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BACKGROUND: Tuberculous meningitis (TBM) is difficult to diagnose. We investigated whether a 3-gene host response signature in blood can distinguish TBM from other brain infections. METHODS: The expression of 3 genes (dual specificity phosphatase 3 [DUSP3], guanylate-binding protein [GBP5], krupple-like factor 2 [KLF2]) was analyzed by RNA sequencing of archived whole blood from 4 cohorts of Vietnamese adults: 281 with TBM, 279 with pulmonary tuberculosis, 50 with other brain infections, and 30 healthy controls. Tuberculosis scores (combined 3-gene expression) were calculated following published methodology and discriminatory performance compared using area under a receiver operator characteristic curve (AUC). RESULTS: GBP5 was upregulated in TBM compared to other brain infections (P < .001), with no difference in DUSP3 and KLF2 expression. The diagnostic performance of GBP5 alone (AUC, 0.74; 95% confidence interval [CI], .67-.81) was slightly better than the 3-gene tuberculosis score (AUC, 0.66; 95% CI, .58-.73) in TBM. Both GBP5 expression and tuberculosis score were higher in participants with human immunodeficiency virus (HIV; P < .001), with good diagnostic performance of GBP5 alone (AUC, 0.86; 95% CI, .80-.93). CONCLUSIONS: The 3-gene host signature in whole blood has the ability to discriminate TBM from other brain infections, including in individuals with HIV. Validation in large prospective diagnostic study is now required.
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Tuberculosis Meníngea , Humanos , Tuberculosis Meníngea/diagnóstico , Tuberculosis Meníngea/sangre , Tuberculosis Meníngea/genética , Masculino , Femenino , Adulto , Persona de Mediana Edad , Proteínas de Unión al GTP/genética , Factores de Transcripción de Tipo Kruppel/genética , Diagnóstico Diferencial , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/sangre , Biomarcadores/sangre , Adulto Joven , Vietnam , Curva ROCRESUMEN
Peptide therapeutics offer numerous advantages in the treatment of diseases and disorders of the central nervous system (CNS). However, they are not without limitations, especially in terms of their pharmacokinetics where their metabolic lability and low blood-brain barrier penetration hinder their application. Targeted nanoparticle delivery systems are being tapped for their ability to improve the delivery of therapeutics into the brain non-invasively. We have developed a family of mannosylated glycoliposome delivery systems for targeted drug delivery applications. Herein, we demonstrate via in vivo distribution studies the potential of these glycoliposomes to improve the utility of CNS active therapeutics using dynantin, a potent and selective dynorphin peptide analogue antagonist of the kappa opioid receptor (KOR). Glycoliposomal entrapment protected dynantin against known rapid metabolic degradation and ultimately improved brain levels of the peptide by approximately 3-3.5-fold. Moreover, we linked this improved brain delivery with improved KOR antagonist activity by way of an approximately 30-40% positive modulation of striatal dopamine levels 20 min after intranasal administration. Overall, the results clearly highlight the potential of our glycoliposomes as a targeted delivery system for therapeutic agents of the CNS.
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Dinorfinas , Péptidos , Receptores Opioides kappa/antagonistas & inhibidores , Cuerpo Estriado/metabolismo , Dopamina , Dinorfinas/química , Dinorfinas/farmacocinética , Dinorfinas/farmacología , Humanos , Liposomas , Péptidos/química , Péptidos/farmacocinética , Péptidos/farmacologíaRESUMEN
The two-machine permutation flow shop scheduling problem with buffer is studied for the special case that all processing times on one of the two machines are equal to a constant c. This case is interesting because it occurs in various applications, for example, when one machine is a packing machine or when materials have to be transported. Different types of buffers and buffer usage are considered. It is shown that all considered buffer flow shop problems remain NP-hard for the makespan criterion even with the restriction to equal processing times on one machine. However, the special case where the constant c is larger or smaller than all processing times on the other machine is shown to be polynomially solvable by presenting an algorithm (2BF-OPT) that calculates optimal schedules in O(nlogn) steps. Two heuristics for solving the NP-hard flow shop problems are proposed: (i) a modification of the commonly used NEH heuristic (mNEH) and (ii) an Iterated Local Search heuristic (2BF-ILS) that uses the mNEH heuristic for computing its initial solution. It is shown experimentally that the proposed 2BF-ILS heuristic obtains better results than two state-of-the-art algorithms for buffered flow shop problems from the literature and an Ant Colony Optimization algorithm. In addition, it is shown experimentally that 2BF-ILS obtains the same solution quality as the standard NEH heuristic, however, with a smaller number of function evaluations.
