Gemcitabine and Edelfosine for the Treatment of Pediatric Cancer”

Departamento de Tecnología y Química Farmacéuticas

Facultad de Farmacia y Nutrición

Universidad de Navarra

TESIS DOCTORAL

“COMBINATORIAL NANOMEDICINE MADE OF SQUALENOYL-GEMCITABINE AND EDELFOSINE FOR THE TREATMENT OF PEDIATRIC CANCER”

Carlos Rodríguez Nogales

Pamplona, 2020

Departamento de Tecnología y Química Farmacéuticas

Facultad de Farmacia y Nutrición

Universidad de Navarra

TESIS DOCTORAL

“COMBINATORIAL NANOMEDICINE MADE OF SQUALENOYL-GEMCITABINE AND EDELFOSINE FOR THE TREATMENT OF PEDIATRIC CANCER”

Trabajo presentado por Carlos Rodríguez Nogales para obtener el grado de Doctor

Fdo. Carlos Rodríguez Nogales

Pamplona, 2020

UNIVERSIDAD DE NAVARRA

FACULTAD DE FARMACIA Y NUTRICIÓN

Departamento de Tecnología y Química Farmacéuticas

DÑA. MARÍA JOSÉ BLANCO PRIETO, Doctora en Farmacia y Catedrática del Departamento de Tecnología y Química Farmacéuticas de la Universidad de Navarra y, D. PATRICK COUVREUR, profesor emérito de la Universidad Paris-Saclay y miembro de la Academia de las Ciencias en Francia, certifican:

Que el presente trabajo, titulado “Combinatorial nanomedicine made of squalenoyl-gemcitabine and edelfosine for the treatment of pediatric cancer”, presentado por D. CARLOS RODRÍGUEZ NOGALES, para optar al grado de Doctor en Farmacia, ha sido realizado bajo su dirección en el Departamento de Tecnología y Química Farmacéuticas de la Universidad de Navarra. Considerando finalizado el trabajo autorizan su presentación a fin de que pueda ser juzgado y calificado por el Tribunal correspondiente.

Y para que así conste, firma la presente:

Fdo: Dra. María José Blanco Prieto Dr. Patrick Couvreur

Pamplona, 2020

Esta tesis doctoral se ha llevado a cabo gracias a becas otorgadas para la formación del personal investigador de la Asociación Española Contra el Cáncer (AECC) y la Asociación de familiares y amigos de pacientes con neuroblastoma (NEN, Nico contra el Cáncer).

A mis padres, Ángel y Pilar

La belleza, Luminoso instante Entre la rutina y la nada Espuma del tiempo.

“Estelas” (Instantáneas, de A.L. Rodríguez)

AGRADECIMIENTOS

Quisiera agradecer de la manera más breve posible a todos aquellos que me han acompañado a lo largo de este trabajo y que han hecho posible su consecución.

En primer lugar, me gustaría dar las gracias a la Universidad de Navarra y en particular a la Facultad de Farmacia y Nutrición y al Departamento de Tecnología y Química Farmacéuticas, por haberme permitido realizar esta tesis doctoral. No quisiera olvidarme de la Facultad de Farmacia de la Universidad Complutense de Madrid, donde di los primeros pasos de mi formación. Gracias, también, a la Asociación Española contra el Cáncer (AECC) y a la Asociación de familiares y amigos de pacientes con neuroblastoma (NEN, Nico contra el Cáncer), por darme la oportunidad de aportar mi granito de arena en la lucha contra el cáncer infantil. Gracias Joaquín (Molí) por confiar en mí.

Por supuesto doy las gracias a mi directora de tesis, María Blanco, por brindarme la oportunidad de trabajar y crecer profesionalmente junto a ti. Gracias por tu constancia, preocupación y trabajo; pero sobre todo por compartir mis éxitos y fracasos a lo largo de estos años. Gracias también a mi co-director Patrick Couvreur. Travailler avec toi a été un véritable honneur pour moi. Tes connaissances, ton dévouement et ton leadership m’ont inspiré jour après jour et je ne l` oublierai jamais. Merci beaucoup et bonne chance.

Me gustaría agradecer profundamente a las personas que han participado en este proyecto de investigación y han puesto todo su empeño y dedicación para que saliera adelante: Simona Mura (Université Paris-Sud), Silvia Irusta (Universidad de Zaragoza), Haritz Moreno (CIMA), Carolina Zandueta (CIMA), Azucena Aldaz (CUN) y Rosa Noguera (INCLIVA). De manera significativa, quisiera dar las gracias a Víctor Sebastián (Universidad de Zaragoza) por su gran implicación, disposición para ayudar siempre y por sus magníficas fotos de microscopía; a Fernando Lecanda (CIMA), por confiar en nosotros y motivarnos en la última fase de la tesis. Por último, a Didier Desmaële (Université Paris-Sud). Merci beaucoup Didier, de me laisser prendre mes repas avec toi, prendre un café avec toi et faire de la chimie organique avec toi et de ne jamais abandonner et toujours chercher des solutions.

Quiero dar las gracias a todos los profesores e investigadores del departamento por vuestra ayuda. Del mismo modo, gracias a los doctorandos galénicos Ana, Jorge, Cristian, Alba; pero también a los del otro ala; Leire, Diego, Nicolás, Itziar y muchos más que me dejo. Muchas gracias Hugo, por decirme siempre lo que piensas, por tu ayuda y afecto dentro y fuera del

laboratorio y por donar tus eritrocitos para este trabajo. Por otro lado, quisiera agradecer también el apoyo recibido dentro y fuera del Institut Galien a mis compañeros de la Universidad Paris-Sud y en especial a Arnaud, Federica, Raúl y Flavio. Me gustaría agradecer el apoyo y cariño recibido por mis compañeros y compañeras de equipo Elisa, Edurne I., Yolanda, Simón, Edurne L., Pablo, Souhaila y, especialmente, a Laura por caminar conmigo desde el primer momento hasta el final y compartir los momentos más difíciles.

Gracias a los míos aquí: Violeta, Belén, Manuel, Iratxe, Íñigo, Mónica y Maite; por estar siempre a mi lado cuando lo he necesitado. Vio, sé que mi hogar estará siempre donde tú estés. Sin duda, gracias/mila esker a mis compañeros de viaje musical, Mikel y Eneko, por ayudarme a poner banda sonora a esta tesis. Gracias también a todos mis amigos y amigas de Pozuelo de Alarcón, San Lorenzo del Escorial y de Madrid por estar ahí siempre para mí desde la distancia (creo que sois demasiados para citaros aquí).

Por último, gracias de todo corazón a mi familia y especialmente, a mi padre Ángel, mi madre Pilar, y mi hermano Jaime, porque sin vosotros esto no hubiera sido posible. Gracias por apoyarme, educarme y quererme como soy. Gracias a mi abuela Pilar, por preguntarme hasta el último instante cuándo regresaría a Madrid. Tuviste que dejarnos en este periodo pero vino Ana, a la que también le doy las gracias por revolucionar y llenar de alegría a nuestra familia.

A TODOS, GRACIAS

TABLE OF CONTENTS

ABBREVIATIONS ...... 1

INTRODUCTION 3 Nanomedicines for pediatric cancers

ABSTRACT ...... 6

1. INTRODUCTION ...... 7 1.1. Chemotherapeutic toxicity in pediatric cancer ...... 7 1.2. Physiology-based pharmacokinetic models (PBPK) ...... 8 1.3. Nanomedicines for pediatric cancers ...... 8

2. BLOOD CANCERS ...... 10 2.1. Leukemias ...... 10 2.2. Hodgkin and non-Hodgkin lymphoma ...... 12 2.3. Clinical nanomedicines in blood cancers ...... 13

3. CANCERS OF THE CENTRAL NERVOUS SYSTEM ...... 16

3.1. Nanotechnology and blood-brain-barrier ...... 16 3.2. Gliomas ...... 17 3.3. Medulloblastoma ...... 18

4. NON-CENTRAL NERVOUS SYSTEM EMBRYONAL TUMORS ...... 19 4.1. Neuroblastoma ...... 19 4.2. Retinoblastoma ...... 20

5. BONE TUMORS AND SOFT TISSUE SARCOMAS ...... 21

5.1. Osteosarcoma ...... 21 5.2. Ewing Sarcoma ...... 23

6. KEY MILESTONES IN PEDIATRIC CANCER NANOMEDICINE ...... 25

7. CONCLUDING REMARKS ...... 26

ACKNOWLEDGMENTS ...... 26 REFERENCES ...... 27

HYPOTHESIS AND OBJECTIVES ...... 41

CHAPTER I 45 A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl- lysophospholipid edelfosine ABSTRACT ...... 48 1. INTRODUCTION ...... 49 2. MATERIAL AND METHODS...... 50 2.1. Materials ...... 50 2.2. Preparation of nanoassemblies ...... 51 2.3. UHPLC–MS/MS method ...... 51 2.4. Electron microscopy imaging ...... 52 2.5. XPS assays ...... 52 2.6. Hemolysis evaluation ...... 52 2.7. Cell culture ...... 53

3. RESULTS AND DISCUSSION ...... 53

3.1. Design and characterization of supramolecular squalene based nanoassemblies ...... 53 3.2. Supramolecular conformation ...... 56 3.3. Element distribution ...... 58 3.4. Colloidal stability ...... 59 3.5. Hemolytic activity ...... 60 3.6. Anticancer activity ...... 61

4. CONCLUSIONS ...... 62

ACKNOWLEDGMENTS ...... 63

REFERENCES ...... 63

SUPPORTING MATERIAL ...... 68

Synthesis of 4-(N)-Trisnorsqualenoyl-Gemcitabine (SQ-Gem) ...... 68 UHPLC/MS/MS Parameters...... 69

CHAPTER II 71 Squalenoyl-gemcitabine/edelfosine nanoassemblies: anticancer activity in pediatric cancer cells and pharmacokinetic profile in mice ABSTRACT ...... 74 1. INTRODUCTION ...... 75

2. MATERIALS AND METHODS ...... 76

2.1. Materials ...... 76 2.2. Preparation and characterization of squalenoyl NAs ...... 76 2.3. Cell culture ...... 77 2.4. Cell internalization of SQ-Gem/EF NAs ...... 77 2.5. Cytotoxicity studies ...... 78 2.6. Pharmacokinetic studies ...... 78 2.7. Statistical analysis ...... 78

3. RESULTS ...... 79

3.1. Nanoprecipitation allowed for an easy preparation of SQ-Gem/EF NAs of 50 nm ...... 79 3.2. SQ-Gem/EF NAs were efficiently internalized by patient-derived metastatic pediatric OS cells ...... 79 3.3. Gem and EF free acted synergistically against OS and NB cells .. 82 3.4. SQ-Gem/EF NAs showed improved anticancer activity in OS and NB cells ...... 83 3.5. SQ-Gem/EF NAs favorably modified the pharmacokinetic profile of EF and Gem ...... 83

4. DISCUSSION ...... 85

5. CONCLUSION ...... 86

ACKNOWLEDGEMENTS ...... 87

REFERENCES ...... 87

SUPPORTING MATERIAL ...... 91

Characterization of squalenoyl NAs ...... 91 Quantification of Gem, SQ-Gem and EF in plasma ...... 92 Pharmacokinetic profile of SQ-Gem/EF NAs after a single peroral administration ...... 94

CHAPTER III 97 Squalenoyl-gemcitabine/edelfosine combination nanomedicine for pediatric osteosarcoma

ABSTRACT ...... 100

1. INTRODUCTION ...... 101

2. MATERIALS AND METHODS ...... 102

2.1. Materials ...... 102 2.2. Formulation of squalenoyl NAs ...... 102

2.3. Cell culture and cytotoxicity assays ...... 103 2.4. Pharmacokinetic studies ...... 103 2.5. In vivo efficacy studies ...... 104 2.6. Clinical chemistry and hematology analysis ...... 104 2.7. Statistical analysis ...... 104

3. RESULTS ...... 105

3.1. SQ-Gem/EF NAs displayed better anticancer activity in c-Fos overexpressing OS cells P1.15 over p53+/- Oss ...... 105 3.2. SQ-Gem/EF NAs drastically diminished the exposure of the total Gem pool in the blood stream in comparison with SQ-Gem NAs ...... 106 3.3. SQ-Gem/EF NAs delayed the progression of the primary OS tumor growth in a P1.15 orthotopic model ...... 107 3.4. SQ-Gem/EF NAs caused regression of lung metastases of P1.15 OS bearing mice ...... 108 3.5. SQ-Gem/EF NAs showed a suitable safety profile in vivo after eight administrations ...... 109

4. DISCUSSION ...... 110

5. CONCLUSIONS ...... 112

ACKNOWLEDGEMENTS ...... 113

REFERENCES ...... 113

GENERAL DISCUSSION ...... 117

REFERENCES ...... 129

GENERAL CONCLUSIONS ...... 137

CONCLUSIONES GENERALES ...... 141

ANNEX I 145 Therapeutic opportunities in neuroblastoma using nanotechnology

Abbreviations

ABBREVIATIONS

ALK Anaplastic lymphoma kinase ALL Acute lymphoblastic leukemia AML Acute myeloid leukemia ara-C Cytarabine AUC Area under the curve BBB Blood-brain barrier BChol-red CholEsteryl BODIPY 542/563 CI Combination index Cl Systemic clearance CNS Central nervous system Cs-corrected STEM Aberration corrected scanning transmission electron microscopy DAPI 4’,6-diamidino-2-phenylindole DDS Drug delivery systems DLS Dynamic light scattering DOX Doxorubicin DRI Dose-reduction index EF Edelfosine EPR Enhanced permeability and retention FA Fraction affected GD2 Disialoganglioside 2 Gem Gemcitabine HAADF High angle annular dark field detector HDL High-density lipoprotein HL Hodgkin lymphoma

IC50 Inhibitory concentration 50 LLOQ Lower limit of quantification MDR Multi-drug resistance MIBG Metaiodobenzylguanidine

Abbreviations

MRI Magnetic resonance imaging MTD Maximum tolerated dose NA Nanoassembly Nab-PTX Albumin-bound paclitaxel NB Neuroblastoma NHL Non-Hodgkin lymphoma NP Nanoparticle OS Osteosarcoma PBPK Physiology-based pharmacokinetic PBS Phosphate buffer saline PDI Polydispersity index PEG Poly (ethylene glycol) SD Standard deviation SEM Standard error of the mean siRNA Small interfering RNA SQ-COOH Squalenic acid SQ-Gem Squalenoyl-gemcitabine TEM Transmission electron microscopy UHPLC/MS/MS Ultra-high performance liquid chromatography tandem mass spectroscopy Vss Volume of distribution at steady state XPS X-ray photoelectron spectroscopy

INTRODUCTION

Nanomedicines for pediatric cancers

Introduction

Nanomedicines for pediatric cancers

Carlos Rodríguez-Nogalesa,b, Yolanda González-Fernándeza, Azucena Aldazc, Patrick Couvreurd*, María J. Blanco-Prietoa,b*

a Pharmacy and Pharmaceutical Technology Department, University of Navarra, Pamplona 31008, Spain b Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain

c Department of Pharmacy, Clínica Universidad de Navarra, Pamplona 31008, Spain

d Institut Galien Paris-Sud, UMR CNRS 8612, Université Paris-Sud, Université Paris- Saclay, Châtenay-Malabry Cedex 92296, France

Conflict of interest: authors have no conflict of interest.

*Correspondence to: M.J. Blanco-Prieto, Department of Chemistry and Pharmaceutical Technology, School of Pharmacy, Universidad de Navarra, C/Irunlarrea 1, 31008 Pamplona, Spain. Tel: +34 948425679, Fax: 34 948425740. E-mail: [email protected] Prof Patrick Couvreur, Université Paris-Saclay, CNRS, Institut Galien Paris Sud, 92296, Châtenay-Malabry France. Tel: +33 1 46835396, Fax: 34 948425740. E-mail: [email protected]

ACS Nano 12 (2018) 7482-7496 DOI: 10.1021/acsnano.8b03684

Nanomedicines for pediatric cancers

ABSTRACT Chemotherapy protocols for childhood cancers are still problematic due to the high toxicity associated with chemotherapeutic agents, and incorrect dosing regimens extrapolated from adults. Nanotechnology has demonstrated significant ability to reduce toxicity of anticancer compounds. Improvement in the therapeutic index of cytostatic drugs, makes this strategy an alternative to common chemotherapy in adults. However, the lack of nanomedicines specifically for pediatric cancer care raises a medical conundrum. This review highlights the current state and progress of nanomedicine in pediatric cancer and discusses the real clinical challenges and opportunities.

Keywords: Pediatric cancer, nanoparticles, liposomes, drug delivery systems, chemotherapy, leukemia, glioma, neuroblastoma, osteosarcoma.

Vocabulary: Physiology-based pharmacokinetic models, a mathematical framework similar to traditional pharmacokinetic models but including physiological knowledge; targeted nanomedicines, surface-modified nanocarriers with antibodies, aptamers or peptides among others that display active targeting towards specific tissues; nanoparticle pegylation; a strategy that endows stealth properties to nanoparticles for improving the efficiency of drug delivery; blood-brain barrier, a biological barrier at brain capillaries with highly selective permeability that protects the central nervous system; xenograft tumor model, a cancer disease animal model based on the transplantation of cancer cells from one species to another.

Introduction

1. INTRODUCTION

Over the last twenty years, impressive progress has been made in the treatment of children with cancer due to advances in surgery, radiotherapy, chemotherapy and early diagnosis. Nevertheless, after domestic accidents, cancer still remains the second leading cause of death in children and adolescents1,2 and their perspectives are still poor when compared to adult cancer victims.3 The search for novel therapeutic strategies to treat cancer is, therefore, particularly important in the case of pediatric cancers. Independently of sex and age, the most representative cancer types in children and adolescents are mainly hematological cancers, while central nervous system (CNS) tumors are the second main priority for pediatric oncologists in terms of diagnosis and incidence. Following brain tumors, non-CNS embryonal tumors are the third most frequently diagnosed cancer, while bone tumors and soft tissue sarcomas conclude the list of the most prevalent tumors in children4–6 (Fig. 1). As the field of nanotechnology research continues to progress towards ameliorating cancer therapeutics, this review summarizes our current understanding of its impact in the pediatric population and provides an outline of previous research in in vitro and in vivo pediatric models as well as taking account of the clinical perspectives.

1.1. Chemotherapeutic toxicity in pediatric cancer Within the overall therapeutic options to manage pediatric cancer, chemotherapy emerges as the last opportunity for children with poor prognosis. Although the anti-tumor efficacy of common cytostatic drugs such as anthracyclines or alkylating agents is well-proven, their clinical use normally entails a risk of toxicity which is augmented in the pediatric population. Unfortunately, the intense doses of cytostatic drugs administered to these children to attain a long-term cure are still unbalanced and lead to poor tolerance as well as late toxicities. In this sense, the occurrence of side effects such as cardiotoxicity, cutaneous reactions, necrosis or lymphoid and myeloid suppression, sometimes leads to the abandonment of first-line treatments.7,8 In addition, chemotherapy exposure may cause subsequent related neoplasms, cardiovascular complications or future cognitive dysfunctions.9 On the other hand, despite considerable progress in our understanding of pharmacokinetics in pediatric patients, some erroneous methods for drug dose selection are still used in these patients. The main reason is the lack of studies about drug disposition in pediatric patients, due to ethical or economic causes or feasibility issues. On the few occasions when clinical pharmacokinetics is used in clinical practice, the limitations of dosing algorithms to predict drug exposition are self-evident. Extrapolation from adult dosing is a common method for dose selection in pediatric patients. This can be based on age, as in Fried’s method (valid up to 2 years of age) or Young’s method (for children from 2 to 12 years of age), on weight (as in Clark’s method), or on an allometric method (which uses the ratio children’s weight divided by adult’s weight, all raised to the

Nanomedicines for pediatric cancers

power of ¾).10 However, all these algorithms only consider the children’s development and growth, without considering maturation in different children’s developmental stages. Their main drawback is the lack of proportionality between weight, age or body surface area and enzymatic development and maturation (ontogeny).11 In addition, around 70% of drugs used in children have not been appropriately studied in pediatric patients, which makes it necessary to use other methods that permit the extrapolation of pharmacokinetic and pharmacodynamic data obtained in adults to pediatric patients. All these facts support the need for physiology-based pharmacokinetic models (PBPK), as well as the design of nanoformulations for better control of drug delivery and biodistribution in children.

1.2. Physiology-based pharmacokinetic models (PBPK) PBPK integrate data from different sources, including ontogenesis of metabolizing and transporters routes, to predict the ADME process (absorption, distribution, metabolism, and elimination) in pediatric patients. PBPK give a mechanistic quantitative framework which, when relevant physiological properties of the target population are available, allows extrapolation of the information from another patient population to that population. Information incorporated in these models includes drug parameters and system parameters for a specific population.12 PBPK models have been developed for analgesic drugs, metilxantins, anesthetic drugs and anticancer drugs, such as docetaxel.13 They have demonstrated utility in finding solutions for pharmacotherapy optimization in pediatric patients. Precisely this should be crucial in the oncology field, given the chemosensitivity and curability of most prevalent tumors in the pediatric population, such as acute lymphoblastic leukemia (ALL), in which common errors in cytostatic drug dosage have fatal consequences. In this context, when comparing chemotherapy doses expressed as a body surface product between children and adults, the exposure to cytotoxic drugs tends to be higher in the pediatric population. Marsoni et al. observed that 16 out of 17 antineoplastic agents analyzed displayed a dose quotient between children and adults of 1.5 mg/m2.14 This behavior has been erroneously interpreted in routine clinical practice as showing better tolerance to chemotherapy side effects at younger ages, leading to negative consequences. Therefore, it is crucial to bear in mind the different pharmacokinetic profiles of distinct pediatric subgroups to avoid under or over-dosing.

1.3. Nanomedicine for pediatric cancers Drug delivery systems (DDS) have been widely studied with the aim of enhancing and preserving the clinical potential of drugs by improving their protection, absorption, penetration and distribution. This issue is crucial in the case of cytostatic drugs and has resulted in the development of several formulations based on polymer, metallic or lipid

Introduction

nanoparticles (NP), liposomes, micelles, nanofibers, and many other nanomaterials loaded with chemotherapeutics (Fig. 1). These nanosized suspensions are able to increase the therapeutic index of loaded drugs and improve tumor targeting, thus modifying drug pharmacokinetics and tissue distribution.15–17 Drug targeting can be activated by surface ligand engineering18 or by the design of stimuli-responsive nanodevices.19,20 And even if limited to head and neck or Kaposi tumors, nanometric particles may also display some inherent passive accumulation in tumor tissue, which is known as the enhanced permeability and retention (EPR) effect, an effect that is not as well understood as was initially thought.21,22

Figure 1. Nanomedicines as therapeutic strategies for the most relevant pediatric tumors. Nanoparticles (NPs), liposomes, dendrimers or micelles, among other drug delivery systems, may overcome common limitations derived from chemotherapy in central nervous system (CNS) cancers, blood cancers, non-CNS embryonal tumors and musculoskeletal tumors. The encapsulation of cytotoxic drugs in NPs confers sustained drug release, better tissue accumulation, bioavailability enhancement and improvement of the therapeutic index.

It is important to bear in mind that children are not just ‘small adults’ and that their unique physiology is an extra incentive for the design of nanomedicines to improve therapeutics in the pediatric population. Incorrect dosing regimens extrapolated from adults worsen the chemotherapeutic toxicity that is, in fact, reported to be more severe in children than in adults per se. And nanotechnology has demonstrated potential applicability in the field to ameliorate the therapeutic index of cytostatic agents by decreasing their toxicity. The use of anticancer nanomedicines in the pediatric population may therefore represent one step forward in avoiding problems associated with drug dose selection, as well as, the chemotherapeutic associated toxicity. However, the success of

Nanomedicines for pediatric cancers

these nanotherapies resides in taking into consideration some key aspects such as the safety of the nanocarriers or the tumor microenvironment.23 Moreover, the arrival of novel therapies is hampered by common translational barriers in the pediatric research. These aspects are summarized in Fig. 2.

Figure 2. Schematic illustration of the proposed approaches to improve therapeutics in pediatric cancer management.

2. BLOOD CANCERS Hematologic malignancies account for about 40% of all pediatric cancers. ALL and acute myeloid leukemia (AML) occasion an abnormal blood cell increase that leads to an outcome that is fatal without treatment. On the other hand, Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) are a malignant proliferation of lymphocytes that generally arises from the lymph nodes of the immune system.

