Down-regulation of protein kinase C isoforms in irritant contact dermatitis

Protein kinase C (PKC) isoforms are important in cell signal transduction associated with regulation of cell proliferation and differentiation. In this study, alterations of PKC isoform levels in irritant patch test reactions were detected by Western immunoblots. 4 chemically and structurally different irritants, 4% and 8% hydrochloric acid (HCl); 1% and 2% sodium hydroxide (NaOH); 20% and 40% nonanoic acid (NON) in 1-propanol; and 5% and 10% sodium dodecyl sulfate (SDS) were applied on the backs of mice in Finn Chambers and fixed with surgical dressings. The patches were kept on the skin for 24 h and removed. 24 h after removal, mild erythema with thickening of skin without vesicles was observed in all irritated skin, except that 4% HCl and 1% NaOH treated skin showed unremarkable skin reaction. No visible skin reaction was detected in vehicle-treated skin. PKC isoform alpha, beta, gamma, and delta levels in irritated skin revealed a 10% to 65% decrease compared to vehicle treated skin. These results indicate that in HCl, NaOH, NON and SDS-induced irritation, activation of the PKC related cell signal transduction cascade may be involved, and that PKC mediated events may be a common phenomenon in irritant contact dermatitis.     

Influence of formulation on the in vitro transdermal penetration of flutrimazole

Flutrimazole (1-[(2-fluorophenyl)(4-fluorophenyl)phenylmethyl]-1 H-imidazole, CAS 119006-77-8, UR-4056) is a new wide spectrum local imidazolic antifungal agent that has already been formulated as a dermal cream (FDC). A comparative study was carried out of the release of flutrimazole from two emulsions in which the drug has been incorporated differently: one dissolved in the oily phase (E24) and the other dispersed in the aqueous formulation phase (E25). Based on the E25 formulation, two more dermal creams were prepared, E27 with benzyl alcohol and E28 with diazolidinyl urea as preservative agents. A comparative study of transdermal penetration including E27, E28, FDC (reference 1% flutrimazole dermal cream) and 1% flutrimazole hydroalcoholic solution was also performed. An amount of the sample dosage form containing 10 mg of flutrimazole was applied to a Franz type cell. The penetration membrane used was cellulose acetate in the release studies and human skin provided by a plastic surgery clinic in the transdermal penetration study. The amount released after 7 h was 36.3 +/- 4.9 micrograms when flutrimazole was dissolved (E24) and 35.9 +/- 5.3 micrograms when flutrimazole was dispersed (E25). Although the differences were not significant, the cream with dispersed flutrimazole was selected for further penetration studies due to its better stability observed in previous studies. The amounts of drug penetrated after 44 h were 31.3, 41.5, 38.3 and 186.5 micrograms for E27, E28, FDC dermal creams and topical hydroalcoholic solution, respectively. The solution showed a statistically significant difference (p < 0.05) from the other formulations, however, no differences were observed between the dermal cream formulations. No differences were neither obtained between the different dermal creams when the amount of drug retained in the skin was compared. This allows to assert that the excipients used do not have different influences on transdermal penetration. In all cases, the mean quantity penetrated in relation to the dose applied was at most 0.5%. These results allow to infer that flutrimazole shows scarce transdermal penetration. Further, the amount of flutrimazole retained per gram of skin is more than 100 times the MIC per gram obtained in previous in vitro studies. It may be assumed that the topical application of the new formulations assayed would allow to obtain a good therapeutic response.     

Percutaneous penetration of 2-phenoxyethanol through rat and human skin

2-Phenoxyethanol applied in methanol was absorbed (64 +/- 4.4% at 24 hr) through unoccluded rat skin in vitro in the static diffusion cell with ethanol/water as receptor fluid. By comparison (43 +/- 3.7% in 24 hr) was absorbed in the flow-through diffusion system with tissue culture medium as receptor fluid. 2-Phenoxyethanol applied in methanol was absorbed (59.3 +/- 7.0% at 6 hr) through unoccluded human skin in vitro in the flow-through diffusion cell with tissue culture medium. With both unoccluded cells, 2-phenoxyethanol was lost by evaporation but occlusion of the static cell reduced evaporation and increased total absorption to 98.8 +/- 7.0%. Skin, post mitochondrial fraction, metabolized phenoxyethanol to phenoxyacetic acid at 5% of the rate for liver. Metabolism was inhibited by 1 mM pyrazole, suggesting involvement of alcohol dehydrogenase. However, first-pass metabolism of phenoxyethanol to phenoxyacetic acid was not detected during percutaneous penetration through viable rat skin in the flow-through system. First-pass metabolism in the skin does not therefore have an influence on systemic availability of dermally absorbed phenoxyethanol. These measures of phenoxyethanol absorption through rat and human skin in vitro agree well with those obtained previously in vivo.     

