Proliposomes as a drug delivery system to decrease the hepatic first-pass metabolism: Case study using a model drug

European Journal of Pharmaceutical Sciences 64 (2014) 26–36

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Proliposomes as a drug delivery system to decrease the hepatic first-pass metabolism: Case study using a model drug Sarala Yanamandra a, Natarajan Venkatesan b, Veeran Gowda Kadajji c, Zhijun Wang a, Manish Issar d, Guru V. Betageri a,c,⇑ a

College of Pharmacy, Western University of Health Sciences, Pomona, CA 91766, United States Chicago College of Pharmacy, Midwestern University, Downers Grove, IL 60515, United States Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA 91766, United States d College of Osteopathic Medicine of the Pacific, Western University of Health Sciences, Pomona, CA 91766, United States b c

a r t i c l e

i n f o

Article history: Received 28 February 2014 Received in revised form 15 August 2014 Accepted 18 August 2014 Available online 23 August 2014 Keywords: Proliposomes Lovastatin Bioavailability First-pass metabolism In vivo

a b s t r a c t Objective of the present study was to develop a proliposomal formulation to decrease the hepatic firstpass metabolism of a highly metabolized drug. Lovastatin was chosen as the model drug. Proliposomes were prepared by mixing different ratios of phospholipids such as soy phosphatidylcholine (SPC), hydrogenated egg phosphatidylcholine (HEPC) and dimyristoyl phosphatidylglycerol (DMPG) individually with drug and cholesterol in an organic solvent. Proliposomal powder was obtained following evaporation of the solvent. The proliposomal powder was either filled into capsules or compressed into tablets. Physical characterization, in vitro drug transport studies and in vitro dissolution of formulations and pure drug was carried out. In vitro transport across the membrane was evaluated using parallel artificial membrane permeability assay (PAMPA). The extent of drug released from various proliposomal formulations in the first 30 min was 85%, 87% and 96% with DMPG, SPC and HEPC containing formulations respectively, while the pure drug formulation showed 48% drug release in the same period. In vivo studies were carried out in male Sprague–Dawley rats. Following single oral administration of the selected formulation (F9), a relative bioavailability of 162% was achieved compared to pure lovastatin. The study demonstrated that proliposomes can be used as a drug delivery system to decrease the hepatic first-pass metabolism. Ó 2014 Published by Elsevier B.V.

1. Introduction Oral route is the most preferred route of administration. Efficacy of an orally administered drug is dependent on its oral bioavailability, which in turn is dependent on the dissolution, extent of absorption and first-pass metabolism (Custodio et al., 2008). Oral absorption of a drug is fundamentally dependent on that drug’s aqueous solubility or intestinal drug solubilization and gastrointestinal permeability (Amidon et al., 1995). Amidon et al. first proposed the biopharmaceutical classification system (BCS) in 1995 that classified drug substances based on their aqueous solubility and intestinal permeability into four classes (Amidon et al., 1995). Any factor impacting the dissolution characteristics of these drugs would have a profound impact on their bioavailability, for example drugs such as carabamazepine, ⇑ Corresponding author at: Graduate College of Biomedical Sciences, Western University of Health Sciences, Pomona, CA 91766, United States. Tel.: +1 909 469 5682; fax: +1 909 469 8750. E-mail address: [email protected] (G.V. Betageri). http://dx.doi.org/10.1016/j.ejps.2014.08.008 0928-0987/Ó 2014 Published by Elsevier B.V.

ketoprofen and verapamil. The inability of a drug to go into solution is sometimes a more important limitation to its overall rate of absorption than its ability to permeate the intestinal mucosa (Horter and Dressman, 2001). For drugs that cross the intestinal mucosa easily, the onset of drug levels is dictated by the time taken by the dosage form to release the drug content. Thus, poor bioavailability of poorly water soluble molecules that are not permeation-rate limited can be attributed to dissolution-rate kinetics (Merisko-Liversidge and Liversidge, 2008). Solubility issues affect the delivery of many existing drugs. Compared to highly soluble compounds, low drug solubility often manifests itself in a host of in vivo consequences that include decreased bioavailability, increased chance of food effect, incomplete release from the dosage form and higher inter-patient variability (Williams et al., 2013). Classical approach to deal with the solubility issue is to generate salts of poorly soluble drugs or pro-drugs to improve the solubility while retaining the biological activity (Fiese and Hagen, 1986). Other strategies to resolve the solubility problems of drugs include formulation approaches such as pH adjustment (Williams et al., 2013), use of cosolvents (Zhao et al., 2013), use of surfactants

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(Li et al., 2013), complexation (Patel and Rajput, 2009), micronization (Han et al., 2013) and novel drug delivery approaches like liposomes (Kaluderovic et al., 2012), microspheres (Hu et al., 2011), nanoparticles (Tran et al., 2013) etc. In our study, we followed the approach of using proliposomes as a carrier for the delivery of a BCS class II drug. Proliposomes are dry, free flowing powder formulations which upon reconstitution in aqueous environment forms liposomes (Payne et al., 1986). The objectives of our present study were to (a) develop and characterize the proliposomal formulation (b) investigate the effectiveness of proliposomes as a delivery system to improve oral bioavailability by decreasing hepatic first-pass metabolism. Lovastatin a BCS class II drug which has low solubility and high permeability with extensive first-pass metabolism was selected as a model drug for this study (Henwood and Heel, 1988; Suresh et al., 2007). Lovastatin, an inactive lactone, upon oral ingestion is hydrolyzed to the active metabolite b-hydroxyacid (lovastatin acid). The conversion of lovastatin to its active metabolite takes place in the liver. This makes it an ideal candidate to see if the drug upon formulating into proliposomes can reduce the first-pass metabolism. This can be determined by measuring the increased levels of lovastatin and decreased levels of the active metabolite in the blood. 2. Materials and methods 2.1. Materials Dimyristoyl phosphatidylglycerol (DMPG) was obtained from Genzyme pharmaceuticals (Cambridge, MA, USA). Soy phosphatidylcholine (SPC) was obtained from Avanti polar lipids (Alabaster, AL, USA), hydrogenated egg phosphatidylcholine (HEPC) from NOF Corporation (Tokyo, Japan). Cholesterol and magnesium stearate were obtained from Spectrum chemical and laboratory products (Los Angeles, CA, USA). Silicified microcrystalline cellulose (SMCC), microcrystalline cellulose (MCC) and sodium starch glycolate were gifted by JRS Pharma (Patterson, NY, USA). Lovastatin was obtained from TCI America (Portland, OR, USA), lovastatin hydroxy acid and simavastatin hydroxy acid was obtained from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada), simvastatin was obtained from Sigma Aldrich (Munich, Germany). PAMPA plates were obtained from Millipore (Billerica, MA, USA). Sprague–Dawley rats were obtained from Harlan Incorporated (Indianapolis, IN, USA) and rat plasma was obtained from Valley Biomedical (Winchester, VA, USA). All other materials and solvents used in the study were of HPLC grade and were obtained from EMD (Billerica, MA, USA). 2.2. Methods 2.2.1. Preparation of proliposomes Proliposomal formulations containing lovastatin were prepared using lipids such as DMPG, SPC, HEPC and cholesterol. The details of the formulation composition are summarized in Table 1. Solvent evaporation method was used to prepare proliposomes. Briefly, to prepare DMPG proliposomes, lipid, cholesterol and lovastatin were dissolved in chloroform. In the case of SPC and HEPC formulations, lipid and cholesterol were dissolved in ethanol followed by the addition of the lovastatin and the resultant dispersion was adsorbed onto SMCC. Solvent was removed under a stream of nitrogen to obtain a proliposomal powder. This proliposomal powder was then subjected to vacuum desiccation to remove any residual solvent and passed through the sieve (250 lm; 60 mesh) to obtain free flowing proliposomal powder. The prepared formulation was either filled into capsules or compressed into tablets for further evaluation.

