Eudragit-Based Nanosuspension of Poorly Water-Soluble Drug: Formulation and In Vitro–In Vivo Evaluation

The present study was performed to investigate potential of Eudragit RLPO-based nanosuspension of glimepiride (Biopharmaceutical Classification System class II drug), for the improvement of its solubility and overall therapeutic efficacy, suitable for peroral administration. Nanoprecipitation method being simple and less sophisticated was optimized for the preparation of nanosuspension. Physicochemical characteristics of nanosuspension in terms of size, zeta potential, polydispersity index, entrapment efficiency (% EE) and in vitro drug release were found within their acceptable ranges. The size of the nanoparticles was most strongly affected by agitation time while % EE was more influenced by the drug/polymer ratio. Differential scanning calorimetry and X-ray diffraction studies provided evidence that enhancement in solubility of drug resulted due to change in crystallinity of drug within the formulation. Stability study revealed that nanosuspension was more stable at refrigerated condition with no significant changes in particle size distribution, % EE, and release characteristics for 3 months. In vivo studies were performed on nicotinamide–streptozotocin-induced diabetic rat models for pharmacokinetic and antihyperglycaemic activity. Nanosuspension increased maximum plasma concentration, area under the curve, and mean residence time values significantly as compared to aqueous suspension. Oral glucose tolerance test and antihyperglycaemic studies demonstrated plasma glucose levels were efficiently controlled in case of nanosuspension than glimepiride suspension. Briefly, sustained and prolonged activity of nanosuspensions could reduce dose frequency, decrease drug side effects, and improve patient compliance. Therefore, glimepiride nanosuspensions can be expected to gain considerable attention in the treatment of type 2 diabetes mellitus due to its improved therapeutic activity.KEY WORDS: diabetes mellitus, glimepiride, nanoprecipitation, poloxamer, sustained release     

In Vitro Mutational Analysis of the β2 Adrenergic Receptor,an In Vivo Surrogate Odorant Receptor

Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse β2-adrenergic-receptor (mβ2AR), robustly traffics to the plasma membrane. We set out to characterize mβ2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged mβ2AR (mβ2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mβ2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mβ2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mβ2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mβ2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mβ2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs.     

In Vivo and Ex Vivo Evaluation of L-Type Calcium Channel Blockers on Acid β-Glucosidase in Gaucher Disease Mouse Models

Gaucher disease is a lysosomal storage disease caused by mutations in acid β-glucosidase (GCase) leading to defective hydrolysis and accumulation of its substrates. Two L-type calcium channel (LTCC) blockers—verapamil and diltiazem—have been reported to modulate endoplasmic reticulum (ER) folding, trafficking, and activity of GCase in human Gaucher disease fibroblasts. Similarly, these LTCC blockers were tested with cultured skin fibroblasts from homozygous point-mutated GCase mice (V394L, D409H, D409V, and N370S) with the effect of enhancing of GCase activity. Correspondingly, diltiazem increased GCase protein and facilitated GCase trafficking to the lysosomes of these cells. The in vivo effects of diltiazem on GCase were evaluated in mice homozygous wild-type (WT), V394L and D409H. In D409H homozygotes diltiazem (10 mg/kg/d via drinking water or 50–200 mg/kg/d intraperitoneally) had minor effects on increasing GCase activity in brain and liver (1.2-fold). Diltiazem treatment (10 mg/kg/d) had essentially no effect on WT and V394L GCase protein or activity levels (<1.2-fold) in liver. These results show that LTCC blockers had the ex vivo effects of increasing GCase activity and protein in the mouse fibroblasts, but these effects did not translate into similar changes in vivo even at very high drug doses.     

