Memoria para optar al título de Químico Farmacéutico / En este estudio se determinó la biodisponibilidad relativa para la bioequivalencia del producto similar Zeite® de Laboratorios Recalcine S.A., producto de prueba, con Glivec® fabricado por Laboratorios Novartis AG, producto de referencia; ambas formulaciones orales de Imatinib de 400 mg. Para este estudio doble ciego, cruzado y randomizado se seleccionaron 24 voluntarios sanos; de los cuales, 17 voluntarios finalizaron el estudio. Los niveles plasmáticos de Imatinib, después de la administración de una sola dosis, se determinaron esencialmente por la técnica HPLC descrita por Velpandian y colaboradores, 2004, modificada en el Laboratorio. Para este efecto la técnica fue validada de acuerdo a los parámetros siguientes: especificidad, exactitud, precisión y linealidad. Para evaluar la biodisponibilidad se compararon los siguientes parámetros farmacocinéticos de los productos Zeite® y Glivec®: área bajo la curva ABC (concentración plasmática versus tiempo) y concentración máxima (Cmáx). Los resultados de los parámetros farmacocinéticos fueron: ABC0→∞ 18,5154 ± 6,4942 y 20,8107 ± 6,1045 μg/h/mL; ABC0→24 12,9506 ± 4,4824 μg/h/mL y 14,6367 ± 3,6799 μg/h/mL; Cmáx 1215,12 ± 410,96 ηg/mL y 1189,42 ± 293,70 ηg/mL, para A y B, respectivamente. Todas estas diferencias resultaron ser estadísticamente no significativas, p>0.05. Los intervalos de confianza para el cuociente A/B fueron: ABC0→∞ 91,27 % a 99,40 %; ABC0→24, 89,31 % a 98,73 % y Cmáx 97,64 % a 102,03 %. El rango de confianza recomendado por la FDA es 80% a 125 % con una probabilidad de bioequivalencia del 100%. Todos los parámetros cinéticos para la bioequivalencia (Cmáx, ABC0→24, ABC0→∞) ensayados se encontraron dentro de este rango; por lo tanto, se concluye que Zeite® 400 mg, A (test), es un genérico bioequivalente e intercambiable con Glivec® 400 mg, B (referencia). / This study determined the relative bioavailability to demonstrate the bioequivalence of the similar product Zeite® of Laboratories Recalcine S.A., test product, and Glivec® of Laboratories Novartis AG, reference product. For this double blind, crossed and randomizaded study which include 24 healthy volunteers, which received one dose of 400 mg Imatinib. The Imatinib plasmatic levels, was determinated essentially for the HPLC technique described by Velpandian et al., 2004. This technique was validated respect to its specificity, accuracy, precision and linearity. The pharmacokinetics parameters compared were: area under the curve AUC, (plasmatic concentration vs time) and maximum concentrations (Cmax). The results of the pharmacokinetics parameters of samples A and B were: AUC0→∞ 18,5154 ± 6,4942 y 20,8107 ± 6,1045 μg/h/mL; AUC0→24 12,9506 ± 4,4824 μg/h/mL y 14,6367 ± 3,6799 μg/h/mL; Cmáx 1215,12 ± 410,96 ηg/mL and 1189,42 ± 293,70 ηg/mL, respectively. Significant differences between the kinetic parameters values of both products, p>0.05 were not observed. The confidence intervals for the rate A/B were: AUC0→∞ 91,27 % to 99,40 %; AUC0→24, 89,31 % to 98,73 % and Cmáx 97,64 % to 102,03 %. The range recommended by FDA is 80 % to 125 % with a bioequivalence probability of 100 %. All the kinetic parameters for the bioequivalence (Cmáx, AUC0→24, AUC0→∞) were included in this range; therefore, Zeite® 400 mg, (A test), is a generic product bioequivalent and interchangeable with Glivec®, (B reference)

Philadelphia positive (Ph+) Acute Lymphoblastic Leukaemia (ALL) is characterised by the presence of the BCR-ABL fusion gene, which encodes a protein tyrosine kinase with aberrant activity. Imatinib, a chemical Bcr-Abl inhibitor, is rarely effective in Ph+ ALL patients as a single agent. In this study, insight into molecular and signalling changes occurring in Ph+ ALL during Imatinib therapy were investigated using cDNA microarrays. An optimal microarray assay was established to examine the gene expression changes in leukaemic cells from Ph+ ALL patients treated with Imatinib. Over 500 genes with ≥1.5-fold up- or down-regulation were identified. Based on gene ontology and novelty to Bcr-Abl signalling, six genes were selected and expression changes in five of these genes (PKCε, PINK1, SPRY2, ATF4 and PECAM1) confirmed by real time RT-PCR in Imatinib treated primary Ph+ ALL cells or the SUP-B15 cell line. The functional role of Protein Kinase C epsilon (PKCε) in response to Imatinib was further investigated using the Ph+ lymphoid and myeloid cell lines, SUP-B15 and K562. Detection of Imatinib-induced apoptosis by annexin V and PI staining demonstrated that SUP-B15 cells were less sensitive to Imatinib compared to K562 cells. PKCε mRNA was 50-fold higher in Ph+ ALL cells than Ph+ myeloid