Vitamin D3 seems more appropriate than D2 to sustain adequate levels of 25OHD: a pharmacokinetic approach.

BACKGROUND/OBJECTIVES: The superiority of cholecalciferol (D3) over ergocalciferol (D2) in sustaining serum 25-hydroxy vitamin D (25OHD) levels is controversial. To compare D2 with D3 we performed a single-blind, placebo-controlled randomized trial spanning 11 weeks. SUBJECTS/METHODS: Healthy volunteers (n=33, aged 33.4±6 years) were divided into three groups (n=11, each): D2, D3 and placebo. Treatment started with a loading dose (100,000 IU) followed by 4800 IU/day (d) between d7 and d20 and follow-up until d77. Serum samples were obtained at baseline and at days 3, 7, 14, 21, 35, 49, 63 and 77. RESULTS: Baseline 25OHD values in the D2 group were lower than those in the D3 and placebo groups (P<0.01). Placebo 25OHD levels never changed. As after the loading dose both D2 and D3 groups had reached similar 25OHD levels, we tested equivalence of the area under the concentration × time curve (AUC) between d7 and d77. The AUC was 28.6% higher for D3 compared with D2, and both were higher with respect to placebo. At d77, D2 25OHD levels were higher than those at baseline, but similar to placebo; both were lower than D3 (P<0.04). According to raw data, the elimination half-life of 25OHD was 84 and 111 days under D2 and D3 supplementation, respectively; after subtracting the placebo values, the corresponding figures were 33 and 82 days. CONCLUSIONS: D2 and D3 were equally effective in elevating 25OHD levels after a loading dose. In the long term, D3 seems more appropriate for sustaining 25OHD, which could be relevant for classic and non-classic effects of vitamin D.

Effects of enoxaparin preparations on thrombin generation and their correlation with their anti-FXa activity.

OBJECTIVE: Anticoagulant effect of LMWHs is monitored by anti-factor Xa (anti-FXa) activity assay. Since this test has several limitations, the aim of this study was to explore the activity of two LMWHs by thrombin generation assay (TG, which presents an overall picture of hemostatic balance) and its correlation with their anti-FXa activity. METHODS: In an open-label, randomized cross-over study, 40 mg of two enoxaparins, the original branded formulation (R) and another one, also marketed in Argentina (T), were daily injected subcutaneously, for 7 days, to 20 healthy volunteers, with a 7-day washout interval. Blood samples were collected before treatment and 180 minutes after the injection on days 3 and 7. TG in platelet-poor plasma activated with tissue factor was assessed by lag time (LT), time to peak (TTP), peak (PTG), and endogenous thrombin potential (ETP). Anti-FXa and anti-FIIa activities, free tissue factor pathway inhibitor (free TFPI), tissue plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), and euglobulin lysis time (ELT) were also assayed. RESULTS: The mean (SD) anti-FXa (UI/ml) for T and R increased on days 3 and 7. LT and TTP were significantly prolonged by both LMWHs, with no differences between them. The mean ETP (nmol/L) for T and R at 3 and 7 days after treatment were significantly reduced when compared with basal values (p = 0.001 for all). On day 3, a significant correlation was shown between the variables describing TG and anti-FXa for T and R, without differences between them, for LT (r: 0.516 and 0486), ETP (r: 0.532 and 0.574), PEAK (r: 0.482 and 0.501), and TTP (r: 0.577 and 0.503), respectively. This correlation was also significant on day 7. Anti-FIIa activity and free TFPI increased significantly at 3 and 7 days for both LMWHs, without differences between them. R and T decreased ELT and PAI-1, but had no effect on t-PA. There were no differences between both LMWHs in routine hemostatic tests. No adverse events were reported. CONCLUSIONS: Correlation between TG and anti-FXa activity was good. Both enoxaparins induced similar change of coagulation parameters, with a significant increase in fibrinolytic activity.

Dual effect of nitric oxide on uterine prostaglandin synthesis in a murine model of preterm labour.

