Diss. (sammanfattning) Stockholm : Karol. Inst., 2002. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Kentucky, 2004. / Title from document title page (viewed Jan. 6, 2005). Document formatted into pages; contains viii, 88p. : ill. Includes abstract and vita. Includes bibliographical references (p. 80-86).

Des bioconjugués squalénisés du semaxanib et du sunitinib , deux inhibiteurs puissants de l'angiogénèse et de tyrosine kinases, ont été synthétisés avec un bras espaceur pH sensible de type hémiaminal . Les prodrogues sont préparées selon une séquence en trois étapes impliquant : (i) la N- alkylation avec du chlorométhoxy - triisopropylsilane ; (ii) désilylation ; et (iii) acylation avec l'acide trisnorsqualenique . Ces prodrogues squalénisées ont la capacité de s'auto-assembler en nanoparticules en milieu aqueux sans nécessité de tensioactif . Les agrégats de taille nanométrique ont été caractérisés par diffraction dynamique de la lumière et microscopie électronique à transmission , et semblent être stables dans l'eau sur plusieurs jours. Les études biologiques in vitro ont montré que les nanoparticules de sunitinib sont cytotoxiques contre la lignée de cellules ombilicale veineuse endothéliale humaine ( HUVEC), qui est impliqué dans la formation de vaisseaux de la tumeur. / Squalenoyl conjugates of semaxanib and sunitinib, two potent antiangiogenic (pyrrolyl)methylidenyl-substituted oxindole multitarget tyrosine kinase inhibitors, were synthesized with a hemiaminal-based pH-sensitive linker. The prodrugs were prepared according to a three-step sequence involving (i) N-alkylation with chloromethoxy-triisopropylsilane; (ii) desilylation; and (iii) acylation with trisnorsqualenic acid. These squalenoyl prodrugs were found to selfassemble into nanoassemblies in aqueous media without the need for any surfactant. The nanosized aggregates were characterized by dynamic light scattering and transmission electron microscopy, and appeared to be stable in water for several days, as determined by particle-size measurement. In vitro biological studies showed that squalenoyl sunitinib nanoassemblies are notably cytotoxic against the human umbilicalvein endothelial cell line (HUVEC), which is involved in the tumor vessel formation.

The oleaginous yeasts have an ability to accumulate an increased number of lipids, under certain circumstances. These microbial lipids differentiate in the number of fat acids present, which enables their wide application in biotechnological industry. This master’s thesis is aimed on lipid production, number of the fat acid groups present, and squalene production by Metschnikowia yeasts, based on the cultivating conditions. Biomass and lipid production was observed in separate cultivation media, with the addition of the different waste substrates. Production properties were observed by method of the gas chromatography. For the squalene production observation, a HLPC method was chosen. All production groups were able to accumulate lipids on the waste substrate, although in different values. These values were very individual, especially in the areas of the specific groups and growth on the given substrate. The lipid composition was different, which was caused by differences in the waste substrates. With regards to the squalene production – the yeasts from the Metschnikowia family were not able to produce squalene in the presence of the terbinafine and its increasing concentration. Therefore, the same procedure was chosen, as it was for the Yarrowia lipolytica yeast, with the difference in the sterol synthesis, however squalene was still not produced this way.

Squalene synthase (EC 2.5.1.2.1; SQS) is located at a branch point in the isoprenoid pathway and catalyzes the condensation of two molecules of farnesyl diphosphate to form squalene. SQS activity contributes to the formation of triterpenes and sterols, including phytosterols, brassinosteroids, cholesterol, and in potato plants, steroidal glycoalkaloids (SGAs). These compounds have diverse functions in the plant. SGAs are defense compounds that deter feeding by potato pests. The wild potato Solanum chacoense accumulates higher amounts of SGAs than cultivated potato and some of its accessions produce leptines, a rare class of SGAs that is toxic to Colorado potato beetle. Unlike most eukaryotes, higher plants have more than one gene coding for SQS. Three sqs gene homologs were isolated from S. chacoense, sqs1Sc, sqs2Sc, and sqs4Sc, that have 74 to 83% identity at the amino acid level. Some of the amino acid differences between sqs isoforms are likely to affect enzyme activity. Each of the three genes contained an intron in the 3'UTR. This feature may have a role in the nonsense-mediated decay of incomplete sqs mRNAs. A partial SQS polypeptide retaining catalytic activity but lacking the membrane anchoring domain could adversely affect a cell with the randomly distributed accumulation of squalene. The mRNA of sqs1Sc and sqs2Sc was detected in all tissues whereas sqs4Sc transcript was limited to bud tissue. The sqs2Sc transcript was less uniformly distributed in the plant than sqs1Sc and accumulated most abundantly in floral tissue. The results demonstrate that the three sqs genes have different patterns of gene expression and encode proteins with different primary structures indicating distinct roles in plant squalene metabolism. / Master of Science

