Problems of emerging antibiotic resistance are becoming a serious threat worldwide, and at the same time, the interest to develop new antimicrobials has declined. There is consequently a need for efficient methods to develop new treatments that minimize the risk of resistance development and that are effective on infections caused by resistant strains. Based on in silico mathematical models, describing the time course of exposure (Pharmacokinetics, PK) and effect (Pharmacodynamics, PD) of a drug, information can be collected and the outcome of various exposures may be predicted. A general model structure, that characterizes the most important features of the system, has advantages as it can be used for different situations. The aim of this thesis was to develop Pharmacokinetic-Pharmacodynamic (PKPD) models describing the bacterial growth and killing after mono- and combination exposures to antibiotics and to explore the predictive ability of PKPD-models across preclinical experimental systems. Models were evaluated on data from other experimental settings, including prediction into animals. A PKPD model characterizing the growth and killing for a range of E. coli bacteria strains, with different MICs, as well as emergence of resistance, was developed.  The PKPD model was able to predict results from different experimental conditions including high start inoculum experiments, a range of laboratory and clinical strains as well as experiments where wild-type and mutant bacteria are competing at different drug concentrations. A PKPD model, developed based on in vitro data, was also illustrated to have the capability to replicate the data from an in vivo study. This thesis illustrates the potential of PKPD models to characterize in vitro data and their usage for predictions of different types of experiments. The thesis supports the use of PKPD models to facilitate development of new drugs and to improve the use of existing antibiotics.

Liposomal ciprofloxacin (Lipo-CPFX) is being developed for inhalation, with a goal of sustaining the therapeutic activity, compared to unformulated ciprofloxacin (CPFX). However, the kinetics and mechanism of its sustained local lung retention and pharmacological activity are yet to be fully characterized. This project hypothesized that Lipo-CPFX enables slower and sustained lung epithelial transport and uptake, compared to CPFX, thereby producing prolonged local pharmacological actions. The human bronchial epithelial Calu-3 cells were used as monolayers to characterize the kinetics and mechanism of transport and/or uptake, and to assess the effects of such slow kinetics for Lipo-CPFX on its inhibition against lipopolysaccharide (LPS)-induced proinflammatory IL-8 release. The transport fluxes for Lipo-CPFX across the highly restricted Calu-3 cell monolayers was transepithelial electrical resistance-independent, which suggested predominant transcellular transport. Compared to CPFX, Lipo-CPFX showed 6-18 times slower transport, while the flux was increased with increasing concentration proportionally without saturation. Its unaltered transport by cellular energy depletion, transport inhibition by a reduced temperature (4 oC) and endocytosis/lipid fusion inhibitors, filipin and LysoPC, and increased transport by excess empty liposomes collectively suggested cell energy-independent, lipid bilayer fusion mechanisms for the Lipo-CPFX transport across the Calu-3 cells. Likewise, Lipo-CPFX showed 2-4 fold lower cellular uptake than CPFX, proportional to concentration. Lipo-CPFX exhibited significant inhibitory activities at ≥ 0.01 mg/mL on LPS-induced IL-8 release from the Calu-3 cells, which was equipotent to CPFX. Upon 24 h pre-incubation, Lipo-CPFX caused 36.9 and 47.5 % inhibition at 0.01 and 0.05 mg/mL, respectively, while CPFX failed to do so. However, the effect was negated upon repeated wash of the mucosal cell surface, speculating the importance of cell membrane-associated drug/formulation on the inhibitory activities for Lipo-CPFX. Upon 24 h transport, Lipo-CPFX retained 79.0 % of the 4 µg dose on the mucosal cell surface, which was 1.9-times greater than 40.7 % for CPFX. As a result, when LPS was added at 24 h of the transport, Lipo-CPFX was still capable of causing 60.1 % inhibition, as its sustained local anti-inflammatory activity; CPFX however also exhibited equipotent inhibition, by virtue of comparable cellular drug uptake/transport.