BACKGROUND AND PURPOSE: Maternal infections are one of the main causes of adverse developmental outcomes including embryonic resorption and preterm labour. In this study a mouse model of inflammation-associated preterm delivery was developed, and used to study the relationship between nitric oxide (NO) and prostaglandins (PGs). EXPERIMENTAL APPROACH: The murine model of preterm labour was achieved by assaying different doses of bacterial lipopolysaccharides (LPS). Once established, it was used to analyse uterine levels of prostaglandins E(2) and F(2α) (by radioimmunoassay), cyclooxygenases (COX) and NOS proteins (by Western blot) and NO synthase (NOS) activity. Effects of inhibitors of COX and NOS on LPS-induced preterm labour were also studied. In vitro assays with a nitric oxide donor (SNAP) were performed to analyse the modulation of prostaglandin production by NO. KEY RESULTS: Lipopolysaccharide increased uterine NO and PG synthesis and induced preterm delivery. Co-administration of meloxicam, a cyclooxygenase-2 inhibitor, or aminoguanidine, an inducible NOS inhibitor, prevented LPS-induced preterm delivery and blocked the increase in PGs and NO. Notably, the levels of NO were found to determine its effect on PG synthesis; low concentrations of NO reduced PG synthesis whereas high concentrations augmented them. CONCLUSIONS AND IMPLICATIONS: An infection-associated model of preterm labour showed that preterm delivery can be prevented by decreasing PG or NO production. NO was found to have a dual effect on PG synthesis depending on its concentration. These data contribute to the understanding of the interaction between NO and PGs in pregnancy and parturition, and could help to improve neonatal outcomes.

Management and triage of women with human papillomavirus infection in follow-up for low-grade cervical disease: association of HPV-DNA and RNA-based methods.

Type-specific persistent infection with Human Papillomavirus (HPV) is a significant risk factor for the development of cervical diseases. Persistent infection could be further refined by a sequencing approach to detect early cervical lesions that are at high risk of developing an invasive squamous cervical cancer. The aim of the present study is to investigate the clinical utility of detecting mRNA transcripts of HPV oncogenes E6/E7 by using a Real-time NASBA technology (mRNA test) and to identify women with low-grade cytological disease but with an increased risk of developing high-grade cervical abnormalities or invasive squamous cervical cancer. Our preliminary results show that E6/E7 is detected in only a subset of HR-HPV-positive cases. Since viral persistence is considered to be the true precursor of neoplastic progression, only the detection of E6/E7 mRNA can identify the infection which is more likely to persist and induce neoplasia in future. For these reasons we believe that this test would be useful for the characterization of women with HR-HPV DNA positivity who should be effectively treated because at high-risk of developing a high grade cervical lesion or an invasive squamous cervical cancer.

The mechanisms involved in the action of metformin in regulating ovarian function in hyperandrogenized mice.

The aim of this study was to investigate the mechanisms by which N,N'-dimethylbiguanide metformin (50 mg/100 g body weight (BW) in 0.05 ml of water, given orally with a cannula) prevents the ovarian disorders provoked by the hyperandrogenization with dehydroepiandrosterone (DHEA) in prepuberal BALB/c mice. The injection of DHEA (6 mg/100 g BW in 0.1 ml of oil) for 20 consecutive days re-creates a mouse model that resembles some aspects of the human polycystic ovary syndrome (PCOS). The treatment with DHEA increased ovarian oxidative stress because it enhanced lipid peroxidation (LPO) and diminished both catalase (CAT) activity and glutathione (GSH) content. Therefore, the treatment with DHEA diminished both ovarian nitric oxide synthase (NOS) activity and prostaglandin E (PGE) production. When metformin was administered together with DHEA, the ovarian GSH content, NOS activity and PGE production did not differ when compared with controls. However, metformin was not able to prevent the effect of DHEA on ovarian LPO or CAT activity. Finally, DHEA increased the ovarian protein expressions of inducible NOS (iNOS), inducible cyclooxygenase (COX2) and the phosphorylated AMP-dependent kinase alpha (AMPK-alpha) (Thr172). Metformin administered together with DHEA was able to prevent the increase of ovarian iNOS and COX2 expressions and to enhance the activation of phosphorylated AMPK-alpha expression.

