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Bioavailability of seocalcitol
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 233–242
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Bioavailability of seocalcitol II: Development and characterisation of self-microemulsifying drug delivery systems (SMEDDS) for oral administration containing medium and long chain triglycerides b,∗ ¨ Mette Grove a,b , Anette Mullertz , Jeanet Løgsted Nielsen c , a Gitte Pommergaard Pedersen a
Pharmaceutical Formulation, LEO Pharma A/S, Industriparken 55, DK-2750 Ballerup, Denmark Department of Pharmaceutics, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark c Department of Pharmacokinetics, LEO Pharma A/S, Industriparken 55, DK-2750 Ballerup, Denmark b
a r t i c l e
i n f o
a b s t r a c t
Article history:
By constructing ternary phase diagrams it was possible to identify two self-microemulsifying
Received 28 September 2005
drug delivery systems (SMEDDS) containing either medium chain triglycerides (MC-
Received in revised form 22
SMEDDS) or long chain triglycerides (LC-SMEDDS), with the same ratio between lipid, sur-
November 2005
factant and co-surfactant. The SMEDDS ended up having a composition of 25% lipid, 48%
Accepted 20 February 2006
surfactant and 27% co-surfactant, MC-SMEDDS: viscoleo, cremophor RH40, akoline MCM and
Published on line 2 May 2006
LC-SMEDDS: sesame oil, cremophor RH40, peceol. Upon dilution with water both SMEDDS resulted in clear to bluish transparent microemulsions with a narrow droplet size of 30 nm.
Keywords:
The industrial usefulness of the developed SMEDDS was evaluated with regard to bioavail-
SMEDDS
ability and chemical stability using the vitamin D analogue, seocalcitol, as model compound.
Oral bioavailability
The absorption and bioavailability of seocalcitol in rats were approximately 45% and 18%,
Lipid-based formulations
respectively, from both the MC-SMEDDS and LC-SMEDDS indicating similar in vivo behavior
Oral absorption
of the two formulations, despite the difference in nature of lipid component. There was
Poorly soluble drug substances
no improvement in bioavailability by the use of SMEDDS, compared to the bioavailability
Stability
achieved from simple MCT and LCT solutions (22–24%) (Grove, M., Pedersen, G.P., Nielsen,
Seocalcitol
J.L., Mullertz, A., 2005. Bioavailability of seocalcitol. I. Relating solubility in biorelevant media with oral bioavailability in rats-effect of medium and long chain triglycerides. J. Pharm. Sci. 94, 1830–1838.). After 3 months’ storage at accelerated conditions (40 ◦ C/75% RH), a decrease in concentration of seocalcitol of 10–11% was found in MC-SMEDDS and LC-SMEDDS compared with a degradation of less than 3% for the simple lipid solutions of MCT and LCT. In this study the simple lipid solutions seem to be a better choice compared with the developed SMEDDS due to a slightly higher bioavailability and a better chemical stability of seocalcitol. © 2006 Elsevier B.V. All rights reserved.
∗
Corresponding author. Tel.: +45 35306440; fax: +45 35306030. ¨ E-mail address: [email protected] (A. Mullertz). 0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.02.005
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1.
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Introduction
An increasing number of potential drug substances discovered by the pharmaceutical industry are poorly soluble in water, but have a high permeability and are therefore classified as class 2 drug substances according to the Biopharmaceutical Classification System (BCS) (Amidon et al., 1995). For the class 2 drug substances the bioavailability is often low and variable due to an insufficient dissolution process in the gastrointestinal tract, hence it will be beneficial to dose this kind of drug substances in their soluble form. For drug substances with sufficient lipophilicity, lipid-based drug delivery systems e.g. lipid solution, lipid emulsion, microemulsion, dry emulsion, Self Emulsifying Drug Delivery System (SEDDS) or Self Microemulsifying Drug Delivery System (SMEDDS) could be possible formulation approaches. Lipid-based drug delivery systems have gained considerable interest after the commercial success of Sandimmune NeoralTM (Cyclosporine A), Fortovase (Saquinavir) and Norvir (Ritonavir). Much attention has been on SMEDDS/SEDDS; an increase in bioavailability was found for L-365,260 (Lin et al., 1991), WIN 54954 (Charman et al., 1992), Ro 15-0778 (Shah et al., 1994), ontazolast (Hauss et al., 1998), halofantrine (Khoo et al., 1998) and danazol (Porter et al., 2004) when administered in self-emulsifying systems, compared to solid dosage forms. SMEDDS are defined as isotropic mixtures of lipid, surfactant, co-surfactant and drug substance that rapidly form a microemulsion upon mixing with water. A narrow droplet size distribution is often seen with a droplet size typically less than 50 nm (Gursoy and Benita, 2004). The self-emulsification process occurs spontaneously because the free energy required to form the microemulsion is either low and positive or negative (Constantinides, 1995). The self-emulsification process was shown to be specific to the nature of the lipid/surfactant pair, the surfactant concentration, the ratio between lipid and surfactant (Pouton, 1985; Wakerly et al., 1987) and only specific pharmaceutical excipient combinations can lead to efficient self-emulsifying systems (Charman et al., 1992; Shah et al., 1994). Drug substances with adequate solubility in lipid/surfactants blends are candidates for this formulation concept (Gershanik and Benita, 2000). The SMEDDS are believed to be superior compared with lipid solutions due to the presence of surfactants in the formulations leading to a more uniform and reproducible bioavail-
ability as seen for cyclosporine (Mueller et al., 1994). The surfactants act by dispersing the lipid formulation in the gastrointestinal tract (GIT) upon dilution with the gastrointestinal fluid (Stuchlik and Zak, 2001). This results in the formation of fine droplets providing a large surface area for pancreatic lipase to hydrolyse triglycerides and thereby promoting a rapid release of the drug substance and/or the formation of mixed micelles containing the drug substance (Tarr and Yalkowsky, 1989). The small droplets formed in contact with the gastrointestinal fluid may also be responsible for transporting the drug substance through the unstirred water layer to the gastrointestinal membrane for absorption (de Smidt et al., 2004). In the literature, medium chain triglycerides (MCT) have been preferred in SMEDDS due to the higher fluidity, better solubility properties and self-emulsification ability compared with long chain triglycerides (LCT) (Charman et al., 1992; Shah et al., 1994), as well as a better chemical stability of drug substance in MCT due to the purity of the lipid and the lack of double bonds, that can catalyse oxidation. The two lipids are differently transported in the body: MCT is directly transported by the portal blood to the systemic circulation (Porter and Charman, 1997), whereas LCT is transported in the intestinal lymphatics. Lipid-based drug delivery systems containing LCT are likely to enhance the lymphatic transport of a lipophilic drug substance (Palin and Winson, 1984; Caliph et al., 2000) and as the lymphatic transport circumvents the liver, the first-pass metabolism of a drug substance may be reduced (Porter and Charman, 1997). SMEDDS containing either MCT or LCT have been studied with halofantrine (Khoo et al., 1998) and danazol (Porter et al., 2004) and in both cases LC-SMEDDS were found to give the highest bioavailability. Studies comparing the bioavailability of drug substance from SMEDDS and other lipid-based drug delivery systems e.g. pure lipid solutions are still limited in number and until now most studies have been comparing self-emulsifying systems with solid dosage forms (Lin et al., 1991; Charman et al., 1992; Shah et al., 1994). MC-SMEDDS and LC-SMEDDS have been compared in two studies with halofantrine (Khoo et al., 1998) and danazol (Porter et al., 2004). However, in these studies the SMEDDS contain different amounts of lipid and surfactant, which makes the comparison complicated, since more than one variable exists. In a study with vitamin E a SMEDDS was found to give rise to a higher bioavailability compared with a lipid solution, but more lipid was dosed in the SMEDDS, which makes the comparison difficult (Julianto et al., 2000).
Fig. 1 – Chemical structure of seocalcitol and tritium labeled seocalcitol, 3 H-seocalcitol.
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The aim of the current study was to develop, characterize and compare the suitability of two SMEDDS containing either MCT or LCT. In order to make a direct comparison between the two SMEDDS possible, criteria of success were that they should have the same ratio between lipid/surfactant/co-surfactant, be monophasic at room temperature and have the same droplet size distribution after dispersion in water. Cremophor RH40 was used as surfactant in both cases, whereas the cosurfactant was chosen to resemble the lipid component with regard to chain length. The bioavailability of a poorly soluble model drug substance from the two developed SMEDDS, was determined in rats, and compared to the bioavailability from simple MCT and LCT solutions (Grove et al., 2005). In order to further evaluate the industrial usefulness of SMEDDS, the chemical stability of seocalcitol in the formulations, filled into hard gelatin capsules, was determined. The model drug substance used in the study was seocalcitol, a class 2 drug substance, with the chemical structure shown in Fig. 1. Tritium labeled seocalcitol was used in the bioavailability study thereby also enabling an estimation of oral absorption based on the presence of radioactive material in blood. Log P for seocalcitol is 4.8 and the solubility in MCT and LCT is 5.3 and 1.7 mg/g, respectively.
2.
Materials and methods
2.1.
Chemicals
Seocalcitol and tritium labeled seocalcitol (3 H-seocalcitol, specific activity of 0.844 MBq/g and radiochemical purity of 97%) were synthesized at LEO Pharma A/S. Viscoleo (MCT) was purchased from ICI Espana, Spain and Sesame oil (LCT) was purchased from Henry Lamotte GmbH, Germany. The fatty acid composition of the triglycerides in MCT and LCT is as specified in Ph.Eur. Cremophor RH40 (macrogol 40 glycerol hydroxystearate) was purchased from BASF, Germany. Akoline MCM (a mixture of mono, di- and triglycerides of octanoic and decanoic acids) was a gift from Karlshamns, Sweden ´ and Peceol (glycerol monooleate) was a gift from Gattefosse, France. Hard gelatin capsules, posilock size 4, were a gift from Shionogi Qualicaps Sa, Japan, butylhydroxyanisole (BHA) was purchased from Rhone Poulenc Sante (France), Hypnorm® (fentanyl 0.2 mg/mL, fluanisone 10 mg/mL) was purchased from Janssen, Belgium, and Dormicum (Midazolam 5 mg/mL) from Roche, Switzerland. Heparin 10.000 IU/mL was produced at LEO Pharma A/S. Isotonic sterile NaCl was purchased at Dilab, Sweden. The water was obtained from a Milli-Q-water purification system (Millipore, MA, USA). Pico-AquaTM and Pico Flour 40TM were purchased from Packard. All other chemicals were of analytical grade.