Estradiol induction of retinoic acid receptors in human breast cancer cells

Retinoic acid inhibits proliferation and steroid receptor gene expression in human breast cancer cell lines. Retinoic acid receptors (RAR)alpha, -beta, and -gamma are expressed in these cells and the expression of RAR alpha is significantly greater in estrogen receptor (ER)-positive cells. This study was undertaken to determine whether the same relationship between RAR alpha and ER gene expression was present in human breast cancers and to explore the possibility that the higher level of RAR alpha in ER-positive cells was due to estrogen regulation of RAR alpha gene expression. RAR alpha and ER mRNA expression were determined by Northern blot analysis in 116 primary breast tumors; 94 (81%) tumors were ER-positive and of these 87 (93%) were also RAR alpha-positive. The coexpression of ER and RAR alpha was statistically significant (P = 0.0052 by chi 2 contingency analysis). There was also a positive correlation (by linear regression analysis) between the levels of expression of ER and RAR alpha mRNA (r2 = 0.251, P = 0.0001), which confirmed the relationship previously documented in breast cancer cell lines and suggested that RAR alpha expression may be modulated in breast cancer in vivo by estrogens acting via the ER. The ability of estradiol to regulate RAR alpha gene expression was examined in vitro using T-47D cells which had been rendered sensitive to estrogen by repeated passage in steroid-depleted medium. Estradiol increased RAR alpha gene expression, but not that of RAR beta or RAR gamma, in a concentration-dependent manner, with the effect being maximal at 10(-10) M and less marked at higher concentrations. The effect was rapid, being detectable 1 h after and maximal 6 h after treatment with 10(-10) M estradiol. Co-treatment of cells with estradiol and antiestrogens (tamoxifen or ICI 164384, 4 x 10(-7) M for 6 h) inhibited the estradiol induction of RAR alpha gene expression, demonstrating that the effect was ER mediated. The estradiol sensitivity of the effect was underscored by the demonstration that addition of untreated serum to cells growing under steroid-depleted conditions was sufficient to induce maximal RAR alpha gene expression. This effect was totally abolished by addition of ICI 164384. In summary, the demonstration that estradiol increased RAR alpha mRNA levels in breast cancer cells supports the hypothesis that the correlation between RAR alpha and ER gene expression in breast tumors and breast cancer cell lines is due to estradiol augmentation of RAR alpha gene expression.     

Pharmacodynamics of immediate and delayed effects of paclitaxel: role of slow apoptosis and intracellular drug retention

The kinetics of the time-dependent antitumor effects of paclitaxel are not fully understood; some literature reports indicate a higher activity by prolonging treatment durations, whereas other reports indicate no enhancement under in vitro conditions. The present study was designed to address this controversy and to determine the mechanism of the higher cytotoxicity associated with longer treatment durations. Six human epithelial cancer cell lines (bladder RT4, breast MCF7, pharynx FaDu, ovarian SKOV3, and prostate PC3 and DU145) were used. To determine whether the higher activity observed for the longer treatment durations is due to a delayed exhibition of drug effects and/or a reflection of cumulative effects that required a continuous drug exposure, cells were treated with paclitaxel for 3-96 h and then either: (a) immediately processed for drug effect measurement; or (b) washed, incubated in drug-free medium, and processed for drug effect measurement at 96 h. The overall drug effect (i.e., combination of cytostatic and apoptotic effects) was determined by the sulforhodamine B assay, which measures the cellular protein. In addition, to determine whether apoptosis occurs with a time delay, apoptosis was measured in cells that were collected immediately after drug treatment for various durations or in cells that were treated with drugs for 3 h but collected at later time points. Apoptosis was determined using agarose gel electrophoresis and by measuring the cytoplasmic DNA-histone complex using ELISA. The contribution of the intracellularly retained drug to the delayed drug effect was studied by characterizing the kinetics of cellular drug uptake and efflux and by examining the effect of removal of the intracellularly retained drug. All six cell lines showed similar results, as follows: (a) paclitaxel produced cytotoxicity that was exhibited immediately after treatment (immediate effect) and after treatment was terminated (delayed effect); (b) the immediate and delayed effects showed different pharmacodynamics. The immediate effect increased with treatment duration and drug concentration. For the delayed effect, all treatments produced the same maximum effect at 96 h, although treatments for < or = 12 h showed higher IC50s than longer treatments, whereas treatments for > or = 24 h showed indistinguishable IC50s; (c) treatment for as brief as 3 h was sufficient to induce apoptosis, which occurred with a lag time of about 24 h, although longer treatments produced a greater extent of apoptosis; (d) The intracellular and extracellular concentrations reached an equilibrium at approximately 5 h, which rules out slow and/or insufficient uptake as the cause of the lower effects at shorter treatment times (i.e., < 24 h); (e) upon removal of drug-containing medium, the amount of drug retained intracellularly was about 10% of the applied dose and was reduced to approximately 0.5% after three successive washes, separated by 3-h equilibration periods; and (f) the delayed effect of the 3-h treatment was largely due to the drug retained intracellularly, whereas the delayed effect of the 24 h treatment was independent of the drug retained intracellularly. In conclusion, in human epithelial cancer cells, paclitaxel-induced cytotoxicity occurred after termination of drug treatment, which was partly due to the slow manifestation of apoptosis and partly due to the significant amount of drug retained intracellularly. Based on these findings and recognizing that some previous studies measured the immediate effect whereas the other studies measured the delayed effect, we propose that the conflicting data in the literature regarding the effect of treatment duration on paclitaxel activity under in vitro conditions are in part due to the different pharmacodynamics of the immediate and delayed drug effects. Furthermore, differences in the delayed effects for treatments of < 24 h and the minimal differences for treatments of > or = 24 h indicate that th     

