Ustekinumab for the treatment of moderate-to-severe plaque psoriasis in paediatric patients (≥ 6 to < 12 years of age): efficacy, safety, pharmacokinetic and biomarker results from the open-label CADMUS Jr study.

BACKGROUND: Limited options are available for treatment of paediatric psoriasis. OBJECTIVES: To evaluate the efficacy and safety of ustekinumab in paediatric patients with psoriasis (≥ 6 to < 12 years of age). METHODS: CADMUS Jr, a phase III, open-label, single-arm, multicentre study, evaluated ustekinumab in paediatric patients with moderate-to-severe plaque psoriasis. Patients received weight-based dosing of ustekinumab (< 60 kg: 0·75 mg kg-1 ; ≥ 60 to ≤ 100 kg: 45 mg; > 100 kg: 90 mg) administered by subcutaneous injection at weeks 0 and 4, then every 12 weeks through week 40. Study endpoints (all at week 12) included the proportions of patients achieving a Physician's Global Assessment score of cleared/minimal (PGA 0/1) and ≥ 75%/90% improvement in Psoriasis Area and Severity Index (PASI 75/90), and change in Children's Dermatology Life Quality Index (CDLQI). Serum ustekinumab concentrations, antidrug antibodies and cytokine levels were measured through week 52. Safety was evaluated through week 56. RESULTS: In total, 44 patients (median age 9·5 years) received at least one dose of ustekinumab. Three patients discontinued the study agent through week 40. At week 12, 77% of patients achieved PGA 0/1, 84% achieved PASI 75 and 64% achieved PASI 90 response. The mean change in CDLQI was -6·3. Trough serum ustekinumab concentrations reached steady state at weeks 28-52. The incidence of antidrug antibodies was 10% (n = 4). Mean serum concentrations of interleukin-17A/F and interleukin-22 were significantly reduced at weeks 12 and 52. Overall, 34 patients (77%) had at least one adverse event and three (7%) had a serious adverse event. CONCLUSIONS: Ustekinumab effectively treated moderate-to-severe psoriasis in paediatric patients, and no new safety concerns were identified. What is already known about this topic? Ustekinumab is approved for use in adolescents (≥ 12 to < 18 years of age) and adults (≥ 18 years) with moderate-to-severe psoriasis. What does this study add? Ustekinumab effectively treats moderate-to-severe psoriasis in paediatric patients (≥ 6 to < 12 years of age), with no new safety concerns. Linked Comment: Reich. Br J Dermatol 2020; 183:606-607.

Extension of ustekinumab maintenance dosing interval in moderate-to-severe psoriasis: results of a phase IIIb, randomized, double-blinded, active-controlled, multicentre study (PSTELLAR).

BACKGROUND: Phase III studies showed that some patients maintained response for ≥ 6 months following ustekinumab discontinuation. OBJECTIVES: To assess clinical responses with extended ustekinumab maintenance dosing intervals. METHODS: Adults with moderate-to-severe plaque psoriasis received ustekinumab at weeks 0, 4 and 16 during open-label treatment. Patients achieving a week-28 Physician's Global Assessment (PGA) score of cleared/minimal (PGA = 0/1) were randomized 1 : 4 to group 1 [approved every 12 weeks (q12 wk) maintenance] or group 2 (q12-24 wk; response-based dosing determined by time to loss of PGA = 0/1). Key end points included the number of visits with PGA = 0/1 (primary end point) and ≥ 75% improvement in Psoriasis Area and Severity Index (PASI 75) between weeks 88 and 112, and PGA/PASI responses between weeks 28 and 112. RESULTS: Overall, 378 patients achieved PGA = 0/1 at week 28 and were randomized to group 1 (n = 76) or group 2 (n = 302). Patients in group 1 had numerically greater mean numbers of visits with PGA = 0/1 than group 2 and also with PASI 75 from week 88 to 112. A higher proportion of patients in group 1 (55%) than group 2 (39%) had PGA = 0/1 at all seven visits from week 88 to 112. Maintenance of response was observed with dose-interval extension beyond q12 wk in a subset of patients. Extending the dosing interval did not affect antibody development or safety. CONCLUSIONS: Efficacy was better maintained among week-28 PGA responders randomized to continue q12 wk ustekinumab vs. extending maintenance dosing based on clinical response, although some patients maintained high levels of efficacy with up to q24 wk dosing.