2.2.
Development of SMEDDS
A series of mixtures were prepared with varying ratio of lipid, surfactant and co-surfactant. The components were weighted into glass vials and mixed at 50 ◦ C using a stirring rate of 500 rpm, until the components were perfectly dissolved. The mixtures were cooled to 37 ◦ C and 1 g was transferred to a beaker where 250 mL of 37 ◦ C water was added under gen-
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tle stirring of 50 rpm. The dispersions were visually inspected and characterised for droplet size. A mixture was defined to be a suitable SMEDDS, if (1) rapid self-emulsification (within 1 min) was obtained after dispersion in 37 ◦ C water, followed by the formation of a clear transparent microemulsion giving a droplet size of less than 100 nm measured by photon correlation spectroscopy (PCS) and (2) mono-phasic mixture was obtained after storage at room temperature for 1 month.
2.3.
Droplet size analysis
The droplet size of the microemulsions was measured by means of PCS, using a Zetasizer 3000 (Malvern Instruments, UK). The samples were measured at 37 ◦ C after addition of 250 mL 37 ◦ C water to 1 g of mixture. No further treatment of the samples was necessary.
2.4.
Construction of ternary phase diagrams
Ternary phase diagrams were constructed for both MCSMEDDS and LC-SMEDDS. SMEDDS giving a droplet size of less than 100 nm after addition of 250 mL 37 ◦ C water to 1 g of SMEDDS were marked in a solid line area. In addition, monophasic SMEDDS were identified by visual inspection after 1 month’s storage at ambient temperature and marked in a dotted line area. To be able to identify identical compositions of MCSMEDDS and LC-SMEDDS, the two individual ternary phase diagrams were superimposed. In this way, an area was identified containing mono-phasic SMEDDS, with either MCT or LCT, resulting in the formation of clear transparent microemulsions with a droplet size of less than 100 nm. Within this area SMEDDS with the same ratio between lipid/surfactant/cosurfactant were identified.
2.5.
Selection of SMEDDS for further investigation
Based on the superimposed ternary phase diagrams one MCSMEDDS and one LC-SMEDDS were chosen for further investigation. Each had a composition of 25% lipid, 48% surfactant and 27% co-surfactant. The following investigations were performed with these two SMEDDS: (a) dispersion characteristics of SMEDDS containing dissolved seocalcitol; (b) chemical and physical stability testing of SMEDDS filled in hard gelatin capsules and (c) oral absorption and bioavailability of seocalcitol upon administration of the formulations in rats.
2.6. Dispersion characteristics of SMEDDS containing dissolved seocalcitol MC-SMEDDS and LC-SMEDDS containing dissolved seocalcitol were prepared. The amount of seocalcitol dissolved in the SMEDDS was 80% of the solubility in the lipid component corresponding to 1.1 mg/g in the MC-SMEDDS and 0.3 mg/g in the LC-SMEDDS. About 1 g of SMEDDS was transferred to a beaker, where 250 mL of 37 ◦ C water was added under gentle stirring of 50 rpm. The dispersions were visually inspected for the formation of a clear transparent microemulsion and for precipitation of seocalcitol. Furthermore, the samples were characterised by PCS.
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2.7.
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Stability of seocalcitol in formulations
The MC-SMEDDS and the LC-SMEDDS as well as MCT and LCT solutions containing seocalcitol were filled into hard gelatine capsules using a capsule filling apparatus (Hibar) and a band sealing machine (Quali-seal Capsule Sealing Machine, Laboratory Model). The capsules were packed in tropicalised blister foil 191 (Alcan Packaging Singen GmbH) and stored for 3 months at 5 ◦ C, 25 ◦ C 60% relative humidity (RH) and 40 ◦ C 75% RH. For comparison MCT and LCT solutions were filled into hard gelatin capsules and packed and stored under the same conditions as the MC-SMEDDS and the LC-SMEDDS. Each capsule had a concentration of 5 g seocalcitol. Butylhydroxyanisole in a concentration of 0.02% w/w was added to the formulations in order to stabilize the oxidation sensitive drug substance. The chemical stability and the following physical parameters were tested: disintegration, appearance and smell and content of water in the capsule shell.
2.8.
HPLC analysis of seocalcitol in formulations
The concentration of seocalcitol in the formulations was determined by a validated reversed-phase HPLC method consisting of a Hitachi Module L-7100 with a Spark Holland autosampler, column oven (25 ◦ C), a Hitachi Detector L-7400 set at 264 nm and data were processed by using Millennium Chromatography Manager version 32 (Waters Associates, Milford, MA, USA). About 100 L formulation was mixed with 6 mL of a mixture of acetonitrile and 0.01 M diammonium hydrogenphosphate (60:40) (v/v). About 20 mL n-heptane was added and the solution was mixed for 15 min on a mechanical shaker. After phase separation, the lower phase was collected and placed at 50 ◦ C for 15–18 h. The samples were injected into the chromatographic system after cooled to room temperature. The analytical column was a Superspher RP-18, 75 mm × 4 mm i.d. 4 m (Merck) and the injection volume was 100 L. The mobile phase was acetonitrile and phosphate buffer pH 6.5 (60:40) (v/v) and the flow rate was 1.2 mL/min. The retention time of seocalcitol was approximately 6 min and the quantification was performed using peak heights for seocalcitol in the chromatograms from test and reference solutions, the lower limit of quantification being 0.13 g/g.
2.9. Preparation of oral 3 H-seocalcitol formulations to bioavailability study Two lipid-based formulations, one MC-SMEDDS (25% viscoleo, 48% cremophor RH40, 27% akoline MCM) and one LC-SMEDDS (25% sesame oil, 48% cremophor RH40, 27% peceol), were prepared by dissolving 3 H-seocalcitol in the formulation, resulting in a concentration of 3 H-seocalcitol of 31 g/mL with a radioactivity of 26 MBq/mL and a radio chemical purity of 97%. The SMEDDS were produced on the day prior to dosing and stored at 5 ◦ C until dosing.
2.10.
Bioavailability study of 3 H-seocalcitol in rats
All surgical and experimental procedures were reviewed and approved by the local Animal Experimentation Ethics Committee. Male Sprague-Dawley rats weighing 280–320 g
Table 1 – Composition of the SMEDDS (%w/w) selected for bioavailability assessment in rats Excipient Viscoleo Akoline MCM Cremophor RH40 Sesame oil Peceol
MC-SMEDDS
LC-SMEDDS
25 27 48
48 25 27
The concentration of 3 H-seocalcitol in the formulations was 31 g/mL equal to 26 MBq/mL.
(Møllegaard Breeding center, Lille Skensved, Denmark) maintained on a standard feeding and water ad libitum were included in the study. During the acclimatization, two rats were housed in each cage maintained at 22 ± 2 ◦ C with a 50% relative humidity, an air change of 15 changes per hour and a 12-h light–dark cycle. During the experiment, the animals were anesthetized for the duration of the surgery by subcutaneous injection of 2.7 mL/kg of a solution consisting of Hypnorm® , Dormicum and water (1:1:2). The day prior to dosing the right carotid artery was cannulated with a tygon catheter (0.40 mm i.d., 0.79 mm OD, Dilab, Sweden). The catheter was exteriorized at the back of the neck and connected to a swivel apparatus designed to allow free animal movement and computerised blood sampling (Dilab, Sweden). The apparatus enabled automatic, consecutive blood sampling from the same animal. After the surgery, the animals received isotonic NaCl containing 25 U/mL heparin through the catheter to stabilize the liquid balance of the animal. Two oral formulations were administered: MC-SMEDDS and LCSMEDDS. Table 1 shows the compositions of the SMEDDS. Each rat received 1.5 mL formulation/kg corresponding to 47 g 3 Hseocalcitol/kg (equal to 40 MBq/kg) and 0.8 mL/kg lipid. After dosing the rats received water resulting in a total volume of administration of 1 mL. Blood samples of 250 L were collected at 0 (predose), 0.5, 1, 1.5, 1, 2.5, 3, 5, 7 and 9 h following oral administration. Serum was prepared and kept at −80 ◦ C until analysis. A total of four rats received each formulation. The dose of lipid (the amount of co-surfactant is calculated within the total amount of lipid in the SMEDDS) as well as dose of seocalcitol administered is the same as in a previous study (Grove et al., 2005) studying the bioavailability of seocalcitol from solutions of MCT and LCT.
2.11.
HPLC analysis of 3 H-seocalcitol in serum
The concentration of 3 H-seocalcitol in serum was determined using the radio-HPLC method described earlier (Grove et al., 2005). In brief, a reversed-phase HPLC-system with cooling on the autosampler (8 ◦ C), online vacuum degassing, column oven (40 ◦ C) (all Waters Associates, Milford, MA, USA), was added a Packard Radiomatic Flow scintillation Analyzer using Pico-AquaTM (Packard) as scintillator. The analytical column was a Symmetri C8, 50 mm × 2.1 mm i.d. 3.5 m (Waters) and the absorbance was measured at 264 nm. The quantification of 3 H-seocalcitol was performed using a calibration curve of spiked blank serum, where the lower limit of quantification was 0.5 ng/mL.
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2.12. Preparation of samples for total liquid scintillation counting
sons the number of animals used in the present study was minimized.
Duplicates of serum samples (5 L) were mixed with 6 mL of Pico-FlourTM 40 (Packard) in glass scintillation vials and left overnight, before they were measured for 5 min by liquid scintillation counting (LSC) together with representative blank samples, using Packard Tri-Carb 2900 TR with automatic quench correction by an external method.
2.14.
2.13.