Metformin prevents embryonic resorption induced by hyperandrogenisation with dehydroepiandrosterone in mice.

The present study examined the mechanism by which metformin prevents dehydroepiandrosterone (DHEA)-induced embryonic resorption in mice. Treatment with DHEA (6 mg/100 g bodyweight, 24 and 48 h post implantation) induced 88 +/- 1 % embryonic resorption and the diminution of both serum oestradiol (E) and progesterone (P) levels. However, when metformin (50 mg/kg bodyweight) was given together with DHEA, embryo resorption (43 +/- 3% v. 35 +/- 5% in controls) and both serum E and P levels were not significantly different from controls. Glucose and insulin levels were increased in the DHEA-treated mice but when metformin was administered together with DHEA these parameters were similar to control values. Treatment with DHEA increased ovarian oxidative stress and diminished uterine nitric oxide synthase (NOS) activity; however, when metformin was administered together with DHEA, both ovarian oxidative stress and uterine NOS activity were not different from controls. Metformin treatment did not modify the percentage of CD4(+) and CD8(+) T cells from both axillar and retroperitoneal lymph nodes but prevented the increase of serum tumour necrosis factor +/- produced in DHEA-treated mice. These results show that metformin acts in DHEA-induced embryonic resorption in mice by modulating endocrine parameters, ovarian oxidative stress and uterine NOS activity.

Nitric oxide (NO) inhibits prostaglandin E2 9-ketoreductase (9-KPR) activity in human fetal membranes.

Nitric oxide (NO) synthesized by fetal membranes may act either directly inhibiting myometrium contractility or indirectly interacting with tocolytic agents as prostaglandins (PGs). Here we examined if NO could modulate prostaglandin E(2) 9-ketoreductase (9-KPR) activity in human fetal membranes (HFM). 9-KPR is the enzyme that converts PGE(2) into PGF(2alpha), the main PGs known to induce uterine contractility at term. Chorioamnion explants obtained from elective caesareans were incubated with aminoguanidine (AG), an iNOS inhibitor, or NOC-18, a NO donor. NOC-18 (2mM) increased PGE(2) production and diminished PGF(2alpha) synthesis in HFM. AG presented the opposite effect. When we evaluated the activity of 9-KPR by the conversion of [(3)H]-PGE(2) into [(3)H]-PGF(2alpha) and 13,14-dihidro-15-keto prostaglandin F(2alpha) (the PGF(2alpha) metabolite), we found that NOC-18 inhibited 9-KPR activity. Interestingly, AG did not elicit any effect on 9-KPR but l-NAME, a non-selective NOS inhibitor, significantly increased its activity. Our data suggests that exogenous NO inhibits 9-KPR activity in HFM, thus modulating the synthesis of important labor mediators as PGF(2alpha).

Effects of cyclooxygenase inhibitor pretreatment on nitric oxide production, nNOS and iNOS expression in rat cerebellum.

1. The therapeutic effect of nonsteroidal anti-inflammatory drugs (NSAIDs) is thought to be due mainly to its inhibition of cyclooxygenase (COX) enzymes, but there is a growing body of research that now demonstrates a variety of NSAIDs effects on cellular signal transduction pathways other than those involving prostaglandins. 2. Nitric oxide (NO) as a free radical and an agent that gives rise to highly toxic oxidants (peroxynitrile, nitric dioxide, nitron ion), becomes a cause of neuronal damage and death in some brain lesions such as Parkinson and Alzheimer disease, and Huntington's chorea. 3. In the present study, the in vivo effect of three NSAIDs (lysine clonixinate (LC), indomethacine (INDO) and meloxicam (MELO)) on NO production and nitric oxide synthase expression in rat cerebellar slices was analysed. Rats were treated with (a) saline, (b) lipopolysaccharide (LPS) (5 mg kg(-1), i.p.), (c) saline in combination with different doses of NSAIDs and (d) LPS in combination with different doses of NSAIDs and then killed 6 h after treatment. 4. NO synthesis, evaluated by Bred and Snyder technique, was increased by LPS. This augmentation was inhibited by coadministration of the three NSAIDs assayed. None of the NSAIDs tested was able to modify control NO synthesis. 5. Expression of iNOS and neural NOS (nNOS) was detected by Western blotting in control and LPS-treated rats. LC and INDO, but not MELO, were able to inhibit the expression of these enzymes. 6. Therefore, reduction of iNOS and nNOS levels in cerebellum may explain, in part, the anti-inflammatory effect of these NSAIDs and may also have importance in the prevention of NO-mediated neuronal injury.