Pre-stroke physical activity is associated with fewer post-stroke complications, lower mortality and a better long-term outcome.

BACKGROUND AND PURPOSE: Physical activity is associated with a reduced incidence of first-time stroke. However, few studies have examined the effect of pre-stroke physical activity on post-stroke complications and clinical outcomes. METHODS: A total of 39 835 cases of stroke registered in the nationwide stroke registry system of Taiwan between 2006 and 2009 were analyzed according to five levels of severity as determined by National Institutes of Health Stroke Scale score upon hospital admission. Pre-stroke physical activity was defined in the Taiwan Stroke Registry as dedicated leisure-time physical activity for at least 30 min/day for 3 days/week for more than 6 months. A Cox model was used to compare complications and outcomes between active and inactive groups. RESULTS: The active and inactive groups were similar in age distribution and stroke type distribution, but the active group had better National Institutes of Health Stroke Scale scores upon admission. The active group also had significantly fewer post-stroke complications. Active patients had lower hospital mortality and better functional outcomes upon discharge as per the modified Rankin Scale. Improved functional status in the active group was significant at 1, 3 and 6 months post-stroke. CONCLUSION: Dedicated leisure-time physical activity for at least 30 min/day, at least three times per week for more than 6 months was associated with decreased stroke severity, fewer post-stroke complications, lower mortality and better outcomes.

Long-term efficacy and safety of ustekinumab, with and without dosing adjustment, in patients with moderate-to-severe psoriasis: results from the PHOENIX 2 study through 5 years of follow-up.

BACKGROUND: Evaluation of the dosing flexibility and long-term efficacy of biological agents is limited. OBJECTIVES: To evaluate the long-term efficacy and safety of ustekinumab with and without dosing adjustment in the 5-year PHOENIX 2 study. METHODS: Patients were randomized to placebo or ustekinumab (45 or 90 mg) at weeks 0, 4, then every 12 weeks; patients receiving placebo crossed-over at week 12. Dosing adjustments were permitted at/beyond week 28 for early adjusters (weeks 28 or 40 per response); late adjusters (during long-term extension per investigator judgement); and nonadjusters (maintained randomized treatment throughout the study). Efficacy and safety were evaluated through weeks 244 and 264, respectively. RESULTS: In the overall population, 70% (849 of 1212) of ustekinumab-treated patients completed treatment through week 244, with high proportions of patients responding to the 45-mg and 90-mg doses, respectively: 75% improvement in Psoriasis Area and Severity Index (PASI 75) (76·5% and 78·6%) and PASI 90 (50·0% and 55·5%). Approximately 20% of patients were early adjusters, 30% were late adjusters and 50% were nonadjusters. Approximately half of the late adjusters initiated adjustments after already achieving PASI 75. Improved response was generally observed following dosing adjustments. Through week 264, safety event rates did not increase and event rates were generally comparable between dose groups and between patients with and without dosing adjustment. CONCLUSIONS: Treatment with ustekinumab for up to 5 years was safe and effective. Improved response was generally demonstrated following dosing adjustments; further investigations are required to quantify actual incremental benefits. The results also suggest that some patients may desire treatment goals beyond PASI 75.

Long-term safety of ustekinumab in patients with moderate-to-severe psoriasis: final results from 5 years of follow-up.

