Antithrombotic potential of Lippia alba: A mechanistic approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Lippia alba (Mill.) N.E.Br. ex Britton & P. Wilson is traditionally used in Brazil as an adjunct in the relief of mild anxiety, as an antispasmodic, and as an antidyspeptic. This medicinal species was included in the Phytotherapeutic Form of the Brazilian Pharmacopeia 2nd edition (2021) and has already been described as the most used medicinal plant in a study with patients from an Anticoagulation Clinic in Brazil. Meanwhile, no studies were found that support the safety of the use of L. alba in patients using anticoagulants, a drug with several safety limitations. AIM OF THE STUDY: Provide scientific evidence to ensure the safety of the concomitant use of L. alba and warfarin and support the management of these patients by evaluating its in vitro anticoagulant effect and chemical composition. And, as a timely complementation, evaluate the potential of this medicinal species in the development of new antithrombotics. METHODS: The chemical profile of L. alba derivatives was analyzed by chromatographic methods such as Ultra-Performance Liquid Chromatography (UPLC) coupled with electrospray ionization mass spectrometry (ESI-MS), qualitative UPLC using Diode-Array Detection, and Thin Layer Chromatography. The anticoagulant activity was evaluated by the innovative Thrombin Generation Assay by Calibrated Automated Thrombogram method and using traditional coagulometric tests: prothrombin time, activated partial thromboplastin time, and plasma fibrinogen measurement. RESULTS: Extracts and fractions prolonged the coagulation time in all the tests and reduced thrombin formation in thrombin generation assay. Coagulation times with the addition of ethanloic extract (2.26 mg/mL) was 17.78s, 46.43s and 14.25s respectively in prothrombin time, activated partial thromboplastin time and fibrinogren plasma measurement. In thrombin generation test, this same extract showed ETP as 323 nM/min compared to control (815 nM/min) with high tissue factor and 582 nM/min compared to control (1147 nM/min) using low tissue factor. Presence of flavonoids, phenylpropanoids, and triterpenes were confirmed by chromatographic methods and 13 compounds were identified by UPLC-ESI-MS. Based on these results and on the scientific literature, it is possible to propose that phenylpropanoids and flavonoids are related to the anticoagulant activity observed. CONCLUSION: The results demonstrate the in vitro anticoagulant activity of L. alba, probably due to the activation of intrinsic and extrinsic pathways. It is concluded, then, that there is a potential for interaction, which needs to be further studied, between L. alba and warfarin. Also, this medicinal species shows a great potential for use in the development of new antithrombotics.

p-Aminobenzamidine attenuates cardiovascular dysfunctions in spontaneously hypertensive rats.

AIMS: Diminazene aceturate, a putative ACE2 activator, is susceptible to cleavage resulting in the formation of p-aminobenzamidine (PAB). This study aimed to investigate the effects of PAB in addressing cardiovascular dysfunctions in spontaneously hypertensive rats (SHR). MAIN METHODS: Acute effects of PAB on mean arterial pressure (MAP), heart rate (HR), and aortic (AVC) and mesenteric vascular conductance (MVC) were evaluated in anesthetized SHR. Isolated aortic rings and the Langendorff technique were used to investigate the acute and chronic effects of PAB in the artery and heart. Chronic treatment with PAB (1 mg/kg, gavage) was carried out for 60 days. During this period, systolic blood pressure (SBP) and HR were measured by tail-cuff plethysmography. After the treatment, the left ventricle was collected for histology analyses, western blotting, and ACE2 activity. KEY FINDINGS: Bolus infusion of PAB acutely reduced MAP and increased both AVC and MVC in SHR. Additionally, PAB induced coronary and aorta vasodilation in isolated organs from Wistar and SHR in an endothelial-dependent manner. The chronic PAB treatment in SHR significantly attenuated the increase of SBP and improved the aorta vasorelaxation induced by acetylcholine and bradykinin-induced coronary vasodilation. In addition, chronic treatment with PAB attenuated the cardiomyocyte hypertrophy and extracellular matrix deposition in hearts from SHR. PAB did not alter the protein expression of the AT1, AT2, Mas, ACE, ACE2, or ACE2 activity. SIGNIFICANCE: PAB induced beneficial effects on cardiovascular dysfunctions induced by hypertension, suggesting that this molecule could be used in the development of new drugs for the treatment of cardiovascular diseases.

