Probiotic for pathogen-specific Staphylococcus aureus decolonisation in Thailand: a phase 2, double-blind, randomised, placebo-controlled trial.

BACKGROUND: Decolonisation is considered a valuable means to reduce Staphylococcus aureus infection rates. However, previous topical strategies targeting the nose or skin had little success, and oral antibiotic-based decolonisation is ill advised because of eradication of the microbiota and development of antibiotic resistance. We previously showed that the probiotic Bacillus subtilis significantly diminished S aureus at the main intestinal colonisation site via specific bacterial interaction in mice; in this study, we tested this probiotic approach to control S aureus colonisation in humans. METHODS: We did a single-centre, phase 2, double-blind, randomised, placebo-controlled trial in adults from the Songkhla region of Thailand who were colonised by S aureus. Eligible participants were adults (aged ≥18 years) without history of intestinal disease, antibiotic treatment, or hospital admission within the previous 90 days. Participants were excluded if they were pregnant, breastfeeding, taking probiotics, or had diarrhoea. Participants were allocated (1:1) to groups by computer randomisation in blocks of four, and research coordinators were masked to group allocation. Participants received 250 mg of probiotic B subtilis MB40 or placebo once per day for 30 days and S aureus colonisation was determined after the last dose was received. The primary outcome was colonisation by S aureus (continuous, mean decrease in colony-forming-unit count) in the intestine (by faecal counts) and nares (by nasal swabs) after intervention (30-day regimen of B subtilis probiotic). This trial is registered with the Thai Clinical Trials Registry, TCTR20210128003. FINDINGS: The trial was done between Jan 29 and June 30, 2021, with enrolment taking place from Jan 29 to April 6, 2021. 115 participants were colonised by S aureus, either in the intestine (n=84), nose (n=50), or both (n=19), and were randomly assigned to treatment (n=55) and placebo groups (n=60). Oral probiotic B subtilis resulted in significant reduction of S aureus in stool (96·8%; p<0·0001) and nose (65·4%; p=0·0002). There were no differences in adverse effects or significant microbiome changes between the intervention and placebo groups. INTERPRETATION: B subtilis probiotic eliminated more than 95% of the total S aureus colonising the human body without altering the microbiota. This probiotic strategy offers several key advantages over presently used decolonisation strategies for potential use in people with chronic or long-term risk of S aureus infection. Furthermore, by establishing a defining role of the intestinal colonisation site, our findings call for revisiting fundamental notions about S aureus colonisation. FUNDING: National Research Council of Thailand and US National Institutes of Health.

Effect of dietary coated granules containing garlic oil diallyl disulphide and diallyl trisulphide on performance, in vitro digestibility and gastrointestinal functionality in laying hens.

This experiment was conducted to investigate the effects of dietary supplementation with coated granules (CG) on performance, in vitro digestibility and the gastrointestinal functionality in laying hens. A total of 40 Hisex Brown laying hens (36 weeks of age) were randomly divided into five equal groups; one served as a control and the other four were actual experimental groups supplemented with 0.75, 1.5, 3 or 6 g CG per day. All the hens were fed restrictively with target feed intake of 100 g basal diet/hen per day. There were no differences in egg production or egg weight among the groups, but feed conversion ratio was significantly improved (linearly, p < 0.05) with the dose level of CG in diet. Dietary CG increased the jejunal weight (linear and quadratic terms, p < 0.05) but decreased the ileal weight (linearly, p < 0.05). There was mostly statistical interaction between dietary CG and specific activity of intestinal digestive enzymes with similar patterns for dietary CG treatments (p < 0.05). A positive linear correlation was observed with in vitro protein digestibility by using the crude enzyme extract from dietary CG supplementation. Dietary CG decreased the caecal Escherichia coli population while the Lactobacillus spp.: E. coli ratio increased (quadratic fit, p < 0.05). CG supplementation, on the other hand, significantly altered intestinal morphology by increasing the height of duodenal and ileal villi (linearly, p < 0.05). Also, duodenal antioxidant capacity observed via remaining reducing power improved linearly (p < 0.05). This suggests that CG, unlike garlic oils without encapsulation, may be a good candidate for feed supplementation in commercial egg production. It could be included up to 6 g CG per day without any adverse effects on performance, which may relate to improving nutrient digestibility and better utilization of limited feed intake when using a low amount of diet or other observations in this study.

