Free-Energy Landscape and Rate Estimation of the Aromatic Ring Flips in Basic Pancreatic Trypsin Inhibitors Using Metadynamics.

Aromatic side chains (phenylalanine and tyrosine) of a protein flip by 180° around the Cß-Cγ axis (χ2 dihedral of the side chain), producing two symmetry-equivalent states. The study of ring flip dynamics with nuclear magnetic resonance (NMR) experiments helps to understand local conformational fluctuations. Ring flips are categorized as slow (milliseconds and onward) or fast (nanoseconds to near milliseconds) based on timescales accessible to NMR experiments. In this study, we investigated the ability of the infrequent metadynamics approach to estimate the flip rate and discriminate between slow and fast ring flips for eight individual aromatic side chains (F4, Y10, Y21, F22, Y23, F33, Y35, and F45) of the basic pancreatic trypsin inhibitor. Well-tempered metadynamics simulations were performed to estimate the ring-flipping free-energy surfaces for all eight aromatic residues. The results indicate that χ2 as a standalone collective variable (CV) is not sufficient to obtain computationally consistent results. Inclusion of a complementary CV, such as χ1(Cα-Cß), solved the problem for most residues and enabled us to classify fast and slow ring flips. This indicates the importance of librational motions in ring flips. Multiple pathways and mechanisms were observed for residues F4, Y10, and F22. Recrossing events were observed for residues F22 and F33, indicating a possible role of friction effects in ring flipping. The results demonstrate the successful application of infrequent metadynamics to estimate ring flip rates and identify certain limitations of the approach.

Efficacy of Boron Neutron Capture Therapy in Primary Central Nervous System Lymphoma: In Vitro and In Vivo Evaluation.

BACKGROUND: Boron neutron capture therapy (BNCT) is a nuclear reaction-based tumor cell-selective particle irradiation method. High-dose methotrexate and whole-brain radiation therapy (WBRT) are the recommended treatments for primary central nervous system lymphoma (PCNSL). This tumor responds well to initial treatment but relapses even after successful treatment, and the prognosis is poor as there is no safe and effective treatment for relapse. In this study, we aimed to conduct basic research to explore the possibility of using BNCT as a treatment for PCNSL. METHODS: The boron concentration in human lymphoma cells was measured. Subsequently, neutron irradiation experiments on lymphoma cells were conducted. A mouse central nervous system (CNS) lymphoma model was created to evaluate the biodistribution of boron after the administration of borono-phenylalanine as a capture agent. In the neutron irradiation study of a mouse PCNSL model, the therapeutic effect of BNCT on PCNSL was evaluated in terms of survival. RESULTS: The boron uptake capability of human lymphoma cells was sufficiently high both in vitro and in vivo. In the neutron irradiation study, the BNCT group showed a higher cell killing effect and prolonged survival compared with the control group. CONCLUSIONS: A new therapeutic approach for PCNSL is urgently required, and BNCT may be a promising treatment for PCNSL. The results of this study, including those of neutron irradiation, suggest success in the conduct of future clinical trials to explore the possibility of BNCT as a new treatment option for PCNSL.

Selective N-Arylation of p-Aminophenylalanine in Unprotected Peptides with Organometallic Palladium Reagents.

The selective N-arylation of p-aminophenylalanine in polypeptides with pre-formed palladium oxidative addition complexes is described. The depressed pKa of the aniline NH2 group enables chemoselective C-N bond formation on peptides containing multiple other aliphatic amino groups at lysines or the N-terminus via Curtin-Hammett control under mild conditions. Using palladium complexes derived from electron-poor aryl halides, p-aminophenylalanine is fully arylated in aqueous buffer in as little as one hour at micromolar concentrations. A complementary protocol using the non-nucleophilic, organic base 1,5-diazabicyclo(4.3.0)non-5-ene (DBN), expands the substrate scope to tolerate electron-rich functional groups provides up to 97 % conversion. These procedures enable the chemoselective conjugation of functionally diverse small molecule pharmaceuticals to p-aminophenylalanine containing derivatives of cell-penetrating peptides.

