Nanocarriers for drug targeting and improved bioavailability

Università degli Studi di CagliariScienze e Tecnologie Farmaceutiche

PhD Program - ciclo XXV

PhD candidate: Francesco CorriasPhD program coordinator: Prof. Elias Maccioni

Supervisor: Dr. Francesco Laianno accademico 2012 – 2013

SSD Chim/09

Acknowledgements Dr. Francesco Corrias was financed by Regione Autonoma della Sardegna under the Master and Back Program. Reference code: PRR-MAB-A2011-18833

Liposomes for the delivery of the NK3 receptor agonist senktide to the central nervous system ___________________ 1

Introduction 1

Receptor-mediated transcytosis (RMT) 5

Transferrin receptor 5

Lactoferrin receptor 6

Senktide and NK3 receptors 9

Abbreviations 13

Materials & methods 13

Materials 13

Liposomes preparation 14

Liposomes characterization (phisycochemical characterization: mean size, ZP, PI) 14

Incorporation efficiency 15

Morphological characterization 15

In vivo brain microdialysis experiments 15

Animals and experimental groups 15

Surgery 16

Analytical procedure 16

Histology 16

Senktide tissue distribution study 16

LC/MS analysis 17

Statistics 17

Results and discussion 18

Liposome preparation and characterization 18

In vivo brain microdialysis 21

Responsiveness of NAc shell DA to senktide delivered by liposomes conjugated to mAb OX26 21

Blockade of NAc shell DA increase senktide-mediated by NK3 receptor antagonist (SB222200) 21

Responsiveness of NAc shell DA to senktide delivered by liposomes conjugated to Lactoferrin 21

Senktide tissue distribution study 22

Senktide distribution in brain tissue after different administration 22

Senktide distribution in brain and liver 22

Discussion 22

References 28

Nanosuspensions ___________________________________ 40

Introduction 40

Preparation methods 41

Top down technologies 43

Media milling 43

High pressure homogenization (HPH) 46

Bottom up technologies 50

Precipitation by liquid solvent-antisolvent method 50

Sonoprecipitation 51

Evaporative precipitation techniques 51

Nanosuspension pharmacokinetic properties 52

Oral administration route 52

References 56

Nanosuspension improves tretinoin photostability and delivery to the skin _________________________________ 64

Introduction 64

Materials and Methods 68

Materials 68

Nanoemulsion preparation 68

Nanosuspension preparation 68

Nanosuspension and nanoemulsion characterization 68

HPLC method 69

Photodegradation study 69

In vitro skin penetration and permeation studies 69

Results and discussion 71

Conclusion 76

Formulation strategy and evaluation of nanocrystal piroxicam orally disintegrating tablets manufacturing by freeze-drying ____________________________________ 78

Introduction 78

Materials and methods 81

Materials 81

PRX polymorphic form preparation 81

PRX polymorphic form solubility 81

PRX/poloxamer 188 physical mixture preparation 82

Coarse suspension preparation 82

Nanosuspension preparation 82

ODT preparation 82

Analytical characterization 82

In vitro dissolution studies 83

UV analysis 83

Particle size measurement 83

Statistical analysis of data 83

Results and discussion 84

Bulk PRX characterization 84

Nanosuspension and ODT preparation and particle size measurement 84

Analytical characterization 85

In vitro dissolution studies 87

Formulation and evaluation of quercetin nanosuspension incorporated maltodextrin fast - dissolving films ____________________________________ 96

Introduction 96

Materials and methods 98

Materials 98

Quercetin nanosuspension preparation 98

Particle size and zeta potential measurements 98

Film preparation 98

Film thickness 99

Tensile properties 99

Transmission electron microscopy (TEM) 100

In vitro dissolution studies 100

Results and discussion 101

Reference 104

1

Liposomes for the delivery of the NK3 receptor agonist senktide to the central nervous system

Introduction

Historically, the first demonstration of the presence of a barrier able to limit the penetration of circulating compounds into the central nervous system (CNS) parenchyma was obtained by Paul Ehrlich. In a historical experiment he observed that dyes (e.g., trypan blue), injected intravenously in animals, stained all organs with the exception of the brain and spinal cord (Ehrlich, 1885). Later, Edwin Goldmann, an Ehrlich’s student, injecting directly the dyes into the spinal fluid of the brain, found that in this case the brain became dyed while the rest of the body remained dye-free. Thus, he first hypothesized that the capillaries of the brain were the base of the physiological separation between the CNS and the rest of the body (Goldmann, 1913). This hypothesis was confirmed only in the 1960s with the development of the

electron microscope with which the anatomical differences between brain and peripheral capillary endothelial cells were demonstrated. The so-called Blood Brain Barrier (BBB) is primarily composed of the brain endothelial cells. Other structures such as extracellular base membrane, adjoining pericytes, perivascular astrocytes and microglia constitute the neurovascular unit that is an integral part of the BBB, contributing in the formation of a physical and metabolic barrier, which seems to be of primary importance in the maintenance of brain functionality (Fig. 1).The human brain contains about 100 billion capillaries that develop approximately 650 km and a corresponding surface area of about 20 m2 (W Kamphorst,

2002) (Pardridge, 2003). Brain capillary endothelia are very thin cells, roughly 40% lower than peripheral capillaries, since the luminal and abluminal membranes are only separated by about 500 nm or less.

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The CNS endothelial cells are anatomically different from capillary endothelial cells of the rest of the body and are characterized by the lack of fenestration, the presence of only few pinocytotic vesicles (Abbott,

2005) (Stewart, 2000) and high numerical density of mitochondria (Claudio et al.,

1989) (Oldendorf et al., 1977), roughly five times greater than in peripheral endothelia, necessary for the multiple energy-dependent processes supporting the nutrient uptake and protection of the brain. Adjacent endothelial cells have specialized junctional regions: adherens junctions (AJs) and tight junctions (TJs), considered as the primary physical barrier limiting paracellular movement of solutes, ions and water (Hawkins and Davis, 2005)

(Persidsky et al., 2006). In particular, TJs (Bernacki et al., n.d.) (Wolburg and

Lippoldt, 2002) (Jin et al., 2010) are located on the most apical site of the cleft between brain endothelial cells (Fig. 2) and

are essentially formed by three different integral protein types (occludin, claudins, and junctional adhesion molecules [JAMs]. TJs are organized in complex parallel, interconnected, transmembranal and cytoplasmatic strands forming a zipper-like seal between adjacent endothelial cells.The permeability of the brain capillary endothelium forming the BBB is about two orders of magnitude lower than that of endothelia in peripheral organs and therefore, the brain is probably one of the least accessible organs to circulating compounds. Although the BBB tends to protect the CNS from endogenous and exogenous potentially toxic substances, penetration of circulating compounds essential for its correct metabolism is also required. Several transport routes exist for the permeation of different molecules (both endogenous and exogenous) across the BBB (Fig. 3) (Abbott and Romero, 1996)

(Pardridge, 2005) (Pardridge, 2007) and,

in general, they can be classified as: - Paracellular aqueous pathway- Transcellular lipophilic pathway- Transport proteins or Carrier

mediated transport (CMT)- Receptor mediated transcytosis

(RMT)- Adsorptive mediated transcytosis

(AMT)- Cell mediated transcytosis

Compounds like ethanol, nicotine, benzodiazepine, steroid hormones and other small lipid soluble substances can passively diffuse through the BBB following a concentration gradient-dependent process. These substances enter the brain transcellularly by dissolving in lipid of the endothelial cell plasma membrane. The ability to cross the BBB by passive transcellular diffusion is only possible for substances having molecular weight less than 500 Da, unionized form, partition coefficient value (log P) close to

Fig. 1. Schematic representation of the blood-brain barrier (BBB) and other components of a neurovascular unit

Interneuron

Capillary

Neuron

Tight Junction

Endothelial cell

Pericyte

Perivascular macrophage

Basal lamina

Microglia

Astrocyte

3

Fig. 2. Schematic diagram of the BBB structure that comprises the tight and adherens junctions. The tight junctions consist of occludin, claudins, and junctional adhesion molecules (JAM). The tight junctions also consist of several accessory proteins necessary to form structural support, including the zonula occluden (ZO) proteins, AF6, 7H6, cingulin, and others. Most of the tight junction components (ZO proteins, claudin, and occludin) have the ability to bind to actin cytoskeleton in brain endothelial cells. The adherens junctions consist of vascular endothelial cadherin (VE-cadherin) and catenin proteins, provide structural integrity and attachment between the cells, and are necessary for formation of tight junctions

ᵞActin �laments

CiugulirAF6, 7H6, etc

Catenins

α-actintin

Vinculin α

ß

ZO-1

ZO-3

ZO-2

p120

ᵞα

ßp120

PECAM-1

JAM (-A,-B,-C)

Claudins(-3,-5,-12)

Occludin

Adherensjunction

VE-Cadherin

Intercellular clefts

TightjunctionZO-1

ZO-3

ZO-2

4

a.Paracellular aqueous pathway

Water-soluble agents

Blood

Brain

b.Transcellular lipophilic pathway

Lipid-soluble agents

c.Transport proteins

Glucose, amino acids, nucleosides

d.E�ux pumps

f.Adsorptive transcytosis

Albumin, other plasma proteins

g.Cell mediated transcytosis

Continuous membrane

Monocytes liposomes

MonocytesMicrogliaAstrocyte

Perivascular macrophage

NeuronNeuronNeuron

Liposomes

Tight junction

Endothelium

Pericyte

e.Receptor-mediated transcytosis

Insulin, transferrin

++

++

+ -

+ -- +

- +

-

--

-

++

++

+ -

+ -- +

- +

++

++

+

+ +

+

- - - - - - -

++

++

+

+ +

+

Fig. 3. Transport routes across the BBB. Pathways “a” to “f ” are commonly for solute molecules; and the route “g” involves monocytes, macrophages and other immune cells and can be used for any drugs or drugs incorporated liposomes or nanoparticles

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2 and cumulative number of hydrogen bonds not more than 10 (Pardridge,

2003) (Camenisch et al., 1998) (Chen

and Liu, 2012)(Cohen and Bangham,

1972) (van de Waterbeemd et al., 1998).

Receptor-mediated transcytosis (RMT)Different macromolecules can enter the intact CNS using a specific transcytotic mechanism connected to the presence of specific receptors highly expressed in specialized areas, rich on clathrin and known as coated pits, present on the luminal side of the plasma membrane. Transcytosis of molecules at the BBB is an energy-requiring/ATP-dependent transport process (Simionescu et al.,

2002). Molecules such as transferrin, albumin, insulin, insulin growth factor, low-density lipoproteins, ceruloplasmin, and others have a specific receptor on the BBB and enter the brain by receptor-

mediated transcytosis (RMT). The first RMT step involves the binding of a solute (ligand) with its receptor (Fig. 3). The formation of the ligand/receptor complex triggers the pits invagination into the cytoplasm and the formation of a clathrin-coated vesicle. This coated vesicle, or early endosome, contains the ligand/receptor complex and moves through the cytoplasm of the endothelial cell losing its clathrin coating and maturing to secondary endosome. After acidification of the secondary endosome, the ligand will dissociate from the receptor that is then recycled to the apical surface. The secondary endosome may fuse with a lysosome, to form the secondary lysosome, or can cross the entire cell and pour out its contents to the CNS parenchyma (exocytosis) without having been modified (Simionescu et al., 2002) (Broadwell et

al., 1988) (Mellman, 1996).

Transferrin receptorThe human transferrin (Tf ) is a monomeric glycoprotein (apo-Tf ) that contains 679 amino acids with a molecular weight of around 80 kDa including two specific high affinity Fe(III) binding sites. Transferrin can transport one (monoferric Tf ) or two (diferric Tf ) iron atoms as well as divalent cations, such as gallium or manganese. The cellular uptake of iron, required for cellular processes and enzymatic reactions, occurs through a transcytosis of iron-loaded Tf mediated by the transferrin receptor (TfR) (Pardridge et al., 1987)

(Moos and Morgan, 2000). However, there are still many open questions about the actual extent of Tf transcytosis through the BBB. Different studies have shown that iron uptake by the brain exceeds that of Tf (Crowe and Morgan,

1992) (Morgan and Moos, 2002). The TfR is a homodimer (180 kDa) type II transmembrane associated glycoprotein

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consisting of two identical monomers (760 amino acids, molecular weight 90 -- 95 kDa) joined by two intermolecular disulfide bonds. Each monomer contains a short intracellular N-terminal domain (61 amino acids), a single-pass transmembrane domain (28 amino acids), and a large extracellular C-terminal domain (671 amino acids) that holds the Tf-binding site. TfR is ubiquitously expressed at low levels on the surface of normal cells (Thorstensen and Romslo, 1993) (Gatter

et al., 1983) (Omary et al., 1980). Its expression is elevated, however, on cells with a high proliferation rate including the basal epidermis and intestinal epithelium cells, placental trophoblasts and a variety of human cancers cells, (Li and Qian,

2002) (Sutherland et al., 1981). High TfR expression has also been observed on nonproliferating cells including endocrine pancreas, luminal membranes of the breast, hepatocytes, Kupffer cells of the liver,

tubules of the kidney, and brain endothelial cells (Moos and Morgan, 2000) (Gatter

et al., 1983) (Harel et al., 2011).

Lactoferrin receptorLactoferrin (Lf ), an 80-kDa single-chain glycoprotein, has been found in human various tissues and cells and performs multiple physiological functions such as facilitating iron absorption, modulating immune system, prompting embryonic development and inducing cell proliferation (Lönnerdal and Iyer,

1995) (Nichols et al., 1987) (Ward et

al., 1999). The realization of biological activity of Lf depends of the specific lactoferrin receptor (LfR) on the surface of the target cells. Different researches have demonstrated that different target tissues and cells express their own LfR and that the characteristics and functions of LfR vary among different cell types (Suzuki and Lönnerdal, 2002) (Suzuki

et al., 2005). LfR have been reported to be anchored to the plasma membrane of target cells (small intestine, liver, brain, lymphocytes, monocytes, platelets and so on) by glycophosphatidylinositol and to act as a mediator between Lf and target cells (Birgens, 1991) (Cox et al., 1979)

(Legrand et al., 1992) (Leveugle et al.,

1996) (Maneva et al., 1993) (Ziere et al.,

1993). In general, the biological role of the LfR-mediated mechanism has not yet been fully elucidated, but it has well known that in Parkinson’s disease, neurons have been found to accumulate high concentrations of human Lf (Leveugle et al., 1996) and it was demonstrated that Lf crosses the BBB through a brain LfR mediated transcytosis mechanism. It has been calculated that more than 98% low molecular weight drugs and about 100% high molecular weight compounds do not cross the BBB. Natural, recombinant or synthesized peptides

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and proteins, small-interfering RNA (siRNA), monoclonal antibodies and gene therapeutics with a well-established activity to CNS receptors do not readily permeate into brain parenchyma due to the presence of the BBB (Pardridge,

2006) (Skaper, 2008). Considering that many brain-associated diseases are undertreated by effective therapies and bearing in mind the narrow number of drugs able to cross the BBB, drug transport to the CNS is a priority for the academic and industrial researchers. Therefore, several strategies have been developed for delivery therapeutics to the brain, using both invasive and noninvasive techniques. Invasive methods include disruption of the BBB (Deli, 2009) (Garcia-Garcia et al.,

2005) (Neuwelt et al., 1979), convection-enhanced delivery (Bobo et al., 1994), intracerebro and intracerebro-ventricular infusion (Chauhan, 2002) (Chamberlain

et al., 1997) and use of implants (Guerin

et al., 2004) (Wang et al., 2002). Invasive strategy presents many limitations like high costs for anesthesia and hospitalization, low drug diffusion into the brain parenchyma, enhancement of tumor dissemination after TJs disruption, lack of efficacy and others (Gabathuler, 2010). Noninvasive techniques and, in particular, the use of nanocarriers have shown superior patient compliance, higher efficacy, and, in many cases, safety in comparison with the invasive ones (Garcia-Garcia et al.,

2005) (Wong et al., 2012) (Invernici et

al., 2011) (Yang, 2010). Nevertheless, design of effective strategies requires a deep understanding of the anatomy and physiology of CNS barriers, in particular the BBB, to achieve a method possessing efficacy and safety with regards to its impact on the overall protective function of the BBB. In the last decade, a great deal of interest has been devoted to the development of nanocarriers able to deliver

drugs to the brain. These drug delivery systems include different colloidal carriers (i.e., micelles, liposomes, nanoparticles etc) that have been properly tailored to target drugs to the CNS. Among these, liposomes have been studied largely as potential “magic bullet” capable of circumventing the BBB (Barbu et al., 2009) (Celia et

al., 2011). Liposomes are spherical, self-closed structures formed by one or several concentric lipid bilayers with an aqueous phase inside, which were first proposed and tested as a drug delivery system in the early 1970s. Since then, they have been adopted as the vehicle of choice for drug delivery and targeting, due to their very unique and attractive biological and physical chemical properties (Gregoriadis,

1995). Liposomes are biocompatible and can both entrap and protect water-soluble (hydrophilic) molecules in their internal water compartment and water-insoluble (hydrophobic) into the lipid

8

bilayers. Moreover, they provide a unique opportunity to deliver pharmaceuticals into cells or even inside individual cellular compartments. Indeed, in order to facilitate the association and interaction with the target site, size, charge and surface properties of liposomes can be easily changed by simply adding new ingredients to the lipid mixture before liposome preparation and/or by variation of preparation methods (Torchilin,

2005). For all these reasons liposomes, opportunely prepared and functionalized, have been considered as nanocarriers for brain drug targeting in several pathologies after intravenous administrations. To deliver liposomes to the brain, they must be firstly modified into long circulation vesicles by decreasing particle size (< 100 nm) or by linking polyethylene glycol (PEG) chains to their surface. Then, they have to be specifically designed to release the encapsulated drug in target site

(Béduneau et al., 2007) (Laquintana

et al., 2009). To target PEG-liposomes specifically to the brain, they have to be coupled with various ligands, such as transferrin, insulin, lactoferrin etc., whose receptors are highly expressed on the BBB. The attachment of specific molecules to the liposomal surface-granted PEG chains allows vesicles to be actively taken up by the target cells and, therefore, they can enter the brain by an RMT mechanism. Essentially, this approach comprises four steps:

1) Drug loading (small molecules, peptides, gene, etc.) into the liposomes.2) Conjugation of specific ligands, capable of targeting the receptors (transferrin receptors [TfR], insulin receptors [IR], Lactoferrin receptor [LfR] etc.) at the BBB, with the liposomes surface. Endogenous ligands (transferrin, lactoferrin) and modified endogenous ligands or antibodies

are used.3) Transcytosis of the liposomes through the BBB, triggered by the ligand binding with the receptor, and their accumulation into the brain parenchyma.4) For properly designed liposomal nanocarriers, liposomes cross neuronal and nuclear neuronal membrane leading to their (or their content) accumulation in the neuronal cytoplasm or nucleus.Tf has been used as a homing device to target carriers to different cells, in particular brain endothelial cells (Ulbrich

et al., 2009) (van Rooy et al., 2011). However, Tf-modified carriers must compete for TfR binding site with the endogenous Tf present in high amount in plasma (2.6 mg/ml), i.e., an amount many-fold greater than the dissociation constant (KD) of the BBB TfR (5.6 ± 1.4 nM) (Pardridge et al., 1987). Therefore, on the whole, circulating transferring heavily saturates the BBB transferrin

9

receptor. For this reason, other molecules capable of binding TfR were tested and monoclonal antibodies (mAb) have been largely studied. Jefferies et al. identified a mouse mAb (OX26) that can react in vitro and in vivo with rat TfR but cannot block Tf binding because it reacts in a binding site different from that of Tf (Jefferies et

al., 1985) (Jefferies et al., 1984). Friden et al. first developed a drug delivery system for brain release of methotrexate, used as a model drug, by means of the mAb OX26 as a carrier (a vector) (Friden et al., 1991). They demonstrated that accumulation of radiolabeled methotrexate in the rat brain parenchyma, after intravenous administration, is greatly enhanced when the drug is conjugated to OX-26. The same group of researchers used an intraocular fetal forebrain septal area tissue transplant model to compare the ability of nerve growth factor (NGF) alone or conjugated to OX26 (OX26-NGF) to protect

cholinergic neurons in rat (Granholm et

al., 1994). Huwyler et al. first developed immunoliposomes, liposomes with antibody attached to their surface, for brain delivery of radiolabeled daunomycin mediated by the binding of OX26 to the rat transferrin receptor (Huwyler et al.,

1996). They demostrated that OX26 PEG-immunoliposomes determined an increase of the permeability of the BBB to daunomycin, leading to an increase of the brain tissue accumulation, compared to free daunomycin. Receptor-mediated transcytosis across the BBB was also explored by means of lactoferrin-conjugated liposomes. As the expression of lactoferrin receptor on microvessels and neurons is increased under certain pathological conditions, such as Parkinson’s disease and Alzheimer’s disease, the delivery system with lactoferrin attachment may be very efficient. Starting from these assumptions, Chen et al.

prepared lactoferrin-modified procationic liposomes (by using a cholesterol carbamate with a disulfide bond inside) as a potential brain drug delivery vector (Chen et al.,

2011) (Chen et al., 2010). The results indicated that lactoferrin-modified procationic liposomes, in comparison with conventional or procationic liposomes, improved performance in the uptake efficiency.