BACKGROUND: Long-term safety evaluations of biologics are needed to inform patient management decisions. OBJECTIVES: To evaluate the safety of ustekinumab in patients with moderate-to-severe psoriasis treated for up to 5 years. METHODS: Safety data were pooled from four studies of ustekinumab for psoriasis. Rates of adverse events (AEs), serious AEs (SAEs) and AEs of interest [infections, nonmelanoma skin cancers (NMSCs), other malignancies and major adverse cardiovascular events (MACE)] per 100 patient-years (PY) of follow-up were analysed by ustekinumab dose (45 or 90 mg) and by year of follow-up (years 1-5) to evaluate the dose response and impact of cumulative exposure. Observed rates of overall mortality and other malignancies were compared with those expected in the general U.S. population. RESULTS: Analyses included 3117 patients (8998 PY) who received one or more doses of ustekinumab, with 1482 patients treated for ≥4 years (including 838 patients ≥5 years). At year 5, event rates (45 mg, 90 mg, respectively) for overall AEs (242·6, 225·3), SAEs (7·0, 7·2), serious infections (0·98, 1·19), NMSCs (0·64, 0·44), other malignancies (0·59, 0·61) and MACE (0·56, 0·36) were comparable between dose groups. Year-to-year variability was observed, but no increasing trend was evident. Rates of overall mortality and other malignancies were comparable with those expected in the general U.S. population. CONCLUSIONS: No dose-related or cumulative toxicity was observed with increasing duration of ustekinumab exposure for up to 5 years. Rates of AEs reported in ustekinumab psoriasis trials are generally comparable with those reported for other biologics approved for the treatment of moderate-to-severe psoriasis.

Cardiovascular safety of ustekinumab in patients with moderate to severe psoriasis: results of integrated analyses of data from phase II and III clinical studies.

BACKGROUND: Patients with psoriasis are believed to be at an increased risk of cardiovascular (CV) morbidity, and the effect of biological agents on CV safety is not fully understood. OBJECTIVES: To evaluate the effect of ustekinumab on CV events using detailed analyses of pooled data from the phase II/III clinical studies of its use in moderate to severe psoriasis. METHODS: The incidence of major adverse CV events [MACE: myocardial infarction (MI), stroke or CV death] is reported. Meta-analyses using risk difference and odds ratio estimates are presented based on data collected during the placebo-controlled period of ustekinumab trials. The cumulative numbers of events and rates of MIs and strokes over time were compared with those expected in the psoriasis and/or general populations. RESULTS: During the placebo-controlled period (12/20 weeks), five MACE were reported in 1582 ustekinumab-treated patients [0·3%; 95% confidence interval (CI) 0·1-0·7%] compared with no events in 732 placebo-treated patients (0·0%; 95% CI 0·0-0·5%). MACE rates were stable over time during both the controlled and uncontrolled study periods, with 19 of 3117 ustekinumab-treated patients (0·6%) experiencing 21 events for a combined event rate per 100 patient-years of follow-up of 0·44 (95% CI 0·27-0·67) through up to 3 years. Standardized incidence ratios for comparison of ustekinumab clinical data with external data sources ranged from 0·34 to 0·52, suggesting no increased risk of MI or stroke in ustekinumab-treated patients compared with the general U.S. and psoriasis populations. CONCLUSIONS: The totality of available clinical data suggests neither a detrimental nor a beneficial effect of ustekinumab on serious CV events. Additional data are needed to define the net effect of ustekinumab on CV events.

Inhibition of HIV replication in acute and chronic infections in vitro by a Tat antagonist.

The human immunodeficiency virus-1 (HIV-1) trans-activator Tat is an attractive target for the development of antiviral drugs because inhibition of Tat would arrest the virus at an early stage. The drug Ro 5-3335 [7-chloro-5-(2-pyrryl)-3H-1,4-benzodiazepine-2(H)-one], inhibited gene expression by HIV-1 at the level of transcriptional trans-activation by Tat. The compound did not inhibit the basal activity of the promoter. Both Tat and its target sequence TAR were required for the observed inhibitory activity. Ro 5-3335 reduced the amount of cell-associated viral RNA and antigen in acutely, as well as in chronically infected cells in vitro (median inhibition concentration 0.1 to 1 micromolar). Effective inhibition of viral replication was also observed 24 hours after cells were transfected with infectious recombinant HIV-1 DNA. The compound was active against both HIV-1 and HIV-2 and against 3'-azido-3'-deoxythymidine (AZT)-resistant clinical isolates.

Apoptotic effect of Helicobacter pylori on oesophageal squamous-cell carcinoma cells in vitro.

