Andreza Rochelle

Vale Morais

Francisco Humberto

Xavier Júnior

Éverton Do

Nascimento Alencar

Philippe M Loiseau

Gillian Barratt

Hatem Fessi

Abdelhamid Elaissari

Sócrates Tabosa Do Egito
email: socratesegito@gmail.com

Nanotecnologia Farmacêutica

E Sócrates Tabosa Do Egito

N Kumar

A H Nandi

S Sardar

S Das

K Kumar

V Pandey

M Ravidas

V N Jha

N Das

C S Verma

D Lal

S Verma

T J Walsh

E J Anaissie

D W Denning

R Herbrecht

D P Kontoyiannis

K A Marr

V A Morrison

B H Segal

W Huang

Z Zhang

X Han

J Tang

J Wang

F Gaboriau

M Cheron

L Leroy

P Wasko

R Luchowski

K Tutaj

W Grudzinski

P Adamkiewicz

W I Gruszecki

Mol Pharm

B Brime

P Frutos

P Bringas

A Nieto

M P Ballesteros

G Frutos

J Antimicrob

Eryvaldo Sócrates
email: socrates@ufrnet.br

Tabosa Do Egito

MICROEMULSION SYSTEM CONTAINING AMPHOTERICIN B FOR LEISHMANIASIS TREATMENT CHAPTER II In-vitro and in-vivo antileishmanial activity of inexpensive Amphotericin B formulations: heated Amphotericin B and Amphotericin B-loaded microemulsion SECTION II FREEZE-DRYING AS STRATEGY FOR IMPROVE EMULSIFIED SYSTEMS STABILITY CHAPTER III Freeze-drying of emulsified systems: A review……………………………. CHAPTER IV Application of design of experiment to optimize the freeze drying process of microemulsion systems

Keywords: Amphotericin B, microemulsion, visceral leishmaniasis, lyophilization drug delivery systems, Leishmania, lyophilization, amphotericin B, colloidal nanocarriers

J'ai appris que l'on mesure le succès non pas par la situation que l'on a atteinte dans la vie, mais par les obstacles que l'on a surmontés pour essayer de réussir. » Booker T. Washington

RESUMO

A leishmaniose visceral é uma doença tropical negligenciada que pode ser fatal se não tratada. A Anfotericina B (AmB) é eficiente no tratamento desta doença, porém o seu elevado custo ou sua alta toxicidade torna necessário o desenvolvimento de novos sistemas terapêuticos para solucionar tais inconvenientes. Nesse contexto, pode-se utilizar o aquecimento da formulação micelar de AmB aquecida (A-AmB), e o desenvolvimento de uma microemulsão (ME) contendo AmB (MEAmB).

Adicionalmente, é desejada a remoção da água desses sistemas microemulsionados a fim de diminuir instabilidades relacionadas à contaminação microbiológica e a hidrólise. Desta forma, o objetivo deste trabalho foi desenvolver e avaliar a atividade e toxicidade in vitro e in vivo da A-AmB e da MEAmB contra Leishamania donovani (LV9), além de otimizar um sistema microemulsionado liofilizado contendo AmB. Caracterizações da reologia, do tamanho e da morfologia da gotícula mostraram que a MEAmB apresentou tamanhos médios de 35 nm, um comportamento Newtoniano e uma morfologia esférica. A caracterização da A-AmB mostrou a formação de superagregados, que, são menos tóxicos que os outros estados de agregação. Análises in vitro, tanto para a forma amastigota axênica como para a intramacrofágica mostraram que as atividades da A-AmB e da MEAmB foram semelhantes ao do Ambisome®. Além disso, foi observado um alto índice de seletividade da A-AmB e da MEAmB comparada a formulação não aquecida. Adicionalmente, essas duas formulações, quando comparadas ao Ambisome®, mostraram elevada atividade contra cepas AmB resistentes. Essas formulações foram testadas in vivo a fim de avaliar sua atividade e toxicidade. Os resultados não apresentaram diferenças significativas entre as atividades das amostras contendo AmB, e, com relação a toxicidade, não mostraram diferenças expressivas capazes de causar uma disfunção renal ou hepática. Portanto, tanto a A-AmB como a MEAmB podem ser usados como alternativa no tratamento contra LV9, apresentando a vantagem sobre o Ambisome® devido aos menores custos de suas produções. Por conseguinte, a fim de liofilizar o sistema microemulsionado, foi realizado um planejamento experimental completo, no qual o observou-se que MEs com menores tamanhos de gotículas foram obtidos quando utilizado maltose como crioprotetor na concentração de 5 %, com congelamento a -80 ºC e por um período de liofilização de 24 h. Além disso, foi observado que a ME contendo AmB não teve mudanças significativas quanto ao conteúdo do fármaco, quando comparado o produto antes e após o processo de liofilização. Desta forma, a ME, em sua forma liofilizada, pode manter a estabilidade do sistema perante os danos que poderiam ser causados pela quantidade de água.

Palavras-chave: Anfotericina B, microemulsão, leishmaniose viceral, liofilização

Université Paris Sud for all support in the in vivo and in vitro experiments of my thesis, as well as for all knowledge that I learned in his lab. I am grateful to the Laboratoire d'Automatique et de Génie des Procédés -Université Lyon 1, under Dr. Hatem Fessi and Dr. Abdelhamid Elaissari coordinations, for help me to improve my expertise in freeze-drying technique.

I would like to thank my laboratory mates from LASID in Brazil, in particular M.Sc. Renata Rutckeviski, M.Sc. Christian Melo and M.Sc. Gutemberg Felipe. Furthermore, I am deeply grateful to my laboratory mates and friends, Dr. Junior Xavier, for all the advising and friendship during these last 10 years, Dr. Éverton Alencar who has always been my best lab mate and best friend for his love, help and friendship.

I am thankful to my former lab mates from The Université Paris Sud, Rachel Oliveira and Felix Sauvage for all the help when I arrive in the lab. Without their support I would not be able to adapt so smoothly to the lab. Not to mention wonderful PhD students from the same division that I had the experience to share scientific meetings and coffee breaks. I am greatful to my friends from Brazil Raquel Pinheiro, Samara Carvalho, Janaína Goes, Paulo Arthur, Lucianne Silva, Andreza Dayanne and Jellika Patrícia for not allowing me to give up and for laugh a little harder, cry a little less, and smile a lot more. Also I am thank Karídia Melo for standing by me and sharing failures and success for more than 8 years of my life. I am thankful to coworkers and friends from FACENE for teaching me to be a better teacher and reach my goals.

Nonetheless, I would like to thank other dear friends I've met in France, who were my support while I was away from my family during the development of this research, the Brazilian crew: Michele Lima, Ernandes Tenório, Kellen Tijoe, Eloísa Berbel, Bruno Caetano and Bárbara Fonseca. Any Taylor, the most brasilian a mexican person could be. Also, my dearest and the best friend I could always trust and depend on, William Herrera for being always by my side while I was there. I could not leave out to mention the friends Henrique Marcelino, André Leandro e Júnior Xavier that I had already known before France, but it was there that our friendship has become stronger.

I would like to thank Marília Ricarte for her support and being by my side in the final months of my doctorate studies and during the writing of this document, for her caring, support and comfort during a difficult time.

Most importantly, I would like to thank my family for the unconditional love, support, encouragement and kind gestures. My dear loving parents Hildo and Ione, my sisters Débora, my brother Júnior, Naga (my sister's dog) and my puppy Maggie. Different types of emulsified systems. Figure 2.

Freeze-drying cycle, showing shelf, product and condenser temperatures, and chamber pressure.. Influence of the type and concentration of CP on the droplet size of the ME formulations before the freeze-drying process.

LIST OF TABLES

Section I Table 1.

Current VL treatments and their main properties Chapter II Table 1.

In vitro evaluation of the cytoxicity and the anti-leishmanial activity on L. donovani WT and AmB-R.

Table 2.

In-vivo antileishmanial activity and acute toxicity of H-AmB and ME-AmB compared to M-AmB and AmBisome®. Mice were treated intravenously at 1 mg/kg equivalent AmB at Day 8, Day 10 and Day 12 post-infection.

Section II Chapter III Table 1.

Cryoprotectants used for freeze-drying of emulsified systems with their molecular formula, molar mass and collapse temperature.

Table 2.

Freeze-drying conditions for different emulsified systems.

Chapter IV Table 1.

Experimental levels of independent variables of the DOE for ME droplet size evaluation after freeze-drying process.

Table 2. DOE for the freeze-drying process of the Mes for each CP type.

Table 3.

Statistical Analysis (ANOVA) of ME-MT and ME-LT samples.

Table 4.

Physicochemical characterization of the ME-MT before and after the freeze-drying process (FDP).

Table 5.

Physicochemical characterization of ME with AmB containing 5 % of MT before and after freeze-drying process.

General Introduction

Leishmaniasis

Leishmaniasis is a vector-borne zoonotic disease caused by a protozoa parasite from one of over 20

Leishmania species that have dogs as a common reservoir and humans as an incidental hosts (1).

Despite being present worldwide, this disease is neglected because it is associated with poverty due to factors such as malnutrition, poor housing, migration, deforestation, irrigation schemes, urbanization and the building of dams. Furthermore, the existing programs to control leishmaniasis are not sufficient and there is no important investment in the development of new drugs (2).

This parasite is transmitted by the bite of an infected female sandfly (over 90 species) belonging to the genus Phlebotomus and Lutzomyia. These sandflies are more often found in tropical and temperate regions, which, together with the risk factors mentioned above, explains why Brazil, Ethiopia, India, Somalia South Sudan and Sudan account for more than 90% of the 50 000 to 90 000 new cases of visceral leishmaniasis, that are reported worldwide each year (3).

The Leishmania parasite is an obligate intracellular protozoon with two different morphological stages: a promastigote flagellar form and an amastigote form. The infection occurs when an infected sandfly regurgitates promastigotes into the mammalian host during blood feeding. Thereafter, the promatigotes are phagocytized by macrophages and other types of mononuclear phagocytic cells in which they differentiate into amastigotes form that multiply until the host cell bursts and then proceed to infect other macrophages. Many factors, including ones concerning the parasites and the host, influence the clinical manifestations and form of the disease. Sandflies become infected by ingesting the amastigotes while feeding on an infected person. The transformation of amastigotes to promastigotes occurs in the sandfly's gut, from which the promastigotes migrate to the proboscis, ready to infect another mammalian host (4).

The parasite may survive for decades in asymptomatic infected people or can induce symptoms ranging from localized ulcers to fatal infections depending on the parasite cell and organ tropism, which characterize the clinical form of the disease. There are three main forms: visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL). CL, the most common form of leishmaniasis, is caused by two Leishmania species:

Leishmania major and L. tropica. The parasites remain at the site of insect vector bite, causing localized symptoms such as ulcers on the skin. In MCL, usually considered as a subset of CL, the Current treatment for leishmaniasis is based mainly on pentavalent antimonials, Miltefosine, micellar Amphotericin-B (M-AmB) and Liposomal Amphotericin-B (L-AmB). Thje advantages and disadvantages of these different therapies are listed in Table 1.

Pentavalent antimonials were first choice for leishmaniasis treatment for many years due to their low price, despite their variable efficacy; however due to its dangerous side effects the patients needed be monitored, and sometimes the treatment had to be suspended. Furthermore, the resistance of Leishmania against pentavalent antimonials has increased. Miltefosine (hexadecylphosphocholine) is the first effective oral treatment against VL and the most frequently used antileishmanial drug on the market, however its teratogenicity poses a problem, as does the rapid emergence of resistance. Amphotericin B (AmB) is highly active against Leishmania and is the drug of choice to treat immunosuppressed patients. There are different formulations of AmB on the market, the main ones being M-AmB (Fungizone®) and L-AmB (Ambisome®). M-AmB, which contains deoxycholate to solubilize the drug, has good activity, but is highly toxic, requiring slow IV administration, whereas L-AmB has the advantage of lower side effects, however its high cost is a limiting factor for its use in poor countries (4, 6). AmB is a potent drug which has a good antileishmanial activity without showing many cases of resistance in the field. However its toxicity and the price of some AmB-based formulations render it unfeasible for use in many situations. Strategies to overcome these drawbacks would be of a great value to improve leishmaniasis treatment.

Amphotericin B

AmB has been used for a long time as the major antifungal drug to treat systemic infections. It possesses a wide spectrum of activity, so that it remains almost 100 % effective as a a first-line therapy. This drug is also been used for treatment of VL (7).

AmB was extracted from a strain of Streptomyces nodosus in the 1950s (8). Its amphoteric structure is composed of a hydrophilic polyhydroxyl chain and a mycosamine sugar with a lipophilic polyene hydrocarbon chain (Figure 1). AmB has low solubility in water, which leads low bioavailability by oral route, so that its use is limited to the intravenous route and local application. However, this makes the drug an ideal candidate for incorporation into lipid-based preparations (9). Different aggregation states of AmB can be observed, depending on the concentration. In water AmB exists as a mixture of water-soluble monomers and oligomers (the most toxic form) with insoluble aggregates. The toxicity of the micellar formulation is related to the different aggregate forms of AmB. Monomeric AmB interacts with ergosterol in membranes, forming ion channels, whereas soluble self-associated oligomers cause damage to sterol-free and cholesterol-containing membranes and are susceptible to oxidation, thereby enhancing their toxicity. As well as these two forms, super-aggregates has been shown to be less toxic because they are less susceptible to oxidative degradation, and in mammalian cells, it is less selective for the cholesterol membrane. This form is obtained by heating M-AmB (11).

Both the therapeutic and the toxic effects of AmB are related to its interactions with sterols in biological membranes, such as ergosterol from fungal and parasite cells and cholesterol from mammalian cells. A complex of eight AmB molecules hydrophobically interacting via the polyene chain with eight sterol molecules forms a pore which leads the leakage of electrolytes and other cell components, causing acidification of the interior with precipitation of the cytoplasm, leading the cell death (12).

Different approaches have been applied to decrease the toxicity of AmB. Since the Leishmania parasite is an obligate intracellular found within macrophages of the reticuloendothelial system, this presents a major challenge in the treatment of leishmaniasis, since traditional antileishmanial drugs are not readily able to penetrate inside the macrophages to kill the parasite. Therefore this disease is suited for drug delivery therapy, in which the delivery systems are able to accumulate in specific locations (spleen, liver, bone morrow, dermis) after administration. Lipid-based nanocarriers are able to penetrate into macrophages and delivery the drug within the cell in order to reach the therapeutic concentration necessary to kill the protozoa. Therefore the main strategy is to target the drugs directly to mononuclear phagocyte system system (hepatic macrophages), where Leishmania parasites accumulate (4, 13). Therefore, new AmB lipid-based formulations have been proposed for the VL treatment.

These lipid formulations have some advantages over native AmB, such as improving the solubility of the drug, protection of the drug from enzymatic degradation and/or host immune factor inactivation; improving its bioavailability and pharmacokinetic properties; preventing uncontrolled drug leakage by the association of AmB with the carrier; and facilitating the uptake of the complex by the cells of the mononuclear phagocyte system in order to targeted the drug to desired sites of infection (12).

Developments in nanotechnology have provided new approaches to the treatment of VL treatment using lipid formulation in order to decrease the toxicity while maintaining the efficacy and decreasing the product cost. In addition to the formulations which are already on the market such as AmB deoxycholate, Abelcet ® and Amphocil ® or Amphotec ® and Ambisome ® , AmB-loaded emulsified systems have been developed.

Microemulsion

A microemulsion (ME) is a emulsified system which was first studied by Hoar and Schulman in 1943 (14). The ME is a thermodynamically stable system comprised of two immiscible liquids (e.g water and oil) stabilized by a mixtureof surfactants. This system is clear, stable, isotropic and possesses low surface tension and small droplet size. It has been used as a drug delivery system improving both drug absorption and permeation (15).

MEs are generally formed spontaneously, without any significant energy input. They can be classified as water-in-oil, oil-in-water and bicontinuous. Their advantages over conventional emulsions are the thermodynamically stability and the minimum energy requirement for formation, ease of manufacturing, enhanced drug solubilization and improved bioavailability, and as a result they have widespread applications as colloidal drug delivery systems for drug targeting and controlled release (16).

Drug release from ME can be described by two models. The first one takes drug diffusion through the droplet as the rate-limiting step of drug release. The other one considers the rate-determining step of drug release to be passage through the interfacial barrier between the droplet and the surrounding medium (17).

However the presence of free water in MEs can destabilize the system by hydrolysis, drug leakage or microbiological contamination. To overcome these drawbacks, a drying technique, such as freeze-drying, could be used to increase the shelf-life of the preparations.

Freeze-drying

Freeze-drying (FD), also known as lyophilization, is a dehydration technique that transforms the water in the sample from the ice state directly to the gas state at low temperatures and pressures.

There are three main steps: freezing, primary drying and secondary drying. These steps ensure the water removal, however specific conditions that includes temperature, time and pressure are necessary for the success of the process (18). However, during these drying steps as the sample may undergo physical or chemical damage, making it necessary to use cryoprotectants. These compounds can prevent the stresses that occur in lipid bilayers during freeze-drying (19).

Therefore, it is important to evaluate the various parameters which can influence the process and lead to an acceptable freeze-dried product, such as the nature of the surfactants, the solubility and partitioning characteristics of the drug, the type and concentration of cryoprotectants and the time and temperature of each step (20).

Previous study by our research team

AmB has been one of the themes of the research group run by Prof. Sócrates Egito at Laboratório de Sistemas Dispersos (LASID)-UFRN since his PhD studies (11, 21-33). The strategies used by this group to improve AmB therapy are loading into emulsified systems, such as microemulsions, and changing the aggregate state of micellar Amphotericin B (M-AmB) by heating (H-AmB).

The formulation (ME-AmB) used in this work was developed, characterized and evaluated for its toxicity and antifungal activity by Silveira et. al. (2016) and Damasceno et. al. (2012) (29, 33). This work focused on the development of ME-AmB for ocular application and systemic fungal infections, in order to increase its efficacy and decrease its toxicity. Its physicochemical properties, polarized light microscopy, droplet size, drug-loading efficiency, minimum inhibitory concentration against fungal strains and microemulsion toxicity and in-vitro toxicity against red blood cells were all evaluated. The results showed that the presence of AmB within the system did not cause significant changes in the physicochemical properties of the microemulsion compared with the unloaded system. Furthermore, AmB molecules were strongly and individually bound to the microemulsion droplets. The in-vitro assay showed that ME-AmB had high activity against Candida strains and showed lower toxicity against human red blood cells compared with the commercial product (M-AmB). Therefore, the microemulsion is an eligible drug carrier for Amphotericin B and has potential applications against fungal infections.

