International Journal of Pharmaceutics 478 (2015) 193–201
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Preparation and characterization of spironolactone-loaded nanoemulsions for extemporaneous applications François Hallouard a , Gilles Dollo a,b, * , Nolwenn Brandhonneur a , Fabien Grasset c,d, Pascal Le Corre a,b a
Université de Rennes I, Laboratoire de Pharmacie Galénique, Biopharmacie et Pharmacie Clinique, Rennes, France Centre Hospitalo-Universitaire de Rennes, Pôle Pharmacie, Rennes, France Université de Rennes I, Institut des Sciences Chimiques de Rennes, UMR/CNRS 6226, Rennes, France d CNRS, UMI 3629CNRS/Saint-Gobain, Laboratory for Innovative Key Materials and Structures-Link, National Institute of Material Science (NIMS), GREEN/ MANA Room 512, 1-1 Namiki, 305-0044 Tsukuba, Japan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 October 2014 Received in revised form 5 November 2014 Accepted 8 November 2014 Available online 20 November 2014
In neonates as well as in adults having swallowing difficulty, oral medication is given through a nasogastric tube making liquid formulations preferable. In this study, we present the high potential of nanometric emulsions formulated by spontaneous surfactant diffusion, as extemporaneous formulations of hydrophobic drug. Spironolactone used as hydrophobic drug model, was incorporated in oil before formulation at a concentration of 13.5 mg/g oil. Then, all formulations were evaluated from pharmacotechnical and clinical standpoints, for their use in hospital or community pharmacy. The strength of this new liquid formulation lies on the simplicity, efficiency and reproducibility of their low energy process as on clinical aspects: high dose uniformity, facility to be administered through in nasogastric tube without any retention and a stability of 2 months at least compatible for an extemporaneous use. Moreover, this emulsion presented spironolactone content of 3.75 mg/ml among the most concentrated formulations published. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Nano-emulsions Nasogastric tube Spironolactone Spontaneous surfactant diffusion Swallowing difficulty
1. Introduction Spironolactone is a specific aldosterone antagonist which is used as a potassium sparing diuretic (Sweetman, 2009). Spironolactone is therefore interesting in the treatment of primary hyperaldosteronism or for the management of heart failure in both adults and infants. This drug is also used to treat refractory edema reducing, for example, lung congestion in premature infants (Atkinson et al., 1988). In neonates like in adult having swallowing difficulty (mostly in neurology, gastroenterology, geriatric and reanimation departments), oral medication is given through a nasogastric tube making liquid formulations preferable. These liquid formulations should preferably have a minimal spironolactone content of 3 mg/ml to minimize the extra water-load to kidneys (Kaukonen et al., 1997). However, this drug content should
* Corresponding author at: Laboratoire de Pharmacie Galénique, Biopharmacie et Pharmacie Clinique, Faculté de Pharmacie, Université de Rennes I, 2, avenue du Pr Léon Bernard, F-35043 Rennes, France. Tél.: +33 2 23 23 48 02; fax: +33 2 23 23 48 46. E-mail address:
[email protected] (G. Dollo). http://dx.doi.org/10.1016/j.ijpharm.2014.11.046 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
be lower than 5 mg/ml to be handled during patient dosing adjustment. Therefore, the spironolactone-loaded preparation volume is between 5 and 100 ml. Currently, there is no commercially available oral liquid preparation (Agence nationale de sécurité du médicament et des produits de santé (ANSM)) (ANSM, 2014) due to the poor water-solubility of spironolactone (28 mg/ml at 25 C) (Sutter and Lau, 1975). Various extemporaneous preparations were therefore developed. Poor water solubility has been firstly solved by using high osmolality syrups as suspending agents (Allen and Erickson, 1996; Mathur and Wickman, 1989; Nahata et al., 1993) or high amount of cosolvents (Pramar et al., 1992) both approaches being not recommended especially for neonates (Leff and Roberts, 1987). In addition, suspensions like tablets showed incomplete oral behavior, slow dissolution rate and a risk of degradation during storage (Clarke et al., 1977; Laouini et al., 2011; Levy, 1962). Then, approaches using cyclodextrins (Kaukonen et al., 1997; Soliman et al., 1997), nanocapsules (Limayem Blouza et al., 2006), nanoparticles (Dong et al., 2009) or liposomes (Laouini et al., 2011) were developed. Spironolactone solubilization into nanoparticles may be particularly interesting by improving the dissolution rate and protecting the drug from degradation by confining it within these