Cellular distribution and gene regulation of estrogen receptors alpha and beta in the rat pituitary gland

The pituitary gland is a heterogeneous tissue comprised of several hormone secreting and supporting cells, most of which are targeted by estrogens. Estrogen-induced changes in the pituitary are presumably mediated via the classical estrogen receptor, ER alpha. However, a novel receptor, ER beta, and pituitary-specific truncated estrogen receptor products (TERPs) were recently identified. The objectives of this study were to examine the distribution of these receptors in the rat pituitary and compare their regulation by estradiol in Sprague-Dawley and the estrogen-sensitive Fischer 344 rats. Pituitary cryosections were subjected to immunocytochemistry for specific cell types, followed by in situ hybridization for ER alpha or ER beta. ER alpha was expressed by approximately 45% of the lactotrophs and melanotrophs, 35% of the corticotrophs and folliculo-stellate cells, and 25% of the gonadotrophs. The expression of ER beta showed a similar pattern but was generally lower than ER alpha. In two cell types, melanotrophs and gonadotrophs, ER beta expression was significantly lower than ER alpha. In the second experiment, pituitary sections were immunostained for ER alpha, followed by in situ hybridization for ER beta. Only a minute population (6-10%) of either anterior or intermediate lobe cells coexpressed ER alpha and ER beta. In the next experiment, Fischer 344 and Sprague-Dawley rats were injected with oil or estradiol for 24 h. Total RNA from dissected anterior and posterior (neurointermediate) pituitaries was subjected to RT-PCR for ER alpha, ER beta, or TERPs. Interestingly, ER alpha and ER beta were unchanged by estradiol in either lobe of the pituitary. In contrast, estradiol increased pituitary TERP messenger RNA levels 4- to 7-fold. A 20-kDa TERP protein was detected by Western blots in the pituitary but not the uterus. There were no differences in the estradiol-induced expression of any of the receptors between the two strains of rats. We conclude that: 1) ER beta is expressed in all anterior and intermediate lobe cell types examined, albeit at a lower level than ER alpha; 2) no more than 10% of pituitary cells coexpress ER alpha and ER beta; and 3) estradiol markedly increases TERP messenger RNA levels but does not alter the expression of ER alpha or ER beta. We propose that estrogen receptor heterogeneity contributes to the diversity of pituitary cell responsiveness to estrogens.     

alpha-Melanocyte stimulating hormone inhibits tumour necrosis factor-alpha stimulated intercellular adhesion molecule-1 expression in normal cutaneous human melanocytes and in melanoma cell lines

alpha-Melanocyte stimulating hormone (alpha-MSH) was found significantly to reduce tumour necrosis factor-alpha (TNF-alpha) stimulated upregulation of intercellular adhesion molecule-1 (ICAM-1) in normal adult cutaneous melanocytes. The maximum inhibitory response to alpha-MSH was obtained at around 10(-10) mol/L alpha-MSH when cells were coincubated with alpha-MSH and TNF-alpha for 24 h. alpha-MSH had little or no effect on basal ICAM-1 expression in melanocytes and the effects of alpha-MSH could be mimicked with 3-isobutyl-1-methylxanthine (IBMX). Preliminary data in three human melanoma cell lines also showed alpha-MSH and forskolin to be effective in significantly reducing TNF-alpha stimulated ICAM-1 expression over 24 h. The extent of the inhibition varied from cell line to cell line and was greatest in those cells with the highest number of alpha-MSH receptors. These data suggest that alpha-MSH has the ability to oppose the action of the pro-inflammatory cytokine TNF-alpha on melanocytes and melanoma cells.     