Table 1 Formulation composition of proliposomes. Formulation code

Lipid

Drug (in parts)

Lipid (in parts)

Cholesterol (in parts)

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

DMPG DMPG SPC SPC SPC HEPC HEPC HEPC SPC HEPC

1 1 1 1 1 1 1 1 1 1

0.45 0.9 2 1 0.5 2 1 0.5 0.45 0.45

0.05 0.1 0 0 0 0 0 0 0.05 0.05

2.2.2. Physical characterization of proliposomes The proliposomal powder was dispersed in nanopure water and hydrated by mixing gently for 5 min at room temperature. The liposomes formed after hydration was observed through an optical microscope (Nikon Eclipse Ti-Series, Nikon instruments Inc., Melville, NY, USA). The sample was also subjected to Cryo-TEM analysis. Proliposomal tablet formulation was hydrated using 10 ml of distilled water (37 °C) for about 20 min. The sample was then centrifuged at 10,000 rpm for 5 min to separate the excipients from the liposomes (supernatant). The liposomal dispersion was transferred to an eppendorf tube. A drop of the liposomal dispersion was placed onto a carbon with copper grid (Lacey FormvarÒ). The samples were then frozen using Vitrobot Mark IV (FEI Company, Eindhoven, Netherlands). The prepared samples were observed under a Cryo-TEM (JEOL-JEM 1230 Electron Microscope; JEOL, Tokyo, Japan). Proliposomes were hydrated in nanopure water as described above to obtain liposomes. The resultant dispersion was then analyzed for size distribution using Malvern Zetasizer ZS90 (Malvern Instruments, Worcestershire, UK). 2.2.3. Compression of proliposomal powder into tablets The proliposomal powder formulations containing different compositions were compressed into tablets using Cadmach mini rotary tablet press (Cadmach Machinery Co. Pvt. Ltd., Gujarat, India). Sodium starch glycolate (3% of the weight of proliposome powder in each tablet) and magnesium stearate (0.5% of the weight of proliposome powder in each tablet) were used as tablet excipients. Tablets containing pure drug without any lipids were also compressed using the same composition and quantity of tablet excipients as used in the proliposomal tablets. 2.2.4. Tablet weight variation, breaking force and disintegration time Weight variation of the prepared tablets was carried out on 20 tablets. Tablet breaking force was determined using a tablet hardness tester (Varian-VK200, Cary, NC, USA). Disintegration time was determined using a tablet disintegrating apparatus (Electrolab, Mumbai, India). 2.2.5. Analysis of lovastatin A reversed phase HPLC method as reported in the United States Pharmacopoeia (USP) monograph for lovastatin tablets (USP, 2007) was used with slight modification. A Waters HPLC system (2487/ 600/717) with UV–visible detector was used for the analysis (Waters, San Diego, CA, USA). The mobile phase consisted of acetonitrile:phosphate buffer (pH 4.0):methanol (5:3:1), set at a flow rate of 1.2 ml/min with a sample injection volume of 50 ll. The chromatographic separation was performed on a Symmetry C-18 column (3.5 lm, 75  4.6 mm) preceded by a pre-column of similar material. The column assembly was enclosed within a column oven whose temperature was maintained at 45 °C. The column

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eluate was monitored on a UV–VIS detector set at 230 nm. A calibration curve was established in the concentration range of 1–70 lg/ml. The method was validated for inter- and intra-day variations. 2.2.6. Assay of lovastatin The assay of lovastatin in proliposomal powder formulation, pure drug tablets and proliposomal tablets was carried out as per the method reported in the USP monograph for lovastatin tablets (USP, 2007). Briefly, the tablets were ground and an accurately weighed portion of the powder that was equivalent to 40 mg of lovastatin was taken into a 200 ml volumetric flask. To this, 150 ml of the dissolving solvent (water with glacial acetic acid, 0.33% with pH 4.0) was added and sonicated for 20 min and then diluted to volume. The above solution (20 ml) was then added to 100 ml of volumetric flask, diluted with dissolving solvent and mixed thoroughly. A portion of this solution was centrifuged and the supernatant was injected into the HPLC. 2.2.7. In vitro dissolution studies Dissolution studies were conducted using USP type I apparatus (basket) when capsules were evaluated and USP type II apparatus (paddle) was used when tablets were evaluated (VK7000, Varian Inc., Cary, NC, USA; attached to an auto sampler VK8000, Varian Inc., Cary, NC, USA). In vitro dissolution was first carried out in phosphate buffer (pH 7.0). Based on the data, the dissolution medium chosen for the study was 900 ml of phosphate buffer (pH 7.0) with 2% w/v sodium dodecyl sulfate (USP, 2007). Dissolution studies were carried out under non-sink conditions. Each experiment was performed in triplicates and the samples were withdrawn at fixed time intervals up to 4 h. Volume replenishment was not done for the withdrawn volume and appropriate adjustments were used in the calculation to compensate the loss. Cumulative percent of the drug released from the tablets was calculated and mean of the three samples was used in the data analysis. In order to study the effect of compression of the proliposomal formulation into a tablet, proliposomal powder was also filled in size ‘0’ capsules and subjected to in vitro dissolution testing to obtain the percent drug released from the capsules. The results were compared to see if there were any changes in the dissolution profiles. 2.2.8. Parallel artificial membrane permeability assay (PAMPA) PAMPA studies were carried out using the following protocol. The 96-well filter plate (Millipore, Billerica, MA, USA) was used as permeation receptor and the 96-well receiver plate was used as the permeation donor. A 1% solution (w/v) of lecithin in dodecane was prepared and the mixture was sonicated to ensure complete dissolution. Lecithin/dodecane (5 ll) was then carefully added into each well of the donor plate. Immediately after the application of the artificial membrane, 150 ll of the proliposomal suspension was added into each well of the donor plate. Phosphate buffer (pH 7.0; 300 ll) was added to each well of the receptor plate. Subsequently, the drug-filled donor plate was carefully placed into the receptor plate, ensuring that the lower side of the membrane was in contact with the buffer in all wells. The whole donor-receptor assembly was then covered by a lid to avoid any evaporation and was incubated at room temperature. Following incubation, samples were collected from the donor and the receptor component at predetermined time intervals and were analyzed for lovastatin concentration using the validated HPLC method. The flux J (lg/cm2/h) at steady state of the mass transport (dc) per unit time (dt) related to the area (A) was calculated using the following formula as reported earlier (Kanzer et al., 2010; Frank et al., 2012).

