La prise en charge des patients atteints de cancer fait intervenir le pharmacien hospitalier dans la préparation des médicaments anticancéreux injectables. Afin de limiter les coûts de cette prise en charge médicamenteuse, une des alternatives consiste à optimiser leur préparation en prenant en compte la stabilité physico-chimique des anticancéreux : 1-en utilisant les reliquats générés lors de la préparation. 2- en évaluant la possibilité de fabriquer à l’avance les préparations d’anticancéreux. L’absence de données de stabilité de ces médicaments nous a conduits à l’évaluer sur le pemetrexed et le methotrexate afin de répondre à cette double problématique et d’étudier le gain de coût associé.Nous avons démontré, au cours de ce travail, la stabilité des reliquats de pemetrexed-Alimta® pendant14 jours permettant ainsi leur réutilisation. Nous avons également montré la stabilité de solutions de méthotrexate conditionnées en seringues pendant 28 jours.Une des conséquences de ces études est la réalisation d’économie pour la sécurité sociale par l’optimisation de la gestion des reliquats d’anticancéreux. Cette économie représente environ 10 % du budget annuel des anticancéreux injectables soit 750 k€. Nous avons montré au cours de ce travail l’apport de la chimie analytique et organique ainsi que l’apport de la pharmacie dans la mise en place et l’exploitation des études de stabilité sur les médicaments anticancéreux injectables. / Hospital pharmacists are involved in the management of cancer patients through preparation of intravenous anticancer drugs. To limit the costs of the chemotherapies, an alternative is to optimize their preparation by considering the physicochemical stability of anticancer drugs. This can be done by using the leftovers generated during the preparation and by preparing in advance the devices to be used for administration. The lack of data on the stability of anticancer drugs has led us to evaluate pemetrexed and methotrexate with the aim of answering both the above issues. In parallel, we studied the associated cost savings.In this thesis, we have demonstrated the stability of leftovers of pemetrexed-Alimta® for 14 days allowing their use for subsequent preparations. We also showed the stability of solutions of methotrexate packaged in syringes for 28 days.One consequence of these studies is its translation in cost-savings for our Healthcare Funding Organization by managing leftover anticancer drugs. These cost-savings (about 750 k€) represent about 10 % of the total annual expenditure of anticancer drugs. Our work illustrates the contribution of analytical and organic chemistry and the role pharmacists can have in improving costs by performing stability studies of anticancer drugs.

Unidad de práctica para optar al título de Químico Farmacéutico / Autor no autoriza el acceso a texto completo de su documento / En el presente trabajo se realizaron análisis para la Validación de las metodologías analíticas: Análisis de determinación Endotoxinas Bacterianas y Análisis de test esterilidad, utilizadas en el laboratorio de Microbiología, Departamento de Operaciones Analíticas del laboratorio farmacéutico Synthon Chile. Estos análisis fueron dirigidos a tres productos farmacéuticos estériles. Para el Análisis de Determinación Endotoxinas Bacterianas se realizó una evaluación de potenciación o inhibición que ejerce el producto sobre el reactivo LAL, con el que se validó la técnica de coagulación GEL-CLOT. Mientras que en el Análisis de Test de Esterilidad, se realizó la técnica de filtración por membrana en la que se buscó neutralizar la actividad bacteriostática y/o fungistática del fármaco. Para esto se inocularon las muestras con cepas estandarizadas de la colección American Type Culture Collection (ATCC) con una carga de microorganismos ≤ 100 unidades formadoras de colonias (ufc), indicadas por la Farmacopea Europea. Los resultados obtenidos de este proceso de validación fueron dispares para los distintos fármacos utilizados. Ondansetron fue el que presentó los mejores resultados, logrando ser validados ambos métodos de análisis (esterilidad y endotoxinas). Mientras que en los otros dos fármacos no alcanzó a finalizar la validación, esto debido a resultados no satisfactorios y falta de muestras respectivas para el reanálisis

