In vitro and in vivo evaluation of topical formulations of Spantide II

The purpose of this study was to develop and evaluate topical formulations of Spantide II, a neurokinin-1 receptor (NK-1R) antagonist, for the treatment of inflammatory skin disorders. Spantide II lotion and gel was formulated with and without n-methyl-2-pyrrolidone (NMP) as a penetration enhancer. The release of Spantide II from gels was evaluated using microporous polyethylene and polypropylene membranes in a Franz Diffusion cell setup. In vitro percutaneous absorption of Spantide II from lotion and gel formulations was evaluated using the above setup by replacing the membranes with hairless rat skin. The in vivo anti-inflammatory activity of Spantide II formulations was evaluated in an allergic contact dermatitis (ACD) mouse model. Among different gels studied, PF127 gel showed highest (70-fold) release of Spantide II compared with hydroxypropyl methylcellulose (HPMC) and hydroxypropyl cellulose (HPC) gels. Lotion and gel formulations with or without NMP showed no detectable levels of Spantide II in the receiver compartment of the Franz diffusion cell until 24 hours. However, Spantide II showed significant retention in epidermis and dermis from lotion and gel formulations at 24 hours. The dermal levels increased ≈3.5- and 2-fold when the lotion and gel formulations contained NMP as compared with the formulation with no NMP (P<.05). The in vivo studies indicated that Spantide II formulations with NMP were effective in significantly reducing ACD response, similar to dexamethasone (0.5 mM). In conclusion, Spantide II was stable as a topical formulation and delivered to target skin tissue (epidermis and dermis) for the treatment of ACD. In addition this study supports the role of cutaneous neurosensory system in modulating inflammatory responses in the skin. Published: October 31, 2005     

In vivo,ex vivo,and in vitro studies on apelin's effect on myocardial glucose uptake

Apelin is an endogenous peptide hormone recently implicated in glucose homeostasis. However, whether apelin affects glucose uptake in myocardial tissue remains undetermined. In this study, we utilized in vivo, ex vivo and in vitro methods to study apelin's effect on myocardial glucose uptake. Pyroglutamated apelin-13 (2 mg/kg/day) was administered to C57BL6/J mice for 7 days. In vivo myocardial glucose uptake was measured by FDG-PET scanning, and GLUT4 translocation was assessed by immunofluorescence imaging. For in vitro studies, differentiated H9C2 cardiomyoblasts were exposed to pyroglutamated apelin-13 (100 nM) for 2 h. To test their involvement in apelin-stimulated myocardial glucose uptake, the energy sensing protein kinase AMPK were inhibited by pharmacologic inhibition (compound C) and RNA interference. IRS-1 phosphorylation was assessed by western blotting using an antibody directed against IRS-1 Ser-789-phosphorylated form. We found that apelin increased myocardial glucose uptake and GLUT4 membrane translocation in C57BL6/J mice. Apelin was also sufficient to increase glucose uptake in H9C2 cells. Apelin-mediated glucose uptake was significantly decreased by AMPK inhibition. Finally, apelin increased IRS-1 Ser-789 phosphorylation in an AMPK-dependent manner. The results of our study demonstrated that apelin increases myocardial glucose uptake through a pathway involving AMPK. Apelin also facilitates IRS-1 Ser-789 phosphorylation, suggesting a novel mechanism for its effects on glucose uptake.     

Differential permeation of propranolol enantiomers across human skin in vitro from formulations containing an enantioselective excipient.

