Controlled Release Matrix Tablets of Olanzapine: Influence of Polymers on the <Emphasis Type="Italic">In Vitro</Emphasis> Release and Bioavailability

Controlled-release (CR) tablet formulation of olanzapine was developed using a binary mixture of Methocel® K100 LV-CR and Ethocel® standard 7FP premium by the dry granulation slugging method. Drug release kinetics of CR tablet formulations F1, F2, and F3, each one suitably compressed for 9-, 12-, and 15-kg hardness, were determined in a dissolution media of 0.1 N HCl (pH 1.5) and phosphate buffer (pH 6.8) using type II dissolution apparatus with paddles run at 50 rpm. Ethocel® was found to be distinctly controlling drug release, whereas the hardness of tablets and pH of the dissolution media did not significantly affect release kinetics. The CR test tablets containing 30% Methocel® and 60% Ethocel® (F3) with 12-kg hardness exhibited pH-independent zero-order release kinetics for 24 h. In vivo performance of the CR test tablet and conventional reference tablet were determined in rabbit serum using high-performance liquid chromatography coupled with electrochemical detector. Bioavailability parameters including C_max, T_max, and AUC_0–48 h of both tablets were compared. The CR test tablets produced optimized C_max and extended T_max (P < 0.05). A good correlation of drug absorption in vivo and drug release in vitro (R² = 0.9082) was observed. Relative bioavailability of the test tablet was calculated as 94%. The manufacturing process employed was reproducible and the CR test tablets were stable for 6 months at 40 ± 2°C/75 ± 5% relative humidity. It was concluded that the CR test tablet formulation successfully developed may improve tolerability and patient adherence by reducing adverse effects.Key words: bioavailability, controlled release, Ethocel®, olanzapine     

Cholesterol absorption status and fasting plasma cholesterol are modulated by the microsomal triacylglycerol transfer protein −493 G/T polymorphism and the usual diet in women

An important inter-individual variability in cholesterol absorption has been reported. It could result from polymorphisms in genes coding for proteins involved in the absorption process and in interaction with dietary intakes. To assess whether the extent of cholesterol absorption or synthesis is modified in adult women according to the −493 G/T polymorphism in the microsomal triglyceride transfer protein gene (MTP) and/or the habitual diet. Cholestanol and sitosterol, as well as desmosterol and lathosterol, surrogate markers of cholesterol absorption or synthesis, respectively, were analyzed in the fasting plasma of 69 middle-aged women under a Western-type diet (WD) and after 3 months on a low-saturated fat, low-cholesterol/Mediterranean-type diet (LFLCD). Genotypes for MTP −493G/T polymorphism were determined. Under an usual WD, subjects homozygous for the MTP −493 T allele exhibited higher (P < 0.05) fasting serum concentrations of cholestanol (199.0 ± 30.0 vs. 133 ± 7.4 × 10² mmol/mol cholesterol) and lathosterol (188.7 ± 21.8 vs. 147.6 ± 9.1 × 10² mmol/mol cholesterol), as well as total cholesterol (7.32 ± 0.22 vs. 6.63 ± 0.12 mmol/l) compared to G carrier subjects. After 3 months on a LFLCD, level of absorption markers decreased in TT subjects with no change in synthesis ones, leading to values comparable to those measured in G carriers. The lowering of plasma total and LDL cholesterol due to dietary change was 2.4- and 2.3-fold greater in TT women than in G carriers. The polymorphism −493G/T in MTP modulates the level of cholesterol absorption but not synthesis in women under a WD, an effect abolished under a prudent LFLCD.     

Kinetic and Thermodynamic Characterization of Glucoamylase from Colletotrichum sp. KCP1

