Gene Therapy for Mucopolysaccharidosis Type II-A Review of the Current Possibilities.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder based on a mutation in the IDS gene that encodes iduronate 2-sulphatase. As a result, there is an accumulation of glycosaminoglycans-heparan sulphate and dermatan sulphate-in almost all body tissues, which leads to their dysfunction. Currently, the primary treatment is enzyme replacement therapy, which improves the course of the disease by reducing somatic symptoms, including hepatomegaly and splenomegaly. The enzyme, however, does not cross the blood-brain barrier, and no improvement in the function of the central nervous system has been observed in patients with the severe form of the disease. An alternative method of treatment that solves typical problems of enzyme replacement therapy is gene therapy, i.e., delivery of the correct gene to target cells through an appropriate vector. Much progress has been made in applying gene therapy for MPS II, from cellular models to human clinical trials. In this article, we briefly present the history and basics of gene therapy and discuss the current state of knowledge about the methods of this therapy in mucopolysaccharidosis type II.

Glycosylation of Therapeutic Proteins: A Critical Quality Attribute.

Glycosylation is a common posttranslational modification of therapeutic proteins. The glycosylation pattern is dependent on many parameters such as the host cell line or the culture conditions. N- and O-linked glycans usually play a great role on the stability, safety, and efficacy of the drug. For this reason, glycosylation is considered as a critical quality attribute of therapeutic glycoproteins, and a thorough characterization should be performed, as well as a systematic control for each batch produced. This chapter gives a short presentation of the structure of glycans commonly found on recombinant therapeutic proteins, and their role on the properties of the drug, in terms of stability, pharmacokinetics, safety, and efficacy. Lastly, the use of mass spectrometry for the analysis of glycoproteins is briefly described.

Glyco-engineered HEK 293-F cell lines for the production of therapeutic glycoproteins with human N-glycosylation and improved pharmacokinetics.

N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient's bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4 and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties.

Development and Application of a Physiologically-Based Pharmacokinetic Model to Predict the Pharmacokinetics of Therapeutic Proteins from Full-term Neonates to Adolescents.

Physiologically-based pharmacokinetic (PBPK) modelling provides an integrated framework to predict the disposition of small molecule drugs in children and is increasingly being used for dose recommendation and optimal design of paediatric studies and in regulatory submissions. Existing paediatric PBPK models can be adopted to describe the disposition of therapeutic proteins (TPs) in children by incorporating information on age-related changes of additional physiological and biological parameters (e.g. endogenous IgG, neonatal Fc receptor, lymph flow). In this study, physiological parameters were collated from literature and evaluated for any age-dependent trends. The age-dependent physiological parameters were used to construct a paediatric PBPK model for TPs. The model was then used to predict the pharmacokinetics of recombinant human erythropoietin (EPO), infliximab, etanercept, basiliximab, anakinra and enfuvirtide in paediatric subjects. The developed paediatric PBPK model predicted the drug concentration-time profiles reasonably well in full-term neonates (clinical PK data only available for EPO), infants, children and adolescents with the ratios of predicted over observed clearance values within 1.5-fold and 25 out of 26 clearance predictions were within 0.8- to 1.25-fold of the observed values. The clinically reported data are required to further assess the predictive accuracy of PK for Fc-containing proteins in term-born children younger than 2 months. This study demonstrates the ability of PBPK models accounting for age-dependent changes in relevant parameters to predict the pharmacokinetics of different types of TPs in paediatrics. The information gained from the PBPK models described here can facilitate our understanding of the complexities of TPs' disposition during growth and development.

Simulated in vitro infant gastrointestinal digestion of yak milk fat globules: A comparison with cow milk fat globules.