This work tested the hypothesis that a stereospecific topical formulation could be used to engineer differential permeation rates for each enantiomer of an applied racemate across human skin in vitro. Racemic and enantiomerically pure R or S propranolol HCI were formulated with cellulose tris(3,5-dimethyl phenyl carbamate) (CDMPC) and applied to excised human skin using side-by-side Franz-type diffusion cells. When the pure enantiomers were used, there was a marked difference between the penetration rates of R and S propranolol (flux ratio: 2.06; P = 0.04). When racemic propranolol was used, the difference was reduced, although still statistically significant (flux ratio: 1.2; P = 0.08), particularly in view of the differential activities of the two enantiomers. Control experiments, in which no CDMPC was present, produced equal permeation rates. The results can be rationalised in terms of differential adsorption onto CDMPC within the vehicle, whereby S-propranolol is preferentially bound relative to R-propranolol. This causes an imbalance in the apparent donor phase concentrations that (in accordance with Fickian diffusion laws and thermodynamic activity) gives rise to differences in permeation rates. The diminished differential observed when the racemate was used, rather than individual enantiomers, is less easily rationalised. In this work, it was the permeation of the eutomer (S-propranolol) that was retarded, although the general principle of stereoselectively retarded skin permeation has been established.     

Preparation of matrine ethosome,its percutaneous permeation in vitro and anti-inflammatory activity in vivo in rats

The aim of this work was to evaluate the preparation of matrine ethosome and the percutaneous permeation in vitro and the anti-inflammatory activity in vivo in the rat skin. The matrine ethosomes were prepared by the ethanol injection-sonication method. The particle size of the ethosomes was measured by a laser particle-size analyzer, and the entrapment efficiency was detected by ultracentrifugation. The anti-inflammatory activity in vivo of the matrine formulations was determined by a reflection spectrophotometer. In this study, we found that the average particle size of matrine ethosomes was in the range of 50–200?nm with a narrow distribution, and the entrapment efficiency was in the range of 40–90%. Compared with other formulations, matrine ethosomes had the largest 24-hour accumulative permeation quantity (60.5%) and with no permeation lag time. Matrine ethosomes were able to make the induced erythema disappear more rapidly than the nonethosomes formulations of matrine. This study reveals that the average particle size of matrine ethosomes decreases with the increase of ethanol concentration and increases with the increase of phospholipid concentration, while the entrapment efficiency increases with the increase of the concentration of both ethanol and phospholipid. Matrine ethosomes can increase the percutaneous permeation of matrine in the experiment in vitro and improve the anti-inflammatory activity of matrine in vivo in rat skin.     

Clearance of radiation-induced apoptotic lymphocytes: ex vivo studies and an in vitro co-culture model

Lymphocytes are very sensitive to radiation. Our aim was to test the possibility of detecting apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure. Our in vitro data confirmed the dose-time-effect relationships involved in radiation-induced apoptosis. The detection of in vivo induction of apoptosis in circulating lymphocytes after exposure of animals to radiation appears to depend critically on the technique used to measure apoptosis. Among the different techniques we investigated, mitochondrial modification was the most appropriate; they allowed establishment of dose-time-effect relationships when animals were observed for 72 h. A model of in vitro phagocytosis of apoptotic lymphocytes by macrophages was developed to mimic clearance of apoptotic cells occurring in vivo. Together, our data show that mitochondrial labeling may make it possible to detect ex vivo radiation-induced apoptosis of lymphocytes before macrophage ingestion occurs. We propose the measurement of apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure.     

Tri-block co-polymer nanocarriers for enhancement of oral delivery of felodipine: preparation,in vitro characterization and ex vivo permeation