Nanoencapsulated Doxorubicin Prevents Mucositis Development in Mice.

Doxorubicin (DOX), a chemotherapy drug successfully used in the therapy of various types of cancer, is currently associated with the mucositis development, an inflammation that can cause ulcerative lesions in the mucosa of the gastrointestinal tract, abdominal pain and secondary infections. To increase the safety of the chemotherapy, we loaded DOX into nanostructured lipid carriers (NLCs). The NLC-DOX was characterized by HPLC, DLS, NTA, Zeta potential, FTIR, DSC, TEM and cryogenic-TEM. The ability of NLC-DOX to control the DOX release was evaluated through in vitro release studies. Moreover, the effect of NLC-DOX on intestinal mucosa was compared to a free DOX solution in C57BL/6 mice. The NLC-DOX showed spherical shape, high drug encapsulation efficiency (84.8 ± 4.6%), high drug loading (55.2 ± 3.4 mg/g) and low average diameter (66.0-78.8 nm). The DSC and FTIR analyses showed high interaction between the NLC components, resulting in controlled drug release. Treatment with NLC-DOX attenuated DOX-induced mucositis in mice, improving shortening on villus height and crypt depth, decreased inflammatory parameters, preserved intestinal permeability and increased expression of tight junctions (ZO-1 and Ocludin). These results indicated that encapsulation of DOX in NLCs is viable and reduces the drug toxicity to mucosal structures.

Essential oils of Taxandria fragrans and Melaleuca alternifolia have effective antidermatophytic activities in vitro and in vivo that are antagonised by ketoconazole and potentiated in gold nanospheres.

The investigation of the effects of three essential oils (EOs) from Taxandria fragrans (FRA), Melaleuca alternifolia (TTO) and Boswellia serrata (IF), alone and combined with ketoconazole (KTZ), and their functionalised gold nanoparticles (AuNP) against Trichophyton interdigitale both in vitro and in vivo indicated that EOs presented activity against T. interdigitale. The combination of EOs and KTZ was antagonistic. FRA, TTO, gold nanoparticles capped with T. fragrans (AuNPFRA) and gold nanoparticles capped with M. alternifolia (AuNPTTO) presented antidermatophytic activity in vivo, with the capacity to reduce fungal burden and to preserve tissue architecture; however, combination treatment with KTZ increased fungal burden and caused tissue damage. The combination of EO with KTZ exhibited antagonistic activity and was histologically harmful. In contrast, FRA, TTO, AuNPFRA and AuNPTTO are promising treatments for dermatophytosis.

Retinoic acid-loaded solid lipid nanoparticles surrounded by chitosan film support diabetic wound healing in in vivo study.

Repair of tissue damaged in diabetic wounds is essential to minimize the cases of amputation of the limbs in millions of diabetic people around the world. Although the all-trans retinoic acid (ATRA) is described as a potential wound healing agent, however its effects are controversial due to adverse reactions that may impair the wound healing during the treatment schedules. Our aim was to design and characterize an ATRA-loaded solid lipid nanoparticles surrounded by chitosan film to promote an ATRA controlled release and to evaluate its effectiveness in promoting wound healing in a diabetic mouse model. The SLN-ATRA were developed using biocompatible lipids without using organic solvent. The SLN-ATRA had high drug entrapment efficiency (98.0 %) and low polydispersity index (PDI) and average diameter, respectively, 0.24 ± 0.02 and 83.0 ± 6 nm. The transmission electron microscope (TEM) image presented that the SLN-ATRA were homogeneous in size and had spherical structures. The incorporation of SLN-ATRA in the chitosan films propitiated a homogeneous distribution of the drug and a controlled drug release. Furthermore, in vivo assay proved that chitosan films containing SLN-ATRA accelerated the closure of wounds of diabetic mice when compared to the control chitosan films without ATRA. SLN-ATRA chitosan films also reduced leukocyte infiltrate in the wound bed, improved collagen deposition, and reduced scar tissue. No sign of skin irritation was observed. These results indicated that SLN-ATRA surrounded in chitosan films are a promising candidate to treat diabetic wounds, improving tissue healing.