Extracellular glucoamylase of Colletotrichum sp. KCP1 produced through solid state fermentation was purified by two steps purification process comprising ammonium sulphate precipitation followed by gel permeation chromatography (GPC). The Recovery of glucoamylase after GPC was 50.40 % with 19.3-fold increase in specific activity. The molecular weight of enzyme was found to be 162.18 kDa by native-PAGE and was dimeric protein of two sub-units with molecular weight of 94.62 and 67.60 kDa as determined by SDS-PAGE. Activation energy for starch hydrolysis was 26.45 kJ mol⁻¹ while temperature quotient (Q₁₀) was found to be 1.9. The enzyme was found to be stable over wide pH range and thermally stable at 40–50 °C up to 120 min while exhibited maximum activity at 50 °C with pH 5.0. The pKa₁ and pKa₂ of ionisable groups of active site controlling V_max were 3.5 and 6.8, respectively. V_max, K_m and K_cat for starch hydrolysis were found to be 58.82 U ml⁻¹, 1.17 mg (starch) ml⁻¹ and 449 s⁻¹, respectively. Activation energy for irreversible inactivation (E_a(d)) of glucoamylase was 74.85 kJ mol⁻¹. Thermodynamic parameters of irreversible inactivation of glucoamylase and starch hydrolysis were also determined.     

Thermally Triggered Mucoadhesive In Situ Gel of Loratadine: β-Cyclodextrin Complex for Nasal Delivery

The aim of the present study was to increase the solubility of an anti-allergic drug loratadine by making its inclusion complex with β-cyclodextrin and to develop it’s thermally triggered mucoadhesive in situ nasal gel so as to overcome first-pass effect and consequently enhance its bioavailability. A total of eight formulations were prepared by cold method and optimized by 2³ full factorial design. Independent variables (concentration of poloxamer 407, concentration of carbopol 934 P, and pure drug or its inclusion complex) were optimized in order to achieve desired gelling temperature with sufficient mucoadhesive strength and maximum permeation across experimental nasal membrane. The design was validated by extra design checkpoint formulation (F9) and Pareto charts were used to help eliminate terms that did not have a statistically significant effect. The response surface plots and possible interactions between independent variables were analyzed using Design Expert Software 8.0.2 (Stat Ease, Inc., USA). Faster drug permeation with zero-order kinetics and target flux was achieved with formulation containing drug: β-cyclodextrin complex rather than those made with free drug. The optimized formulation (F8) with a gelling temperature of 28.6 ± 0.47°C and highest mucoadhesive strength of 7,676.0 ± 0.97 dyn/cm² displayed 97.74 ± 0.87% cumulative drug permeation at 6 h. It was stable for over 3 months and histological examination revealed no remarkable damage to the nasal tissue.     

Development of Controlled-Release Matrix Tablet of Risperidone: Influence of Methocel®- and Ethocel®-Based Novel Polymeric Blend on <Emphasis Type="Italic">In Vitro</Emphasis> Drug Release and Bioavailability

Controlled-release (CR) matrix tablet of 4 mg risperidone was developed using flow bound dry granulation–slugging method to improve its safety profile and compliance. Model formulations F1, F2, and F3, consisting of distinct blends of Methocel® K100 LV-CR and Ethocel® standard 7FP premium, were slugged. Each batch of granules (250–1,000 μm), obtained by crushing the slugs, was divided into three portions after lubrication and then compressed to 9-, 12-, and 15-kg hard tablets. In vitro drug release studies were carried out in 0.1 N HCl (pH 1.2) and phosphate buffer (pH 6.8) using a paddle dissolution apparatus run at 50 rpm. The CR test tablet, containing 30% Methocel® and 60% Ethocel® (F3) with 12-kg hardness, exhibited pH-independent zero-order release kinetics for 24 h. The drug release rate was inversely proportional to the content of Ethocel®, while the gel layer formed of Methocel® helped in maintaining the integrity of the matrix. Changes in the hardness of tablet did not affect the release kinetics. The tablets were reproducible and stable for 6 months at 40 ± 2°C/75 ± 5% relative humidity. Risperidone and its active metabolite, 9-hydroxyrisperidone, present in the pooled rabbit’s serum, were analyzed with HPLC-UV at λ_max 280 nm. The CR test tablet exhibited bioequivalence to reference conventional tablet in addition to the significantly (p < 0.05) optimized peak concentration, C_max, and extended peak time, T_max, of the active moiety. There was a good association between drug absorption in vivo and drug release in vitro (R² = 0.7293). The successfully developed CR test tablet may be used for better therapeutic outcomes of risperidone.KEY WORDS: controlled release, dry granulation slugging method, risperidone     

Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome‐blocking complexes

Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington''s, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer''s disease models, nematodes with muscle or pan-neuronal Aβ_1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72–86% (P < 10⁻⁶). In worms expressing Aβ_1–42, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10⁻⁶). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity.     

