Formulation,Pharmacokinetic, and Efficacy Studies of Mannosylated Self-Emulsifying Solid Dispersions of Noscapine

Purpose

To formulate hydroxypropyl methylcellulose-stabilized self-emulsifying solid dispersible carriers of noscapine to enhance oral bioavailability.

Methods

Formulation of noscapine (Nos) self-emulsifying solid dispersible microparticles (SESDs) was afforded by emulsification using an optimized formula of Labrafil M1944, Tween-80, and Labrasol followed by spray-drying with hydroxypropyl methylcellulose (HPMC), with and without mannosamine (Mann-Nos_SESDs and Nos_SESDs respectively); self-microemulsifying liquid dispersions (SMEDDs) with and without mannosamine (Mann-Nos_SMEDDs and Nos_SMEDDs respectively) were also prepared. SMEDDs and SESDs were characterized for size, polydispersity, surface charge, entrapment efficiency, in vitro permeability, in vitro release kinetics, and oral pharmacokinetics in Sprague-Dawley rats (10 mg/kg p.o). The antitumor efficacy of Mann-Nos_SESDs on the basis of chemosensitization to cisplatin (2.0 mg/kg, IV) was investigated in a chemorefractory lung tumor Nu/Nu mouse model up to a maximal oral dose of 300 mg/kg.

Results

The oil/surfactant/co-surfactant mixture of Labrafil M1944, Tween-80, and Labrasol optimized at weight ratios of 62.8:9.30:27.90% produced stable self-microemulsifying dispersions (SMEDDs) at a SMEDD to water ratio of 1–3:7–9 parts by weight. SMEDDs had hydrodynamic diameters between 231 and 246 nm; surface charges ranged from -16.50 to -18.7 mV; and entrapment efficiencies were between 32 and 35%. SESDs ranged in size between 5.84 and 6.60 μm with surface charges from -10.62 to -12.40 mV and entrapment efficiencies of 30.96±4.66 and 32.05±3.72% (Nos_SESDs and Mann-Nos_SESDs respectively). Mann-Nos_SESDs exhibited saturating uptake across Caco-2 monolayers (P_app = 4.94±0.18 × 10⁻⁶ cm/s), with controlled release of 50% of Nos in 6 hr at pH 6.8 following Higuchi kinetics. Mann-Nos_ SESDs was 40% more bioavailable compared to Nos_SESDs; and was effective in sensitizing H1650 SP cells to Cisplatin in vitro and in an orthotopic lung tumor model of H1650 SP origin.

Conclusions

Mannosylated noscapine self-emulsifying solid dispersions (Mann-Nos_SESDs) are bioavailable and potentiate the antineoplastic effect of cisplatin-based chemotherapy in cisplatin-resistant NSCLC.     

Structural and Functional Studies of Phosphoenolpyruvate Carboxykinase from Mycobacterium tuberculosis

Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn²⁺ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn²⁺ and Mg²⁺ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe²⁺ in the presence of Mn²⁺ or Mg²⁺ cations. In contrast, simultaneous presence of Fe²⁺ and Mn²⁺ or Mg²⁺ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn²⁺ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.     

The mycobacterial thioredoxin peroxidase can act as a one-cysteine peroxiredoxin

Thioredoxin peroxidase (TPx) has been reported to dominate the defense against H(2)O(2), other hydroperoxides, and peroxynitrite at the expense of thioredoxin (Trx) B and C in Mycobacterium tuberculosis (Mt). By homology, the enzyme has been classified as an atypical 2-C-peroxiredoxin (Prx), with Cys(60) as the "peroxidatic" cysteine (C(P)) forming a complex catalytic center with Cys(93) as the "resolving" cysteine (C(R)). Site-directed mutagenesis confirms Cys(60) to be C(P) and Cys(80) to be catalytically irrelevant. Replacing Cys(93) with serine leads to fast inactivation as seen by conventional activity determination, which is associated with oxidation of Cys(60) to a sulfinic acid derivative. However, in comparative stopped-flow analysis, WT-MtTPx and MtTPx C93S reduce peroxynitrite and react with TrxB and -C similarly fast. Reduction of pre-oxidized WT-MtTPx and MtTPx C93S by MtTrxB is demonstrated by monitoring the redox-dependent tryptophan fluorescence of MtTrxB. Furthermore, MtTPx C93S remains stable for 10 min at a morpholinosydnonimine hydrochloride-generated low flux of peroxynitrite and excess MtTrxB in a dihydrorhodamine oxidation model. Liquid chromatography-tandem mass spectrometry analysis revealed disulfide bridges between Cys(60) and Cys(93) and between Cys(60) and Cys(80) in oxidized WT-MtTPx. Reaction of pre-oxidized WT-MtTPx and MtTPx C93S with MtTrxB C34S or MtTrxC C40S yielded dead-end intermediates in which the Trx mutants are preferentially linked via disulfide bonds to Cys(60) and never to Cys(93) of the TPx. It is concluded that neither Cys(80) nor Cys(93) is required for the catalytic cycle of the peroxidase. Instead, MtTPx can react as a 1-C-Prx with Cys(60) being the site of attack for both the oxidizing and the reducing substrate. The role of Cys(93) is likely to conserve the oxidation equivalents of the sulfenic acid state of C(P) as a disulfide bond to prevent overoxidation of Cys(60) under a restricted supply of reducing substrate.     