Lipolysis products released during digestion exert positive metabolic impacts on the nutrition of newborns. However, the lipolysis behavior of yak milk lipids during digestion remains unknown. In this study, the simulated in vitro infant gastrointestinal digestion of cow, yak and standardized yak milk fat globules the same size as those from cow milk (Cow MF, Yak MF and Yak SMF) were compared. Although Cow MF showed a higher lipolysis rate at the beginning of gastric digestion, Yak MF and Yak SMF exhibited a higher lipolysis level during later gastrointestinal digestion. Higher hydrolysis efficiency of yak milk lipids was due to their lipid properties, including their composition and structure. Furthermore, yak milk lipids released more unsaturated fatty acids than Cow MF throughout digestion. This study highlights the crucial role of lipid characteristics in the efficient digestion of milk lipids and provides new insight for the design of yak milk infant diets.

Safety, Pharmacokinetics and Pharmacodynamics of TNHH, a Novel Targeted Neutrophil-Inhibitory Hirulog Hybrid Glycoprotein, in Healthy Volunteers.

BACKGROUND: Targeted neutrophil inhibitory-hirulog (TNHH) is a novel hybrid glycoprotein that may be a potential drug candidate for acute ischaemic stroke. OBJECTIVE: The aim of this study was to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics of TNHH in healthy volunteers and thereby determine the dose range for future clinical studies. METHODS: This randomized, placebo-controlled study was a single ascending dose design with dose levels of 0.05-1.8 mg/kg (n = 4-6 active, 2 placebos per cohort) in 68 participants. In the TNHH 0.2-1.8 mg/kg and control cohorts, pharmacokinetic and pharmacodynamic blood samples were collected over 168 h after intravenous (IV) administration. TNHH occupancy in peripheral blood neutrophils and blood coagulation were evaluated as the markers of target engagement. RESULTS: Two subjects withdrew from the trial before administration of the study treatment, 66 subjects are included in the data analysis. TNHH was well tolerated in all dose regimens. In total, five mild, self-limiting adverse events (AEs) were observed in 4 of the 66 study subjects. Dose-proportional increases in maximum plasma concentration (Cmax) and area under the curve (AUC0-t) of TNHH were observed. Traces of TNHH were excreted in urine. The elimination half-life (t½) ranged from 0.6 to 1.3 h in the eight groups with ascending dose levels. TNHH combined with CD11b/CD18 quickly achieved > 90% receptor occupancy in groups with doses above 0.2 mg/kg. The Cmax and AUC of binding TNHH with CD11b/CD18 increased with the dose. A significant prolongation with dose was observed on thrombin time (TT), and weak influences were observed on prothrombin time (PT) and activated partial thromboplastin time (APTT). CONCLUSION: TNHH was well-tolerated following IV infusion. The pharmacokinetic and pharmacodynamic characteristics of TNHH indicate that it merits clinical trials. It is recommended that the single dose of TNHH should be 1.0 mg/kg in future studies, and the expected effect may be achieved after 5-7 days of continuous administration. TRIAL REGISTRATION: The study is registered at http://www.chictr.org.cn as ChiCTR-TQR-14004752.

Two compartment pharmacokinetic model describes the intra-articular delivery and retention of rhprg4 following ACL transection in the Yucatan mini pig.

Treatment of the injured joint with rhPRG4 is based on recent observations that inflammation diminishes expression of native PRG4. Re-establishing lubrication between pressurized and sliding cartilage surfaces during locomotion promotes the nascent expression of PRG4 and thus intra-articular (IA) treatment strategies should be supported by pharmacokinetic evidence establishing the residence time of rhPRG4. A total of 21 Yucatan minipigs weighing ∼55 kg each received 4 mg of 131 I-rhPRG4 delivered by IA injection 5 days following surgical ACL transection. Animals were sequentially euthanized following IA rhPRG4 at 10 min (time zero), 24, 72 h, 6, 13 and 20 days later. The decay of the 131 I-rhPRG4 was measured relative to a non-injected aliquot and normalized to the weight of cartilage samples, menisci and synovium, and known cartilage volumes from each compartment surface obtained from representative Yucatan minipig knees. Decay of 131 I-rhPRG4 from joint tissues best fit a two-compartment model with an α half-life (t1/2α ) of 11.28 h and ß half-life (t1/2ß ) of 4.81 days. The tibial and femoral cartilage, meniscii, and synovium retained 7.7% of dose at 24 h. High concentrations of rhPRG4 were found in synovial fluid (SF) that was non-aspiratable and resided on the articular surfaces, removable by irrigation, at 10 min following 131 I-rhPRG4 injection. Synovial fluid K21 exceeded K12 and SF t1/2ß was 28 days indicating SF is the reservoir for rhPRG4 following IA injection. Clinical Significance: rhPRG4 following IA delivery in a traumatized joint populates articular surfaces for a considerable period and may promote the native expression of PRG4. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:386-396, 2019.