Another formulation of ME-AmB was developed and characterized by Silva et al (2013) (31) for the oral route. This AmB-loaded ME based on pseudoternary phase diagrams was able to improve the apparent solubility of the drug. Moreover, it was observed that part of the AmB in the MEs was aggregated and could act as an AmB reservoir carrier, explaining its low toxicity to macrophagelike cells. Therefore, this formulation may be considered as a promising carrier for AmB for oral delivery.

Our research group has recent results concerning the other alternative formulation proposed in this thesis: H-AmB. Silva-Filho et al. (2012) (11) evaluated how an aqueous M-AmB could be restructured after heating treatment. Physicochemical studies were performed, as well as an in-vitro pharmacotoxicity assay using red blood cells from human donors and Candida parapsilosis. The results of a spectrophotometric study revealed a blue shift of the aggregate peak after the heating procedure. Furthermore, they showed that the heated micellar AmB was less toxic to mammalian cells while keeping its activity against fungal cells. Therefore, it is a simple, inexpensive, and safe way for the treatment of systemic fungal infections. Siqueira et al. (2014) (32) continued these studies by evaluating the behavior of H-AmB after a freeze-drying process, examining their physicochemical and biological properties by spectrophotometry, antifungal activity and red blood cell toxicity assay. It was observed that H-AmB could be freeze-dried and could retain its physicochemical properties, activity and low toxicity, and was therefore a suitable method to increase the long-term stability of H-AmB systems.

Presentation of the Thesis Work

In the light of these observations, the aim of the work described in this thesis was to develop lipid formulations containing Amphotericin B as microemulsions (MEAmB) and as its heated micellar form (AmB-H). Furthermore, freeze-drying of MEAmB in order to diminish the water content and thereby increase drug stability was also optimized.

In view of the importance of these subjects, an overview highlighting leishmaniasis, microemulsion, AmB and lyophilization was written and published. It is presented here as the first chapter of this manuscript.

.

CHAPTER I Freeze-dried microemulsion containing amphotericin B for leishmaniasis treatment: An overview

Introduction

Leishmaniasis is a neglected tropical disease caused by 20 different species belonging to the genus Leishmania, which is transmitted by the bite of a female mosquito of the genus Phlebotomus and Lutzomyia, commonly known as sandflies 1,2 . In vertebrates, this parasite multiplies inside the cells of the mononuclear phagocyte system after acquiring intracellular growth ability. Moreover, it is capable of escaping the host immune response due to complement activation. The clinical manifestations of leishmaniasis recognized by the World Health Organization are divided into three main forms: cutaneous, mucocutaneous and visceral (VL), the latter being the most severe form 1,3 .

Conventional treatment of VL is based on parenteral administration of salts of pentavalent antimony. However, parasite resistance to these drugs and their high toxicity have restricted their use to some areas and conditions. When this treatment is not effective and/or in patients seropositive to the human immunodeficiency virus, second-line agents, such as Amphotericin B (AmB), have been used. AmB interacts with ergosterol and cholesterol, which are presented as a cell membrane component in Leishmania and mammalian species, respectively. For this reason the toxic effects of AmB are exerted not only in the parasites, but also in humans 4 . AmB, a natural antibiotic obtained from Streptomyces nodosus cultures 5,6 , can present different aggregation states in aqueous media: a monomeric form, water-soluble oligomers and nonwater-soluble aggregates, the first being the least toxic form against mammalian cells 7 . Besides these, a recent study revealed that the AmB within the micelle system, Fungizone ® , can exists as a super-aggregate form by the condensation of monomeric AmB with its aggregates, showing larger size, increased stability and efficacy, and low toxicity against mammalian cells 8 . Currently, as well as the traditional AmB-sodium deoxycholate micelle system (Fungizone ® ), there are several pharmaceutical dosage forms of AmB available in the market: i.e. liposomal AmB (AmBisome), AmB lipid complex (ABLC ABELCET ® ) and AmB colloidal dispersion (Amphocil ® ). However, the high cost of such products makes their use prohibitive 9 . Therefore, the development of novel and inexpensive AmB dosage forms has become mandatory to spread its use to the treatment of Leishmaniasis and other neglected diseases 10 .

In this context, an alternative to reduce the AmB adverse effects could be the development of an oil-in-water microemulsion (ME) system. MEs are thermodynamically stable, transparent and isotropic systems comprised by hydrophilic and lipophilic phases and stabilized by surfactants 11,12 .

Furthermore, these systems are able to solubilize drugs in their internal phase or at their interface 13,14 . In this way, therapeutic efficacy can be enhanced while minimizing adverse effects 15 .

However, the presence of water in the ME system may decrease its stability due to the hydrolysis 16 and to microbial contamination 17 , which can lead to loss of biological activity of the drug. Thus, drying procedures may be useful in order to decrease the moisture and thereby, increase the stability of the system 18 .

Freeze-drying, also called lyophilization, is a dehydration method capable of removing water by vacuum sublimation 19 . Thereby, it is not necessary to use high temperatures for drying, which could destabilize the system or degrade thermosensitive drugs such as AmB 20 .

The purpose of this manuscript is to overview the use of AmB containing ME for the treatment of Leishmaniasis. Additionally, the ability of this system be freeze-dried was evaluated.

Therefore, aspects such as Leishmaniasis, AmB, ME, freeze-drying and studies from the literature and from our group addressing these issues are discussed.

Leishmaniasis

Leishmania is an intracellular protozoan that causes a complex of diseases called Leishmaniasis, which is classified into visceral (VL), cutaneous and mucocutaneous forms 2, 3 . Among them, the VL is the most severe one, because if left untreated it can be fatal. According to the World Health Organization (WHO), approximately 1.3 million of new cases are annually detected, provoking 20,000 to 30,000 deaths. The number of new patients worldwide suffering from VL each year is estimated in 300,000, and over 20,000 deaths are reported annually. Leishmaniasis is widespread on almost all continents, but highly prevalent (90 % of cases) in six countries: Bangladesh, Brazil, Ethiopia, India, South Sudan and Sudan 21 .

Leishmaniasis is a public health problem associated with poor housing, population displacement, malnutrition and HIV-Leishmania co-infection. Concerning VL, the poor access to diagnosis and treatment is one of the most important drawbacks to its control 22 . Furthermore, there is no satisfactory drug treatment so far, due to the inherent high drug toxicity, the drug resistance and the high cost of drugs that present low-toxicity.

The bite of infected female Phlebotomine ssp. ("Old world" -Africa, Asia, Europe) and Lutzomyia spp.(New world -Americas) sandflies transmits leishmaniasis to humans and to animals 23 . The parasite in the promastigote form enters the vertebrate host when the sandfly is feeding; then it is phagocytosed by macrophages in which it transforms into an amastigote form and reproduces.

When the host cell becomes overwhelmed by parasites, the amastigotes are released and infect other macrophages and finally reach the circulation and infect the organs. The amastigote-infected blood is sucked by uninfected sandflies. Moreover, the amastigote forms are metamorphosed into promastigote ones in the insect gut to subsequently infect a new host 24 .

The usual immune response to VL is to kill the intracellular parasites by increasing the level of reactive oxygen metabolites or nitric oxide by activation of the infected macrophages through the antigen presentation, with consequent activation of Th1 and production of cytokines (IL-2, ITF-γ).

However, in VL infections remarkable interference with the cell signaling is observed. The complex immune response pathways were well described by Gupta et al. 25 .

However, sometimes the intracellular amastigote forms escape from the immune system, obtains nutrients from the host cell and proliferates in it. Additionally, some strategies are used by the amastigote forms to survive and adapt their life in the inhospitable environment of the phagolysosome of the macrophage in which the pH rage from 4.5 to 5.0. For instance, they can interact with surface molecules such as lipophosphoglycan and proteases (H+-ATPase, transporters 26 , GP63 metalloproteases, which disable the key microbicide pathways, disturbing the metabolism and the signaling pathways to provide the parasite growth, and induction of DNA methylation in order to turn off genes controlling microbicidal pathways 27 .

Anti-leishmanial chemotherapy is related to the parasites' biology. Drugs with a suitable molecular weight and pK a can be designed to be accumulated into the phagosome and to be taken up by the amastigote form in the low pH environment 26 . The current Leishmaniasis treatment is based on five group of drugs 28 , pentavalent antimonials, miltefosine, paromomycin, pentamidine and AmB. The first introduced treatment against Leishmaniasis was based on the antimonials in the early 1900s 29 . The pentavalent antimonials, commercially available as Glucantime ® and Pentostam ® , were first used between 1935 and 1945 and have been extensively used as the first-line treatment 30 . Nevertheless, an increase in clinical resistance has been reported 31 . On the other hand, Miltefosine (hexadecylphosphocholine [HePC]) was initially used for treatment of cutaneous metastases of breast cancers. Nowadays, it is the first oral drug on the market effective against VL.

However, this drug has a long half-life in the body and is very expensive 32 . Paromomycin, an intramuscular drug used to treat intestinal infections, has been recently approved for Leishmaniasis treatment because it has bactericidal activities. However, it has shown serious adverse reactions, such as renal and ototoxicity. Furthermore, its administration by the parenteral route proved to be less effective than AmB for cutaneous leishmaniasis. Pentamidine, on the other hand, is less active than other drugs. For this reason, it has been used as an alternative treatment for both visceral and cutaneous leishmaniasis, although this engenders an increase in the treatment failure against Leishmaniasis 33,[START_REF] Steckel | Effect of cryoprotectants on the stability and aerosol performance of nebulized aviscumine, a 57-kDa protein[END_REF] .

AmB has been quite effective against fungal infections caused by Candida albicans and Aspergillus fumigatus. Its use as leishmanicidal dates from 1959 [START_REF] Tandel | Preparation and evaluation of cilnidipine microemulsion[END_REF] . Despite being very effective, Fungizone ® (the micellar delivery system for AmB containing deoxycholate), has severe side effects [START_REF] Abdelwahed | Investigation of nanocapsules stabilization by amorphous excipients during freeze-drying and storage[END_REF] . Consensus recommendations from the WHO recognized in 2010 AmBisome ® , the liposomal form of AmB, as the first choice for VL. However, the treatment remains quite expensive, even with the reduced price negociated by the WHO ($18 per 50-mg ampoule) [START_REF] Mazerski | Effect of the modifications of ionizable groups of amphotericin B on its ability to form complexes with sterols in hydroalcoholic media[END_REF] .

The onset of resistance to the current chemotherapeutics is a major concern for the current Leishmaniasis treatment. Although the major resistance mechanisms are not elucidated, there are some studies about how the Leishmania develop resistance to these chemotherapies. For example, the antimonials, which are the most affordable drug treatment, have been revealed to be the most vulnerable to resistance. The mechanisms of drug resistance are related to a decrease of drug uptake and an increase in the efflux mechanism, which reduce the drug concentration inside the parasite by the inhibition of the drug activation, the inactivation of the active drug and the gene amplification [START_REF] Steckel | Effect of cryoprotectants on the stability and aerosol performance of nebulized aviscumine, a 57-kDa protein[END_REF][START_REF] Pestana | Oil-in-water lecithin-based microemulsions as a potential delivery system for amphotericin B. Colloids and surfaces B[END_REF] . The Pentamidine-resistant promastigote is also able to reduce the drug uptake and increase the drug efflux 39 . The resistance for Miltefosine is associated to the suppression of oxidative stressinduced programmed cell death and, probably, to the increase of the gene expression (FeSODA and SIR2) 40 . Studies regarding Paromycim-resistant Leishmania have shown that the binding of the drug to the parasite cell surface is reduced 41 . Some AmB-unresponsive cases encountered in India were explained by the absence of ergosterol in the membranes of resistant Leishmania and the upregulated AmB efflux and reactive oxygen species scavenging machinery 42 . Therefore, all these issues concerning drug resistance revealed that the development of new therapies is an urgent need. The major challenge in the treatment of Leishmaniasis is the ability of the drug to penetrate inside the macrophages. Since nanocarriers have the ability to penetrate into these cells, their combination with antileishmanial drugs would be a promising approach in Leishmaniasis treatment. As well as the ability to overcome biological barriers, nanocarriers can release the drug inside the macrophage, leading to a local high drug concentration that kills the parasite 43 .

Nanotechnology has brought promising results for the treatment of Leishmaniasis. There are numerous studies involving drug delivery systems for this disease. Among them, colloidal carriers are the most experimentally investigated 44 . Examples of these nanocarriers include liposomes 45 , emulsified systems (emulsions, nanoemulsions and MEs) [46][47][48] , solid lipid nanoparticles 49 , polymeric nanoparticles 50 and polymeric drug conjugates 51 . Such systems are able to improve drug bioavailability and to reduce drug toxicity, improving the selectivity, enhancing the treatment efficacy, increasing the drug solubilization, modulating the drug pharmacokinetics and protecting the drug from degradation 43 .

The development of nanostructured carriers applied to the drugs currently used in

Leishmaniasis has gained notoriety. AmB has been the main drug incorporated in these colloidal carries, since it has an excellent cure rate 52 .

In our work, ME was chosen as the nanostructured carrier due to the aforementioned advantages, besides their relatively easy handling compared with other formulations such as liposomes. Indeed, these system usually do not use organic solvent in their production, has an impact for the environment, and for ease of manufacturing and scale-up 53 . Moreover, ME systems would allow targeting of AmB to the cells and organs affected with VL (liver, spleen and bone marrow).

Amphotericin B

AmB is an antifungal and antiprotozoal drug that was originally isolated from Streptomyces nodosus 5,6 . Chemically, this molecule is considered a macrolide due to its large macrocyclic lactone ring composed of 37 carbons in the main chain. This molecule contains a heptaene from the carbon 20 to the carbon 33, providing a non-polar characteristic to AmB 54 . The opposite side of the molecule provides polarity due to the presence of seven hydroxyl groups and one carboxyl group directly attached to the main cycle. As a result, this molecule has amphiphilic properties 55 . As well as the carboxyl group (pK a 5.5), AmB has a primary amine group (pK a 10) attached to the mycosamine ring (3-Amino-3,6-dideoxy-β-D-mannopyranose). This amino sugar group is connected to the main macrocyclic chain by an o-glycoside linkage at the carbon 19. The anionic carboxylic acid and the cationic protonated amino group result in a zwitterion, providing the molecule with its amphoteric properties 56 . Figure 1 shows the chemical structure of AmB.

Figure 1

AmB is known for interacting with sterol molecules, such as cholesterol and ergosterol, the latter being present in fungal and Leishmania cell membranes [START_REF] Pestana | Oil-in-water lecithin-based microemulsions as a potential delivery system for amphotericin B. Colloids and surfaces B[END_REF] . AmB interaction with ergosterol in fungal cells was the primary suggested information that led to studies of AmB's mechanism of action. It is suggested that the AmB may act on several different targets in fungal cells leading to their disruption. The first and most accepted mechanism of action is that the AmB binds to the ergosterol molecules on the fungal cell membrane and form pores, which increase the permeability of the membranes to small cations, promoting depletion of intracellular ions and cell death 57, 58 .

Another possible mechanism is the induction of ergosterol sequestration resulting in membrane disruption 59 .

Although pore formation is a well established mechanism, Sokol-Anderson et al. showed that without oxidative damage the pre-lethal ionic leakage would not be sufficient to kill fungal cells 60 . It is now known that the AmB acts in the intracellular level inducing an oxidative burst.

Possible mechanisms are its action as a pro-oxidant, inducing the formation of reactive oxygen species (ROS) that cause protein, DNA and membrane damage. In order to this to happen, it is necessary for the cell to take up AmB by interaction with ergosterol. Another possible mechanism is the interference of the ROS generated with the cells' mitochondrial activity since these reactive species are part of the respiratory chain 59 .

Although more extensive studies on the AmB mechanism of action have been conducted in fungal cells, the mechanism of action involving the interaction with ergosterol and the formation of pores that result in osmotic changes and cell lysis has been confirmed to happen in Leishmania, as well as oxidative damage 61 . Additionally, it has been reported that the interaction of AmB with human cholesterol in macrophages inhibits the binding of Leishmania donovani promastigotes to human macrophage 62 .

Various studies have shown that L. donovani promastigotes can develop resistance to AmB in order to reduce the drugs' lethal effect. Purkait et al. 63 and Mbongo et al. 64 studied different strains of L. donovani in order to investigate the mechanisms of resistance. Purkait and his coworkers investigated the intracellular ROS levels for AmB-resistant and AmB-sensitive promastigotes. Their study showed that the ROS level of the AmB-sensitive strain was approximately 3-fold greater than that of the AmB-resistent strain. The authors suggested that resistant strains have upregulated ROS scavenging machinery and also enhanced AmB efflux.

Mbongo and co-workers 64 observed that the AmB-sensitive strains had ergosterol as the major sterol in their membrane, whereas the AmB-resistant strain membrane sterol composition showed no ergosterol, containing cholesta-5, 7, 24-trien-3β -ol as the major sterol. Changes in the sterol chains, such as methylation, would decrease the AmB affinity to the membrane. Since the interaction of AmB with ergosterol leads to the formation of pores and cell disruption, the reduction of ergosterol would decrease the AmB effectiveness on Leishmania membranes 64,65 . It was also observed that the membrane fluidity is fairly well correlated to the parasite resistance 42 . Indeed, AmB-resistant cells presented higher membrane fluidity, mainly due to the presence of the cholesta-5, 7, 24-trien-3β -ol instead of the ergosterol 64 . However, it is noteworthy that both AmB-resistant and AmB-sensitive promastigotes strains were infective for macrophages in vitro, whereas the AmB-sensitive strain was infective only in vivo 64 . Therefore, it is still unclear whether the AmBresistant strains represent a health issue, because the study showed reduced infectivity due to the change of the sterol composition and the decreased ability to infect macrophages. AmB has a broad antifungal spectrum of action, including Candida spp., Aspergillus spp., and other filamentous fungi [START_REF] Abdelwahed | Investigation of nanocapsules stabilization by amorphous excipients during freeze-drying and storage[END_REF] . However, the high nephrotoxicity induced by the AmB micelle system, Fungizone ® , has limited its use as second-line treatment when first course with azoles or echinocandins (regarding patients' conditions and specific infection) fails to eliminate the infections [66][67][68] . AmB has been used to treat VL in cases of Leishmania resistance to antimonial drugs. However, its toxicity can limit the treatment due to the severe side-effects and poor patient compliance 69 . The infusion of Fungizone ® can be accompanied by shaking, fever, vomiting, tachypnea and may require co-administration of antipyretics, antihistamines, antiemetics and hydrocortisone to reduce the side effects [START_REF] Abdelwahed | Investigation of nanocapsules stabilization by amorphous excipients during freeze-drying and storage[END_REF] .

