Objetivo: Avaliar a farmacocinética da vildagliptina em animais sadios e diabéticos, através da análise dos níveis plasmáticos totais e livres teciduais, empregando-se a técnica de microdiálise. Metodologia: A doença foi induzida nos animais através da administração de 42mg/kg de aloxano através da via intravenosa (i.v.). A vildagliptina foi administrada nas doses de 50 mg/kg (n = 6) e 75 mg/kg (n = 6) via i.v. nos animais diabéticos e na dose de 50 mg/kg (n = 6) nos animais sadios. As concentrações plasmáticas foram quantificadas por CLAE-EM-EM em método desenvolvido e validado. A ligação às proteínas plasmáticas foi determinada por microdiálise, assim como a avaliação tecidual. As sondas de microdiálise foram calibradas in vitro através de diálise e retrodiálise e in vivo utilizando retrodiálise. Para determinação das concentrações teciduais, uma segunda metodologia foi desenvolvida e validada em CLAE-EM-EM. Avaliações compartimentais (software Scientist ®) e não compartimentais (software Excel ®) foram realizadas. Resultados e Discussão: A ligação as proteínas plasmáticas apresentou um valor médio de 9,44 % ± 3,23, condizente com valores encontrados na literatura. Os valores de Ke, clearance, tempo de meia vida, MRT e VDss não apresentaram diferença estatística significativa entre as diferentes doses administradas nos animais diabéticos e entre os animais sadios. As calibrações in vitro por diálise e retrodiálise apresentaram uma recuperação média de 30%, sem diferença estatística entre as duas metodologias empregadas (α = 0,05). A recuperação in vivo também apresentou o mesmo valor médio de recuperação. A penetração tecidual do fármaco em animais diabéticos para as diferentes doses estudadas apresentou mesmo valor nos tecidos estudados, uma média de 0,20. A penetração tecidual semelhante no animal diabético pode ser devido ao dano similar entre os órgãos sofrido durante a indução da doença. Já os animais sadios apresentaram penetração tecidual similar no músculo sem diferença estatística significativa em relação aos diabéticos, entretanto no fígado foi observada uma penetração quarenta e quatro vezes inferior a observada no músculo. Essa disparidade pode ser atribuída a diferença de expressão de proteínas transportadoras no fígado do animal diabetico quando comparado ao sadio. O perfil farmacocinético plasmático foi semelhante entre os dois grupos avaliados, sendo que os parâmetros não diferiram estatisticamente (α = 0,05). Foi empregado o modelo de dois compartimentos para prever as concentrações teciduais. A previsão supõe concentrações superiores as encontradas experimentalmente, contradizendo dados de literatura. Esses dados são inéditos na literatura e demostram a importância da determinação do fármaco em tecidos alvo, uma vez que nem sempre modelos matemáticos conseguem prever a realidade fisiológica. Conclusões: As metodologias analíticas para quantificação da vildagliptina em microdialisado e plasma foram desenvolvidas e validadas, seguindo os requisitos do FDA. O perfil farmacocinético plasmático foi adequadamente descrito pelo modelo de 2 compartimentos. Os perfis teciduais obtidos nesse trabalho podem contribuir para o melhor entendimento dos mecanismos farmacológicos envolvidos e contribuir para futura otimização de terapias. / Objective: To evaluate the pharmacokinetics of vildagliptin in healthy and diabetic animals using a microdialysis technique. Methodology: Diabetes was induced in animals by administration of 42 mg/kg of alloxan intravenously (iv). Vildagliptin was administered intravenously as 50 mg/kg (n = 6) and 75 mg/kg doses (n = 6) in the diabetic animals and as a 50 mg/kg dose (n = 6) in healthy animals. Plasma concentrations were quantified by a HPLC-MS-MS method developed and validated. The plasma protein binding was determined by microdialysis and tissue evaluation. Microdialysis probes were calibrated in vitro using dialysis and retrodialysis and in vivo using retrodialysis. A second method was developed and validated using HPLC-MS-MS to determine tissue concentrations. Results and Discussion: Mean plasma protein was 9.44% ± 3.23, consistent with values reported in the literature. The values of Ke, clearance, half-life, MRT and Vdss showed no statistical difference between the