2.1. Leukemias The severe treatment-related toxicity of both chemotherapy and hematopoietic stem cell transplantation means that surveillance of cardiac toxicity, infectious diseases and other fatal outcomes is needed.24,25 Besides this, leukemia relapses are still incurable in most cases and pediatric oncologists have called for nanomedicines to be made available, in the hope that these will provide new opportunities. In this view, dexamethasone, a glucocorticoid frequently used in chemotherapy to induce apoptosis of B and T lymphocytes, was encapsulated in poly(ethylene glycol) (PEG) and poly(ε-caprolactone) amphiphilic block copolymer nanoassemblies in order to improve the drug therapeutic

Introduction

index.26 The NPs developed were biocompatible, non-toxic, and increased the efficacy of dexamethasone in mice by increasing the drug’s half-life, allowing the authors to make arguments for the long-term use of encapsulated dexamethasone in ALL children. With respect to the therapeutic management of pediatric AML, methotrexate-gold NPs have been shown to improve the therapeutic index of this antimetabolite. NPs proved to be non-toxic in normal hematopoietic cells and displayed an augmented selective drug delivery of methotrexate against THP-1 AML cell line, achieving a pronounced anticancer potency. Their in vivo efficacy and safety was later evaluated in a murine xenotransplant model of primary human AML. Methotrexate-gold NPs ameliorated leukemic cell suppression without added toxicity in comparison to the control group and the free methotrexate.27 However, such nanoformulation would certainly deserve more detailed pre-clinical toxicological investigations to elucidate the elimination pathway after repeated administration, as well as, the absence of cell and tissue accumulation. Bearing in mind that the relevance of pharmacogenetics in methotrexate response in children is still controversial28 and that no full consensus in protocols about the management of methotrexate toxicities has been achieved,29 the encapsulation of methotrexate may represent a reliable advantage in the pediatric population. The use of targeted therapies against leukemic cells is still a major challenge for researchers, since cytotoxic agents must discern between malignant and normal circulating blood cells. In this sense, Basha et al. underlined the necessity of preserving the viability of healthy cells and postulated the design of reconstituted high-density lipoprotein (HDL) NPs to effectively target HDL/SR-B1 receptors, overexpressed in malignant lymphocytes.30 On the other hand, Matthay et al. investigated antibody- liposomes in pediatric leukemia cell lines 3 decades ago and reported the possible clinical benefits derived from their enhanced intracellular penetration.31 For instance, anti-CD33 antibodies targeting ligands against AML cells were coupled to lipid NPs,32 triggering a high NP uptake by CD33 positive cells. Moreover, the clinical potential of anti-CD33 for improving immune targeting of AML cells has been confirmed and finally, gemtuzumab ozogamicin (Mylotarg) has recently been approved by the FDA for children of 2 years of age and older, with relapsed or refractory CD33-positive AML.33 The above-mentioned approaches merely represent proof of the vast literature on NPs (e.g. refs. 34,35) but it is convenient to consider that in many cases, the distinction between adult and childhood leukemia research can be ambiguous. The majority of these publications have reported the nanomedicines’ potential to improve the therapeutic efficacy of antitumor drugs and to minimize toxicities in pediatric leukemia. However, it is difficult to elucidate if the proposed formulations are clinically relevant or provide a specific benefit for affected children as compared to common therapeutic extrapolation from adults. In the best-case scenario, the animal models used are based on rodents xenografted with pediatric leukemic cells that cannot be examined for late toxicities. In addition, physicians’ main concern is still the achievement of a global consensus in the chemotherapy dose adjustment in children. In fact, the progress made in the last decade

Nanomedicines for pediatric cancers

has increased substantially the survival rates, so that the novel therapeutic strategies have to demonstrate strong evidence in order to attract pediatric oncologists’ attention and lead to modification of the established protocols. Importantly, some recent articles have reported the challenges faced when registering childhood cancers, both in terms of their incidence, and as regards our understanding of the reasons for survival.6,36 In any case, unlike adults, children are still at a disadvantage since more than the 50% of patients treated with an anthracycline will develop cardiac abnormalities during long-term follow- up, while 10% of children exposed to a cumulative anthracycline dose greater than 300 mg/m2 will develop congestive heart failure.37,38 In addition, a recent study reported the adult neurocognitive performance in childhood leukemia survivors in relation to intrathecal methotrexate.39 Although this kind of CNS-directed prophylactic chemotherapy prevents relapses and increases survival, the study revealed that childhood leukemia survivors displayed significant late neurocognitive sequelae. At the moment, the most realistic opportunities for these children reside specifically in the consolidation of best-in-class nanocarriers loaded with anthracyclines, methotrexate or taxanes among others. Indeed, when encapsulated into NPs or liposomes, those drugs have been shown to decrease both cardiac and neurological toxicity because they do not accumulate in those tissues.37

2.2. Hodgkin and non-Hodgkin lymphoma Nanotechnology has been also requested to challenge refractory and aggressive lymphomas, and the narrow relationship with ALL has supported still further the development of nanomedicines to combat overall lymphoid malignancies. The use of anthracyclines such as DOX in lymphomas is becoming compulsory given its high antitumor efficacy; however, its prolonged use entails several risks. In order to improve anthracycline safety, researchers have attempted to load this molecule into nanocarriers since the encapsulation of DOX was shown to dramatically decrease cardiac toxicity.40 Although survival rates have increased significantly following the implementation of the French protocol LMB 96, HL and NHL survivors after treatments are at risk of having secondary leukemias, breast cancer, myelosuppression, late cardiac complications and endocrine sequelae.41–43 The principal challenge for pediatric oncologists is nowadays to lower the aggressiveness of both radiotherapy and chemotherapy in order to avoid the above-mentioned late complications. Nanomedicines could help to maintain therapeutic concentrations of cytotoxic drugs below the toxic threshold for long periods, ameliorating the future outlook of survivors. Within this context, the encapsulation of anthracyclines could be an optimal therapeutic strategy for administering long-term therapies without compromising children´s health. The surface singularity of malignant lymphoid tissue has prompted researchers to design targeted nanomedicines for increasing drug selectivity. This could be optimal in diffuse tumors such as lymphomas, especially when common chemotherapy is unable to achieve a complete remission. Rituximab integrates the first generation of antibodies

Introduction

approved for B-cell antigen CD20, a well-known target moiety for NHL cells. Thus, Wu et al. conjugated the fab fragment of rituximab to DOX loaded liposomes.44 In vivo biodistribution assays demonstrated a high liposome accumulation in the tumor tissue, suggesting this formulation as a potential candidate for NHL children’s care. Similar results were obtained by conjugation of anti-CD20 antibodies to DOX-coated polymer active carbon NPs,45 DOX-mesoporous silica NPs46 and albumin bound-paclitaxel NPs.47 The application of targeted nanomedicines as non-viral nanovectors for small interfering RNA (siRNA) delivery was also explored for B cell malignancies. The poor stability in biological fluids and the low intracellular penetration of siRNAs hamper their therapeutic use, making the use of nanocarriers necessary.48 In this sense, Weinstein et al. prepared CD-38 targeted lipid NPs loaded with siRNAs against cyclin D1, a tumorigenic factor overexpressed in Mantle cell lymphoma cells. Coating NPs with anti-CD38 monoclonal antibodies permitted specific NP uptake by these cells, inducing efficacious gene silencing. Moreover, the administration of this nanoformulation demonstrated prolonged survival of tumor-bearing mice with no observed toxicity.49

Unlike leukemia, lymphomas are solid tumors and there is a need to deliver drugs selectively toward malignant lymphoid tissues. Further preclinical studies are expected to enable these nanotechnology-based approaches to proceed to clinical trials.

2.3. Clinical nanomedicines in blood cancers Within the nanomedicines approved for clinical trials in cancer, mainly liposomes and polymeric micelles, only a few have been approved for clinical research in the pediatric population, and among these, the majority belongs to hematological malignancies rather than to other pediatric cancers.50 The reason probably arises from the high prevalence of these diseases in children. The different nanoformulations ongoing in clinical trials corresponding to pediatric leukemia and lymphoma are shown in Table 1. Liposomes were used as drug carriers in most of the studies because they are probably one of the safest nanoformulations reported to date and they can encapsulate distinct drugs in their phospholipid bilayers or aqueous cores. In brief, although data related to children are still limited concerning the clinical studies using vincristine sulfate liposomal formulation (Marqibo), the first reports are encouraging. The good tolerability makes Marqibo a solid candidate to replace the use of bulk vincristine in the future.51,52 Pediatric oncologists argue that the use of these liposomes will allow for dose intensification with the aim to ameliorate survival outcomes with reduced or comparable neurotoxicity in comparison to free vincristine. Liposomal daunorubicin is another nanomedicine being investigated. Phase I-II studies have generated satisfying results concerning the pharmacokinetic profile of encapsulated daunorubicin in comparison with its free counterpart.53–55 Although the comparison with investigations in adults showed no substantial differences in pharmacokinetics with respect to children, a longer follow up in children would be necessary to determine the ultimate role of these liposomes in avoiding cardiovascular complications in the future. In that sense, it has been reported that the myocardium is

Nanomedicines for pediatric cancers

characterized by tight capillary junctions, which would prevent liposomal anthracyclines from crossing the capillaries into the myocardial tissue,37 thus suggesting that there may be lower cardiotoxicity in the future. Taking this setting into consideration, the International Berlin-Frankfurt-Münster Study Group performed a randomized phase III study with liposomal daunorubicin added to FLAG (fludarabine + high-dose cytarabine + G-CSF). Although overall survival and toxicity were similar compared to FLAG alone, the combination with daunorubicin liposomes improved early treatment response in pediatric relapsed AML.56 Intrathecal liposomal cytarabine (ara-C) (DepoCyt) administration has been also approved in children with leukemia relapsing to CNS.57,58 The slow continuous release of ara-C results in a lower peak of ara-C levels and longer duration of exposure compared with standard ara-C.59 No permanent adverse neurological sequelae have been observed in combination with dexamethasone and intrathecal prednisolone succinate.60 However, larger prospective trials are still needed before liposomal ara-C can be used as first-line prevention of relapse. As seen in table 1, some clinical trials were terminated or the recruitment was suspended. The reasons for assays interruption are sometimes unavailable and may result from unexpected toxicological events, economic/logistic reasons or absence of treatment improvement.

Introduction

Table 1 Liposomal-based therapies undergoing clinical investigation for leukemia and lymphomas

Formulation Drug (trade Age Type of Current Code and NP size name) (years) cancer status (update year) Phase I 1-21 2017 NCT02879643 (recruiting) Relapsed ALL Phase I/II All 2014 NCT00144963 (completed) Non-PEGylated Vincristine Phase II 2016 0-21 NCT02518750 liposomes; sulfate (recruiting) 100 nm (Marqibo) Relapsed Phase I 0-21 leukemia and 2014 NCT00933985 lymphoma (terminated) Phase II 2012 All NCT00038207 (completed) Phase III 2016 0-21 High-risk HL NCT01026220 (not recruiting) Lymphomatous Phase II 2016 3-31 (NHL) NCT02393157 (recruiting) meningitis

Non-PEGylated High-risk ALL Phase III 2012 multivesicular Intrathecal 1-18 (leukemic (recruitment NCT00991744 liposomal ara-C meningitis) suspended) matrix; (Depocyte) 3-30 μm Lymphomatous Phase I 1-21 and leukemic 2010 NCT00003073 meningitis (unknown)

Non-PEGylated ara-C- Recurrent Phase I/II 2017 liposomes; Daunorubicin 1-21 NCT02642965 leukemia (not recruiting) 100 nm (CPX-351) Non-PEGylated Daunorubicin Phase III 2016 liposomes; 0- 17 AML NCT02724163 (Daunoxome) (recruiting) 45 nm PEGylated DOX Phase I/II liposomes; hydrochloride All Relapsed HL 2016 NCT00006029 90 nm (Doxil) (completed) Non-PEGylated DOX >18 Phase II 2011 liposomes; NHL NCT01009970 (Myocet) months (unknown) 100-200 nm Non-PEGylated Phase I 2011 liposomes; Annamycin 1-21 Leukemia NCT00430443 (terminated) 30 nm ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; ara-C, cytarabine; DOX, doxorubicin; HL, Hogdkin lymphoma; NHL, non-Hodgkin lymphoma; NP, nanoparticle; PEG, poly(ethylene glycol).

Nanomedicines for pediatric cancers

3. CANCERS OF THE CENTRAL NERVOUS SYSTEM After leukemias, brain tumors are the second most common type of childhood cancer. Brain tumor cells in children are mainly neurons and glial cells that give rise to astrocytomas/gliomas, medulloblastomas and ependymomas.

3.1. Nanotechnology and blood-brain-barrier The blood-brain barrier (BBB) is located in brain capillaries (Fig. 3) and present fenestrated or continuous endothelial cells which are strongly linked by tight junctions and surrounded by astrocytes, pericytes and the continuous basement membrane.61,62 The BBB limits the transit of many molecules and protects the CNS from circulating agents such as xenobiotics or hydrophilic cytostatic drugs.63,64 Additionally, it results in a dramatic restriction of the antitumor drug arsenal which exacerbates the prognosis of affected children. Some pediatric CNS neoplasms have, however, been reported to exhibit BBB breakdown and therefore increased drugs permeability of the BBB.65 Nevertheless, standard cytostatic agents are unable to reach the tumor tissue and effect cures in these patients. This therapeutic failure could be due to the spread of malignant invasive tumor cells beyond the areas of BBB disruption.66,67 In that sense, chemotherapy could be delivered only in the necrotic tumor center with the disrupted BBB and not in the periphery of the growing tumor. Notably, data collected about the penetrability of drugs into the CNS have shown that the BBB permeability between adults and children is similar, at least after 4 months. Interestingly, there are strategies focused on temporarily disturbing the BBB to deliver more drug inside the brain.68,69 In view of the lack of awareness of chemotherapeutic failure in pediatric brain tumors, surface-functionalized nanomedicines with the possible ability to cross the BBB are becoming a valuable option. Over the past few years, nanotechnology has made significant progress in carrying chemotherapeutic drugs inside the CNS.70 The main materials investigated for nanomedicines in high-grade gliomas are gold, lipids and proteins conjugated with endothelial cell-penetrating molecules.71 Besides chemo-electric charge or particle size, another crucial aspect that determines the BBB passage is the particle surface.72 For instance, our group, among others, has reported that non-ionic surfactants such as Tween80 are decisive to achieve better brain uptake.73 Moreover, the existence of nutrient transporters in BBB cells such as insulin receptor, LAT-1, GLUT-1, transferrin receptor and lipoprotein receptors has prompted researchers to consider them as portals to carry drugs inside the brain stem (Fig. 3). For targeting high-grade gliomas, transferrin, Angiopep-2, Apolipoprotein E-derived peptide and many other ligands have been conjugated to NPs, obtaining interesting outcomes.74–76 However, these experiments have been conducted with the aim of treating adult high-grade gliomas, and presumably pediatric brain tumors might not meet similar criteria77–79 so that these approaches should be further assessed in suitable pediatric brain tumor models.

Introduction

In general, the amount of NPs able to translocate intact BBB remains still limited to less than 1%/gr tissue of the injected dose,80 except when the tumor growth result in a sufficient breakdown of the BBB. In this case, anticancer compounds delivered to the brain may reach higher drug concentrations since nanoencapsulation can avoid drug degradation and favor the extravasation through EPR effect, which may constitute a significant improvement as compared to the drug free treatment.

Figure 3. Nanomedicines for crossing the blood-brain barrier (BBB) in pediatric brain tumors. The illustration shows transferrin (green) decorated nanoparticles (NP) (blue) loaded with cytotoxic drugs against a high-grade pediatric glioma. (1) NP ligands are recognized by nutrient receptors overexpressed in the endothelium of brain capillaries, triggering receptor mediated endocytosis of NPs. (2) NPs extravasate into central nervous system (CNS) by eluding endothelial cells, pericytes and astrocytes being further taken- up by malignant cells. (3) Internalized NPs release their cytotoxic drug content leading to tumor cell death.

3.2. Gliomas Compared to non-malignant astrocytomas, high-grade astrocytomas such as glioblastoma multiforme, diffuse intrinsic pontine glioma or anaplastic astrocytoma have a markedly poorer prognosis.79 Unfortunately, many anticancer drugs have shown only moderate success in clinical trials, including those which presented a suitable response in adult glioma, such as temozolomide.78 Mueller and Chang remarked that pediatric high-grade gliomas are biologically different from their adult counterpart.81 The benefit of treating the pediatric population specifically with NPs results from the impossibility of accomplishing high-risk invasive procedures or giving high doses of cytostatic drugs and radiation. The infant population is, indeed, more vulnerable than adults to this kind of procedure, which may lead to premature deaths or late toxicities.

Nanotechnology research for gliomas includes a wide range of studies regarding drug/gene delivery and brain-targeting, as well as, tumor imaging applications,82-85 but

Nanomedicines for pediatric cancers

the literature lacks pre-clinical experiments concerning the use of nanomedicines specifically for pediatric glioma treatment. In such a paper by Li et al., carbon dots conjugated with transferrin were used for DOX delivery and pediatric glioma application.86 In vitro studies performed in SJGBM2 cells, a human-derived pediatric glioblastoma multiforme cell line, revealed marked cell uptake enhancement when DOX was encapsulated into the functionalized carbon dots. Blank transferrin carbon dots are supposed to be non-toxic in vitro, but the safety of this material for brain delivery in children is highly questionable since the fate of carbon dots in the brain parenchyma remains unanswered. Despite being higher in incidence, the available pediatric astrocytoma/glioma cell lines is very limited, probably because some of these tumors have been difficult to culture.87 This has probably hindered their investigation and may explain the absence of nanotechnology studies in comparison with standard pediatric brain cancers such as medulloblastoma.

3.3. Medulloblastoma After astrocytomas, this malignant embryonal tumor of the cerebellum is one of the most common brain tumors in children under 10 years.88 With the aim of improving therapeutics in this kind of tumor, some versatile nanomedicine platforms have been proposed. A recent proof is the design of glutathion tumor responsive albumin-NPs containing cisplatin.89 Cross-linked glutathion in NPs permitted, via redox, the cell- specific modulation of cisplatin delivery in DAOY cells, a standard pediatric medulloblastoma cell line,87 increasing the cytotoxic action of cisplatin in vitro. However, in vivo the question still remains if it is profitable to merely prolong the half-life of a cytostatic drug/gene if the drug is only distributed in the peripheral compartments and not in the CNS. At least, permeability studies on in vitro BBB models, which are of particular concern in pediatric CNS tumors, should be mandatory to further elucidate the potential of these approaches. The clinical investigation of liposomes has been extensively carried out in the last few decades, which has led regulatory agencies to approve DOX-liposomes (Doxil), the first nano-drug which has reached the market. Subsequently, liposomal DDS have also been proposed for multiple purposes, including brain drug delivery. In this regard, PEGylated liposomal DOX has been tested in combination with radiofrequency ablation therapy.90 Although tumor prognosis in old mice was found to be dissimilar to young mice, these studies demonstrated that radiofrequency ablation therapy combined with DOX loaded liposomes could overcome tumor progression regardless of tumor environment or age. Furthermore, due to encouraging results in clinical trials gathered mainly in hematologic tumors,57,58 ara-C-liposomes have also entered clinical trials in children with relapsed medulloblastoma.91 Clinicians have reported a prolonged progression-free survival after their intrathecal administration. Recently, albumin-bound-rapamycin NPs for use in combination with temozolomide and irinotecan in the pediatric and adolescent population

Introduction

have entered Phase I clinical trials (NCT02975882) for recurrent CNS neoplasms and other solid tumors. Bearing in mind that the main limitation that leads to therapeutic failure is the incapacity of cytostatic drugs to effectively reach the brain tumor tissue, we need to leverage the active targeting of NPs in order to carry antitumor drugs across the BBB and achieve a marked antitumor effect.

4. NON-CENTRAL NERVOUS SYSTEM EMBRYONAL TUMORS Although there are also embryonal tumors that arise from the brain or the musculoskeletal tissue, the following malignancies are grouped in this section because of their close similarity. These cancers are frequently diagnosed in children under 5 years and originate from the developing embryonal cells that constitute miscellaneous tissues and organs such as the peripheral nervous system (neuroblastoma), the eye (retinoblastoma) or the kidney (Wilms tumor).

4.1. Neuroblastoma Peripheral neuroblastic tumors are classified histologically as neuroblastoma, ganglioneuroblastoma and ganglioneuroma, neuroblastoma being the most clinically relevant variant due to its malignancy and heterogeneous behavior.92 Generalized cytotoxicity that leads to severe side effects is often derived from the use of potent alkylators such as cisplatin.93 In order to dispose of that issue, Zhen et al. prepared casein NPs to encapsulate cisplatin.94 The in vivo experiments determined a significant anticancer efficacy and toxicity improvement with encapsulated cisplatin. Nevertheless, Ghaghada et al. postulated that more data were required about NP interaction with tumor tissue in order to gain preclinical insights and better knowledge about their real advantages.95 The evaluation of a liposomal contrast agent in an orthotopic kidney capsule model of neuroblastoma made it possible to establish that the liposome cell uptake in pediatric solid tumors such as neuroblastoma was heterogeneous and not governed by tumor geometry. The aggressiveness of high-risk neuroblastoma calls into question the nanomedicines’ potential in eradicating tumor relapses. Nanoencapsulated alkylators have, indeed, proven drug prolongation in the organism, as well as drug safety and efficacy improvement. Notwithstanding, many of these nanoformulations have been tested only in vitro, and in vivo studies are mainly from xenograft tumor models that do not properly reproduce childhood neuroblastoma. Thus, the preclinical assessment of NPs for translation to clinical trials should certainly be improved. Standardization of animal models is mandatory96,97 in order to predict NPs response, and it should take into account the age differences, tumor environment, immune responses and late sequelae present in children.

Nanomedicines for pediatric cancers

Children with high-risk neuroblastoma are at present given anti-disialoganglioside 2 mAb therapy due to the abundant levels of this cell surface antigen.98 This target has drawn attention not only as an immunotherapy approach99 but also for developing immune-targeting strategies, allowing tumor-specific delivery of cytostatic drugs for upgrading current therapies. For instance, porous silica NPs surface was coated with anti- disialoganglioside 2 antibodies to facilitate the targeted delivery of microRNA-34a.100 The tumor-specific over-expression of microRNA-34a induced the activation of caspase- mediated apoptotic pathways, leading to a significant decrease of tumor growth in neuroblastoma bearing mice. Several other experiments have been further performed by using NPs to target disialoganglioside 2 in neuroblastoma cells. In these studies, antibodies against this receptor were conjugated to liposomes,101 iron102 and gold103 NPs before their administration. In vitro and in vivo assays confirmed the greater anti- proliferative efficiency, the improved accumulation in tumor tissues and the specificity of these antibody-conjugated NPs, compared to their non-decorated NPs counterparts. Peripheral neuroblastic tumors are still challenging owing to their heterogeneity. Knowledge gained from genomic discoveries has shown that mutational burden increases on relapse.104,105 Thus, if personalized treatments represent opportunities for high-risk neuroblastoma children, the use of targeted nanomedicines like the ones described above might be required to improve therapeutics. Such approaches may be optimal in relapsed neuroblastoma even if, as with other embryonal tumors, extreme variability and poor understanding of the disease still restrain new therapeutic attempts.

4.2. Retinoblastoma Enucleation is the definitive treatment for intraocular retinoblastoma before the tumor spreads, while if there is a chance to save the patient’s sight, clinicians recur to cryotherapy, thermotherapy, laser photocoagulation, external beam radiation and chemotherapy, among other methodologies.106 The limitations of chemotherapy have attracted some attention in the pharmaceutical field, given that DDS might reduce the number of intravitreal or subconjunctival injections, thus improving local and systemic toxicity. On a preliminary note, it is worth stressing that safe biocompatible materials are mandatory in the design of nanocarriers for cytostatic ocular delivery in children. With the aim of overcoming the rapid clearance of free carboplatin from the eyes, Ahmed et al. leveraged the high biocompatibility of proteins and loaded carboplatin in apo- transferrin and lactoferrin based nanocarriers.107 Carboplatin NPs exhibited a higher cell uptake, unlike soluble carboplatin, probably boosted by endocytosis mediate receptors. Notably, nanoformulations have been administered not only locally but also systemically,108 this bringing a certain degree of controversy about which is the best route of administration. Even though local therapies have demonstrated a substantial systemic toxicity reduction, intra-arterial chemotherapy is attracting interest nowadays. Thus, the

Introduction

current era of retinoblastoma management entails intra-arterial chemotherapy plus additional intravitreal chemotherapy.109,110 More studies are needed to elucidate the best route of administering nanomedicines, with a view to ameliorating globe salvage in eyes with advanced retinoblastoma in children. The aforementioned studies suggest an up-and-coming role of NPs for the local administration and non-invasive therapies of retinoblastoma. Interestingly, nanoencapsulated carboplatin for ocular administration is currently under clinical trials, but only in young adults with advanced retinoblastoma. Preliminary results show a sustained-release behavior of carboplatin in retina and vitreous without systemic side effects.111 In light of the previous example, it is questionable if we should promote clinical research that can be translated into health opportunities not only in retinoblastoma but also in overall childhood cancers. Increasing the resources to achieve satisfactory submissions to ethics committees has to be encouraged mainly in the case of nanomedicines.

5. BONE TUMORS AND SOFT TISSUE SARCOMAS Musculoskeletal tumors are a heterogeneous group of diseases with a low global incidence. Among them, Osteosarcoma (OS) and Ewing sarcoma are the most common malignant bone tumors diagnosed.

5.1. Osteosarcoma Although OS accounts for only 5% of all childhood cancers,112 due to its aggressive progression together with the high tendency of primary OS to metastasize to the lungs, it is responsible for 9% of all cancer-related deaths in children.113 Methotrexate has been one of the key drugs in OS therapy since the early 1970s.114 However, despite its great efficacy, several drawbacks are associated with this antimetabolite, such as severe toxicity and rapid blood clearance. To avoid the need for rescue and to increase the drug half-life, methotrexate was encapsulated into poly(lactic-co-glycolic acid)-layered double hydroxide NPs and, in comparison with free methotrexate, this formulation showed higher efficacy for inhibiting OS tumor growth, and improved survival rates in vivo.115 The need for multi-agent therapy for the treatment of resistant tumors such as OS is widely known and accepted. The complementary cellular targets of DOX and edelfosine, an alkyl-lysophospholipid that had previously shown anti-OS activity led our group to evaluate whether their combination would be synergistic against OS cells. The combination of DOX-containing-lipid NPs and edelfosine-containing-lipid NPs showed a synergistic activity against cancer cells and, on the other hand, the encapsulated DOX

Nanomedicines for pediatric cancers

could revert cellular drug resistance mechanisms and increase the effectiveness comparatively to the non-encapsulated drugs.116 The strong investment in clinical trials in Europe and in the USA to amend the standards of chemotherapy agents’ combinations and schedules has not improved the survival rates that were achieved in the last decades. Dose-response curve for some cytostatic drugs such as ifosfamide have reached the maximum;117 however, bearing in mind the pre-clinical results obtained with nanomedicines in OS, some of these nanoformulations could emerge in clinical practice as an implement to augment further the therapeutic index of the chemotherapeutic agents. Finally, the market authorization of liposomal mifamurtide (Mepact) in 2009 as a second line treatment for children with OS118 represented an encouraging advance, although this is an immunostimulant nanomedicine and not a cytotoxic per se. Bisphosphonates are commonly used drugs for the treatment of bone resorption disorders because they display a pyrophosphate-like structure able to coordinate to calcium ions present in the hydroxyapatite matrix of the bone.119 In this context, bisphosphonate-bound polymeric NPs have been developed to deliver chemotherapeutics to bone tumors.120–122 The higher efficacy of DOX when targeted by means of bisphosphonate-bound NPs was confirmed in vivo and very importantly, no sign of DOX- induced-cardiac toxicity was observed.120 The authors reported that, at short time after administration, the targeted and non-targeted carriers predominantly distributed close to the tumor areas in OS xenografts; however, only bisphosphonate-NP remained present in the tumor bulk after 7 days. The authors suggest that the initial uptake of non-targeted NP could be caused by a passive targeting owing to the EPR effect, but only the binding of bisphosphonate to hydroxyapatite could keep the NPs in the tumor area. Clearly, the knowledge of tumor cell epitopes has allowed the development of targeted nanovehicles able to selectively deliver antitumor compounds in the diseased area. The use of bisphosphonates,120–122 aptamers,123 folate124 or hydroxyapatite125 (Fig. 4) has been shown to produce an increase in the effectiveness of the NPs treatments, while maintaining drug safety levels. Notably, the lung is the site where the majority of relapses occur and thereby, nanomedicines targeted to the lungs are called to overcome micro or gross metastases by enhancing the therapeutic levels of cytotoxic agents, without causing dose-limiting damage in children’s marrow. Thus, some oncologists suggest that the use of the inhalatory route could be optimal.126,127

Introduction

Figure 4. Ligand targeted strategies for bone tumor selective drug delivery (green). (1A) NPs (nanoparticles) can be functionalized with folic acid to interact with folate receptors overexpressed in OS (osteosarcoma) cells. (1B) Bisphosphonate-decorated-NPs display high affinity to bone matrix by coordination with calcium ions of hydroxyapatite crystals. (1C) CD133 aptamers conjugated to NPs can bind the cluster of differentiation of the transmembrane glycoprotein CD133, present in OS cancer stem cells. (2) Hydroxyapatite based NPs have affinity to bone tissue since hydroxyapatite is a natural bone mineral composed of calcium phosphate crystals and several other trace elements.