Bacterial infections are becoming progressively more difficult to treat due to antibiotic resistance and the decreasing rate at which new antibiotics are brought to market. The Turos laboratory has attempted to tackle this problem for the last 15 years with the discovery of N-thiolated β-lactams leading to the design of polyacrylate nanoparticles to deliver these and other drugs. Chapter 1 discusses many reported instances of utilizing different types of polymeric nanoparticles to deliver antibiotics. Poly(lactic-co-glycolic acid) (PLGA), poly(alkyl cyanoacrylate) (PACA), poly(styrene-co-butylacrylate), and chitosan are the main polymers used to make nanoparticles. Chapter 2 describes the synthesis, antibacterial activity, and mechanism of action of N-acyl ciprofloxacins, which have demonstrated in vitro bioactivity against Staphyloccocus aureus, methicillin-resistant Staphylococcus aureus, Bacillus anthracis, Enterococcus faecalis, Bartonella, and E. coli. Antimicrobial activity was found to diminish with increasingly lipophilic acyl groups of the derivatives. The N-acyl ciprofloxacins were found to utilize the same mechanism of action as the parent drug, ciprofloxacin, however, several exhibited lower mutation frequencies. Chapter 3 discusses the use of the N-acyl ciprofloxacins as probes for experimentation on the poly(vinyl benzoate) nanoparticles. These compounds were incorporated into poly(vinyl benzoate) nanoparticles, also designed in the Turos laboratory, to determine the effects of the lipophilic acyl groups on drug loading and drug release. N-acyl ciprofloxacins with higher lipophilicities (predicted logP values) experienced higher drug loading than the less lipophilic counterparts. However, the nanoparticles were unable to release large amounts of entrapped drug. N-acyl ciprofloxacins with increased hydrophilicity were found to either not be incorporated at all, or in incredibly small amounts. Drug release studies of these drugs indicate that possible the hydrophobic compounds that were associated with the nanoparticles were done so via adsorption onto the surface resulting in a burst release of drug. The work is concluded in Chapter 4, followed by experimental procedures and spectral data in Chapters 5 and 6.

Σκοπός της μελέτης ήταν να καθορισθεί εάν η μονοθεραπεία με σιπροφλοξασίνη σε υψηλές δόσεις (400 mg x 3φορές/ημέρα ενδοφλεβίως), είναι αποδεκτή ως αρχική εμπειρική αντιμικροβιακή θεραπεία σε εμπύρετα επεισόδια ουδετεροπενικών ασθενών με αιματολογικά νοσήματα σε σχέση με τον καθιερωμένο συνδυασμό κεφταζιντίμης+αμικασίνης, και επιπρόσθετα να διερευνηθεί εάν η χορήγηση σιπροφλοξασίνης από του στόματος (750 mg x 2 φορές/ημέρα) μπορεί αποτελεσματικά να υποκαταστήσει την ενδοφλέβια χορήγηση 72 ώρες μετά την έναρξη αυτής. Σε προοπτική τυχαιοποιημένη μελέτη μελετήθηκαν συνολικά 123 εμπύρετα ουδετεροπενικά επεισόδια, σε 61 από τα οποία χορηγήθηκε σιπροφλοξασίνη και σε 62 ο συνδυασμός κεφταζιντίμης+αμικασίνης. Επί του συνόλου σε 96 (78,1%) η υποκείμενη νόσος ήταν οξεία λευχαιμία, σε 24 (19,5%) μη-Hodgkin λέμφωμα και σε 3 (2,4%) απλαστική αναιμία. Επιτυχής κλινική ανταπόκριση στο τέλος της θεραπείας χωρίς τροποποίηση αυτής παρατηρήθηκε σε 30/61 (49,2%) επεισόδια στην ομάδα της σιπροφλοξασίνης και σε 31/62 (50%) στην ομάδα της κεφταζιντίμης+αμικασίνης (p=1,00). Σε 40/61 (65,6%) επεισόδια στην ομάδα της σιπροφλοξασίνης η ενδοφλέβια θεραπεία αντικαταστάθηκε με από του στόματος μετά από 4,3±069 ημέρες, με