Ameliorative effect of betulin from Betula platyphylla bark on scopolamine-induced amnesic mice.

Alzheimer's disease (AD) is a neurodegenerative disease induced by cholinergic neuron damage or amyloid-beta aggregation in the basal forebrain region and resulting in cognitive disorder. We previously reported on the neuroprotective effects of Betula platyphylla bark (BPB) in an amyloid-beta-induced amnesic mouse model. In this study, we obtained a cognitive-enhancing compound by assessing results using a scopolamine-induced amnesic mouse model. Our results show that oral treatment of mice with BPB and betulin significantly ameliorated scopolamine-induced memory deficits in both passive avoidance and Y-maze tests. In the Morris water maze test, administration of BPB and betulin significantly improved memory and cognitive function indicating the formation of working and reference memories in treated mice. Moreover, betulin significantly increased glutathione content in mouse hippocampus, and the increase was greater than that from betulinic acid treatment. We conclude that BPB and its active component betulin have potential as therapeutic, cognitive enhancer in AD.

ent-kaurane and ent-pimarane diterpenes from Siegesbeckia pubescens inhibit lipopolysaccharide-induced nitric oxide production in BV2 microglia.

The extract of Siegesbeckia pubescens herb and its chemical constituents were tested for the ability to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 microglia. The methanol extract and the 90% MeOH fraction of S. pubescens effectively attenuated lipopolysaccharide-induced nitric oxide production. Several steps of chromatography yielded eight ent-kaurane diterpenes (1-8) and one ent-pimarane diterpene (9) from the 90% MeOH fraction. Among these compounds, compounds 2-9 showed significant inhibitory effect on lipopolysaccharide-induced nitric oxide production in BV2 microglia. Compounds 3 and 9 concentration-dependently decreased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), supported by quantitative real time polymerase chain reaction (PCR) and Western blot analysis. These results suggest that ent-kaurane and ent-pimarane diterpenes isolated from S. pubescens are expected to be potential candidates against neuroinflammation-related disease.

Incorporation of monodisperse oligoethyleneglycol amino acids into anticonvulsant analogues of galanin and neuropeptide y provides peripherally acting analgesics.

Delivery of neuropeptides into the central and/or peripheral nervous systems supports development of novel neurotherapeutics for the treatment of pain, epilepsy and other neurological diseases. Our previous work showed that the combination of lipidization and cationization applied to anticonvulsant neuropeptides galanin (GAL) and neuropeptide Y (NPY) improved their penetration across the blood-brain barrier yielding potent antiepileptic lead compounds, such as Gal-B2 (NAX 5055) or NPY-B2. To dissect peripheral and central actions of anticonvulsant neuropeptides, we rationally designed, synthesized and characterized GAL and NPY analogues containing monodisperse (discrete) oligoethyleneglycol-lysine (dPEG-Lys). The dPEGylated analogues Gal-B2-dPEG(24), Gal-R2-dPEG(24) and NPY-dPEG(24) displayed analgesic activities following systemic administration, while avoiding penetration into the brain. Gal-B2-dPEG(24) was synthesized by a stepwise deprotection of orthogonal 4-methoxytrityl and allyloxycarbonyl groups, and subsequent on-resin conjugations of dPEG(24) and palmitic acids, respectively. All the dPEGylated analogues exhibited substantially decreased hydrophobicity (expressed as logD values), increased in vitro serum stabilities and pronounced analgesia in the formalin and carrageenan inflammatory pain assays following systemic administration, while lacking apparent antiseizure activities. These results suggest that discrete PEGylation of neuropeptides offers an attractive strategy for developing neurotherapeutics with restricted penetration into the central nervous system.

Cyclic analogs of galanin and neuropeptide Y by hydrocarbon stapling.

Hydrocarbon stapling is an effective strategy to stabilize the helical conformation of bioactive peptides. Here we describe application of stapling to anticonvulsant neuropeptides, galanin (GAL) and neuropeptide Y (NPY), that are implicated in modulating seizures in the brain. Dicarba bridges were rationally introduced into minimized analogs of GAL and NPY resulting in increased α-helical content, in vitro metabolic stability and n-octanol/water partitioning coefficient (logD). The stapled analogs retained agonist activities towards their respective receptors and suppressed seizures in a mouse model of epilepsy.