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Algoritmos , HeurísticaRESUMEN
The addictive nature of nicotine is likely the most significant reason for the continued prevalence of tobacco smoking despite the widespread reports of its negative health effects. Nicotine vaccines are an alternative to the currently available smoking cessation treatments, which have limited efficacy. However, the nicotine hapten is non-immunogenic, and successful vaccine formulations to treat nicotine addiction require both effective adjuvants and delivery systems. The immunomodulatory properties of short, non-natural peptide sequences not found in human systems and their ability to improve vaccine efficacy continue to be reported. The aim of this study was to determine if small "non-natural peptides," as part of a conjugate nicotine vaccine, could improve immune responses. Four peptides were synthesized via solid phase methodology, purified, and characterized. Ex vivo plasma stability studies using RP-HPLC confirmed that the peptides were not subject to proteolytic degradation. The peptides were formulated into conjugate nicotine vaccine candidates along with a bacterial derived adjuvant vaccine delivery system and chitosan as a stabilizing compound. Formulations were tested in vitro in a dendritic cell line to determine the combination that would elicit the greatest 1L-1ß response using ELISAs. Three of the peptides were able to enhance the cytokine response above that induced by the adjuvant delivery system alone. In vivo vaccination studies in BALB/c mice demonstrated that the best immune response, as measured by nicotine-specific antibody levels, was elicited from the conjugate vaccine structure, which included the peptide, as well as the other components. Isotype analyses highlighted that the peptide was able to shift immune response toward being more humorally dominant. Overall, the results have implications for the use of non-natural peptides as adjuvants not only for the development of a nicotine vaccine but also for use with other addictive substances and conventional vaccination targets as well.
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Nicotina/inmunología , Trastornos Relacionados con Sustancias/inmunología , Tabaquismo/inmunología , Vacunas de Subunidad/farmacología , Adyuvantes Inmunológicos/farmacología , Animales , Cromatografía Líquida de Alta Presión , Sistemas de Liberación de Medicamentos , Haptenos/efectos de los fármacos , Haptenos/inmunología , Humanos , Inmunidad/efectos de los fármacos , Inmunidad/inmunología , Interleucina-1beta/genética , Ratones , Nicotina/metabolismo , Péptidos/inmunología , Péptidos/farmacología , Trastornos Relacionados con Sustancias/genética , Trastornos Relacionados con Sustancias/patología , Trastornos Relacionados con Sustancias/prevención & control , Tabaquismo/genética , Tabaquismo/prevención & control , Vacunas Conjugadas/inmunología , Vacunas Conjugadas/farmacología , Vacunas de Subunidad/inmunologíaRESUMEN
BACKGROUND: Nattokinases/Subtilisins (EC 3.4.21.62) belong to the second large family of serine proteases, which gain significant attention and play important role in many biotechnology processes. Thus, a number of nattokinases/subtilisins from various Bacillus species, especially from B. subtilis strains, extensively have been investigated to understand their biochemical and physical properties as well as to improve the production for industrial application. The purpose of this study was to clone a nattokinase gene from Bacillus subtilis strain VTCC-DVN-12-01, enhance its production in B. subtilis WB800, which is deficient in eight extracellular proteases and characterize its physicochemical properties for potential application in organic synthesis and detergent production. RESULTS: A gene coding for the nattokinase (Nk) from B. subtilis strain VTCC-DVN-12-01 consisted of an ORF of 1146 nucleotides, encoding a pre-pro-protein enzyme (30-aa pre-signal peptide, 76-aa pro-peptide and 275-aa mature protein with a predicted molecular mass of 27.7 kDa and pI 6.6). The nattokinase showed 98-99% identity with other nattokinases/subtilisins from B. subtilis strains in GenBank. Nk was expressed in B. subtilis WB800 under the control of acoA promoter at a high level of 600 mg protein per liter culture medium which is highest yield of proteins expressed in any extracellular-protease-deficient B. subtilis system till date. Nk was purified to homogeneity with 3.25 fold purification, a specific