Discovery of novel non-peptidic β-alanine piperazine amide derivatives and their optimization to achiral,easily accessible,potent and selective somatostatin sst1 receptor antagonists

Structural simplification of the core moieties of obeline and ergoline somatostatin sst₁ receptor antagonists, followed by systematic optimization, led to the identification of novel, highly potent and selective sst₁ receptor antagonists. These achiral, non-peptidic compounds are easily prepared and show promising PK properties in rodents.     

Fitness of Mycobacterium tuberculosis Strains of the W-Beijing and Non-W-Beijing Genotype

Background

Multidrug resistant tuberculosis (MDR-TB) is a major threat for global tuberculosis control. The W-Beijing Mycobacterium tuberculosis genotype has been associated with drug resistance. Elucidation of the mechanisms underlying this epidemiological finding may have an important role in the control of MDR-TB. The aim of this study was to evaluate the fitness of drug-susceptible and MDR M. tuberculosis strains of the W-Beijing genotype compared with that of Non-W-Beijing strains.

Methodology/Principal Findings

Fitness of M. tuberculosis strains was determined by evaluating the difference in the growth curves obtained in the MGIT960 automated system and assessing the competitive growth capacity between W-Beijing and non-W-Beijing strains. The W-Beijing MDR strains had a significant longer lag phase duration compared to the other strains but did not present a significant fitness cost. When grown in competition they had an advantage only in medium containing 0.1% Tween 80.

Conclusions/Significance

It was not possible to confirm a selective advantage of W-Beijing strains to grow, except for differences in their resistance to Tween 80. Further studies are needed to elucidate the putative advantage of W-Beijing strains compared to other genotypes.     

DNA-binding properties of the recombinant high-mobility-group-like AT-hook-containing region from human BRG1 protein

The hBRG1 protein, a central ATPase of the human switching/sucrose non-fermenting (SWI/SNF) remodeling complex, has a catalytic ATPase domain, an AT-hook motif and a bromodomain. Bromodomains, found in many chromatin-associated proteins, recognize N-acetyl-lysine in histones and other proteins. The AT-hook motif, first described in the high-mobility group of non-histone chromosomal proteins HMGA1/2, is a DNA-binding motif. The AT-hook binds to the AT-rich DNA sequences in the minor groove of B-DNA in a non-sequence specific manner. AT-hook motifs have been identified in many other DNA-binding proteins. In this study we cloned and purified a fragment of hBRG1 encompassing the AT-hook region and the bromodomain. Nuclear magnetic resonance (NMR) and circular dichroism (CD) analyses show that the recombinant domains are structured. The functionality of subdomains was checked by assessing their interactions with N-acetylated peptides from histones and with DNA. Isothermal titration calorimetric (ITC) analysis demonstrates that the primary micromolar interaction is through the AT-hook motif. The AT-hook region binds to linear DNA by unwinding it. These properties resemble the characteristics of the HMGA1/2 proteins and their interaction with DNA.     

