Correlation of lactulose-to-mannitol ratios in plasma and urine for intestinal permeability assessment in pigs.

OBJECTIVE: The lactulose-to-mannitol ratio test is a test to assess the disorders associated with gut permeability. The test requires an oral administration of the mixture of lactulose and mannitol and urine collection. The urinary ratio of lactulose to mannitol is an indicator of intestinal permeability. Due to the complexity of urine collection in animal studies, plasma exposure ratios of lactulose to mannitol compared to their urinary concentration ratios were evaluated following an oral administration of the sugar mixture in pigs. ANIMALS: 10 pigs were orally dosed with a solution of lactulose and mannitol mixture. PROCEDURES: Plasma samples were collected at predose, 10 and 30 minutes and 2, 4, and 6 hours postdosing, and cumulated urinary samples were collected at 6 hours for liquid chromatography-mass spectrometry analysis. The ratios of pharmacokinetic parameters of lactulose to mannitol and the plasma sugar ratios at a single time point or the mean values of several time points were compared to their urinary sugar ratios. RESULTS: The results revealed that the lactulose-to-mannitol ratios of AUC0-6h, AUCextrap, and Cmax were correlated to the urinary sugar ratios, and the plasma sugar ratios of a single time point at 2, 4, or 6 hours and the mean values of those time points were also appropriate to replace their urinary ratios in pigs. CLINICAL RELEVANCE: Following an oral administration of lactulose and mannitol mixture, blood collection, and assay can be an option for assessing intestinal permeability, especially in animal studies.

Co-rotating twin screw process for continuous manufacturing of solid crystal suspension: A promising strategy to enhance the solubility, permeation and oral bioavailability of Carvedilol.

Background: In the current work, co-rotating twin-screw processor (TSP) was utilized to formulate solid crystal suspension (SCS) of carvedilol (CAR) for enhancing its solubility, dissolution rate, permeation and bioavailability using mannitol as a hydrophilic carrier. Methods: In-silico molecular dynamics (MD) studies were done to simulate the interaction of CAR with mannitol at different kneading zone temperatures (KZT). Based on these studies, the optimal CAR: mannitol ratios and the kneading zone temperatures for CAR solubility enhancement were assessed. The CAR-SCS was optimized utilizing Design-of-Experiments (DoE) methodology using the Box-Behnken design. Saturation solubility studies and in vitro dissolution studies were performed for all the formulations. Physicochemical characterization was performed using differential scanning calorimetry , Fourier transform infrared spectroscopy, X-ray diffraction studies, and Raman spectroscopy analysis. Ex vivo permeation studies and in vivo pharmacokinetic studies for the CAR-SCS were performed. Stability studies were performed for the DoE-optimized CAR-SCS at accelerated stability conditions at 40 ºC/ 75% RH for three months. Results: Experimentally, the formulation with CAR: mannitol ratio of 20:80, prepared using a KZT of 120 ºC at 100 rpm screw speed showed the highest solubility enhancement accounting for 50-fold compared to the plain CAR. Physicochemical characterization confirmed the crystalline state of DoE-optimized CAR-SCS. In-vitro dissolution studies indicated a 6.03-fold and 3.40-fold enhancement in the dissolution rate of optimized CAR-SCS in pH 1.2 HCl solution and phosphate buffer pH 6.8, respectively, as compared to the pure CAR. The enhanced efficacy of the optimized CAR-SCS was indicated in the ex vivo and in vivo pharmacokinetic studies wherein the apparent permeability was enhanced 1.84-fold and bioavailability enhanced 1.50-folds compared to the plain CAR. The stability studies showed good stability concerning the drug content. Conclusions: TSP technology could be utilized to enhance the solubility, bioavailability and permeation of poor soluble CAR by preparing the SCS.

A Semi-Physiological Three-Compartment Model Describes Brain Uptake Clearance and Efflux of Sucrose and Mannitol after IV Injection in Awake Mice.

