Novel semisolid SNEDDS based on PEG-30-di-(polyhydroxystearate): Progesterone incorporation and in vitro digestion.

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IJP 14753 1–11 International Journal of Pharmaceutics xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

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Pharmaceutical nanotechnology

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Novel semisolid SNEDDS based on PEG-30-di-(polyhydroxystearate): Progesterone incorporation and in vitro digestion

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Tamer H. Hassan, Karsten Mäder *

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Department of Pharmaceutics and Biopharmaceutics, Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck Str. 4, D-06120 Halle (Saale), Germany

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 November 2014 Received in revised form 12 March 2015 Accepted 16 March 2015 Available online xxx

The aim of this work is to study the digestibility of PEG-30-di-(polyhydroxystearate) (Cithrol1 DPHS) and its semisolid novel self-nanoemulsifying drug delivery systems (SNEDDS). Furthermore, the SNEDDSmediated solubility enhancement of the poorly water-soluble drug Progesterone was evaluated in different media. Additionally, the impact of digestion on Progesterone solubilization was investigated in vitro by a pancreatin digestion assay. The Progesterone-loaded semisolid self-nanoemulsifying formulation (F2) was comprehensively characterized by photon correlation spectroscopy (PCS), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and Fourier transform infrared spectroscopy (FTIR). SNEDDS were able to enhance the equilibrium solubility of Progesterone at various media. Only a minor part of Cithrol1 DPHS was digested by pancreatin (less than 6%). Furthermore, protection of Progesterone against digestion-mediated precipitation was observed. Therefore, DPHS containing SNEDDS are attractive candidates for the development of bio robust drug delivery systems for the oral delivery of poorly soluble drugs. ã 2015 Published by Elsevier B.V.

Keywords: Semisolid SNEDDS Cithrol1 DPHS pH-stat Progesterone HPTLC Lipid digestion Selfemuslifying

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1. Introduction

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Self-nanoemulsifying drug delivery systems (SNEDDS) are lipid formulations that have drawn considerable attention and ultimately therapeutical and commercial success in the oral delivery of poorly water-soluble drugs (PWSDs). They provide the PWSDs in the form of solubilized nanodispersions (Williams et al., 2013). Accordingly, the rate-limiting step of PWSDs dissolution is bypassed. Nevertheless, SNEDDS are typically filled in soft gelatin capsules, which might cause the following problems: interaction with the capsule shell, instability, higher production cost and

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Abbreviations: 12-HSA, 12-hydroxystearic acid; CEH, carboxyl ester hydrolase; DPHS, Cithrol1 DPHS; DSC, differential scanning calorimetry; ESR, electron spin resonance; FaSSIF, fasted state simulated intestinal fluid; FeSSIF, fed state simulated Intestinal fluid; FTIR, Fourier transform infrared spectroscopy; HPLC, high performance liquid chromatography; HPTLC, high performance thin-layer chromatography; HS15, Kolliphor1 HS 15; MCM, Capmul1 MCM; PCS, photon correlation spectroscopy; PDI, polydispersity index; PEG, polyethylene glycol; PLA2, phospholipase A2; PLPR2, pancreatic lipase-related protein 2; PWSDs, poorly water-soluble drugs; PXRD, powder X-ray diffraction; rpm, revolutions per minute; SD, standard derivation; SDM, solvent displacement method; SNEDDS, selfnanoemulsifying drug delivery systems; USP, United States pharmacopeia; UTM, Ultra-Turrax1 method. * Corresponding author. Tel.: +49 345 55 25167; fax: +49 345 55 27029. E-mail address: [email protected] (K. Mäder).

possible drug precipitation (Chen et al., 2010; Gullapalli, 2010; Serajuddin et al., 1986; Zhang and DiNunzio, 2012). Therefore, alternative formulation strategies, e.g., the inclusion of SNEDDS into a solid or semisolid dosage form are desirable; nonetheless, very challenging. One approach is the incorporation of the liquid system into a hydrophilic polymer matrix such as PEG (Li et al., 2009) or the use of solid polymeric amphipiles such as Poloxamer 188 (Shah and Serajuddin, 2012). Another approach is the use of semisolid or solid lipids such as Acconon1 C-44 (Patel et al., 2012b), Acconon1 C-50 (Patel et al., 2012a), Cithrol1 DPHS (Hassan et al., 2014), Gelucire1 50/13 (Patel et al., 2012a) and Gelucire1 44/14 (Breitkreitz et al., 2013; Fernandez et al., 2008). Other approaches include extrusion/spheronization (Abdalla and Mäder, 2007), melt granulation (Nanda Kishore et al., 2014), adsorption to solid carriers (Agarwal et al., 2013), spray and freeze drying (Singh et al., 2013), rotary evaporation (Myers and Shively, 1992) and Q2 fluid-bed coating (Krupa et al., 2014). Semisolid SNEDDS are more advantageous than the liquid ones. They have higher viscosity that usually afford high drug stability (Singh et al., 2014) and may protect the PWSDs against precipitation upon minor change in the product temperature. In our previous study, we have reported the novel use of PEG-30-di(polyhydroxystearate) as a lipophilic excipient in the formulation of SNEDDS (Hassan et al., 2014). We have successfully developed semisolid SNEDDS based on Cithrol1 DPHS (DPHS), Capmul1 MCM

http://dx.doi.org/10.1016/j.ijpharm.2015.03.044 0378-5173/ ã 2015 Published by Elsevier B.V.

Please cite this article in press as: Hassan, T.H., Mäder, K., Novel semisolid SNEDDS based on PEG-30-di-(polyhydroxystearate): Progesterone incorporation and in vitro digestion. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.03.044

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T.H. Hassan, K. Mäder / International Journal of Pharmaceutics xxx (2015) xxx–xxx