This study aimed to prepare, optimize and characterize novel felodipine-loaded polymeric nanomicelles, using a pluronic mixture of F127 and P123. Thin-film hydration method was adopted for the preparation of different polymeric nanomicelles (T1–T12) according to a 4¹.3¹ full factorial design. Factors studied were: Pluronic®:drug ratio (P:D ratio) (10, 20, 30 and 40?w/w) and percent of hydrophilic polymer (F127%) (33.33%, 50% and 66.67% w/w). Optimization criteria were to maximize transmittance percent (T%) and entrapment efficiency percent (EE%) and to minimize particle size (PS) and polydispersity index (PDI). The optimized formulation was further characterized by DSC, FTIR and ¹H NMR studies. It was also subjected to stability testing and ex vivo permeation using rabbit intestines. Spherical nanomicelles of particle size ranging from 26.18 to 87.54?nm were successfully obtained. The optimized formulation was found to be the already prepared formulation T12 (P:D ratio of 40 and 66.67% F127) with suitable T% and EE% of 95.12% and 91.75%, respectively. DSC, FTIR and ¹H NMR studies revealed felodipine (FLD) incorporation within T12 nanomicelles. T12 enhanced the ex vivo intestinal permeation of FLD when compared to a drug suspension and showed good stability. Therefore, pluronic nanomicelles could be promising for improved oral delivery of FLD.     

Investigating superiority of novel bilosomes over niosomes in the transdermal delivery of diacerein: in vitro characterization,ex vivo permeation and in vivo skin deposition study

Skin is considered the most accessible organ of the body because of its underlying capillary network. However, stratum corneum (SC), the upper most layer of skin, represents major diffusional barrier for most drugs. Hence, the use of edge activators (EAs) in designing novel elastic vesicles is hypothesized to impart their lipid bilayer with ultra-flexibility to trespass SC by high self-optimizing deformability. To confirm this hypothesis, this work aimed at developing novel bilosomes by modulating conventional niosomal composition using different bile salts as EAs and investigating their superiority over niosomes for transdermal delivery of diacerein (DCN), as model drug. Bilosomes were prepared by thin film hydration (TFH) technique according to full 3¹.2² factorial design to select the optimal formulation using Design-Expert^® software. The optimal bilosomes (B6) showed nanosized vesicles (301.65?±?17.32?nm) and 100.00?±?0.00 % entrapment efficiency. Ex vivo permeation studies and in vivo evaluation revealed that B6 exhibited superior permeation and drug retention capacity compared to the conventional niosomal formulation and drug suspension. Furthermore, B6 was subjected to in vivo histopathological study using male Wistar rats which ensured its safety for topical application. Overall, the results confirmed the hypothesized superiority of bilosomes over niosomes for enhancing DCN flux across the skin.     

The serotonergic antidepressant nefazodone inhibits the serotonin transporter: In vivo and ex vivo studies

Nefazodone HCl (Serzone^®) is a new antidepressant with a chemical structure unrelated to selective serotonin reuptake inhibitors (SSRIs), tricyclics, tetracyclics, or monoamine oxidase inhibitors (MAOIs). Nefazodone is active in a number of preclinical tests for antidepressant activity and shows clinical efficacy in the treatment of depression with a more favorable side-effect profile than the structurally similar antidepressant trazodone. Previous studies have shown that nefazodone is a potent antagonist of 5-HT_2A receptors and binds to the serotonin transporter in vitro and in vivo. Nefazodone also binds to the norepinephrine transporter in vitro and in acute ex vivo studies. To further investigate the ability of nefazodone to modify serotonergic transmission, the ability of systemically administered nefazodone to inhibit the serotonin transporter was assessed by investigating the ability of nefazodone to prevent p-chloroamphetamine- (PCA) induced depletions of cortical 5-HT concentrations. In addition, the ability of acute and subchronic nefazodone administration to inhibit ex vivo [³H]-5-HT uptake was assessed. Acute administration of nefazodone (30, 100, and 150 mg/kg) antagonized PCA-induced depletion of cortical 5-HT concentrations in a dose-dependent manner at 1,2, and 3 hours post-treatment. This effect was directly correlated with serum nefazodone concentrations. Both 100 mg/kg and 150 mg/kg of nefazodone were equipotent with fluoxetine (10 mg/kg) over the course of the experiment with respect to sparing of 5-HT depletion. Acute administration of nefazodone (100 and 150 mg/kg s.c.) significantly increased the K_m, for [³H]-5-HT uptake in rat cortical synaptosomes from 60 nmol/L in controls to 230 and 242 nmol/L in nefazodone-treated rats, respectively. Subchronic administration of nefazodone (100 and 150 mg/kg, s.c., b.i.d. × 5.5 days) reduced [³H]-5-HT uptake by 24% and 29%, respectively. Sub-chronic dosing with fluoxetine (5 mg/kg, s.c., b.i.d. × 5.5 days) reduced [³H]-5-HT uptake by 65% These experiments confirm and extend previous reports concerning the ability of nefazodone to inhibit the 5-HT transporter in vivo.     

