In situ biodistribution and residency of a topical anti-inflammatory using fluorescence lifetime imaging microscopy.

BACKGROUND: GSK2894512 is a topically delivered investigational drug being developed for treatment of atopic dermatitis and psoriasis. OBJECTIVES: To investigate, in a phase I clinical trial, the spatial biodistribution and residency of GSK2894512 within the epidermis and dermis of healthy human participants noninvasively using fluorescence lifetime imaging microscopy (FLIM). METHODS: Two topical drug formulations containing GSK2894512 1% were applied to the right and left forearms of six participants for seven consecutive days, followed by seven days of observation for residency. FLIM images were obtained daily throughout the study, approximately every 24 h. During the treatment phase of the study, images were collected from each participant pretreatment, reflecting the residual dose from the previous day. Three punch biopsies from each participant of one formulation was obtained from the treated region during the post-treatment follow-up period between days 8 and 14 for comparison with FLIM results. RESULTS: Cellular and subcellular features associated with different epidermal and dermal layers were visualized noninvasively, down to a depth of 200 µm. Results yielded three-dimensional maps of GSK2894512 spatial distribution and residency over time. This fluorescence data provided a marker that was used as a monitor for day-to-day variance of drug presence and residency postapplication. CONCLUSIONS: The results suggest FLIM could be a viable alternative to skin biopsies without the usual patient discomfort and limitations, thereby enabling the direct measurement of skin distribution through longitudinal monitoring. These results are the first step in establishing the unique capabilities that multiphoton imaging could provide to patients through noninvasive drug detection.

Effects of Intracerebroventricularly (ICV) Injected Ghrelin on Cardiac Inducible Nitric Oxide Synthase Activity/Expression in Obese Rats.

The aim of this study was to examine the effects of ghrelin on regulation of cardiac inducible nitric oxide synthase (iNOS) activity/expression in high fat (HF), obese rats.For this study, male Wistar rats fed with HF diet (30% fat) for 4 weeks were injected every 24 h for 5 days intracerebroventricularly (ICV) with ghrelin (0.3 nmol/5 µl) or with an equal volume of phosphate buffered saline (PBS). Control rats were ICV injected with an equal volume of PBS. Glucose, insulin and nitric oxide (NO) concentrations were measured in serum, while arginase activity and citrulline concentrations were measured in heart lysate. Protein iNOS and regulatory subunit of nuclear factor-κB (NFκB-p65), phosphorylation of enzymes protein kinase B (Akt) at Ser(473), and extracellular signal-regulated kinases 1/2 (ERK1/2) at Tyr(202)/Tyr(204) were determined in heart lysate by Western blot. For gene expression of iNOS qRT-PCR was used.Results show significantly (p<0.01) higher serum NO production in ghrelin treated HF rats compared with HF rats. Ghrelin significantly reduced citrulline concentration (p<0.05) and arginase activity (p<0.01) in HF rats. In ghrelin treated HF rats, gene and protein expression of iNOS and NFκB-p65 levels were significantly (p<0.05) increased compared with HF rats. Increased phosphorylation of Akt (p<0.01) and decreased (p<0.05) ERK1/2 phosphorylation were detected in HF ghrelin treated rats compared with HF rats hearts.Results from this study indicate that exogenous ghrelin induces expression and activity of cardiac iNOS via Akt phosphorylation followed by NFκB activation in HF rats.

Functionally active toll-like receptor 3 on human primary and metastatic cancer cells.

Toll-like receptor 3 (TLR3) is a member of Toll-like receptors who recognize structurally conserved molecules derived from pathogens and trigger the immune response. To clarify if TLR3, is expressed in certain tumour cell lines and whether it is functional and what is the response of these cell lines to different concentrations of poly I:C treatment, we have screened SW480, SW620, FaDu and Detroit 562 cell lines using real-time PCR, flow cytometry and ELISA. We have shown that all these cell lines express TLR3 on mRNA and protein level but it is only functional in Detroit 562 cell line since only in these cells poly I:C treatment triggered the IL-6 secretion. In addition, poly I:C treatment inhibited cell growth and triggered up-regulation of IL-12p40, IL-8 and Il-1alpha in Detroit 562 cell line. By using annexin-V apoptosis detection kit, we have found that poly I:C triggers apoptosis in Detroit 562 cell line. We have found here that based on the results of TLR3 functionality there is a huge difference between FaDu and Detroit 562 cell lines which are of the same origin (pharynx) but FaDu is primary and Detroit 562 metastatic carcinoma. Our study also shows that Detroit 562 cell line could be a good model for cancer therapy research and development as it is responsive to TLR3 agonists which consequently drives it to apoptosis.