Niosomal Gel of Lornoxicam for Topical Delivery: In vitro Assessment and Pharmacodynamic Activity

Lornoxicam is a potent oxicam class of non steroidal anti-inflammatory agent, prescribed for mild to moderate pain and inflammation. Niosomal gel of lornoxicam was developed for topical application. Lornoxicam niosomes (Lor-Nio) were fabricated by thin film hydration technique. Bilayer composition of niosomal vesicles was optimized. Lor-Nio dispersion was characterized by DSC, XRD, and FT-IR. Morphological evaluation was performed by scanning electron microscopy (SEM). Lor-Nio dispersion was incorporated into a gel using 2% w/w Carbopol 980 NF. Rheological and texture properties of Lor-Nio gel formulation showed suitability of the gel for topical application. The developed formulation was evaluated for in vitro skin permeation and skin deposition studies, occlusivity test and skin irritation studies. Pharmacodynamic activity of the Lor-Nio gel was performed by carragenan-induced rat paw model. Optimized Lor-Nio comprised of Span 60 and cholesterol in a molar ratio of 3:1 with 30 μM dicetyl palmitate as a stabilizer. It had particle size of 1.125 ± 0.212 μm (d₉₀), with entrapment efficiency of 52.38 ± 2.1%. DSC, XRD, and IR studies showed inclusion of Lor into niosomal vesicles. SEM studies showed spherical closed vesicular structure with particles in nanometer range. The in vitro skin permeation studies showed significant improvement in skin permeation and skin deposition for Lor-Nio gel (31.41 ± 2.24 μg/cm², 30.079 ± 1.2 μg/cm²) over plain lornoxicam gel (7.37 ± 1.27 μg/cm², 6.6 ± 2.52 μg/cm²). The Lor-Nio gel formulation showed enhanced anti-inflammatory activity by exhibiting mean edema inhibition (87.69 ± 1.43%) which was significantly more than the plain lornoxicam gel (53.84 ± 2.21%).KEY WORDS: anti-inflammatory activity, lornoxicam, niosomes, rheology, texture analysis     

Novel Simvastatin Inhalation Formulation and Characterisation

Simvastatin (SV), a drug of the statin class currently used orally as an anti-cholesterolemic via the inhibition of the 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase, has been found not only to reduce cholesterol but also to have several other pharmacological actions that might be beneficial in airway inflammatory diseases. Currently, there is no inhalable formulation that could deliver SV to the lungs. In this study, a pressurised metered-dose inhaler (pMDI) solution formulation of SV was manufactured, with ethanol as a co-solvent, and its aerosol performance and physico-chemical properties investigated. A pMDI solution formulation containing SV and 6% w/w ethanol was prepared. This formulation was assessed visually and quantitatively for SV solubility. Furthermore, the aerosol performance (using Andersen Cascade impactor at 28.3 L/min) and active ingredient chemical stability up to 6 months at different storage temperatures, 4 and 25°C, were also evaluated. The physico-chemical properties of the SV solution pMDI were also characterised by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and laser diffraction. The aerosol particles, determined using scanning electron microscopy (SEM), presented a smooth surface morphology and were spherical in shape. The aerosol produced had a fine particle fraction of 30.77 ± 2.44% and a particle size distribution suitable for inhalation drug delivery. Furthermore, the short-term chemical stability showed the formulation to be stable at 4°C for up to 6 months, whilst at 25°C, the formulation was stable up to 3 months. In this study, a respirable and stable SV solution pMDI formulation for inhalation has been presented that could potentially be used clinically as an anti-inflammatory therapy for the treatment of several lung diseases.Key Words: lung inflammation, pMDI, pressurised metered dose inhaler, simvastatin     

The effect of amphotericin B on the water and nonelectrolyte permeability of thin lipid membranes