J¼

dc 1  dt A

2.2.9. In vitro stability of proliposomes Stability of prepared proliposomes upon dispersion in gastric and intestinal pH was studied. The proliposomal tablet (1) was dropped into 200 ml of 0.1 N HCl (pH 1.2) or phosphate buffer (pH 6.8) maintained at 37 °C and stirred at 50 rpm over a magnetic stirrer. Samples were withdrawn at time intervals of 15, 30, 60, 90 and 120 min in the case of 0.1 N HCl and 15, 30, 60, 90, 120, 180 and 240 min in the case of phosphate buffer. The withdrawn samples were diluted 1:10 with the respective media at 37 °C and the samples were observed under an optical microscope (Nikon Eclipse Ti-DH, Nikon, Japan) and measured for their particle size using a dynamic light scattering technique (Nano ZS, ZEN3600, Malvern Instruments, Malvern, Worcestershire, UK). To study the effect of bile salt on proliposomal formulations, a stock solution of sodium taurocholate solution (40 mM) was prepared and used as the bile salt solution. Proliposomal formulation equivalent to 10 mg of lovastatin was taken and hydrated with 10 ml of phosphate buffer solution pH 7 to get 1 mg/ml of drug solution. This solution was further diluted with equal volume of the prepared bile salt solution and incubated at room temperature for 30 min. Following incubation, the samples were subjected to centrifugation (10,000 rpm/5 min/5 °C) (Eppendorf 5415R, Eppendorf, Hamburg, Germany). The clear supernatant was collected and appropriately diluted with the dissolving solvent (as described in Section 2.2.6) and analyzed for lovastatin using an HPLC method. The amount of drug leached out of liposomes in the presence of bile slats was calculated to see the effect of bile salts on the formulations. 2.2.10. Determination of molecularly dissolved drug The amount of drug dissolved in molecular form was determined using a ultrafiltration experiment (Fischer et al., 2011). Nanosep 100 K Omega centrifugal devices, (Pall Life Sciences, Port Washington, NY, USA) were used. Hydrated proliposomal formulation containing known concentration of lovastatin was poured in the filter unit, and the tube was centrifuged at 10,000 rpm/25 °C/ 5 min (Eppendorf 5415R, Eppendorf, Hamburg, Germany). The amount of drug in the filtrate and the supernatant was estimated using an HPLC method. 2.2.11. Differential scanning calorimetric (DSC) analysis Differential scanning calorimetric (DSC) studies were performed to evaluate the physical state of the drug in the formulation. DSC profiles of pure lovastatin, lovastatin–SPC proliposomal powder, lovastatin–SPC proliposomal tablet, excipients used in the tablet formulation and cholesterol were carried out using a DSC-Q2000 connected to a RC-40 refrigeration unit (TA Instruments, New Castle, DE, USA). Samples were weighed into aluminum pan ranging between 3 and 8 mg. The pan was sealed manually. Empty aluminum pan was weighed and used as reference pan. Both, the sample and reference pan were placed into the DSC furnace. The samples were run under a stream of nitrogen. The samples were exposed to a heat-cool-heat cycle. The pan was maintained at a temperature of 40 °C and was ramped to 200 °C at 20 °C/min. The cooling cycle was ramped at 10 °C/min. DSC of the tablet formulation was carried out by cutting the tablet into small pieces so as to weigh 3–8 mg of the sample and the same tablets were also crushed into powder using a glass mortar and pestle to see if there was any change due to grinding the tablet into a powder. 2.2.12. Estimation of lovastatin in plasma using LC/MS/MS Stock solution of 1 mg/ml of lovastatin and 1 mg/ml lovastatin hydroxy acid were prepared separately by dissolving them individually in acetonitrile. Using the stock solution, working solutions containing a mixture of lovastatin and lovastatin hydroxy acid were

S. Yanamandra et al. / European Journal of Pharmaceutical Sciences 64 (2014) 26–36

prepared in the range of 10, 20, 50, 100, 200, 500 and 1000 ng/ml. Plasma samples were spiked using this working standard solution. Quality control samples were prepared at low (10 ng/ml), medium (50 ng/ml) and high (100 ng/ml) concentrations. A liquid–liquid extraction method was employed for the extraction of lovastatin, its metabolite lovastatin hydroxy acid and their corresponding internal standards (simvastatin and simvastatin hydroxy acid). Briefly, working standard and internal standard solution (10 ll each) were added to 100 ll of plasma. The tube was mixed well using a vortex mixer. To this, 100 ll of 0.1 N HCl was added. The resultant mixture was then centrifuged at 10,000 rpm for 2 min at room temperature followed by the addition of 800 ll of diethylether and was centrifuged again at 10,000 rpm for 3 min at room temperature. The organic supernatant was transferred to a glass tube and evaporated to dryness at room temperature under a stream of nitrogen. Finally, the residue was reconstituted in 100 ll of 50% methanol in water, vortex mixed and centrifuged for 2 min at 10,000 rpm at room temperature and the supernatant was filled into an HPLC vial for analysis. Analysis was carried out using a Zorbax SB-C18 column (Agilent, Foster City, CA, USA) (particle size 5 lm; 2.1  150 mm) with the corresponding guard column (12.5  2.1 mm) maintained at room temperature. A mixture of 0.1% formic acid and acetonitrile (80:20) was used as the mobile phase. Sample injection was carried out at 10 ll while the mobile phase flow rate was set at 0.35 ml/min. The detector used was triple-quadrupole LC–MS mass spectrometer (AP1 3200, Framingham, MA, USA). The mass ion spectrophotometer was operated in the negative ion mode for the first 5 min and then switched to the positive mode for the rest 5 min of the run. Linearity and range was evaluated using freshly prepared spiked plasma samples and the calibration curves were constructed using seven non-zero standard points covering the range of 1–100 ng/ml. In addition a blank (non-spiked sample) was run to discard the presence of any interference. The ratios of corresponding peak heights of lovastatin to simvastatin and lovastatin hydroxy acid to simvastatin hydroxy acid were calculated. Precision and accuracy between inter-and intra-day samples were carried out. 2.2.13. Pharmacokinetic study Jugular vein-cannulated male Sprague–Dawley rats (225–250 g body weight) were used in the study. The rats were divided into 2 groups of 4 animals in each. All animals were fasted overnight prior to the day of experiment with free access to water. Using an oral gavage, group 1 animals were administered with a suspension (0.5 ml) of pure lovastatin at a dose of 10 mg/kg (one animal died during the study) and group 2 animals were administered with proliposomal formulation (F9) of equal dose. Pure lovastatin and proliposomal suspension were both prepared by suspending the pure drug and proliposomes in 1% w/v methylcellulose solution. After administration of the drug, 0.3 ml of blood samples were collected from pre-cannulated jugular vein at 0, 0.5, 1, 2, 4, 6 and 24 h into a heparinized eppendorf tube. After the terminal sample collection, the animals were sacrificed by administering a lethal dose of isoflurane. The collected blood samples were centrifuged at 10,000 rpm/4 °C for 5 min (Eppendorf 5415R, Hamburg, Germany). Plasma was separated and stored at 80 °C until analysis. Animals had free access to food and water 4 h following oral dosing. The animal study protocol was approved by the IACUC, Western University of Health Sciences, Pomona, California. 2.2.14. Statistical analysis Statistical analysis of the dissolution profiles was carried out by two-way analysis of variance (ANOVA) and that of PAMPA was carried out by one-way analysis of variance (ANOVA). Tukey’s multiple comparison tests was used as the post hoc test.