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particles or oil droplets (Couvreur et al., 2002; Laouini et al., 2011). In addition, it is worth noting that nanometric particles or oil droplets also induce stabilization against sedimentation or creaming and so prevent from heterogeneous preparation, in so far as the particles/droplets are solely under the influence of the Brownian motion (Anton et al., 2008). In hospital or community pharmacy, formulation process have to be simple, quick, reproducible, requiring few personal/materials and compatible to the good manufacturing practices (GMP) or adapted GMP according to the considering state. Besides, storage stability beyond the month is not necessary for extemporaneous preparation due to treatment personalization and the frequent prescription changes. A promising technology lies to formulate liquid preparation of hydrophobic drugs is in the low-energy nano-emulsification of drug loaded oils (Anton et al., 2008; Li et al., 2013). The nanoemulsions are generated through a spontaneous emulsification method, which is a simple, quick, and efficient alternative to obtain extemporaneous liquid preparation. The purpose of the present study is to investigate the potential of emulsification process to develop liquid preparation of hydrophobic drugs for hospital applications. For this purpose, we developed new formulations of spironolactone-loaded nano-emulsions made by low-energy emulsification process. Spironolactone was chosen as common hydrophobic drug model. Then, all formulations were evaluated from pharmacotechnical and clinical standpoints, for their use in hospital applications. 2. Materials and methods 2.1. Materials Micronized spironolactone (batch 1104544443) was purchased from Inresa, Bartenheim, France. Oils (oleoyl polyoxyl-6 glycerides, Labrafil M 1944CS1; medium chain triglycerides, Labrafac lipophile WL 13491 and propylene glycol dicaprylate/dicaprate, Labrafac PG1) were kindly gifted from Gattefossé, Saint-Priest, France. Another oil (caprylic/capric triglycerides, Miglyol 812N1)
was kindly gifted from Cremer, Witten, Germany. Nonionic surfactants (polyoxyl 35 castor oil, Kolliphor ELP1; polyoxyl 15 hydroxystearate, Kolliphor HS 151 and polyoxyl 40 hydrogenated castor oil, Kolliphor RH 401), were kindly gifted from BASF, Ludwigshafen, Germany. Chemicals from analytical grade used were as follows: ethanol (99.9%, Fischer Scientific, Leicestershire, UK), ethyl acetate (99.98%, Fischer Scientific, Leicestershire, UK) and acetonitrile (>99.9%, Fischer Scientific, Leicestershire, UK). Radiopaque polyurethane nasogastric tubes (reference AL514, batch 14A27) were purchased by Cair LGL, Civrieux d’azergues, France. Distilled water was obtained using an Autostill 4000x1 system (Jencons, Franklin, TN, USA). 2.2. Methods 2.2.1. Emulsion formulation First, various amounts of oil and nonionic surfactants were magnetically stirred at 50 rpm for 30 s at a controlled temperature of (25 1) C. Their respective proportion is a critical parameter that allows the precise control of the nano-emulsion size distribution and polydispersity. Once homogeneous, this mixture was added to distilled water and magnetically stirred at 200 rpm until a bluish and translucent suspension was obtained (achieved within a few seconds). We described the mechanism upon which the method is based in our previous work (Hallouard et al., 2011). The formulation parameters were rationalized through (i) the surfactant/oil weight ratio: SOR = 100 wsurfactant/(wsurfactant + woil) and (ii) the surfactant–oil/water weight ratio: SOWR = 100 wsurfactant+oil/(wsurfactant+oil + wwater). The value of the SOWR was kept constant at 40% throughout this study since its influence on the nano-emulsion formation is negligible (it only influences the droplet concentration (Hallouard et al., 2011). All formulations were prepared in triplicate. 2.2.1.1. Spironolactone solubility studies. The criteria for selecting oil for pharmaceutical use are the lack of toxicity, the absence of drug degradation in the selected oil and a high capacity for the oil to dissolve the drug in question. Thence, a solubility study of
Fig. 1. Montage for emulsion administration study through a nasogastric tube (left). All purges were collected and their volume measured in order to determine their spironolactone content by UV–vis spectrophotometer (bottom right). Distal end of the nasogastric tube presenting lateral openings (top right).
