Biomedical subjects
Publications and source records attributed to M O Moraes.
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We compare the clinical and histological data with the immunological status of a borderline leprosy patient who experienced an erythema nodosum leprosum (ENL) reaction followed by a reversal reaction (RR) after 12 weeks of anti-inflammatory treatment (pentoxifylline, PTX, 1200 mg daily). Skin biopsies, serum and blood samples were collected sequentially during the reactional episodes. At the outset of RR, the patient's lymphocytes secreted interferon (IFN) -gamma and there was a positive lymphoproliferative test in response to Mycobacterium leprae, which had been absent during ENL. The lepromin reaction reversed from negative (0 mm) at diagnosis, to positive (3 mm) 3 months after the development of RR. Tumour necrosis factor (TNF) -alpha levels in the serum decreased after 1 week of treatment and increased slightly thereafter. The immunohistochemical data for ENL showed a diffuse dermal and hypodermal infiltrate composed of mononuclear cells and neutrophils, while RR was characterized by an epithelioid granulomatous infiltrate with a marked presence of gammadelta T cells. Reverse transcription-polymerase chain reaction showed a mixed cytokine profile characterized by the expression of TNF-alpha, IFN-gamma, interleukin (IL) -6, IL-10 and IL-12 mRNA in the skin, which persisted throughout the development of ENL and RR lesions. IL-4 mRNA, first detected after 7 days of PTX treatment, was still present during RR. The results suggest the emergence of an initial Th0-like cytokine profile in ENL, typical of a state of immunoactivation, before conditions optimal for the appearance of an antigen-specific cell-mediated immune response and gammadelta T-cell migration are created.
The discovery that angiogenesis is a key condition for the growth of a tumor beyond a millimeter or two, brings about a new approach in the treatment of tumors using drugs able to inhibit the formation of new blood vessels. Also, it has been realized that antiangiogenic drugs can be useful in the treatment of other pathological processes, now classified as angiogenesis-dependent diseases. Initially, cartilage was considered as a possible natural source of antiangiogenic compounds due to its known avascular nature. To date, a number of in vitro and in vivo studies have suggested the existence of antiangiogenic and antitumor compounds in bovine and shark cartilage. However, the potential usefulness of shark cartilage in the treatment of cancer and other angiogenesis-dependent diseases have not been totally accepted due to (i) unsatisfactory patient outcome in clinical trials that have used shark cartilage in cancer patients, (ii) the lack of data that correlates bioavailability with pharmacological effects using oral shark cartilage. Thus, the objective of this review is to describe the main basic and clinical investigations reported in the literature, in which the antiangiogenic and/or antitumor properties of shark cartilage or of its extracts were evaluated. Possible explanations for conflicting results are discussed as well.
Several angiogenic inhibitors have been obtained from shark cartilage, some of these are currently in clinical trials for assessment of safety and therapeutic efficacy in humans. Still, shark cartilage taken orally is commonly used in alternative and complimentary medicine for various ailments including serious diseases such as cancer. However, only few studies of oral shark cartilage have demonstrated pharmacological effects in experimental animals or patients, to indicate safe doses with sufficient bioavailability. In the present study we demonstrated the antiangiogenic properties of oral shark cartilage in the rabbit cornea model. Slow-release, polymethylmetacrylate pellets containing basic fibroblast growth factor (bFGF) were surgically implanted in the rabbit cornea to stimulate neovascularization scored by stereo microscopy. Powdered shark cartilage (PSC; commercial product) was tested orally along with a water-soluble fraction (WSF) of this cartilage product which was tested by local application. Animals were treated with oral dosages of 100 mg/kg PSC or 200 mg/kg thalidomide as positive control. Pellets containing WSF (50, 100 or 200 microg/pellet) or bFGF-inhibitor pentosan polysulfate were implanted adjacent to the bFGF pellet. Oral shark cartilage inhibited bFGF-induced angiogenesis, as did oral thalidomide, in this in vivo model. WSF and pentosan polysulfate was shown to block neovascularization in the cornea when applied locally. This study demonstrates that in the rabbit, oral shark cartilage appears to produce systemic levels of angiogenesis inhibitors that can exert their effect at the cornea.