Biophysical and structural investigation of the regulation of human GTP cyclohydrolase I by its regulatory protein GFRP.

GTP Cyclohydrolase I (GCH1) catalyses the conversion of guanosine triphosphate (GTP) to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). BH4 functions as co-factor in neurotransmitter biosynthesis. BH4 homeostasis is a promising target to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). Dependent on the relative cellular concentrations of effector ligands, BH4 and phenylalanine, GFRP binds GCH1 to form inhibited or activated complexes, respectively. We determined high-resolution structures of the ligand-free and -bound human GFRP and GCH1-GFRP complexes by X-ray crystallography. Highly similar binding modes of the substrate analogue 7-deaza-GTP to active and inhibited GCH1-GFRP complexes confirm a novel, dissociation rate-controlled mechanism of non-competitive inhibition to be at work. Further, analysis of all structures shows that upon binding of the effector molecules, the conformations of GCH1 or GFRP are altered and form highly complementary surfaces triggering a picomolar interaction of GFRP and GCH1 with extremely slow koff values, while GCH1-GFRP complexes rapidly disintegrate in absence of BH4 or phenylalanine. Finally, comparing behavior of full-length and N-terminally truncated GCH1 we conclude that the disordered GCH1 N-terminus does not have impact on complex formation and enzymatic activity. In summary, this comprehensive and methodologically diverse study helps to provide a better understanding of the regulation of GCH1 by GFRP and could thus stimulate research on GCH1 modulating drugs.

Accessing p-Hydroxycinnamic Acids: Chemical Synthesis, Biomass Recovery, or Engineered Microbial Production?

p-Hydroxycinnamic acids (i. e., p-coumaric, ferulic, sinapic, and caffeic acids) are phenolic compounds involved in the biosynthesis pathway of lignin. These naturally occurring molecules not only exhibit numerous attractive properties, such as antioxidant, anti-UV, and anticancer activities, but they also have been used as building blocks for the synthesis of tailored monomers and functional additives for the food/feed, cosmetic, and plastics sectors. Despite their numerous high value-added applications, the sourcing of p-hydroxycinnamic acids is not ensured at the industrial scale except for ferulic acid, and their production cost remains too high for commodity applications. These compounds can be either chemically synthesized or extracted from lignocellulosic biomass, and recently their production through bioconversion emerged. Herein the different strategies described in the literature to produce these valuable molecules are discussed.

Application of achiral-chiral two-dimensional HPLC for separation of phenylalanine and tryptophan enantiomers in dietary supplement.

This study developed a two-dimensional heart-cutting LC method for the separation of amino acid enantiomers. Two approaches for achiral separation of amino acids, phenylalanine and tryptophan, were selected. Amino acids were separated on C18 or hydrophilic interaction liquid chromatography (HILIC) columns in first dimension after their enantiomer separation on a teicoplanin chiral column in second dimension. Mobile phases for both separation systems were optimized by testing different types of organic modifiers, concentrations of ion-pair agent (sodium 1-octanesulfonate), and ionic modifier (ammonium acetate). The resolution of enantiomers higher than 1.5 for both amino acids was achieved using a C18-teicoplanin coupled column separation system with mobile phases methanol/2 mM sodium 1-octanesulfonate (10:90 and 75:25, step gradient between achiral and chiral columns, respectively). The lower resolution of amino acid enantiomers (RS ˃ 0.9), but higher column efficiency, was achieved on a HILIC-teicoplanin separation system with mobile phases acetonitrile/50 mM ammonium acetate (90:10 and 80:20, step gradient between achiral and chiral columns, respectively). The developed heart-cutting 2D-LC methods were validated in terms of linearity, limit of detection, limit of quantification, precision, and accuracy. The results suggested that the developed methods were applicable for the simultaneous determination of amino acid enantiomers in the dietary supplement. The sample contained l-phenylalanine and l-tryptophan.

Natural Products: Review for Their Effects of Anti-HBV.