PURPOSE: To evaluate a three-compartmental semi-physiological model for analysis of uptake clearance and efflux from brain tissue of the hydrophilic markers sucrose and mannitol, compared to non-compartmental techniques presuming unidirectional uptake. METHODS: Stable isotope-labeled [13C]sucrose and [13C]mannitol (10 mg/kg each) were injected as IV bolus into the tail vein of awake young adult mice. Blood and brain samples were taken after different time intervals up to 8 h. Plasma and brain concentrations were quantified by UPLC-MS/MS. Brain uptake clearance (Kin) was analyzed using either the single-time point analysis, the multiple time point graphical method, or by fitting the parameters of a three-compartmental model that allows for symmetrical exchange across the blood-brain barrier and an additional brain efflux clearance. RESULTS: The three-compartment model was able to describe the experimental data well, yielding estimates for Kin of sucrose and mannitol of 0.068 ± 0.005 and 0.146 ± 0.020 µl.min-1.g-1, respectively, which were significantly different (p < 0.01). The separate brain efflux clearance had values of 0.693 ± 0.106 (sucrose) and 0.881 ± 0.20 (mannitol) µl.min-1.g-1, which were not statistically different. Kin values obtained by single time point and multiple time point analyses were dependent on the terminal sampling time and showed declining values for later time points. CONCLUSIONS: Using the three-compartment model allows determination of Kin for small molecule hydrophilic markers with low blood-brain barrier permeability. It also provides, for the first time, an estimate of brain efflux after systemic administration of a marker, which likely represents bulk flow clearance from brain tissue.

Absorption, metabolism and excretion of once-weekly somapacitan, a long-acting growth hormone derivative, after single subcutaneous dosing in human subjects.

Somapacitan is a reversible albumin-binding growth hormone (GH) derivative in clinical development for once-weekly administration in patients with adult GH deficiency (AGHD) and children with GH deficiency (GHD). To date, the use of somapacitan in AGHD or severe AGHD has been approved in the USA and Japan, respectively. This study (ClinicalTrials.gov, NCT02962440) investigated the absorption, metabolism and excretion, as well as the pharmacokinetics (PK), of tritium-labelled somapacitan ([3H]-somapacitan). Seven healthy males received a single subcutaneous dose of 6 mg somapacitan containing [3H]-somapacitan 20 MBq. Blood, serum, plasma, urine, faeces, and expired air were collected for radioactivity assessment. Metabolites were identified and quantified in plasma and urine collected. The PK of plasma components were determined, and the radioactive peaks of the most abundant plasma metabolites and urine metabolites were selected for analysis. Twenty-eight days after dosing, 94.0% of the administered dose was recovered as [3H]-somapacitan-related material, most of which was excreted in urine (80.9%); 12.9% was excreted in faeces, and an insignificant amount (0.2%) was exhaled in expired air. PK properties of [3H]-somapacitan-related material appeared to be consistent across plasma, serum and blood. Three abundant plasma metabolites (P1, M1 and M1B) and two abundant urine metabolites (M4 and M5) were identified. The total exposure of intact somapacitan accounted for 59% of the total exposure of all somapacitan-related material, P1 accounted for 21% and M1 plus M1B accounted for 12%. M4 and M5 were the most abundant urine metabolites and accounted for 37% and 8% of the dosed [3H]-somapacitan radioactivity, respectively. No intact somapacitan was found in excreta. Two subjects had six adverse events (AEs); all were mild in severity and unlikely to be related to trial product. The majority of dosed [3H]-somapacitan (94%) was recovered as excreted metabolites. Urine was the major route for excretion of somapacitan metabolites, followed by faeces, and exhalation in expired air was negligible. The low molecular weights of identified urine metabolites demonstrate that somapacitan was extensively degraded to small residual fragments that were excreted (fully biodegradable). The extensive metabolic degradation and full elimination of metabolites in excreta were the major clearance pathways of somapacitan and the key elements in its biological fate. A single dose of 6 mg somapacitan (containing [3H]-somapacitan) in healthy male subjects was well tolerated with no unexpected safety issues identified.

Spray-Congealing and Wet-Sieving as Alternative Processes for Engineering of Inhalation Carrier Particles: Comparison of Surface Properties, Blending and In Vitro Performance.