This paper reports the effects of amphotericin B, a polyene antibiotic, on the water and nonelectrolyte permeability of optically black, thin lipid membranes formed from sheep red blood cell lipids dissolved in decane. The permeability coefficients for the diffusion of water and nonelectrolytes (P_DDi) were estimated from unidirectional tracer fluxes when net water flow (J_w) was zero. Alternatively, an osmotic water permeability coefficient (P_f) was computed from J_w when the two aqueous phases contained unequal solute concentrations. In the absence of amphotericin B, when the membrane solutions contained equimolar amounts of cholesterol and phospholipid, P_f was 22.9 ± 4.6 µsec^-1 and P _DDHDH2O was 10.8 ± 2.4 µsec^-1. Furthermore, P_DDi was < 0.05 µsec^-1 for urea, glycerol, ribose, arabinose, glucose, and sucrose, and σ_i, the reflection coefficient of each of these solutes was one. When amphotericin B (10^-6 M) was present in the aqueous phases and the membrane solutions contained equimolar amounts of cholesterol and phospholipid, P _DDHDH2O was 18.1 ± 2.4 µsec^-1; P_f was 549 ± 143 µsec^-1 when glucose, sucrose, and raffinose were the aqueous solutes. Concomitantly, P_DDi varied inversely, and σ_i directly, with the effective hydrodynamic radii of the solutes tested. These polyene-dependent phenomena required the presence of cholesterol in the membrane solutions. These data were analyzed in terms of restricted diffusion and filtration through uniform right circular cylinders, and were compatible with the hypothesis that the interactions of amphotericin B with membrane-bound cholesterol result in the formation of pores whose equivalent radii are in the range 7 to 10.5 A.     

Skeletal muscle myofibrillar and sarcoplasmic protein synthesis rates are affected differently by altitude-induced hypoxia in native lowlanders

As a consequence to hypobaric hypoxic exposure skeletal muscle atrophy is often reported. The underlying mechanism has been suggested to involve a decrease in protein synthesis in order to conserve O₂. With the aim to challenge this hypothesis, we applied a primed, constant infusion of 1-¹³C-leucine in nine healthy male subjects at sea level and subsequently at high-altitude (4559 m) after 7–9 days of acclimatization. Physical activity levels and food and energy intake were controlled prior to the two experimental conditions with the aim to standardize these confounding factors. Blood samples and expired breath samples were collected hourly during the 4 hour trial and vastus lateralis muscle biopsies obtained at 1 and 4 hours after tracer priming in the overnight fasted state. Myofibrillar protein synthesis rate was doubled; 0.041±0.018 at sea-level to 0.080±0.018%⋅hr⁻¹ (p<0.05) when acclimatized to high altitude. The sarcoplasmic protein synthesis rate was in contrast unaffected by altitude exposure; 0.052±0.019 at sea-level to 0.059±0.010%⋅hr⁻¹ (p>0.05). Trends to increments in whole body protein kinetics were seen: Degradation rate elevated from 2.51±0.21 at sea level to 2.73±0.13 µmol⋅kg⁻¹⋅min⁻¹ (p = 0.05) at high altitude and synthesis rate similar; 2.24±0.20 at sea level and 2.43±0.13 µmol⋅kg⁻¹⋅min⁻¹ (p>0.05) at altitude. We conclude that whole body amino acid flux is increased due to an elevated protein turnover rate. Resting skeletal muscle myocontractile protein synthesis rate was concomitantly elevated by high-altitude induced hypoxia, whereas the sarcoplasmic protein synthesis rate was unaffected by hypoxia. These changed responses may lead to divergent adaptation over the course of prolonged exposure.     

A novel medium for the development of in vitro cell culture system from Penaeus monodon

Lack of a valid shrimp cell line has been hampering the progress of research on shrimp viruses. One of the reasons identified was the absence of an appropriate medium which would satisfy the requirements of the cells in vitro. We report the first attempt to formulate an exclusive shrimp cell culture medium (SCCM) based on the haemolymph components of Penaeus monodon prepared in isosmotic seawater having 27 ‰ salinity. The SCCM is composed of 22 amino acids, 4 sugars, 6 vitamins, cholesterol, FBS, phenol red, three antibiotics, potassium dihydrogen phosphate and di-sodium hydrogen phosphate at pH 6.8–7.2. Osmolality was adjusted to 720 ± 10 mOsm kg⁻¹ and temperature of incubation was 25 ºC. The most appropriate composition was finally selected based on the extent of attachment of cells and their proliferation by visual observation. Metabolic activity of cultured cells was measured by MTT assay and compared with that in L-15 (2×), modified L-15 and Grace’s insect medium, and found better performance in SCCM especially for lymphoid cells with 107 % increase in activity and 85 ± 9 days of longevity. The cells from ovary and lymphoid organs were passaged twice using the newly designed shrimp cell dissociation “cocktail”.     