Thermally Induced Denaturing Energetics of Human Blood Plasma Albumin by Differential Scanning Calorimetry (DSC) as an Indicator for Breast Cancer Diagnosis in Female Patients.

Cancerous invasion yields unusual metabolisms providing a significant amount of peptide albuminomes that modulate albumin stability via binding. The study aimed at the investigation of the thermal stability of human plasma albumin with breast cancer of various stages by means of differential scanning calorimetry (DSC). Blood plasma was drawn from 11 female breast cancer patients and 50 healthy volunteers of homogeneous demographics. Plasma samples and their albumin-enriched fractions were subjected to DSC scanning between 37 and 90°C at 5°C/min rate. For normal blood plasma, a characteristic signature of DSC tracing was observed. And, the deconvolution of DSC thermograms revealed the recognition of thermal transition of albumin. It was found that denaturing temperature of albumin increased with increasing breast cancer staging which implied the increase in albuminome/peptide abundance produced by cancerous invasion. The analysis of albumin denaturing energetics based on rational approximation of the simple Lumry-Eyring model demonstrated that thermal transition of free albumin and albuminome-bound form attained energetic levels expressed as apparent activation energy (Eapp ± s.e.) of 132.68 ± 14.21 and 46.76 ± 8.42 Kcal/mol corresponded to the schemes dominated by irreversible alteration and by reversible unfolding, respectively. Thus, Eapp value may indicate the degrees of cancerous invasion. It was proposed that Eapp may be used as an indicator to diagnose and assess the prognosis of breast cancer.

Statistical optimization of bambara groundnut protein isolate-alginate matrix systems on survival of encapsulated Lactobacillus rhamnosus GG.

Encapsulation may protect viable probiotic cells. This study aims at the evaluation of a bambara groundnut protein isolate (BGPI)-alginate matrix designed for encapsulating a probiotic Lactobacillus rhamnosus GG. The response surface methodology was employed to gain the optimal concentrations of BGPI and alginate on encapsulation efficiency and survival of encapsulated cells. The capsules were prepared at the optimal combination by the traditional extrusion method composed of 8.66% w/v BGPI and 1.85% w/v alginate. The encapsulation efficiency was 97.24%, whereas the survival rates in an acidic condition and after the freeze-drying process were 95.56% and 95.20%, respectively-higher than those using either BGPI or alginate as the encapsulating agent individually. The designed capsules increased the probiotic L. rhamnosus GG survival relative to free cells in a simulated gastric fluid by 5.00 log cfu/ml after 3 h and in a simulated intestinal fluid by 8.06 log cfu/ml after 4 h. The shelf-life studies of the capsules over 6 months at 4 °C and 30 °C indicated that the remaining number of viable cells in a BGPI-alginate capsule was significantly higher than that of free cells in both temperatures. It was demonstrated that the BGPI-alginate capsule could be utilized as a new probiotic carrier for enhanced gastrointestinal transit and storage applied in food and/or pharmaceutical products.

Theoretical Aspects of Differential Scanning Calorimetry as a Tool for the Studies of Equilibrium Thermodynamics in Pharmaceutical Solid Phase Transitions.

Although differential scanning calorimetry (DSC) is a non-equilibrium technique, it has been used to gain energetic information that involves phase equilibria. DSC has been widely used to characterize the equilibrium melting parameters of small organic pharmaceutical compounds. An understanding of how DSC measures an equilibrium event could make for a better interpretation of the results. The aim of this mini-review was to provide a theoretical insight into the DSC measurement to obtain the equilibrium thermodynamics of a phase transition especially the melting process. It was demonstrated that the heat quantity obtained from the DSC thermogram (ΔH) was related to the thermodynamic enthalpy of the phase transition (ΔH (P) ) via: ΔH = ΔH (P) /(1 + K (- 1)) where K was the equilibrium constant. In melting, the solid and liquefied phases presumably coexist resulting in a null Gibbs free energy that produces an infinitely larger K. Thus, ΔH could be interpreted as ΔH (P). Issues of DSC investigations on melting behavior of crystalline solids including polymorphism, degradation impurity due to heating in situ, and eutectic melting were discussed. In addition, DSC has been a tool for determination of the impurity based on an ideal solution of the melt that is one of the official methods used to establish the reference standard.