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OBJECTIVE: The aim of this study was to investigate the safety and tolerability of a novel anti-folate drug, pemetrexed, in the setting of a phase I clinical trial in patients with non-HIV related central nervous system lymphoma (CNSL). METHODS: In this multicenter, open-label, phase I dose finding clinical trial, pemetrexed was investigated as a single agent treatment for primary or secondary CNSL. RESULTS: A total of 18 patients were enrolled between January 2009 and November 2014. The mean age was 64.6 years old (range: 47-79). The ratio of male to female was 1:1. One out of six patients experienced a dose limiting toxicity (DLT) at dose level 1 (600mg/m2). There were no DLTs among the four patients enrolled at dose level 2 (900m/m2). Two of six patients experienced a DLT at dose level 3 (1200mg/m2). The MTD was therefore determined to be 900mg/m2. Overall, pemetrexed was well tolerated but toxicities were seen and need to be monitored. All patients experienced at least one type of toxicity of any grade. Most patients (92.9%) experienced at least one type of neurological toxicity. Grade-3 toxicities included confusion, speech impairment, and psychosis. Twelve patients (85.7%) experienced at least one bone marrow type of toxicity of any grade. These toxicities included anemia (78.6%), thrombocytopenia (57.1%), neutropenia (50%), leukocytopenia (42.9%), and lymphopenia (42.9%). Four patients experienced either grade-3 (14.3%) or grade-4 (14.3%) neutropenia. Three patients experienced grade-3 leukopenia (21.4%). One patient experienced grade-3 lymphopenia (7.1%) and two patients experienced grade-4 lymphopenia (14.3%). Twelve patients (85.7%) experienced at least one metabolic type of toxicity of any grade. A majority of these were also grade-1 or 2, with the exception of hypophosphatemia (grade-4), hyperglycemia (grade-3) and increased ALT (grade-3), increased AST (grade-3) and increased creatinine phosphokinase (CPK) (grade-4). Constitutional and gastrointestinal symptoms were seen in >60% of patients. These consisted mainly of fatigue, constipation, nausea, and anorexia. Musculoskeletal symptoms were seen in greater than 60% of patients. Less common adverse events included pain (<60%), infection (<40%), dermatologic, ocular/visual, and pulmonary/upper respiratory (<30%). The average number of cycles on treatment for all patients was 5.5 cycles. 14 patients were evaluated for response to treatment by neuroimaging (MRI) while on treatment. Of these, four patients (28.6%) showed a complete response (CR). Of those patients, 2 patients achieved this response after only 2 doses, and 2 patients after a total of 8 doses. 5 patients (35.7%) showed a partial response (PR) and four patients (28.6%) achieved stable disease (SD). The overall response rate (ORR) was determined at 92.9% (SD, PR and CR combined). CONCLUSIONS: Given this data, pemetrexed is a powerful drug and feasible alternative to existing treatment options; however, certain toxicities need to be closely monitored. Further studies are needed to assess the efficacy of pemetrexed in a larger cohort of patients with CNSL.

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Since its discovery, it was appreciated that the antifolate pemetrexed had multiple targets within folate metabolism. This laboratory was instrumental in showing that pemetrexed elicited its primary action as a thymidylate synthase inhibitor. Unusual for an antifolate, pemetrexed showed significant clinical activity against malignant pleural mesothelioma and non-small cell lung cancer. Accordingly, the FDA recently issued first-line approvals for pemetrexed in these diseases, leading us to question whether the effects of pemetrexed on other folate-dependent targets could explain this atypical clinical activity of the drug. Studies in this dissertation showed that in addition to thymidylate synthase inhibition, pemetrexed was also an inhibitor of aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate- dependent enzyme of de novo purine synthesis. Consequent of AICART inhibition, pemetrexed caused robust activation of a key energy-sensing regulatory enzyme of the PI3K-AKT signal transduction pathway, AMP-activated protein kinase (AMPK). AMPK activation resulted from xx accumulation of the AMP-mimetic, ZMP, behind the AICART block. Constituents of the PI3K- AKT cascade are frequently deregulated in human carcinomas, uncoupling nutrient supply from proliferative capacity. Therefore, interventions that reinstate control over aberrant signaling along this axis, such as AMPK activation, are of significant cancer therapeutic interest. The cellular consequences of AMPK activation in response to pemetrexed were assessed. In particular, effects on the downstream target of PI3K-AKT signaling, the mammalian target of rapamycin complex 1 (mTORC1), were studied. Unlike targeted mTORC1 inhibitors, such as rapamycin and its analogs, pemetrexed-mediated activation of AMPK also signaled to mTOR- independent controlling elements of protein and lipid synthesis, highlighting additional benefits of AMPK activating agents that extend beyond effects on mTOR signaling. We therefore propose that the unusual activity of pemetrexed in mesothelioma and non-small cell lung cancer is due in part to effects on signaling processes downstream of AMPK activation. These findings present a novel approach to AMPK activation secondary to an AICART block, define pemetrexed as a molecularly targeted agent, and ultimately extend the utility of antifolates beyond their traditional function.