The novel phosphoramidate derivatives of NSAID 3-hydroxypropylamides: synthesis, cytostatic and antiviral activity evaluations.

The target phosphoramidates 5a-e were prepared in one step from 3-hydroxypropyl derivatives 3a-e of nonsteroidal anti-inflammatory drugs (fenoprofen, ketoprofen, ibuprofen, indomethacin, diclofenac). The products 3a-e and 5a-e were evaluated for their cytostatic and antiviral activity against malignant tumour cell lines and normal human fibroblasts (WI 38). All phosphoramidate derivatives 5a-e possess significantly greater inhibitory activities than the corresponding 3-hydroxypropyl derivatives 3a-e, whereby compound 5a showed the most potent inhibitory activities against cervical, pancreatic and colon carcinoma cell lines (IC(50)=5-7 microM).

Synthesis and in vitro antitumor effect of diclofenac and fenoprofen thiolated and nonthiolated polyaspartamide-drug conjugates.

This paper reports the synthesis and antiproliferative effects of new thiomer-diclofenac and fenoprofen conjugates, hydrophilic, bioadhesive, polymeric prodrugs, as well as antiproliferative effects of diclofenac, fenoprofen and a series of previously described polymer-fenoprofen conjugates on five tumor cell lines. Thiolated and nonthiolated polyaspartamides were the chosen polymeric components. Drug-loading ranged from 5.6 to 22.4%, and the amount of SH groups ranged from 6.9 to 45.6micromol g(-1). Tensile studies demonstrated a clear correlation between the amount of thiol and the mucoadhesive properties of the conjugates. The growth-inhibitory activity of the tested polymer-drug conjugates demonstrates that polyaspartamide-type polymers, especially thiolated polymers, enable inhibition of tumor cell growth with significantly lower doses of the active substance.

The temperature dependence of intracellular pH in isolated frog skeletal muscle: lessons concerning the Na(+)-H+ exchanger.

We used 31P NMR to investigate the temperature-dependence of intracellular pH (pHi) in isolated frog skeletal muscles. We found that Ln[H+i] is a linear function of 1/Tabs paralleling those of neutral water (i.e., H+ = OH-) and of a solution containing the fixed pH buffers of frog muscle cytosol. This classical van't Hoff relationship was unaffected by inhibition of glycolysis and was not dependent upon the pH or [Na+] in the bathing solution. Insulin stimulation of Na(+)-H+ exchange shifted the intercept in the alkaline direction but had not effect on the slope. Acid loading followed by washout resulted in an amiloride-sensitive return to the (temperature dependent) basal pHi. These results show that the temperature dependence of activation of Na(+)-H+ exchange is similar to that of the intracellular buffers, and suggest that constancy of [H+]/ [OH-] with changing temperature is achieved in the short term by intracellular buffering and in the long term by the set-point of the Na(+)-H+ exchanger. Proton activation of the exchanger has an apparent standard enthalpy change (delta H degree) under both control and insulin-stimulated conditions that is similar to the delta H degree of the intracellular buffers and approximately half of the delta H degree for the dissociation of water. Thus, the temperature-dependent component of the standard free-energy change (delta F degree) is unaffected by insulin stimulation, suggesting that changes in Arrhenius activation energy (Ea) may not be a part of the mechanism of hormone stimulation.

Estimation of oral drug absorption in man based on intestine permeability in rats.