Assessment of the Dissociation Energetics of Some Selected Ligand Drugs Bound on Human Serum Albumin by Differential Scanning Calorimetry.

Drug-protein binding may play a role in the thermal energetics of protein denaturation and could lead to the determination of its equilibrium dissociation parameter. The aim of this study was to assess the energetics of a drug that was bound to human serum albumin (HSA) during thermal denaturation. Drugs that were bound at a single high-affinity primary binding site on HSA, including diazepam and ibuprofen, were employed. Commercial HSA was treated with charcoal to remove stabilizers and adjusted to 20% w/v in a pH 7.4 buffered solution. Serial concentrations of individual drugs up to 0.16 mmole/g-protein were added to the cleaned HSA solutions whereas diazepam was added to a commercial HSA solution. Samples were subjected to differential scanning calorimetry (DSC) set to run from 37 to 90°C at 3.0°C/min. Binding of the drug slightly increased the denaturing temperature of the cleaned HSA due to a shift in the equilibrium toward the native protein bound with the drug. Diazepam depressed the denaturing temperature of the commercial HSA by competing with the stabilizers already bound to the primary site of the HSA. This yielded not only the HSA-stabilizer but also the HSA-diazepam type complexes that exhibited a different denaturation process. A rational approximation of the Lumry-Eyring protein denaturation model was used to treat the DSC endotherms. The approximated scheme: [Formula: see text] was successfully fitted to the data. It was used to determine the dissociation parameters for diazepam and ibuprofen bound to the HSA. These results were comparable to those obtained from other methods.

The role of caprylate ligand ion on the stabilization of human serum albumin.

Sodium caprylate was added to a pharmaceutical-grade human serum albumin (HSA) to stabilize the product. In this study we have aimed to establish how caprylate ligand protects HSA from thermal degradation. The fatty acid stabilizer was first removed from commercial HSA by charcoal treatment. Cleaned HSA was made to 10% w/v in pH 7.4 buffered solutions and doped with sodium caprylate in serial concentrations up to 0.16 mmol/g-protein. These solutions as well as a commercial HSA, human serum, and enriched-albumin fraction were subjected to differential scanning calorimetry (DSC) within the temperature range of 37-90°C at a 5.0°C/min scanning rate. The globular size of the cleaned HSA solutions was measured by dynamic light scattering. The denaturing temperatures for albumin with sodium caprylate and a commercial one were significantly higher than for albumin only. It was found that the protein globules of cleaned HSA were not as stable as that of the native one due to aggregation, and the caprylate ion may reduce the aggregation by enlarging the globules' electrical double layer. A rational approximation of the Lumry-Eyring protein denaturation model was used to treat DSC denaturing endotherms. The system turned from irreversible dominant Scheme: N (k3K)→ P to reversible dominant Scheme:N (k1)→ P with the increase in caprylate concentration from null to ~0.08 mmol/g-protein. It was postulated that the caprylate ligand may decrease the rate of reversible unfolding as it binds to the IIIA domain which is prone to reversible unfolding/refolding and causes further difficulty for irreversible denaturation which, in turn, HSA can be stabilized.

Effectiveness of Artocarpus lakoocha extract, poloxamer 407, on Enterococcus faecalis in vitro.

