A pragmatic approach for engineering porous mannitol and mechanistic evaluation of particle performance

The importance of mannitol has increased recently as an emerging diluent for orodispersible dosage forms. The study aims to prepare spray dried mannitol retaining high porosity and mechanical strength for the development of orally disintegrating tablets (ODTs).Aqueous feed of d-mannitol (10% w/v) comprising ammonium bicarbonate, NH₄HCO₃ (5% w/v) as pore former was spray dried at inlet temperature of 110–170 °C. Compacts were prepared at 151 MPa and characterized for porosity, hardness and disintegration time. Particle morphology and drying mechanisms were studied using thermal (HSM, DSC and TGA) and polymorphic (XRD) methods.Tablet porosity increased from 0.20 ± 0.002 for pure mannitol to 0.53 ± 0.03 using fabricated porous mannitol. Disintegration time dropped by 50–77% from 135 ± 5.29 s for pure mannitol to 75.33 ± 2.52–31.67 ± 1.53 s for mannitol 110–170 °C. Hardness increased by 150% at 110 °C (258.67 ± 28.89 N) and 30% at 150 °C (152.70 ± 10.58 N) compared to pure mannitol tablets (104.17 ± 1.70 N). Increasing inlet temperature resulted in reducing tablet hardness due to generation of ‘micro-sponge’-like particles exhibiting significant elastic recovery. Impact of mannitol polymorphism on plasticity/elasticity cannot be ruled out as a mixture of α and β polymorphs formed upon spray drying.     

Pulmonary delivery of pyrazinamide-loaded large porous particles

We have improved the aerodynamic properties of pyrazinamide loaded large porous particles (PZA-LPPs) designed for pulmonary delivery. To overcome the segregation of the different components occurring during the spray drying process and to obtain homogeneous LPPs, spray drying parameters were modified to decrease the drying speed. As a result, good aerodynamic properties for lung delivery were obtained with a fine particle fraction (FPF) of 40.1 ± 1.0%, an alveolar fraction (AF) of 29.6 ± 3.1%, a mass median aerodynamic diameter (MMAD_aer) of 4.1 ± 0.2 μm and a geometric standard deviation (GSD) of 2.16 ± 0.16. Plasma and epithelial lining fluid (ELF) concentrations of pyrazinamide were evaluated after intratracheal insufflation of PZA-LPPs (4.22 mg kg⁻¹) into rats and compared to intravenous administration (iv) of a pyrazinamide solution (5.82 mg kg⁻¹). The in vivo pharmacokinetic evaluation of PZA-LPPs in rats reveals that intratracheal insufflation of PZA-LPPs leads to a rapid absorption in plasma with an absolute bioavailability of 66%. This proves that PZA-LPPs dissolve fast upon deposition and that PZA crosses efficiently the lung barrier to reach the systemic circulation. PZA concentrations were 1.28-fold higher in ELF after intratracheal administration than after iv administration and the ratio of ELF concentrations over plasma concentrations was 2-fold greater. Although these improvements are moderate, lung delivery of PZA appears an interesting alternative to oral delivery of the molecule and should now be tested in an infected animal model to evaluate its efficacy against Mycobacterium tuberculosis.     

Formulation of pH responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids

Nucleic acids have the potential to be used as therapies or vaccines for many different types of disease, but delivery remains the most significant challenge to their clinical adoption. pH responsive peptides containing either histidine or derivatives of 2,3-diaminopropionic acid (Dap) can mediate effective DNA transfection in lung epithelial cells with the latter remaining effective even in the presence of lung surfactant containing bronchoalveolar lavage fluid (BALF), making this class of peptides attractive candidates for delivering nucleic acids to lung tissues. To further assess the suitability of pH responsive peptides for pulmonary delivery by inhalation, dry powder formulations of pH responsive peptides and plasmid DNA, with mannitol as carrier, were produced by either spray drying (SD) or spray freeze drying (SFD). The properties of the two types of powders were characterised and compared using scanning electron microscopy (SEM), next generation impactor (NGI), gel retardation and in vitro transfection via a twin stage impinger (TSI) following aerosolisation by a dry powder inhaler (Osmohaler™). Although the aerodynamic performance and transfection efficacy of both powders were good, the overall performance revealed SD powders to have a number of advantages over SFD powders and are the more effective formulation with potential for efficient nucleic acid delivery through inhalation.     