Predicting the fraction of an oral dose absorbed in humans is of considerable interest at an early stage of a research program in the pharmaceutical industry. Models described in the literature to predict the oral absorption in man include: the permeability in Caco-2 cells, absorption from a perfused segment of rat intestinal lumen and uptake into everted rings. The present study used an isolated and vascularly perfused rat small intestine to determine the permeability values of eleven compounds across the intestinal epithelium. A good correlation was obtained between the permeability values determined in this model and the proportion of an oral dose absorbed in humans. Compared to the other models, the present one could allow the appearance in the artificial bloodstream and the intestinal metabolism of a compound to be studied simultaneously.

Elevating intracellular free Mg2+ preserves sensitivity of Na(+)-K+ pump to ATP at reduced temperatures in guinea pig red blood cells.

Red cells of hibernating species have a higher relative rate of Na(+)-K+ pump activity at low temperature than the red cells of a mammal with a typical sensitivity to cold. The kinetics of ATP stimulation of the Na(+)-K+ pump were determined in guinea pig and ground squirrel red cells at different temperatures between 5 and 37 degrees C by measuring ouabain-sensitive K+ influx at different levels of ATP. In guinea pig cells, elevation of intracellular free Mg2+ to 2 mmol.1-1 by use of the divalent cation ionophore A23187 caused the apparent affinity of the pump for ATP to increase with cooling to 20 degrees C, rather than to decrease, as occurs in cells not loaded with Mg2+. In ground squirrel cells raising intracellular free Mg2+ had little effect on apparent affinity of the pump for ATP at 20 degrees C. ATP affinity rose slightly with cooling both in Mg(2+)-enriched and in control ground squirrel cells. Increased intracellular free Mg2+ in guinea pig cells stimulated Na(+)-K+ pump activity so that at 20 degrees C the pump rate was the same in the Mg(2+)-enriched guinea pig and control ground squirrel cells. Pump activity in Mg(2+)-enriched guinea pig cells at 5 degrees C was significantly improved but still lower than pump activity in control cells from ground squirrel. Thus, loss of affinity of the Na(+)-K+ pump for ATP that occurs with cooling in cold-sensitive guinea pig red cells can be, at least partially, prevented by elevating cytoplasmic free Mg2+. Conversely, in ground squirrel red cells natural rise of free Mg2+ may in part account for the preservation of the ATP affinity of their Na(+)-K+ pump with cooling.

A comparison of effect of temperature on phosphorus metabolites, pH and Mg2+ in human and ground squirrel red cells.

1. 31P NMR spectra were obtained at temperatures ranging from 2 to 30 degrees C from freshly drawn human (cold-sensitive) and ground squirrel (cold-tolerant) red cells. The concentration of ATP was also determined by luciferin-luciferase assay over the same temperature range. 2. The concentration of ATP as determined by NMR or by the luciferin-luciferase assay did not change with temperature in either species. The absolute concentration of ATP in human cells determined by NMR was not significantly different from the total ATP determined enzymatically. 3. The concentration of 2,3-diphosphoglycerate was higher and that of pyridine nucleotides lower in human than in ground squirrel red cells. This species difference was independent of temperature. 4. Intracellular pH, as determined from the positions of the NMR peaks of 2- and 3-phosphates of diphosphoglycerate, became more alkaline as the temperature was lowered. 5. Free intracellular magnesium, determined from the difference in the positions of the peaks for alpha- and beta-phosphorus of ATP, increased in the ground squirrel red cells and decreased in the human red cells with cooling from 30 to 2 degrees C. Total magnesium, as determined by atomic emission spectroscopy, did not change with temperature in red cells of either species. 6. The intensities of all phosphorus metabolite signals from the ground squirrel cells increased with decreasing temperature, while those from the human cells were unaffected. Since chemical shift anisotropy in the presence of magnesium is a powerful spin-lattice relaxation mechanism for phosphates, this is additional evidence for the temperature dependence of free magnesium concentration in the ground squirrel cells. 7. We conclude that there is no difference in phosphorus metabolites or intracellular pH which could account for the differential cold sensitivity in human and ground squirrel red cells. We suggest that, in the cold-tolerant red cells from the ground squirrel, magnesium is released from binding sites as the temperature is lowered. The change in free intracellular Mg2+ may account at least in part for the unusually low temperature sensitivity of the Na(+)-K+ pump in the red cells of this species.

