Ionic Liquid Forms of Weakly Acidic Drugs in Oral Lipid Formulations: Preparation, Characterization, in Vitro Digestion, and in Vivo Absorption Studies

Article abstract of DOI:10.1021/acs.molpharmaceut.7b00442

This study aimed to transform weakly acidic poorly water-soluble drugs (PWSD) into ionic liquids (ILs) to promote solubility in, and the utility of, lipid-based formulations. Ionic liquids (ILs) were formed directly from tolfenamic acid (Tolf), meclofenamic acid, diclofenac, and ibuprofen by pairing with lipophilic counterions. The drug-ILs were obtained as liquids or low melting solids and were significantly more soluble (either completely miscible or highly soluble) in lipid based, self-emulsifying drug delivery systems (SEDDS) when compared to the equivalent free acid. In vivo assessment of a SEDDS lipid solution formulation of Tolf didecyldimethylammonium salt and the same formulation of Tolf free acid at low dose (18 mg/kg, where the free acid was soluble in the SEDDS), resulted in similar absorption profiles and overall exposure. At high dose (100 mg/kg), solution SEDDS formulations of the Tolf ILs (didecyldimethylammonium, butyldodecyldimethylammonium or didecylmethylammonium salts) were possible, but the lower lipid solubility of Tolf free acid dictated that administration of the free acid was only possible as a suspension in the SEDDS formulation or as an aqueous suspension. Under these conditions, total drug plasma exposure was similar for the IL formulations and the free acid, but the plasma profiles were markedly different, resulting in flatter, more prolonged exposure profiles and reduced C_max for the IL formulations. Isolation of a weakly acidic drug as an IL may therefore provide advantage as it allows formulation as a solution SEDDS rather than a lipid suspension, and in some cases may provide a means of slowing or sustaining absorption. The current studies compliment previous studies with weakly basic PWSD and demonstrate that transformation into highly lipophilic ILs is also possible for weakly acidic compounds.

Full text of DOI:10.1021/acs.molpharmaceut.7b00442

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Article
Ionic liquid forms of weakly acidic drugs in oral lipid formulations:
preparation, characterization, in vitro digestion and in vivo absorption studies
Yasemin Sahbaz, Tri-Hung Nguyen, Leigh Ford, Claire L. McEvoy,
Hywel D. Williams, Peter J. Scammells, and Christopher J.H. Porter
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00442 • Publication Date (Web): 27 Sep 2017
Downloaded from http://pubs.acs.org on September 28, 2017
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Molecular Pharmaceutics
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Ionic liquid forms of weakly acidic drugs in oral lipid
formulations: preparation, characterization, in vitro digestion and
in vivo absorption studies
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Yasemin Sahbaz†‡, TriꢀHung Nguyen‡, Leigh Ford†, Claire L. McEvoy‡, Hywel D.