A comparison between spray drying and spray freeze drying for dry powder inhaler formulation of drug-loaded lipid-polymer hybrid nanoparticles

Lipid-polymer hybrid nanoparticles - polymeric nanoparticles enveloped by lipid layers - have emerged as a potent therapeutic nano-carrier alternative to liposomes and polymeric nanoparticles. Herein we perform comparative studies of employing spray drying (SD) and spray freeze drying (SFD) to produce inhalable dry-powder form of drug-loaded lipid-polymer hybrid nanoparticles. Poly(lactic-co-glycolic acid), lecithin, and levofloxacin are employed as the polymer, lipid, and drug models, respectively. The hybrid nanoparticles are transformed into micro-scale nanoparticle aggregates (or nano-aggregates) via SD and SFD, where the effects of (1) different excipients (i.e. mannitol, polyvinyl alcohol (PVA), and leucine), and (2) nanoparticle to excipient ratio on nano-aggregate characteristics (e.g. size, flowability, aqueous reconstitution, aerosolization efficiency) are examined. In both methods, PVA is found more effective than mannitol for aqueous reconstitution, whereas hydrophobic leucineis needed to achieve effective aerosolization as it reduces nano-aggregate agglomeration. Using PVA, both methods are equally capable of producing nano-aggregates having size, density, flowability, yield and reconstitutibility in the range ideal for inhaled delivery. Nevertheless, nano-aggregates produced by SFD are superior to SD in terms of their aerosolization efficiency manifested in the higher emitted dose and fine particle fraction with lower mass median aerodynamic diameter.     

Through-vial impedance spectroscopy of critical events during the freezing stage of the lyophilization cycle: The example of the impact of sucrose on the crystallization of mannitol

The aim of this work was to evaluate the application of through-vial impedance spectroscopy in the measurement of eutectic crystallization during the freezing stage of the lyophilisation cycle.Impedance measurements of various sugar solutions (mannitol 5%, 10% and 15% w/v, sucrose 5% w/v and mannitol 5% w/v, and sucrose 5% w/v solutions) were taken during a freeze–thaw cycle, over a frequency range 10–10⁶ Hz with a scan interval of 1.5 min, using measurement vials with externally attached electrodes connected to a high resolution impedance analyzer.Estimates for the electrical resistance of the mannitol solutions record the exothermic crystallization of mannitol at a temperature of −24 °C during the temperature ramp down stage of the freezing cycle, which is in close agreement with the off-line DSC measurement of −22 °C. The freezing profile of a 5% mannitol solution with 5% sucrose (a component that does not crystallize in the frozen solution) demonstrated the inhibition of mannitol crystallization (with the implication that the product will then require sub-T_g′ freezing and drying).The work suggests a role for through-vial impedance spectroscopy in the concurrent development of the product formulation and freeze drying cycle without the uncertainty introduced when using off-line date to define the critical process parameters.     

Superparamagnetic iron oxide nanoparticles (SPIONs)-loaded Trojan microparticles for targeted aerosol delivery to the lung

Targeted aerosol delivery to specific regions of the lung may improve therapeutic efficiency and minimise unwanted side effects. Targeted delivery could potentially be achieved with porous microparticles loaded with superparamagnetic iron oxide nanoparticles (SPIONs)—in combination with a target-directed magnetic gradient field. The aim of this study was to formulate and evaluate the aerodynamic properties of SPIONs-loaded Trojan microparticles after delivery from a dry powder inhaler. Microparticles made of SPIONs, PEG and hydroxypropyl-β-cyclodextrin (HPβCD) were formulated by spray drying and characterised by various physicochemical methods. Aerodynamic properties were evaluated using a next generation cascade impactor (NGI), with or without a magnet positioned at stage 2. Mixing appropriate proportions of SPIONs, PEG and HPβCD allowed Trojan microparticle to be formulated. These particles had a median geometric diameter of 2.8 ± 0.3 μm and were shown to be sensitive to the magnetic field induced by a magnet having a maximum energy product of 413.8 kJ/m³. However, these particles, characterised by a mass median aerodynamic diameter (MMAD) of 10.2 ± 2.0 μm, were considered to be not inhalable. The poor aerodynamic properties resulted from aggregation of the particles. The addition of (NH₄)₂CO₃ and magnesium stearate (MgST) to the formulation improved the aerodynamic properties of the Trojan particles and resulted in a MMAD of 2.2 ± 0.8 μm. In the presence of a magnetic field on stage 2 of the NGI, the amount of particles deposited at this stage increased 4-fold from 4.8 ± 0.7% to 19.5 ± 3.3%. These Trojan particles appeared highly sensitive to the magnetic field and their deposition on most of the stages of the NGI was changed in the presence compared to the absence of the magnet. If loaded with a pharmaceutical active ingredient, these particles may be useful for treating localised lung disease such as cancer nodules or bacterial infectious foci.     