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3. Results and discussion The use of proliposomal formulation represents an alternative approach to the liposomal formulation. Stability problems associated with the liposomal formulations that include aggregation, susceptibility to hydrolysis and oxidation can be avoided by developing a proliposomal formulation. The use of proliposomal formulations is well known in the pharmaceutical field for delivering bioactive agents by various routes of administration such as intravenous (Sarbolouki et al., 2000), subcutaneous (Supersaxo et al., 1991), nasal (Jung et al., 2000), vaginal (Pavelic et al., 2001) and through the skin (Chung, 1999). Among various routes of administration, oral route is the most advantageous because of its versatility, safety and patient comfort. Many attempts were made to deliver bioactive agents encapsulated in liposomes through oral route of administration mainly through Peyer’s patches for the delivery of vaccines (Chen et al., 1996). Very few successful attempts have been made so far to develop oral proliposomal formulations. Recently, proliposomal formulations have been developed successfully for the oral delivery of both water soluble and water insoluble drugs to enhance their oral bioavailability (Hiremath et al., 2009; Agnihotri et al., 2010). In the present study, Proliposomes were studied as a drug delivery system to decrease hepatic first-pass metabolism. Lovastatin was chosen as the model drug. 3.1. Formulation of proliposomes SPC, HEPC and DMPG were the three lipids used in the present investigation. Cholesterol was added to the formulation since it was reported to increase the bilayer rigidity of liposomes and prevent leakage (Liu et al., 2008). After the solvent was completely evaporated, unlike DMPG formulations, SPC and HEPC formulations were observed to be sticky and were difficult to proceed with tablet compression. Earlier studies in our lab reported the preparation of proliposomes without the use of a carrier (Hiremath et al., 2009). In the case of SPC we attribute the stickiness to the low liquid–crystalline temperature (Tc) of the lipid and the nature of phosphatidylcholine in itself. However, in the case of HEPC the Tc is much higher (ca 50 °C) and it was surprising to see that the proliposomal formulation still turned to be sticky. This may be due to a decrease in the Tc of the lipid in the presence of the drug. To overcome the stickiness and to obtain a tablet, SMCC was added to the above formulations which resulted in free flowing powder formulations that were easy to compress into tablets with addition of other tableting excipients. In order to ensure that compression of the proliposomal formulation did not affect the dissolution, the same formulation was filled into capsules and subjected to in vitro dissolution. 3.2. Physical characterization of proliposomes Proliposomes upon hydration and when observed under an optical microscope, confirmed the formation of spherical structures of liposomes. Cryo-TEM studies were performed in order to observe the morphological characteristics of the hydrated proliposomal formulation. Hydrated proliposomal formulation formed oligolamellar structures (Fig. 1a) and multilamellar liposomes (Fig. 1b). The average particle size upon hydration of the proliposomal formulations were found to be 1.36 ± 0.28 lm for SPC containing formulations, 0.982 ± 0.15 lm for HEPC containing formulations and 1.03 ± 0.17 lm for DMPG containing formulations. This indicates that upon hydration, proliposomal formulations form multilamellar liposomes which are typically in the micron size range. The advantage of multilamellar liposomes is

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S. Yanamandra et al. / European Journal of Pharmaceutical Sciences 64 (2014) 26–36

that they exhibit high encapsulation efficiency for water insoluble compounds. 3.3. Tablet weight variation, breaking force and disintegration time Results of the tablet weight variation, breaking force and disintegration of all the prepared tablet formulations are presented in Table 2. Weight of tablets in case of all the proliposomal formulations was within +10% range of the target weight as shown in Table 2. Disintegration time of the tablets containing higher amount of lipids were a little longer as compared to those with lower amount. This difference in disintegration time with higher lipid containing formulation could be attributed to the increased hydrophobicity with increased lipid content. 3.4. Assay of lovastatin Assay values of all the prepared lovastatin formulations are presented in Table 2. Assay values show that the amount of drug

Fig. 1. Cryo-TEM image of proliposomal formulation (SPC) following hydration: (a) liposomes inside liposomes (oligolamellar vesicles); (b) multilamellar vesicles (bar = 50 nm).

