Irache, J.M. (Juan Manuel)
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- Effect of topical berberine in murine cutaneous leishmaniasis lesions(Oxford University Press, 2022) Calvo, A. (Alba); Larrea, E. (Esther); Sanmartin-Grijalba, C. (Carmen); Moreno, E. (Esther); Gonzalez-Peñas, E. (Elena); Espuelas, S. (Socorro); Aldalur, I. (Irati); Irache, J.M. (Juan Manuel)Objectives: More effective topical treatments remain an unmet need for the localized forms of cutaneous leishmaniasis (CL). The aim of this study was to evaluate the efficacy and safety of a topical berberine cream in BALB/c mice infected with Leishmania major parasites. Methods: A cream containing 0.5% berberine-β-glycerophosphate salt and 2.5% menthol was prepared. Its physicochemical and stability properties were determined. The cream was evaluated for its capacity to reduce lesion size and parasitic load as well as to promote wound healing after twice-a-day administration for 35 days. Clinical biochemical profile was used for estimating off-target effects. In vitro time-to-kill curves in L. major-infected macrophages and skin and plasma pharmacokinetics were determined, aiming to establish pharmacokinetic/pharmacodynamic relationships. Results: The cream was stable at 40°C for 3 months and at 4°C for at least 8 months. It was able to halt lesion progression in all treated mice. At the end of treatment, parasite load in the skin was reduced by 99.9% (4 log) and genes involved in the wound healing process were up-regulated compared with untreated mice. The observed effects were higher than expected from in vitro time-to-kill kinetic and plasma berberine concentrations, which ranged between 0.07 and 0.22 μM. Conclusions: The twice-a-day administration of a topical berberine cream was safe, able to stop parasite progression and improved the appearance of skin CL lesions. The relationship between drug plasma levels and in vivo effect was unclear.
- Gamma interferon loaded onto albumin nanoparticles: in vitro and in vivo activities against Brucella abortus(American Society of Microbiology, 2007) Espuelas, S. (Socorro); Gamazo, C. (Carlos); Irache, J.M. (Juan Manuel); Segura, S. (S.)The aim of this study was to evaluate the activity of gamma interferon (IFN-gamma) when it was either adsorbed onto or loaded into albumin nanoparticles. Brucella abortus-infected macrophages and infected BALB/c mice were selected as the models for testing of the therapeutic potentials of these cytokine delivery systems, in view of the well-established role of IFN-gamma-activated macrophages for the control of Brucella sp. infections. Whereas the encapsulation of IFN-gamma inside the matrix of nanoparticles completely abrogated its activity, adsorbed IFN-gamma increased by 0.75 log unit the bactericidal effect induced by RAW macrophages activated with free IFN-gamma, along with a higher level of production of nitric oxide. In infected BALB/c-mice, IFN-gamma adsorbed onto nanoparticles was also more active than free cytokine in reducing the number of bacteria in the spleens, and the effect was mediated by an increased ratio of IFN-gamma-secreting (Th1) to interleukin-4-secreting (Th2) cells. Overall, albumin nanoparticles would be suitable as carriers that target IFN-gamma to macrophages and, thus, potentiate their therapeutic activity.
- Molecular buckets: cyclodextrins for oral cancer therapy(Future Science, 2012) Ruiz-Gaton, L. (Luisa); Agüeros, M. (Maite); Huarte, J. (Judit); Espuelas, S. (Socorro); Calleja, P. (Patricia); Irache, J.M. (Juan Manuel)The oral route is preferred by patients for drug administration due to its convenience, resulting in improved compliance. Unfortunately, for a number of drugs (e.g., anticancer drugs), this route of administration remains a challenge. Oral chemotherapy may be an attractive option and especially appropriate for chronic treatment of cancer. However, this route of administration is particularly complicated for the administration of anticancer drugs ascribed to Class IV of the Biopharmaceutical Classification System. This group of compounds is characterized by low aqueous solubility and low intestinal permeability. This review focuses on the use of cyclodextrins alone or in combination with bioadhesive nanoparticles for oral delivery of drugs. The state-of-the-art technology and challenges in this area is also discussed.