(MCM) and Kolliphor1 HS 15 (HS15). All formulations were able to produce nanodispersions with an average droplet size diameter below 25 nm. Although formulations were semisolid at room temperature, they have shown high molecular mobility upon dispersion in aqueous media at body temperature, which is very essential for the in vivo performance of the SNEDDS. Oral administration of lipids stimulates the secretion of gastric lipase from the gastric mucosa, with the consequent secretion of pancreatic lipase and co-lipase from the pancreas along with other esterases such as phospholipase A2 (PLA2), carboxyl ester hydrolase (CEH) and pancreatic lipase related protein 2 (PLPRP2). Most of the lipid excipients are esters such as glycerides (MCM), polyethylene glycol esters of fatty acids (HS15 and DPHS), polysorbates and phospholipids. Ester bonds are generally potential substrates to lipolytic enzymes. For example, triglycerides are hydrolyzed into two free fatty acids and 2-monoacylglycerides (N'Goma et al., 2012). The breakdown products are then incorporated in the bile mixed micelles. However, lipid digestion might decrease the solubilization capacity of the carrier, and – as a consequence – drug precipitation might occur. Therefore, in vitro lipid digestion studies, using biorelevant dissolution media containing enzymes and naturally occurring surfactants such as bile salts and lecithin, are recommended to evaluate the performance of SNEDDS (Christophersen et al., 2014; Fatouros and Mullertz, 2008; Phan et al., 2014; Thomas et al., 2012). The most important methods that are used to evaluate the in vitro lipid digestibility are related to pH-stat measurements and high performance thin layer chromatography (HPTLC) combined with spectrodensitometry (Fatouros and Mullertz, 2008; Williams et al., 2012b). The pH-stat method is the simplest and the most widely used method for evaluation of in vitro lipid digestion. However, this method relies on the ionization state of the fatty acids. The titration should be carried out at pH values, which are at least 2 units higher than the pKa of the acid. Long chain fatty acids (e.g., C16 and C18) might be underestimated due to their higher apparent pKa values and partial localization in more lipophilic environments, which makes them non accessible for titration. Therefore, back titration at higher pH values is sometimes used to minimize these problems (Williams et al., 2012b). HPTLC combined with spectrodensitometry provides a better overview of all digestion products such as fatty acids, monoglycerides, diglycerides, triglycerides as well as other digestion products (e.g., PEG esters). On the other side, pH-stat combined with the back titration method provides only the total fatty acid concentration (Sek et al., 2001, 2002). A direct measurement of lipid digestion by optical methods is difficult due to the complex and turbid nature of pancreatin. However, a continuous monitoring of digestion induced translocation of model compounds has been described using electron spin resonance (ESR) (Abdalla and Mäder, 2009; Noack et al., 2012). The beauty of a direct measurement is counterbalanced by the fact that ESR requires paramagnetic molecules and therefore no real drugs can be monitored. Progesterone was selected as a PWSD model with a log P of 3.87 (Bard et al., 2008). Progesterone is a steroid that is used in the treatment of many gynecological disorders (McAuley et al., 1996). It occurs in 2 polymorphic forms: form 1 (a-form) and form 2 (b-form) (Araya-Sibaja et al., 2014). Oral Progesterone administration is associated with poor bioavailability because of its very low water solubility as well as extensive hepatic first-pass metabolism (Sitruk-Ware et al., 1987). Accordingly, Progesterone is a good candidate for SNEDDS-mediated bioavailability enhancement. The aim of this study is to evaluate the digestibility of the novel PEG-30-di-(polyhydroxystearate) based SNEDDS and their excipients, with particular interest on DPHS digestibility. The assessment was done by means of an in vitro pancreatin digestion assay under both fasted (FaSSIF) and fed (FeSSIF) states. Both pH-stat

combined with back titration and HPTLC combined with spectrodensitometry were used in the digestibility evaluation. Furthermore, the Progesterone loading was studied in the single excipients (DPHS, MCM and HS15) as well as in their SNEDDS mixtures. Moreover, the SNEDDS-mediated enhancement of Progesterone equilibrium solubility was evaluated in different media. The droplet size distributions were measured by static or dynamic light scattering. Differential scanning calorimetry (DSC) and Fourier transform infrared spectroscopy (FTIR) were used to determine the possible SNEDDS–Progesterone interactions. Powder X-ray diffraction (PXRD) was used to evaluate the physical state of the Progesterone-loaded SNEDDS.

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2. Materials and methods

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2.1. Materials

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Cithrol1 DPHS was kindly provided by Croda GmbH, Nettetal, Germany. Kolliphor1 HS 15 was a gift from BASF AG, Ludwigshafen, Germany. Capmul1 MCM was kindly supplied by Abitec Corporation, Janesville, WI, USA. Progesterone, porcine bile extract and porcine pancreatin powder (4xUSP specification activity) were purchased from Sigma–Aldrich, Steinheim, Germany. Phospholipon1 90G (purified phosphatidylcholine from soybean lecithin) was purchased from Lipoid, Ludwigshafen, Germany. Titrisol1 (standard sodium hydroxide solution, 1 M) was purchased from Merck, Darmstadt, Germany. Acetonitrile HPLC was purchased from VWR, Darmstadt, Germany. All other materials were of analytical grade and were used as received.

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2.2. Formulation preparation

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DPHS, MCM and HS15 were molten together and mixed (Table 1). The mixtures were cooled down to room temperature and allowed to equilibrate for 24 h at 23 C prior to further analysis (Hassan et al., 2014).

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2.3. In vitro lipid digestion

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2.3.1. Used media Simulated intestinal fluids are composed of Sørensen’s phosphate buffer pH 6.8 (35.6 mM potassium dihydrogen phosphate and 31.1 mM disodium monohydrogen phosphate dihydrate), 150 mM sodium chloride and 5 mM calcium chloride. FaSSIF was supplemented with 5 mM bile extract and 1.25 mM phospholipids while FeSSIF was supplemented with 15 mM bile extract and 3.75 mM phospholipids. As an enzyme source, 450 U/ml of porcine pancreatin was used (Abdalla et al., 2008).

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2.3.2. Preparation of Cithrol1 DPHS dispersions Two methods were used to prepare DPHS dispersions: the Ultra-Turrax1 method (UTM) and the solvent displacement method (SDM).

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2.3.2.1. Ultra-Turrax1 method (UTM). Molten DPHS was mixed with Sørensen’s phosphate buffer pH 6.8 using a rotor-stator mixer (Ultra-Turrax1 T18 basic, IKA, Staufen, Germany) operated at 24,000 rpm for 5 min at room temperature.