PURPOSE: Traditionally, α-lactose monohydrate is the carrier of choice in dry powder inhaler (DPI) formulations. Nonetheless, other sugars, such as D-mannitol, have emerged as potential alternatives. Herein, we explored different particle engineering processes to produce D-mannitol carriers for inhaled delivery. METHODS: Wet-sieving and spray-congealing were employed as innovative techniques to evaluate the impact of engineering on the particle properties of D-mannitol. To that end, the resulting powders were characterized concerning their solid-state, micromeritics and flowability. Afterwards, the engineered carrier particles were blended with inhalable size beclomethasone dipropionate to form low dose (1 wt%) DPI formulations. The in vitro aerosolization performance was evaluated using the NEXThaler®, a reservoir multi-dose device. RESULTS: Wet-sieving generated D-mannitol particles with a narrow particle size distribution and spray-congealing free-flowing spherical particles. The more uniform pumice particles with deep voids and clefts of wet-sieved D-mannitol (Pearl300_WS) were beneficial to drug aerosolization, only when used in combination with a ternary agent (10 wt% of 'Preblend'). When compared to the starting material, the spray-congealed D-mannitol has shown to be promising in terms of the relative increase of the fine particle fraction of the drug (around 100%), when used without the addition of ternary agents. CONCLUSIONS: The wet-sieving process and the related aerosolization performance are strongly dependent on the topography and structure of the starting material. Spray-congealing, has shown to be a potential process for generating smooth spherical particles of D-mannitol that enhance the in vitro aerosolization performance in binary blends of the carrier with a low drug dose.

Deletion of the Imprinted Phlda2 Gene Increases Placental Passive Permeability in the Mouse.

Genomic imprinting, an epigenetic phenomenon that causes the expression of a small set of genes in a parent-of-origin-specific manner, is thought to have co-evolved with placentation. Many imprinted genes are expressed in the placenta, where they play diverse roles related to development and nutrient supply function. However, only a small number of imprinted genes have been functionally tested for a role in nutrient transfer capacity in relation to the structural characteristics of the exchange labyrinthine zone. Here, we examine the transfer capacity in a mouse model deficient for the maternally expressed Phlda2 gene, which results in placental overgrowth and a transient reduction in fetal growth. Using stereology, we show that the morphology of the labyrinthine zone in Phlda2-/+ mutants is normal at E16 and E19. In vivo placental transfer of radiolabeled solutes 14C-methyl-D-glucose and 14C-MeAIB remains unaffected at both gestational time points. However, placental passive permeability, as measured using two inert hydrophilic solutes (14C-mannitol; 14C-inulin), is significantly higher in mutants. Importantly, this increase in passive permeability is associated with fetal catch-up growth. Our findings uncover a key role played by the imprinted Phlda2 gene in modifying placental passive permeability that may be important for determining fetal growth.

Stress Impairs Skin Barrier Function and Induces α2-3 Linked N-Acetylneuraminic Acid and Core 1 O-Glycans on Skin Mucins in Atlantic Salmon, Salmo salar.

The skin barrier consists of mucus, primarily comprising highly glycosylated mucins, and the epithelium. Host mucin glycosylation governs interactions with pathogens and stress is associated with impaired epithelial barrier function. We characterized Atlantic salmon skin barrier function during chronic stress (high density) and mucin O-glycosylation changes in response to acute and chronic stress. Fish held at low (LD: 14-30 kg/m3) and high densities (HD: 50-80 kg/m3) were subjected to acute stress 24 h before sampling at 17 and 21 weeks after start of the experiment. Blood parameters indicated primary and secondary stress responses at both sampling points. At the second sampling, skin barrier function towards molecules was reduced in the HD compared to the LD group (Papp mannitol; p < 0.01). Liquid chromatography-mass spectrometry revealed 81 O-glycan structures from the skin. Fish subjected to both chronic and acute stress had an increased proportion of large O-glycan structures. Overall, four of the O-glycan changes have potential as indicators of stress, especially for the combined chronic and acute stress. Stress thus impairs skin barrier function and induces glycosylation changes, which have potential to both affect interactions with pathogens and serve as stress indicators.

Optimal Monitoring of Weekly IGF-I Levels During Growth Hormone Therapy With Once-Weekly Somapacitan.