Investigations on the effect of antimicrobial drugs on platelet aggregation in vitro and ex vivo

The influence of ten betalactam antibiotics and ten other antibiotics on platelet aggregation induced by ADP or adrenaline was investigated in vitro. In concentrations of 900 mg/l most antimicrobial drugs exerted a moderate inhibition that was not seen in concentrations of 90 mg/l that better corresponded to therapeutic levels. The influence on the platelets of a single large intravenous dose was also tested using eight antimicrobial drugs, viz. benzylpenicillin, ampicillin, carbenicillin, piperacillin, cloxacillin, cefuroxime, cefotaxime and erythromycin. Each drug was given to five healthy volunteers but none caused any significant inhibition of platelet aggregation. The second wave of aggregation persisted after administration of the drugs and it was even seen after the administration of such a high dose as 10 g of carbenicillin.     

Toll-like receptor adaptor signaling molecule MyD88 on intervertebral disk homeostasis: in vitro, ex vivo studies

MyD88 is an adapter protein that links toll-like receptors (TLRs) and Interleukin-1 receptors (IL-1Rs) with downstream signaling molecules. The MyD88 has been found to be an essential mediator in the development of osteoarthritis in articular cartilage. However, the role of the MyD88 pathway has yet to be elucidated in the intervertebral disk (IVD). Using in vitro techniques, we analyzed the effect of MyD88 pathway-specific inhibition on the potent inflammatory and catabolic mediator LPS and IL-1 in bovine and human nucleus pulposus (NP) cells by assessing matrix-degrading enzyme expression, including matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family). We also analyzed inhibition of MyD88 in the regulation of inducible nitric oxide synthase and TLR-2. Finally, we used an ex vivo organ culture model to assess the effects of MyD88 inhibitor (MyD88i) on catabolic factor-induced disk degeneration in mice lumbar disks. In bovine NP cells, MyD88i potently antagonizes LPS- or IL-1-mediated induction of cartilage-degrading enzyme production, including MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. MyD88i also attenuates the LPS- or IL-1-mediated induction of iNOS and TLR-2 gene expression. Our ex vivo findings reveal inhibition of MyD88 via counteraction of IL-1-mediated proteoglycan depletion. The findings from this study demonstrate the potent anti-inflammatory and anti-catabolic effects of inhibition of MyD88 pathway inhibition on IVD homeostasis, suggesting a potential therapeutic benefit of a MyD88i in degenerative disk disease in the future.     

Comparative in vitro and ex vivo studies on the bactericidal activity of Tetraclean, a new generation endodontic irrigant, and sodium hypochlorite

The aim of this study was to compare the efficacy of a new generation endodontic irrigant, Tetraclean, to the widely used sodium hypochlorite. Tetraclean combines a powerful detergent effect with a strong antimicrobial efficacy, whereas sodium hypochlorite has several drawbacks and is sometimes ineffective in preventing microbial-mediated endodontic failure. The bactericidal activity of both irrigants against Enterococcus faecalis, the most commonly isolated species from root canals of teeth with post-treatment disease, was assessed i) in vitro, according to the European Standard lines for the evaluation of the bactericidal activity of chemical disinfectants, and ii) with an ex vivo model of extracted and decoronated human teeth, infected with E. faecalis and subsequently irrigated with either of the irrigants. Both irrigants display very similar bactericidal activity against E. faecalis in vitro. However, the ex vivo model shows that only in the teeth irrigated with Tetraclean did the bacterial burden gradually drop until no bacteria were detectable a few days post-irrigation. Vice versa, in the teeth irrigated with sodium hypochlorite, the drop in the bacterial burden was rapid but temporary and most of the teeth were colonized again by 48 hours post-irrigation.     