5.2. Ewing sarcoma The Ewing sarcoma is the second most malignant bone tumor in pediatric patients, appearing not only in bone but also in extra-skeletal soft tissue sites.128,129 Even though a systemic treatment is the most suitable approach for tackling tumor metastasis, local treatment of the tumor area is also necessary to avoid the metastatic spread. Thus, the irinotecan produg SN-38 has been loaded onto polymeric nanofibers to avoid a suboptimal tumor distribution of the free drug, which was responsible for failure in clinical trials.130 After subcutaneous implantation in the tumor-resection bulk, SN-38 was totally distributed in the tumor of nude mice, providing evidence of the suitability of polymeric nanofibers for the local control of potential metastatic tumors. Importantly, 95% of Ewing sarcoma tumors present a chromosomal translocation that leads to the formation of the oncogenic transcription factor EWS-FLI1, responsible for the oncogenic and metastatic processes.131 This drove several researchers to nanoencapsulate siRNA against the oncogene, and they reported effective oncogene expression knockdown in vitro and in vivo.132,133 For instance, poly (isobutyl cyanoacrylate) NPs with a chitosan corona were biotinylated, allowing the NPs to be functionalized with avidinylated anti- CD99 antibody. The authors observed that the chitosan-pluronic corona allowed the NPs to remain longer into the blood circulation, whereas, the inhibition of the EWS-FLI1 gene

Nanomedicines for pediatric cancers

expression was higher when the siRNA was loaded onto immunoNPs, compared to the NPs without the ligand. Abraxane, paclitaxel-bound albumin NPs already approved for breast, metastatic pancreatic and non-small cell lung cancers, was tested by the Pediatric Preclinical Testing Program (PPTP) against Ewing sarcoma, OS and rhabdomyosarcoma xenograft models.134 The PPTP in vivo experiments gave evidence of the powerful activity of Abraxane against Ewing sarcoma and rhabdomyosarcoma models expressing secreted acidic cysteine rich glycoprotein SPARC. This nanomedicine is now in Phase I/II clinical trials, indicated for various pediatric solid tumors, incl. OS, Ewing sarcoma or neuroblastoma) (code NCT01962103). Faced with the impossibility of condensing all the studies reviewed for this article, the following chart (Fig. 5) gives a global perspective of the current state of the art of nanotechnology approaches for pediatric cancers.

Figure 5. Nanomedicines for pediatric cancers: an estimation of the state of the art. Prevalence and 5-years survival rate percentages include also low-grade tumors but survival rates decrease dramatically in the case of aggressive tumors, which are of particular concern in this review. Research reviewed on nanotechnology approaches for childhood cancers in the last 5 years (%) is matched with their global burden of prevalence and 5-year survival rate (gross data estimations refer to children and adolescents between 0-19 years in the last decade) 1,2,4–6. Global overview of current state of the art shows a deficit of research on CNS tumors in spite of their high prevalence and mortality, whereas blood cancers and non-CNS embryonal tumors feature a balanced quotient. Finally, although bone tumors are the least prevalent, the literature contains a wide range of studies.

Introduction

6. KEY MILESTONES IN PEDIATRIC CANCER NANOMEDICINE The urgent need for pediatric investigation plans when companies request approval for medicines drove the FDA to grant a law about pediatric exclusivity research in the Modernization Act enactment of 1997 (Fig. 6). Even if those incentives have been updated and reauthorized subsequently, since the market approval of Doxil in 1995, several anticancer nanomedicines have been authorized for their use in adults but not in the pediatric population. Although a specific protocol for pediatric clinical research already exists, which was standardized and regulated in Europe and updated in the USA (FDA Amendments Act) in 2007, there is still a lack of compliance and collaboration between different institutions that hampers the inclusion of children in clinical trials.135– 139 Notwithstanding, some nanoformulations have recently reached phases III in pediatric clinical trials (see Table 1) and in 2009, liposomal mifamurtide was approved for pediatric osteosarcoma.118 Regarding the use of off-label nanomedicines, ferumoxytol, a superparamagnetic iron oxide NP approved in 2009 by the FDA for the treatment of iron deficiency anemia in adults was used as a contrast agent for clinical pediatric imaging.140 More recently, it has been clinically reported to be effective as a magnetic resonance contrast agent in cancer staging of children with solid tumors.141

Figure 6. Historical timeline in the field of cancer nanomedicine and clinical research incentives in children.

Nanomedicines for pediatric cancers

7. CONCLUDING REMARKS Pediatric cancer management remains elusive and the survival rates are still low. Despite recent progress in nanotechnology applications for treating adult cancers, such approaches are not well developed for use in pediatric oncology. Childhood cancers are commonly treated by applying combinations of highly toxic chemotherapeutic agents. These agents were originally designed to treat adult cancers, and adult treatment regimens were modified for use in pediatrics. Since combination chemotherapy has proven to be effective in controlling cancer progression, its use in pediatric cancer therapy has changed little over the last twenty years. Nevertheless, since children are growing and developing, they may well respond differently to chemotherapy drugs. Moreover, if a child becomes resistant to a drug and he or she is on the maximum tolerable dose, there is no scope to still increase the dose further without toxic side effects. Sadly, long-term survivors can experience lifelong health impacts from their treatment: about 70% of childhood cancer survivors suffer side effects, especially cardiovascular, which may even also include secondary cancers.

Bearing in mind our current knowledge about late toxicities and the standstill in improvement of the survival rate, the severe imbalance between adults and children, as far as the use of nanomedicines is concerned, should draw our attention. The main obstacle is progression to clinical trials. The inclusion of children in the therapeutic programs is, indeed, hampered by current pediatric clinical trial protocols, especially in relation to ethical issues. Moreover, the clinical bottleneck in children is exacerbated given that in vivo pre-clinical proofs are sometimes insufficient to reproduce correctly the disease or the age differences in children while late toxicities are not evaluated, which is of particular concern in pediatric cancer. More active collaboration between technologists, pediatric oncologists and clinicians is mandatory to design appropriate strategies. One reason to be optimistic is that at present, more than twenty years of experience with nanomedicines in the clinic endorses their potential for improving therapeutics in childhood cancers, which means that considerable progress is likely in the near future.

ACKNOWLEDGMENTS

Asociacion Española Contra el Cancer (AECC) (CI14142069-́ BLAN) and Fundacion Caja Navarra/Obra Social La Caixa are gratefully acknowledged.

Introduction

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Nanomedicines for pediatric cancers

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HYPOTHESIS AND OBJECTIVES

Hypothesis and objectives

HYPOTHESIS AND OBJECTIVES Cancer nanomedicine has been widely investigated in recent decades with the aim of enhancing the therapeutic potential of antitumor drugs. Nonetheless, current nanotechnology treatments are mainly only available in adult cancer protocols and not for their use in the pediatric population. Our research is principally focused on pediatric osteosarcoma (OS), whose importance resides in its dismal prognosis. The role of chemotherapy is crucial for the survival of metastatic or refractory patients. On many occasions the current chemotherapeutic regimens are not well tolerated and prove inefficient to effect cures. This points to the need for novel therapeutic approaches such as nanotechnology to improve the implementation of current therapeutic protocols. In this context, squalenization technology has achieved promising outcomes for the treatment of a great variety of tumors. Among squalenoyl prodrugs, squalenoyl-gemcitabine (SQ- Gem) was chosen for the construction of a novel multidrug nanocomposite in combination with edelfosine (EF), an alkyl-lysophopholipid with proven anticancer activity.

Having in mind the above-mentioned precedents, the hypothesis of the present thesis is:

Considering the amphiphilic nature of SQ-Gem and EF, we propose that the co-assembly of both anticancer compounds, with complementary molecular targets, could lead to the formation of a new combinatorial nanomedicine, with no need to use polymers, lipids or surfactants. Their easy fabrication process, high drug content and multitherapeutic antitumor activity may be optimal for improving therapies in childhood cancers like OS.

Based on this premise the main goal was to design, develop and characterize SQ-Gem/EF nanoassemblies (NAs), and to evaluate their in vitro and in vivo efficacy against OS models.

To that end, the following partial objectives were defined:

1. To design and formulate nanocomposites made of SQ-Gem and EF, and characterize their physico-chemical features. 2. To evaluate the biological effects of the developed NAs against commercial and patient derived pediatric tumor cells in vitro, and to elucidate their pharmacokinetic profile in mice. 3. To evaluate the in vivo efficacy and safety of SQ-Gem/EF NAs against an aggressive OS orthotopic murine model.

CHAPTER I

A unique multidrug nanomedicine made of squalenoyl- gemcitabine and alkyl-lysophospholipid edelfosine

Chapter I

A unique multidrug nanomedicine made of squalenoyl- gemcitabine and alkyl-lysophospholipid edelfosine

C. Rodríguez-Nogalesa,b, V. Sebastiánc,d, S. Irustac,d, D. Desmaëlee, P. Couvreure*, M.J. Blanco-Prietoa,b*

a Chemistry and Pharmaceutical Technology Department, Universidad de Navarra, Pamplona 31008, Spain b Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain c Department of Chemical and Environmental Engineering & Institute of Nanoscience of Aragon (INA), University of Zaragoza, Zaragoza 50018, Spain

d Networking Research Center on Bioengineering, Biomaterials and Nanomedicine, CIBER-BBN, Madrid 28029, Spain.

e Institut Galien Paris-Sud, UMR CNRS 8612, Université Paris-Sud, Université Paris- Saclay, Châtenay-Malabry Cedex 92290, France

Conflict of interest: Authors have no conflict of interest.

European Journal of Pharmaceutics and Biopharmaceutics 144 (2019) 165-173 DOI: 10.1016/j.ejpb.2019.09.017

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine

ABSTRACT Among anticancer nanomedicines, squalenoyl nanocomposites have obtained encouraging outcomes in a great variety of tumors. The prodrug squalenoyl-gemcitabine has been chosen in this study to construct a novel multidrug nanosystem in combination with edelfosine, an alkyl-lysophopholipid with proven anticancer activity. Given their amphiphilic nature, it was hypothesized that both anticancer compounds, with complementary molecular targets, could lead to the formation of a new multitherapy nanomedicine. Nanoassemblies were formulated by the nanoprecipitation method and characterized by dynamic light scattering, transmission electron microscopy and X-ray photoelectron spectroscopy. Because free edelfosine is highly hemolytic, hemolysis experiments were performed using human blood erythrocytes and nanoassemblies efficacy was evaluated in a patient-derived metastatic pediatric osteosarcoma cell line. It was observed that these molecules spontaneously self-assembled as stable and monodisperse nanoassemblies of 51 ± 1 nm in a surfactant/polymer free-aqueous suspension. Compared to squalenoyl-gemcitabine nanoassemblies, the combination of squalenoyl-gemcitabine with edelfosine resulted in smaller particle size and a new supramolecular conformation, with higher stability and drug content, and ameliorated antitumor profile.

Keywords: Cancer, chemotherapy, multidrug nanomedicine, nanoassemblies, squalene, gemcitabine, edelfosine, pediatric osteosarcoma

Chapter I

1. INTRODUCTION The current limitations of conventional chemotherapy have prompted the implementation of nanomedicine to improve cancer treatment. Interest is now focusing on nanocarriers to administer antitumor agents and exploit their intrinsic potential for providing more effective and safer treatments [1]. These nanosized suspensions are able to improve the therapeutic index of encapsulated drugs by modifying drug pharmacokinetics and tissue distribution [2-4]. Nonetheless, most of these nanotherapies are failing to reach clinical stages. We may anticipate that some of the reasons for this failure are linked to the so- called drug “burst release” together with low drug entrapment rates. These and other limitations are sometimes detrimental when it comes to achieving a correct therapeutic window and can even trigger limiting toxicities in cancer patients [5]. Until these issues are resolved, nanotechnology approaches will be unlikely to offer therapeutic progress in comparison to free drug administration. In order to address these limitations, a new technology based on the “squalenoylation” concept was developed [6-9]. The bioconjugation of drugs to squalenic acid (SQ-COOH), a nontoxic and biocompatible lipid squalene-derived molecule, leads to the formation of squalenoyl prodrugs able to self-assemble into supramolecular structures to form stable nanocomposites. In this way, each drug molecule is covalently linked to a SQ-COOH chain, which ensures high drug loading. As it is a prodrug, drug burst release from the nanoparticles (NPs) is avoided. An additional advantage is that neither polymers nor surfactants are needed to form the NPs. Among the squalenoyl nanoassemblies (NAs) investigated, considerable activity has centered on squalenoyl-gemcitabine (SQ-Gem) NAs [10-12]. Gemcitabine (Gem) is a nucleoside analogue that is very active against various tumors, including pancreatic cancer, non-small-cell lung cancer and ovarian cancer [13,14]. The antitumor efficacy of this molecule is, however, restricted owing to the rapid extra- and intracellular deamination, as well as to resistances mainly associated with nucleoside transporter HENT1 downregulation [15]. SQ-Gem NAs have shown improved anti-cancer activity compared to free Gem in murine tumor models of pancreas or leukemia. This improved therapeutic index was attributed to reduced drug metabolization, which resulted in higher blood concentration, better biodistribution and increased intracellular penetration, through a mechanism independent of the HENT1 transporter [16,17]. In order to further improve the above-mentioned nanoformulation we took into consideration some aspects. On the one hand, cancer treatment is often inefficient when using a single anticancer drug and several multitherapy approaches have been proposed [18]. We suggest that the co-encapsulation of another drug with a distinct therapeutic target may enhance the anticancer potential of the SQ-Gem NAs. On the other hand, since squalenoyl prodrugs display an amphiphilic structure that allows them to self-assemble into nanocomposites, it was hypothesized that to achieve a successful multidrug approach, the additional drug should possess an amphiphilic character too [19].

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine

We selected the amphiphilic drug edelfosine (EF), an alkyl-lysophospholipid with proven anticancer activity in several tumors [20-22]. Unlike Gem, exerting anticancer activity through DNA synthesis inhibition, EF acts on a completely different pharmacological target. Although the exact cell death mechanisms are not fully elucidated, EF is reported to selectively accumulate in tumor cell membranes, inducing Fas receptor clustering and activation on lipid rafts, triggering cellular apoptosis [23-24]. Moreover, we have previously demonstrated that the nanoencapsulation of EF considerably augmented the therapeutic index in comparison with its free counterpart when administered in vivo [25,26].

Here, we investigate whether the physical mixture of these two compounds (i.e. SQ- Gem and EF) could lead to the formation of a functional multidrug nanomedicine (Fig. 1A). The hydrodynamic properties of this nanoformulation were analyzed by dynamic light scattering (DLS) and cryo-Transmission electron microscopy (TEM). In order to elucidate the new structure and composition of the new co-assembly, TEM UHPLC/MS/MS and X-ray photoelectron spectroscopy (XPS) analyses were performed. The hemolysis activity of encapsulated EF was analyzed using human blood erythrocytes and the anticancer activity was tested in patient-derived metastatic pediatric osteosarcoma cells.

2. MATERIALS AND METHODS 2.1. Materials Gemcitabine hydrochloride was purchased from Carbosynth, Ltd. (Compton, UK). Edelfosine was obtained from Apointech (Salamanca, Spain). The synthesis of squalenoyl acid and squalenoyl-gemcitabine and 1,10,2-tris-nor-squalenic acid was performed as previously described [9] (see SI for further information). Ethanol absolute was purchased from Panreac Química (Barcelona, Spain). Phosphotungstic acid hydrate and Triton X- 100 were purchased from Sigma Aldrich (Barcelona, Spain). Amicon Ultra-15 10 kD MWCO filter devices were provided by Millipore (Cork, Ireland) and all reagents employed for mass spectroscopy (gradient grade for liquid chromatography) were obtained from Merck (Barcelona, Spain). Celltiter 96® aqueous one solution cell proliferation assay (MTS) was purchased from Promega (Alcobendas, Spain). Heat inactivated fetal bovine serum, Trypsin-EDTA and penicillin/streptomycin and alpha- MEM cell medium were provided by Gibco® (Invitrogen Inc., Carlsbad, USA). Phosphate Buffer Saline (PBS) was purchased from Lonza (Verviers, Belgium). Tissue cell culture 96-well plate was provided by Corning Inc. (New York, USA).

Chapter I

2.2. Preparation of nanoassemblies Squalenoyl NAs were prepared using the nanoprecipitation method. Briefly, a solution of SQ-Gem in ethanol (200 μL at 11.6 μmol mL-1) alone or mixed with a solution of EF (200 μL at 11.6 μmol mL-1) was added dropwise under stirring (500 rpm) into 2 mL of distilled water. Nanoprecipitation of the SQ-Gem NAs or SQ-Gem/EF NAs occurred spontaneously. Ethanol was completely evaporated in a vacuum using a Rotavapor (Buchi R-210/215, Buchi Corp., Canada) to obtain an aqueous suspension of SQ-Gem NAs or SQ-Gem/EF NAs. SQCOOH NAs (200 μL at 18.75 μmol mL-1) and SQCOOH NAs loaded with EF (SQCOOH/EF NAs) at a SQCOOH: EF molar ratio of 2:1 (200 μL at 9.375 μmol mL-1 of EF), were prepared employing the same procedure as described above for SQ-Gem NAs and SQ-Gem/EF NAs. Mean particle size (Z-average) and polydispersity index (PDI) of the NAs were determined at 25ºC by DLS (Zetasizer Nano ZS Malvern; Malvern Instruments SA, UK) after 1:15 dilution in ultrapure water. Surface charge of the NPs was characterized by measuring the zeta potential with laser Doppler velocimetry (Zetasizer Nano ZS Malvern; Malvern Instruments SA, UK) and using the Smoluchowski equation. Drug recoveries from the NAs were quantified by Ultra-high performance liquid chromatography-tandem mass spectrometer (UHPLC–MS/MS) after washing the formulations with Amicon Ultra- 15 10 kD MWCO filter devices (3x10 mins, 4ºC, 5.000 r.p.m).

2.3. UHPLC–MS/MS method An UHPLC/MS/MS method based on modifications of Estella-Hermoso de Mendoza et al. for EF [27] and of Khoury et al. for SQ-Gem [28] was optimized to perform the simultaneous determination of SQ-Gem and EF. The UHPLC system was composed of an Acquity UPLCTM system (Waters Corp., Milford, MA, USA). Separation was carried TM out on an Acquity UPLC BEH C18 column (50mm×2.1mm, 1.7m; Waters Corp., Milford, MA, USA) with a gradient elution, using a mobile phase starting from 30% of a 1% formic acid aqueous solution and 70% of methanol (for further details see SI).

Triple-quadrupole tandem mass spectrometric detection was performed on an AcquityTM TQD mass spectrometer (Waters Corp., Milford, MA, USA) with an electrospray ionization (ESI) interface. The mass spectrometer operating in a positive mode was set up for multiple reaction monitoring (MRM) to monitor the transition of m/z 524.3→104.2 for EF and the transition of m/z 646.7→112 for the SQ-Gem. Data acquisition and analysis were performed using the MassLynxTM (for further details see SI).

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine

2.4. Electron microscopy imaging The morphology of the squalenoyl NAs was characterized by TEM, using negative staining, or by cryo-TEM, using cryogenic freezing. Electron microscopy images were recorded on a T20-FEI Tecnai thermoionic microscope operated at an acceleration voltage of 200 kV. Aberration corrected scanning transmission electron microscopy (Cs-corrected STEM) images were acquired using a high angle annular dark field detector (HAADF) in a FEI XFEG TITAN electron microscope operated at 300 kV and equipped with a CETCOR Cs-probe corrector from CEOS. Fast Fourier Transform (FFT) analysis was used to shed light on the internal structure and assembly of the NPs.

Negative stained samples were prepared by dropping 20 µl of sample in carbon coated copper grids (200 mesh), dried at room temperature and stained with a negative staining agent (phosphotungstic acid). The grid preparation of cryogenically immobilized samples required an extremely rapid sample freezing to achieve a vitreous state. A thin film vitrified specimen was prepared with a Vitrobot (FEI) in melting ethane. Afterwards, the sample holder with the specimen grid was maintained under cryogenic conditions with liquid nitrogen.

2.5. XPS assays XPS analysis was performed with an AXIS Supra (Kratos Tech.). The spectra were excited by a monochromatic AlK source (1486.6 e.V) run at 15 kV and 15 mA. The photoelectron takeoff angle (angle of the surface with the direction in which the photoelectrons are analyzed) was 90°. For the individual peak regions, a pass energy of 20 eV was used. Peaks were analyzed with CasaXPS software. The atomic concentration of each element was calculated by integration of each element's main peak, taking into account the corresponding atomic sensitivity factors (ASF). The composition of the material was analyzed at different depths after sputtering the surface of the sample for a certain time using a gas cluster ion source. Argon ion clusters of mean size 1000 atoms and energy 10kV were used for etching.

2.6. Hemolysis evaluation Erythrocytes were separated from human blood plasma by centrifugation (4000 x g, for 7 min), washed with PBS 3-times and diluted with 50 ml of PBS. Then, 0.5 ml of this suspension was added to 1.5 ml of free and nanoformulations of EF. Incubation with PBS served as negative control and with Triton X-100 as positive control. After 1h, the samples were centrifuged (4000 x g, for 7 min) and the supernatants' absorbance was measured in an Agilent 8453 UV-visible spectrophotometer (Agilent, CA, USA) at 540 nm.

Chapter I

2.7. Cell culture The cell line used throughout the study was a metastatic pediatric osteosarcoma cell line derived from a patient treated at the Clínica Universidad de Navarra, coded as 531M. This cell line was maintained in alpha-MEM cell medium, supplemented with 10% of fetal bovine serum and 1% penicillin/streptomycin in an incubator at 37ºC and a humidified atmosphere with 5% carbon dioxide. For the in vitro cytotoxicity assays 531M cells were plated on 96-well plates at a density of 10,000 cells per well, 24 h before the addition of different concentrations of free and nanoparticulate Gem and EF in triplicate wells. After 72 h of incubation with the different formulations, the medium was withdrawn and 100 μL of complete medium containing 15 % v/v of MTS was added to each well. Absorbance was measured 3 h later in a microplate reader (iEMS reader MF, Labsystem, Helsinki, Finland) at a test wavelength of 492 nm with the reference wavelength set at 690 nm. The concentration of drug required to inhibit cell growth by 50% (IC50) was estimated by fitting the dose response curve to a sigmoidal function using the GraphPad Prism version 5.0 for Windows (GraphPad Software, San Diego, CA, USA).

3. RESULTS AND DISCUSSION 3.1. Design and characterization of supramolecular squalene based nanoassemblies SQ-Gem/EF NAs were prepared by nanoprecipitation from an ethanolic solution as described in the Materials and Methods. By modifying the ratio of SQ-Gem and EF in the squalene-based NAs, it was possible to select the best nanoformulation in terms of size distribution. As seen in Fig. 1B and 1C, although initial concentrations of EF led to high PDI values, the particle population homogeneity was again recovered at equimolar concentrations. Interestingly, the mean particle size was considerably decreased from 121 ± 14 nm for SQ-Gem NAs to 50 ± 1 nm for SQ-Gem/EF NAs. However, when EF concentrations surpassed equimolarity, the mean particle size barely changed, whereas the uniformity of the particle population rapidly worsened as it was observed at the ratio SQ-Gem/EF 1:2. Of note, SQCOOH NAs and SQCOOH NAs loaded with EF were also prepared in similar conditions. As observed for SQ-Gem/EF NAs, a very similar particle size reduction was noticed from 99 ± 5 nm for SQCOOH NAs to 47 ± 2 nm for SQCOOH/EF NAs. Finally, based on DLS data showing that the equimolecular mixture of both components (i.e., SQ-Gem and EF) led to optimal colloid properties, it was suggested that EF completely co-assembled with SQ-Gem. To verify this, NAs were washed using a method previously described to efficiently separate unloaded EF from lipid NPs [20-22] and afterwards quantified by UHPLC/MS/MS. Results showed EF recoveries close to 100%, confirming the complete encapsulation/insertion of EF into SQ- Gem. Unlike EF, SQ-Gem is completely insoluble in aqueous medium, and therefore there is also no loss of product during the formulation process.

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine

Figure 1. (A) Diagram of the formation of the new NAs. The physical mixture of SQ-Gem and EF leads to the formation of SQ-Gem/EF NAs. (B) Effect of the SQ-Gem:EF molar ratio on the particle size and PDI. (C) Size distribution by intensity peaks corresponding to SQ-Gem/EF NAs (green), SQ-Gem NAs (red) and to a mixture of SQ-Gem and EF at a molar ratio 1:0.41 (blue), and 1:2 (black).

With respect to the changes in the particle surface charge, the results show that the incorporation of EF in the NAs brought about a slight increase in the zeta potential values (Table 1). SQ-Gem NAs displayed zeta potentials of -17.73 ± 1.26 mV which was in agreement with previous studies [6,29]. On the other hand, designed and optimized SQ- Gem/EF NAs (ie., at a SQ-Gem:EF ratio of 1:1) showed a zeta potential value of -12.56 ± 1.15 mV. Of note, EF polar head presents a negatively charged phosphate group but also a positively charged quaternary amine group, making the ionic balance neutral [30]. As Table 1 shows, independent preparations (n ≥ 5) of the NAs demonstrated this nanoformulation as highly reproducible, giving nearly identical results for each sample. Moreover, as confirmed by UHPLC/MS/MS, this approach allowed to obtain NAs with very high overall drugs content of 67.28 wt %, containing both Gem (22.49 wt %) and EF (44.73 wt %). This is a signiﬁcant improvement in terms of drug loading in comparison with common drug delivery systems, which often display much lower drug loading capacity. Such is the case with previously reported lipid nanoformulations loaded with Gem [31] or EF [25] accounting for a drug loading of 7.3 and 2.26 wt % respectively. The high drug payload of SQ-Gem/EF NAs may thus help to attain a correct therapeutic window and to avoid infra/overdosing that might provoke toxicities [3]. And given that the SQ-Gem/EF NAs were built with only drugs or prodrugs, possible adverse effects derived from the incorporation of polymers, surfactants or other colloidal stabilizers may be avoided [32,33].

