Derivation of Pluripotent Stem Cells with In Vivo Embryonic and Extraembryonic Potency.

Of all known cultured stem cell types, pluripotent stem cells (PSCs) sit atop the landscape of developmental potency and are characterized by their ability to generate all cell types of an adult organism. However, PSCs show limited contribution to the extraembryonic placental tissues in vivo. Here, we show that a chemical cocktail enables the derivation of stem cells with unique functional and molecular features from mice and humans, designated as extended pluripotent stem (EPS) cells, which are capable of chimerizing both embryonic and extraembryonic tissues. Notably, a single mouse EPS cell shows widespread chimeric contribution to both embryonic and extraembryonic lineages in vivo and permits generating single-EPS-cell-derived mice by tetraploid complementation. Furthermore, human EPS cells exhibit interspecies chimeric competency in mouse conceptuses. Our findings constitute a first step toward capturing pluripotent stem cells with extraembryonic developmental potentials in culture and open new avenues for basic and translational research. VIDEO ABSTRACT.

Porcine Pluripotent Stem Cells Established from LCDM Medium with Characteristics Differ from Human and Mouse Extended Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) have unlimited self-renewal and multifunctional development potential in vitro. Porcine PSCs are highly desirable due to the conserved characteristics between pigs and humans. Extended PSCs (EPSCs) are additionally capable of differentiating into embryonic (Em) and extraembryonic (E×Em) parts. Here, we employed the LCDM culture system (consisting of human LIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride), which can establish EPSCs from humans and mice, to derive and maintain stable porcine PSCs (pLCDM) from in vivo blastocysts. Transcriptome analysis revealed the unique molecular characteristics of pLCDMs compared with early-stage embryos. Meanwhile, the parallels and differences in the transcriptome features among pLCDMs, human EPSCs, and mouse EPSCs were carefully analyzed and evaluated. Most noteworthy, the trophoblast lineage differentiation tendency of pLCDMs was clarified by inducing trophoblast-like cells and trophoblast stem cells (TSCs) in vitro. Further research found that 2 of the small molecules in LCDM culture system, (S)-(+)-dimethindene maleate (DiM) and minocycline hydrochloride (MiH), probably play a crucial role in promoting trophoblast lineage differentiation potential of pLCDMs.

Green Solvents for Eco-Friendly Synthesis of Dimethindene: A Forward-Looking Approach.

Dimethindene is a selective histamine H1 antagonist and is commercially available as a racemate. Upon analyzing the synthetic pathways currently available for the industrial preparation of dimethindene, we set up a sustainable approach for the synthesis of this drug, switching from petroleum-based volatile organic compounds (VOCs) to eco-friendly solvents, such as 2-methyltetrahydrofuran (2-MeTHF) and cyclopentyl methyl ether (CPME) belonging to classes 3 and 2, respectively. Beyond decreasing the environmental impact of the synthesis (E-factor: 24.1-54.9 with VOCs; 12.2-22.1 with 2-MeTHF or CPME), this switch also improved the overall yield of the process (from 10% with VOCs to 21-22% with 2-MeTHF or CPME) and remarkably simplified the manual operations, working under milder conditions. Typical metrics applied at the first and second pass, according to the CHEM21 metrics toolkit, were also calculated for the whole synthetic procedure of dimethindene, and the results were compared with those of the classical procedure.

Etv5 safeguards trophoblast stem cells differentiation from mouse EPSCs by regulating fibroblast growth factor receptor 2.

Previous studies have demonstrated that transcription factor Etv5 plays an important role in the segregation between epiblast and primitive endoderm at the second fate decision of early embryo. However, it remains elusive whether Etv5 functions in the segregation between inner cell mass and trophectoderm at the first cell fate decision. In this study, we firstly generated Etv5 knockout mouse embryonic stem cells (mESCs) by CRISPR/Cas9, then converted them into extended potential stem cells (EPSCs) by culturing the cells in small molecule cocktail medium LCDM (LIF, CHIR99021, (S)-(+)-dimethindene maleate, minocycline hydrochloride), and finally investigated their differentiation efficiency of trophoblast stem cells (TSCs). The results showed that Etv5 knockout significantly decreased the efficiency of TSCs (CDX2+) differentiated from EPSCs. In addition, Etv5 knockout resulted in higher incidence of the differentiated cells with tetraploid and octoploid than that from wild type. Mechanistically, Etv5 was activated by extracellular-signal-regulated kinase (ERK) signaling pathway; in turn, Etv5 had a positive feedback on the expression of fibroblast growth factor receptor 2 (FGFR2) which lies upstream of ERK. Etv5 knockout decreased the expression of FGFR2, whose binding with fibroblast growth factor 4 was essentially needed for TSCs differentiation. Collectively, the findings in this study suggest that Etv5 is required to safeguard the TSCs differentiation by regulating FGFR2 and provide new clues to understand the specification of trophectoderm in vivo.