Impact of P-Glycoprotein on Intestinal Absorption of an Inhibitor of Apoptosis Protein Antagonist in Rats: Mechanisms of Nonlinear Pharmacokinetics and Food Effects.

PURPOSE: This study was designed to investigate the effects of P-glycoprotein (P-gp) expressed in the intestine on the nonlinear pharmacokinetics (PK) of T-3256336, an inhibitor of apoptosis protein inhibitor, and food effects on its bioavailability in rats. METHODS: To investigate the factors that contribute to nonlinear PK of T-3256336 in the intestine and liver, rats double-cannulated in the portal vein and femoral artery (PS rats) were used. FaFg (Fa, absorption ratio; Fg, intestinal availability) and hepatic availability (Fh) were simultaneously evaluated based on the difference between the portal and systemic blood area under the concentration-time curve (AUC). Elacridar was used as a P-gp inhibitor to assess the impact of P-gp on the intestinal absorption. RESULTS: After oral administration of T-3256336 to PS rats at 3 and 30 mg/kg, FaFg value increased with dose escalation, whereas Fh value was nearly constant. Moreover, co-administration of elacridar resulted in a 5-fold increase in the FaFg value at 3 mg/kg. The AUC value of T-3256336 under fed conditions was 3-fold lower than that under fasted conditions. This food effect on the oral bioavailability (BA) was reduced by concomitant administration of elacridar. CONCLUSION: P-gp expressed in the intestine would cause nonlinear PK and a food effect on BA of T-3256336 in rats.

Targeted transport of nanocarriers into brain for theranosis with rabies virus glycoprotein-derived peptide.

For successful theranosis of brain diseases, limited access of therapeutic molecules across blood-brain barrier (BBB) needs be overcome in brain delivery. Currently, peptide derivatives of rabies virus glycoprotein (RVG) have been exploited as delivery ligands to transport nanocarriers across BBB and specifically into the brain. The targeting peptides usually conjugate to the nanocarrier surface, and the cargoes, including siRNA, miRNA, DNA, proteins and small molecular chemicals, are complexed or encapsulated in the nanocarriers. The peptide ligand of the RVG-modified nanocarriers introduces the conjugated targeted-delivery into the brain, and the cargoes are involved in disease theranosis. The peptide-modified nanocarriers have been applied to diagnose and treat various brain diseases, such as glioma, Alzheimer's disease, ischemic injury, protein misfolding diseases etc. Since the targeting delivery system has displayed good biocompatibility and desirable therapeutic effect, it will raise a potential application in treating brain diseases.

RVG29-modified docetaxel-loaded nanoparticles for brain-targeted glioma therapy.