Developing In Vitro–In Vivo Correlation of Risperidone Immediate Release Tablet

The present study was aimed to predict the absorption profile of a risperidone immediate release tablet (IR) and to develop the level A in vitro–in vivo correlation (IVIVC) of the drug using the gastrointestinal simulation based on the advanced compartmental absorption and transit model implemented in GastroPlus™. Plasma concentration data, physicochemical, and pharmacokinetic properties of the drug were used in building its absorption profile in the gastrointestinal tract. Since the fraction absorbed of risperidone in simulation was more than 90% with low water solubility, the drug met the criteria of class II of the Biopharmaceutics Classification System. The IVIVC was developed based on the model built using the plasma data and the in vitro dissolution data in several dissolution media based on the Japanese Guideline for Bioequivalence Studies of Generic Products. The gastrointestinal absorption profile of risperidone was successfully predicted. A level A IVIVC was also successfully developed in all dissolution media with percent prediction error for Cmax and the area under the curve less than 10% for both reference and test drug.Key words: GastroPlus™, immediate release tablet, in vitro–in vivo correlation, risperidone     

In Vitro–In Vivo Correlation of Efavirenz Tablets Using GastroPlus®

The aim of the present work was to use GastroPlus™ software for the prediction of pharmacokinetic profiles and in vitro–in vivo correlation (IVIVC) as tools to optimize the development of new generic medications. GastroPlus™ was used to simulate the gastrointestinal compartment and was based on the advanced compartmental absorption and transit model. Powder dissolution and efavirenz tablet dissolution studies were carried out to generate data from which correlation was established. The simulated plasma profile, based on the physicochemical properties of efavirenz, was almost identical to that observed in vivo for biobatches A and B. A level A IVIVC was established for the dissolution method obtained for the generic candidate using the Wagner–Nelson (r² = 0.85) and for Loo–Riegelman models (r² = 0.92). The percentage of fraction absorbed indicated that 0.5% sodium lauryl sulfate may be considered a biorelevant dissolution medium for efavirenz tablets. The simulation of gastrointestinal bioavailability and IVIVC obtained from immediate-release tablet formulations suggests that GastroPlus™ is a valuable in silico method for IVIVC and for studies directed at developing formulations of class II drugs.KEY WORDS: bioavailability, computational simulation, efavirenz, GastroPlus™, in vivo–in vitro correlation     

Pseudomonas putida AlkA and AlkB Proteins Comprise Different Defense Systems for the Repair of Alkylation Damage to DNA – In Vivo,In Vitro,and In Silico Studies

Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada) response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS) or N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i) the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii) the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB), characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB), PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems against alkylating agents of exo- and endogenous origin.     

Gasping Generation in Developing Swiss–Webster Mice In Vitro and In Vivo

During hypoxia the respiratory network produces gasping in vivo and in vitro. To understand the mechanisms involved in such response and to validate in vitro findings, correlative studies are necessary. During perinatal age gasping generation is robust and then declines during postnatal development, possibly due to changes in either the rhythm generator (the pre-Bötzinger complex, PBC) and/or its motor outputs. We tested this hypothesis by recording respiratory response to hypoxia in vivo and in vitro during postnatal development. We found that postnatal age influences: (1) The hypoxia-induced pattern change in the PBC bursts, (2) The coupling between the PBC and the XII nucleus during prolonged hypoxia and (3) The ability of mice to gasp and autoresuscitate from hypoxic conditions. We conclude that the inability of mice to gasp during late postnatal development might be determined by a progressive uncoupling between the respiratory rhythm generator and its motor outputs in hypoxia.     

In Vitro Evolution and Affinity-Maturation with Coliphage Qβ Display

The Escherichia coli bacteriophage, Qβ (Coliphage Qβ), offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV). DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb) SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.     