Williams+, Peter J. Scammells†* and Christopher J. H. Porter‡§*
‡ Drug Delivery, Disposition and Dynamics and † Medicinal Chemistry, Monash Institute of
Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia
+
Capsugel R&D Australia, Monash Institute of Pharmaceutical Sciences, Monash
University, 381 Royal Parade, Victoria 3052, Australia
§
ARC Centre of Excellence in Convergent BioꢀNano Science and Technology, Monash
Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia
*
Corresponding authors
Christopher J.H. Porter chris.porter@monash.edu
Peter J. Scammells: peter.scammells@monash.edu
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ABSTRACT
This study aimed to transform weakly acidic poorly water soluble drugs (PWSD) into ionic
liquids (ILs) to promote solubility in, and the utility of, lipidꢀbased formulations. Ionic liquids
(ILs) were formed directly from tolfenamic acid (Tolf), meclofenamic acid, diclofenac and
ibuprofen by pairing with lipophilic counterions. The drugꢀILs were obtained as liquids or
low melting solids and were significantly more soluble (either completely miscible or highly
soluble) in lipid based, selfꢀemulsifying drug delivery systems (SEDDS) when compared to
the equivalent free acid. In vivo assessment of a SEDDS lipid solution formulation of Tolf
didecyldimethyl ammonium salt and the same formulation of Tolf free acid at low dose (18
mg/kg, where the free acid was soluble in the SEDDS), resulted in similar absorption profiles
and overall exposure. At high dose (100 mg/kg), solution SEDDS formulations of the Tolf
ILs (didecyldimethyl ammonium, butyldodecyldimethyl ammonium or didecylmethyl
ammonium salts) were possible, but the lower lipid solubility of Tolf free acid dictated that
administration of the free acid was only possible as a suspension in the SEDDS formulation
or as an aqueous suspension. Under these conditions, total drug plasma exposure was similar
for the IL formulations and the free acid, but the plasma profiles were markedly different,
resulting in flatter, more prolonged exposure profiles and reduced Cmax for the IL
formulations. Isolation of a weakly acidic drug as an IL may therefore provide advantage as it
allows formulation as a solution SEDDS rather than a lipid suspension, and in some cases
may provide a means of slowing or sustaining absorption. The current studies compliment
previous studies with weakly basic PWSD and demonstrate that transformation into highly
lipophilic ILs is also possible for weakly acidic compounds.
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Keywords: ionic liquid, drug delivery, lipid formulation, SEDDS, in vitro digestion, lipolysis;
poorly water soluble drug
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INTRODUCTION
Increased emphasis on potency, and the application of contemporary drug discovery screening
methods, continues to result in the increasingly frequent identification of drug candidates with
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high lipophilicity and poor water solubility. The low water solubility of these drugs, often
combined with high dose requirements, poses a major challenge for oral bioavailability due to
poor absorption from the gastrointestinal (GI) tract. Several formulation approaches have
been developed to enhance apparent drug solubility in the GI tract and to promote the
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absorption of poorly water soluble drugs (PWSD). Lipid formulations provide one such
approach and SelfꢀEmulsifying Drug Delivery Systems or SEDDS are perhaps the most
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commonly applied class of oral lipid formulation. SEDDS typically comprise drug dissolved
in a mixture of lipids, surfactants and coꢀsolvents, combinations of excipients that are chosen
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such that they emulsify spontaneously on contact with the GI fluids after oral administration.
SEDDS formulations enhance oral absorption of PWSD via two major mechanisms. Firstly,
since drug is usually preꢀdissolved in the formulation, traditional dissolution is avoided.
Secondly, integration of the SEDDS formulation into endogenous lipid digestion processes
leads to trafficking of coadministered PWSD into intestinal mixed micelles. Although this
approach has been shown to be highly effective in a number of cases, a limitation is the
identification of drugs with sufficient solubility in formulation lipids to allow the prospective
dose to be dissolved in a quantity of formulation that can be readily filled into (ideally) a
single capsule. Although lipid suspension formulations are possible, and may be effective,
lipid solution formulations have additional advantages with respect to dissolution, and
processing advantages such as increased physical stability, content uniformity and ease of
capsule filling.
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For PWSD, many of which are inherently hydrophobic, low lipid solubility usually arises as a
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result of strong intermolecular forces and high crystallinity in the solid state. Here we
explore the potential benefits of transformation of PWSD drugs, and in particular weakly
acidic PWSDs, into ionic liquids (IL), with lower intermolecular forces and reduced
crystallinity as a means of increasing lipid solubility and therefore increasing the applicability
of SEDDS formulations.
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ILs are defined as low melting organic salts (melting points below 100 ºC) which generally
consist of bulky cations and/or anions with more charge delocalisation than simple ionic
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, 5
species. They are often referred to as “designer solvents” as the cation and anion can be
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varied widely to tune the physicochemical properties. Accordingly, these multifunctional
solvents have been the subject of widespread interest in a range of areas; for example as
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, 7, 8
alternate green solvents, electrolytes or functional materials.