A liquid chromatographic atmospheric pressure chemical ionization tandem mass spectrometric method is described for the determination of 21-hydroxydeflazacort in human plasma using dexamethasone 21-acetate as an internal standard. The procedure requires a single diethyl ether extraction. After evaporation of the solvent under a nitrogen flow, the analytes are reconstituted in the mobile phase, chromatographed on a C18 reversed-phase column and analyzed by mass spectrometry via a heated nebulizer interface where they are detected by multiple reaction monitoring. The method has a chromatographic run time of less than 5 min and a linear calibration curve with a range of 1-400 ng ml(-1) (r>0.999). The between-run precision, based on the relative standard deviation for replicate quality controls, was < or =5.5% (10 ng ml(-1)), 1.0% (50 ng ml(-1)) and 2.7% (200 ng ml(-1)). The between-run accuracy was +/-7.1, 3.8 and 4.8% for the above concentrations, respectively. This method was employed in a bioequivalence study of two DFZ tablet formulations (Denacen from Marjan Industria e Comercio, Brazil, as a test formulation, and Calcort from Merrell Lepetit, Brazil, as a reference formulation) in 24 healthy volunteers of both sexes who received a single 30 mg dose of each formulation. The study was conducted using an open, randomized, two-period crossover design with a 7-day washout interval. The 90% confidence interval (CI) of the individual geometric mean ratio for Denacen/Calcort was 89.8-109.5% for area under the curve AUC(0-24 h) and 80.7-98.5% for Cmax. Since both the 90% CI for AUC(0-24 h) and Cmax were included in the 80-125% interval proposed by the US Food and Drug Administration, Denacen was considered bioequivalent to Calcort according to both the rate and extent of absorption.
Ten compounds derived from plants indigenous to Northeast Brazil were examined for antiproliferative effects on human cells in vitro. The effects of these phytochemicals on cell growth were determined by the MTT microtitre assay with 3-day continuous drug exposure. Three human cell lines were used: CEM leukaemia, SW1573 lung tumour and CCD922 normal skin fibroblasts. Four active compounds were found with IC(50) values less than 10 microg/mL in the two cancer cell lines. Oncocalyxones A and C, both 1,4-anthracenediones from Auxemma oncocalyx (Boraginaceae), showed cytotoxicity with mean IC(50) values of 0.8-2, 7-8 and 12-13 microg/mL against CEM, SW1573 and CCD922, respectively. One diterpene and one flavonoid, both from Egletes viscosa (Compositae), were also active. 12-Acetoxy-hawtriwaic acid lactone was cytotoxic with mean IC(50) values of 6, 10 and 10 microg/mL, respectively. 4,5-Dihydroxy-3,3,7, 8-tetramethoxy flavone (ternatin) was only growth-inhibitory with mean IC(50) values of 2, 1 and 10 microg/mL, respectively. These four most active compounds were examined further for their effects on DNA integrity and on DNA synthesis. All but ternatin caused substantial DNA damage and marked inhibition of 5-bromo-2'-deoxyuridine incorporation within 24 h. This study demonstrated the antiproliferative activity of four novel phytochemicals, three of which are DNA-reactive and inhibit DNA synthesis. Further studies are warranted to evaluate these compounds for antitumour potential.