Cette thèse porte sur l’influence de la formulation de méthode de nanoprecipitation sur la taille et la structure interne de particules de dérivés squalénés. Ces composés introduits en 2006 par P. Couvreur sont basés sur l’association de principe actif au groupe squalène pour obtenir des propriétés d’auto-assemblage. L’augmentation de l’efficacité thérapeutique des nanoparticules par rapport au principe actif libre et la versatilité de fonctionnalisation ont ouvert une nouvelle stratégie pour les applications biomédicales. Cependant, la compréhension des mécanismes de formation des particules et de leur structure interne reste incomplète. Ces caractéristiques (taille et structure) pouvant influencer l’activité thérapeutique, il est crucial en vue d’une application médicale de déterminer les paramètres clef de la formation des nanoparticules. Dans cette thèse, les particules de squalène deoxycitidine ont été caractérisées par diffusion de neutrons et de rayons X aux petits angles et par cryo-microscopie électronique. Dans une première partie, nous avons identifié les paramètres optimums de formulation pour la nanoprecipitation. Dans une deuxième partie, l’étude de la nature du solvant organique sur la nanoprecipitation a mis en évidence pour certains solvants un mécanisme de nucléation croissance. Les effets de solvants sont cependant faibles par rapport à ce qui est classiquement observé pour les polymères. La troisième partie basée sur la cryo-TEM des particules oriente vers un mécanisme décrit pour les autres cubosomes d’attachements inter lamellaires pour les structures internes cubiques. Enfin, à l’issue de ce travail, nous avons étendu le protocole de nanoprecipitation au D α- Tocophérol Succinate en tentant de stabiliser la phase cristalline hexagonale présente dans le diagramme de phase binaire eau/ α-TOS. / This thesis investigates the influence of the formulation on the size and the internal structure of particles obtained by nanoprecipitation of squalene derivatives. These compounds were introduced in 2006 by P. Couvreur's team as part of a process called squalenoylation. Based on the association of drug with the squalene group it confers to these molecules self-assembling properties and it increased therapeutic efficacy. The particles are prepared by the solvent displacement method ("Ouzo" effect) and were characterized by neutron and X-ray scattering at small angles and by cryo-electron microscopy.We have highlighted the necessary preparation parameters for the reproducible preparation of cubosomes in a dimater of hundred nanometres. A study on the effect of the nature of the organic solvent on Squalene deoxycitidine has highlighted for some of them (isopropanol and DMSO) a nucleation mechanism that could be followed by growth (ethanol and acetone). TEM cryo-microscopy analysis suggests that cubic internal structures form by the mechanism of interlamellar attachments. Finally, we extended the nanoprecipitation method to Dα-Tocopherol Succinate, a derivative of Vitamin E

Supernatant protein factor (SPF) is a 46-kDa cytosolic protein that stimulates squalene monooxygenase, which catalyses the second committed step in cholesterol biosynthesis. The mechanism by which SPF stimulates this enzyme is not understood and the goal of these studies was to see if SPF affected cholesterol synthesis in cultured cells. Rat supernatant protein factor-like protein (SPF2) shares 77% sequence identity with human SPF. In my studies SPF2 also stimulated squalene monooxygenase in vitro and incubation of SPF2 with protein kinase A (PKA) and C increased its activity by about 2-fold, as shown earlier with SPF. GTP and GDP prevented the stimulation of squalene monooxygenase by SPF2, suggesting that binding of these nucleotides inhibits SPF2. This inhibition could be prevented by the addition of -tocopherol, although -tocopherol alone had no effect on SPF2 activity in vitro. Expression of human SPF in hepatoma cells, which lack expression of endogenous SPF, increased cholesterol synthesis by 2-fold and addition of dibuytrylcAMP, a PKA activator, to these cells yielded an additional 62% increase whereas addition of a PKA inhibitor completely blocked the ability of SPF to stimulate cholesterol synthesis. To further confirm a role for phosphorylation in the regulation of SPF, substitution of alanine for serine-289 (a putative PKA recognition site) reduced the PKA-mediated activation of SPF in vitro by 50%, as measured with microsomal squalene monooxygenase and completely blocked the ability of SPF to stimulate cholesterol synthesis in hepatoma cells. In further structure-function studies, deletion of the carboxy-terminal Golgi-dynamics domain greatly increased the ability of SPF to stimulate squalene monooxygenase in microsomes, but, paradoxically prevented SPF from stimulating cholesterol synthesis in cell culture. Addition of brefeldin A, which disrupts Golgi formation, also abolished the ability of SPF to stimulate cholesterol synthesis, supporting a role for the Golgi in SPF function. Since squalene monooxygenase is not thought to be rate-limiting with regard to cholesterol synthesis, the possibility that SPF might stimulate other enzymes in the cholesterol biosynthetic pathway was investigated. The substitution of 14Cmevalonate for 14C-acetate completely blocked an SPF-induced 1.5-fold increase in squalene synthesis, suggesting that SPF stimulated mevalonate synthesis at HMGCoA reductase. 2,3-Oxidosqualene synthesis from 14C-mevalonate remained elevated (1.3-fold) with mevalonate demonstrating that SPF also stimulated squalene monooxygenase in hepatoma cells. SPF did not increase HMG-CoA reductase or squalene monooxygenase enzyme levels in cells, indicating that SPF directly activated these enzymes. Indeed, addition of purified recombinant SPF to rat liver microsomes stimulated HMG-CoA reductase by about 1.5-fold. These results reveal that SPF directly stimulates HMG-CoA reductase, the rate-limiting step of the cholesterol biosynthetic pathway, as well as squalene monooxygenase, and, coupled with the ability of PKA-mediated phosphorylation to regulate SPF activity, suggest a new means by which cholesterol synthesis can be rapidly modulated in response to hormonal and environmental signals.