However, until recently, the potential benefits of ILs in drug delivery have not been well
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explored. The excellent solvent properties of ILs provide the opportunity to more readily
10ꢀ16
dissolve active pharmaceutical ingredients (API) when compared to traditional excipients,
and the tunable solvent properties of ILs create the potential to tailor an IL to a particular
drug. We have also recently demonstrated that formulations containing ILs as excipients may
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provide for sustained drug release following oral administration. In addition to exploiting
ILs as formulation excipients, the possibility of generating ILs from ionisable drugs (weakly
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acidic or weakly basic) to improve drug properties also exists.
These latter studies have
mostly addressed the advantages of drugꢀILs in overcoming the issue of polymorphism in the
solid crystalline state or in decreasing melting point in order to increase aqueous solubility
and dissolution rate. In contrast, our recent focus has been on using drugꢀILs to promote drug
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solubility (and therefore drug loading) in lipidꢀbased (non-aqueous) formulations (LBFs).
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For example, we recently showed significant enhancement of nonꢀaqueous solubility of drugꢀ
ILs formed from the weakly basic PWSD cinnarizine and itraconazole and a range of
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lipophilic counterions. Promisingly, lipophilic drugꢀILs in combination with LBFs resulted
in up to 20ꢀfold increases in oral exposure when compared to formulations containing the
maximum quantity of the equivalent free base form. Similar results have been reported for the
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poorly water soluble weak base atazanavir. We have also applied this approach to a range of
weak bases where low water solubility was not the primary motivation for conversion to a
LBF, but instead where LBF might be preferred to achieve a fast onset of action, facilitate
2
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taste masking or simply to provide alternate patient preference options. To this point,
however, the use of ionic liquids (or more broadly, lipophilic salts, where the latter definition
allows for ionic complexes with lipophilic counterions, but where where the melting point of
the salt is higher than the ionic liquid definition of 100 °C) to promote the solubility of
weakly acidic drugs in lipid based formulations, has not been described.
Herein we investigate the applicability of drug transformation into lipophilic salts or ILs, in
order to promote LBF utility, for the weakly acidic drugs, tolfenamic acid (Tolf),
meclofenamic acid (Mec), diclofenac (Diclof) and ibuprofen (Ibu). IL forms of the acidic
drugs were synthesised with a range of lipophilic counterions, the ILs characterised and their
ability to increase lipid solubility assessed. Model lipidic SEDDS formulations containing
TolfꢀILs (didecyldimethyl ammonium, butyldodecyldimethyl ammonium and didecylmethyl
ammonium salts) were further evaluated using in vitro lipid digestion models to profile
patterns of drug solubilisation during formulation digestion. Finally, in vivo drug absorption
profiles were obtained for Tolf after oral administration of SEDDS formulations containing
Tolf ILs in solution and compared to control formulations containing Tolf as a suspension of
the free acid.
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Figure 1 The weakly acidic drugs (shown as carboxylates) employed in this study (Tolf, Mec,
Diclof and Ibu) and the lipophilic cations employed to form ILs.
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EXPERIMENTAL SECTION
Materials
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Tolfenamic acid, meclofenamic acid, meclofenamic sodium salt, diclofenac sodium salt,
ibuprofen sodium salt, benzalkonium chloride, didecyldimethyl ammonium bromide, N,Nꢀ
dimethyldodecylamine, octadecylamine, 1ꢀbutanol, soybean oil, sodium taurodeoxycholate
(NaTDC), porcine pancreatin extract (8 X USP specification activity), 4ꢀbromophenylboronic
acid were purchased from SigmaꢀAldrich (St. Louis, MO, USA). Spermine was ordered from
Alfa Aesar. Diclofenac acid, didecylmethylamine and dimethyldioctadecyl ammonium
bromide were obtained from TCI (Tokyo, Japan). Lipoid E PC S (lecithin for egg, ~99% pure
phosphatidylcholine (PC) was purchased from Lipoid GmBH, Ludwigshafen, Germany.