BACKGROUND: The relationship between Helicobacter pylori (Hp) infection and oesophageal squamous-cell carcinoma (ESCC) risk is still inconclusive. Our previous study found an inverse association between the two, but its mechanism is still unknown. Thus, we conducted in vitro studies to clarify the issue. MATERIALS AND METHODS: One ESCC (CE 81T/VGH) cell line was co-cultured with Hp, using one gastric adenocarcinoma (AGS) cell line as the control. Hp-induced cell apoptosis was determined by flow cytometry, terminal transferase-mediated dUTP nick end labelling (TUNEL) assay and staining; caspase-3 protein expressions in cell lysates were detected by Western immunoblot. RESULTS: Increased apoptosis was found in CE 81T/VGH, but not in AGS cells, by flow cytometry and TUNEL assay after being co-cultured with Hp at the multiplicity of infection of 1/100 (but not at 1/400) for 36 h. The amount of activated caspase-3 (17/19 kDa) also increased in CE 81T/VGH, but not in AGS cells, after co-culturing with Hp at MOI of 1/100 for 36 h. The results were confirmed by triplicate experiments in which the different apoptotic assays remained consistent. CONCLUSIONS: Our study provides indirect evidence of the inverse association between Hp infection and ESCC risk, which is possibly due to Hp-induced apoptosis in ESCC cells. A further in vivo study is necessary to confirm our findings.

Effects of age and gender on intravenous midazolam premedication: a randomized double-blind study.

BACKGROUND: Given the potentially important effects that age and gender may have on midazolam premedication, this study aimed at determining if these factors alter anxiety, sedation, and cardiorespiratory outcomes when administering two different doses of i.v. midazolam. METHODS: After randomization, patients were premedicated 1 h before surgery with either i.v. midazolam 0.02 or 0.06 mg kg(-1) depending on their age and gender group. Levels of anxiety and sedation, heart rate, respiratory rate (RR), mean blood pressure (MBP), and oxygen saturation (Sp(O2)) were measured before and 15 min after midazolam administration. RESULTS: A higher level of preoperative anxiety was more often observed in women than in men, and in young than in older patients. The female or younger patients showed significant anxiolytic benefits from midazolam. A deeper sedation level was found in men compared with women. Forty-two of 45 patients (93.3%) with excessive sedation received midazolam 0.06 mg kg(-1). The elderly patients receiving midazolam 0.06 mg kg(-1) showed significant reductions in MBP, RR, and Sp(O2). Of the patients with an Sp(O2)<90%, 72.7% had received midazolam 0.06 mg kg(-1). CONCLUSIONS: Age and gender differences in neuropsychological and physiological responses after midazolam premedication were evident. Midazolam is effective for producing sedation and anxiolysis at a dose of 0.02 mg kg(-1), with minimal effects on cardiorespiration and oxygen saturation to patients. Dosage adjustments based on these covariates are, therefore, necessary.

Human antibody and antigen response to IncA antibody of Chlamydia trachomatis.

The high prevalence of C. trachomatis worldwide has underscored the importance of identifying specific immunogenic antigens in facilitating diagnosis as well as vaccine development. The aim of this study is to evaluate IncA antibody and antigen production in natural human infections. Our temporal expression study showed that IncA transcription and protein expression could be detected as early as 4 hours after the start of infection. Antibody responses could be detected in urine and genital swab samples from C. trachomatis-positive patients. It is especially interesting to note that the IncA antigen could be detected in urine. In conclusion, we have identified IncA as an important antigen in human. The potential applicability of the IncA antibody or antigen in the diagnosis as well as to vaccine development for C. trachomatis is also discussed.

15-Deoxy-Delta(12,14)-prostaglandin J(2) suppresses IL-6-induced STAT3 phosphorylation via electrophilic reactivity in endothelial cells.