cells. In SUP-B15 cells, Imatinib upregulated PKCε mRNA but the protein was reduced by proteolytic cleavage. Inhibition of caspases showed that this cleaved product was not required for Imatinib induced-apoptosis. The treatment of SUP-B15 and primary Ph+ ALL cells with TAT-εV1-2 peptide, a specific inhibitor of PKCε, increased Imatinib-induced apoptosis. While the forced overexpression of PKCε in K562 cells reduced Imatinib-induced apoptosis. This increased expression of PKCε was associated with the increase of survival and anti-apoptotic proteins, Akt and Bcl-2. In summary, Gene expression profiling of Ph+ ALL cells during Imatinib therapy identified PKCε as an Imatinib responsive gene. A novel role of PKCε in Ph+ ALL response to imatinib is proposed. Experimental data presented in this thesis indicate that PKCε mediates pro-survival/anti-apoptosis signals in Ph+ ALL thereby reducing Imatinib-induced death. Thus, targeting PKCε during Imatinib therapy may be beneficial for the future treatment of Ph+ ALL.

Thesis (Ph.D.)--Victoria University (Melbourne, Vic.), 2009.

Zugl.: Berlin, Humboldt-Univ., Diss., 2005 u.d.T.: Na, Il-Kang: Zur Behandlung der chronischen myeloischen Leukämie mit dem selektiven bcr-abl Tyrosinkinaseinhibitor Imatinib

The natural history of CML has been transformed in recent years by the introduction of Glivec[superscript TM] (imatinib mesylate), an ABL kinase inhibitor, which provides the new treatment paradigm for chronic phase CML. While the majority of patients with CP-CML respond very well to imatinib, there are approximately 15% of patients who fail to respond, or respond suboptimally. While the major cause of secondary imatinib resistance can be attributable to kinase domain mutations, the underlying cause of primary resistance is yet to be elucidated. Utilizing the phosphorylation of the adaptor protein Crkl, an immediate downstream partner of BCRABL, as a surrogate measure of BCR-ABL kinase activity, a large interpatient variation in the degree of imatinib induced kinase inhibition achieved in-vitro, was observed in previously untreated CP-CML patients. The observed in-vitro sensitivity was a good predictor of molecular response in patients treated with 600mg imatinib as front line therapy. Furthermore, analysis of the in-vivo reduction in p-Crkl mediated measured in blood cells in response to imatinib over the first 28 days of therapy, revealed that patients with higher % reductions respond significantly better over a two year period, than those with lower % reductions. Using 14-C labelled imatinib, it was demonstrated that this intrinsic sensitivity correlated to the amount of drug which was retained within the target haemopoietic cell, and furthermore, that a critical determinant of the active influx of imatinib, was the functional activity of the human organic cation transporter -1 (OCT-1), as determined by a prazosin (potent inhibitor of OCT-1) inhibition assay. Patients with high OCT-1 Activity had superior molecular responses when compared to those with low OCT-1 Activity, but in those patients who could tolerate increased imatinib dose, these inferior responses could be largely overcome. In contrast, Nilotinib, a more potent second generation tyrosine kinase inhibitor, is not dependent on OCT-1 for influx, making it a possible treatment choice for patients with low OCT-1 Activity. Both imatinib and nilotinib interact with the efflux transporters ABCB1, and ABCG2. In combination studies imatinib results in a significantly increased intracellular concentration of nilotinib, most likely through interaction with these efflux transporters. Furthermore, commonly used therapies such as proton pump inhibitors also interact with ABCB1 and ABCG2, and demonstrable changes in intracellular drug concentrations were observed in-vitro with concomitant administration of these agents and imatinib or nilotinib at clinically relevant concentrations. In conclusion, these data demonstrate that the degree of kinase inhibition mediated in-vitro and in-vivo by imatinib, is a critical determinant of subsequent molecular response. This intrinsic sensitivity to imatinib induced kinase inhibition is related to the activity of the OCT-1 protein. This protein is not involved in the transport of nilotinib, suggesting it as a possible treatment alternative in those patients with low OCT-1 Activity. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1319077 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine; and, Institute of Medical and Veterinary Science, Division of Haematology, 2005. / Title from title page of source document; viewed 19 July 2005. Bibliography: p. 157-184 of source document. Also available in print.