Oxidative stress contributes to silica nanoparticle-induced cytotoxicity in human embryonic kidney cells

In order to elucidate the nanoparticle-induced cytotoxicity and its mechanism, the effects of 20 and 50 nm silica nanoparticles on cultured human embryonic kidney (HEK293) cells were investigated. Cell viability, mitochondrial function, cell morphology, reactive oxygen species (ROS), glutathione (GSH), thiobarbituric acid reactive substance (TBARS), cell cycle and apoptosis were assessed under control and silica exposed conditions. Exposure to 20 or 50 nm SiO₂ nanoparticles at dosage levels between 20 and 100 μg/ml decreased cell viability in a dose-dependent manner. Median lethal dose (LD₅₀) of 24 h exposure was 80.2 ± 6.4 and 140.3 ± 8.6 μg/ml for 20 and 50 nm SiO₂ nanoparticles, respectively. Morphological examination revealed cell shrinkage and nuclear condensation after SiO₂ nanoparticle exposure. Increase in intracellular ROS level and reduction in GSH content were also observed in SiO₂ nanoparticle-exposed HEK293 cells. Increase in the amount of TBARS suggested an elevated level of lipid peroxidation. Flow cytometric analysis showed that SiO₂ nanoparticles can cause G2/M phase arrest and apoptotic sub-G1 population increase in a dose-dependent manner. In summary, exposure to SiO₂ nanoparticles resulted in a dose-dependent cytotoxicity in cultured HEK293 cells that was associated with increased oxidative stress.     

Reduced Renal Clearance of Cefotaxime in Asians with a Low-Frequency Polymorphism of OAT3 (SLC22A8)

Organic anion transporter 3 (OAT3, SLC22A8), a transporter expressed on the basolateral membrane of the proximal tubule, plays a critical role in the renal excretion of organic anions including many therapeutic drugs. The goal of this study was to evaluate the in vivo effects of the OAT3-Ile305Phe variant (rs11568482), present at 3.5% allele frequency in Asians, on drug disposition with a focus on cefotaxime, a cephalosporin antibiotic. In HEK293- Flp-In cells, the OAT3-Ile305Phe variant had a lower maximum cefotaxime transport activity, V_max, [159 ± 3 nmol*(mg protein)^- 1/min (mean ± SD)] compared with the reference OAT3 [305 ± 28 nmol*(mg protein)^? 1/min, (mean ± SD), p < 0.01], whereas the Michaelis-Menten constant values (K_m) did not differ. In healthy volunteers, we found volunteers that were heterozygous for the Ile305Phe variant and had a significantly lower cefotaxime renal clearance (CL_R; mean ± SD: 84.8 ± 32.1 mL/min, n = 5) compared with volunteers that were homozygous for the reference allele (158 ± 44.1 mL/min, n = 10; p = 0.006). Furthermore, the net secretory component of cefotaxime renal clearance (CL_sec) was reduced in volunteers heterozygous for the variant allele [33.3 ± 31.8 mL/min (mean ± SD)] compared with volunteers homozygous for the OAT3 reference allele [97.0 ± 42.2 mL/min (mean ± SD), p = 0.01]. In summary, our study suggests that a low-frequency reduced-function polymorphism of OAT3 associates with reduced cefotaxime CL_R and CL_sec .©2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:3451–3457, 2013     

Through-Vial Impedance Spectroscopy of the Mechanisms of Annealing in the Freeze-Drying of Maltodextrin: The Impact of Annealing Hold Time and Temperature on the Primary Drying Rate