επιτυχή ανταπόκριση στο 75%(30/40) αυτών. Επί του συνόλου των επεισοδίων στις μικροβιολογικά αποδεδειγμένες λοιμώξεις κυριαρχούσαν οι Gram-θετικοί μικροοργανισμοί σε ποσοστό 56,8%, ενώ οι Gram-αρνητικοί αποτελούσαν το 43,2%. Η ανταπόκριση στην θεραπεία με σιπροφλοξασίνη στις μικροβιολογικά αποδεδειγμένες λοιμώξεις (βακτηριαιμίες και άλλες λοιμώξεις) ήταν 40,0%, στις κλινικά αποδεδειγμένες λοιμώξεις 50% και στα επεισόδια πυρετού αγνώστου αιτιολογίας 55,2%, έναντι 41,2%, 43,8% και 58,6% αντίστοιχα στην δεύτερη ομάδα. Ειδικότερα στις βακτηριαιμίες από gram-θετικά παθογόνα επιτυχής έκβαση παρατηρήθηκε στο 20% των επεισοδίων στην ομάδα της σιπροφλοξασίνης και στο 12,5% στην ομάδα του συνδυασμού. Στη διάρκεια της μελέτης παρατηρήθηκαν 6 επιλοιμώξεις μόνο στην ομάδα της σιπροφλοξασίνης και 4 επαναλοιμώξεις μόνο στην ομάδα του συνδυασμού κεφταζιντίμης/αμικασίνης, (p=0,006). Οι επιλοιμώξεις ήταν δύο μυκητιασικές πνευμονίες, μια λοίμωξη ουροποιητικού από P. aeruginosa, και τρεις βακτηριαιμίες από P. Aeruginosa, St. viridans και Enterococcus spp.. Οι επαναλοιμώξεις αφορούσαν δύο βακτηριαιμίες μία από P. aeruginosa και μια από St. Aureus, μία λοίμωξη ουροποιητικού από E. coli και μία γαστρεντερίτιδα από Ε. faecalis. Ο αριθμός των θανάτων στις πρώτες 72 ώρες ήταν 2 για την ομάδα της σιπροφλοξασίνης και 1 για την ομάδα κεφταζιντίμης+αμικασίνης. Επιπλέον 11 ασθενείς ακόμη (7 vs 4, p=0,363) κατέληξαν στη φάση της ουδετεροπενίας. Επί του συνόλου 12/14 θανάτους οφείλονταν σε λοίμωξη. Από τη στατιστική ανάλυση των δεδομένων προκύπτει ότι η παρατεταμένη και σοβαρή ουδετεροπενία αποτελεί σημαντικό παράγοντα για την ανταπόκριση στη θεραπεία. Στην ομάδα της σιπροφλοξασίνης ουδετεροπενία με ουδετερόφιλα <100/μl για περισσότερο από 14 ημέρες επηρεάζει εξαιρετικά ισχυρά την ανταπόκριση στη θεραπεία (p=0,003), ενώ στην ομάδα του συνδυασμού κεφταζιντίμης+αμικασίνης η διαφορά αυτή, παρότι υπάρχει, δεν εμφανίζεται στατιστικά σημαντική (p=0,07). Με ανάλυση λογαριθμικής παλινδρόμησης καθορίσθηκαν παράγοντες που κατά την έναρξη του εμπυρέτου επεισοδίου και την διάρκεια αυτού ασκούν σημαντική επίδραση στην ανταπόκριση στη θεραπεία, και επομένως είναι δυνατόν να χαρακτηρίσουν τον ασθενή ως “χαμηλού κινδύνου” για την εμφάνιση επιπλοκών. Θετική επίδραση στην ανταπόκριση στη θεραπεία κατά την έναρξη του πυρετού έχουν η απουσία κλινικής λοίμωξης, ο αριθμός των ουδετεροφίλων >100/μl, η αιματολογική κακοήθεια σε ύφεση, το διάστημα από την προηγηθείσα αντινεοπλασματική χημειοθεραπεία να είναι > από 10 ημέρες από την έναρξη αυτής και το περιβάλλον εμφάνισης της λοίμωξης να είναι αυτό της κοινότητας (εξωνοσοκομειακή λοίμωξη). Κατά τη διάρκεια του επεισοδίου οι παράγοντες αυτοί είναι η άνοδος των ουδετεροφίλων >100/μl και >500/μl, η διάρκεια της ουδετεροπενίας <100/μl να είναι <14ημέρες, καθώς και η διάρκεια της ουδετεροπενίας <500/μl να είναι επίσης <14ημέρες. Τέλος αναλύοντας τα επεισόδια με ουδετερόφιλα <100/μl για περισσότερο από 14ημέρες είχαν 2,75 φορές πιθανότητα επιτυχούς έκβασης εάν έπαιρναν το συνδυασμό κεφταζιντίμης+αμικασίνης (p=0,038, odds ratio=2,75). Ανεπιθύμητες ενέργειες παρατηρήθηκαν σε 9/61 (14,7%) επεισόδια της ομάδας της σιπροφλοξασίνης και σε 10/62 (16,1%) της ομάδας κεφταζιντίμης+αμικασίνης (p=1,00), χωρίς να απαιτηθεί σε κανένα διακοπή της θεραπείας. Συμπερασματικά φαίνεται ότι η μονοθεραπεία με υψηλές δόσεις σιπροφλοξασίνης είναι εξ’ ίσου αποτελεσματική και ασφαλής με τον καθιερωμένο συνδυασμό κεφταζιντίμης+αμικασίνης, ως εμπειρική θεραπεία