Extracellular production of a thermostable Cellvibrio endolytic ß-agarase in Escherichia coli for agarose liquefaction.

Four GH16 family ß-agarases (GH16A, GH16B, GH16C, and GH16D), originated from an agarolytic bacterium Cellvibrio sp. KY-GH-1, were expressed in an Escherichia coli system and their activities were compared. Only GH16B (597 amino acids, 63.8 kDa), with N-terminal 22-amino acid signal sequence, was secreted into the culture supernatant and demonstrated a robust endolytic agarose hydrolyzing activity for producing neoagarotetraose (NA4) and neoagarohexaose (NA6) as end products. The optimal temperature and pH for the enzyme activity were 50 °C and 7.0, respectively. The enzyme was stable up to 50 °C and over a pH range of 5.0-8.0. The kinetic parameters, including Km, Vmax, kcat, and kcat/Km, of GH16B ß-agarases for agarose were 14.40 mg/mL, 542.0 U/mg, 576.3 s-1, and 4.80 × 106 s-1 M-1, respectively. The addition of 1 mM MnCl2 and 15 mM tris(2-carboxyethyl)phosphine enhanced the enzymatic activity. When agarose or neoagaro-oligosaccharides were used as substrates, the end products of enzymatic catalysis were NA4 and NA6, whereas agaropentaose was produced along with NA4 and NA6 when agaro-oligosaccharides were used as substrates. Treatment of 9%[w/v] melted agarose with the enzyme (1.6 µg/mL) under continuous magnetic stirring at 50 °C for 14 h resulted in efficient agarose liquefaction into NA4 and NA6. Purification of NA4 and NA6 from the enzymatic hydrolysate (9%[w/v] agarose, 20 mL) via Sephadex G-15 column chromatography yielded ~ 650 mg NA4/~ 900 mg NA6 (i.e., ~ 85.3% of the theoretical maximum yield). These findings suggest that the recombinant thermostable GH16B ß-agarase is useful for agarose liquefaction to produce NA4 and NA6.

Chemical constituents of Alnus firma and their inhibitory activity on lipopolysaccharide-induced nitric oxide production in BV2 microglia.

A methanolic extract of ALNUS FIRMA barks (Betulaceae) significantly attenuated nitric oxide production in lipopolysaccharide-stimulated BV2 microglia. Two new compounds, characterized as 4-hydroxy-2,6-dimethoxyphenyl 6'- O-syringoyl- beta- D-glucopyranoside (1) and 4-hydroxy-2,6-dimethoxyphenyl 6'- O-vanilloyl- beta- D-glucopyranoside (2), were isolated from the barks of A. FIRMA using bioactivity-guided fractionation, together with two known phenolic glycosides (3, 4) and 11 known diarylheptanoids (5- 15). Among these compounds, 1- 4 and 6- 11 showed significant inhibitory effect on lipopolysaccharide-induced nitric oxide production in BV2 microglial cells at concentrations ranging from 10 microM to 100 microM.

Ocular translational science: A review of development steps and paths.

Developing successful drug delivery methods is challenging for any tissue, and the eye is no exception. Translating initial concepts into advanced technologies treating diseases in preclinical models and finally into functional and marketable products for humans can be particularly daunting. While referring to specific ophthalmic companies and products, this review considers key exchanges that lead to successful translation. By building on basic science discoveries in the academic setting, applied science can perform proof-of-concept work with simple, benchtop experiments. Eventually, simple models need to be translated to more robust ones where cells, tissues, and entire organisms are incorporated. Successful translation also includes performing due diligence of the intellectual property, understanding the market needs, undertaking clinical development, meeting regulatory requirements, and eventually scale up manufacturing. Different stages of the translation can occur in different environments, including moving from academia to industry, from one company to another, or between veterinary and human applications. The translation process may also rely on contract organizations to move through the complex landscape. While the path to a commercial, marketable product may not look the same each time, it is important to design a development plan with clear goals and milestones to keep on track.

Treatment of corneal chemical alkali burns with a crosslinked thiolated hyaluronic acid film.