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spironolactone was carried out in the following oils: Labrafil M 1944CS1, Labrafac PG1, Labrafac lipophile WL 13491 and Miglyol 812N1. Excess amounts of spironolactone were dissolved in 10 ml of oil. The samples were agitated for 1 min at room temperature with a vortex and then, stored at (25 0.1) C under mechanic agitation for 7 days. The suspensions were centrifuged (ultra-centrifugation Optima MAX-XP1, Beckman Coulter, Brea, CA, USA) twice at 10,000 g for 15 min to remove drug excess. The centrifuged samples were solubilized in ethanol (or ethanol/ethyl acetate 10/90 for Labrafil M 1944CS1) and finally analyzed using an UV–vis spectrophotometer (Specord 2051, AnalytikJena, Jena, Germany) at wavelength of 240 nm. Calibration curve of spironolactone at 240 nm in ethanol and ethanol/ethyl acetate (10/90) are available in supplement data. Volumes of spironolactone loaded oils were adjusted by an eVol XR1 dispensing system (Thermo scientific, Waltham, MA, USA). 2.2.1.2. Surfactant optimization. The influence of surfactant on emulsion mean size and polydispersity was determined with Kolliphor ELP1, Kolliphor HS 151 and Kolliphor RH 401. Surfactant amount in formulation varied in order to modify emulsion SOR from 10 to 70% (10, 15, 20, 25, 30, 40, 50, 60 and 70). 2.2.2. Formulation characterization
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2.2.3. Emulsion administration per nasogastric tube The nasogastric tube was first rinsed with 20 ml of distilled water according to hospital care practice (Fig. 1). Then, 5 or 10 ml of emulsion was introduced in the tube followed by 5 purges with distilled water (the first time with 15 ml then with 10 ml). The residues were collected, dissolved in ethanol/acetonitrile (10/90) and analyzed at 237 nm to determine the spironolactone amount in each collected residues. Another study of emulsion administration (5 ml) per nasogastric tube was performed after cutting the distal end part of this tube eventually responsible of a siphon effect. Each measurement and analysis was repeated three times. 2.2.4. Stability of native and spironolactone loaded nano-emulsions In vitro stability of native or spironolactone-loaded nanoemulsions was checked during 2 months (0, 7, 14, 28 and 60 days). The nano-emulsions were stored at (25 0.1) C protected from light. Nano-emulsion variations in pH, osmolarity, size, and PDI at 3 different SOR, using Labrafac PG1 or Labrafac lipophile WL 13491 as oil were determined as a function of time. Each measurements was the mean of three prepared emulsions. 3. Results and discussion The main challenge for this study is to achieve simple, quick and reproducible process to prepare extemporaneous and
2.2.2.1. Size and zeta potential. Hydrodynamic diameters and Polydispersity index (PDI) were obtained by dynamic light scattering with a Zetasizer NanoZS1 (Malvern, Brookhaven, UK). The helium/neon laser, 4 mW, operated at 633 nm, with the scatter angle fixed at 173 , and the temperature was maintained at 25 C. PDI is a measure of the broadness of a size distribution derived from the cumulant analysis of DLS data according to ISO 13321:1996; for a single Gaussian population with standard deviation, s and mean size, xPCS; thus, PDI = s 2 = x2PCS is the relative variance of the distribution. In other words, it shows the quality of the dispersion. Values 0.1 reflect a very good monodispersity and quality of the nanoparticulate suspensions. Zeta potential measurements were performed with the same apparatus. Measurements were performed in triplicate for each. 2.2.2.2. Viscosity. Viscosities of native oils, spironolactone saturated oils and emulsions were measured with a rotational viscometer (R/S3+ CPS P11, Brookfield, Harlow, UK) during 3 min at 25 C. The gap between the fixed bottom and the upper measuring plate was adjusted at 5 mm. The rotational speed of the plate was kept constant at 50 rpm. Each analysis was repeated three times. 