ATP dependence of Na(+)-K+ pump of cold-sensitive and cold-tolerant mammalian red blood cells.

1. The ATP concentration of intact, cold-tolerant (ground squirrel) red cells and cold-sensitive (guinea-pig and human) red cells was monitored by use of the firefly tail, luciferin-luciferase assay. ATP kinetics of the pump in intact red blood cells was investigated by altering cell [ATP] by progressive depletion of ATP in the presence of 2-deoxy-D-glucose and then by measurement of ouabain-sensitive K+ influx at each level of [ATP] at various temperatures between 37 and 5 degrees C. Na(+)-K(+)-ATPase activity of broken membranes was also determined in parallel experiments using ouabain-sensitive release of 32P from [gamma-32P]ATP as a measure of activity. 2. Without depletion, there is no immediate decrease in [ATP] of intact cold-sensitive cells at low temperature (5 degrees C) at times when there are marked differences in the activities of the Na(+)-K+ pump of cold-tolerant and cold-sensitive cells. 3. At 37 degrees C Na(+)-K(+)-ATPase of all three species exhibited two components of ATP dependence at 37 degrees C, one with high velocity, low affinity, the other with low velocity, high affinity. Affinities of both components rose with cooling. 4. A similar, two component pattern was observed in intact guinea-pig and human red cells at 37 degrees C, except that the segment corresponding to the high affinity component had an apparent Km (Michaelis-Menten constant) 3- to 4-fold higher than that of the broken membrane preparation. 5. Cooling intact guinea-pig and human red cells decreased the apparent affinity of the high velocity, low affinity component for ATP, so that at 20 degrees C the value of Km approached or exceeded the levels of physiological ATP concentration. Below 20 degrees C only one component with values corresponding to that of the low velocity, high affinity component could be observed. 6. In intact ground squirrel cells only the low affinity, high velocity component was apparent between 37 and 5 degrees C. Its affinity for ATP rose with cooling between 37 and 5 degrees C.

Membrane transport of potassium ions in erythrocytes of the American black bear, Ursus americanus.

1. Membrane transport of K ions was investigated in red blood cells of bears by methods of measurement of unidirectional isotopic fluxes. 2. Unlike red cells of dogs, red cells of bears exhibited a significant, though small, component of ouabain-sensitive K influx. 3. Ouabain-insensitive K influx, as in other carnivore cells, was activated by swelling and inhibited by shrinkage. Swelling-induced K influx was dependent upon presence of chloride ions but was not inhibited by furosemide or bumetanide. 4. Ouabain-sensitive K influx was largest with ATP and with high concentration of Na in the cell, but it persisted in the absence of cytoplasmic Na or ATP. It was also resistant to the drug, harmaline, at a concentration that in other cells fully inhibits ouabain-sensitive K influx. 5. It was concluded that under such adverse conditions ouabain-sensitive K influx represents another mode of the Na/K pump not fully described elsewhere. 6. Also, as in low K red cells of sheep and goat, apparent absence of Na/K pump activity in carnivore red cells may represent suppression rather than elimination of activity. 7. Ouabain-insensitive K influx showed a seasonal pattern with minima occurring in early winter, earlier than for the minimum observed in Na influx. 8. Ouabain-sensitive K influx tended to be lower in the hibernation season of the bear, but the seasonal pattern was not consistent.

[Intrauterine extraction of intrauterine devices using the BMK-2 extractor of our design]. / Intrauterina ekstrakcija intrauterinih ulozaka ekstraktorom sopstvene konstruckcije BMK-2.

The so-called "lost" IUD's cannot be removed by common vaginocervical traction. The author extracts them by the intrauterine BMK-extractor (Instrumntalia, Zagreb) of his own construction. There were 150 extractions with no complication, performed by the use of the BMK-2 extractor in period from August 1, 1985 to December 31, 1988.