Pharmacokinetic analysis
Pharmacokinetic parameters were determined using WinNonLin Professional version 3.3 (Pharsight Corp., Mountain View, CA, USA). Maximum serum concentration (Cmax ) and time (Tmax ), at which Cmax occurred, were found by visual inspection of the 3 H-seocalcitol serum concentration–time data from individual animals. z was calculated by logarithmic-linear regression of the serum concentration–time curve, and serum half-life (T1/2 ) was calculated as ln 2/z . The area under the curve (AUC) was calculated by non-compartmental analysis to the final measurable sample (AUC0→t ) using the linear-log trapezoidal method. The linear trapezoidal rule was used up to Cmax , and the logarithmic trapezoidal rule was used after Cmax . The logarithmic trapezoidal rule is used, as the serum concentration after Cmax decreased exponentially. In the concentration range 0.6–50 g/kg, seocalcitol display linear kinetics (Internal report, LEO Pharma A/S). By using radioactive seocalcitol it was possible to measure both parent compound and any degradation and/or metabolites entering the systemic circulation. Any radioactive substance determined in the blood could only originate from the dosed 3 H-seocalcitol. Therefore, an estimation of the oral absorption of 3 H-seocalcitol based on total radioactivity measurements in serum (3 H-seocalcitol, degradation products and metabolites) was calculated as the percent ratio of the AUCtotal radioactivity, p.o., 0→9. divided by AUCtotal radioactivity, i.v., 0→9 with correction of the actual dose of radioactivity. The figure of total absorption is interesting as it makes it possible to determine whether absorption and/or metabolism is responsible for the bioavailability obtained of a dosed drug substance in a given formulation. The oral bioavailability (F) was defined as the percentage of the administered 3 H-seocalcitol dose that was absorbed from the formulation in to the systemic circulation. F was calculated based on the concentration of 3 H-seocalcitol in serum quantified by radio-HPLC. The oral bioavailability of 3 H-seocalcitol was calculated as the percent ratio of AUC3H-seocalcitol, p.o., 0→9 and AUC3H-seocalcitol, i.v., 0→9, with correction of the actual dose of 3 H-seocalcitol. In order to calculate the absorption and the bioavailability the AUC obtained following an intravenous administration of 3 H-seocalcitol determined in a previous study was used (Grove et al., 2005). The use of IV data from another study is not ideal, but justified as similar experimental conditions were applied i.e. same experimental procedure with regard to handling of the animals and operation of the carotid artery; same batch of rats, same batch of radio labeled 3 H-seocalcitol and same staff. Due to ethical rea-
Statistical analysis
The data for Cmax , Tmax T1/2 , AUC and bioavailability (F) were logarithmically transformed in order to normalise variations. Hereby, a one way analysis of variance was used followed by pairwise comparisons adjusted for multiplicity by the Tukey method. In general, a 5% level of significance was used. The results are presented as means (untransformed data) ± their standard deviations (±S.D.).
3.
Results
3.1.
Construction of ternary phase diagrams
Figs. 2 and 3 present the ternary phase diagrams for MCT and LCT, respectively. In both figures the solid line encloses areas, where a microemulsion with a droplet size of less than 100 nm is formed upon addition of 250 mL water to 1 g SMEDDS. The dotted line areas represent areas, where the mixtures of lipid/surfactant/co-surfactant (before addition of water) are mono-phasic after storage for 1 month at room temperature. Mono-phasic mixtures were aimed to mimic production technical considerations. The overlapping small area for the two lipids, where microemulsions are formed with both MCT and LCT, and where the SMEDDS are mono-phasic, is shown in Fig. 4. SMEDDS within the area in Fig. 4 fulfils both the criterion of being mono-phasic after storage for 1 month at room temperature and as well as being a microemulsion with a droplet size less than 100 nm upon dispersion in water. Within this area, SMEDDS with a composition of 25% lipid, 48% surfactant and 27% co-surfactant (Table 1) were chosen for further investigation to compare the suitability of MCT and LCT, respectively.
Fig. 2 – The ternary phase diagram for MCT with cremophor RH40 and akoline MCM. The solid line area represents the area where a microemulsion is formed upon addition of 250 mL water to 1 g of SMEDDS and the dotted line areas represent the areas where the SMEDDS are mono-phasic.
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Fig. 3 – The ternary phase diagram for LCT with cremophor RH40 and peceol. The solid line area represents the area where a microemulsion is formed upon addition of 250 mL water to 1 g of SMEDDS and the dotted line area represents the area where the SMEDDS are mono-phasic.
3.2. Dispersion characteristics of SMEDDS containing dissolved seocalcitol Upon dispersion with water the MC-SMEDDS and the LCSMEDDS containing seocalcitol had the same appearance as those without seocalcitol; the solutions were clear and slightly bluish and exhibited a monomodal droplet size distribution with a droplet size of 29 ± 1 and 30 ± 1 nm, respectively. There was no precipitation of seocalcitol upon dilution with water (1:250), indicating that the microemulsions formed are capable of keeping seocalcitol solubilised.
3.3. Absorption and bioavailability study of 3 H-seocalcitol in rats Tritium labeled seocalcitol was used in the study in order to determine the total absorption of radioactivity e.g. 3 H-
Fig. 4 – The area in the phase diagram represents microemulsions with either MCT or LCT as lipid phase, cremophor RH40 as surfactant and akoline MCM or peceol as co-surfactant obtained after addition of 250 mL water to 1 g of SMEDDS giving a droplet size of less than 100 nm. The SMEDDS in the area are mono-phasic at room temperature.
seocalcitol and 3 H-seocalcitol related compounds in the serum samples. The 3 H-seocalcitol related compounds covers degradation products e.g. generated in the intestine before absorption and metabolites. The absorption data represents the percentage of dosed tritium labeled material absorbed to the systemic circulation, as the entire radioactivity appearing in the blood originates from 3 H-seocalcitol. The absorption data are interesting as it suggest the percentage of radioactivity remaining in e.g. the intestine, as it can be anticipated that the amount of drug substance distributed to organs and tissue will be similar after dosing the oral and the IV formulation due to comparable dosages. However, data providing this kind of information is rarely published. The level of absorption is the same for the two SMEDDS, being 44% for the MC-SMEDDS and 45% for the LC-SMEDDS (Table 2). The part of the radioactivity presumably not being absorbed may have precipitated in the
Table 2 – Pharmacokinetic parameters (mean ± S.D.) following single oral administration of 47 g/kg of 3 H-seocalcitol formulated as MC-SMEDDS and LC-SMEDDS to rats (n = 4) Pharmacokinetic parameter
MC-SMEDDS
Based on 3 H-seocalcitol concentration Tmax (h) Cmax (ng/mL) T1/2 (h) AUC3H-seocalcitol, 0→9 (ng/mL h−1 ) Bioavailability, F (%)
1.9 3 3.4 14 18
Based on total radioactivity measurement AUCtotal radioactivity, 0→9 (ng-eqvi/mL h−1 ) Absorption (%)
54 ± 4 44 ± 3
± ± ± ± ±
0.5 1 0.4 3 4
LC-SMEDDS 2.1 3 3.1 13 18
± ± ± ± ±
0.8 1 0.1 3 4
55 ± 3 45 ± 3
MCT 1.5 5 2.6 18 24
± ± ± ± ±
0.4 1 0.5 3 4
57 ± 9 47 ± 7
LCT 1.6 4 2.7 17 22
± ± ± ± ±
0.3 1 0.5 5 6
55 ± 3 45 ± 2
In order to calculate the absorption and oral bioavailability, the AUC-values obtained after an intravenous administration of 3 H-seocalcitol in a previous study with 3 H-seocalcitol were used (Grove et al., 2005). For comparison, the pharmacokinetic parameters for MCT and LCT solutions are shown as well (dose 47 g/kg of 3 H-seocalcitol) (Grove et al., 2005). There was no statistical significantly difference between the formulations.
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Table 3 – Percentage degradation of seocalcitol (5 g per capsule) after storage for 3 months at 5 ◦ C, 25 ◦ C 60% RH and 40 ◦ C 75% RH dissolved in four lipid formulations filled into hard gelatin capsules and packed in tropicalised blister foil Storage condition 5 ◦C 25 ◦ C, 60% RH 40 ◦ C, 75% RH
Fig. 5 – Serum concentration–time profiles (mean values, n = 4) following oral administration of 47 g/kg (equal to 40 MBq/mL) of 3 H-seocalcitol to male rats. Two formulations were tested: MC-SMEDDS (䊉) and LC-SMEDDS (×). The serum concentration–time profiles found in a previous study, where the same amount of seocalcitol was dosed in a MCT () and a LCT solution (), are also shown (Grove et al., 2005).
intestine, which indicates that solubility related problems is associated with absorption. The pharmacokinetic parameters obtained after oral administration of 3 H-seocalcitol from MCSMEDDS and LC-SMEDDS are listed in Table 2. For comparison Table 2 also contains pharmacokinetic parameters obtained after oral administration of 47 g 3 H-seocalcitol/kg dosed in MCT and LCT solutions, respectively (Grove et al., 2005). The serum concentration of 3 H-seocalcitol versus time profiles are shown in Fig. 5 for all four formulations. The bioavailability of 3 H-seocalcitol is found to be 18% for both the MC-SMEDDS and LC-SMEDDS formulations. As expected, the absorption value is higher than the bioavailability (F), because the bioavailability calculation is based solely on the concentration of 3 Hseocalcitol in serum. The discrepancy between the absorption and bioavailability values may be a figure of a degradation of drug substance taking place in the intestine before absorption and/or a metabolism taking place before systemic circulation is reached. The absorption, bioavailability, Tmax , T1/2 and Cmax were not statistically different for any of the four formulations. However, there was a trend towards an enhanced bioavailability from MCT and LCT solutions compared with the SMEDDS.
3.4.
Stability of seocalcitol in formulations
No change in any of the physical parameters (disintegration, appearance and smell, content of water in the capsule shell) tested was observed for the four formulations: MC-SMEDDS, LC-SMEDDS, MCT and LCT solution. The chemical stability of seocalcitol after storage for 3 months is shown in Table 3. The stability of seocalcitol seems to be much better in the simple lipid solutions compared with the SMEDDS. A pronounced decrease in concentration of seocalcitol of 10–11% is seen in both of the SMEDDS compared with less than 3% degradation in the pure lipid solutions after storage at 40 ◦ C and 75% RH for 3 months.
4.