Intermittent exposure of medulloblastoma cells to topotecan produces growth inhibition equivalent to continuous exposure

Camptothecin analogues such as topotecan increase the number of covalent topoisomerase I-DNA complexes, which, in turn, have been proposed to initiate apoptosis. If induction of apoptosis by the camptothecins is, in fact, dependent on the formation of topoisomerase I-DNA complexes, then it would be of clinical relevance to identify schedules of exposure to the camptothecins that maximize the formation of these complexes but minimize the total amount of the drug administered. The time and dose dependence of topoisomerase I-DNA complex formation was determined by incubating Daoy pediatric medulloblastoma cells in vitro with topotecan at concentrations equivalent to those achievable in the plasma clinically (10, 50, or 200 nM) and measuring the number of complexes present in cells incubated for 15 min to 48 h with the drug. Regardless of the concentration of topotecan used, covalent topoisomerase I-DNA complexes were maximal within 15 min following addition of the lactone form of topotecan to the tissue culture medium. After 2 h of exposure to topotecan, complexes had decreased from maximum to approximately half of that value. Few, if any, complexes were detectable with topotecan incubations of 24-48 h. Growth inhibition studies showed that the IC50s of topotecan for the Daoy cell line (2.2 x 10(-9) M) and also for a second pediatric medulloblastoma cell line, SJ-Med3 (3.6 x 10(-9) M), exposed to topotecan 8 h daily for 5 days or continuous exposure were equivalent. The decrease in topoisomerase I-DNA complexes between 15 min and 1 h was consistent with a pH-dependent re-equilibration of topotecan to the less active hydroxyacid form of the drug. The decrease in complexes after a 2-48-h incubation with the drug was attributable neither to biological inactivation of topotecan as shown by sequential growth inhibition studies nor to a decrease in amount of topoisomerase I in the drug-treated cells. Indirect immunofluorescence labeling of topoisomerase I in Daoy cells incubated for 48 h with 10 nM topotecan showed a redistribution of nucleolar topoisomerase I. We are currently evaluating the antitumor effect of intermittent repetitive exposures to topotecan in mice bearing Daoy cells as a xenograft. The clinical utility of each effective schedule of exposure will depend on whether the therapeutic index of repetitive intermittent exposure to the drug is more or less favorable than the therapeutic index of continuous exposure.     

Immunohistochemical localization of estradiol and progesterone receptors in human uterus throughout pregnancy: expression in endometrial blood vessels

Although progesterone and estrogens are essential to maintain human pregnancy after implantation, the localization of their specific receptors in different uterine cell types during pregnancy has not been investigated. We studied uteri (n = 40) obtained during the first 3 months of pregnancy (n = 21) and in late pregnancy (n = 9) as well as from women 5-14 weeks pregnant (n = 10) who had received the antiprogestagen RU 38486 (Roussel-UCLAF) to induce cervical dilation. Frozen tissues were processed for indirect immunocytochemical staining with specific monoclonal antibodies against estrogen receptors (ER; Abbott Laboratories) and progesterone receptors (PR; Li 417). Specific staining for steroid receptors was only detected in the nucleus. In the endometrium, PR staining remained fairly constant throughout pregnancy, whereas ER staining was initially weak and then undetectable. PR was widely expressed in stromal cells and in spiral arterial wall cells, whereas ER was expressed in scattered stromal cells and arterial cells. Both PR and ER were absent from glandular epithelium, contrasting with the secretory activity during the first trimester. Spiral arteries of the endometrium and myometrial smooth muscle cells showed intense PR and moderate ER staining in early pregnancy. The progesterone antagonist RU 38486 (mifepristone), given in early pregnancy at a dose of 200 mg, caused a marked increase in ER staining and a smaller increase in PR staining in stromal cells, whereas the glandular epithelium remained negative for both ER and PR (except for one and two specimens, respectively). We conclude the following. 1) Stromal cells retain PR despite the high progesterone levels during pregnancy, in keeping with the role of progesterone in stromal decidualization. The absence of PR from the secretory glandular epithelium suggests a paracrine link between decidualized stromal cells and epithelial cells. 2) Significant PR down-regulation by progesterone during pregnancy occurs only in epithelial cells of the endometrium. 3) In contrast, the absence or low level of ER staining in the various cell types of the endometrium during gestation concurs with the known effect (down-regulation) of steroid hormones on ER mRNA or protein levels. The increase in ER in human decidua after RU 38486 treatment indicates that the main cause of the low ER levels is progesterone secretion. 4) The intense PR staining in smooth muscle cells of spiral arteries during early pregnancy suggests that progesterone is essential for modulating blood flow during pregnancy.     

Determinants of trimetrexate lethality in human colon cancer cells

We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 microM TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 microM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > or = 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (< or = 20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 microM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24-48 h in both cell lines. With 1 and 10 microM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.     

Furazolidone disposition after intravascular and oral dosing in the channel catfish

1. The pharmacokinetics, tissue distribution and excretion of the nitrofuran drug furazolidone have been examined in the channel catfish. [14C]Furazolidone was administered by intravascular or oral routes in a single dosage of 1 mg/kg body weight. 2. A two-compartment pharmacokinetic model best described parent furazolidone concentrations in the plasma after intravascular dosing. Elimination of parent compound was extremely rapid, with a terminal half-life of 0.27h and total body clearance of 1901 ml/h/kg. 3. After oral dosing, furazolidone concentrations in the plasma were highest at 1 h and were below the limit of determination (< 20 ng/ml) at 5 h. The oral bioavailability of parent furazolidone administered in solution was 58%, compared with 28% in a feed mixture. 4. Concentrations of furazolidone and its metabolites were highest in the excretory tissues and lowest in the muscle after oral dosing. Parent furazolidone comprised 10% of the total 14C in the muscle at 8 h and was not detectable (< 1 ng/g) at 24 h; total 14C concentrations declined from 274 to 59 ng furazolidone equiv./g between 8 and 168 h. Non-extractable (bound) residues comprised 18% of total 14C in muscle at 8 h and 33% at 168h. 5. Renal excretion was the primary route of elimination of 14C residues and accounted for nearly 55% of the oral dose.     

