Cyclodextrin-mediated Crystallization of Acid β-glucosidase in Complex with Amphiphilic Bicyclic Nojirimycin Analogues

Cyclodextrin-based host-guest chemistry has been exploited to facilitate co-crystallization of recombinant human acid β-glucosidase (β-glucocerebrosidase, GlcCerase) with amphiphilic bicyclic nojirimycin analogues of the sp2-iminosugar type. Attempts to co-crystallize GlcCerase with 5-N,6-O-[N′-(n-octyl)iminomethylidene]nojirimycin (NOI-NJ) or with 5-N,6-S-[N′-(n-octyl)iminomethylidene]-6-thionojirimycin (6S-NOI-NJ), two potent inhibitors of the enzyme with promising pharmacological chaperone activity for several Gaucher disease-associated mutations, were unsuccessful probably due to the formation of aggregates that increase the heterogeneity of the sample and affect nucleation and growth of crystals. Cyclomaltoheptaose (β-cyclodextrin, βCD) efficiently captures NOI-NJ and 6S-NOI-NJ in aqueous media to form inclusion complexes in which the lipophilic tail is accommodated in the hydrophobic cavity of the cyclooligosaccharide. The dissociation constant of the complex of the amphiphilic sp2-iminosugars with βCD is two orders of magnitude higher than that of the corresponding complex with GlcCerase, allowing the efficient transfer of the inhibitor from the βCD cavity to the GlcCerase active site. Enzyme-inhibitor complexes suitable for X-ray analysis were thus grown in the presence of βCD. In contrast to what was previously observed for the complex of GlcCerase with the more basic derivative, 6-amino-6-deoxy-5-N,6-N-[N′-(n-octyl)iminomethylidene]nojirimycin (6N-NOI-NJ), the β-anomers of both NOI-NJ and 6S-NOI-NJ were seen in the active site, even though the α-anomer was exclusively detected both in aqueous solution and in the corresponding βCD:sp2-iminosugar complexes. Our results further suggest that cyclodextrin derivatives might serve as suitable delivery systems of amphiphilic glycosidase inhibitors in a biomedical context.Ministerio de Ciencia e Innovación CTQ2007-61180/PPQ, SAF2010-15670, CTQ2010-15848Junta de Andalucía P08-FQM-03711Fondo Europeo de Desarrollo Regional 03122, ISSG-CT-2007-03719

A Plant-Derived Recombinant Human Glucocerebrosidase Enzyme—A Preclinical and Phase I Investigation

Gaucher disease is a progressive lysosomal storage disorder caused by the deficiency of glucocerebrosidase leading to the dysfunction in multiple organ systems. Intravenous enzyme replacement is the accepted standard of treatment. In the current report, we evaluate the safety and pharmacokinetics of a novel human recombinant glucocerebrosidase enzyme expressed in transformed plant cells (prGCD), administered to primates and human subjects. Short term (28 days) and long term (9 months) repeated injections with a standard dose of 60 Units/kg and a high dose of 300 Units/kg were administered to monkeys (n = 4/sex/dose). Neither clinical drug-related adverse effects nor neutralizing antibodies were detected in the animals. In a phase I clinical trial, six healthy volunteers were treated by intravenous infusions with escalating single doses of prGCD. Doses of up to 60 Units/kg were administered at weekly intervals. prGCD infusions were very well tolerated. Anti-prGCD antibodies were not detected. The pharmacokinetic profile of the prGCD revealed a prolonged half-life compared to imiglucerase, the commercial enzyme that is manufactured in a costly mammalian cell system. These studies demonstrate the safety and lack of immunogenicity of prGCD. Following these encouraging results, a pivotal phase III clinical trial for prGCD was FDA approved and is currently ongoing.ClinicalTrials.gov NCT00258778

Regulation of Cytokine Signaling by B Cell Antigen Receptor and Cd40-Controlled Expression of Heparan Sulfate Proteoglycans