TM
®
Maisine
35ꢀ1 (Gattefossé, SaintꢀPriest, France); Kolliphor EL (BASF Corporation,
Ludwigshafen, Germany); 1.0 M sodium hydroxide (Ajax Finechem Pty Ltd, Sydney,
Australia) were obtained from the indicated suppliers. Acetonitrile, ethanol, petroleum spirits,
chloroform, dichloromethane, methanol, ethyl acetate, diethyl ether were purchased from
Merck (Bayswater, Australia) and used without any preꢀtreatment. All other chemicals and
solvents were of analytical purity or high performance liquid chromatography (HPLC) grade.
Drug and Counterion properties
a
Molecular
weight (Da)
pKaꢀ1
(acid)
pKaꢀ1
(base)
Drug/counterion
cLogP
5.49
cLogD7.4
2.26
Tolfenamic acid
261.71
296.15
3.9
(
Tolf)
Meclofenamic acid
(Mec)
3
.8
6.09
2.82
Diclofenac (Diclof)
Ibuprofen (Ibu)
296.15
206.29
4.0
4.9
4.26
3.84
1.10
1.34
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Butylamine
NH Bu)
73.14
10.2
0.2
10.2
0.2
0.70
2.48
4.25
6.92
ꢀ1.45
8.17
ꢀ1.91
ꢀ0.13
1.65
4.32
ꢀ8.77
5.40
(
3
Octylamine
(NH Oct)
129.25
185.36
269.52
1
3
Dodecylamine
NH Dodec)
(
3
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Octadecylamine
NH Octadec)
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(
3
202.35
10.8b
Spermine
(
10.2 )
Didecylmethylamine
NHDec Me)
311.60
1
0.3
(
2
a
Data for quaternary ammonium counterions not generated since they have a permanent
positive charge
pKaꢀ2 for spermine in parentheses.
Data calculated using JChem for Excel (Ver. 16.4.1100.717), ChemAxon Ltd.
b
Characterisation
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The H and C NMR spectra of the purified products were recorded in CDCl (Cambridge
3
3 6
Isotope Laboratories Inc., 99.8% D), CD OD (Sigma Aldrich, 99.8% D) or DMSOꢀd
(Cambridge Isotope Laboratories Inc., 99.9% D) on a Bruker Avance III Ultrashield Plus
spectrometer at 400.13 and 100.62 MHz, respectively. High resolution mass spectra were
obtained using a Waters LCT Premier XE timeꢀofꢀflight mass spectrometer fitted with an
electrospray (ESI) ion source and controlled with MassLynx software version 4.5. Low
resolution mass spectra were obtained using an Agilent 1200 Single Quad LCMS.
For XRD analysis was performed on a Shimadzu XRDꢀ7000 diffractometer (Shimadzu
Scientific Instruments, Japan) with Cu Kꢀalpha radiation. The applied voltage and current
were 40 kV and 35 mA respectively. Samples were mounted on a stainless steel sample
holder and were scanned in continuous mode between 2° and 40° (2θ), with a step size of
0.02° and a scanning speed of 2 s/step. A divergent slit width of 1° was employed for the
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beam source, and a scattering slit width of 1° and receiving slit width of 0.3 mm were used
for the detector.
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General Methods for Drug-IL Preparation
Tolfenamic acid didecyldimethylammonium salt (Tolf•NDec Me )
2
2
Tolf (2.11 g, 8.06 mmol) was suspended in saturated sodium bicarbonate (100 mL) and 2M
NaOH (20 mL) was added. The solution was stirred until all of the Tolf dissolved and a clear
solution was obtained. Didecyldimethylammonium bromide (3.28 g, 8.06 mmol) was
completely dissolved in a mixture of water (50 mL) and methanol (20 mL). The two solutions
were mixed and oil droplets were immediately formed in the aqueous medium. The reaction
mixture was stirred further for 30 min at room temperature. The oil phase was extracted with
dichloromethane (3 x 100 mL) and the combined organic phases were washed with distilled
water (3 x 100 mL) until a negative AgNO
3
test was obtained. The organic phase was dried
(
over anhydrous MgSO ), filtered and evaporated to afford the desired product that was dried
4
under high vacuum.
