About 40% of compounds identified through high-throughput or combinatorial screening approaches are poorly water soluble (1–3).
They are usually difficult to formulate and often exhibit a low oral bioavailability, which may result in delays in development
or cause them to be dropped from the pipeline (4). Scientists have developed and proposed various formulation techniques to
improve the solubility and bioavailability of poorly water-soluble compounds (5). One of the most common techniques entails
using various solubilizing agents, including surfactants. Gattefossé's Labrasol (saturated polyglycolyzed C8 -C10 glyceride) surfactant is widely used as a powerful agent for increasing the solubility and bioavailability of low-solubility
However, despite its high solubilizing capacity, Labrasol cannot keep drugs solublized when a neat drug–Labrasol formulation
is diluted with an aqueous medium. Drugs often precipitate out of the formulation after dilution, thus causing low bioavailability
(7, 8). Such precipitation in vivo may reduce drug concentration needed in the aqueous phase for immediate action, thus resulting in a delayed or reduced efficacy
(9, 10). Drug precipitation in vivo is a predominant cause of low oral bioavailability for poorly water-soluble compounds (11).
Formulators have made significant efforts to inhibit or reduce drug precipitation from Labrasol formulations. One common approach
is to mix Labrasol with pharmaceutical excipients, such as hydrophobic oils, lipids, surfactants, and cosolvents, to form
microemulsions or emulsion systems (12–15). Drugs can be held tightly in the more hydrophobic cores of the emulsion systems
than in Labrasol alone upon dilution. This characteristic inhibits drug precipitation. Despite a significant improvement in
inhibiting drug precipitation, these Labrasol formulations are usually complicated and involve multiple components. The formulations
also may raise concerns about drug loading, scale-up, and cost.
The aim of this study was to inhibit drug precipitation from Labrasol formulations by identifying and incorporating a precipitation
inhibitor into them. Two of Johnson and Johnson's poorly water-soluble compounds were selected as model compounds. Pluronic
F127 was identified and added as a precipitation inhibitor into Labrasol formulations. Compound precipitation from Labrasol
formulations with or without Pluronic F127 was assessed by a parallel microscreening precipitation method using 96-well plates
and a Tecan robot.
Materials and methods
Two poorly water-soluble compounds (Compounds A and B) were obtained from the Johnson and Johnson Pharmaceutical Research
and Development compound collection. The molecular weights of Compound A and B were 425.51 g/mol and 676.77 g/mol, respectively.
Compound A had aqueous solubility of 1 μg/mL at neutral pH, and the solubility of Compound B was < 0.2 μg/mL at pH 7.4. Both
compounds were thus typical of Biopharmaceutics Classification System Class II compounds. One of the factors that led to the
poor oral bioavailability of both compounds was their low aqueous solubility.
Formulation excipients and biorelevant media.
Pluronic F127 and Labrasol were obtained from BASF and Gattefossé, respectively. Polyethylene glycol (PEG) 400, 1-methyl-2-pyrrolidinone
(NMP), and pyrene were purchased from Sigma-Aldrich. Simulated intestine fluid (SIF) (pH 7.4) was prepared according to the
US Pharmacopeia, but no enzymes were added.
In vitro precipitation method.
The in vitro drug precipitation upon dilution in an aqueous medium was assessed as described in the literature (10, 16, 17). The stock
solutions of the tested compounds and the excipients, including Labrasol, were first prepared in n-propanol at concentrations of 5 and 20 mg/mL, respectively. Then, according to formulation design, the compounds and each
excipient solution at various volume ratios were automatically dispensed into each well of a 96-well microtiter plate (Scienceware,
Bel-Art Products) by a Tecan robot (GENESIS Workstation). After vortexing for homogeneous mixing, the microtiter was then
placed in a centrifugal vacuum evaporator (HT-4X, GeneVac) and run for 2 h to remove n-propanol from the plates. After solvent removal, 120 μL of SIF was added to each well of the plate. Following 24-h incubation
at room temperature, aqueous solution was filtered through a 0.2-μm 96-well polyvinylidene fluoride (PVDF) filter plate, and
drug aqueous concentration in the filtrates was determined using high-performance liquid chromatography (HPLC).
An HP1100 HPLC instrument with an autosampler module for a 96-well microtiter plate (Agilent) was used to analyze compound
concentration. In the HPLC assay for Compound A, a C18 column (50 × 4.6 mm, 2.5 μm, Thermo BDS hypersil, 35 °C) and a mobile
phase containing 40% acetonitrile and 60% ammonium formate buffer (pH 3.3) (by volume) were used. The flow rate was controlled
at 1 mL/min with an injection volume of 20 μL. The effluent was detected for the compound concentration at a wavelength of
270 nm. The run time was 4 min per sample. In the HPLC assay for Compound B, 20 μL of the sample were injected into a Waters
Xterra RP18 column (150 mm × 4.6 mm, 3.5 μm) (Waters). The sample was eluted at 30 °C at 1 mL/min by a mobile phase consisting
of 70% (v/v) acetonitrile with 0.1% trifluoroacetic acid (TFA) and 30% (v/v) water with 0.1% TFA. Compound B's concentration
was measured at an ultraviolet wavelength of 245 nm. The retention time of the compound was 2.5 min during a total 4.25-min
run time per sample. None of the excipients used in the study interfered with both assays.
Compound solubility in neat excipients.
The solubility of Compounds A and B in neat excipients, such as Labrasol, PEG 400, and NMP, was determined by a shake-flask
method. Excess compound solids were suspended in 1 mL of tested excipients, and the suspensions were shaken at 37 °C for two
to five days. Aliquots were withdrawn and filtered through a 0.2-μm PVDF filter. The filtrate was diluted with acetonitrile,
and the compound concentration in the filtrate was analyzed by a corresponding HPLC method. Equilibrium solubility was determined
when the concentration of the compound in the suspension did not increase further with incubation time.
Pyrene vibrational emission measurement.
The authors prepared 2-mg/mL solutions of Labrasol, Pluronic F127, and the mixture of Pluronic F127 and Labrasol by dissolving
the appropriate amount of substances in SIF. Pyrene solution (0.05 mg/mL) was prepared in acetone. Subsequently, 10 μL of
pyrene solution was added to the appropriate volume of Labrasol, Pluronic F127, and their mixture to achieve a final pyrene
concentration of 6 × 10–7 M. Samples were incubated at 37 °C in a shaker for 1 h, followed by an overnight shaking at room temperature. Fluorescence
excitation spectra of pyrene were obtained using a Hitachi F-4500 fluorescence spectrophotometer. The vibrational-emission
spectrum of pyrene comprising the formulations from 350 to 400 nm was measured at an excitation wavelength of 340 nm. The
excitation and emission-band slits were 4 and 2 nm, respectively. The intensity ratio of I333/I338 was calculated.