AIM: The antiviral activities of Artocarpus lakoocha (A. lakoocha) extract have been reported in a number of studies; however, data regarding its antibacterial capability are limited. The aim of the present study was to examine the effectiveness of A. lakoocha extract, poloxamer 407, on Enterococcus faecalis (E. faecalis). METHODS: The effect of the antimicrobial activity of A. lakoocha extract and A. lakoocha extract, poloxamer 407, against E. faecalis was investigated. The antibacterial efficacy of A. lakoocha extract, poloxamer 407, against E. faecalis was compared to calcium hydroxide in a tooth model. RESULTS: The minimal inhibitory concentration and minimal bactericidal concentration (MBC) of A. lakoocha extract against E. faecalis were 0.39 ± 0 mg/mL and 3.12 ± 0 mg/mL, respectively; the MBC of calcium hydroxide against E. faecalis was 31.25 ± 0 mg/mL. In the tooth model, the E. faecalis count in all groups significantly decreased as the depth into the dentin increased compared to the control. There were no significant differences between 4% A. lakoocha extract poloxamer and UltraCal XS at any time period (P > 0.05) with one exception: at a depth of 0.6 mm on day 5, UltraCal XS had a significantly greater colonization than 4% A. lakoocha extract poloxamer. CONCLUSION: A. lakoocha extract, poloxamer 407, might be a useful alternative for antimicrobial medication in endodontic treatment. However, controlled clinical studies to evaluate its efficacy are needed.

Effect of pharmacist participation in the health care team on therapeutic drug monitoring utilization for antiepileptic drugs.

OBJECTIVE: To compare the proportions of appropriate TDM utilization regarding the indication, sampling time, and application of the measured drug levels of antiepileptic drugs (AEDs) between the pre-intervention period and pharmacist intervention period. MATERIAL AND METHOD: The baseline evaluation and pharmacist intervention study of TDM use for phenytoin, carbamazepine, or valproic acid were conducted at a medical teaching hospital in Southern Thailand. TDM requests, interpretation and dosage adjustment recommendations were mainly responsible by residents. In the intervention period, each of the three-step TDM process was assessed by the pharmacist for appropriateness and a suggestion provided if necessary prior to a final recommendation made by the resident. The criteria for appropriateness of TDM for AEDs were developed and validated by two neurologists. The present study included 44 TDM tests (22 patients) during the baseline period and 43 tests (27 patients) during the intervention period. The proportions of appropriate TDM utilization between the two periods were compared using Chi-square test. RESULTS: In the baseline period, proportions of appropriately performed TDM were: indication (63.6%), sampling time (47.7%), and application of drug levels (63.6%). Pharmacist intervention significantly increased the proportions of appropriate indication (97.7%, p = 0.001), sampling time (79.1, p = 0.0023), and applications (83.7%, p = 0.0293). There were 12 tests (27.3%) and 29 tests (67.4%) (p = 0.0001) during the baseline and the intervention period, respectively, that met all 3 criteria of appropriate TDM use. Sixteen requests without indication found in the baseline period was reduced to one in the intervention period, and thus reduced the unnecessary cost by 90%. Of 59 steady-state drug levels, 34 (57.6%, p = 0.0005) significantly correlated with clinical responses. CONCLUSION: Pharmacist intervention significantly improved appropriateness of TDM use, and substantially reduced unnecessary costs. Using a screening checklist including the indication, sampling time and data needed for proper interpretation of the results can help improve the appropriateness of TDM utilization.

Behavior of freezable bound water in the bacterial cellulose produced by Acetobacter xylinum: an approach using thermoporosimetry.

The aim of the study is to examine thermal behavior of water within reticulated structure of bacterial cellulose (BC) films by sub-ambient differential scanning calorimetry (DSC). BC films with different carbon source, either manitol (BC (a)) or glycerol (BC (b)), were produced by Acetobacter xylinum using Hestrin and Shramm culture medium under static condition at 30 +/- 0.2 degrees C for 3 days. BC samples were characterized by electron scanning microscopy and X-ray diffraction spectroscopy. The pore analysis was done by B.H.J. nitrogen adsorption. The pre-treated with 100% relative humidity, at 30.0 +/- 0.2 degrees C for 7 days samples were subjected to a between 25 and -150 degrees C-cooling-heating cycle of DSC at 5.00 degrees C/min rate. The pre-treated samples were also hydrated by adding 1 mul of water and thermally run with identical conditions. It is observed that cellulose fibrils of BC (a) were thinner and reticulated to form slightly smaller porosity than those of BC (b). They exhibited slightly but non-significantly different crystalline features. The freezable bound water behaved as a water confinement within pores rather than a solvent of polymer which is possible to use thermoporosimetry based on Gibb-Thomson equation to approach pore structure of BC. In comparison with nitrogen adsorption, it was found that thermoporosimetry underestimated the BC porosity, i.e., the mean diameters of 23.0 nm vs. 27.8 nm and 27.9 nm vs. 33.9 nm for BC (a) and BC (b), respectively, by thermoporosimetry vs. B.H.J. nitrogen adsorption. It may be due to large non-freezable water fraction interacting with cellulose, and the validity of pore range based on thermodynamic assumptions of Gibb-Thomson theory.