ουδετεροπενικών ασθενών “χαμηλού κινδύνου” με πυρετό. Επιπρόσθετα στους ασθενείς που ανταποκρίθηκαν, έχει το πλεονέκτημα της από του στόματος χορήγησης για τη συμπλήρωση της αγωγής, ενώ συγχρόνως στερείται νεφροτοξικότητας και ωτοτοξικότητας. Παρόλα αυτά ιδιαίτερα σημαντικό είναι κατά την επιλογή της εμπειρικής αντιμικροβιακής θεραπείας, να λαμβάνεται υπόψη το μικροβιακό φάσμα που επικρατεί και η ανθεκτικότητα των μικροοργανισμών που απομονώνονται στο συγκεκριμένο νοσοκομείο. / The aim of the present study was to compare administration of ciprofloxacin, given initially at the higher intravenous dose 400 mg three times a day for at least 72 hours, followed by oral administration 750 mg twice a day, with the standard combination regimen of ceftazidime plus amikacin as empiric treatment in patients with febrile neutropenia. In a prospective study, a total of 123 febrile neutropenic patients were randomized: 61 in the ciprofloxacin group and 62 in the ceftazidime plus amikacin group. In 78,1% of the patients acute leukemia was the underlying disease, another 19,5% of the patients suffered from high-grade non-Hodgkin’s lymphoma, and the remaining 2,4% of the patients suffered from aplastic anemia. The frequency of successful clinical response without modification at the end of therapy was almost identical for ciprofloxacin [49,2% (30/61 patients)] compared with that for ceftazidime plus amikacin [50% (31/62 patients)], (p=1,00). For 40/61 (65,6%) patients, it was possible to switch from parenteral ciprofloxacin to the oral after a mean of 4,3±069 days, and the response was successful for 30/40 (75%) patients. Gram-positive organisms accounted for 56,8% of all organisms isolated. The response to therapy in ciprofloxacin group was 40,0% for the microbiologically documented infections, 50% for the clinically documented infections and 55,2% for the episodes with fever of unknown origin, compared with 41,2%, 43,8% and 58,6% respectively in ceftazidime plus amikacin group. The efficacies of the regimens against gram-positive bacteremias were 20% for the ciprofloxacin group and 12,5% for the combination group. Superinfections were seen in 6 episodes in ciprofloxacin group and 4 episodes of reinfection in ceftazidime plus amikacin group (p=0,006). 3 patients (2 of ciprofloxacin group and 1 of combination group) died within 72 hours of randomization. Another 11 patients (7 vs 4 respectively, p=0,363) died before resolution of neutropenia. Of the total 12/14 patients died because of infection. The analysis of the data shown that the prolonged and severe granulocytopenia is a critical factor for the successful outcome. In ciprofloxacin group the neutropenia with neutrophils <100/μl for 14 days or more is significantly associated with the response to treatment (p=0,003). In the combination group this association is not significant (p=0,07). Logistic regression analyses were performed to estimate the probability of success and to identify “low risk” neutropenic patients. The covariates could be assessed at the onset of fever and during treatment as well. Among the tested covariates, the following variables were significant predictors of outcome at the onset of fever: absence of signs of clinically documented infection, neutrophils >100/μl, primary disease in remission, fever developing more than 10 days from the recent course of chemotherapy and outpatients status before the onset of fever. The significant predictors of outcome