Analytical investigation of ternary mixture of phenylephrine hydrochloride, dimetindene maleate and benzalkonium chloride using validated stability indicating HPLC-DAD method.

A stability-indicating high performance liquid chromatography method with diode array detection (HPLC-DAD) was developed and validated for simultaneous determination of phenylephrine hydrochloride (PHR), dimetindene maleate (DMD) and benzalkonium chloride (BZM) in nasal drops and gel dosage forms. Effective liquid chromatographic separation was accomplished by employing Venusil XBP Cyano column (4.6 × 250 mm, 5 µm particle size) with gradient elution of the mobile phase consisting of buffer solution of potassium dihydrogen phosphate (0.025 M) and sodium 1-butane sulfonate (SBS) (0.025 M) (adjusted to pH 6.0) and acetonitrile. Peak areas of PHR, DMD and BZM at 271, 256 and 206 nm, respectively were measured and correlated to their concentrations. Peaks of PHR and DMD eluted at retention times 3.76 and 9.06 min, respectively, while BZM eluted as a couple of peaks at 11.88 and 12.51 min. The proposed HPLC procedure was carefully validated in terms of system suitability, linearity, ranges, precision, accuracy, specificity, robustness, detection and quantification limits. The linearity range for both PHR and BZM was 10-400 µg/mL and DMD was 5-300 µg/mL with correlation coefficients >0.9999. The studied compounds were subjected to stress conditions of neutral, acidic and alkaline hydrolysis, oxidation and thermal degradation. Good resolution of the three compounds from their forced degradation products proves specificity and stability-indicating merits of the proposed method. In addition, resolution of the three drugs under investigation from some pharmaceutical compounds of different medicinal categories showed the high specificity of the described method.

Adhesion Prevention to Polypropylene Meshes Using Combined Icodextrin Four Percent and Dimetindene Maleate.

BACKGROUND: The use of surgical meshes in ventral hernia repair has significantly reduced hernia recurrence rates. However, when placed intraperitoneally prosthetic materials can trigger the development of peritoneal adhesions. The present experimental study evaluated the combined icodextrin 4% and dimetindene maleate treatment in preventing peritoneal adhesion formation to polypropylene and titanium-coated polypropylene meshes. MATERIALS AND METHODS: Sixty female white rabbits were divided into four groups. A 2 × 2 cm piece of mesh was fixed to intact peritoneum in all animals through a midline laparotomy. A lightweight polypropylene mesh was implanted in groups 1 and 2 and a titanium-coated polypropylene mesh in groups 3 and 4. Groups 2 and 4 were treated, intraoperatively, with intravenous dimetindene maleate (0.1 mg/kg) and intraperitoneal solution of icodextrin 4% (20 mL/kg) and for the next 6 d with dimetindene maleate intramuscularly. The observation period lasted 15 d. Adhesion scores, percentage of mesh affected surface, tissue hydroxyproline levels, and tissue histopathology were examined. RESULTS: All animals in group 1 and 57% of animals in group 3 presented postoperative adhesions. The combination of antiadhesives significantly reduced the extent and severity of adhesions as well as the hydroxyproline levels in groups 2 and 4 compared with groups 1 and 3. On microscopic evaluation, animals in group 1 exhibited higher inflammation scores compared with group 2, whereas animals in groups 2 and 4 had better mesotheliazation compared with groups 1 and 3. CONCLUSIONS: The combined administration of icodextrin 4% and dimetindene maleate reduces the extent and severity of adhesions and may be successfully used to prevent adhesion formation after mesh intraperitoneal placement.

Life of lesions in eosinophilic cellulitis (Wells' syndrome)-a condition that may be missed at first sight.