evaluated doses in diabetic animals and between healthy animals (α = 0.05). Calibrations in vitro by dialysis and retrodialysis showed mean recovery of 30%, with no statistical difference between the two methodologies. Mean recovery in vivo also showed the same value. The tissue penetration of the drug in diabetic animals for the different doses studied showed the same value in both tissues studied, an mean of 0.20. The tissue penetration similar in diabetic animals could be due to the similar damage suffered between organs during induction of the disease. The healthy animals showed similar muscle penetration, compared with diabetics animals, although the liver showed a penetration forty four times lower than muscle. This discrepancy can be attributed to differential expression of transporter proteins in the liver of diabetic animals, when compared to the healthy group. The plasma pharmacokinetic profile was similar between the investigated groups, and the parameters did not differ. The two-compartment model was employed to describe the data and used to predict the concentration in the tissues. This is the first study to present these tissue profiles, which presented concentrations below the estimated by the model. These data demonstrate the importance of determining the drug inside the target tissue, as the mathematical models sometimes cannot predict physiology. Conclusions: The analytical methods for the quantification of vildagliptin in microdialysate and plasma were developed and validated by following the requirements of the FDA. The plasma pharmacokinetic profile was correctly described by the model of two compartmental models. The novel tissue profiles obtained in this study may contribute to a better understanding of the pharmacological mechanisms involved and contribute to optimization of future therapies.

abstract: Patients with schizophrenia have impaired cognitive flexibility, as evidenced by behaviors of perseveration. Cognitive impairments may be due to dysregulation of glutamate and/or loss of neuronal plasticity in the medial prefrontal cortex (mPFC). The purpose of these studies was to examine the effects of mGluR5 positive allosteric modulators (PAMs) alone and in combination with the NMDAR antagonist MK-801, a pharmacological model of schizophrenia. An operant-based cognitive set-shifting task was utilized to assess cognitive flexibility, in vivo microdialysis procedures to measure extracellular glutamate levels in the mPFC, and diolistic labeling to assess the effects on dendritic spine density and morphology in the mPFC. Results revealed that chronic administration of the mGluR5 PAM CDPPB was able to significantly reduce the effects of chronically administered MK-801 on both behavioral perseveration and glutamate neurotransmission. Results also showed that CDPPB had no evidence of an effect on dendritic spine density or morphology, but the mGluR5 negative allosteric modulator fenobam caused significant increases in spine density and the frequency of occurrence of spines with smaller head diameters. Conclusions include that CDPPB is able to reverse MK-801 induced cognitive deficits as well as alterations in mPFC glutamate neurochemistry. The culmination of these studies add further support for targeting mGluR5 with PAMs as a novel mechanism to alleviate cognitive impairments in patients with schizophrenia. / Dissertation/Thesis / Doctoral Dissertation Psychology 2014

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-08-22T12:25:47Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Bedor_Noely_Tese.pdf: 3627031 bytes, checksum: bffd788a8d2405e29a360eb20d7a089c (MD5) / Made available in DSpace on 2016-08-22T12:25:47Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Bedor_Noely_Tese.pdf: 3627031 bytes, checksum: bffd788a8d2405e29a360eb20d7a089c (MD5) Previous issue date: 2016-02-29 / FACEPE / CAPEs / A olanzapina é um antipsicótico atípico cujo efeito colateral ganho de peso pode ser a causa de não adesão ao tratamento. Não se sabe qual o mecanismo fisiológico que explicaria essa relação, daí a necessidade de pesquisas na área da farmacocinética (PK) e da farmacodinâmica (PD). Assim, o objetivo da tese buscou entender melhor a farmacocinética da OLZ através de estudos em humanos e