during treatment were: increasing neutrophils count >100/μl, increasing neutrophils count >500/μl, neutrophils <100/μl for less than 14 days and neutrophils <500/μl for less than 14 days as well. Furthermore episodes with 14 days or more of neutropenia <100/μl treated with ceftazidime plus amikacin had response rates 2,75 times higher compared to episodes treated with ciprofloxacin (p=0,038, odds ratio=2,75). Adverse events were mostly self-limited and were observed in 9 (14,7%) ciprofloxacintreated patients and 10 (16,1%) patients who were receiving the combination. In summary, this trial suggests that high-dose ciprofloxacin is therapeutically equivalent to the routine regimen of ceftazidime plus amikacin in “low risk” febrile neutropenic patients and has the advantages of intravenous and oral administration, without nepfro- and ototoxicity. However, it is very important that before an empirical therapy is chosen each hospital determine bacteriologic predominance and perform resistance surveillance.

Steroidal estrogens and antibiotics used in veterinary and human medicine are detected in livestock manure or sewage sludge and biosolids. Biodegradation is an important process by which estrogenic compounds are removed from organic amendments, but antibiotics have been shown to impede microbial communities. Although both compounds are often present in these media, the fate of estrogens in association with antibiotics has not been previously studied. In this study, both rates of tetracycline (40 and 200 mg kg-1) in liquid swine manure induced a lag phase of 40 to 50 days prior to the onset of a log phase of estrone and 17 β-estradiol mineralization, and tetracycline at 200 mg kg-1 significantly reduced maximum mineralization of estrone and 17 β-estradiol in manure. In soils amended with a high rate of manure, penicillin at 200 mg kg1 also significantly decreased maximum mineralization of estrone and 17 β-estradiol relative to soils free of antibiotics. Estrogen mineralization almost always significantly decreased in the order of: manure > soil amended with a low rate of manure = soil > soil amended with a high rate of manure. In order to examine the relationship between physical and chemical parameters of media and estrogen mineralization, sewage sludge and biosolid samples with vastly different characteristics were selected for a study of 17 β-estradiol and 17 α-ethinylestradiol mineralization in the presence of ciprofloxacin, an antibiotic commonly used to treat urinary and intestinal infections in humans. Ciprofloxacin was persistent in all media, as less than 0.05% mineralization was observed over 133 d. Despite this persistence, no significant effect of ciprofloxacin addition on 17 β-estradiol or 17 α-ethinylestradiol mineralization was observed at 133 days. Consistent with its chemical structure, maximum mineralization of 17 α-ethinylestradiol was always less than that of 17 β-estradiol, indicating resistance to microbial degradation. PCA analysis indicated that total nitrogen, ammonium-nitrogen and total carbon demonstrated a positive association with respiration and maximum mineralization of 17 β-estradiol, but a negative association with 17 α-ethinylestradiol maximum mineralization. Sorption of 17 α-ethinylestradiol was greater than 17 β-estradiol in all media, limiting maximum mineralization of 17 α-ethinylestradiol.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

München, Techn. Univ., Diss., 2004.