abstract: The majority of chronic myeloid leukemia (CML) and some of acute lymphocytic leukemia (ALL) cases are associated with possessing the BCR-Abl fusion protein from an oncogenic translocation, resulting in a constantly active form of Abl and rapid proliferation. CML and ALL cells that possess the BCR-Abl fusion protein are known as Philadelphia chromosome positive (Ph+). Currently, Imatinib (selective Abl inhibitor) is used as therapy against CML and ALL. However, some patients may have malignancies which show resistance to Imatinib. Previous work displays that the transformation of progenitor B cells with the v-Abl oncogene of Abelson murine leukemia virus results in cell cycle progression, rapid proliferation, and potentially malignant transformation while preventing any further differentiation. Progenitor B cells transformed with the temperature-sensitive form of the v-Abl oncogene have served as a model to study cellular response to Imatinib treatment. After some manipulation, very few cells were forced to progress to malignancy, forming tumor in vivo. These cells were no long sensitive to v-Abl inactivation, resembling the Imatinib resistant ALL. Autophagy is the process by which proteins and organelles are broken-down and recycled within the eukaryotic cell and has been hypothesized to play a part in cancer cell survival and drug-resistance. LC3 processing is a widely accepted marker of autophagy induction and progression. It has also been shown that Imatinib treatment of Ph+ leukemia can induce autophagy. In this study, we examined the autophagy induction in response to v-Abl inactivation in a Ph+-B-ALL cell model that shows resistance to Imatinib. In particular, we wonder whether the tumor cell line resistant to v-Abl inactivation may acquire a high level of autophagy to become resistant to apoptosis induced by v-Abl inactivation, and thus become addicted to autophagy. Indeed, this tumor cell line displays a high basal levels of LC3 I and II expression, regardless of v-Abl activity. We further demonstrated that inhibition of the autophagy pathway enhances the tumor line's sensitivity to Imatinib, resulting in cell cycle arrest and massive apoptosis. The combination of autophagy and Abl inhibitions may serve as an effective therapy for BCR-Abl positive CML. / Dissertation/Thesis / M.S. Biological Design 2011

L’érythropoïèse est le processus aboutissant à la production des hématies à partir d’une cellule souche hématopoïétique. La différenciation érythroïde implique des changements morphologiques en partie liés à la perte d’expression membranaire du récepteur à activité tyrosine kinase de classe III, c-Kit. En réponse à son ligand, le SCF, c-Kit est activé puis internalisé et dégradé par la voie du protéasome, via l’ubiquitine E3-ligase c-Cbl, ou par la voie lysosomale suite à une endocytose. Dans la première partie de ce travail, nous avons pu mettre en évidence qu’en absence de SCF et en réponse à un inhibiteur de tyrosine kinase, l’imatinib, les érythroblastes cultivés ex vivo perdent l’expression membranaire de c-Kit et accélèrent leur entrée en différenciation terminale. Au vu de ces observations, nous avons cherché à comprendre les mécanismes impliqués. Sur un modèle de cellules érytholeucémiques dépendantes de l’érythropoïétine, mais exprimant de manière endogène c-Kit, nous avons montré que l’imatinib induit une internalisation du récepteur ainsi que sa dégradation par la voie lysosomale et de manière indépendant de c-Cbl. De plus, nous avons montré que cet effet est réversible et que l’imatinib ne bloque pas la réexpression de c-Kit après son internalisation en réponse au SCF. Des marquages métaboliques ont permis de montrer que l’imatinib ne modifie ni la synthèse ni la maturation de c-Kit et que le profil phospho-tyrosine des cellules traitées à l’imatinib est globalement inchangé. Enfin, nous avons montré que la fixation de l’imatinib à la poche catalytique de c-Kit est indispensable à son internalisation, et par conséquent à sa dégradation. Il apparait donc que l’imatinib lève l’auto-inhibition de c-Kit, qui semble