PURPOSE: The study objective was to test the utilization of a crosslinked, thiolated hyaluronic acid (CMHA-S) film for treating corneal chemical burns. METHODS: Burns 5.5mm in diameter were created on 10 anesthetized, male New Zealand white rabbits by placing a 1N NaOH soaked circular filter paper onto the cornea for 30s. Wounds were immediately rinsed with balanced salt solution (BSS). CMHA-S films were placed in the left inferior fornix of five injured and five uninjured animals. Five animals received no treatment. At 0h, 48h, 96h, and on day 14 post chemical burn creation, eyes were evaluated by white light imaging, fluorescein staining, and optical coherence tomography (OCT). Corneal histology was performed using H&E and Masson's Trichrome stains. RESULTS: Image analysis indicated biocompatible CMHA-S treatment resulted in significant decreases in the areas of corneal opacity at 48h, 96h, and on day 14 postoperatively. A significant increase in re-epithelialization was seen 14days post injury. CMHA-S treated corneas showed significantly less edema than untreated burns. No pathological differences were observed in corneal histological samples as a result of CMHA-S treatment. CONCLUSIONS: CMHA-S films facilitate re-epithelialization and decrease the area of corneal opacity in our corneal alkali burn rabbit model.

A marine analgesic peptide, Contulakin-G, and neurotensin are distinct agonists for neurotensin receptors: uncovering structural determinants of desensitization properties.

Neurotensin receptors have been studied as molecular targets for the treatment of pain, schizophrenia, addiction, or cancer. Neurotensin (NT) and Contulakin-G, a glycopeptide isolated from a predatory cone snail Conus geographus, share a sequence similarity at the C-terminus, which is critical for activation of neurotensin receptors. Both peptides are potent analgesics, although affinity and agonist potency of Contulakin-G toward neurotensin receptors are significantly lower, as compared to those for NT. In this work, we show that the weaker agonist properties of Contulakin-G result in inducing significantly less desensitization of neurotensin receptors and preserving their cell-surface density. Structure-activity relationship (SAR) studies suggested that both glycosylation and charged amino acid residues in Contulakin-G or NT played important roles in desensitizing neurotensin receptors. Computational modeling studies of human neurotensin receptor NTS1 and Contulakin-G confirmed the role of glycosylation in weakening interactions with the receptors. Based on available SAR data, we designed, synthesized, and characterized an analog of Contulakin-G in which the glycosylated amino acid residue, Gal-GalNAc-Thr10, was replaced by memantine-Glu10 residue. This analog exhibited comparable agonist potency and weaker desensitization properties as compared to that of Contulakin-G, while producing analgesia in the animal model of acute pain following systemic administration. We discuss our study in the context of feasibility and safety of developing NT therapeutic agents with improved penetration across the blood-brain barrier. Our work supports engineering peptide-based agonists with diverse abilities to desensitize G-protein coupled receptors and further emphasizes opportunities for conotoxins as novel pharmacological tools and drug candidates.

The effects of Betula platyphylla bark on amyloid beta-induced learning and memory impairment in mice.

Alzheimer's disease (AD) is closely associated with amyloid ß (Aß)-induced neurotoxicity and oxidative stress in the brain. Betula platyphylla, which has been used to treat various oxidative-stressed related diseases, has recently received attention for its preventive activity on age-related neurodegenerative diseases. In this study, we attempted to investigate the effects of B. platyphylla bark (BPB-316) on Aß(1-42)-induced neurotoxicity and memory impairment. Oral treatment using BPB-316 significantly attenuated Aß-induced memory impairment which was evaluated by behavioral tests including the passive avoidance, Y-maze and Morris water maze test. BPB-316 also inhibited the elevation of ß-secretase activity accompanying the reduced Aß(1-42) levels in the hippocampus of the brain. Furthermore, BPB-316 significantly decreased the acetylcholinesterase activity and increased the glutathione content in the hippocampus. In addition, we confirmed that the expression of both cAMP responsive element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in the hippocampus of Aß(1-42)-injected mice were markedly upregulated by the treatment of BPB-316. Our data suggest that the extracts of B. platyphylla bark might be a potential therapeutic agent against AD.