Senktide and NK3 receptorsThe tachykinin peptide family comprises three neuropeptides — substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) — that are widely expressed in both the central and peripheral nervous system (Ideker et al., 2001). The key characteristic of the tachykinins is a highly conserved carboxy-terminal aminoacid sequence Phe–X–Gly–Leu–Met–NH2, where X is either a branched aliphatic residue (for NKA and NKB) or an aromatic

10

residue (Ideker et al., 2001). SP, NKA and NKB mediate their effects through three distinct types of G-protein coupled receptors (GPCRs) — the NK1, NK2 and NK3 receptors, respectively — that have a similar gene structure (Ideker et al., 2001)

(Kitano et al., 2004). This family of receptors comprise a group of recognized druggable targets and have recently become of interest with regard to schizophrenia. Schizophrenia is a severe, disabling and lifelong condition that affects 1% of the population. It is traditionally characterized by positive (psychotic) symptoms, such as delusions, hallucinations and paranoia, and negative symptoms, such as anhedonia, anergia, avolition, flat affect and loss of spontaneity. However, cognitive impairment (for example, attention deficits, working memory deficits and deficits in executive function) is now also recognized as a key hallmark of the disease (Butcher, 2005) (Grabowski, 2004). The

aetiology of schizophrenia is not known, but it is generally accepted that both genetic and environmental factors are important in the development and clinical manifestation of this disorder (Butcher,

2005). A range of pharmacological treatments that are relatively effective in providing symptomatic relief are now available. Although diverse in nature and chemical structure, all currently approved antipsychotic drugs share the trait of reducing dopaminergic function by at least two mechanisms — either dopamine D2 receptor antagonism or partial agonism (Booth and Zemmel, 2004). Many evidences suggest the clinical importance of NK3 receptor on dopamine function. NK3 receptors are almost exclusively found in the central nervous system and spinal cord (Ideker et al., 2001). In particular, they are consistently detected in areas that include cortical regions such as frontal, parietal and cingulate cortex, various nuclei

of the amygdala, the hippocampus and, most importantly, in midbrain structures such as the substantia nigra, ventral tegmental area and raphe nuclei (van der

Greef et al., 2004) (Steuer et al., 2003). Data obtained after lesion technique indicate that NK3 receptors, more precisely, are located exactly on the surface of dopamine cells of this areas. (van der

Greef et al., 2004) (Steuer et al., 2003). Over the last years, encouraging preclinical and clinical evaluation of NK3 antagonist (e.g. osanetant, talnetant) has led to the hypothesis that NK3 receptors play an important role in the pathophysiology of psychoses and conditions characterized by an elevated release of DA (Spooren et al.,

2005) (Meltzer and Prus, 2006) (Dawson

and Smith, 2010). Thus, tachykinin NK3 receptors have become of interest for the treatment of schizophrenia and a large number of novel selective NK3receptor antagonists have been developed as

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antipsychotics (Spooren et al., 2005). Senktide (S; Succinyl-Asp-Phe-Me-Phe-Gly-Leu-Met-NH2) is a synthetic peptide selective NK3 receptor agonist unable to cross the BBB (Fig. 4). A directly administration of senktide in ventral tegmental area or in the substantia nigra by icv originate an intense stimulation of NK3 receptors that lead to a high dopaminergic response at the level of nucleus accumbens shell (Nac shell) mediated by D2 receptors and subsequently to a reproduction of the same pathological condition of several symptom of schizophrenia. In particular, it has been observed that the responsiveness of NAc shell DA to senktide (0.1 nmol/5µl icv) is completely abolished by pre-treatment with a novel NK3 antagonist (SB222200), showing his potential antipsychotic activity (De Luca et al., 2011) (Fig. 5).As already mentioned, senktide is unable to cross BBB and this property represents a

boundary to further neuropharmacological studies, but, on the other hand, it is an excellent model molecule for the study of brain delivery. For these reasons, the objective of the present study was the development of liposomes to which anti-transferrin-monoclonal antibodies (TfR-mAb, Ox26) or lactoferrin was bounded to transport senktide across the BBB. Senktide was loaded in liposomes for iv administration and in vivo microdialysis studies were performed to estimate the responsiveness of NAc shell DA to senktide as a consequence of its delivery across BBB. Moreover, in order to study the potential antipsychotics activity of the NK3 antagonist SB222200, we will study his ability to completely abolishes the stimulation of DA transmission in NAc shell produced by senktide.

.

Fig. 4. Senktide structure

HOOC

O

O

COOH

NH2NH

NH

N

NH

NH

NH

O O

SMe

O O O

12

Fig. 5. Effector systems of NK3 receptors

Dopamine

Ventral tegmental area (dopamine cell) Nucleus accumbens

Hyper-NK3 state = hyper-dopaminergic state

Normalized NK3 state = normalized dopaminergic state

DopamineD2 receptor

NKB NK3 receptor NK3 receptorantagonist

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AbbreviationsBBB, blood-brain-barrierCNS, central nervous systemDA, dopamineDSPC, Distearoylphosphatidylcholine DSPE-PEG, polyethylene glycol-distearoylphosphatidylethanolamine DSPE-PEG, polyethylene glycol-distearoylphosphatidylethanolamine maleimideHPLC, high-performance liquid chromatographicv, intracerebroventriculariv, intravenous LC/MS, Liquid chromatography – mass spectrometryLf, Lactoferrin LfR, Lactoferrin receptorLOx26 empty OX26 conjugated ImmunoliposomesOx26, monoclonal antibody to the transferrin receptorPI, polydispersity indexRMT, receptor mediated trans-cytosis

S, senktideSL empty stealth liposomesSLOx26 OX26 conjugated Immunoliposomes SLLf Lactoferrin conjugated liposomesSSL Stealth liposomesTEM, Transmission Electron MicroscopyTf, transferrinTfR, transferrin receptorZP, zeta potential

Materials & methods

MaterialsDistearoylphosphatidylcholine (DSPC) and polyethylene glycol-distearoylphosphatidylethanolamine-(DSPE-PEG) were purchased from Lipoid (Ludwigshafen, Germany). DSPE-PEG-maleimide was purchased from NOF corporation (Tokyo, Japan). Cholesterol, 2-iminothiolane (Traut’s reagent), Lactoferrin, Sepharose CL-4B, 10 nm gold-labeled rabbit antimouse secondary antibody, acetonitrile and uranyl acetate were purchased from Sigma (Milan, Italy). Centriprep-30 (molecular weight cut-off: 30,000) concentrators were from Amicon (Billerica, Massachusetts). Anti-rat transferrin receptor OX26 mAb was from AbD Serotec (Kidlington, UK). Senktide was from Tocris Bioscience (Bristol, UK). The NK3 antagonist SB222200 was provided by Roche ( Basel, Switzerland).

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Liposomes preparationLiposomes were prepared using the thin-film hydration method. DSPC (5.2 µmol), cholesterol (4.5 µmol), DSPE-PEG (0.3 µmol) and, for the preparation of immunoliposomes (SLOx26) or lactoferrin conjugated liposomes (SLLf ), DSPE-PEG-maleimide (0.18 µmol), were dissolved in a chloroform/methanol (2:1) mixture. The solvent was evaporated under reduced pressure at room temperature. The vacuum was applied for 6 h to ensure total removal of solvent trace. The obtained lipid film was hydrated under mechanical stirring with a 100 µg/ml senktide phosphate buffer solution at 65°C. Obtained liposomes were subjected to five freeze-thaw cycles (-80°C, 65°C) and extruded through a 200 nm (9 cycles), 100 nm (9 cycles) and 50 nm (5 cycles) pore size polycarbonate membrane using an Avanti Mini-Extruder (Avanti Polar Lipids, Alabaster, AL, USA). For the preparation of immunoliposomes

(SLOx26), OX26 antibody was thiolated by reaction with iminothiolane. In a typical experiment, iminothiolane (32 µg; 0.23 µmoles) is reacted with OX26 (1 mg; 0.0058 µmoles) in 3 mL of borate buffer solution adjusted at pH 8.1. 4mM solution of EDTA is added to chelate divalent metals eventually present in the solution. The mixture is stirred for 2 hours at room temperature. Thiolated OX26 solutions were concentrated and the buffer exchanged with phosphate buffer solution using a Centriprep-30 concentrator (molecular weight cut-off: 30,000). Finally, purified thiolated OX26 antibody were incubated with maleimide grafted liposomes overnight at room temperature. Lactoferrin conjugated liposomes (SLLf ) were prepared with the same method. Thiolated Lf was prepared using a iminothiolane and lactoferrin concentration of 0,09 µmol. SLOx26 and SLLf were separated from senktide and

free mAb or Lf by a Sephadex CL-4B gel filtration chromatography using phosphate buffer as eluent.

Liposomes characterization (phisycochemical characterization: mean size, ZP, PI)The average diameter, polydispersity index (PI) and zeta potential of the samples were determined by Photon Correlation Spectroscopy (PCS) using a Zetasizer nano-ZS (Malvern Instrument, UK). Samples were backscattered by a helium-neon laser (633 nm) at an angle of 173° and a constant temperature of 25°C. The instrument systematically and automatically adapts to the sample by adjusting the intensity of the laser and the attenuator of the photomultiplier, thus ensuring reproducibility of the experimental measurement conditions. The PI was used as a measure of the width

15

of the size distribution. PI less than 0.2 indicates a homogenous and monodisperse population. Zeta potential was estimated using the Zetasizer nano-ZS by means of the M3-PALS (Phase Analysis Light Scattering) technique, which measures the particle electrophoretic mobility in a thermostated cell. All the samples were analyzed 24 h after their preparation.

Incorporation efficiencyIncorporation efficiency (E%), expressed as the percentage of the encapsulated drug respect to the amount of senktide initially used in the liposomes preparation, was determined by liquid chromatography coupled to mass spectrometry (LC/MS, see LC/MS analysis paragraph). Senktide incorporated into liposomes was separated from unincorporated senktide by gel filtration (Sepharose CL-4B). Senktide LC/MS analysis was performed after vesicles disruption using methanol.

Morphological characterizationTransmission electron microscopy (TEM) of liposomes and immunoliposomes was performed. Liposomes were deposited for 2 min on formvar coated 200 mesh copper grids and negatively stained with 2,5% uranyl acetate for 2 min, washed with water, drained and observed at 80 kV with a TECNAI 12 (FEI) TEM. For demonstration of antibody presence on the immunoliposome surface, immunoliposomes were allowed to react ‘‘on grid’’ with 10 nm gold-labeled rabbit antimouse secondary antibody for 120 min. Excess of antibody was washed with PBS, immuliposomes were then stained and observed as below.

In vivo brain microdialysis experiments

Animals and experimental groupsMale Sprague-Dawley rats (Harlan, Italy) weighing 250-275g upon arrival, were housed in groups of six per cage with standard food (MIL topi e ratti, GLP diets, Stefano Morini, S. Polo D’Enza, RE, Italy) and water ad libitum, for at least one week in the central animal room, under constant temperature (23 °C), humidity (60%) and a 12 h light/dark cycle (light from 8 a.m. to 8 p.m.). Animals were divided into 10 experimental groups: 1) S iv (0.1 mg/kg); 2) S icv (0.2 nmol/5µl); 3) SL (empty stealth liposomes, 0.1 ml/kg iv); 4) SSL (S loaded stealth liposomes, 0.1 mg/kg iv); 5) SLOx26 (S loaded OX26 conjugated immunoliposomes, 10 µg/kg iv); 6) LOx26 (empty OX26 conjugated immunoliposomes, 1 ml/kg iv); 7) SB (SB22220, NK3 receptor antagonist, 3

16

mg/kg iv); 8) SB + SLOx26 (3 mg/kg iv + 10 µg/kg iv); 9) SLLf (S loaded lactoferrin conjugated liposomes, 10 µg/kg iv); 10) animals for senktide distribution study in brain and liver tissue. All procedures and experiments were carried out in an animal facility according to Italian (D.L. 116/92 and 152/06) and European Council directives (609/86 and 63/2010) and in compliance with the approved animal policies by the Ethical Committee for Animal Experiments (CESA, University of Cagliari) and the Italian Department of Health.

SurgeryRats were anaesthetized with 300 mg/kg intraperitoneal (ip) of chloral hydrate (Carlo Erba, Italy) and placed in a stereotaxic apparatus. The skull was exposed and a small hole drilled to expose the dura on one side; this was removed and a microdialysis probe was inserted vertically at the level of

the NAc shell (A+2.2, L+1.0 from bregma, V-7.8 from dura) according to the atlas of Paxinos and Watson (De Luca et al., 2012)

(Paxinos and Watson, 1998) (Fig. 6). In the same session, a polyethylene catheter was inserted in the right jugular vein; one group was also implanted with icv cannula at the level of the lateral ventricle (A-0.9; L+1.5 from bregma, V-3.2 from dura).

Analytical procedureOn the day following surgery, the probes were connected to an infusion pump and perfused with Ringer’s solution (147 mM NaCl, 4 mM KCl, 2.2 mM CaCl2) at a constant rate of 1 µl/min. Dialysate samples (10 µl) were taken every 10 min and injected into an HPLC equipped with a reverse phase column (LC-18 DB, 15 cm, 5 µm particle size, Supelco) and a coulometric detector (ESA, Coulochem II, Bedford, MA) to quantify DA. The first electrode of the detector was set at + 130

mV (oxidation) and the second at - 175 mV (reduction). The composition of the mobile phase was: 50 mM NaH2PO4, 0.1 mM Na2-EDTA, 0.5 mM n-octyl sodium sulfate, 15% (v/v) methanol, pH 5.5. The sensitivity of the assay for DA was 5 fmol/sample.

HistologyAt the end of the in vivo microdialysis experiments, rats were transcardially perfused with 50 ml saline and 150 ml of a 10% formaldehyde solution. The probes were removed and the brains were cut by a vibratome in serial coronal slices and the location of the probes was reconstructed and referred to the atlas of Paxinos and Watson (1998) (Paxinos and Watson,

1998).

Senktide tissue distribution study The brain and liver were post-mortem

 

Fig. 6. Schematic representation of the localization of microdialysis probes dialysing portion within the NAc shell and core (according to Paxinos and Watson, 1998). CPU, Caudate putamen; co, sh, core and shell of the NAc; cc, corpus callosum; ca, anterior commisure

17

collected from treated animals and kept at -80° C until analysis. 0.5 g of sample were individually weighed, placed in a falcon tube and homogenized in 5 mL phosphate buffer at pH 7.4 by means of an ultrasonic disruptor. The matrix thus obtained was acidified with 2 mL perchloric acid (0.1 M) centrifuged at 13000 rpm and the surnatant was filtered with Acrodisc Syringe Filters with 0,45 µm PTFE Membrane (Life Science) and subjected to chromatographic analysis by LC/MS.

LC/MS analysis The amount of encapsulated senktide as well as brain and liver levels after in vivo experiments were determined by liquid chromatography coupled to mass spectrometry (LC/MS). A Varian 1200 L triple-quadrupole tandem mass spectrometer (Palo Alto, CA, USA) together with a ProStar 410 autosampler, two ProStar 210 (Varian Inc., CA, USA)

pumps and a 1200 L triple-quadrupole mass spectrometer (Varian Inc., CA, USA) was used with an electrospray ionization source (ESI). The Varian MS workstation version 6.7 software was used for data acquisition and processing. Chromatographic separation was performed on a Phenomenex Column Synergi MAX-RP 80A (4.6 mm × 150 mm i.d., 4 µm) (Phenomenex Italy, Castel Maggiore (Bo), Italy). The mobile phase consisted of (A) acetonitrile 50% (v/v) containing 0.1% trifluoroacetic acid and (B) double distilled water 50% (v/v) containing 0.1% trifluoroacetic acid reaching 80% of A in 15 min. The mobile phase, previously degassed with high-purity helium, was pumped at a flow rate of 0.3 ml/min, the injection volume was 10 µl and total run time 15 min. Liposomes preparation were diluted ESI was operated in the positive ion mode. The electrospray capillary potential was set at 149V, the

needle at 5000V, and the shield at 600V. Nitrogen, at 48 mTorr and 375°C, was used as a drying gas for solvent evaporation. Full-scan spectrum was obtained in the ranges of 100-1000 atomic mass unit (amu) for senktide, scan time of 0.75 amu, scan width of 0.70 amu, and detector at 1450V. For ESI the atmospheric pressure ionization (API) housing was kept at 50°C. Senktide was detected in the single ion monitoring mode (SIM) observing the sodium and potassium adducts at mass 865 and 881 m/z respectively. The scan time was 1 sec, and the detector multiplier voltage was set to 2000V, with an isolation width of m/z 1.2 for quadrupole 1.

StatisticsStatistical analysis was carried out by Statistica for Windows. Difference in the levels of extracellular DA between groups were analysed by ANOVA analysis of variance for repeated measures. Results

18

from treatments showing significant overall changes were subjected to post hoc Tukey’s test. Basal values were the means of three consecutive samples differing by no more than 10%. Difference in the tissue distribution of senktide were analysed by T-test. It was determined that p<0.05 was statistically significant.