Gliomas are the most common malignant brain tumor, but treatment is limited by the blood-brain barrier (BBB), especially for chemotherapeutic drugs. Although some chemotherapy drugs can pass through the BBB, many of these agents are toxic to normal brain tissue. To maximize therapeutic effects, chemotherapeutic drugs must accumulate at the glioma site. In this study, a specific ligand (the RVG29 peptide) that can combine with acetylcholine receptors was conjugated to polyethylene glycol-modified poly-(d,l-lactide-co-glycolide) (PEG-PLGA) to develop a targeted carrier; preparation of the targeted docetaxel nanoparticles (DTX-NPs) was performed by the nanoprecipitation method. The NPs were approximately 110 nm and had smooth surfaces. Enzyme-linked immunoassay results showed that the amount of receptor on the surface of glioma cells was 2.04-fold higher than that of nonmalignant cells, which may promote accumulation of RVG29-modified NPs at the targeting site. NPs showed targeting properties for glioma cells compared with the non-targeting NPs in an in vitro cellular uptake test. Targeted NPs also showed better BBB penetration in an in vitro model. In vivo tests indicated that RVG29-PEG-PLGA-NPs could selectively accumulate in intracranial glioma tissue. In conclusion, these results indicated that the RVG29-modified NPs have potential efficacy for glioma therapy.

ADPase CD39 Fused to Glycoprotein VI-Fc Boosts Local Antithrombotic Effects at Vascular Lesions.

BACKGROUND: GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric GPVI-Fc (Revacept) binds to GPVI binding sites on plaque collagen. As expected, it did not increase bleeding in clinical studies. GPVI-Fc is a potent inhibitor of atherosclerotic plaque-induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing GPVI-Fc to the ectonucleotidase CD39 (fusion protein GPVI-CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion-directed dual antiplatelet therapy that is expected to lack systemic bleeding risks. METHODS AND RESULTS: GPVI-CD39 effectively stimulated local ADP degradation and, compared with GPVI-Fc alone, led to significantly increased inhibition of ADP-, collagen-, and human plaque-induced platelet aggregation in Multiplate aggregometry and plaque-induced platelet thrombus formation under arterial flow conditions. GPVI-CD39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride-induced arterial thrombosis, GPVI-CD39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo. CONCLUSIONS: GPVI-CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances GPVI-Fc-mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk-benefit ratio.

N-Glycoprofiling Analysis for Carbohydrate Composition and Site-Occupancy Determination in a Poly-Glycosylated Protein: Human Thyrotropin of Different Origins.

Human thyrotropin (hTSH) is a glycoprotein with three potential glycosylation sites: two in the α-subunit and one in the ß-subunit. These sites are not always occupied and occupancy is frequently neglected in glycoprotein characterization, even though it is related to folding, trafficking, initiation of inflammation and host defense, as well as congenital disorders of glycosylation (CDG). For the first time N-glycoprofiling analysis was applied to the site-occupancy determination of two native pituitary hTSH, in comparison with three recombinant preparations of hTSH, a widely used biopharmaceutical. A single methodology provided the: (i) average N-glycan mass; (ii) mass fraction of each monosaccharide and of sulfate; and (iii) percent carbohydrate. The results indicate that the occupancy (65%-87%) and carbohydrate mass (12%-19%) can be up to 34%-57% higher in recombinant hormones. The average glycan mass is 24% lower in pituitary hTSH and contains ~3-fold fewer moles of galactose (p < 0.005) and sialic acid (p < 0.01). One of the two native preparations, which had the smallest glycan mass together with the lowest occupancy and GalNAc, sulfate, Gal and sialic acid contents, also presented the lowest in vivo bioactivity and circulatory half-life. The methodology described, comparing a recombinant biopharmaceutical to its native equivalent, can be applied to any physiologically or clinical relevant glycoprotein.

Co-ingesting milk fat with micellar casein does not affect postprandial protein handling in healthy older men.

