Effects of molecular weights on the physico-pharmaceutical properties of poly-L-glutamic acid-cytarabine conjugates

In order to obtain some informations about the effect of molecular weight on the release rate of drug from drug carrier, two types of poly-L-glutamic acid (PLGA)-cytarabine (ara-C) conjugates, PLGA-ara-C:I and PLGA-ara-C:II, were synthesized using two types of PLGA having different average molecular weight, 43,000 and 77,800, respectively. The PLGA-ara-C conjugates were synthesized by mixed anhydride method and found to be covalently linked. Both types of conjugates charged negatively at biological pH. The pH-dependent release rate of ara-C was observed in both cases, and the release rate was accelerated in basic, acidic conditions (the k values were 0.015 day^?1 at pH 7.0, 0.024 day^?1 at pH 5.0, and 0.059 day^?1 at pH 9.0 in the case of PLGA-ara-C:I) and in the presence of protease. The time required for the release of 16.5% of ara-C from PLGA-ara-C:I were 8 hr and 144 hr in the presence and absence of protease, respectively. Although both types of conjugates showed similar drug substitution ratio, they showed different release rates. Between the two types of conjugates, PLGA-ara-C:II showed the faster release rate (0.030 vs 0.042 day^?1 in pH 7.4 phosphate buffer solution at 37°C) and the smaller activation energy for the release of drug (12.5 vs 7.7 Kcal/mol) than PLGA-ara-C:I. The characteristic effect of molecular weight on the release rates of PLGA-ara-C conjugates suggests that the drug release rate might be effectively controlled over a prolonged period of time by the combined use of the different types of PLGA-ara-C conjugates having different molecular weights.     

Acylation of Exenatide by Glycolic Acid and its Anti-Diabetic Activities in db/db Mice

Purpose

To prepare acylated exenatide analogues and investigate their biological properties for guiding the development of PLGA formulations of exenatide.

Methods

The acylated exenatide analogues were prepared by reaction with glycolic acid (GA), one constitutional unit of PLGA, and characterized by HPLC-MS/MS and Circular Dichroism (CD). The pharmacokinetic properties and anti-diabetic activities were studied in SD rats and db/db mice, respectively.

Results

Structural characterizations of the acylated products showed that one to four glycolic acids (GAs) were connected to the primary amine groups of exenatide, and there was a conversion of α-helix to β-sheet to some extent. Pharmacokinetic studies in SD rats revealed that acylated exenatides had a similar Tmax with that of the prototype drug, whereas the Cmax and the AUC values of the adducts were significantly decreased. Biological activity tests demonstrated that exenatide and acylated exenatide analogues had similar in vivo antidiabetic activities in terms of controlling blood glucose concentration, HbA1c level, body weight and food intake.

Conclusions

These findings suggest that GA conjugated exenatide had no influence on the peptide efficacy, therefore it’s not necessary to inhibit exenatide acylation in PLGA formulations during the peptide release process.     

Zinc Protoporphyrin Polymeric Nanoparticles: Potent Heme Oxygenase Inhibitor for Cancer Therapy

Purpose

Oxidation therapy is an antitumor strategy in which, apoptosis or necrosis is caused by either excess delivery of reactive oxygen species (ROS) as an oxidant or anti-oxidant inhibition. Heme oxygenase (HO) is an anti-oxidant enzyme that plays an important role in cell growth and proliferation. The purpose of this study was to prepare poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with zinc protoporphyrin (ZnPP) to deliver the HO inhibitor into tumor.

Methods

PLGA NPs were prepared using nanoprecipitation technique and their characteristics were optimized by Box-Behnken experimental design. Scanning electron microscopy and in vitro studies consisting of drug release, HO inhibitory effect, cytotoxicity and cellular uptake followed by in vivo biodistribution and blood cytotoxicity were carried out. Internalization of coumerin-6 loaded NPs by PC3 cells was visualized by confocal laser scanning microscopy beside quantitatively analysis.

Results

NPs average size, entrapment efficiency and drug loading were 100.12?±?5.345 nm, 55.6%?±?2.49 and 7.98%?±?0.341 respectively. Equal HO inhibitory effect of NPs compared to free ZnPP was observed. The IC₅₀ value of ZnPP-NPs for PC3 human prostate cancer cells was found to be 2.14?±?0.083 μM.