CONTEXT: Somapacitan is a long-acting growth hormone (GH) in development for once-weekly treatment of GH deficiency (GHD). Optimal monitoring of insulin-like growth factor-I (IGF-I) levels must account for weekly IGF-I fluctuations following somapacitan administration. OBJECTIVE: To develop and assess the reliability of linear models for predicting mean and peak IGF-I levels from samples taken on different days after dosing. DESIGN: A pharmacokinetic/pharmacodynamic model was used to simulate IGF-I data in adults and children following weekly somapacitan treatment of GHD. SETTING AND PATIENTS: 39 200 IGF-I profiles were simulated with reference to data from 26 adults and 23 children with GHD. INTERVENTION(S): The simulated dose range was 0.02 to 0.12 mg/kg for adults and 0.02 to 0.16 mg/kg for children. Simulated data with >4 average standard deviation score were excluded. MAIN OUTCOME MEASURE(S): Linear models for predicting mean and peak IGF-I levels based on IGF-I samples from different days after somapacitan dose. RESULTS: Robust linear relationships were found between IGF-I sampled on any day after somapacitan dose and the weekly mean (R2 > 0.94) and peak (R2 > 0.84). Prediction uncertainties were generally low when predicting mean from samples taken on any day (residual standard deviation [RSD] ≤ 0.36) and peak from samples taken on day 1 to 4 (RSD ≤ 0.34). IGF-I monitoring on day 4 and day 2 after dose provided the most accurate estimate of IGF-I mean (RSD < 0.2) and peak (RSD < 0.1), respectively. CONCLUSIONS: Linear models provided a simple and reliable tool to aid optimal monitoring of IGF-I by predicting mean and peak IGF-I levels based on an IGF-I sample following dosing of somapacitan. A short visual summary of our work is available (1).

LC-MS/MS-based in vitro and in vivo investigation of blood-brain barrier integrity by simultaneous quantitation of mannitol and sucrose.

BACKGROUND: Understanding the pathophysiology of the blood brain-barrier (BBB) plays a critical role in diagnosis and treatment of disease conditions. Applying a sensitive and specific LC-MS/MS technique for the measurement of BBB integrity with high precision, we have recently introduced non-radioactive [13C12]sucrose as a superior marker substance. Comparison of permeability markers with different molecular weight, but otherwise similar physicochemical properties, can provide insights into the uptake mechanism at the BBB. Mannitol is a small hydrophilic, uncharged molecule that is half the size of sucrose. Previously only radioactive [3H]mannitol or [14C]mannitol has been used to measure BBB integrity. METHODS: We developed a UPLC-MS/MS method for simultaneous analysis of stable isotope-labeled sucrose and mannitol. The in vivo BBB permeability of [13C6]mannitol and [13C12]sucrose was measured in mice, using [13C6]sucrose as a vascular marker to correct for brain intravascular content. Moreover, a Transwell model with induced pluripotent stem cell-derived brain endothelial cells was used to measure the permeability coefficient of sucrose and mannitol in vitro both under control and compromised (in the presence of IL-1ß) conditions. RESULTS: We found low permeability values for both mannitol and sucrose in vitro (permeability coefficients of 4.99 ± 0.152 × 10-7 and 3.12 ± 0.176 × 10-7 cm/s, respectively) and in vivo (PS products of 0.267 ± 0.021 and 0.126 ± 0.025 µl g-1 min-1, respectively). Further, the in vitro permeability of both markers substantially increased in the presence of IL-1ß. Corrected brain concentrations (Cbr), obtained by washout vs. vascular marker correction, were not significantly different for either mannitol (0.071 ± 0.007 and 0.065 ± 0.009 percent injected dose per g) or sucrose (0.035 ± 0.003 and 0.037 ± 0.005 percent injected dose per g). These data also indicate that Cbr and PS product values of mannitol were about twice the corresponding values of sucrose. CONCLUSIONS: We established a highly sensitive, specific and reproducible approach to simultaneously measure the BBB permeability of two classical low molecular weight, hydrophilic markers in a stable isotope labeled format. This method is now available as a tool to quantify BBB permeability in vitro and in vivo in different disease models, as well as for monitoring treatment outcomes.

How is small bowel permeability in endometriosis patients? a case control pilot study.