Chapter I

All the preparations were further characterized by cryo-TEM analysis. The different nanoformulations displayed spherical shape and suitable colloid characteristics, in good agreement with DLS data (Fig. 2). Again, SQ-Gem/EF NAs (Fig. 2B) show a substantial particle diameter reduction compared to SQ-Gem NAs (Fig. 2A). The particle size reduction towards ca. 50 nm resulted from the insertion of EF into SQ-Gem NAs, suggesting that the amphiphilic nature of EF allowed the co-assembly with SQ-Gem by means of molecular interactions between the two molecules. Importantly, such particle size reduction could improve NPs biodistribution. For instance, particles under 10 nm were reported to be rapidly filtered by the kidneys, whereas particles larger than 100 nm undergo an enhanced liver uptake [34,35]. Cabral et al. postulated that although the NP size may not affect the particle tumor distribution in hyperpermeable tumors, only NPs smaller than 70 nm, which is the case of SQ-Gem/EF NAs, were able to accumulate efficiently in poorly permeable tumors such as human pancreatic adenocarcinoma [36]. Bearing in mind that Gem is given in chemotherapy for pancreatic cancer, the use of specifically designed SQ-Gem/EF NAs of around 50 nm might improve the pharmacological efficacy in this type of poorly permeable tumor, by means of the so called Enhanced Permeability and Retention (EPR) effect. In any case, the EPR effect in solid tumors is not as well understood as initially thought [37] and further experiments in vivo are mandatory to elucidate the real behavior of SQ-Gem/EF NAs.

Table 1 Colloidal characteristics of squalene-based nanocomposites

% NAs size (nm)1 PDI1 ζ (mv)2 % Gem3 % EF3 total3 SQ-Gem 121 ± 14 0.1 ± 0.02 -17.73 ±1.26 40.7 0 40.7 SQ-Gem/EF 51 ± 1 0.12 ± 0.03 -12.56 ± 1.15 22.49 44.73 67.28 SQCOOH 99 ± 5 0.12 ± 0.01 -29.8 ± 2.91 0 0 0 SQCOOH/EF 47 ± 2 0.267 ± 0.03 -23.76 ± 5.17 0 39.57 39.57 1Determined by DLS. 2Zeta potential. 3 % = MW drug/MW nanocomposites.

Figure 2. Morphological characterization of squalene-based nanocomposites. Cryogenic transmission electron microscopy of SQ-Gem (A), SQ-Gem/EF (B), SQ-COOH (C) and SQ-COOH/EF (D) nanocomposites.

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine

3.2. Supramolecular conformation Negatively stained TEM analysis also revealed striking morphological differences between SQ-Gem and SQ-Gem/EF NAs. As shown in Fig. 3, the surface of SQ-Gem NAs showed a supramolecular organization consisting of inverse hexagonal phases, sometimes surrounded by an amorphous layer. This 2–D organization of SQ-Gem molecules could be observed directly depending on the particle arrangement with respect to the microscopy sight angle (Fig. 3A-C). This was confirmed by the geometric projection of the NAs. Such supramolecular organization has been already previously described by X- ray diffraction studies [6] but here we provide solid evidence of the particle conformation by TEM, which represents an original approach.

Figure 3. Transmission electron microscopy of SQ-Gem NAs. Morphological analysis vs. tilt angle (A-C) and 2-D inverse hexagonal phase macromolecular organization (D, E). Supramolecular organization diagram, Fourier transform image spectrum analysis and mean lattice spacing measurement of SQ-Gem NAs (F).

Very importantly, this supramolecular organization was no longer observed with the multidrug SQ-Gem/EF NAs, which revealed a round shape and the TEM images suggested a multilamellar onion-like structure at this resolution scale (Fig. 4AC). In fact, Fourier transform analysis performed on SQ-Gem and SQ-Gem/EF NAs also detected a different periodicity, as indicated by the image spectrum analysis (Fig. 3F and 4F). The calculated mean lattice spacing between SQ-Gem/EF NAs layers was 3.6 ± 0.1 nm, instead of 3.2 ± 0.2 nm for SQ-Gem NAs, which would also explain the distinct conformation and the size differences. This structural relaxation is considered to be boosted by their self-assembly mode and structure (i.e. lamellar instead of hexagonal). In this case and bearing in mind that the mean-lattice spacing can be independent of the

Chapter I

number of assembled layers in terms of size, the larger distance between adjacent planes found in the SQ-Gem/EF NAs was correlated with a particle shrinking. These findings strongly support the notion that the SQ-Gem/EF NAs were structurally different from the SQ-Gem NAs. However, at this image resolution and given the reduced size of the NAs, it was not possible to elucidate the morphological features of SQ-Gem/EF NAs thoroughly. Therefore, to go into further detail about the supramolecular organization of SQ-Gem/EF NAs, the image resolution was improved by analyzing SQ- Gem/EF NAs with high angle annular dark field (HAADF) high resolution scanning transmission electron microscopy (STEM). Although this imaging technique is very aggressive to organic materials due to the high voltages inflicted on the samples, image acquisitions of SQ-Gem/EF NAs were obtained with a greater resolution thanks to the assistance of a negative staining agent. With this technique, we were able to confirm the multilamellar assembly of SQ-Gem/EF NAs, displaying a concentric or spiral disposition of the layers (Fig. 4DE). Changes in the topological structure of amphiphilic molecules can play an important role in tuning self-assembled morphologies [38]. The loss of the native supramolecular structure of SQ-Gem led to an amorphous particle conformation, as already observed when SQ-Gem was co-encapsulated with the lipophilic compound iso-combretastatin A4 [39]. In another study, Caron et al. formulated nanocomposites of SQ-Gem monophosphate instead of SQ-Gem, resulting in the formation of unilamellar vesicles [40]. Conversely, the new SQ-Gem/EF NAs structure displayed physical resemblance to the onion-like multigeometry assembly of polymeric amphiphilic compounds described by Zhang et al [41] and Wang et al. [42].

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine

Figure 4. Transmission electron microscopy of SQ-Gem/EF NAs (A-C). High angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) of SQ-Gem/EF NAs (D, E) (the signal corresponding to the organic material is inverted in comparison with TEM). Supramolecular organization diagram, Fourier transform image spectrum analysis and mean lattice spacing measurement of SQ-Gem NAs (F).

3.3. Element distribution The distribution of elements throughout SQ-Gem/EF NAs was analyzed by XPS. Survey spectra measured at the surfaces (before sputtering) of SQ-Gem/EF NAs, SQ-Gem NAs and free EF samples showed peaks corresponding to carbon, oxygen and nitrogen. Beside those elements, the peak belonging to the fluorine atom of Gem appeared at 687 eV. [43]. The related KLL fluorine Auger peak could also be observed (Fig. 5A). On the other hand, EF showed characteristic peaks related to the presence of phosphorus in the molecule. The presence of EF on the surface of SQ-Gem/EF NAs was demonstrated by a P 2p peak clearly observed in the spectrum (Fig. 5A). The binding energy values for P 2p were 133.4 and 133.6 eV for EF and SQ-Gem/EF NAs respectively. These values corresponded to the organic phosphate groups [44]. The similar binding energies found for both samples showed an identical chemical environment, i.e., there was no chemical interaction between EF phosphate groups and SQ-Gem.

In order to verify the presence of EF in the inner part of SQ-Gem/EF NAs, depth profiling was used to study the distribution of elements throughout the particle. Since the concentration of each molecule could be related to the atomic concentration of fluorine (SQ-Gem) or phosphorus (EF), the P/F atomic ratio was calculated, and its variation with etching time is shown in Fig. 5B. Etching was continued up to a point that allowed us to ensure that most of the particles had the inner part exposed. The depth profile showed

Chapter I

that P/F atomic ratio increases from around 0.6 on the particle surface up to more than 2 after 35 s etching, corresponding to inner parts of the NAs. Thus, EF concentration was shown to be around four times the SQ-Gem concentration after etching, suggesting an enrichment in this compound inside the NAs. These results indicated only a slight increase of SQ-Gem concentration on the particles surface, considering that the ratio P/F was expected to be theoretically closer to 0 under equimolecular concentrations. Nevertheless, the increment of the phosphorus signal detected in a concentric multilamellar system is thought to be boosted by the higher element exposure corresponding to the inner layers. On the other hand, these results are also in agreement with UHPLC/MS/MS analysis, showing complete encapsulation of EF in the NA. A matter of concern was that EF could be only surfacting or coating the particle as a result of its phospholipid nature. Nevertheless, with XPS depth profiling analysis we were able to remove SQ-Gem/EF NAs’ outer layers and detect the presence of EF in inner areas. It should be borne in mind that the physico-chemical properties of SQ-Gem confer an intrinsic ability to self-assemble and form stable NAs in aqueous medium, as observed in many other squalenoyl NAs [45,46]. In this sense, SQ-Gem might tend to accommodate itself at the particle surface with the purpose of stabilizing the colloid dispersion, in this way displacing EF towards the inner parts.

Figure 5. Molecular tracking (X-ray photoelectron spectroscopy assays) of the fluorine (F) of SQ-Gem and the phosphorus (P) of EF. (A) F 1s and P 2p core level regions of 1) SQ-Gem/EF NAs; 2) SQ-Gem NAs and 3) free EF. (B) P/F atomic ratio obtained by depth profile of SQ-Gem/EF NAs.

3.4. Colloidal stability SQ-Gem/EF NAs and SQ-Gem NAs were analyzed for changes in the hydrodynamic diameter and PDI over 60 days. SQ-Gem/EF NAs formulation was found to be stable throughout the experiment, demonstrating that the incorporation of EF did not trigger NA disruption. Indeed, as shown in Fig. 6A, no appreciable changes in the particle size were observed given that the mean particle diameter was augmented from approximately 50

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine up to 58 nanometers over two months. According to this, the PDI parameter of particle homogeneity value (Fig. 6B) only showed a slight increase and remained below the accepted values (PDI <0.3) [47]. On the other hand, SQ-Gem NAs showed colloid stability for approximately only 10 days, considering that mean particle size and PDI started to surpass desirable values at that time. These results indicated that SQ-Gem/EF NAs were stable with no signs of particle disruption, agglomeration or precipitation, probably due to the presence of a complementary amphiphilic compound such as EF in the NAs. Nie et al. suggested that a regular multilamellar structure at the proper concentration ratio of components is a key factor for optimal stability [48]. The colloidal properties of the NAs were evaluated in this experiment at 4ºC with regard to future administrations. This might facilitate handling and a hypothetical storage of the multidrug NAs at 2-8ºC (5ºC ± 3ºC for drug substances intended for storage in a refrigerator according to the ICH guideline Q1A(R2)). Moreover, a lyophilization process involving the incorporation of cryoprotectants or problems associated with NP reconstitution is not strictly required.

Figure 6. SQ-Gem NAs (red) and SQ-Gem NAs (green) stored at 4ºC are measured for particle size (A) and PDI (B) variations for 60 days (n≥3, data: mean ± SD).

3.5. Hemolytic activity Hemolysis represents one of the main side effects associated with EF treatment. It is the consequence of the amphipathic structure of this compound [49], making necessary its encapsulation in drug delivery systems. In order to evaluate if SQ-Gem/EF NAs could reduce this toxicity, hemolytic experiments were performed using human blood erythrocytes. In vitro results showed that free EF was 100% hemolytic at a concentration of 10 µg/ml, while the same concentration of EF loaded onto SQ-Gem presented only 6% of hemolysis. It was concluded that SQ-Gem/EF NAs were able to protect erythrocytes from EF associated hemolysis. The low effect produced could be attributed to the scant presence of EF attached to the particle surface, as indicated in the XPS analysis section.

Chapter I

An effective reduction of this toxicity represents a crucial safety aspect that determines the utility of squalenoyl nanocarriers for administering EF without compromising the patient´s health. Although SQ-Gem/EF NAs could be also administered intraperitoneally, orally or locally among others, it is important to consider that intravenously administered squalenoyl NAs have been reported to disassemble, being incorporated into endogenous lipoproteins [10]. However, the physico-chemical features found in the SQ-Gem/EF NAs suggest that they might avoid an interaction with lipoproteins, remaining longer in the circulation in their native conformation. Future experiments in vivo will shed light on the tumor accumulation of SQ-Gem/EF NAs and as well as the hypothetical role of lipoproteins as endogenous drug delivery systems.

3.6. Anticancer activity Cell proliferative assays revealed that these treated patient-derived cells were quite resistant to anticancer drugs due to their marked aggressive behavior. As seen in Fig. 7A, free EF showed an IC50 value of 69 ± 1 μM. Conversely, free Gem treatment displayed an initial higher anticancer activity but reached a plateau at 50 % of cell viability, hindering IC50 calculation. SQ-Gem NAs bypassed the plateau and displayed an IC50 value of 22 ± 3 μM. Importantly for our study, the insertion of EF in the NAs was not detrimental to the native antitumor activity of SQ-Gem NAs. SQ-Gem/EF NAs showed an IC50 value of 20 ± 2 μM for each compound (molar ratio 1:1), displaying more antitumor activity than SQ-Gem NAs, especially once the threshold of 50% of cell viability was crossed (Fig. 7B). The slight IC50 improvement of the SQ-Gem/EF NAs over the single SQ-Gem NAs in vitro may be explained by the fact that part of 531M cell population remained more sensitive to Gem than to EF (Fig. 7A, first part of dark blue curve vs light blue curve). In this sense, the combination of free EF and free Gem at a molar ratio 1:1 also indicated that the antitumor action of EF is more visible under the threshold of 50% of cell viability. In addition, SQCOOH/EF NAs was found to reduce the IC50 value of EF from 69 ± 1 μM to 60.5 ± 2 μM, which confirmed the suitability of the co-encapsulation of EF with squalenoyl compounds. Of note, SQCOOH NAs was not observed to interfere in cell dead mechanisms given that no signs of cytotoxicity were observed below the threshold of 100 µM (Fig. 7B). The interest in using a treated patient-derived cell line as an in vitro model relies on the tumor resistance to conventional cytostatic agents that often leads to therapeutic failure in high-risk or relapsed patients. In this sense, we have not only provided solid evidence concerning the correct design for SQ-Gem/EF NAs but also confirmed the therapeutic potential of anticancer nanomedicines. Future in vivo experiments that consider drug pharmacokinetics, biodistribution and NAs interaction with the primary and metastatic tumor will shed light on the real clinical profits of this new multidrug nanomedicine.

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine

Figure 7. Cell viability assays. 531M cells were exposed to SQ-Gem/EF NAs*, SQ-Gem NAs, free Gem, free EF, SQCOOH/EF NAs, free Gem + free EF* and SQCOOH NAs treatments for 72h (n≥3, data: mean ± SD). *X axis concentrations (µM) correspond to the individual amount of each molecule at a molar ratio 1:1.

4. CONCLUSIONS The aim of this study was to design and characterize SQ-Gem/EF NAs usable as multitherapy nanodevices in cancer therapy. The results of this study show that the alkyl- lysophospholipid EF was able to co-assemble with the pro-drug SQ-Gem to form NAs with no need to use any stabilizer. We provide insights into an easy formulation process for this nanomedicine with drugs only by leveraging their amphiphilic nature. At a molecular ratio of 1:1, NAs displayed reproducible and homogeneous formulation batches of 51 ± 1 nm. This is a considerable particle size reduction over SQ-Gem NAs, and also provides a substantial increment in the drug content, up to 67.28 wt %. This co- assembly led to a new supramolecular arrangement, showing a multilamellar concentric conformation. SQ-Gem NAs have previously shown great anticancer activity in various tumor models. An important fact that reinforces the combination with EF is that it acts not only as a second antitumor agent but also as a NP stabilizer. In vitro, the SQ-Gem/EF NAs suppressed the hemolytic activity of EF, a major side effect of this molecule, and presented a better antitumor profile than SQ-Gem NAs. In this work we have designed and formulated a novel multidrug nanomedicine. Its biocompatible nature and high drug content make it a suitable pre-clinical candidate for cancer therapy.

Chapter I

ACKNOWLEDGEMENTS

Asociación Española Contra el Cáncer (AECC) (CI14142069BLAN) (Spain) and Fundación Caja Navarra/Obra Social La Caixa are gratefully acknowledged. CIBER- BBN is an initiative funded by theVI Spanish National R&D&i Plan 2008–2011 , Iniciativa Ingenio 2010, Consolider Program, CIBER Actions (Spain) and financed by the Instituto de Salud Carlos III (Spain) with assistance from the European Regional Development Fund.

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[38] J. Wang, X. Wang, F. Yang, H. Shen, Y. You, D. Wu, Effect of topological structures on the self-assembly behavior of supramolecular amphiphiles., Langmuir. 31 (2015) 13834–13841. [39] A. Maksimenko, M. Alami, F. Zouhiri, J.-D. Brion, A. Pruvost, J. Mougin, A. Hamze, T. Boissenot, O. Provot, D. Desmaële, P. Couvreur, Therapeutic modalities of squalenoyl nanocomposites in colon cancer: an ongoing search for improved efficacy., ACS Nano. 8 (2014) 2018–2032. [40] J. Caron, E. Lepeltier, L.H. Reddy, S. Lepêtre-Mouelhi, S. Wack, C. Bourgaux, P. Couvreur, D. Desmaële, Squalenoyl gemcitabine monophosphate: synthesis, characterisation of nanoassemblies and biological evaluation, European J. Org. Chem. 2011 (2011) 2615–2628.

Chapter I

[41] K. Zhang, L. Gao, Y. Chen, Z. Yang, Onion-like microspheres with tricomponent from gelable triblock copolymers, J. Colloid Interface Sci. 346 (2010) 48–53. [42] Z. Wang, H. Wang, M. Cheng, C. Li, R. Faller, S. Sun, S. Hu, Controllable multigeometry nanoparticles via cooperative assembly of amphiphilic diblock copolymer blends with asymmetric architectures, ACS Nano. 12 (2018) 1413- 1419. [43] M. Parsian, G. Unsoy, P. Mutlu, S. Yalcin, A. Tezcaner, U. Gunduz, Loading of Gemcitabine on chitosan magnetic nanoparticles increases the anti-cancer efficacy of the drug., Eur. J. Pharmacol. 784 (2016) 121–128. [44] K.S. Siow, L. Britcher, S. Kumar, H.J. Griesser, Deposition and XPS and FTIR analysis of plasma polymer coatings containing phosphorus, plasma process. Polym. 11 (2014) 133–141. [45] A. Maksimenko, F. Dosio, J. Mougin, A. Ferrero, S. Wack, L.H. Reddy, A.-A. Weyn, E. Lepeltier, C. Bourgaux, B. Stella, L. Cattel, P. Couvreur, A unique squalenoylated and nonpegylated doxorubicin nanomedicine with systemic long- circulating properties and anticancer activity., Proc. Natl. Acad. Sci. U. S. A. 111 (2014) E217-226.

[46] F. Dosio, L.H. Reddy, A. Ferrero, B. Stella, L. Cattel, P. Couvreur, Novel nanoassemblies composed of squalenoyl−paclitaxel derivatives: synthesis, characterization, and biological evaluation, bioconjug. Chem. 21 (2010) 1349– 1361. [47] M. Danaei, M. Dehghankhold, S. Ataei, F. Hasanzadeh Davarani, R. Javanmard, A. Dokhani, S. Khorasani, M. Mozafari, Impact of particle size and polydispersity index on the clinical applications of lipidic nanocarrier systems, Pharmaceutics. 10 (2018) 57. [48] S. Nie, X. Zhang, R. Gref, P. Couvreur, Y. Qian, L. Zhang, Multilamellar Nanoparticles Self-assembled from opposite charged blends: insights from mesoscopic simulation, J. Phys. Chem. C. 119 (2015) 20649–20661. [49] J. Kötting, N.W. Marschner, W. Neumüller, C. Unger, H. Eibl, Hexadecylphosphocholine and octadecyl-methyl-glycero-3-phosphocholine: a comparison of hemolytic activity, serum binding and tissue distribution., Prog. Exp. Tumor Res. 34 (1992) 131–142.

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine

SUPPORTING MATERIAL Synthesis of 4-(N)-Trisnorsqualenoyl-Gemcitabine (SQ-Gem) Trisnorsqualenoic acid was previously synthetized from squalene by a four-step synthesis (Van Tamelen, E.E: Curphey, T.J. Tetrahedron Lett. 1962, 121-124) (Fig. S1). The coupling of the squalene chain on Gem was achieved by simple amide bond formation using the mixed anhydride of squalenic acid with ethyl chloroformate. To do this, to a mixture of Trisnorsqualenoic acid (2.27 mmol) and triethylamine (2.83 mmol) in anhydrous THF (9 mL) cooled at 0°C, ethyl chloroformate (2.06 mmol) was added dropwise. The mixture was stirred at 0°C for 15 min and a solution of gemcitabine base (Gem) (1.89 mmol) and triethylamine (2.83 mmol) in anhydrous DMF (3 ml) was added dropwise. The reaction mixture was stirred for 72 h at 25ºC and then concentrated in vacuum. Aqueous sodium hydrogen carbonate was added and the mixture was extracted with ethyl acetate (3 x 50 ml). The combined extracts were washed with water, dried over MgSO4, and concentrated under reduced pressure. The final product was purified by chromatography on silica gel eluting with 1 to 5% methanol in dichloromethane to give pure 4-N-trisnorsqualenoyl-gemcitabine (m/z: 646) as an amorphous white solid. 1H NMR (300 MHz, CDCl3) δ: 9.15 (broads, 1 H, NHCO), 8.21-8.19 (d, 1 H, J = 7.5 Hz, H6), 7.51-7.48 (d, 1 H, J = 7.5 Hz, H5), 6.18 (t, 1 H, J =7.0 Hz, H1’), 5.30-5.09 (m, 5 H, HC=C(CH3)), 4.51-4.48 (m, 1 H, H3’), 4.04-3.94 (m, 2 H, H4’ and H5’), 3.91 (d, 1H , J = 10.5 Hz, H5’), 2.56-2.54 (m, 2 H, NHCOCH2), 2.33 (m, 2 H, NHCOCH2CH2), 2.04- 1.98 (m, 16 H, CH2), 1.67-1.61 (s, 3H, C=C(CH3)2), 1.59 (s, 15 H, C=C(CH3)CH2);

Figure S1. Multistep synthesis of squalenic acid and conjugation to gemcitabine (Gem) to form squalenoyl- gemcitabine (SQ-Gem).

Chapter I

UHPLC/MS/MS parameters Column temperature was maintained at 50ºC and the flow rate was set at 0.5 mL/min. The autosampler was conditioned at 4ºC and the injection volume was 2µL using partial loop mode for sample injection. As seen in Table S1, the gradient elution method was set at 5 mins (0.5 ml/min) using a mobile phase starting with 30% of a 1% formic acid aqueous solution and 70% of methanol. The following optimized MS parameters employed were: 2.5 kV capillary voltage, 65 V cone voltage, 120ºC source temperature and 350ºC desolvation temperature. Nitrogen was used for the desolvation and as cone gas at a flow rate of 650 and 50 L/h, respectively. Argon was used as the collision gas. Under these conditions, edelfosine (EF) and squalenoyl-gemcitabine (SQ-Gem) were eluted at 2.61 (524.5 >104.2) and 2.81 min (646.7 >112), respectively.

Figure S2. (A) UHPLC/MS/MS chromatograms of squalenoyl-gemcitabine (SQ-Gem) and edelfosine (EF) and (B) calibration curves of both compounds (R2= 0.998).

Table S1 Gradient elution parameters

%B Flow %A Time (1% formic acid (mL/min) (Methanol) aqueous solution) Initial 0.5 70.0 30.0

1.00 0.5 70.0 30.0 3.00 0.5 90.0 10.0 4.00 0.5 90.0 10.0 4.50 0.5 70.0 30.0 5.00 0.5 70.0 30.0

CHAPTER II

Squalenoyl-gemcitabine/edelfosine nanoassemblies: anticancer activity in pediatric cancer cells and pharmacokinetic profile in mice

Chapter II

Squalenoyl-gemcitabine/edelfosine nanoassemblies: anticancer activity in pediatric cancer cells and pharmacokinetic profile in mice

C. Rodríguez-Nogalesa,b, S. Murac, P. Couvreurc*, M.J. Blanco-Prietoa,b*

a Chemistry and Pharmaceutical Technology Department, Universidad de Navarra, Pamplona 31008, Spain

b Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona 31008, Spain c Université Paris-Saclay, CNRS, Institut Galien Paris Sud, 92296, Châtenay-Malabry, France.

Conflict of interest: authors have no conflict of interest.

Manuscript submitted to journal

(under revision, January 2020)

Squalenoyl-gemcitabine/edelfosine nanoassemblies: pharmacokinetic profile in mice and anticancer activity in pediatric cancer cells

ABSTRACT Despite the great advances accomplished in the treatment of pediatric cancers, recurrences and metastases still exacerbate prognosis in some aggressive solid tumors such as neuroblastoma and osteosarcoma. In view of the poor efficacy and toxicity of current chemotherapeutic treatments, we propose a single multitherapeutic nanotechnology-based strategy by co-assembling in the same nanodevice two amphiphilic antitumor agents: squalenoyl-gemcitabine and edelfosine. Homogeneous batches of nanoassemblies were easily formulated by the nanoprecipitation method. Their anticancer activity was tested in pediatric cancer cell lines and pharmacokinetic studies were performed in mice. In vitro assays revealed a synergistic effect when gemcitabine was co-administered with edelfosine. Squalenoyl-gemcitabine/edelfosine nanoassemblies were found to be capable of intracellular translocation in patient-derived metastatic pediatric osteosarcoma cells and showed a better antitumor profile than squalenoyl-gemcitabine nanoassemblies alone. The intravenous administration of this combinatorial nanomedicine in mice exhibited a controlled release behavior of gemcitabine and diminished edelfosine plasma peak concentrations. These findings make it a suitable pre-clinical candidate for childhood cancer therapy.

Keywords: pediatric cancer, chemotherapy, neuroblastoma, osteosarcoma, nanomedicine, gemcitabine, edelfosine.

Chapter II

1. INTRODUCTION Nanotechnology has been widely applied to medicine in the last few decades with the aim of enhancing the therapeutic potential of antitumor compounds. Improving drug protection from degradation, absorption, body distribution and cell penetration has become crucial in clinical practice, and several nanocarriers loaded with chemotherapeutics have been developed to this end [1–3]. Unfortunately, these therapies are to date essentially available in adult therapeutic protocols only, and not for use in pediatrics [4]. This is the case for tumors with a very poor prognosis such as osteosarcoma (OS) and neuroblastoma (NB). OS arises from the mesenchymal cells of growing bones and presents very low survival rates in children and adolescents due to its metastatic behavior [5]. Even though the global incidence of this musculoskeletal tumor is only 4-5 cases per million persons per year, it is responsible for 9% of child cancer deaths [6]. NB is an embryonal tumor of the peripheral nervous system, normally originating in the adrenal glands [7]. The 5-year survival rates remain below 70%, and it is responsible for 15% of pediatric cancer deaths due to its heterogeneous and metastatic behavior [8].