Moderate- and high-intensity exhaustive exercise in the heat induce a similar increase in monocyte Hsp72

This study examined the relationship between exhaustive exercise in the heat at moderate and high intensities on the intracellular heat shock protein 72 (iHsp72) response. Twelve male subjects cycled to exhaustion at 60 and 75 % of maximal oxygen uptake in hot conditions (40 °C, 50 % RH). iHsp72 concentration was measured in monocytes before, at exhaustion and 24 h after exercise. Rectal temperature, heart rate and oxygen uptake were recorded during exercise. Volitional exhaustion occurred at 58.9 ± 12.1 and 27.3 ± 9.5 min (P < 0.001) and a rectal temperature of 39.8 ± 0.4 and 39.2 ± 0.6 °C (P = 0.002), respectively, for 60 and 75 %. The area under the curve above a rectal temperature of 38.5 °C was greater at 60 % (17.5 ± 6.6 °C min) than 75 % (3.4 ± 4.8 °C min; P < 0.001), whereas the rate of increase in rectal temperature was greater at 75 % (5.1 ± 1.7 vs. 2.2 ± 1.4 °C h⁻¹; P < 0.001). iHsp72 concentration increased similarly at exhaustion relative to pre-exercise (P = 0.044) and then increased further at 24 h (P < 0.001). Multiple regression analysis revealed no predictor variables associated with iHsp72 expression; however, a correlation was observed between exercise intensities for the increase in iHsp expression at exhaustion and 24 h (P < 0.05). These results suggest that iHsp72 expression increased in relation to the level of hyperthermia attained and sustained at 60 % and the higher metabolic rate and greater rate of increase in core temperature at 75 %, with the further increase in iHsp72 concentration 24 h after exercise reinforcing its role as a chaperone and cytoprotective agent.     

Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV

Human dipeptidylpeptidase IV (hDPPIV) is an enzyme that is in hydrolase class and has various roles in different parts of human body. Its deficiency may cause some disorders in the gastrointestinal, neurologic, endocrinological and immunological systems of humans. In the present study, hDPPIV enzyme was expressed on Spodoptera frugiperda (Sf9) cell lines as a host cell, and the expression of hDPPIV was obtained by a baculoviral expression system. The enzyme production, optimum multiplicity of infection, optimum transfection time, infected and uninfected cell size and cell behavior during transfection were also determined. For maximum hDPPIV (269 mU mL⁻¹) enzyme, optimum multiplicity of infection (MOI) and time were 0.1 and 72 h, respectively. The size of infected cells increased significantly (P < 0.001) after 24 h post infection. The results indicated that Sf9 cell line was applicable to the large scale for hDPPIV expression by using optimized parameters (infection time and MOI) because of its high productivity (4.03 mU m L⁻¹ h⁻¹).     

Preparation and Characterization of Stable pH-Sensitive Vesicles Composed of α-Tocopherol Hemisuccinate

The current study aims to develop a stable pH-sensitive drug delivery system. First, cleavable polyethylene glycol-α-tocopherol hemisuccinate (PEG-THS) was synthesized. Conventional pH-sensitive vesicles composed of the Tris salt of α-tocopherol hemisuccinate (THST) were then prepared using the detergent removal technique. The vesicles had a mean particle size of (163.8 ± 5.5) nm and a zeta potential of −74.5 ± 6.4 mV. The THST vesicles were then modified using PEG-THS or uncleavable PEG-cholesterol (PEG-CHOL) (THST/PEG-lipids, 100:6 molar ratio). The mean vesicle particle size and absolute zeta potential decreased with increasing PEG-THS proportion. When the pH was decreased, the vesicle particle size and calcein release rate increased. The THST vesicles were initially Ca²⁺-unstable but exhibited significantly improved stability after modification with PEG-THS, especially at PEG-lipid ratios above 6%. Incubation in an acid serum increased the calcein release rate of conventional THST vesicles to 45 ± 1.98% at 10 min. However, the release rate of the PEG-CHOL vesicles remained low. The calcein release rate of PEG-THS vesicles was between those of conventional and PEG-CHOL-V. Therefore, PEG-THS can protect vesicles in serum and reconstitute their pH sensitivity in acidic conditions. Cleavable PEG-THS can be used in stable pH-sensitive preparations without loss of pH sensitivity. Free calcein and conventional vesicles eliminated from the plasma soon after injection, as well as the half-life (t_1/2) and area under the curve of PEG-THS-V encapsulating calcein, were dramatically increased. This phenomenon indicates that the use of PEG-lipid derivatives has gained a favorably long circulation effect in mice.Key words: cleavage, long circulation, PEG-α-tocopherol hemisuccinate, pH-sensitive, vesicles     