Formulations MCT
LCT
0.2 0.1 1.6
0.2 0.4 2.6
MC-SMEDDS 0.8 5.8 10.4
LC-SMEDDS 5.9 8.4 11.3
Discussion
In order to compare the capability of the formulations to keep the drug substance in solution after entering the GIT before absorption, it is of interest to study the bioavailability from different lipid-based formulations containing the drug substance in the dissolved state. The present study with SMEDDS distinguishes from previous studies by having the same concentration of cremophor RH40 in both MC-SMEDDS and LC-SMEDDS as well as the same concentration of lipid phase. Furthermore, the bioavailability enhancing potential of MC-SMEDDS and LCSMEDDS can be directly compared with data obtained in a previous study (Grove et al., 2005) where the bioavailability of seocalcitol from pure oil solutions of MCT and LCT was determined as the same amount of lipid was dosed.
4.1.
Construction of ternary phase diagrams
The ternary phase diagrams (Figs. 2 and 3) show that a larger microemulsion area was achieved when using MCT instead of LCT. This is due to the difference in polarity between the lipids, where the more hydrophobic LCT is more difficult to emulsify. This is in accordance with findings of Deckelbaum et al. (1990) showing that MCT is more soluble and have a higher mobility in the lipid/water interfaces than LCT associated with a more rapid hydrolysis of MCT (Deckelbaum et al., 1990). In general, when using LCT, a higher concentration of cremophor RH40 was required to form microemulsions compared with MCT. The overlapping area in Fig. 4, where microemulsions with both MCT and LCT are formed, is relatively small, illustrating the difficulties in developing both MC-SMEDDS and LC-SMEDDS with the same surfactant and the same ratio between lipid, surfactant and co-surfactant. The relatively high concentration of surfactant required in this study to form microemulsions with both MCT and LCT is in accordance with other studies, where the use of high concentrations of surfactants was found to be necessary to achieve fast and efficient self-emulsification (Shah et al., 1994; Khoo et al., 1998; Kommuru et al., 2001; Kang et al., 2004). A good relation between the visual observations and the achieved droplet sizes was observed. In the microemulsion area clear to bluish transparent microemulsions with droplet sizes less than 100 nm were formed compared with white emulsion-like dispersions with droplet sizes above 100 nm outside this area. The droplet sizes achieved in the microemulsion areas were all within the range between 20 and 40 nm, well below 100 nm, indicating that if microemulsions are
240
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 233–242
formed the resulting droplet size is very small due to the thermodynamical stability of these systems.
4.2. Absorption and bioavailability study of 3 H-seocalcitol in rats 4.2.1.
MC-SMEDDS versus LC-SMEDDS
Upon oral administration of the MC-SMEDDS and LC-SMEDDS to male rats, the absorption and bioavailability of 3 Hseocalcitol were approx. 45% and 18%, respectively, for both SMEDDS (Table 2). The two SMEDDS were developed as to have the same characteristics in terms of composition, appearance and droplet size, however, the used MCT and LCT lipids do have different physico-chemical characteristics and also a different in vivo fate and therefore a difference in absorption and bioavailability of seocalcitol could be anticipated. The results, however, indicate that despite the difference in nature of the lipids, the processes of dispersion, distribution, trafficking of 3 Hseocalcitol in the GIT lumen, results in the same bioavailability of 3 H-seocalcitol for both SMEDDS. An equal solubility has been found for seocalcitol in simulated intestinal media containing either medium or long chain lipolytic products (Grove et al., 2005), which supports the hypothesis that an equal solubilising capacity is seen in vivo. For danazol and halofantrine the bioavailability from LCSMEDDS was found to be superior compared with MCSMEDDS, which made the authors suggest that MCT has a reduced solubilising capacity for some lipophilic drug substances compared with LCT based formulations (Khoo et al., 1998; Porter et al., 2004). However, in these studies the SMEDDS contain different amounts of lipid and surfactant, which makes the comparison more complicated since more than one variable exists. Taking the present study into account it seems to be drug dependent whether MCT or LCT based formulations will give rise to the highest bioavailability.
4.2.2.
SMEDDS versus lipid solutions
It is important to consider the amount of lipid dosed when comparing lipid-based formulations, as the lipid itself may be responsible for enhancement in bioavailability. Vitamin E showed a two-fold increase in bioavailability from a SMEDDS compared to a lipid solution (Julianto et al., 2000) however, as more lipid was dosed in the SMEDDS than the lipid solution, it is difficult to draw a firm conclusion. In the present studies the rats were dosed equal amounts of lipid from either SMEDDS or lipid solutions (0.8 mg/kg), and the results (Table 2) can therefore be directly compared. The MCT or LCT lipid solutions (Grove et al., 2005), showed a slightly higher bioavailability of seocalcitol (22–24% in comparison with 18%) than was found for the SMEDDS, even though there was no significant difference. The SMEDDS were expected to give rise to a higher bioavailability compared to a lipid solution, due to more rapid and uniform distribution of the drug substance in the GIT (Mueller et al., 1994). The large surface area obtained after administration of the SMEDDS increases the hydrolysis of triglycerides and promotes a rapid release of the drug substance (Humberstone and Charman, 1997) and/or the drug substance may be absorbed directly from the small droplets of the microemulsion (de Smidt et al., 2004). The pure lipid
solutions are dependent on the emulsification by endogenous surfactants (e.g. bile salt and phospholipids) before the onset of triglyceride hydrolysis. However, the obtained results are not in agreement with this theory, since no significant difference was found between bioavailability of 3 H-seocalcitol from SMEDDS and lipid solutions. The presence of surfactants in the SMEDDS does not seem to provide an enhancement in GI solubilisation of seocalcitol over that provided by the lipid itself, even though a rather high concentration of surfactant is present in the SMEDDS. This is in agreement with studies conducted by Porter et al. showing the same bioavailability of danazol when dosed in either LCT solution or LC-SMEDDS in dogs (Porter et al., 2004). When studying SMEDDS the animal model may be important to consider due to the dependency of the dynamic processes responsible for the emulsification process. Rats were chosen in the present study due to the low cost and relatively facile accessibility and rats have previously been used as animal model in bioavailability assessment of SEDDS (Kim and Ku, 2000; Bravo Gonzalez et al., 2002; Kim et al., 2002; Yang et al., 2004), hence none of these studies have compared a lipid solution with a SMEDDS. However, the amount of fluid present in the stomach of the rat might be insufficient to emulsify the dose of SMEDDS administered. Titrating the SMEDDS with water forms a very viscous structure, probably consisting of bicontinuous structures, until the ratio between SMEDDS and water exceeds factor 2 (1:2) where an aqueous solution is formed. This phenomenon supports the hypothesis that a certain level of GI fluid has to be present in order for the selfmicroemulsifation process to occur. In terms of the pure lipid solutions the lipid phase is not miscible with the GI fluid and the absence of surfactant in these formulations avoids the formation of high viscous gel-like structures. The influence of the animal model on the bioavailability of formulations being dependent on dynamic processes in the GIT has not yet been studied, therefore it would be of interest to study the bioavailability of seocalcitol from SMEDDS in a larger animal e.g. the mini-pig or dog.
4.3.
Stability of seocalcitol in formulations
To obtain a product with a shelf life of 2 years at 25 ◦ C a degradation of drug substance concentration of approximately 7% within 3 months is acceptable at the condition 40 ◦ C 75% RH representing accelerated stability condition (Pope, 1980). For the SMEDDS this criterion is not met, as a decrease of 10–11% is observed at this condition, whereas for the simple lipid solutions the degradation of seocalcitol was found to be less than 3% (Table 3). Seocalcitol is sensitive to oxidation why butylhydroxyanisole was added to the formulations in a concentration of 0.02% w/w. However, the concentration of antioxidant in the formulations was the same after 3 months’ storage indicating that the decrease in seocalcitol concentration may be ascribed to another process than oxidation or a combination of different reactions. Unfortunately, seocalcitol is not degraded to one or a few compounds but rather a large number of different compounds all present at very low concentration. Seocalcitol is acid labile and may be sensitive for excipients containing acid impurities. Since the stability of seocalcitol is acceptable in the pure lipid solutions and not in the SMEDDS, the
european journal of pharmaceutical sciences
increased degradation seen in the SMEDDS must be ascribed to the surfactant (cremophor RH40) and/or the co-surfactant (akoline MCM and peceol). The surfactant and co-surfactants in the SMEDDS are mixtures of products that may contain small amounts of impurities from the manufacturing process possibly leading to the observed degradation of seocalcitol. To further identify the cause of instability of seocalcitol, stability studies should be performed in each excipient separately. There is not much literature investigating the chemical stability of drug substances in SMEDDS even though this is a very important aspect from an industrial point of view. An example of a poor chemical stability was seen for paclitaxel, showing a degradation of 20% after storage at 37 ◦ C for 24 h when formulated in SEDDS (Gursoy et al., 2003). The poorer stability of drug substances in SMEDDS may partly explain the limited number of marketed products based on this formulation concept.
4.4.
Industrial relevance of developed SMEDDS
In order to evaluate the industrial relevance of formulating seocalcitol in the SMEDDS developed in the present study, the chemical stability as well as the bioavailability enhancing potential of seocalcitol needs to be taken into consideration. When comparing the SMEDDS to the lipid solutions, this study shows that the lipid solutions seem to be a better choice. The degradation of seocalcitol in the SMEDDS is higher compared with the lipid solutions (Table 3) and the large decrease in concentration of seocalcitol in the SMEDDS makes the formulations unsuitable for commercial use as a sufficient shelf life will not be obtainable. The bioavailability of 3 H-seocalcitol is slightly higher from the lipid solutions compared with the SMEDDS and from a production point of view the lipid solutions are also preferable, because less excipients are required. All together the simple lipid solutions seem to be superior for the formulation of seocalcitol compared with the corresponding more advanced SMEDDS developed in the present study.