Endometriosis is a chronic inflammatory disease of women of reproductive age. Small bowel (SB) permeability and lipopolysaccharides (LPS) could play a role in the perduration of low grade inflammation status and the pathogenesis of endometriosis. To clarify this hypothesis, we measured SB permeability through plasma values of LPS and urinary secretion of lactulose (La), mannitol (Ma) and their ratio (L/M) in patients with endometriosis compared with healthy controls (HC). Eight patients and 14 HC entered the study. SB permeability was evaluated by high-performance liquid chromatography of urine concentrations of La and Ma. Plasma levels of LPS were measured in the blood. Moreover, a nutritional, gastroenterological, quality of life evaluation was performed through validates questionnaires and complete gynaecological evaluations. The statistical analysis of the obtained data did not show differences in anthropometric and nutritional characteristics and gastrointestinal functional disease in the two groups. Patients reported higher levels of pelvic chronic pain (3.87 ± 2.99 vs 0.15 ± 0.55; pe = 0.001) and significantly higher LPS plasma levels (0.529 ± 0.11 vs 0.427 ± 0.08; p value = .027) than HC. Our results indicate that intestinal permeability is abnormal in endometriosis patients, and it might play a role in the pathogenesis of this chronic disease.

Modeling hyperosmotic blood-brain barrier opening within human tissue-engineered in vitro brain microvessels.

As the majority of therapeutic agents do not cross the blood-brain barrier (BBB), transient BBB opening (BBBO) is one strategy to enable delivery into the brain for effective treatment of CNS disease. Intra-arterial infusion of the hyperosmotic agent mannitol reversibly opens the BBB; however, widespread clinical use has been limited due to the variability in outcomes. The current model for mannitol-induced BBBO assumes a transient but homogeneous increase in permeability; however, the details are poorly understood. To elucidate the mechanism of hyperosmotic opening at the cellular level, we developed a tissue-engineered microvessel model using stem cell-derived human brain microvascular endothelial cells (BMECs) perturbed with clinically relevant mannitol doses. This model recapitulates physiological shear stress, barrier function, microvessel geometry, and cell-matrix interactions. Using live-cell imaging, we show that mannitol results in dose-dependent and spatially heterogeneous increases in paracellular permeability through the formation of transient focal leaks. Additionally, we find that the degree of BBB opening and subsequent recovery is modulated by treatment with basic fibroblast growth factor. These results show that tissue-engineered BBB models can provide insight into the mechanisms of BBBO and hence improve the reproducibility of hyperosmotic therapies for treatment of CNS disease.

Measurement of intestinal permeability using lactulose and mannitol with conventional five hours and shortened two hours urine collection by two different methods: HPAE-PAD and LC-MSMS.

Urinary excretion of two orally-administered non-metabolizable sugars, lactulose and mannitol, is a valuable marker for evaluating intestinal permeability. Usually this test involves a time consuming procedure of about 5 hour's urine collection, which makes the test incompatible to some extent. As the results are expressed as the ratio of lactulose and mannitol recovered in urine within certain time, it may be possible to get similar result despite the reduced urine collection time of 2 hours. Moreover, different laboratories do the test by different methods, which make the results incomparable between laboratories. Here, we are also trying to find the correlation between results from most commonly used methods: HPAE-PAD and LC-MSMS. The lactulose: mannitol (LM) test was performed in a cohort of Bangladeshi infants considered at-risk for environmental enteropathy. 208 urine specimens from 104 (52 male and 52 female) infants were collected at 2 and 5 hours after LM solution administration and were tested for lactulose and mannitol by two different methods, one HPAE-PAD platform and another LC-MSMS platform. Median age of the children was 15.0 months (range 6.9 to 25.8 months) and their mean weight-for-age z-score was -0.92. A higher percentage of lactulose and mannitol recovery was found in 5 hours urine collection than in the corresponding 2 hours by both HPAE-PAD and LC-MSMS method, but when results were expressed as lactulose to mannitol ratio (LMR) there was no significant difference between 2 and 5 hours urine collection in both HPAE-PAD (P = 0.138) and LC-MSMS (P = 0.099) method. LMR based on 2 hours urine collection correlated well with LMR based on traditional 5 hours urine collection (Spearman's correlation coefficient 0.578 and 0.604 respectively for HPAE-PAD and LC-MSMS). In future, LM test to assess intestinal permeability in children can be simplified by shortening the urine collection time from 5 hours to 2 hours.

Effect of USP Induction Ports, Glass Sampling Apparatus, and Inhaler Device Resistance on Aerodynamic Patterns of Fluticasone Propionate-Loaded Engineered Mannitol Microparticles.