Rooting affects the photosystem II activity: in vitro and ex vitro studies on energy hybrid sorrel

Rumex tianschanicus × Rumex patientia is a high-biomass-yielding plant suitable for fuel and biogas production. The protocol of the hybrid sorrel micropropagation was used to study the changes in the photosystem II (PSII) activity as well as to analyse the ultrastructure of the chloroplasts. The lowest effective PSII quantum yield [Y(II)] and an apparent electron transport rate of PSII [ETR(II)] were observed for adventitious shoots that had been regenerated in vitro, before rooting. These fluorescence parameters were higher and similar for both the leaves of the same adventitious shoots that had been rooted under in vitro conditions and for the shoots that had been acclimated and grown in ex vitro conditions. The analysis indicated that the PSII activity strongly depends on the formation of properly functioning roots and that in vitro or ex vitro culture conditions are, at least to some degree, less important. TEM analysis revealed that chloroplasts from plants rooted in vitro were sufficiently mature and acclimatization processes have less impact on their development. This is the first report concerning the analysis of PSII activity and the ultrastructure of the chloroplasts at all of the stages of micropropagation, i.e. adventitious shoot formation in vitro, rooting in vitro and acclimation to ex vitro conditions. It strongly indicated that rooting under in vitro conditions, rather than the acclimation to ex vitro conditions, plays a key role in the development of a completely functional photosynthetic apparatus in hybrid sorrel.     

Correlation between rheological properties, in vitro release, and percutaneous permeation of tetrahydropalmatine

The aim of the present work was to investigate the influence of formulation factors including different grades of Carbopol® matrices and penetration enhancers on the percutaneous permeation of tetrahydropalmatine (THP), rheological properties, and in vitro release; and the correlation behind rheological properties, in vitro release, and percutaneous permeation. Transdermal penetration of THP through excised rabbit skin and in vitro release of THP across transparent Cellophane® were performed by vertical Franz diffusion cell. Rheological analyses were proceeded in terms of “steady flow tests”, “oscillation stress sweep”, and “creep recovery”. The result of percutaneous penetration of THP indicated that, the emulgel prepared with Carbopol® 971P (Cp 971P) as the matrix and N-methyl-2-pyrrolidone (NMP) as the penetration enhancer had the highest cumulative permeation amount (118.19 μg/cm²). All the experimental data showed a good fit to the Casson model in viscosimetric studies no matter what the types of matrices or the kinds of penetration enhancers were. The release profile fitted the zero-order release kinetics model with Cp 971P as the matrix without any penetration enhancers. However, when adding penetration enhancers, in vitro release of THP presented anomalous (non-Fickian) release kinetics. Clarifying the relationship behind percutaneous permeation of THP, rheological properties, and in vitro release will provide us with profound insights and facilitate the design of specific emulgel.KEY WORDS: Carbopol®, emulgel, in vitro release, rheological properties, THP     

Time-lapse imaging of the dynamics of CNS glial-axonal interactions in vitro and ex vivo

Background

Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize the various stages of myelination.

Methodology/Principal Findings

We took two approaches to follow myelination over time: i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. We demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, we provide evidence from time-lapse fluorescence microscopy and confocal microscopy that the oligodendrocytes'' cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs, which then extend longitudinally along the axons. We predict from this model that these spiral cuffs must extend over each other first before extending to form internodes of myelin.

Conclusion

These experiments show the feasibility of visualizing the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualization of an entire internodal length of myelin and the ex vivo approach validating the in vitro data.     