2.2.2.3. Drug loading. Spironolactone loading in oil droplets was assessed by subtracting drug content in emulsion with the one in emulsion water phase. For drug content in emulsion, spironolactone was dissolved in ethanol/acetonitrile (10/90). For drug content in emulsion water phase, emulsion was filtered through a tangential filter having polysulphone membranes with molecular weight cutoff around 50 kD (MicroKros1, Spectrum Laboratories, Rancho Dominguez, USA) in order to separate the water phase from the lipophilic phase of this emulsion. Then, the water phase was analyzed by UV–vis spectrophotometer (Specord 2051, AnalytikJena, Jena, Germany) at wavelength of 237 nm. Each measurement was repeated three times. The reference samples were blank emulsions undergoing the same drug extraction process. Calibration curves of spironolactone at 240 nm in water or at 237 nm in ethanol/acetonitrile (10/90) are supplement data. Volumes were adjusted during extraction and spironolactone measurement by an eVol XR1 dispensing system (Thermo scientific, Waltham, MA, USA).
Fig. 2. Schematic representation of the nano-emulsion generating process (top), and of the nano-droplets morphology (bottom). This illustration was inspired from Hallouard et al. (2011).
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Fig. 3. Influence of oils and surfactants on emulsion size and polydispersity. These emulsions were formulated with one of three different native oils (Labrafil M 1944 CS1, Labrafac PG1 and Labrafac WL 13491) and a non-ionic surfactant: Kolliphor ELP1 (filled circles), Kolliphor RH 401 (open circles) and Kolliphor HS151 (filled squares). SOWR was kept constant at 40%. Each point was in triplicate.
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homogeneous liquid spironolactone-containing formulations in hospital or community pharmacy. These formulations should preferably have a minimal spironolactone content of 3 mg/ml to minimize the extra water-load to kidneys (Kaukonen et al., 1997). Indeed, recommended neonatal dosing is 1–3 mg/kg/days divided every 12 to 24 h (Taketomo, 2012). However, this drug content should be lower than 5 mg/ml to be handled during patient dosing adjustment. In addition, these formulations should use only pharmaceutical and compatible excipients for an oral administration. 3.1. Emulsions formulation The simplicity of the nano-emulsion generating process is illustrated in Fig. 2. Once the organic and aqueous phases were in contact, the nonionic surfactants firstly solubilized in oil underwent a very rapid diffusion toward water. This resulted in a demixing of the lipophilic molecules in the form of nanometricscaled droplets, immediately stabilized by the surfactant molecules. The morphology of the resulting droplets (bottom part of Fig. 2) was a spironolactone loaded oily core surrounded by a nonionic surfactant layer, thus developing a hairy surface due to the PEG moiety of the amphiphiles. For all oil/surfactant combination, we showed that the hydrodynamic size (dh) and PDI were correlated with the surfactant/oil weight ratio (SOR). These relationships between SOR and dh or PDI are illustrated in Fig. 3. Above a SOR of 50%, emulsion size and polydispersity undergo a rise. This can be explained as the consequence of a too massive diffusion of surfactant molecules toward the water resulting in a reduction in their diffusion speed. This reduced diffusion speed suggests a partial fusion of oil droplets before their stabilization by the diffused surfactant molecules. Such
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Table 1 Maximum solubility of spironolactone in different oils. The oil densities are according their inspection certificates, 0.943, 0.946, 0.920 and 0.940 for Miglyol 812 N, Labrafac W 1349, Labrafac PG and Labrafil M 1944CS respectively. Oil
Concentration (mg/g of oil) Concentration (mg/ml of oil)
13.210 0.171 Labrafac W1349 Labrafac PG 14.567 0.115 Labrafil M 1944CS 7.071 0.098
12.457 0.162 13.401 0.106 6.647 0.085