The aggressiveness and complex biology of these tumors are especially visible in high- risk or relapsed patients, where current therapies are still insufficient to effect cures [9,10]. Hence, we propose the implementation of combinatorial nanomedicines made solely with antitumor agents, with an optimal safety profile and prepared by an easy manufacture process. Our approach is based on the squalenoylation concept [11] and consists in the combination of two amphiphilic compounds: (i) the squalenoyl- gemcitabine (SQ-Gem) pro-drug, able to spontaneously form stable nanoassemblies (NAs) with a size around 120 nm [12], and (ii) the alkyl-lysophospholipid edelfosine (EF), which belongs to a new generation of antitumor drugs [13,14]. Gemcitabine (Gem) is a nucleoside analogue with proven anticancer activity in several tumors. It is given as a second-line treatment in pediatric OS and is currently in Phase II/III clinical trials for NB (NCT00407433). Its chemical conjugation to squalenic acid protects this cytotoxic drug from a rapid metabolization in vivo, one of its main drawbacks in the clinic [15], thereby considerably improving the therapeutic index of the molecule [16,17]. Unlike Gem and many other common cytotoxic drugs, EF acts by interfering with the lipid metabolism and signaling through lipid rafts, among other apoptotic pathways, triggering selective tumor cell death [18]. However, its use in the clinic has been hampered by gastrointestinal and hematological toxicity, making the encapsulation of EF in drug delivery systems mandatory [19].

The equimolar mixture of SQ-Gem and EF has recently been reported to form stable NAs with no need for using polymers or surfactants as transporter material [20]. Not only this co-assembly was able to preserve EF from hemolytic activity but also modified the physico-chemical features of SQ-Gem NAs (e.g., lower mean diameter, distinct supramolecular organization, higher drug content and stability). In the present study, we investigated the biological activity of SQ-Gem/EF NAs in NB and OS cells. Pharmacokinetic studies were performed after a single administration of SQ-Gem/EF

Squalenoyl-gemcitabine/edelfosine nanoassemblies: pharmacokinetic profile in mice and anticancer activity in pediatric cancer cells

NAs in mice. We highlighted the therapeutic potential of SQ-Gem/EF NAs to treat pediatric solid tumors such as NB or OS.

2. MATERIALS AND METHODS

2.1. Materials Gemcitabine (Gem) hydrochloride was purchased from Carbosynth, Ltd. (Compton, UK). Edelfosine was obtained from R. Berchtold (Biochemisches Labor, Bern, Switzerland). The synthesis of squalenoyl-gemcitabine (SQ-Gem) obtained by the chemical conjugation of Gem to squalenic acid has been previously reported by our group [11,12]. Ethanol absolute was purchased from Panreac Química (Barcelona, Spain). Phosphotungstic acid hydrate and paraformaldehyde were purchased from Sigma Aldrich (Barcelona, Spain). Celltiter 96® aqueous one solution cell proliferation assay (MTS) was purchased from Promega (Alcobendas, Spain). Heat inactivated fetal bovine serum (FBS), trypsin-EDTA, penicillin/streptomycin, Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM)-alpha medium and phosphate buffer saline (PBS) were provided by Gibco® (Invitrogen Inc., Carlsbad, USA). McCoy's 5A cell medium, CholEsteryl BODIPY 542/563 (BChol-red), 4’,6-diamidino-2-phenylindole (DAPI) and 8-well Nunc™ Lab-Tek™ II chamber slides were purchased from Thermo Fisher Scientific (Massachusetts, USA).

2.2. Preparation and characterization of squalenoyl NAs SQ-Gem/EF NAs were formulated by nanoprecipitation method as previously reported [20]. Briefly, a solution of SQ-Gem in ethanol alone or mixed with EF at equimolar concentrations was added dropwise into 2 mL of distilled water in a ratio ethanol/water 1:5 under stirring. Ethanol was evaporated in vacuum using a Rotavapor (Buchi R- 210/215, Buchi Corp., Canada) to obtain an aqueous suspension of NAs at a final concentration of 1.16 μmol mL-1 per each compound. SQ-Gem/EF NAs loaded with the orange-red dye CholEsteryl BODIPY 542/563 (BChol-red) (2% w/w) were prepared employing the same procedure (for further details see SM). Mean particle size expressed in Z-average and polydispersity index (PDI) of the NAs were determined by dynamic light scattering (DLS) (Zetasizer Nano ZS Malvern; Malvern Instruments SA, UK) after 1:20 dilution in ultrapure water. Likewise, surface charge of the NAs was characterized by measuring the zeta potential with laser Doppler velocimetry (Zetasizer Nano ZS Malvern; Malvern Instruments SA, UK). Aberration corrected scanning transmission electron microscopy (Cs-corrected STEM) images were obtained using a high angle annular dark field detector (HAADF) in a FEI XFEG TITAN electron microscope operated at 300 kV and equipped with a CETCOR Cs-probe

Chapter II

corrector from CEOS. Negative stained samples were stained with phosphotungstic acid in carbon coated copper grids (200 mesh).

2.3. Cell culture The human immortalized OS cell line U-2 OS (ATCC®HTB96™) was purchased from the American Type Culture Collection (Sigma–Aldrich). The thrice cloned NB cell line SH- SY5Y (ATCC®CRL-226™) was obtained from the laboratory of Prof. Rosa Noguera (University of Valencia-INCLIVA, Valencia, Spain). A metastatic pediatric OS cell line termed 531M was obtained from a patient treated at the Clínica Universidad de Navarra (Pamplona, Spain). 531M, SH-SY5Y and U-2 OS cell lines were cultured in MEM-alpha, DMEM and McCoy's 5A cell medium respectively, supplemented with 10% of heat inactivated FBS and 1% penicillin/streptomycin and maintained in a humidified atmosphere at 37 ºC with 5% carbon dioxide.

2.4. Cell internalization of SQ-Gem/EF NAs In order to measure cell uptake kinetics of SQ-Gem/EF NAs, 531M cells were seeded on 12-well plates at a density of 100,000 cells per 1 mL/well for 24 h to achieve 80% confluence. The fluorescently-labeled NAs and free BChol-red 2% w/w were then added at a final concentration of 10 μM (Eq. to EF and SQ-Gem) to each well. After incubation at different time intervals (0.5, 2, 4, 8 and 24 h), cells were washed with PBS, trypsinized and centrifuged for 5 mins at 200 g. Samples were analyzed on a BD Accuri C6 flow cytometer with an argon laser as light source of 488 nm wavelength. The mean cell fluorescence intensity was determined in a total of 12,000 cells using the BD Accuri CFlow Plus software (Accuri Cytometers, Ltd., UK). For detection of BChol-red fluorescence the emission spectrum was measured at 540-590 nm. For the evaluation of NAs cell internalization and localization, 531M cells were cultured on 8-well Nunc™ Lab-Tek™ II chamber slide systems at a density of 30,000 cells per 0.5 mL/well for 24 h to achieve approximately 50-60% confluence. Cells were then treated for different time periods (0.5, 2, 4, 8 and 24 h) with fluorescently labeled SQ-Gem/EF NAs (BChol-red 2% w/w) or with free BChol-red 2% w/w at a concentration of 20 μM (Eq. to EF and SQ-gem). After incubation with treatments, cells were washed with PBS and incubated with 4% paraformaldehyde for 10 min at 4ºC and DAPI for 5 mins. Cells were imaged with a LSM 800 confocal fluorescence microscope (Zeiss, Madrid, Spain) equipped with a 63X oil immersion lens. Excitation wavelength was set at 543 nm and detection of BChol-red was performed through a 560 nm filter.

Squalenoyl-gemcitabine/edelfosine nanoassemblies: pharmacokinetic profile in mice and anticancer activity in pediatric cancer cells

2.5. Cytotoxicity studies For the in vitro cytotoxicity assays the cell lines SH-SY5Y, U-2 OS were plated on 96- well plates at a density of respectively 5,000 and 2,000 cells per 100 μL/well, 24 h before the addition of different concentrations of Gem, EF, SQ-Gem NAs and SQ-Gem/EF NAs in triplicate wells (n ≥ 3). After 72 h incubation, complete medium containing 15 % v/v of MTS was added to each well. Absorbance was measured 2-4 h later in a microplate reader (iEMS reader MF, Labsystem, Helsinki, Finland) at a test wavelength of 492 nm and 690 nm for the reference wavelength. The concentration of drug required to inhibit cell proliferation by 50% (IC50) was calculated using GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). For drug synergy evaluation, Gem was combined with EF at a constant combination ratio of equipotent concentrations in SH-SY5Y and U-2 OS cell lines. Cell viability at 72 h was analyzed with each treatment alone and in combination using the same procedure described above. The dose-reduction index (DRI) and combination index (CI) were calculated according to the Chou-Talalay method [21] in order to determine the interactive effect of drugs.

2.6. Pharmacokinetic studies 6-8-week-old female athymic nude mice were obtained from Envigo (Harlan Laboratory). Procedures involving animal handling and care were approved by the Animal Care and Ethics Committee of the University of Navarra (n°:084-14). To measure the pharmacokinetics, a single dose of SQ-Gem/EF NAs (the molar equivalent of 30 mg/kg of EF at a ratio 1:1) or the free drug equimolar mixture (i.e., Gem + EF) in a 5% dextrose solution was intravenously injected via tail vein (12 mice per group). At various time points (5’, 15’, 30’, 1, 2, 4, 8, 24 and 48 h), blood was collected from the submandibular vein in EDTA surface-coated tubes (n= 3 at each time point). The simultaneous quantification of Gem, SQ-Gem and EF in mouse plasma was performed using a UHPLC/MS/MS method previously optimized (see SM for further details). Pharmacokinetic calculations were performed by non-compartmental analysis using PKSolver add-in program for pharmacokinetics and pharmacodynamics data analysis [22].

2.7. Statistical analysis Statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software, San Diego, CA, USA). Statistical comparison between different groups was performed using an ANOVA One-Way with Tukey post-hoc correction. Data were reported as mean ± SD and statistical significance levels were defined as *P <0.05.

Chapter II

3. RESULTS

3.1. Nanoprecipitation allowed for an easy preparation of SQ-Gem/EF NAs of 50 nm The proposed therapy relies on the co-assembly of the amphiphilic compounds SQ-Gem and EF (Fig. 1A) by nanoprecipitation from an ethanolic solution. All the nanoformulations exhibited a monomodal size distribution with only one population peak (Fig. 1B) and a mean particle size of 50 ± 4 nm (for further details see Fig. S1A and B), as confirmed by DLS and HAADF-STEM (Fig. 1C). This narrow particle size distribution correlated with a PDI of 0.124 ± 0.035. The surface charge of the SQ-Gem/EF NAs ranged from -11 to -14 mV, indicating that they were negatively electrostatically stabilized. Considering the molecular weights of Gem and EF (Fig. 1A), the equimolecular co-assembly resulted in a drug loading of 67.28 % (w/w of total drug content).

Figure 1. Characterization of the squalenoyl-gemcitabine/edelfosine nanoassemblies (SQ-Gem/EF NAs). (A) Molecular structures of SQ-Gem and EF. (B) Size distribution by intensity of NAs determined by dynamic light scattering (DLS). (C) High angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) representative micrograph of a multilamellar SQ-Gem/EF NA.

3.2. SQ-Gem/EF NAs were efficiently internalized by patient-derived metastatic pediatric OS cells The uptake of BChol-red SQ-Gem/EF NAs (size of 53 nm) (Fig. S1C) by patient treated- derived metastatic pediatric OS cells was investigated. It was observed by flow cytometry that after 0.5 h incubation, NAs started to associate with 531M cells (Fig. 2). Negative control group data indicated only a baseline cell fluorescence with the non-treated cells (Fig. 2B, green line) and a minor association of free BChol-red (Fig. 2B, blue line). In fact, the cell-associated fluorescence of BChol-red NAs increased during the first 8 h incubation (5.7-fold times vs free BChol-red) followed by a plateau until 24 h (Fig. 2A).

Squalenoyl-gemcitabine/edelfosine nanoassemblies: pharmacokinetic profile in mice and anticancer activity in pediatric cancer cells

Figure 2. Flow cytometry studies. (A) Time course of fluorescence accumulation in 531M cells exposed to fluorescently labeled squalenoyl-gemcitabine/edelfosine nanoassemblies (SQ-Gem/EF NAs) and free CholEsteryl BODIPY 542/563 (BChol-red). Normalized data with respect to the non-treated cells group represented as mean and SD from triplicate independent experiments is shown after incubation at different time intervals (0.5, 2, 4, 8 and 24 h). (B) Fluorescence intensity plot signals of cells incubated with free BChol-red, SQ-Gem/EF NAs, free BChol-red and cell medium only at 0.5 and 8 h.

Cells were also observed by confocal fluorescence microscopy with the aim of localizing the BChol-red SQ-Gem/EF NAs within the cells. Fluorescence images confirmed a time-dependent accumulation pattern of fluorescent NAs, with an appreciable initial phase of accumulation starting at 2-4 h (Fig. 3A). The minor fluorescence signal vs time after incubation with the negative controls (i.e. free dye and untreated cells) excluded any possibility of fluorescent artifact internalization, as shown in Fig. 3B and D. In accordance with flow cytometry assays, the maximum fluorescence intensity was observed between 8 and 24 h. Bright field and nucleus staining merged images demonstrated the intracellular localization of the red fluorescence signal which was still more clearly visible at higher magnification (Fig. 3C).

Chapter II

Figure 3. Confocal images (40 X magnification) of 531M cells after incubation with (A) CholEsteryl BODIPY 542/563 (BChol-red) squalenoyl-gemcitabine/edelfosine nanoassemblies (SQ-Gem/EF NAs), (B) BChol-red free at different time points (0.5, 2, 4, 8, 24 h). (C) 63X magnification of 531M cells after 24 h incubation with BChol-red SQ-Gem/EF NAs. (D) Non-treated cells. (a, BChol-red; b, DAPI; c, merge (BChol-red +DAPI); d, merge (bright field + DAPI + BChol-red). The fluorescence of the cholesterol analogue BChol-red was detected at 560 nm.

Squalenoyl-gemcitabine/edelfosine nanoassemblies: pharmacokinetic profile in mice and anticancer activity in pediatric cancer cells

3.3. Gem and EF free acted synergistically against OS and NB cells In order to evaluate whether the antitumor effect of EF was synergistic with that of Gem, SH-SY5Y and U-2 OS cells were exposed to the individual or combined drugs at a constant equipotent ratio. Patient-treated derived cell line 531M was excluded from these studies due to the inability of Gem to kill beyond 50% of the cell population [20]. As shown in Table 1, the antitumor activity of compound EF was in the micromolar range according to its IC50 value, being 1.71 and 25 µM for SH-SY5Y and U-2 OS, respectively. By contrast, Gem displayed a cytostatic activity at nanomolar concentrations with a characteristic cell viability plateau at 20-25 % (Fig. S4). Calculated IC50 values were 19 nM for U-2 OS and 28 nM for SH-SY5Y cell lines.

Table 1

Synergy analysis for Gem and EF combination therapy at the IC50 on U-2 OS and SH-SY5Y cell lines after 72 h of treatment Single treatment Combinative treatment*

Cell line IC50 Gem IC50 EF IC50 Gem IC50 EF CI (FA 0.5) (nM) (μM) (nM) (μM) U-2 OS 19 ± 3 25 ± 4 6 ± 1 13.5 ± 2.78 0.94 ± 0.04 SH-SY5Y 28 ± 17 1.71 ± 0.37 8 ± 2 1.29 ± 0.22 0.95 ± 0.13 *Drug ratio Gem (nM):EF (μM) for U-2 OS=1:2, and for SH-SY5Y=5:1.

Bearing in mind the distinct pharmacological activities of both drugs in these cells, different combinative ratios were investigated to achieve the highest DRIs. As a result, combinative IC50 values at optimized ratios are also shown in Table 1. When combining Gem and EF, the CI to inhibit 50% of cells was 0.94 ± 0.04 for U-2 OS and 0.95 ± 0.13 for SH-SY5Y cells. No additive or antagonistic interactions were observed for this combination in any of the conditions studied against NB (Fig. 4A) and OS (Fig. 4B) cells. Plots depicting CI values vs FA (fraction of inhibited viability) showed the occurrence of synergistic effects with CI values below 1 in all affected fractions.

Figure 4. Combination index (CI) analysis. Graphical representation of experimental CI values for gemcitabine (Gem) and edelfosine (EF), in SH-SY5Y cells (A) and U-2 OS cells (B) at different cell death fractions (FA). CI >1 antagonism, CI=1 additivity, CI <1 slight synergism, CI <<1 strong synergism.

Chapter II

3.4. SQ-Gem/EF NAs showed improved anticancer activity in OS and NB cells The anticancer activity of SQ-Gem/EF NAs was evaluated in SH-SY5Y and U-2 OS cells. For comparison purposes, SQ-Gem NAs (121 ± 14 nm size) (Fig. S1D) and a freshly prepared mixture of SQ-Gem NAs + free EF were used as controls. Contrary to EF, SQ- Gem NAs were efficacious at nanomolar concentrations. Nevertheless, comparison of the dose-response curves revealed that the equimolecular co-assembly of EF into SQ-Gem improved its efficacy, by decreasing the IC50 from 77 to 58 nM (24%) (Fig. 5A) and from 126 to 117 (7%) (Fig. 5B) in SH-SY5Y and U-2 OS cells, respectively. Of note, the anticancer efficacy improvement of SQ-Gem/EF NAs was more pronounced when compared to the mixture SQ-Gem NAs + free EF, this being 36% and 13% for SH-SY5Y and U-2 OS cells, respectively. As seen in the graphs, this effect was more visible in the SH-SY5Y cell line than in the U-2 OS, resulting statistically significant in comparison not only to SQ-Gem NAs but also to the physical mixture SQ-Gem NAs +free EF (Fig. 5A).

Figure 5. Cell viability assays in (A) SH-SY5Y and (B) U-2 OS cell lines. Graphical representation of a

MTS assay (n ≥ 3, data: mean ± SD) and statistical analysis of the IC50s (n ≥ 3, data: mean ± SD, *P value <0.05, **P value <0.01). Cells were exposed to squalenoyl-gemcitabine/edelfosine nanoassemblies (SQ- Gem/EF NAs), SQ-Gem NAs and SQ-Gem NAs + free EF for 72 h. Concentrations (nM) correspond to the individual amount of each drug molecule at a molar ratio 1:1.

3.5. SQ-Gem/EF NAs favorably modified the pharmacokinetic profile of EF and Gem Here, we studied whether the co-assembly of SQ-Gem and EF was able to improve the drug pharmacokinetic profile of EF and Gem in vivo. Fig. 6 depicts the plasma concentration vs time of drugs after a single-dose i.v. administration. Pharmacokinetic parameters estimated after non-compartmental analysis of experimental data are shown

Squalenoyl-gemcitabine/edelfosine nanoassemblies: pharmacokinetic profile in mice and anticancer activity in pediatric cancer cells in Table 2. As seen in Fig. 6, SQ-Gem/EF NAs modified the pharmacokinetic profile comparatively to free EF by reducing its exposure in blood plasma during the first 2 hours. Drug plasma levels were balanced after 4 hours at a concentration around 10 μg/mL, once steady-state was reached. According to this, pharmacokinetic analysis of EF in blood plasma showed a Cmax of 46 μg/mL for EF from SQ-Gem/EF NAs and 54 μg/mL for free EF. Average AUC0-∞ values calculated at steady-state were 554 (EF from SQ- Gem/EF NAs) and 626 μg·h/mL (free EF), correlating with steady-state volume of distribution (Vss) values of 2903 and 2622 mL/kg, respectively. On the other hand, SQ- Gem/EF NAs exhibited a sustained release of Gem with a Cmax 4.5-times lower than of free Gem (18 µg/mL vs 4 µg/mL). Notwithstanding, the AUC0-∞ values were similar (9- 11 μg·h/mL), since free Gem presented an elimination half-life 5-times lower (0.35 vs 1.87 h). In that sense, the Vss of Gem released from SQ-Gem/EF NAs was 7.5-times higher than with the free drug. (4770 vs 634 mL/kg). Moreover, the presence of SQ-Gem increased considerably the total pool of Gem (SQ-gem + Gem), being detectable in blood plasma after 8 h.

Figure 6. Plasma concentrations of gemcitabine (Gem), squalenoyl-gemcitabine (SQ-Gem) and edelfosine (EF) after a single intravenous (i.v.) injection (via tail vein) of squalenoyl-gemcitabine/edelfosine nanoassemblies (SQ-Gem/EF NAs) and the free drug mixture (i.e., Gem + EF) (30 mg/kg Eq. EF). The values are the mean and SD (n= 3).

Chapter II

Table 2 Pharmacokinetic parameters of squalenoyl-gemcitabine (SQ-Gem), gemcitabine (Gem) or edelfosine (EF), in mice treated i.v. with either SQ-Gem/EF nanoassemblies (NAs) or the mixture Free Gem + Free EF (30 mg/kg EQ of EF) in athymic nude female mice SQ-Gem/EF NAs Free Gem + Free EF Parameter EF SQ-Gem Gem EF Gem t1/2 (h) 38 ± 13 0.86 ± 0.39 1.87 ± 0.31 39 ± 11 0.35 ± 0.06 Cmax (μg/mL) 46 ± 8 80 ± 20 4 ± 0.77 54 ± 6 18.2 ± 0.87

AUC 0-∞ (μg·h/mL) 554 ± 104 28 ± 7 9 ± 2 626 ± 118 11 ± 2 Cl_obs (mL.kg /h) 55 ± 10 1368 ± 336 1660 ± 340 48 ± 8 1367 ± 285 Vss_obs (mL/kg) 2903 ± 435 658 ± 328 4770 ± 2081 2622 ± 306 634 ± 21 t1/2 (hours), terminal elimination half-life; Cmax (micrograms per milliliter), maximum observed plasma concentration; AUC 0-inf_obs (micrograms hour per milliliter), Area Under the curve from 0 extrapolated to infinite time; Cl_obs (milliliters per hour), systemic clearance; Vss, volume of distribution at steady-state (milliliters per kilogram). Values of SQ-Gem, Gem and EF were calculated considering a dose of 37 mg/kg, 15 mg/kg and 30 mg/kg, respectively.

4. DISCUSSION

High-dose chemotherapy regimens still remain the last opportunity for children with aggressive cancers. Nanotechnology research in the field of cancer treatment represents an alternative approach able to increase the effectiveness of these therapies while decreasing their associated side effects [4,23]. However, complex design and architecture of many proposed nanomedicines as well as problems associated with their industrial scale-up hamper their translation from research to clinic [24,25]. This paper shows how we successfully formulated several homogenous batches of multidrug nanodevices of 50 nm (Fig. S1B) by the association between two antitumor drugs only (i.e. SQ-Gem and EF), without the need of any stabilizing agent using a simple procedure which can be easily scaled up.

Using the cell line 531M as an in vitro model, we determined how SQ-Gem/EF NAs were able to penetrate progressively patient-treated derived metastatic pediatric OS cells. Intact SQ-Gem NAs have been reported to be located mostly in the cell membrane and not be internalized by endocytosis due to their inverted hexagonal supramolecular organization structure [26]. Interestingly, multilamellar SQ-Gem/EF NAs (Fig. 1C) presented a clear intracellular cytoplasmic localization near the nucleus (Fig. 3C) where Gem and EF exert their action. We have to bear in mind that the intracellular penetration of Gem is dependent on the nucleoside transporter hENT1, down-regulated in resistant cells [27]. As a consequence, we have previously demonstrated that Gem was only able to kill half of the cell population whereas SQ-Gem/EF NAs bypassed this resistance (IC50 >36 µM vs 20 µM) [20]. The co-administration of EF and Gem showed only a modest synergy (CI >0.5<1) at the different drug effect levels studied in two pediatric cancer cell lines (Fig. 4). Valdez

Squalenoyl-gemcitabine/edelfosine nanoassemblies: pharmacokinetic profile in mice and anticancer activity in pediatric cancer cells et al. suggested that this synergy might operate at a mitochondrial level [28] since, as previously reported, Gem inhibits mitochondrial DNA via thymidine kinase, whereas EF accumulates and subsequently disrupts the mitochondrial membrane potential [29]. The results herein obtained suggest that combination therapy with the free drugs could be useful to reduce the Gem dose required to maintain treatment efficacy while reducing its toxicity. Of note, EF was found to be substantially more active in the SH-SY5Y cells in comparison with the U-2 OS (IC50 1.71 vs 25 μM) and other cancer cells previously reported [30,31]. This boosted effect could be triggered by the specific alteration in neurons of the de novo biosynthesis of phosphatidylcholine, the inhibition of the MAPK/ERK mitogenic pathway or the above-mentioned mitochondrial disruption at a membrane level by EF [29,32,33]. In vitro biological assessment also showed that the equimolecular insertion of EF into SQ-Gem led to an improved anticancer activity despite the negligible effect of EF on cells at nanomolar concentrations (Fig. 5). The lower cytotoxicity observed with the mixture SQ-Gem NAs + free EF in comparison with SQ- Gem NAs was thought to be likely due to the impairment of the macrostructure of SQ- Gem NAs by the physical mixture of EF. According to this, the incorporation of EF in a SQ-Gem NAs dispersion provoked a fast particle disruption as observed by DLS. In previous studies we have reported how we delivered tailored therapeutic concentrations of orally administered EF loaded into solid lipid nanoparticles [34,35]. In our study, SQ-Gem/EF NAs showed a similar EF oral absorption compared with EF free (Fig. S3). Notwithstanding, it was shown that i.v. administered SQ-Gem/EF NAs decreased plasma concentrations of EF in its initial decay phase, where erythrocytes are more exposed to the detergent action of EF at that peak concentration (Fig. 6). Therefore, a major advantage of this strategy lies in the possibility to administer i.v. the hemotoxic compound EF. On the other hand, the i.v. injection of SQ-Gem/EF NAs not only smoothly prolonged the plasma concentrations of the total Gem pool but also exhibited a less abrupt Gem release than in the case of previously reported SQ-Gem NAs [16,36]. This denotes a unique biodistribution of SQ-Gem/EF NAs, probably influenced by their changes in size, structure or lipophilicity. For instance, given the analogy of EF with some sphingolipids present in endogenous lipoproteins [37] and their high affinity for SQ-Gem [17], we believe that SQ-Gem/EF NAs escape from them, remaining co-assembled for a longer time. This higher systemic clearance of the total Gem pool (SQ-Gem + cleaved Gem) by SQ-Gem/EF NAs could be required in clinical practice to prevent Gem associated myelosuppression and vascular side effects [38,39].