Inhibition of adhesion molecules by budesonide on a human epithelial cell line (lung carcinoma)

Inhaled corticosteroids in the treatment of asthma have been shown to produce marked reductions in the number of inflammatory cells (mainly mast cells and eosinophils) and their products at bronchial level (such as cytokines). Recently, it has been demonstrated that epithelial cells express ICAM-1/CD54 in allergic patients both during natural allergen exposure and after allergen challenge. We have previously demonstrated that deflazacort (a systemic steroid) reduces the expression of ICAM-1 on conjunctival epithelial cells. The present study aimed to evaluate the effects exerted by budesonide on adhesion molecule expression by a human epithelial cell line (lung carcinoma: DM) and on soluble ICAM-1. Budesonide was added at concentrations corresponding to 10(-8), 10(-7), and 10(-6) mol/l in cultured epithelial cells, either in the absence of any stimulus or in the presence of interferon-gamma (IFN-gamma) at 500 U/ml. After 24 h of incubation, cytofluorometric analysis was performed for ICAM-1 and CD29/VLA beta 1. The 24-h supernatants of the same cultures were collected and then evaluated for soluble ICAM-1 (sICAM-1). The results showed that budesonide inhibits ICAM-1 and CD29 basal expression on the cells studied (P < 0.05): budesonide was effective in a dose-dependent manner. In addition, budesonide reduced surface ICAM-1 upregulation induced by IFN-gamma at 500 U/ml (P < 0.05). Finally, cell cultures with budesonide showed decreased levels of soluble ICAM-1 in basal condition, but not after IFN-gamma stimulation.     

Comparative effects of felodipine ER, amlodipine and nifedipine GITS on 24 h blood pressure control and trough to peak ratios in mild to moderate ambulatory hypertension: a forced titration study

OBJECTIVE: To evaluate the 24 h antihypertensive efficacy and duration of action of felodipine extended release (ER) in comparison with two other long acting dihydropyridine calcium antagonists, amlodipine and nifedipine gastrointestinal therapeutic system (GITS), in patients with mild to moderate essential hypertension substantiated by ambulatory blood pressure (BP) monitoring. DESIGN: Randomized, forced titration, parallel group study. Clinic BP was measured at every patient's visit, and 24 h ambulatory BP was monitored at baseline and at the end of each dose-titration period. SETTING: Single centre: hypertension research unit in Quebec City, Quebec. PATIENTS: There were 89 patients enrolled into the study. Eighty-four eligible patients were randomized, and 83 completed the study and were included in the final efficacy analysis. INTERVENTIONS: Following a two-to four-week washout period (baseline), patients were randomly allocated to receive felodipine ER 5 mg, amlodipine 5 mg or nifedipine GITS 30 mg for four weeks (low dose). All study patients had their daily dose doubled to felodipine ER 10 mg, amlodipine 10 mg or nifedipine GITS 60 mg for a further four weeks (high dose). MAIN RESULTS: Significant (P < 0.001) and similar changes from baseline in clinic BP were observed in all treatment groups for low and high doses. Ambulatory BP profiles showed comparable blood pressure reductions with felodipine ER and amlodipine, and a trend towards a lesser reduction with nifedipine GITS during 24 h, daytime and night-time periods. BP loads were similarly reduced with the three treatments. Trough to peak ratios (T:Ps) were calculated from 24 h ambulatory BP curves according to two different approaches: for diastolic and systolic BP, respectively, the global approach produced T:Ps of 0.49 and 0.50 with felodipine ER 5 mg; 0.50 and 0.34 with felodipine ER 10 mg; 0.70 and 0.60 with amlodipine 5 mg; 0.88 and 0.82 with amlodipine 10 mg; 0.65 and 0.55 with nifedipine GITS 30 mg; 0.68 and 0.53 with nifedipine GITS 60 mg. T:Ps in the individual approach were 0.07 and 0.10 with felodipine ER 5 mg; 0.23 and 0.31 with felodipine ER 10 mg; 0.22 and 0.31 with amlodipine 5 mg; 0.45 and 0.58 with amlodipine 10 mg; 0.27 and 0.31 with nifedipine GITS 30 mg; and 0.24 and 0.40 with nifedipine GITS 60 mg. CONCLUSION: There was no evidence in this study of a difference among felodipine ER, amlodipine and nifedipine GITS in lowering ambulatory or clinic BP. Treatment based on ambulatory BP may be preferable to treatment guided by T:Ps because ambulatory BP is firmly established as a predictor of cardiovascular risk. Furthermore, there is no consensus on how to calculate T:Ps, and different methods of calculation may give divergent results.     