The effect of Enalapril on PGI(2) and NO levels in hypertensive patients.

The effects of the angiotensin-converting enzyme inhibitors (ACEIs), may be partially mediated by the kinins' paracrine influence. Their actions may be exerted through nitric oxide and prostacyclin (PGI(2)) synthesis stimulation. The aim of our study was to determine whether the antihypertensive effect of Enalapril correlated with the increment in the plasmatic levels of NO and PGI(2) in essential moderate hypertensive patients. Normalization of blood pressure was observed in 20 patients, four on the 28th day, 15 on the 42th day and one on the 56th day. Enalapril-respondent subjects showed increased nitrate/nitrite levels on the 14th day (30% increment), on the 28th day (64%), on the 42th day (93.5%) and on the 56th day (96.2%) compared with basal levels, but they did not modify the circulating 6-keto PGF(1 alpha) levels. Four non-respondent patients showed a diminution in nitrate/nitrite and 6-keto PGF(1 alpha) circulating levels along the treatment. We conclude that the administration of 5-30 mg of Enalapril increases circulating NO metabolites in respondent-essential hypertensive subjects. The lack of responsiveness to the treatment may be related to the presence of risk factors such as those linked to an increase of oxidative stress. Finally, we consider that the evaluation of circulating NO may represent a predictive of the response to Enalapril in essential hypertensive patients.

[Effect of nonsteroidal antiinflammatory drugs on colonic lipoxygenase and cyclooxygenase activities from patients with colonic neoplasia]. / Acción de los antiinflamatorios no esteroideos sobre la actividad lipooxigenasa y ciclooxigenasa colónica de pacientes con neoplasia de colon.

Lysine clonixinate (LC) is a nonsteroidal anti-inflammatory drug (NSAID) with good gastrointestinal tolerance. Treatment with LC at levels equivalent to those found in plasma following therapeutic doses resulted in significant inhibition of both cyclooxygenase 2 (COX-2) and production of 5 hydroxy-eicosatetraeonic acid (5-HETE) and slightly affected levels of cyclooxygenase 1 (COX-1) in in vitro studies carried out on human tissues. This study deals with the in vivo effect of the drug on human colon segments. Experiment 1: Five patients about to undergo hemicholectomy due to colon neoplasia were treated preoperatively with a continuous infusion of LC, to achieve a steady-state concentration between 4 and 6 mg/ml. Human colon segments from the five patients and from another five control patients receiving no treatment with [14C]-arachidonic acid were incubated. Human colon segments treated with LC showed significant inhibition of PGE2, the only prostaglandin (PG) synthesised by the tissue, as well as of 5-HETE. Experiment 2: Fifteen patients received an i.v. bolus of LC 100 mg (n1 = 5); LC 200 mg (n2 = 5) or indomethacin (INDO) 50 mg (n3 = 5). Both doses of LC showed greater inhibition of PGE2 synthesis than the INDO bolus. Both NSAIDs studied proved to have different effects on the production of 5-HETE; while treatment with LC elicited significant inhibition, levels with INDO remained unchanged. Western blotting analysis showed expression of both COX isoforms in colon segments, COX-2 levels being 20% higher. Both types of in vivo studies conducted continuous infusion and i.v. bolus, revealed that LC exerted significant inhibition of basal synthesis of PGE2 and 5-HETE.

Antispasmodic/analgesic associations in primary dysmenorrhea double-blind crossover placebo-controlled clinical trial.