Pemetrexed (PTX), an antifolate drug, has been approved by the US FDA for first line therapy of mesothelioma and non-small cell lung cancer. In addition to its primary site of action on thymidylate synthase (TS), PTX also inhibits the second folate-dependent enzyme of purine biosynthesis aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The accumulation of the substrate for AICART, ZMP, in PTX-inhibited cancer cells leads to activation of AMP-activated protein kinase (AMPK) with subsequent inhibition of mammalian target of rapamycin (mTOR) and hypophosphorylation of its downstream targets responsible for protein synthesis and cell proliferation. Inhibitors of mTORC1 like Rapamycin and its analogs (rapalogs) have only partial effects on tumor cells as they do not inhibit mTORC2, which phosphorylates Akt subsequently relieving the inhibition of mTORC1, thus leading to poor cytotoxicity by rapalogs. AMPK exerts control on mTORC1 kinase activity and PTX mediated activation of AMPK leads to its subsequent downregulation and hence, would be expected to have a therapeutic interaction with direct mTOR inhibitors. AZD8055, an ATP-competitive inhibitor of mTOR kinase, potently inhibits both mTORC1 and mTORC2 and therefore, can overcome the feedback mechanism(s) limiting the action of rapalogs to cytostatic effects. To study the effects of AMPK activation and mTOR inhibition pharmacologically, we performed growth suppression assays using pemetrexed, AICAR, RAD001, and AZD8055. The effect of inhibition of mTOR with these drugs was assessed by examining the dephosphorylation of mTORC1 substrates S6K1 and 4E-BP1, as single agents and in combination, at their 50% inhibitory concentrations (IC50) by western blotting. Our data suggested that AMPK activation via PTX mediated AICART inhibition in combination with direct mTOR inhibition by AZD8055 has a synergistic interaction on the proliferation of NSCLC cells in culture. Inhibition of mTOR endogenously by pemetrexed, along with direct pharmacological inhibition of mTOR prevents the feedback circuit which may compromise the therapeutic efficacy of rapamycin analogs. Pemetrexed and AZD8055, as single agents, demonstrated inhibitory activity on phosphorylation events of mTORC1 substrates. This activity was markedly increased by combining both the drugs. Our findings suggest that direct inhibitors of mTOR enhance the effects of activators of AMPK. These effects appear to be mediated via combined effects on mTORC1. Taken together, the combination of catalytic site mTOR inhibitors and pemetrexed is a promising therapeutic strategy and calls for further preclinical and clinical investigations.

The tumor suppressor TP53 is the most frequently altered gene in human cancers. The growth-promoting complex, mTORC1 plays a part of the oncogenic profile caused by dysfunctional p53. mTORC1 sits downstream of AMPK and other crucial tumor suppressors/oncogenes, PTEN, LKB1, and Akt. The antifolate pemetrexed was found by this laboratory to activate AMPK via the inhibition of the enzyme AICART in de novo purine synthesis. This work presents a mechanism of mTORC1 activation with p53 loss, as well as of mTORC1 inhibition by pemetrexed-induced AMPK. We have found that mTORC1 activity was substantially upregulated by the loss or mutation of p53. This activation involves the loss of TSC2 from lysosomal membranes, the site of mTORC1 activation by Rheb. We demonstrate that loss of lysosomal TSC2 increased the levels of lysosomal Rheb. Control of mTORC1 was restored by overexpression of TSC2, which correlated with decreased lysosomal Rheb. Surprisingly, pemetrexed-activated AMPK did not phosphorylate TSC2 because of an accumulation of nonfunctional p53, and a subsequent decrease in TSC2 mRNA. Accordingly, lysosomal TSC2 decreased, however, the levels of lysosomal Rheb decreased. Future studies will question whether the robust Raptor phosphorylation by pemetrexed is involved in this decrease in lysosomal Rheb. AMPK activation by pemetrexed also significantly increased the translocation of AMPK to the nucleus, and we will explore the function of this nuclear AMPK. Overall, these findings present a mechanism involved in the oncogenic signaling of mTORC1 with loss of p53 and offer insight into how pemetrexed reinstates control.

Thymidylate synthase (TS), encoded by the ThyA gene, is essential for the growth and survival of Mycobacterium tuberculosis and therefore is a potential drug target. Thymidylate synthase binds both a substrate, 2'-deoxyuridine-5'monophosphate (dUMP) as well as a cofactor, (6R,S)-5,10-methylenetetrahydrofolate (mTHF), providing the ability to inhibit a single target by two separate classes of molecules. 5'-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) is a very tight binding mechanism based inhibitor, shown to have a Ki of 2nM for Mtb TS. Pemetrexed and Raltitrexed are both anti-folates, targeting the cofactor binding site of thymidylate synthase. The x-ray crystal structures of Mycobacterium tuberculosis thymidylate synthase were solved in the binary complexes ThyA-dUMP and ThyA-FdUMP at 2.5 A and 2.4 A resolutions, respectively. The ternary complex, ThyA-dUMP-Pemetrexed was solved to a resolution of 1.7 A. The enzyme is comprised of 8 alpha-helices as well as 23% of the protein formed by beta-sheets, including the dimer interface which is a beta-sandwich. Examination of the dUMP binding site allowed the identification of key conserved residues that play a role in ligand binding and catalysis. Comparison of the dUMP-Pemetrexed ternary complex with that of the human crystal structure shows two fewer interactions in the Mtb enzyme. One is due to the replacement of a Met with a Val which doesn't allow hydrophobic interactions with the ring system of Pemetrexed, and the other is the replacement of an Asn with a Trp, depriving the Mtb protein of a hydrogen bond at the N7 of the pyrrolo ring. A spectrophotometric assay that monitored DHF formation was used to determine the inhibition of Pemetrexed and Raltitrexed on Mtb TS. Both were verified as noncompetitive inhibitors, and Pemetrexed was found to have an IC50 of 17muM and a Ki of 16.8muM, while Raltitrexed had an IC50 of 3.5muM and a Ki of 3.2muM.

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