Use of chitosan as pharmaceutical excipient in ocular drug delivery systems: Sterilization and pharmacokinetics.

The use of chitosan as a pharmaceutical excipient in the ocular field is already established. Nevertheless, some aspects related to its ocular administration, such as sterilization and excipient's pharmacokinetics, remain unclear. So, in this study, we evaluated those two relevant aspects, related to chitosan administration in eye. We used chitosan-based ocular inserts (CI) as formulation model. CI were produced by solvent/casting method and sterilized by saturated steam. Sterilization was confirmed by direct inoculation of inserts in suitable microbiological growth media. Physicochemical characterization of inserts before and after sterilization was performed. Results suggested that, although steam sterilization changed the arrangement of the matrix, the heat and the humidity did not modify the structure of the main polymeric chain. Pharmacokinetics of CI radiolabeled with technetium-99m (99m Tc) was assessed by scintigraphic images and ex vivo biodistribution study, after ocular administration in male Wistar rats. Scintigraphic and images analysis and ex vivo biodistribution study showed that the insert remained mainly in the eye until 6 hr after administration and its degradation products began to migrate to the abdominal cavity after 18 hr. Together, these data represent an important step forward the manufacturing and the clinical application of CI in the ophthalmic field.

Thrombin generation test with the calibrated automated thrombogram and anticoagulant activity of Mentha crispa.

: Evaluate the in-vitro effect of Mentha crispa extract on blood coagulation, compare the conventional coagulometric tests with thrombin generation test (TGT), and study the qualitative micromolecular composition of M. crispa. Extract of M. crispa was incubated with plasma and used in the coagulometric tests: prothrombin and activated partial thromboplastin times, fibrinogen, and TGT. A phytochemical prospection was performed to evaluate the chemical composition of this extract. The extract was efficient in prolonging prothrombin time and activated partial thromboplastin time, and reducing fibrinogen levels and TGT parameters, indicating that the extract of M. crispa inhibited the intrinsic and extrinsic pathways of blood coagulation. The results obtained in TGT are in agreement with the results of conventional coagulometric tests and the in-vitro anticoagulant activity of M. crispa suggests that its use by patients using oral anticoagulants deserves caution.

Chitosan/hydroxyethyl cellulose inserts for sustained-release of dorzolamide for glaucoma treatment: In vitro and in vivo evaluation.

Eye drops containing hydrophilic drugs are commonly used to reduce intraocular pressure (IOP) in glaucoma patients, but compliance to the treatement is commonly reduced by frequent dosing and eventual systemic side effects. Sustained-release drug delivery systems, such as ocular inserts, can reduce dosing, limit systemic exposure, reduce side effects, and, then, improve patient adherence to therapy. Here, we developed and evaluated chitosan/hydroxyethyl cellulose-based ocular inserts for sustained release of dorzolamide, a hydrophilic drug. Dorzolamide inserts (DI) were produced by solvent/casting method and characterized by various physicochemical techniques. Pharmacokinetics studies were performed using scintigraphic images and ex vivo biodistribution. The effectiveness of inserts was tested in glaucomatous rats. Characterization studies showed that the drug strongly interacted with the polymeric matrix, but in vitro results showed that DI took only 3 h to release 75% of dorzolamide entraped. However, scintigraphic images and ex vivo biodistribution studies revealed that more than 50% of 99mTc-dorzolamide remained in the eye after 18 h of DI administration, while only about 30% of the drug remained in the eye after drops instilation. DI exerted significant hypotensive effect for two weeks, after single administration, while IOP values remained high in placebo and untreated groups. Eye drops were effective only during the treatment period. Only DI treatment prevented retinal ganglion cells death. Altogether, these findings evidenced the potential application of polymeric-based inserts for sustained release of dorzolamide in glaucoma management.

New mesalamine polymeric conjugate for controlled release: Preparation, characterization and biodistribution study.