Eosinophilic cellulitis is an inflammation of, until now, unknown etiology that was first described by George Wells in 1971. Its dominating histological hallmarks are so-called "flame figures" and an eosinophilic infiltrate. Here, we report the case of a 46-year-old man who initially presented with excoriated papules that were histologically interpreted as consistent with "arthropod reactions." Later on, the clinical presentation changed to erythematous plaques, partially with cockade-like aspects. At this time, new biopsies were performed showing a superficial and deep perivascular lymphocytic and heavily eosinophilic infiltrate and flame figures, thus allowing to establish the diagnosis of Wells' syndrome. Under treatment with oral prednisolone and dapsone, the patient showed a rapid improvement of the condition. The presented case demonstrates both the clinical and histopathologic life of lesions of Well's syndrome in the course of the disease from unspecific to distinctive. The need for repeated biopsies is discussed. Current understanding of the pathogenesis of Wells' syndrome and its correlating histological features are elucidated.

Dimetindene-Is the minimum toxic dose for children too strict?

Dimetindene is a sedating antihistamine indicated for the symptomatic treatment of allergic conditions. Dimetindene is marketed among others under the trade name Fenistil (oral solution). Toxicity data are limited, and there is no consensus on the dose at which children require hospitalization. Objective is to determine the potentially toxic dose in children. Data in children with age up to 15 years were obtained from hospital discharge reports. Of 139 paediatric hospital discharge reports, 23 cases (16.5%) were excluded because of uncertain ingestion. In 116 children (46 boys and 70 girls, mean age 2 years and 9 months ± 1 year and 1 month), the majority of children developed no symptoms (87 children, 75%, mean age 3 years±1 year) and the remaining 29 children (25%, mean age 2 years and 11 months ± 1 year and 3 months) developed only mild and spontaneously resolving symptoms of poisoning after a dose of 0.82 ± 0.32 mg/kg b.w. (range 0.26-1.82 mg/kg). In 98% of all cases, hospitalized children were observed for a maximum 24 h, and their condition did not require specific treatment. In conclusion, the prognosis for accidental dimetindene poisoning in children appears to be good and the minimum toxic dose has been determined to be 0.5 mg/kg b.w.

Identification and determination of ketotifen hydrogen fumarate, azelastine hydrochloride, dimetindene maleate and promethazine hydrochloride by densitometric method.

Conditions for determination of: ketotifen hydrogen fumarate, azelastine hydrochloride, dimetindene maleate and promethazine hydrochloride by densitometric method in substances and pharmaceuticals were provided. Maximum wavelenghts were: 228 nm for ketotifen hydrogen fumarate, 295 nm for azelastine hydrochloride, 265 nm for dimetindene maleate and 255 nm for promethazine hydrochloride. The limits of quantification were in the ranges of 0.2-5 microg/spot. The statistical data showed adequate accuracy and precision of developed methods.

Topical bioequivalence: Experimental and regulatory considerations following formulation complexity.

Documenting topical bioequivalence can be an extremely complex process, which is intrinsically dependent on the formulation technological features. According to EMA guideline, for simple formulations, BE may be demonstrated by documenting the qualitative (Q1), quantitative (Q2), microstructure (Q3) and performance (Q4) equivalence. Nevertheless, when addressing complex semisolids, equivalence regarding local availability should also be demonstrated. The purpose of this study is to pursue this strategy using two opposite scenarios: a simple dimetindene maleate 1 mg/g gel formulation and a diclofenac diethylammonium 23.2 mg/g emulgel, representing a complex formulation. For both formulations, Q1/Q2 test (TP) and reference products (RP) were used. Rheology, in vitro release (IVRT) and in vitro permeation methods (IVPT) were developed and validated for both products. For the dimetindene formulation, equivalence pertaining to Q4 was established. However, high variability was observed for some rheology endpoints, especially for the different RP batches. Therefore, equivalence could not be established for Q3 as per EMA requirements. Can some rheology endpoints be waived? Can we establish reasonable criteria that are overall feasible for generic manufacturers and at the same time safe for the patient? An attempt was made to propose a wider acceptance range based on the inter-batch variability of the RP. For that, the rationale presented in the EMA guideline on bioequivalence for highly variable products was used. For the diclofenac formulation, Q3 equivalence was likewise not established. Q4 equivalence was only found for some batch combinations and when applying a wider acceptance criterion (75-133%). Furthermore, IVPT equivalence also failed to be demonstrated. Nevertheless, since the TP displays an equivalent pharmacokinetic profile compared to the RP, the observed Q3, Q4 and local availability differences are not expected to be clinically significant. This study draws attention to an effective search to determine the most appropriate strategy for assessing topical bioequivalence on a case-by-case basis.