Conclusion

In conclusion, ZnPP loaded PLGA NPs could exhibit enough HO inhibitory effect against cancer cells to be considered as a promising candidate for cancer treatment investigation.     

MDMA administration during adolescence exacerbates MPTP-induced cognitive impairment and neuroinflammation in the hippocampus and prefrontal cortex

Rationale

We have recently shown that chronic exposure to 3,4-methylenedioxymethamphetamine (MDMA, “ecstasy”) of adolescent mice exacerbates dopamine neurotoxicity and neuroinflammatory effects elicited by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the substantia nigra and striatum at adulthood.

Objectives

The present study investigated whether the amplification of MPTP effects by previous treatment with MDMA extends to the limbic and cortical regions and consequently affects cognitive performance.

Methods

Mice received MDMA (10 mg/kg, twice a day/twice a week) for 9 weeks, followed by MPTP (20 mg/kg?×?4 administrations), starting 2 weeks after MDMA discontinuation. Complement type 3 receptor (CD11b) and glial fibrillary acidic protein (GFAP) were evaluated by immunohistochemistry in both the hippocampus and the medial prefrontal cortex (mPFC) to measure microglia and astroglia activation. These neurochemical evaluations were paired with an assessment of cognitive performance by means of the novel object recognition (NOR) and spontaneous alternation tasks.

Results

MPTP administration to MDMA-pretreated mice elicited a stronger activation of CD11b and GFAP in both the hippocampus and the mPFC compared with either substance administered alone. Furthermore, NOR performance was lower in MDMA-pretreated mice administered MPTP compared with mice that received either substance alone.

Conclusions

These results demonstrate that MDMA–MPTP negative interactions extend to the limbic and cortical regions and may result in cognitive impairment, providing further evidence that exposure to MDMA may amplify the effects of later neurotoxic insults.     

One-Step Production of Protein-Loaded PLGA Microparticles via Spray Drying Using 3-Fluid Nozzle

Purpose

The aim of this study was to investigate the potential of using a spray-dryer equipped with a 3-fluid nozzle to microencapsulate protein drugs into polymeric microparticles.

Methods

Lysozyme and PLGA were used as a model protein and a model polymer, respectively. The effects of process and formulation variables, such as i) the type of organic solvent, ii) the feeding rate ratio of the outer PLGA-containing feed solution to inner lysozyme-containing feed solution, and iii) the mass ratio of PLGA to protein, on the properties (morphology, internal structure, protein surface enrichment and release profiles) of the spray dried microparticles were investigated to understand protein microencapsulation and particle formation mechanisms.

Results

The spherical, condensed microparticles were obtained with D₅₀ of 1.07–1.60 μm and Span in the range of 0.82–1.23. The lysozyme surface content decreased upon different organic solvents used as follows: acetonitrile?>?acetone?>?dichloromethane. Additionally, the lysozyme surface enrichment decreased slightly when increasing the feeding rate ratio of the outer feed solution to the inner feed solution from 4:1 to 10:1. Furthermore, it was observed that there was a correlation between the degree of burst release and the lysozyme surface enrichment, whereas the lysozyme loading content had no substantial impact on the release kinetics.

Conclusions

The present work demonstrates the potential of spray dryer equipped with a 3-fluid nozzle in microencapsulation of proteins into PLGA matrices with different characteristics by varying process and formulation parameters.     