We studied 125 patients with primary dysmenorrhea in a prospective randomized double-blind crossover study. After an admission pretreatment period without medication, the patients completed three consecutive randomized treatment phases with lysine clonixinate 125 mg plus propinox 10 mg or paracetamol 500 mg plus hyoscine N-butylbromide 10 mg or placebo, according to a fixed-dose schedule of 1 tablet every 6 h, 3 days before onset of menses and for 5 days thereafter. Changes in menstrual pain intensity and duration, amount of bleeding measured according to the number of daily pads used and concomitant symptoms were assessed on the fifth day of each cycle. Every night, the patients recorded the average intensity of menstrual pain during the first 4 days of menstruation in a diary The follow-up visit carried out at day 5 showed significant reduction in pain intensity with both active treatments vs. the other two phases: baseline: 2.72 +/- 0.61; placebo: 1.85 +/- 0.87; lysine clonixinate plus propinox 1.36 +/- 0.81, and paracetamol plus hyosine N-butylbromide: 1.45 +/- 0.87. The patients' diaries showed increasingly lower pain intensities starting from day 1 with the three treatments. Active treatments revealed significantly higher analgesic efficacy from the outset compared with baseline and placebo; however, only the lysine clonixinate plus propinox combination reached a statistically significant difference by days 3 and 4. No changes in duration or intensity of menstrual bleeding or in the incidence of adverse effects were observed during the four study periods.

[Effects of lysine clonixinate on platelet function. Comparison with other non-steroidal anti-inflammatory agents]. / Acción del clonixinato de lisina sobre la función plaquetaria. Comparación con otras drogas antiinflamatorias no esteroideas.

One of the mechanisms of action of non steroid antiinflammatory drugs (NSAIDs) consists of inhibition of prostaglandin synthesis. This explains many of the pharmacological effects and adverse events observed in medical practice. Administration of NSAIDs to patients with hemostatic disorders or perioperative conditions entails the risk of bleeding due to inhibition of platelet function. This study deals with platelet changes induced by lysine clonixinate vs diclofenac, ibuprofen and aspirin in classical tests such as platelet count, platelet factor 3 (PF3) activity and platelet aggregation with various inductors and more recent procedures such as P-selectin measurement by flow cytometry. Unlike control drugs, lysine clonixinate did not induce changes in platelet count or function when administered to healthy volunteers at the commonly used therapeutic doses.

In vivo and in vitro effects of lysine clonixinate on nitric oxide synthase in LPS-treated and untreated rat lung preparations.

Recent studies have shown that some nonsteroidal antiinflammatory drugs (NSAIDS) inhibited the inducible NO synthase (iNOS) without direct effect on the catalytic activity of this enzyme. This study was conducted to investigate the in vitro and in vivo effects of lysine clonixinate (LC) and indomethacin (INDO) on NOS activity in rat lung preparation. LC is a drug with antiinflammatory, antipyretic, and analgesic action. In the in vitro experiments, rats were injected with saline or lipopolysaccharide (LPS) and killed 6 h after treatment. Lung preparations were incubated with LC at 2.3 x 10(-5) M or 3.8 x 10(-5) M. The minimum concentration did not modify NOS activity in control or LPS-treated rats but the maximum dose inhibited increased NO production induced by LPS. Furthermore, INDO at 10(-6) M had no effect on enzymatic activity in control or LPS-treated rats. In the in vivo experiments, 40 mg/kg of LC were injected ip. Such a dose did not affect basal production of NO. When LC and LPS were injected simultaneously 6 h before sacrifice, a significant decrease in LPS-induced NOS activity was observed. INDO 10 mg/kg injected in control animals had no effect on NOS activity and did not block LPS induced stimulation of NO production when injected simultaneously. Finally, when LC (40 mg/kg) was injected 3 h after LPS, the enzymatic activity remained unchanged. Expression of iNOS was detected by Western blotting in rats treated with LPS plus 4, 10, 20, and 40 mg/kg of LC. The lowest dose was the only one showing no effect on LPS-induced increase of iNOS. In short, LC is a NSAID with inhibitory action on the expression of LPS-induced NOS, effect that was not seen with INDO in our experimental conditions.

Stem cell transplantation from HLA-matched related donor for Fanconi's anaemia: a retrospective review of the multicentric Italian experience on behalf of AIEOP-GITMO.