nécessaire pour son maintien à la membrane. Dans la seconde partie de ce travail, nous nous sommes intéressés aux changements morphologiques subis par les nucléoles, lieu de la biogenèse des ribosomes, au cours de différenciation des érythroblastes. L’étude de la taille et du potentiel prolifératif des cellules, ainsi que l’analyse morphologique des nucléoles, nous a permis de confirmer que la réduction de taille des cellules est contemporaine d’un ralentissement de leur prolifération ainsi que de la réduction du volume et de la surface du composé granulaire (CG), « matrice » du nucléole. En microscopie électronique, nous montrons la persistance des CG en fin de maturation. Enfin, nous avons également étudié l’évolution des nucléoles dans un contexte pathologique de syndromes myélodysplasiques de faible risque, qui se caractérisent par une hématopoïèse inefficace. Nous observons que les cellules pathologiques immatures ont des CG plus volumineux que les cellules normales immatures, et qu’au cours de la différenciation, la morphologie des nucléoles est identique entre les cellules normales et pathologiques. En conclusion, ce travail a permis de décrire 1) le mécanisme d’internalisation d’un récepteur à activité tyrosine kinase de classe III, c-Kit par l’imatinib et 2) la morphologie du nucléole au cours de la différenciation érythroïde normale et pathologique des syndromes myélodysplasiques de faible risque. / Erythropoiesis is the process leading to the production of red blood cells from hematopoietic stem cell. The erythroid differentiation involves morphological cell changes, in part related to the loss of membrane expression of the type III receptor tyrosine kinase, c-Kit. In response to its ligand SCF, c-Kit is activated, then internalized and degraded by the proteasome pathway via the E3 ubiquitin ligase c-Cbl, or by the lysosomal pathway, after endocytosis. In the first part of this work, we demonstrated that in the absence of SCF and in response to tyrosine kinase inhibitor, imatinib, erythroblasts cultured ex vivo, lose membrane expression of c-Kit and accelerate their terminal differentiation. In view of these observations, we sought to understand the mechanisms involved. On an erythropoietin dependent cell line expressing c-Kit at the membrane, we showed that imatinib induces receptor internalization and degradation by the lysosomal pathway, independently of c -Cbl. Furthermore, we showed that this effect is reversible and that imatinib does not block the c-Kit re-expression after its internalization, in response to SCF. Metabolic labelling showed that imatinib does not alter synthesis or maturation of c -Kit and that the phospho-tyrosine profile of cells treated with imatinib is generally unchanged. Finally, we showed that the binding of imatinib to the catalytic pocket of c-Kit is essential for its internalization, and therefore its degradation. So, it appears that imatinib removes c-Kit self-inhibition, which seems necessary to its retention at the membrane. In the second part of this work, we studied the morphological changes of nucleoli, the site of ribosome biogenesis, during erythroid differentiation. We showed that the reduction of cell size takes place at the same time than reduction of cell proliferation and reduction of surface and volume of the Granular Compound (GC), the “matrix” of the nucleolus. Moreover, we showed by electronic microscopy, the persistence of GC at the end of maturation. Finally, we also studied the evolution of nucleoli in a pathological context of low risk myelodysplastic syndromes, which are characterized by ineffective hematopoiesis. We observed that immature pathological cells have larger GC than immature normal cells, but that during differentiation, the morphology of nucleoli is identical between normal and pathological cells. In conclusion, this work has allowed us to describe 1) the mechanisms of internalization of a class III receptor tyrosine kinase, c-Kit by imatinib and 2) the morphology of the nucleolus during normal and pathological low risk myelodysplastic syndromes of erythroid differentiation.