Acknowledgements The authors wish to thank ATV (The Danish Academy of Technical Sciences) for financial support. The radiolabeled compound was kindly synthesized by Dr. Gunnar Grue-Sørensen, LEO Pharma A/S. Rie Andreasen, LEO Pharma A/S, is thanked for skilful help with animal surgery and dosing. Analysis & Stability, LEO Pharma A/S, is thanked for help with the stability study. Jonas Wiedemann, LEO Pharma A/S, is thanked for help with the statistical analysis.
references
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available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Bioavailability of seocalcitol II: Development and characterisation of self-microemulsifying drug delivery systems (SMEDDS) for oral administration containing medium and long chain triglycerides b,∗ ¨ Mette Grove a,b , Anette Mullertz , Jeanet Løgsted Nielsen c , a Gitte Pommergaard Pedersen a
Pharmaceutical Formulation, LEO Pharma A/S, Industriparken 55, DK-2750 Ballerup, Denmark Department of Pharmaceutics, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark c Department of Pharmacokinetics, LEO Pharma A/S, Industriparken 55, DK-2750 Ballerup, Denmark b
a r t i c l e
i n f o
a b s t r a c t
Article history:
By constructing ternary phase diagrams it was possible to identify two self-microemulsifying
Received 28 September 2005
drug delivery systems (SMEDDS) containing either medium chain triglycerides (MC-
Received in revised form 22
SMEDDS) or long chain triglycerides (LC-SMEDDS), with the same ratio between lipid, sur-
November 2005
factant and co-surfactant. The SMEDDS ended up having a composition of 25% lipid, 48%
Accepted 20 February 2006
surfactant and 27% co-surfactant, MC-SMEDDS: viscoleo, cremophor RH40, akoline MCM and
Published on line 2 May 2006
LC-SMEDDS: sesame oil, cremophor RH40, peceol. Upon dilution with water both SMEDDS resulted in clear to bluish transparent microemulsions with a narrow droplet size of 30 nm.
Keywords:
The industrial usefulness of the developed SMEDDS was evaluated with regard to bioavail-
SMEDDS
ability and chemical stability using the vitamin D analogue, seocalcitol, as model compound.
Oral bioavailability
The absorption and bioavailability of seocalcitol in rats were approximately 45% and 18%,
Lipid-based formulations
respectively, from both the MC-SMEDDS and LC-SMEDDS indicating similar in vivo behavior
Oral absorption
of the two formulations, despite the difference in nature of lipid component. There was
Poorly soluble drug substances
no improvement in bioavailability by the use of SMEDDS, compared to the bioavailability
Stability
achieved from simple MCT and LCT solutions (22–24%) (Grove, M., Pedersen, G.P., Nielsen,
Seocalcitol
J.L., Mullertz, A., 2005. Bioavailability of seocalcitol. I. Relating solubility in biorelevant media with oral bioavailability in rats-effect of medium and long chain triglycerides. J. Pharm. Sci. 94, 1830–1838.). After 3 months’ storage at accelerated conditions (40 ◦ C/75% RH), a decrease in concentration of seocalcitol of 10–11% was found in MC-SMEDDS and LC-SMEDDS compared with a degradation of less than 3% for the simple lipid solutions of MCT and LCT. In this study the simple lipid solutions seem to be a better choice compared with the developed SMEDDS due to a slightly higher bioavailability and a better chemical stability of seocalcitol. © 2006 Elsevier B.V. All rights reserved.
∗
Corresponding author. Tel.: +45 35306440; fax: +45 35306030. ¨ E-mail address: [email protected] (A. Mullertz). 0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.02.005
234
1.
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 233–242
Introduction
An increasing number of potential drug substances discovered by the pharmaceutical industry are poorly soluble in water, but have a high permeability and are therefore classified as class 2 drug substances according to the Biopharmaceutical Classification System (BCS) (Amidon et al., 1995). For the class 2 drug substances the bioavailability is often low and variable due to an insufficient dissolution process in the gastrointestinal tract, hence it will be beneficial to dose this kind of drug substances in their soluble form. For drug substances with sufficient lipophilicity, lipid-based drug delivery systems e.g. lipid solution, lipid emulsion, microemulsion, dry emulsion, Self Emulsifying Drug Delivery System (SEDDS) or Self Microemulsifying Drug Delivery System (SMEDDS) could be possible formulation approaches. Lipid-based drug delivery systems have gained considerable interest after the commercial success of Sandimmune NeoralTM (Cyclosporine A), Fortovase (Saquinavir) and Norvir (Ritonavir). Much attention has been on SMEDDS/SEDDS; an increase in bioavailability was found for L-365,260 (Lin et al., 1991), WIN 54954 (Charman et al., 1992), Ro 15-0778 (Shah et al., 1994), ontazolast (Hauss et al., 1998), halofantrine (Khoo et al., 1998) and danazol (Porter et al., 2004) when administered in self-emulsifying systems, compared to solid dosage forms. SMEDDS are defined as isotropic mixtures of lipid, surfactant, co-surfactant and drug substance that rapidly form a microemulsion upon mixing with water. A narrow droplet size distribution is often seen with a droplet size typically less than 50 nm (Gursoy and Benita, 2004). The self-emulsification process occurs spontaneously because the free energy required to form the microemulsion is either low and positive or negative (Constantinides, 1995). The self-emulsification process was shown to be specific to the nature of the lipid/surfactant pair, the surfactant concentration, the ratio between lipid and surfactant (Pouton, 1985; Wakerly et al., 1987) and only specific pharmaceutical excipient combinations can lead to efficient self-emulsifying systems (Charman et al., 1992; Shah et al., 1994). Drug substances with adequate solubility in lipid/surfactants blends are candidates for this formulation concept (Gershanik and Benita, 2000). The SMEDDS are believed to be superior compared with lipid solutions due to the presence of surfactants in the formulations leading to a more uniform and reproducible bioavail-
ability as seen for cyclosporine (Mueller et al., 1994). The surfactants act by dispersing the lipid formulation in the gastrointestinal tract (GIT) upon dilution with the gastrointestinal fluid (Stuchlik and Zak, 2001). This results in the formation of fine droplets providing a large surface area for pancreatic lipase to hydrolyse triglycerides and thereby promoting a rapid release of the drug substance and/or the formation of mixed micelles containing the drug substance (Tarr and Yalkowsky, 1989). The small droplets formed in contact with the gastrointestinal fluid may also be responsible for transporting the drug substance through the unstirred water layer to the gastrointestinal membrane for absorption (de Smidt et al., 2004). In the literature, medium chain triglycerides (MCT) have been preferred in SMEDDS due to the higher fluidity, better solubility properties and self-emulsification ability compared with long chain triglycerides (LCT) (Charman et al., 1992; Shah et al., 1994), as well as a better chemical stability of drug substance in MCT due to the purity of the lipid and the lack of double bonds, that can catalyse oxidation. The two lipids are differently transported in the body: MCT is directly transported by the portal blood to the systemic circulation (Porter and Charman, 1997), whereas LCT is transported in the intestinal lymphatics. Lipid-based drug delivery systems containing LCT are likely to enhance the lymphatic transport of a lipophilic drug substance (Palin and Winson, 1984; Caliph et al., 2000) and as the lymphatic transport circumvents the liver, the first-pass metabolism of a drug substance may be reduced (Porter and Charman, 1997). SMEDDS containing either MCT or LCT have been studied with halofantrine (Khoo et al., 1998) and danazol (Porter et al., 2004) and in both cases LC-SMEDDS were found to give the highest bioavailability. Studies comparing the bioavailability of drug substance from SMEDDS and other lipid-based drug delivery systems e.g. pure lipid solutions are still limited in number and until now most studies have been comparing self-emulsifying systems with solid dosage forms (Lin et al., 1991; Charman et al., 1992; Shah et al., 1994). MC-SMEDDS and LC-SMEDDS have been compared in two studies with halofantrine (Khoo et al., 1998) and danazol (Porter et al., 2004). However, in these studies the SMEDDS contain different amounts of lipid and surfactant, which makes the comparison complicated, since more than one variable exists. In a study with vitamin E a SMEDDS was found to give rise to a higher bioavailability compared with a lipid solution, but more lipid was dosed in the SMEDDS, which makes the comparison difficult (Julianto et al., 2000).
Fig. 1 – Chemical structure of seocalcitol and tritium labeled seocalcitol, 3 H-seocalcitol.
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 233–242
The aim of the current study was to develop, characterize and compare the suitability of two SMEDDS containing either MCT or LCT. In order to make a direct comparison between the two SMEDDS possible, criteria of success were that they should have the same ratio between lipid/surfactant/co-surfactant, be monophasic at room temperature and have the same droplet size distribution after dispersion in water. Cremophor RH40 was used as surfactant in both cases, whereas the cosurfactant was chosen to resemble the lipid component with regard to chain length. The bioavailability of a poorly soluble model drug substance from the two developed SMEDDS, was determined in rats, and compared to the bioavailability from simple MCT and LCT solutions (Grove et al., 2005). In order to further evaluate the industrial usefulness of SMEDDS, the chemical stability of seocalcitol in the formulations, filled into hard gelatin capsules, was determined. The model drug substance used in the study was seocalcitol, a class 2 drug substance, with the chemical structure shown in Fig. 1. Tritium labeled seocalcitol was used in the bioavailability study thereby also enabling an estimation of oral absorption based on the presence of radioactive material in blood. Log P for seocalcitol is 4.8 and the solubility in MCT and LCT is 5.3 and 1.7 mg/g, respectively.
2.
Materials and methods
2.1.
Chemicals
Seocalcitol and tritium labeled seocalcitol (3 H-seocalcitol, specific activity of 0.844 MBq/g and radiochemical purity of 97%) were synthesized at LEO Pharma A/S. Viscoleo (MCT) was purchased from ICI Espana, Spain and Sesame oil (LCT) was purchased from Henry Lamotte GmbH, Germany. The fatty acid composition of the triglycerides in MCT and LCT is as specified in Ph.Eur. Cremophor RH40 (macrogol 40 glycerol hydroxystearate) was purchased from BASF, Germany. Akoline MCM (a mixture of mono, di- and triglycerides of octanoic and decanoic acids) was a gift from Karlshamns, Sweden ´ and Peceol (glycerol monooleate) was a gift from Gattefosse, France. Hard gelatin capsules, posilock size 4, were a gift from Shionogi Qualicaps Sa, Japan, butylhydroxyanisole (BHA) was purchased from Rhone Poulenc Sante (France), Hypnorm® (fentanyl 0.2 mg/mL, fluanisone 10 mg/mL) was purchased from Janssen, Belgium, and Dormicum (Midazolam 5 mg/mL) from Roche, Switzerland. Heparin 10.000 IU/mL was produced at LEO Pharma A/S. Isotonic sterile NaCl was purchased at Dilab, Sweden. The water was obtained from a Milli-Q-water purification system (Millipore, MA, USA). Pico-AquaTM and Pico Flour 40TM were purchased from Packard. All other chemicals were of analytical grade.