In vivo tissue cholesterol efflux is reduced in carriers of a mutation in APOA1

Atheroprotection by high density lipoprotein (HDL) is considered to be mediated through reverse cholesterol transport (RCT) from peripheral tissues. We investigated in vivo cholesterol fluxes through the RCT pathway in patients with low plasma high density lipoprotein cholesterol (HDL-c) due to mutations in APOA1. Seven carriers of the L202P mutation in APOA1 (mean HDL-c: 20 ± 19 mg/dl) and seven unaffected controls (mean HDL-c: 54 ± 11 mg/dl, P < 0.0001) received a 20 h infusion of ¹³C₂-cholesterol (¹³C-C). Enrichment of plasma and erythrocyte free cholesterol and plasma cholesterol esters was measured. With a three-compartment SAAM-II model, tissue cholesterol efflux (TCE) was calculated. TCE was reduced by 19% in carriers (4.6 ± 0.8 mg/kg/h versus 5.7 ± 0.7 mg/kg/h in controls, P = 0.02). Fecal ¹³C recovery and sterol excretion 7 days postinfusion did not differ significantly between carriers and controls: 21.3 ± 20% versus 13.3 ± 6.3% (P = 0.33), and 2,015 ± 1,431 mg/day versus 1456 ± 404 mg/day (P = 0.43), respectively. TCE is reduced in carriers of mutations in APOA1, suggesting that HDL contributes to efflux of tissue cholesterol in humans. The residual TCE and unaffected fecal sterol excretion in our severely affected carriers suggest, however, that non-HDL pathways contribute to RCT significantly.     

Temperature sensitivity of the low-moisture-content limit to negative seed longevity--moisture content relationships in hermetic storage

• Background and Aims The negative logarithmic relationship between orthodox seed longevity and moisture content in hermetic storage is subject to a low-moisture-content limit (m_c), but is m_c affected by temperature?• Methods Red clover (Trifolium pratense) and alfalfa (Medicago sativa) seeds were stored hermetically at 12 moisture contents (2–15 %) and five temperatures (–20, 30, 40, 50 and 65 °C) for up to 14·5 years, and loss in viability was estimated.• Key Results Viability did not change during 14·5 years hermetic storage at −20 °C with moisture contents from 2·2 to 14·9 % for red clover, or 2·0 to 12·0 % for alfalfa. Negative logarithmic relationships between longevity and moisture contents >m_c were detected at 30–65 °C, with discontinuities at low moisture contents; m_c varied between 4·0 and 5·4 % (red clover) or 4·2 and 5·5 % (alfalfa), depending upon storage temperature. Within the ranges investigated, a reduction in moisture content below m_c at any one temperature had no effect on longevity. Estimates of m_c were greater the cooler the temperature, the relationship (P < 0·01) being curvilinear. Above m_c, the estimates of C_H and C_Q (i.e. the temperature term of the seed viability equation) did not differ (P > 0·10) between species, whereas those of K_E and C_W did (P < 0·001).• Conclusions The low-moisture-content limit to negative logarithmic relationships between seed longevity and moisture content in hermetic storage increased the cooler the storage temperature, by approx. 1·5 % over 35 °C (4·0–4·2 % at 65 °C to 5·4–5·5 % at 30–40 °C) in these species. Further reduction in moisture content was not damaging. The variation in m_c implies greater sensitivity of longevity to temperature above, compared with below, m_c. This was confirmed (P < 0·005).     