Twenty-seven consecutive Italian patients with Fanconi's anaemia (FA) underwent stem cell transplantation (SCT) from an HLA-matched related donor in 10 Italian centres of the Associazione Italiana Ematologia ed Oncologia Pediatrica (AIEOP), Gruppo Italiano di Trapianto di Midollo Osseo (GITMO). Twenty-two patients (81.5%) were conditioned with low-dose (median 20 mg/kg) cyclophosphamide (Cy) and thoraco-abdominal or total body irradiation (median dose 500 cGy), five patients (18.5%) with high-dose Cy (median 120 mg/kg). Graft-vs.-host disease (GVHD) prophylaxis was carried out with cyclosporin A in 26 cases; methotrexate (MTX) was added in eight cases. One patient received MTX alone. The median follow-up was 36 months. Ninety-two percent of patients (25 out of 27) engrafted, grade II and III acute GVHD occurred in 28% and 8% of patients, respectively, with chronic GVHD in 12.5%. Conditioning-related toxicity was mild: 4% of patients had grade III mucositis, 7.4% had grade II haemorrhagic cystitis, 14.8% had grade III liver toxicity and 11.1% had grade III renal toxicity. Transplant-related mortality at 12 months was 19.2%, survival at 36 months was 81.5%, with a median Karnofsky score of 100%. No late tumours occurred after a mean follow-up of the survivors of 5 years. None of the studied variables significantly affected the survival, including conditioning regimen, acute GVHD and clinical non-haematological phenotype. Among the studied variables, only conditioning regimens containing high-dose Cy and the presence of genital abnormalities were significantly (P < 0.05) associated with an increased rate of acute GVHD. Our study demonstrates that the Italian FA patients undergoing SCT from an HLA-matched related donor have a very good outcome. These patients, when compared with others of different ethnic origin who underwent allogeneic bone marrow transplantation, showed a less severe non-haematological phenotype, raising the possibility that this milder phenotype may have, at least in part, contributed to the outcome. Our data may provide a useful tool for further studies aiming to correlate genotype with phenotype.

[Multimedia methods for the teaching of pharmacology]. / Métodos multimediales en la enseñanza de la farmacología.

Pharmacology is by definition a subject of integration. Students take on a passive role during the learning process and do not count with image aids that could foster understanding, fixation and evocation. Therefore, a hypermedia pharmacology CD ROM program was developed. Such a program includes the following features: hypertext format set up as a network of nodes and arcs, multimedia technology, highly interactive and customized navigation. The prototype thoroughly develops cholinergic neurotransmission pharmacology in its historical, anatomic, physiological, biochemical, pharmacological and therapeutic aspects. The program is divided into three modules; namely, information, exercises and evaluation. Each network node may contain a text, hypertext, images, 3D animation and experimental videos. Information resources include the navigation conceptual map for the chosen node, a track record to go back or forward immediately, the possibility of adding text or images, a text search function, images, animation and videos to find such objects in the different nodes together with the possibility of printing any of the contents. Apart from the information framework, the model has manifold useful integration exercises to assess the learning improvement and prompt the student accordingly to review the topic whenever mistakes exceed a certain amount. This program promotes knowledge integration and building through association of contents. To access to the program's demo, consult the following page.

Acción del clonixinato de lisina sobre la función plaquetaria. Comparación con otras drogas antiinflamatorias no esteroideas. / [Effects of lysine clonixinate on platelet function. Comparison with other non-steroidal anti-inflammatory agents]

One of the mechanisms of action of non steroid antiinflammatory drugs (NSAIDs) consists of inhibition of prostaglandin synthesis. This explains many of the pharmacological effects and adverse events observed in medical practice. Administration of NSAIDs to patients with hemostatic disorders or perioperative conditions entails the risk of bleeding due to inhibition of platelet function. This study deals with platelet changes induced by lysine clonixinate vs diclofenac, ibuprofen and aspirin in classical tests such as platelet count, platelet factor 3 (PF3) activity and platelet aggregation with various inductors and more recent procedures such as P-selectin measurement by flow cytometry. Unlike control drugs, lysine clonixinate did not induce changes in platelet count or function when administered to healthy volunteers at the commonly used therapeutic doses.