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Table 1 Composition of the PEG-30-di-(polyhydroxystearate) SNEDDS formulations (m%). Formulation code

Cithrol1 DPHS

Capmul1 MCM

Kolliphor1 HS 15

F1 F2 F3

20 26.67 30

20 13.33 10

60 60 60


109 110 111 112 113 114 115 116 117 118 119

123 124 125 126 127 128 129 130 131 132 133

136 137 138

141 142 143 144 145 146 147 148

150 151 152

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2.3.2.2. Solvent displacement method (SDM). Molten DPHS was dissolved in acetone to yield 10% m/m solution. Double distilled water was heated to 50 C. The organic solution was added dropwise using a syringe to the double distilled water under agitation (1000 rpm). The produced milky emulsion was then allowed to reach the room temperature under agitation until all acetone was vaporized. 2.3.3. Droplet size measurement The droplet size distribution of DPHS dispersions was measured using static laser diffraction (Mastersizer 2000, Malvern Instruments, Worcestershire, UK). Samples were added until an average laser obscuration of about 8% was obtained. The refractive index was 1.45 and the absorption was 0.001. The stirrer speed was set to 1200 rpm without sonication. The droplet size distribution of HS15 micellar dispersion (1% m/V in Sørensen’s phosphate buffer pH 6.8) was assessed using dynamic light scattering as described in Section 2.3.4. 2.3.4. pH-stat method The digestion experiment was carried out under fasted and fed state conditions. One percent m/V dispersion of each excipient or formulation was prepared in 15 ml FaSSIF or FeSSIF medium. In the case of DPHS dispersions, the respective volume that yield 1% m/V of DPHS in FaSSIF or FeSSIF media was deducted from the contribution volume of the buffer to the respective medium. The respective medium was heated to 371 C in a water bath and stirred at 700 rpm. The temperature was maintained constant during the experiment using a thermocouple. For the blank samples, lipids were replaced by a respective volume of Sørensen’s phosphate buffer pH 6.8. A pH electrode connected to an autotitrator (DL 21, Mettler Toledo, Giessen, Germany) was placed into the liquid. The start pH was adjusted to 6.8. The digestion was initiated by the addition of pancreatin powder equivalent to 450 U/ml of pancreatic lipase activity. The autotitrator kept the pH constant at 6.8 throughout the experiment by adding 0.1 M sodium hydroxide (Titrisol1). The cumulative consumption of sodium hydroxide was recorded over 120 min (Noack et al., 2012). 2.3.5. Back titration method The pH-stat titration was performed as described above for 120 min. At the end of the titration process, the pH was raised from 6.8 to 9 and the required volume of sodium hydroxide was recorded. 2.3.6. Lipid analysis by high performance thin layer chromatography (HPTLC) combined with spectrodensitometry The pH-stat titration was performed as described above. At a predetermined time schedule, 100 ml were withdrawn and diluted with 900 ml of a chloroform and methanol (1:1 v/v) mixture. The organic mixture stops the enzymatic reaction and causes the precipitation of the enzymes. The mixture was then centrifuged at 12,045 g for 10 min in an Eppendorf centrifuge (MiniSpin, Eppendorf AG, Hamburg, Germany) and the supernatant was filled into glass vials and stored in the dark at 20 C for further investigation. The HPTLC analysis was performed according to Klein (2013); Oidtmann, (2013); Rübe et al. (2006) and Sek et al. (2001). 12Hydroxystearic acid (12-HSA) standard solutions were prepared. Samples and standards were separated simultaneously on each plate to avoid inter-assay variations. Standard 12-HSA solutions corresponding to 0.1–2 mg were applied together with 10 ml of samples on silica gel plates (HPTLC Silica gel 60, Merck, Darmstadt, Germany) using an automatic TLC Sampler 4 (CAMAG, Muttenz, Switzerland). The separation was performed by HPTLC (AMD 2, CAMAG, Muttenz, Switzerland) employing an 11-step gradient

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based on hexane and ethyl acetate (Table 2). Afterwards, the plates were submerged in a copper sulfate solution (15% copper sulfate pentahydrate, 8% phosphoric acid (85% m/V) and 5% methanol) for 20 s. Residual liquid was removed and the plates were heated in an oven at 150 C for 20 min. The stained spots on the plate were quantified by spectrodensitometry at 675 nm (CAMAG TLC Scanner 3, CAMAG, Muttenz, Switzerland). The optical densities were converted into masses using a calibration curve.

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2.4. Incorporation of Progesterone

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2.4.1. Progesterone loading Progesterone equilibrium solubility was assessed in DPHS, HS15, MCM as well as the SNEDDS at 50 C. Progesterone was added in excess to 5 g of the corresponding lipid in sealed tubes. Samples were incubated in an end over end mixer at 50 1 C for 24 h. Afterwards, they were centrifuged for 5 min (Avanti J-20XP centrifuged equipped with JLA-16.250 rotor, Beckman Coulter, Miami, USA) at 12,045 g at 40 C. The supernatant was diluted with acetonitrile to a suitable concentration range then centrifuged again for 5 min and analyzed by HPLC.

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2.4.2. Progesterone equilibrium solubility Progesterone equilibrium solubility measurements were carried out in the following media: double distilled water, 0.1 N HCl, phosphate buffer pH 6.8 USP and simulated intestinal fluids in both fasted (FaSSIF) and fed state (FeSSIF) as well as blank dispersion media containing 1% m/V of the SNEDDS. Progesterone was added in excess to 5 ml of the dispersion medium in sealed tubes. Samples were incubated in an end over end mixer at 37 1 C for 24 h. Afterwards, they were centrifuged for 5 min in an Eppendorf centrifuge (MiniSpin, Eppendorf AG, Hamburg, Germany) at 12,045 g. The supernatant was filtered through a 0.2 mm Millipore filter, diluted with acetonitrile to an appropriate concentration range then centrifuged again for 5 min and analyzed by HPLC (Abdalla et al., 2008).

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2.4.3. Droplet size distribution F2 dispersions with or without Progesterone (15–27 mg/g) were analyzed for droplet size distribution by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) with a back scattering angle of 173 . One percent dispersion m/V of F2 was prepared in 10 ml of double distilled water (20 mg/ g), 0.1 N HCl (27 mg/g) or phosphate buffer pH 6.8 USP (15 mg/g). The dispersions were incubated in a preheated 37 1 C end over end mixer rotating at 10 rpm. After complete dispersions, samples were measured at 37 C after equilibration time of 20 min. The viscosity of the samples were determined at 37 C prior to the analysis as described in (Hassan et al., 2014). Each sample was measured 3 times with 12–17 runs over 10 s. The measurement position was fixed in the middle of the cuvette. Z-average

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Table 2 HPTLC gradients used for the separation of long chain lipids. Step

Migration distance (mm)

Hexane (% v/v)

Ethyl acetate (% v/v)