present in each formulation depends on its composition. Assay value was higher with formulation containing higher amount of lipids. This clearly indicates the increased affinity of the drug towards the lipid. 3.5. In vitro dissolution studies In vitro dissolution in phosphate buffer (pH 7.0) resulted in less than 1% of drug release in the case of pure lovastatin and about 6% with proliposomal formulation (Fig. 2). Dissolution of the selected formulation in phosphate buffer (pH 7.0) was very poor. In order to have a discriminating dissolution media, 2% w/v sodium dodecyl sulfate was added to the phosphate buffer (pH 7.0). In vitro dissolution studies were performed in 900 ml of phosphate buffer containing 2% w/v sodium dodecyl sulfate (pH 7.0) for lovastatin using an USP type II dissolution apparatus. The ideal ratio of drug and lipid for optimum dissolution was determined by dissolution studies of proliposomal formulations comprised of various lipid:drug ratios of 2:1, 1:1 and 0.5:1. The in vitro dissolution profiles of these formulations are shown in Figs. 3–7. The amount of lovastatin released from pure drug (powder) formulation was 49% in 0.5 h which increased to 92% at the end of 4 h. In the case of DMPG proliposomal formulations the percentage of lovastatin released from F1 was 85% in 0.5 h which increased to 99% by the end of 2 h and remained the same until the end of the study period. Formulation F2 showed 84% and 102% at the end of 0.5 and 2 h respectively. There was no significant difference between the dissolution profiles of F1 and F2 (Fig. 3). The ANOVA analysis of dissolution profile for pure lovastatin powder and F1 (p < 0.05 at the end of 0.5, 1 and 2 h), pure lovastatin powder and F2 (p < 0.05 at the end of 0.5, 1, 2 and 3 h) proved that incorporation of DMPG had a significant influence on the dissolution of lovastatin from proliposomal formulation. However, there was no significant difference between the dissolution profiles of F1 and F2, and pure lovastatin powder at the end of 4 h, indicating that the extent of drug release remained same in spite of the incorporation of DMPG. Also there was no significant difference between the dissolution profiles of F1 and F2 indicating that increase in the drug to lipid ratio had no influence on the release of lovastatin from proliposomal formulations. The rapid dissolution of DMPG containing formulations as compared to SPC and HEPC containing formulation may be attributed to the increased ionization of DMPG as compared to poor ionization of SPC and HEPC at pH 7.0. In the case of SPC proliposomal formulations (F3–F5), amount of lovastatin released increased from 32% to 77% as the concentration of lipid decreased from 2:1 (F3) to 0.5:1 (F5) (Fig. 4). The ANOVA analysis of dissolution profile for pure lovastatin powder and F5 (p < 0.05 at the end of 0.5 h) proved that incorporation of SPC had a significant influence on the release of lovastatin from proliposomes. However, there was no significant difference in the dissolution profiles of F3 and F4, and the pure lovastatin powder at any time point indicating that increase in the drug to lipid ratio had no influence on the release of lovastatin from proliposomal formulations. Moreover, dissolution profile of formulation F5 with lower lipid content showed a significant increase (p < 0.05) in the percent drug release at the end of 0.5 h as compared to the formulations that had a higher drug to lipid ratio. Similar observation was made with HEPC proliposomal formulations (Fig. 4), which, showed an increase in the amount of lovastatin released from 50% to 78% as the concentration of lipid decreased from 2:1 (F6) to 0.5:1 (F8), although the increase in the dissolution profiles of F6–F8, and pure lovastatin powder were not significant (p > 0.05). However, in all the proliposomal formulations, irrespective of the lipid to drug ratios, complete drug release was achieved within 2 h, while in the case of pure drug formulation it took more than 4 h. The in vitro dissolution studies suggested that there is a significant increase (p < 0.05) in the

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S. Yanamandra et al. / European Journal of Pharmaceutical Sciences 64 (2014) 26–36 Table 2 Physicochemical evaluation of proliposomal tablets. Formulation code

Tablet weight (mg)

Weight variation (mean ± SD) (mg)

Tablet breaking force (mean ± SD) (N)

Disintegration time (min)

Assay (mean ± SD) %

Pure lovastatin tablets F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

290 90 90 550 525 412 555 525 412 412 412

294.5 ± 4.5 89.5 ± 3.8 90.4 ± 2.9 558 ± 8.5 532.8 ± 4.9 419 ± 9.3 556.8 ± 3.6 538 ± 6.4 422 ± 4.8 427 ± 3.2 418 ± 4.6

103 ± 13 96 ± 3 103 ± 4 119 ± 23 110 ± 18 112 ± 16 121 ± 20 103 ± 23 109 ± 15 108 ± 17 105 ± 14

6 6.5 7 9 7 5 11 7 6 6 6

92.2 ± 0.3 91.4 ± 2.6 88.2 ± 0.5 84.3 ± 0.1 83.4 ± 2.2 89.7 ± 0.4 78.3 ± 0.1 90 ± 1.2 86.3 ± 0.1 85.8 ± 1.4 88.6 ± 2.7

120

10

Pure Lovastatin F1

8

6 80

% Drug released

% Drug released

100

4

2

0 0

0.5

1

1.5

2

2.5

3

3.5

4

60

Plain lovastatin powder F5 (SPC: drug - 0.5:1) 40

F4 (SPC: drug - 1:1)

Time (h)

F3 (SPC: drug - 2:1)

Fig. 2. In vitro dissolution of pure lovastatin and formulation F1 in phosphate buffer (pH 7.0) (n = 3 mean ± SD).

120

F9 (SPC+ CH) : drug - 0.5:1 20

0 0

1

2

3

4

Time (h) 100

Fig. 4. In vitro dissolution of SPC tablet formulations (n = 3 mean ± SD).

% Drug released

80

60

Plain lovastatin powder F1 (DMPG + CH) : drug - 0.5:1

40

F2 (DMPG + CH) : drug - 1:1 20

0 0

1

2

3

4

Time (h) Fig. 3. In vitro dissolution of DMPG tablet formulations (n = 3 mean ± SD).

dissolution with incorporation of lipids in the formulation indicating that phospholipids enhance the dissolution of the poorly soluble drug in the chosen dissolution medium. Although incorporation of phospholipids resulted in an increase in the dissolution of lovastatin, our study indicated that increase in the concentration of lipid in the formulation did not result in increase in the amount of drug released in the first 0.5 h and this

was observed irrespective of the type of phospholipid used in the formulation. This might be either because of higher affinity of the drug towards the lipid which might not have allowed it to get released in the dissolution medium or high lipid content that might have hindered the partition of lovastatin from the proliposomal formulation into the dissolution medium. Compared to SPC and HEPC formulations that does not contain cholesterol incorporated in them (F5 and F8) to those formulations that had cholesterol (F9 and F10) showed a higher drug release. Fig. 6 represents the comparison of drug release profile of pure formulations and proliposomal formulations F1, F9 and F10 that have a drug lipid ratio of 1:0.5 and a lipid to cholesterol ratio of 9:1. Incorporation of cholesterol in the formulation might have reduced the affinity between the lipid and drug that resulted in an increased drug release. Dissolution profile of the proliposomal formulation (F9) filled into capsules showed a comparable dissolution profile to that of the tablet formulation prepared using the same proliposomal formulation (F9) (Fig. 7). 3.6. Parallel artificial membrane permeability assay (PAMPA) PAMPA is a non-cell based permeability model that provides an estimate of the passive transcellular permeability because it lacks

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S. Yanamandra et al. / European Journal of Pharmaceutical Sciences 64 (2014) 26–36

120

120

100

% Drug released

100

% Drug released

80

80

60

Plain lovastatin tablets F9 (SPC+CH): drug 0.5:1 -Tablet

40

F9 (SPC+CH): drug 0.5:1 -Capsule

60

Plain lovastatin powder

20

F8 (HEPC: drug - 0.5:1) 0

F7 (HEPC: drug - 1:1)

40

0

1

2

F6 (HEPC: drug - 2:1) 20

0 2

3

4

Time (h) Fig. 5. In vitro dissolution of HEPC tablet formulations (n = 3 mean ± SD).