Recently, biochemical, cell biological, and genetic studies have converged to reveal that integral membrane heparan sulfate proteoglycans (HSPGs) are critical regulators of growth and differentiation of epithelial and connective tissues. As a large number of cytokines involved in lymphoid tissue homeostasis or inflammation contain potential HS-binding domains, HSPGs presumably also play important roles in the regulation of the immune response. In this report, we explored the expression, regulation, and function of HSPGs on B lymphocytes. We demonstrate that activation of the B cell antigen receptor (BCR) and/or CD40 induces a strong transient expression of HSPGs on human tonsillar B cells. By means of these HSPGs, the activated B cells can bind hepatocyte growth factor (HGF), a cytokine that regulates integrin-mediated B cell adhesion and migration. This interaction with HGF is highly selective since the HSPGs did not bind the chemokine stromal cell–derived factor (SDF)-1α, even though the affinities of HGF and SDF-1α for heparin are similar. On the activated B cells, we observed induction of a specific HSPG isoform of CD44 (CD44-HS), but not of other HSPGs such as syndecans or glypican-1. Interestingly, the expression of CD44-HS on B cells strongly promotes HGF-induced signaling, resulting in an HS-dependent enhanced phosphorylation of Met, the receptor tyrosine kinase for HGF, as well as downstream signaling molecules including Grb2-associated binder 1 (Gab1) and Akt/protein kinase B (PKB). Our results demonstrate that the BCR and CD40 control the expression of HSPGs, specifically CD44-HS. These HSPGs act as functional coreceptors that selectively promote cytokine signaling in B cells, suggesting a dynamic role for HSPGs in antigen-specific B cell differentiation

Evolution of Plant-Made Pharmaceuticals

The science and policy of pharmaceuticals produced and/or delivered by plants has evolved over the past twenty-one years from a backyard remedy to regulated, purified products. After seemingly frozen at Phase I human clinical trials with six orally delivered plant-made vaccines not progressing past this stage over seven years, plant-made pharmaceuticals have made a breakthrough with several purified plant-based products advancing to Phase II trials and beyond. Though fraught with the usual difficulties of pharmaceutical development, pharmaceuticals made by plants have achieved pertinent milestones albeit slowly compared to other pharmaceutical production systems and are now at the cusp of reaching the consumer. Though the current economic climate begs for cautious investment as opposed to trail blazing, it is perhaps a good time to look to the future of plant-made pharmaceutical technology to assist in planning for future developments in order not to slow this technology’s momentum. To encourage continued progress, we highlight the advances made so far by this technology, particularly the change in paradigms, comparing developmental timelines, and summarizing the current status and future possibilities of plant-made pharmaceuticals

Memory Influences Visual Cognition across Multiple Functional States of Interactive Cortical Dynamics

No embargo requiredMemory supports a wide range of abilities from categorical perception to goal-directed behavior, such as decision-making and episodic recognition. Memory activates fast and surprisingly accurately and even when information is ambiguous or impoverished (i.e., showing object constancy). This paper proposes the multiple-state interactive (MUSI) account of object cognition that attempts to explain how sensory stimulation activates memory across multiple functional states of neural dynamics, including automatic and strategic mental simulation mechanisms that can ground cognition in modal information processing. A key novel postulate of this account is ‘multiple-function regional activity’: The same neuronal population can contribute to multiple brain states, depending upon the dominant set of inputs at that time. In state 1, the initial fast bottom-up pass through posterior neocortex happens between 95 ms and ~200 ms, with knowledge supporting categorical perception by 120 ms. In state 2, starting around 200 ms, a sustained state of iterative activation of object-sensitive cortex involves bottom-up, recurrent, and feedback interactions with frontoparietal cortex. This supports higher cognitive functions associated with decision-making even under ambiguous or impoverished conditions, phenomenological consciousness, and automatic mental simulation. In the latest state so far identified, state M, starting around 300 to 500 ms, large-scale cortical network interactions, including between multiple networks (e.g., control, salience, and especially default mode), further modulate posterior cortex. This supports elaborated cognition based on earlier processing, including episodic memory, strategic mental simulation, decision evaluation, creativity, and access consciousness. Convergent evidence is reviewed from cognitive neuroscience of object cognition, decision-making, memory, and mental imagery that support this account and define the brain regions and time course of these brain dynamics

Glycosylation and functionality of recombinant β-glucocerebrosidase from various production systems

Synopsis The glycosylation of recombinant β-glucocerebrosidase, and in particular the exposure of mannose residues, has been shown to be a key factor in the success of ERT (enzyme replacement therapy) for the treatment of GD (Gaucher disease). Macrophages, the target cells in GD, internalize β-glucocerebrosidase through MRs (mannose receptors). Three enzymes are commercially available for the treatment of GD by ERT. Taliglucerase alfa, imiglucerase and velaglucerase alfa are each produced in different cell systems and undergo various post-translational or post-production glycosylation modifications to expose their mannose residues. This is the first study in which the glycosylation profiles of the three enzymes are compared, using the same methodology and the effect on functionality and cellular uptake is evaluated. While the major differences in glycosylation profiles reside in the variation of terminal residues and mannose chain length, the enzymatic activity and stability are not affected by these differences. Furthermore, the cellular uptake and in-cell stability in rat and human macrophages are similar. Finally, in vivo studies to evaluate the uptake into target organs also show similar results for all three enzymes. These results indicate that the variations of glycosylation between the three regulatory-approved β-glucocerebrosidase enzymes have no effect on their function or distribution