Après administration intraveineuse, un nanovecteur va interagir avec de nombreuses molécules endogènes, notamment celles présentes dans la circulation sanguine. En fonction de la composition chimique du nanovecteur, ces molécules vont conférer à celui-ci une signature spécifique qui va orienter sa biodistribution et sa reconnaissance par certaines cellules de l’organisme. Plusieurs études sur l'identification des protéines adsorbées à la surface des nanovecteurs ont été menées alors que moins d'attention a été consacrée à l'interaction avec les lipoprotéines (LPs). Or, un nombre élevé de récepteurs aux LPs a été observé dans les cellules à croissance rapide et des études ont démontré que certaines cellules cancéreuses surexpriment ces récepteurs. De ce fait, l’utilisation des LPs comme vecteurs de médicaments anticancéreux a été proposée afin de favoriser le ciblage des cellules tumorales. Ce projet de thèse repose sur l’utilisation d’un bioconjugué (SQGem) issu du couplage chimique de la gemcitabine (Gem), une molécule anticancéreuse, au squalène (SQ) (un lipide naturel et précurseur de la biosynthèse du cholestérol), dont l’auto-organisation sous forme de nanoparticules à préalablement été décrite au laboratoire. Nous avons pu mettre en évidence la capture et le transport spontané de la SQGem par les LPs plasmatiques. Les résultats in vitro et in vivo que nous avons obtenus démontrent parfaitement que l’association préférentielle de la SQGem aux LPs corrèle fortement avec la quantité de cholestérol présente dans ces derniers. De plus, les simulations in silico effectuées ont révélé l’incorporation de SQGem dans le noyau hydrophobe des LPs. Par la suite, cette interaction spontanée a été mise à contribution pour effectuer le ciblage indirect des cellules cancéreuses ayant une expression élevée de récepteurs aux LPs, ce qui a été confirmé in vitro sur cellules ainsi qu’in vivo sur un modèle de tumeur expérimentale chez la souris. L’ensemble de ces résultats suggère l’originalité de notre approche, basée sur le ciblage des tumeurs de manière indirecte via les LPs qui constituent ainsi des « vecteurs » endogènes de SQGem. La « squalénisation » évite ainsi la préparation fastidieuse de vecteurs à base de LPs reconstitués et représente, par l’utilisation des LPs endogènes, une stratégie innovante et potentiellement révolutionnaire dans le traitement expérimental du cancer. / The in vivo fate of intravenously injected nanoparticles is strongly affected by their interactions with the blood components. Several studies have focused on the identification of proteins adsorbed at the surface of nanoparticles whereas less attention has been devoted to the interaction with lipoproteins (LPs). Interestingly, LPs have been previously described as excellent carriers for delivery of anticancer drugs due to their particularly high receptor-mediated uptake on several cancer cell lines. In this PhD project, we focused on a bioconjugate obtained by covalent linkage of the anticancer drug gemcitabine (Gem) to squalene (SQ) (a natural lipid and precursor of the cholesterol’s biosynthesis) whose ability to spontaneously self-assemble in the form of nanoparticles has been previously described in our laboratory. We have demonstrated that this conjugation enables the spontaneous capture and transport of the SQGem by circulating lipoproteins. In vitro and in vivo experiments revealed a preeminent affinity of SQGem towards cholesterol-rich LP particles and in silico simulations further displayed their incorporation into the hydrophobic core of LPs. Such spontaneous interaction allowed for indirect targeting of cancer cells with high expression of LP receptors, which was confirmed both in vitro and in vivo in an experimental tumor model in mice. To the best of our knowledge, the use of squalene to induce drug insertion into LPs for indirect cancer cell targeting is a novel concept in drug delivery. It represents a flexible, highly versatile platform that would enable efficient drug delivery by simply exploiting endogenous lipoproteins without the need for complex nanoparticles surface functionalization or artificial lipoproteins production.