P0 (protein zero) mutation S34C underlies instability of internodal myelin in S63C mice

P0 constitutes 50–60% of protein in peripheral nerve myelin and is essential for its structure and stability. Mutations within the P0 gene (MPZ) underlie a variety of hereditary neuropathies. MpzS63C transgenic mice encode a P0 with a serine to cysteine substitution at position 34 in the extracellular domain of mature P0 (P0S34C), associated with the hypomyelinating Déjérine-Sottas syndrome in human. S63C mice develop a dysmyelinating neuropathy, with packing defects in peripheral myelin. Here, we used x-ray diffraction to examine time-dependent packing defects in unfixed myelin. At ∼7 h post-dissection, WT and S63C(+/+) myelin showed native periods (175 Å) with the latter developing at most a few percent swollen myelin, whereas up to ∼50% of S63C(+/−) (mutant P0 on heterozygous P0 null background) or P0(+/−) myelin swelled to periods of ∼205 Å. In the same time frame, S63C(−/−) myelin was stable, remaining swollen at ∼210 Å. Surprisingly, treatment of whole S63C(−/−) nerves with a reducing agent completely reverted swollen arrays to native spacing and also normalized the swollen arrays that had formed in S63C(+/−) myelin, the genotype most closely related to the human disorder. Western blot revealed P0-positive bands at ∼27 and ∼50 kDa, and MALDI-TOF mass spectrometry showed these bands consisted of Ser³⁴-containing peptides or P0 dimers having oxidized Cys³⁴ residues. We propose that P0S34C forms ectopic disulfide bonds in trans between apposed Cys³⁴ side chains that retard wrapping during myelin formation causing hypomyelination. Moreover, the new bonds create a packing defect by stabilizing swollen membrane arrays that leads to demyelination.     

Relative Bioavailability of Chlorothiazide from Mucoadhesive Compacts in Pigs

The relative bioavailability of chlorothiazide from mucoadhesive polymeric compacts is compared to commercial oral suspension in pigs. A single-dose randomized study was conducted in 12 healthy pigs that are 9–10 weeks old. After overnight fasting, pigs were divided into two groups of six animals. To the first group, a reference product containing 50 mg of chlorothiazide suspension, and in the second group, test product (mucoadhesive compacts) chlorothiazide (50 mg) was administered with 75 mL of water via gastric tubes. Blood samples were collected between 0 to 24 h using catheters inserted into the jugular vein. Plasma was separated by protein precipitation, and chlorothiazide concentrations were determined using a high-performance liquid chromatography method. The mean T_max and the C_max of chlorothiazide following the administration of oral suspension and mucoadhesive compacts were 0.58 ± 0.20 h and 682.97 ± 415.69 ng/mL and 2.17 ± 0.98 h and 99.42 ± 124.08 ng/mL, respectively. The K_el and T_1/2 of chlorothiazide were found to be 1.06 ± 0.28 h⁻¹ and 0.70 ± 0.21 h from suspension and 0.95 ± 1.11 h⁻¹ and 2.05 ± 1.90 h from the compacts, respectively. The T_max of mucoadhesive compacts were significantly longer (p < 0.05; 2.17 h) than the reference products (0.58 h), whereas the C_max of compacts were significantly lower (99 ng/mL) than the reference product (683 ng/mL; p < 0.05). The area under the curve (AUC) of compacts accounts only 50.15% (404.32 ± 449.93 ng h/mL) of the reference product’s AUC (806.27 ± 395.97 ng h/mL). The relative bioavailability of the compacts was lower than that of the suspension, and this may be due to the narrow window of absorption for chlorothiazide.Key words: bioavailability, chlorothiazide, mucoadhesive compacts, pigs     

Partial rescue of Rett syndrome by ω-3 polyunsaturated fatty acids (PUFAs) oil

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with ω-3 PUFAs-containing fish oil (DHA: 72.9 ± 8.1 mg/kg b.w./day; EPA: 117.1 ± 13.1 mg/kg b.w./day; total ω-3 PUFAs: 246.0 ± 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F₂-dihomo-isoprostanes, F₃-isoprostanes, F₄-neuroprostanes, and F₂-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the ω-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Electronic supplementary material

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