Results and discussion

Liposome preparation and characterization Liposomes (see Fig.7 for schematic representation) were prepared using the film hydration method and repeated extrusion through 200, 100 and 50 nm pore size polycarbonate membrane. Six different formulations have been prepared using the components reported in Table 1. SSL were prepared using (DSPC), DSPE-PEG and cholesterol. For the preparation of SLOx26 and SLLf part of DSPC has been substituted with the lipid linker DSPE-PEG maleimide. The introduction of DSPE-PEG maleimide in the vesicle structure is required to link the homing device (OX26 or Lf ) to the liposomes bilayer. In order to bind the linker with a thioether bond, OX26 and Lf have been thiolated. In the case of OX26 a mAb/iminothiolane 1:40 molar ratio has been

used to ensure an average thiolation of one primary ammine per mAb (Huwyler et al.,

1996). As previously reported, thiolation of OX26 does not interfere with TfR recognition (Kang et al., 1995). Since the maleimide group slowly hydrolyses when in contact with water, it was essential to proceed for the preparation of SLOx26 and SLLf quickly. The senktide encapsulation efficiency (E%) was determined by LC/MS after separation of unincorporated senktide by gel filtration. The introduction of OX26 mAb or Lf little influence the E% compared to that of SSL. Reproducible E% values of 34.5±2.2, 32.5±2.5 and 30.3±2.1 were obtained for SSL, SLOx26 and SLLf respectively.

Formulations

Components SSLmol

SLmol

LOx26mol

SLOx26mol

SLLf mol

LLfmol

DSPC 5.02 5.02 5.20 5.20 5.20 5.20

Cholesterol 4.50 4.50 4.50 4.50 4.50 4.50

DSPE- PEG 0.30 0.30 0.30 0.30 0.30 0.30

DSPE- PEG Maleimide - - 0.18 0.18 0.18 0.18

Senktide 0.12 - 0.12 -

-

0.12

OX26 - - 0.002 0.002 - -

-

Lactoferrin - - - 0.09 0.09

Table 1. Liposomes composition. SSL (senktide loaded stealth liposomes), SL (empty Stealth liposomes), SLOx26 (senktide loaded OX26 conjugated liposomes), LOx26 (empty OX26 conjugated liposomes), SLLf (senktide loaded lactoferrin conjugated liposomes), LLf (empty lactoferrin conjugated liposomes)

19

Lactoferrin

Lactoferrin

SSL

SLOx26SLLf

OX26 Mab

OX26 Mab

Target: Transferrin receptor TfRTarget: Lactoferrin receptor LfR

PEG

Senktide

Fig.7. Schematic representation of liposomes

20

In table 2 the mean diameter, polydispersion index and Z potential of different formulations are reported. All six liposome formulations showed a mean diameter around 120 nm, as determined by Photon Correlation Spectroscopy (PCS). It is important that the liposomes mean diameter do not exceed this value in order to ensure a good circulation of the nanocarriers through the tighter rat brain blood vessels which is about 5 µm and to prevent obstructions (Villringer

et al., 1994). Polydispersion index (0,131 for SSL, 0,134 for SLOx26 and 0,185 for SLLf ) demonstrated a good monodisperse size distribution for all the formulations. Slightly negative Z potential values (near zero) for the three types of liposomes suggesting a modest stability. To overcome possible liposomes aggregations, formulations were tested 24h after their preparation. In table 2 the mean diameter, polydispersion index and Z potential of

empty liposomes are also reported. Empty liposomes showed a smaller diameter compared to that of corresponding loaded formulations. Polydispersion index and Z potential values of empty liposomes were similar of that of loaded formulations.TEM analysis (Fig.8) was performed to investigate the liposomes morphology, to confirm their mean diameter determined by Photon Correlation Spectroscopy (PCS) and to verify the linkage between OX26 and the liposomes bilayer. Unfortunately, in normal conditions, surface-linked OX26 mAb is not detected by electron microscopy, so we use an anti IgG secondary antibody carrying at its distal end a 10 nm gold nanoparticles. This second antiboby specifically react which anti-transferrin-monoclonal antibodies OX26. After negatively stain with uranyl acetate and after purification, 10 nm gold nanoparticle appear in micrography like a small, spherical shadows attached onto

liposome surface indicating the presence of OX26 and his occurred bond with the liposomes surface.

SSL

SLOx26

SLLf

SL

LOx26

LLf

Mean size (nm)

129,3 ± 5,9

120,6 ± 2,1

123,7 ± 4,9

112,3 ± 3,6

109,7 ± 1, 8

117,8 ± 7,2

PI

0,131 ± 0,005

0,134 ± 0,018

0,185 ± 0,024

0,115 ± 0,037

0,177 ± 0,002

0,163 ± 0,088

ZP

- 2,02 ± 0,24

- 0,85 ± 1,52

- 3,62 ± 0,99

- 1,37 ± 0,83

- 2,22 ± 0,65

- 0,94 ± 0,37

Table 2. Liposome physicochemical characterization of SSL (senktide loaded stealth liposomes), SL (empty Stealth liposomes), SLOx26 (senktide loaded OX26 conjugated liposomes), LOx26 (empty OX26 conjugated liposomes), SLLf (senktide loaded lactoferrin conjugated liposomes), LLf (empty lactoferrin conjugated liposomes)

21

In vivo brain microdialysisBasal values of DA in the NAc shell were: 52±6 (N=34). Values were expressed as fmoles/10-µl sample (mean±SEM).

Responsiveness of NAc shell DA to senktide delivered by liposomes conjugated to mAb OX26Figure 9 shows the effect of senktide administration by liposome conjugated to OX26 (SLOx26, 10 µg/kg iv) on NAc shell dyalisate DA. The figure also shows the comparison between SLOx26 and the administration of senktide iv, icv, by stealth liposome and the effect of empty stealth liposome and empty LOx26. Two-way ANOVA showed a significant effect of group [F(5,21)=11.85, p<0.001] and a significant group x time interaction [F(90,378)=1.5, p<0.005]. Tuckey’s post hoc test revealed differences between senktide administered icv and by LOx26 and all the

other groups. Interestingly, the DA increase observed after SLOx26 administration overlapped the increase induced by the icv infusion of S.

Blockade of NAc shell DA increase senktide-mediated by NK3 receptor antagonist (SB222200)Figure 10 shows the blockade of the effect of senktide on dialysate DA by the NK3 receptor antagonist SB222200 (SB, 3 mg/kg iv). Two-way ANOVA showed a significant effect of group [F(2,13)=16.6, p<0.001] and a significant group x time interaction [F(36,234)=1.7, p<0.005]. Tuckey’s post hoc test revealed differences between senktide administered ICV and by LOx26 and all the other groups.

Responsiveness of NAc shell DA to senktide delivered by liposomes conjugated to Lactoferrin

Figure 11 shows the central effect of senktide delivered by liposomes conjugated to Lactoferrin compared to S icv or loaded in stealth liposomes (SSL). Two-way ANOVA showed a significant effect of group [F(2,9)=18.23, p<0.001] and a significant group x time interaction [F(60,270)=2.06, p<0.0001]. Tuckey’s post hoc test revealed differences between senktide administered by SLLf and all the other groups.

Fig. 8. Gold modified Ox-26 liposomes: TEM

22

Senktide tissue distribution study

Senktide distribution in brain tissue after different administrationFigure 12 shows the distribution of senktide in brain after the administration of S iv, loaded in stealth liposomes (SSL) or administered by SLOx26 or SLLf. One-way ANOVA showed a significant effect of group [F(3,24)=60.93, p<0.0001]. Tuckey’s post hoc test revealed differences between SLOx26 and SLLf and all the other groups.

Senktide distribution in brain and liver Figure 13 shows the distribution of senktide in brain or liver tissue of rats. One-way ANOVA showed a significant effect of group [F(3,8)=2.23, p<0.0001]. Tuckey’s post

hoc test revealed differences between liver and brain senktide distribution after the

administration by SLOx26 and SSLf. DiscussionThe main finding of this study is that senktide, delivered to the CNS by liposomes conjugated to the specific homing devices anti-transferrin-monoclonal antibody (SLOx26) and lactoferrin (SLLf ), are able to increase dopamine transmission in the NAc shell. This finding indicates that both SLOx26 and SLLf formulations represent an effective way to deliver senktide across the BBB. In general, the value of brain uptake of a drug is equally dependent on two factors: the drug ability to permeate through the BBB and his plasma AUC. Senktide is a peptide with a high hydrophilicity that prevents its penetration through the BBB by passive diffusion. Moreover, BBB does not present specific carrier protein nor receptor able to transport senktide by CMT or RMT. These are the reasons why senktide cannot permeate the BBB to reach the brain parenchyma. However, senktide is able to increase NAc

23

shell if injected directly into the brain (icv) (Sandoval-Guzmán and Rance, 2004). In fact, senktide acts as the endogenous ligand neurokinin B (NKB) that stimulates DA neurons by high NK3 receptor occupancy at the VTA level. This results in an increased DA release at the level of DA terminal regions such as the NAc and a high level of DA D2 receptor occupancy post-synaptically (Spooren et al., 2005). For this reason, it has been hypothesized a clinical efficacy of NK3 receptor antagonists for psychoses or “hyper dopaminergic state”. NK3 receptor antagonists prevents NKB from binding to the NK3 receptor. Accordingly, the release of DA at the level of DA terminal regions (e.g. NAc) and post-synaptic D2 receptor occupancy is significantly reduced. This study aimed to overcome BBB by different senktide loaded liposomes formulated using OX26 or Lf as homing devices. The ability of senktide to promote the DA release in the NAc shell

when administered icv and the comparison of this effect with the administration of the free drug iv or loaded into stealth liposomes and SLOx26 and SLLf iv has been evaluated (figure 9-11). Interestingly, the iv administration of senktide loaded OX26 conjugated liposome exerted an increase of NAc shell dyalisate DA which overlapped the increase probuced by the icv administration of senktide (Fig 9) from 20 to 180 min after the injection. This effect was mediated by NK3 receptor, in fact it was blocked by the administration of SB222200 30 min prior to the SLOx26 administration (Fig 10). Similar results were obtained by the administration of senktide loaded SLLf (Fig 11). However, the increase of DA was delayed (190-300 min after injection) and this effect was probably mediated by a different kinectic of the Lf receptor compared to TfR. The increase of DA in the NAc shell is mediated by the action of senktide as

agonist at VTA NK3 receptors. The results obtained show that the administration of SLOx26 encapsulated senktide produces a stimulation of DA transmission in the shell of the NAc comparable to that obtained by administration of senktide directly into the brain (icv). The fact that the increase of DA transmission in shell observed by us after administration of liposomes conjugated to OX26 is similar to that achieved by administration of S icv, shows, albeit in an indirect way, that the senktide has exceeded the BBB and for this it is able to exert its action in the CNS. The senktide produces a release of DA in the shell because it binds to NK3 receptors localized at the level of the VTA on mesolimbic dopaminergic neurons. Furthermore, the fact that the administration of NK3 antagonist (SB222200) completely abolishes the stimulation of DA transmission in shell produced by senktide, provides a confirmation of the central action of

24

senktide and of its effectiveness if properly formulated as in the case of SLOx26. The in vivo senktide up-take in brain and liver tissues was examined in rats 3 hours after administration (figure 12, 13). For the determination of the amount of senktide accumulated in tissues, the brain and the liver were post-mortem collected from treated animals and treated as previously reported. After administration of free peptide iv and peptide loaded stealth liposomes (SSL) iv, no measurable senktide was found in the brain (figure 12). As previously reported, the high hydrophilic nature of senktide and the absence of specific carrier protein nor receptor able to transport this peptide by CMT or RMT, prevent his accumulation in the brain parenchima. One of the characteristic of SSL is they ability to increase the circulation times in the bloodstream increasing thus the plasma AUC, a prerequisite factor to enable a drug accumulation in the brain.

However, as for free senktide, stealth liposomes cannot permeate through the BBB because a lack of specific transport mechanism and, as demonstrated in literature, their brain permeability-surface area is 0 (Huwyler et al., 1997). After administration of SLOx26 and SLLf the amount of senktide found in the brain parenchima is 0.0110±0.0012 and 0.0092±0.0005 µg per g of tissue, respectively. This corresponds of about the 0,12% and 0.09% of the injected dose, a values similar to that reported in literature for small molecules delivered to the brain using immunoliposomes. The higher up-take of senktide obtained 3 hours after the administration of SLOx26 is probably due to a different kinectic of the TfR receptor compared to LfR. The senktide up-take in liver expressed as µg/g of tissue, was approximately 75 and 100-fold higher than in brain for SLOx26 and SLLf, respectively. TfR and LfR was identified

in the brain microvessel but also in other tissues like monocytes, lymphocytes, liver, mammary epithelial cells, spleen, intestine etc (Chen et al., 2010). Many studies also showed that lactoferrin could bind to different receptors on hepatocytes including low-density lipoprotein receptor-related protein receptors, and the asialoglycoprotein receptors. Moreover, the high level of senktide found in liver can be explained considering the abundant expression of TfR and LfR on hepatocyte plasma membranes. In particular the higher level of senktide found in liver after administration of SLLf could be the result of the high affinity of this tissue for Lf. Indeed, very rapid Lf clearance (93% of the dose) at 5 min after iv injection, was found to be mainly a result of association of lactoferrin to the liver, in particular to the parenchymal cells (Ziere et al., 1992). The liver uptake of OX26 is conversely, about 3 times lower than measured for Lf (Ji et al.,

25

S (0.1 mg/kg iv)

SSL (0.1 mg/kg iv)SLOx26 (0.5 µ g/kg iv)

S (0.2 nmol/5 µ l icv)

SL (1 ml/kg iv)

LOx26 (1 ml/kg iv)

Fig. 9. Responsiveness of NAc shell DA to senktide delivered by SLOx26

0 60 120 180

100

150

200

250

*

**

*

*

* * *

*

* *

*

#

#

#

**

*

*

Time (min)

DA

leve

ls (%

of b

asal

)

26

SLOx26 (0.5 µ g/kg iv)SB( 3 mg/kg iv)

SB (3 mg/kg iv) + SLOx26 (0.5 µ g/kg iv)

Fig. 10. Blockade of NAc shell DA increase senktide-mediated by NK3 receptor antagonist (SB222200)

0 60 120 180

100

150

200

250

Time (min)

DA

leve

ls (%

of b

asal

)

*

**

*

*

* * *

*

**

*

#

# #

*

*

*

*

#

#

##

#

*

27

Time (min)

DA

leve

ls (%

of b

asal

)

0 60 120 180 240 300

100

150

200

* *

**

*

#

#

#

#

Fig. 11. Responsiveness of NAc shell DA to senktide delivered by SLLf

S (0.1 mg/kg iv)SSL (0.1 mg/kg iv)SLLf (0.5 µ g/kg iv)

28

2006). Moreover, although the presence of PEG on the surface of vesicles increases circulation times in the bloodstream delaying the opsonisation process and the liposomes up-take by macrophage cells in the liver and spleen (mononuclear phagocytic system or RES), however this process occur. As already mentioned, and according to our results, we can say that the formulations studied here are an effective method for the administration of peptide drugs for tachykinin receptor agonists or not. Further nanoformulations made for other animal species or humans, for example linked to a monoclonal antibody specific for the man (eg. HIR, monoclonal antibody to the insulin receptor) could represent a valid system for the delivery of molecules potentially useful in therapy, but until now not used because of their inability to cross the BBB.In conclusion, the present observations indicate that both SLOx26 and SLLf

formulations represent an effective way to deliver senktide across the BBB probably by a RMT (receptor mediated transport). According to their efficacy in term of effectiveness and rapidity of action, both formulations could be used for different therapeutic requirements.

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Béduneau, A., Saulnier, P., Benoit, J.-

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Nanosuspensions Introduction

The advent of combinatorial chemistry and high throughput screening in drug development programs has led to a generation of molecules with poor aqueous solubility and poor bioavailability profile. In recent years, the number of drug candidates defined as having low solubility has increased, and about 70% of this new drugs have shown poor aqueous solubility (Ku and Dulin, 2010). Moreover, approximately 40% of the marketed immediate release oral drugs are categorized as practically insoluble (<100 µg/ml) (Takagi et al., 2006). Poor aqueous solubility is one of the major hurdles in the development of new compounds into oral dosage forms, since dissolution is the first step in the absorption of the drugs. The limited dissolution rate, in turn, frequently lead up to a low bioavailability of orally administered drugs, and compounds with

aqueous solubility lower than 100 µg/ml generally present dissolution-limited absorption (Hörter and Dressman,

2001). Poorly soluble molecules have been successfully formulated by employing a variety of techniques such as: solubilization in surfactant solutions; use of cosolvents; pH adjusted solutions; emulsions; liposomes; complexation with cyclodextrins; and solid dispersions (Kesisoglou et al., 2007) (Patravale et al.,

2004). However, most of these techniques require a large amount of additives limiting their use from the safety perspective. Moreover, when drugs are insoluble in both aqueous and organic media (the so-called ‘brick dust drugs’), these approaches are almost ineffective. For all these reasons, in the last decades, drug nanocrystal technology has been strongly studied and developed and without doubt, it represents one of the highlights in pharmaceutical field. This technology, in general, allow to

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generate nanosuspensions, a dispersions of drug crystals in a liquid medium (typically water), with size below 1µm, stabilized by surfactants or polymers (Fig. 1). Nanosuspensions provide an alternative method to formulate poorly soluble compounds and their pharmaceutical benefits include improvement in formulation performance, such as enhanced dissolution velocity and saturation solubility, reproducibility of oral absorption, improved dose-bioavailability, proportionality and increased patient compliance via reduction of number of oral units to be taken (Müller et al.,

2001) (Rabinow, 2005). In particular, nanocrystal serves as ideal delivery system for oral drugs having the dissolution velocity as rate limiting step for absorption. In addition, nanocrystals can be injected intravenously as aqueous nanosuspensions (Rabinow et al., 2007) and also employed in ocular (Pignatello et al., 2002) and

pulmonary delivery (Jacobs and Müller,

2002). Another remarkable aspect is how fast these nanocrystals entered the pharmaceutical market. It took about 25 years for the liposomes to appear in pharmaceutical products on the market (around 1990, e.g. Alveofact from Dr. Thomae GmbH). It was less than 10 years for the nanocrystals, having the first patent applications filed at the beginning of the 1990s (Keck and Müller, 2006), and the first product Emend® on the market in 2000. All these advantages have so tremendous impacts on promoting drug nanocrystals successfully from experimental researches to patients that several products have been launched into market (Table 1).