In this study, the effects of 15d-PGJ(2) were investigated in IL-6-activated endothelial cells (ECs). 15d-PGJ(2) was found to abrogate phosphorylation on tyr705 of STAT3 in IL-6-treated ECs, in a dose- and time-dependent manner, but did not inhibit serine phosphorylation of STAT3 and the upperstream JAK2 phosphorylation. Other PPAR activators, such as WY1643 or ciglitazone, had no effect upon IL-6-induced STAT3 phosphorylation. Additionally, neither orthovanadate nor l-NAME treatment reverses the inhibition of STAT3 phosphorylation by 15d-PGJ(2). Otherwise, the effect of 15d-PGJ(2) requires the alpha,beta-unsaturated carbonyl group in the cyclopentane ring. A 15d-PGJ(2) analog, 9,10-Dihydro-15d-PGJ(2), which lack alpha,beta-unsaturated carbonyl group showed no increase in ROS production and no effect in inhibition of IL-6-induced STAT3 phosphorylation. The electrophilic compound, acrolein, mimics the inhibition effect of 15d-PGJ(2). Among the antioxidants, only NAC and glutathione reversed the effects of 15d-PGJ(2). NAC, glutathione and DTT all reversed the inhibition of STAT3 phosphorylation when preincubated with 15d-PGJ(2). The inhibition of ICAM-1 gene expression by 15d-PGJ(2) was abrogated by NAC and glutathione in IL-6-treated ECs. Taken together, these results suggest that 15d-PGJ(2) inhibits IL-6-stimulated phosphorylation on tyr705 of STAT3 dependent on its own electrophilic reactivity in ECs.

Upregulation of heme oxygenase-1 by Epigallocatechin-3-gallate via the phosphatidylinositol 3-kinase/Akt and ERK pathways.

Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated by various phytochemicals and we examined the ability of Epigallocatechin-3-gallate (EGCG), the major constituent of green tea, to upregulate HO-1 expression in endothelial cells (ECs). We demonstrate that EGCG induces HO-1 expression in a concentration- and time-dependent manner. Furthermore, EGCG-mediated HO-1 induction was abrogated in the presence of actinomycin D and cycloheximide, indicating that this upregulation of HO-1 occurred at the transcriptional level. EGCG also upregulates Nrf2 levels in nuclear extracts and increases ARE-luciferase activity. Furthermore, EGCG is the most potent inducer of HO-1 expression of the different green tea constituents that we analyzed, but had no detectable cytotoxic effects over the 25-100 microM dosage range. The inhibition of intracellular ROS production by N-acetylcysteine (NAC), glutathione (GSH), superoxide dismutase (SOD), catalase and the mitochondrial complex I inhibitor, rotenone, results in a decrease in EGCG-dependent HO-1 expression. In addition, we determined that tyrosine kinase is involved in EGCG induction of HO-1 as this is abrogated by genistein. ECs treated with EGCG exhibit activation of Akt and ERK1/2. In addition, pharmacological inhibitors of phosphatidylinositol 3-kinase and MEK1/2, which are upstream of Akt and ERK1/2, respectively, attenuate EGCG-induced HO-1 expression. On the other hand, pretreatment of these cells with EGCG exerts significant cytoprotective effects against H2O2, suggesting that the induction of HO-1 is an important component in the protection against oxidative stress. Hence, EGCG is a novel phytochemical inducer of HO-1 expression and we further identify the principal underlying mechanisms involved in this process.

Resveratrol suppresses IL-6-induced ICAM-1 gene expression in endothelial cells: effects on the inhibition of STAT3 phosphorylation.

Resveratrol, a polyphenolic phytoaxelin present in red wine, has been suggested to protect against atherosclerosis and cardiovascular disease because of its antioxidant effects. Intercellular adhesion molecule (ICAM-1), induced by cytokines, has been hypothesized to play a role in the early events during atherosclerosis. In this study we tested the effects of resveratrol upon both IL-6-induced ICAM-1 gene expression and its underlying signaling pathways in endothelial cells (ECs). Resveratrol was found to inhibit both TNFalpha- and IL-6-induced ICAM-1 gene expression at the promoter, transcriptional and protein levels. Resveratrol also abrogates the tyr705 phosphorylation of STAT3 in IL-6-treated ECs, in a dose- and time-dependent manner. Although quercetin had similar effects, resveratrol showed higher inhibitory properties following 2-4 h pretreatments. Resveratrol has been shown to induce the activity of endothelial nitric oxide synthase (eNOS) and increase NO production. Consistent with this, the treatment of ECs with a NO donor (SNAP) reduces IL-6-induced STAT3 phosphorylation. Conversely, exposure of ECs to a NOS inhibitor reversed the effects of resveratrol upon IL-6-induced STAT3 phosphorylation. Furthermore, ECs transfected with constitutively active Rac1 (RacV12) showed increases in ICAM-1 promoter activity, intracellular reactive oxygen species (ROS) levels and STAT3 phosphorylation, and these increases were attenuated by resveratrol treatment. In summary, we demonstrate for the first time that resveratrol inhibits IL-6-induced ICAM-1 gene expression, in part, by interfering with Rac-mediated pathways via the attenuation of STAT3 phosphorylation. This study therefore provides important new insights that may contribute to the proposed beneficial effects of resveratrol in endothelial responses to cytokines during inflammation.