Hepatitis B is a global infectious disease, seriously endangering human health. Currently, there are mainly interferons and nucleoside analogues treatment of hepatitis B in the clinic, which have certain therapeutic effects on hepatitis B, but their side effects and drug resistance are increasingly prominent. Therefore, it is urgently needed to discover and develop new anti-HBV drugs, especially natural products, which have novel, high efficiency, and low toxicity anti-HBV compounds with novel antiviral mechanisms. In this manuscript, the natural products (polysaccharides and 165 compounds) with the activity of antihepatitis B virus are discussed according to their chemical classes, including 14 phenylpropanoids, 8 flavonoids,12 xanthones, 13 anthroquinones, 47 terpenoids, 6 alkaloids, 15 enediynes, 11 aromatics, 18 phenylalanine dipeptides compounds, and 13 others. In addition, the anti-HBV mechanism and targets of natural product were also discussed. The aim of this review is to report new discoveries about anti-HBV natural products and to provide reference for researchers.

Electrostatic interactions regulate the release of small molecules from supramolecular hydrogels.

Supramolecular hydrogels have great potential as biomaterials for sustained delivery of therapeutics. While peptide-based supramolecular hydrogels have been developed that show promise for drug delivery applications, the high cost of production has limited their widespread adoption. Low molecular weight (LMW) supramolecular hydrogels are emerging as attractive and inexpensive alternatives to peptide-based hydrogels. We recently reported novel cationic fluorenylmethyloxycarbonyl-modified phenylalanine (Fmoc-Phe) hydrogels for localized and sustained in vivo release of an anti-inflammatory agent for functional pain remediation. In an effort to further elucidate design principles to optimize these materials for delivery of a variety of molecular agents, we herein report a systematic examination of electrostatic effects on the release of cargo molecules from Fmoc-Phe derived hydrogels. Specifically, we interrogate the release of cationic, anionic, and neutral cargo molecules from a series of cationic and anionic Fmoc-Phe derived hydrogels. We observed that cargo was readily released from the hydrogels except when the cargo and hydrogel network had complementary charges, in which case the cargo was highly retained in the network. These results demonstrate that the electrostatic characteristics of both the hydrogel network and the specific cargo are critical design parameters in the formulation of LMW supramolecular hydrogel systems in the development of next-generation materials for drug delivery applications.

Efficient optimization of high-dose formulation of novel lyophilizates for dry powder inhalation by the combination of response surface methodology and time-of-flight measurement.

Inhalation of proteins/peptides has recently received attention as various biopharmaceuticals have emerged on the market. Novel lyophilisates for dry powder inhalation (LDPIs), which are aerosolized by air impact, have been reported and LDPIs are considered an attractive option for the pulmonary administration of biopharmaceuticals. However, desirable disintegration and aerosolization properties have been unavailable in high-dose formulations, which has been a critical issue. This study aimed to investigate high-dose LDPIs and their optimization. In the present study, lysozyme (Lysoz) was used as a stable model protein and formulated with various amino acids. Furthermore, response surface methodology (RSM) and time-of-flight measurement were applied for efficient optimization. Superior disintegration and aerosolization properties were confirmed in the LDPIs with phenylalanine (Phe) and leucine (Leu). RSM results revealed that 0.5 mg/vial of Phe and 1.0 mg/vial of Leu are the optimal quantities for high-dose formulation. Based on these optimum quantities, high-dose LDPI formulations were prepared and the maximum formulable quantity of Lysoz with acceptable inhalation performance was confirmed to be 3.0 mg/vial. The results suggest that LDPI can cover the milligram-order pulmonary administration of proteins/peptides. LDPIs are expected to have biopharmaceutical applications.

Honey as Source of Nitrogen Compounds: Aromatic Amino Acids, Free Nucleosides and Their Derivatives.