activity of 12.7 U/mg, and a recovery of 54.17%. The purified Nk was identified by MALDI-TOF mass spectrometry through three peptides, which showed 100% identity to corresponding peptides of the B. subtilis nattokinase (CAC41625). An optimal activity for Nk was observed at 65 °C and pH 9. The nattokinase was stable at temperature up to 50 °C and in pH range of 5-11 and retained more than 85% of its initial activity after incubation for 1 h. Mg2+ activated Nk up to 162% of its activity. The addition of Triton X-100, Tween 20, and Tween 80 showed an activation of Nk up to 141% of its initial activity but SDS strongly inhibited. The enzyme was highly resistant to organic solvents. CONCLUSIONS: Our findings demonstrated that an eight-protease-gene-deficient Bacillus subtilis WB800 could overproduce the nattokinase from B. subtilis VTCC-DVN-12-01. Due to high resistance to detergents and organic solvents of this nattokinase, it could be potentially applied in organic synthesis and detergent production.
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Bacillus subtilis/enzimología , Bacillus subtilis/genética , Proteínas Bacterianas/biosíntesis , Detergentes/metabolismo , Subtilisinas/biosíntesis , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación de Organismos , Electroforesis en Gel de Poliacrilamida , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solventes/metabolismo , Subtilisinas/química , Subtilisinas/genética , Subtilisinas/metabolismoRESUMEN
Many traditional cancer treatments such as radiation and chemotherapy are known to induce cellular DNA damage as part of their cytotoxic activity. The cGAS-STING signaling axis, a key member of the DNA damage response that acts as a sensor of foreign or aberrant cytosolic DNA, is helping to rationalize the DNA-damaging activity of these treatments and their emerging immunostimulatory capacity. Moreover, cGAS-STING, which is attracting considerable attention for its ability to promote antitumor immune responses, may fundamentally be able to address many of the barriers limiting the success of cancer immunotherapy strategies, including the immunosuppressive tumor microenvironment. Herein, we review the traditional cancer therapies that have been linked with cGAS-STING activation, highlighting their targets with respect to their role and function in the DNA damage response. As part of the review, an emerging "chemoimmunotherapy" concept whereby DNA-damaging agents are used for the indirect activation of STING is discussed as an alternative to the direct molecular agonism strategies that are in development, but have yet to achieve clinical approval. The potential of this approach to address some of the inherent and emerging limitations of cGAS-STING signaling in cancer immunotherapy is also discussed. Ultimately, it is becoming clear that in order to successfully employ the immunotherapeutic potential of the cGAS-STING axis, a balance between its contrasting antitumor and protumor/inflammatory activities will need to be achieved.
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OBJECTIVES: We investigate the anticancer activity and human stimulator of interferon genes pathway activation by a new hydrated-prenylated tetraoxygenated xanthone, garcicowanone I (1) and two known xanthones (2 and 3) that were isolated from the root bark of Garcinia cowa Roxb. ex Choisy. METHODS: The anticancer activity of each compound was evaluated by sulforhodamine B assay in immortalized cancer cell lines. Stimulator of interferon genes pathway activation was assessed by western blot analysis using human THP-1-derived macrophages. The production of pro-inflammatory cytokines from these macrophages was also evaluated via enzyme-linked immunosorbent assay. KEY FINDINGS: Both compounds 1 and 3 displayed moderate inhibitory effects on the cancer cells, including a cisplatin-resistant cell line, with IC50 values in the range of 10-20 µM. All three xanthones activated the stimulator of interferon genes, as evidenced by phosphorylation of tank-binding kinase 1, the stimulator of interferon genes protein and interferon regulatory factor 3. Furthermore, treatment of these macrophages with compounds 1-3 led to the production of pro-inflammatory cytokines, including interleukin 6, tumour necrosis factor α and interleukin 1ß. CONCLUSIONS: In conclusion, the isolated xanthones, including the novel garcicowanone I, displayed promising anticancer and immunomodulatory activity that warrants further research.