Age-dependent and lobe-specific spontaneous hyperplasia in the brown Norway rat prostate

PURPOSE: Response of quiescent (Q) and total tumor cells in solid tumors to neutron irradiation with three different cadmium (Cd) ratios was examined. The role of Q cells in tumor control was also discussed. METHODS AND MATERIALS: C3H/He mice bearing SCC VII tumors received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells. Thirty minutes after intraperitoneal injection of sodium borocaptate-10B (BSH), or 3 h after oral administration of dl-p-boronophenylalanine-10B (BPA), the tumors were irradiated with neutrons, or those without 10B-compounds were irradiated with gamma rays. This neutron irradiation was performed using neutrons with three different cadmium (Cd) ratios. The tumors were then excised, minced, and trypsinized. The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis-blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total (P + Q) tumor cells was determined from tumors that were not pretreated with BrdU. The sensitivity to neutrons was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency). RESULTS: Without 10B-compounds, the MN frequency in Q cells was lower than that in the total cell population. The sensitivity difference between total and Q cells was reduced by neutron irradiation. Relative biological effectiveness (RBE) of neutrons compared with gamma rays was larger in Q cells than in total cells, and the RBE values for low-Cd-ratio neutrons tended to be larger than those for high-Cd-ratio neutrons. With 10B-compounds, MN frequency for each cell population was increased, especially for total cells. This increase in MN frequency was marked when high-Cd-ratio neutrons were used. BPA increased the MN frequency for total tumor cells more than BSH. Nevertheless, the sensitivity of Q cells treated with BPA was lower than that in BSH-treated Q cells. This tendency was clearly observed in high-Cd-ratio neutrons. CONCLUSION: From the viewpoint of enhancing the Q-cell sensitivity, tumors should be irradiated with high-Cd-ratio neutrons after BSH administration. However, normal tissue reaction remains to be examined because of its low tumor-to-normal tissue and tumor-to-blood biodistribution ratios.     

Analysis of autoantibody epitopes on steroid 21-hydroxylase using a panel of monoclonal antibodies

A panel of five mouse monoclonal antibodies (MAbs) to human recombinant steroid 21-hydroxylase (21-OH) were produced, characterized, and used to study the interaction of 21-OH autoantibodies (AAbs) with different epitopes on human 21-OH. AAbs in patients with isolated autoimmune Addison's disease, autoimmune polyglandular syndromes types I and II, and 21-OH antibody-positive patients without overt Addison's disease (25 patients in total) were studied. Four MAbs were IgG1 subclass, one was IgG2a, and all had kappa light chains. The affinities of four of the antibodies were in the range 2.0 x 10(8) M(-1) to 7.0 x 10(8) M(-1), and the affinity of the other was 2.3 x 10(7) M(-1) 21-OH MAbs did not cross-react with 17alpha-hydroxylase (17alpha-OH)) or P450 side chain cleavage enzyme. Studies using a series of 21-OH fragments allowed the identification of short stretches of amino acids (AA) that were involved in forming the MAb binding sites. AA 391-405, defined as epitope region (ER) 1, were found to be important for binding of M21-OH1 and M21-OH2, AA 406-411 (ER2) were important for M21-OH3 and M21-OH4 binding, and AA 335-339 (ER3) for M21-OH5 binding. In addition, MAb Fab or F(ab')2 fragments were used to study 21-OH AAb epitopes in competition experiments. These investigations demonstrated that 21-OH AAbs recognize similar epitopes to the MAbs, with ER2 and ER3 being part of two distinct major epitopes, and ER 1 being part of a minor epitope. Mixtures of M21-OH antibody Fab or F(ab')2 fragments caused almost complete inhibition (80%-95%) of AAb binding in 24 out of 25 sera, and in the case of the remaining serum, the effect was marked but incomplete (67% inhibition). There were no major differences between the binding characteristics of AAbs from patients with different forms of autoimmune adrenal disease. All five 21-OH MAbs reacted with human adrenal tissue in an immunofluorescence test, but only M21-OH1 and M21-OH2 reacted with bovine adrenal tissue in these experiments. None of the MAbs reacted with human ovarian tissue in an immunofluorescence test. Overall, these studies indicate that 21-OH AAbs bind to at least three different epitopes in the C-terminal part of 21-OH, and two of these epitopes appear to be human 21-OH specific.     