animal, visando otimização na posologia, empregando correlações de PK/PD. Foi realizado um estudo de bioequivalência utilizando comprimidos teste e referência 10 mg. Em seguida, para evidenciar possíveis variações em alguns parâmetros de PK e buscar dados de PD, realizou-se um estudo de PK plasmática e microdiálise cerebral (MD) em animais. Paralelamente, foram preparadas sondas de MD em laboratório e realizada uma comparação com as sondas comercias, através de diálise e retrodiálise in vitro. Para o estudo com MD, foi realizada a cirurgia estereotáxica para implantação da sonda e punção da artéria e veia femoral, para administração da dose e coleta de amostras plasmáticas. O estudo de bioequivalência foi do tipo aberto, aleatório, cruzado 2 x 2, com 28 voluntários sadios. O método de quantificação da OLZ, em plasma humano por CL-EM/EM, mostrou-se simples, rápido e sensível com o tempo de corrida de 2 min. Os parâmetros farmacocinéticos em humanos Cmáx, Tmáx, ASC0-t e ASC0-∞ obtidos para a formulação teste e referência foram de 11,47±3,65; 12,50±3,73 ng·mL-1; 4,50±1,85; 4,30±2,37 h; 240,97±78,46 e 283,12±94,84 ng·(mL·h)-1, respectivamente, sendo os medicamentos bioequivalentes. As amostras de plasma de animais foram analisadas por CL-EM/EM e de acordo com disposição cinética, o modelo bicompartimental foi o que melhor se ajustou aos dados experimentais. Os parâmetros farmacocinéticos obtidos através de Excel, Winnolin e Pksolver foram considerados próximos. O local de inserção da sonda foi confirmado através de histologia. Os resultados da recuperação por diálise e retrodiálise para as sondas comercial e de laboratório foram 19,27±10,11; 19,42%±6,54; 23,06±2,36; 24,59±3,14, respectivamente. Portanto, as mesmas podem ser intercambiáveis. Por fim, essa tese colaborou com a formação de recursos humanos para inserir no grupo de pesquisa uma técnica pouco utilizada no Brasil, microdiálise cerebral, além de publicação de artigo em periódico internacional. / The olanzapine is an atypical antipsychotic, which confers effects as weight gain, which may be the cause of non-compliance. It is not known what the physiological mechanism that is related to weight gain, hence it is necessary improve the research in pharmacokinetics (PK) and pharmacodynamics (PD). The aim of the thesis sought to better understand the pharmacokinetics of OLZ through studies in humans and animals, aimed at optimizing the dosage regimen using PK / PD correlation. It conducted a bioequivalence study using test and reference tablets 10 mg. Then, to understand possible variations in some parameters PK and PD data fetch, it was performed a plasma PK study and brain microdialysis (MD) in animals. At the same time, MD probes were prepared at the laboratory and compared with commercial probe by dialysis and retrodialysis in vitro. For the MD study, stereotaxic surgery was performed to implant the MD probe and puncture of the femoral artery and vein was performed to dosing and collecting of plasma samples. The bioequivalence study was open, randomized, crossover 2x2 with 28 healthy participants. The method of quantification of OLZ in human plasma by LC-MS/MS developed and validated is simple, fast and sensitive with analysis time (2 min). The human pharmacokinetic parameters Cmax, Tmax, AUC0-t e AUC0-∞ obtained for the test and reference formulation were de 11.47±3.65, 12.50 ± 3.73 ng·mL-1, 4.50 ± 1.85, 4.30 ± 2.37 h, 240.97±78.46 e 283.12 ± 94.84 ng·(mL·h)-1, considering the formulations bioequivalent. Plasma samples of animals were analyzed by LC-MS / MS and according to kinetic disposition, the two-compartment model was the best fit to the experimental data. Pharmacokinetic parameters obtained from Excel, Winnolin and Pksolver were considered approximate. The insertion of the probe into the striatum of the brain of animals was confirmed by histological sections. The results for recovery by dialysis and retrodialysis for commercial and laboratory probes were 19.27% ± 10.11; 19.42% ± 6.54; 23.06% ± 2.36; 24.59% ± 3.14. Therefore, they can be interchangeable. Finally, this thesis collaborated with the training of human resources to insert in the research group, a technique not widely used in Brazil, besides publishing scientific paper in an international journal.