PACE: Pharmacists use the power of communication in paediatric asthma

Background Paediatric asthma is a public health burden in Australia despite the availability of national asthma guidelines. Community pharmacy interventions focusing on paediatric asthma are scarce. Practitioner Asthma Communication and Education (PACE) is an evidence-based program, developed in the USA for general practice physicians, aimed at addressing the issues of poor clinician-patient communication in the management of paediatric asthma. This program has been shown to improve paediatric asthma management practices of general practitioners in the USA and Australia. The development of a PACE program for community pharmacists will fill a void in the current armamentarium for pharmacist-patient care. Objectives To adapt the educational program, PACE, to the community pharmacy setting. To test the feasibility of the new program for pharmacy and to explore its potential impact on pharmacists’ communication skills and asthma related practices. Setting Community pharmacies located within the Sydney metropolitan. Method The PACE framework was reviewed by the research team and amended in order to ensure its relevance within the pharmacy context, thereby developing PACE for Pharmacy. Forty-four pharmacists were recruited and trained in small groups in the PACE for Pharmacy workshops. Pharmacists’ satisfaction and acceptability of the workshops, confidence in using communication strategies pre- and post-workshop and self-reported behaviour change post workshop were evaluated. Main Outcome Measure Pharmacist self-reported changes in communication and teaching behaviours during a paediatric asthma consultation. Results All 44 pharmacists attended both workshops, completed pre- and post-workshop questionnaires and provided feedback on the workshops (100 % retention). The participants reported a high level of satisfaction and valued the interactive nature of the workshops. Following the PACE for Pharmacy program, pharmacists reported significantly higher levels in using the communication strategies, confidence in their application and their helpfulness. Pharmacists checked for written asthma self-management plan possession and inhaler device technique more regularly, and provided verbal instructions more frequently to paediatric asthma patients/carers at the initiation of a new medication. Conclusion This study provides preliminary evidence that the PACE program can be translated into community pharmacy. PACE for Pharmacy positively affected self-reported communication and education behaviours of pharmacists. The high response rate shows that pharmacists are eager to expand on their clinical role in primary healthcare.     

Effects of Antibiotic Physicochemical Properties on Their Release Kinetics from Biodegradable Polymer Microparticles

Purpose

This study investigated the effects of the physicochemical properties of antibiotics on the morphology, loading efficiency, size, release kinetics, and antibiotic efficacy of loaded poly(DL-lactic-co-glycolic acid) (PLGA) microparticles (MPs) at different loading percentages.

Methods

Cefazolin, ciprofloxacin, clindamycin, colistin, doxycycline, and vancomycin were loaded at 10 and 20 wt% into PLGA MPs using a water-in-oil-in water double emulsion fabrication protocol. Microparticle morphology, size, loading efficiency, release kinetics, and antibiotic efficacy were assessed.

Results

The results from this study demonstrate that the chemical nature of loaded antibiotics, especially charge and molecular weight, influence the incorporation into and release of antibiotics from PLGA MPs. Drugs with molecular weights less than 600 Da displayed biphasic release while those with molecular weights greater than 1,000 Da displayed triphasic release kinetics. Large molecular weight drugs also had a longer delay before release than smaller molecular weight drugs. The negatively charged antibiotic cefazolin had lower loading efficiency than positively charged antibiotics. Microparticle size appeared to be mainly controlled by fabrication parameters, and partition and solubility coefficients did not appear to have an obvious effect on loading efficiency or release. Released antibiotics maintained their efficacy against susceptible strains over the duration of release. Duration of release varied between 17 and 49 days based on the type of antibiotic loaded.

Conclusions

The data from this study indicate that the chemical nature of antibiotics affects properties of antibiotic-loaded PLGA MPs and allows for general prediction of loading and release kinetics.     

Developmental alterations in anxiety and cognitive behavior in serotonin transporter mutant mice

Rationale

A promoter variant of the serotonin transporter (SERT) gene is known to affect emotional and cognitive regulation. In particular, the “short” allelic variant is implicated in the etiology of multiple neuropsychiatric disorders. Heterozygous (SERT^+/?) and homozygous (SERT^?/?) SERT mutant mice are valuable tools for understanding the mechanisms of altered SERT levels. Although these genetic effects are well investigated in adulthood, the developmental trajectory of altered SERT levels for behavior has not been investigated.

Objectives

We assessed anxiety-like and cognitive behaviors in SERT mutant mice in early adolescence and adulthood to examine the developmental consequences of reduced SERT levels. Spine density of pyramidal neurons was also measured in corticolimbic brain regions.

Results

Adult SERT^?/? mice exhibited increased anxiety-like behavior, but these differences were not observed in early adolescent SERT^?/? mice. Conversely, SERT^+/? and SERT^?/? mice did display higher spontaneous alternation during early adolescence and adulthood. SERT^+/? and SERT^?/? also exhibited greater neuronal spine densities in the orbitofrontal but not the medial prefrontal cortices. Adult SERT^?/? mice also showed an increased spine density in the basolateral amygdala.

Conclusions

Developmental alterations of the serotonergic system caused by genetic inactivation of SERT can have different influences on anxiety-like and cognitive behaviors through early adolescence into adulthood, which may be associated with changes of spine density in the prefrontal cortex and amygdala. The altered maturation of serotonergic systems may lead to specific age-related vulnerabilities to psychopathologies that develop during adolescence.     