Thermal behavior of water in the selected starch- and cellulose-based polymeric hydrogels.

In a polymer-water matrix, freezable water is depressed due to either porosity confinement or interaction. The aim of the study was to examine water crystallization/melting depression by sub-ambient differential scanning calorimetry. The selected starch- and cellulose-based polymers including pre-gelatinized starch (PS), sodium alginate, sodium starch glycolate, hydroxypropylmethyl cellulose (HPMC), sodium carboxymethyl cellulose, and croscarmellose sodium were employed. The pre-treated with ambient humidity (85-100% relative humidity, at 30.0+/-0.2 degrees C for 10 days) and with excess water (hydrogels) samples were subjected to between 25 and -150 degrees C cooling-heating cycle at 5.00 degrees C/min rate. The volume fractions of hydrogels were measured by light scattering technique. It was observed that all polymers but PS and HPMC with ambient humidity presented freezable water in two distinct fractions namely bound water where crystallizing/melting temperature was depressed and bulk water. The water transition in samples with various contents exhibited the pattern as a polymer solution, thus rather than confinement, the depression was due to interaction. The volume fraction-melting temperature data derived from endotherms of hydrogels were successfully fitted to Flory's model (r(2): 0.934-0.999). The Flory's interaction parameters (chi(1)) were found to vary between 0.520 and 0.847. In addition, the smaller the value of chi(1), the larger melting was depressed, i.e., stronger affinity for water.

ATR-FTIR characterization of transport properties of benzoic acid ion-pairs in silicone membranes.

A novel technique based on Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy was used to study the transport of benzoic acid ion-pairs/salts in silicone membranes. The benzoic acid ion-pairs were prepared using various counter-ions with different degrees of lipophilicity, e.g. triethylamine (TA), diethylamine (DE), tert-butylamine (t-BA), 2-amino-2-methyl-propanol (AMP), and 2-amino-2-methyl-propanediol (AMPD). Silicone membrane, treated or untreated with propylene glycol (PG), was placed on the surface of a ZnSe crystal and the transport solution was applied to the upper surface of the membrane. A mathematical model, based on Fick's second law describing the build up of permeant concentration at the membrane/crystal interface with time was applied to determine diffusion coefficients. Absorption due to the acid (1700 cm(-1)) or benzoate anion (1555 cm(-1)) was observed at different regions without the interference from PG or silicone membrane. Benzoate anion, a charged species, was observed to permeate the membrane. The permeation of benzoate anion from sodium benzoate and polar ion-pairs of AMP and AMPD was very low in contrast to their high-saturated concentrations in PG as compared to the t-BA ion-pair. This indicated that benzoate anion preferentially permeates the membrane as an ion-pair rather than a single anion; otherwise its permeation should correspond to its concentration in PG instead of the lipophilicity of the ion-pairs. Additionally, the diffusion coefficient values of benzoic acid and benzoate anions through the treated and untreated membranes were not statistically different.

Ambroxol Lozenge Bioavailability : Part II - Analysis of Additional Systemic AvailabilityPart I of this article was published in Clin Drug Invest 2003; 23 (4): 273-80.