Cumulative conc. (µg/ml)

6

1

4

Fig. 7. In vitro dissolution of SPC containing proliposomal tablets vs capsules (n = 3 mean ± SD).

F10 (HEPC + CH) :drug - 0.5:1

0

3

Time (h)

120

(a)

5

J = 0.763 ± 0.064

4 3 2 1 0 0

4

2

6

8

Time (h) 100

% Drug released

80

60

Plain lovastatin tablets F1 (DMPG + CH) : drug - 0.5:1

40

Cumulative conc. (µg/ml)

6

(b)

5

J = 0.978 ± 0.071

4 3 2 1 0

F9 (SPC + CH) : drug - 0.5:1

0

2

4

20

0

1

2

3

4

Time (h) Fig. 6. Comparison of in vitro dissolution profile of selected proliposomal tablet formulations (n = 3 mean ± SD).

transporter and pore mediated permeability. An adequate lipophilicity (log P) is necessary for a drug to travel across the phospholipid membranes by passive diffusion (Balimane et al., 2006). Effect of different phospholipids on permeability of lovastatin in PAMPA model is shown in Fig. 8. The cumulative drug concentration vs time was plotted to obtain the flux value (J). Estimation of flux has been reported to be more appropriate in cases where the drug is not fully dissolved (Kanzer et al., 2010). The figure does not

Cumulative conc. (µg/ml)

4

0

6

8

Time (h)

F10 (HEPC + CH) : drug - 0.5:1

(c) J = 1.088 ± 0.122

3

2

1

0 0

2

4

6

8

Time (h) Fig. 8. Permeation curve of lovastatin from selected formulations: (a) DMPG containing formulation F-1; (b) SPC containing formulation F-9 and (c) HEPC containing formulation F-10. Flux (J) (lg/cm2/h) was calculated based on the slope (n = 4 mean ± SD).

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S. Yanamandra et al. / European Journal of Pharmaceutical Sciences 64 (2014) 26–36 Table 3 Z-average and PDI of formulation F-9 following in vitro stability studies in 0.1 N HCl (pH 1.2) and phosphate buffer (pH 6.8). Time (min)

15 120 240

In 0.1 N HCl (pH 1.2)

In phosphate buffer (pH 6.8)

Z-average (mean ± SEM) (n = 3)

PDI (mean ± SEM) (n = 3)

Z-average (mean ± SEM) (n = 3)

PDI (mean ± SEM) (n = 3)

238.77 ± 10.32 192.33 ± 2.94 –

0.43 ± 0.01 0.33 ± 0.03 –

448.50 ± 33.17 – 238.60 ± 12.30

0.70 ± 0.04 – 0.57 ± 0.03

Fig. 9. Optical microscope pictures of proliposomal formulation (F9) following exposure to (a) pH 1.2 (15 min); (b) pH 1.2 (2 h); (c) pH 6.8 (15 min) and (d) pH 6.8 (4 h) (bar = 50 lm).

3.7. In vitro stability of the drug and molecular solubility Proliposomal formulation (F9) upon exposure to pH 1.2 up to 2 h and pH 6.8 up to 4 h showed a decrease in particle size over time (Table 3). However, the shape and structure of the vesicle seemed to be intact as observed under the microscope (Fig. 9).

5

4

% Drug leached

include the permeation details of pure drug (control) since it was difficult to prepare a solution form of lovastatin without using an organic solvent. PAMPA study allows only a maximum concentration of 5% DMSO to dissolve the drug compounds which was not sufficient enough for the pure drug. Flux was found to be 1.088 ± 0.122 for DMPG containing formulation (F1), while it was 0.978 ± 0.071 for SPC containing formulation (F9). HEPC containing formulation gave a flux value of 0.763 ± 0.064. There was no statistically significant difference between formulation F1 and F9 (Student’s t-test; p > 0.05). The results give a preliminary insight of the proliposomal formulation. However, a more detailed study using higher analytical techniques may be required to understand the mechanism in which the flux takes place. In order to achieve high permeability across PAMPA a high log P is required. Proliposomal formulations in general provide a high log P due to the presence of lipids in the formulation. However, in addition to the high log P a ‘fluid’ state of the lipid may also have a role in permeability across PAMPA.

3

2

1

0 F1

F9

F10

Fig. 10. Stability of selected proliposomal formulations (F1, F9 and F10) in the presence of bile salts.

The study was carried out for 2 h in pH 1.2 and 4 h in pH 6.8 considering the maximum duration the formulation can be in the stomach or intestine under normal physiological conditions. The difference in initial particle size in different media may be attributed to their ionic concentration and pH. The same formulation when hydrated in deionized water showed particle size in micron range (Section 3.2). This indicates that the hydration media may

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S. Yanamandra et al. / European Journal of Pharmaceutical Sciences 64 (2014) 26–36

Fig. 11. DSC profiles of (a) lovastatin, (b) proliposomal powder formulation (F9), (c) proliposomal tablet formulation (F9), (d) excipients (without lipids) and (e) cholesterol.

play a role in determining the particle size. Exposure to bile salt containing media showed that formulation F10 had the lowest leaching among the three formulations (Fig. 10). The decreased leaching from formulation F10 may be due to the hydrogenation of the lipid. The amount of drug dissolved at molecular level was found to be less than 1% for F1 and F9 while no drug was detected with F10. This could be the reason for low flux with formulation F10. The drug seems to be either in an ‘incorporated state’ or ‘micelle state’ within the lipid formulation which is not clear from the present study.

3.8. DSC analysis DSC studies of pure lovastatin showed a sharp melting point at 172.44 °C. The drug when subjected to heat-cool-heat cycle showed a peak again at 172.44 °C. Fig. 11a shows that apart from a melting endotherm, a glass transition peak and a recrystallization peak were also observed in the thermogram indicating that the pure drug was in crystalline form. The drug containing proliposomal powder formulation (F9) showed a peak at 172.40 °C (Fig. 11b) which is the melting endotherm, indicating that the drug

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S. Yanamandra et al. / European Journal of Pharmaceutical Sciences 64 (2014) 26–36

in the formulation remained in the crystalline form. Proliposomal tablet formulation (F9) exhibited similar endothermic peak as observed in the proliposomal powder formulation (Fig. 11c). There was no endothermic or exothermic peak observed with the nonlipid excipients, which included SMCC, MCC, and magnesium stearate and sodium starch glycolate (Fig. 11d). Cholesterol showed an endothermic peak at 148.79 °C (Fig. 11e), while rest of the excipients did not show any. These results clearly indicate that the drug in the formulation was present in a crystalline form.