Responses of blood pressure and lactate levels to various aquatic exercise movements in postmenopausal women.

AIM: Middle-aged and elderly women represent the main attending group in head-out aquatic exercise (HOAE). Blood pressure (BP) significantly increases both during water immersion and aquatic walking. Based on risk concerns, it is important to evaluate BP responses in postmenopausal women doing HOAE. The aim of this study was to determine BP, lactate levels, and rating of perceived exertion (RPE) changes associated with performing 3 different movements at 3 levels of exercise intensity in water. METHODS: Twelve postmenopausal women (59.9±0.6 years old) participated in 3 aquatic trials involving running (RU), rocking (RO), and scissor kicks (SK) on separate days. Systolic BP, mean arterial pressure (MAP), lactate levels, RPE, and motion cadence were measured at rest; upon reaching 50%, 65%, and 80% of heart rate reserve for 6 minutes; and 10 and 30 minutes after exercise. RESULTS: Under similar RPE responses at 3 levels of intensity, SK resulted in higher systolic BP, MAP, and lactate levels than RO at 10 minutes after exercise (P<0.05) and the lowest motion cadence (P<0.05). RO resulted in the lowest MAP and diastolic BP responses during exercise (P<0.05). RU resulted in lower responses of lactate levels at high exercise intensity (P<0.05). CONCLUSION: RO resulted in lower diastolic BP and MAP responses compared with RU and SK during exercise. These findings suggest that RO movement in aquatic exercises is more suitable for people at high risk for cardiovascular disease.

Attainment of therapeutic fluoride levels in serum without major side effects using a slow-release preparation of sodium fluoride in postmenopausal osteoporosis.

The bioavailability, biochemical effects, and safety of a slow-release preparation of sodium fluoride were examined. In 8 normal volunteers, a single administration of slow-release sodium fluoride (25 mg) caused a slow rise and gradual decline in serum fluoride concentration, thus avoiding sharp peaks produced by a rapid-release preparation. In 37 patients with postmenopausal osteoporosis, serum fluoride concentration was kept within the "therapeutic window" (95-100 ng/ml) during long-term intermittent sodium fluoride (slow-release) therapy (25 mg twice/day, given for 3 months in each 5-month cycle over five cycles). Serum fluoride was also kept within the therapeutic window in 64 patients who took sodium fluoride (slow release) continuously over 12 months. Serum osteocalcin concentration increased progressively during fluoride treatment (correlation coefficient of 0.88, p less than .001 for the relationship between serum osteocalcin and duration of therapy). Side effects to slow-release sodium fluoride therapy, assessed in 101 patients at two study sites, were minor and included diarrhea in 2 patients, nausea in 2 patients, abdominal pain and cramping in 2 patients, foot pain in 2 patients, and joint pain in 6 patients. Thus, slow-release sodium fluoride confers desired level of fluoride in serum, while providing safety of usage.

Effect of cAMP elevating agents on carbachol-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells.