The study aims to investigate the impact of annealing hold time and temperature on the primary drying rate/duration of a 10% (w/v) solution of maltodextrin with an emphasis on how the mechanisms of annealing might be understood from the in-vial measurements of the ice crystal growth and the glass transition. The electrical impedance of the solution within a modified glass vial was recorded between 10 and 10⁶ Hz during freeze-drying cycles with varying annealing hold times (1–5 h) and temperatures. Primary drying times decreased by 7%, 27% and 34% (1.1, 4.3 and 5.5 h) with the inclusion of an annealing step at temperatures of –15 °C, –10 °C and –5 °C, respectively. The glass transition was recorded at approximately –16 °C during the re-heating and re-cooling steps, which is close to the glass transition (T_g′) reported for 10% (w/v) maltodextrin and therefore indicates that a maximum freeze concentration (~ 86%, w/w, from the Gordon–Taylor equation) was achieved during first freezing, with no further ice being formed on annealing. This observation, coupled to the decrease in electrical resistance that was observed during the annealing hold time, suggests that the reduction in the drying time was because of improved connectivity of ice crystals because of Ostwald ripening rather than devitrification. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1799–1810, 2014     

Chemical composition,antiproliferative and antioxidant properties of lipid classes in ordinary and dark muscles from chub mackerel (Scomber japonicus)

This study investigated to compare lipid profiles in ordinary and dark muscles from chub mackerel and to examine antiproliferative and antioxidative properties of lipid classes. The average levels of neutral lipids (NL), glycolipids (GL), and phospholipids (PL) in ordinary muscle were 92.32 ± 0.19%, 5.10 ± 0.48%, and 2.58 ± 0.46%; in dark muscle were 96.88 ± 0.15%, 2.59 ± 0.36%, and 0.54 ± 0.29%, respectively. The fatty acid composition indicated that PL had a higher percentage of PUFA (especially 22:6n?3) with lower percentages of SFA and MUFA compared to NL and GL (p < 0.05). The main ion peaks of GL in ordinary and dark muscles showed that monocharged and bischarged molecular ion were presented at m/z 876.9 and 438.8, respectively. In MTT assay, inhibition of AGS and HT-29 cell proliferation was greatest with the 0.5 and 1.0 mg mL^?1 GL treatments. The GL of ordinary muscle with 0.05 mg mL^?1 concentrations markedly decreased the levels of reactive oxygen species (ROS) induced by H₂O₂ compared to the control (p < 0.05). From our results, GL might have antiproliferative and antioxidant properties based on protective effect against the production of intracellular ROS.     

Preparation of estradiol chitosan nanoparticles for improving nasal absorption and brain targeting

The estradiol(E₂)-loaded chitosan nanoparticles (CS-NPs) were prepared by ionic gelation of chitosan with tripolyphosphate anions (TPP). The CS-NPs had a mean size of (269.3 ± 31.6) nm, a zeta potential of +25.4 mV, and loading capacity of E₂ CS-NPs suspension was 1.9 mg ml⁻¹, entrapment efficiency was 64.7% on average. Subsequently, this paper investigated the levels of E₂ in blood and the cerebrospinal fluid (CSF) in rats following intranasal administration of E₂ CS-NPs. E₂-loaded CS-NPs were administered to male Wister rats either intranasally or intravenously at the dose of 0.48 mg kg⁻¹. The plasma levels achieved following intranasal administration (32.7 ± 10.1 ng ml⁻¹; t_max 28 ± 4.5 min) were significantly lower than those after intravenous administration (151.4 ± 28.2 ng ml⁻¹), while CSF concentrations achieved after intranasal administration (76.4 ± 14.0 ng ml⁻¹; t_max 28 ± 17.9 min) were significantly higher than those after intravenous administration (29.5 ± 7.4 ng ml⁻¹ t_max 60 min). The drug targeting index (DTI) of nasal route was 3.2, percent of drug targeting (DTP%) was 68.4%. These results showed that the E₂ must be directly transported from the nasal cavity into the CSF in rats. Finally, compared with E₂ inclusion complex, CS-NPs improved significantly E₂ being transported into central nervous system (CNS).     