The present investigation is to study the effect of two different induction ports (IP), i.e., USP IP and USP-modified IP equipped with andersen cascade impactor on in vitro aerodynamic performance along with the impact of USP-modified glass sampling apparatus on delivered dose uniformity of fluticasone propionate (FP) dry powder inhaler (DPI). FP DPI was fabricated by spray drying technique using engineered mannitol microparticles (EMP) with different force controlling agents, i.e., leucine and magnesium stearate. Additionally, commercially available two DPI inhaler devices namely Handihaler® and Breezhaler® were used to aerosolize the FP blends. Spherical smooth surface of EMP showed good powder flow properties and acceptable percentage content uniformity (> 95%). Amounts of FP deposited in cascade assembly using USP-modified IP with the Breezhaler® device was significantly higher (1.32-fold) as compared with the Handihaler® device. Moreover, USP-modified IP showed better deposition as compared with USP IP. Additionally, both inhaler devices showed a satisfactory delivered dose (> 105%) for FP using modified glass sampling apparatus at a flow rate of 60 L/min for 2 s. It was interesting to note that not only formulation properties but also IP geometry and device resistance have significant impact on DPI deposition pattern. This study is a first detailed account of aerodynamic performance of FP using USP-modified IP and USP-modified glass sampling apparatus. Thus, it can be of potential importance for both the academic and industry perspective.

Levothyrox® New and Old Formulations: Are they Switchable for Millions of Patients?

In France, more than 2.5 million patients are currently treated with levothyroxine, mainly as the marketed product Levothyrox®. In March 2017, at the request of French authorities, a new formulation of Levothyrox® was licensed, with the objective of avoiding stability deficiencies of the old formulation. Before launching this new formulation, an average bioequivalence trial, based on European Union recommended guidelines, was performed. The implicit rationale was the assumption that the two products, being bioequivalent, would also be switchable, allowing substitution of the new for the old formulation, thus avoiding the need for individual calibration of the dosage regimen of thyroxine, using the thyroid-stimulating hormone level as the endpoint, as required for a new patient on initiating treatment. Despite the fact that both formulations were shown to be bioequivalent, adverse drug reactions were reported in several thousands of patients after taking the new formulation. In this opinion paper, we report that more than 50% of healthy volunteers enrolled in a successful regulatory average bioequivalence trial were actually outside the a priori bioequivalence range. Therefore, we question the ability of an average bioequivalence trial to guarantee the switchability within patients of the new and old levothyroxine formulations. We further propose an analysis of this problem using the conceptual framework of individual bioequivalence. This involves investigating the bioavailability of the two formulations within a subject, by comparing not only the population means (as established by average bioequivalence) but also by assessing two variance terms, namely the within-subject variance and the variance estimating subject-by-formulation interaction. A higher within individual variability for the new formulation would lead to reconsideration of the appropriateness of the new formulation. Alternatively, a possible subject-by-formulation interaction would allow a judgement on the ability, or not, of doctors to manage patients effectively during transition from the old to the new formulation.

A multi-chamber microfluidic intestinal barrier model using Caco-2 cells for drug transport studies.

This paper presents the design and fabrication of a multi-layer and multi-chamber microchip system using thiol-ene 'click chemistry' aimed for drug transport studies across tissue barrier models. The fabrication process enables rapid prototyping of multi-layer microfluidic chips using different thiol-ene polymer mixtures, where porous Teflon membranes for cell monolayer growth were incorporated by masked sandwiching thiol-ene-based fluid layers. Electrodes for trans-epithelial electrical resistance (TEER) measurements were incorporated using low-melting soldering wires in combination with platinum wires, enabling parallel real-time monitoring of barrier integrity for the eight chambers. Additionally, the translucent porous Teflon membrane enabled optical monitoring of cell monolayers. The device was developed and tested with the Caco-2 intestinal model, and compared to the conventional Transwell system. Cell monolayer differentiation was assessed via in situ immunocytochemistry of tight junction and mucus proteins, P-glycoprotein 1 (P-gp) mediated efflux of Rhodamine 123, and brush border aminopeptidase activity. Monolayer tightness and relevance for drug delivery research was evaluated through permeability studies of mannitol, dextran and insulin, alone or in combination with the absorption enhancer tetradecylmaltoside (TDM). The thiol-ene-based microchip material and electrodes were highly compatible with cell growth. In fact, Caco-2 cells cultured in the device displayed differentiation, mucus production, directional transport and aminopeptidase activity within 9-10 days of cell culture, indicating robust barrier formation at a faster rate than in conventional Transwell models. The cell monolayer displayed high TEER and tightness towards hydrophilic compounds, whereas co-administration of an absorption enhancer elicited TEER-decrease and increased permeability similar to the Transwell cultures. The presented cell barrier microdevice constitutes a relevant tissue barrier model, enabling transport studies of drugs and chemicals under real-time optical and functional monitoring in eight parallel chambers, thereby increasing the throughput compared to previously reported microdevices.