1 2 3 4 5 6 7 8 9 10 11

20 25.1 30.2 35.3 40.4 45.5 50.6 55.7 60.8 65.9 71

70 73 76 79 82 85 88 91 94 97 100

30 27 24 21 18 15 12 9 6 3 0


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229 230 231 232 233 234 235 236 237

239 240 241 242 243 244 245 246 247 248 249 250 251

253 254 255 256 257 258 259 260 261 262 263 264 265

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diameters and polydispersity indices (PDI) were determined by the cumulant analysis software of the instrument (Zetasizer Software 6.32, Malvern Instruments, Worcestershire, UK). 2.4.4. Differential scanning calorimetry (DSC) DSC investigations were performed using a nitrogen purged Netzsch DSC 200 (Netzsch-Gerätebau GmbH, Selb, Germany). All samples (approx. 15 mg) were accurately weighed in aluminum pans and hermetically sealed. Small sample weight (approx. 0.3 mg) was used in case of pure Progesterone. DSC thermograms were generated by cooling the samples to 20 C. The samples were then equilibrated for 3 min and the heating thermograms at a rate of 5 K/min up to 200 C were recorded. 2.4.5. Powder X-ray diffraction (PXRD) Samples were measured using a powder X-ray diffractometer (STADI MP, Stoe, Darmstadt, Germany) equipped with a curved Ge (1 11) monochromator (CoKa radiation, l = 0.178896 nm) operated at 40 KV and 30 mA and an image plate detector at room temperature. The samples were scanned from 5 (2u) to 123 (2u) with a step size of 0.03 (2u) and a count time of 2000 s per step. 2.4.6. Fourier transform infrared spectroscopy (FTIR) Samples were measured using a FTIR spectrometer (IFS 28, Bruker, Ettlingen, Germany) equipped with an ATR attachment (Thermo Spectra Tech, Connecticut, USA), a Fresnel ZnSe crystal (angle of incidence 45 , diameter 20 mm) and DTGS detector. Each spectrum (680–4000 cm1) was collected with 32 scans and a resolution of 2 cm1. 2.4.7. Possible drug precipitation upon digestion Progesterone was incorporated at 70% of its saturation solubility in the formulations (34 mg/g F1; 38.4 mg/g F2; 45.2 mg/g F3) in order to maintain a constant thermodynamic activity across all formulations. Progesterone was dissolved in the molten SNEDDS at 50 C. Progesterone-loaded SNEDDS were cooled down to room temperature and allowed to equilibrate for 24 h prior to the digestion study. The lipid formulations were accurately weighed into sealed glass tubes. Afterwards, the freshly prepared FaSSIF or FeSSIF was added. One percent m/V was allowed to disperse prior to initiation of digestion in an end over end mixer rotating at a rate of 10 rpm at 37 1 C. A dispersion volume of 7.5 ml was used in all experiments. The digestion was initiated by the addition of pancreatin powder equivalent to 450 U/ml of pancreatic lipase activity. 200 ml samples were withdrawn at regular time intervals and centrifuged at 12,045 g for 5 min in an Eppendorf centrifuge (MiniSpin, Eppendorf AG, Hamburg, Germany) in order to separate the micellar phase from precipitated drug, calcium salts of fatty acids and precipitated parts of pancreatin. An aliquot of the supernatant was diluted with acetonitrile and centrifuged again for 5 min at 12,045 g. The organic supernatant was filled into a vial for HPLC analysis. 2.4.8. HPLC method The HPLC system consisted of a Jasco model PU-1580 pump equipped with LG-1580-04 quaternary gradient unit, degasser, AS 1559 intelligent autosampler, UV 1559 intelligent UV/vis detector. Purospher1 Star RP-18 endcapped (5 mm) column (Merck, Darmstadt, Germany) was used as an analytical column. Acetonitrile and double distilled water in a ratio 70:30 v/v were used as the mobile phase at a flow rate of 1 ml/min. The oven temperature was adjusted to 40 C. Standard Progesterone solutions (5–250 mg/ml) were prepared in acetonitrile. 10 ml were injected and the column effluent was monitored at a wavelength of 240 nm. The retention time for Progesterone was found to be 3.9 min.

2.5. Statistical analysis

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Unless otherwise stated, results are represented as mean SD (n = 3). F-test was performed to check if the samples have an equal variance. Significance between the mean values was tested using Student’s t-test. Probability values P < 0.05 were considered statistically significant.

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3. Results and discussion

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3.1. In vitro lipid digestion

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Oral administration of lipids stimulates the secretion of several lipolytic enzymes such as lipases (gastric and pancreatic), PLA2, CEH and PLPRP2. These lipolytic enzymes hydrolyze the ester bonds in many lipid excipients (N'Goma et al., 2012). The digestion products are mostly fatty acids and partial glycerides. Fatty acids are titrated by means of a pH-stat method. The rate and extent the titration volume needed to keep the pH constant reflects the degree of lipid digestibility.

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3.1.1. Digestibility of the excipients

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3.1.1.1. Preparation and droplet size of excipient dispersions. Lipase enzymes work on the oil/water interface, which is highly affected by the droplet size. Therefore, the droplet size distributions of the excipient dispersions (1% m/V in Sørensen’s phosphate buffer pH 6.8) were evaluated by static or dynamic laser diffraction (Table 3). MCM formed unstable dispersions in Sørensen’s phosphate buffer pH 6.8, which have a very broad droplet size distribution and is dependent on the agitation speed. Furthermore, MCM droplets coalescence occurred after stopping the agitation. Therefore, it was difficult to evaluate its particle size distribution. HS15 formed stable micellar dispersions with an average micellar size of about 11 nm. Compared to MCM and HS15, DPHS was difficult to be dispersed in the buffer by simple agitation. DPHS is semisolid at room temperature and does not completely melt at 37 C. Therefore, high shear stress was applied using Ultra-Turrax1 (Ultra-Turrax1 method, UTM). The produced dispersion had a wide droplet size distribution with an average size of about 232 mm. In order to reduce the droplet size and the polydispersity of the dispersion, another preparation method was developed (solvent displacement method, SDM). The SDM resulted in about 13 times reduction of DPHS average droplet size and decrease in the polydispersity of the dispersion (Fig. S1 of the Supplementary data). DPHS dispersions prepared by both methods were evaluated for their digestibility.