A diferenciação entre processos infecciosos e inflamatórios representa um grande desafio na área de diagnóstico. A complexação do antimicrobiano ciprofloxacino (CIP) com o tecnécio (99mTc-CIP) vem sendo estudada com objetivo de desenvolver um radiofármaco com alta especificidade no diagnóstico de infecções, baseado em seu amplo espectro de atividade antibacteriana. O mecanismo de ação do fármaco consiste na ligação deste com a enzima ADN-girase da bactéria, permitindo sua ligação à bactéria ativa e a obtenção de imagens devido a fótons emitidos pelo 99mTc. Tendo em vista que o local de formação do complexo envolve os mesmos grupos funcionais responsáveis pela ligação do fármaco à enzima, sua eficácia pode ser prejudicada. A diferenciação entre processos infecciosos e inflamatórios representa um grande desafio na área de diagnóstico. A complexação do antimicrobiano ciprofloxacino (CIP) com o tecnécio (99mTc-CIP) vem sendo estudada com objetivo de desenvolver um radiofármaco com alta especificidade no diagnóstico de infecções, baseado em seu amplo espectro de atividade antibacteriana. O mecanismo de ação do fármaco consiste na ligação deste com a enzima ADN-girase da bactéria, permitindo sua ligação à bactéria ativa e a obtenção de imagens devido a fótons emitidos pelo 99mTc. Tendo em vista que o local de formação do complexo envolve os mesmos grupos funcionais responsáveis pela ligação do fármaco à enzima, sua eficácia pode ser prejudicada. O objetivo deste trabalho foi avaliar as condições ideais para ligação do 99mTc a duas quinolonas (CIP e pefloxacino), utilizando diferentes agentes redutores (SnCl2 e FSA), pH da solução e temperatura de incubação. A complexação dos fármacos ao radioisótopo 99mTc foi avaliada através do controle radioquímico, utilizando cromatografia em camada delgada. Posteriormente, testou-se sua atividade antimicrobiana in vitro utilizando diferentes microorganismos. O agente redutor FSA não foi eficiente na formação do complexo, resultando em uma concentração alta de Tc livre (37%). A formulação com Sn aumentou a formação de colóide com aquecimento (100 ºC). A complexação dos antimicrobianos ao Tc impediu sua ligação à ADN-girase. A taxa de ligação variou de 10% (filtração) a 16% (centrigugação). Além disso, verificou-se boa correlação entre a formação de colóide e quantidade de radiação ligada à bactéria em ambos os testes. Esta perda de atividade vem de encontro a alguns estudos clínicos relatados na literatura, que revelam a não diferenciação entre processos infecciosos e processos inflamatórios pelas quinolonas marcadas com 99mTc. / Differentiation between inflammation and infection represents a major challenge in clinical diagnostics. Several studies using ciprofloxacin (CIP), a broad spectrum antimicrobial agent, complexed with technetium (99mTc) have been reported in order to evaluate its capacity to diagnose infections. CIP mechanism of action involves its binding with bacterial DNA-gyrase during the growth phase and the acquisition of images due to 99mTc radioactivity. The fact that the sites of complexation are the same as those involved in bacterial binding it may affect its antimicrobial activity. The objective of the present work was to evaluate the ideal conditions for the complexation of CIP and pefloxacin with 99mTc using stannous chloride and formamidinesulfinic acid (FSA) as reducing agents, different pH and temperatures. The efficiency of complexation was monitored using radiochemical control as well as thin layer chromatography. Furthermore the antimicrobial activity of both complexes was evaluated in vitro. FSA was not adequate as a reducing agent and SnCl2 was better at room temperature than using heat (100 oC). The complexes did not bound well to the bacterias with binding efficiencies ranging from 10 (filtration method) to 16% (centrifugation method). Besides that, a good correlation between colloid formation (impurity) and radioactivity bound to bacterias was found. This decrease in bacterial activity was previously reported in some articles suggesting that quinolones complexed with 99mTc are not suitable to diagnose infections in the presence of inflammation.