Modulating Protein Release Profiles by Incorporating Hyaluronic Acid into PLGA Microparticles Via a Spray Dryer Equipped with a 3-Fluid Nozzle

Purpose

The purpose of this study was to modulate the release profiles of the model protein drug from spray dried poly(DL-lactic-co-glycolic acid) (PLGA) microparticles by incorporating hyaluronic acid (HA) in the formulation.

Methods

Bovine serum albumin (BSA)-loaded PLGA microparticles with or without HA were prepared using a spray dryer equipped with a 3-fluid nozzle. The effects of HA on the surface tension and the rheological behavior of the inner feed solution were investigated. The physicochemical properties of the resulting microparticles were characterized using scanning electron microscopy (SEM), laser diffraction (LD), confocal laser scanning microscopy (CLSM) and X-ray photoelectron spectroscopy (XPS). Circular dischoism (CD) was used to characterize conformational integrity of BSA released from the microparticles.

Results

Spherical microparticles with D₅₀ of 5–10 μm were obtained. Addition of HA in inner feed solutions increased the feed viscosity, but with no influence on the surface tension. All inner feed solutions showed non-Newtonian shear thinning behavior and the rheological properties were not time dependent. The CLSM and XPS analyses suggested a core-shell like structure of the microparticles when HA was incorporated. The release profiles of BSA were extended and the initial burst releases were suppressed with an increase in HA in the microparticle formulations. In addition, HA seemed to protect BSA from degradation upon the spray-drying process.

Conclusions

The present work demonstrates the potential of HA to modulate protein release profile from PLGA microparticle formulations produced via spray drying using 3-fluid nozzle.     

ABC Transporters in Multi-Drug Resistance and ADME-Tox of Small Molecule Tyrosine Kinase Inhibitors

The past decade has seen tremendous efforts in the research and development of new chemotherapeutic drugs using target-based approaches. These efforts have led to the discovery of small molecule tyrosine kinase inhibitors (TKIs). Following the initial approval of imatinib by the US FDA in 2001, more than 15 TKIs targeting different tyrosine kinases have been approved, and numerous others are in various phases of clinical evaluation. Unlike conventional chemotherapy that can cause non-discriminating damage to both normal and cancerous cells, TKIs attack cancer-specific targets and therefore have a more favorable safety profile. However, although TKIs have had outstanding success in cancer therapy, there has been increasing evidence of resistance to TKIs. The enhanced efflux of TKIs by ATP-binding cassette (ABC) transporters over-expressed in cancer cells has been found to be one such important resistance mechanism. Another major drawback of TKI therapies that has been increasingly recognized is the extensive inter-individual pharmacokinetic variability, in which ABC transporters seem to play a major role as well. This review covers recent findings on the interactions of small molecule TKIs with ABC transporters. The effects of ABC transporters on anticancer efficacy and the absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) of the small molecule TKIs are summarized in detail. Since TKIs have been found to not only serve as substrates of ABC transporters, but also as modulators of these proteins via inhibition or induction, their influence upon ABC transporters and potential role on TKI-drug interactions are discussed as well.     

Phosphatidylinositol 4,5-bisphosphate depletion fails to affect neurosteroid modulation of GABAA receptor function

Rationale

Neurosteroids and likely other lipid modulators access transmembrane sites on the GABA_A receptor (GABA_AR) by partitioning into and diffusing through the plasma membrane. Therefore, specific components of the plasma membrane may affect the potency or efficacy of neurosteroid-like modulators. Here, we tested a possible role for phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that governs activity of many channels and transporters, in modulation or function of GABA_ARs.

Objectives

In these studies, we sought to deplete plasma-membrane PIP2 and probe for a change in the strength of potentiation by submaximal concentrations of the neurosteroid allopregnanolone (3α5αP) and other anesthetics, including propofol, pentobarbital, and ethanol. We also tested for a change in the behavior of negative allosteric modulators pregnenolone sulfate and dipicrylamine.

Methods

We used Xenopus oocytes expressing the ascidian voltage-sensitive phosphatase (Ci-VSP) to deplete PIP2. Voltage pulses to positive membrane potentials were used to deplete PIP2 in Ci-VSP-expressing cells. GABA_ARs composed of α1β2γ2L and α4β2δ subunits were challenged with GABA and 3α5αP or other modulators before and after PIP2 depletion. KV7.1 channels and NMDA receptors (NMDARs) were used as positive controls to verify PIP2 depletion.