The potential of dimetindene maleate inducing resistance to blast fungus Magnaporthe oryzae through activating the salicylic acid signaling pathway in rice plants.

BACKGROUND: Rice blast disease (Magnaporthe oryzae) is considered the most destructive rice disease all over the world. Dimetindene maleate is used in medication against allergic reactions in humans. Dimetindene maleate used to induce systemic acquired resistance (SAR) in rice (Oryza sativa L.) in order to protect rice plants from blast disease. RESULTS: Dimetindene maleate was not effective against fungus linear growth in vitro. In glasshouse conditions, dimetindene maleate significantly improved resistance at 25, 50, 125, 250, 500 and 1000 mg L-1 concentrations. Leaf blast severity reached 14.18% on plants treated with the most effective concentration of 125 mg L-1 compared with control plants. In field conditions during both seasons (2016 and 2017), 125 mg L-1 dimetindene maleate decreased the disease severity to 1.1% and 2.7%, respectively, after 30 days of treatment. Also, grain yield was increased to 13.27 and 12.90 t ha-1 in 2016 and 2017 seasons, respectively. Moreover, dimetindene maleate induces some of the indicators for salicylic acid and jasmonic acid pathways via gene expression. These genes include OsWRKY45, OsNPR1, AOS2, JAMYB and PBZ1 (OsPR10), recording 15.14-, 16.47-, 5.3-, 5.37- and 5.1-fold changes, respectively, 12-h postinoculation. CONCLUSION: The results overview investigated the effectiveness of dimetindene maleate for increasing rice resistance to blast disease through inducing SAR in rice plants under glasshouse and field conditions, which could be through the SA defense pathway by expression of genes (OsWRKY45 and OsNPR1). © 2021 Society of Chemical Industry.

Targeted Use of Placebo Effects Decreases Experimental Itch in Atopic Dermatitis Patients: A Randomized Controlled Trial.

Evidence from pain research shows that the effectiveness of active pharmacological treatments can be enhanced by placebo effects. The "open drug administration" is superior to "hidden drug administration.​" In a randomized controlled trial, we aimed to show that the targeted use of placebo effects increases the efficacy of an antihistamine (dimetindene) infusion in participants with atopic dermatitis. We openly infused dimetindene (drug) in full sight with information (intervention group 1: OPEN-DRUG+INST), openly infused drug with an additional classical conditioning learning experience (intervention group 2: OPEN-DRUG+INST+COND) or infused drug without any information or sight (i.e., hidden administration (control group 1: HIDDEN-DRUG)). Control group 2 received a placebo infusion (saline) declared as dimetindene and also experienced the conditioning experience (PLAC+INST+COND). Itch was experimentally induced with histamine via a skin prick test. Outcome was assessed at the subjective (primary end point: experimental itch intensity, numeric rating scale), and objective level (secondary end point: wheal size, mm2 ). Experimental-induced itch intensity decreased in all groups but at different rates (P < 0.001). The groups with the open administration, whether it was dimetindene or placebo, had significantly stronger reductions in itch compared to the HIDDEN-DRUG group (OPEN-DRUG+INST+COND: P < 0.001; OPEN-DRUG+INST: P = 0.009; and PLAC+INST+COND: P < 0.001). Additional drug conditioning mediated via expectation led to a stronger reduction of itching (P = 0.001). Results on wheal size were similar (P = 0.048), however, no significant difference between the HIDDEN-DRUG group and the PLAC+INST+COND group (P = 0.967) was found. We conclude that specifically generated targeted placebo effects can significantly increase the action of a drug (dimetindene) and should be used in clinical practice.

Identification of a novel selective H1-antihistamine with optimized pharmacokinetic properties for clinical evaluation in the treatment of insomnia.