Fragmented particles containing octreotide acetate prepared by spray drying technique for dry powder inhalation.

Dry powder inhalers (DPIs) have been proposed as an alternative administration route for protein and peptide drugs. However, DPI particles are easy to aggregate due to the strong interactions between the particles, leading to poor aerosolization performance. In this study, fragmented particles containing octreotide acetate (OA) were prepared by spray drying technique for dry powder inhalation, which were expected to decrease the particle-particle interaction by reducing the contact sites. Mannitol and ammonium carbonate were used as protein stabilizer and fragment-forming agent, respectively. The obtained fragmented particles presented larger particle size, lower density, better dispersibility, and well in vitro aerodynamic behavior (emitted dose > 97%, fine particle fraction ≈ 40%). The circular dichroism spectrum results indicated that OA maintained the stability throughout the spray drying process. The relative bioavailability of dry powder inhalation (DPI) compared with subcutaneous injection of commercial product was up to 88.0%, demonstrating the feasibility of DPI for OA delivery. These results confirmed that the proposed fragmented particles had great potential for pulmonary delivery of protein and peptide drugs in a painless, rapid, and convenient manner.

Additional Common Bean in the Diet of Malawian Children Does Not Affect Linear Growth, but Reduces Intestinal Permeability.

Background: Chronic malnutrition, as manifested by linear growth faltering, is pervasive among rural African children. Improvements in complementary feeding may decrease the burden of environmental enteric dysfunction (EED) and thus improve growth in children during the critical first 1000 d of development. Objective: We tested the hypothesis that systematically including common bean or cowpea into complementary feeding would reduce EED and growth faltering among children in rural Malawi. Methods: This was a double-blind clinical trial in which children 12-23 mo of age were randomly assigned to receive complementary feeding with 1 of 3 foods: roasted cowpea or common bean flour, or an isoenergetic amount of corn-soy blend as a control food for 48 wk. Children aged 12-23 mo received 155 kcal/d and thereafter until 35 mo received 200 kcal/d. The primary outcomes were change in length-for-age z score (LAZ) and improvements in a biomarker of EED, the percentage of lactulose (%L) excreted as part of the lactulose:mannitol dual-sugar absorption test. Anthropometric measurements and urinary %L excretion were compared between the 2 intervention groups and the control group separately with the use of linear mixed model analyses for repeated measures. Results: A total of 331 children completed the clinical trial. Compliance with the study interventions was excellent, with >90% of the intervention flour consumed as intended. No significant effects on LAZ, change in LAZ, or weight-for-length z score were observed due to either intervention legume, compared to the control. %L was reduced with common bean consumption (effect estimate was -0.07 percentage points of lactulose, P = 0.0007). The lactulose:mannitol test was not affected by the legume intervention. Conclusion: The addition of common bean to complementary feeding of rural Malawian children during the second year of life led to an improvement in a biomarker of gut health, although this did not directly translate into improved linear growth. This trial was registered at clinicaltrials.gov as NCT02472301.

Effects of surfactant-based permeation enhancers on mannitol permeability, histology, and electrogenic ion transport responses in excised rat colonic mucosae.

Surfactant-based intestinal permeation enhancers (PEs) are constituents of several oral macromolecule formulations in clinical trials. This study examined the interaction of a test panel of surfactant-based PEs with isolated rat colonic mucosae mounted in Ussing chambers in an attempt to determine if increases in transepithelial permeability can be separated from induction of mucosal perturbation. The aim was to assess the effects of PEs on (i) apparent permeability coefficient (Papp) of [14C]-mannitol (ii) histology score and (iii) short-circuit current (ΔIsc) responses to a cholinomimetic (carbachol, CCh). Enhancement ratio increases for Papp values followed the order: C10 > C9 = C11:1 > a bile salt blend > sodium choleate > sucrose laurate > Labrasol® >C12E8 > C12 > Cremophor® A25 > C7 > sucrose stearate > Kolliphor® HS15 > Kolliphor® TPGS. Exposures that increased the Papp by ≥2-fold over 120 min were accompanied by histological damage in 94% of tissues, and by a decreased ΔIsc response to CCh in 83%. A degree of separation between the increased Papp of [14C]-mannitol and histological damage and diminution of the ΔIsc response to CCh was observed at selected concentrations of Labrasol®. Overall, this surfactant-based PE selection caused transcellular perturbation at similar concentrations to those that enhanced permeability.