OBJECTIVE: To examine whether or not the early dissolution/absorption in the oral cavity of lozenge administration contributes to superior bioavailability of ambroxol compared with the commercially available tablet. METHODS: 24-hour plasma level pharmacokinetic profiles of 20 healthy volunteers receiving oral administration of identical single doses (30mg) of lozenges and a commercially available tablet with a 1-week washout period were collected. The data were analysed by a non-compartmental model with a statistical moment and mean transit time concept. The mean transit times obtained after lozenge and tablet administration were compared. RESULTS: The variance in absorption phase was significantly higher than that in the distribution/elimination phase in the pharmacokinetic profiles of the lozenge, suggesting additional absorption processes. The mean transit time of the tablet was significantly greater than that of the lozenge, by 7.69 hours with a 90% confidence interval (CI) of 3.11, 12.27. Early drug dissolution/absorption in the oral cavity and gastrointestinal absorption was successfully modelled to the pharmacokinetic profiles after lozenge administration. The additional availability of the drug to systemic circulation was mainly due to complete dissolution in the oral cavity prior to absorption as well as to oral mucosal transport. Between the two processes, dissolution was proposed to be a limiting step, since oral mucosal absorption was at a very high rate. The estimated average dissolution rate constant (90% CI) in first-order fashion was 0.13h(-1) (0.08, 0.32). CONCLUSION: Absorption rates between lozenge and tablet could be differentiated with the aid of the mean transit time concept. However, estimation of oral mucosal absorption was not possible because the blood sampling intervals were not sufficiently frequent.

Ambroxol lozenge bioavailability : an open-label, two-way crossover study of the comparative bioavailability of ambroxol lozenges and commercial tablets in healthy thai volunteers.

OBJECTIVE: To compare the bioavailability of two 15mg ambroxol lozenges with a commercial 30mg ambroxol tablet. DESIGN: Open-label, two-way crossover study. METHOD: Each formulation was randomly administered to 20 healthy Thai volunteers (ten male and ten female) with a 1-week washout period between formulations. After administration, serial blood samples were collected over a 24-hour period and the plasma concentration of ambroxol was subsequently measured using high performance liquid chromatography with ultraviolet detection after liquid-liquid extraction. Pharmacokinetic parameters were analysed by a noncompartmental pharmacokinetic model and compared between formulations using analysis of variance with a significance level of 0.05. RESULTS: The point estimates (90% CI) of the area under the plasma concentration-time curve (AUC) and peak plasma concentration (C(max)) ratios between lozenge and commercial tablet were 1.07 (0.89 to 1.28) and 1.20 (1.04 to 1.40), respectively. The point estimate (90% CI) of the difference between formulations for time to C(max) was 0.40 (-0.20 to 1.00). CONCLUSION: The two formulations under test were not bioequivalent based on the stipulated bioequivalence criteria. The bioavailability from the ambroxol lozenge might be better, since the 90% CI of the AUC(0-infinity) fell outside the bioequivalence range, and its range was narrower. The difference in rate of absorption was not conclusive because ambroxol was delivered from the lozenge by two parallel processes, namely absorption via oral and gastrointestinal mucosa. The additional oral mucosal absorption might not only contribute more absorption but also introduce variability compared with that of tablet administration. The relative importance of oral versus gastrointestinal mucosal absorption of ambroxol from the lozenge formulation, and the clinical significance of this, requires further study.

Thermal porosity analysis of croscarmellose sodium and sodium starch glycolate by differential scanning calorimetry.

The aim of the study was to demonstrate the applicability of differential scanning calorimetry (DSC) on porosity analysis for cellulose and starch. Croscarmellose sodium (CCS) and sodium starch glycolate (SSG) were allowed to sorb moisture in 85%, 90%, 95%, and 100% relative humidity (RH) at 40 degrees C for 24 hours. The pretreated samples were then subjected to DSC running temperature ranging from 25 degrees C to -50 degrees C at a cooling rate of 10 degrees C/min. The cooling traces of water crystallization, if present, were transformed to porosity distribution via capillary condensation using Kelvin's equation. The porosity analysis of CCS and SSG was also done using nitrogen adsorption as a reference method. It was found that sorbed water could not be frozen (in cases of 85% and 90% RH) until the moisture content exceeded a cutoff value (in cases of 95% and 100% RH). The nonfreezable moisture content was referred to tightly bound, plasticizing water, whereas the frozen one may be attributed to loosely bound water condensation in pore structure of CCS and SSG surfaces. Not only capillary condensation but also the tightly bound, nonfreezable monolayer water lying along the inner pores of the surface contributed to porosity determination. Good agreement with less than 5% deviation of mean pore size was observed when the results were compared with nitrogen adsorption. The narrower pore size distributions, however, were obtained because of the limitations of the technique. It was concluded that pore analysis by DSC could be successful. Further research needs to be done to account for limitations and to extend the applicability of the technique.