Preparation methods

Now a day exists basically two main approaches to produce drug nanosuspensions: the so called ‘bottom up’ and ‘top down’ (Fig. 2).Top-down processes consist of particle size reduction of large drug particles into smaller particles using various wet milling techniques such as media milling, microfluidization and high pressure homogenization. No harsh solvents are used in top down techniques; moreover, this technology can be employed for all insoluble drugs including “brick dust drugs”.The bottom-up process is broadly called a precipitation process, because the principle here is to precipitate drug particles from a supersaturated solution of the drug in the presence of a stabilizer. Various adaptations of this approach include: solvent–anti-solvent method; supercritical fluid processes; spray drying and emulsion–solvent evaporation (Date and Patravale, 2004) (Rabinow,

Fig. 1. Schematic representation from micro to nano size

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Product/Company Drug compound Nano -sizing approach

Administration route

Date of FDA approval

Rapamune®/Wyeth Sirolimus Top – down media milling

Oral 2000

Emend®/Merck Aprepitant Top – down media milling

Oral 2003

Tricor®/Abbott Feno�brate Top – down media milling

Oral 2004

Megace® ES/Par Pharma

Megestrol acetate Top – down media milling

Oral 2005

Tridlide™/Skye Pharma

Feno�brate Top – down HPH

Oral 2005

Invega Sustenna/Johnson

& Johnson

Paliperidone palmitate

Top – down Media milling

Injection 2009

Table 1. Key characteristics of available commercial drug products based on drug nanoparticle technology

Attrition - for example,

milling

Agglomeration

Dissolution

Nucleating and crystal growth

Water molecule Water - surfactant interaction

ba

Fig. 2. Nanosuspension preparation techniques

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2004). Top down technologiesMedia millingWet ball milling WBM (also referred as pearl milling or bead milling) is by far the most frequently used production method for drug nanocrystals in the pharmaceutical industry. Around the 1990s Gary Liversidge and his colleagues from Sterling Drug Inc./Eastman Kodak have applied a wet media-based milling technique, adapted from the paint and photographic industry, to reduce the particle size of poorly water-soluble drugs; (Liversidge and Conzentino,

1995). This process has evolved since and eventually became well known as NanoCrystal® technology in the pharmaceutical industry and is to date the most successful nanosizing approach with currently 5 products on the market (Merisko-Liversidge and Liversidge,

2011). The milling procedure itself is rather

simple; therefore this process can be basically performed in almost every lab. The easiest way of doing WBM is through low energy ball milling (LE-WBM) using a jar filled with milling media (often just very simple glass beads.This system is charged with coarse drug substance, preferably in micronized form, which is suspended in a dispersion medium (normally water or a buffer) containing at least one stabilizing agent. By moving the beads either with an electric stirrer (Fig. 3A), a magnetic stirrer, or by moving the whole jar, with a roller plate or a mixer (Fig. 3B), the milling beads can get in touch with drug particles. In the LE-WBM process, a combination of cleavage, abrasion and fractures due to the strong collisions between drug crystals and the beads can be assumed as the main mechanism of size reduction (Hennart et al., 2012). The process generally yields very fine particles with a narrow size distribution when it is

Fig. 3A – 3B. Setup for low energy wet ball milling. (A) Vial filled with milling beads, suspension and a magnetic bar placed on a magnetic stirrer plate, the beads are moved by the rotating magnetic bar inside the vial. (B) Plastic bottle (small picture lower right) filled with milling beads and suspension moved by a standard mixer, the whole system is moved.

44

performed long enough.However, the relatively low energy input used in this techniques leads to very long milling times of several days (Liversidge

and Conzentino, 1995) (Merisko-

Liversidge et al., 1996).Alternative milling procedures based on high energy processes had to be developed in order to make this process more desirable for industrial pharmaceutical applications. The NanoCrystalTM process in its current form is based on such a high energy wet ball milling process (HE-WBM) (Merisko-

Liversidge and Liversidge, 2008). A necessary prerequisite for HE-WBM is the availability of suitable equipment. The manufacturers for milling equipment had to develop equipment with sufficiently high power densities for the improved processes (Fig. 4).Today, HE-WBM can be regarded as a standard procedure to produce nanosuspensions. Due to the much higher

power density, the production times are significantly reduced. Normally, the drug needs to be exposed to the high energy for about 30–120 min in order to achieve a nanosuspension of good quality (Merisko-

Liversidge and Liversidge, 2011). In this case, the comminution is a result of shear stresses and compression forces inside the milling chamber (Kwade, 1999). As for LE-WBM, drug particles are reduced in size by abrasion and cleavage mechanisms (Hennart et al., 2012). It is obvious that high energy mills require special milling media which has to be properly selected based on the material of the inner surfaces of the mill, the agitator types and other factors. The use of glass beads or zirconium oxide milling beads can lead to significant contamination of the nanosuspension caused by the abrasion either of the milling beads or parts of the milling chamber (Hennart et al., 2010) (Juhnke et al.,

2012). Initially, impurities caused by

abrasion were one of the major obstacles for a broader acceptance of WBM. Therefore, a major milestone for the broad acceptance of the milling process was the introduction of zirconium stabilized with yttrium beads or highly crosslinked polystyrene beads as milling media (Kesisoglou et

al., 2007) (Merisko-Liversidge et al.,

2003). This milling media shows elastic deformation, thereby the formation of cracks and abrasion from beads is reduced. This leads to product qualities which allow the use of nanosuspensions even for parenteral administration (Merisko-

Liversidge and Liversidge, 2011). In the early nineties, there was no equipment available to produce nanosuspensions at very small scale. Hence, it was difficult to use this formulation approach for discovery purposes. Today, even high-energy mills are available for small-scale production of nanosuspensions. Several research groups have reported ways to use

45

Recirculation chamber

Coolant

Milling shaft

Milling media

Screen - retaining milling media chamber

Nanocrystals

Milling chamber

Motor

Charged with drug, water and stabilizer

Large drug crystals

Fig. 4. Schematic representation of the media milling process. The milling chamber charged with polymeric media is the active component of the mill. The mill can be operated in a batch or recirculation mode. A crude slurry consisting of drug, water and stabilizer is fed into the milling chamber and processed into a nanocrystalline dispersion. The typical residence time generated for a nanometer-sized dispersion with a mean diameter of <200nm is 30–120 min.

46

existing planetary ball mills (Fig. 5A-5B) with modified sample holders which can be used to process several nanosuspensions at the same time (Juhnke et al., 2010) (Van

Eerdenbrugh et al., 2009). With the commercial availability of suitable equipment for small-scale production up to the commercial scale production, wet ball milling can be regarded as scalable approach. This aspect has definitely helped for broader acceptance of this rather complex technology (Merisko-

Liversidge and Liversidge, 2011). The versatility of wet ball milling is certainly another, if not the most important aspect for the success of this technology. Almost any poor soluble drugs can be processed with wet media milling (Cooper, 2010). Interestingly, most particle sizes reported for nanosuspensions prepared by wet ball milling are in the range between 100 and 300 nm, irrespectively whether LE-WBM or HE-WBM was used. All these aspects

have opened the possibility to use wet ball milling as a platform technology for formulating poorly soluble compounds.

High pressure homogenization (HPH)The application of HPH as particle size reduction method requires the availability of special equipment and it cannot be tested with a system as simple as “beads in a beaker”. The use of homogenizers was already described for the production of liposomes and emulsion systems (Brandl

et al., 1990) (Collins-Gold et al., 1990), moreover, high pressure homogenizers can also be used for the production of solid lipid nanoparticles or nanostructured lipid carriers (Müller et al., 2000) (Müller

et al., 2002) (Müller et al., 2011). The possibility to employ the production equipment for various formulation approaches (multi-purpose production lines) is an important advantage, as it

is rather costly to establish production lines in-house. In this technology we can recognize three basic processes used: Microfluidizer technology (IDD-PTM technology) based on the jet stream principle; piston-gap homogenization either in water (Dissocubes® technology, SkyePharma) or alternatively in water-reduced/non-aqueous media (Nanopure® technology, prev. PharmaSol GmbH, Berlin, now Abbott Laboratories). The Microfluidizer technology (Fig. 6) is based on the jet stream principle and can generate small particles by a frontal collision of two fluid streams in a Y-type or Z-type chamber under pressures up to 1700 bar. The jet streams lead to particle collision, shear forces and cavitation forces (Microfluidizer®, Microfluidics Inc.). Often a relatively high number of cycles (50–100 passes) are necessary to obtain sufficient particle size reduction. SkyePharma Canada Inc. (formerly Canadian company

Fig. 5A. Planetary ball mill

47

Material and balls fall back down

Lifting plates carrying the material up the side of the SAG mill

Direction of rotation of mill

Fig. 5B. Schematic representation of the inside of an agate jar in a planetary ball mill

48

Research Triangle pharmaceuticals (RTP)) uses this principle for their Insoluble Drug Delivery-Particles (IDD-PTM) technology to achieve the production of submicron particles of poorly soluble drugs (Keck and

Müller, 2006). The Dissocubes® technology was developed by Müller and co-workers by employing piston-gap homogenizers (e.g. APV Gaulin/Rannie homogenizers) (Fig. 7). The technology was acquired by SkyePharma PLC in 1999. This approach consists in forcing a drug dispersed in an aqueous surfactant solution (macrosuspension) by a piston under pressure (up to 4000 bar, typically 1500–2000 bar) through a tiny gap (5–20 µm). Previously, this coarse suspension is pre-milled passing several times through the HPH at low pressure (about 500 bar). Later, the applied pressure is increased step-wise from 10% to 100% in order to avoid clogging of the narrow homogenization gap. This explains the

importance of the pre-mixing procedure for deagglomeration and wetting purposes, especially when relatively coarse material is processed. The resulting high streaming velocity of the suspension causes an increase in the dynamic pressure. This is compensated by a reduction in the static pressure below the vapor pressure of the aqueous phase; hence, water starts boiling forming gas bubbles. These gas bubbles collapse immediately when the liquid leaves the homogenization gap (=cavitation). The drug particles are reduced in size due the high power of the shockwaves caused by cavitation together with shear forces and collision.Another approach using the piston-gap homogenizer is the Nanopure® technology. This technology uses a primary dispersion medium, non-aqueous liquids, oils, liquid and solid (melted) PEG, or water reduced media (e.g. glycerol–water, ethanol–water mixtures), and optionally homogenization

at low temperatures. In general, several homogenization cycles are needed to reach the minimal particle size. The number of passes (homogenization cycles) depends on many factors. Thereby, the employed drug delivery technology defines the type of homogenizer as well as the process conditions (e.g. IDD-PTM technology, Dissocubes® or the Nanopure® technology) (Keck and Müller, 2006) (Shegokar

and Müller, 2010). Additional factors determining the process efficiency include size of the starting material, hardness and amount of the drug and maximum pressure that can be reached by the machine. In general, higher pressure leads to faster particle size reduction (Dumay et

al., 2013) (Kluge et al., 2012). The size of the impaction zone and the corresponding volume are important factors, as they determine proportionally the power density of the equipment. HPH is less prone in generating process impurities

49

Ending particlesending size: 74 microns

Starting particlesstarting size: 500 microns

Outlet reservoircooling jacket

Intensi�er pump constant pressure

Inlet reservoir Fixed geometrypatented interactionchamber

Pressure gaugePressure to 40,000 psi

Fig. 6. Microfluidizer technology

50

as consequence of abrasion and wearing of the equipment compared to WBM. Although high pressure homogenizers consist mainly of steel parts, the impurity levels found in nanosuspensions prepared via HPH processes are considerably low. Abrasion and wearing of HPH equipment can occur when extremely hard material is processed in piston-gap homogenizers. HPH is a scalable process, which is applied not only in the pharmaceutical but also in the cosmetics and food industry (Dumay et al., 2013). Today high pressure homogenizers are available from ml-scale to large production scale (Keck and

Müller, 2006). There is no general rule, but it seems that HPH is the method of choice for relatively soft materials with tendency to smear when processed with others methods, such as WBM. Finally, HPH and WBM can be defined by far the most industrial relevant technologies to produce drug nanocrystals and it is

represented by the six different commercial pharmaceutical products based on nanosizing approaches already approved.

Bottom up technologiesBottom-up processes have some advantages if compared with top-down technology. This advantages include that these are low energy processes, require simple instruments, are less expensive and can be operated at a low temperature, making them particularly suitable for thermolabile drugs (Rasenack et al., 2004). The particle size obtained by bottom-up technology has a narrow size distribution, unlike other top-down processes (Rasenack

et al., 2004) (Zhang et al., 2008). The bottom-up process is broadly called a precipitation process, because the principle here is to precipitate drug particles from a supersaturated solution of the drug. The precipitation can be induced by processes that further increase the supersaturation

in the system, such as evaporation of the solvent, reduction of temperature or by mixing it with an antisolvent. In the last 20 years, a number of research studies have been published based on precipitation technology for preparation of nanoparticles of organic drug substances. Broadly, these studies can be classified in four categories: precipitation by liquid solvent–antisolvent addition, precipitation in presence of supercritical fluid, precipitation by removal of solvent and precipitation in presence of high energy processes.

Precipitation by liquid solvent-antisolvent methodAmong the various precipitation techniques, nanoprecipitation by liquid solvent antisolvent addition has been the most reported. This is because of the fact that it is the simplest and the most cost effective method. List and Sucker in 1988 first reported preparation of “Hydrosol” by

Forcer

Homogenisedproduct

Homogenisedproduct

Seat

Gap ≈ 0.1mm

Fig. 7. Piston gap homogenizer

51

controlled precipitation method (Gao et

al., 2012). Later, Soliqs (Abbott GmbH & Co.KG, Ludwigshafen, Germany) developed the NanoMorph® technology for preparing nanosuspensions of amorphous form stable throughout the shelf life (Gao et al., 2012). Precipitation has been applied for years to prepare submicron particles within the last decade (Patravale

et al., 2004). Typically, the drug is firstly dissolved in a solvent. Then this solution is mixed with a miscible antisolvent in the presence of surfactants. Rapid addition of a drug solution to the antisolvent (usually water) leads to sudden supersaturation of drug in the mixed solution, and generation of ultrafine crystalline or amorphous drug solids. This process involves two phases: nuclei formation and crystal growth. When preparing a stable suspension with the minimum particle size, a high nucleation rate but low growth rate is necessary. Both rates are dependent on temperature:

the optimum temperature for nucleation might lie below that for crystal growth, which permits temperature optimization. The solvent for preparing the drug solution can be any organic solvent like acetone, ethanol, methanol, isopropanol (IPA), N-methylpyrrolidone (NMP), etc., a cosolvent or mixture of cosolvents like polyethylene glycols (PEGs), propylene glygol (PG), buffer system of particular pH (Dong et al., 2009) (Douroumis

and Fahr, 2007) (Raghavan et al., 2003)

(Wang et al., 2005). Often a solvent is selected which gives the highest solubility for the chosen compound, however, the interaction of stabilizer with solvent needs to be considered as well. The antisolvent used in this process is often an aqueous solution of some stabilizer (s). However, sometimes both the solvent and antisolvent can be organic solvent with sufficient miscibility (Zhang et al., 2006) (Zhu et

al., 2010).

Mixing solvent and antisolvent is the simplest method for preparing nanoparticles. However, it has been observed that the addition of some external factors (such as ultrasonic wave) or undertaking the precipitation process in some altered environment (inside a freeze dryer, spray dryer or with high gravity reactive precipitation) often result in a smaller particle size or narrower particle size distribution (PSD). The lower particle size obtained by these modified methods is a result of either faster mixing to affect the nucleation stage or by arresting particle growth.

SonoprecipitationUltrasonic sound has been used by various researchers to induce crystallization (Kumar et al., 2009) (Li et al., 2003)

(Louhi-Kultanen et al., 2006) (Luque de

Castro and Priego-Capote, 2007). The ultrasonic energy can be introduced simply

52

by dipping a probe sonicator in a vessel kept under stirring for mixing a solvent with an antisolvent (Dhumal et al., 2008). The ultrasonic source can also be fitted with a mixing device (Beck et al., 2010). Sonication increases micromixing, reduces particle growth and agglomeration, and it is possible to obtain spherical amorphous particle with uniform size distribution (Dhumal et al., 2008) (Xia et al., 2010). The particle size is dependent on sonication duration and intensity, horn length, depth of horn immersion and cavitation. As reported, it was possible to obtain a particle size as low as 80 nm (Dhumal et al., 2008)

(Nishida, 2004) (Xia et al., 2010).

Evaporative precipitation techniquesEvaporative precipitation into aqueous solution (EPAS) is another modified solvent antisolvent precipitation method, which was first proposed by a research

group at the University of Texas, Austin (Sarkari et al., 2002). In this process, the drug is dissolved in a low-boiling-point solvent and heated above its boiling point. Thereafter, the heated solution is sprayed into a heated aqueous medium with stabilizer.EPAS was used for preparing nanocrystals of several active pharmaceutical ingredients (APIs). A relatively recent report has described a evaporative method for the preparation of nanosuspension (EPN) by simply mixing solvent/antisolvent, followed by quick evaporation of the solvent in rotary vacuum evaporator to obtain drug nanoparticles (Kakran et al.,

2010).

Nanosuspension pharmacokinetic properties

It is well known that many characteristics of drug nanoparticle systems, such as particle size, surface properties and so on determine their release, absorption, distribution and targeting ability, which further influences the in vivo fates of nanoparticles (Müller

and Wallis, 1993) (Brigger et al., 2002). Studies on drug nanocrystals have also disclosed the similar findings.

Oral administration routeDrug absorption in the gasto intestinal tract (GIT) is considered to involve a dissolution step of the drug from formulation into aqueous luminal fluids followed by transporting the drug across the GI epithelium. The dissolution is considered as the rate determining process in the oral delivery (Dressman and Reppas,

2000) (Wang and Thanou, 2010). Poorly

53

soluble compounds tend to be eliminated from the GIT before they completely dissolve. Biopharmaceutically acceptable formulations for poorly soluble drugs, mainly belong to the biopharmaceutics classification system (BCS) II and IV compounds (Fig. 8), are a challenge because slow and erratic dissolution prevent rapid and complete absorption of these compounds, especially for drugs mainly absorbed in a narrow window in the GIT. In general, drugs possessing a poor solubility exhibit simultaneously a very low dissolution velocity. For this class of drug, despite gut permeability and fast uptake of the drug from the gut lumen, the blood levels will be low because the drug does not dissolve sufficiently fast. At the same time, drug elimination from the blood takes place leading to this low drug levels. In addition, the low concentration gradient between lumen and blood leads to relatively slow drug diffusion from the gut

to the blood.Many reports suggest that drug nanocrystals have many positive effects on the oral drug delivery of poorly soluble drugs, generally, including increased maximum plasma concentration (Cmax), reduced time to maximum plasma concentration (Tmax), enhanced area under the blood concentration–time curve (AUC) and reduced fasted/fed variability.The mechanisms responsible of the improved absorption could be majorly summarized as two points (Gao et al.,

2012):1. improved solubility and

dissolution rate2. bioadhesion to the intestinal

wallIn general, drugs possessing a poor solubility (saturation solubility, cs) exhibit simultaneously a very low dissolution velocity. The Noyes–Whitney law describing the dissolution velocity of a

solid particle can explain this behavior.

dQ/dt: dissolution velocityD: diffusion coefficientA: drug powder surface areaCs: drug concentration in diffusion layer or saturation solubilityC: drug concentration in surrounding liquidh: diffusion layer

This equation describes the solid particle dissolution velocity dQ/dt which depends on the surface area A and on the ratio (cs – c)/h, where cs represents the drug concentration in diffusion layer, c is the bulk concentration of the drug in the surrounding liquid and h the diffusional distance above the drug particle surface. Micronisation is a very simple traditional approach to increase the dissolution

10.1

1

10

100

10 100 250 1,000 10,000 100,000

Volume required to dissolve the highest dose (mL)

Biopharmaceutical Classi�cation System

I- High solubility- High permeability

II- Low solubility- High permeability

III- High solubility- Low permeability

IV- Low solubility- Low permeability

Perm

eabi

lity

(1x1

0-6 c

m p

er s)