Severe toxicity is usually attributed to the interaction of AmB aggregates with cholesterol molecules in human cells. AmB is able to self-associate and generate different aggregated forms. In its monomeric form this molecule is able to interact only with ergosterol and thus exhibit its pharmacological effect. Aggregated structures such as dimers are suggested to also be able to interact with cholesterol-rich membranes present in human cells and cause side-effects. Lambing et al. 70 and Huang et al. 71 suggested that dimerization is a necessary event for AmB interaction with cholesterol. On the other hand, studies have shown that heat-induced AmB superaggregates are able to reduce toxicity against human cells, possibly by reducing the affinity to cholesterol-rich membranes and acting as a reservoir by releasing monomers to the media 8,72 .

Regarding the stability of AmB, few studies have reported on its chemical stability in formulations. In particular, the identity and toxicological safety of AmB degradation products and the kinetics of their formation in various environmental conditions and drug delivery systems have not been described. Studies have demonstrated that the AmB toxicity is reduced in lipid-based delivery systems 73,74 . However, it is unclear whether reduced toxicity is only due to the reduced dimer and AmB-aggregate concentrations or if the changes in the pattern and rate of degradation product formation can play a role in the toxicity.

Because Fungizone ® was the first approved formulation containing AmB for intravenous use, it is considered the traditional formulation of AmB. This micellar system was developed in order to improve drug solubility in water. Due to the acute and long-term side effects of this formulation, other formulations containing AmB have been studied. Nowadays, it is also known that the reduced side-effects of lipid formulations of AmB enables the use of this drug for prophylaxis 75 . Currently, several lipid-base formulations are available and approved to treat leishmaniasis in different countries, such as the liposomal formulation (AmBisome ® ) and the lipid other complexes (Amphotec ® and Abelcet ® ). However, their high cost of such formulations limits their use, especially in emerging and underdeveloped countries. AmBisome ® was the first drug approved by the U.S. Food and Drug Administration (FDA) for VL in the United States 76 . This approval was a consequence of increasing cases of resistance to antimonials in parts of Europe, India, East Africa and South America. In Brazil, a study compared different treatment regimens using AmBisome ® . Patients were children younger than 12 years old.

The study showed 100% success in a regimen consisting of 2mg/kg on days 1-10 (total dose, 20 mg/kg) 77 . The Brazilian government recommends a regimen with AmBisome ® consisting of 3mg/kg on days 1-7 (total dose, 21 mg/kg) or 4mg/kg on days 1-5 (total dose, 20 mg/kg) 78 .

However, this treatment is suggested only in patients with renal insufficiency due to its high cost.

The WHO suggested a single dose of liposomal AmB (AmBisome) (10mg/kg) as first line treatment of VL in the South-East Asia region. The treatment was suggested on the Regional Technical Advisory Group (WHO) meeting in 2009 and later endorsed by the expert committee meeting in Geneva, 2010 79 .

A clinical study performed by Mondal and co-workers 80 evaluated the feasibility of the suggested treatment from WHO in patients from rural areas in Bangladesh. The study was conducted with 398 patients diagnosed with VL. After the patient's consent and some exclusions due to other criteria, 298 patients received complete treatment consisting of 10mg/kg of liposomal AmB as a single dose. Patients were discharged and returned for follow-up visits on day 30 and day 180 after treatment. After 30 days, 36 patients had not attained the necessary increase in the hemoglobin concentrations (part of initial cure protocol). However, they did not show any sign of active disease. On the additional follow-up visits (day 60 and day 90), they were considered initially cured. On the other hand, between days 30 and day 180, 7 patients relapsed and were retreated. The overall cure rate was 97%. The study provided an important assessment on the feasibility of singledose treatment with AmBisome for rapid elimination of Leishmaniasis as recommended by the WHO. It is noteworthy that the most common adverse events during and 2 hours after treatment were fever with rigor, fever, vomiting and hypotension. In addition, the incidence of adverse events was 47% during treatment and 63% after 2 hours of treatment.

Although the commercially available liposomal formulation of AmB have shown positive results in the treatment of Leishmaniasis, the occurrence of toxicity and the high cost of the product still limits its use. Therefore, further research regarding the development of new drug delivery systems containing AmB, such as nanoparticles, nanoemulsions and MEs would be a suitable alternative to obtain safe formulations to deliver this drug in a controlled way and to protect the human cells against the AmB toxicity, since it has been proved that some of these systems are capable of reducing AmB toxicity when compared to AmBisome 81-83 .

Microemulsion

Different carriers have been used as drug delivery systems to treat infectious diseases; e.g. MEs 48, 84, 85 , nanoemulsions 86 , nanoparticles [87][88][89][90][91] , solid lipid systems [92][93][94][95][96][97] , carbon nanotubes 98, 99 , liposomes 100-105 , micelles 102, 106-111 and dendrimers 112 . Among them, MEs show interesting properties for delivering drugs to Leishmania parasites. MEs are colloidal optically transparent systems, with thermodynamic stability, low viscosity and surface tension, small droplet size, composed of two immiscible liquids (water and oil), stabilized by an interfacial film of a surfactant, usually in combination with a co-surfactant [113][114][115][116][117][118] . These systems, first described by Hoar and Schulman 11 , can also be classified as three major microstructures: swollen micellar (oil-in-water, O/W), reverse micelles (water-in-oil, W/O) and bicontinuous 119,120 . Other types of microstructures formed in MEs are cubic, cylinder-like (such as rod-micelles or reverse micelles), plane-like (e.g., lamellar structures) and hexagonal structures 121,122 , depending on the concentration, nature and arrangement of the molecules in the formulation 123 .

MEs differ from emulsions by their smaller droplet size as a result of the low interfacial tension 124,125 . The dispersed phase occurs as very small droplets (10 -100 nm) that hardly scatter light in the visible wavelength domain, explaining their tendency to appear transparent. Moreover, these systems present a spontaneous formation and thermodynamic stability due to a relatively large amount of surfactant stabilizing their interfacial area 126 . The low interfacial tension compensates the dispersion entropy; hence the system is thermodynamically stable 127 and forms spontaneously under a specific set of composition and environmental conditions 124 .

Surfactants play an important role in ME development due to their self-association into a variety of equilibrium phases when dispersed in water or in non-aqueous solvents 127,128 . During film formation, the type of system formed is dependent on the surfactants' affinity for water or oil 127, 128 . Hydrophilic lipophilic balance (HLB), the critical packing parameter (CPP) and the solubility parameters approaches have been proposed to rationalize surfactant selection for ME production. HLB takes into account the relative contribution of the hydrophilic and the hydrophobic fragments of the surfactant molecule 127,129,130 . CPP relates the preferred geometry adopted by the surfactant to form particular aggregates 115 . The size and shape of the ME are mainly governed by the curvature free energy of the interface between water and oil, the bending elastic constant and the curvature of the surfactant film. In the solubility parameter theory, two chemical compounds are infinitely soluble and capable of formation of ME when the chemical intermolecular forces are equivalents between them [131][132][133][134][135] . ME formation has been explained by the following three theories: interfacial or mixed film theory, solubilization theory and thermodynamic theory 115,136,137 . The interfacial or mixed film theory considers that the interfacial film can be considered duplex in nature (region bounded by water by one side and oil by the other) with an inner and an outer interfacial tension acting independently to form the ME spontaneously due to the low interfacial tension, which tends to zero 138 . The solubilization theory is based in the principle that MEs are swollen micelles in which either the water or the oil are solubilized in reverse and normal micelles, respectively 139,140 . The thermodynamic theory considers that the free energy must be negative to form thermodynamically stable MEs, which depend on the reduction of the surface tension of the oilwater interface by the surfactants and the change in the entropy of the system 11,137,141 .

Although MEs may form spontaneously, external factors can be used to overcome kinetic barriers, thereby reducing the time of formation of these systems. Some factors that can accelerate and facilitate the formation of the ME system are the order of component addition, the application of mechanical agitation and the use of ultrasounds or heat. Moreover, ME can be formed by phase inversion and phase titration methods 126 .

The ME system has many advantages as a drug delivery system, including enhancement of drug solubility, high stability and small droplet size, which provides a large surface area improving the absorption or the permeation ability of the drugs through membranes compared to the conventional formulations 115,119,127,142 . Due to the ability to enhance the bioavailability of poorly soluble drugs by maintaining them as a molecular dispersion, MEs can promote an effective control and sustained release of their active agent 127,143 . Moreover, MEs can store drugs in their internal phase, protecting them against digestive enzymes and from light (photosensitive drugs) 144,145 .

Additionally, they are easy to manufacture (not process-dependent) and scale-up 120,146,147 .

MEs have been used by different routes of administration, such as percutaneous, oral, topical, transdermal, ocular and parenteral 116,148 . In particular, in parenteral administration, ME improves the drug residence in the blood circulation and may reduce the drug adverse effects 149 . Additionally, this system causes minimum immune reactions or fat embolism in contrast to emulsions 150 . ME has been an efficient carrier for AmB with potential fungicide and fungistatic applications against histoplasmosis, aspergillosis and candidiasis 151,152 , as well as, for protozoan infections such as visceral leishmaniasis 153 . In this context, a recent study carried out by our research team aimed to use an oil-in-water (o/w) ME system to improve the solubility of AmB for the oral route 84 . Different nonionic surfactants were tested to evaluate their solubilization ability in combination with several oils. AmB-loaded MEs with mean droplet sizes of about 120 nm were successfully produced. These systems were able to improve the drug solubility up to 1000-fold.

Cytotoxicity studies revealed limited toxicity against macrophages, suggesting that this formulation could be a suitable carrier for the AmB that would also reduce its in-vivo toxicity. Some years ago

Gupta et al. developed an o/w ME containing quercetin based in a pseudoternary diagram study for

Leishmaniasis treatment in hamster models 48 . They showed that clove oil/Tween 20 ® /water can form a very stable (temperature and salt resistant) o/w ME system with reasonably low droplet dimension. A significant improvement in the pharmacological efficacy of the drug was observed.

Although the vehicle showed some hepatotoxic and nephrotoxic effects, the drug-loaded ME seemed to function better against experimental leishmaniasis in hamster models than the free drug.

In another study, Brime et al demonstrated the in vivo efficacy of a new AmB o/w lecithinbased ME compared to the Fungizone ® . The study was conducted in immunocompetent and neutropenic murine models of systemic candidiasis 154 . The doses administered (1, 2 and 3 mg/kg) were well tolerated due to their reduced toxicity. The AmB-loaded ME mice treated group had a longer survival time than the infected mice without treatment. According to the authors, the ME presented better results than the Fungizone ® in an established infection in both immunocompetent and neutropenic mice.

Recently, a topical ME containing AmB was investigated for the treatment of invasive fungal infections 155 . The influence of the surfactant and co-surfactant mass ratio on the ME formation was evaluated. Also, the permeation of the ME through excised rat skin was studied. The authors produced an optimized formulation consisting of 0.1% (w/w) AmB, 5% (w/w) Isopropyl Myristate and 35% (w/w) surfactant (Tween 80: Propylene glycol (3:1)). The ME showed an average droplet size of 84.2 nm, a polydispersity index of 0.164, a pH of 7.36 and a conductivity of 229 µS. The formulation was found to be stable at 2-8 °C and at room temperature, for the period of three months. Also, it exhibited a 2-fold higher drug permeation when compared to the plain drug solution. The in-vitro anti-fungal activity against Trichophyton rubrum fungal species have shown that the developed ME showed a higher zone of inhibition of fungal growth (5.1mm after 72h) due to its high fluidity compared to other ME systems.

AmB in a lipid-based preparation as a ME was developed by Darole et al. (2008). Its toxicity was also evaluated in comparison to the commercial formulation Fungizone ® 156 .

Pseudoternary phase diagrams were constructed to identify areas of existence of ME composed of glyceryl monooleate as oil phase and polyethylene glycol (40)-stearate and polyethylene glycol (15)-hydroxystearate as surfactants. The average droplet size of the ME was 84 nm. The ME showed a 92% decrease in the hemolysis of human red blood cells (RBCs) in vitro when compared with the commercially available Fungizone ® . The LD 50 in mice was estimated to be 3.4 mg/kg. The authors concluded that the formulation would be a safe and effective alternative to classical AmB therapy. In a similar study, an AmB-loaded ME was developed in order to increase its efficacy and decrease its toxicity compared to Fungizone ® 157 . On this case, the AmB-loaded ME showed an average size of 300 nm. The selectivity for fungal cells was maintained after the incorporation of the drug in the ME and this system had the same efficacy of Fungizone ® against C. albicans.

Additionally, the ME showed lower leakage of hemoglobin and K + from the RBCs compared to Fungizon ® .

Patents involving the formation of AmB-loaded ME have also been developed. A stable ME composition including of AmB with additives suitable for intravenous administration was disclosed.

The formulation was designed to provide safety, to improve stability and to be produced by an efficient, simple and rapid process 158,159 .

Considering the high cost of Ambisome ® and the high toxicity of Fungizone ® , in our work 160 (data not shown) the strategy to improve the therapeutic index of AmB with inexpensive ingredients was to produce a biocompatible ME, which was prepared with phosphate buffer pH 7.4, Tween 80 ® , Lipoid S100 ® (Soybean phosphatidylcholine) and Mygliol 810 ® . The AmB load content was 5 mg/mL. The ME droplet size measured by Dynamic Light Scattering was about 40 nm. The spherical shape of the system was confirmed by the Transmission Electron Microscopy.

Additionally, the rheological analysis showed low viscosity and Newtonian behavior of the ME system.

Our group has also evaluated the biological activity of this AmB-ME formulation against Leishmania donovani LV9 160 (TO BE PUBLISHED SOON 1). The overall result allowed us to conclude that the IC 50 on axenic amastigotes for the AmB-ME was 0.2 µM, and the IC 50 on intramacrophagic amastigotes growing in J774 cells was 0.81 µM. The AmB-ME selectivity Index was higher than Anforicin B ® (a Brazilian product similar to Fungizone ® ). Furthermore, the AmB-ME showed a high activity against AmB-resistant amastigotes in comparison with Ambisome ® . The in-vivo efficacy was evaluated in Balb/C mice infected with Leishmania donovani LV9 after IV administration of the different formulations (three doses of 1 mg/kg AmB). The AmB-ME showed a reduction of the parasite burden of 78% compared to 72% for the Anforicin B ® . The in-vivo toxicity was determined by the changes in total serum cholesterol, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatinine and urea concentrations. The results did not show important differences among the samples analyzed. Furthermore, they were not able to cause kidney or liver dysfunction. Therefore, the ME containing AmB developed in our work seems to be an inexpensive alternative formulation for this molecule, showing good efficacy and selectivity for the Leishmania donovani and merits further investigation.

Freeze-drying

Many pharmaceutical industries have used this technique as a method to obtain more stable products, since the aqueous media, present in large amount in colloidal systems, may cause some chemical and microbiological reactions that can destabilize them 161 . Freeze-drying is a dehydration technique capable of bringing the product to an anhydrous or almost anhydrous state, using low temperatures and pressures 162 . This process consists of three consecutive stages: (1°) freezing (solidification of the water), (2°) primary drying (ice sublimation) and (3°) secondary drying (desorption of water) 19 . The first step is of utmost importance since the ice crystal formation can induce physicochemical changes, as well as droplets aggregation 163 . The temperatures to which the samples are exposed must be below the glass transition temperature (Tg). If this is not the case, during the subsequent step, the sample will collapse, losing its integrity and stability 164 . Furthermore, the freezing rate should be carefully chosen, since different water crystals can be formed, influencing the primary and the secondary drying in different ways. Large crystals are produced in small quantities when a slow freezing is used. On the other hand, fast freezing is able to form small and numerous ice crystals 165 .

After freezing, the next step, the primary drying, is the most time-and energy-consuming, because the sublimated water vapor has to be continuously removed from the sample, maintaining the freeze-dryer chamber pressure below the vapor pressure of the ice, with subsequent condensation on a cooling coil 20 . Therefore, the temperature during this step should be optimized in order to reduce the cost of the process. The freezing rate influences the length of this step, because if the process was conducted at the fast rate, the small ice crystals formed will hinder the movement of the water vapor 163 . Furthermore, the higher the sample temperature, the lower the time required for the primary drying. However, the structure of the product could be lost if its temperature reaches values above the Tg, leading to a collapse that affects the quality of the freeze-dried product 164 .

The unfrozen water that was not removed by sublimation will be removed by desorption from the solute phase during the secondary drying, once the shelf temperature has been increased and the chamber pressure reduced. As for the primary drying, the temperature of the sample during this step must be below the Tg in order to prevent the product from collapsing. This step is also influenced by the freezing rate. Since the adsorption-desorption equilibrium is the most important factor during the secondary drying, the large surface area of samples submitted to a fast freezing procedure will favors water desorption and reduce the time for this step 166 .

At the end of the freeze-drying process the moisture content can be reduced to levels below 1% 19 . However, the system may undergo some damage during these steps, resulting in the loss of its properties, such as the increase in the interaction between the system components, which can lead to aggregation of the droplets and change in the ME pH 18 .

The system integrity can be protected against these drawbacks and destabilization by proper selection of cryoprotectant molecules that are capable of reducing the stresses caused by the temperature reduction and the dehydration 167 .

Cryoprotectants are able to bind to the surfactants, avoiding droplet aggregation when the water separates from the other components during the freezing process 168 . These compounds are also able to decrease the interaction between the hydrocarbon chains of the phospholipids during the drying steps 169 , as well as to replace the hydrogen bonds with water 165 . Although the mechanism of action of cryoprotectants is not yet well understood, the theory currently used to explain their action is the ability of these molecules to form hydrogen bonds that replace water during the sublimation 167 The main cryoprotectant molecules used are carbohydrates, such as trehalose, sorbitol, glucose, mannitol, sucrose, fructose and lactose 170 . The concentrations used are dependent on factors such as temperature and rate of cooling and freezing 171 . The concentration of the molecule reaches a limit of stabilization, whereas above this limit the cryoprotectant destabilizes the system 18 .

An acceptable freeze-dried product has to maintain the same physical and chemical properties of the system before and after reconstitution. Therefore, it is important to analyze all the parameters that affect the final freeze-dried product 172 . Several important factors must be considered during the lyophilization; for example, the thickness of the frozen layer, the formulation, the type of container, the equipment and the process variables such as the shelf temperature, the chamber pressure, the time (freezing, primary and secondary drying), and the nature and concentration of the cryoprotectant 19 .

Freeze-drying is an excellent drying method for dehydrating biological products and drug delivery systems with high water content 19 . Examples of such systems are MEs; however the lyophilization process has been rarely studied for these systems. Moreno et al. freeze-dried a ME system using 5% of mannitol as cryoprotectant, freezing at -40ºC for 2 h, and drying for 24 h (primary drying) and 48 h (secondary drying). The process did not interfere on the parameters studied for these formulations (macroscopic aspects, droplet size distribution, residual moisture, viscosity and rheological behavior). Furthermore, the freeze-dried MEs showed better stability than the non-lyophilized ones 173 . This work also evaluated the efficacy of the AmB-ME against Candida strains, its safety 174 and its pharmacokinetics parameters 154 . The freeze-dried ME maintained their antifungal activity and reduced toxicity. The system also demonstrated good efficacy in animals and showed biphasic elimination from the blood.