Recent approaches in depression therapeutics include triple reuptake inhibitors, drugs that target three monoamine systems. Using in vivo electrophysiological and microdialysis techniques, the effects of 2- and 14-day treatments of escitalopram, nomifensine and the co-administration of these two drugs (TRI) were examined in male Sprague-Dawley rats. Short- and long-term TRI administration decreased NE firing and had no effect on DA neurons. Normal 5-HT firing rates were maintained after 2-day TRI administration compared to the robust inhibitory action of selective serotonin reuptake inhibitors (SSRIs). Escitalopram treatment enhanced the tonic activation of the 5-HT1A receptors given the increase in firing observed following WAY100635 administration. Nomifensine treatment enhanced tonic activation of the α2–adrenoceptors following idazoxan administration. TRI treatment caused a robust increase in extracellular DA levels that was in part mediated by a serotonergic contribution. Therapeutic effects of the drugs examined in this study may be due to the enhancement of 5-HT, NE and/or DA neurotransmission.

Chronic pain is a significant healthcare problem. It is disabling and diminishes quality of life. Opioids, such as morphine, remain a primary pharmacologic management for chronic pain. Opioids act at mu opioid receptors (MOPr) in the rostroventral medial medulla (RVM) to produce their analgesic effect. The RVM is a critical relay in pain inhibitory and facilitatory pathways of pain modulation. Furthermore, chronic inflammatory pain, produced by CFA hindpaw injection, leads to adaptive changes in the RVM that change the balance of these pathways and increase the potency of opioids. MOPr are known to produce their effects via Gi/o proteins. Pretreatment of several pain modulatory regions with pertussis toxin (PTX) effectively attenuates the antinociceptive effects of MOPr agonists, such as DAMGO. In the RVM, PTX effectively reduced DAMGO stimulated GTPãS binding in uninjured rats. However, despite their effective inactivation of Gi/o proteins, PTX did not diminish the antinociceptive effects of DAMGO in the RVM of uninjured rats. In contrast, in rats with a chronic inflammatory injury, PTX completely abolished the antinociceptive effects of DAMGO. These results suggest a transition from Gi/o independent to Gi/o dependent mechanisms following CFA treatment. In addition, the anti-hyperalgesic effects of DAMGO were not inhibited by PTX, suggesting that DAMGO produces anti-hyperalgesia and antinociception by different mechanisms. In the RVM, MOPr are present both postsynaptically and presynaptically. Postsynaptic MOPr are thought to produce antinociception by activating GIRK channels, resulting in hyperpolarization and inhibition of pain facilitatory neurons. Indeed, inhibition of GIRK channels in the RVM, via microinjection of tertiapin-Q, attenuated the antinociceptive effects of DAMGO in uninjured rats, providing the first behavioral evidence that MOPr agonists produce analgesia via this proposed mechanism. Interestingly, however, tertiapin-Q did not block the anti-hyperalgesic effects of DAMGO, nor did it diminish the antinociceptive effects of DAMGO in the contralateral hindpaw of CFA treated rats. Furthermore, these differential effects of tertiapin-Q in the uninjured and injured rats are not the result of transcriptional down regulation of GIRK channels in the RVM. Finally, tertiapin-Q alone in the RVM produced a modest antinociception in uninjured rats, providing the first evidence of constitutive GIRK channel activity in the RVM and demonstrating a role for these in pain modulation. Presynaptic MOPr are thought to produce antinociception by decreasing GABA release onto pain inhibitory neurons. Indeed, microdialysis studies demonstrated that levels of GABA release were decreased in response to DAMGO perfused into the RVM, as well as to high potassium after perfusion of DAMGO. However, they were not decreased in rats after CFA treatment. This suggests that chronic inflammatory injury alters the presynaptic actions of MOPr agonists in the RVM. Interestingly, levels of GLU release where not altered by DAMGO in uninjured or injured rats. Moreover, basal levels of GLU and GABA were also unaltered by CFA treatment. In conclusion, although MOPr mediate their antinociceptive effects in other pain modulatory regions via Gi/o proteins, this is not the case in the RVM during an uninjured state. However, MOPr-induced antinociception transitions from Gi/o independent to Gi/o dependent mechanisms after CFA treatment. Additionally, these results support both the presynaptic and the postsynaptic postulates by which MOPr agonists are thought to produce their analgesic effects. However, although CFA treatment alters the activity of neurons in the RVM and promotes changes that result in an enhanced anti-hyperalgesic and antinociceptive response to DAMGO in the RVM, neither the postsynaptic nor the presynaptic mechanism, in isolation, seem to account for this enhancement.