The aim of this study was to investigate in what ways in vivo anti-inflammatory treatment affects cytokine mRNA expression in situ in both erythema nodosum leprosum and reversal reaction patients. Serial biopsies were collected from the patients undergoing leprosy reactions before and during pentoxifylline (n = 7) or thalidomide (n = 3) treatment for erythema nodosum leprosum and prednisone (n = 3) for reversal reaction. Clinical evolution of the skin lesion was assessed during the study and semiquantitative reverse transcription-polymerase chain reaction was used to investigate cytokine mRNA expression at the lesion site. Results showed expression of interferon-gamma, interleukin-6, interleukin-10, interleukin-12 p40, and tumor necrosis factor-alpha in all patients tested at the onset of reactional episodes, but interleukin-4 mRNA was rarely detected in the lesions (n = 4). Follow-up analysis showed that, irrespective of the drugs used, tumor necrosis factor-alpha mRNA was diminished in 10 of the 13 patients tested. A concomitant decrease of mRNA accumulation was also observed for interferon-gamma (nine of 11 patients), interleukin-6 (nine of 11), and interleukin-12 p40 (six of eight). An inhibitory effect on interleukin-10 mRNA was likewise seen after thalidomide and pentoxifylline, but not subsequent to prednisone treatment. The data also demonstrated that cytokine mRNA inhibition correlates to the resolution of the inflammatory response in situ (n = 10), whereas the persistence/enhancement of cytokine message expression after treatment was associated with worsening of the skin condition, as seen in three erythema nodosum leprosum patients whose maintenance of local inflammation was accompanied by the appearance/persistence of interleukin-4 gene expression in situ subsequent to anti-inflammatory treatment. In summary, the participation of cytokines in leprosy inflammatory episodes seems to be directly associated with the patients' clinical evolution following therapy for reaction.
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Stannous ion (Sn) has been employed in nuclear medicine and in food industry. We described that Stannous Chloride (SnCl2) inactivation effect in Escherichia coli is mediated by a Fenton-like reaction. The effect of SnCl2 was studied through: (i) the alteration of plasmid topology in neutral and acidic pH by gel electrophoresis; and (ii) the transformation efficiency of an wild type E. coli strain. Treatment of plasmid DNA pUC 9.1 with SnCl2, at pH 7.4, results in DNA single-strand breaks (SSB), in a dose-dependent manner. Addition of sodium benzoate partly inhibited the DNA damage, while EDTA completely abolishes DNA-SSB. Furthermore, the ability of the plasmid to transform E. coli was reduced. At pH 1.3, SnCl2 exerts a protective effect on plasmid against HCI depurination. Our results suggest the generation of ROS, such as *OH by a Fenton-like reaction, close to the site of the lesions due to a possible complexation of stannous ion to DNA.
The present work showed that glutamate decreased hippocampal cell viability in a dose-dependent manner. While no significant effect was observed after cell exposure to 0.1 mM glutamate, cell incubation for 0.5 h caused a progressive decrease of cell viability, which at 5 mM concentration reached 68% as compared to controls. No further effect was observed in the presence of 10 mM glutamate. While nerve growth factor (NGF) at the dose of 0.5 ng/ml presented no effect, it significantly reduced glutamate cytotoxicity at a higher dose (1 ng/ml) increasing the cell viability to 66%. Similarly, cell viabilities in the presence of the ganglioside GM, (5 and 10 ng/ml) after glutamate exposure were 19 and 73%, respectively. A dose-response relationship was observed after cell incubation with vitamin E (0.5 and 1 mM) which resulted in cell viability of the order of 34 and 70%, respectively. Surprisingly, a potentiation of the effect was observed after the association of NGF (0.5 ng/ml) plus ganglioside GM1 (5 ng/ml) or vitamin E (0.5 mM) plus ganglioside GM1 (5 ng/ml), after pre-incubation with glutamate. In these conditions, significantly higher viabilities were demonstrated (66 and 71% for the two associations, respectively) as compared to each one of the compounds alone (NGF 0.5 ng/ml--29.5%; ganglioside GM1 5 ng/ml--19.4%). However, no potentiation was seen after the association of NGF plus vitamin E on glutamate pre-exposed cells. These results showed a cytoprotective effect of ganglioside GM1, NGF and vitamin E on the glutamate-induced cytotoxicity in rat hippocampal cells.