Cyclopia Extracts Act as ERα Antagonists and ERβ Agonists,In Vitro and In Vivo

Hormone replacement therapy associated risks, and the concomitant reluctance of usage, has instigated the search for new generations of estrogen analogues that would maintain estrogen benefits without associated risks. Furthermore, if these analogues display chemo-preventative properties in breast and endometrial tissues it would be of great value. Both the selective estrogen receptor modulators as well as the selective estrogen receptor subtype modulators have been proposed as estrogen analogues with improved risk profiles. Phytoestrogen containing extracts of Cyclopia, an indigenous South African fynbos plant used to prepare Honeybush tea may serve as a source of new estrogen analogues. In this study three extracts, P104, SM6Met, and cup-of-tea, from two species of Cyclopia, C. genistoides and C. subternata, were evaluated for ER subtype specific agonism and antagonism both in transactivation and transrepression. For transactivation, the Cyclopia extracts displayed ERα antagonism and ERβ agonism when ER subtypes were expressed separately, however, when co-expressed only agonism was uniformly observed. In contrast, for transrepression, this uniform behavior was lost, with some extracts (P104) displaying uniform agonism, while others (SM6Met) displayed antagonism when subtypes were expressed separately and agonism when co-expressed. In addition, breast cancer cell proliferation assays indicate that extracts antagonize cell proliferation in the presence of estrogen at lower concentrations than that required for proliferation. Furthermore, lack of uterine growth and delayed vaginal opening in an immature rat uterotrophic model validates the ERα antagonism of extracts observed in vitro and supports the potential of the Cyclopia extracts as a source of estrogen analogues with a reduced risk profile.     

In Vitro and In Vivo Neuronal Electrotaxis: A Potential Mechanism for Restoration?

Electrical brain stimulation used to treat a variety of neurological and psychiatric diseases is entering a new period. The technique is well established and the potential complications are well known and generally manageable. Recent studies demonstrated that electrical fields (EFs) can enhance neuroplasticity-related processes. EFs applied in the physiological range induce migration of different neural cell types from different species in vitro. There are some evidences that also the speed and directedness of cell migration are enhanced by EFs. However, it is still unclear how electrical signals from the extracellular space are translated into intracellular actions resulting in the so-called electrotaxis phenomenon. Here, we aim to provide a comprehensive review of the data on responses of cells to electrical stimulation and the relation to functional recovery.     

Latency Associated Peptide Has In Vitro and In Vivo Immune Effects Independent of TGF-β1

Latency Associated Peptide (LAP) binds TGF-β1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-β1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-β1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-β1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation.     

Functional Analyses of Escherichia coli MutS-β Clamp Interaction In Vitro and In Vivo

Escherichia coli MutS is a highly conserved mismatch repair (MMR) protein that plays a key role in recognizing DNA mismatches and the early steps of MMR. Previous studies revealed an interaction between MutS and the replicative protein β clamp, but it remains unclear whether the interaction functions during the process of MMR. In order to provide insight into the significance of this interaction, Far Western, Surface plasmon resonance and cell survival/mutagenesis assays were used to determine its possible influences on the in vitro and in vivo properties of MutS. The results show that a quintuple mutation of MutS residues 812–816 (MutS^βC), or single alanine substitution mutation of MutS residues M813 or L815 completely blocks binding of MutS to β clamp. Wild type β clamp interferes with DNA binding by MutS. When treated with the base analog 2-aminopurine, MutS^βC confers more mutations and less cellular growth rate in the mutS-deficient strain than the wild type MutS. These data indicate that the MutS-β interaction has functional consequences during MMR in E. coli.     

Saccharomyces cerevisiae–Based Platform for Rapid Production and Evaluation of Eukaryotic Nutrient Transporters and Transceptors for Biochemical Studies and Crystallography