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Garcinia , Xantonas , Humanos , Garcinia/química , Xantonas/farmacología , Xantonas/química , Extractos Vegetales/farmacología , Extractos Vegetales/química , Línea Celular , Interferones , Estructura MolecularRESUMEN
Perhaps the greatest limitation for the continually advancing developments in cancer immunotherapy remains the immunosuppressive tumor microenvironment (TME). The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) axis is an emerging immunotherapy target, with the resulting type I interferons and transcription factors acting at several levels in both tumor and immune cells for the generation of adaptive T cell responses. The cGAS-STING axis activation by therapeutic agents that induce DNA damage, such as certain chemotherapies, continues to be reported, highlighting the importance of the interplay of this signaling pathway and the DNA damage response in cancer immunity/immunotherapy. We have developed a multi-targeted mannosylated cationic liposomal immunomodulatory system (DS) which contains low doses of the chemotherapeutic cytarabine (Ara-C). In this work, we show that entrapment of non-cytotoxic doses of Ara-C within the DS improves its ability to induce DNA double strand breaks in human ovarian and colorectal cancer cell lines, as well as in various immune cells. Importantly, for the first time we demonstrate that the DNA damage induced by Ara-C/DS translates into cGAS-STING axis activation. We further demonstrate that Ara-C/DS-mediated DNA damage leads to upregulation of surface expression of immune ligands on cancer cells, coinciding with priming of cytotoxic lymphocytes as assessed using an ex vivo model of peripheral blood mononuclear cells from colorectal cancer patients, as well as an in vitro NK cell model. Overall, the results highlight a broad immunotherapeutic potential for Ara-C/DS by enhancing tumor-directed inflammatory responses.
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Glycosylated NPs, including liposomes, are known to target various receptors involved in cellular carbohydrate transport, of which the mannoside binding receptors are attracting particular attention for their expression on various immune cells, cancers, and cells involved in maintaining central nervous system (CNS) integrity. As part of our interest in NP drug delivery, mannosylated glycoliposomal delivery systems formed from the self-assembly of amphiphilic neoglycolipids were developed, with a C12-alkyl mannopyranoside (ML-C12) being identified as a lead compoundcapable of entrapping, protecting, and improving the delivery of structurally diverse payloads. However, ML-C12 was not without limitations in both the synthesis of the glycolipids, and the physicochemical properties of the resulting glycoliposomes. Herein, the chemical syntheses of a novel series of mannosylated neoglycolipids are reported with the goal of further improving on the previous ML-C12 glyconanoparticles. The current work aimed to use a self-contingent strategy which overcomes previous synthetic limitations to produce neoglycolipids that have one exposed mannose residue, an aromatic scaffold, and two lipid tails with varied alkyl chains. The azido-ending carbohydrates and the carboxylic acid-ending lipid tails were ligated using a new one-pot modified Staudinger chemistry that differed advantageously to previous syntheses. The formation of stable neoglycoliposomes of controllable and ideal sizes (≈100-400 nm) was confirmed via dynamic light scattering (DLS) experiments and transmission electron microscopy (TEM). Beyond chemical advantages, the present study further aimed to establish potential improvements in the biological activity of the neoglycoliposomes. Concanavalin A (Con A) agglutination studies demonstrated efficient and stable cross-linking abilities dependent on the length of the linkers and lipid tails. The efficacy of the glycoliposomes in improving cytosolic uptake was investigated using Nile Red as probe in immune and cancer cell lines. Preliminary ex vivo safety assessments showed that the mannosylated glycoliposomes are hemocompatible, and non-immunogenic. Finally, using a model peptide therapeutic, the relative entrapment capacity and plasma stability of the optimal glycoliposome delivery system was evaluated and compared to the previous neoglycoliposomes. Overall, the new lead glycoliposome showed improved biological activity over ML-C12, in addition to having several chemical benefits including the lack of stereocenters, a longer linker allowing better sugar availability, and ease of synthesis using novel one-pot modified Staudinger chemistry.
