The human colon adenocarcinoma Caco-2 and Madin-Darby canine kidney epithelial cell lines provide in vitro tools to assess a drug's permeability and transporter interactions during discovery and ...development. The cells, when cultured on semiporous filters, form confluent monolayers that model the intestinal epithelial barrier for permeability, transporter and drug-interaction assays. The applications of these assays in pharmaceutical research include qualitative prediction and ranking of absorption, determining mechanism(s) of permeability, formulation effects on drug permeability, and the potential for transporter-mediated drug-drug interactions. This review focuses on recent examples of Caco-2 and Madin-Darby canine kidney cells assays for drug permeability including transfected and knock-down cells, miniaturization and automation, and assay combinations to better understand and predict intestinal drug absorption.
Highlights • Prevalence of genitourinary syndrome of menopause (GSM) was investigated in a multicentric national study. • Prevalence ranged from 65% to 84% in women 1–6 years after menopause. • ...Vaginal dryness and objective assessment of dry vagina were the most common findings. • Most of the women did not have a previous diagnosis of GSM. • Most of the women with a previous diagnosis received some kind of treatment but almost all still had GSM.
Experimental models of permeability in animals, excised tissues, cell monolayers, and artificial membranes are important during drug discovery and development as permeability is one of several ...factors affecting the intestinal absorption of oral drug products. The utility of these models is demonstrated by their ability to predict a drug’s
in vivo
intestinal absorption. Within the various permeability models, there are differences in the performance of the assays, along with variability in animal species, tissue sources, and cell types, resulting in a variety of experimental permeability values for the same drug among laboratories. This has led to a need for assay standardization within laboratories to ensure applicability in the drug development process. Method suitability provides a generalized approach to standardize and validate a permeability model within a laboratory. First, assay methodology is optimized and validated for its various experimental parameters along with acceptance criteria for the assay. Second, the suitability of the model is demonstrated by a rank order relationship between experimental permeability values and human extent of absorption of known model compounds. Lastly, standard compounds are employed to classify a test drug’s intestinal permeability and ensure assay reproducibility and quality. This review will provide examples of the different aspects method suitability for
in situ
(intestinal perfusions),
ex vivo
(everted intestinal sacs, diffusion chambers), and
in vitro
(cell monolayers, artificial membranes) experimental permeability models. Through assay standardization, reference standards, and acceptance criteria, method suitability assures the dependability of experimental data to predict a drug’s intestinal permeability during discovery, development, and regulatory application.
Summary
Anti‐Müllerian hormone (AMH) is a dimeric glycoprotein, a member of the transforming growth factor (TGF) superfamily. It is produced exclusively in the gonads and is involved in the ...regulation of follicular growth and development. In the ovary AMH is produced by the granulosa cells of early developing follicles and seems to be able to inhibit the initiation of primordial follicle growth and FSH‐induced follicle growth. As AMH is largely expressed throughout folliculogenesis, from the primary follicular stage towards the antral stage, serum levels of AMH may represent both the quantity and quality of the ovarian follicle pool. Compared to other ovarian tests, AMH seems to be the best marker reflecting the decline of reproductive age. AMH measurement could be useful in the prediction of the menopausal transition. It could also be used to predict poor ovarian response and possibly the prognosis of in vitro fertilization (IVF) cycles. AMH has been shown to be a good surrogate marker for polycystic ovary syndrome (PCOS). Finally, its use as a marker for granulosa cell tumours has been proposed. A clearer understanding of its role in ovarian physiology may help clinicians to find a role for AMH measurement in the field of reproductive medicine.
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► Copper nanoparticles on nanosized magnetic support as catalyst for alkyne synthetic transformations. ► Commercially available silica coated maghemite (MagSilica) is used as ...magnetic support. ► The nanocatalyst is easily recovered and reused by means of an external magnet. ► High versatility of the catalyst for high atom economy alkyne coupling and cycloaddition reactions.
A versatile and magnetically recoverable catalyst consisting of copper nanoparticles on silica coated maghemite nanoparticles (MagSilica®) is presented. The catalyst has been prepared under mild conditions by fast reduction of anhydrous CuCl2 with lithium sand and a catalytic amount of DTBB (4,4’-di-tert-butylbiphenyl) as electron carrier, in the presence of the magnetic support. The catalyst has been fully characterized and its performance in different coupling and cycloaddition reactions of terminal alkynes has been studied. This new copper-based catalyst has shown to be very efficient and easily reusable in the Glaser alkyne dimerization reaction in THF, the multicomponent Huisgen 1,3-dipolar cycloaddition reaction in water and the three-component synthesis of propargylamines under solvent free conditions.
