH. These reactions occurred at both pH 5 and pH 7 but were favored at acidic pH. Moreover, our data shows that Sb(V) is reduced more slowly in the form of meglumine antimonate than in its non-complexed form, indicating that the complexation of Sb(V) tends to slow down the rate of its reduction. In conclusion, our data supports the hypothesis that Sb(V) is reduced in vivo by T(SH)2 within Leishmania parasites and by Cys or Cys-Gly within the acidic compartments of mammalian cells.

Antimony(V) complex formation with adenine nucleosides in aqueous solution.

Despite the clinical use of pentavalent antimonial drugs for over half a century, their mode of action against leishmaniasis remains poorly understood. In this paper, we investigated the ability of Sb(V) to form in aqueous solution complexes with adenine nucleosides and deoxynucleosides, using circular dichroism (CD) and (1)H and (13)C NMR spectroscopies. We report that the ribonucleosides, adenosine (A) and adenosine monophosphate (AMP), form in water complexes with Sb(V), as evidenced by the changes induced in their CD spectra. On the other hand, 2'-deoxyadenosine (dA) did not show such a change. CD titration of the ribonucleosides with Sb(V) suggests the formation of 1:2 Sb(V)-nucleoside complexes. NMR analysis indicates that Sb(V) binds to the sugar moiety at the 2' position. Furthermore, the incubation of the antimonial drug, meglumine antimonate, with adenosine at 37 degrees C led to the transfer of Sb(V) from its original ligand to the nucleoside molecule, at acidic pH (pH 5), but not at neutral pH (7.2). Our data therefore suggests that the formation of such complexes may take place in vivo within the acidic cell compartments, including the phagolysosome of macrophage in which Leishmania resides.

Schistosomidal activity of oxamniquine encapsulated in stericallly stabilized liposomes / Atividade esquistosomicida da oxamnique encapsulada em lipossomas estabilizados estericamente

A droga esquistosomicida, oxamniquine (OXA), foi encapsulada em lipossomas estabilizados estericamente (LOXA) e avaliada em camundongos infectados por cinco semans com schistosoma mansoni. LOXA, quando administrada por via intraperitoneal, mostrou a mesma eficiência na reduçäo do número de parasitos que OXA livre. Este dado estabelece a eficiência de LOXA, mas indica também que a encapsulaçäo da OXA em lipossomas estabilizados estericamente näo resultou no aumento de sua atividade. O fato de LOXA ser mais eficiente for via intraperitoneal que por via subcutânea sugere que os lipossomas estabilizados estericamente têm que entrar rapidamente na circulaçäo sangüínea para atingir o parasito