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Tobacco smoking continues to be a global epidemic and the leading preventable cause of cancer and cardiovascular disease. Nicotine vaccines have been investigated as an alternative to currently available smoking cessation strategies as a means to increase rates of success and long-term abstinence. Recently, we demonstrated that a mucosal nicotine vaccine was able to induce robust mucosal and systemic antibodies when delivered heterologously using intranasal and intramuscular routes. Herein, we investigated the neutralization ability of the anti-nicotine antibodies using both intranasal and intracardiac nicotine challenges. Combining the extraction of lyophilized organ samples with RP-HPLC methods, we were able to recover between 47% and 56% of the nicotine administered from the blood, brain, heart, and lungs up to 10 min after challenge, suggesting that the interaction of the antibodies with nicotine forms a stable complex independently of the route of vaccination or challenge. Although both challenge routes can be used for assessing systemic antibodies, only the intranasal administration of nicotine, which is more physiologically similar to the inhalation of nicotine, permitted the crucial interaction of nicotine with the mucosal antibodies generated using the heterologous vaccination route. Notably, these results were obtained 6 months after the final vaccination, demonstrating stable mucosal and systemic antibody responses.
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Cancer treatment remains unsatisfactory with high rates of recurrence and metastasis. Immunomodulatory agents capable of promoting cellular antitumor immunity while inhibiting the local immunosuppressive tumor microenvironment could greatly improve cancer treatment. We have developed a multi-targeted mannosylated cationic liposome delivery system containing muramyl dipeptide (DS) and low doses of the chemotherapeutic agent cytarabine (Ara-C). Immunomodulation of primary immune cells and immortalized cancer cell lines by Ara-C/DS was assessed by measuring cytokine levels and surface marker expression. As a proof of concept, the generation of targeted cellular immunity was investigated in the context of responses to viral antigens. This report is the first demonstrating that Ara-C combined with DS can modulate immune responses and revert immunosuppression as evidenced by increased IFN-γ and IL-12p40 without changes in IL-10 in peripheral blood mononuclear cells, and increased CD80 and decreased CD163 on immunosuppressive macrophages. Furthermore, Ara-C/DS increased MHC class I expression on cancer cells while increasing the production of antigen-specific IFN-γ+ CD8+ T cells in viral peptide-challenged lymphocytes from both humans and vaccinated mice. Taken together, these results are the first to document immunomodulatory properties of Ara-C linked with recognition of antigens and potentially the generation of antitumor immune memory.
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Citarabina , Liposomas , Animales , Linfocitos T CD8-positivos , Inmunidad Celular , Inmunomodulación , Leucocitos Mononucleares , RatonesRESUMEN
Dynantin is a potent and selective synthetic polypeptide kappa opioid receptor antagonist which has potential antidepressant and anxiolytic-like therapeutic applications, however its clinical development has been hampered by plasma stability issues and poor penetration of the blood brain barrier. Targeted liposome delivery systems represent a promising and non-invasive approach to improving the delivery of therapeutic agents across the blood brain barrier. As part of our work focused on targeted drug delivery, we have developed a novel mannosylated liposome system. Herein, we investigate these glycoliposomes for the targeted delivery of dynantin to the central nervous system. Cholesterol was tested and optimized as a formulation excipient, where it improved particle stability as measured via particle size, entrapment and ex vivo plasma stability of dynantin. The in vitro PRESTO-TANGO assay system was used to confirm that glycoliposomal entrapment did not impact the affinity or activity of the peptide at its receptor. Finally, in vivo distribution studies in mice showed that the mannosylated glycoliposomes significantly improved delivery of dynantin to the brain. Overall, the results clearly demonstrate the potential of our glycoliposomes as a targeted delivery system for therapeutic agents of the central nervous system.