5. CONCLUSION In this study, we evaluated in vitro and in vivo a combinatorial nanomedicine designed by the single enrichment of SQ-Gem with the alkyl-lysophopholipid agent EF. Multitherapy of Gem and EF proved to be a profitable combinatorial strategy. SQ- Gem/EF NAs diffused intracellularly in metastatic OS cells and displayed an improved anticancer efficacy over SQ-Gem NAs and the mixture SQ-Gem NAs + free EF in NB

Chapter II

and OS cells. Their main benefit resulted from an outstanding modification of the pharmacokinetic profiles of the biologically active compounds Gem and EF, this representing a step ahead towards further investigation in pre-clinical models of diseases such as NB or OS. The urgent need for realistic approaches in the pediatric cancer field supports make this a promising alternative to current treatments.

ACKNOWLEDGEMENTS Asociación Española Contra el Cáncer (AECC) (CI14142069BLAN), Fundación Caja Navarra/Obra Social La Caixa and Asociación de familiares y amigos de pacientes con neuroblastoma (NEN, Nico contra el cáncer) are gratefully acknowledged for the financial support.

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Squalenoyl-gemcitabine/edelfosine nanoassemblies: pharmacokinetic profile in mice and anticancer activity in pediatric cancer cells

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[20] C. Rodríguez-Nogales, V. Sebastián, S. Irusta, D. Desmaële, P. Couvreur, M.J. Blanco-Prieto, A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine, Eur. J. Pharm. Biopharm. 144 (2019) 165– 173. [21] T.C. Chou, P. Talalay, Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors, Adv. Enzyme Regul. 22 (1984) 27–55. [22] Y. Zhang, M. Huo, J. Zhou, S. Xie, PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel, Comput. Methods Programs Biomed. 99 (2010) 306–314. [23] C. Rodriguez-Nogales, R. Noguera, P. Couvreur, M.J. Blanco-Prieto, Therapeutic opportunities in neuroblastoma using nanotechnology, J. Pharmacol. Exp. Ther. 370 (2019) 625-635. [24] P. Grodzinski, M. Kircher, M. Goldberg, A. Gabizon, Integrating Nanotechnology into Cancer Care, ACS Nano. 13 (2019) 7370–7376. [25] M. Hwang, E.J. Lee, S.Y. Kweon, M.S. Park, J.Y. Jeong, J.H. Um, S.A. Kim, B.S. Han, K.H. Lee, H.J. Yoon, Risk assessment principle for engineered nanotechnology: In food and drug, Toxicol. Res. 28 (2012) 73–79. [26] L. Bildstein, C. Dubernet, V. Marsaud, H. Chacun, V. Nicolas, C. Gueutin, A. Sarasin, H. Bénech, S. Lepêtre-Mouelhi, D. Desmaële, P. Couvreur, Transmembrane diffusion of gemcitabine by a nanoparticulate squalenoyl prodrug: an original drug delivery pathway, J. Control. Release. 147 (2010) 163–70. [27] J.R. Mackey, R.S. Mani, M. Selner, D. Mowles, J.D. Young, J.A. Belt, C.R. Crawford, C.E. Cass, Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines, Cancer Res. 58 (1998) 4349–4357.

[28] B.C. Valdez, A.R. Zander, G. Song, D. Murray, Y. Nieto, Y. Li, R.E. Champlin, B.S. Andersson, Synergistic cytotoxicity of gemcitabine, clofarabine and edelfosine in lymphoma cell lines., Blood Cancer J. 4 (2014) e171. [29] S.C. Lim, K.R. Parajuli, S.I. Han, The alkyllysophospholipid edelfosine enhances TRAIL-mediated apoptosis in gastric cancer cells through death receptor 5 and the mitochondrial pathway, Tumor Biol. 37 (2016) 6205–6216. [30] M.Á. Aznar, B. Lasa-Saracíbar, A. Estella-Hermoso de Mendoza, M.J. Blanco- Prieto, Efficacy of edelfosine lipid nanoparticles in breast cancer cells, Int. J. Pharm. 454 (2013) 720–726.

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[31] A. Estella-Hermoso de Mendoza, V. Préat, F. Mollinedo, M.J. Blanco-Prieto, In vitro and in vivo efficacy of edelfosine-loaded lipid nanoparticles against glioma, J. Control. Release. 156 (2011) 421–426. [32] H. Marcucci, L. Paoletti, S. Jackowski, C. Banchio, Phosphatidylcholine biosynthesis during neuronal differentiation and its role in cell fate determination, J. Biol. Chem. 285 (2010) 25382–25393. [33] A.H. Van Der Luit, M. Budde, P. Ruurs, M. Verheij, W.J. Van Blitterswijk, Alkyl- lysophospholipid accumulates in lipid rafts and induces apoptosis via raft- dependent endocytosis and inhibition of phosphatidylcholine synthesis, J. Biol. Chem. 277 (2002) 39541–39547. [34] Y. González-Fernández, H.K. Brown, A. Patiño-García, D. Heymann, M.J. Blanco-Prieto, Oral administration of edelfosine encapsulated lipid nanoparticles causes regression of lung metastases in pre-clinical models of osteosarcoma, Cancer Lett. 430 (2018) 193–200. [35] A.E.H. De Mendoza, M.A. Campanero, J. De La Iglesia-Vicente, C. Gajate, F. Mollinedo, M.J. Blanco-Prieto, Antitumor alkyl ether lipid edelfosine: Tissue distribution and pharmacokinetic behavior in healthy and tumor-bearing immunosuppressed mice, Clin. Cancer Res. 15 (2009) 858–864.

[36] A. Maksimenko, M. Alami, F. Zouhiri, J.-D. Brion, A. Pruvost, J. Mougin, A. Hamze, T. Boissenot, O. Provot, D. Desmaële, P. Couvreur, Therapeutic modalities of squalenoyl nanocomposites in colon cancer: an ongoing search for improved efficacy., ACS Nano. 8 (2014) 2018–32. [37] J. Iqbal, M.T. Walsh, S.M. Hammad, M.M. Hussain, sphingolipids and lipoproteins in health and metabolic disorders, trends Endocrinol. Metab. 28 (2017) 506–518. [38] C.A. Dasanu, Gemcitabine: Vascular toxicity and prothrombotic potential, Expert Opin. Drug Saf. 7 (2008) 703–716.

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Chapter II

SUPPORTING MATERIAL Characterization of squalenoyl NAs Analysis of particle size distribution by number showed that the majority of squalenoyl- gemcitabine/edelfosine nanoassemblies (SQ-Gem/EF NAs) ranged between 20 and 70 nm (Fig. S1A). In order to evaluate the reproducibility and robustness of the preparation method independent batches were formulated on different days. Z-average size of 9 samples (Fig. S1B) showed a coefficient of variation of 8.07 % and the standard error of the mean was 1.36. When initial concentrations were augmented 5 times according to the therapeutic in vivo requirements (i.e., from 1.16 to 5.8 μmol mL-1), the colloidal properties of the NAs barely changed. Fluorescent SQ-Gem/EF NAs (CholEsteryl BODIPY 542/563 2% w/w) displayed nearly identical hydrodynamic properties than their non-labeled counterparts with a mean particle size diameter of 53 nm, PDI of 0.141 (Fig. S1C) and zeta potential of -14.7 mV. SQ-Gem NAs displayed a mean particle size of 121 nm (Fig. S1D), PDI of 0.1 and zeta potential of -14 mV.

Figure S1. (A) Size distribution by number of the squalenoyl-gemcitabine/edelfosine nanoassemblies (SQ- Gem/EF NAs) and (B) mean particle size plot of 9 freshly prepared formulations. Size distribution by intensity of (C) fluorescent SQ-Gem/EF NAs and (D) SQ-Gem NAs.

Squalenoyl-gemcitabine/edelfosine nanoassemblies: pharmacokinetic profile in mice and anticancer activity in pediatric cancer cells

Quantification of Gem, SQ-Gem and EF in plasma A UHPLC/MS/MS method was optimized to perform the simultaneous quantification of gemcitabine (Gem), SQ-Gem and EF in mouse plasma. 2’-deoxycytidine and platelet- activating factor-16 were used as internal standards. Methanol (HPLC grade), formic acid, platelet activating factor-16 and tetrahydrouridine were obtained from Merck (Barcelona, Spain). 2’-deoxycytidine, and acetone (HPLC grade) were purchased from Sigma Aldrich (Barcelona, Spain). Sample processing Tetrahydrouridine (10 mg/mL) was added to each collected mice blood sample to prevent Gem ex vivo deamination into inactive uracil derivative 2’2’-difluorodeoxyuridine. Blood samples were centrifuged (2000 g for 10 min at 4ºC) to separate the plasma and stored at -80ºC until analysis. For in vivo plasma extraction and quantitation of SQ-Gem and Gem, 1980 μL of a methanol/acetone (1:20) and 1% v/v formic acid solution containing the internal standard (2’-deoxycytidine 0.25 μg/mL) was added to 20 μL of plasma sample. Mixture was vortex-mixed 2 min and then centrifuged at 20,000 g for 20 min at 4°C. 100 μL of the supernatant was then diluted in 900 μL of pure acetone and transferred to an injection vial for analysis. For in vivo plasma extraction and quantitation of EF, 80 μL of an acetonitrile/methanol (1:1) and 1% v/v formic acid solution containing the internal standard (platelet-activating factor-16 16.6 μg/mL) was added to 40 μL of plasma sample. Mixture was vortex-mixed 2 min and then centrifuged at 20,000 g for 20 min at 4°C. 40 μL of supernatant was then diluted with 40 μL of pure methanol and transferred to an injection vial for analysis. UHPLC/MS/MS method The UHPLC system was composed of an Acquity UPLCTM system (Waters Corp., Milford, MA, USA). The extracted samples, maintained at 4°C, were TM chromatographically separated using an Acquity UPLC BEH C18 column (50mm×2.1mm, 1.7m; Waters Corp., Milford, MA, USA) with a gradient elution. The injection volume was 2 µL using partial loop mode for sample injection. Initial conditions consisted of mobile phase A (1% formic acid in water) and mobile phase B (methanol) with a column temperature of 50°C and a flow rate of 0.5 mL/min. The gradient conditions ramped from 70% B to 85% B between 0 and 1.0 min and maintained up to 3.0 min; then ramped to 90% up to min 4.0 and finally ramped back to 70% until min 5.0 for re-equilibration. Under these conditions Gem and 2’-deoxycytidine displayed a mean retention time of 0.31 and 0.34 min, while platelet-activating factor-16, EF, and SQ-Gem showed a mean retention time of 2.69, 2.74 and 3.24 min, respectively (Fig. S2). Triple-quadrupole tandem mass spectrometric detection was performed on an AcquityTM TQD mass spectrometer (Waters Corp., Milford, MA, USA) with an electrospray ionization (ESI) interface. The following optimized MS parameters employed were: 2.5 kV capillary voltage, 65 V cone voltage, 120 ºC source temperature and 350ºC desolvation temperature. Nitrogen was used for the desolvation and as cone

Chapter II

gas at a flow rate of 650 and 50 L/h, respectively. Argon was used as the collision gas. The mass spectrometer operating in a positive mode was set up for multiple reaction monitoring (MRM). Analytes were monitored using a pair value cone voltage (V) /collision energy (eV) set at 40/25, 30/20, 60/30, 30/20 and 65/45 for Gem, 2’- deoxycytidine, EF, platelet-activating factor-16 and SQ-Gem, respectively. Monitored MRM transitions were m/z 264/112, 228/112, 524.5/104.2 or 184.2, 552.2/184.2 and 646.7/112 for the above list of compounds, respectively. Under these conditions drug recoveries in plasma calculated ranged between 80% and 100%. For the three compounds to be quantified, linear regression with 1/X2 weighing was used for calibrations in plasma samples. Lower limit of quantification (LLOQ) values were selected according to signal/noise ratio >10, coefficient of variation <15% and linearity R2 >0.990, obtained in independent experiments. Thus, calibration curves ranged from 0.05 to 125 μg/mL for SQ-Gem (R2= 0.994) and from 1 to 100 μg/mL for EF (R2= 0.995) and Gem (R2= 0.996).

Squalenoyl-gemcitabine/edelfosine nanoassemblies: pharmacokinetic profile in mice and anticancer activity in pediatric cancer cells

Figure S2. Mass chromatograms of mice plasma samples spiked with (A) edelfosine (EF), (B) platelet activating factor-16, (C) gemcitabine (Gem), (D) squalenoyl-gemcitabine (SQ-Gem) and (E) 2’- deoxycytidine.

Pharmacokinetic profile of SQ-Gem/EF NAs after a single peroral administration 8 week-old female athymic nude mice were obtained from Envigo (Harlan Laboratory). Procedures involving animal handling and care were approved by the Animal Care and Ethics Committee of the University of Navarra (n°: 084-14). A total of 18 healthy mice were randomly assigned into two groups. To measure the pharmacokinetics, a single dose of SQ-Gem/EF NAs (the molar equivalent of 30 mg/kg of EF at a ratio 1:1) or the free drug equimolecular mixture (i.e., Gem + EF) in a 5% dextrose solution was perorally

Chapter II

administered (9 mice per group). At various time points (30’, 1, 2, 4, 8, 24, 48 and 72 h), blood was collected in EDTA surface-coated tubes (n= 3 at each time point). Samples were processed and quantified as pointed in the section above. Very low concentrations of SQ-Gem and Gem could be found in plasma along the time interval of 0-2 hours after the oral administration of both treatments (Fig. S3). After 2 hours, these concentrations were below the quantification limits of the technique (LLOQ= 1 µg/mL for Gem and 0.05 µg/mL for SQ-Gem), not being sufficient for the calculation of pharmacokinetic parameters. Concentrations in plasma vs time for co-assembled and free EF proved to be constant after 48-72 h, displaying a similar Cmax and AUC0-72h around 3-4 μg/mL and 179 μg·h/mL, respectively.

Figure S3. Plasma concentrations of gemcitabine (Gem), squalenoyl-gemcitabine (SQ-Gem) and edelfosine (EF) after a single peroral administration of squalenoyl-Gemcitabine/edelfosine nanoassemblies (SQ-gem/EF NAs) and the free drug mixture (i.e., Gem + EF) (30 mg/kg Eq. EF). The values are the mean and SD (n= 3).

Squalenoyl-gemcitabine/edelfosine nanoassemblies: pharmacokinetic profile in mice and anticancer activity in pediatric cancer cells

FIGURE S4:

Figure S4. Graphical representation of a MTS assay (data: mean ± SD of triplicate wells). SH-SY5Y and U-2 OS cells were exposed to gemcitabine (Gem), edelfosine (EF) and their combination for 72 h. *Drug ratio Gem (nM):EF (μM) for U-2 OS=1:2, and for SH-SY5Y=5:1.

CHAPTER III

Squalenoyl-gemcitabine/edelfosine combination nanomedicine for pediatric osteosarcoma

Chapter III

Squalenoyl-gemcitabine/edelfosine combination nanomedicine for pediatric osteosarcoma

C. Rodríguez-Nogalesa,b, H. Morenob,c, C. Zanduetab,c, D. Desmaëled, F. Lecandab,c, P. Couvreurd*, M.J. Blanco-Prietoa,b*

a Chemistry and Pharmaceutical Technology Department, Universidad de Navarra, Pamplona, Spain

b IdiSNA, Navarra Institute for Health Research, Pamplona, Spain c Programme in Solid Tumours, Division of Oncology, Centre for Applied Biomedical Research (CIMA), CIBERONC, University of Navarra, Pamplona, Spain

d Université Paris-Saclay, CNRS, Institut Galien Paris Sud, 92296, Châtenay-Malabry, France.

Conflict of interest: authors have no conflict of interest.

Paper under preparation

Squalenoyl-gemcitabine/edelfosine combination nanomedicine for pediatric osteosarcoma

ABSTRACT

Survival rates in pediatric osteosarcoma are poor owing to chemoresistance and their high propensity to form lung metastasis. The introduction of combinatorial nanomedicines in current therapeutic protocols can ameliorate the perspectives in high-risk patients. In this study, squalenoyl-gemcitabine/edelfosine nanoassemblies of 50 nm were tested with the aim of improving the therapeutic index of gemcitabine and the alkyl-lysophospholipid edelfosine. Pharmacokinetic profile of squalenoyl-gemcitabine/edelfosine nanoassemblies after a single retro-orbital plexus administration in mice revealed a higher systemic clearance of the total gemcitabine pool in comparison with squalenoyl- gemcitabine nanoassemblies, which correlated with a better tolerability and lower toxic profile after multiple administrations. For their in vivo pre-clinical assessment in an orthotopic osteosarcoma tumor model, c-Fos overexpressing P1.15 cells were intratibially injected in athymic nude mice. In comparison with the control groups, the combinatorial nanomedicine was found to decrease the primary tumor growth kinetics and to reduce the number of lung metastases. Our findings support the candidature of squalenoyl- gemcitabine/edelfosine nanoassemblies as a potential pediatric osteosarcoma therapy.

Keywords: osteosarcoma, chemotherapy, pediatric cancer, metastasis, nanomedicine, gemcitabine, edelfosine.

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1. INTRODUCTION

Pediatric osteosarcoma (OS) is a primary osseous malignancy, predominantly affecting patients younger than 20 years [1]. It commonly originates from primitive bone-forming mesenchyme of the knee region, between the distal femoral and proximal tibial metaphyseal area [2]. Children and adolescents diagnosed with OS in 2020 are still treated with chemotherapeutic drug regimens that have remained mostly unmodified since the consolidation of methotrexate, doxorubicin, and cisplatin in the early 1980s [3]. A complete tumor resection together with adjuvant chemotherapy allows for a successful eradication of localized OS; however, lung metastasis represents the most common fatal outcome with dismal prognosis [4]. Even though current protocols have brought an overall 5-year survival around 70-80%, these rates decrease to 20% in patients with metastasis [5,6]. Pediatric OS is characterized by a high multi-complex genomic instability. Cancer cells often present a high predisposition to germline mutations in the pRb and p53 tumor-suppressor genes as well as oncogene overexpression of c-Met, c- Myc and c-Fos, among others [7–9].

Having in mind that radiation and immunotherapy have had a limited role in the treatment of OS to date, conventional chemotherapy represents the last chance for high- risk patients [10]. To prevent tumor recurrence current protocols include the administration of high doses of triple-drug chemotherapy combinations. Unfortunately, on many occasions these regimens entail poor tolerance, resistance, long-term toxicities and morbidity, and are especially risky in children. This situation requires active research on novel therapeutic approaches [11,12].

Among the miscellaneous applications of nanotechnology, cancer nanomedicine has emerged in clinical practice to improve the therapeutic index of chemotherapeutic drugs in adults, but this is still not available for the pediatric population [13,14]. With regard to pediatric OS, Gemcitabine (Gem) (Gemzar®) is a second-line antineoplastic agent given in pediatric OS alone or in combination with docetaxel [15]. Although it is extremely active against a wide variety of tumors, this nucleoside analogue (2',2'- difluorodeoxycytidine) is rapidly inactivated via cytidine deaminase [16]. Squalenoyl- gemcitabine nanoassemblies (SQ-Gem NAs) have been reported to dramatically increase the low half-life of Gem in vivo, yielding an improved efficacy in several tumor models [17,18]. Since these treatments are often inefficient when a single anticancer drug is used, the insertion of the alkyl-lysophospholipid edelfosine (EF) within SQ-Gem was recently proposed in order to construct a novel combinatorial nanomedicine. EF is a pro-apoptotic drug that does not damage the DNA integrity like Gem; nonetheless, it presents some drawbacks such as hemolysis and gastrointestinal toxicity [19,20]. Its anticancer activity mainly relies on the activation of the Fas/CD95 cell death receptor via lipid rafts or inhibition of the MAPK/ERK and the Akt/protein kinase B survival pathways [21,22]. We previously reported that the amphiphilic structure of SQ-Gem and EF allowed for their complete equimolecular co-assembly by an easy nanoformulation method, resulting

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in the formation of stable multilamellar NAs of 50 nm [23]. This combination favorably modified the physico-chemical characteristics and anticancer profile in vitro of SQ-Gem, and has prompted their pre-clinical validation in vivo.

All in all, the goal of this nanotechnology approach is to overcome high-grade OS tumors while preserving the inherent toxicity of Gem and EF. For the in vivo pre-clinical testing, an aggressive OS mouse tumor model was designed by the orthotopical inoculation of murine OS cells in the tibiae of athymic nude mice. These cells grow intratibially with a short latency period and are prone to metastasize to the lungs after few weeks. SQ-Gem/EF NAs were well tolerated and delayed the progression of the primary tumor as well as its metastatic lung dissemination. These results demonstrate the possibilities of combinatorial nanomedicine to overcome the obstacles of current chemotherapy.

2. MATERIALS AND METHODS

2.1. Materials Edelfosine (EF) was obtained from R. Berchtold (Biochemisches Labor, Bern, Switzerland). Gemcitabine (Gem) hydrochloride was purchased from Carbosynth, Ltd. (Compton, UK). Ethanol absolute was purchased from Panreac Química (Barcelona, Spain). Celltiter 96® aqueous one solution cell proliferation assay (MTS) and D-luciferin was purchased from Promega (Alcobendas, Spain). Heat inactivated fetal bovine serum, trypsin-EDTA, penicillin/streptomycin, alpha-Minimum Essential medium and Phosphate Buffer Saline (PBS) were provided by Gibco® (Invitrogen Inc., Carlsbad, USA). Difco™ Dextrose was obtained from BD Biosciences (Madrid, Spain).

2.2. Formulation of squalenoyl NAs 4-(N)-Tris-nor-squalenoyl-gemcitabine (SQ-Gem) was synthesized by linking the 1,1′,2- tris-nor-squalenic acid covalently to the amino group of the heterocyclic ring of Gem [17]. SQ-Gem/EF NAs and SQ-Gem NAs were formulated by nanoprecipitation method as previously described [23]. A solution of SQ-Gem in ethanol alone or mixed with a solution of EF at equimolar concentrations was added dropwise into 2 mL of distilled stirring water. Ethanol was evaporated in vacuum using a Rotavapor (Buchi R-210/215, Buchi Corp., Canada) to obtain an aqueous suspension of NAs. Mean particle size expressed in Z-average and polydispersity index (PDI) of the NAs was determined at 25ºC by DLS (Zetasizer Nano ZS Malvern; Malvern Instruments SA, UK). Surface charge of the NAs was characterized by measuring the zeta potential with laser Doppler velocimetry (Zetasizer Nano ZS Malvern; Malvern Instruments SA, UK).

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2.3. Cell culture and cytotoxicity assays The P1.15 clonal OS cell line was cultured from a c-Fos/AP-1 murine transgenic OS [24]. Primary OS cells (p53+/- OSs) were obtained from a spontaneous OS developed in p53+/- knock-out mouse. All cell lines were maintained in alpha-MEM cell medium, supplemented with 10% of inactivated fetal bovine serum and 1% penicillin/streptomycin in an incubator at 37ºC with a humidified atmosphere and 5% carbon dioxide. For the in vitro cytotoxicity assays p53+/- OS and P1.15 cell lines were plated on 96- well plates at a density of 1,000 cells per well, 24 h before the addition of different treatments in triplicate wells for 72 h. After incubation with the different formulations, 100 μL of complete medium containing 15 % v/v of MTS was added to each well. Absorbance was measured 3 h later in a microplate reader (Labsystem, Helsinki, Finland) at a test wavelength of 492 nm and 690 nm for the reference wavelength. The concentration of drug required to inhibit cell proliferation by 50% (IC50) was calculated using GraphPad Prism software (San Diego, CA, USA).

2.4. Pharmacokinetic studies 6-8-week-old female athymic nude mice were obtained from Harlan Laboratory (Barcelona, Spain). Procedures involving animal handling and care were approved by the Animal Care and Ethics Committee under license CEEA/031-18 (CIMA, Pamplona, Spain). A total of 24 healthy mice were randomly assigned into two groups. To measure the pharmacokinetics, a single dose of SQ-Gem/EF NAs or SQ-Gem NAs at 58 µmol/kg (the molar equivalent at a ratio 1:1 that corresponds to 30 mg/kg of EF) in a dextrose 5% solution; were injected via retro-orbital plexus (12 mice per group). Afterwards, blood was collected in EDTA surface-coated tubes (n= 3 at each time point) at various time points (5’, 15’, 30’, 1, 4, 8, 24 and 48 h). Pharmacokinetic calculations were performed by non-compartmental analysis using PKSolver add-in program for pharmacokinetics and pharmacodynamics data analysis [25].

Simultaneous quantification of Gem, SQ-Gem and EF in mouse plasma was performed using a UHPLC/MS/MS method previously described by our team (See SM Chapter II). The UHPLC system was composed of an Acquity UPLCTM system (Waters Corp., Milford, MA, USA). Samples were chromatographically separated using an Acquity TM UPLC BEH C18 column (50mm×2.1mm, 1.7m; Waters Corp., Milford, MA, USA). Triple-quadrupole tandem mass spectrometric detection was performed on an AcquityTM TQD mass spectrometer (Waters Corp., Milford, MA, USA) with an electrospray ionization (ESI) interface. Calibration curves ranged from 0.05 to 125 μg/mL for SQ-gem (R2= 0.994) and from 1 to 100 μg/mL for EF (R2= 0.995) and Gem (R2= 0.996).

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2.5. In vivo efficacy studies

P1.15 cultured cells were harvested and diluted in PBS to a final concentration of 20 x 103 cells per 5 μL. Intratibial injections were performed on 6-8-week-old female athymic nude mice (CEEA/031-18). Studies were performed at n= 7 per group, allocated randomly into experimental groups. Four days after tumor cell inoculation, animals received via retro-orbital plexus either SQ-Gem/EF NAs, SQ-Gem NAs, free Gem at 38 µmol/kg (the molar equivalent at a ratio 1:1 that corresponds to 20 mg/kg of EF) or dextrose 5% (Dex 5%) vehicle control twice per week during the experiment up to eight administrations.

P1.15 cells are genetically modified to express the luciferase gene, and the efficacy of the treatments was assessed by monitoring the primary tumor growth by means of bioluminescence imaging every week. Animals were anesthetized and inoculated with 50 μL of 15 mg/mL D-luciferin. Images were taken during 1 min with a PhotonIMAGER™ imaging system (Biospace Lab) and analyzed using M3Vision software (Biospace Lab). Photon flux was calculated by using a region of interest or by delineating the mouse for whole-body bioluminescence quantification. Bioluminescence signals were normalized with values obtained from day 0. Histological analyses of the lungs recovered in the necropsy on day 32 were performed following fixation in 4% paraformaldehyde/phosphate-buffered saline. Paraffin sections were prepared for hematoxylin and eosin staining and Ki-67 immunohistochemistry. Aperio ImageScope software (Leica Biosystems) was used for histological visualization of the lungs in order to evaluate the presence of microscopic metastases.