- Protective passive immunity in escherichia coli ETEC-challenged neonatal mice conferred by orally immunized dams with nanoparticles containing homologous outer membrane vesicles(MDPI AG, 2020) Gamazo, C. (Carlos); Pastor, Y. (Yadira); Matías, J. (Jose); Irache, J.M. (Juan Manuel)Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of illness and death in mammals, including neonatal, recently weaned pigs and infant human beings. We have previously shown that outer membrane vesicles (OMV) obtained from ETEC serotypes encapsulated into zein nanoparticles, coated with a Gantrez-mannosamine polymer conjugate (OMV-NP), were immunogenic in mice and sows. In the present study, we show that pups from vaccinated mice were protected against ETEC F4 serotype challenge through maternal passive immunization. OMV from F4 cultures were collected and characterized. Two-week-pregnant BALB/c mice were orally immunized with a single dose of vesicles (0.2 mg) either free (OMV) or encapsulated into nanoparticles (OMV-NP). Evaluation of the antibodies in serum (IgG1, Ig2a or IgA) and feces (IgA) of dams immunized with OMV-NP revealed an enhancement of specific immunogenicity. The antibody response conferred by the nanoparticle adjuvant was also correlated with IL-6 and IL-10 splenic levels. Each mother was allowed to feed her progeny for one week. Suckling pups presented specific IgA in feces demonstrating their passive immunization through colostrum intake. Two weeks after the pups were born, they were infected orally with a single dose of F4 E. coli (1.2 × 108 CFU/pup). Results showed that 70% of the pups from dams immunized with OMV-NP were protected. In contrast, 80% of the pups from dams immunized with free OMV died as a result of the experimental challenge. These findings support the use of zein nanoparticles coated with a Gantrez-mannosamine shield as adjuvant delivery system for the oral immunization during pregnancy to confer immunity to the offspring through maternal immunization
- Humoral immune response in hens naturally infected with Salmonella Enteritidis against outer membrane proteins and other surface structural antigens(EDP Sciences, 2004) Ochoa-Repáraz, J. (Javier); Sesma, B. (Begoña); Gamazo, C. (Carlos); Alvarez, M. (Miguel); Renedo, M.J. (María Jesús); Irache, J.M. (Juan Manuel)A simple procedure for obtaining surface exposed antigens of Salmonella Enteritidis is described. A heat treatment of whole bacteria in saline solution induced the release of small membrane vesicles containing outer membrane components as well as surface appendage components, such as fimbriae and flagellin. The characterization of the structural components of this extract, called HE, was established by SDS-PAGE and immunoblotting using polyclonal and monoclonal specific antibodies. Five major groups of proteins were identified: flagellin, porins, OmpA, SEF21 and SEF14 fimbriae. The immunogenicity of these proteins was studied by immunoblotting with serum samples from naturally infected hens. Flagellin, porins, OmpA, SEF14 and SEF21 fimbriae were immunogenic in the S. Enteritidis infected hens (frequency of reactants: 47.3, 97.3, 64.7, 50.0 and 60.8%, respectively); porins also reacted with sera from non infected hens (66.7%). The immunogenicity of these antigens in infected birds provide promise that they may serve as components of an effective subcellular vaccine for poultry salmonellosis.