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Encéfalo/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Manosa/metabolismo , Antagonistas de Narcóticos/metabolismo , Receptores Opioides kappa/antagonistas & inhibidores , Receptores Opioides kappa/metabolismo , Administración Intranasal , Animales , Encéfalo/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Liposomas , Manosa/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Antagonistas de Narcóticos/administración & dosificaciónRESUMEN
Situs inversus totalis is a condition where there is a transposition of all internal organs from their normal anatomical location. This infrequent and rare congenital condition has been described in several species of mammals. Dextorcardia is a series of conditions associated with an abnormal congenital positioning of the heart, and is often associated with situs inversus totalis. Here we report a case of situs inversus totalis and two cases of dextrocardia identified in Sprague Dawley rats during gross necropsy evaluations at both the Health Sciences North Research Institute (HSNRI) in Canada and Finlay Institute of Vaccine Research and Production in Cuba. The intent of this report is to share our findings and aid in the accumulation of data on these rare conditions.
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Despite the increased risks of cancers and cardiovascular related diseases, tobacco smoking continues to be prevalent in the population due largely in part to the addictive nature of nicotine. Nicotine vaccines are an attractive alternative to the current smoking cessation options but have yet to be successful enough in clinical trials to reach the market due to a lack of neutralizing antibodies and inconsistent results. Using AFPL1 derived from the Cuban meningococcal vaccine as an adjuvant, we have previously published promising results with an intranasally administered nicotine vaccine. In order to examine the immunogenicity and safety of this vaccine in mice we set up a pilot trial administering the vaccine either intranasally, intramuscularly or utilizing both routes simultaneously and evaluated immune responses and clinical symptoms throughout the duration of the vaccination protocol and post-mortem. These data further demonstrate the ability of the AFPL1 nicotine conjugate vaccine to be a safe and potential candidate for clinical use.
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Adyuvantes Inmunológicos/farmacología , Nicotina/inmunología , Proteolípidos/inmunología , Vacunación , Vacunas Conjugadas/inmunología , Animales , Anticuerpos/inmunología , Femenino , Ratones Endogámicos BALB C , Membrana Mucosa/efectos de los fármacos , Membrana Mucosa/inmunología , Músculos/efectos de los fármacos , Tamaño de los Órganos/efectos de los fármacos , Bazo/efectos de los fármacosRESUMEN
Tobacco smoking is the cause of 20% of Canadian deaths per year. Nicotine vaccines present a promising alternative to traditional smoking cessation products, but to date, no vaccine has been able to move through all phases of clinical trials. We have previously demonstrated that the AFPL1-conjugate nicotine vaccine does not induce systemic or immunotoxicity in a mouse model and that a heterologous vaccination approach is more advantageous than the homologous routes to inducing mucosal and systemic anti-nicotine antibodies. The purpose of this study was to confirm the safety profile of the vaccine in a repeat-dose toxicity study. The heterologous vaccination strategy was again used, and Sprague Dawley rats were administered a dose five times greater than in our previous studies. Physiological conditions, food and water consumption, body temperature, injection site inflammation, relative weights of organs, histopathology, and blood chemistry and hematology were evaluated during the course of the vaccination period to determine the safety of the vaccine. The AFPL1-conjugate nicotine vaccine did not induce clinically relevant changes or induce symptoms that would be associated with toxicity, making it a promising candidate for future investigations.
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In an attempt to develop effective and safe anticancer agents, we designed, synthesized and examined 23 novel quinacrine (QC) derivatives by combining the 9-aminoacridine scaffold and the [1,3]thiazinan-4-ones group. Most of these hybrids showed strong anticancer activities, among which 3-(3-(6-chloro-2-methoxyacridin-9-ylamino)propyl)-2-(thiophen-2-yl)-1,3-thiazinan-4-one (25; VR151) effectively killed many different cancer cell types, including eight breast cancer cell lines with different genetic background, two prostate cancer and two lung cancer cell lines. In contrast, compound 25 is less effective against non-cancer cells, suggesting it may be less toxic to humans. Our data showed that cancer cells are arrested in S phase for a prolonged period due to the down-regulation of DNA replication, leading to eventual cell death. We have also shown that the S phase arrest may be resulted by the down-regulation of cyclin A coupled with the continued up-regulation of cyclin E, which coincide with the down-regulation of mTor-S6K and mTor-4EBP1 pathways.