Mesalamine (5-ASA) consists of the first-line therapy for the treatment of ulcerative colitis; however, it has low bioavailability, can cause several systemic adverse events, and has low treatment adherence due to the inconvenient dosing scheme. In this work, a new drug delivery system consisting of chondroitin sulfate linked to 5-ASA was synthesized using a carbodiimide as conjugating agent. The system was characterized by spectroscopic techniques (UV, ATR-FTIR, XRD, and NMR 1H) and thermal analysis (TG/DTG and DSC), suggesting the conjugation between the drug and the polymer. The in vitro release and the corresponding kinetics were also evaluated, revealing that approximately 40% of the drug linked was released at pH9 for up to 50h, following Higuchi's model. The conjugate did not show cytotoxicity for the human monocytic cell line at the doses tested, and an in vivo biodistribution study showed that the conjugate remained in the lower GIT for up to 8h with no uptake in the upper GIT. These data corroborate with the radiation found per segment of GIT and in blood. For this last test the conjugate was radiolabeled with Technetium-99m to allow the scintigraphy evaluation and radiation quantification. In conclusion, the polymeric conjugate was successfully synthesized and demonstrated a mucoadhesiveness on the colon as desired, thus supporting its potential use in the treatment of ulcerative colitis.

Poloxamer 407 (Pluronic(®) F127)-based polymeric micelles for amphotericin B: In vitro biological activity, toxicity and in vivo therapeutic efficacy against murine tegumentary leishmaniasis.

In the present study, a Poloxamer 407-based amphotericin B (AmpB)-containing polymeric micelles system (AmpB/M) was employed in the treatment of Leishmania amazonensis-infected BALB/c mice. Initially, the in vitro antileishmanial activity (IC50 value) of AmpB/M and B-AmpB/M (empty micelles) against stationary promastigotes and amastigotes-like forms of the parasites was determined, and results were of 1.83 ± 0.4 and 22.1 ± 0.7 µM, respectively, for the promastigotes, and of 2.27 ± 0.5 and 33.98 ± 2.6 µM, respectively, for the amastigotes-like. The cytotoxic concentration (CC50) values of these products were also evaluated, and we found the results of 119.5 ± 9.6 and 134.7 ± 10.3 µM, respectively. With these values, the selectivity index (SI) was calculated and results were of 65.3 and 5.4, respectively, for the promastigotes, and of 59.3 and 3.96, respectively, for the amastigotes-like of the parasites. Free AmpB showed IC50 values of 1.2 ± 0.3 and 2.5 ± 0.5 µM for the promastigotes and amastigotes-like, respectively, whereas the CC50 value was of 9.5 ± 0.4 µM. The SI values of this drug were of 7.9 and 3.8, respectively, for the promastigote and amastigote-like stages of the parasites. After, animals were infected and received saline or were treated subcutaneously with free AmpB, AmpB/M or B-AmpB/M. In the results, free AmpB-treated and infected mice showed reductions in their body weight, which were associated with hepatic and renal damage; however, no organic alteration was observed in the AmpB/M-treated animals. In addition, these animals showed significant reductions in their lesion average size and in the parasite burden in all evaluated infected tissue and organs, when compared to the other groups; as well as significantly higher levels of antileishmanial IFN-γ, IL-12, GM-CSF and nitrite, which were associated with low production of IL-4, IL-10 and IgG1 isotype antibodies. In conclusion, this AmpB/M system could be considered as an alternative for future studies in the treatment of tegumentary leishmaniasis.

Antileishmanial activity and mechanism of action from a purified fraction of Zingiber officinalis Roscoe against Leishmania amazonensis.

In recent years, considerable attention has been given to identify new antileishmanial products derived from medicinal plants, although, to date, no new effective compound has been recently applied to treat leishmaniasis. In the present study, the antileishmanial activity of a water extract from Zingiber officinalis Roscoe (ginger) was investigated and a purified fraction, named F10, was identified as responsible by this biological activity. The chemical characterization performed for this fraction showed that it is mainly composed by flavonoids and saponins. The water extract and the F10 fraction presented IC50 values of 125.5 and 49.8 µg/mL, respectively. Their selectivity indexes (SI) were calculated and values were seven and 40 times higher, respectively, in relation to the value found for amphotericin B, which was used as a control. Additional studies were performed to evaluate the toxicity of these compounds in human red blood cells, besides of the production of nitrite, as an indicator of nitric oxide (NO), in treated and infected macrophages. The results showed that both F10 fraction and water extract were not toxic to human cells, and they were able to stimulate the nitrite production, with values of 13.6 and 5.4 µM, respectively, suggesting that their biological activity could be due to macrophages activation via NO production. In conclusion, the present study shows that a purified fraction from ginger could be evaluated in future works as a therapeutic alternative, on its own or in association with other drugs, to treat disease caused by L. amazonensis.