140

Plasma drug conc. (ng/ml)

120

3.9. Estimation of lovastatin in plasma using LC/MS/MS Lovastatin and lovastatin hydroxy acid extracted from the plasma showed well resolved chromatographic peaks in the chromatogram. The blank plasma after extraction contained no significant interference peaks. The relation between lovastatin concentration and peak area ratios of lovastatin to its internal standard simvastatin and lovastatin hydroxy acid to its internal standard simvastatin hydroxy acid was linear from 1 to 100 ng/ml (y = 0.081x  0.0798, r2 = 0.9987 and y = 0.3479x + 0.2012, r2 = 0.9994 respectively). Precision and accuracy were determined based on the calibration curve.

Plain lovastatin formulation

100

Proliposomal formulation

80

60

40

20

0 0

4

8

12

16

20

24

Time (h) Fig. 13. Mean plasma concentration of lovastatin hydroxy acid following oral administration of pure drug (n = 3, mean ± SD) and proliposomal formulation (n = 4, mean ± SD) to Sprague Dawley rats.

3.10. Pharmacokinetic study The mean plasma concentration–time profiles of lovastatin and lovastatin hydroxy acid respectively, after oral administration of proliposomal formulation (F9) and the pure lovastatin at a dose of 10 mg/kg as lovastatin are shown in Figs. 12 and 13. Tables 4 and 5 show the pharmacokinetic parameters obtained based on Figs. 12 and 13. From Tables 4 and 5, one can see that the Tmax of lovastatin from pure lovastatin formulation was 0.5 h whereas for proliposomal formulation it was 2 h. The Cmax of pure lovastatin was 140 ng/ml which was 3 times lower compared to Cmax of proliposomal formulation which was 432 ng/ml. The systemic exposure (AUC0–24) of oral proliposomal formulation was also higher compared to the systemic exposure obtained from oral administration of pure lovastatin. This increase in the overall exposure of the proliposomal formulation corresponds to an increase in its relative bioavailability which was 162% when compared to pure lovastatin. Based on the fact that lovastatin has low bioavailability due to its increased hepatic first-pass metabolism, the increased AUC by proliposomal formulation indicates an increase in the systemic exposure of lovastatin with the proliposomal formulation. There was no corresponding increase in the plasma concentration of its metabolite, lovastatin hydroxy acid indicating that the proliposomal 700

Plasma drug conc. (ng/ml)

600 500

Plain lovastatin formulation

400

Proliposomal formulation 300 200 100 0 0

4

8

12

16

20

24

Time (h) Fig. 12. Mean plasma concentration of lovastatin following oral administration of pure drug (n = 3, mean ± SD) and proliposomal formulation (n = 4, mean ± SD) to Sprague Dawley rats.

Table 4 Pharmacokinetic parameters of lovastatin proliposomal and pure drug formulation.

a

PK parameters

Pure drug formulation (n = 3 mean ± SD)

Proliposomal formulation (F9) (n = 4 mean ± SD)

Tmax (h) Cmax (ng/ml) AUC0–24 (ng/ml/h) Relative bioavailabilitya (%)

0.5 140.54 ± 56.57 797.14 –

2 432.76 ± 93.07 1293.42 162.26

Relative bioavailability = [AUCproliposome]/[AUCpure lovastatin].

Table 5 Pharmacokinetic parameters of lovastatin hydroxy acid from proliposomal and pure drug formulation. PK parameters

Pure drug formulation (n = 3 mean ± SD)

Proliposomal formulation (F9) (n = 4 mean ± SD)

Tmax (h) Cmax (ng/ml) AUC0–24 (ng/ml/h)

0.5 78.01 ± 23.23 252.49

1 40.69 ± 7.93 121.54

formulation might be subjected to a lower degree of first-pass metabolism. Tmax of lovastatin hydroxy acid from pure lovastatin was 0.5 h whereas that from proliposomal formulation was 2 h. The Cmax of lovastatin hydroxy acid from pure lovastatin and proliposomal formulation was 78 and 40 ng/ml, respectively. The initial spike with proliposomal formulation (Fig. 12) may be attributed to the free form of the drug in the formulation or due to the formation of oligolamellar structures which are capable of releasing the drug faster as compared to multilamellar vesicles. This observed difference as longer Tmax and higher Cmax of proliposomal formulation as opposed to a shorter Tmax and lower Cmax for pure lovastatin could be due to slower release of lovastatin from the proliposomal formulation in vivo which avoids the firstpass metabolism. However, the hypothesis warrants further mechanistic studies. The reason for shorter Tmax with pure drug is due to its high permeability (BCS class II). Hence, when the drug is administered in the pure form, it is readily absorbed and rapidly metabolized leading to decreased blood levels within a short period of time. This clearly supports our theory that proliposomal formulation can improve dissolution of the drug, leading to better absorption and minimizing the hepatic first-pass metabolism. The

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decreased amount of the active metabolite in the systemic circulation is an indication of the proliposomal formulation playing a role in the reduction of the hepatic first-pass metabolism. Though the study is not intended for the drug lovastatin, we chose this as a model to show that the formation of an active metabolite by first-pass metabolism can be reduced using our formulation approach. Findings from this study shows that proliposomal approach can be used to deliver oral drugs effectively which undergo significant first-pass metabolism. 4. Conclusions The free-flowing proliposomal powder formulation of the present investigation could be incorporated into the capsules or compressed into tablets after processing with other conventional excipients commonly used in the manufacture of capsules and tablets. In order to protect the proliposomal formulation from the harsh gastric condition, we propose that the formulation to be enteric coated. Proliposomal formulations resulted in an increase in the bioavailability of lovastatin, which appears to be due to the combined effect of improved dissolution and minimized firstpass metabolism of lovastatin. We believe that the results obtained demonstrate the use of proliposomal formulations for improved oral delivery of lipophilic drugs which undergoes first-pass metabolism. Acknowledgements We thank Dr. Basavaraj Siddalingappa and Dr. Vijay Nekkanti for useful discussions. We also would like to thank Ms. Charlene Wilke, Bioimaging facility, Northwestern University, Evanston, IL for her help with Cryo-TEM images and Dr. Mary Leonard, Midwestern University for her help with Optical microscopy images. A part of this work was supported from a start-up research grant to NV, by the Chicago College of Pharmacy, Midwestern University, IL. References Agnihotri, S.A., Soppimath, K.S., Betageri, G.V., 2010. Controlled release application of multilamellar vesicles: a novel drug delivery approach. Drug Deliv. 17, 92– 101. Amidon, G.L., Lennernas, H., Shah, V.P., Crison, J.R., 1995. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 12, 413–420. Balimane, P.V., Han, Y.H., Chong, S., 2006. Current industrial practices of assessing permeability and p-glycoprotein interaction. AAPS J. 8, E1–E13. Chen, H., Torchilin, V., Langer, R., 1996. Lectin-bearing polymerized liposomes as potential oral vaccine carriers. Pharm. Res. 13, 1378–1383. Chung, S.J., 1999. Future drug delivery research in South Korea. J. Control. Release 62, 73–79. Custodio, J.M., Wu, C.Y., Benet, L.Z., 2008. Predicting drug disposition, absorption/ elimination/transporter interplay and the role of food on drug absorption. Adv. Drug Deliv. Rev. 60, 717–733. Fiese, E.F., Hagen, T.A., 1986. Preformulation. In: Lachman, L., Liberman, H.A., Kanig, J.L. (Eds.), The Theory and Practice of Industrial Pharmacy, third ed. Varghese Publishing House, Bombay, pp. 171–196.