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Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Diseño de Fármacos , Quinacrina/análogos & derivados , Tiazinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Estructura Molecular , Quinacrina/síntesis química , Quinacrina/química , Quinacrina/farmacología , Relación Estructura-Actividad , Tiazinas/síntesis química , Tiazinas/químicaRESUMEN
The therapeutic application of peptide-based drugs is significantly limited by the rapid proteolytic degradation that occurs when in blood. Encapsulation of these peptide structures within a delivery system, such as liposomes, can greatly improve both stability and target delivery. As part of our work focused on novel ambiphilic mannosylated neoglycolipids as targeted drug delivery systems, we have developed a C14-alkyl-mannopyranoside that forms self-assembled monodisperse liposomes. Herein, these glycoliposomes are investigated as a potential method to improve the plasma stability of peptide-based drugs. Reversed phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) methods were developed to assess the in vitro plasma stability of two structurally diverse peptides, including the kappa opioid receptor selective antagonist dynantin, and the NOD2 innate immune receptor ligand muramyl dipeptide (MDP). The RP-HPLC methods developed were able to resolve the peptides from background plasma contaminants and provided suitable response levels and linearity over an appropriate concentration range. Both compounds were found to be significantly degraded in rat plasma. Increasing degrees of both entrapment and stabilization were noted when dynantin was combined with the C14-alkyl-mannopyranoside in increasing peptide:glycoside ratios. The combination of MDP with the glycolipid also led to peptide entrapment, which greatly improved the plasma stability of the peptide. Overall, the results clearly indicate that the stability of peptide-based structures, which are subject to degradation in plasma, can be greatly improved via entrapment within C14-alkyl-mannopyranoside-bearing glycoliposomes.
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Sistemas de Liberación de Medicamentos , Nanopartículas/administración & dosificación , Péptidos/administración & dosificación , Péptidos/farmacocinética , Animales , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Estabilidad de Medicamentos , Dinorfinas/administración & dosificación , Dinorfinas/sangre , Dinorfinas/farmacocinética , Femenino , Glucolípidos/administración & dosificación , Glucolípidos/química , Técnicas In Vitro , Liposomas/administración & dosificación , Liposomas/química , Nanopartículas/química , Antagonistas de Narcóticos/administración & dosificación , Antagonistas de Narcóticos/sangre , Antagonistas de Narcóticos/farmacocinética , Péptidos/sangre , Estabilidad Proteica , Proteolisis , Ratas , Ratas Sprague-Dawley , Receptores Opioides kappa/antagonistas & inhibidores , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Tobacco smoking is recognized as a global pandemic resulting in 6 million deaths per year. Despite a variety of anti-smoking products available to aid with tobacco cessation, the majority of people who attempt to quit smoking relapse within 6 months due to the addictive nature of nicotine. An immunotherapy approach could offer a promising treatment option by inducing a potent selective antibody response against nicotine in order to block its distribution to the brain and its addictive effects in the central nervous system. Our nicotine vaccine candidate was administered intranasally using the Neisseria meningitidis serogroup B Adjuvant Finlay Proteoliposome 1 (AFPL1) as a part of the delivery system. This system was designed to generate a robust immune response by stimulating IL-1ß production through Toll-like receptor 4 (TLR4), a potent mechanism for mucosal immunity. The vaccine induced high antibody titers in mice sera in addition to inducing mucosal antibodies. The efficacy of our vaccine was demonstrated using in vivo challenge experiments with radioactive [(3)H]-nicotine, followed by an analysis of nicotine distribution in the lung, liver, blood and brain. Our results were encouraging as the nicotine concentration in the brain tissue of mice vaccinated with our candidate vaccine was four times lower than in non-vaccinated controls; suggesting that the anti-nicotine antibodies were able to block nicotine from crossing the blood brain barrier. In summary, we have developed a novel nicotine vaccine for the treatment of tobacco addiction by intranasal administration and also demonstrated that the AFPL1 can be used as a potential adjuvant for this vaccine design.