This study demonstrated that polymorphonuclear neutrophils (PMN) participate in the acute inflammatory response in leprosy as effector cells. Lepromatous patients present intense infiltrate of neutrophils in reactional (ENL) lesions. Circulating PMN of nonreactional patients, healthy donors, and reactional patients were purified and analyzed in vitro. The study confirmed the short lifespan of these cells in culture with progressive changes characteristic of apoptosis. Apoptosis was greatly accelerated in ENL patients as shown by cellular morphology, later confirmed by qualitative and quantitative analysis of fragmented DNA. It was observed that neutrophils stimulated with lipopolysaccharide, Mycobacterium leprae, and lipoarabinomannan secrete interleukin-8 and tumor necrosis factor alpha (TNF-alpha). Thalidomide, a drug known to inhibit TNF-alpha synthesis on monocytes, also exerted an inhibitory effect on TNF-alpha secretion in neutrophils. These data suggest that PMN can participate in the regulation of the immune response in leprosy and can contribute to the amplification of TNF-alpha production at the site of ENL lesion.
Leprosy patients during the natural course of the disease may develop reactional episodes, namely reversal reaction (RR) and erythema nodosum leprosum (ENL). Immunological events described as occurring during RR indicate up-regulation of the immune response, whereas in ENL the events are not fully understood. The aim of this study was to analyse the in vivo pattern of cytokine gene expression in the reactional states of leprosy. Peripheral blood mononuclear cells (PBMC, n = 14) and tissue samples (n = 17) obtained from patients with ENL and RR were obtained and assayed by RT-PCR. PBMC obtained from unreactional patients (n = 15) and normal individuals (n = 5) were also assessed. Expression of interferon (IFN)gamma, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL)-2Rp55, perforin and IL-1beta mRNA in PBMC were detected mostly in ENL/RR patients, but not in unreactional patients. Likewise, cytokines such as IL-6, IL-8, tumour necrosis factor (TNF)alpha and TNFbeta were also present in reactional and tuberculoid patients as opposed to lepromatous leprosy (BL/LL). Interestingly, the majority of ENL/RR patients showed messages for IL-6, IL-10, IL-12 and TNFalpha in the skin. IFNgamma was detected in 84.6% (ENL) and 100% (RR) of the patients, whereas IL-4 was detected only in few individuals (38.5 and 25%, respectively). Although mRNA expression and protein levels may be different, the data reported in this study suggest a cytokine mRNA profile that seems to be indistinguishable for RR and ENL. In addition, it shows up-regulation of immuno-inflammatory cytokines in the blood and tissue of the same patient examined before and during reaction. Furthermore, it is suggested that this pattern of response results from an immunological reactivation that might lead to an acute inflammatory response in both reactional episodes.
To develop a new method for quantifying fluoconazole in human plasma and to compare the bioavailability of two fluconazole capsule formulations, an open, randomized, two-period crossover study with a one-week washout interval was conducted in 24 healthy volunteers. Plasma samples were obtained up to 168 hours after drug administration and the serum fluconazole concentrations were analyzed using electrospray tandem mass spectrometry coupled to liquid chromatography using multiple reaction monitoring mode. The pharmacokinetic parameters obtained for fluconazole after the administration of each formulation included the Area under the curve (AUC)(0-168h), AUC(0-infinity), Cmax, Cmax/AUC(0-168h), Tmax, elimination rate constant (Ke), and half-life (T1/2). Within- and between-run imprecision was less than 2.3% and 8.2%, respectively. Inaccuracy within and between runs was -1.5% and -9.7%, respectively. The pharmacokinetic parameters for bioequivalence showed a normal distribution, and the variance of AUC(0-168h), AUC(0-infinity), and Cmax were homoscedastic. The geometric mean for the Fluconal/Zoltec (Fluconal; Libbs Farmacêutica Ltda, São Paulo, Brazil; Zoltec; Laboratórios Pfizer Ltda., São Paulo, Brazil) individual percent ratio was 94.9% for AUC(0-168h), 94.7% for AUC(0-infinity), 80.1% for Cmax, 102.6% for Ke, 97.5% for T1/2, and 0.93 for Tmax (arithmetic mean of individual differences). We have developed a method in which liquid chromatography is coupled with electrospray tandem mass spectrometry to improve the pharmacokinetic analysis of fluconazole. Because the 90% CI AUC is within the interval proposed for the Food and Drug Administration, we concluded that Fluconal is bioequivalent to Zoltec in terms of absorption. The CV was 27.5% for the Cmax parameter, indicating that fluconazole's absorption rate is highly variable. The European Union Regulatory Agency accepts an interval of 70-143%, and because the 90% CI for Cmax is within the interval proposed for the European Union agency, we conclude that Fluconal is bioequivalent to Zoltec for the rate of absorption.