Experiments on the freeze-drying process were also performed for MEs developed by our group 175 (data not shown). Since this system has a large volume of aqueous phase, the surfactants Tween 80 ® and Lipoid S100 ® or the medium chain triglycerides may undergo hydrolysis, as well as being susceptible to drug leakage and microbial growth. Therefore, water removal might improve the stability of the ME, enhancing its shelf life. However, a large number of freeze-drying factors may influence the quality of the final product. For this reason, we decided to use a 2 3 full factorial design with three replicated points to optimize the parameters: freezing temperature, freeze-drying time, type and concentration of cryoprotectants.

Design of experiment (DOE) is a tool used to improve the quality of products and processes , that is able to evaluate a large number of variables using a reduced number of samples through matrix combinations between the levels of the variables investigated. Moreover, it is possible to identify the variables and their interactions that influence on the parameters of interest, and optimize multiple variables 176,177 .

The results showed that maltose presented the best protective effect. It was possible to observe in the DOE using this cryoprotectant that the freezing was the only studied parameter that had a significant effect on the droplet size of the freeze-dried ME. This is probably because of the separation of ice from the concentrated solution, which can cause interaction among the droplets, and, consequently, their fusion 19 .

The response surface analysis showed that the concentration of 5% of maltose led to the lowest droplet size 175 (TO BE PUBLISHED SOON 2). In addition, the best quality product was obtained by freezing the sample at around -88°C and freeze-drying for 24 hours. The optimized conditions were then evaluated by comparing the physicochemical characteristics of the system before and after the freeze-drying process. The changes observed in the freeze-dried ME did not impact on the overall properties of this system, which could be administered by the parenteral route.

The same optimized parameters were used to freeze-dry the AmB-loaded ME. The results observed were similar to the system without the drug. Moreover, the drug content remained the same as that before freeze-drying. Therefore, this process did not induce drug degradation.

The ME morphology (with and without AmB), examined by transmission electron microscopy and scanning electron microscopy, was found to be similar before and after the freezedrying process. However, the droplet size was slightly increased after reconstitution of the system to the original volume with vortex stirring. During the lyophilization, the phosphatidylcholine present in the ME returned to the dry state. Therefore, in order to reform the ME droplets to the same size, the reconstituted ME must be heated above its transition temperature of 52°C.

The influence of the heating on the phase behavior of the ME system reconstituted from a lyophilized state was also studied 178 (TO BE PUBLISHED SOON). The changes in the droplet size and the transparency were evaluated. The use of vortex stirring followed by heating was able to reconsitute the ME after the lyophilization process, since at 60°C a phase transition from emulsion to ME occurred and at approximately 65°C the system showed a great transparency and a small droplet size. Indeed, by heating, the nonionic surfactant becomes more hydrophobic; therefore, less lipophilic surfactant is needed to reach the optimum HLB 179 . Furthermore, with increasing temperature the spontaneous curvature of the membrane becomes negative, which favors the formation of O/W structures 180 .

Conclusion

VL is a parasitic disease that can be fatal. It affects numerous countries, especially in emerging and undeveloped areas. The resistance to traditional antimonials, the toxicity of the micellar AmB formulation and the high cost of the liposomal AmB formulation have hindered the eradication of VL. Thus, the development of a less toxic and more affordable system containing AmB has been pursued. This review shows that promising studies have been carried out in order to obtain an affordable and effective ME system containing AmB to treat VL. In-vitro studies from our group have shown that the ME containing AmB was more effective against AmB-resistant strain of Leishmania donovani than the commercially available liposomal AmB formulation. In-vivo studies also showed a good profile of efficacy for the ME system. The literature suggests that the freezedrying is as an effective technique to improve the stability of drugs and pharmaceutical products.

Preliminary studies from our group aiming to improve the AmB-containing MEs' stability have shown that the freeze-drying MEs require optimum conditions and parameters to guarantee the success of the entire process. Our studies revealed that the freeze-drying technique was an excellent method to obtain a dried AmB-containing ME that was easy to redisperse. Despite the recent developments aiming to improve the treatment of VL, further studies with the freeze-dried ME containing AmB are still necessary in order to obtain all necessary information to establish this system as a new pharmaceutical product to treat VL. However, the formulations available on the market have a high cost, high toxicity and/or are subject to resistance from the parasite. Therefore, the work described in Chapter II was designed to find new ways to deliver AmB with low cost of production and low toxicity, which would make it possible for the populations at risk to have access to an affordable, efficient and safe formulation.

Therefore, two systems were produced: a microemulsion containing this drug and a system derived by heating the commercially available AmB micellar form. These systems were characterized for their physic-chemical properties and their toxicity and activity were evaluated in vitro and in vivo.

This work was carried out in Paris with the collaboration of Professor Philippe Loiseau (UMR 8076 CNRS BioCIS), responsible for the team "Chimiothérapie Antiparasitaire".

In-vitro and in-vivo

INTRODUCTION

Leishmaniasis is a parasitic infectious disease caused by obligate intra-macrophage protozoa of the Leishmania species, transmitted to humans via the bite of female sand flies of the genera Phlebotomus and Lutzomyia. This disease is widespread in tropical and subtropical areas as three different clinical manifestations: mucocutaneous, cutaneous, and visceral leishmaniasis (VL), also known as kala-azar, which is fatal if untreated (1).

Pentavalent antimonials are the most commonly used drugs to treat VL; however, they are no longer recommended due to their toxicity and the emergence of drug resistance (2). Amphotericin B (AmB) and miltefosine are the two alternative drugs able to replace antimonials, although neither drug is completely safe (3).This work is focused on the exploitation of AmB as low-cost and low-toxic formulations. AmB has been used as a second-line drug for leishmaniasis treatment (4). Despite its high efficacy, toxic effects, such as cardiotoxicity and nephrotoxicity, limit its successful therapeutic use. The AmB selectivity and toxicity level depend on its aggregation state, which can exist as monomeric, aggregated and super-aggregated states. In order to reduce the side effects credited to AmB, several lipid formulations have been developed. These systems prevent the AmB self-aggregation and slowly release AmB monomers to the surrounding medium (5). An example of success of the lipid-based approach is the liposomal AmB formulation (AmBisome ® ), which is less toxic than Fungizone ® , the commercial available micellar AmB (M-AmB). Nevertheless, AmBisome ® is not widely available due to its cost.

To summarize, the current visceral leishmaniasis treatment remains limited by the issues of drug resistance, toxicity and high cost (6). The therapeutic properties and pharmacological profile of AmB can be optimized by modifying the aggregation state of the drug (7) and by developing new drug delivery systems.

M-AmB has good availability and relatively low cost; hence it is the most widely used AmB-based product. However, the presence of AmB in the aggregated state allows the formation of ion channels in membranes containing cholesterol, which causes toxicity (8). Studies have shown that the heating of M-AmB is capable of inducing a new type of aggregate, called super-aggregates, which is less selective for the cholesterol in the mammalian cell membranes. Therefore, the toxicity can be significantly reduced without loss of activity (5, 7, 9, 10). The molecular rearrangement that yields super-aggregate structures can be achieved by appropriate heating of M-AmB (5, 11).

An alternative formulation without the drawbacks of cost and toxicity could be a microemulsion (ME). Microemulsions are anisotropic, clear, small droplet sized and thermodynamically stable drug delivery systems, comprised of an oil and aqueous phase, stabilized by surfactants (12). The ME would be able to carry AmB (ME-AmB) in its dispersed phase, increasing its solubilization and bioavailability, and decreasing its toxicity (13, 14).

The aim of this work was to investigate Heated M-AmB (H-AmB) and ME-AmB in comparison with M-AmB and AmBisome ® with regard to their in-vitro and in-vivo safety and antileishmanial activity against Leishmania donovani, in order to improve the therapeutic index of AmB while using inexpensive ingredients.

MATERIALS AND METHODS

Chemicals

Micellar AmB (Anforicin B ® ) was a gift from Cristália (Itapira, Brazil). Liposomal AmB (AmBisome ® ) was purchased from Gilead (Foster City, USA). Miglyol ® 812, used as the oil phase of the ME, was obtained from CONDEA Chemie GMBH (Hamburg, Germany).

Lipoid ® S100, used as surfactant, was purchased from LIPOID GMBH (Ludwigshafen, Germany). The AmB, used to load the ME, Tween ® 80, used as surfactant, Sodium Phosphate Monobasic and Sodium Phosphate dibasic, used to prepare the phosphate buffer pH 7.4, were all acquired from Sigma Aldrich Inc (St. Louis, USA).

Sample preparation

AmBisome ® was prepared according to the manufacturer's instructions. The commercial M-AmB was prepared by adding 10 mL of water for injection into the vial containing 50 mg of AmB (final concentration of 5x10 -3 M), followed by vortex shaking until dissolution. In order to obtain the H-AmB, the M-AmB was heated at 70°C for 20 min.

In order to prepare the ME, 68 % (w/w) of phosphate buffer pH 7.4, 14.7 % (w/w) of Tween ® 80, 6.3 % (w/w) of Lipoid ® S100 and 11 % (w/w) of Miglyol ® 812 were weighed. They were mixed under magnetic stirring followed by 3 cycles of probe sonication (80 watts power output) in a Branson Digital Sonifier S-250 (Branson Ultrasonic Corporation -Danbory, USA) for 1.5 min and followed by 3 min in a 1210E-MTH Bransonic Ultrasonic bath (Branson Ultrasonic Corporation -Danbory, USA). To incorporate AmB into the ME, solid AmB was added to a final concentration of 5x10 -3 M. Then, the system was alkalinized with NaOH 1M until complete AmB solubilization. After that the loaded ME was neutralized by addition of HCl 1M to a final pH of 7.4. Afterwards, the MEAmB was filtered in 22µm membrane.

M-AmB and H-AmB characterization

AmB molecules exhibit absorbance bands correlated to their aggregation state in the UV/Vis range of 300 to 450 nm. A PerkinElmer Lambda 25 UV/VIS spectrometer (Waltham, MA, USA) was used to analyze AmB-containing samples in 1cm path length cuvettes. Samples were diluted in pure water and analyzed immediately after dilution. M-AmB and H-AmB were diluted to 5x10 -6 M of AmB (5, 13).

Circular dichroism (CD) spectra were recorded with a Jasco J-180 dichrograph (Easton, MD, USA). Samples were diluted in water to 5.10 -5 M AmB 24h before analysis.

Measurements were conducted at room temperature using a 0.1 cm path length cuvette (15). Results are expressed as ΔƐ (differential molar absorption dichroic coefficient).

Microemulsion characterization

Droplet size distribution analyses were carried out by Dynamic Light Scattering (DLS) using a Malvern-Zetasizer Nano ZS (Malvern, UK). Transmission electron microscopy (TEM) was used to investigate the ME morphology. The MEs was observed after staining with 2% phosphotungstic acid, using a JEOL 1400 apparatus (SamXPlus, France), operated at 80kV as the acceleration voltage, equipped with a high resolution CCD Gatan digital camera (SC1000 Orius, France). Before performing both types of analysis, the MEs were diluted 1:20 with water.

The rheological properties of the MEs were evaluated by a Haake Rheo Stress 600 rheometer equipped with 35mm cone-plate geometry (Thermo Scientific, USA). The flow curve and the viscosity curve of the samples were performed by controlled shear rate rotation tests (from 0.1 s-1 to 1000 s-1).

The aggregation state of the AmB in the ME-AmB was also evaluated by spectrophotometry and CD as described above.

In vitro antileishmanial activity

The promastigote stages of Leishmania donovani (MHOM/IN/80/DD8) wild-type and of the AmB-resistant (AmB-R) strain obtained from the wild-type parasites by in-vitro drug pressure were grown in M-199 medium supplemented with 40 mM HEPES, 100 μM adenosine, 0.5 mg/L haemin, and 10% heat-inactivated fetal bovine serum (FBS) at 26 °C in a dark environment under an atmosphere of 5 % CO 2 . Parasites in their logarithmic phase of growth were used for all experiments. Promastigotes were grown at 37 °C in 5% CO 2 atmosphere for 24 hours before treatment for differentiation. The axenic amastigotes were generated by differentiation of promastigotes. To achieve this, a 1 × 10 6 promastigote suspension was diluted in 5mL of axenic amastigote media (1 X M-199, 40 mM HEPES, 100 μM adenosine, 0.5 mg/L haemin, 2 mM CaCl 2 , 2 mM MgCl 2 and 20% FBS) and the pH was adjusted to pH 6.

Evaluation of the in vitro antileishmanial activity on axenic amastigote stage

Axenic amastigotes were suspended at 5 x 10 6 cells/mL in a final volume of 200 µL.

Serial dilutions from 100 to 0.39 µg AmB/mL was performed by distributing AmBisome ® , M-Amb, H-AmB, ME-AmB, and ME in 96-well plates. Triplicates were used for each concentration. The viability of the axenic amastigote stage was assessed using the trypan blue exclusion method after a 3-day incubation period at 37 °C, in the dark, under a 5% CO 2 atmosphere. Parasite growth was determined by using SYBR Green I (Invitrogen, France), a dye whose fluorescence is greatly enhanced upon contact with the parasite DNA. The plates were frozen 3 times and the DNA was extracted from the plates according to the Direct PCR (cell) protocol (Viagen Biotech Inc, Eurogentec, France). Into the PCR plate 10µL of Lysate and 40 µL of direct PCR (cell) supplemented with SYBR green I (5 µL of SYBR Green I / 10 mL of lysis buffer) were added. Untreated parasites were used as control. The fluorescence was directly evaluated on a Mastercycler ® ep realplex real-time PCR system (Eppendorf, France). The program was as follows: 90 °C, 1 min and ramp time of 5 min to obtain 10 °C, during this period the fluorescence was obtained in continue and in a hold step at 10 °C. The curves of fluorescence were analyzed and the fluorescence at 10°C was used to determine the concentrations inhibiting parasite growth by 50 % (IC 50 ). Indeed, the IC 50 , the concentration inhibiting the parasite growth by 50%, was determined using the software in line at http://www.antimalarial-icestimator.net/MethodIntro.htm.

Evaluation of the in vitro antileishmanial activity on intramacrophagic amastigotes

RAW 264.7 cells (mouse leukemic monocyte macrophage cells) cultured in DMEM with L-glutamine (Life technologies) and 10% FBS (Life technologies) were suspended to yield 1.5 x 10 5 cells/mL in 96-well plate, in a final volume of 100 µL. After a 24 h incubation period, cells were infected by a suspension of 1 x 10 6 Leishmania donovani axenic amastigotes/mL in DMEM, and incubated for 24 h at 37 °C in the dark and under a 5 % CO 2 atmosphere. The free parasites were, then, eliminated by washing and intramacrophagic amastigotes were treated with the same AmB samples and concentrations as those in the assay on axenic amastigotes. After 48 h of incubation under RAW 264.7 cells maintained in DMEM supplemented with 10% of heat-inactivated FBS (Life technologies) were seeded to early confluence in a 96-well plate at density of 7500 cell/well and incubated in 5 % CO 2 at 37 °C for 24 h in a final volume of 100 µL.

Thereafter, M-AmB, H-AmB, ME-AmB, AmBisome ® and ME were added to achieve final AmB concentrations from 0.1 to 108 µM and incubated for a further 24 h under the same conditions. In order to estimate background absorbance due to light scattering, wells without cells, but containing the same concentration of the samples, were analyzed. The cytotoxicity of the formulations was evaluated using the MTS [3-(4,5-dimethyl-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay, whereupon 20 μL of MTS solution were added into the wells and incubated for further 2 h. Finally, the absorbance was measured using a 492-nm high-pass filter in a Multiskan MS microwell plate reader (Labsystem, Ramat-Gan, Israel) and the IC 50 for each formulation was calculated. For this assay 4 replicates were performed.

In vivo toxicity assay

Blood samples taken from the mice from the antileishmanial activity experiment were kept at room temperature for about 30 min and, then, centrifuged at 3,500 g for 10 min. In order to evaluate the renal and liver toxicity, serum was harvested from each blood sample and total serum cholesterol (CHO), alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanineaminotransferase (ALT), creatinine (CREA) and urea (URE) concentrations were determined by commercially available kits according to the manufacturer's instruction.

Statistical analysis

Statistical analyses were performed by using analysis of variance or Kruskal-Wallis tests (nonparametric), followed by the Tukey′s or the Dunn's multiple comparison test. GraphPad Prism5 version (GraphPad Software Inc., La Jolla, CA) was used to perform the analysis. Significance was established for a P value < 0.05.

RESULTS

M-AmB and H-AmB characterization

The UV-Vis spectrum for M-AmB (Figure 1a) showed four main peaks at 329, 367, 388 and 408 nm. The first peak is credited to the aggregated form of AmB whereas the one at 408 is ascribed to the monomeric form (15). After heat treatment it was observed that H-AmB (Figure 1a) presented similar peaks to M-AmB regarding the monomeric form, but the aggregate-related peak underwent a blue shift and became centered on 323 nm in this preparation. The UV-Vis results were in accordance with those observed by circular dichroism (Figure 1c), in which the doublet related to the AmB aggregation was also blueshifted for H-AmB.

Microemulsion characterization

The produced ME was optically clear with a mean droplet size of 22 nm for the unloaded ME and 36 nm for the ME containing AmB, with polydispersity indexes of 0.170 and 0.594, respectively, indicating that the addition of AmB led to slightly larger particles. TEM revealed that the droplets were spherical in shape even after the addition of AmB (Figure 2). Furthermore, the average size was similar to the DLS results. The rheological analysis revealed that both the unloaded ME and the ME containing AmB (Figure 3) showed a linear relationship between shear stress and shear rate, which is characteristic of a Newtonian material. The apparent viscosity of the unloaded ME and ME containing AmB were 0.0623 Pas ± 0.0101 and 0.1141 Pas ± 0.0049, respectively, which is low because the ME was of the oil-in-water type. The low viscosity would ensure good syringeability as well as ease of mixing with intravenous fluids using minimum mechanical agitation. The AmB molecule showed to be in both aggregated and monomeric forms (Figure 1b and1d), since peaks at 323 nm (aggregated form), 367, 388 and 408 nm (monomeric form) were observed. All these results: the small spherical droplet size, the transparency and the low viscosity, allow these samples to be characterized as a true ME system. The incorporation of AmB did not affect these parameters.

Toxicity of the formulations

The CC 50 was evaluated in vitro for each formulation (Table 1). M-AmB showed high toxicity towards RAW cells (CC 50 4.5 ± 0.4 µM). However, when this formulation was heated this value increased approximately 8-folds to 39.8± 3.2 µM, indicated a decrease in toxicity. In fact, in this study, the toxicity of H-AmB was similar to that of AmBisome ® . On the other hand, although the incorporation of the AmB into the ME decreased the CC 50 of the formulation compared to the unloaded ME, the cytotoxic effect of ME-AmB (CC 50 9.0 ± 0.3) was slightly lower than that of M-AmB.