- Assesment of β-lapachone loaded in lecithin-chitosan nanoparticles for the topical treatment of cutaneous leishmaniasis in L. major infected BALB/c mice(Elsevier, 2015) Larrea, E. (Esther); Sanmartin-Grijalba, C. (Carmen); Font, M. (María); Schwartz, J. (Juana); Conde, I. (Iosune); Espuelas, S. (Socorro); Moreno-Amatria, E. (Esther); Irache, J.M. (Juan Manuel)Abstract Patients affected by cutaneous leishmaniasis need a topical treatment which cures lesions without leaving scars. Lesions are produced not only by the parasite but also by an uncontrolled and persistent inflammatory immune response. In this study, we proposed the loading of β-lapachone (β- LP) in lecithin-chitosan nanoparticles (NP) for targeting the drug to the dermis, where infected macrophages reside, and promote wound healing. The loading of β-LP in lecithin-chitosan NP was critical to achieve important drug accumulation in the dermis and permeation through the skin. In addition, it did not influence the drug antileishmanial activity. When topically applied in L. major infected BALB/c mice, 2 β-LP NP achieved no parasite reduction but they stopped the lesion progression. Immuno-histopatological assays in CL lesions and quantitative mRNA studies in draining lymph nodes confirmed that β-LP exhibited anti-inflammatory activity leading to the downregulation of IL-1β and COX-2 expression and a decrease of neutrophils infiltrate.
- Poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as innate immune system activators(Elsevier, 2011) Mansilla, C. (Cristina); Camacho, A.I. (A.I.); Souza, J. (Juliana) de; Gamazo, C. (Carlos); Tamayo, I. (Ibai); Esparza, I. (Irene); Irache, J.M. (Juan Manuel); Raquel; Lasarte, J.J. (Juan José)Adjuvant research is being oriented to TLR-agonists, but complement activation has been relatively unexplored. In previous studies it was demonstrated that poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (PVMA NPs) used as adjuvant differentially activate dendritic cells through toll like receptors (TLR) stimulation, however, a high dose of these NPs was used. Now, we demonstrated a dose-response effect, with a concentration as low as 20μg/mL able to stimulate TLR2 and TLR4 transfected dendritic cells. In addition, we investigated whether PVMA NPs are able to exploit also the immunomodulatory benefits of complement activation. Results indicated that the hydroxylated surface of these NPs highly activated the complement cascade, as measured by adsorption studies and a complement fixation bioassay. Stable binding of C3b to NPs was confirmed as indicated by lability to SDS treatment after washing resistance. Complement consumption was confirmed as the lytic capacity of complement exposed to NPs was abolished against antibody-sensitized sheep erythrocytes, with a minimal inhibitory concentration of 50μg NPs, equivalent to a surface of 1cm(2). On the contrary, nanoparticles prepared with poly(lactic-co-glycolic acid) (PLGA), used as a reference, did not consume complement at a concentration ≥3mg NPs (≥40cm(2)). Complement consumption was inhibited when PVMA NPs were cross-linked with diamino groups (1,3-diaminopropane), indicating the role of hydroxyl groups as responsible of the phenomenon. These results favour a model whereby PVMA NPs adjuvant activate complement on site to attract immature antigen presenting cells that are activated through TLR2 and TLR4.
- Polymeric carriers for amphotericin B: in vitro activity, toxicity and therapeutic efficacy against systemic candidiasis in neutropenic mice(Oxford University Press, 2003) Campanero, M.A. (Miguel Angel); Espuelas, S. (Socorro); Gamazo, C. (Carlos); Barratt, G. (G.); Legrand, P. (P.); Cheron, M. (M.); Irache, J.M. (Juan Manuel); Appel, M. (M.)Objective: To study the toxicity and activity of two new amphotericin B formulations: poly(ε-caprolactone) nanospheres coated with poloxamer 188 (AmB-NP) and mixed micelles with the same surfactant (AmB-MM). Materials and methods: The toxicity of these formulations was evaluated in erythrocytes, J774.2 macrophages and LLCPK1 renal cells, as well as in mice. Activity was determined in clinical isolates and in neutropenic mice. Mice were made neutropenic with 5-fluorouracil, infected with Candida albicans and treated with the antifungal formulations for three consecutive days. AmB association in cells and accumulation in kidneys and liver of animals was quantified by HPLC. Results: Both formulations decreased between 8- and 10-fold the MIC of the polyene against clinical isolates of C. albicans. However, their activity was lower than or equal to that of AmB-deoxycholate when it was assessed against C. albicans-infected macrophages. When given as a single intravenous dose in mice, AmB-MM and AmB-NP had an LD50 of 9.8 and 18.6 mg/kg, respectively, compared with 4 mg/kg for AmBdeoxycholate. Comparison of residual infection burdens in the liver and kidneys showed that AmB-deoxycholate (0.5 mg/kg) was more effective and faster in eradicating yeast cells than polymeric formulations. This fact can be related to a lower AmB accumulation inside macrophages and in liver and kidneys (about 1.5 mg drug/g tissue) of mice, compared with those detected for AmB-deoxycholate (4 mg drug/g). Overall, the efficacy of these formulations at 2 mg/kg was equal to that of AmB-deoxycholate at 0.5 mg/kg. Conclusions: AmB-MM and AmB-NP decreased the in vivo antifungal activity of AmB, and higher concentrations were therefore necessary to obtain a similar therapeutic effect. However, these higher concentrations were achievable owing to the reduced toxicity of these formulations.