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3.1.1.2. pH-stat method. Fig. 1 shows the consumption of sodium hydroxide during the in vitro digestion of the excipients in both FaSSIF and FeSSIF media. The titration curve has normally 2 phases. The first phase (5–15 min) shows a high initial consumption of sodium hydroxide, corresponding to a high output of free fatty acids. The second phase shows a slow, constant increase in the free

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Table 3 Droplet size distribution data of Cithrol1 DPHS and Kolliphor1 HS 15 dispersions. Excipient

d (0.1) d (0.5) d (0.9) D [4,3] (mm) (mm) (mm)

Cithrol1 DPHS–UTMa Cithrol1 DPHS–SDMa Kolliphor1 HS 15b

23.4 5.2

208.2 13.6

470.8 35.5

Z-average PDI (nm)

232.4 mm 18.3 mm 10.1 nm 11.1

0.014

UTM is the Ultra-Turrax1 method and SDM is the solvent displacement method. a Determined by static laser diffraction. b Determined by dynamic laser diffraction.


328 329 330 331

335 336 337 338 339 340 341

344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366

368 369 370 371 372

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fatty acids output. For blank digestion media, the digestion speed was approximately the same in fasted and fed state conditions. However, the total consumption of sodium hydroxide was higher for the blank FeSSIF compared to the blank FaSSIF. This effect can be attributed to the higher content of both phospholipids and bile salts in FeSSIF compared to FaSSIF. Phospholipids are substrates to PLA2, PLPRP2 and CEH. In addition, bile salts clean the lipid–water interface by incorporation of the digestion products, especially long chain monoglycerides and fatty acids, into mixed micelles. Otherwise, accumulation of the digestion products at the interface would inhibit the action of pancreatic lipase. Furthermore, phospholipids digestion is enhanced when incorporated in bile salts mixed micelles (N'Goma et al., 2012). HS15, being a PEG ester of 12-hydroxystearic acid, is susceptible to digestion. It is reported that lipase has no significant activity on PEG esters digestion compared to PLPRP2 and CEH (Fernandez et al., 2008). Furthermore, incomplete digestion of HS15 was reported due to its inhibitory effect on the lipolytic enzymes (Christiansen et al., 2010; Klein, 2013). The digestion of HS15 was significantly higher in FeSSIF medium compared to FaSSIF (Fig. 1) due to the higher content of phospholipids and bile salts. The MCM samples showed the highest rate and extent of digestion in both FaSSIF and FeSSIF media. In contrast, the sodium hydroxide consumptions of the DPHS dispersions prepared by UTM were comparable to those of the blank media (control) (Fig. 1). The titration curves of DPHS dispersions prepared by SDM did also match with the control (blank) in the FaSSIF medium. However, a small degree of digestibility was observed under FeSSIF conditions (Fig. 2). The pH-stat titration method is strongly sensitive to the degree of fatty acids ionization, which is governed by their pKa, pH and the ionic strength of the media. The apparent pKa of fatty acids is dependent on their phase behavior, chain length as well as the bile salt concentration (Klein, 2013; Sek et al., 2002; Small et al., 1984).

5

Fig. 2. Fatty acid concentrations determined by pH-stat titration during the in vitro digestion of Cithrol1 DPHS in both FaSSIF and FeSSIF media (mean SD, n = 3).

Medium chain fatty acids have lower pKa values than long chain ones. This leads to a higher degree of ionization at pH 6.8. In addition, they are more water-soluble compared to the long chains fatty acids. Consequently, medium chain fatty acids are easily detected by the pH-stat method while long chain fatty acids are often underestimated. Hence, other methods should be used to reevaluate the digestibility of long chain lipid excipients (DPHS and HS15).

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3.1.1.3. Back titration method. Back titration is a potential option to reevaluate the digestibility of long chain lipids (Fernandez et al., 2008). Due to their higher apparent pKa values, long chain fatty acids are not completely ionized at pH 6.8 (Fukuda et al., 2001; Maeda et al., 1992). Therefore, the pH of the digestion medium was raised from 6.8 to 9 at the end of the pH-stat titration. The required volume of sodium hydroxide was recorded (Table 4). The difference in sodium hydroxide consumption between excipients and blank medium reflects the degree of underestimation by the pH-stat method. In both FaSSIF and FeSSIF media, the required sodium hydroxide volume for all excipients was significantly higher compared to the blank media (t-test, a = 0.05). Furthermore, the

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Table 4 Volume of sodium hydroxide (ml) required to raise the pH from 6.8 to 9 at the end of pH-stat titration (mean SD, n = 3).

Fig. 1. Fatty acid concentrations determined by pH-stat titration during the in vitro digestion of the excipients in both FaSSIF and FeSSIF media (mean, n = 3).

Blank media Cithrol1 DPHS–UTM Cithrol1 DPHS–SDM Kolliphor1 HS 15 Capmul1 MCM

FaSSIF

FeSSIF

6.04 0.27 6.49 0.09 6.67 0.09 6.36 0.10 6.55 0.04

5.15 0.17 5.40 0.06 5.82 0.06 5.48 0.04 5.77 0.03

UTM is the Ultra-Turrax1 method and SDM is the solvent displacement method.


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spectrodensitometry was used (Sek et al., 2001). Under our experimental conditions, the recovery rate of MCM digestion products (with chain lengths of C8 and C10) was low and highly variable due to evaporation during the processing of the plate at 150 C. These problems do not occur with long chain lipids (C16, C18 and higher). 12-Hydroxystearic acid (12-HSA) is the main digestion product of HS15 and DPHS. Therefore, calibration curves of standard 12-HSA solutions were prepared and used to quantify the fatty acid released during their digestion process. The relation between the mass of 12-HSA and the area under the peak was linear until saturation was achieved. Afterward, a change in the slope was observed. Therefore, the calibration curves were fitted as Hill slope (Fig. S2 of the Supplementary data). The results of HPTLC–spectrodensitometry analysis are presented in Fig. 3. The release rate of 12-HSA was faster in the first 30 min. Afterwards, a slower 12-HSA release was observed. The digestibility of both DPHS and HS15 was underestimated by pH-stat combined with back titration method in both media (Table 5). The digestibility of HS15 was higher than DPHS despite its lower ester value (125 mg KOH/g for DPHS compared to 60 mg KOH/g for HS15). The difference in droplet size and molecular weight could attribute to this finding. Furthermore, DPHS is an ABA block copolymer. The middle block (B) is the hydrophilic poly(ethylene oxide) while the two outer blocks (A) are poly(12-HSA). Most lipolytic enzymes act on the lipid/water interface. Due to the DPHS interfacial packing, the lipophilic part (poly(12-HSA)) is mainly shielded in the oil core from the action of the lipolytic enzymes while few ester bondages are available at the interface. In addition, after cleavage at the interface the produced poly(12-HSA) is expected to be slowly digested due to its relatively low polarity. As expected, DPHS dispersions prepared by SDM showed higher 12-HSA release compared to dispersions prepared by UTM. However, both dispersions showed less than 6% of the expected fatty acid release (Table 5). The incomplete digestion of long chain lipids was reported (N'Goma et al., 2012; Williams et al., 2012a,b). Furthermore, the digestion rate was higher in FeSSIF compared to the FaSSIF. Because of their low polarity, long chain fatty acids are accumulated on the interface with the consequent deactivation of lipolytic enzymes. Consequently, the incorporation of long chain fatty acids into the bile salts mixed micelles is essential for long chain lipids digestion (N'Goma et al., 2012). Therefore, the digestibility of long chain lipids is more sensitive to bile salts concentration than medium chain ones (Sek et al., 2002).