1
Yield 95%. H NMR (CDCl
3
) δ 8.09 (dd, J = 7.7, 1.5 Hz, 1H), 7.32 (dd, J = 7.8, 0.9 Hz, 1H),
7
2
δ
6
3
.16–7.07 (m, 2H), 7.01–6.93 (m, 2H), 6.73–6.69 (m, 1H), 3.38–3.34 (m, 4H), 3.31 (s, 6H),
1
3
3
.38 (s, 3H), 1.60 (br s, 4H), 1.26–1.22 (m, 28H), 0.88 (2 x t, J = 6.9, 6H). C NMR (CDCl )
172.9, 145.8, 143.5, 135.2, 132.2, 130.1, 127.6, 126.5, 123.8, 121.8, 117.3, 117.1, 114.2,
3.6, 51.0, 31.9, 29.5, 29.4, 29.3, 29.2, 26.3, 22.8, 22.7, 15.1, 14.2. HRMS +ve calcd
26.3787 found 326.3792 –ve calcd 260.0478 found 260.0477.
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Tolfenamic acid didecylmethylammonium salt (Tolf•NHDec Me)
2
A mixture of Tolf (1.55 g, 5.92 mmol) and didecylmethylamine (2.27 mL, 5.91 mmol) in
methanol (50 mL) was stirred at room temperature for 30 min. The methanol was evaporated
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(
rotavap) and the resultant orange oil was dried further under high vacuum.
1
Yield 98%. H NMR (MeODꢀd
4
) δ 7.95 (dd, J = 7.6, 1.6 Hz, 1H), 7.28 (dd, J = 7.6, 1.2 Hz,
1H), 7.17–7.21 (m, 1H), 7.02–7.10 (m, 2H), 6.97 (dd, J = 8.4, 0.8 Hz 1H), 6.70–6.74 (m, 1H)
2
.99–3.03 (m, 4H), 2.77 (s, 3H), 2.35 (s, 3H), 1.65–1.72 (m, 4H), 1.23–1.37 (m, 30H), 0.89 (2
13
x t, J = 6.8 Hz, 6H). C NMR (MeODꢀd
4
)
δ
175.8, 147.3, 143.8, 136.3, 133.1, 129.3, 127.9,
1
24.0, 122.2, 119.8, 118.6, 115.3, 57.1 (2C), 40.3, 33.0 (2C), 30.6 (2C), 30.5 (2C), 30.4 (2C),
30.2 (2C), 27.7 (2C), 25.2 (2C), 23.7 (2C), 15.2, 14.5 (2C). HRMS +ve calcd 312.3630 found
12.3635 –ve calcd 260.0478 found 260.0489.
3
Tolfenamic acid butyldodecyldimethylammonium salt (Tolf•NBuDodecMe2)
The precursor, butyldodecyldimethylammonium bromide was initially synthesized from 1ꢀ
bromobutane and N,Nꢀdimethyldodecylamine according to a slightly modified literature
17
procedure.
A mixture of N,Nꢀdimethyldodecylamine (3.88 g, 5.00 mL, 18.2 mmol) and 1ꢀbromobutane
2.99 g, 2.34 mL, 21.8 mmol) in toluene (60 mL) were stirred for 1 h at room temperature
(
under nitrogen. The reaction was then refluxed overnight. The solution was cooled to room
temperature and the toluene was evaporated in vacuo. The resultant yellow oily solid was
triturated with diethyl ether (200 mL) for 2 h and washed further with diethyl ether (3 x 100
mL). Diethyl ether was removed by decantation and the resultant product was dried under
high vacuum to afford butyldodecyldimethylammonium bromide as a white solid.