The results of the evaluation of the toxicity in vivo are given in Table 2. There were no significant changes (p>0.05) among the samples for the two transaminase activities levels (AST and ALT) and the creatinine levels. However, all treatments caused a significant increase (P<0.05) in the urea level except for mice treated with unloaded ME. A significant decrease in this parameter was also observed in the mice treated with the ME and the ME-AmB compared to the AmBisome ® . Furthermore, it is noteworthy that heating of M-AmB slightly decreased the blood urea levels. The ALP value increased significantly with all treatments compared to the untreated group. Nevertheless, the ALP level for the mice treated with the AmBisome ® was significantly lower than that of the mice treated with the ME-AmB. The CHO results showed no difference between the ME, the AmBisome ® and the untreated group. However, M-AmB, H-AmB and ME-AmB caused a significant decrease in this parameter, while the lowest values were observed in the mice treated with the ME-AmB.

Antileishmanial activity

The activity of the formulations against Leishmania donovani was evaluated in vitro and in vivo. The in-vitro assays were performed on both Wild-type (WT) and AmB-R intramacrophagic and axenic amastigotes (Table 1). All formulations, except ME (unloaded microemulsion), showed high activity against WT parasites. ME-AmB and H-AmB yielded lower IC 50 values than the AmBisome ® for both amastigote stages. As far as the activity on axenic amastigotes was concerned, all the formulations exhibited lower IC 50 compared to the intramacrophage amastigote. However, the IC 50 of the AmBisome ® did not change significantly (1.73 µM and 1.76 µM).

The H-AmB showed a higher IC 50 than the M-AmB, when the Leishmania was within macrophage, and similar antileishmanial activity (IC 50 = 0.05 µM) against the axenic stage.

However, the selectivity index for the H-AmB was approximately 3-fold higher than that of the M-AmB. Moreover, the ME-AmB showed a better selectivity index than the M-AmB, although it remained lower than the AmBisome ® . Some clinical isolates of Leishmania donovani have shown resistance against the AmB (18). Therefore, the issue of AmB resistance should be considered early in the development of any new AmB delivery system. With this in mind, the formulations were evaluated against both AmB-resistant axenic and intramacrophagic amastigotes. The AmBisome ® was active against AmB-resistant intramacrophage amastigotes, but it did not show any activity on AmB-resistant axenic amastigote. On the other hand, the other formulations were active in a range from 0.8 to 4 µM on both axenic and intramacrophagic amastigotes. The drug pressure to get the AmB-R line was previously carried out using M-AmB. Indeed, the IC 50 value of M-AmB on the AmB-R line was about 30 times higher than those of the WT, both on axenic and intramacrophage amastigotes. Interestingly, the IC 50 values of H-AmB, AmBisome® and ME-AmB on the AmB-R intramacrophage amastigotes were only about twice as high as those on the WT. These results suggest that these formulations could be useful for the treatment of AmB-R leishmaniasis.

The in-vivo antileishmanial evaluation was performed on the Leishmania donovani/Balb/C mice model. The percentage of parasite burden reduction in vivo is shown in the Table 2. Whereas the unloaded ME alone did not show good activity in vivo, M-AmB, H-AmB, ME-AmB and AmBisome ® all reduced the parasite burden greatly, with no significant difference between the formulations. No sign of acute toxicity was observed in the mice and the biological parameters did not show significant toxicity, as shown in Table 2.

DISCUSSION

The MEs prepared in this study showed a spherical shape and a small droplet size, which is in agreement with other studies that describe these systems as having diameters less than 150 nm (12, 14). Although the addition of AmB to the ME increased its droplet size and polydispersity index, probably due to the large molecule size and its deposition on the ME interface, to the heterogeneous distribution of AmB in ME or to the formation of other nanostructures as a result of interaction between AmB and components of the ME's.

Nevertheless, the ME-AmB droplet size remained within the acceptable range. Such small droplets within MEs produce only weak scattering of visible light, and as a result the system is transparent, as observed macroscopically in this work. Moreover, the probability of embolus formation after injection of MEs is insignificant due to their small droplet size (12).

Since the nanosized droplets do not interact with each other, the resulting MEs have low viscosity (19). The viscosity analysis is of utmost importance since the parenteral administration of very viscous samples could be painful for the patient. Furthermore, it is well known that the syringeability is affected by the viscosity of the parenteral formulations (20). The ME and ME-AmB showed a constant low viscosity and proportionality between shear stress and shear rate, for which they were deemed to behave as Newtonian fluids.

As a consequence, they can be considered suitable for the intravenous route. This low viscosity ensures ease of mixing with intravenous fluids with minimum mechanical agitation as well as good syringeability and, therefore, can ensure good patient compliance. Spectrophotometric studies revealed that after the heating process, superaggregates are formed from M-AmB, manifested as a blue-shift in the electronic spectra as well as a decrease on the dichroic doublet, which is also blue-shifted in the spectra of H-AmB (15). When AmB was loaded into the ME, a pattern similar to H-AmB was observed.

Since the system was not heated in order to form AmB super-aggregates, the peak at 323 nm probably represents the association of the AmB with the ME-oil phase. Pham et al, (2014) (21) have shown blue-shifts in the AmB dichroic doublet when the molecule is loaded into liposomes. Our results are in agreement with Silva et al (2013) (22), who have developed ME systems to carry AmB and have demonstrated peaks of AmB aggregates and monomers at the same time. However, the aggregate peak is not the same as one observed in our work, supporting the theory of that such a peak is a result of a complex formed between the molecule and the system, which for instance depends on the used raw material, mainly the surfactants. Larabi et al (2004) (23) reported studies in which absorption spectrum and circular dichroism varied according to the proportion of lipid in the formulation. Knowing the aggregation state of the molecule inside the carrier is very important since it influences the drug efficacy and toxicity (5, 9, 13, 15, 24).

The treatment of visceral leishmaniasis was dominated by pentavalent antimonials for a long time. However due to their toxicity and adverse side effects, these drugs have progressively been replaced as the first-line treatment by the AmB, which is considered as the best existing drug against this disease. Among the different commercial formulations, Fungizone ® (a mixture of AmB with deoxycholate in a phosphate buffer) is the most frequently used. However, the liposomal formulation is the most effective and safe, despite its high cost (4).

In this work, we have proposed two new formulations of AmB which retain its activity while reducing its toxicity, and are furthermore inexpensive, with costs accessible for patients from the tropical endemic countries and easy to manufacture. Both H-AmB and ME-AmB were thoroughly characterized and shown to be appropriate for parenteral use against visceral leishmaniasis. In order to investigate the antileishmanial activity of these formulations, their IC 50 in axenic and intramacrophage amastigote were evaluated in vitro, as well as the percentage of reduction of parasite burden in vivo. In addition, the cytotoxicity of the formulations was examined in vitro, allowing calculation of the Selectivity Index, while the toxic effects in vivo were analyzed by assessment of renal and liver function, because the most serious side effect of AmB is nephrotoxicity (25).

Both in-vitro and in-vivo experiments with the WT line demonstrated that, as expected, all formulations containing AmB were effective against Leishmania donovani. On the other hand, despite a moderate effect in-vitro, unloaded ME did not show significant invivo antileishmanial activity, proving that it was the addition of AmB that produced this effect. Some differences in the in-vitro effectiveness were observed when comparing the axenic and intramacrophagic amastigotes. The axenic amastigote assay allowed us to evaluate an intrinsic activity on parasites, whereas the intramacrophage amastigote assay demonstrates the ability of the active drug to be delivered through the macrophage membrane, the parasitophorous vacuole membrane and finally into the parasite (26, 27). It is noteworthy that AmBisome ® was not active against AmB-R axenic amastigotes while the other formulations did have some activity. This result cannot be a result of the ME diameter being less than that of AmBisome ® (35 vs 80 nm, respectively) since H-AmB, AmBisome® and ME-AmB had similar activities on the WT intramacrophage amastigotes. This observation could be related to the composition of the AmB-R membranes, poor in sterols, more fluid than the WT membranes and, therefore, more stable and less sensitive to a liposomal effect. With the intramacrophagic amastigotes, this difference was not observed since all the formulations after macrophage phagocytosis released AmB within the parasitiphorous vacuole, probably after fusing with lysosomes. Indeed, AmBisome® is a very stable formulation with cholesterol and a high-transition-temperature phosphatidylcholine making a very impermeable membrane and phosphatidylglycerol forming an electrostatic complex with AmB. It is possible that parasite-derived enzymes are necessary to release the AmB from the liposomes and that the AmB-R parasites have a different enzyme profile and are less efficient at this. On the other hand, AmB may be less strongly associated in M-AmB, H-AmB and ME-AmB. Further studies could be focused on the ME 78ehavior within the infected macrophages through confocal studies.

M-AmB showed the lowest selectivity index, while H-AmB presented the highest index. Therefore, the heating process was capable of considerably reducing the cytotoxicity of AmB in vitro as demonstrated by the CC 50 values. This result is supported by Gaboriau et al. (1997) (28) and Petit et al. (1998) (10) whose have found the mild heating as a simple way to decrease the toxicity of micellar AmB systems. In addition, the therapeutic window between the IC 50 (1.53 µM) and CC 50 (39.8 µM) of H-AmB is wider than that of M-AmB, thus improving its safety. It is interesting to mention that H-AmB have shown to favor drug uptake by macrophage-like cell line (9). The authors suggest that this fact could be interesting since the macrophage could act as a reservoir, releasing monomeric AmB on the infection site and, thus, improving antiparasitic effect (9). ME-AmB (selectivity index = 10.23) also showed a better selectivity compared to M-AmB. When the selectivity index is greater than 10, it is generally, considered that the pharmacological efficacy is not due to the in-vitro cytotoxicity (29), therefore, the incorporation of AmB in ME systems was able to improve the effectiveness of the drug.

The potential of the new formulations (H-AmB and ME-AmB) was confirmed by the in-vivo studies, which showed no significant differences in their ability to reduce the parasite burden compared to the AmBisome ® . In addition, both formulations demonstrated minimal cytotoxicity in vivo.

The biochemical evaluation of kidney and liver parameters showed that the untreated infected mice had lower values than those with no parasite burden (reference values were found in (30). Some studies have described a reduction of the serum cholesterol concentrations as a function of the splenic parasite burden, since this organ is responsible for cholesterol biosynthesis (31). An overview of all the biochemical results showed that despite some values, which were statistically different, there was a minimal variation among the groups, showing that none of the formulations induced toxicity at the doses used in this study. Therefore, ME-AmB and H-AmB were considered safe regarding the limited damage to macrophage and the in vivo evaluation. Furthermore, ME-AmB, M-AmB and H-AmB showed efficiency against AmB-R intramacrophagic amastigotes. AmB-unresponsive cases have been reported at the Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Bihar, India. These drug-resistant cases are to be expected due to the very high frequency of AmB use (18).

Since visceral leishmaniasis is fatal, the development of new systems, which do not demonstrate drug resistance is very important to ensure that suitable treatment would be available. Therefore, these two inexpensive alternative formulations for AmB showing good efficacy and selectivity for Leishmania donovani merit further investigation. Unfortunately, it was not possible to verify the in-vivo efficacy of the formulations on an AmB-R/ mice model since the AmB-R parasites are poorly infectious for Balb/c mice (32).

CONCLUSION

These experiments show a novel approach to AmB therapy, addressing both the cost of formulations and the AmB-resistance. The formulations H-AmB and ME-AmB proved to be very successful in the treatment of a Leishmania donovani Balb/c mouse model. Both in-vivo and in-vitro evaluations showed good efficacy and low toxicity for these formulations.

Since the method of preparing the H-AmB is just the heating of the reconstituted M-AmB for a few minutes, it appears to be a low cost alternative to the commercially available products. The cost of producing MEs is also lower than that of producing the liposomal AmB, since the components are commonly used pharmaceutical excipients rather than expensive phospholipids. Therefore, the formulation developed in this work is a promising way to reduce toxicity while maintaining the efficacy of AmB. * There was no significant difference in comparison to untreated (P>0.05) ** There was significant difference in comparison to AmBisome ® (P<0.05) a P > 0.05 b P < 0.05 BRIEFING Since the results shown in the previous chapter demonstrated that a microemulsion containing Amphotericin B (MEAmB) has a good activity against Leishmania donovani and low toxicity, it is important to think about the fate of the formulation during storage.

The high water content (63%) in the MEAmB makes the formulation susceptible to the drawbacks of high humidity, such as microbial contamination and hydrolysis. A way of solving this problem could be to remove the water through a drying process that does not use high temperatures, since AmB is a thermolabile drug.

Lyophilization, also known as freeze-drying, is a process that removes the frozen water from a material by sublimation using low temperatures, which has already been used to improve the stability of emulsified systems. However, an in-depth study on lyophilization of emulsified systems is required since these systems are not easily freeze-dried due to the components present in their formulation and to the process, which can cause damage such as drug loss, precipitation, loss of system structure, coalescence and phase separation.

Several factors may influence the outcome of freeze-drying;, including the thickness of the frozen layer, composition and concentration of the product, type and concentration of cryoprotectant, the equipment and parameters of the lyophilization process, such as time and temperature (freezing, primary and secondary drying). For this reason, it is important to use appropriate techniques to identify the variables that influence the process and to choose the best parameters to obtain the optimal response. Design of experiment (DOE) is a tool used to improve the quality of products and processes, able to evaluate a high number of variables using a reduced number of samples. Therefore, DOE was employed in this study to determine the best parameters for the lyophilization of AmB-loaded microemulsions in order to increase their stability.

experimental decision-making based on studies already carried out and to avoid unnecessary experiments, thereby increasing the probability of success. Therefore, a review about freeze-drying of emulsified systems was carried out to assist the development of a freeze-dried microemulsion containing AmB. This review helped to define the types of cryoprotectant to be used in the experimental study and their concentration range, as well as the time and temperature ranges to be tested. Amphotericin B (AmB) is the drug of choice for the treatment in immunodeficient patients affected by systemic fungal infections, as well as local treatment of fungal infections in the eye. (5, 6) However, due to the high incidence of adverse drug reactions, i.e. cardiotoxicity and hepatotoxicity of conventional formulations,(7) AmB has been incorporated into a number of colloidal drug delivery systems, including MEs in order to improve its therapeutic index. (8, 9) Generally, pharmaceutical MEs are produced with water as the continuous phase, which carries some risks such as microbiological contamination, degradation by hydrolysis and loss of pharmacological activity of the drug. A possible solution to these problems consists of the use of freeze-drying process, also known as lyophilization. Freeze-dried products have good stability and are easy to transport and store.(10) However, the freeze-drying process for MEs has rarely studied in been studied in pharmaceutical technology due to the difficulties that arise during freezing of colloidal systems. Lyophilization consists in removing water by sublimation through three steps: freezing, primary drying and secondary drying. (11) During this process stress may be generated that could destabilize the colloidal structure of the MEs, in particular the freezing stage, during which the crystallization of ice may produce mechanical stress.

INTRODUCTION

However, cryoprotectants (CP) can be used in order to protect these systems against damage.(12) Among the compounds that can exert cryoprotective effects, carbohydrates are interesting because are chemically innocuous and can be easily vitrified during freezing, supporting their use as CPs in the freeze-drying process.( 9)

The freeze-drying process has been used for several years in the pharmaceutical and biotechnology industries, leading to products destined for various administration routes such as parenteral, oral, nasal or pulmonary. However, the freeze-drying of colloidal systems is delicate due to the tendency of the droplets to interact and damage caused by the freezing process, which can increase the droplet size and destabilize the system. (10) In fact, there are very few reports in the literature of the successful freezedrying of colloidal systems such as ME.

The quality of the final freeze-dried product can be influenced by several factors relating to the formulation, to the container, to the equipment and to the freeze-drying process. (10) Therefore, the process efficiency must be optimized through adjustment of these factors and process monitoring is crucial in order to obtain a product with the desired quality. It is necessary to select suitable freeze-drying parameters, evaluate the effects of these parameters and their possible interactions with the entire process.

Therefore, Design of Experiment (DOE) has been used for the optimization, modeling and characterization of the freeze-drying process of some pharmaceutical products that are not colloidal systems. (13) DOE is a statistical approach used to determine the influence of several independents variables on the dependent variable of the process. The optimal design allows the time and cost of the experimentation to be reduced, as well as improving the process yield.( 14) Therefore, this method is used much more often than the One Factor at a Time Method, which is time-consuming and expensive because it requires a large number of experiments and does not examine interactions between the variables. (15) The response surface methodology is a technique of DOE that combines mathematics and statistics to analyze the relative significance of different parameters, finding the optimal working conditions, by combining a small number of variables, resulting in fewer experiments. (16) The aim of this work was to develop a freeze-dried ME product as a model to design freeze-dried ME systems containing drugs and thereby improve their stability.

Therefore, AmB was used as a model drug incorporated within the ME. A DOE approach was used in order to establish the ideal CP content and the technical procedure to produce freeze-dried colloidal systems. Important parameters, which can influence the droplet size of this system, such as concentration and type of CP, freezing temperature and freeze-drying time were also evaluated. Corporation, NY, USA). Previous experiments revealed that this dilution process has no influence on the DLS measurements.

MATERIALS AND METHODS

Materials

Differential Scanning Calorimetry (DSC)

In order to characterize the type of system (water-in-oil, bicontinuous or oil-inwater), (17) the thermal behavior of the ME and its components were analyzed by DSC using a DSC-60 Shimadzu (Shimadzu Scientific Instruments, Kyoto, Japan). The samples (5-15mg) were weighed into aluminum pans. A temperature ratio of 5 °C/min, a nitrogen atmosphere at the flow of 50 ml/min and cooling at a pre-determined rate from 25 °C to -40 °C (for 10 min) followed by heating at a constant scanning rate up to 110 °C were used. An auto-cooling system TAC-60i (Shimadzu Scientific Instruments, Kyoto, Japan) was used to decrease the temperature during the cooling curve.

DOE methodology for freeze-drying conditions

In order to determine the optimum parameters for the freeze-drying process, the DOE methodology was performed for ME systems for which no changes in droplet size were observed after CP incorporation. The CP type and concentrations, the freezing temperature and the freeze-drying time were chosen as independent variables. The ME droplet size after freezedrying process, on the other hand, was chosen as the dependent output response variable. The experimental levels of independent variables for ME droplet size after freeze-drying are given in Table 1.