- Poly(Anhydride) Nanoparticles Act as Active Th1 Adjuvants through Toll-Like Receptor Exploitation(American Society of Microbiology, 2010) Ochoa-Repáraz, J. (Javier); Mansilla, C. (Cristina); Gamazo, C. (Carlos); Tamayo, I. (Ibai); Irache, J.M. (Juan Manuel); Lasarte, J.J. (Juan José)The mechanisms that underlie the potent Th1-adjuvant capacity of poly(methyl vinyl ether-co-maleic anhydride) nanoparticles (NPs) were investigated. Traditionally, polymer NPs have been considered delivery systems that promote a closer interaction between antigen and antigen-presenting cells (APCs). Our results revealed that poly(anhydride) NPs also act as agonists of various Toll-like receptors (TLRs) (TLR2, -4, and -5), triggering a Th1-profile cytokine release (gamma interferon [IFN- ], 478 pg/ml versus 39.6 pg/ml from negative control; interleukin-12 [IL-12], 40 pg/ml versus 7.2 pg/ml from negative control) and, after incubation with dendritic cells, inducing a 2.5- to 3.5-fold increase of CD54 and CD86 costimulatory molecule expression. Furthermore, in vivo studies suggest that NPs actively elicit a CD8 T-cell response. Immunization with empty NPs resulted in a significant delay in the mean survival date (from day 7 until day 23 postchallenge) and a protection level of 30% after challenge against a lethal dose of Salmonella enterica serovar Enteritidis. Taken together, our results provide a better understanding of how NPs act as active Th1 adjuvants in immunoprophylaxis and immunotherapy through TLR exploitation.
- Casein nanoparticles in combination with 2-hydroxypropyl-β-cyclodextrin improves the oral bioavailability of quercetin(Elsevier BV, 2019) Morales, J. (Jorge); Gonzalez-Navarro, C.J. (Carlos Javier); Larrañeta, E. (Eneko); Peñalva, R. (Rebeca); Esparza, I. (Irene); Irache, J.M. (Juan Manuel)The aim of this work was to optimize the preparative process of quercetin loaded casein nanoparticles as well as to evaluate the pharmacokinetics of this flavonoid when administered orally in Wistar rats. Nanoparticles were obtained by coacervation after the incubation of casein, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) and quercetin in an aqueous environment. Then, nanoparticles were purified and dried. The resulting nanoparticles displayed a size of 200 nm with a negative zeta potential and a payload of about 32 μg/mg. Release studies showed a zero-order kinetic, suggesting a mechanism based on erosion of the nanoparticle matrix. For the pharmacokinetic study, quercetin was orally administered to rats as a single dose of 25 mg/kg. Animals treated with quercetin-loaded casein nanoparticles displayed higher plasma levels than those observed in animals receiving the solution of the flavonoid (control). Thus, the relative oral bioavailability of quercetin when administered as casein nanoparticles (close to 37%) was found to be about 9-times higher than the oral solution of the flavonoid in a mixture of PEG 400 and water. In summary, the combination of casein and 2-hydroxypropyl-β-cyclodextrin produces nanoparticles that may be a good option to load quercetin for both nutraceutical and pharmaceutical purposes.