2.2.
Development of SMEDDS
A series of mixtures were prepared with varying ratio of lipid, surfactant and co-surfactant. The components were weighted into glass vials and mixed at 50 ◦ C using a stirring rate of 500 rpm, until the components were perfectly dissolved. The mixtures were cooled to 37 ◦ C and 1 g was transferred to a beaker where 250 mL of 37 ◦ C water was added under gen-
235
tle stirring of 50 rpm. The dispersions were visually inspected and characterised for droplet size. A mixture was defined to be a suitable SMEDDS, if (1) rapid self-emulsification (within 1 min) was obtained after dispersion in 37 ◦ C water, followed by the formation of a clear transparent microemulsion giving a droplet size of less than 100 nm measured by photon correlation spectroscopy (PCS) and (2) mono-phasic mixture was obtained after storage at room temperature for 1 month.
2.3.
Droplet size analysis
The droplet size of the microemulsions was measured by means of PCS, using a Zetasizer 3000 (Malvern Instruments, UK). The samples were measured at 37 ◦ C after addition of 250 mL 37 ◦ C water to 1 g of mixture. No further treatment of the samples was necessary.
2.4.
Construction of ternary phase diagrams
Ternary phase diagrams were constructed for both MCSMEDDS and LC-SMEDDS. SMEDDS giving a droplet size of less than 100 nm after addition of 250 mL 37 ◦ C water to 1 g of SMEDDS were marked in a solid line area. In addition, monophasic SMEDDS were identified by visual inspection after 1 month’s storage at ambient temperature and marked in a dotted line area. To be able to identify identical compositions of MCSMEDDS and LC-SMEDDS, the two individual ternary phase diagrams were superimposed. In this way, an area was identified containing mono-phasic SMEDDS, with either MCT or LCT, resulting in the formation of clear transparent microemulsions with a droplet size of less than 100 nm. Within this area SMEDDS with the same ratio between lipid/surfactant/cosurfactant were identified.
2.5.
Selection of SMEDDS for further investigation
Based on the superimposed ternary phase diagrams one MCSMEDDS and one LC-SMEDDS were chosen for further investigation. Each had a composition of 25% lipid, 48% surfactant and 27% co-surfactant. The following investigations were performed with these two SMEDDS: (a) dispersion characteristics of SMEDDS containing dissolved seocalcitol; (b) chemical and physical stability testing of SMEDDS filled in hard gelatin capsules and (c) oral absorption and bioavailability of seocalcitol upon administration of the formulations in rats.
2.6. Dispersion characteristics of SMEDDS containing dissolved seocalcitol MC-SMEDDS and LC-SMEDDS containing dissolved seocalcitol were prepared. The amount of seocalcitol dissolved in the SMEDDS was 80% of the solubility in the lipid component corresponding to 1.1 mg/g in the MC-SMEDDS and 0.3 mg/g in the LC-SMEDDS. About 1 g of SMEDDS was transferred to a beaker, where 250 mL of 37 ◦ C water was added under gentle stirring of 50 rpm. The dispersions were visually inspected for the formation of a clear transparent microemulsion and for precipitation of seocalcitol. Furthermore, the samples were characterised by PCS.
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2.7.
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Stability of seocalcitol in formulations
The MC-SMEDDS and the LC-SMEDDS as well as MCT and LCT solutions containing seocalcitol were filled into hard gelatine capsules using a capsule filling apparatus (Hibar) and a band sealing machine (Quali-seal Capsule Sealing Machine, Laboratory Model). The capsules were packed in tropicalised blister foil 191 (Alcan Packaging Singen GmbH) and stored for 3 months at 5 ◦ C, 25 ◦ C 60% relative humidity (RH) and 40 ◦ C 75% RH. For comparison MCT and LCT solutions were filled into hard gelatin capsules and packed and stored under the same conditions as the MC-SMEDDS and the LC-SMEDDS. Each capsule had a concentration of 5 g seocalcitol. Butylhydroxyanisole in a concentration of 0.02% w/w was added to the formulations in order to stabilize the oxidation sensitive drug substance. The chemical stability and the following physical parameters were tested: disintegration, appearance and smell and content of water in the capsule shell.
2.8.
HPLC analysis of seocalcitol in formulations
The concentration of seocalcitol in the formulations was determined by a validated reversed-phase HPLC method consisting of a Hitachi Module L-7100 with a Spark Holland autosampler, column oven (25 ◦ C), a Hitachi Detector L-7400 set at 264 nm and data were processed by using Millennium Chromatography Manager version 32 (Waters Associates, Milford, MA, USA). About 100 L formulation was mixed with 6 mL of a mixture of acetonitrile and 0.01 M diammonium hydrogenphosphate (60:40) (v/v). About 20 mL n-heptane was added and the solution was mixed for 15 min on a mechanical shaker. After phase separation, the lower phase was collected and placed at 50 ◦ C for 15–18 h. The samples were injected into the chromatographic system after cooled to room temperature. The analytical column was a Superspher RP-18, 75 mm × 4 mm i.d. 4 m (Merck) and the injection volume was 100 L. The mobile phase was acetonitrile and phosphate buffer pH 6.5 (60:40) (v/v) and the flow rate was 1.2 mL/min. The retention time of seocalcitol was approximately 6 min and the quantification was performed using peak heights for seocalcitol in the chromatograms from test and reference solutions, the lower limit of quantification being 0.13 g/g.
2.9. Preparation of oral 3 H-seocalcitol formulations to bioavailability study Two lipid-based formulations, one MC-SMEDDS (25% viscoleo, 48% cremophor RH40, 27% akoline MCM) and one LC-SMEDDS (25% sesame oil, 48% cremophor RH40, 27% peceol), were prepared by dissolving 3 H-seocalcitol in the formulation, resulting in a concentration of 3 H-seocalcitol of 31 g/mL with a radioactivity of 26 MBq/mL and a radio chemical purity of 97%. The SMEDDS were produced on the day prior to dosing and stored at 5 ◦ C until dosing.
2.10.
Bioavailability study of 3 H-seocalcitol in rats
All surgical and experimental procedures were reviewed and approved by the local Animal Experimentation Ethics Committee. Male Sprague-Dawley rats weighing 280–320 g
Table 1 – Composition of the SMEDDS (%w/w) selected for bioavailability assessment in rats Excipient Viscoleo Akoline MCM Cremophor RH40 Sesame oil Peceol
MC-SMEDDS
LC-SMEDDS
25 27 48
48 25 27
The concentration of 3 H-seocalcitol in the formulations was 31 g/mL equal to 26 MBq/mL.
(Møllegaard Breeding center, Lille Skensved, Denmark) maintained on a standard feeding and water ad libitum were included in the study. During the acclimatization, two rats were housed in each cage maintained at 22 ± 2 ◦ C with a 50% relative humidity, an air change of 15 changes per hour and a 12-h light–dark cycle. During the experiment, the animals were anesthetized for the duration of the surgery by subcutaneous injection of 2.7 mL/kg of a solution consisting of Hypnorm® , Dormicum and water (1:1:2). The day prior to dosing the right carotid artery was cannulated with a tygon catheter (0.40 mm i.d., 0.79 mm OD, Dilab, Sweden). The catheter was exteriorized at the back of the neck and connected to a swivel apparatus designed to allow free animal movement and computerised blood sampling (Dilab, Sweden). The apparatus enabled automatic, consecutive blood sampling from the same animal. After the surgery, the animals received isotonic NaCl containing 25 U/mL heparin through the catheter to stabilize the liquid balance of the animal. Two oral formulations were administered: MC-SMEDDS and LCSMEDDS. Table 1 shows the compositions of the SMEDDS. Each rat received 1.5 mL formulation/kg corresponding to 47 g 3 Hseocalcitol/kg (equal to 40 MBq/kg) and 0.8 mL/kg lipid. After dosing the rats received water resulting in a total volume of administration of 1 mL. Blood samples of 250 L were collected at 0 (predose), 0.5, 1, 1.5, 1, 2.5, 3, 5, 7 and 9 h following oral administration. Serum was prepared and kept at −80 ◦ C until analysis. A total of four rats received each formulation. The dose of lipid (the amount of co-surfactant is calculated within the total amount of lipid in the SMEDDS) as well as dose of seocalcitol administered is the same as in a previous study (Grove et al., 2005) studying the bioavailability of seocalcitol from solutions of MCT and LCT.
2.11.
HPLC analysis of 3 H-seocalcitol in serum
The concentration of 3 H-seocalcitol in serum was determined using the radio-HPLC method described earlier (Grove et al., 2005). In brief, a reversed-phase HPLC-system with cooling on the autosampler (8 ◦ C), online vacuum degassing, column oven (40 ◦ C) (all Waters Associates, Milford, MA, USA), was added a Packard Radiomatic Flow scintillation Analyzer using Pico-AquaTM (Packard) as scintillator. The analytical column was a Symmetri C8, 50 mm × 2.1 mm i.d. 3.5 m (Waters) and the absorbance was measured at 264 nm. The quantification of 3 H-seocalcitol was performed using a calibration curve of spiked blank serum, where the lower limit of quantification was 0.5 ng/mL.
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2.12. Preparation of samples for total liquid scintillation counting
sons the number of animals used in the present study was minimized.
Duplicates of serum samples (5 L) were mixed with 6 mL of Pico-FlourTM 40 (Packard) in glass scintillation vials and left overnight, before they were measured for 5 min by liquid scintillation counting (LSC) together with representative blank samples, using Packard Tri-Carb 2900 TR with automatic quench correction by an external method.
2.14.
2.13.