1

Fig. 3. Digestion induced formation of 12-hydroxystearic acid from Cithrol DPHS and Kolliphor1 HS 15 in both FaSSIF and FeSSIF media analyzed by HPTLC – spectrodensitometry (mean SD, n = 3). 427 428 429 430 431 432 433 434 435

consumption of sodium hydroxide was lower in FeSSIF compared to FaSSIF. DPHS dispersions prepared by SDM showed a higher sodium hydroxide consumption compared to those prepared by UTM. The difference in the droplet size contributes to this result. Furthermore, the results also proved the underestimation of MCM digestibility by pH-stat method at pH 6.8. 3.1.1.4. Lipid analysis by high performance thin layer chromatography (HPTLC) combined with spectrodensitometry. In order to determine precisely the extent of excipients digestion, HPTLC combined with

Table 5 Comparison between the fatty acids concentration determined by both pH-stat method and HPTLC–spectrodensitometry after 120 min of digestion (mean, n = 3) of 1% (m/V) Kolliphor1 HS15 and Cithrol1 DPHS dispersions in FaSSIF and FeSSIF. Excipient

FaSSIF Kolliphor1 HS15 Cithrol1 DPHS–UTM Cithrol1 DPHS–SDM FeSSIF Kolliphor1 HS15 Cithrol1 DPHS–UTM Cithrol1 DPHS–SDM

Fatty acids available for digestion (mM)a

Fatty acids determined by HPTLC Fatty acids determined by pH-stat Percentage of fatty acids detected by Digestion (%) method (mM) method (mM)b pH-stat method (%)

10.69

2.42

0.80

33.1

22.6

22.28

0.63

0.45

71.4

2.8

22.28

0.95

0.63

66.3

4.3

10.69

2.51

0.87

34.7

23.5

22.28

0.90

0.25

27.8

4.0

22.28

1.26

0.70

55.6

5.7

UTM is the Ultra-Turrax1 method and SDM is the solvent displacement method. a Fatty acids estimation is based on the ester values of both Cithrol1 DPHS (125 mg KOH/g) and Kolliphor1 HS 15 (60 mg KOH/g). b Calculations are based on the titrated fatty acids obtained during the pH-stat method and the back titration method. The fatty acids titrated during blank media digestion were subtracted from the results.


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Table 7 Equilibrium solubility of Progesterone in excipients and formulations at 50 C (mean SD, n = 3).

Fig. 4. Fatty acid concentrations determined by pH-stat titration during the in vitro digestion of the semisolid SNEDDS formulations in both FaSSIF and FeSSIF media (mean, n = 3). 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494

3.1.2. Digestibility of the formulations DPHS formulations (SNEDDS) showed a significant increase in sodium hydroxide consumption compared to blank media (Fig. 4). Both DPHS and HS15 are slowly and incompletely digested (Table 5). On the other hand, based on its ester values (270 mg KOH/g) and pH-stat combined with back titration digestibility data, MCM showed complete digestibility in FaSSIF (99.7%) and FeSSIF (100.1%). Accordingly, the source of fatty acids, available for pH-stat titration, is mainly MCM. Therefore, a decreasing tendency of the overall fatty acids available for titration was observed with decreasing the MCM contents in the formulations and the digestibility of the SNEDDS did not significantly changed in either FaSSIF or FeSSIF media. Based on the fatty acids released during the excipients digestion and their ester values, an estimation of expected fatty acids release during the SNEDDS dispersions digestion was calculated (Table 6). According to the calculations,

Excipient/formulation

Progesterone solubility (mg/g)

Cithrol1 DPHS Kolliphor1 HS 15 Capmul1 MCM F1 F2 F3

35.5 2.3 55.7 1.2 104.7 3.4 49.0 2.1 54.8 2.7 64.6 3.3

an incomplete digestion of DPHS formulations was observed and only about 30–40% of the possible ester groups were cleaved under the experimental conditions. This result was surprising because lipases work on the oil/water interface, which is strongly dependent on the droplet size. Due to the very small size (below 25 nm) of the SNEDDS formulations (Hassan et al., 2014), a high digestibility of the formulations was expected due to their larger interfacial surface area. An explanation to this result could depend on the difference in the interfacial packing between the single excipient and formulations. In case of formulations, PEG loops are projecting on the interface. Therefore, a steric hindrance could be developed on the interface that hinders the lipase anchoring and lipase activation. Feeney et al. (2014) have reported the resistance of stealth (PEGylated) nanoparticles to the pancreatin-mediated digestion due to the formation of a steric barrier on the nanoparticles surface. The inhibitory effect was found to be dependent on the molecular weight and the packing density of the surface PEG.

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3.2. Incorporation of Progesterone

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3.2.1. Progesterone loading The maximum solubility of the drug in its formulation limits the maximum drug load. Table 7 shows the equilibrium solubility of Progesterone in excipients and formulations. DPHS is semisolid at room temperature and is not completely molten at 37 C. Therefore, the measurements were performed at 50 C. The Progesterone solubility in MCM was significantly higher than the other excipients. Progesterone solubility in DPHS was lower than HS15. Surprisingly, increasing the ratio of DPHS in the formulations resulted in an increase in Progesterone solubility. The reason could be the enhanced mobility of DPHS in the formulation compared to its mobility alone as observed in our previous study (Hassan et al., 2014).