The effects of increases in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on carbachol-induced generation of inositol phosphates (IPs) and increases in intracellular Ca2+ ([Ca2+]i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). The cAMP elevating agents, cholera toxin (CTX) and forskolin, induced concentration- and time-dependent cAMP formation with half-maximal effects (-logEC50) at concentrations of 7.6 +/- 1.3 g/ml and 4.8 +/- 0.9 M, respectively. Forskolin caused a concentration-dependent inhibition of carbachol-induced increase in [Ca2+]i with half-maximal inhibition (-logEC50) at 5.2 +/- 0.7 M. Pretreatment of TSMCs with either CTX (10 micrograms/ml, 4 h), forskolin (10-100 microM, 30 min), or dibutyryl cAMP (1 mM, 30 min) inhibited carbachol-stimulated Ca2+ mobilization and IPs accumulation. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration-response curve of carbachol without changing the EC50 values. After treatment with forskolin for 24 h, carbachol-induced IPs accumulation and Ca2+ mobilization were close to those of control group. SQ-22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, 10 microM], an inhibitor of adenylate cyclase, and HA-1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, 50 microM], an inhibitor of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit carbachol-induced IPs accumulation. Moreover, the inactive analogue of forskolin, 1,9-dideoxy forskolin, did not inhibit these responses evoked by carbachol, suggesting that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The KD and Bmax values of the muscarinic receptor (mAChR) for [3H]-N-methyl scopolamine binding were not significantly changed by forskolin treatment for 30 min and 24 h, suggesting that the inhibitory effect of forskolin is distal to the mAChR. The locus of this inhibition was further investigated by examining the effect of forskolin treatment on AIF4(-)-stimulated IPs accumulation in canine TSMCs. The AIF4(-)-induced response was inhibited by forskolin, supporting the notion that G protein(s) are directly activated by AIF4- and uncoupled to phospholipase C by forskolin treatment. We conclude that cAMP elevating agents inhibit carbachol-stimulated generation of IPs and Ca2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca2+]i are very early events in the activation of mAChRs, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of cAMP on tracheal smooth muscle formation.

Enhanced calcium bioavailability from a solubilized form of calcium citrate.

An improved formulation of calcium citrate with higher aqueous solubility and bioavailability was sought. Mixtures of calcium hydroxide and citric acid, with a calcium to citrate molar ratio ranging from 0.67-1.5, dissolved rapidly in water, creating a metastably supersaturated solution. The presence of an excess of citrate in the mixture delayed the precipitation of calcium citrate and kept calcium in solution longer. Thus, the mixture with a calcium to citrate molar ratio of 1.25, containing 500 mg elemental calcium, dissolved in 300 mL water within 2 min and could be kept in solution for 1 h at a wide pH range between 2 and 7. Intestinal calcium absorption, measured from the increment in urinary calcium during the second 2 h following an oral calcium load (500 mg) in 15 normal subjects was significantly higher from the mixtures (calcium to citrate molar ratios of 1.5 and 1.25) than from tricalcium dicitrate. The fractional calcium absorption, obtained from fecal recovery of radiocalcium after oral administration of 500 mg calcium prelabeled with 47Ca in 11 normal subjects, was also higher for the mixture with a calcium to citrate molar ratio of 1.25. The most efficient calcium absorption was obtained with the mixture of calcium hydroxide and citric acid with a calcium to citrate molar ratio of 1.25. The increment in urinary calcium after an oral load with this mixture was 62.4% greater than that obtained with tricalcium dicitrate [0.138 +/- 0.056 (+/- SD) vs. 0.085 +/- 0.086 mg/dL glomerular filtrate; P less than 0.05]. The fractional calcium absorption was 88.4% higher (0.324 +/- 0.107 vs. 0.172 +/- 0.061; P less than 0.05). This mixture provided the highest concentration of ionic calcium, indicating that calcium (rather than calcium-citrate complex) is the fraction absorbed from the intestinal tract. This study, therefore, suggests that a liquid calcium preparation formulated from the mixture of calcium hydroxide and citric acid is more effective than a solid preparation of tricalcium dicitrate in providing soluble and bioavailable calcium.

Assessment of the pathogenetic role of physical exercise in renal stone formation.