Multiple thioredoxin-mediated routes to detoxify hydroperoxides in Mycobacterium tuberculosis

Drug resistance and virulence of Mycobacterium tuberculosis are in part related to the pathogen's antioxidant defense systems. KatG(-) strains are resistant to the first line tuberculostatic isoniazid but need to compensate their catalase deficiency by alternative peroxidase systems to stay virulent. So far, only NADH-driven and AhpD-mediated hydroperoxide reduction by AhpC has been implicated as such virulence-determining mechanism. We here report on two novel pathways which underscore the importance of the thioredoxin system for antioxidant defense in M. tuberculosis: (i) NADPH-driven hydroperoxide reduction by AhpC that is mediated by thioredoxin reductase and thioredoxin C and (ii) hydroperoxide reduction by the atypical peroxiredoxin TPx that equally depends on thioredoxin reductase but can use both, thioredoxin B and C. Kinetic analyses with different hydroperoxides including peroxynitrite qualify the redox cascade comprising thioredoxin reductase, thioredoxin C, and TPx as the most efficient system to protect M. tuberculosis against oxidative and nitrosative stress in situ.     

Structural and Kinetic Properties of Nonglycosylated Recombinant Penicillium amagasakiense Glucose Oxidase Expressed in Escherichia coli

The gene coding for Penicillium amagasakiense glucose oxidase (GOX; β-d-glucose; oxygen 1-oxidoreductase [EC 1.1.3.4]) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. Recombinant Escherichia coli expression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence. When transformed into E. coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml of E. coli culture containing more than 60% inactive GOX. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml⁻¹ in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol. Reactivation followed first-order kinetics and was optimal at 10°C. The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography. Up to 12 mg of active GOX could be purified from a 1-liter E. coli culture. Circular dichroism demonstrated similar conformations for recombinant and native P. amagasakiense GOXs. The purified enzyme has a specific activity of 968 U mg⁻¹ and exhibits kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native GOX from P. amagasakiense. In contrast to the native enzyme, recombinant GOX is nonglycosylated and contains a single isoform of pI 4.5. This is the first reported expression of a fully active, nonglycosylated form of a eukaryotic, glycosylated GOX in E. coli.Glucose oxidase (GOX; β-d-glucose; oxygen 1-oxidoreductase [EC 1.1.3.4]) is a hydrogen peroxide-generating flavoprotein catalyzing the oxidation of β-d-glucose to d-glucono-1,5-lactone. GOX is used in the food industry for the removal of glucose from powdered eggs, as a source of hydrogen peroxide in food preservation, for gluconic acid production, and in the production of beer and soft drinks, in which its reaction serves an antioxidant function (10, 39, 42). GOX is also used extensively for the quantitative determination of d-glucose in samples such as blood, food, and fermentation products (10, 39, 49). The enzyme has been purified from both Aspergillus niger (45) and Penicillium spp. (33), with A. niger NRRL3 being the most widely used strain for industrial-scale production (11). A problem with utilizing GOX from its native source is the presence of impurities such as catalase, cellulase, and amylase, which may impair some of its applications. To overcome these difficulties and to simplify the stringent purification procedures, which are relatively expensive, A. niger GOX has been cloned and expressed in Saccharomyces cerevisiae as a highly glycosylated form (17).The most frequent employment of GOX has been in biosensors, in which the biochemical event of glucose oxidation is detected by electrochemical, thermometric, or optical techniques. The most interesting possibilities appear to lie in electron transfer reactions, with artificial electron acceptors or mediators being used to transfer information from the enzyme to the electrode (49). The electrical communication between GOX and the electrode and thereby its biosensor performance are hampered by the protein-bound carbohydrate moiety of the enzyme (1, 15), which most probably impedes electron tunneling through the enzyme (32). Almost complete (24, 27) or partial (15, 32) deglycosylation of GOX is possible, but the procedure is expensive and complicated. A more efficient and effective way of obtaining nonglycosylated GOX would be to express the enzyme in a prokaryotic host. This would also enable the properties and efficiency of GOX to be improved for its use in biosensors by protein engineering techniques (49). As a first step towards this objective, GOX from Penicillium amagasakiense was cloned and expressed in Escherichia coli. GOX from P. amagasakiense was selected since the enzyme has a higher turnover rate and a better affinity for β-d-glucose than its A. niger counterpart (30, 33).In this study, we describe the cloning and expression of the gene encoding P. amagasakiense GOX and the refolding, purification, and characterization of the nonglycosylated recombinant enzyme. The activity of the recombinant GOX, expressed in the form of insoluble inclusion bodies, was reconstituted, and the active enzyme was shown to possess properties and secondary structure composition similar to those of native P. amagasakiense GOX. This is the first reported expression of a fully active nonglycosylated form of a eukaryotic glycosylated GOX in a prokaryote, which enabled us to demonstrate that in contrast to previous assumptions (4, 9, 47) the protein-bound carbohydrate moiety is not essential for the correct folding of GOX.