Analogs of the known H(1)-antihistamine R-dimethindene with suitable selectivity for key GPCRs, P450 enzymes and hERG channel were assessed for metabolism profile and in vivo properties. Several analogs were determined to exhibit diverse metabolism. One of these compounds, 10a, showed equivalent efficacy in a rat EEG/EMG model to a previously identified clinical candidate and a potentially superior pharmacokinetic profile as determined from a human microdose study.

10-year-old girl with severe edema caused by adder bite.

We report a case of acute hemorrhagic edema in a 10-year-old girl caused by adder bite. Therapy consisted of analgesics, antihistamines, corticosteroids. Antivenum serum was applied in a single dose due to severe spreading of the edema. The patient recovered almost completely after 10 days of treatment.

Permeability alterations after surgical trauma in normal rabbit peritoneum.

BACKGROUND: To investigate whether surgical trauma in a rabbit adhesion formation model and the administration of normal saline (N/S), icodextrin (ID) and/or dimetindene maleate (DM) changes the permeability of the normal rabbit parietal peritoneum. MATERIALS AND METHODS: A total of 45 female rabbits were operated on for adhesion formation and were euthanized 10 days later. In some rabbits, ID or N/S was instilled intraabdominally during operation, whereas in others DM was infused intravenously. In others, ID plus DM or no agent was used. Specimens were obtained postoperatively and were mounted between Ussing chambers. Amiloride was used to investigate Na(+) channels. Transmesothelial resistance (R(TM)) was determined as a permeability indicator. RESULTS: Amiloride increased the R(TM) of both surfaces. Surgical trauma increased R(TM) and partially inhibited the effect of amiloride. ID and N/S increased R(TM) and inhibited the effect of amiloride. Use of DM did not change R(TM) and did not inhibit the effect of amiloride. Use of ID plus DM slightly increased R(TM), but the effect of amiloride was blocked. CONCLUSIONS: Surgical trauma impairs the permeability of the normal rabbit parietal peritoneum. ID or N/S surmounted this effect, but DM did not, suggesting that surgical trauma is a diffuse process. Antiadhesion measures influence peritoneal physiology.

Prevention of postoperative adhesion formation by individual and combined administration of 4 per cent icodextrin and dimetindene maleate.

BACKGROUND: To date, no single method has been successful in eliminating peritoneal adhesion formation after major abdominal surgery. This study evaluated the individual and possible synergistic effect of a local intraperitoneal barrier, 4 per cent icodextrin, and an intravenously administered antihistamine drug, dimetindene maleate, in the prevention of adhesion development following surgical trauma. METHODS: De novo experimental adhesions were induced by standardized trauma of the peritoneum and large bowel in 120 New Zealand White rabbits. The animals were randomized into four groups receiving intraperitoneal saline, intraperitoneal 4 per cent icodextrin (60 ml), intravenous dimetindene maleate (0.1 mg/kg) and 4 per cent icodextrin-dimetindene in combination (n = 30 per group). Ten days later, adhesion scores and incidence were assessed by two independent surgeons. and surface area by computer-aided planimetry. RESULTS: Treatment with either icodextrin or dimetindene maleate significantly reduced adhesion scores and increased the incidence of adhesion-free animals in an equipotent manner. The effect of combined treatment on severity, incidence and surface area of adhesions was more pronounced than that of each drug administered separately. CONCLUSION: Combined administration of 4 per cent icodextrin and dimetindene maleate may be used safely and efficaciously to prevent surgically induced adhesions.

Analytical method for simultaneously measuring ex vivo drug receptor occupancy and dissociation rate: application to (R)-dimethindene occupancy of central histamine H1 receptors.