The effects of lignan-riched extract of Shisandra chinensis on amyloid-ß-induced cognitive impairment and neurotoxicity in the cortex and hippocampus of mouse.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruits of Schisandra chinensis (Trucz.) Baill. (Schisandraceae) which have been used as a tonic especially for kidney yin deficiency in Chinese traditional medicine are recently receiving attention for its preventive activity on age-related neurodegenerative diseases. A variety of studies demonstrated the cognitive-enhancing effects of Schisandra chinensis through animal tests and also in clinical trials. AIM OF STUDY: In this study, we attempted to investigate the effects of the lignan-riched extract of Schisandra chinensis fruits (ESP-806) on neurotoxicity and memory impairment induced by Aß1-42 injection in mice. MATERIALS AND METHODS: The fruits of Schisandra chinensis were extracted with the mixture of n-hexane:ethanol (9:1), which is riched with bioactive dibenzocyclooctadiene lignans, schizandrin, gomisin N, wuweigisu C. After oral treatment of ESP-806 (100 mg/kg body weight) followed by injection of Aß1-42 (2 µg/mouse, i.c.v.), novel object recognition and passive avoidance tests were evaluated. To verify the cognition enhancing effects of ESP-806, we examined the effects of ESP-806 on the activities of ß-secretase and acetylcholinesterase, and the contents of Aß and the reduced glutathione within the cortex and hippocampus of Aß-injected mice. RESULTS: Oral treatment of ESP-806 (100 mg/kg body weight) significantly attenuated Aß1-42-induced memory impairment evaluated by behavioral tests. Furthermore, the treatment of ESP-806 attenuated the elevation of ß-secretase activity accompanying the reduced level of Aß1-42 in the cortex and hippocampus of the brain. ESP-806 also significantly inhibited the acetylcholinesterase activity in the hippocampus and increased the content of the reduced glutathione in the cortex and hippocampus of mouse brain. CONCLUSIONS: These data suggested that the extract of Schisandra chinensis fruits riched with dibenzocyclooctadiene lignans may be useful in the prevention and treatment of Alzheimer's disease.

Prickly pear cactus (Opuntia ficus indica var. saboten) protects against stress-induced acute gastric lesions in rats.

The protective activity of prickly pear cactus (Opuntia ficus indica var. saboten) fruit juice and its main constituent, betanin, were evaluated against stress-induced acute gastric lesions in rats. After 6 h of water immersion restraint stress (WIRS), gastric mucosal lesions with bleeding were induced in Sprague-Dawley rats. Pretreatment of a lyophilized powder containing O. ficus indica var. saboten fruit juice and maltodextrin (OFSM) and betanin significantly reduced stress lesions (800-1600 mg/kg). Both OFSM and betanin effectively prevented the decrease in gastric mucus content as detected by alcian blue staining. In addition, OFSM significantly suppressed WIRS-induced increases in the level of gastric mucosal tumor necrosis factor-α and myeloperoxidase (MPO). Betanin alone was only effective in decreasing MPO. These results revealed the protective activity of OFSM against stress-induced acute gastric lesions and that betanin may contribute to OFSM's gastric protective activity, at least in part. When OFSM and betanin were taken together, OFSM exerted gastroprotective activity against stress-induced gastric lesions by maintaining gastric mucus, which might be related to the attenuation of MPO-mediated damage and proinflammatory cytokine production.

Synthesis and applications of polyamine amino acid residues: improving the bioactivity of an analgesic neuropeptide, neurotensin.

Conjugated polyamines are potential carriers for biotherapeutics targeting the central nervous system. We describe an efficient synthesis of a polyamine-based amino acid, lysine-trimethylene(diNosyl)-spermine(triBoc) with Dde or Fmoc orthogonal protecting groups. This nonnatural amino acid was incorporated into a neurotensin analogue using standard Fmoc-based protocols. The analogue maintained high affinity and agonist potency for neurotensin receptors and exhibited dramatically improved analgesia in mice. Our work provides a basis for use of polyamine amino acids in polypeptides.

Glycosylated neurotensin analogues exhibit sub-picomolar anticonvulsant potency in a pharmacoresistant model of epilepsy.