Entwicklung der Vaskularisierung und des Fasergehaltes im Knochenmark von CML-Patienten während der Behandlung mit Imatinib Gesteigerte Gefäßneubildung und retikuläre Fibrose sind zwei morphologische Veränderungen im Knochenmark von CML-Patienten, welche mit einem schlechteren Krankheitsverlauf assoziiert sind. Imatinib, ein selektiver Tyrosinkinaseinhibitor, hemmt die angiogenen und fibrogenen Substanzen VEGF und PDGF. Diese Dissertation untersuchte den Einfluss von Imatinib auf die Vaskularisierung und den retikulären Fasergehalt im Knochenmark von Patienten mit chronischer myeloischer Leukämie (CML). Es wurden 67 repräsentative Knochenmarkbiopsien von insgesamt 19 Patienten, behandelt in Multicenter-Studien an der Universitätsklinik Leipzig, eingeschlossen. Davon waren zehn Patienten bereits mit IFN-α + Cytarabin vorbehandelt worden. Neun Patienten mit neudiagnostizierter CML erhielten Imatinib als Ersttherapie. Knochenmarkbiopsien zu den Zeitpunkten t0 (vor Therapiebeginn), t1 (6-15 Monate) und t2 (21-36 Monate) nach Behandlungsstart mit Imatinib wurden untersucht. Weiter wurde eine Kontrollgruppe aus 19 KM-Biopsien gebildet, welche ohne pathologischen Befund waren. Während der Behandlung mit Imatinib kam es bei der Mehrzahl der Patienten zu einer Normalisierung der Gefäßdichte im Knochenmark. Der Fasergehalt im Knochenmark der CML-Patienten sank bei allen Patienten auf Normwerte ab. Dies zeigt den positiven Einfluss von Imatinib auf die morphologischen Veränderungen Vaskularisierung und Faserdichte im Knochenmark von CML-Patienten. Ein Einfluss der Vorbehandlung mit IFN-α + Cytarabin auf die Dynamik der morphologischen Parameter während der Behandlung mit Imatinib war nicht nachweisbar.

Les modifications physiopathologiques induites par l’obésité sont susceptibles de modifier la pharmacocinétique d’un grand nombre de médicaments. Toutes les étapes peuvent-être touchées : modification des compartiments de l’organisme, de la fonction rénale et de l’expression des protéines du métabolisme. L’intensité des variations induites reste difficile à anticiper en l’absence d’études spécifiques, ce qui pose également la question du choix du meilleur estimateur de poids dans l’expression du schéma posologique du médicament chez le sujet obèse. Nous avons évalué l’impact de différents descripteurs de poids sur les variabilités inter-individuelles de trois médicaments couramment utilisés en diabétologie, dans la douleur et en cancérologie. Une modélisation a été faite en pharmacocinétique de population (Monolix®) avec une évaluation des descripteurs de poids (poids de masse maigre, poids idéal, poids total, surface corporelle…). Pour la metformine (n=105 patients), molécule fortement hydrophile, CL/F et V/F augmentent avec le poids. Le poids de masse maigre est le meilleur descripteur de poids pour les deux paramètres et permet d’abaisser de manière importante la variabilité inter-individuelle. Dans le cas de la morphine (n=31), aucun descripteur n’est corrélé à CL/F ou V/F dans une population de sujets atteints d’obésité morbide. L’analyse de l’imatinib (n=54), de lipophilie plus importante, et de son métabolite actif montre que le poids idéal est la covariable la plus performante. Son impact n’explique cependant qu’une part minime de la variabilité de CL/F et ne remet pas en cause le principe d’une dose fixe. Les résultats confirment l’intérêt des méthodes de pharmacocinétique de population dans l’évaluation des covariables morphologiques, et indiquent qu’il n’existe pas de descripteur de poids universel. Ces résultats doivent nous encourager à évaluer systématiquement l’impact de l’obésité avec des modèles de population. Dans certains cas, des études de confirmation comparant différents schémas devraient-être mises en place. / Pharmacokinetic of drugs may be altered by pathophysiological changes associated with obesity: renal function, body compartments, expression of metabolism proteins. Real impact may be difficult to appreciate due to small number of specific studies. What is the best size-descriptor for optimal dosing in obesity remains a question of importance. Impact of different size descriptors was studied for three drugs currently used in diabetology, pain and oncology. Population pharmacokinetic modeling was done using Monolix® software to evaluate different covariates (LBW, TBW, BSA, …). CL/F and Vd/F of metformin, highly hydrophilic drug, increases positively with body weight (n=105). LBW was the best size-descriptor leading to substancial decrease in the between-subject variability BSV. No size-descriptors showed significant impact for morphine (n=31). Ideal body weight is the best size-descriptor for imatinib and its main metabolite (n=54), lipophilic drug. It explains only a small part of BSV and fixed dosing stays justified. Population PK analysis are the most formal assessment of morphological covariates and no single size descriptor can described all variabilities. Results must lead us to more systematic population PK analysis. Confirmation studies comparing different dosing regimens have to be done.