Antileishmanial activity of standardized fractions of Stryphnodendron obovatum (Barbatimão) extract and constituent compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Stryphnodendron obovatum Benth. is a Brazilian tree used to treat skin ulceration, promote wound healing, and inhibit the growth of protozoa, including Trypanosoma and Leishmania species. Bioguided fractionation of the ethanol extract of S. obovatum stem bark was performed, and antileishmanial and antioxidant activities of the standardized fractions were analyzed. MATERIALS AND METHODS: Stationary-phase Leishmania amazonensis promastigotes, murine macrophages, and human red blood cells (RBCs) were exposed to plant extract, standardized fractions or isolated compounds for 48 h at 37 °C to evaluate their antiparasitic activity and cytotoxicity. The 2,2-diphenyl-1-picryl-hidrazyl assay was used to evaluate antioxidant activity. RESULTS: The S. obovatum extract and fractions showed antileishmanial and antioxidant activity; however, the organic fraction (OF) showed the best efficacy. We identified gallic acid, gallocatechin, epigallocatechin, catechin, and epigallocatechin gallate in the OF fraction. These compounds effectively inhibited L. amazonensis activity, with gallic acid, gallocatechin, and epigallocatechin gallate showing the highest selectivity. Furthermore, the evaluated compounds had no significant effect on murine macrophages and human RBCs. CONCLUSIONS: The compounds present in the S. obovatum plant bark ethanol extract may provide an alternative therapeutic approach for L. amazonensis treatment.

Propolis--based chitosan varnish: drug delivery, controlled release and antimicrobial activity against oral pathogen bacteria.

BACKGROUND: Dental caries is the most prevalent oral disease in several Asian and Latin American countries. It is an infectious disease and different types of bacteria are involved in the process. Synthetic antimicrobials are used against this disease; however, many of these substances cause unwarranted undesirable effects like vomiting, diarrhea and tooth staining. Propolis, a resinous substance collected by honeybees, has been used to control the oral microbiota. So, the objective of this study was to develop and characterize sustained-release propolis-based chitosan varnish useful on dental cariogenic biofilm prevention, besides the in vitro antimicrobial activity. METHODS: Three formulations of propolis - based chitosan varnish (PCV) containing different concentrations (5%, 10% and 15%) were produced by dissolution of propolis with chitosan on hydro-alcoholic vehicle. Bovine teeth were used for testing adhesion of coatings and to observe the controlled release of propolis associated with varnish. It was characterized by infrared spectroscopy, scanning electron microscopy, casting time, diffusion test in vitro antimicrobial activity and controlled release. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were tested for the main microorganisms involved in the cariogenic biofilm through the microdilution test in 96-well plates. RESULTS: The formulations presented a tooth surface adherence and were able to form films very fast on bovine tooth surface. Also, propolis-based chitosan varnishes have shown antimicrobial activity similar to or better than chlorhexidine varnish against all oral pathogen bacteria. All microorganisms were sensitive to propolis varnish and chitosan. MIC and MBC for microorganisms of cariogenic biofilme showed better results than chlorhexidine. Propolis active components were released for more than one week. CONCLUSION: All developed formulations turn them, 5%, 10% and 15% propolis content varnish, into products suitable for clinical application on dental caries prevention field, deserving clinical studies to confirm its in vivo activity.

An optimized nanoparticle delivery system based on chitosan and chondroitin sulfate molecules reduces the toxicity of amphotericin B and is effective in treating tegumentary leishmaniasis.