In vitro stability and ex vivo absorption of thymol monoglucosides in the porcine gut

Thymol α-D-glucopyranoside (TαG) and thymol β-D-glucopyranoside (TβG) are believed to have different kinetic behaviours in the porcine gut than its parent aglycon thymol. However, recently, it was shown that concentrations of both glucosides decreased rapidly in the stomach and proximal small intestine following oral supplementation to piglets as did thymol. Yet, the stability of thymol glucosides in gut contents and their absorption route remains obscure. Therefore, a series of in vitro incubations were performed, simulating the impact of pH, digestive enzymes, bacterial activity and mucosal extracts on stability of these glucosides. Their absorption mechanisms were investigated using the Ussing chamber model in the presence or the absence of inhibitors of sodium-dependent glucose linked transporter 1 and lactase phlorizin hydrolase. Both glucosides remained intact at physiological pH levels in the presence of digestive enzymes. Recoveries from TαG and TβG were below 90% when incubated with small intestinal homogenates from the distal jejunum or from all sampled sites, respectively. However, no aglycon could be detected in these samples. Bacterial inoculum of the small intestine, on the other hand, hydrolysed TβG quickly with up to 44% of free aglycon appearing. TαG proved more resistant to porcine gastro-intestinal bacterial glucosidases with only trace amounts (<1%) of free thymol at the end of the incubations. Electrophysiological measurements in Ussing chambers did not suggest active transport of the glucosides. Mucosal TαG and TβG concentrations were unchanged between start and end of the absorption measurements. Additionally, no TαG and only a very limited amount of TβG were retrieved from the serosal side. Tissue associated concentrations, although marginal (<1% of luminal concentration), were mainly as intact glucoside or as aglycon for TαG and TβG, respectively. Addition of both inhibitors significantly increased the amount of intact glucosides retrieved from the mucosal tissues as compared to controls. In conclusion, bacterial hydrolysis was identified as the most important source of TβG loss, whereas TαG seemed less prone to degradation or absorption in these in vitro and ex vivo models.     

The in vitro use of the hair follicle closure technique to study the follicular and percutaneous permeation of topically applied drugs

Recent studies on follicular permeation emphasise the importance of hair follicles as diffusion pathways, but only a limited amount of data are available about the follicular permeation of topically applied drugs. This study examines the use of a hair follicle closure technique in vitro, to determine the participation of hair follicles in transdermal drug penetration. Various substances, with different lipophilicities, were tested: caffeine, diclofenac, flufenamic acid, ibuprofen, paracetamol, salicylic acid and testosterone. Diffusion experiments were conducted with porcine skin, the most common replacement material for human skin, in Franz-type diffusion cells over 28 hours. Different experimental settings allowed the differentiation between interfollicular and follicular permeation after topical application of the test compounds. A comparison of the apparent permeability coefficients of the drugs demonstrates that the percutaneous permeations of caffeine and flufenamic acid were significantly higher along the hair follicles. In the cases of paracetamol and testosterone, the follicular pathway appears to be of importance, while no difference was found between interfollicular and follicular permeation for diclofenac, ibuprofen and salicylic acid. Thus, the hair follicle closure technique represents an adequate in vitro method for gaining information about follicular or percutaneous permeation, and can replace in vivo testing in animals or humans.     

In vitro and ex vivo permissivity of hepatocytes for Leishmania donovani

Using models of ex vivo infection of murine, rat, and human primary hepatocytes by Leishmania donovani, we showed that hepatocytes are permissive for Leishmania at a low level. We then modeled the in vitro infection of a human hepatoma-derived cell line to examine the parasite's capability to proliferate and to cause direct damage to hepatocytes. Results showed that L. donovani can infect hepatocytes, but do not massively proliferate. This slight infection under our experimental conditions resulted in limited damage to hepatocytes. These results bring into question a possible role for hepatocytes as a parasite reservoir during latent infection.