Mannitol clearance for the determination of glomerular filtration rate-a validation against clearance of 51 Cr-EDTA.

We studied the agreement between plasma clearance of mannitol and the reference method, plasma clearance of 51 Cr-EDTA in outpatients with normal to moderately impaired renal function. Forty-one patients with a serum creatinine <200 µmol l-1 entered the study. 51 Cr-EDTA clearance was measured with the standard bolus injection technique and glomerular filtration rate (GFR) was calculated by the single-sample method described by Jacobsson. Mannitol, 0·25 g kg-1 body weight (150 mg ml-1 ), was infused for 4-14 min and blood samples taken at 1-, 2-, 3- and 4-h (n = 24) or 2-, 3-, 3·5- and 4-h after infusion (n = 17). Mannitol in serum was measured by an enzymatic method. Plasma clearance for mannitol and its apparent volume of distribution (Vd) were calculated according to Brøchner-Mortensen. Mean plasma clearance (±SD) for 51 Cr-EDTA was 59·7 ± 18·8 ml min-1 . The mean plasma clearance for mannitol ranged between 57·0 ± 20·1 and 61·1 ± 16·7 ml min-1 and Vd was 21·3 ± 6·2% per kg b.w. The between-method bias ranged between -0·23 and 2·73 ml min-1 , the percentage error between 26·7 and 39·5% and the limits of agreement between -14·3/17·2 and -25·3/19·9 ml min-1 . The best agreement was seen when three- or four-sample measurements of plasma mannitol were obtained and when sampling started 60 min after injection. Furthermore, accuracy of plasma clearance determinations was 88-96% (P30) and 41-63% (P10) and was highest when three- or four-sample measurements of plasma mannitol were obtained, including the first hour after the bolus dose. We conclude that there is a good agreement between plasma clearances of mannitol and 51 Cr-EDTA for the assessment of GFR.

Mannitol and the blood-labyrinth barrier.

BACKGROUND: Characterization of the blood labyrinth barrier (BLB) is extremely important to determine whether the BLB can be manipulated pharmacologically. However, experiments to investigate the BLB are technically difficult to perform. In this report, we demonstrated a unique method of controlling the BLB, and established the pharmacokinetics of gentamicin in perilymph, cerebrospinal fluid (CSF) and blood with and without mannitol. STUDY DESIGN: Controlled animal research project. METHODS: Permeability of the BLB and the blood brain barrier (BBB) to gentamicin with and without mannitol was studied by collecting 175 samples from 44 guinea pigs using concentrations relevant to human clinical situations. Samples were taken from two groups of 22 animals, with each animal undergoing sampling at a different time after administration of either 10 mg/ml gentamicin (4 mg/kg) (Gardena, CA) alone or gentamicin with 20% mannitol (250 mg/kg) (Mallinckrodt Inc., KY). The sample times varied from 0.5 to 17.5 h post-infusion. Samples were also taken from 4 animals as negative controls after administration of normal saline. Our goal was to simultaneously assess the pharmacokinetics of gentamicin in each of three different fluid samples in the same animal. Thus at the pre-determined post-infusion sampling time, each animal was sampled once for perilymph, CSF, and blood before being euthanized. Each animal contributed to a single time point on the subsequent pharmacokinetic curves with more than one animal per time point. RESULTS: Mannitol increased the rate of entry and egress of gentamicin through BLB significantly (p = 0.0044) but the effects on the BBB did not reach statistical significance (p = 0.581). Mannitol did not alter renal clearance of gentamicin from the blood (p = 0.433). The concentration of gentamicin in perilymph and CSF was always significantly lower than in blood. CONCLUSIONS: Mannitol administration transiently increases the permeability of the BLB. Potential clinical benefits may accrue from selected timing of administration of osmotic agents such as mannitol augmenting the rate of entry and egress of compounds such as gentamicin into and out of perilymph.