Mucoadhesive microparticles as potential carriers in inhalation delivery of doxycycline hyclate: a comparative study

The present work compares and evaluates the suitability of different polymer-based microparticles for inhalation delivery of doxycycline hyclate. Mucoadhesive polymers, such as sodium carboxymethyl cellulose, sodium alginate, polyvinyl alcohol, polyvinylpyrrolidone, starch, and carbopol were selected as carriers for inhalation delivery. Microparticles were prepared by spray drying and evaluated in terms of yield, moisture content, morphology, tapped density, encapsulation efficiency, in vitro mucoadhesion, thermal properties and in vitro aerosolization performance. Additionally, the cytotoxicity of the microparticles on H1299 human alveolar cell line was examined. Smooth spherical to collapsed doughnut shaped particles were formed. They exhibited tap densities of 0.202–0.502 g/cm³ and mass median aerodynamic diameter of 3.74–6.54 μm. Mucoadhesion was highest in case of carbopol-based microparticles. Drug release from these microparticles exhibited biphasic Fickian type of diffusion. Only at the highest concentration of microparticles (1 mg/mL) less than 90% cell viability was seen in DX loaded sodium alginate microparticles (DXSA, 87.2%), starch microparticles (DXST, 85.1%) and carbopol microparticles (DXCP, 82.7%) preparations after 48 h of exposure to alveolar cells. The results clearly indicate that sodium carboxymethyl cellulose-based microparticles may serve as an ideal carrier for inhalation delivery of doxycycline hyclate.     

Folate-mediated poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) nanoparticles for targeting drug delivery

A novel targeting drug delivery system (TDDS) has been developed. Such a TDDS was prepared by W₁/O/W₂ solvent extraction/evaporation method, adopting poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] as the drug carrier, folic acid (FA) as the targeting ligand, and doxorubicin (DOX) as the model anticancer drug. The average size, drug loading capacity and encapsulation efficiency of the prepared DOX-loaded, folate-mediated P(HB-HO) nanoparticles (DOX/FA–PEG–P(HB-HO) NPs) were found to be around 240 nm, 29.6% and 83.5%. The in vitro release profile displayed that nearly 50% DOX was released in the first 5 days. The intracellular uptake tests of the nanoparticles (NPs) in vitro showed that the DOX/FA–PEG–P(HB-HO) NPs were more efficiently taken up by HeLa cells compared to non-folate-mediated P(HB-HO) NPs. In addition, DOX/FA–PEG–P(HB-HO) NPs (IC₅₀ = 0.87 μM) showed greater cytotoxicity to HeLa cells than other treated groups. In vivo anti-tumor activity of the DOX/FA–PEG–P(HB-HO) NPs showed a much better therapeutic efficacy in inhibiting tumor growth, and the final mean tumor volume was 178.91 ± 17.43 mm³, significantly smaller than normal saline control group (542.58 ± 45.19 mm³). All these results have illustrated that our techniques for the preparing of DOX/FA–PEG–P(HB-HO) NPs developed in present work are feasible and these NPs are effective in selective delivery of anticancer drug to the folate receptor-overexpressed cancer cells. The new TDDS may be a competent candidate in application in targeting treatment of cancers.     

Dry Powder Aerosols Generated by Standardized Entrainment Tubes From Drug Blends With Lactose Monohydrate: 2. Ipratropium Bromide Monohydrate and Fluticasone Propionate

The objectives of this study were: systematic investigation of dry powder aerosol performance using standardized entrainment tubes (SETs) and lactose-based formulations with two model drugs; mechanistic evaluation of performance data by powder aerosol deaggregation equation (PADE). The drugs (IPB and FP) were prepared in sieved and milled lactose carriers (2% w/w). Aerosol studies were performed using SETs (shear stresses τ_s = 0.624-13.143 N/m²) by twin-stage liquid impinger, operated at 60 L/min. PADE was applied for formulation screening. Excellent correlation was observed when PADE was adopted correlating FPF to τ_s. Higher τ_s corresponded to higher FPF values followed by a plateau representing invariance of FPF with increasing τ_s. The R² values for PADE linear regression were 0.9905-0.9999. Performance described in terms of the maximum FPF (FPF_max: 15.0–37.6%) resulted in a rank order of ML-B/IPB > ML-A/IPB > SV-A/IPB > SV-B/IPB > ML-B/FP > ML-A/FP > SV-B/FP > SV-A/FP. The performance of IPB was superior to FP in all formulations. The difference in lactose monohydrate carriers was less pronounced for the FPF in IPB than in FP formulations. The novel PADE offers a robust method for evaluating aerodynamic performance of dry powder formulations within a defined τ_s range.     