The effects of moderate physical exercise (performed on a bicycle ergometer to 70-75% of maximum oxygen consumption) without fluid replenishment on urinary chemistries and crystallization of kidney stone-forming substances were compared to those of rest in six normal subjects. Moderate physical exercise significantly decreased urinary pH [from 6.35 +/- 0.32 (+/-SD) to 5.79 +/- 0.33; P less than 0.05] and citrate [from 121.1 +/- 63.5 to 88.2 +/- 44.4 mg/6-h period from initiation of physical exercise; P less than 0.05 (630 +/- 331 to 459 +/- 231 mumol/6 h)], owing to induced metabolic acidosis. The total renal excretion of stone-forming constituents decreased [for example, calcium from 31.2 +/- 15.8 to 21.4 +/- 6.5 mg/6 h (0.8 +/- 0.4 to 0.5 +/- 0.2 mmol/6 h), phosphorus from 155 +/- 42 to 127 +/- 27 mg/6 h (5.01 +/- 1.4 to 4.1 +/- 0.9 mmol/6 h), and uric acid from 172 +/- 60 to 117 +/- 13 mg/6 h (1.0 +/- 0.4 to 0.7 +/- 0.1 mmol/6 h), each P less than 0.05], probably due to extracellular volume contraction (from sweating) and enhanced renal tubular reabsorption. However, the urinary concentration of stone-forming constituents significantly increased during and after moderate exercise because of the fall in urinary volume from 847 +/- 312 to 290 +/- 36 ml/6 h (P less than 0.01). Thus, urinary calcium oxalate saturation increased significantly from 2.62- to 6.68-fold saturation (P less than 0.01). The urinary undissociated uric acid concentration significantly rose [from 31.6 +/- 24.8 to 125.7 +/- 60.3 mg/L (0.19 +/- 0.15 to 0.76 +/- 0.36 mmol/L; P less than 0.01)], due to higher total uric acid concentration and reduced urinary pH. The saturation of calcium phosphate (brushite) did not change significantly, because the rise in urinary calcium concentration was compensated for by reduced phosphate dissociation (from lower urinary pH). The propensity for spontaneous precipitation of calcium oxalate was greater after exercise, as less soluble oxalate was required to elicit nucleation of calcium oxalate [58.0 +/- 21.2 to 49.0 +/- 16.4 mg/L (644 +/- 236 to 544 +/- 182 mumol/L); P less than 0.05]. The results suggest that moderate physical exercise, without increased fluid intake to compensate for excessive sweating, may cause the crystallization of uric acid and calcium oxalate in urine and may enhance the risk of the formation of renal stones composed of these salts.

Photoaffinity labeling of beef liver monoamine oxidase-B by 4-fluoro-3-nitrophenyl azide.

4-Fluoro-3-nitrophenyl azide (FNPA) competitively inhibited beef liver monoamine oxidase-B (MAO-B) in the dark (Ki = 2.8 microM). Upon irradiation in the presence of FNPA, a concentration-dependent photoinactivation of MAO-B was observed. The kinetic analysis showed that the photoinactivation of MAO-B resulted in a decrease in Vmax but no change in Km. This result suggests that an irreversible linkage may be formed between the enzyme and the photolyzed FNPA. When [3H]FNPA was photoirradiated with the purified MAO-B, a single radioactive band associated with MAO-B was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The photo-dependent incorporation could be protected by phenylethylamine, the substrate for MAO-B, in a concentration-dependent manner. Complete tryptic-chymotryptic digestion of [3H]FNPA-labeled MAO-B resulted in three radioactive peaks on Sephadex G-25 column chromatography. With the same digestion and separation procedures, only one major radioactive peak was observed for the [3H]pargyline-labeled MAO-B, and its elution volume was different from that of [3H]FNPA-labeled peptides. These results suggest that, upon photolysis, FNPA may incorporate into a region in the active site of MAO-B which may be different from the pargyline binding site--the FAD prosthetic group of the enzyme.

Variability of tobramycin pharmacokinetics in cystic fibrosis.

This study determined the extent and impact of tobramycin pharmacokinetic variability in cystic fibrosis patients. Twenty patients were hospitalized twice and the tobramycin half-life, volume of distribution and clearance were determined during Weeks 1 and 2 of both admissions. A difference (P less than 0.05) existed between Weeks 1 and 2 of each admission, but not between admissions, for the clearance and half-life. No difference existed between weeks or admissions for the volume of distribution. No significant correlations existed between weeks within an admission for the half-life and clearance. There was a significant correlation for the volume of distribution between Weeks 1 and 2 of the second admission but not for the first admission. The percents of coefficient of variation and ranges were large. With dosing regimens derived from previously determined factors, "within admission" predicted peaks and troughs would result in 60 and 35% of patients outside the therapeutic range for Admissions 1 and 2, respectively. "Between admission" predictions would result in 65 and 75% of patients outside the therapeutic range. We conclude that considerable variability exists and recommend weekly determinations of serum concentrations and dosing adjustments.