Fig. 8. The biopharmaceutics classification system

dQ

dt=

D A (CS - C)

h

54

h n

hm hn< hm

velocity by enlarging the drug surface area. The particle size of normally sized drug powders (approximately being in the range 20–100 µm) is reduced to a size in a range of approximately l–10 µm, the mean diameter being typically in the range somewhere between 2 and 5 µm. However, many new drugs coming from synthesis or biotechnological processes exhibit such a low solubility (and related dissolution velocity) that the increase in surface area, allowed by micronisation technique, is not large enough to achieve a sufficiently high dissolution velocity leading to therapeutic blood levels. Therefore, the next consequent step was taken, going from micronisation to nanonisation that means transferring the drug microparticles to drug nanoparticles. Drug nanoparticles possess sizes of approximately 10–1000 nm, most production methods yield a main diameter somewhere between 200 and 400 nm. Nanonisation has an additional effect

compared to micronisation, it increases not only the surface area A, but also simultaneously the saturation solubility cs. The solubility of normally sized powders is a compound specific constant, depending only on the temperature and the solvent. This changes when one goes below a size of approximately 1 µm. The dissolution pressure increases due to the strong curvature of the particles leading to an increase in cs, the theoretical background being provided by the Ostwald–Freundlich equations (Brittain et al., 1991). According to Noyes–Whitney, this leads to a further increase in dQ/dt in addition to the gain by an increased surface area.Moreover, according to the Prandl equation, the diffusion layer h is reduced for small particles (Muller et al., 1998) (Fig. 9).The simultaneous increase in saturation solubility cs and decrease in h leads to an increased concentration gradient (cs – c)/h,

thus enhancing the dissolution velocity in addition to the surface effect. Therefore, the drug nanocrystals are a smart delivery system, a universal principle, which can be applied to any drug because any drug can be diminuted to nanocrystals. The increase in the saturation solubility leads to the formation of a supersaturated solution compared to the solubility of normally sized drug powders (size »1µm). So, when drugs are administered as nanocrystal formulations, a high drug concentration gradient between GIT and blood vessel will markedly improve absorption and result in a high bioavailability (Fig. 10).As said before, another important aspect is the mucoadhesion. It is generally known that nanoparticles possess general mucoadhesion to biological mucosa including GI mucosa (Ponchel et al.,

1997). This interaction also plays an important role in the enhancement of oral bioavailability. There are four general

Fig. 9. Schematic representation of the diffusion layer of solid particles of different diameter in a dissolution process

55

Blood vesselAbsorption

2 - 5 µm

Dissolved molecules

Mucous membranae

10 - 1000 nm

Di�usion layer

Fig. 10. Differences in diffusion and absorption by both micro and nano particles

56

theories of mucoadhesion mechanisms of nanocrystals or more in general of nanoparticles: the electronic theory (electrostatic attraction forces between the surfaces of particles and mucus), the adsorption theory (secondary forces such as hydrogen and van der Waals bonds between the surfaces of particles and mucus), the diffusion theory (interpenetration and physical entanglement of the protein of the mucus and polymer chains) and the trapping theory (retention of nanoparticles by the uneven mucosa surface). Because of mucoadhesion to GI mucosa, drugs can be released exactly at the absorption sites. This leads to a higher concentration gradient and also a prolonged retention time (Li et

al., 2009).Poorly soluble drugs often exhibit increased or accelerated absorption when they are administered with food. This can be attributed to the enhancement of the dissolution rate in the GIT caused by many

factors such as delayed gastric emptying, increased bile secretion, larger volume of the gastric fluid, increased gastric pH (for acidic drugs), and increased splanchnic blood flow (Jinno et al., 2006). In general, the fasted/fed variation will be dangerous for drugs with a narrow therapeutic window. When poorly soluble drugs are formulated as uniform nanosuspensions, this variation may be minimized. The reason is that the dissolution rate of nanocrystals is fast enough even under the fasted condition. Then, the absorptions both in fasted and fed state might be a permeability-limited progress, and the absorption difference resulting from variable dissolution between the two conditions will be eliminated.

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64

Introduction

Among the multiple, complex functions of mammalian skin, the most important is to form an effective barrier between the ‘inside’ and the ‘outside’ of the organism (Fig. 1). The skin has evolved defensive mechanisms that give it physical, immunological, metabolic and UV-protective barriers to allow it to inhibit attacks by microbes, toxic chemicals, UV radiation and particulate matter (including nanoparticles, which may occur in the natural environment). Skin consists of two main layers (Fig. 2). The underlying dermis contains a variety of cell types, nerves, blood vessels and lymphatics embedded in a dense network of connective tissue. Above the dermis and separated from it by the basement membrane, the epidermis is composed mostly of layers of stratified keratinocytes, where the SC cells or corneocytes are

bathed in a protein-rich envelope with an outer lipid envelope, surrounded by an extracellular lipid matrix. Interspersed amongst the keratinocytes in the viable epidermis are cells with roles such as melanin production (melanocytes), sensory perception (Merkel cells) and immunological function (Langerhans and other cells). In addition to the structured cellular components of skin, there are appendages including the pilosebaceous units (hair follicles and associated sebaceous glands), apocrine and eccrine sweat glands. In particular, the SC represents the principal barrier against the percutaneous penetration of chemicals and microbes and is capable of withstanding mechanical forces. It is further involved in the regulation of water release from the organism and into the atmosphere, known as transepidermal water loss (TEWL). The SC forms a continuous sheet of protein-enriched cells (corneocytes) connected

Nanosuspension improves tretinoin photostability and delivery to the skinPublished in International Journal of Pharmaceutics 458, 104-109, 2013

65

Sweat gland pore

Basement membrane

Sweat gland duct

Touch receptor

Capillary

Subcutaneous layer

Dermis

Epidermis

Hair shaft

Fig. 2. Skin structure

Barrier to water loss Stratum corneum

Epidermis

Prevention of excessive water loss and dessication; disrupted barrier leads to increased transepidermal water loss

Physical assaults(mechanical injury, UV-irradiation)

Microbial assaults(bacteria, fungus, virus)

Chemical assaults(irritants, allergens)

Fig. 1. Functions of the epidermal ‘inside-outside’ and ‘outsideinside’ barrier.

66

by corneo desmosomes and embedded in an intercellular matrix enriched in non-polar lipids and organized as lamellar lipid layers. In particular, corneocytes originate from keratinocytes after a process of keratinisation, in which the cell differentiates and moves upward from the basal layer (stratum basale), through the stratum spinosum and stratum granulosum, arriving finally to stratum corneum. On reaching the SC, cells become anucleated and flattened and are eventually sloughed off (Bouwstra and Honeywell-

Nguyen, 2002). Although the SC is recognized as the most important physical barrier, the nucleated epidermal layers are also significant in barrier function. A low to moderate increase in TEWL occurs after removal of the SC by tape-stripping, whereas loss of the entire epidermis leads to a severe disturbance in barrier function. Loss of the SC and parts of the granular layers in staphylococcal scalded skin

syndrome are not life-threatening (Elias

et al., 1977). In contrast, suprabasal and subepidermal blistering diseases like pemphigus vulgaris, toxic epidermal necrolysis (Lyell syndrome) and severe burns (intra as well as subepidermal blistering) are lifethreatening when large areas of the body are involved.Tretinoin (TRA) (Fig. 3) is the all-trans-retinoic acid form of vitamin A, commonly commercially formulated in cream and gel forms for the treatment of acne vulgaris, due to its ability to regulate epithelial cell growth and differentiation, sebum production and collagen synthesis (Zouboulis, n.d.).Topical TRA is also used to reduce rhytids and photoaging (Darlenski et al., 2010). However, as all topical retinoids, TRA may cause xerosis, irritation, erythema, and desquamation due to hyperproliferative effect and increased cell turnover of the epidermal keratinocytes. Moreover, its

topical use is limited by its poor water solubility and photolability (Manconi,

2003) (Ourique et al., 2011). Indeed, TRA is known to be susceptible to degradation by light, heat, and oxidizing agents and, under light exposure, it is rapidly isomerized, forming 13-cis and 9- cis retinoic acids (Brisaert and Plaizier-

Vercammen, 2000) (Manconi, 2003). In order to overcome all these drawbacks, nanoparticulate drug delivery systems, which include liposomes, niosomes, nanocapsules, solid lipid nanoparticles, and nanoemulsions, have been used widely (Manconi, 2003) (Sinico et al., 2005)

(Manconi et al., 2006) (Shah et al.,

2007) (Ourique et al., 2011). Among these approaches, vesicular carriers have been investigated to the main extent and incorporation of TRA in vesicular formulations has been largely studied to circumvent the undesirable effects of the drug, to maximize its accumulation into

Fig. 3. Trans retinoic acid structure

H3C CH3

CH3

CH3 CH3 OH

O

67

the skin, and to prevent its fast degradation. In particular, our group has demonstrated that different vesicular carriers (liposomes, niosomes, and penetration enhancer-containing vesicles) are able to reduce TRA photodegradation (Manconi, 2003) as well as to strongly improve new born pig skin hydration and cutaneous retention of this drug (Sinico et al., 2005) (Manconi

et al., 2006) (Manconi et al., 2011). However, crucial factors that need to be considered for topical formulation and design of nanomedicines also include drug loading, stability, reproducibility, and cost of manufacturing. Nanosuspensions, sub-micron colloidal dispersions of drug particles stabilized by surfactants, polymers or a mixture of both, have high drug loading, low incidence of side effects due to excipients, high reproducibility, and low cost (Patravale et al., 2004). Owing to the increased surface-to-volume ratio of the nanocrystals, especially for particle

size below 1 µm, an increase in saturation solubility and very fast dissolution rate can be obtained (Müller and Peters,

1998). Therefore, the aims of this work were to improve cutaneous targeting and photostability of tretinoin by using nanosuspension formulation of TRA (nanoS). To this purpose, the TRA skin permeation and deposition were studied in vitro by diffusion experiments through new born pig skin while TRA photostability was investigated by irradiating samples with UV light, set at 366 nm. As an appropriate comparison, an O/W nanoemulsion (nanoE) was also prepared and tested. Indeed, nanoemulsions have shown to be promising drug delivery systems with practical role for pharmaceutical, cosmetic, and chemical industry applications. Moreover, they have shown to be particularly useful as vehicle for dermal and transdermal delivery especially of hydrophobic

compounds (Shakeel et al., 2012) (Wu

et al., 2013). NanoS was prepared using precipitation method, meanwhile TRA nanoE was obtained by sonication. Formulations were characterized by photo correlation spectroscopy for mean size and size distribution, DSC for studying TRA cristallinity and melting point, and by transmission electron microscopy for morphological studies. Dermal and transdermal delivery of both TRA systems were tested in vitro by using Franz diffusion cells, working in nonocclusive conditions. At the end of the experiments, TRA amount delivered into/through the skin was detected by a HPLC method. The drug photostability was investigated also in comparison with a TRA methanolic solution, used as a further control.

68

Materials and Methods

MaterialsSoybean lecithin was obtained from Galeno (Italy). Glycerol, trans-retinoic acid (TRA), isopropyl myristate, and all the other products were of analytical grade and were purchased from Aldrich, Milan, Italy.

Nanoemulsion preparationComposition of nanoE is shown in Table 1. The nanoE was prepared as previously reported (Lai et al., 2008). Briefly, soybean lecithin, TRA, and isopropyl myristate were blended under constant stirring. Then the water phase (water and glycerol) was added and the mixture homogenized for 5 min using an Ultra Turrax T-25 basic (Ika®-Werke). At the end, the formed bi-phasic system was sonicated by a probe sonicator Soniprep (MSE, Crowley) for 60 cycles of 10 s at constant amplitude. Nanoemulsion was prepared under yellow light and then

stored in the dark at 4 ± 1 °C.

Nanosuspension preparationComposition of nanoS is shown in Table 1. Nanosuspension was obtained in accordance with the precipitation method by liquid solvent–antisolvent addition (Sinha et al., 2013). TRA and soybean lecithin were dissolved in methanol. The organic phase was added dropwise into deionized water under magnetic stirring at room temperature. The organic solvent was removed at room temperature, under vacuum using a Buchi rotavapor R200 equipped with a vacuum pump membrane MZ-2c (Vacuubrand-Wertheim, Germany). The absence of methanol residues was checked by HPLC. The final volume of the nanosuspension was adjusted to 20 ml with distilled water. Nanosuspension was prepared under yellow light and then stored in the dark at 4 ± 1 °C.

Nanosuspension and nanoemulsion characterizationThe average diameter and polydispersity index (P.I.) of the samples were determined by Photon Correlation Spectroscopy (PCS) using a Zetasizer nano-ZS (Malvern Instrument, UK). Samples were backscattered by a helium–neon laser (633 nm) at an angle of 173° and a constant temperature of 25 °C. The instrument systematically and automatically adapts to the sample by adjusting the intensity of the laser and the attenuator of the photomultiplier, thus ensuring reproducibility of the experimental measurement conditions. The P.I. was used as a measure of the width of the size distribution: the less its value the more homogenous and monodisperse is the sample. Zeta potential was estimated using the Zetasizer nano-ZS by means of the M3-PALS (Phase Analysis Light Scattering) technique, which measures

69

the particle electrophoretic mobility in a thermostated cell. All the samples were analyzed 24 h after their preparation. A long-term stability study of formulations stored at 4 ± 1 °C was performed by monitoring formulations average size, polydispersity, and surface charge over 90 days. The DSC curves of TRA and TRA nanoS were recorded on a Perkin Elmer DSC 6 differential scanning calorimeter, calibrated with indium, at the heating rates of 10 °C/min. The thermal behavior was studied by heating 2 mg samples in aluminium crimped pans under nitrogen gas flow within the temperature range 50–350 °C.

HPLC methodTRA content was quantified at 350 nm using a chromatograph Alliance 2690 (Waters, Milan, Italy), equipped with a photodiode array detector and a computer integrating apparatus (Empower 3). The

column was a Symmetry C18 (3.5 µm, 4.6 mm × 100 mm, Waters), and the mobile phase was a mixture of acetonitrile, water, and acetic acid (84.5:15:0.5, v/v), eluted at a flow rate of 1.2 ml/min. A standard calibration curve was built up by using working, standard solutions. Calibration graphs were plotted according to the linear regression analysis, which gave a correlation coefficient value (R2) of 0.999.

Photodegradation studyThe degradation study of tretinoin was carried in triplicate as previously reported (Manconi, 2003) by using a UV lamp set at 366 nm (Min UVIS, Desaga, GmbH, Germany). TRA methanolic solution, nanosuspension and nanoemulsion (2 ml in a glass flask) were exposed to UV radiation from a 30 W lamp (366 nm) for 1 h at a fixed distance of 10 cm. At regular time intervals (every 10 min), samples were first stirred and then 100 µl of the dispersion

was withdrawn, diluted with methanol and finally analyzed by HPLC in order to quantify TRA concentration. Experiments were carried out at 25 ± 1 °C on samples with an initial TRA concentration of 350 µg/ml.

In vitro skin penetration and permeation studiesExperiments were performed non-occlusively using Franz diffusion vertical cells with an effective diffusion area of 0.785 cm2, and new born pig skin. One day old Goland Pietrain hybrid pigs (~1.2 kg) were provided by a local slaughterhouse. The skin, stored at −80 °C, was pre-equilibrated in physiological solution (0.9%, w/v of NaCl) at 25 °C, 24 h before the experiments. Skin specimens (n = 12 per formulation) were sandwiched securely between donor and receptor compartments of the Franz cells, with the stratum corneum (SC) side facing the donor compartment.

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The receptor compartment was filled with 5.5 ml with 5.5 ml of a hydroalcoholic solution (ethanol/PBS 50:50, v/v), which was continuously stirred with a small magnetic bar and thermostated at 37 °C throughout the experiments to reach the physiological skin temperature (i.e. 32 °C). One hundred microliters of TRA nanosuspension or nanoemulsion were placed onto the skin surface. At regular intervals, up to 8 h, the receiving solution was withdrawn and replaced with an equivalent volume of pre-thermostated (37 °C) fresh hydroalcoholic solution, to ensure sink conditions. Withdrawn receiving solutions were analyzed by HPLC for drug content. After 8 h, the skin surface of specimens was gently washed (3 times) with 1 ml of distilled water, then dried with filter paper. The SC was removed by stripping with adhesive tape Tesa® AG (Hamburg, Germany). The method was previously validated by histological

examination of stripped skin (Sinico et

al., 2005). Each piece of the adhesive tape was firmly pressed on the skin surface and rapidly pulled off with one fluent stroke. Ten stripping procedures were performed consecutively. Epidermis was separated from dermis with a surgical sterile scalpel. Tape strips, epidermis, and dermis were cut and placed each in a flask with methanol and then sonicated for 4 min in an ice bath to extract the drug. The tapes and tissue suspension was centrifuged for 10 min at 10,000 rpm, and the supernatant was then filtered and assayed for drug content by HPLC.

Transmission electron microscopy (TEM)Characterization of the external morphology of TRA nanosuspension was determined using transmission electron microscopy (TEM). A drop of nanosuspension and an equal volume of an

aqueous 1% uranyl acetate solution were adsorbed on the surface of copper grid and dried at room temperature for 24 h. The sample was transferred into a TEM (Tecnai 12, FEI) operating at 120 kV. 2.9. Statistical analysis of data Data analysis was carried out with the software package R, version 2.10.1. Results are expressed as the mean ± standard deviation. Multiple comparisons of means (Tukey test) were used to substantiate statistical differences between groups, while Student’s t-test was applied for comparison between two samples. Significance was tested at the 0.05 level of probability (p).