Amphotericin B (AmpB) is active against leishmaniasis, but its use is hampered due to its high toxicity observed in patients. In this study, a nanoparticles-delivery system for AmpB (NQC-AmpB), containing chitosan and chondroitin sulfate molecules, was evaluated in BALB/c mice against Leishmania amazonensis. An in vivo biodistribution study, including biochemical and toxicological evaluations, was performed to evaluate the toxicity of AmpB. Nanoparticles were radiolabeled with technetium-99m and injected in mice. The products presented a similar biodistribution in the liver, spleen, and kidneys of the animals. Free AmpB induced alterations in the body weight of the mice, which, in the biochemical analysis, indicated hepatic and renal injury, as well as morphological damage to the kidneys of the animals. In general, no significant organic alteration was observed in the animals treated with NQC-AmpB. Mice were infected with L. amazonensis and treated with the nanoparticles or free AmpB; then, parasitological and immunological analyses were performed. The NQC-AmpB group, as compared to the control groups, presented significant reductions in the lesion size and in the parasite burden in all evaluated organs. These animals presented significantly higher levels of IFN-γ and IL-12, and low levels of IL-4 and IL-10, when compared to the control groups. The NQC-AmpB system was effective in reducing the infection in the animals, and proved to be effective in diminishing the toxicity evoked by AmpB, which was observed when it was administered alone. In conclusion, NQC-AmpB could be considered a viable possibility for future studies in the treatment of leishmaniasis.

Antileishmanial activity and cytotoxicity of Brazilian plants.

Leishmaniasis is a major public health problem, and the alarming spread of parasite resistance has increased the importance of discovering new therapeutic products. The present study aimed to investigate the in vitro leishmanicidal activity from 16 different Brazilian medicinal plants. Stationary-phase promastigotes of Leishmania amazonensis and murine macrophages were exposed to 44 plant extracts or fractions for 48 h at 37°C, in order to evaluate their antileishmanial activity and cytotoxicity, respectively. The most potent extracts against L. amazonensis were the hexanic extract of Dipteryx alata (IC50 of 0.08 µg/mL), the hexanic extract of Syzygium cumini (IC50 of 31.64 µg/mL), the ethanolic and hexanic extracts of leaves of Hymenaea courbaril (IC50 of 44.10 µg/mL and 35.84 µg/mL, respectively), the ethanolic extract of H. stignocarpa (IC50 of 4.69 µg/mL), the ethanolic extract of Jacaranda caroba (IC50 of 13.22 µg/mL), and the ethanolic extract of J. cuspidifolia leaves (IC50 of 10.96 µg/mL). Extracts of D. alata and J. cuspidifolia presented higher selectivity index, with high leishmanicidal activity and low cytotoxicity in the mammalian cells. The capacity in treated infected macrophages using the extracts and/or fractions of D. alata and J. cuspidifolia was also analyzed, and reductions of 95.80%, 98.31%, and 97.16%, respectively, in the parasite burden, were observed. No nitric oxide (NO) production could be observed in the treated macrophages, after stimulation with the extracts and/or fractions of D. alata and J. cuspidifolia, suggesting that the biological activity could be due to mechanisms other than macrophage activation mediated by NO production. Based on phytochemistry studies, the classes of compounds that could contribute to the observed activities are also discussed. In conclusion, the data presented in this study indicated that traditional medicinal plant extracts present effective antileishmanial activity. Future studies could focus on the identification and purification of the antileishmanial compounds within these plants for analysis of their in vivo antileishmanial activity.

Novel targeting using nanoparticles: an approach to the development of an effective anti-leishmanial drug-delivery system.