The effect of neonatal manganese (Mn) exposure in a 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease was investigated. Pregnant Wistar rats were given drinking water with 10,000ppm of Manganese (MnCl2.4H2O) from the time of conception until weaning on the 21st day after delivery. Control rats consumed tap water. Three days after the birth, other groups of neonatal rat pups were pretreated with desipramine (20mg/kg ip 1h) prior to bilateral ICV administration of 6-OHDA or its vehicle, saline-ascorbic (0.1%) (control). Two months after the birth, striatal dopamine and homovanilic acid efflux measured by an in vivo microdialysis method were reduced in rats lesioned with 6-OHDA. Co-exposure to perinatal Mn did not modify neurotransmission alterations. However, there were prominent abnormalities in behavioral testing in rats perinatally exposed to Mn and treated neonatally with 6-OHDA. These findings demonstrate that although Mn did not further damage neurotransmitter activity in the neostriatum, ontogenetic exposure to Mn enhances the behavioral toxicity to 6-OHDA.

Study Design: Controlled laboratory study. Objectives: To determine the time course of dexamethasone sodium phosphate (Dex-P) iontophoresis delivery to underlying tissues using microdialysis. Background: The efficacy of iontophoresis at delivering Dex-P through the skin is unknown in humans because of the lack of minimally invasive measurement techniques. Methods: Sixty-four healthy male participants (age = 24.4 ± 3.3 yrs, height = 71.8 ± 2.5 in, weight = 181.8 ± 26.1 lbs) were randomly assigned into one of six groups: 1) 1 mA current, 1 mm probes depth ; 2) 1 mA current, 4 mm probes depth; 3) 2 mA current, 1 mm probes depth; 4) 2 mA current, 4 mm probes depth; 5) in vivo retrodialysis; and 6) skin perfusion flowmetry. Microdialysis probes assess the combined recovery (Dextotal) of Dex-P, dexamethasone (Dex) and its metabolite. In vivo calibration of the microdialysis probes occurred via retrodialysis. Laser Doppler flowmetry assessed skin perfusion. Results: There was no difference of Dextotal between current intensities (P = 0.99) but a greater amount of Dextotal was recovered by the 1 mm probe (P < 0.0001) compared to the 4 mm probe. Peak means for the 1 and 2 mA at 1 mm were 10.8 ± 8.1 and 7.7 ± 5.5 μg/ml and at 4mm being 2.0 ± 0.8 and 1.3 ± 0.9 μg/ml, respectively. Skin perfusion rapidly increased during both current intensity treatments, but significantly decreased before the conclusion of the 1 mA treatment (P < 0.0001). Peak skin perfusion was 741.4 ± 408.7% and 711.6 ± 260.8% baseline for 1 and 2 mA intensities, respectively. Conclusion: Iontophoresis delivery of Dex-P was successful measured in vivo through human skin. Significant concentrations of Dextotal were found regardless of current intensity. Though current induced vasodilation occurred, it did not significantly affect the tissue accumulation of Dextotal.

No description available.