Neurotensin (NT) is an endogenous neuropeptide involved in a variety of central and peripheral neuromodulatory effects. Herein we show the effects of site-specific glycosylation on the in vitro and in vivo pharmacological properties of this neuropeptide. NT analogues containing O-linked disaccharides (beta-melibiose and alpha-TF antigen) or beta-lactose units linked by a PEG(3) spacer were designed and chemically synthesized using Fmoc chemistry. For the latter analogue, Fmoc-Glu-(beta-Lac-PEG(3)-amide) was prepared. Our results indicate that the addition of the disaccharides does not negatively affect the sub-nanomolar affinity or the low-nanomolar agonist potency for the neurotensin receptor subtype 1 (NTS1). Interestingly, three glycosylated analogues exhibited sub-picomolar potency in the 6 Hz limbic seizure mouse model of pharmacoresistant epilepsy following intracerebroventricular administration. Our results suggest for the first time that chemically modified NT analogues may lead to novel antiepileptic therapies.

Anticonvulsant Met-enkephalin analogues containing backbone spacers reveal alternative non-opioid signaling in the brain.

Prosthesis of non-critical parts of a polypeptide backbone is an attractive strategy to simplify bioactive peptides. This approach was applied to an opioid neuropeptide, Met-enkephalin, in which two adjacent Gly2-Gly3 residues were replaced with a series of non-peptidic backbone spacers varying in length and/or physicochemical properties. The backbone spacers did not affect the overall structural properties of the analogues, but they did dramatically reduce their affinities and agonist activities toward delta- and mu-opioid receptors. Molecular modeling suggested that the decrease of the affinity of Met-enkephalin to delta-opioid receptor could be accounted for by the loss of a single hydrogen bond. Remarkably, the analogues containing the most isostere spacers retained potent antinociceptive and anticonvulsant properties that were comparable to that of the endogenous peptide. This unexpected high in vivo potency could not be accounted for by an increase in metabolic stability. Moreover, the antiepileptic activity could not be reversed by opioid receptor antagonists. In summary, the results obtained with the analogues containing backbone spacers suggest a novel mechanism for seizure control in the brain that involves alternative non-opioid signaling.

Design, synthesis, and characterization of high-affinity, systemically-active galanin analogues with potent anticonvulsant activities.

Galanin is an endogenous neuropeptide that modulates seizures in the brain. Because this neuropeptide does not penetrate the blood-brain barrier, we designed truncated galanin analogues in which nonessential amino acid residues were replaced by cationic and/or lipoamino acid residues. The analogues prevented seizures in the 6 Hz mouse model of epilepsy following intraperitoneal administration. The most active analogue, Gal-B2 (NAX 5055), contained the -Lys-Lys-Lys(palmitoyl)-Lys-NH(2) motif and exhibited high affinity for galanin receptors (K(i) = 3.5 nM and 51.5 nM for GalR1 and GalR2, respectively), logD = 1.24, minimal helical conformation and improved metabolic stability. Structure-activity-relationship analysis suggested that cationization combined with position-specific lipidization was critical for improving the systemic activity of the analogues. Because the anticonvulsant activity of galanin is mediated by the receptors located in hippocampus and other limbic brain structures, our data suggest that these analogues penetrate into the brain. Gal-B2 may lead to development of first-in-class antiepileptic drugs.

Photoaffinity labeling identifies the substrate-binding site of mammalian squalene epoxidase.

Squalene epoxidase (SE) catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) in order to identify the location of the substrate-binding site and the roles of key residues in catalysis. Truncated 50-kDa rrSE was purified and photoaffinity labeled by competitive SE inhibitor (Ki=18.4 microM), [(3)H]TNSA-Dza. An 8-kDa CNBr/BNPS-skatole peptide was purified and the first 24 amino acids were sequenced by Edman degradation. The sequence PASFLPPSSVNKRGVLLLGDAYNL corresponded to residues 388-411 of the full-length rat SE. Three nucleophilic residues (Lys-399, Arg-400, and Asp-407) were labeled by [(3)H]TNSA-Dza. Triple mutants were prepared in which bulky groups were used to replace the labeled charged residues. Purified mutant enzymes showed lower enzymatic activity and reduced photoaffinity labeling by [(3)H]TNSA-Dza. This constitutes the first evidence as to the identity of the substrate-binding site of SE.