Applicability of combination with tirapazamine in boron neutron capture therapy

SCC VII tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating cells. After injection of tirapazamine (TPZ), a bioreductive agent, combined with sodium borocaptate-10B (BSH) or dl-p-boronophenylalanine-10B (BPA) administration, the tumors were irradiated with thermal neutrons, and then isolated and incubated with cytochalasin-B (a cytokinesis blocker). The micronucleus (MN) frequency in cells without BrdU labeling (quiescent (Q) cells) was determined by means of immunofluorescence staining for BrdU, and that for total cells was obtained from tumors not pretreated with BrdU. Even when no 10B-compound was administered, TPZ increased the MN frequency of tumor cells including Q cells, resulting in reduction of the difference in MN frequency between total and Q cells, mainly by increasing the MN frequency of Q cells. TPZ increased the MN frequency of Q cells when combined with BPA administration, but TPZ showed no apparent effect on each cell population when combined with BSH. Namely, TPZ reduced the difference in MN frequency between total and Q cells caused by 10B-compound administration, especially when BPA was administered. From the viewpoint of the overall cell killing effect in boron neutron capture therapy (BNCT), combination with TPZ appeared to be useful in BPA-BNCT, but not in BSH-BNCT.     

Synergistic interaction between cisplatin and gemcitabine in vitro

2',2'-Difluorodeoxycytidine (dFdC; gemcitabine) is a new antineoplastic agent that is active against ovarian carcinoma, non-small-cell lung carcinoma, and head and neck squamous cell carcinoma. cis-diamminedichloroplatinum (CDDP; cisplatin) is used commonly for the treatment of these tumors. Because the two drugs have mechanisms of action that might be complementary, we investigated a possible synergism between dFdC and CDDP on growth inhibition. The combination was tested in the human ovarian carcinoma cell line A2780, its CDDP-resistant variant ADDP and its dFdC-resistant variant AG6000, the human head and neck squamous cell carcinoma cell line UMSCC-22B, and the murine colon carcinoma cell line C26-10. The cells were exposed to dFdC and CDDP as single agents and to combinations in a molar ratio of 1:500 for 1, 4, 24, and 72 h with a total culture time of 72 h. Synergy was evaluated using the multiple drug effect analysis. In A2780 and ADDP cells, simultaneous exposure to the drugs for 24 and 72 h resulted in synergism, but shorter exposure times were antagonistic. No synergism was found in the UMSCC-22B and C26-10 cell lines at prolonged simultaneous exposure. However, a preincubation with CDDP for 4 h followed by a dFdC incubation for 1, 4, 24, and 72 h was synergistic in all cell lines except C26-10 cells. A 4-h preincubation with dFdC followed by an incubation with the combination for 20 and 68 h was synergistic in all cell lines. Initial studies of the mechanism of interaction concentrated on the effect of CDDP on dFdCTP accumulation and DNA strand break formation. In all cell lines, CDDP failed to increase dFdCTP accumulation at 4- or 24-h exposure to dFdC; in two cell lines, CDDP even tended to decrease dFdCTP accumulation. Neither dFdC nor CDDP caused more than 25% double strand break formation, whereas in the combination, CDDP even tended to decrease this type of DNA damage. The synergistic interaction between the two drugs is possibly the result of dFdC incorporation into DNA and/or CDDP-DNA adduct formation, which may be affected by each other.     

Pharmacokinetics of a new reversible proton pump inhibitor, YH1885, after intravenous and oral administrations to rats and dogs: hepatic first-pass effect in rats

The pharmacokinetics of YH1885 were evaluated after intravenous (iv) and oral administrations of the drug to rats and dogs. The reason for the low extent of bioavailability (F) of YH1885 after oral administration of the drug to rats and the absorption of the drug from various rat gastrointestinal (GI) segments were also investigated. After iv administration of YH1885, 5-20 mg kg(-1), to rats, the pharmacokinetic parameters of YH1885 seem to be independent of the drug at the dose ranges studied. After oral administration of YH1885, 50-200 mg kg(-1), to rats, the area under the plasma concentration-time curve from time zero to 12 or 24 h (AUC(0-12 h) or AUC(0-24 h)) was proportional to the oral dose of the drug, 50-100 mg kg(-1), however, the AUC(0-24 h) value at 200 mg kg(-1) increased with less proportion to the dose increase (324, 689, and 815 microg x min mL(-1) for 50, 100, and 200 mg kg(-1), respectively) due to the poor water solubility of the drug. This was proved by the considerable increase in the percentages of the oral dose remaining in the entire GI tract as unchanged YH1885 at 24 h (11.8, 15.3, and 42.8% for 50, 100, and 200 mg kg(-1), respectively). The F value after oral administration of YH1885 to rats was relatively low; the value was approximately 40% at the oral dose of 50 and 100 mg kg(-1). The reason for the low F in rats was investigated. The liver showed the highest metabolic activity for YH1885 based on an in vitro rat tissue homogenate study; hence, the liver first-pass effect was estimated. The value of AUC after intraportal administration of the drug, 5 mg kg(-1), was approximately 70% (116 versus 163 microg x min mL(-1)) of that after iv administration of the drug, 5 mg kg(-1), to rats; the liver first-pass effect of YH1885 in rats was estimated to be approximately 30%. The total body clearance of YH1885 after iv administration of the drug, 5-20 mg kg(-1), to rats were considerably lower than the cardiac output of rats, indicating that the lung and/or heart first-pass effect of YH1885 could be negligible in rats. After oral administration of YH1885, 50 and 100 mg kg(-1), to rats, the F value was approximately 40%, and approximately 15% of the oral dose was recovered from the entire GI tract as unchanged YH1885 at 24 h, and 30% of the oral dose disappeared with the liver first-pass effect. Therefore, the remainder, approximately 15% of the oral dose, could have disappeared with the small intestine first-pass effect and/or degradation of the drug in the GI tract. YH1885 was absorbed from ileum, duodenum, and jejunum of rat, however, YH1885 was under the detection limit in plasma when the drug was instilled into the rat stomach and large intestine. After iv administration of YH1885, 5-20 mg kg(-1), to dogs, the pharmacokinetic parameters of YH1885 also seemed to be independent of the drug at the dose ranges studied. However, after oral administration of YH1885, 0.5 and 2 g per whole body weight, to dogs, the AUC(0-10 h) values were not significantly different (96.8 versus 98.2 microg x min mL(-1)) and this could be due to the poor water-solubility of the drug. YH1885 was not detected in the urine after both iv and oral administration of the drug to both rats and dogs.     