The content of selected major nitrogen compounds including nucleosides and their derivatives was evaluated in 75 samples of seven varieties of honey (heather, buckwheat, black locust, goldenrod, canola, fir, linden) by targeted ultra-high performance liquid chromatography-diode array detector - high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QqTOF-MS) and determined by UHPLC-DAD. The honey samples contained nucleosides, nucleobases and their derivatives (adenine: 8.9 to 18.4 mg/kg, xanthine: 1.2 to 3.3 mg/kg, uridine: 17.5 to 51.2 mg/kg, guanosine: 2.0 to 4.1 mg/kg; mean amounts), aromatic amino acids (tyrosine: 7.8 to 263.9 mg/kg, phenylalanine: 9.5 to 64.1 mg/kg; mean amounts). The amounts of compounds significantly differed between some honey types. For example, canola honey contained a much lower amount of uridine (17.5 ± 3.9 mg/kg) than black locust where it was most abundant (51.2 ± 7.8 mg/kg). The presence of free nucleosides and nucleobases in different honey varieties is reported first time and supports previous findings on medicinal activities of honey reported in the literature as well as traditional therapy and may contribute for their explanation. This applies, e.g., to the topical application of honey in herpes infections, as well as its beneficial activity on cognitive functions as nootropic and neuroprotective, in neuralgia and is also important for the understanding of nutritional values of honey.

Biochemical Pathway of Benzyl Nitrile Derived from l-Phenylalanine in Tea (Camellia sinensis) and Its Formation in Response to Postharvest Stresses.

Volatiles affect tea (Camellia sinensis) aroma quality and have roles in tea plant defense against stresses. Some volatiles defend against stresses through their toxicity, which might affect tea safety. Benzyl nitrile is a defense-related toxic volatile compound that accumulates in tea under stresses, but its formation mechanism in tea remains unknown. In this study, l-[2H8]phenylalanine feeding experiments and enzyme reactions showed that benzyl nitrile was generated from l-phenylalanine via phenylacetaldoxime in tea. CsCYP79D73 showed activity for converting l-phenylalanine into phenylacetaldoxime, while CsCYP71AT96s showed activity for converting phenylacetaldoxime into benzyl nitrile. Continuous wounding in the oolong tea process significantly enhanced the CsCYP79D73 expression level and phenylacetaldoxime and benzyl nitrile contents. Benzyl nitrile accumulation under continuous wounding stress was attributed to an increase in jasmonic acid, which activated CsCYP79D73 expression. This represents the first elucidation of the formation mechanism of benzyl nitrile in tea.

l-Phenylalanine Alters the Privileged Secondary Metabolite Production in the Marine-Derived Fungus Trichoderma erinaceum F1-1.

The effects of a single-amino-acid culture strategy on secondary metabolite production in the marine-derived fungus Trichoderma erinaceum F1-1 were investigated by culturing the fungus in GPY medium supplemented or not supplemented with l-phenylalanine. A suite of secondary metabolites, including seven terpenoids (1-7) and one polyketide (8), among which are four new compounds, harziandione A (1), cyclonerodiols A and B (3, 4), and trichodermaerin A (6), were isolated from the GPY medium without l-phenylanine, whereas 18 aromatic compounds (9-26), including six new compounds, trichoderolides A-F (9, 10, and 14-17), were isolated from the culture grown in the GPY medium with l-phenylalanine. The structures of the new compounds were determined by high-resolution mass spectrometry, NMR spectroscopic analysis, optical rotation calculations, chemical methods, and X-ray crystallography. Compounds 10, 12, 13, and 26 exhibited cytotoxic activities against MDA-MB-435 human melanocyte cancer cells. Compound 26 was cytotoxic to A549 adenocarcinomic human alveolar basal epithelial cells.

Hepatitis C Virus Helicase Binding Activity Monitored through Site-Specific Labeling Using an Expanded Genetic Code.