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Results and discussion

Composition of nanoemulsion (nanoE) and nanosuspension (nanoS) formulations are listed in Table 1, where the amount of the different compounds is expressed as % (w/w). As shown in the table, nanoE was prepared by using isopropyl mirystate as lipidic phase, and soybean lecithin as stabilizer. Both formulations, nanoE and nano S, were loaded with the same amount of TRA (0.035%, w/w) and stabilized with the same surfactant in different concentrations (1.2% and 0.0035%, respectively).PCS diameter (Z-AVE), polydispersity index (PI), and Z potential of nanoE and nanoS were determined just after preparation and are reported in Table 2. Freshly prepared nanoE showed a Z-AVE of 175 nm (0.09 PI) and a Z potential of −69.7 mV, while freshly prepared nanoS presented a Z-AVE of 324 nm (0.24 PI)

and a Z potential of −53.2 mV. As can be seen, both formulations, nanoE and nanoS, showed very highly negative zeta potential values that should ensure high stability against aggregation phenomena. Stability was checked by measuring average diameter, polydispersity index and Z potential of formulations stored at 4 ± 1 °C for 90 days. As shown by the Table 2, freshly prepared nanoE was nicely homogeneously dispersed with reduced particle size and PI, in comparison with the nanoS that was slightly less homogeneously disperse. However, Table 2 clearly shows that nanoemulsion (nanoE) was less stable than the nanoS. Indeed, after only one day on storage, the average diameter of nanoE increased from 175 nm to 344 nm, reaching a mean size of 745 nm after 90 days. The low stability of this formulation was also confirmed by the increasing of polydispersity index value from 0.09 to 0.69. By contrast, nanoS maintained

similar mean diameter and polydispersity index values during all the checked period (~330 nm and 0.25 PI). Nanosuspension data were confirmed by TEM images (Fig. 4).To evaluate the influence of the preparation method on the TRA degree of cristallinity and melting point, DSC characterization of nanoS was also performed. As reported in literature (Caviglioli et al., 2006), two polymorphic forms of TRA are known: form I and form II. The thermal behaviour of form I shows an endothermic peak at 153.39 °C, due to the solid state transition from the monoclinic to the triclinic structure, which melts at 184.58 °C; while form II shows only the endothermic peak at 183.7 °C. The commercial trans-retinoic acid used in this study to produce nanocrystals agrees with form I. The thermic profile comparison between commercial and nanoS TRA evidenced that nanosuspension process

Formulations

Compounds % (w/w) NE NS

TRA 0,035 0,035

Isoprpyl myristate 10 -

Glycerol 2,26 -

Soybean lecithin 1,2 0,0035

Water 86,5 99,9

Table 1. Sample composition

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produced trans-retinoic acid form II. Since the two polymorphs show comparable solubility values, the solid state transition that occurred during nanosuspension preparation did not affect drug behaviour, which was mainly influenced by the reduced particle size (Caviglioli et al.,

2006). The low stability of the nanoE could be the consequence of the choice of the stabilizer, the soybean lecithin, which was used here to have nanoS and nanoE with the same additive. Indeed, while the small amount of this phospholipid mixture was capable of highly stabilizing the nanoS, the nanoE showed aggregation phenomena on storage. Before testing nanoS for the (trans)dermal delivery of TRA, we evaluated the influence of the nanosuspension on TRA photostability, which was studied by irradiating TRA nanoS with UV light (λ=366 nm) in comparison with nanoE a TRA methanolic solution (controls). HPLC analyses showed that TRA

photodegradation in methanol is very fast and, after only 10 min of UV exposure, a great amount of drug was degraded. Actually, after 1 h of UV irradiation, only approximately 27% of the initial TRA concentration was still present. On the contrary, when nanosuspension (nanoS) and nanoemulsion (nanoE) were tested for the same period of time, the 83% and 52% of the initial drug concentration was found to be stable. Fig. 5 clearly shows that a good linear correlation was obtained by plotting tretinoin concentration as a function of time during the 1 hour irradiation with UV light of the methanolic solution (R2 = 0.954). The same linearity was obtained studying the photodegradation of the drug in nanoS and nanoE (R2 = 0.925 and R2 = 0.953 respectively). Therefore, as shown previously, the photodegradation of TRA can be treated as a zero order kinetic in all the studied samples (Manconi, 2003). Photodegradation rate constants (k) and

half-lives (t1/2) of TRA in the different formulations exposed to UV light for 1 h are shown in Table 3. TRA in methanol solution showed a half-life time of about 0.4 h that became more than two fold higher in nanoE (t1/2 = 0.9 h), and, surprisingly, even 7.5-fold higher with nanoS (t1/2 = 3 h). Therefore, nanosuspensions were able to significantly improve TRA photostability in comparison with nanoE and methanol solution. This behaviour can be explained as a consequence of the TRA nanocrystal structure. In fact, here, only the external TRA molecules of the nanocrystals can be irradiated and, therefore, degraded (Del

Rosso et al., 2012). On the contrary, in both nanoE and methanolic solution, TRA is molecularly disperse in a solvent (isopropylmiristate and methanol, respectively) and, therefore, more exposed to the UV rays that can reach and degrade TRA molecules more easily. Moreover, it is well known that reactions at molecular

nano E nano S

MD (nm) PI ZP (mV) MD (nm) PI ZP (mV)

D0 175 ± 2,1 0,09 ± 0,01 - 69,7 ± 1,3 324 ± 1,5 0,24 ± 0,01 - 53,2 ± 1,0

D1 344 ± 16 0,38 ± 2,1 - 76,6 ± 0,3 307 ± 1,8 0,17 ± 0,01 - 57,4 ± 0,5

D7 427 ± 28 0,42 ± 0,02 - 57,6 ± 0,5 329 ± 11 0,28 ± 0,01 - 48,4 ± 0,3

D14 460 ± 6,1 0,45 ± 0,01 - 62,2 ± 0,7 343 ± 7,6 0,28 ± 0,01 - 46,4 ± 0,9

D30 545 ± 33 0,58 ± 0,1 - 60,9 ± 0,5 322 ± 5,1 0,26 ± 0,02 - 47,8 ± 1,9

D60 1060 ± 33 0,84 ± 0,02 - 55,7 ± 0,2 316 ± 3,2 0,22 ± 0,04 - 47,7 ± 0,3

D90 745 ± 82 0,69 ± 0,08 - 59,9 ± 1,1 330 ± 8,5 0,27 ± 0,01 - 49,3 ± 1,2

Table 2. Mean size (Z-AVE), polydispersity index (PI) and Z potential (ZP) of TRA nanoemulsion and nanosuspension monitored during a 90-day period on storage at 4 ± 1 °C

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level are faster and easier in solution. The slower photodegradation rate of TRA in nanoE, when compared to the methanol solution, can be ascribed to the protection of the lecithin layer around the oil droplets. TRA delivery into and through newborn pig skin was evaluated in vitro under non occlusive conditions, using Franz vertical diffusion cells. During this study, percutaneous delivery of TRA from nanoS was compared to that from nanoE, used as control. Comparison of data obtained from nanoS and nanoE (Fig. 6) highlights a different TRA delivery into and through the pig skin with the two formulations. As expected, the nanoE led to a higher drug deposition and transdermal delivery than nanoS. However, differences of drug deposition in the three main skin strata (SC, epidermis and dermis) were not statistically significant (p > 0.05) while a strong and statistically significant difference was detected in the amount of

permeated drug: nanoE led to a mean value of ≈6.5% of the applied drug delivered to the receiver, whereas using nanoS only a ≈0.4% (p < 0.01) T of the applied TRA was found. Comparison of data obtained from nanoS and nanoE underlines the influence of the different formulations on the in vitro drug availability; nanoE is useful to improve both drug skin accumulation and transdermal delivery. On the contrary, nanoS is able to especially favour TRA accumulation into the skin and at the same time to reduce drug delivery to the systemic circulation. Indeed, using nanoS, TRA was mostly deposited in the stratum corneum (6.86 ± 1.10%) while a lower accumulation in the epidermis (3.09 ± 0.81%) and a minimal deposition in dermis (1.65 ± 0.19%) and receptor fluid (0.28 ± 0.10%) were found. On the contrary, using nanoE, the highest drug delivery was found in in the stratum corneum (6.51 ± 0.82%) and receptor fluid (6.64 ± 1.80%)

and a smaller deposition in epidermis (4.34 ± 1.12%) and dermis (2.26 ± 1.35%), respectively. Therefore, the results highlight the nanoS capability to favour TRA retention in the skin layers while minimizing transdermal delivery of the drug, responsible of its side effects. These results can be explained considering that poor solubility of solid crystals, like TRA, is generally linked to a slow dissolution rate. Thus, after permeation through a biological membrane of the few drug molecules in solution, further dissolution of the active crystals is not fast enough to replace the permeated molecules. As a result, the rate-limiting step for absorption of such drugs (so-called class II drugs of the biopharmaceutical classification system) is the dissolution rate (Desai et al., 1996) (Kasim et al., 2004) (Martinez and

Amidon, 2002). In this case, in contrast, finely divided and uniformly suspended TRA nanocrystals possess an increased

Fig. 4. TEM images of TRA nanosuspension

   

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dissolution rate due to their large surface area and increased saturation solubility. Solid drug dissolves in the vehicle, diffuses through the vehicle to the skin, establishes local phase equilibrium with the outer layer of skin and finally, in the case of TRA, thanks to the larger concentration gradient penetrates the skin forming a depot in the lipophilic SC from which it diffuses. On the other hand, when a nanoemulsion is applied on the skin, two consecutive physical events may become rate-limiting steps in cutaneous permeation: the drug release from the vehicle and its penetration through the cutaneous barrier. These two processes are closely related, and both depend on the physicochemical properties of drug, vehicle, and barrier. As for nanoemulsion cutaneous behaviour, previous research indicated that the permeation process of lipophilic molecules was consistent with a skin-controlled mechanism (Fang et al., 2003). Differences

between trend of release rates and fluxes can be attributed to the SC/vehicle partition coefficient. The degree of partitioning of the drug into the SC depends on relative affinity for the vehicle and for the intercellular environment. In this study, the higher drug permeability may be due to the soy lecithin (used in highly greater amount than in nanoS, Table 1) and the oily phase, which taken together act as penetration enhancers. Furthermore, as shown for microemulsions, the small particle size of the nanoE makes it an excellent carrier for promoting in vitro skin TRA permeation (Gupta et al., 2012). Overall results show that nanosuspensions are eligible for the use as suitable nanomedicine for dermal delivery of poorly soluble drugs such as tretinoin. Nanosuspensions are almost exclusively composed of the drug nanoparticles with small amounts of biocompatible and safe surfactants, such as the soy lecithin used in this work. This

leads to a highly fast dissolution process that favours drug penetration into the skin. Moreover, although only few toxicological data are available at the moment, no side effects are known or expected from nanocrystal topically applied. Indeed, as suggested by (Müller et al., 2011), each solid macro/micro-particle applied on the skin will convert to nanocrystals during its dissolution process and, up to now, no intolerability has been reported (Müller et al., 2011). Therefore, the same should happen when nanocrystals are applied directly on the skin. Moreover, nanosuspensions show additional advantages over other colloidal carriers such as simple, reproducible, and scalable preparation methods.

Time (min)

NE

NS

In MeOH

% T

RA

In %

TR

A

00

102030405060708090

100110

0

0,5

1

1,5

2

2,5

3

3,5

40

4,5

5

10 20 30 40 50 60 70

Fig. 5. Photodegradation of TRA in methanolic solution, nanoemulsion and nanosuspension after 1 h of UV irradiation ( λ = 366 nm)

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Formulations

Kinetic order - K 0

a t(1/2)b (h)

Methanolic solution First 72,83 0,4

Nanoemulsion

Zero

48,31

0,9

Nanosuspension

Zero

16,46

3

Table 3. Photodegradation rate constants (K0) and half-lives (t1/2) of TRA methanolic solution, TRA nanoemulsion and TRA nanosuspension exposed to UV light for 1 h

Fig. 6. Cumulative amount of TRA retained into and permeated through pig skin layers (SC, stratum corneum; Ep, epidermis; D, dermis) after 8-h non-occlusive treatment with TRA nanoemulsion (nanoE) and nanosuspension (nanoS). Each value is the mean ± SD (n = 12)

FormulationsNE NS

SC %

EP %

DE %

% T

RA

0

2

4

6

8

10

12

14

16

18

20

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ConclusionOn the whole, this work has shown the high potentiality of nanosuspensions in dermal drug delivery of TRA. Indeed, nanoS has been demonstrated to be able to localize the drug into the pig skin in vitro with a very low transdermal drug delivery, which is responsible of systemic side effects of this drug. Moreover, the nanoS has shown to give comparable cutaneous TRA delivery as the nanoemulsion, which strongly enhanced in vitro transdermal drug delivery. Furthermore, the application of nanosuspension in topical TRA delivery has the advantage of increasing photostability of the drug also in comparison with the nanoemulsion. In conclusion, results of this work highlight that nanosuspension formulation approach could be a useful tool in the design of new TRA nanomedicines for treatment of skin diseases.

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Formulation strategy and evaluation of nanocrystal piroxicam orally disintegrating tablets manufacturing by freeze-drying

Introduction

For the past one decade, there has been an enhanced demand for more patient-friendly and compliant dosage forms. As a result, the demand for developing new technologies has been increasing annually (Shyamala and Narmada,

2002). Since the development cost of a new drug molecule is very high, efforts are now being made by pharmaceutical companies to focus on the development of new drug dosage forms for existing drugs with improved safety and efficacy together with reduced dosing frequency, and the production of more cost effective dosage forms. For most therapeutic agents used to produce systemic effects, the oral route still represents the preferred way of administration, owing to its several advantages and high patient compliance compared to many other routes (Valleri

et al., 2004). Tablets and hard gelatin

capsules constitute a major portion of drug delivery systems that are currently available. However, many patient groups such as the elderly, children, and patients who are mentally retarded, uncooperative, nauseated, or on reduced liquid-intake/diets have difficulties swallowing these dosage forms. Those who are traveling or have little access to water are similarly affected (Hanawa et al., 1995) (Mallet, 1996)

(Porter, 2001). To fulfill these medical needs, pharmaceutical technologists have developed a novel oral dosage form known as Orally Disintegrating Tablets (ODTs) (Fig. 1).US Food and Drug Administration Center for Drug Evaluation and Research (CDER) and Italian and European Pharmacopeia defines an ODT as “a solid dosage form containing medicinal substances, which disintegrates rapidly, usually within a matter of seconds, when placed upon the tongue” (Manach et al.,

79

2004). The significance of these dosage forms is highlighted by the adoption of the term, “Orodispersible Tablet”, by the European Pharmacopoeia which describes it as a tablet that can be placed in oral cavity where it disperses rapidly before swallowing (“European Directorate for

quality of Medicines,” 1998). The major claim of these formulations, compared with conventional oral forms, are increased bioavailability and faster onset of action. Dispersion in saliva in oral cavity causes pregastric absorption from some formulations in those cases where drug dissolves quickly. Buccal, pharyngeal and gastric regions are all areas of absorption for many drugs (Yarwood, 1990). Any pregastric absorption avoids first pass metabolism and can be a great advantage in drugs that undergo a great deal of hepatic metabolism. Furthermore, safety profiles may be improved for drugs that produce significant amounts of toxic metabolites

mediated by first-pass liver metabolism and gastric metabolism, and for drugs that have a substantial fraction of absorption in the oral cavity and pregastric segments of GIT (Pfister and Ghosh, 2005). ODTs can be used easily in children who have lost their primary teeth but do not have full use of their permanent teeth (Mizumoto et al.,

2005). Recent market studies indicate that more than half of the patient population prefers ODTs to other dosage forms (Deepak, 2004) and most consumers would ask their doctors for ODTs (70%), purchase ODTs (70%), or prefer ODTs to regular tablets or liquids (>80%) (Brown, 2001). ODT products have been developed for numerous indications ranging from migraines (for which rapid onset of action is important) to mental illness (for which patient compliance is important for treating chronic indications such as depression and schizophrenia) (Ghosh et al., 2005). The performance

of ODTs depends on the technology used during their manufacture. The necessary property of such tablets is the ability to disintegrate rapidly and disperse or dissolve in saliva, thereby obviating the need for water.Various technologies have been developed to formulate ODT, the most known are: freeze drying, moulding, sublimation, spray drying, mass extrusion and finally direct compression. Independently of the technologies used for they production, an ideal ODT should meet the following criteria:• does not require water for oral administration yet disintegrates and dissolves in oral cavity within a few seconds• has sufficient strength to withstand the rigors of the manufacturing process and post-manufacturing handling• allow high drug loading• has a pleasant mouth feel• is insensitive to environmental conditions

 

Fig. 1. Oral disintegrating tablet (ODT)

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such as humidity and temperature• is adaptable and amenable to existing processing and packaging machineries• is cost-effective.Piroxicam (PRX) (Fig. 2) is a non-steroidal anti-inflammatory drug characterized by slow absorption after oral administration because of its poorly water solubility (Amidon et al., 1995; Graf, 1985). Due to adverse side effects associated with its oral administration, such as gastric irritation, there is a considerable interest in developing new formulations to improve oral mucosa drug absorption. During the last years, our research group has been studying nanocrystals as tool to improve drug dissolution rate and, thus, bioavailability of poorly soluble drugs. In particular, it was demonstrated an improved dissolution rate for PRX nanosuspension incorporated in orally disintegrating tablets (ODT) in comparison with the corresponding coarse formulation (Lai et

al., 2011).In this work, we carried out a study on a new formulation strategy aimed to obtain nanocrystal Orally Disintegrating Tablets (ODT) of piroxicam with a drug dissolution profile faster than those of the ODTs currently available on the market. For this purpose, we focused our attention on the influence of ODT excipients, on the enhancement of the PRX dissolution rate and, therefore, on its direct absorption through the oral mucosa. The absorption in this site allows the drug to reach the systemic circulation bypassing the gastrointestinal tract, thus avoiding the first-pass metabolism of the liver. In particular, starting from the same PRX nanosuspension formulation, we prepared and studied ODT by using three different excipients: xanthan gum, gelatin, and crosscarmellose. Xanthan gum, a polysaccharide secreted by the bacterium Xanthomonas campestris and

gelatin, a derived of collagen obtained from various animal by-products, are commonly used in food, pharmaceuticals, and cosmetic products as additive, gelling agent, rheology modifier, stabilizer, and binding agents. Croscarmellose sodium, or sodium CMC, is a cross-linked polymer of carboxymethylcellulose sodium. It is a white, fibrous, free-flowing powder, FDA-approved disgregant, commonly used in pharmaceutical formulations to facilitate the breakup of a tablet in the gastro intestinal tract after oral administration. PRX nanocrystals were prepared using a high pressure homogenization technique (HPH) (Keck and Müller, 2006) and poloxamer 188 was used as a stabilizer. PRX nanosuspension ODT were prepared using a freeze-drying technology (Corveleyn and Remon, 1998; Owen

et al., 2000; Sugimoto et al., 2006). Characterization of PRX nanocrystal ODT was carried out by different

Fig. 2. Piroxicam structureOH

N NH

N

S

O

O O

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techniques: infrared spectroscopy (FTIR), X-ray powder diffractometry (XRPD), differential scanning calorimetry (DSC), photon correlation spectroscopy (PCS). Dissolution study of PRX nanosuspension ODT was performed in distilled water (pH 5.5) and was compared to that of PRX coarse suspension ODT, PRX/poloxamer 188 physical mixture, bulk PRX samples and a PRX commercial ODT (Feldene Fast®).

Materials and methods

MaterialsPluronic F68 (poloxamer 188) was a gift from BASF AG (Ludwigshafen, Germany). Maltodextrin (DE 39) having a Dextrose Equivalent (DE) equal to 39 was kindly supplied by Roquette (France). Piroxicam (PRX), poly(ethylenglycol) 4000 (PEG 4000), xanthan gum (XG) molecular weight approximately 3 x 105 g/mol, gelatin, croscarmellose, citric acid, aspartame, mannitol and high-performance liquid chromatography (HPLC)-grade methanol were purchased from Sigma–Aldrich (Milan, Italy). All the other compounds were of analytical grade and used as received from Sigma–Aldrich (Milan, Italy).

PRX polymorphic form preparationAll the crystalline forms of piroxicam were

prepared following literature methods as previously reported (Vrečer et al., 1991;

Vrečer et al., 2003). Commercial piroxicam agrees with form I (white prismatic crystals). Briefly, form II (white needle) was crystallised from commercial piroxicam hot saturated absolute ethanol solution. Form III (white powder) was obtained by spray drying. The monohydrate form (yellow powder) was obtained by dissolving commercial piroxicam in acetone and by slowly adding distilled water until the appearing of a yellow precipitate, which was filtrated and dried.

PRX polymorphic form solubilityThe solubility of the different PRX polymorphic forms was determined in different media: pure water, water with different concentrations of poloxamer 188, maltodextrin, PEG4000 and xanthan gum (or gelatin or croscarmellose) water solution at the same PRX/additives ratios

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of the ODT formulation. PRX solubility was also determined in citric acid and/or aspartame and/or mannitol water solutions. An excess of drug was added to the medium in screw capped tubes (10 ml) and stirred at 25 °C for 48 h. Each sample was centrifuged, then 0.2 ml of the clear supernatant was diluted with methanol and analysed by UV.

PRX/poloxamer 188 physical mixture preparationPhysical mixture was prepared by blending PRX and poloxamer 188 in an agata mortar until a homogeneous mixture was obtained, using the same drug/surfactant ratio (w/w) of corresponding ODT formulations (Table 1).

Coarse suspension preparationDrug coarse suspensions were prepared dispersing PRX in a poloxamer 188 bidistilled water solution using an Ultra

Turrax T25 basic (IKA, Werke) for 1 min at 8000 rpm (Table 1).