Pharmacokinetic analysis
Pharmacokinetic parameters were determined using WinNonLin Professional version 3.3 (Pharsight Corp., Mountain View, CA, USA). Maximum serum concentration (Cmax ) and time (Tmax ), at which Cmax occurred, were found by visual inspection of the 3 H-seocalcitol serum concentration–time data from individual animals. z was calculated by logarithmic-linear regression of the serum concentration–time curve, and serum half-life (T1/2 ) was calculated as ln 2/z . The area under the curve (AUC) was calculated by non-compartmental analysis to the final measurable sample (AUC0→t ) using the linear-log trapezoidal method. The linear trapezoidal rule was used up to Cmax , and the logarithmic trapezoidal rule was used after Cmax . The logarithmic trapezoidal rule is used, as the serum concentration after Cmax decreased exponentially. In the concentration range 0.6–50 g/kg, seocalcitol display linear kinetics (Internal report, LEO Pharma A/S). By using radioactive seocalcitol it was possible to measure both parent compound and any degradation and/or metabolites entering the systemic circulation. Any radioactive substance determined in the blood could only originate from the dosed 3 H-seocalcitol. Therefore, an estimation of the oral absorption of 3 H-seocalcitol based on total radioactivity measurements in serum (3 H-seocalcitol, degradation products and metabolites) was calculated as the percent ratio of the AUCtotal radioactivity, p.o., 0→9. divided by AUCtotal radioactivity, i.v., 0→9 with correction of the actual dose of radioactivity. The figure of total absorption is interesting as it makes it possible to determine whether absorption and/or metabolism is responsible for the bioavailability obtained of a dosed drug substance in a given formulation. The oral bioavailability (F) was defined as the percentage of the administered 3 H-seocalcitol dose that was absorbed from the formulation in to the systemic circulation. F was calculated based on the concentration of 3 H-seocalcitol in serum quantified by radio-HPLC. The oral bioavailability of 3 H-seocalcitol was calculated as the percent ratio of AUC3H-seocalcitol, p.o., 0→9 and AUC3H-seocalcitol, i.v., 0→9, with correction of the actual dose of 3 H-seocalcitol. In order to calculate the absorption and the bioavailability the AUC obtained following an intravenous administration of 3 H-seocalcitol determined in a previous study was used (Grove et al., 2005). The use of IV data from another study is not ideal, but justified as similar experimental conditions were applied i.e. same experimental procedure with regard to handling of the animals and operation of the carotid artery; same batch of rats, same batch of radio labeled 3 H-seocalcitol and same staff. Due to ethical rea-
Statistical analysis
The data for Cmax , Tmax T1/2 , AUC and bioavailability (F) were logarithmically transformed in order to normalise variations. Hereby, a one way analysis of variance was used followed by pairwise comparisons adjusted for multiplicity by the Tukey method. In general, a 5% level of significance was used. The results are presented as means (untransformed data) ± their standard deviations (±S.D.).
3.
Results
3.1.
Construction of ternary phase diagrams
Figs. 2 and 3 present the ternary phase diagrams for MCT and LCT, respectively. In both figures the solid line encloses areas, where a microemulsion with a droplet size of less than 100 nm is formed upon addition of 250 mL water to 1 g SMEDDS. The dotted line areas represent areas, where the mixtures of lipid/surfactant/co-surfactant (before addition of water) are mono-phasic after storage for 1 month at room temperature. Mono-phasic mixtures were aimed to mimic production technical considerations. The overlapping small area for the two lipids, where microemulsions are formed with both MCT and LCT, and where the SMEDDS are mono-phasic, is shown in Fig. 4. SMEDDS within the area in Fig. 4 fulfils both the criterion of being mono-phasic after storage for 1 month at room temperature and as well as being a microemulsion with a droplet size less than 100 nm upon dispersion in water. Within this area, SMEDDS with a composition of 25% lipid, 48% surfactant and 27% co-surfactant (Table 1) were chosen for further investigation to compare the suitability of MCT and LCT, respectively.
Fig. 2 – The ternary phase diagram for MCT with cremophor RH40 and akoline MCM. The solid line area represents the area where a microemulsion is formed upon addition of 250 mL water to 1 g of SMEDDS and the dotted line areas represent the areas where the SMEDDS are mono-phasic.
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Fig. 3 – The ternary phase diagram for LCT with cremophor RH40 and peceol. The solid line area represents the area where a microemulsion is formed upon addition of 250 mL water to 1 g of SMEDDS and the dotted line area represents the area where the SMEDDS are mono-phasic.
3.2. Dispersion characteristics of SMEDDS containing dissolved seocalcitol Upon dispersion with water the MC-SMEDDS and the LCSMEDDS containing seocalcitol had the same appearance as those without seocalcitol; the solutions were clear and slightly bluish and exhibited a monomodal droplet size distribution with a droplet size of 29 ± 1 and 30 ± 1 nm, respectively. There was no precipitation of seocalcitol upon dilution with water (1:250), indicating that the microemulsions formed are capable of keeping seocalcitol solubilised.
3.3. Absorption and bioavailability study of 3 H-seocalcitol in rats Tritium labeled seocalcitol was used in the study in order to determine the total absorption of radioactivity e.g. 3 H-
Fig. 4 – The area in the phase diagram represents microemulsions with either MCT or LCT as lipid phase, cremophor RH40 as surfactant and akoline MCM or peceol as co-surfactant obtained after addition of 250 mL water to 1 g of SMEDDS giving a droplet size of less than 100 nm. The SMEDDS in the area are mono-phasic at room temperature.
seocalcitol and 3 H-seocalcitol related compounds in the serum samples. The 3 H-seocalcitol related compounds covers degradation products e.g. generated in the intestine before absorption and metabolites. The absorption data represents the percentage of dosed tritium labeled material absorbed to the systemic circulation, as the entire radioactivity appearing in the blood originates from 3 H-seocalcitol. The absorption data are interesting as it suggest the percentage of radioactivity remaining in e.g. the intestine, as it can be anticipated that the amount of drug substance distributed to organs and tissue will be similar after dosing the oral and the IV formulation due to comparable dosages. However, data providing this kind of information is rarely published. The level of absorption is the same for the two SMEDDS, being 44% for the MC-SMEDDS and 45% for the LC-SMEDDS (Table 2). The part of the radioactivity presumably not being absorbed may have precipitated in the
Table 2 – Pharmacokinetic parameters (mean ± S.D.) following single oral administration of 47 g/kg of 3 H-seocalcitol formulated as MC-SMEDDS and LC-SMEDDS to rats (n = 4) Pharmacokinetic parameter
MC-SMEDDS
Based on 3 H-seocalcitol concentration Tmax (h) Cmax (ng/mL) T1/2 (h) AUC3H-seocalcitol, 0→9 (ng/mL h−1 ) Bioavailability, F (%)
1.9 3 3.4 14 18
Based on total radioactivity measurement AUCtotal radioactivity, 0→9 (ng-eqvi/mL h−1 ) Absorption (%)
54 ± 4 44 ± 3
± ± ± ± ±
0.5 1 0.4 3 4
LC-SMEDDS 2.1 3 3.1 13 18
± ± ± ± ±
0.8 1 0.1 3 4
55 ± 3 45 ± 3
MCT 1.5 5 2.6 18 24
± ± ± ± ±
0.4 1 0.5 3 4
57 ± 9 47 ± 7
LCT 1.6 4 2.7 17 22
± ± ± ± ±
0.3 1 0.5 5 6
55 ± 3 45 ± 2
In order to calculate the absorption and oral bioavailability, the AUC-values obtained after an intravenous administration of 3 H-seocalcitol in a previous study with 3 H-seocalcitol were used (Grove et al., 2005). For comparison, the pharmacokinetic parameters for MCT and LCT solutions are shown as well (dose 47 g/kg of 3 H-seocalcitol) (Grove et al., 2005). There was no statistical significantly difference between the formulations.
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Table 3 – Percentage degradation of seocalcitol (5 g per capsule) after storage for 3 months at 5 ◦ C, 25 ◦ C 60% RH and 40 ◦ C 75% RH dissolved in four lipid formulations filled into hard gelatin capsules and packed in tropicalised blister foil Storage condition 5 ◦C 25 ◦ C, 60% RH 40 ◦ C, 75% RH
Fig. 5 – Serum concentration–time profiles (mean values, n = 4) following oral administration of 47 g/kg (equal to 40 MBq/mL) of 3 H-seocalcitol to male rats. Two formulations were tested: MC-SMEDDS (䊉) and LC-SMEDDS (×). The serum concentration–time profiles found in a previous study, where the same amount of seocalcitol was dosed in a MCT () and a LCT solution (), are also shown (Grove et al., 2005).
intestine, which indicates that solubility related problems is associated with absorption. The pharmacokinetic parameters obtained after oral administration of 3 H-seocalcitol from MCSMEDDS and LC-SMEDDS are listed in Table 2. For comparison Table 2 also contains pharmacokinetic parameters obtained after oral administration of 47 g 3 H-seocalcitol/kg dosed in MCT and LCT solutions, respectively (Grove et al., 2005). The serum concentration of 3 H-seocalcitol versus time profiles are shown in Fig. 5 for all four formulations. The bioavailability of 3 H-seocalcitol is found to be 18% for both the MC-SMEDDS and LC-SMEDDS formulations. As expected, the absorption value is higher than the bioavailability (F), because the bioavailability calculation is based solely on the concentration of 3 Hseocalcitol in serum. The discrepancy between the absorption and bioavailability values may be a figure of a degradation of drug substance taking place in the intestine before absorption and/or a metabolism taking place before systemic circulation is reached. The absorption, bioavailability, Tmax , T1/2 and Cmax were not statistically different for any of the four formulations. However, there was a trend towards an enhanced bioavailability from MCT and LCT solutions compared with the SMEDDS.
3.4.
Stability of seocalcitol in formulations
No change in any of the physical parameters (disintegration, appearance and smell, content of water in the capsule shell) tested was observed for the four formulations: MC-SMEDDS, LC-SMEDDS, MCT and LCT solution. The chemical stability of seocalcitol after storage for 3 months is shown in Table 3. The stability of seocalcitol seems to be much better in the simple lipid solutions compared with the SMEDDS. A pronounced decrease in concentration of seocalcitol of 10–11% is seen in both of the SMEDDS compared with less than 3% degradation in the pure lipid solutions after storage at 40 ◦ C and 75% RH for 3 months.
4.