To produce large quantities of high quality eukaryotic membrane proteins in Saccharomyces cerevisiae, we modified a high-copy vector to express membrane proteins C-terminally-fused to a Tobacco Etch Virus (TEV) protease detachable Green Fluorescent Protein (GFP)-8His tag, which facilitates localization, quantification, quality control, and purification. Using this expression system we examined the production of a human glucose transceptor and 11 nutrient transporters and transceptors from S. cerevisiae that have not previously been overexpressed in S. cerevisiae and purified. Whole-cell GFP-fluorescence showed that induction of GFP-fusion synthesis from a galactose-inducible promoter at 15°C resulted in stable accumulation of the fusions in the plasma membrane and in intracellular membranes. Expression levels of the 12 fusions estimated by GFP-fluorescence were in the range of 0.4 mg to 1.7 mg transporter pr. liter cell culture. A detergent screen showed that n-dodecyl-ß-D-maltopyranoside (DDM) is acceptable for solubilization of the membrane-integrated fusions. Extracts of solubilized membranes were prepared with this detergent and used for purifications by Ni-NTA affinity chromatography, which yielded partially purified full-length fusions. Most of the fusions were readily cleaved at a TEV protease site between the membrane protein and the GFP-8His tag. Using the yeast oligopeptide transporter Ptr2 as an example, we further demonstrate that almost pure transporters, free of the GFP-8His tag, can be achieved by TEV protease cleavage followed by reverse immobilized metal-affinity chromatography. The quality of the GFP-fusions was analysed by fluorescence size-exclusion chromatography. Membranes solubilized in DDM resulted in preparations containing aggregated fusions. However, 9 of the fusions solubilized in DDM in presence of cholesteryl hemisuccinate and specific substrates, yielded monodisperse preparations with only minor amounts of aggregated membrane proteins. In conclusion, we developed a new effective S. cerevisiae expression system that may be used for production of high-quality eukaryotic membrane proteins for functional and structural analysis.     

In Vivo Validation of In Silico Predicted Metabolic Engineering Strategies in Yeast: Disruption of α-Ketoglutarate Dehydrogenase and Expression of ATP-Citrate Lyase for Terpenoid Production

Background

Engineering of the central carbon metabolism of Saccharomyces cerevisiae to redirect metabolic flux towards cytosolic acetyl-CoA has become a central topic in yeast biotechnology. A cell factory with increased flux into acetyl-CoA can be used for heterologous production of terpenoids for pharmaceuticals, biofuels, fragrances, or other acetyl-CoA derived compounds. In a previous study, we identified promising metabolic engineering targets in S. cerevisiae using an in silico stoichiometric metabolic network analysis. Here, we validate selected in silico strategies in vivo.

Results

Patchoulol was produced by yeast via a heterologous patchoulol synthase of Pogostemon cablin. To increase the metabolic flux from acetyl-CoA towards patchoulol, a truncated HMG-CoA reductase was overexpressed and farnesyl diphosphate synthase was fused with patchoulol synthase. The highest increase in production could be achieved by modifying the carbon source; sesquiterpenoid titer increased from glucose to ethanol by a factor of 8.4. Two strategies predicted in silico were chosen for validation in this work. Disruption of α-ketoglutarate dehydrogenase gene (KGD1) was predicted to redirect the metabolic flux via the pyruvate dehydrogenase bypass towards acetyl-CoA. The metabolic flux was redirected as predicted, however, the effect was dependent on cultivation conditions and the flux was interrupted at the level of acetate. High amounts of acetate were produced. As an alternative pathway to synthesize cytosolic acetyl-CoA, ATP-citrate lyase was expressed as a polycistronic construct, however, in vivo performance of the enzyme needs to be optimized to increase terpenoid production.

Conclusions

Stoichiometric metabolic network analysis can be used successfully as a metabolic prediction tool. However, this study highlights that kinetics, regulation and cultivation conditions may interfere, resulting in poor in vivo performance. Main sites of regulation need to be released and improved enzymes are essential to meet the required activities for an increased product formation in vivo.     