Introduktion: Mikrodialys används för att studera metabola förändringar i olika vävnader. Vid mikrodialys sätts en kateter i muskeln vilket inducerar en traumafas, som kan påverka frisläpp av substanser. En 120 minuters stabiliseringstid har föreslagits så att metabola förändringar p.g.a. traumat ska normaliseras och ej påverka resultatet. En lång stabiliseringstid leder dock till att dessa studier är tidskrävande och därför även dyra och svårgenomförda. Syfte: Att undersöka och fastställa lämplig stabiliseringsperiod för mikrodialys av serotonin och glutamat i massetermuskeln hos friska individer samt hos patienter med myofascial TMD. Material och metod: Intramuskulär mikrodialys utfördes och dialysat samlades in på 15 friska kontroller samt 15 patienter med myofascial TMD för analys av serotonin och glutamat. För att tillåta vävnaden återhämta sig från traumafasen utvärderades en 120-minuters stabiliseringsperiod, där de första 20 minuterna utgjorde ursprunglig baseline och de sista 20 minuterna stabiliserad baseline. Resultat: Ingen signifikant förändring av serotonin och glutamat observerades över tid för kontroll-gruppen (P>0,05). För TMD-gruppen sågs däremot såg en signifikant sänkning av serotoninhalten over tid (P<0,001) följt av en signifikant ökning mellan tidpunkten T100-120 och T120-140 (P<0,001). För glutamat sågs en reduktion vid tidpunkten T20-40 jämför med det ursprungliga baselinevärdet (P<0,05). Konklusion: Resultaten antyder att 20 minuters stabiliseringsperiod är tillräcklig för friska individer vid mikrodialys av serotonin och glutamat i masseter muskeln. Hos patienter med myofacial TMD är glutamat-nivåerna stabiliserade efter 40 minuter. Serotonin nivåerna är däremot inte stabiliserade efter 120 minuter, vilket tyder på en spontan ökning av intramuskulär serotonin 2 timmar efter införandet av katetern. / Introduction: Microdialysis is a technique used to study metabolic changes in tissues. When performing microdialysis, a catheter is inserted into the muscle inducing a trauma phase, which may affect the release of substances. A 2-hour stabilization period has been suggested to allow tissues to recover from metabolic changes following the trauma. A long stabilization period however, makes these studies time-consuming and thus expensive.Aim: To investigate the necessary stabilization period for microdialysis of serotonin and glutamate in the masseter muscle in healthy subjects and in patients with myofascial TMD.Material and Methods: Intramuscular microdialysis was carried out in 15 patients with myofascial TMD and 15 healthy controls to collect serotonin and glutamate. To allow the tissue to recover following the probe insertion, a 120-min stabilization period was evaluated where the first 20 min served as the zero baseline and the last 20 min as the stabilized baseline. Results: No significant alterations of serotonin or glutamate were observed over the 2-hour period for the controls (P>0.05). For the TMD group, a significant decrease of serotonin was observed over time (P<0.001) followed by a significant increase between T100-120 and T120-140 (P<0.001). For glutamate, a significant reduction was observed at T20-40 compared with the zero baseline (P<0.05). Conclusion: A 20-min stabilization period is sufficient for healthy subjects for microdialysis of serotonin and glutamate in the masseter muscle. In patients with myofascial TMD, glutamate levels are stabilized after 40 minutes. Serotonin levels are not stabilized after 2 hours indicating a spontaneous increase of serotonin.

In this thesis, fluid characteristics and sampling efficiency of high molecular weight cut-off microdialysis are presented, with the aim of improving the understanding of microdialysis sampling mechanisms and its performance regarding extraction efficiency of biological fluid and biomarkers. Microdialysis is a well-established clinical sampling tool for monitoring small biomarkers such as lactate and glucose. In recent years, interest has raised in using high molecular weight cut-off microdialysis to sample macro molecules such as neuropeptides, cytokines and proteins. However, with the increase of the membrane pore size, high molecular weight cut-off microdialysis exhibits drawbacks such like unstable catheter performance, imbalanced fluid recovery, low and unstable molecule extraction efficiency, etc. But still, the fluid characteristics of high molecular weight cut-off microdialysis is rarely studied, and the clinical or in vitro molecule sampling efficiency from recent studies vary from each other and are difficult to compare.   Therefore, in this thesis three aspects of high molecular weight cut-off microdialysis have been explored. The first, the fluid characteristics of large pore microdialysis has been investigated, theoretically and experimentally. The results suggest that the experimental fluid recovery is in consistency with its theoretical formula. The second, the macromolecule transport behaviour has been visualized and semi-quantified, using an in vitro test system and fluorescence imaging. The third, two in vitro tests have been done to mimic in vivo cerebrospinal fluid sampling under pressurization, using native and differently surface modified catheters. As results, individual protein/peptide extraction efficiencies were achieved, using targeted mass spectrometry analysis. In summary, a theory system of the fluid characteristics of high molecular weight cut-off microdialysis has been built and testified; Macromolecular transport of microdialysis catheter has been visualized; In vivo biomolecules sampling has been simulated by well-defined in vitro studies; Individual biomolecular extraction efficiency has been shown; Different surface modifications of microdialysis catheter have been investigated. It was found that, improved sampling performance can be achieved, in terms of balanced fluid recovery and controlled protein extraction efficiency.