Table 1

The levels of the variables CP concentration and freeze-drying time were chosen in accordance with the values commonly found on the literature. On the other hand, the freezing temperature used was selected according to the temperature attained by the use of liquid nitrogen (-196 ºC -the minimum temperature), the temperature produced by a regular freezer (Frost Free 260 (Brastemp, São Paulo, Brazil) (-20 ºC -the maximum temperature) and the temperature produced by a ultra-freezer Glacier NU-9438 ULT Freezer (NuAire,Inc., Minnesota, U.S.A.) (-80 ºC -which is the DOE replicated point). A two-level three-factor fullfactorial design with three replicated points leading to 8 experimental randomizations runs was performed for each CP (Table 2).

Table 2

Freeze-drying was performed using a Christ Alpha 1-2 LD freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany). After the freeze-drying process, the MEs were reconstituted by adding the same amount of water as that lost during the process, followed by vortex mixing and one cycle of sonication (40 watts power output for 1.5 min), after which the droplet size was measured by DLS.

The effect of the studied variables was graphically and statistically interpreted using the Statistic software version 7.0 (StatSoft Inc., Oklahoma, USA). Based on the statistical results, the parameters that would produce a dry powder ME after freezedrying and that would present the smallest droplet size values after reconstitution were determined. Thereafter, the ME systems that showed the best aspect and concentration of CP, and the best freezing temperature and the freeze-drying time, was produced and thermodynamically characterized for their pH, conductivity and droplet size before and after the freeze-drying process. The optimized formulation was then used for further studies, including drug loading.

Incorporation of AmB to the ME system

AmB was incorporated, as a model drug, into the optimal ME system determined by the DOE methodology. The aim was to observe the efficiency of the freeze-drying process on the production of dried ME systems containing drugs. The production of a dried powder for this system was verified and pH, conductivity and droplet size before and after freeze-drying were measured. Furthermore, the influence of drug loading on the thermodynamic stability of the ME was evaluated.

The incorporation of AmB was performed by adding the drug to the ME to a final concentration of 2 x 10 -3 M, under continuous stirring. In order to improve the AmB dissolution, the pH of the ME was increased (pH ≥ 10.0) by the addition of sodium hydroxide solution (NaOH 1 N). Afterwards, the pH was reduced using hydrochloric acid solution (HCl 1 N) to physiological pH (pH 7.4).( 8)

Spectrophotometric analysis

A spectrophotometric assay was performed to analyze and compare the amount of AmB before and after the freeze-drying process, and to evaluate its stability. The experiment was carried out using a Biochrom Libra S32 UV-VIS spectrophotometer (Biochorom US, Massachusetts, USA), at the wavelength of 405 nm, by measuring the absorbances of the reference solution (methanol) and of the ME with and without AmB.

The assay was performed in triplicate. The ME samples were first diluted with DMSO:

methanol (1:9) at the ratio of 1:10 and, then, 50 µL of this solution was diluted with 20 ml of methanol. The drug loading efficiency was calculated by comparing the AmB concentration before and after centrifugation (Centrifuge 5410, Eppendorf, Hamburg, Germany), which was performed in triplicate by centrifuging 1 ml of ME with and without AmB at 14.000 g for 20 min to precipitate any insoluble content. Moreover, an aliquot of 100 µL of the supernatant of each sample was used to evaluate the AmB content as previously described.

Statistical Analysis

The statistical tests used in the experiments were the Analysis of Variance (ANOVA) performed to analyze the statistical significance between 3 groups, followed by Dunnet post-test, to compare with the control or Bonferroni to compare all samples.

Finally, a paired t-test was used in case of 2 unpaired groups. P values less than 0.05 (p<0.05) were considered to indicate significance.

RESULTS AND DISCUSSION

Characterization of the ME system before adding the CP

Macroscopic aspect, pH evaluation, isotropy and conductivity analysis

The ME, a homogeneous and clear yellowish product that appeared dark under cross-polarized light microscopy (no birefringence), was classified as an isotropic system. Its pH value was 7.1 ± 0.09, which is considered physiologically acceptable.

In general, MEs can be divided into 3 types: water-in-oil (W/O), bicontinuous, and oil-in-water (O/W).( 18) A correlation between ME structure and electrical conductivity has been demonstrated and can be used as a tool to assess its properties. (19) The electrical conductivity of the ME studied was 832 ± 22 µS cm -1 . It is well known that O/W MEs have conductivity values similar to the aqueous phase. (20, 21) This high value is probably due to the large volume percentage of water in the system, from which can be inferred that the ME is of the O/W type. (22, 23) could release water on conversion to the anhydrous crystal form and thereby cause phase separation in the cryo-concentrated portion of the frozen samples, resulting in the loss of CP activity and destabilization of the system. (15, 29) Formulations in which a dry powder were generated, ME containing MN 20%

(w/w) and all samples of MEs containing MT and LT, were reconstituted with the same amount of water as that lost during the freeze-drying process and presented a Winsor IV ME system. Moreover, the droplet sizes of these samples were evaluated and a Pareto's Chart to investigate the standardized effect of the independent variables and their interaction with the ME droplet size was constructed. The results for MEs containing MT showed that the "square freezing" is the most important factor influencing the droplet size. The negative coefficients for the square freezing variables indicated an unfavorable or antagonistic effect on the droplet size. Therefore, the closer to the lower limit (-1 level) of the square freezing temperature (-20 ºC), larger the droplet sizes. In other words, the higher the freezing temperatures, the larger the ME droplet sizes (Figure 3a). (30) Freezing is the stage at which most of the water is separated from the solutes to form ice and concentrated solution. (11) In the specific case of MEs, the "concentrated solution" is composed of oil, surfactants and buffer salts. Increasing the concentration may enhance the interaction between the droplets, leading to their aggregation or fusion and increasing the droplet sizes.( 25)

The Pareto's Chart analysis of the MEs containing LT (Figure 3b) showed positive coefficients for model components (x 2 , x 3 , x 2 x 3 , x 1 x 3 ) showed a synergistic or favorable effect on the droplet size. Additionally, the CP concentration was the most significant variable for droplet size variation.(30)

Date et al (31) The statistical significance, evaluated using the Fisher's F-test and ANOVA, showed, based on probability values inferior than 0.05, that it was indicated for the quadratic term of freezing in MEs containing MT. On the other hand, MEs containing LT presented statistical significance for concentration, freeze-drying, interaction between them, and interaction between freezing and freeze-drying. Furthermore, the ANOVA results (Table 3) for both MEs showed that the calculated F value was found to be greater than the tabulated F value at the 5% level, indicating that the treatment combinations are significant. Moreover, concerning the lack of fit and pure error, the calculated F < tabulated F demonstrated that the model was predictive. (16) The value of the coefficient of determination for MEs containing MT and LT were, respectively, R 2 = 0.9236 and R 2 = 0.9120, indicating that 92.36% and 91.2% of the response variability elucidated by the analysis of response surface diagrams, which was able to evaluate the relative significance of all factors involved in the process (Figure 5). Throughout this analysis it was possible to determine the optimum operational conditions for the system and the process. (32) It was observed that the ME droplet size decreased with the reduction on the freeze-drying time and MT concentration in a linear way. However, when freezing time and other independent variables were compared, no direct linear relationship among them was observed. Nevertheless, it was possible to identify the smaller droplet size values of ME-MT at -80 ºC of freezing temperature (Figure 5a and Figure 5b).

The response surface analysis revealed that the droplet size increased with increasing LT concentration and freeze-drying time. In fact, the smallest droplet size was found at the central point of freezing temperature (Figure 5c and Figure 5d).

Figure 5

Therefore, the best parameters for producing small droplets for freeze-dried MEs using either MT or LT as CP were a concentration of 5%, a freezing temperature of -80 ºC and a freeze-drying time of 24 h. Since at this concentration ME-LT become rubbery few minutes after the freeze-drying process and at a higher concentration the CP could not be dissolved in the system, MT was chosen as the best CP for this ME.

Characterization of the ME before and after freeze-drying

The general physicochemical characteristics of the ME-MT at 5% showed some changes before and after the freeze drying process. Before freeze drying, the ME-MT presented a droplet size of 52 nm ± 0.7, a pH value of 7.1 ± 0.09 and a conductivity 123 value of 832.9 µS cm -1 ± 22.1. After the freeze drying process and subsequent dispersion with water, its macroscopic appearance remained homogeneous and a clear yellowish product was obtained. However its pH, conductivity values and droplet size were slightly different (Table 4).

Table 4

The pH value decreased, probably due crystallization of the buffer salts during the freezing drying process. (11) However, even at this pH (6.86), the ME-MT is physiologically acceptable.

The reduction on the droplet size of the ME-MT might be due to the cosurfactant role that CP could play. The short poly-hydroxyl chain-alcohol could be adsorbed and intercalated into the interfacial film of the ME after the freeze-drying process and reconstitution, thereby decreasing its surface tension. (33, 34) However, at this droplet size (20.13 nm) the sample remains within the ME droplet size range of 10 -100 nm. (35) The decrease of conductivity just after CP addition and before the freeze drying process was probably due to its protective mechanism that enhances the viscosity of the system due to the interaction between the hydroxyl from the CP and the water molecules. In fact, it is well known that the crystallization of ice can be suppressed by the increase on the viscosity of the medium, which limits the mechanical damage. (25) Therefore, a decrease in the amount of ions can induce a reduction in the conductivity value of the ME-MT.

On the other hand, the increase of this conductivity value in the reconstituted ME-MT can be explained because when the water is removed during the drying process, the ions from the buffer remain in the sample. Thus, when the water was added to (Table 4 and Table 5). In fact, the ionization of functional groups on AmB (carboxyl and/or amino groups) may increase the amount of ions in the system, thereby increasing the electrical conductivity. (37) The increase in the droplet size may be explained by the partitioning of AmB molecules into the oil phase since it is insoluble in water, increasing the oily phase volume. Furthermore, due its amphiphilic properties, the molecule might be located in the system interface, also increasing the droplet size.( 38)

A standard curve (data not shown) with increasing concentrations of AmB was constructed in order to determine the amount of AmB in the ME. The method showed to be robust, efficient and sensitive. Thus, the linear equation (y = 1.2565x -0.0257 and R 2 = 0.9992) could fit more than 99% of the experimental data. Therefore, the methodology may be confidently used for quantitative analysis of AmB. The AmB content of the ME remained unchanged after the freeze-drying process (p > 0.05).

Therefore, MT at 5% was effective in preventing ME diameter changes and loss of AmB.

Although the AmB loading efficiency (encapsulation of the drug) was quite low (24.4%), the freeze-drying process did not change the concentration of entrapped drug (p > 0.05). Therefore, no loss of drug occurred during the process.

MT was an efficient CP avoiding drug loss, probably as a result of its ability to form hydrogen bonds to phospholipid head groups and replacing the water by reducing the gel to liquid crystalline phase transition that occurs when the dried phospholipids are rehydrated. In this way, the MT did not lead to inhomogeneous rearrangement of phospholipids that could result in particle aggregation and loss of incorporated drug into the aqueous medium.( 10) research teams, who have looked at features of M-AmB and H-AmB such as the AmB aggregation state, demonstrating that H-AmB contained superaggregated and monomeric forms (5). The morphology examined by cryo-transmission electron microscopy showed pleiomorphic cobweb structures and laser diffraction revealed a mean particle size of approximately 300 nm (6).

-1 -1 -1 + 1 -1 -1 -1 + 1 -1 + 1 + 1 -1 -1 -1 + 1 + 1 -1 + 1 -1 + 1 + 1 + 1 + 1 + 1
Fluorescence studies showed similar stability to disaggregation by addition of the surfactant sodium dodecyl sulfate, providing the groundwork for future fluorescence characterization of the interactions of M-AmB and H-AmB with cell membranes (7). The interaction between AmB formulations and lipid monolayers has been studied, showing a large disruptive effect for both M-AmB and H-AmB (8). The effect of the aggregation state of AmB on the toxicity of the formulation was examined in detail, indicating that the smaller the aggregation size fraction the higher the toxicity determined by hemolysis (9). Finally, it was were observed in vitro and in vivo that heat treatment preserved the activity of M-AmB against fungal cells (5, 10, 11) and parasites (12, 13), while reducing toxicity probably as result of its increased uptake by macrophages (14).

Furthermore, the serum pharmacokinetics, tissue distribution, and renal toxicity of AmB were determined after administration to rabbits. M-AmB caused serum creatinine concentrations to rise compared with baseline while H-AmB showed no difference. The AmB area under the concentration-time curve after H-AmB administration was significantly lower than that of M-AmB.

On the other hand, total body clearance of AmB was significantly greater for H-AmB. Kidney, lung and spleen AmB concentrations were higher after H-AmB administration than M-AmB. However, liver AmB concentrations were significantly higher after H-AmB administration (15).

As mentioned above, one of the main aims of this work was to test the activity of these two systems, MEAmB and H-AmB. The experimental studies were carried out at the Institut Galien Paris Sud at the Université Paris Sud XI, in Châtenay Malabry, France, under the supervision of Dr. Gillian Barratt and Prof. Philippe Loiseau.

There is not much information in the literature about the use of these two systems for leishmaniasis treatment. In this current work, it was observed that both strategies were very successful in vitro and in vivo for the treatment of this disease since both of them revealed an efficacy that was not significantly different from Fungizone ® , whereas they showed much lower toxicity. These results are in agreement with the literature mentioned previously. In addition, it should be emphasized once the materials used to prepare the formulations are inexpensive and, as far as H-AmB is concerned it is not necessary to buy ingredients to prepare the formulation since its preparation involves only the heating of a formulation that is already available in the market.

The second section of this thesis addresses the freeze-drying of emulsified systems. The experimental study was carried out partly in the LASID guided by Prof. Sócrates, and partly at the Laboratoire d'Automatique et de Génie des Procédés (LAGEP), Lyon, under the guidance of Prof.

Hatem Fessi and Dr. Abdelhamid Elaissari.

Firstly, a comprehensive literature survey was made of the most interesting findings about freezedrying of emulsified systems. This survey was focused on the databases compiled by ISI Web of Knowledge and PUBMED. Publications were selected for having one of these following keywords in the abstract: "emulsion", "nanoemulsion", "microemulsion", "cryoprotectant", "freeze-drying"

and "lyophilization". The aim of this survey was to summarize and provide an update of knowledge on this subject.

Emulsified systems are very important since they are able to transport active molecules, increase the drug solubility and stability as well as improve the pharmacokinetics and decrease the adverse effects of some drugs. These systems are comprised of oil, surfactants and water. The presence of this aqueous phase may lead to microbial contamination of the system, as well as some physicalchemical instability: degradation by hydrolysis and drug loss. Freeze-drying is a good removal water technique; however it should be thoroughly studied with respect to the many parameters that influence the quality of the dry powder and the reconstituted product. These parameters are related to the product, the container, the equipment and the process, including the time and temperature of each freeze-drying step (freezing, primary-drying and secondary drying), and type and concentration of cryoprotectant.

As a result of the literature survey, the experimental work plan was developed. The literature search revealed that for emulsified systems the main cryoprotectants used are sugars at concentrations from 5% to 20%, the freezing temperatures range from -20 °C to -196 °C (liquid nitrogen) and the freeze-drying time ranges from 24h to 72h. Faced with the problem of choosing suitable parameters on many variables, the second chapter of the second section is about the development of a freezedried microemulsion containing AmB based on optimization process by design of experiment. The results showed that the best conditions were 5 % of maltose as cryoprotectant, freezing at -80 ºC and freezing drying for 24 h. The amount of AmB did not change after freeze-drying and the other parameters such size, pH and conductivity remained acceptable. Therefore, the process did not influence ME properties greatly and would be a good strategy to increase the stability of such products.

Work described the freeze-drying of microemulsions is not frequent in the literature. Moreno et al (2001) (16) and Brime, et al (2002, 2003 and 2004) (17-19) have reported that the microemulsion conserved its original physical-chemical properties after the lyophilization process. The freeze-dried systems were found to be more stable than their nonlyophilized counterparts. In addition, the efficacy against Candida strains and pharmacokinetics were evaluated. The activity was maintained and the toxicity was reduced compared with traditional formulations on the market; moreover no histologically demonstrable renal lesions or changes in blood chemistry were observed.

Future perspectives

This work was designed to advance the development of new strategies to combat visceral leishmaniasis using an old drug, AmB, while overcoming some drawbacks such as adverse effect and cost. Two alternative systems have been studied and discussed: the heating of AmB deoxycholate and incorporation of AmB into a microemulsion . Furthermore, during this work we developed a method to dry the microemulsion without losing its properties, which represents a major challenge as far as dispersed systems are concerned. However, further studies are needed in

INTRODUCTION

Since the beginning of my undergraduate studies, I have been working on the topic of nanostructured systems and the use of natural products to produce them. During the PhD, I entered into collaboration with other researchers, working on Copaiba oil and Bullfrog oil.

The review article "Microemulsion systems containing bioactive natural oils: an overview on the state of the art" was published in collaboration and it concerns the same subject as this thesis, microemulsions. In addition to being produced with synthetic oils, these systems can be produced with natural and vegetable oils, in which the therapeutic activity is due to the presence of these natural products.

In addition to microemulsions, other emulsified systems, such as emulsions and nanoemulsions, can also be produced using natural oils, in order to treat a particular disease according to the therapeutic characteristic of the oil chosen to be part of the formulation. The article "Chemical Characterization and Antimicrobial Activity Evaluation of Natural Oil Nanostructured

Emulsions" emphasizes the use of the Copaiba oil as an antimicrobial agent in emulsion systems.

On the other hand, the article "New Trends on Antineoplastic Therapy Research: Bullfrog (Rana catesbeiana Shaw) Oil Nanostructured Systems" discusses the development of a nanoemulsion containing bullfrog oil as anti-tumoral treatment.

The design of experiment used as a tool in this thesis to optimize thee freeze-drying process can also be used to develop nanostructured systems containing natural oils. The article "Experimental design approach applied to the development of chitosan coated poly(isobutylcyanoacrylate) nanocapsules encapsulating copaiba oil" illustrates nanocapsule production from an experimental design evaluating the pH of the polymerization media, temperature of production and concentration of chitosan.

plan d'expérience a donc été mis en oeuvre afin d'optimiser la lyophilisation de nos formulations.

Ainsi, les objectifs de ce travail ont été de développer des formulations H-AmB et ME-AmB et d'évaluer leur activité sur Leishmania donovani (souche LV9) ainsi que leur toxicité in-vitro et invivo et, de plus, de mettre au point une formulation lyophilisée de Me-AmB.

La formulation H-AmB a été préparée en chauffant la forme conventionnelle micellaire à 70°C

FinallyFigure 1 . 1 .

 11 Figure 1. Structure of Amphotericin B showing the numbering of carbon atoms in the molecule (Molecular Mass 924.08 g/mol).

Figure 2 .

 2 Figure 2.Transmission electronic microscopy of ME (a) and ME containing AmB (b).

Figure 3 .

 3 Figure 3.Rheological behavior of ME and ME containing AmB Section II

Figure 3 .Figure 4 1 .