Development and characterization of an orodispersible film containing drug nanoparticles

In this study, a novel orodispersible film (ODF) containing drug nanoparticles was developed with the goal of transforming drug nanosuspensions into a solid dosage form and enhancing oral bioavailability of drugs with poor water solubility. Nanosuspensions were prepared by high pressure homogenization and then transformed into ODF containing drug nanoparticles by mixing with hydroxypropyl methylcellulose solution containing microcrystalline cellulose, low substituted hydroxypropylcellulose and PEG-400 followed by film casting and drying. Herpetrione, a novel and potent antiviral agent with poor water solubility that extracted from Herpetospermum caudigerum, was chosen as a model drug and studied systematically. The uniformity of dosage units of the preparation was acceptable according to the criteria of Japanese Pharmacopoeia 15. The ODF was disintegrated in water within 30 s with reconstituted nanosuspensions particle size of 280 ± 11 nm, which was similar to that of drug nanosuspensions, indicating a good redispersibility of the fast dissolving film. Result of X-ray diffraction showed that HPE in the ODF was in the amorphous state. In the in vitro dissolution test, the ODF containing HPE nanoparticles showed an increased dissolution velocity markedly. In the pharmacokinetics study in rats, compared to HPE coarse suspensions, the ODF containing HPE nanoparticles exhibited significant increase in AUC_0–24h, C_max and decrease in T_max, MRT. The result revealed that the ODF containing drug nanoparticles may provide a potential opportunity in transforming drug nanosuspensions into a solid dosage form as well as enhancing the dissolution rate and oral bioavailability of poorly water-soluble drugs.     

Optimization of mead production using Response Surface Methodology

The main aim of the present work was to optimize mead production using Response Surface Methodology. The effects of temperature (x₁: 20–30 °C) and nutrients concentration (x₂: 60–120 g/hL) on mead quality, concerning the final concentrations of glucose (Y₁), fructose (Y₂), ethanol (Y₃), glycerol (Y₄) and acetic acid (Y₅), were studied. Twelve operational conditions were tested. No delays and moods were observed during fermentations. The second order polynomial models determined produced satisfactory fittings of the experimental data with regard to glucose (R² = 0.646, p = 0.001), ethanol (R² = 0.741, p = 0.049), glycerol (R² = 0.899, p = 0.002), fructose (R² = 0.902, p = 0.033) and acetic acid (R² = 0.913, p = 0.001). The optimum extraction conditions determined in order to maximize the combined responses were 24 °C and a nutrients concentration of 0.88 g/L. The mead produced under these conditions had the following characteristics: ethanol concentration of 10.2%, acetic acid 0.54 g/L, glycerol 7.8 g/L, glucose 1.8 g/L and fructose 2.5 g/L. These values were in agreement with the predicted and were within the safe limit established for acetic acid and the recommended range for glycerol. Furthermore, the residual sugars concentration was also low, decreasing the possibility of occurring undesirable refermentations.     

Principles of rational design of thermally targeted liposomes for local drug delivery

Drug release from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes occurs close to the main transition temperature T_m = 41 °C. The exact release temperature can be adjusted by additional lipids, which shift T_m. A major issue is drug leakage at 37 °C. We here describe a novel approach with improved drug retention yet rapid release. To obtain spherical, smooth liposomes we included: i) 2 mol% cholesterol, to soften bilayers (Lemmich et al 1997), ii) lipids, which due to their spontaneous curvature stabilize the negative and positive curvatures of the inner and outer leaflets of unilamellar liposomes. In addition to differential scanning calorimetry (DSC) and fluorescence spectroscopy, the lipid mixtures were analyzed by a Langmuir balance for their elastic properties and lipid packing, aiming at high elasticity modulus C_S^− 1. Maxima in C_S^− 1 coincided with minima in the free energy of lateral mixing. These liposomes have reduced drug leakage, yet retain rapid release.From the Clinical EditorThis paper reports the development of optimized DPPC liposomes for drug delivery, with reduced drug leakage but maintained rapid release.     