Fischer, S.M., Brandl, M., Fricker, G., 2011. Effect of the non-ionic surfactant Poloxamer 188 on passive permeability of poorly soluble drugs across Caco-2 cell monolayers. Eur. J. Pharm. Biopharm. 79, 416–422. Frank, K.J., Rosenblatt, K.M., Westedt, U., Holig, P., Rosenberg, J., Magerlein, M., Fricker, G., Brandl, M., 2012. Amorphous solid dispersion enhances permeation of poorly soluble ABT-102: true supersaturation vs. apparent solubility enhancement. Int. J. Pharm. 437, 288–293. Han, X., Ghoroi, C., Dave, R., 2013. Dry coating of micronized API powders for improved dissolution of directly compacted tablets with high drug loading. Int. J. Pharm. 442, 74–85. http://dx.doi.org/10.1016/j.ijpharm.2012.08.004. Henwood, J.M., Heel, R.C., 1988. Lovastatin: a preliminary review of its pharmacodynamic properties and therapeutic use in hyperlipidaemia. Drugs 36, 429–454. Hiremath, P.S., Soppimath, K.S., Betageri, G.V., 2009. Proliposomes of exemestane for improved oral delivery: formulation and in vitro evaluation using PAMPA, Caco-2 and rat intestine. Int. J. Pharm. 380, 96–104. Horter, D., Dressman, J.B., 2001. Influence of physicochemical properties on dissolution of drugs in the gastrointestinal tract. Adv. Drug Deliv. Rev. 46, 75– 87. Hu, Y., Wang, J., Zhi, Z., Jiang, T., Wang, S., 2011. Facile synthesis of 3D cubic mesoporous silica microspheres with a controllable pore size and their application for improved delivery of a water-insoluble drug. J. Colloid Interface Sci. 363, 410–417. http://dx.doi.org/10.1016/j.jcis.2011.07.022. Jung, B.H., Chung, B.C., Chung, S.J., Lee, M.H., Shim, C.K., 2000. Prolonged delivery of nicotine in rats via nasal administration of proliposomes. J. Control. Release 66, 73–79. Kaluderovic, G.N., Dietrich, A., Komeera, H., Kuntsche, J., Mader, K., Mueller, T., Paschke, R., 2012. Liposomes as vehicles for water insoluble platinum-based potential drug: 2-(4-(tetrahydro-2H-pyran-2-yloxy)-undecyl)-propane-1,3diamminedichloroplatinum(II). Eur. J. Med. Chem. 54, 567–572. http:// dx.doi.org/10.1016/j.ejmech.2012.06.004. Kanzer, J., Tho, I., Flaten, G.E., Magerlein, M., Holig, P., Fricker, G., Brandl, M., 2010. In-vitro permeability of screening of melt extrudate formulations containing poorly water-soluble drug compounds using the phospholipid vesicle-based barrier. J. Pharm. Pharmacol. 62, 1591–1598. Li, M., Qiao, N., Wang, K., 2013. Influence of sodium lauryl sulfate and Tween 80 on carbamazepine-nicotinamide cocrystal solubility and dissolution behaviour. Pharmaceutics 5, 508–524. http://dx.doi.org/10.3390/pharmaceutics5040508. Liu, R., Cannon, J.B., Paspal, S.Y.L., 2008. Liposomes in solubilization. In: Liu, R. (Ed.), Water-insoluble Drug Formulation, second ed. CRC Press, Boca Raton, Florida, pp. 375–416. Merisko-Liversidge, E.M., Liversidge, G.G., 2008. Drug nanoparticles: formulating poorly water-soluble compounds. Toxicol. Pathol. 36, 43–48. Patel, S.G., Rajput, S.J., 2009. Enhancement of oral bioavailability of cilostazol by forming its inclusion complexes. AAPS PharmSciTech 10, 660–669. http:// dx.doi.org/10.1208/s12249-009-9249-7. Pavelic, Z., Skalko-Basnet, N., Schubert, R., 2001. Liposomal gels for vaginal drug delivery. Int. J. Pharm. 219, 139–149. Payne, N.I., Timmins, P., Ambrose, C.V., Ward, M.D., Ridgway, F., 1986. Proliposomes: a novel solution to an old problem. J. Pharm. Sci. 75, 325–329. Sarbolouki, M.N., Parsaee, S., Kosary, P., 2000. Mixed micelle proliposomes for preparation of liposomes containing amphotericin B, in-vitro and ex-vivo studies. PDA J. Pharm. Sci. Technol. 54, 240–246. Supersaxo, A., Hein, W.R., Steffen, H., 1991. Mixed micelles as a proliposomal, lymphotropic drug carrier. Pharm. Res. 8, 1286–1291. Suresh, G., Manjunath, K., Venkateswarlu, V., Satyanarayana, V., 2007. Preparation, characterization, and in vitro and in vivo evaluation of lovastatin solid lipid nanoparticles. AAPS PharmSciTech 8, E162–E170. http://dx.doi.org/10.1208/ pt0801024. Tran, P.H., Tran, T.T., Lee, B.J., 2013. Enhanced solubility and modified release of poorly water-soluble drugs via self-assembled gelatin-oleic acid nanoparticles. Int. J. Pharm. 455, 235–240. http://dx.doi.org/10.1016/j.ijpharm.2013.07.025. United States Pharmacopoeia, 2007. Lovastatin Tablets, p. 2502. Williams, H.D., Trevaskis, N.L., Charman, S.A., Shanker, R.M., Charman, W.N., Pouton, C.W., Porter, C.J., 2013. Strategies to address low drug solubility in discovery and development. Pharmacol. Rev. 65, 315–499. http://dx.doi.org/10.1124/ pr.112.005660. Zhao, Y., Liu, X., Wang, J., Zhang, S., 2013. Insight into the cosolvent effect of cellulose dissolution in imidazolium-based ionic liquid systems. J. Phys. Chem. B 117, 9042–9049. http://dx.doi.org/10.1021/jp4038039.