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Yield 87%. H NMR (DMSOꢀd ) δ 3.27–3.22 (m, 4H), 3.00 (s, 6H), 1.64–1.58 (m, 4H), 1.34–
6
13
1
.24 (m, 20H), 0.93 (t, J = 7.3 Hz, 3H), 0.85 (t, J = 6.7 Hz, 3H). C NMR (CDCl
3
)
δ
62.8,
6
2.6, 49.9, 31.3, 29.0 (2C), 28.9, 28.8, 28.7, 28.5, 25.8, 23.7, 22.1, 21.7, 19.2, 13.9, 13.5.
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The title compound was synthesized using a slight modification of the procedure used in the
synthesis of Tolf acid didecyldimethyl ammonium salt. In this case the reaction was carried
out in water without methanol.
1
Yield 93%. H NMR (DMSOꢀd
6
) δ 7.88 (dd, J = 7.6, 1.6 Hz, 1H), 7.29 (d, J = 7.9, 1H), 7.11–
7
.03 (m, 3H), 6.91 (d, J = 7.8 Hz, 1H), 6.66–6.62 (m, 1H), 3.24–3.19 (m, 4H), 2.98 (s, 6H),
2.32 (s, 3H), 1.63–1.57 (m, 4H), 1.33–1.24 (m, 20H), 0.92 (t, J = 7.3 Hz, 3H), 0.85 (t, J = 6.8
13
Hz, 3H). C NMR (CDCl
3
)
δ
173.0, 145.9, 143.3, 135.2, 132.2, 130.3, 127.7, 126.5, 123.3,
1
21.9, 117.3 (2C), 114.2, 63.6, 63.5, 50.9, 31.9, 29.6, 29.5, 29.4, 29.3, 29.2, 26.2, 24.6, 22.7
(2C), 19.6, 15.1, 14.2, 13.6. HRMS +ve calcd 270.3161 found 270.316 –ve calcd 260.0478
found 260.0491.
Tolfenamic acid dimethyldioctadecylammonium salt (Tolf•NMe Octadec )
2
2
The title compound was synthesized by the procedure used in the synthesis of Tolf
didecyldimethylammonium salt.
1
Yield 86%. H NMR (CDCl
3
) δ 8.08 (dd, J = 7.7, 1.5 Hz, 1H), 7.32 (dd, J = 7.9, 1.2 Hz, 1H),
7
2
δ
.16–7.08 (m, 2H), 7.01–6.93 (m, 2H), 6.72–6.68 (m, 1H), 3.37–3.33 (m, 4H), 3.31 (s, 6H),
13
.37 (s, 3H), 1.58 (br s, 4H), 1.31–1.20 (m, 60H), 0.87 (2 x t, 6H, J = 6.9). C NMR (CDCl
3
)
172.9, 145.8, 143.5, 135.2, 132.2, 130.0, 127.5, 126.4, 123.9, 121.8, 117.3, 117.0, 114.2,
63.6, 51.0, 31.9, 29.8, 29.7, 29.6, 29.5, 29.4, 29.2, 26.3, 22.7, 15.1, 14.2. HRMS +ve calcd
5
50.6291 found 550.6307 –ve calcd 260.0478 found 260.0485.
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Tolfenamic acid benzalkonium salt (Tolf•NAlkBnMe2)
The title compound was synthesized using a slight modification of the procedure used in the
synthesis of Tolf didecyldimethylammonium salt. In this case the reaction was carried out in
water only.