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Table 6 Comparison between the estimated and measured fatty acids concentration determined by pH-stat method after 120 min of digestion (mean, n = 3) of the 1% (m/V) SNEDDS dispersions in FaSSIF and FeSSIF. Formulation Predicted amount of fatty acids available for titration based on Fatty acids determined by pH-stat method (mmol)a Percentage formulations digestibility based on ester values (%) the ester values of the excipients (mmol) MCM

DPHS

HS15

Total

FaSSIF F1 F2 F3

144 96 72

32 43 48

206 206 206

382 345 326

143 129 112

37.4 37.4 34.4

FeSSIF F1 F2 F3

144 96 72

32 43 48

206 206 206

382 345 326

128 104 88

33.5 30.1 27.1

a Calculations are based on the titrated fatty acids obtained during the pH-stat method. The fatty acids titrated during blank media digestion were subtracted from the results.


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Fig. 5. Equilibrium solubility of Progesterone in different media in the presence of 1% m/V of different self-nanoemulsifying formulations at 37 C (mean SD, n = 3). 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560

3.2.2. Equilibrium solubility Blank formulations were mixed with different dispersion media (1% m/V) in order to assess the impact of their presence on Progesterone equilibrium solubility (Fig. 5). Furthermore, the solubility of Progesterone in bio-relevant dissolution media (FaSSIF and FeSSIF) was evaluated. Preliminary studies have shown that the solubility of Progesterone in the respective media did not change after 24 h of incubation in an end over end mixer. Therefore, the equilibrium solubility was conducted for 24 h. The solubility of Progesterone was enhanced when incorporated in FaSSIF and FeSSIF media compared to the bio-irrelevant ones. The presence of the SNEDDS, even at low concentration of 1% m/V, successfully enhanced the equilibrium solubility of Progesterone in all media. Despite the nearly same Progesterone solubility in both pH 1.2 and 6.8, a pronounced enhancement of Progesterone equilibrium solubility was observed for the SNEDDS formulations in 0.1 N HCl compared to phosphate buffer pH 6.8. The SNEDDS-mediated Progesterone solubility enhancement in 0.1 N HCl is expected due to the acid values of both DPHS and MCM, which constitute the lipophilic core of the produced nanoemulsions. The acid values of DPHS and MCM is 10 mg KOH/g and 4 mg KOH/g, respectively. Because the free carboxylic groups are protonated at the acidic pH, the polarity of the lipophilic core of the nanoemulsions is decreased with the subsequent increase of the solubilization power to lipophilic drugs. 3.2.3. Droplet size distribution Based on our previous study (Hassan et al., 2014), we have found that the dispersibility of the F1 formulation is pHdependent. On the other hand, both F2 and F3 formulations have shown pH-independent dispersibility. F2 formulation was found to be slightly superior to F3 one in the dispersion time and the mobility studies. Therefore, F2 was selected for the further studies. Progesterone was incorporated in F2 at a level of 15–27 mg/g. The selection of this Progesterone level is based on its equilibrium

solubility assessed in Section 3.2.2. At this level, no Progesterone precipitation is expected upon dilution in the three test media. Furthermore, Progesterone remained in the solubilized state in the SNEDDS and no phase separation or crystallization of the drug was observed upon storage at 23 C for few months. The droplet size distribution of 1% m/V dispersions of F2, with or without centrifugation, in different media at 37 C is summarized in Table 8. F2 formulation showed mono modal volume distributions in all media with an average hydrodynamic diameter of less than 25 nm. The effect of the ionic strength, pH and filtration on the droplet size distribution of F2 was minimal (Hassan et al., 2014). Centrifugation of the samples (12,045 g/5 min) resulted in the reduction of the PDI with a minor change in the Z-average. The lipid recovery after centrifugation ranged from 94% to 99% depending on the dispersion media. Higher lipid recovery was observed in double distilled water and phosphate buffer pH 6.8 media compared to 0.1 N HCl medium. Incorporation of Progesterone did not cause a pronounced change in the average hydrodynamic diameter.

561

3.2.4. Differential scanning calorimetry (DSC) In order to assess the influence of Progesterone incorporation on the physical state and melting behavior of the semisolid SNEDDS (F2), DSC thermograms of Progesterone-loaded SNEDDS, Progesterone and the parent SNEDDS (F2) are shown in Fig. 6. Two levels of Progesterone were tested: 15 mg/g and 50 mg/g. The physical state and the melting point were not affected by incorporation of Progesterone at the level of 15 mg/g. However, a depression in the SNEDDS melting point was observed at the level of 50 mg/g. The reduction of the melting point at the higher Progesterone content was expected as the molecularly dissolved Progesterone may act as a solute or impurity in the semisolid SNEDDS with the consequent depression of their melting point. Progesterone showed a melting endotherm at about 130 C, which can be detected even in low sample weight (0.3 mg). The absence of this characteristic melting endotherm in the Progesterone-loaded SNEDDS is often interpreted in the direction of its molecular solubility. However, Progesterone could also solubilize in the freshly molten SNEDDS during the DSC measurement. Therefore, DSC cannot be used to confirm the solubility of Progesterone in the SNEDDS.

580

3.2.5. Powder X-ray diffraction (PXRD) PXRD was used to evaluate the solid state of the Progesteroneloaded SNEDDS (F2) as well as Progesterone solubility in the SNEDDS. PXRD patterns of Progesterone-loaded SNEDDS, Progesterone and the parent SNEDDS are shown in Fig. 7. The diffraction pattern of SNEDDS indicates the presence of a partial amorphous or liquid and a partial crystalline system with two strong short spacing at 4.6 Å and 3.8 Å (Hassan et al., 2014). The presence of an amorphous or liquid part in the formulation could be advantageous. It is generally well known that crystalline structures are not able to accommodate foreign molecules. Therefore, crystallization leads to drug expulsion (Mehnert and Mäder, 2001; Müller et al.,

601

Table 8 Average droplet size diameter of the nanoemulsion produced by 1% (m/V) dispersions of F2 in different media measured by dynamic light scattering at 37 C (mean, n = 3). Formulation

F2 F2 (centrifuged)b F2 (Progesterone) F2 (Progesterone–centrifuged)b a b

Double distilled water

0.1 N HCl

Z-average (nm)

PDI

Recovery (%)

18.2 17.8 20.1 19.3

0.245 0.15 0.214 0.167

– 97.1 – 98.1

a

Phosphate buffer pH 6.8

Z-average (nm)

PDI

Recovery (%)

21.5 19.1 23.5 20.1

0.299 0.09 0.287 0.122

– 95.1 – 93.5

a

Z-average (nm)

PDI

Recovery (%)a

21.7 21.2 23.2 21.2

0.272 0.114 0.262 0.117

– 98.4 – 99.2

The percentage recovery is calculated based on the concentration measured during the dynamic light scattering measurements before and after centrifugation. 12045 g/5 min.