Micropartícula de quitosana complexada com íons metálicos [Fe(II), Fe(III), Zn(II)] foram preparadas e caracterizadas a fim de se obter sistemas microparticulados para a adsorção de substâncias. A técnica de preparo utilizada foi a secagem por aspersão, através da qual se obteve micropartículas esféricas colapsadas e com superfície rugosa. O tamanho de partícula foi influenciado tanto pela reação de reticulação com glutaraldeído como pela presença e tipo do metal utilizado para a formação do complexo. A adsorção do ciprofloxacino pelas micropartículas desenvolvidas foi estudada em meio aquoso, O estado de equilíbrio foi influenciado pelo tipo de metal presente na micropartícula e pela concentração inicial de ciprofloxacino na solução. Com o intuito de descrever o mecanismo de adsorção, foram utilizados modelos matemáticos de isotermas de Langmuir e Freundlich, sendo que os dados apresentaram um melhor ajuste para a isoterma de Freundlich. Através da análise de modelos de cinética de pesudo-primeira-ordem e pseudo-segunda-ordem, verificou-se que os dados melhor se ajustaram ao modelo de pseudo-segunda-ordem, indicando que o mecanismo de adsorção do ciprofloxacino pelas micropartículas é através de quimissorção. A determinação da capacidade de adsorção do ciprofloxacino, conduzido in vitro, demonstrou que as micropartículas de quitosana-Fe(III) e quitosana-Zn(II) apresentam efetividade de adsorção. Com isso as micropartículas de quitosana-Fe(III) foram encapsuladas por uma matriz de pectina, sendo este utilizado para a adsorção de antimicrobianos residuais presentes no cólon. A estabilidade das esferas foi determinada em meios digestivos simulados, onde se verificou uma estabilidade de 1 h e 5 h nos meios gástricos e intestinal, respectivamente. Quando as esferas foram incubadas no meio colônico, observou-se uma degradação dependente da concentração de pectina. Com o intuito de evitar a adsorção do ciprofloxacino no meio intestinal, as esferas foram revestidas com Eudragit RS. Estudos de adsorção em meio colônico simulado demonstrou que a capacidade de adsorção das micropartículas de quitosana-Fe(III) não é afeta pela encapsulação na matriz de pectina. O sistema desenvolvido demonstra ser promissor para a extração do ciprofloxacino presente no meio colônico simulado. / The aim of this work was to develop, characterize chitosan- metal ion [Fe(II), Fe(III), Zn(II)] microparticle, and evaluate the adsorption capacities of ciprofloxacin by these complexes. The microparticles were prepared by a spray drying method. They showed good sphericity and a roughness surface morphology. The particle size was influenced by crosslinking reaction and by the kind of metal ion onto the microparticle. A batch adsorption system was applied to study the adsorption of ciprofloxacin from aqueous solution by chitosan-metal ion crosslinked microparticle. The adsorption process was fast, and the equilibrium contact times were influenced by the kind of metal ion onto microparticle. The Langmuir and Freundlich adsorption models were used for mathematical description of the adsorption equilibrium, and it was found that experimental data fitted well to Freundlich model. Adsorption models, based on the assumption of the pseudo-first-order and pseudo-second-order mechanism showed that the pseudo-second-order adsorption mechanism is predominant, and the adsorption process appears to be controlled by the chemical reaction. Chitosan-Fe(III) and chitosan-Zn(II) microparticle demonstrated the highest adsorption of ciprofloxacin. Chitosan-Fe(III) microparticle was encapsulated in a pectin matrix. The system was used for the adsorption of colonic residual antibiotics responsible by the emergence of resistance. The stability of the beads was carried out on simulated digestive media. Beads incubated in simulated gastric and intestinal medium were stable for 1 h and 5 h, respectively. When incubated in simulated colonic medium, beads were then degraded by pectinases contained in the medium. Coating with Eudragit RS was needed to prevent the early adsorption of antibiotics in intestinal medium. Adsorption studies in simulated colonic medium show that the adsorption capacity of chitosan-Fe(III) is not modified after encapsulation within pectin beads making the elimination reaching the colon clinically feasible.