phenomenon was already described during the formulation of polymeric nanoparticles by emulsion–diffusion process (Quintanar-Guerrero et al., 1996). Concerning the influence of oil structure on the emulsification process, it is worth to note that all compatible oils for an oral administration were not suitable for this emulsification process. Indeed, such emulsions using long chain triglycerides (olive oil, sunflower oil, linseed oil and rape oils) or of medium chain polyoxyl-glycerides (Caprylocaproyl polyoxyl8 glycerides, Labrasol1, HLB of 12) were destabilized in few minutes (Hallouard, 2012). These results showed the importance of the equilibrium in oil structures between their hydrophilic and lipophilic parts. Labrasol1 seems to have a too predominant hydrophilic part compare to their lipophilic one unlike triglycerides having long fatty acid chains. The optimal HLB of the oily compounds with the 3 nonionic Kolliphor surfactants seems to be near 9 corresponding to Labrafil M 1944CS1 (oleoyl polyoxyl6 glycerides) (Gattefossé, 2010). 3.1.1. Spironolactone oil solubility A spironolactone oil-solubility study was then made using oils used in our emulsification process. The results are summarized in Table 1. Concerning Labrafil M 1944CS1, spironolactone content was determined in a solvent mixture composed of hexane and
Table 2 Characteristics of nanoemulsions formulated using spironolactone-loaded oils (Labrafac PG1 or Labrafac WL 13491), a nonionic surfactant (Kolliphor ELP1) and water.
SOR 25% Hydrodynamic diameter (nm) PDI pH Osmolarity (mOsm/l) j potential (mV) a j potential (mV) b a b
201.800 9.000 0.196 0.025 5.740 0.185 43 1 10.900 0.889 7.500 0.458
Labrafac PG1 SOR 30% 164.900 4.660 0.117 0.024 5.900 0.180 61 1 8.700 0.359 7.200 0.345
SOR 40%
SOR 30%
Labrafac WL 13491 SOR 40%
SOR 50%
115.700 3.85 0.110 0.016 5.840 0.080 104 1 6.800 0.570 5.650 0.687
166.500 3.01 0.164 0.005 5.950 0.192 59 1 29.200 0.907 12.800 0.321
107.800 2.460 0.141 0.005 5.930 0.137 72 1 27.100 0.981 8.220 0.773
65.380 1.730 0.156 0.010 5.910 0.113 112 1 23.300 0.792 7.390 0.523
Formulations using a spironolactone-loaded oil. Formulations using a native oil.
Fig. 4. Viscosities of native or spironolactone loaded oils (A) and different selected emulsions formulated with these last ones (B and C). Native (black) or spironolactone loaded (gold) oils were Labrafac PG1 (filled squares) and Labrafac WL 13491 (filled circles). Emulsions were prepared with Labrafac PG1 (B) or Labrafac WL 13491 (C) as oil and with Kolliphor ELP1 as nonionic surfactant at a SOR of 25 (triangles), 30 (circles), 40 (lozenges) and 50% (squares). Points in gold correspond to viscosities of spironolactone loaded emulsions. Each measurement was repeated three times. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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ethanol (65/35) because this oil is not miscible in ethanol (Gattefossé, 2010) and the drug shows a low solubility in hexane (European Directorate for the Quality of Medicines & HealthCare (EDQM)) (EDQM, 2014). The calibration curves of spironolactone in ethanol or in hexane/ethanol (65/35) used in our study, are available in supplement data. 3.1.2. Influence of spironolactone on emulsions The selected formulations to be used as extemporaneous liquid preparation of spironolactone was a compromise between the amount of drug solubilized in oil, the quality of the dispersion (i.e. low size and PDI) and the amount of surfactant. Therefore, both Labrafac PG1 and Labrafac WL 13491 and the nonionic surfactant Kolliphor ELP1 were selected for the following spironolactoneloaded nano-emulsions preparation. The formulation parameters for spironolactone-loaded emulsions were SOWR = 40% and SOR = 25–40% for Labrafac PG1 and 30–50% Labrafac WL 13491. 3.1.2.1. Emulsions characteristics. Characteristics of selected emulsions are reported in Table 2. The very low PDI values indicate the extreme monodispersity of these spironolactoneloaded nano-emulsions. Emulsions pH was between 5.75 and 6 which is compatible with spironolactone because this drug
presents a less stability in alkaline environment (PharmInfoTech, 2001). Zeta potentials measured on spironolactone-loaded and native nano-emulsions disclosed a significant negative surface charge and the presence of spironolactone increased the negative charges of particle surface. In addition, these potentials also decreased when SOR increased. These results are coherent with the chemical nature of the compounds used: the nonionic surfactant accumulating at surface particle and oil (which can contain free fatty acid) leading negative charges. Indeed, macroglycerides are prepared by saponification of vegetal oils followed by fatty acid esterification with polyethylene glycol. Therefore, these oils contain little proportions of free fatty acids, free glycerol and mono-, di or triglycerides as demonstrated in our previous work (Hallouard et al., 2011). The lack of significant spironolactone influence supposed the absence of this drug in the dispersing phase. To confirm this hypothesis, we showed by UV–vis spectrophotometry the absence of spironolactone in dispersing phase after extraction from emulsion by tangential filtration. 