2.6. Clinical chemistry and hematology analysis

Blood samples of mice were collected in EDTA surface-coated tubes on day 32. Quantification of liver enzymes, creatinine and urea in blood plasma was performed using a Roche Cobas c311analyzer. Blood counting analysis was performed using a Sysmex XT-1800i (Roche Diagnostics).

2.7. Statistical analysis Statistical analysis was performed using GraphPad Prism software (San Diego, CA, USA). Statistical comparison between different groups was performed using an ANOVA One-Way with Tukey post-hoc correction. Data were reported as mean ± s.e.m./SD and statistical significance levels were defined as *P <0.05.

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3. RESULTS

3.1. SQ-Gem/EF NAs displayed better anticancer activity in c-Fos overexpressing OS cells P1.15 over p53+/- OSs

Prior to the in vitro efficacy tests, SQ-Gem NAs and SQ-Gem/EF NAs were characterized (Table 1). SQ-Gem NAs mean particle diameter was measured to be around 116 nm with a unimodal size distribution (PDI: 0.09), zeta potential of −17 mV and drug content of 40.7 %. The particle population of the SQ-Gem/EF NAs was also very uniform, with a mean particle size diameter of 50 nm, and a low PDI of 0.12. The full co-assembly of EF into SQ-Gem also decreased the particle surface charge to -12 mV and incremented the total drug content up to 67.28 % (44% for EF and 22% for Gem). Table 1

Colloidal characteristics of squalenoyl-gemcitabine/edelfosine nanoassemblies (SQ-Gem/EF NAs) and SQ-Gem NAs

NAs size (nm) PDI ζ (mv)1 % Gem2 % EF2 % total2

SQ-Gem 116 ± 10 0.09 ± 0.01 -17 ±1 40.7 0 40.7

SQ-Gem/EF 50 ± 4 0.12 ± 0.03 -12 ± 1 22.49 44.73 67.28

1Zeta potential. 2 %= MW drug/MW nanocomposites.

Two OS cell lines were screened to measure the anticancer activity of the free drugs (i.e. Gem and EF) and their nanoparticulate counterparts. In both cell lines, IC50 values were very similar, although free Gem (1.7 and 2.5 µM) was shown to be strongly active over free EF (59 and 64 µM) in P1.15 and p53+/- OSs, respectively (Fig. 1A). Squalenoyl NAs were 5-12 times less active than Gem free due to the “pro-drug” effect of SQ-Gem at these in vitro conditions and given the low anticancer effects of EF at those concentrations. In fact, the anticancer improvement of SQ-Gem/EF NAs over SQ-Gem NAs was only slight, as observed in Fig. 1B. Notwithstanding, SQ-Gem/EF NAs exhibited a clearly ameliorated antitumor profile in P1.15 cells over p53+/- OSs (Fig. 1B, continued lines vs dotted lines), displaying IC50 values of 11 and 29 µM, respectively.

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Figure 1. In vitro cytotoxicity representative curves of (A) free gemcitabine (Gem) versus free edelfosine (EF) and (B) squalenoyl-gemcitabine nanoassemblies (SQ-Gem NAs) versus SQ-Gem/EF NAs treatments +/- tested on p53 OS and P1.15 cells. IC50 values are the mean ± SD of at least three independent determinations.

3.2. SQ-Gem/EF NAs drastically diminished the exposure of the total Gem pool in the blood stream in comparison with SQ-Gem NAs SQ-Gem/EF NAs and SQ-Gem NAs were injected via retro-orbital plexus in healthy mice to compare their pharmacokinetic profile. Plasmatic concentration-time curves depicted in Fig. 2 showed that the co-assembly between EF and SQ-Gem (blue curve) modified the pharmacokinetic behavior of SQ-Gem NAs only (red curve). AUC0-inf for SQ-Gem from the SQ-Gem NAs treated group was 2.25-fold higher in comparison with SQ-Gem from SQ-Gem/EF NAs (88 versus 39 μg·h/mL) and correlated inversely with a total systemic clearance of 462 instead of 948 mL.kg/h. In addition, Gem released from SQ- Gem NAs displayed a higher cleavage during the first 4 h (grey dotted line versus pink dotted line). However, its AUC0-inf was not much higher (21 versus 17 μg·h/mL) because the presence of Gem from SQ-Gem/EF NAs was prolonged up to 8 h. Other relevant pharmacokinetic parameters are listed in Table 2, including those for EF from SQ-Gem/EF NAs.

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Figure 2. Plasma concentrations of gemcitabine (Gem), squalenoyl-gemcitabine (SQ-Gem) and edelfosine (EF) after a single administration via retro-orbital plexus of SQ-gem/EF nanoassemblies (NAs) or SQ-Gem NAs (58 μmol/kg at a ratio 1:1), as a function of time post-injection. The values are the mean ± SD (n= 3).

Table 2 Comparison of pharmacokinetic parameters of squalenoyl-gemcitabine (SQ-Gem), gemcitabine (Gem) and edelfosine (EF) after the administration via retro-orbital plexus of SQ-Gem nanoassemblies (NAs) or SQ- Gem/EF NAs (58 μmol/kg at a ratio 1:1). The values are the mean ± SD (n= 3) SQ-Gem NAs SQ-Gem/EF NAs Parameter SQ-Gem Gem SQ-Gem Gem EF t1/2 (h) 1.78 ± 0.18 4.49 ± 0.47 2.37 ± 1.44 5 ± 0.6 30 ± 10 Cmax (μg/mL) 49 ± 13 3.3 ± 0.1 79 ± 10 3.65 ± 1.34 42 ± 6 Cl_obs (mL.kg/h) 462 ± 159 689 ± 47 948± 86 856 ± 18 86 ± 7 Vss_obs (mL/kg) 902 ± 280 4526 ± 165 1265 ± 694 7334 ± 1073 3534 ± 788 AUC 0-inf_obs 88 ± 34 21 ± 1 39 ± 4 17 ± 0.38 346 ± 30 (μg·h/mL) t1/2, terminal elimination half-life; Cmax, maximum observed plasma concentration; Cl_obs, systemic clearance; Vss, volume of distribution at steady-state; AUC 0-inf_obs, Area Under the curve from 0 extrapolated to infinite time.

3.3. SQ-Gem/EF NAs delayed the progression of the primary OS tumor growth in a P1.15 orthotopic model

The therapeutic benefit of SQ-Gem/EF NAs was evaluated in a P1.15 orthotopic OS tumor model. Fig. 3A illustrates the growth of the primary bone tumor as hindlimb bioluminescence signal during the course of the study for SQ-Gem/EF NAs, free Gem and Dex 5% (control group). SQ-Gem NAs were found to be sub-lethal after the second administration, meaning an endpoint for this treatment group. Free Gem and SQ-Gem/EF NAs started to show tumor inhibitory effects 13 days after tumor cell inoculation. Representative hindlimb bioluminescence images of the different treatment groups can be observed in Fig. 3B. At day 27, free Gem inhibited the tumor growth, expressed as mean bioluminescence signal value, by around 43 %. This result was not significant compared to the control group due to the high variability encountered in these groups.

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Notwithstanding, the tumor growth was significantly delayed by around 60% (P* <0.05) in mice treated with SQ-Gem/EF NAs (Fig. 3C).

Figure 3. Tumor growth inhibition after multidrug treatment of mice bearing osteosarcoma (OS) cells. All groups received eight intravenous injections in the retro-orbital plexus of either squalenoyl- gemcitabine/edelfosine nanoassemblies (SQ-Gem/EF NAs), free gemcitabine (Gem) or a dextrose 5% solution (Dex 5%) at the dose of 38 μmol/kg at a ratio 1:1. (A) Hindlimb bioluminiscence was regularly measured during the experimental period. The values are the mean ± SD (n= 7) normalized at day 0. (B) Representative bioluminescence images of OS bearing mice 27 days after the intratibial inoculation of P1.15 cells and (C) Hindlimb bioluminiscence on day 27 normalized at day 0. (n= 7, data= mean ± s.e.m., *P <0.05).

3.4. SQ-Gem/EF NAs caused regression of lung metastases of P1.15 OS bearing mice At the end point of the experiment, the presence of metastases in the lungs was further examined by Ki-67 (Fig. 4A) and hematoxylin and eosin staining (Fig. 4B). It was observed that the control group presented more than one nodule per mouse (black arrows) and that some of these macroscopic nodules already presented necrotic tissue (red arrows). As seen in Fig. 4C, six out of seven mice in the control group developed multiple lung metastases. SQ-Gem/EF NAs and free Gem evidenced a major anti-metastatic potential against OS. In that sense, only two and three out of seven mice presented a single metastatic nodule in the SQ-Gem/EF NAs and Free Gem treatment groups, respectively.

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Figure 4. Representative photomicrographs of (A) Ki-67-stained and (B) hematoxylin and eosin-stained histological sections of lungs of a non-treated tumor-bearing mouse, 32 days after the intratibial injection of P1.15 osteosarcoma (OS) cells. (C) Quantification of the number of total metastatic nodules and the percentage of mice presenting macroscopic lung metastases treated with Dextrose 5% (Dex 5%), free gemcitabine (Gem) and squalenoyl-Gem/edelfosine nanoassemblies (SQ-Gem/EF NAs). (D) Body weight loss: (i) Dex 5%, (ii) Gem free, (iii) SQ-Gem/EF NAs. Animals were weighted twice per week in order to detect any weight loss associated with the chemotherapeutic treatment. The values are the mean ± SD (n= 7). Statistical analysis on day 32 was performed using one-way analysis of variance considering 95% confidence interval at significance level P* <0.05.

3.5. SQ-Gem/EF NAs showed a suitable safety profile in vivo after eight administrations SQ-Gem/EF NAs administration in mice at the therapeutic dose of 38 μmol/kg at a molar ratio 1:1 was found to be safe at least up to eight administrations. No weight loss was observed in any of the treatments (Fig. 4D), albeit the mean gain weight of mice receiving free Gem was slowed down from day 21. At day 32, this weight difference reached around 7%, being statistically significant in comparison with the non-treated and SQ-Gem/EF NAs groups (P* <0.05).

As hepatotoxicity and renal damage underlies the clinically reported restrictions to the use of Gem, the contribution of SQ-Gem/EF NAs to inducing these toxicities was evaluated. Thus, Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), creatinine and urea plasmatic parameters were measured (Table 3). These levels were not noticeably higher or significantly different among treatments, indicating the absence of renal or hepatic disruption at the end of the experiment. Only the creatinine levels in the group of free Gem were observed to be lower compared to the non-treated or SQ-Gem/EF NAs group (0.063 versus 0.093 and 0.092 mg/100 mL, respectively). On the other hand, hematological analysis showed a low blood cell count associated with the chemotherapeutic treatment, except for the platelet count. As seen in Table 3, no considerable differences in the white and red blood cell counts were observed among the free Gem and SQ-Gem/EF NAs groups. Although EF is a highly hemolytic compound, the red blood cell count corresponding to the SQ-Gem/EF NAs treatment was found to

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be equal to that of free Gem treatment (7.93 versus 7.94 106/µL), and correlated with hemoglobin concentrations of 12.93 and 13.41 g/dL, respectively. Moreover, the number of neutrophils encountered in mice treated with SQ-Gem/EF NAs was slightly augmented in comparison with mice treated with free Gem (0.70 versus 0.59 103/µL, respectively). Table 3

Evaluation of blood and plasma parameters in osteosarcoma (OS) bearing mice on day 32 after eight retro- orbital plexus injections of either dextrose 5% solution (Dex 5%), free gemcitabine (Gem) or squalenoyl- Gem/edelfosine nanoassemblies (SQ-Gem/EF NAs) at the dose of 38 μmol/kg at a molar ratio 1:1

Parameter Units Dex 5% Gem free SQ-Gem/EF NAs Plasma ALT (UI/L) 20.3 24.2 17.8 AST (UI/L) 202.8 123.4 134.8 Creatinine (mg/100 mL) 0.093 0.063 0.092 Urea (mg/100 mL) 42.1 45.6 37.2 Blood WBC [103/µL] 3.30 1.79 1.74 NEUT [103/µL] 1.65 0.59 0.70 NEUT (%) 54.00 34.53 38.99 RBC [106/µL] 8.82 7.93 7.94 HGB [g/dL] 14.41 12.93 13.41 PLT [103/µL] 604.33 753.5 710.85 The results are expressed as mean, n= 7 mice.

4. DISCUSSION The outlook for children and adolescents with bone sarcomas has not progressed throughout the three past decades. The situation is still discouraging in high-risk patients, who are normally prone to tumor relapses and metastasis [26]. In this paper we shed light on the realistic opportunities offered by a combinatorial nanomedicine made of SQ-Gem and EF for improving current therapies and thus, the quality of life of these patients. In vitro, free Gem and EF treatments each displayed a similar anti-cancer activity in P1.15 and p53+/- OS cells. Gem was in the micromolar range when it is normally nanomolar in other OS cells [27]. This indicated that the cell lines tested still retain drug resistance mechanisms as a consequence of accumulating few oncogenic mutations or chromosomic alterations. Surprisingly, the anticancer activity of SQ-Gem/EF NAs was found to be 2.6 times greater in P1.15 cells than in p53+/- OSs (Fig. 1B) and hence, P1.15 cells were selected for the subsequent in vivo experiments. The explanation for this boosted effect could reside in how the biology of cells affects the SQ-Gem “pro-drug” effect in vitro. c- Fos overexpression in P1.15 osteoblasts is responsible for the development of 100% penetrance skeletal neoplasm. This proto-oncogen is related to the upregulation of fibroblast growth factor receptors, among other tumorigenic mechanisms, which involves an accelerated S-phase entry and a high propensity to migrate and invade other tissues [28,29]. This could lead to an enhanced hydrolization/release of Gem from SQ-Gem and

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a subsequent increased vulnerability to its cytostatic action in these cells in comparison with the p53+/- OSs. SQ-Gem/EF NAs injected via retro-orbital plexus behaved comparably to their i.v. administration via tail vein (see chapter II), demonstrating the suitability of this parenteral route for both free drugs [30] and nanocomposites. The resulting higher systemic clearance of SQ-Gem triggered by the insertion of EF in the NAs led to better tolerance and safety of the SQ-Gem/EF NAs. However, at this dosage we surpassed the maximum tolerated dose (MTD) for SQ-Gem NAs. Around 90% of the mice presented subacute toxicity and died after the second administration. We have to bear in mind that the reported MTD of free Gem in mice is approximately 5 times higher than its equivalent dose of SQ-Gem NAs [31]. This subacute toxicity was thought to be mainly associated with the myelosuppression/hemotoxicity triggered by the long-term exposure of the total Gem pool (SQ-Gem + released Gem) in the blood stream (Fig. 2), in agreement with pre- clinical toxicology data [31–33]. Maksimenko et al. administered 4 administrations of SQ-Gem NAs at 13 mg/kg in athymic nude mice that would correspond to a dose of 5.25 mg/kg of Gem free [34]. However, in our experimental conditions this chemotherapeutic regimen may point to the risk of underdosing, if we take into account that eight administrations of Gem at 10 mg/kg were not very effective in delaying the primary tumor growth. Furthermore, the co-assembly between SQ-Gem and EF is unavoidably equimolecular in our nanoformulation. This made it mandatory to increase the dosage as much as possible (i.e. 20 mg/kg for EF) in order to reach effective concentrations, along the lines determined in previous experiments [35]. Free EF treatment was not included in the in vivo efficacy studies for ethical reasons due to its high hemotoxicity. Therefore, only free Gem treatment could be eligible as the standard treatment control group. This cytostatic drug was considerably more active in vitro than SQ-Gem/EF NAs; notwithstanding, SQ-Gem/EF NAs were more effective at detaining primary tumor progression than free Gem in vivo (Fig. 3). In our study, hindlimb bioluminescence differences were not so marked between groups and hampered the observation of greater antitumor effects resulting from the action of EF or SQ-Gem. This was thought to be likely due to the lower bioluminescence signal of the necrotic inner parts of advanced tumors. On the other hand, metastases in lungs at diagnosis or especially during chemotherapy are one of the worst prognostic factors in pediatric OS [36]. SQ-Gem/EF NAs and free Gem treatments reduced the occurrence of lung metastases in mice from 80% down to 28-42% in favor of the NAs (Fig. 4C). This meant that SQ-Gem/EF NAs were also effective at suppressing the metastatic spread of cancer circulating cells from the primary tumor. Clinical chemistry performed at the end of the in vivo efficacy studies confirmed that SQ-Gem/EF NAs were safe and did not cause any apparent renal or hepatic damage (Table 3). Hematology analysis revealed a common dose/limiting myelosuppression associated with Gem treatment in the clinic [37]. Very importantly, the red blood cell count with the NAs was equal to that of the free Gem group and not much lower than the

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non-treated group. This confirms the low hemolytic profile of SQ-Gem/EF NAs previously reported in vitro [23], suggesting that this strategy holds promise for the parenteral delivery of EF. Moreover, only the free Gem treatment group showed a slowdown in the weight gain of mice from day 21 (Fig. 4D). Creatinine plasma levels were also lower in this group, suggesting that mice may suffer malnutrition, gastrointestinal adverse reactions or diminished metabolic activity, probably caused by the repeated administrations of Gem [38]. The vast majority of nanotechnology approaches for OS treatment to date rely on the anthracycline doxorubicin as a chemotherapeutic drug model [39]. Only two recent papers report the successful encapsulation of Gem in magnetite nanoparticles [40] and liposomes [41]. In the latter case, and encouraged by the suitable results of the combination of Gem/docetaxel in the clinic for high-grade OS [15], Caliskan et al. also proposed the combination of Gem with another antitumor agent that is not a DNA disrupter per se like EF. This group co-loaded Gem with clofazimine in liposomes and determined their improved anticancer activity in comparison to their individual use against OS cells in vitro. However, to our knowledge this is the first time in which a Gem- based nanoformulation (i.e. SQ-Gem/EF NAs) has proved to have anti-OS potential in vivo. An important fact that reinforces this combinatorial nanomedicine approach is that EF is not an antimetabolite or an alkylating agent, which is crucial for the quality of life of pediatric patients. It would be interesting in the future to perform these experiments in a cancer cell line that is more sensitive to the action of EF. For instance, orally administered EF loaded into solid lipid nanoparticles proved to be very efficacious in an ® HOS (ATCC CRL-1543™) orthothopic tumor model [42] but the IC50 of EF in this cell line was 2.72 µM compared to 59 µM in P1.15 cells.

5. CONCLUSIONS The findings reported in this study allow us to postulate the use of SQ-Gem/EF NAs as a reliable alternative to conventional chemotherapy for high-risk OS patients. This combinatorial nanomedicine exhibited a better in vitro cytotoxicity profile against osteoblastic cells overexpressing c-Fos/AP-1 oncogene than in p53 deficient OS cells. Pharmacokinetic studies revealed that the insertion of EF into SQ-Gem drastically modified the exposure of SQ-Gem in the blood stream, which led to better tolerability of the NAs after repeated i.v. administrations. Despite the reduction of the total Gem pool and the worse anticancer profile in vitro over free Gem, SQ-Gem/EF NAs were able to slow the progression of the primary tumor growth in P1.15 orthotopic OS mice in the absence of concomitant adverse effects. Moreover, SQ-Gem/EF NAs diminished the metastatic spread of P1.15 OS cells from the primary tumor to the lungs at a similar rate to free Gem. Further experiments are required to determine the optimal dose schedule of SQ-Gem/EF NAs to better characterize the anticancer potential of EF i.v. administered by means of the use of nanotechnology. In that sense, it would be of interest to test this combinatorial nanomedicine in other tumor models more sensitive to the action of EF.

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ACKNOWLEDGEMENTS Asociación Española Contra el Cáncer (AECC) (CI14142069BLAN) and Fundación Caja Navarra/Obra Social La Caixa are gratefully acknowledged. We would like to gratefully thank the Asociación de familiares y amigos de pacientes con neuroblastoma (NEN, Nico contra el cáncer) for the grants received.

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[17] J.L. Arias, L.H. Reddy, M. Othman, B. Gillet, D. Desmaële, F. Zouhiri, F. Dosio, R. Gref, P. Couvreur, Squalene based nanocomposites: a new platform for the design of multifunctional pharmaceutical theragnostics., ACS Nano. 5 (2011) 1513–21. [18] P. Couvreur, B. Stella, L.H. Reddy, H. Hillaireau, C. Dubernet, D. Desmaële, S. Lepêtre-Mouelhi, F. Rocco, N. Dereuddre-Bosquet, P. Clayette, V. Rosilio, V. Marsaud, J.-M. Renoir, L. Cattel, Squalenoyl nanomedicines as potential therapeutics., Nano Lett. 6 (2006) 2544–8. [19] M. Briglia, A. Fazio, E. Signoretto, C. Faggio, F. Lang, Edelfosine Induced Suicidal Death of Human Erythrocytes, Cell. Physiol. Biochem. 37 (2015) 2221– 2230. [20] W.E. Berdel, U. Fink, J. Rastetter, Clinical phase I pilot study of the alkyl lysophospholipid derivative ET-18-OCH3 1, Lipids. 22 (1987) 967–969. [21] S.F. Teixeira, C.P. Rodrigues, C.J.S. Costa, T.N. Pettinati, R.A. de Azevedo, L.I. Mambelli, S.D. Jorge, R.N. Ramos, E.S. Ferro, J.A.M. Barbuto, A.K. Ferreira, Edelfosine: an antitumor drug prototype, Anticancer. Agents Med. Chem. 18 (2018) 865–874. [22] C. Gajate, F. Mollinedo, Lipid Rafts, Endoplasmic reticulum and mitochondria in the antitumor action of the alkylphospholipid analog edelfosine, Anticancer. Agents Med. Chem. 14 (2014) 509–527.

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GENERAL DISCUSSION

On 17th November of 2020, we will celebrate 25 years of cancer nanomedicine following the commercialization of Doxil® (liposomal-doxorubicin) by the Food & Drug Administration (FDA) for the treatment of AIDS-related Kaposi’s sarcoma [1]. This occurrence gave rise to the subsequent incorporation over the course of these decades of other anti-cancer nanomedicines such as Abraxane® (albumin-bound paclitaxel), Marqibo® (vincristine-loaded liposomes) or DepoCyt® (cytarabine-loaded liposomes) [2]. Nevertheless, the subsequent overoptimism generated about the opportunities that cancer nanomedicine offers for effecting “cures” has been deflated in the last few years. The current skepticism mainly comes from the clinical outcomes concerning the active or passive targeting of nanoparticles. The well-known and controversial EPR (enhanced permeability and retention) effect of nanoparticles is now understood to be limited to only a few solid tumors [3]. Moreover, despite the great advances conducted in active targeting, nanoparticle surface engineering with antibodies or other moieties is not yet producing outstanding benefits [4,5]. So why are there nanomedicines in the market? Terms like toxicity, safety or tolerance may hold the answer to this question. The vehiculization of highly toxic chemotherapeutic agents in nanosystems was made possible on the basis of the nanosized suspensions concept in the early 1960s [6]. Since then, the nano-encapsulation of these drugs has been shown to improve their therapeutic index, thereby reducing chemotherapeutic associated toxicities [2,7]. For example, PEGylated doxorubicin liposomes are reported to diminish the presence of doxorubicin in the cardiac tissue, bypassing the characteristic cardiotoxicity associated with this anthracycline [8]. For all these reasons, our attention was drawn to the fact that there were no nanomedicines for chemotherapeutic drug delivery approved for use in the pediatric population. As underlined in the Introduction to this thesis, this population is more vulnerable to the cytotoxic action of these drugs, as revealed by recent prospective studies in survivors [9]. Only a few nanomedicines have reached phase III clinical trials in this context. Liposomal muramyl tripeptide phosphatidylethanolamine (Mepact®) heads a solitary list of immunomodulatory nanomedicines approved for pediatric cancer management [10]. All these facts call for the design of a new nanomedicine for implementation in pediatric cancers, and more concretely in pediatric osteosarcoma (OS). This musculo-skeletal tumor has a low incidence, but is one of the most lethal cancers in children and adolescents [11], owing to its heterogeneous behavior and high metastatic dissemination rate from the primary tumor (e.g., long bones) to the lungs [12,13].

In the present project we developed a combinatorial nanomedicine based on the unique co-assembly of squalenoyl-gemcitabine (SQ-Gem) and edelfosine (EF) for the treatment of pediatric cancers such as OS. This approach originates from a close collaboration between the Universidad de Navarra and the University Paris-Sud. Briefly, the previously

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described chapters outline linearly the design and physico-chemical characterization of SQ-Gem/EF NAs [14] (Chapter I); their in vitro testing in tumor cells and pharmacokinetic analysis (Chapter II); and finally the in vivo efficacy studies in OS tumor bearing mice (Chapter III).

Among all the possibilities in the field of nanotechnology, combinatorial nanomedicine has recently been taking on considerable importance [15–17]. The main goals of Chapter I were the design, formulation, and physico-chemical characterization of a multidrug nanomedicine made of SQ-Gem and EF. The nucleoside analogue gemcitabine (Gem) is given as a second line treatment in pediatric OS. Although it displays powerful antitumor activity, its half-life is very low [18]. Its linkage to squalenic acid (SQ-COOH) is reported to increase the therapeutic index, being considerably more efficacious in vivo [19]. Thus, it is convenient to understand first the “squalenization technology”, a concept developed by the group of Prof. Patrick Couvreur over these years. This is based on the chemical linking of drugs to a squalene chain, resulting in the formation of amphiphilic pro-drugs able to form nanoassemblies (NAs) spontaneously. Since 2006, this strategy has proved optimal for the nanodelivery of multiple drugs ranging from nucleoside analogues to opioids, leading to publications in high impact factor journals [20–22] and the Best European Inventor Award in 2013 (Fig. 1).

Figure 1. Historical time line of milestones achieved in “squalenization technology”.

On the other hand, we selected EF, an alkyl-lysophospholipid in which the group led by Dra. María J. Blanco had extensive experience [23,24]. Although EF has proven anticancer activity in several tumors, its high hemolytic and gastrointestinal toxicity has

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frustrated its consolidation in the clinic, making its encapsulation in drug delivery systems mandatory [25,26]. At this point, the first issue with the strategy proposed arose, since EF does not display any functional group in its molecular structure, unlike for instance, the amine moiety of Gem, which prevents its covalent linkage to SQ-COOH. Nonetheless, EF is an alkyl-lysophopholipid and possesses an amphiphilic structure similar to SQ-Gem per se (Fig. 2). Therefore, it was hypothesized that the intermolecular interactions between both compounds could be adequate for the construction of the nanocomposite.