The mechanism of unwinding catalyzed by the hepatitis C virus nonstructural protein 3 helicase (NS3h) has been a subject of considerable interest, with NS3h serving as a prototypical enzyme in the study of helicase function. Recent studies support an ATP-fueled, inchworm-like stepping of NS3h on the nucleic acid that would result in the displacement of the complementary strand of the duplex during unwinding. Here, we describe the screening of a site of incorporation of an unnatural amino acid in NS3h for fluorescent labeling of the enzyme to be used in single-molecule Förster resonance energy transfer (FRET) experiments. From the nine potential sites identified in NS3h for incorporation of the unnatural amino acid, only one allowed for expression and fluorescent labeling of the recombinant protein. Incorporation of the unnatural amino acid was confirmed via bulk assays to not interfere with unwinding activity of the helicase. Binding to four different dsDNA sequences bearing a ssDNA overhang segment of varying length (either minimal 6 or 7 base length overhang to ensure binding or a long 24 base overhang) and sequence was recorded with the new NS3h construct at the single-molecule level. Single-molecule fluorescence displayed time intervals with anticorrelated donor and acceptor emission fluctuations associated with protein binding to the substrates. An apparent FRET value was estimated from the binding events showing a single FRET value of ∼0.8 for the 6-7 base overhangs. A smaller mean value and a broad distribution was in turn recorded for the long ssDNA overhang, consistent with NS3h exploring a larger physical space while bound to the DNA construct. Notably, intervals where NS3h binding was recorded were exhibited at time periods where the acceptor dye reversibly bleached. Protein induced fluorescence intensity enhancement in the donor channel became apparent at these intervals. Overall, the site-specific fluorescent labeling of NS3h reported here provides a powerful tool for future studies to monitor the dynamics of enzyme translocation during unwinding by single-molecule FRET.

Thermal Sensitive Liposomes Improve Delivery of Boronated Agents for Boron Neutron Capture Therapy.

PURPOSE: Boron neutron capture therapy (BNCT) has the potential to become a viable cancer treatment modality, but its clinical translation requires sufficient tumor boron delivery while minimizing nonspecific accumulation. METHODS: Thermal sensitive liposomes (TSLs) were designed to have a stable drug payload at physiological temperatures but engineered to have high permeability under mild hyperthermia. RESULTS: We found that TSLs improved the tumor-specific delivery of boronophenylalanine (BPA) and boronated 2-nitroimidazole derivative B-381 in D54 glioma cells. Uniquely, the 2-nitroimidazole moiety extended the tumor retention of boron content compared to BPA. CONCLUSION: This is the first study to show the delivery of boronated compounds using TSLs for BNCT, and these results will provide the basis of future clinical trials using TSLs for BNCT.

Synthesis and Initial Biological Evaluation of Boron-Containing Prostate-Specific Membrane Antigen Ligands for Treatment of Prostate Cancer Using Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a therapeutic modality which has been used for the treatment of cancers, including brain and head and neck tumors. For effective treatment via BNCT, efficient and selective delivery of a high boron dose to cancer cells is needed. Prostate-specific membrane antigen (PSMA) is a target for prostate cancer imaging and drug delivery. In this study, we conjugated boronic acid or carborane functional groups to a well-established PSMA inhibitor scaffold to deliver boron to prostate cancer cells and prostate tumor xenograft models. Eight boron-containing PSMA inhibitors were synthesized. All of these compounds showed a strong binding affinity to PSMA in a competition radioligand binding assay (IC50 from 555.7 to 20.3 nM). Three selected compounds 1a, 1d, and 1f were administered to mice, and their in vivo blocking of 68Ga-PSMA-11 uptake was demonstrated through a positron emission tomography (PET) imaging and biodistribution experiment. Biodistribution analysis demonstrated boron uptake of 4-7 µg/g in 22Rv1 prostate xenograft tumors and similar tumor/muscle ratios compared to the ratio for the most commonly used BNCT compound, 4-borono-l-phenylalanine (BPA). Taken together, these data suggest a potential role for PSMA targeted BNCT agents in prostate cancer therapy following suitable optimization.

Development of national consensus statements on food labelling interpretation and protein allocation in a low phenylalanine diet for PKU.