Objetivo: O objetivo geral deste trabalho foi avaliar o perfil farmacocinético para os compostos enrofloxacino (ENRO) e seu principal metabólito, ciprofloxacino (CIPRO), e cloranfenicol (CAP) em frangos de corte visando subsidiar a tomada de ações para controle de resíduos de medicamentos veterinários em produtos de origem animal e a produção de materiais de referência para fins de controle de qualidade analítica. Métodos: Frangos machos da linhagem Cobb-Vantress foram utilizados nos experimentos para determinação da farmacocinética plasmática e níveis teciduais para os compostos ENRO/CIPRO (10 mg/kg), CAP (100 mg/kg) após administração em dose única oral e administração contínua através da água de consumo simulando as condições reais de produção. As concentrações plasmáticas e teciduais foram determinadas utilizando a técnica de cromatografia líquida acoplada à espectrometria de massas em tandem (LC-MS/MS) através de métodos desenvolvidos e validados in house. Análise farmacocinética nãocompartimental e modelagem compartimental dos dados foram realizadas utilizando o software WinNonlin e NONMEM v.6, respectivamente. Para determinação das concentrações teciduais os animais foram sacrificados em tempos pré-definidos e amostras de plasma, músculo e fígado coletadas para os compostos ENRO/CIPRO e plasma e músculo para CAP. Resultados e Discussão: Três métodos multirresíduos para determinação de medicamentos veterinários incluindo diferentes compostos pertencentes às classes químicas ou terapêuticas previstas nesse projeto foram desenvolvidos utilizando LCMS/ MS com limites de quantificação (LOQ) em acordo com os propósitos de aplicação das metodologias. Além disso, métodos específicos para a análise dos compostos individuais nas amostras de plasma foram desenvolvidos para a determinação dos perfis farmacocinéticos em plasma. Conclusões: com base nos dados gerados foi possível implementar novas metodologias para o controle de resíduos de medicamentos veterinários relevantes na produção de aves de corte, bem como o estudo dos perfis farmacocinéticos e depleção tecidual permitiram o delineamento correto das etapas de planejamento e predição de valores encontrados em amostras experimentais. De forma complementar, os dados gerados permitirão a produção de amostras naturalmente contaminadas contendo os fármacos em questão para a produção de materiais de referência com o objetivo de serem utilizados para controle de qualidade analítico. / Objective: The aim of this study was to evaluate the pharmacokinetic profile for compounds enrofloxacin (ENRO) and its major metabolite, ciprofloxacin (CIPRO), and chloramphenicol (CAP) in broilers in order to support the actions taken to vet drugs residues control in animal products and production of reference materials for purposes of analytical quality control. Methods: Cobb- Vantress male broilers were used in experiments to determine the plasma pharmacokinetics and tissue levels to ENRO / CIPRO (10 mg / kg) and CAP (100 mg / kg) after single oral dose administration and continuous administration compounds through drinking water simulating the production conditions. Plasma and tissue concentrations were determined using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) using inhouse developed and validated methods. The non-compartmental and compartmental pharmacokinetic data modeling was performed using WinNonlin software and NONMEM v.6, respectively. For determination of tissue concentrations, animals were sacrificed at predefined times and plasma, muscle and liver samples collected for ENRO/ CIPRO and plasma and muscle samples to CAP. Results and Discussion: Three mutiresidue methods for determination of veterinary drugs including compounds belonging to same chemical and therapeutic classes in this project were developed using LCMS/ MS with limits of quantification (LOQ) in accordance with the purposes of application of methodologies. Furthermore, specific for analyzing individual compounds in the plasma samples methods have been developed for determination of plasma pharmacokinetic profiles. Conclusions: Based on the data generated was possible to implement new methodologies for control of veterinary drug residues relevant in broiler production, as well as the study of pharmacokinetic profiles and tissue depletion allowed the correct delineation of planning and predicting values found in experimental samples. As a complement, the data generated will enable the production incurred samples containing the drugs for production of reference materials for analytical quality control purposes.