Steroidal glycoalkaloids (SGAs) are secondary metabolites produced by approximately 350 species in the Solanaceae family. SGAs are reported to be important for pest resistance and flavor enhancement at low concentrations but are toxic to humans and other mammals at high concentrations. Studies on sterol / SGA biosynthesis have implicated squalene synthase as a key regulatory enzyme because it catalyzes an irreversible step from the mevalonic acid pathway. However, the regulatory mechanisms of squalene synthase are not yet known. A study was conducted to elucidate the distribution pattern of SGAs and to clone the squalene synthase gene in order to determine a relationship between SGAs and gene expression levels. Solanum chacoense, a wild potato species was used as a model plant from which tissues were harvested at specified developmental stages and analyzed for SGA content. The results from the SGA analysis suggest a qualitative and quantitative tissue- and age-dependent accumulation of SGAs. Regenerative tissues such as, axiliary shoots, flowers and floral buds had the highest levels of 88, 49 and 63 µmole/g DW, respectively. The roots, stems and tubers showed the lowest amounts of SGAs of 1 to 8, 5 to 15 and 7 to 15 µmole/g DW, respectively. Stolons and tubers accumulated higher amounts of α-chaconine (59 to 67%) than α-solanine (61 to 64%) at all developmental stages analyzed. On the other hand, in young expanding, fully expanded, and old senescing leaves where leptine and leptinines tend to dominate, α-solanine and α-chaconine together accounted for only 8 to 15%, 7 to 15%, and 8 to 45%, respectively. Plant organs that showed the highest biosynthetic activity for SGA production also had high levels of transcripts coding for genes of isoprenoid biosynthesis. The results from the cloning and characterization of squalene synthase suggest that the cloned cDNA fragment is a putative S. chacoense squalene synthase gene with an open reading frame / predicted protein precursor of 411 amino acids. The cloned cDNA has high similarity (68-100%) to known plant squalene synthase genes and contains six deduced peptide domains observed in other species. The 3â untranslated regions of floral buds, young leaves (early vegetative stage), and fully expanded leaves (anthesis) were different in length with, 249, 335, and 202 nucleotides, respectively. The Southern blot analysis suggests a single copy gene although the existence of a gene family cannot be ruled out. / Ph. D.

NADPH-cytochrome P450 reductase (CPR) is a flavoprotein containing both FAD and FMN and functions as the electron donor protein for several oxygenase enzymes found on the endoplasmic reticulum of eukaryotic cells, including cytochrome P450s involved in drug metabolism and cholesterol biosynthesis. As many as three enzymes in the cholesterol biosynthetic pathway have been demonstrated, or proposed, to use CPR as a redox partner: squalene monooxygenase, which converts squalene to 2,3-oxidosqualene; lanosterol demethylase, a cytochrome P450 (CYP51); and 7-dehydrocholesterol reductase, the final step in cholesterol synthesis. In yeast CPR can be replaced by the NADH-cytochrome b5 pathway, but this has not been demonstrated in animals or plants. My studies with hepatic cytochrome P450 reductase-null mice have revealed a second microsomal reductase for squalene monooxygenase that was not previously detected. Studies carried out with hepatocytes from CPR-null mice demonstrate that this second reductase is active in whole cells and leads to the accumulation of 24-dihydrolanosterol, indicating that lanosterol demethylation, catalyzed by CYP51, is blocked. These results demonstrate that this second reductase plays a significant role in supporting squalene monooxygenase but not cytochrome P450-mediated reactions. 7-Dehydrocholesterol reductase (E.C. 1.3.1.21) catalyzes the reduction of the 7-8 double bond of 7-dehydrocholesterol to yield cholesterol. It has been suggested that cytochrome-P450 reductase is required for this reaction. My studies show that 7-dehydrocholesterol reductase is enzymatically active in CPR-null microsomes, with activity equal to or greater than that found in preparations from wild-type mice. Mammalian cytochrome b5, which can accept electrons from either cytochrome P450 reductase or NADH-cytochrome b5 reductase, is known to be involved in augmenting some P450-dependent monooxygenase reactions. Cytochrome P450 2E1 has been found to exhibit reasonable rates of turnover via an NADHcytochrome b5 pathway in reconstituted enzyme systems and in heterologous hosts. Using microsomes from hepatic CPR-null mice, I have determined that NADH-dependent CYP2E1 activity in the absence of NADPH-dependent activity constituted approximately 10% of CYP2E1 activity observed in microsomal preparations with NADPH from wild-type mice. However, little or no CYP2E1 activity could be detected in primary hepatocytes isolated from CPR-null mice.