BACKGROUND: In the treatment of phenylketonuria (PKU), there was disparity between UK dietitians regarding interpretation of how different foods should be allocated in a low phenylalanine diet (allowed without measurement, not allowed, or allowed as part of phenylalanine exchanges). This led to variable advice being given to patients. METHODOLOGY: In 2015, British Inherited Metabolic Disease Group (BIMDG) dietitians (n = 70) were sent a multiple-choice questionnaire on the interpretation of protein from food-labels and the allocation of different foods. Based on majority responses, 16 statements were developed. Over 18-months, using Delphi methodology, these statements were systematically reviewed and refined with a facilitator recording discussion until a clear majority was attained for each statement. In Phase 2 and 3 a further 7 statements were added. RESULTS: The statements incorporated controversial dietary topics including: a practical 'scale' for guiding calculation of protein from food-labels; a general definition for exchange-free foods; and guidance for specific foods. Responses were divided into paediatric and adult groups. Initially, there was majority consensus (≥86%) by paediatric dietitians (n = 29) for 14 of 16 statements; a further 2 structured discussions were required for 2 statements, with a final majority consensus of 72% (n = 26/36) and 64% (n = 16/25). In adult practice, 75% of dietitians agreed with all initial statements for adult patients and 40% advocated separate maternal-PKU guidelines. In Phase 2, 5 of 6 statements were agreed by ≥76% of respondents with one statement requiring a further round of discussion resulting in 2 agreed statements with a consensus of ≥71% by dietitians in both paediatric and adult practice. In Phase 3 one statement was added to elaborate further on an initial statement, and this received 94% acceptance by respondents. Statements were endorsed by the UK National Society for PKU. CONCLUSIONS: The BIMDG dietitians group have developed consensus dietetic statements that aim to harmonise dietary advice given to patients with PKU across the UK, but monitoring of statement adherence by health professionals and patients is required.

Amphiphilic bromelain-synthesized oligo-phenylalanine grafted with methoxypolyethylene glycol possessing stabilizing thermo-responsive emulsion properties.

A thermo-responsive amphiphile was developed from oligo-phenylalanine [oligo(Phe)]. The hydrophobic moiety of the amphiphile, oligo(Phe) was synthesized via reverse hydrolysis catalyzed by bromelain in dimethyl sulfoxide and dioxane solutions. The production of oligo(Phe) increased by 80.7% by screening suitable reaction conditions. The average degree of polymerization of oligo(Phe) was determined to be four by 1H NMR. By grafting with aldehyde-ended methoxypolyethylene glycol (mPEG), oligo(Phe) was converted to amphiphilic oligo(Phe)-mPEG. The surface tension of oligo(Phe)-mPEG solution increased with decreasing chain length of the mPEG moiety. Cytotoxicity studies showed oligo(Phe)-mPEGs are biocompatible. On varying temperature, a reversible phase transition of oligo(Phe)-mPEG solutions could be observed. N-octane-in-water emulsions and 0.5% beta-carotene containing squalene-in-water emulsions stabilized by oligo(Phe)-mPEGs occurred at 25 °C but de-emulsification took place at >40 °C. Emulsification could be restored once the separated mixture cooled and re-homogenized. The emulsification/de-emulsification cycling could be repeated many times. The time required for de-emulsification decreased with elevated temperature but increased with a reduced concentration of oligo(Phe)-mPEGs and a reduction in the chain length of the mPEG moiety.

Discovery of a novel cathepsin inhibitor with dual autophagy-inducing and metastasis-inhibiting effects on breast cancer cells.