In this study, the authors assessed the bioequivalence of two fluoxetine tablet formulations in 24 healthy volunteers of both sexes who received a single 20 mg dose of each fluoxetine formulation, and a new sensitive method for the quantification of fluoxetine and norfluoxetine in human plasma was developed. The study was conducted using an open, randomized, two-period crossover design with a 4-week washout interval. Plasma samples were obtained over a 672-hour period. Plasma fluoxetine and norfluoxetine concentrations were analyzed by combined liquid chromatography coupled to mass spectrometry (LC-MS) with positive ion electrospray ionization using selected ion recording (SIR). Kolmogorov-Smirnov's test, histograms, probit plots, and the correlation between norfluoxetine AUC(0-infinity) and fluoxetine AUC(0-infinity) were used to analyze the population distribution. The limit of quantification was 0.15 ng.ml-1 and 0.50 ng.ml-1 for both fluoxetine and norfluoxetine, respectively. Within- and between-run imprecision was less than 13% and 17%, respectively. The pharmacokinetic parameters obtained for fluoxetine and norfluoxetine after the administration of each formulation included AUC(0-672 h), AUC(0-infinity), Cmax, Cmax/AUC(0-672 h), tmax, t1/2, and Ke. The AUC values for fluoxetine were not consistent with a normal distribution, reflecting the existence of two different populations (poor and extensive metabolizers). The mean pharmacokinetic parameters for extensive fluoxetine metabolizers were 27.0 ng ml-1 for Cmax, 2064.0 ng h ml-1 for AUC(0-infinity), and 85.4 h t1/2. The mean pharmacokinetic parameters for norfluoxetine (in extensive metabolizers only) were 2532.0 ng h ml-1 for AUC(0-infinity) and 8.4 ng ml-1 for Cmax. For fluoxetine bioequivalence, the 90% CI of the individual ratio geometric mean for Psiquial/Prozac (including both extensive and poor metabolizers) was 101.6% to 121.1% for AUC(0-672 h) and 86.1% to 102.6% for Cmax. For norfluoxetine, the 90% CI of the individual ratio geometric mean for Psiquial/Prozac (including both extensive and poor metabolizers) was 90.3% to 108.3% for AUC(0-672 h) and 84.5% to 106.3% for Cmax. The new method developed (LC-MS) presented high sensitivity, specificity, and short chromatographic run for the quantification of both fluoxetine and norfluoxetine in human plasma. Since both 90% CI for AUC and Cmax geometric mean ratios were included in the 80% to 125% interval proposed by the U.S. Food and Drug Administration, Psiquial was considered bioequivalent to Prozac according to both the rate and extent of absorption. The finding that there were no significant differences in the bioequivalence assessed by either fluoxetine or norfluoxetine pharmacokinetic parameters indicates that future bioequivalence trials may be performed by quantifying fluoxetine only.
The bioequivalence of two different formulations containing roxithromycin (SPE-712-1). Oral suspension 300 mg/15 mL as test formulation and Rotram, tablets 300 mg as reference formulation, both by Schering Plough S.A., Brazil) was evaluated in 24 healthy volunteers of both sexes (12 male and 12 female). The study was conducted open with randomized two-period crossover design and a 14-day washout period. Each subject received 300 mg of each roxithromycin formulation. Plasma samples were obtained over a 72-hour interval and roxithromycin concentrations were analyzed by combined LC-MS/MS with positive ion electrospray ionization using selected ion monitoring method. From the plasma roxithromycin concentration vs time curves the following pharmacokinetic parameters were obtained: AUC(0-72 h), AUC(0-infinity), Cmax, t1/2 ratios and tmax individual differences. The 90% for confidence interval (CI) of geometric mean SPE-712-L/Rotram individual percent ratio were 105.0-128.3% for AUC(0-72 h), and 78.4-96.9 for Cmax. Although this 90% CI were marginally outside the interval proposed by the Food and Drug Administration, the probability assessed by the two-one sided West for ratios was included in the 0.8-1.25 interval, as we concluded that SPE-712-L oral suspension formulation was bioequivalent to Rotram tablet formulation for the extent and rate of absorption.