We introduce a novel experimental method to determine both the extent of ex vivo receptor occupancy of administered compound and its dissociation rate constant (k4). [Here, we reference k4 as the rate of offset of unlabeled ligand in convention with Motulsky and Mahan (1)]. We derived a kinetic rate equation based on the dissociation rate constant for an unlabeled compound competing for the same site as a labeled compound and describe a model to simulate fractional occupancy. To validate our model, we performed in vitro kinetics and ex vivo occupancy experiments in rat cortex with varying concentrations of (R)-dimethindene, a sedating antihistamine. Brain tissue was removed at various times post oral administration, and histamine H1 receptor ligand [3H]-doxepin binding to homogenates from drug-treated or vehicle-treated rats was measured at multiple time points at room temperature. Fractional occupancy and k4 for (R)-dimethindene binding to H1 receptors were calculated by using our proposed model. Rats dosed with 30 and 60 mg/kg (R)-dimethindene showed 42% and 67% occupancy of central H1 receptors, respectively. These results were comparable to occupancy data determined by equilibrium radioligand binding. In addition, drug k4 rate determined by using our ex vivo method was equivalent to k4 determined by in vitro competition kinetics (dissociation half-life t(1/2) approximately 30 min). The outlined method can be used to assess, by simulation and experiment, occupancy for compounds based on dissociation rate constants and contributes to current efforts in drug optimization to profile antagonist efficacy in terms of its kinetic drug-target binding parameters. Data described by the method may be analyzed with commercially available software. Suggested fitting procedures are given in the appendix.

The basophil activation test in the diagnosis of allergy: technical issues and critical factors.

BACKGROUND: The basophil activation test (BAT) is a widely validated and reliable tool especially for the diagnosis of hymenoptera venom allergy. Nevertheless, several pitfalls have to be considered and outcomes may differ because of diverse in-house protocols and commercially available kits. We aimed to identify the factors that may influence results of the CD63-based BAT. METHODS: Basophil responses to monoclonal anti-IgE (clone E124.2.8) and bee and wasp venom were determined by BAT based on CD63. The effect of stimulating factors such as, IL-3, cytochalasin B and prewarming of the samples was investigated. Furthermore, we compared two different flow cytometer systems and evaluated the influence of storage time, different staining protocols and anti-allergic drugs on the test results. RESULTS: Interleukin-3 enhanced the reactivity of basophils at 300 pM, but not at 75 and 150 pM. Prewarming of samples and reagents did not affect basophil reactivity. CD63 expression assayed after storage time of up to 48 h showed that basophil reactivity already started to decline after 4 h. Basophils stained with HLA-DR-PC5 and CD123-PE antibodies gated as HLA-DR(neg)/CD123(pos) cells showed the highest reactivity. No effect on test outcomes was observed at therapeutic doses of dimetindene and desloratadine. Finally, slight differences in the percentage of activated basophils, depending on the cytometer system used, were found. CONCLUSION: Basophil activation test should be performed as early as possible after taking the blood sample, preferably within 4 h. In contrast to the skin test, BAT can be performed in patients undergoing treatment with antihistamines. For reasons of multiple influencing factors, BAT should be performed only at validated laboratories.

Determination of dimethindene enantiomers in pharmaceuticals by capillary electrophoresis with carboxyethyl-beta-cyclodextrin.

Cyclodextrin-mediated capillary zone electrophoresis in a hydrodynamically closed separation system with suppressed electroosmotic flow and UV absorbance photometric detection was developed for a high effective separation and quantitation of dimethindene enantiomers in various pharmaceutical formulations (solution, gel, capsules). The running buffer consisted of epsilon-aminocaproic acid (20 mmol/l) adjusted to pH 4.5 with acetic acid, negatively charged carboxyethyl-beta-cyclodextrin (2.5 mg/ml), serving as chiral selector, and 0.1% (w/v) methylhydroxyethylcellulose, serving as an electroosmotic flow suppressor. The proposed method was successfully validated appraising parameters of sensitivity, linearity, precision, accuracy/recovery and robustness and it is useful for routine use.

Are antihistamines effective in children? A review of the evidence.

BACKGROUND AND AIMS: During the last decades, much attention has been paid to off-label and unlicensed prescriptions in paediatrics. However, on-label prescribing can also cause health issues. In this paper, the case of first-generation H1-antihistamines is investigated, notably the range of indications for which products are licensed in different European countries and the evidence base (or lack thereof) for each indication, as well as reported adverse drug reactions. METHODS: Review of the Summary of Product Characteristics of first-generation H1-antihistamines with a focus on paediatric use. This is plotted against the evidence available in the literature. RESULTS: This investigation shows a large variability in labelled indications and licensing ages when compared in five different European countries. Moreover, most of the indications are not based on clinical trials evaluating efficacy and safety of these drugs in children. CONCLUSIONS: Many of the licensed indications of first-generation antihistamines do not appear to be evidence based.