Nanosuspension preparationNanosuspension was prepared using bulk PRX. PRX was dispersed in a poloxamer 188 bidistilled water solution using an Ultra Turrax T25 basic for 1 min at 8000 rpm. The obtained coarse suspension was sonicated for 1 h and then homogenised at high pressure (three cycles at 500 bar and 30 cycles at 1500 bar) using an Emulsiflex C5 apparatus (Avestin, Ottawa, Canada). Nanosuspensions were prepared with the same drug/surfactant ratio (w/w) of corresponding ODT formulations (Table 1).

ODT preparationPEG4000, maltodextrins and Xanthan gum or gelatin or croscarmellose, were dissolved into the previously prepared nanosuspension or coarse suspension

(Table 1). An amount of this suspension containing 20 mg of PRX placed in a PVC blister, was frozen at -20 °C and was freeze-dried overnight with an initial shelf temperature of -10 °C rising 20 °C at a pressure of 0.5 mbar using a freeze-dryer apparatus (Criotecnica, Rome, Italy).

Analytical characterizationFT-IR spectra were recorded using a Perkin Elmer (MA, USA) FT-IR Spectrometer “Spectrum One” in a spectral region between 4000 and 600 or 450 cm-1 for solid or liquid compounds, respectively, and analyzed by transmittance technique. Solid samples were mixed in a mortar with KBr (1:100) and pressed in a hydraulic press (14 tons) to small tablets, while one drop of liquid samples was placed between two windows of sodium chloride. The powder X-ray diffraction patterns were recorded with a Rigaku Miniflex diffractometer with a Ni-filtered CuKα

Table 1. Composition of PRX ODT Formulations

Components % (w/w) ODT - Xanthan ODT -

Gelatin

ODT -Croscaramellose

PRX 2.5 2.5 2.5

Poloxamer 188 1.5 1.5 1.5

PEG 4000 1 1 1

DE 39 20 20 20

Xanthan 2 - -

Gelatin - 2 -

Croscarmellose - - 2

H2O 73.0 73.0 73.0

83

radiation detector (λ=1.5405 Å) operating at a voltage of 30 kV and a current of 15 mA in the 2θ range from 3° to 60° with a scan angular speed of 2°/min and a scan step time of 2.00s. The DSC studies were carried out with a Perkin Elmer DSC 6 differential scanning calorimeter calibrated with indium. Samples were scanned from 50°-350°C at the heating rates of 10°C/min in aluminium crimped pans under nitrogen gas flow.

In vitro dissolution studiesIn vitro dissolution studies were performed according to the United States Pharmacopeia (USP) by means of the rotating basket method (Erweka apparatus). The dissolution media was 1000 ml of distilled water kept at 37 ± 0.1 °C and at rotation speed of 100 ± 2 rpm. At preselected time intervals, 1 ml sample was withdrawn (replaced with 1 ml of pre-thermostated fresh dissolution

medium), filtered through polycarbonate membranes (0.45 µm, Millipore), diluted with ethanol (1:5) and analysed by UV for PRX content. Dissolution tests were performed in triplicate.

UV analysisQuantitative determination of PRX was performed by UV spectroscopy. The stock standard solution of PRX (1 mg/ml) was prepared by dissolving the drug in ethanol and storing at 4 °C. A standard calibration curve (peak area of PRX vs. known drug concentration) was built up using standard solutions (0.5–100 µg/ml) prepared by dilution of the stock standard solution with ethanol. Calibration graphs were plotted according to the linear regression analysis, which gave a correlation coefficient value (R) of 0.999. Sample preparation and analyses were performed at room temperature.

Particle size measurementThe average diameter and polydispersity index (PI) of the nanocrystals were determined by photon correlation spectroscopy (PCS) using a Zetasizer nano (Malvern Instrument, UK). Samples were backscattered by a helium–neon laser (633 nm) at an angle of 173° and a constant temperature of 25 °C. The PI was used as a measure of the width of the size distribution. Zeta potential was estimated using the Zetasizer nano by means of the M3-PALS (Phase Analysis Light Scattering) technique, which measures the particle electrophoretic mobility in a thermostated cell. All the samples were analyzed 24 h after their preparation.

Statistical analysis of dataData analysis was carried out with the software package R, version 2.10.1. Results are expressed as the mean ± standard deviation. Multiple comparisons of means

84

(Tukey test) were used to substantiate statistical differences between groups, while Student’s t-test was used for comparison between two samples. Significance was tested at the 0.05 level of probability (p).

Results and discussion

Bulk PRX characterizationAs previously reported, all the crystalline piroxicam forms were prepared and characterized by XRPD, FTIR and DSC (Lai et al., 2011). A clear identification of the PRX polymorphs is achieved by XRPD in the range between 3° and 40° 2Ɵ. In fact, form I shows intense diffraction peaks at 8.58°, 17.66°and 27.36° (2 Ɵ); form II at 8.94° and 38.22° (2 Ɵ); form III at 8.70° and 12.66° (2 Ɵ), while only the monohydrate form exhibits a peak at 26.08° (2 Ɵ). Piroxicam (polymorphs and monohydrate) FTIR spectra exhibit significant differences in the range between 3400 and 3300 cm_1 where bands for OH and NH stretchings can be found, and in the 1700–1100 cm_1 range in which there are C=O, C=N, SO2 (asymmetric and symmetric) stretchings. DSC analysis of form I shows a melting point at 203°C,

while the thermal behavior of form II shows an endothermic peak at 201°C with a shoulder at 202°C. Form III melting point is 202°C and the monohydrate shows an endothermic peak at 131°C, corresponding to the loss of water, followed by another endothermic peak at 202°C. The comparison of each PRX polymorph analytical data with those of bulk PRX allowed us to confirm that the utilized commercial piroxicam presents a crystalline form which agrees with form I.

Nanosuspension and ODT preparation and particle size measurement In a previous article we produced different formulations of PRX nanosuspensions (water suspension of PRX nanocrystals) by HPH using bulk PRX and different amounts of poloxamer 188 as stabilizer. In this work, to formulate the ODTs, we selected the nanosuspension with 1.5%

85

of poloxamer 188 (calculated as % in the final ODT formulation) that had shown the lowest PCS average diameter. Selected operational conditions for nanosuspensions preparation were 30 homogenization cycles at 1500 bar, which were proceeded by 3 cycles at 500 bar as a kind of pre-milling. PRX nanocrystals PCS diameter (Z-AVE) and Z potential were respectively 414 nm (0.40 PI) and -18.1±0.4 mV, determined just 24 h after the last homogenization cycle. ODT are dried products of a water-soluble mixture with drug. The liquid nanosuspension was transformed into ODT by means of the lyophilisation method using different components as excipients (PEG4000, maltodextrins and xanthan gum or gelatin or crosscaramellose, table 1) (Thakur and Kashi, 2011). The mixture of the water-soluble excipients and the drug nanosuspension was placed in blister pockets and freeze-dried to remove the water by sublimation (seen section 2).

Different cryoprotectants, typically water-soluble sugars, have been generally added to the nanosuspension prior to lyophilisation to protect them from freezing damage, due to ice formation, and to minimise the particle size growth during lyophilisation (Van Eerdenbrugh et al., 2008). As previously reported, the presence of the stabilizer (poloxamer 188), used in the nanosuspension preparation, reduced PRX particle aggregation after ODT rehydration (Lai et al., 2011, 2009). PCS diameter (Z-AVE) and Z potential of PRX particles were determined just after ODT disaggregation in water (few seconds) and are reported in table 2. As it can be seen in table 2, the lyophilisation process little influences the PRX particle size and Z potential for all ODT formulation (Mauludin et al., 2009a).

Analytical characterizationRecently, PRX ODT and the corresponding

coarse suspension were characterized by XRPD, FTIR and DSC techniques (Lai et al., 2011). In order to avoid the analytical contribute of ODT formulation components, nanocrystals of PRX stabilized by poloxamer188 were also characterized. Former study allowed us to establish that a polymorphic transition took place during the high pressure homogenization; the polymorphic transition was also evident by simple visual inspection: starting PRX bulk powder was white, conversely, the obtained nanosuspension and relatives ODT formulations showed a bright yellow color (Fig. 3).In fact, in the prepared PRX nanocrystal ODT the presence of monohydrate (yellow powder) and form III piroxicam mixture was proved. Since in the present study we prepared PRX ODT by using new excipients useful to improve drug solubility, we carried out a new analytical characterization, but taking into account

ZAVE (nm) P.I. ZP (mV)

PRX nanocrystals 414.3± 21.1 0.40± 0.02 - 18.1± 0.4

ODT Xanthan 509.3±20.3 0.42 ±0.07 - 17.4±0. 7

ODT Gelatin 532.5 ± 25.6 0.44 ±0.07 - 19.8 ±0.5

ODT Crosscaramellose 524.3 ± 19.7 0.43 ±0.09 - 21.8 ±0.3

Table 2. PCS average diameter (Z-AVE), polydispersity index (PI) and zeta potential (ZP) of starting PRX nanocrystals and PRX nanocrystals after ODT disaggregation in water.

86

I II III Monohydrate ODT

Xanthana ODT

croscarmellose

ODT

gelatin

2the

ta

8.58 8.94 8.70 9.94 -

- -

11.62

12.48

13.24

13.96

14.42

10.06 - 11.76 12.06(#) 12.02(#) 12.00(#)

- - 12.66 - -

- -

17.66 - 17.80

13.78

14.30

14.82

16.34

- - -

-

19.66,

19.90,

20.28

18.40 - -

- -

- 22.98 24.62 21.20 21.48(#) 21.48(#) 21.42(#)

26.72 27.36 27.70

- - 26.08 26.34(#) 26.34(#) 26.34(#)

- - 28.00

28.96

27.98

29.52

30.28

28.06(^)

28.32(^)

28.26(^)

29.82(#)

27.98(#)

28.96(^)

- 38.22 - - - -

Table 3. Comparison of the 2θ degree value between each PRX polymorph forms and PRX nanocrystal ODTs, (*)=relative to form I, (^)=relative to form III, (#)=relative to monohydrate form

87

the previously obtained results. The ODT diffraction patterns are not crystalline due to the high percentage of amorphous components; however, some peaks imputable to PRX and poloxamer (19.1° and 23.3° 2θ) are evident. In Table 3, comparison between XRPD peaks of piroxicam polymorphic forms and that of all the prepared ODT, is shown. In ODT formulations FTIR spectra, the poloxamer 188 absorption bands due to OH (3400 cm-1) and C-O stretchings (1112-1108 cm-1) are evident. Furthermore, it can be also observed: C=O stretching in the range 1643-1633 cm-1, OH bending typical of monohydrate form at 1602 cm-1, C=N stretching in the range 1556-1530 cm-1, SO2 asymmetric stretching in the range 1354 and 1331 cm-1 and SO2 symmetric stretching at 1151 cm-1.Moreover, in the region between 3600 and 3200 cm-1, the croscarmellose ODT spectrum showed also a peak at 3343

cm-1 characteristic of PRX form III N-H stretching (Table 4), overlapped to the large OH stretching. As in the previously studied ODT, the XRPD and FTIR analyses allowed us to confirm the presence of monohydrate and form III mixture in the new ODT. The thermal behavior of bulk piroxicam shows an onset temperature of 202°C and a melting peak at 203°C while poloxamer 188 onset temperature is at 51°C and the melting peak at 55°C. Due to their amorphous state, all the ODT thermograms do not exhibit significant thermal events besides that belonging to poloxamer. The solid state of PRX commercial ODT (Feldene Fast®) was also investigate. As shown in the 4000-2800 cm1 and 1700-1100 cm-1 regions reported in figure 4, bulk PRX (Form I) and Feldene FTIR spectra are compared. In commercial ODT, the NH stretching at 3338 cm-1 and the absence of the absorption band at 1602

cm-1 (characteristic of monohydrate form) suggested the presence of bulk PRX form I. Due to the amorphous diffraction pattern of Feldene and ODT, a XPRD study is not significant.

In vitro dissolution studiesDrugs like PRX (Biopharmaceutics Classification System class II drugs), characterized by poor solubility and high permeability, show an oral absorption rate strongly dependent on dissolution rate. In order to achieve superior dissolution rate and, therefore, higher oral absorption, in this work we prepared three ODTs (starting from the same nanosuspension and containing PEG4000 and maltodextrins), which differed each other for the nature of only one excipient: xanthan gum or gelatin or croscarmellose. To study the influence of these different excipients on the PRX dissolution properties, dissolution tests were performed in distilled water according

 

Fig. 3. White bulk powder PRX ODT (left) and bright yellow ODT with PRX nanosuspension (right)

88

Form I Form II Form III Monohydrate ODT xanthana

ODT croscarmellose

ODTGelatin

OH - - - 3451 3391 3411 3041

NH 3338 3393 3343

3323

3377 Overlapped with OH

3343 Overlapped with OH

C=O 1630 1642 1634 1642 1643 1633 1643

OH - - - 1599 1602 1607 1602

C=N 1530 1530 1531 Overlapped with OH

1531 1534 1553

SO 2 as. 1351 1354 1354 1331 1332 1332 1332

SO 2 s. 1181 1180 1184 1160 1152 1150 1151

% T

1700 1600

a

b

1500 1400cm-1

1300 1200 1100

% T

4000 3800

a

b

3600 3400cm-1

3200 3000 2800

Table 4. Comparison of the 2q degree value between each PRX polymorph forms and PRX nanocrystal ODTs, (*)=relative to form I, (^)=relative to form III, (#)=relative to monohydrate form

Fig. 4. FTIR spectra comparison (4000-2800 cm-1 and 1700-1100 cm-1 regions) PRX (Form I) and Feldene

89

to the United States Pharmacopeia (Figure 5 a-d) (Ambrogi et al., 2007)

(Tantishaiyakul et al., 1999). Dissolution rate of the PRX nanosuspension ODT was compared with those of PRX coarse suspension ODT, PRX/poloxamer 188 physical mixture, bulk PRX samples and a PRX commercial ODT (Feldene Fast®). All samples contained 20 mg of PRX. Very similar dissolution profiles for bulk and physical mixture were obtained since, as previously shown, the presence of the surfactant at the tested concentration did not increase the PRX solubility (Lai et al.,

2011). ODT formulations prepared using coarse PRX showed a faster release than bulk PRX and physical mixture of PRX/poloxamer 188. To elucidate this result, we performed a study on the variation of the PRX solubility using the same PRX/excipient ratio as in the ODT formulation. Addition of PEG4000, poloxamer 188, xanthan gum or croscarmellose did not

give any significant variation of the PRX solubility. By contrast, the addition of maltodextrin or gelatin determined, respectively, a little (17.02 ± 0.45 mg/l) or great increase in the PRX solubility (94.78 ± 2.38 mg/l, Table 5). PRX solubility was also determined in citric acid and/or aspartame and/or mannitol water solutions because these are the excipients, together with gelatin, of the commercial PRX formulation Feldene Fast® (Table 5). Since the amount of every Feldene Fast® component is not precisely known, we prepared these solutions with the same PRX:excipient ratios reported in Zydis patent, where the preparation procedure of PRX ODT is reported (Owen et al.,

2000). PRX coarse suspension ODT with xanthan gum did not reach the dissolution rate of the same ODT prepared with gelatin or croscarmellose (figure 5 a-c). This result can be due to the fact that croscarmellose is a super disgregant and

that the PRX solubility is significantly increased by the presence of gelatin. Nevertheless, all ODT formulations prepared using PRX nanocrystals showed a drug dissolution rate higher than ODT prepared using coarse PRX. As previously demonstrated by XRPD and FTIR studies, the homogenization process led to a polymorphic transition from the form I (bulk commercial PRX) to the form III and monohydrate form nanocrystals. Indeed, this is because homogenization generates high energetic forces, which lead not only to drug particle break down to the nanometer range but also to a change in crystal structure (i.e., partial or total amorphisation) that could further enhance the solubility (R.H. Muller et al., 2003). Probably, the main reason of this behavior could be the consequence of the particle dissolution followed by recrystallization. However, in this study, the solubility of the different PRX polymorphic forms

Solubility mg/l

PRX (form I) 14.33 ± 0.60

PRX/Maltodextrin 17.02 ± 0.45

PRX/Gelatin 94 . 78 ± 2 . 38

Feldene Fast ® 184. 09 ±4.68

PRX/Mannitol 55 . 34 ± 1 . 49

PRX/Aspartame 58. 36 ±2 . 09

PRX/Citric Acid 54 . 89 ± 2 . 12

Prx+Mannitol+ CitricAcid 58 . 21 ± 2 . 52

Table 5. PRX solubility in different ODT excipients water solutions

90

increased only slightly from bulk PRX (form I) to monohydrate, to form II and to form III (Table 6).Therefore, the variation of the PRX polymorphic form was not the factor responsible for the higher dissolution rate of ODT nanocrystals or, at least, it was not the most important one. So, we have to conclude that, as predicted in the Noyes–Whitney (Noyes and Whitney, 1897) dissolution model, the improvement in piroxicam dissolution rate was mainly caused by the increased surface-to-volume ratio due to the submicron dimension of the drug particles (Kocbek et al., 2006)

(Lai et al., 2011) (Mauludin et al.,

2009b). Dissolution rate of the different PRX nanocrystal ODT formulations was also compared with that of the commercial formulation Feldene Fast®. The PCS diameter (Z-AVE) of PRX particles determined just after Feldene Fast®

disaggregation in water (few seconds) is

1482 nm (0,92 P.I), a value higher than that of PRX obtained from ODT formulation (xanthan, gelatin, croscarmellose) (Table 2). Xantan gum containing PRX ODT showed a very interesting dissolution profile, but slower than that of Feldene Fast® (figure 5d). This behavior can be explained considering the great increase of PRX solubility in the presence of gelatin (a Feldene Fast® excipient).Both gelatin-ODT and, especially, croscarmellose-ODT showed a dissolution profile much faster than the commercial formulation (figure 5d). In particular, it can be noticed that after five minutes, the amount of PRX dissolved from both gelatin- or croscarmellose-ODT was almost twofold higher than Feldene Fast®, with dissolution rate from croscarmellose-containing formulation a little bit higher than the gelatin-ODT. ODTs are designed to easily and rapidly disintegrate in the oral cavity rather than to

be swallowed whole. In this way, the faster drug dissolution in the mouth might be enhanced leading to promoted absorption through the oral mucosa. This is a very important point since the aim of this work was to prepare formulations capable of improving oral mucosa piroxicam absorption for a rapid systemic distribution bypassing the gastrointestinal tract. These interesting data can be explained by different mechanisms. In the case of croscarmellose-ODT the higher dissolution rate compared to that of the commercial formulation is determined by the good disgregant activity of croscarmellose that allows the ODT quick break up with consequent release of the submicron sized PRX particles with an increased surface-to-volume ratio. Gelatin-ODT, like Feldene Fast®, showed an improved PRX solubility due to the presence of gelatin. Therefore, in this case, the increased PRX dissolution rate in comparison with the

PRX polymorphic form

Solubility (mg/l)

Form I ( Bulk PRX) 14.33±0.60

Form II 16.72±1.50

Form III 16.98±2.09

Monohydrate Form 16.52±1.25

Table 6. PRX polymorphic form solubility in water

91

commercial formulation can be only ascribed to the increased drug surface area due to the nanosize of the PRX particles. In conclusion, results of this work confirm that the improvement of PRX dissolution rate using ODT is mainly caused by the increased surface-to-volume ratio due to the submicron dimension of the drug particles (Kocbek et al., 2006; Lai et al.,

2011; Mauludin et al., 2009b). However, a further important result obtained here emphasizes the importance of the proper choice of ODT excipients.