Increasing evidence has implicated TNF-alpha as a pivotal molecule involved in the systemic inflammatory manifestations of ENL, an acute inflammatory complication that may occur in the chronic course of leprosy. In the present study, the mechanism of action of pentoxifylline (PTX) as an alternative therapy for management of leprosy reactions has been evaluated. The effect of PTX on TNF-alpha production was examined in leprosy patients at the protein level and at the transcriptional level as well. Treatment of ENL patients with PTX (1200 mg daily) ameliorated the systemic symptoms and favoured the evolution of reactional leprosy lesions. Serum TNF-alpha was assayed before and during treatment with PTX in 15 patients. The increased TNF-alpha levels seen in the circulation during the reaction were dramatically reduced within 3-7 days of therapy. No significant effect on serum IL-6 was noted. In vitro TNF-alpha production was assayed upon culture stimulation with Mycobacterium leprae. A reduction of inducible TNF-alpha in peripheral blood mononuclear cells (PBMC) was seen after 1-2 weeks of in vivo administration of PTX. Furthermore, no effect of the drug on IL-10 secretion was detected in these cultures. A kinetic analysis of the expression of TNF-alpha and IL-6 mRNA at the site of leprosy lesion was performed in six reactional patients by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The amount of TNF-alpha mRNA was increased in the tissue during ENL compared with before the reaction, and decreased thereafter following treatment for reaction (either PTX or thalidomide). These data suggest that PTX inhibits TNF-alpha production in ENL patients both in vivo and in vitro, and it may be useful in the treatment of leprosy patients undergoing ENL.
OBJECTIVE: To compare the bioavailability of 2 sertraline tablets formulations (Tolrest from Laboratórios Biosintética, and Zoloft from Laboratórios Pfizer, Brazil) in 24 healthy volunteers of both sexes (12 male and 12 female) who received a single 50 mg dose of each sertraline formulation. MATERIAL AND METHODS: The study was conducted open with randomized two-period crossover design and a 14-day washout period. Plasma samples were obtained over a 96-hour interval and sertraline concentrations were analyzed by combined reversed phase liquid chromatography and tandem mass spectrometry (LC-MS-MS) with positive ion electrospray ionization using selected ion monitoring method. From the plasma sertraline concentration vs. time curves the following pharmacokinetic parameters were obtained: AUC(0-96h), AUC(0-infinity), Cmax, Cmax/AUC(0-96h), Tmax, ke, and t(1/2). RESULTS: Pharmacokinetic parameters presented normal distribution according to Probit' s plot and Kolmogorov Smirnov's test, and the variance of AUC(0-96h), AUC(0-infinity) or Cmax were homoscedastic. Geometric mean Tolrest/Zoloft individual percent ratio was 95.22% for AUC(0-96h), 99.87% for Cmax, 100.4% for AUC(0-infinity), 103.6% for Ke, 96.0% for t(1/2) and 93.7% for Tmax. CONCLUSION: Since the 90% CI for both Cmax and AUC(0-96h) mean ratio were within the 80-125% interval proposed by the Food and Drug Administration, it was concluded that Tolrest was bioequivalent to Zolof for both extent and rate of absorption in a single dose administration.
The present work shows that gangliosides prevent glutamate cytotoxicity in PC12 cells. Similarly, ouabain (1 and 5 micrograms/ml) protected cells against glutamate-induced toxicity in a dose-dependent manner. The cytoprotection observed with ouabain was not maintained after its association with gangliosides and glutamate. Also, the effects of gangliosides in blocking glutamate cytotoxicity did not persist in the presence of arachidonic acid. The results indicate that a higher degree of sodium pump inhibition is necessary for ouabain cytotoxicity to be manifested, and that a partial enzyme inhibition may actually be protective to glutamate and/or arachidonic acid-induced toxicity in PC12 cells.