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Fig. 6. Heating DSC thermograms of formulation F2, Progesterone-loaded F2 and Progesterone. 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635

2000). The PXRD pattern of Progesterone conformed to the diffraction pattern of the orthorhombic Progesterone form 1 (Araya-Sibaja et al., 2014). Progesterone-loaded SNEDDS (15 mg/g and 50 mg/g) retained the same diffraction pattern of the parent SNEDDS and did not show any diffraction pattern related to Progesterone. However, under our experimental condition the solubility of Progesterone in the SNEDDS could not be confirmed because of its low concentration. A physical mixture between microcrystalline cellulose and Progesterone at the same concentration (15 mg/g) did also not show the strong diffraction pattern of the Progesterone due to the low amount of the drug (insufficient sensitivity) (Fig. S3 of the Supplementary data). 3.2.6. Fourier transform infrared spectroscopy (FTIR) FTIR was used to study the interaction between the SNEDDS and drug. FTIR spectra of F2, Progesterone and Progesterone-loaded F2 are shown in Fig. 8. FTIR spectra are typically composed of two regions: the fingerprint region (below 1500 cm1) and the function group region (above 1500 cm1). Progesterone fingerprint region conformed to the Progesterone polymorph 1 (Araya-Sibaja et al., 2014). Furthermore, its FTIR spectrum showed 3 strong peaks at 1615 cm1 (C¼C stretch), 1661 cm1 (conjugated cyclic C¼O stretch) and 1698 cm1 (non-conjugated aliphatic methyl C¼O stretch) as well as broad band at 2975–2850 cm1 corresponding

Fig. 7. Wide angle powder X-ray diffractograms of formulation F2, Progesteroneloaded F2 and Progesterone.

9

to the skeleton C—H stretch (Zoppetti et al., 2007). The strong peaks in the SNEDDS was assigned according to the guidelines developed by Guillén and Cabo (1997) for lipids FTIR (Guillén and Cabo, 1997). Three strong peaks were observed at 2922 cm1 (CH2, asymmetric stretch), 2854 cm1 (CH2, symmetric stretch) and 1733 cm1 (C¼O, ester stretch). Additionally, a broad peak at 3474 5.4 cm1 (OH) was observed. Progesterone-loaded SNEDDS showed a little deviation in the SNEDDS fingerprint region and a significant (t-test, a = 0.05, n = 3) shift in the OH region (3461 0.3 cm1). This shift could be due to the intermolecular H-bonding between Progesterone and the SNEDDS and can prove Progesterone solubilization in the SNEDDS. There was no pronounced difference in the FTIR spectra between both Progesterone concentrations.

636

3.2.7. Possible drug precipitation upon digestion Orally administered SNEDDS should be able to keep the incorporated PWSD in the solubilized form throughout the gastrointestinal tract. However, lipid digestion usually changes their solubilization capacity and drug precipitation sometimes occurs (Pouton, 2006). Therefore, the influence of the digestion on the drug solubilization and the possible precipitation should be studied in both fasted and fed state. Fig. 9 demonstrates that Progesterone remained almost completely dissolved after the digestion of 1% m/V of the formulations in both FaSSIF and FeSSIF. A small amount of Progesterone starts to precipitate at the beginning of the digestion process. The maximum precipitation has been seen after 15 min of digestion in both media. The reason could be the higher digestibility of the formulation (mainly the MCM part) that is usually observed at this period (the first phase of the pH-stat titration diagram, Fig. 4). The precipitation rate was lower in FeSSIF compared to FaSSIF. Afterwards, an increase in the solubilization capacity of the formulations was observed. DPHS formulations are composed mainly of long chain lipids (DPHS and HS15). Long chain lipids have lower polarity and show slower, incomplete digestibility (Table 5) compared to medium chain lipids (Sek et al., 2002). Consequently, they have higher capability to maintain the solubilization capacity of the accompanied PWSDs when incorporated into the bile mixed micelles, even in lower concentrations (Porter et al., 2007). More than 80% (FaSSIF) and 90% (FeSSIF) of Progesterone remained in the solubilized form after 4 h of digestion. The difference between DPHS formulations was not pronounced.

650

Fig. 8. FTIR spectra of formulation F2, Progesterone-loaded F2 and Progesterone.


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Fig. 9. Percentage of Progesterone which remained dissolved after the digestion of 1% (m/V) of the self-nanoemulsifying formulations in both FaSSIF and FeSSIF media (mean SD, n = 3). 677

4. Conclusion

678

697

The digestibility of PEG-30-di-(polyhydroxystearate) is for the first time explored. An incomplete (

Novel semisolid SNEDDS based on PEG-30-dipolyhydroxystearate: development and characterization.

SNEDDS Containing Poorly Water Soluble Cinnarizine; Development and in Vitro Characterization of Dispersion, Digestion and Solubilization.

The effect of oestrogen and progesterone on the incorporation of tritiated thymidine in mouse uteri in vitro.

Progesterone in the uterus. X. Dependence of the in vitro progesterone metabolism on progesterone binding in rat uterus.

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Progesterone in the uterus. VI. On the question of the in vitro progesterone metabolism in human endometrium and myometrium.

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Progesterone synthesis by luteal mitochondria in vitro.

Measuring ribonucleotide incorporation into DNA in vitro and in vivo.

Progesterone inhibits in vitro fetal membrane weakening.

In vitro evaluation on neutral detergent fiber and cellulose digestion by post-ruminal microorganisms in goats.

Liposome-Based in Vitro Evolution of Aminoacyl-tRNA Synthetase for Enhanced Pyrrolysine Derivative Incorporation.

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Collaborative investigation of broth microdilution and semisolid agar dilution for in vitro susceptibility testing of Candida albicans.

Impact of salt and lipid type on in vitro digestion of emulsified lipids.

Influence of buffer pH and raw corn starch addition on in vitro fiber digestion kinetics.

A novel hemidesmosomal plaque component: tissue distribution and incorporation into assembling hemidesmosomes in an in vitro model.

A novel transcriptomics based in vitro method to compare and predict hepatotoxicity based on mode of action.

Effects of human chorionic gonadotropin preparations on amino acid uptake and incorporation into protein in vitro.

Effect of folate and folinate on 3H-thymidine incorporation by transforming human lymphocytes in vitro.