The study reported here aimed to develop an optimized nanoparticle delivery system for amphotericin B (AmpB) using a polyelectrolyte complexation technique. For this, two oppositely charged polymers presenting anti-leishmanial activity - chitosan (Cs) and chondroitin sulfate (ChS) - were used: Cs as a positively charged polymer and ChS as a negatively charged polymer. The chitosan (NQ) nanoparticles, chitosan-chondroitin sulfate (NQC) nanoparticles, and chitosan-chondroitin sulfate-amphotericin B (NQC-AmpB) nanoparticles presented a mean particle size of 79, 104, and 136 nm, respectively; and a polydispersity index of 0.2. The measured zeta potential of the nanoparticles indicated a positive charge in their surface, while scanning and transmission electron microscopy revealed spherical nanoparticles with a smooth surface. Attenuated total reflectance-Fourier transform infrared spectroscopy analysis showed an electrostatic interaction between the polymers, whereas the release profile of AmpB from the NQC-AmpB nanoparticles showed a controlled release. In addition, the Cs; ChS; and NQ, NQC, and NQC-AmpB nanoparticles proved to be effective against promastigotes of Leishmania amazonensis and Leishmania chagasi, with a synergistic effect observed between Cs and ChS. Moreover, the applied NQ, NQC, and NQC-AmpB compounds demonstrated low toxicity in murine macrophages, as well as null hemolytic activity in type O(+) human red blood cells. Pure AmpB demonstrated high toxicity in the macrophages. The results show that cells infected with L. amazonensis and later treated with Cs, ChS, NQ, NQC, NQC-AmpB nanoparticles, or pure AmpB presented with a significant reduction in parasite number in the order of 24%, 31%, 55%, 66%, 90%, and 89%, respectively. The data presented indicate that the engineered NQC-AmpB nanoparticles could potentially be used as an alternative therapy to treat leishmaniasis, mainly due its low toxicity to mammals' cells.

Antiglaucomatous effects of the activation of intrinsic Angiotensin-converting enzyme 2.

PURPOSE: To evaluate the effects of the activation of endogenous angiotensin-converting enzyme 2 (ACE2) using the compound diminazene aceturate (DIZE) in an experimental model of glaucoma in Wistar rats. METHODS: DIZE (1 mg/kg) was administered daily, either systemically or topically, and the IOP was measured weekly. To examine the role of the Mas receptor in the effects of DIZE, the Ang-(1-7) antagonist A-779 was co-administered. Drainage of the aqueous humor was evaluated by using scintigraphy. The analysis of ACE2 expression by immunohistochemistry and the counting of retinal ganglion cells (RGCs) were performed in histologic sections. Additionally, the nerve fiber structure was evaluated by transmission electron microscopy. RESULTS: The systemic administration and topical administration (in the form of eye drops) of DIZE increased the ACE2 expression in the eyes and significantly decreased the IOP of glaucomatous rats without changing the blood pressure. Importantly, this IOP-lowering action of DIZE was similar to the effects of dorzolamide. The antiglaucomatous effects of DIZE were blocked by A-779. Histologic analysis revealed that the reduction in the number of RGCs and the increase in the expression of caspase-3 in the RGC layer in glaucomatous animals were prevented by DIZE. This compound also prevented alterations in the cytoplasm of axons in glaucomatous rats. In addition to these neuroprotective effects, DIZE facilitated the drainage of the aqueous humor. CONCLUSIONS: Our results evidence the pathophysiologic relevance of the ocular ACE2/Ang-(1-7)/Mas axis of the renin-angiotensin system and, importantly, indicate that the activation of intrinsic ACE2 is a potential therapeutic strategy to treat glaucoma.

In vitro release and characterization of chitosan films as dexamethasone carrier.

Chitosan, a biodegradable and biocompatible polysaccharide, is a potentially useful material in various fields. We produced mono and bilayer chitosan films containing dexamethasone as a drug carrier for controlled release. The chitosan drug-loaded films were produced by a casting/solvent evaporation technique using 2 wt% acetic acid solution and distilled water and they were dried at room temperature. These films were characterized by release and swelling studies, DSC and ATR-FTIR. The total profile for water absorption was similar for the types of films developed. ATR-FTIR analysis showed little change in the band position of the O--H and N--H stretching from dexamethasone and chitosan, respectively. DSC analysis from bilayer film indicates that the dexamethasone peak was shifted from 256 to 240 degrees C. These results suggested an interaction between hydroxyl and amino groups of chitosan and hydroxyl groups of dexamethasone. In the drug release studies it was observed 89.6% release from the monolayer film in 8h and 84% from the bilayer film in 4 weeks. These results suggested that the chitosan sheet prepared in this study is a promising delivery carrier for dexamethasone.