BACKGROUND Controlled-rate freezing of ovarian cortical tissue for preservation of fertility among young women facing chemo- or radio-therapy is a widely accepted procedure. To improve the method for ...cryopreservation of ovarian tissue, particularly the stroma, we carried out a systematic comparison of vitrification versus slow programmed freezing. METHODS Ovarian tissue from 20 women, donated during Caesarean section, was used for parallel comparison of survival and detailed light and electron microscopic (EM) morphology of oocytes, granulosa cells and ovarian stroma after freezing (slow freezing and vitrification), thawing and 24-h culture. Using tissue obtained from the same patient, we compared four cryopreservation protocols and fresh tissue. The cryoprotectants used in slow freezing were 1,2-propanediol (PrOH)-sucrose and ethylene glycol (EG)-sucrose. For vitrification, tissues were incubated for 5 or 10 min in three solutions containing a combination of dimethyl sulphoxide (DMSO), PrOH, EG and polyvinylpyrrolidone (PVP). RESULTS Cryopreservation using controlled-rate freezing and vitrification preserved the morphological characteristics of ovarian tissue generally well. As revealed by morphological analysis, particularly EM, the ovarian stroma was significantly better preserved after vitrification than after slow freezing (P < 0.001). The follicles were similarly preserved after all freezing methods. CONCLUSIONS Vitrification using a combination of PrOH, EG, DMSO and PVP was comparable to slow freezing in terms of preserving follicles in human ovarian tissue. Ovarian stroma had significantly better morphological integrity after vitrification than after controlled-rate freezing.
Epithelial cells are involved in the regulation of innate and adaptive immunity in response to different stresses. The purpose of this study was to investigate if alkali-injured corneal epithelia ...activate innate immunity through the nucleotide-binding oligomerization domain-containing protein (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway. A unilateral alkali burn (AB) was created in the central cornea of C57BL/6 mice. Mice received either no topical treatment or topical treatment with sodium butyrate (NaB), β-hydroxybutyric acid (HBA), dexamethasone (Dex), or vehicle (balanced salt solution, BSS) quater in die (QID) for two or five days (d). We evaluated the expression of inflammasome components including NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1, as well as the downstream cytokine interleukin (IL)-1β. We found elevation of NLRP3 and IL-1β messenger RNA (mRNA) transcripts, as well as levels of inflammasome component proteins in the alkali-injured corneas compared to naïve corneas. Treatment with NLRP3 inhibitors using NaB and HBA preserved corneal clarity and decreased NLRP3, caspase-1, and IL-1β mRNA transcripts, as well as NLRP3 protein expression on post-injury compared to BSS-treated corneas. These findings identified a novel innate immune signaling pathway activated by AB. Blocking the NLRP3 pathway in AB mouse model decreases inflammation, resulting in greater corneal clarity. These results provide a mechanistic basis for optimizing therapeutic intervention in alkali injured eyes.
Autologous haematopoietic stem cell transplantation (HSCT) with CD34(+) cell selection has recently been used in the treatment of refractory Crohn's disease, showing good safety and promising ...efficacy. We investigated the safety and efficacy of HSCT with unselected peripheral blood stem cells (PBSCs) in moderate-severe refractory Crohn's disease.
Four patients (three male, one female; age range 26-45 years) with active moderate-severe Crohn's disease (median Crohn's Disease Activity Index (CDAI) 319, range 272-345), refractory or intolerant to multiple drugs including infliximab, were enrolled.
Unselected PBSCs were collected after mobilisation with cyclophosphamide (CTX) 1.5 g/m2 and granulocyte-colony stimulating factor (G-CSF) 10 microg/kg. The conditioning regimen included CTX 50 mg/kg on days -5 to -2 and rabbit anti-thymocyte globulin (ATG) 2.5 mg/kg on days -4 to -2.
Primary endpoints were toxicity and clinical remission (CDAI<150) at 3 months. Secondary endpoints were clinical and endoscopic response at 3 months and toxicity, clinical and endoscopic remission at 12 months.
No improvement or slight deterioration was observed following mobilisation (median CDAI 339, range 258-404). At the third month, the primary endpoint of clinical remission was achieved in all patients, with a median CDAI of 91 (range 56-102), and complete endoscopic remission was achieved in 2/3 patients. After a median follow-up of 16.5 months, 3/4 patients maintained both clinical and endoscopic remission, despite withdrawal of all drugs, and complete fistula closure was observed in all affected patients. No deaths or life-threatening infection occurred. Unexpected adverse events included a perianal abscess after mobilisation in one patient, pleural and pericardial effusions in another and BK virus-related macrohaematuria in another, all rapidly resolved with conservative treatment.
Autologous HSCT with unselected PBSC appears to be safe and can induce and maintain remission in previously refractory Crohn's disease patients.
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