BACKGROUND & AIM: Dietary protein digestion and absorption plays an important role in modulating postprandial muscle protein synthesis. The impact of co-ingesting other macronutrients with dietary protein on protein digestion and absorption and the subsequent muscle protein synthetic response remains largely unexplored. This study investigated the impact of co-ingesting milk fat with micellar casein on dietary protein-derived amino acid appearance in the circulation and the subsequent postprandial muscle protein synthetic response in healthy older men. METHODS: Twenty-four healthy, older males (age: 65 ± 1 y, BMI: 25.7 ± 0.5 kg/m2) received a primed continuous infusion of L-[ring-2H5]-phenylalanine and L-[1-13C]-leucine and ingested 20 g intrinsically L-[1-13C]-phenylalanine and L-[1-13C]-leucine-labeled casein with (PRO + FAT; n = 12) or without (PRO; n = 12) 26.7 g milk fat. Plasma samples and muscle biopsies were collected in both the postabsorptive and postprandial state. RESULTS: Release of dietary protein-derived phenylalanine into the circulation increased following protein ingestion (P < 0.001) and tended to be higher in PRO compared with PRO + FAT (Time × Treatment P = 0.076). No differences were observed in dietary protein-derived plasma phenylalanine availability (52 ± 2 vs 52 ± 3% in PRO vs PRO + FAT, respectively; P = 0.868). Myofibrillar protein synthesis rates did not differ between treatments, calculated using either the L-[ring-2H5]-phenylalanine (0.036 ± 0.003 vs 0.036 ± 0.004 %/h after PRO vs PRO + FAT, respectively; P = 0.933) or L-[1-13C]-leucine (0.051 ± 0.004 vs 0.046 ± 0.004 %/h, respectively; P = 0.480) tracer. In accordance, no differences were observed in myofibrillar protein-bound L-[1-13C]-phenylalanine enrichments between treatments (0.018 ± 0.002 vs 0.014 ± 0.001 MPE, respectively; P = 0.173). CONCLUSION: Co-ingesting milk fat with micellar casein does not impair protein-derived phenylalanine appearance in the circulation and does not modulate postprandial myofibrillar protein synthesis rates. CLINICAL TRIAL REGISTRATION NUMBER: NCT01680146 (http://www.clinicaltrials.gov/).

A Method for Determining the Content of Glycoproteins in Biological Samples.

The glycoprotein purified from the mycelium extract of Tremella fuciformis was marked with iodine through the iodine substitution reaction. The content of iodine, which is indicative of the amount of the marked tremella glycoprotein (ITG), was detected with Inductively coupled plasma mass spectrometry (ICP-MS). The method was found to be stable, sensitive, and accurate at detecting the content of iodine-substituted glycoprotein, and was used in the quantitative analysis of biological samples, including blood and organs. Different biological samples were collected from rats after oral administration of ITG, and were tested for iodine content by ICP-MS to calculate the amount of ITG in the samples. The results suggested that ICP-MS is a sensitive, stable, and accurate method for detection of iodinated glycoproteins in blood and organs.

Extracellular domain shedding influences specific tumor uptake and organ distribution of the EGFR PET tracer 89Zr-imgatuzumab.

Preclinical positron emission tomography (PET) imaging revealed a mismatch between in vivo epidermal growth factor receptor (EGFR) expression and EGFR antibody tracer tumor uptake. Shed EGFR ectodomain (sEGFR), which is present in cancer patient sera, can potentially bind tracer and therefore influence tracer kinetics. To optimize EGFR-PET, we examined the influence of sEGFR levels on tracer kinetics and tumor uptake of EGFR monoclonal antibody 89Zr-imgatuzumab in varying xenograft models. Human cancer cell lines A431 (EGFR overexpressing, epidermoid), A549 and H441 (both EGFR medium expressing, non-small cell lung cancer) were xenografted in mice. Xenografted mice received 10, 25 or 160 µg 89Zr-imgatuzumab, co-injected with equal doses 111In-IgG control. MicroPET scans were made 24, 72 and 144 h post injection, followed by biodistribution analysis. sEGFR levels in liver and plasma samples were determined by ELISA. 89Zr-imgatuzumab uptake in A431 tumors was highest (29.8 ± 5.4 %ID/g) in the 160 µg dose group. Contrary, highest uptake in A549 and H441 tumors was found at the lowest (10 µg) 89Zr-imgatuzumab dose. High 89Zr-imgatuzumab liver accumulation was found in A431 xenografted mice, which decreased with antibody dose increments. 89Zr-imgatuzumab liver uptake in A549 and H441 xenografted mice was low at all doses. sEGFR levels in liver and plasma of A431 bearing mice were up to 1000-fold higher than levels found in A549, H441 and non-tumor xenografted mice. 89Zr-imgatuzumab effectively visualizes EGFR-expressing tumors. High sEGFR levels can redirect 89Zr-imgatuzumab to the liver, in which case tumor visualization can be improved by increasing tracer antibody dose.