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60
1
Yield 92%. H NMR (CDCl
3
) δ 8.14 (dd, J = 7.7, 1.5 Hz, 1H), 7.51–7.30 (m, 6H), 7.17–7.08
(m, 2H), 7.01–6.93 (m, 2H), 6.73–6.70 (m, 1H), 4.86 (s, 2H), 3.35–3.31 (m, 4H), 3.22 (s, 6H),
1
3
2
δ
1
.33 (s, 3H), 1.67 (br s, 2H), 1.31–1.20 (m, 21H), 0.88 (t, J = 6.9 Hz, 3H). C NMR (CDCl )
3
173.1, 146.0, 143.4, 135.2, 133.1, 132.3, 130.5, 130.3, 129.1, 127.7, 127.6, 126.5, 123.6,
22.0, 117.4, 117.3, 114.3, 67.6, 63.4, 49.5, 32.0, 31.9, 29.7, 29.6, 29.5, 29.4 (3C), 29.3, 26.3,
22.7, 15.1, 14.2. HRMS +ve calcd 332.3317 found 332.3321 –ve calcd 260.0478 found
60.0477.
2
Tolfenamic acid butylammonium salt (Tolf•NH Bu)
3
To the clear solution of Tolf acid (109.1 mg, 0.42 mmol) in methanol (25 mL) was added to
nꢀbutylamine (30.5 mg, 41.2 µL, 0.42 mmol) and the reaction mixture was stirred at room
temperature for 30 min. The methanol was evaporated in vacuo and the resultant white solid
product was dried further under high vacuum.
1
Yield >95%. H NMR (DMSOꢀd ) δ 8.38 (br s), 7.92 (dd, J = 7.7, 1.7 Hz, 1H), 7.31 (dd, J =
6
8.1, 0.7 Hz, 1H), 7.18–7.14 (m, 1H), 7.11 (t, J = 8.0 Hz, 1H), 7.04 (dd, J = 8.2, 0.8 Hz, 1H),
6.99 (dd, J = 7.9, 0.7 Hz, 1H), 6.69 (m, 1H), 2.79 (t, J = 8.0 Hz, 2H), 2.29 (s, 3H), 1.58–1.51
13
(
m, 2H), 1.37–1.28 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H). C NMR (CDCl
3
)
δ
175.3, 147.5,
1
41.9, 135.6, 132.6, 132.2, 129.8, 126.9, 124.2, 120.6, 118.2, 117.2, 114.3, 39.7, 30.2, 19.9,
5.1, 13.5. HRMS –ve calcd 260.0478 found 260.0489.
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Tolfenamic acid octylammonium salt (Tolf•NH Oct)
3
The title compound was synthesized by the procedure used for the synthesis of Tolf
butylammonium salt.
0
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Yield > 95%. H NMR (DMSOꢀd
6
) δ 8.15 (br s), 7.90 (dd, J = 7.7, 1.7 Hz, 1H), 7.30 (dd, J =
8.1, 0.7 Hz, 1H), 7.17–7.13 (m, 1H), 7.10 (t, J = 8.0 Hz, 1H), 7.03 (dd, J = 8.2, 0.8 Hz, 1H),
6.97 (dd, J = 7.9, 0.7 Hz, 1H), 6.69–6.65 (m, 1H), 2.77 (t, J = 8.0 Hz, 2H), 2.29 (s, 3H), 1.58–
13
1
1
2
3
.50 (m, 2H), 1.30–1.22 (m, 10H), 0.84 (t, J = 6.8 Hz, 3H). C NMR (CDCl ) δ 175.3, 147.5,
41.9, 135.6, 132.5, 132.2, 129.7, 126.8, 124.1, 120.5, 118.4, 117.2, 114.3, 40.1, 31.8, 29.2,
9.1, 28.6, 26.8, 22.7, 15.1, 14.2. HRMS +ve calcd 130.1596 found 130.1598 –ve calcd
260.0478 found 260.0491.
Tolfenamic acid dodecylammonium salt (Tolf•NH Dodec)
3
The title compound was synthesized by the procedure used for the synthesis of Tolf
butylammonium salt.