Covalent inhibition of recombinant human carboxylesterase 1 and 2 and monoacylglycerol lipase by the carbamates JZL184 and URB597

Carboxylesterase type 1 (CES1) and CES2 are serine hydrolases located in the liver and small intestine. CES1 and CES2 actively participate in the metabolism of several pharmaceuticals. Recently, carbamate compounds were developed to inhibit members of the serine hydrolase family via covalent modification of the active site serine. URB597 and JZL184 inhibit fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively; however, carboxylesterases in liver have been identified as a major off-target. We report the kinetic rate constants for inhibition of human recombinant CES1 and CES2 by URB597 and JZL184. Bimolecular rate constants (k_inact/K_i) for inhibition of CES1 by JZL184 and URB597 were similar [3.9 (±0.2) × 10³ M^?1 s^?1 and 4.5 (±1.3) × 10³ M^?1 s^?1, respectively]. However, k_inact/K_i for inhibition of CES2 by JZL184 and URB597 were significantly different [2.3 (±1.3) × 10² M^?1 s^?1 and 3.9 (±1.0) × 10³ M^?1 s^?1, respectively]. Rates of inhibition of CES1 and CES2 by URB597 were similar; however, CES1 and MAGL were more potently inhibited by JZL184 than CES2. We also determined kinetic constants for spontaneous reactivation of CES1 carbamoylated by either JZL184 or URB597 and CES1 diethylphosphorylated by paraoxon. The reactivation rate was significantly slower (4.5×) for CES1 inhibited by JZL184 than CES1 inhibited by URB597. Half-life of reactivation for CES1 carbamoylated by JZL184 was 49 ± 15 h, which is faster than carboxylesterase turnover in HepG2 cells. Together, the results define the kinetics of inhibition for a class of drugs that target hydrolytic enzymes involved in drug and lipid metabolism.     

Mechanism of Protein Stabilization by Trehalose During Freeze-Drying Analyzed by In Situ Micro-Raman Spectroscopy

Raman investigations were performed in situ during freeze-drying of two model proteins, lysozyme and chymotrypsinogen. The structures of proteins dissolved in 0–30 wt % solutions of trehalose in D₂O were monitored with the fingerprint (800–1800 cm^? 1) spectrum, simultaneously with freezing, ice sublimation, and water desorption analyzed in the O-D stretching (2200–2700 cm^? 1) region. In the absence of trehalose, the main changes were detected at the end of primary drying, and correspond to distortion and disordering of secondary structures. A stabilizing effect of trehalose was evidenced in primary and secondary drying stages. Raman images were calculated after freezing and primary drying, providing the distributions of trehalose, water, and protein which occur during the first two stages of the lyophilization cycle. Raman images show a slight heterogeneity in the degree of protein denaturation at the end of primary drying, in relation with the structure of the frozen product observed during freezing. The ability of trehalose to make the protein more rigid was determined as responsible for the protein stabilization during a lyophilization cycle. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2484–2494, 2013     

Toxicological evaluation of pH-sensitive nanoparticles of curcumin: Acute,sub-acute and genotoxicity studies

Research in nanotoxicology is still in nascent stages. This hampers the design of appropriate regulatory policies for these beneficial nano-drug delivery systems thus affecting their routine employment as therapeutics. Establishing the entire toxicological profile is thus indispensable for proving the human safety of nanocarriers, which was the primary objective of the current investigation. The developed curcumin loaded polymeric nanoparticles of Eudragit® S100 were subjected to various toxicological evaluations which included acute-toxicity study, sub-acute-toxicity study (28 days) and various genotoxicity studies like in vivo Micronucleus assay, in vivo Chromosomal Aberration assay and in vivo Comet assay. The formulation was found to be non-toxic at the dose equivalent to 2000 mg/kg of body weight of curcumin in the acute-toxicity study. Sub-acute-toxicity study proved the safety of the formulation for prolonged administration at the commonly used therapeutic dose of 100 mg/kg of body weight of curcumin and at twice the therapeutic dose. Genotoxicity studies proved the cellular safety of the developed formulation at the therapeutic dose, and even at doses equivalent to thrice the therapeutic dose. Thus the developed curcumin loaded polymeric nanoparticles of Eudragit® S100 were found to be safe for oral administration for a short as well as a prolonged duration.