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We studied the expression of the osteogenic and antinociceptive C-terminal histone H4-related peptide fragments, H4-(89-102) (OGP) and H4-(86-100), respectively, within various rat tissues and isolated alveolar macrophages (AM) by radioimmunoassay (RIA). OGP was located mainly within the bone marrow, spleen, thymus, and lungs whereas H4-(86-100) was more concentrated within the bone marrow, lymph nodes, spinal cord, pituitaries and thymus. The expression pattern of the two peptides showed similarities with the tissue expression pattern of the histone H4 mRNA variant H4-v.1. In rat AM, OGP and H4-(86-100) levels were significantly stimulated (2.6- and 1.9-fold, respectively) by LPS (1 microg/ml), along with H4-v.1 mRNA (4.1-fold), but not whole histone H4 (1.1-fold) nor total histone H4 mRNA (1.1-fold). The results suggest that H4-v.1 mRNA may play a role in the synthesis of the naturally occurring peptides H4-(86-100) and OGP via the alternative translation product H4-(84-102), but not whole histone H4.
Asunto(s)
Histonas/química , Histonas/genética , Macrófagos Alveolares/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , ARN Mensajero/metabolismo , Animales , Especificidad de Anticuerpos , Northern Blotting , Cromatografía Líquida de Alta Presión , Histonas/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos Alveolares/química , Macrófagos Alveolares/efectos de los fármacos , Masculino , Especificidad de Órganos , Fragmentos de Péptidos/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Radioinmunoensayo , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Novel analogues of the minimal antinociceptive histogranin (HN) fragment Gly(7)-Gln-Gly-Arg(10), in which amino acids in positions 8, 9, and 10 were replaced by lipophilic amino acids and corresponding d-amino acid residues in combination with N- to C-terminal cyclization, were synthesized and tested in various animal models of pain. All synthetic compounds were potent and efficacious analgesics in the mouse writhing test. Cyclic [-Gly-Ala-Tyr-d-Arg-] (9) and cyclic [-Gly p-Cl-Phe-Tyr-d-Arg-] (10) were the most potent analgesics, being 17 and 135 times as potent as HN, respectively (AD(50) of 1.37 and 0.17 nmol/mouse icv, as compared with 23 nmol/mouse for HN). The times of action of compounds 9 and 10 were also much improved with half-maximal effects still being observed 60 min and >90 min after their administration, respectively, as compared with 8.1 min for the parent peptide HN-(7-10) and 22.1 min for HN. At analgesic doses, compounds 9 and 10 were devoid of motor effect as assessed by the mouse rotarod assay. As already observed with HN, compounds 9 (10 nmol/rat; i.t.) and 10 (0.5 nmol/rat; i.t.) were effective in blocking persistent inflammatory pain in the formalin test and hyperalgesia induced by intraplantar administration of complete Freund adjuvant. In addition, the analgesic effects evoked by compounds 9 (10 nmol/mouse; icv) and 10 (1 micromol/kg; i.v.) in the mouse writhing test and compound 9 (10 nmol/mouse; icv) in the mouse tail flick assay were similarly antagonized by the dopamine D(2) receptor antagonist raclopride (1 nmol/mouse; icv) but not the opiate antagonist naloxone (1 nmol/mouse; i.c.v). Finally, the various cyclic compounds competed with the binding of [(3)H]raclopride in rat brain membrane preparations. Their ability to compete with the binding of the D(2) ligand correlated well with their potency in alleviating pain in the mouse writhing test (r = 0.95). These results indicate that the analgesic activity of the minimal active core in HN can be improved by changes that favor its interaction with the dopamine D(2) receptor.