Drug resistance and cancer cells metastasis have been the leading causes of chemotherapy failure and cancer-associated death in breast cancer patients. In present, various active molecules either exhibiting novel mechanism of action such as inducing autophagy or inhibiting metastasis have been developed to address these problems. However, the compounds exhibiting such dual functions have rarely been described. Previous work in our group showed that TSA, as a synthetic analog of asperphenamate, induced autophagic cell death in breast cancer cells instead of apoptosis. Furthermore, the target enzyme of TSA was predicted to be cathepin L (Cat L) by natural product consensus pharmacophore strategy. Accumulated evidences have shown that cathepsins are closely associated with migration and invasion of breast cancer cells. It seemed likely that TSA-like molecules may possess the dual functions of inducing autophagy and inhibiting metastasis. Therefore, sixty optically active derivatives were firstly designed and synthesized by replacing the A-ring moiety of TSA with other substituted-phenyl sulfonyl groups. Further cathepsin inhibitory activity assay showed that (S, S) and (S, R) isomers displayed no activity against four kinds of cathepsins (L, S, K, B), while all derivatives tested were inactive toward K and B subtypes. Compound 6a with meta-bromo substituent displayed the greatest inhibitory activity, and its inhibitory capability against Cat L and S was 3.9 and 11.5-fold more potent than that of TSA, respectively. Molecular docking also exhibited that 6a formed more hydrogen bonds or π-π contacts with Cat L or S than TSA. In order to determine whether 6a could play dual roles, its anti-cancer mechanism was further investigated. On the one hand, MDC staining experiment and western blotting analysis validated that 6a can induce autophagy in MDA-MB-231 cells. On the other hand, its metastatic inhibitory ability was also confirmed by wound healing and transwell chamber experiment.

Cationic Poly(Amino Acid) Vaccine Adjuvant for Promoting Both Cell-Mediated and Humoral Immunity Against Influenza Virus.

Powerful adjuvants to augment vaccine efficacy with a less immunogenic vaccine system are in great demand. In this study, a novel squalene-based cationic poly(amino acid) adjuvant (CASq) that elicits both cellular (Th1) and humoral (Th2) immune responses is developed. CASq is demonstrated to promote cellular uptake of viral antigen and stimulate macrophages, leading to active production of interleukin-12. Furthermore, co-administration of inactivated pdm H1N1 vaccine with CASq significantly increases the generation of antigen-specific antibodies and T cell immune responses in mice, as well as resulting in complete prevention of disease symptoms and protection against lethal infection.

Eficacia y seguridad de los suplementos nutricionales libres de tirosina y fenilalanina para el tratamiento de pacientes menores de 18 años con diagnóstico de tirosinemia tipo I / Efficacy and safety of tyrosine and phenylalanine free nutritional supplements for the treatment of patients under 18 years of age with diagnosis of tyrosinemia type I