Phase IA Clinical Trial Evaluating the Tolerability, Pharmacokinetics, and Analgesic Efficacy of an Intrathecally Administered Neurotensin A Analogue in Central Neuropathic Pain Following Spinal Cord Injury.

We evaluated CGX-1160 in a Phase Ia clinical trial to determine the safety of escalating doses in patients with central neuropathic pain following spinal cord injury (SCI). Our secondary objective was to detect a trend toward analgesic efficacy. Four subjects received 3 consecutive escalating doses of CGX-1160 starting at 25 µg/h over 6 hours until 2 consecutive subjects experienced any adverse effect; 2 of the 4 subjects received 2 sequences of 3 consecutive dose escalations. Maximum tolerated dose was defined by the development of diarrhea (900 µg/h over 6 hours). Cerebrospinal fluid (CSF) and blood were collected for pharmacokinetic (PK) evaluation. The CSF concentration-versus-time data fit to a biexponential PK model, showing a rapid redistribution phase followed by a significantly slower terminal elimination phase. Incorporating an effect site delay into the model improved the fit to the data (concentration producing 50% of the maximum effect [C50 ], 58.7 ug/mL at the site of drug effect). Maximal reduction from the baseline pain intensity was 63%. In summary, CGX-1160 was generally well tolerated when administered intrathecally at doses up to 1000 µg/h. Peak analgesic effect occurred after the peak intrathecal concentration, indicating the presence of an effect site compartment to the PK model to represent the concentration and effect profiles for this unique compound.

Exploring the glycan interaction in vivo: Future prospects of neo-glycoproteins for diagnostics.

Herein the biodistributions and in vivo kinetics of chemically prepared neoglycoproteins are reviewed. Chemical methods can be used to conjugate various mono- and oligosaccharides onto a protein surface. The kinetics and organ-specific accumulation profiles of these glycoconjugates, which are introduced through intravenous injections, have been analyzed using conventional dissection studies as well as noninvasive methods such as single photon emission computed tomography, positron emission tomography and fluorescence imaging. These studies suggest that glycan-dependent protein distribution kinetics may be useful for pharmacological and diagnostic applications.

Farmacorresistencia en epilepsia. Conceptos clínicos y neurobiológicos / Drug resistant epilepsy. Clinical and neurobiological concepts

La epilepsia farmacorresistente es una condición definida por la Liga Internacional contra la Epilepsia como la persistencia de crisis epilépticas a pesar de haber utilizado al menos dos tratamientos con fármacos antiepilépticos apropiados y adecuados. Cerca de un 20-30% de los pacientes con epilepsia van a ser resistentes a los fármacos antiepilépticos, con diferentes patrones de presentación clínica, los cuales están en relación con las bases biológicas de esta enfermedad (resistencia de novo, recaída-remisión y progresiva). La farmacorresistencia en epilepsia impacta negativamente en la calidad de vida y aumenta significativamente el riesgo de muerte prematura. Desde el punto de vista neurobiológico, esta condición clínica es el resultado de la interacción de múltiples variables relacionadas con la enfermedad de base, las interacciones medicamentosas y los aspectos genéticos propios de cada paciente. Gracias a los avances en la investigación farmacogenética y de biología molecular, actualmente se plantean algunas hipótesis que podrían explicar la causa de esta condición y que promueven el estudio de nuevas opciones terapéuticas. En la actualidad, la sobreexpresión de transportadores de membrana, como la glucoproteína P, parece ser uno de los mecanismos más importantes en el desarrollo de la farmacorresistencia en epilepsia. El objetivo de esta revisión es profundizar en los aspectos generales de esta condición clínica, abordando la definición, los aspectos epidemiológicos, los diagnósticos diferenciales y las bases fisiopatológicas (AU)