92

a - Xanthan

20

15

10

5

0

0 20 40 60

time (min)

80 100 120

PRX nanosuspension ODT (Xanthan)Coarse PRX ODT (Xanthan)Bulk PRX/ poloxamer physical mixtureBulk PRXFeldene Fast

mg/

l PR

XFig. 5a. Dissolution profile of xanthan-ODT prepared using PRX coarse and PRX nanocrystals compared to those of bulk PRX, PRX/poloxamer 188 physical mixture and commercial formulation Feldene Fast

93

b - Gelatin

20

15

10

5

0

0 20 40 60

time (min)

80 100 120

PRX nanosuspension ODT (Gelatin)Coarse PRX ODT (Gelatin)Bulk PRX/ poloxamer physical mixtureBulk PRXFeldene Fast

mg/

l PR

X

Fig. 5b. Dissolution profile of gelatin-ODT prepared using PRX coarse and PRX nanocrystals compared to those of bulk PRX, PRX/poloxamer 188 physical mixture and commercial formulation Feldene Fast

94

c - Croscarmellose

20

15

10

5

0

0 20 40 60

time (min)

80 100 120

PRX nanosuspension ODT (Croscarmellose)Coarse PRX ODT (Croscarmellose)Bulk PRX/ poloxamer physical mixtureBulk PRXFeldene Fast

mg/

l PR

XFig. 5c. Dissolution profile of croscaramellose-ODT prepared using PRX coarse and PRX nanocrystals compared to those of bulk PRX, PRX/poloxamer 188 physical mixture and commercial formulation Feldene Fast

95

d

20

15

10

5

0 20 40 60

time (min)80 100 120

PRX nanosuspension ODT (Xanthan)PRX nanosuspension ODT (Gelatin)PRX nanosuspension ODT (Croscarmellose)Feldene Fast

mg/

l PR

X

Fig. 5d. Dissolution profile of crosscaramellose, gelatin and xanthan-ODT prepared using PRX nanocrystals compared to a commercial formulation Feldene Fast

96

Formulation and evaluation of quercetin nanosuspension incorporated maltodextrin fast - dissolving films

Introduction

Quercetin (3,3,4,5,7 - pentahydroxyflavone, Que, Fig. 1), one of the most common dietary polyphenol widely found in plant kingdom including vegetables, fruits, herb medicine and red wine, has been intensively investigated for its anti-inflammatory, antioxidant, antiviral, and anticarcinogenic properties for various animal and human ailments (Askari et al., 2013) (Date et al.,

2011) (Ekström et al., 2011) (Cui et al.,

2008) without any evidences of toxicity, carcinogenicity and genotoxicity related to consumption (Ruiz et al., 2009) (Utesch

et al., 2008) (Harwood et al., 2007). In the western world, the average daily intake of quercetin has been estimated to be between 20 mg and 40 mg, although it can increase up to 500 mg/day in individuals who ingest large quantities of apples, onions and tomatoes (Manach et

al., 2005) (Manach et al., 2004) (Russo

et al., 2012). However, the poor water solubility, short biological half-life, and low oral bioavailability of quercetin hampered its application as a therapeutic agent (Khaled et al., 2003) (Hollman et al.,

1996) (Gugler et al., 1975). Although the increased dissolution has been reported to significantly enhance both bioavailability and clinical effects of quercetin (Khaled

et al., 2003), the relatively high molecular weight with high melting point and low water-solubility still present the main challenges for improving its solubility characteristics (Pool et al., 2013). Fast-dissolving oral delivery systems are solid dosage forms, which disintegrate or dissolve in few minutes when placed in the mouth without drinking or chewing. The first developed fast-dissolving dosage form consisted in tablet form, and the rapid disintegrating properties were obtained through a special process or formulation modifications (Sandri et

Fig.1 Quercetin structure

OHO

OH

OH

OH

OH O

97

al., n.d.). More recently, fast-dissolving films are gaining interest as an alternative to fast-dissolving tablets to definitely eliminate patient’s fear of chocking and overcome patient impediments. Fast-dissolving films are generally constituted of plasticized hydrocolloids or blends made of this compounds that can be laminated by solvent casting or hot-melt extrusion. According to the film forming material characteristics, the manufacture of the dosage forms can present different critical issues. Common problems are caused by foaming during the film formation due to the heating of the material or solvent evaporation, the flaking during the slitting and the cracking in the cutting phase. Furthermore, the films should be stable to moisture overtime. Finally, to facilitate the handling they have to be flexible and exhibit a suitable tensile stress and do not stick to the packaging materials and fingers. The first material employed to produce edible

films was pullulan, a glucan consisting of maltotriose units, produced from starch by the fungus Aureobasidium pullulans (Leathers, 2003). Even if pullulan-based films are easily manufactured, the use of this material is limited by the low availability and high costs. Therefore, in the last years other edible hydrocolloids have been studied as substitute. The film-forming properties of modified starches, gums, cellulose ethers, alginates, polyvinylalcohols, polyvinylpyrrolidones or blends thereof have been consider (Mashru et al., 2005). Among them, maltodextrins (MDX) with a low dextrose equivalent (DE) were proposed to improve the flexibility and reduce the cracking of modified starch-based films. Nevertheless, the application of MDX as the main film forming material has been scantily investigated (Shamekh et al., 2002) and, to our knowledge, no information about the effect of plasticizers on their tensile

properties has been reported.The aim of this work was to investigate the possible use of maltodextrin IT6 (MDX) to prepare fast-dissolving films, loaded by quercetin nanocrystals. Quercetin nanosuspensions (Qn) were prepared using an high pressure homogenizer, meanwhile drug loaded films were obtained drying in a siliconized polyester sheet Qn with the others compounds in a oven at 60 °C. Films were finally cut and packed within sealed aluminium pouches. Qn were characterized by photo correlation spectroscopy for mean size and size distribution and by transmission electron microscopy for morphological studies. On the other hand, Qn loaded films were characterized in term of flexibility, tensile strength and thickness.Finally, dissolution studies in distilled water were performed, comparing release profiles of quercetin loaded films, quercetin raw material and Qn.

98

Materials and methods

MaterialsMaltodextrin having a D.E. equal to 6 (Glucidex® IT6, MDX6) was kindly obtained by Roquette, (F). Glycerin (GLY) and sorbitan monooleate (Span® 80, S80) were purchased from Carlo Erba Reagenti (I) and Croda (E), respectively. Polysorbate 80 (Tween® 80) was purchased from Galeno and Quercetin (≥95%, HPLC, solid) was purchased from Sigma-Aldrich. All solvents were of analytic grade, unless specified.

Quercetin nanosuspension preparationQuercetin nanosuspensions was prepared by high pressure homogenization. 5% (w/w) Que coarse powder was dispersed in water with 1% (w/w) of Tween 80 and disintegrated into microparticles by a high shear homogenizer (Ultra-Turrax®

T25, IKA, Germany) at 12,000 rpm for 5 min. Obtained microsuspension was homogenized at high pressure using a high pressure homogenizer (EmulsiFlex-C5, Avestin Inc., Ottawa, Canada). At first, 5 cycles at 500 bar were conducted as pre-milling step, and then 20 cycles at 1000 bar were run to obtain the nanosuspensions.

Particle size and zeta potential measurementsQuercetin nanosuspensions were analyzed for particle size and zeta potential by photon correlation spectroscopy (PCS) using a Zetasizer Nano ZS (Malvern Instruments, UK). Samples were backscattered by a helium–neon laser (633 nm) at an angle of 173° and a constant temperature of 25°C. The instrument systematically and automatically adapts to the sample by adjusting the intensity of the laser and the attenuator of the photomultiplier, thus ensuring reproducibility of the

experimental measurement conditions. Zeta potential was estimated using the Zetasizer nano-ZS by means of the M3-PALS (Phase Analysis Light Scattering) technique. All the samples were analyzed 24 h after their preparation. A long-term stability study of formulations stored at 4 ± 1°C was performed by monitoring formulations average size, polydispersity, and surface charge over 90 days. The presence of possible aggregated was investigated by using an Accusizer 770 granulometer (P SS Inc., Santa Barbara, USA). The results are expressed as the mean of three determinations.

Film preparationFilms were prepared modifying the solvent casting technique previously described (Cilurzo et al., 2008). Briefly, drug loaded film was obtained by gradually adding MDX6 to a quercetin nanosuspension. Afterwards, GLY and S80 were added in

99

the specific proportion (Table 1). After a rest period of at least 24h, slurries were cast over a silicone release liner by a laboratory-coating unit Mathis LTE-S(M) (CH). Operative conditions: coating rate: 1 m/min; drying temperature: 50 °C; drying time: 20 min; air circulation speed: 1200 rpm. These conditions were set to obtain films having a thickness of about 120 µm (MI 1000 micrometer, ChemInstruments, USA). Film was cut into suitable shape and size as required for testing, packed immediately after the preparation in individual airtight aluminum seal packs and stored at 25 °C until use. Placebo film was also prepared following the same procedure.

Film thicknessFilm thickness was measured by using a MI 1000 µm (ChemInstruments, USA). The accuracy of the instrument was 2.5 µm ± 0.5%. A 10 x 2.5 cm sample of the film was

placed between the anvil and the presser foot of the micrometer, and its thickness was measured in ten different positions. The determination was performed in triplicate.

Film flexibilityThe film flexibility was determined by adapting the ASTM bend mandrel test (D 4338-97). Briefly, a 2 x 3 cm sample was bended over an 8 mm mandrel and examined for cracks over the area of the bend in a strong light. The film was assumed as flexible if no cracks were visible at a 5x magnification.

Tensile propertiesTensile testing was conducted using a texture analyzer AG/MC1 (Acquati, I), equipped with a 5N load cell. The film was cut into 100 x 12.5 mm strips and equilibrated at 25 °C for 1 week. Tensile tests were performed according to ASTM

International Test Method for Thin Plastic Sheeting (D 882-02). Each test strip was longitudinal placed in the tensile grips on the texture analyzer. Initial grip separation was 60 mm and crosshead speed was 500 mm min-1. The test was considered concluded at the film break. Tensile strength, elongation at break, elastic modulus and tensile energy to break were computed to evaluate the tensile properties of the films. Tensile strength (TS) was calculated by dividing the maximum load by the original cross-sectional area of the specimen and it was expressed in force per unit area (MPa). Percent elongation at break (E%) was calculated by dividing the extension at the moment of rupture of the specimen by the initial gage length of the specimen and multiplying by 100 according to the following equation: E% = L - L0 / L0 x100where L0 is the initial gage length of the specimen and L is the length at the

Ingredient

Composition (%, w/w)

Drug loaded �lm

Placebo �lm

Quercetin 4.98 -

MDX6 71.18 74.91

GLY 20.00 21.06

S80 2.84 2.99

TWEEN80 1.00 1.05

Table 1. Composition of placebo and quercentin loaded film

100

moment of rupture. Elastic modulus or Young’s modulus (M) was calculated as the slope of the linear portion of the stress–strain curve. The result was expressed in force per unit area (MPa). Tensile energy to break (TBE) was defined by the area under the stress–strain curve. The value is in units of energy per unit volume of the specimen’s initial gage region. The result was expressed in energy per unit volume. An average of five measurements was taken for each type of specimen.

Transmission electron microscopy (TEM)Characterization of the external morphology of Qn, placebo film and Qn loaded film was determined using transmission electron microscopy (TEM). For nanosuspensions, a drop of preparation and an equal volume of an aqueous 1% uranyl acetate solution were adsorbed on the surface of copper grid and dried at

room temperature for 24 h. The sample was transferred into a TEM (Tecnai 12, FEI) operating at 120 kV. Placebo film and Qn loaded film, instead, were put in an aluminum stub without any reagent and then analyzed with the same instrument.

In vitro dissolution studiesIn vitro dissolution studies on quercetin raw material, Qn and Qn loaded film were performed according to the United States Pharmacopeia (USP) using the rotating basket method (Erweka apparatus). The dissolution media was 1000 ml of distilled water kept at 37 ± 0.1 °C and a rotation speed of 100 ± 2 rpm. At preselected time intervals, 1 ml samples was withdrawn (replaced with 1 ml of pre-thermostated fresh dissolution medium), filtered through polycarbonate membranes (0.45 µm, Millipore) and analysed by HPLC at 367 nm, using a chromatograph Alliance 2690 (Waters, Milan, Italy). The column

was a SunFire C18 (3.5m, 4.6 x 150 mm). The mobile phase was a mixture of acetonitrile, water and acetic acid (94.8:5:0.2, v/v), delivered at a flow rate of 1.0 ml min-1. Dissolution tests were performed in triplicate.

101

Results and discussion

This work aimed to study maltodextrins (MDX) with a low dextrose equivalent as film forming material and their application in the design of oral fast-dissolving film loaded with quercetin nanosuspensions.Qn were characterized in PCS diameter (Z-AVE), polydispersity index (PI) and Z potential just after preparation. Data are reported in Table 2. A long time stability studies was carried out by measuring the same parameters of Qn stored at 4 ± 1 °C for 90 days. Freshly prepared Qn presented a Z-AVE of 753 nm (0.31 PI) and a Z potential of −51,7 mV. This very highly negative zeta potential values should ensure high stability against the possibility of a re-aggregation phenomena. As shown in the Table 2, during the first 30 days, quercetin nanosuspensions maintained similar mean diameter and polydispersity index values. However, after two months of storage, the

dimension and the polidispersity index slightly increased, reaching 851 nm and 0,45 respectively at 90 days. During the checked three months, the Z potential values were constant. Qn PCS diameter was confirmed by TEM images (Fig. 2).TEM images of quercetin nanosuspensions loaded film and placebo film are showed in figure 3a-b. Qn loaded film (fig. 3a) disclosed a rough surface and showed, mixed with film matrix, several quercetin nanocrystals arranged in different position and orientation. An other feature easily recognizable in figure 3a is the porosity of the film. On the other hand, figure 3b showed TEM images of placebo film. In this case is well visible only the porosity of the film, meanwhile there are not traces of the surface roughness of Qn loaded film, probably due to the presence of quercetin nanocrystals.Since critical issues in the development of a fast-dissolving film are mainly related to

its mechanical properties, different studies were carried out. In particular, flexibility and tensile strength were evaluated for both Qn loaded film and placebo film. In a previous work (Cilurzo et al., 2008), particular attention was given to the selection of a proper plasticizer able to provide a suitable ductility and flexibility to MDX films under different types of mechanical stress. PEG 400 as well as the esters of citric acid was excluded because of the lack of miscibility with MDX. Satisfactory results were obtained adding both glycerol (GLY) and propylene glycol (PG) to MDX at different concentrations (16-18-20-22% w/w). In particular flexible films were obtained at GLY or PG content of at least 16% w/w or 18% w/w, respectively. Films prepared by using GLY or PG amount higher than 20% or 22% w/w, respectively, resulted too ductile for handling. Normally, the increase of the plasticizer content caused a decrease of the

Qn 5% MD (nm) P.I. ZP (mV)

Day 0 753 ±8,2 0,31 ± 0,05 - 51,7 ± 1, 3

Day 1 752 ±1,2 0,33 ± 0,03 - 49,4 ± 0,8

Day7 761±3,3 0,30 ± 0,07 - 50,7 ± 0,1

Day14 757 ±5,7 0,36 ± 0,06 - 47,9 ± 1,0

Day30 783±15 0,38 ± 0,02 - 52,8 ± 1, 3

Day60 843± 4,6 0,42 ± 0,02 - 48,6 ± 0,9

Day90 851 ± 3,2 0,45 ± 0,02 - 49,5 ± 1, 6

Table 2. Mean size (Z-AVE), polydispersity index (PI) and Z potential (ZP) of quercetin nanosuspensions stored at 4 ± 1 °C and monitored during 90-day

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Fig. 2. Quercetin nanosuspension TEM images

Fig. 3a-3b. Quercetin nanosuspensions loaded film (3a) and placebo film (3b)

Table 3. Thickness and tensile properties of the films

�ickness (µm)

E% TS (MPa) EM (MPa) TBE (J)

Placebo �lm 128±3 106,9±10,8 5,24±0,33 1,29±0,12 0,356±0,017

Qn loaded �lm

204±1 9,6±0,4 0,65±0,04 0,14±0,00 0,008±0,001

103

elastic modulus (EM), which is an index of stiffness, and the tensile stress (TS). The ductility, that is expressed as elongation at break (E%), increased increasing the plasticizer amount. The toughness, expressed as the tensile energy to break (TBE), resulted reduced only at the highest concentration of GLY. On the basis of this previous results, GLY was chosen as the suitable plasticizer for MDX and the concentration at 20% w/w was selected to formulate and produce both MDX placebo fast-dissolving films and MDX-Qn loaded fast-dissolving films by a modified casting technologies. Moreover, the addition of a surfactant agent was necessary. Benefits were achieved by adding surfactants with a low HLB value. The addition of sorbitan oleate 80 (S80) at 2,84% in Qn loaded film and 2,99% in placebo film caused a slight increase of stiffness. The addition of quercetin nanosuspensions to the basic formulations did not compromise the

film preparation. Indeed, all films resulted flexible and appeared homogeneous and pale yellow. The values of thickness and the film tensile properties are reported in Table 3. Film thickness (128 µm for placebo film and 203 µm for Qn loaded film) was not very homogeneous probably due to the shrinkage of the mixture spread on the silicone release liner selected for the preparation by casting. The presence of the drug significantly modified the tensile properties of the film, determining a relevant decrease in the elastic modulus (EM= 0,14 for Qn loaded film; EM= 1,29 for placebo film) and in consequence an increase of the film stiffness. Results showed in table 3 confirmed that the dispersion of drug crystals (although in nano-meter range) to a film determines an increase of the film fragility, in fact also E% (9,6% against 106,9%), TS (0,65 MPa against 5,24 MPa) and TBE (0,008 J against

0,356 J) show an important decrease when the film are prepared adding quercetin nanosuspensions. This phenomena is probably due at a less homogenous interaction between the film matrix compounds when drug nano crystals are presents. Despite that, the formulations maintained satisfactory flexibility and resistance to elongation for the production and packaging.To study the influence of different formulations on the Que dissolution properties, dissolution tests were performed in distilled water according to the United States Pharmacopeia. The in

vitro release profiles of quercetin loaded film were compared to those of quercetin raw material and Qn (Fig. 4). All tested samples contained 10 mg of Que. Qn loaded film showed a dissolution profile faster than quercetin nanosuspensions and quercetin raw material. In particular, it can be noticed that after five minutes,

Fig 4. Dissolution profiles of quercetin loaded film, quercetin raw material and quercetin nanosuspensions (Qn)

00

20

40

60

80

100quercetin raw material quercetin loaded �lmsQn

5 10 15 20 25 30 35time (min)

% Q

ue

104

the amount of Que dissolved from Qn loaded film, quercetin nanosuspensions and quercetin raw material was 72.73%, 54.00% and 11,34% respectively.The increased surface-to-volume ratio due to the submicron dimension of the drug particles can explain the faster dissolution profiles of quercetin nanosuspensions and Qn loaded film compared with Que raw material. Meanwhile, the better dissolution profile of Qn loaded film compared to the quercetin nanosuspensions, was probably due to the increased quercetin solubility determined by the presence of film components.

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