Permeation characteristics of 8-methoxypsoralen through human skin; relevance to clinical treatment

The permeation characteristics through human skin of 8-methoxypsoralen (8-MOP) and its physical attributes were investigated. The log octanol/water partition coefficient and saturated aqueous solubility of 8-MOP at 32 degrees C were 1-98 and 55.8 micrograms mL-1 respectively, 8-MOP showed Fickian diffusion, with its flux being linearly related to the concentration of drug in the donor solution. The permeability coefficient of 8-MOP through human skin from different concentrations of aqueous solutions and a 2.6 micrograms mL-1 bath lotion (as used in clinics) were statistically identical with mean values of 1.76 +/- 0.12 x 10(-2) and 1.70 +/- 0.32 x 10(-2) cm h-1 respectively (P > or = 0.05). An ethanol/water (1:1 w/v) receptor solution did not improve the clearance of 8-MOP from the dermis when compared with an aqueous vehicle. Complete removal of the stratum corneum by tape stripping from full-thickness membranes produced a threefold increase in the flux of 8-MOP thus suggesting that the main barrier to 8-MOP permeation resides in the stratum corneum although the aqueous epidermal and dermal tissue provide a significant resistance to transdermal drug permeation. The equilibrium uptake of 8-MOP into psoriatic plaques and the 8-MOP aqueous plaque partition coefficient were found to be more than twofold greater than for normal stratum corneum. The absorption of 8-MOP from the total applied topical dose (396 mg) was assessed as approximately 0.25% and only 2.5% of an oral dose, a significant reduction in the possible toxic hazard. The peak concentration of 8-MOP permeating through the skin was observed at about 35 min after limited exposure for 15 min. Our results suggest that following a 15 min bath in the drug solution, there may be a need for an interval of about 20 min before patients are irradiated to ensure the optimization of photosensitizer with UVA irradiation (PUVA) therapy. Alternatively, UV irradiation could be applied at a lower flux over a longer time.     

Effect of liposomal methylprednisolone on heart allograft survival and immune function in rats

Liposomal methylprednisolone (L-MPL) applied in monotherapy prolonged cardiac allograft survival in rats in comparison with the same dosage regimen of drug in solution (Solu-Medrol). The most efficacious treatment consisted of a 2-mg/kg i.v. dose of L-MPL twice a week (group III), producing survival up to 30 days, followed by a 4-mg/kg/week dose of L-MPL (group IV) and a single 2-mg/kg dose of L-MPL (group II). Survival in animals receiving Solu-Medrol as a 2-mg/kg dose twice a week (group V) did not differ from untreated animals. Only daily 4-mg/kg doses of methylprednisolone (MPL) in solution (group VI) were as effective as group III. The concentrations of MPL in liver and spleen were detectable for 26 days after the last dose of L-MPL, showing tissue selective sequestration of drug. Treatment at these low doses did not suppress endogenous corticosterone determined 24 hr or later in plasma. The administration of steroid caused significant immunosuppression in most animals as measured by inhibition of splenocyte blastogenesis induced with phytohemagglutinin. Cellular immunity data did not differ significantly between groups, but alterations occurred at day 14 to 15 after surgery: CD3, CD4 and ratio CD4:CD8 subsets of cells showed minimum values; CD8, CD4CD8, CD25 and white blood cell counts were at maximum at this time. Slight but significant differences between Immunoglobulin M suppression in group II compared to group I or V were found, whereas Immunoglobulin G values were unchanged. The transplantation and treatment with steroid decreased the total body weight of animals but increased weights of internal organs, particularly spleen, similarly for all groups.(ABSTRACT TRUNCATED AT 250 WORDS)