Drug-resistant epilepsy, is a condition defined by the International League Against Epilepsy as persistent seizures despite having used at least two appropriate and adequate antiepileptic drug treatments. Approximately 20-30% of patients with epilepsy are going to be resistant to antiepileptic drugs, with different patterns of clinical presentation, which are related to the biological basis of this disease (de novo resistance, relapsing-remitting and progressive). Drug resistant epilepsy, impacts negatively the quality of life and significantly increases the risk of premature death. From the neurobiological point of view, this medical condition is the result of the interaction of multiple variables related to the underlying disease, drug interactions and proper genetic aspects of each patient. Thanks to advances in pharmacogenetics and molecular biology research, currently some hypotheses may explain the cause of this condition and promote the study of new therapeutic options. Currently, overexpression of membrane transporters such as P-glycoprotein, appears to be one of the most important mechanisms in the development of drug resistant epilepsy. The objective of this review is to deepen the general aspects of this clinical condition, addressing the definition, epidemiology, differential diagnosis and the pathophysiological bases (AU)

Using glyco-engineering to produce therapeutic proteins.

INTRODUCTION: Glycans are increasingly important in the development of new biopharmaceuticals with optimized efficacy, half-life, and antigenicity. Current expression platforms for recombinant glycoprotein therapeutics typically do not produce homogeneous glycans and frequently display non-human glycans which may cause unwanted side effects. To circumvent these issues, glyco-engineering has been applied to different expression systems including mammalian cells, insect cells, yeast, and plants. AREAS COVERED: This review summarizes recent developments in glyco-engineering focusing mainly on in vivo expression systems for recombinant proteins. The highlighted strategies aim at producing glycoproteins with homogeneous N- and O-linked glycans of defined composition. EXPERT OPINION: Glyco-engineering of expression platforms is increasingly recognized as an important strategy to improve biopharmaceuticals. A better understanding and control of the factors leading to glycan heterogeneity will allow simplified production of recombinant glycoprotein therapeutics with less variation in terms of glycosylation. Further technological advances will have a major impact on manufacturing processes and may provide a completely new class of glycoprotein therapeutics with customized functions.

Pharmacokinetics and osteogenic potential of PEGylated NELL-1 in vivo after systemic administration.

Osteoporosis is a skeletal disorder attributable to an imbalance in osteoblast and osteoclast activity. NELL-1, a secretory protein that promotes osteogenesis while suppressing osteoclastic activity, holds potential as an osteoporosis therapy. Recently, we demonstrated that PEGylation of NELL-1 significantly improves its thermostability while preserving its bioactivity in vitro. However, the effect of PEGylation on the pharmacokinetics and osteogenic potential of NELL-1 in vivo have yet to be investigated. The present study demonstrated that PEGylation of NELL-1 significantly increases the elimination half-life time of the protein from 5.5 h to 15.5 h while distributing more than 2-3 times the amount of protein to bone tissues (femur, tibia, vertebrae, calvaria) in vivo when compared to naked NELL-1. In addition, microCT and DXA analyses demonstrated that systemic NELL-PEG therapy administered every 4 or 7 days significantly increases not only femoral and lumbar BMD and percent bone volume, but also new bone formation throughout the overall skeleton after four weeks of treatment. Furthermore, immunohistochemistry revealed increased osteocalcin expression, while TRAP staining showed reduced osteoclast numbers in NELL-PEG groups. Our findings suggest that the PEGylation technique presents a viable and promising approach to further develop NELL-1 into an effective systemic therapeutic for the treatment of osteoporosis.