Results

We found no evidence that PIP2 depletion affected modulation of GABA_ARs by positive or negative allosteric modulators. By contrast, Ci-VSP-induced PIP2 depletion depressed KV7.1 activation and NMDAR activity.

Conclusions

We conclude that despite a role for PIP2 in modulation of a wide variety of ion channels, PIP2 does not affect modulation of GABA_ARs by neurosteroids or related compounds.     

The self-administration of rapidly delivered cocaine promotes increased motivation to take the drug: contributions of prior levels of operant responding and cocaine intake

Rationale

Rapid drug delivery to the brain might increase the risk for developing addiction. In rats, increasing the speed of intravenous cocaine delivery (5 vs. 90 s) increases drug intake and the subsequent motivation to self-administer cocaine. Increased motivation for cocaine could result not only from more extensive prior drug intake and operant responding for drug, but also from neuroplasticity evoked by rapid drug uptake.

Objective

We determined the contributions of prior drug intake and operant responding to the increased motivation for cocaine evoked by rapid delivery. We also investigated the effects of cocaine delivery speed on corticostriatal expression of brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) mRNA.

Methods

Rats self-administered cocaine (0.25 mg/kg/infusion) delivered over 5 or 90 s during short-access (1 h/session; ShA) or long-access (6 h; LgA) sessions. Motivation for cocaine was then assessed by measuring responding under a progressive ratio schedule of reinforcement. Next, BDNF and TrkB mRNA levels were measured in 5- and 90-s rats.

Results

Five-second ShA and 5-s-LgA rats were more motivated for cocaine than their 90-s counterparts. This effect was dissociable from previous levels of drug intake or of operant responding for cocaine. In parallel, only rats self-administering rapid cocaine injections had altered BDNF and TrkB mRNA levels in corticostriatal regions.

Conclusions

Rapid drug delivery augments the motivation for cocaine independently of effects on the levels of drug intake or operant responding for drug. We suggest that rapid delivery might increase the motivation for drug by promoting neuroplasticity within reward pathways. This neuroplasticity could involve increased regulation of BDNF/TrkB.     

TCDD-induced chick cardiotoxicity is abolished by a selective cyclooxygenase-2 (COX-2) inhibitor NS398

Halogenated aromatic hydrocarbons, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are known to cause severe heart defects in avian species. However, the mechanism of TCDD-induced chick cardiovascular toxicity is unclear. In this study, we investigated cyclooxygenase-2 (COX-2) as a possible mechanism of TCDD-induced cardiotoxicity. Fertile chicken eggs were injected with TCDD and a COX-2 selective inhibitor, NS398, and we investigated chick heart failure on day 10. We found that the chick heart to body weight ratio and atrial natriuretic factor mRNA expression were increased, but this increase was abolished with treatment of NS398. In addition, the morphological abnormality of an enlarged ventricle resulting from TCDD exposure was also abolished with co-treatment of TCDD and NS398. Our results suggested that TCDD-induced chick heart defects are mediated via the nongenomic pathway and that they do not require the genomic pathway.     

Sex differences in neurosteroid and hormonal responses to metyrapone in posttraumatic stress disorder

Rationale

Mechanisms contributing to sex differences in the regulation of acute stress responsivity and their effect on the increased incidence of posttraumatic stress disorder (PTSD) in women are poorly understood. The reproductive hormone, progesterone, through conversion to allopregnanolone (ALLO), suppresses the hypothalamic pituitary adrenal (HPA) axis and has potent anxiolytic effects. The potential that progesterone and allopregnanolone reactivity modulate HPA axis responses and account for sex differences in PTSD has not been previously examined.

Objective

The present study examined the effects of sex and PTSD on adrenocorticotropic hormone (ACTH), progesterone, and allopregnanolone responses to metyrapone and whether progesterone and allopregnanolone reactivity could affect the ACTH response in PTSD.

Methods

Healthy medication-free male and premenopausal follicular phase female participants with chronic PTSD (n?=?43; 49 % female) and controls (n?=?42; 50 % female) completed an overnight metyrapone challenge and ACTH, progesterone, and allopregnanolone were obtained by repeated blood sampling.