3.1.2.2. Emulsions viscosities. A viscosity study about the influence of spironolactone incorporation in Labrafac PG1 and Labrafac WL 13491 and their selected emulsions is illustrated in Fig. 4. Non significant differences between native and drug-loaded oil
Fig. 5. Storage stability of native (in black) or spironolactone-loaded (in gold) Labrafil PG1 emulsions. These emulsions were stored in dark at (25 0.1) C during 60 days, n = 3. Emulsions were prepared with Kolliphor ELP1 as nonionic surfactant at a SOR of 25 (triangles), 30 (circles) and 40% (lozenges). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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viscosities were observed explaining, in part at least, why there was no influence of drug on emulsion particle and polydispersity (Figs. 5 and 6 ). Concerning emulsions, there were also no significant viscosity difference between native and drug loaded emulsions. In additions, SOR is correlated with emulsion viscosity independently from the oil nature. These last results could be explained by the fact that oil droplets were dispersed in these concentrated emulsions and so the oil viscosity did not matter (Broughton and Squires, 1938). Surfactant concentration in dispersing phase seems however not to be the only factor explaining this viscosity increase. Indeed, we studied the viscosity of surfactant solutions at the comparable concentration as in our emulsions. Only surfactant concentration corresponding to a SOR of 50% with a SOWR of 40% could be measured by rheometer at 18 mPa s. The other surfactant solutions were too low (meaning below 5 mPa s). This last result confirms the number of oil droplets, increasing with SOR, has also the positive influence on emulsion viscosities (Broughton and Squires, 1938). 3.1.2.3. Emulsions stability. A storage stability was performed for native or spironolactone-loaded selected Labrafac PG1 emulsions (Fig. 5) and Labrafac WL 13491 ones (Fig. 6). Evolutions as a
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function of time of emulsion size, polydispersity, osmolarity and pH were similar between native and drug-loaded emulsions. Emulsions size and polydispersity for both oils were constant during 2 months instead of Labrafac PG1 emulsions with a SOR of 25% which showed a significant polydispersity increase (0.165 0.037–0.235 0.018). Unlike Labrafac WL 13491 emulsions, pH and osmolarity evolutions of Labrafac PG1 emulsions were constant during a month and then varied in acceptable range, for unbuffered formulations, to stay usable. Drug-loaded Labrafac PG1 emulsions is therefore sufficient for clinical applications as extemporaneous preparation which require mainly a stability of 1 month (Afssaps, 2011). The surfactant degradation by hydrolyze of its PEG – castor oil ester link was probably not the origin of these variations. Kolliphor ELP1 hydrolyzes slowly in contact with plasma containing esterase (Li et al., 2011). This increase may be explained by the surfactant desorption from dispersed oil phase surface to aqueous phase. With Labrafac WL 13491 emulsions, such pH decrease associated with osmolarity increase may supposed a microbial development in these formulations. This could be prevented by using conservative despite most of them present some side effects especially by infants (Standing and Tuleu, 2005). In conclusion, the
Fig. 6. Storage stability of native (in black) or spironolactone-loaded (in gold) Labrafil WL 13491 emulsions. These emulsions were stored in dark at (25 0.1) C during 60 days, n = 3. Emulsions were prepared with Kolliphor ELP1 as nonionic surfactant at a SOR of 30 (circles) and 40 (lozenges) and 50% (squares). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Table 3 Spironolactone content of Labrafac PG1 emulsions having a SOR and a SOWR of 30 and 40%, respectively.