 341 Figure 3.Effect of freeze-drying on the phospholipid rearrangement, droplet size and drug leakage Figure 4 Effect of freezing and dehydration on emulsified systems without (a) and with (b) cryoprotectant. The mechanisms of protection are explained by (1) formation of a eutectic mixture (2) the vitrification theory (3) maintaining the special organization and (4) replacement of the water molecules

Figure 3 .

 3 Figure 3.Pareto chart of the effects of independent variables and their interaction on the (A) ME containing MT droplet size (B) ME containing LT droplet size. (L) is the linear and (Q) is the quadratic interaction of variables.

Figure 4 .

 4 Figure 4. Plots of marginal means for CP concentration, freezing temperature and freeze-drying time to verify the effects of each variable on the droplet size of ME containing MT (a) andME containing LT (b).

Figure 5 .

 5 Figure 5. Response surface plot showing the effects of the mutual interactions between two independent variables on the droplets size. The effect of the freezing temperature and CP concentration on the MEs containing MT (a) and MEs containing LT (c). The effect of the freezing temperature and freeze-drying time on the Mes containing MT (b) and MEs containing LT (d).

  Its pathogenesis is characterized by Interleukin (IL)-12 and Interferon (IFN)-γ production by T cells, as well as the presence of IL-1, IL-6, IL-8, IL-15, and tumor necrosis factor (TNF)-α in the spleen and bone marrow. Furthermore, the death of the parasite also involves the complement system. The main innate response to Leishmania is the natural killer (NK)-cell-activated macrophage system. However, the Leishmania parasites have evolved strategies of immune evasion, the principal mechanisms of which being resistance to complement-mediated lysis, impaired macrophage microbicidal function, impaired antigen presentation to T cells, synthesis of immunosuppressive mediators, generalized depression of T-cell responses and expansion of regulatory T-cell population (1).

$ 10 IV

 10 = intravenous administration; IM = intramuscular administration; IL = intralymphatic administration; PO = oral administration. * Definitive cure at 6 months. Based on (4).

Figure 1 :

 1 Figure 1: Chemical structure of amphotericin B (10)

Figure 1 -

 1 Figure 1 -Structure of Amphotericin B showing the numbering of carbon atoms in the molecule

  antileishmanial activity of inexpensive Amphotericin B formulations: heated Amphotericin B and Amphotericin B-loaded microemulsion Running title: Antileishmanial activity of Amphotericin B formulations Andreza Rochelle do Vale Morais a,c , André Leandro Silva b,c , Sandrine Cojean d , Kaluvu Balaraman d,e , Christian Bories d , Sébastien Pomel d , Gillian Barratt c , Eryvaldo Sócrates Tabosa do Egito a,b , Philippe M Loiseau d# a Universidade Federal do Rio Grande do Norte (UFRN), Programa de Pós-graduação em Nanotecnologia Farmacêutica, Rua Gustavo Cordeiro de Farias, SN. Petrópolis. CEP: 59012-570 Natal/RN -Brazil. b UFRN, Programa de Pós-graduação em Biotecnologia (RENORBIO), Av. Senador Salgado Filho, 3000. Campus Universitário. CEP : 59078-970 Natal/RN -Brazil c Université Paris-Sud, Institut Galien Paris-Sud, UMR-CNRS 8612, 5, Rue Jean-Baptiste Clément, 92296 Châtenay-Malabry cedex, France. d Université Paris-Sud, Faculté de Pharmacie, UMR 8076 CNRS BioCIS, Châtenay-Malabry, France e Chemical Biology Lab, Department of Biotechnology, IITM, Technology Madras, Chennai, India # Corresponding author Prof. Philippe M. LOISEAU philippe.loiseau@u-psud.fr Antiparasitic Chemotherapy, UMR 8076 CNRS BioCIS, Faculty of Pharmacy, Université Paris-Sud Rue Jean-Baptiste Clément, F 92290-Chatenay-Malabry

Figure 1 :

 1 Figure 1: Spectroscopy of M-AmB and H-AmB (a) and of ME-AmB (b) and circular

Figure 3 :

 3 Figure 3: Rheological behavior of ME and ME containing AmB.

  Colloidal drug delivery systems are become more attractive in pharmaceutical technology because they are able to control drug release and to improve bioavailability.(1) Microemulsions (MEs), optically transparent systems with low viscosity, are thermodynamically stable dispersions of two immiscible liquids, stabilized by an interfacial film of a surfactant, usually in combination with a cosurfactant.(2) They have shown several advantages over conventional formulations, including enhanced drug solubility, good thermodynamic stability, an increased surface area improving absorption and enhancement of transdermal passage.(3) These properties give MEs potential applications in the food, cosmetic and pharmaceutical industries when solubilization of lipophilic or hydrophilic ingredients is necessary.(4) In pharmaceutics, MEs are used as vehicles to deliver a number of drugs due to their thermodynamic stability, simple preparation and good appearance.(2)

  Miglyol 812 ® was obtained from CONDEA Chemie GmbH (Hamburg, Germany), Lipoid S100® was purchased from LIPOID GMBH (Ludwigshafen, Germany), Tween 80 ® , Mannitol (MN), Glucose (GC), Lactose (LT) Sorbitol (ST) and Maltose (MT) were obtained from Sigma Aldrich Inc (St. Louis, USA), and the Na 2 HPO 4 and the NaHPO 4 , used to produced the phosphate buffer pH 7.4, were purchased from Vetec Química Fina Ltda (Rio de Janeiro, Brazil). Ultra-pure water obtained by a Milli Q water purification system (Merck Millipore, Massachusetts, U.S.A.) was used along the experiments.

Figure 1 :

 1 Figure 1: DSC thermograms of ME and its components.

Figure 2 :

 2 Figure 2: Influence of the type and concentration of CP on the droplet size of the ME formulations

Figure 3 :Figure 4 :

 34 Figure 3: Pareto chart of the effects of independent variables and their interaction on the (A) ME

Figure 5 :

 5 Figure 5: Response surface plot showing the effects of the mutual interactions between two

  pendant 20 minutes. Les MEs ont été préparées en mélangeant un tampon phosphate à pH 7,4, du Tween 80 ® , du Lipoid S100 ® et du Mygliol 812 ® sous agitation magnétique suivie par trois cycles d'ultrasons. Afin d'incorporer l'AmB, la molécule a été ajoutée à la ME sous agitation continue, avec alcalinisation du milieu suivie de neutralisation. H-AmB a été caractérisé par spectrophotométrie UV/vis et dichroïsme circulaire, tandis que pour la ME-AmB la taille de gouttelettes, la morphologie par microscopie électronique de transmission, le comportement rhéologique ainsi les spectres en UV/vis et dichroïsme circulaire ont été déterminés. Des expériences in-vitro et in-vivo ont été menées afin d'évaluer l'efficacité et la toxicité des formulations en comparaison avec les formes commerciales Fungizone ® et AmBisome ® . Avant la lyophilisation des systèmes, des cryoprotectants -mannitol, maltose, glucose, sorbitol ou lactoseont été ajoutés. Ensuite un plan factoriel complet en 2 3 avec trois répétitions au centre a été effectué. ME et ME-AmB ont été caractérisés en termes de taille de gouttelettes, pH, conductivité et contenu en AmB avant et après lyophilisation selon un protocole optimisé. Les déterminations de taille, morphologie et comportement rhéologique ont montré un diamètre moyen de gouttelette de 35 nm, une morphologie sphérique et un comportement newtonien. L'analyse spectrophotométrique d'H-AmB a révélé la formation de superagrégats, moins toxiques que d'autres types d'agrégats. L'activité anti-leishmanienne in-vitro de H-AmB et ME-AmB sur les parasites axéniques ainsi que sur les amastigotes à l'intérieur des macrophages est similaire à celle de l'AmBisome ® . L'indice de sélectivité observé pour H-AmB et ME-AmB est plus grand que celui

  parasite (L. guyanensis, L. panamensis and L. braziliensis) can spread to mucous regions such as the nose, throat, and mouth, where can lead to partial or complete destruction of the mucous membranes. On the other hand, VL occurs when the parasite causes a systemic infection, spreading through various organs, such as liver and spleen, in which it may destroy the immune cells and can be fatal if untreated. Several Leishmania species can cause VL, in the Old World examples are L. donovani and L. infantum and in the New World L. chagasi(5).

VL has a large spectrum of clinical manifestations in reticuloendothelial tissues, involving the spleen, liver, lymph nodes, and bone marrow. Its symptoms include abdominal pain, hepatomegaly, splenomegaly, diarrhea, epistaxis, pancytopenia, cachexia, fever and cough.

Table 1 :

 1 Current VL treatments and their main properties

	Drugs	Administration	Regimen	Efficacy (*)	Resistance	Toxicity	Price of treatment
	Pentavalent antimonials	IV, IM and IL	30 days 20 mg/kg/day	35-95% (dependin g on area)	Common (>60% in Bihar, India)	+++ hepatotoxicity Cardiotoxicity, pancreatitis, nephrotoxicity,	$50-70
			30 days
	Micellar amphotericin-B	IV	1 mg/kg (15mg/kg total dose)	>90%	Laboratory strains	+++ Nephrotoxicity	$100
			5-20 mg/kg
	Liposomal amphotericin B	IV	total dose 4-10 doses over 10 -20	>97%	Not documented	+/ Rigors and infusion chills during	$280
			days
						+
	Miltefosine	PO	28 days 1.5-2.5 mg/day	94-97%	Laboratory strains	Gastrointestinal, hepatotoxicity, nephrotoxicity,	$70
						teratogenicity
				94%
	Paromomycin sulfate	IM	21 days 15 mg/kg/day	(India) 46-85% (Africa, dependin g on	Laboratory strains	+ Nephrotoxicity, ototoxicity, hepatotoxicity
				dose)

Table 1 :

 1 In vitro evaluation of the cytoxicity and the anti-leishmanial activity on L. donovani WT and AmB-R.

						Cytotoxicity
		Intramacrophage amastigotes	Axenic amastigotes		Selectivity index
	Formulations					RAW 264.7 macrophages
		IC 50 (µM) ± SD	IC 50 (µM) ± SD		SI= CC 50 /IC 50
						CC 50 (µM) ±SD
		WT	AmB-R	WT	AmB-R		WT
	M-AmB	0.67 ± 0.25	2.15 ± 0.18	0.05 ± 0.03	1.69 ± 0.04	4.5 ± 0.4	6.7
	H-AmB	1.54 ± 0.34	2.31 ± 0.32	0.05 ± 0.03	0.82 ± 0.03	39.8 ± 3.2	25.8
	AmBisome®	1.76 ± 0.69	3.54 ± 0.10	1.73 ± 0.87	>100	36.5 ± 2.3	20.7
	ME-AmB	0.88 ± 0.26	1.56 ± 0.01	0.22 ± 0.11	3.38 ± 0.08	9.0 ± 0.3	10.2
	ME (equivalent	27.98 ± 2.71	27.34 ± 0.24	6.82 ± 0.54	27.06 ± 0.52	22.8 ± 2.9	0.8
	AmB)

Table 2 :

 2 In-vivo antileishmanial activity and acute toxicity of H-AmB and ME-AmB compared to M-AmB and AmBisome®. Mice were treated intravenously at 1 mg/kg equivalent AmB at Day 8, Day 10 and Day 12 post-infection.

		In vivo			In vivo toxicity
		antileishmanial
		activity
		% Reduction of	a AST	a ALT	b ALP	b CHO	a CREA	b URE
		parasite burden	U/L	U/L		mM	µM
	M-AmB	72	233.0	134.0	225.5	2.515**	19.5	10.70
	H-AmB	78	189.5	171.5	241.0	2.575**	18.0	9.65
	AmBisome ®	83	242.5	167.0	229.0	*2.755	20.0	10.15
	ME-AmB	78	224.5	169.5	246.0**	2.415**	18.5	8.35**
	ME	*33	293.0	181.0	225.0	*2.710	16.0	*7.60**
	Untreated	-	313.0	239.0	199.0**	2.810	18.0	7.40**

AST : aspartate aminotransferase. Normal value : 456 (U/L

(30)

. ALT: alanine aminotransferase. Normal value : 304 (U/L)

(30)

. CHO : Cholesterol. Normal value: 3.5 mM

(30)

. CREA : Creatinine. Normal value: <18 µM

(30)

. URE : Urea. Normal value 7.4 mM. ALP: Alkaline Phosphatase

  also observed that the droplet size of MEs increased with CP

	concentration. Additionally, Abdelwahed et al. (2006) demonstrated that certain amount
	of CP was necessary to effectively preserve the droplet size and ensure a maximum
	stabilization of nanoparticulate systems.(10)
	An empirical second order polynomial equation was developed for the
	response variable, droplet size, in terms of the three independent variables. This
	equation for MEs containing MT is expressed by y = 23.4 +1.31x 1 + 6.11x 1	2 + 1.41x 2 +
	0.96x 3 + 1.61x 1 x 2 + 0.41x 1 x 3 -0.54x 2 x 3 , and for MEs containing LT by y = 30.93 +
	2.04x 1 + 3.00x 1 2 + 9.86x 2 + 4.19x 3 +2.26x 1 x 2 + 3.19x 1 x 3 + 4.16x 2 x 3 , where y represents

the response variable (droplet size), x 1 , x 2 and x 3 are the coded values of the test variables, which are freezing (x 1 ), CP concentration (x 2 ) and freeze-drying time (x 3 ), respectively.

Table 1 :

 1 Experimental levels of independent variables of the DOE for droplet size evaluation of ME after freeze-drying process.

			Level
	Independent variable
		-1	Replicated (0)	+1
	Freezing
		-20	-80	-196
	temperature (ºC)
	CP concentration (% w/w )	5	12.5	20
	Freeze-drying time (h)	24	36	48

Table 2 :

 2 DOE for the freeze-drying process of MEs for each CP type.

	Freeze-drying	CP	Freeze -
	Samples
	temperature	concentration	drying time

Table 3 :

 3 Statistical Analysis (ANOVA) of results of DOE for ME-MT and ME-LT samples.

			ME -MT		ME -LT
	Source	Mean	Calculate	Tabulate	Mean	Calculated	Tabulated
				R 2			R 2
		square	d F	d F	square	F	F
	Regression 81.518	9.9861	5.117 0.9236 25.,3153 12.57359	5.05 0.9120
	Residue	8.163			19.9080
	Lack of fit	9.755	3.7666	19.35	31.2845 11.00275	19.16
	Pure
		2.590			2.8433
	Error
	Total	15.498			135.1116

Table 4 :

 4 Physicochemical characterization of the ME-MT before and after the freezedrying process (FDP).

				Electrical
					Droplet size
	Samples	Appearance	pH	conductivity
					(nm)
				(µS cm -1 )
		Homogeneous
	ME + MT at
		and clear	7.23 ± 0.04	749.1 ± 25.65	48.13 ± 1.57
	5% before FDP
		yellowish
		Homogeneous
	ME + MT at
		and clear	6.86 ± 0.01 *	876.5 ± 14.80 *	20.13 ± 3.30 *
	5% after FDP
		yellowish

Average values ± SD * p < 0.05 compared with the same sample before freeze-drying.

133

Table 5 :

 5 Physicochemical characterization of ME with AmB containing 5% of MT before and after freeze-drying process.Average values ± SD reported * p < 0.05 compared with the same sample before freeze-drying process. * * p > 0.05 compared with the same sample before freeze-drying process.

				Electrical	Droplet	AmB
	Samples	Appearance	pH	conductivity	size	concentration
				(µS cm -1 )	(nm)	(M)
	Before	Homogeneous
			7.59 ±		54.07 ±
	freeze-	and clear		1583 ± 39.55		1.97 x 10 -3 ± 0.03
			0.10		2.35
	drying	yellowish
	After	Homogeneous	7.4 ±		32.30 ±	1.94 x 10 -3 ±
	freeze-	and clear		1639 ± 53.75 *
			0.11 *		4.20 *	0.02 * *
	drying	yellowish

ACKNOWLEDGEMENTS

The construction of this Thesis depended on the knowledge I have gained along a student journey that allowed me to get to this point in my life, and all of that began with the start of my scientific carrier and all the people who were involved on it. Thus, I extend my acknowledgements not only to the people who were present in my life since 2013, but the ones who helped me since the beginning of college in 2006. This was not the work of a 4 years period. I can definitely say this Thesis is, at the present, the work of my life. And it was not only developed through laboratory experiments performed solely by me, but by through a network of collaborators, involving colleagues of the more distinguished academic degrees, undergraduates, master and doctorate students, Doctors. These collaborations happened frequently overseas, through days and nights of work among Brazil and France. I could not start my personal acknowledgments without thanking my advisor in Brazil, Dr. Socrates Egito for his guidance and support since my undergraduate and master studies. Also, I am thankful for the trust on choosing me to do this collaborative study. I would like to thank CAPES COFECUB grant for a 1.5-year Sandwich Doctorate scholarship. I would also like to thank the Graduate Program in Pharmaceutical Nanotechnology and its former and current coordinators for the support and scholarship while I was in Brazil. I would like to thank my advisor in France Dr. Gillian Barratt for all support scientific or not, and for having me in her lab at the Faculté de Pharmacie, from Université Paris Sud. I am grateful for all the knowledge I have got while doing this research. Also, I would like to thank the École doctorale Innovation Thérapeutique, du fondamental à l'appliqué for all support during my research. I am greateful for all collaborators from Université Paris Saclay such as Dr. Nicholas Huangand, Dr. Monique Chéron, Dr. Claire Boulogne, Dr. Pierre Cheminade, Sônia Abreu, Dr. Cynthia Gillet and Dr. François Xavier. Special thanks to Dr. Phillippe Loiseau and his team (Dr. Sébastien Pomel, Dr. Sandrine Cojean, Dr. Christian Bories and Dr. Kaluvu Balaraman), from ACKNOWLEDGEMENTS: The authors wish to thank the CAPES-Cofecub and CNPq for the financial support. Kaluvu Balaraman was recipient of a post-doctoral fellowship grant No 4803-04 from CEFIPRA. The authors would like to acknowledge Dr. Phuong-Nhi Bories, Hôpital Cochin, Paris, for the measurement of the toxicity markers. The authors are grateful to Dr. Claire Boulogne and Dr Cynthia Gillet, IMAGIF Platform, Gif-sur-Yvette for measurement of the TEM, and Nicolas Huangand and Monique Chéron for the help during the rheological and dichroism analysis, respectively. Acknowledgments The authors would like to acknowledge CAPES and CNPq for the financial support, as well as The Drug Development Laboratory and Center of Research on Oil and Gas of the UFRN for access to equipment. The authors are grateful to Glenn Hawes from the University of Georgia -American Language Program, for editing this manuscript.