Results

The increase in ACTH response to metyrapone was higher in PTSD subjects compared to controls and in women compared to men. Contrary to our initial prediction of an inverse relationship, progesterone and allopregnanolone were positively associated with ACTH. Progesterone and allopregnanolone partially mediated the relationship between PTSD and ACTH.

Conclusions

Our findings of increased ACTH to metyrapone in PTSD and in women may reflect heightened hypothalamic CRF hypersecretion. Progesterone and allopregnanolone partially mediated the ACTH response in PTSD. Further characterizing sex differences in these processes will advance our understanding of the pathophysiology of PTSD, and may ultimately lead to better-targeted, more effective treatment.     

3,4-Methylenedioxymethamphetamine (MDMA – Ecstasy) Decreases Neutrophil Activity Through the Glucocorticoid Pathway and Impairs Host Resistance to Listeria Monocytogenes Infection in Mice

Ecstasy is the popular name of the abuse drug 3,4-methylenedioxymethamphetamine (MDMA) that decreases immunity in animals. The mechanisms that generate such alterations are still controversial. Seven independent pharmacological approaches were performed in mice to identify the possible mechanisms underlying the decrease of neutrophil activity induced by MDMA and the possible effects of MDMA on host resistance to Listeria monocytogenes. Our data showed that MDMA (10 mg kg^?1) administration decreases NFκB expression in circulating neutrophils. Metyrapone or RU-486 administration prior to MDMA treatment abrogated MDMA effects on neutrophil activity and NFκB expression, while 6-OHDA or ICI-118,551 administration did not. As MDMA treatment increased the plasmatic levels of adrenaline and noradrenaline, propranolol pre-treatment effects were also evaluated. Propranolol suppressed both MDMA-induced increase in corticosterone serum levels and its effects on neutrophil activity. In a L. monocytogenes experimental infection context, we showed that MDMA: induced myelosuppression by decreasing granulocyte-macrophage hematopoietic progenitors (CFU-GM) in the bone marrow but increased CFU-GM in the spleen; decreased circulating leukocytes and bone marrow cellularity and increased spleen cellularity; decreased pro-inflammatory cytokine (IL-12p70, TNF, IFN-γ, IL-6) and chemokine (MCP-1) production 24 h after the infection; increased the production of pro-inflammatory cytokines and chemokines 72 h after infection and decreased IL-10 levels at all time points analyzed. It was proposed that MDMA immunosuppressive effects on neutrophil activity and host resistance to L monocytogenes rely on NFκB signaling, being mediated by HPA axis activity and corticosterone.     

Phosphorylation of GABAA receptors influences receptor trafficking and neurosteroid actions

Rationale

Gamma-aminobutyric acid type A receptors (GABA_ARs) are the principal mediators of inhibitory transmission in the mammalian central nervous system. GABA_ARs can be localized at post-synaptic inhibitory specializations or at extrasynaptic sites. While synaptic GABA_ARs are activated transiently following the release of GABA from presynaptic vesicles, extrasynaptic GABA_ARs are typically activated continuously by ambient GABA concentrations and thus mediate tonic inhibition. The tonic inhibitory currents mediated by extrasynaptic GABA_ARs control neuronal excitability and the strength of synaptic transmission. However, the mechanisms by which neurons control the functional properties of extrasynaptic GABA_ARs had not yet been explored.

Objectives

We review GABA_ARs, how they are assembled and trafficked, and the role phosphorylation has on receptor insertion and membrane stabilization. Finally, we review the modulation of GABA_ARs by neurosteroids and how GABA_AR phosphorylation can influence the actions of neurosteroids.

Conclusions

Trafficking and stability of functional channels to the membrane surface are critical for inhibitory efficacy. Phosphorylation of residues within GABA_AR subunits plays an essential role in the assembly, trafficking, and cell surface stability of GABA_ARs. Neurosteroids are produced in the brain and are highly efficacious allosteric modulators of GABA_AR-mediated current. This allosteric modulation by neurosteroids is influenced by the phosphorylated state of the GABA_AR which is subunit dependent, adding temporal and regional variability to the neurosteroid response. Possible links between neurosteroid actions, phosphorylation, and GABA_AR trafficking remain to be explored, but potential novel therapeutic targets may exist for numerous neurological and psychological disorders which are linked to fluctuations in neurosteroid levels and GABA_A subunit expression.     