Labrafac PG + spironolactone (g) Surfactant (g) Water (g) Spironolactone content (mg/g oil) Theoretical content (mg/ml emulsion) Experimental content (mg/ml emulsion) Emulsion density at 25 C
Batch 1
Batch 2
Batch 3
2.099 0.899 4.500 13.590 0.002 3.735 0.032 3.743 0.034 0.973
2.097 0.897 4.500 13.590 0.002 3.734 0.031 3.788 0.032 0.974
2.099 0.907 4.500 13.590 0.002 3.731 0.330 3.697 0.030 0.976
best candidate to be used as extemporaneous liquid preparation of spironolactone was Labrafac PG1 emulsions having a SOR and SOWR of 30 and 40%, respectively. 3.2. Emulsions drug-loading We demonstrated by UV spectrophotometer measurements, a dose uniformity of these optimized Labrafac PG1 emulsions due to their nanometric size form preventing creaming or sedimentation such as with micrometric emulsions or suspensions, respectively (Table 3) (Anton et al., 2008). It is worth to note that drug content in oil was lesser than maximal spironolactone content in order to prevent any potential over-drug saturation phenomenon during formulation process. In addition, there was no significant difference between theoretical and experimental drug content showing the good loading-yield of this method. This monodisperse emulsions showed a final drug content of 3.75 mg/ml which successes in our drug content obligation. The literature indicates that liquid spironolactone formulations are usable in the range of concentrations from 1.5 mg/ml to 100 mg/ml (Allen and Erickson, 1996; Bernal et al., 2014; Dong et al., 2009; Kaukonen et al., 1997; Laouini et al., 2011; Limayem Blouza et al., 2006; Mathur and Wickman, 1989; Nahata et al., 1993; Soliman et al., 1997). Nevertheless, besides this interesting drug concentration, our system is the first example of nanometric sized formulation reaching such high concentration. Indeed maximum drug concentration in other described nanometric sized formulations was 1.5 mg/ml (Laouini et al., 2011; Limayem Blouza et al., 2006). Our nano-emulsion was also better than cyclodextrin formulations which presented a drug concentration of 3 mg/ml but a stability of only few days (Kaukonen et al., 1997). Optimized Labrafac PG1 formulation (SOR = 30% and SOWR = 40%) presents also a surfactant concentration of 12% (w/w). With a maximal recommended dosage per day for human use of 400 mg daily (Sweetman, 2009), these emulsions can be administered per os without toxicological incidences. Indeed, the lowest LD doses of the emulsion compounds was for the nonionic surfactant presenting a LD50 by 6.4 or >10 g/kg body weight by rat or cat and rabbit respectively (Rowe et al., 2009). In addition, one of our previous works established in vivo the absence of toxicity when massive amount of this nonionic surfactant was used in the bulk phase (Hallouard
Table 4 Influence of administered emulsion volume and of distal end of nasogastric tube on the emulsion passage through these tubes.
Administered emulsion volume (ml) Cuted distal end of nasogastric tube 1st aliquot (%) 2nd aliquot (%) 3rd aliquot (%) 4th aliquot (%) 5th aliquot (%) Total (%) a
Absence of colloidal aspect.
1
2
3
5 No 95.7 1.50 3.9 1.83