Formulations MCT
LCT
0.2 0.1 1.6
0.2 0.4 2.6
MC-SMEDDS 0.8 5.8 10.4
LC-SMEDDS 5.9 8.4 11.3
Discussion
In order to compare the capability of the formulations to keep the drug substance in solution after entering the GIT before absorption, it is of interest to study the bioavailability from different lipid-based formulations containing the drug substance in the dissolved state. The present study with SMEDDS distinguishes from previous studies by having the same concentration of cremophor RH40 in both MC-SMEDDS and LC-SMEDDS as well as the same concentration of lipid phase. Furthermore, the bioavailability enhancing potential of MC-SMEDDS and LCSMEDDS can be directly compared with data obtained in a previous study (Grove et al., 2005) where the bioavailability of seocalcitol from pure oil solutions of MCT and LCT was determined as the same amount of lipid was dosed.
4.1.
Construction of ternary phase diagrams
The ternary phase diagrams (Figs. 2 and 3) show that a larger microemulsion area was achieved when using MCT instead of LCT. This is due to the difference in polarity between the lipids, where the more hydrophobic LCT is more difficult to emulsify. This is in accordance with findings of Deckelbaum et al. (1990) showing that MCT is more soluble and have a higher mobility in the lipid/water interfaces than LCT associated with a more rapid hydrolysis of MCT (Deckelbaum et al., 1990). In general, when using LCT, a higher concentration of cremophor RH40 was required to form microemulsions compared with MCT. The overlapping area in Fig. 4, where microemulsions with both MCT and LCT are formed, is relatively small, illustrating the difficulties in developing both MC-SMEDDS and LC-SMEDDS with the same surfactant and the same ratio between lipid, surfactant and co-surfactant. The relatively high concentration of surfactant required in this study to form microemulsions with both MCT and LCT is in accordance with other studies, where the use of high concentrations of surfactants was found to be necessary to achieve fast and efficient self-emulsification (Shah et al., 1994; Khoo et al., 1998; Kommuru et al., 2001; Kang et al., 2004). A good relation between the visual observations and the achieved droplet sizes was observed. In the microemulsion area clear to bluish transparent microemulsions with droplet sizes less than 100 nm were formed compared with white emulsion-like dispersions with droplet sizes above 100 nm outside this area. The droplet sizes achieved in the microemulsion areas were all within the range between 20 and 40 nm, well below 100 nm, indicating that if microemulsions are
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formed the resulting droplet size is very small due to the thermodynamical stability of these systems.
4.2. Absorption and bioavailability study of 3 H-seocalcitol in rats 4.2.1.
MC-SMEDDS versus LC-SMEDDS
Upon oral administration of the MC-SMEDDS and LC-SMEDDS to male rats, the absorption and bioavailability of 3 Hseocalcitol were approx. 45% and 18%, respectively, for both SMEDDS (Table 2). The two SMEDDS were developed as to have the same characteristics in terms of composition, appearance and droplet size, however, the used MCT and LCT lipids do have different physico-chemical characteristics and also a different in vivo fate and therefore a difference in absorption and bioavailability of seocalcitol could be anticipated. The results, however, indicate that despite the difference in nature of the lipids, the processes of dispersion, distribution, trafficking of 3 Hseocalcitol in the GIT lumen, results in the same bioavailability of 3 H-seocalcitol for both SMEDDS. An equal solubility has been found for seocalcitol in simulated intestinal media containing either medium or long chain lipolytic products (Grove et al., 2005), which supports the hypothesis that an equal solubilising capacity is seen in vivo. For danazol and halofantrine the bioavailability from LCSMEDDS was found to be superior compared with MCSMEDDS, which made the authors suggest that MCT has a reduced solubilising capacity for some lipophilic drug substances compared with LCT based formulations (Khoo et al., 1998; Porter et al., 2004). However, in these studies the SMEDDS contain different amounts of lipid and surfactant, which makes the comparison more complicated since more than one variable exists. Taking the present study into account it seems to be drug dependent whether MCT or LCT based formulations will give rise to the highest bioavailability.
4.2.2.
SMEDDS versus lipid solutions
It is important to consider the amount of lipid dosed when comparing lipid-based formulations, as the lipid itself may be responsible for enhancement in bioavailability. Vitamin E showed a two-fold increase in bioavailability from a SMEDDS compared to a lipid solution (Julianto et al., 2000) however, as more lipid was dosed in the SMEDDS than the lipid solution, it is difficult to draw a firm conclusion. In the present studies the rats were dosed equal amounts of lipid from either SMEDDS or lipid solutions (0.8 mg/kg), and the results (Table 2) can therefore be directly compared. The MCT or LCT lipid solutions (Grove et al., 2005), showed a slightly higher bioavailability of seocalcitol (22–24% in comparison with 18%) than was found for the SMEDDS, even though there was no significant difference. The SMEDDS were expected to give rise to a higher bioavailability compared to a lipid solution, due to more rapid and uniform distribution of the drug substance in the GIT (Mueller et al., 1994). The large surface area obtained after administration of the SMEDDS increases the hydrolysis of triglycerides and promotes a rapid release of the drug substance (Humberstone and Charman, 1997) and/or the drug substance may be absorbed directly from the small droplets of the microemulsion (de Smidt et al., 2004). The pure lipid
solutions are dependent on the emulsification by endogenous surfactants (e.g. bile salt and phospholipids) before the onset of triglyceride hydrolysis. However, the obtained results are not in agreement with this theory, since no significant difference was found between bioavailability of 3 H-seocalcitol from SMEDDS and lipid solutions. The presence of surfactants in the SMEDDS does not seem to provide an enhancement in GI solubilisation of seocalcitol over that provided by the lipid itself, even though a rather high concentration of surfactant is present in the SMEDDS. This is in agreement with studies conducted by Porter et al. showing the same bioavailability of danazol when dosed in either LCT solution or LC-SMEDDS in dogs (Porter et al., 2004). When studying SMEDDS the animal model may be important to consider due to the dependency of the dynamic processes responsible for the emulsification process. Rats were chosen in the present study due to the low cost and relatively facile accessibility and rats have previously been used as animal model in bioavailability assessment of SEDDS (Kim and Ku, 2000; Bravo Gonzalez et al., 2002; Kim et al., 2002; Yang et al., 2004), hence none of these studies have compared a lipid solution with a SMEDDS. However, the amount of fluid present in the stomach of the rat might be insufficient to emulsify the dose of SMEDDS administered. Titrating the SMEDDS with water forms a very viscous structure, probably consisting of bicontinuous structures, until the ratio between SMEDDS and water exceeds factor 2 (1:2) where an aqueous solution is formed. This phenomenon supports the hypothesis that a certain level of GI fluid has to be present in order for the selfmicroemulsifation process to occur. In terms of the pure lipid solutions the lipid phase is not miscible with the GI fluid and the absence of surfactant in these formulations avoids the formation of high viscous gel-like structures. The influence of the animal model on the bioavailability of formulations being dependent on dynamic processes in the GIT has not yet been studied, therefore it would be of interest to study the bioavailability of seocalcitol from SMEDDS in a larger animal e.g. the mini-pig or dog.
4.3.
Stability of seocalcitol in formulations
To obtain a product with a shelf life of 2 years at 25 ◦ C a degradation of drug substance concentration of approximately 7% within 3 months is acceptable at the condition 40 ◦ C 75% RH representing accelerated stability condition (Pope, 1980). For the SMEDDS this criterion is not met, as a decrease of 10–11% is observed at this condition, whereas for the simple lipid solutions the degradation of seocalcitol was found to be less than 3% (Table 3). Seocalcitol is sensitive to oxidation why butylhydroxyanisole was added to the formulations in a concentration of 0.02% w/w. However, the concentration of antioxidant in the formulations was the same after 3 months’ storage indicating that the decrease in seocalcitol concentration may be ascribed to another process than oxidation or a combination of different reactions. Unfortunately, seocalcitol is not degraded to one or a few compounds but rather a large number of different compounds all present at very low concentration. Seocalcitol is acid labile and may be sensitive for excipients containing acid impurities. Since the stability of seocalcitol is acceptable in the pure lipid solutions and not in the SMEDDS, the
european journal of pharmaceutical sciences
increased degradation seen in the SMEDDS must be ascribed to the surfactant (cremophor RH40) and/or the co-surfactant (akoline MCM and peceol). The surfactant and co-surfactants in the SMEDDS are mixtures of products that may contain small amounts of impurities from the manufacturing process possibly leading to the observed degradation of seocalcitol. To further identify the cause of instability of seocalcitol, stability studies should be performed in each excipient separately. There is not much literature investigating the chemical stability of drug substances in SMEDDS even though this is a very important aspect from an industrial point of view. An example of a poor chemical stability was seen for paclitaxel, showing a degradation of 20% after storage at 37 ◦ C for 24 h when formulated in SEDDS (Gursoy et al., 2003). The poorer stability of drug substances in SMEDDS may partly explain the limited number of marketed products based on this formulation concept.
4.4.
Industrial relevance of developed SMEDDS
In order to evaluate the industrial relevance of formulating seocalcitol in the SMEDDS developed in the present study, the chemical stability as well as the bioavailability enhancing potential of seocalcitol needs to be taken into consideration. When comparing the SMEDDS to the lipid solutions, this study shows that the lipid solutions seem to be a better choice. The degradation of seocalcitol in the SMEDDS is higher compared with the lipid solutions (Table 3) and the large decrease in concentration of seocalcitol in the SMEDDS makes the formulations unsuitable for commercial use as a sufficient shelf life will not be obtainable. The bioavailability of 3 H-seocalcitol is slightly higher from the lipid solutions compared with the SMEDDS and from a production point of view the lipid solutions are also preferable, because less excipients are required. All together the simple lipid solutions seem to be superior for the formulation of seocalcitol compared with the corresponding more advanced SMEDDS developed in the present study.
Acknowledgements The authors wish to thank ATV (The Danish Academy of Technical Sciences) for financial support. The radiolabeled compound was kindly synthesized by Dr. Gunnar Grue-Sørensen, LEO Pharma A/S. Rie Andreasen, LEO Pharma A/S, is thanked for skilful help with animal surgery and dosing. Analysis & Stability, LEO Pharma A/S, is thanked for help with the stability study. Jonas Wiedemann, LEO Pharma A/S, is thanked for help with the statistical analysis.
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