Influence of Suspension Stabilisers on the Delivery of Protein-Loaded Porous Poly (DL-Lactide-co-Glycolide) (PLGA) Microparticles via Pressurised Metered Dose Inhaler (pMDI)

Purpose

This work investigates the feasibility of delivering large (≈ 25 μm) porous poly (lactide-co-glycolide) (PLGA) microparticles containing a model protein via pressurised metered dose inhaler (pMDI).

Methods

Porous PLGA microparticles were prepared by modified double emulsion method as pMDI suspension based systems containing suspension stabilisers in 1,1,1,2,3,3,3-heptafluoropropane (HFA 227). Physical suspension stability was assessed by visual and optical suspension techniques. Aerosolisation characteristics were investigated using aerosol particle sizing, dose delivery through the valve (DTV) and shot weight.

Results

An optimum concentration of suspensions stabiliser was required to achieve physical pMDI suspension stability; values of; 0.0075%w/w PVP K30 or 0.075%w/w PEG 300 were required. Formulations that exhibited good physical stability also showed optimum aerosolisation characteristics. When employing 0.0075% PVP K30 DTV at the start and end of can life was 98.11(±10.01) % and 75.06 (±7.01) % respectively verses values of 37.39 (±11.12) % and 5.57 (±1.72) % without the inclusion of PVP K30.

Conclusion

Porous PLGA microparticles show potential as macromolecule/protein carrier and also to target lower regions of the lungs when prepared as pMDI suspension formulations in HFA 227 using suspension stabilisers to achieve consistent dose delivery through the life of the pMDI, however, inter-relationship between the device and the formulation need to be considered to achieve suitable respiratory delivery.     

Ginsenoside metabolite compound K stimulates glucagon-like peptide-1 secretion in NCI-H716 cells via bile acid receptor activation

Compound K (CK) is a major metabolite of ginsenosides that is absorbed. CK has antidiabetic effects, although the mechanisms underlying the effects of CK have not fully been known. To elucidate the mechanisms underlying the antidiabetic effects of CK, we studied the effects of CK on GLP-1 secretion from NCI-H716 cells, and explored the mechanisms underlying CK-induced GLP-1 secretion. Treatment of NCI-H716 cells with 10, 50, and 100 μM CK significantly increased GLP-1 secretion, and intracellular Ca²⁺ and cAMP levels in a dose-dependent manner. Transfection of NCI-H716 cells with siRNA specific to α-gustducin and siRNA specific to TAS1R3 had no effect on CK-induced GLP-1 secretion and Ca²⁺ increase. However, transfection of NCI-H716 cells with TGR5-specific siRNA significantly inhibited CK-induced GLP-1 secretion and the increase in Ca²⁺ and cAMP levels. Moreover, CK showed human TGR5 agonist activity in CHO-K1 cells transiently transfected with human TGR5. Our data provide a novel mechanism of CK for antidiabetic effects. Moreover, the findings might suggest that CK is a potential agent that has multiple biological functions in the body via GLP-1 secretion and TGR5 activation.     

Computer Modeling Assisted Design of Monodisperse PLGA Microspheres with Controlled Porosity Affords Zero Order Release of an Encapsulated Macromolecule for 3 Months

Purpose

The aim of this study was the development of poly(D,L-lactide-co-glycolide) (PLGA) microspheres with controlled porosity, to obtain microspheres that afford continuous release of a macromolecular model compound (blue dextran).

Methods

PLGA microspheres with a size of around 40 μm and narrow size distribution (span value of 0.3) were prepared with a double emulsion membrane emulsification method. Gene expression programming (GEP) analysis was applied to design and formulate a batch of microspheres with controlled porosity that shows continuous release of blue dextran.

Results

Low porous microspheres with a high loading efficiency were formed at high polymer concentrations (30% w/w in the oil phase) and were characterized with a burst release <10% and a three-phasic release profile of blue dextran. Increasing porosity (10% w/w polymer concentrations), a sustained release of blue dextran was obtained albeit with up to 40% of burst release. The desired formulation, calculated by GEP, resulted in microspheres with 72% loading efficiency and intermediate porosity. Blue dextran was indeed released continuously in almost a zero order manner over a period of 3 months after an initial small burst release of 9%.

Conclusions

By fine-tuning the porosity, the release profile of PLGA microspheres for macromolecules can be predicted and changed from a three-phasic to a continuous release.