Fabrication and In Vitro/In Vivo Performance of Mucoadhesive Electrospun Nanofiber Mats Containing α-Mangostin

This study aimed to fabricate mucoadhesive electrospun nanofiber mats containing α-mangostin for the maintenance of oral hygiene and reduction of the bacterial growth that causes dental caries. Synthesized thiolated chitosan (CS-SH) blended with polyvinyl alcohol (PVA) was selected as the mucoadhesive polymer. α-Mangostin was incorporated into the CS-SH/PVA solution and electrospun to obtain nanofiber mats. Scanning electron microscopy, differential scanning calorimetry, X-ray diffraction, and tensile strength testing were used to characterize the mats. The swelling degree and mucoadhesion were also determined. The nanofiber mats were further evaluated regarding their α-mangostin content, in vitro α-mangostin release, antibacterial activity, cytotoxicity, in vivo performance, and stability. The results indicated that the mats were in the nanometer range. The α-mangostin was well incorporated into the mats, with an amorphous form. The mats showed suitable tensile strength, swelling, and mucoadhesive properties. The loading capacity increased when the initial amount of α-mangostin was increased. Rapid release of α-mangostin from the mats was achieved. Additionally, a fast bacterial killing rate occurred at the lowest concentration of nanofiber mats when α-mangostin was added to the mats. The mats were less cytotoxic after use for 72 h. Moreover, in vivo testing indicated that the mats could reduce the number of oral bacteria, with a good mouth feel. The mats maintained the amount of α-mangostin for 6 months. The results suggest that α-mangostin-loaded mucoadhesive electrospun nanofiber mats may be a promising material for oral care and the prevention of dental caries.KEY WORDS: dental caries, mucoadhesive property, nanofibers, thiolated chitosan, α-mangostin     

Comparative Analysis of αB-Crystallin Expression in Heat-Stressed Myocardial Cells In Vivo and In Vitro

Relationships between αB-crystallin expression patterns and pathological changes of myocardial cells after heat stress were examined in vitro and in vivo in this study using the H₉C₂ cell line and Sprague-Dawley rats, respectively. Histopathological lesions, characterized by acute degeneration, karyopyknosis and loss of a defined nucleus, became more severe in rat hearts over the course of heat stress treatment from 20 min to 100 min. The expression of αB-crystallin in rat hearts showed a significant decrease (P<0.05) throughout the heat stress treatment period, except at the 40 min time point. Likewise, decreased αB-crystallin expression was also observed in the H₉C₂ cell line exposed to a high temperature in vitro, although its expression recovered to normal levels at later time points (80 and 100 min) and the cellular damage was less severe. The results suggest that αB-crystallin is mobilized early after exposure to a high temperature to interact with damaged proteins but that the myocardial cells cannot produce sufficient αB-crystallin for protection against heat stress. Lower αB-crystallin expression levels were accompanied by obvious cell/tissue damage, suggesting that the abundance of this protein is associated with protective effects in myocardial cells in vitro and in vivo. Thus, αB-crystallin is a potential biomarker of heat stress.     

α-Tomatine-Mediated Anti-Cancer Activity In Vitro and In Vivo through Cell Cycle- and Caspase-Independent Pathways

α-Tomatine, a tomato glycoalkaloid, has been reported to possess antibiotic properties against human pathogens. However, the mechanism of its action against leukemia remains unclear. In this study, the therapeutic potential of α-tomatine against leukemic cells was evaluated in vitro and in vivo. Cell viability experiments showed that α-tomatine had significant cytotoxic effects on the human leukemia cancer cell lines HL60 and K562, and the cells were found to be in the Annexin V-positive/propidium iodide-negative phase of cell death. In addition, α-tomatine induced both HL60 and K562 cell apoptosis in a cell cycle- and caspase-independent manner. α-Tomatine exposure led to a loss of the mitochrondrial membrane potential, and this finding was consistent with that observed on activation of the Bak and Mcl-1 short form (Mcl-1s) proteins. Exposure to α-tomatine also triggered the release of the apoptosis-inducing factor (AIF) from the mitochondria into the nucleus and down-regulated survivin expression. Furthermore, α-tomatine significantly inhibited HL60 xenograft tumor growth without causing loss of body weight in severe combined immunodeficiency (SCID) mice. Immunohistochemical test showed that the reduced tumor growth in the α-tomatine-treated mice was a result of increased apoptosis, which was associated with increased translocation of AIF in the nucleus and decreased survivin expression ex vivo. These results suggest that α-tomatine may be a candidate for leukemia treatment.