INTRODUCCIÓN: La tirosinemia tipo 1 (HT-1) es una enfermedad genética infrecuente causada por la mutación del gen FAH. Esta mutación produce escasez de la enzima fumaril-aceto-acetato hidrolasa; responsable del último paso en la vía de degradación de la tirosina. El acúmulo de los metabolitos tóxicos de la tirosina interrumpe severamente el metabolismo intracelular del hígado y riñón; dañando estos órganos. La HT-1 se presenta en uno de cada 100.000 - 120.000 nacimientos alrededor del mundo. El tratamiento de los pacientes con HT-1 está dirigido a controlar los niveles plasmáticos de tirosina y evitar la formación de sus metabolitos tóxicos. El manejo nutricional reduce la ingesta de tirosina y fenilalanina mediante la dieta. El tratamiento farmacológico involucra el uso de nitisinona; la cual bloquea el segundo paso de la vía de degradación de la tirosina. De esta forma, se evita la formación de los metabolitos tóxicos de la tirosina; pero se incrementa el nivel plasmático de tirosina; lo cual produce, a su vez, molestias visuales debidas a la formación de cristales de tirosina. Para mantener el nivel de tirosina dentro del rango normal, la dieta debe estar reducida en tirosina y fenilalanina (precursor de la tirosina). Esto refuerza la importancia del manejo nutricional mediante la restricción de la ingesta de tirosina y fenilalanina en la dieta. Sin embargo, esta dieta podría no cubrir los demás requerimientos nutricionales, energéticos y proteicos de los pacientes con HT-1. Por lo tanto, los suplementos nutricionales serían necesarios para complementar la dieta. En EsSalud se dispone de suplementos nutricionales pediátricos. Sin embargo, estos no están libres de fenilalanina y tirosina. OBJETIVO: el presente dictamen preliminar expone la eficacia y seguridad de los suplementos nutricionales libres de fenilalanina y tirosina, junto con una dieta reducida en tirosina, comparado con una dieta reducida en tirosina para el tratamiento de los pacientes menores de 18 años, con diagnóstico de HT-1. METODOLOGÍA: Tras una búsqueda sistemática de literatura publicada hasta diciembre del 2018, no se encontró evidencia científica que evalúe el uso de suplementos nutricionales libres de fenilalanina y tirosina acompañados de una dieta reducida en tirosina, comparado con un manejo nutricional que involucre solo la dieta reducida en tirosina, en pacientes menores de 18 años con diagnóstico de HT-1. En su lugar, se hallaron tres artículos de recomendación tipo revisión que abordan el manejo nutricional de los pacientes con HT-1. Uno de ellos no menciona los suplementos libres de tirosina y fenilalanina. Los otros dos, mencionan que es necesario añadir estos suplementos a la dieta de todo paciente con HT-1. Sin embargo, esta afirmación no forma parte de las recomendaciones propiamente dichas, y no se mostraron las referencias de la evidencia utilizada para dar esta afirmación. RESULTADOS: Pese a la escasa evidencia científica disponible, la eficacia de los suplementos nutricionales libres de tirosina y fenilalanina tiene sustento biológico razonable. El requerimiento varía desde 3 g/kg/día, aproximadamente, en menores de 2 años hasta 1.5 g/Kg/día a los 10-14 años. Sin embargo, los requerimientos de tirosina y fenilalanina se alcanzan con pequeñas cantidades de proteína natural (0.5 a 1.0 g/Kg/día). Al restringir la ingesta de proteína natural (para evitar el exceso de tirosina y fenilalanina en pacientes con HT-1), se crea un déficit para cubrir el requerimiento proteico global. Si este déficit no se cubre, el paciente tendrá problemas en el crecimiento y desarrollo psicomotor. Por lo tanto, la inclusión de suplementos nutricionales libres de tirosina y fenilalanina en la dieta de los pacientes HT-1 ayuda a cubrir sus requerimientos proteicos, manteniendo las concentraciones plasmáticas de tirosina en el rango saludable. En línea con lo mencionado, el experto en pediatría de la institución, afirma que los suplementos nutricionales libres de tirosina y fenilalanina permiten el adecuado crecimiento y desarrollo neurológico mientras evita la aparición de problemas visuales. Por lo tanto, el experto en pediatría opina que estos suplementos nutricionales son un complemento terapéutico necesario para el tratamiento dietético. Esta posición coincide con la opinión de los expertos internacionales y las revisiones presentadas en el presente dictamen preliminar. CONCLUSIÓN: Con respecto a lo expuesto previamente, el equipo técnico del IETSI valoró los siguientes aspectos: i) La HT-1 es una enfermedad hereditaria, infrecuente, que afecta la calidad de vida del paciente desde edades muy tempranas y está asociada a una alta mortalidad si no es tratada adecuadamente ii) En el contexto de EsSalud existe un vacío terapéutico para la población objetivo, iii) Aunque la evidencia disponible actualmente es limitada, algunas guías mencionan el uso de suplementos nutricionales libres de tirosina y fenilalanina. Además, su uso cuenta con plausibilidad biológica, lo cual va en línea con lo recomendado por el experto clínico. Por todo lo mencionado, el Instituto de Evaluaciones de Tecnologías en Salud e Investigación ­ IETSI, aprueba el uso de suplementos libres de tirosina y fenilalanina para el manejo de pacientes menores de 18 años, con diagnóstico de HT-1, según lo establecido en el Anexo N° 1 La vigencia del presente dictamen preliminar es de dos años a partir de la fecha de publicación. Así, la continuación de dicha aprobación estará sujeta a la evaluación de los resultados obtenidos y de nueva evidencia que pueda surgir en el tiempo.