SECTION I MICROEMULSION SYSTEM CONTAINING AMPHOTERICIN B FOR LEISHMANIASIS TREATMENT

____________________________________________________________________

CHAPTER II

In-vitro and in-vivo antileishmanial activity of inexpensive Amphotericin B formulations: heated Amphotericin B and Amphotericin B-loaded microemulsion Abstract:

Amphotericin B (AmB) is effective against visceral leishmaniasis (VL), but the renal toxicity of the conventional form, mixed micelles with deoxycholate (M-AmB), is often doselimiting, while the less toxic lipid-based formulations such as AmBisome ® are very expensive. Two different strategies to improve the therapeutic index of AmB with inexpensive ingredients were evaluated on this work: (i) the heat treatment of the commercial formulation (H-AmB) and (ii) the preparation of an AmB-loaded microemulsion (ME-AmB). M-AmB was heated to 70°C for 20 min. The resulting product was characterized by UV spectrophotometry and circular dichroism, showing super-aggregates formation. ME-AmB was prepared from phosphate buffer pH 7.4, Tween 80 ® , Lipoid S100 ® and Mygliol 812 ® with AmB at 5 mg/mL. The droplet size, measured by dynamic light scattering, was about 40 nm and transmission electron microscopy confirmed a spherical shape. Rheological analysis showed low viscosity and Newtonian behavior. All the formulations were active in vitro and in vivo against Leishmania donovani (LV9). A selectivity index (CC 50 on RAW/IC 50 on LV9) higher than 10 was observed for ME-AmB, H-AmB and AmBisome ® . Furthermore, no important in vivo toxicity was observed for all the samples.

The in-vivo efficacy of the formulations after IV administration was evaluated in Balb/C mice infected with LV9 (three doses of 1 mg/kg AmB) and no significant difference was observed between H-AmB, M-AmB, ME-AmB and AmBisome ® . In conclusion, these two inexpensive alternative formulations for AmB showing good efficacy and selectivity for Leishmania donovani merit further investigation.

the same conditions, the experiment was stopped and the plate was treated as for the axenic amastigote stage assay. Each experiment was performed in triplicate (16).

Fluorescence was compared to that obtained with untreated infected and uninfected macrophages and untreated axenic parasites used as controls.

In-vivo evaluation

All procedures involving animals were conducted in compliance with the standards for animal experiments and were approved by the local committee for animal care (0858.01/2014, Versailles, France).

In-vivo antileishmanial activity

A suspension of Leishmania donovani (LV9) amastigotes (1x10 7 /100 µL M199 medium supplemented with 40 mM HEPES, 100 μM adenosine and 0.5 mg/L haemin, 10% FBS), obtained from infected hamster spleen, was injected into female BALB/c mice (weighing 20 g) by intravenous route. The mice were randomly sorted into six groups seven days after the infection. Each group was treated with 100 µL of one formulation (AmBisome ® , M-AmB, H-AmB, ME-AmB and ME), administered by intravenous route for 3 alternate days (1mg/kg/day). Samples were diluted prior to use with 5% glucose to give a final AmB concentration of 2x10 -4 M. Animals were sacrificed three days after the end of treatment. Livers and spleens were weighed and drug activity was estimated microscopically by counting the number of amastigotes/500 liver cells in Giemsa stained impression smears to calculate the Leishmania donovani units (LDU) for liver parasite burdens, using the Stauber's formula (17). The mean number of parasites per gram of liver among treatment groups and controls was compared. Three independent counting were performed and the results were expressed as the mean values ± SD. The parasite burden of treatment groups and controls were compared using the Kruskal-Wallis nonparametric analysis of variance test for comparing two groups. Significance was established for a P value < 0.05.

In vitro cytotoxicity assay

SECTION II

Freeze-drying as strategy for improve emulsified systems stability

CHAPTER III

Freeze-drying of emulsified systems: A review

CHAPTER IV Application of design of experiment to optimize the freeze drying process of microemulsion systems

Application of design of experiment to optimize the freeze drying process of microemulsion systems

Andreza Rochelle do Vale Morais

Application of design of experiment to optimize the freeze drying process of microemulsion systems ABSTRACT

The high aqueous content of oil-in-water microemulsion systems may be responsible for instability of the encapsulated molecule due to chemical reaction or microbiological attack. This drawback may be overcome by the use of the freeze-drying process; however this is not easy to optimize for colloidal systems like microemulsions. Design of Experiment can be used to find the optimal working conditions to achieve this goal. The aim of this work was to develop a freezedried microemulsion using Design of Experiment, using Amphotericin B, which is quite unstable the aqueous media, as a drug model. Different types of cryoprotectants were added to the microemulsion and a 2 3 full factorial design with three replicated points was performed.

Microemulsion, unloaded or loaded with Amphotericin B, were fully characterized before and after the freeze-drying process. Microemulsion containing Maltose at 5% as a cryoprotectant frozen at a temperature of -80 ºC using a 24 h of freeze-drying time yielded the best results. It was observed that after the freeze-drying process, the droplet size became smaller and no significant changes in the Amphotericin B content were observed. The microemulsion containing Maltose at 5% are suitable for drug incorporation and the freeze-drying was able to enhance drug stability in the system.

KEYWORDS:

Amphotericin B, Statistical experimental design, Lyophilization, Drug delivery, Dispersed system.

ME preparation

The MEs were prepared by mixing 68% of phosphate buffer pH 7.4, 14.7% of Tween 80 ® , 6.3% of Lipoid S100 ® and 11% of Mygliol 812 ® using magnetic stirring, following by three cycles of sonication (40 watts power output for 1.5 min) using a Vibra Cell 75041 (Bioblock scientific) and ultrasound bath (120 watts power for 3 min) using a USC-1800A (unique). The MN, MT, GC and LT were added to the MEs prior to the freeze-drying process for the DOE study.

Sample characterization

Macroscopic aspect, pH evaluation, isotropy and conductivity analysis

The color and homogeneity of the freeze-dried products (cake) were evaluated with the naked eye. The isotropy of the samples was evaluated by polarized light microscopy using an Olympus BX4 (Olympus Corporation, Tokyo, Japan) apparatus.

The electrical conductivity was measured using a DM-32 conductivity meter (Digicrom Analytical, SP, Brazil), with a cell constant of 0.11 cm -1 . The pH values were measured by a PG-2000 pHmeter (GEHAKA, SP, Brazil). All analyses were performed in triplicate at 25 ± 2 °C.

Dynamic Light Scattering (DLS)

In order to evaluate the influence of the different CP types and concentration on the ME droplet size before the DOE study, MT, MN, GC, LT and ST were added to the MEs at 5%, 10%, 15% and 20% and the droplet size of all preparations was analyzed.

The samples were previously diluted with water in a ratio of 1:20 and their droplet size distribution was evaluated using a DLS, ZetaPlus (Brookhaven Instruments

DSC analysis of ME components

The thermal behavior of the ME and its individual components (phosphate buffer solution alone, oil phase and surfactants), was evaluated by DSC analysis. Figure 1 shows thermal events associated with the ME: a large exothermic peak at -13.54 °C during the cooling curve, which was due to water freezing, following by an endothermic peak at 4.26 °C during the heating curve explained by the melting of water. A second, broad endothermic peak for ME at a temperature between 50 °C and 100 °C with an enthalpy value of -1.57 kJ/g probably represents water loss. It was also possible to observe small exothermic peaks for Tween 80 ® and Miglyol 812 ® , following by only one endothermic peak for Miglyol 812 ® .

Figure 1

Since it represents the freezing of water during the cooling curve, the position of the exothermic peak may indicate the state of water in the ME. In fact, the strong interaction among water and surfactant molecules compared to the weaker interactions among the water molecules themselves or water molecules and phosphate buffer may induce a large change in the temperature at which the water freezes. (24) The exothermic peak on Figure 1 indicates the freezing of water with less molecular interaction with surfactant molecules, since the onset freezing temperature of ME is approximately, at -18 ºC, which is near to that of phosphate buffer solution (-19.09 ºC). Moreover, no peaks characteristic of Lipoid or Tween 80 ® were observed in the ME thermal profile, which suggests not only a strong interaction between the surfactants and other components of the system, but also that they could be located at the internal phase or at the interface of the ME droplet and not in contact with the continuous phase.

Therefore, the results of DSC measurements lead to the conclusion that water is the continuous phase,(3) thus the ME is of the O/W type, confirming the results of the electrical conductivity.

Droplet size evaluation after addition of CP

Based on the hypothesis that ME stability could be modified by the type and concentration of the CP used, while ,on the other hand, the CP could affect both the droplet size distribution and the freeze-drying process, the influence of different CP types and concentrations in the ME systems were evaluated.

Macroscopically, all the ME systems, with and without CP, were clear, isotropic and homogeneous. On the other hand, those containing LT and the MN presented a turbid aspect at 15% (w/w) and at 20% (w/w) , respectively, showing that these additives are not very soluble in the ME system.

The influence of CP addition on the droplet size can be observed in Figure 2.

MEs showed an average diameter of 22 nm that do not change with the addition of MT, MN, GC and LT (p > 0.05). However, in the case of ST, the addition of even a small amount caused an increase in the ME droplet size (28 nm) and with the highest concentration it reached a 62 nm. Those variations, compared with the ME without CP, were statistically significant (p<0.05).

Figure 2

Therefore ST was excluded from the DOE, while the other CPs: MN, MT, GC and LT, at concentrations from 5% (w/w) to 20% (w/w) may be used to protect MEs during the freezing and drying steps required for the freeze-drying process, since they did not cause significant changes in the ME droplet size.

DOE for freeze-drying parameters

Among the samples submitted to the freeze-drying process, those containing MT and LT produced dry powders. Disaccharides, the class to which these CPs belong, are more effective freeze-drying protectants then other types of molecule because they are able to form an amorphous sugar glass. (10) Although the ME-LT at the lower level (5% (w/w) ) produced a powder, it did not appear to be completely dry. This is probably because the concentration of CP was not high enough to completely stabilize the ME.( 25 Only the ME containing MN at 20% (w/w) , frozen at the highest level (-196 ºC), yielded dry powders after freeze-drying, probably because only this MN concentration was sufficient to stabilize the system while the rapid cooling inhibited the MN crystallization.( 28) However, at this concentration, this CP was not soluble in the system and after a period of time at room temperature the dry powder changed to a rubbery state, revealing that MN was not the best choice as CP. The MEs containing MN at the other DOE levels (5% (w/w) and 12.5% (w/w) ) did not produce powders, probably due to the tendency of MN to partly crystallize into MN hemi-hydrate. This could be explained by the previously discussed models.

Table 3 A marginal means chart was plotted to observe the average responses for each level of each factor (Figure 4). It was observed that for ME containing MT (Figure 4a)

when observing the effect of CP concentration at the lowest level of freeze-drying time, the lowest droplet size was observed using the lowest CP level (5% (w/w) of MT), regardless of the freezing temperature. However, at 48 h of freeze-drying time, the droplet size varies depending on the freezing temperature. In fact, freezing at -196 °C generates smaller ME droplet size when 5% (w/w) of MT was used, while for high freezing temperatures (-20 °C) a 20% (w/w) of MT was necessary to achieve the same result. It was also observed that the droplet size increased with the CP concentration when the rapid freezing process (-196 ºC) was used.

The analysis of the marginal means of ME containing LT (Figure 4b) showed that for both the high and the low level of freeze-drying times (48 h and 24 h) the ME droplet size increased with increasing CP concentration, regardless of the freezing temperature. Nevertheless, at the minimum freeze-drying time level (20 h) the freezing temperature of -20 ºC produced a larger droplet size than the one at -196 ºC. Otherwise, the freeze-drying time of 48 h led to a lower droplet size at this longer freezing time.

Both marginal levels of freezing temperature showed decreased droplet size with decreasing CP concentration.

Figure 4

The relationship between the dependent and independent variables was further reconstitute the freeze-dried ME, these ions dissolve in the water and cause the electrical conductivity to increase.

The protective mechanism of the CPs may be also explained by (i) the formation of an eutectic in the presence of water, due the multi-hydroxyl compounds, that leads to the formation of amorphous or imperfect ice crystalloids; (ii) the maintenance of the spatial orientation and distance between the droplets when the ice sublimes during the freeze-drying process, preventing the formation of aggregates; (36) and (iii) the amorphous glass formation into which the cryo-concentrated solution may vitrify at its Tg. The immobilization of the droplets within a glassy matrix of CP can prevent their aggregation and protect them from the mechanical stress of ice crystals.( 27)

Incorporation and freeze-drying of ME containing AmB

The incorporation of AmB into the ME generated a product with a homogeneous and clear yellowish aspect. The addition of MT at 5% did not cause any changes in the ME appearance. However, the freeze-drying process (24 h of freezing at -80 ºC and freeze-drying for 24 h) altered the AmB-ME formulation, although a dry powder is produced. In fact, the reconstituted AmB-ME, which was made by adding the same amount of water as that lost during the freeze-drying process and using a probe sonicator with an iced bath for fast reconstitution, showed changes in its electrical conductivity, pH and droplet size (Table 5). The aforementioned reasons for changes in unloaded ME-MT formulations can also explain such changes in the AmB-ME product.

Table 5

The addition of AmB increased the ME droplet size and the conductivity value

CONCLUSION

Research and development of freeze-dried microemulsion systems has been a challenger for several research teams. In fact, several parameters may affect the system stability during the freeze-drying process. However, this work revealed that DOE can be a successful tool allowing determining the optimal freeze-drying conditions to achieve this task. By taking in account the type and concentration of CP, the freeze-drying time and freezing time temperature on the DOE, and using the droplet size as variable of response, a ME dried product can be produced using a low number of experiments. MT at 5% as CP, -80 ºC of freezing time temperature and 24 h of freeze-drying time were the best parameters to produce a good aspect brick of freeze-drying ME. The incorporation of AmB did not dramatically change the ME physicochemical properties.

Moreover, AmB-ME can became a new antifungal product to treat not only systemic candidiasis, but also leishmaniais infection.

General discussion

The aim of this thesis was to evaluate the activity and toxicity of two formulations: Amphotericin B-loaded microemulsion (ME-AmB) and Heated Micellar Amphotericin B (H-AmB), developed in order to improve antileishmanial therapy while reducing costs and toxicity. A further objective was to to improve the stability of ME-AmB by reducing the amount of free water, and thereby the possibility of microbial contamination and hydrolysis reactions. Hence, the manuscript is divided in two sections.

The first section concerns the in-vitro and the in-vivo evaluation-of the lipid systems: microemulsion and micelles containing AmB, against Leishmania.

Amphotericin B (AmB) is a potent drug for antifungal and antileishmanial therapy which was discovered more than 60 years ago. Its efficacy has not changed very much over the years and not many cases of resistant strains have been reported. However, its toxicity makes it difficult to use and it is necessary to develop strategies to solve this problem.

Some research teams, as well as our group, have also been investigating AmB incorporation into presence and absence of AmB incorporated into the ME system. The results showed that AmB incorporation into the ME was dependent on both the oil phase and the surfactant proportions, reaching a plateau at high contents, and that it influences the droplet size of the microemulsion.

Furthermore, it was observed that increasing the surfactant concentration decreased the droplet size, demonstrating the stabilizing effect of the surfactant, which is a normal observation for MEs.

The other alternative formulation proposed in this thesis, H-AmB, has been investigated by other order to deepen our knowledge of these strategies and to answer some questions that have appeared during the development of this work.

It is necessary to understand some aspects better; for example how AmB is incorporated into the ME. What happens to the ME structure after AmB addition? Are new nanostructures formed?

Where the drug is located? What is its aggregation state? Is the ME-AmB stable? How is the drug released?

Furthermore, it is important to know how these systems act inside the body; that is, their its pharmacokinetics and pharmacodynamics. The good results in AmB-resistant strains emphasize the importance of studying these formulations in greater depth, to understand why AmB loaded in ME is able to destroy resistant strains.

Moreover, more experiments are required to understand some issues related to freeze-dried ME-AmB, such as its stability compared with non-freeze-dried systems, the drug release, its antileishmanial efficacy in vitro and in vivo, the AmB aggregation state and its relationship with biological activity, and the influence of temperature in reconstituting freeze-dried systems.

In the appendices of this manuscript it is possible to find articles describing the use of natural oils to treat fungal and cancer diseases. An alternative approach using natural oils could be interesting for leishmaniasis treatment. There are few studies using natural oils to treat this disease, especially

Copaiba oil. Therefore, the development of dispersed systems containing Copaiba oil could be another possibility for AmB-resistant strains.

The use of AmB for leishmaniasis treatment has been studied, showing Ambisome ® and Fungizone ® as the most frequently used products. However, both have drawbacks: high cost and toxicity respectively. This work presents two alternative products containing AmB that are effective but do not show a high toxicity and whose production cost are lower than those of Ambisome ® .

Fungizone ® is cheap and has a good efficacy; however, it has high toxicity. The heating of this formulation is one alternative to reduce its toxicity. The results showed that the superaggregates present in heated Fungizone are probably the reason for the decrease in the in-vitro and in-vivo toxicity of the commercial product, but the heated product remained effective, as observed by the high selectivity index against Leishmania donovani.

Other promising results were obtained regarding the AmB-loaded microemulsion. Its small droplet size and observed Newtonian behaviour would sanction its use by injection. The AmB toxicity in ME also decreased compared with Fungizone ® , and the efficacy was not significantly different. Therefore, this system is another alternative to treat visceral leishmaniasis.

Furthermore, this work demonstrated the possibility of freeze-drying the microemulsion with or without AmB. Thereby, the storage stability of the system increased, avoiding drug loss due to reactions with water and decreasing the possibility of microbial contamination. The use of design of experiment as a tool was important to find the optimum conditions of cryoprotectant concentration, freezing temperature and freeze-drying time. Therefore, the freeze-dried ME-AmB could be another pharmaceutical dosage form to treat leishmaniasis. However, more studies are necessary in order to evaluate the efficacy of this system after lyophilization. Title : Freeze-drying to increase stability of Amphotericin B-loaded microemulsion for leishmaniasis treatment Keywords : Amphotericin B, microemulsion, visceral leishmaniasis, lyophilization Abstract : Visceral leishmaniasis is a serious parasitic tropical disease that is fatal without treatment. Amphotericin B is effective against the parasites but has doselimiting side-effects in its conventional form (Fungizone®) while a less toxic lipidbased form (AmBisome®) is expensive. The aim of this thesis was to prepare and evaluate two alternative formulations: heattreated Fungizone® (H-AmB) and a microemulsion from low-cost ingredients (MEAmB). These formulations showed activity comparable to that of the commercial products against the parasites, and lower toxicity than Fungizone® to human cells.

Appendix

Furthermore, they showed activity against parasites that were normally resistant to AmB. In an animal model of leishmaniasis, these new formulations were found to be effective and not to cause toxicity. Therefore, they could be used as a cheaper alternative to AmBisome®. In order to improve its stability during transport and storage, a freeze-dried form of MEAmB was optimized. These results are promising for the development of new treatments for leishmaniasis that would be accessible in the countries where the disease is endemic.