Figure 2. Molecular structures of squalenoyl-gemcitabine (SQ-Gem) and edelfosine (EF).

The first step relied on the chemical synthesis of SQ-Gem that was performed in the Institut Galien Department (University of Paris-Sud) under the supervision of Dr. Didier Desmaële by the middle of 2016. The limiting factor here was not the coupling of Gem to SQ-COOH but the previous obtainment of SQ-COOH by means of a tedious multi-step synthesis from squalene, a natural lipid originating from shark liver and some vegetable oils [27]. The nanoprecipitation method was chosen on the basis of our previous experiments, taking advantage of the fact that both compounds were soluble in ethanol [28]. One concern here was that EF could form micelles by itself when collapsing in the aqueous phase, and not interact with SQ-Gem. Interestingly, an inverse correlation was observed between the increase of EF in the formulation and the mean particle size shrinking of SQ- Gem NAs. This caused the appearance of another peak alongside the peak at 120 nm corresponding to SQ-Gem NAs. These particle changes were tracked in terms of particle population homogeneity. After several attempts, it was concluded that at equimolar concentrations, the two population peaks merged into one with an optimal polydispersity index (PDI). Finally, the mean particle size of SQ-Gem NAs was reduced approximately from 120 to 50 nm, corresponding to the de novo formation of SQ-gem/EF NAs. In Chapter I we also worked in close cooperation with the Instituto de Nanociencia de Aragón and especially with Dr. Victor Sebastián, who kindly contributed in the transmission electron microscopy (TEM) section. The characteristic inverse hexagonal supramolecular structure of SQ-Gem NAs was easily visualized in agreement with previous experiments [29]; however, for the morphology analysis of nanocomposites

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around 20-70 nm (i.e., SQ-Gem/EF NAs), it was more difficult to obtain a suitable resolution. Fortunately, we had the chance of using a high resolution TEM that is normally too aggressive for organic samples. We were able to observe a multilamellar structure displaying concentric layers with a determined periodicity, which finally confirmed the conformation of a new type of nanoconstruct. At first, it was thought that this supramolecular organization could be amorphous as in previous studies. In one such case, the complementary compound was lipophilic [30] but not amphiphilic like EF. This, together with similar molecular weights (524 and 646 g.mol-1 for EF and SQ-Gem, respectively) and length of their carbonated chains, may explain the unique supramolecular organization of their co-assembled molecules. The literature does not input abundant evidences about any therapeutic benefit related to this particle size reduction (e.g., from 120 to 50 nm), although the trend in nanotechnology research nowadays is “the smaller the better” [31]. In fact, the International Organization for Standardization (ISO) and the regulatory agencies officially consider that the term “nanoparticle” should be used if the particle’s 3 dimensions are less than 100 nm/0.1 µm [32], which is strictly the case of SQ-Gem/EF NAs. So what improvements could these modifications provide from a physico-chemical point of view? In this study, we demonstrated that whereas SQ-Gem NAs start to lose their colloidal properties after 5-7 days, SQ-Gem/EF NAs were very stable, and did not suffer any size change for at least 2 months. This indicated that the EF may act as a complementary antitumor drug and as an excipient at the same time, which is not very usual in the pharmaceutical field. On the other hand, we wondered if the squalenoyl compound SQ-Gem was a suitable scaffold for EF drug delivery. Given that hemolysis was one of the most limiting factors for the therapeutic use of EF and very easy to measure in vitro, we performed hemolysis studies using human blood erythrocytes. As seen in Fig. 3, SQ-Gem/EF NAs protected EF from hemolysis, confirming a strong interaction between SQ-Gem and EF that even prevented a burst release of EF from the surface of the NAs. At the end of this chapter, in vitro viability tests performed in patient treated- derived metastatic pediatric OS cells coded as 531M (Clínica Universidad de Navarra) determined that the insertion of EF into the SQ-Gem NAs was not detrimental to the inherent anticancer activity of SQ-Gem NAs (the experiments performed in cells will be discussed in depth below).

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Figure 3. Hemolysis experiments performed after the incubation of human blood erythrocytes with PBS (negative control), squalenoyl-gemcitabine/edelfosine nanoassemblies (SQ-Gem/EF NAs), free EF (10 μg/mL) and Tryton X-100 (positive control) for one hour at 25ºC.

The ease, reproducibility and robustness of this approach makes it preferable to other kinds of conventional formulations that have been attempted in the past to achieve the encapsulation of Gem or EF (most which involved liposomes or lipid nanoparticles [33– 36]). With SQ-Gem/EF NAs we avoid the necessity of using toxic organic solvents, surfactants, polymers or any other extra compound in the formulation. In that sense, the scalability of nanomedicines represents a crucial key point. On many occasions, we encounter in the literature complex nanoformulations exhibiting outstanding therapeutic potential that are very difficult to scale up [4,37,38]. This makes them more vulnerable than classic formulations from a translational point of view, accelerating their relegation to the so-called “death valley”. In our case, our combinatorial nanomedicine is exclusively made up of antitumor agents, a fact that could make it more attractive to regulatory agencies, facilitating its progression to clinical stages.

Although the physico-chemical advantages of SQ-Gem/EF NAs over SQ-Gem NAs were demonstrated in Chapter I, there remained many aspects to elucidate before moving on to consider murine tumor models. For that reason, the main goals of Chapter II were the in vitro testing of the NAs in pediatric tumor cell lines as well as their pharmacokinetic analysis in vivo. In the previous chapter, high resistance was reported in the patient treated-derived metastatic pediatric OS 531M cells to either Gem (IC50 ≥36 μM) or EF (IC50 =69 μM) treatments. This resistance is normally associated with the development of the Multi Drug Resistance gene (MDR-1) and pertinent P-glycoprotein overexpression (an ATP-dependent drug efflux pump) [39]. Considering that SQ-Gem NAs are not able to internalize into cells owing to their supramolecular structure [40], it was decided to verify the uptake of SQ-Gem/EF NAs by this resistant strain. Interestingly, we determined by two techniques (i.e., flow cytometry and confocal microscopy) that the fluorescently labeled SQ-Gem/EF NAs were able to penetrate progressively in these cells. The efficient

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internalization of SQ-Gem/EF NAs by resistant tumor cells is sometimes considered to be a key prerequisite for successful drug delivery but it can prove controversial in terms of macrophage uptake [41]. One of the limitations of this formulation resides in the vast pharmacological differences existing between EF, whose IC50 is normally in the micromolar range, with respect to Gem, which is normally nanomolar. In addition, the combinatory index (CI) of these drugs resulted only in a slight synergy. Moreover, we have to take into account that the co-assembly of SQ-Gem and EF is imperiously equimolecular so that the combinatory ratios used with the free drugs were impossible to attain with the NAs. It is important to recall here that 531M cells were removed from the following experiments in vitro given that Gem was not able to kill cells beyond the threshold of 50 % of cell viability. Thus, we decided to include in the in vitro experiments in Chapter II commercial cell lines of neuroblastoma (NB) (SH-SY5Y) and OS (U-2 OS). NB is a solid tumor that is almost exclusive to the pediatric population, which originates from the peripheral neural system [42]. In Annex I we compile the latest therapeutic advances in the management of this highly lethal and aggressive tumor, including the therapeutic opportunities of nanotechnology [43]. This work was conducted in collaboration with Dra. Rosa Noguera (INCLIVA Instituto de Investigación Sanitaria, Valencia) and was endorsed by the grants given by the Asociación de familiares y amigos de pacientes de neuroblastoma (NEN, Nico contra el Cáncer). Similar to what happened with the 531M cells, the IC50 improvement of SQ-Gem/EF NAs versus SQ-Gem in these cells turned out to be only slight (58 to 77 nM and 117 to 126 in SH-SY5Y and U-2 OS cells, respectively). This time, we also included the control SQ-Gem NAs + free EF treatment group, which surprisingly behaved even worse than the SQ-Gem NAs. This could be related to the colloidal impairment of SQ-Gem NAs by the physical mixture of EF. This effect was afterwards observed by dynamic light scattering (DLS), when EF was added directly into a nanosuspension of SQ-Gem NAs. Of note, the IC50 reduction of SQ-Gem/EF NAs over SQ-Gem NAs was statistically significant in SH-SY 5Y cells. Here the EF IC50 was 14 times lower than in U-2 OS, and thus closer to nanomolar concentrations of SQ-Gem. The anticancer mechanisms of EF in NB cells should be further studied with the aim of gaining more knowledge about the sensitivity of particular cancer cells to the action of EF. In line with this, the EF analog perifosine, which also triggers tumor cell apoptosis through the PI3K/Akt/mTOR pathway, among other apoptotic routes, is starting to be given as an experimental or salvage therapy in NB [44]. In the last section of Chapter II we investigated an important issue that would change the therapeutic perspectives of SQ-Gem/EF NAs in vivo: their pharmacokinetic profile. First, we encountered an unexpected experimental challenge in regards to the simultaneous quantification in plasma of Gem, SQ-Gem and EF. Their determination in biological samples rendered unserviceable the UHPLC/MS/MS methodology set up in Chapter I. The reasons were chiefly associated with: (i) differences in polarity/solubility between Gem, SQ-Gem and EF, (ii) the impossibility of using OSTROS in the case of EF because of its phospholipid nature and (iii) a huge matrix effect of the internal standard

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2’-deoxycytidine and especially of Gem. The novelty here with respect to the literature [45,46] is that acetone was used instead of conventional acetonitrile/methanol mixtures in order to extract and inject 2’-deoxycytidine, Gem and SQ-Gem using only 20 µL of mouse blood plasma. With this methodology, the recoveries obtained for Gem were between 80 and 100 % and the lower limit of quantification (LLOQ) was 1 µg/mL, suggesting a future validation of this method. In order to attain better recoveries and a lower LLOQ in the case of Gem, the C-18 column used in this study could be replaced by one with different polarity. In this way, we could obtain late retention times, and thereby reduce the matrix effect of Gem. Likewise, the UHPLC/MS/MS method for the analysis of platelet-activating factor-16 (internal standard) and EF was also modified to detect EF using 40 µL of mouse plasma instead of the 100 µL used in previous experiments [47]. Pharmacokinetics were performed in athymic nude mice in order to predict the behavior of the NAs in future in vivo tumor models (i.e., the mouse lineage used in Chapter III). Unfortunately, the oral route, which was our previous preferential via of administration for EF [48,49], did not report interesting results as regards the absorption of the SQ-Gem/EF NAs. This was thought to be likely due to the following reasons: (i) SQ-Gem/EF NAs disassemble very fast in the gastro-intestinal tract (ii) SQ-Gem/EF NAs does not penetrate the intestinal mucus/enterocytes, (iii) SQ-Gem is not absorbed or is rapidly hydrolyzed by peptidases or other gastrointestinal enzymes. However, the results i.v. were encouraging. In comparison with free Gem, the AUC of the total Gem pool (SQ- Gem + cleavage Gem) was increased and Gem was released in a sustained manner. In comparison with previous reports about SQ-Gem NAs, this indicated that the co-assembly of SQ-Gem with EF considerably modified the exposure of the total Gem pool in the blood circulation (the therapeutic implications of this phenomenon will be discussed in depth below). On the other hand, the EF concentrations in the blood stream were initially diminished in comparison to the free drug, which was considered better in terms of hemotoxicity. Taking into consideration the above-mentioned results, we could conclude in this chapter that the pharmacological differences encountered in vitro between Gem, SQ-Gem and EF might be compensated in vivo. In vitro, free Gem has been reported many times to be more active than SQ-Gem owing to the “pro-drug” effect (the time needed for the hydrolysis of the pro-drug at in vitro conditions); however, SQ-Gem NAs are more effective in murine models since they preserve Gem from a rapid metabolization by the cytidine deaminase [50,51]. Another important fact is the slower clearance, higher AUC and volume of distribution of EF due to its alkyl-lysophospholipid structure in comparison with SQ-Gem and Gem. We thought that this could also balance its reduced anticancer activity exhibited in vitro and motivate the future testing of SQ-Gem/EF NAs in murine tumor models.

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Chapter III, focuses on the therapeutic potential in vivo of the NAs for the treatment of high-grade OS. We thus continued a previous project by our research group supported by grants from the Asociación Española contra el Cancer (AECC). In our earlier studies, orally administered solid lipid nanoparticles loaded with EF showed a great anti-OS activity in vitro and in vivo [52,53]. Likewise, we proposed a pre-clinical in vivo analysis of the SQ-Gem/EF NAs in an aggressive metastatic model of OS, where chemotherapy - and thus drug delivery nanocarriers - could be crucial [54]. In refractory OS tumors, Gem is given alone or combined with docetaxel (675-1250 mg/m2 in perfusion during 60-90 mins) [55]. We thus suggested another combinatorial nano-therapy in which EF displays specific apoptotic mechanisms different to the characteristic mitotic spindle inhibition of taxoids. The first step in Chapter III was to perform an initial in vitro screening of SQ- Gem/EF NAs in OS cell lines. Interestingly, although the free drugs behaved similarly, the NAs were more efficacious in c-Fos overexpressing cells (P1.15) than in p53 deficient OS cells. The P1.15 cells originated from a murine transgenic OS in which c-Fos/c-Jun AP1 box was genetically modified [56]. This gave these cells high aggressiveness and a great capacity to metastasize to the lungs with a short latency time [57]. These cells were orthotopically inoculated in the tibia of athymic nude mice and treated twice per week for around a month (Fig. 4). The primary tumor growth was measured by bioluminescence imaging since P1.15 cells had been previously modified to express the luciferase gene. This work was carried out in collaboration with the research group led by Dr. Fernando Lecanda (Centro de Investigación Médica Aplicada CIMA, Pamplona).

Figure 4. Schematic in vivo experimental design to evaluate the antitumor efficacy of squalenoyl- gemcitabine/edelfosine nanoassemblies (SQ-Gem/EF NAs) in osteosarcoma (OS) bearing mice.

Against the odds, at the dose of 25 mg/kg, SQ-gem NAs alone proved considerably more toxic than SQ-Gem/EF NAs in athymic nude mice due to their different pharmacokinetic behavior. Thus, the administration of SQ-Gem/EF NAs allowed us to increase the dose above the maximum tolerated dose (MTD) for co-assembled SQ-Gem and demonstrated

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again that EF acts as a modulator/excipient for SQ-Gem. We therefore performed a new pharmacokinetic analysis, this time including SQ-Gem NAs. Our results showed that this toxicity was clearly due to the higher presence of SQ-Gem in the blood stream (a two fold increase in the AUC of SQ-Gem NAs in comparison to SQ-Gem/EF NAs). SQ-Gem NAs killed 90 % of mice after the second administration, reducing our control groups only to the non-treated group and the free Gem group (whose reported MTD was approximately 5 times higher than SQ-Gem [58]). SQ-Gem/EF NAs were more effective than free Gem in delaying the primary tumor growth, as confirmed by bioluminescence imaging (60 % versus 43 %, respectively). Moreover, histological analysis of the lungs revealed that the NAs were also effective in detaining the metastatic spread at a similar rate to free Gem. Importantly for us, the renal, hepatic and hematological toxicity was less than or at least similar to that found with the free Gem. Concerning red blood cell suppression, this is crucial for EF because it opens the possibility of hypothetical i.v. administration with this strategy. In Chapter III we stated the necessity of using the highest doses possible in this experiment, which is the current trend in the clinic [59]. However, with the SQ-Gem NAs offside at this dose and the high hemotoxicity of free EF, it is hard to propose controls to validate this strategy more deeply. Considering the first-order elimination kinetics, and using the formula AUC= dose/clearance, since the AUC of SQ-Gem from SQ-Gem NAs was practically double that of the SQ-Gem/EF NAs, perhaps we could diminish the dose from 25 mg/kg to 12.5 mg/kg to attain a new treatment group of SQ-Gem NAs. Of note, this dosage would match with the dose used by Maksimenko et al. in this type of mouse [30], although they performed only four administrations, compared to eight in our case. On the other hand, we could also consider the inclusion of a treatment group based on the mixture free Gem + SQ-COOH/EF NAs, this time at the same dose. SQ-COOH/EF NAs around 50 nm were formulated in Chapter I for physico-chemical comparison purposes. At the present time, we can conclude that SQ-Gem/EF NAs were safe and effective in an aggressive tumor model of OS. The conundrum as regards to the extent to which they represent an improvement over SQ-Gem NAs is more open-ended and gives rise to some questions such as, for instance, is it worth diminishing the systemic clearance of cytotoxic and myelosuppressor drugs (e.g., Gem and SQ-Gem) when treating a solid tumor? Is it worth avoiding the previously reported forced marriage between SQ-Gem and LDL [60], and thereby remaining more time nanoassembled as SQ-Gem/EF NAs? If the hemotoxicity/myelosupression of SQ-Gem NAs could represent a limitation in the clinic, we are now aware that molecular structures such as alkyl-lysophopholipids (and not phospholipids as used in the past to construct SQ-Gem loaded liposomes [61]) could be of interest in order to modulate the biodistribution of SQ-Gem in the blood compartment when administered i.v.. Squalenoylation technology proves extremely effective by augmenting the half-life of drugs. Its outstanding utility with a view to protecting the microvasculature and preventing ischemic damage has been reported in the case of adenosine, with only 10 seconds of half-life (Fig. 1) [21]. However, in the case of cytostatic compounds, we should consider whether a sustained increase in the drug

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exposure in the blood compartment meets the clinic criteria [18,62,63]. Concerning EF, we should ponder in which tumors the pharmacological combination between SQ-Gem and EF could act best. Recently, a new project started in parallel to test drug delivery systems loaded with EF in NB tumors. At the moment, EF has proven to have great activity in SH-SY5Y cells (Chapter II) but also in aggressive N-Myc amplified SK-N- BE-2 cells.

In the final stretch of this project we proposed a new strategy in order to attain active targeting of our NAs towards the bone by means of bisphosphonates. As detailed in the Introduction, these bone resorption inhibitors display a pyrophosphate structure that confers a high affinity to the natural mineral hydroxyapatite present in the bone [9,64]. By the middle of 2019, in Paris-Sud University we started to synthesize a squalenoyl- alendronate compound with the aim of performing a new co-assembly between this bisphosphonate and SQ-Gem or SQ-Gem/EF NAs. Unfortunately, the coupling of alendronic acid to squalenoyl moieties through its primary amine group was impeded by the low solubility of alendronate (Fig. 5A). It was eventually possible to synthesize a squalenoyl-bisphosphonate (similar to alendronate) (Fig. 5B) by means of a synthesis method devised by Dr. Didier Desmaële. Encouragingly, we now know that this compound is soluble in ethanol and is able to form nanocomposites of around 60-100 nm by nanoprecipitation. These results hold promise for the construction of targeted NAs in order to increase selectively their presence in the bone tumor area.

Figure 5. (A) Alendronate crystals formed in acidic media after incubation with HCL 1N for 24 h were one of the multiple strategies carried out to solubilize the alendronic acid. (B) Molecular structure of the synthesized squalenoyl-bisphosphonate.

Improving the quality of life of cancer patients is essential, particularly in the case of the pediatric population [65–68]. In these studies, we have successfully designed SQ- Gem/EF NAs of 50 nm, which proved to be safe and efficacious in a murine OS model.

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The findings gathered in these chapters shed light on the promising future of combinatory nanomedicine to replace current cancer treatments.

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GENERAL CONCLUSIONS

The experimental work accomplished in this thesis was aimed at the development of squalenoyl-gemcitabine/edelfosine nanoassemblies (SQ-Gem/EF NAs) for the treatment of pediatric osteosarcoma (OS). All the findings gathered during this PhD allow us to conclude the following:

1. The compounds squalenic acid and the pro-drug SQ-Gem were successfully synthetized and purified from the natural lipid squalene. The nanoprecipitation method allowed the formulation of monodisperse squalenic acid and SQ-Gem NAs around 100 and 120 nm, respectively.

2. The co-assembly of SQ-Gem and EF at equimolar concentrations led to the formation of SQ-Gem/EF NAs of 50 nm, with a low polydispersity index, negative zeta potential and a high drug content. Several homogeneous batches of these NAs were formulated by nanoprecipitation without the need for any stabilizing agent, using a simple, robust and reproducible procedure that can be easily scaled up.

3. The insertion of EF into SQ-Gem led to a new supramolecular arrangement, showing a multilamellar concentric conformation. The complete co-encapsulation of EF suppressed its associated hemolytic activity in human blood erythrocytes in vitro. Moreover, EF acted not only as a second antitumor agent but also as a nanoparticle stabilizer, confering SQ-Gem/EF NAs a marked stability improvement over SQ-Gem NAs.

4. Multitherapy of Gem and EF proved to be a profitable combinatory strategy since their combination showed a modest synergy against commercial pediatric cancer cells. SQ-Gem/EF NAs were able to penetrate progressively patient-derived metastatic pediatric OS cells. In addition, SQ-Gem/EF NAs displayed an improved anticancer efficacy over SQ-Gem NAs and the mixture SQ-Gem NAs + free EF in vitro. However, these improvements were only slight due to the lower anticancer effects of EF in comparison with SQ-Gem at equimolar concentrations.

5. A new UHPLC/MS/MS methodology was successfully optimized for the simultaneous quantification of Gem, SQ-Gem and EF in mouse plasma. Pharmacokinetic analysis performed in mice indicated that the oral route was not suitable for the administration of SQ-Gem/EF NAs. However, i.v. administered SQ-Gem/EF NAs decreased plasma concentrations of EF in its initial decay phase, where erythrocytes are more exposed to the toxic action of EF. Moreover, SQ- Gem/EF NAs smoothly prolonged the plasma concentrations of the total Gem pool.

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6. Pharmacokinetic studies after the retro-orbital injection of SQ-Gem/EF NAs in mice also revealed that the insertion of EF into SQ-Gem drastically reduced the exposure of the total Gem pool in the blood stream. This augmented considerably their maximum tolerated dose in comparison with SQ-Gem NAs.

7. In an orthotopic murine model of OS induced by the intratibial injection of c-Fos overexpressing P1.15 cells, SQ-Gem/EF NAs were able to efficiently delay the primary tumor growth kinetics in comparison to free Gem, even though Gem treatment was considerably more efficacious in vitro. Furthermore, SQ-Gem/EF NAs diminished the metastatic spread of P1.15 OS cells from the primary tumor to the lungs at a similar rate to free Gem.

8. SQ-Gem/EF NAs showed a suitable safety profile in vivo after eight i.v. administrations at therapeutic concentrations with no associated weight loss, renal or hepatic damage. In addition, the red blood cell count was equal to that of the free Gem treatment, thereby confirming the low hemolytic profile of SQ-Gem/EF NAs and their suitability for the parenteral delivery of EF.

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CONCLUSIONES GENERALES

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CONCLUSIONES GENERALES

El trabajo experimental recopilado en esta memoria se ha centrado en el desarrollo de nanoensamblados (NAs) de escualeno-gemcitabina/edelfosina (SQ-Gem/EF) para el tratamiento del cáncer pediátrico. Los resultados obtenidos nos permiten concluir:

1. Los compuestos ácido escualénico y el pro-fármaco SQ-Gem fueron sintetizados y purificados con éxito a partir del lípido de origen natural escualeno. El método de nanoprecipitación permitió la formulación de NAs monodispersos de ácido escualénico y SQ-Gem de un tamaño aproximado de 100 y 120 nm, respectivamente.

2. El co-ensamblaje de SQ-Gem y EF a concentraciones equimolares dio lugar a la formación de NAs de SQ-Gem/EF de 50 nm, con un bajo índice de polidispersión, potencial zeta negativo y una carga total de fármacos elevada. Varios lotes homogéneos de estos NAs fueron formulados por el método de nanoprecipitación sin la necesidad de agentes estabilizadores, utilizando un procedimiento sencillo, robusto, reproducible y de fácil escalado.

3. La inserción de la EF en SQ-Gem dio lugar a una nueva conformación supramolecular de tipo multilamelar concéntrica. La encapsulación completa de la EF suprimió su actividad hemolítica in vitro sobre eritrocitos humanos. Por otra parte, la EF además de actuar como agente antitumoral, ejerció un papel estabilizador, ya que confirió a los NAs de SQ-Gem/EF mayor estabilidad en comparación a los NAs de SQ-Gem.

4. La combinación de EF y Gem mostró ser ligeramente sinérgica en líneas celulares tumorales pediátricas comerciales. Por otro lado, los NAs de SQ-Gem/EF fueron capaces de penetrar progresivamente las células de OS pediátricas metastásicas derivadas de paciente. Además, mostraron una mayor eficacia antitumoral sobre los NAs de SQ-Gem y la mezcla física de NAs de SQ-Gem + EF libre in vitro. Sin embargo, esta mayor eficacia fue discreta debido a la menor actividad de la EF en comparación con la de SQ-Gem a concentraciones equimolares.

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5. Se puso a punto y se optimizó con éxito una nueva metodología de UHPLC/MS/MS para la cuantificación simultánea de Gem, SQ-Gem y EF en plasma de ratón. El análisis farmacocinético realizado en ratones indicó que la vía oral no era adecuada para la administración de los NAs de SQ-Gem/EF. Sin embargo, la administración i.v. de NAs de SQ-Gem/EF disminuyó las concentraciones plasmáticas de la EF en su fase inicial de eliminación, donde los eritrocitos están más expuestos a la acción tóxica de este fármaco. Además, los NAs de SQ-Gem/EF prolongaron de manera sostenida las concentraciones plasmáticas totales de Gem.

6. Los estudios farmacocinéticos realizados después de la inyección retro-orbital de NAs de SQ-Gem/EF en ratones corroboraron que la inserción de la EF en SQ- Gem reducía drásticamente la exposición sistémica de la Gem total en el torrente sanguíneo, lo que aumentó considerablemente la máxima dosis tolerada en comparación con los NAs de SQ-Gem.

7. Los NAs de SQ-Gem/EF retrasaron el crecimiento del tumor primario en un modelo murino ortotópico de OS inducido por la inyección intratibial de células P1.15 que sobreexpresan el proto-oncogen c-Fos. Además, estos NAs disminuyeron la diseminación de las células tumorales hacia los pulmones.

8. Tras ocho administraciones vía i.v. de los NAs de SQ-Gem/EF a ratones, estos mostraron un perfil de seguridad adecuado, sin que ello conllevase pérdidas de peso ni daño renal o hepático. Además, el recuento de glóbulos rojos en el grupo que recibió los NAs de SQ-Gem/EF fue el mismo que el grupo tratado con Gem libre.

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