1
Yield >95%. H NMR (DMSOꢀd
6
) δ 7.89 (dd, J = 7.7,1.7 Hz, 1H), 7.30 (dd, J = 8.1, 0.7 Hz,
1
6
0
H), 7.16–7.08 (m, 2H), 7.03 (dd, J = 8.2, 0.9 Hz, 1H), 6.96 (dd, J = 7.9, 0.6 Hz, 1H), 6.68–
.64 (m, 1H), 2.76 (t, J = 7.4 Hz, 2H), 2.28 (s, 3H), 1.56–1.48 (m, 2H), 1.27–1.22 (m, 18H),
1
3
3
.85 (t, J = 6.8 Hz, 3H). C NMR (CDCl ) δ 175.6, 147.4, 142.0, 135.6, 132.4, 132.1, 129.5,
126.8, 124.0, 120.2, 118.9, 117.2, 114.3, 39.9, 32.1, 29.8, 29.7 (2C), 29.5 (2C), 29.2, 28.3,
2
6.8, 22.9, 15.1, 14.3. HRMS +ve calcd 186.2222 found 186.2222 –ve calcd 260.0478 found
60.0486.
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Tolfenamic acid octadecylammonium salt (Tolf•NH Octadec)
3
Tolfenamic acid (1.61 g, 6.17 mmol) and octadecylamine (1.66 g, 6.17 mmol) were heated at
o
6
5 C in 50 mL methanol for 2 h, cooled to rt, and the solvent was removed in vacuo. The
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resulting solid was placed under high vacuum to give the desired product as a white solid.
1
Yield 98%. H NMR (MeOD) δ 7.94 (dd, J = 7.8, 1.6 Hz, 1H), 7.29 (dd, J = 7.8 Hz, 1.2 Hz,
1
H), 7.21–7.14 (m, 1H), 7.07 (t, J = 8.0 Hz, 1H), 7.02 (dd, J = 8.0, 1.2 Hz, 1H), 6.98 (dd, J =
.2, 0.8 Hz, 1H), 6.75–6.69 (m, 1H), 2.92–2.86 (m, 2H), 2.35 (s, 3H), 1.68–1.59 (m, 2H),
8
1
3
1.44–1.22 (m, 30H), 0.90 (t, J = 6.8 Hz, 3H). C NMR (CDCl
3
) δ 175.5, 147.4, 142.0, 135.6,
1
2
32.4, 132.2, 129.6, 126.8, 124.0, 120.3, 118.7, 117.2, 114.3, 40.0, 32.1, 29.9, 29.8 (2C),
9.7, 29.5 (2C), 29.2, 28.4, 26.8, 22.9, 15.1, 14.3, 1.2. HRMS +ve mode calcd 270.3161
o
found 270.3156. –ve mode calcd 260.0472 found 260.0480. m.p. 123ꢀ125 C.
Tolfenamic acid spermine salt (Tolf•Spermine)
A mixture of Tolf (0.64 g, 2.45 mmol) and spermine (0.5 mL, 2.45 mmol) in methanol (35
mL) was stirred at room temperature for 30 min. The methanol was evaporated (rotavap) and
the resultant oil slowly crystallised to give an off white waxy solid.
1
Yield 99%. H NMR (MeOD) δ 7.84 (dd, J = 7.8, 1.6 Hz, 1H), 7.19 (dd, J = 7.8, 1.2 Hz, 1H),
7.08 (ddd, J = 8.4, 7.2, 1.8 Hz, 1H), 6.98 (td, J = 8.0, 0.4 Hz, 1H), 6.90 (ddd, J = 13.8, 8.2, 1.0
Hz, 2H), 6.62 (ddd, J = 7.8, 7.2, 1.2 Hz, 1H), 2.70 (t, J = 7.2 Hz, 4H), 2.62 (t, J = 7.2 Hz, 4H),
13
2
.56 (t, J = 6.8 Hz, 3H), 1.64 (quin, J = 7.2 Hz, 4H), 1.52–1.42 (m, 4H). C NMR (MeOD) δ
176.2, 147.2, 143.9, 136.2, 133.1, 132.1, 129.3, 127.9, 123.9, 123.0, 119.6, 118.6, 115.4, 50.0,
47.9, 40.2, 31.0, 27.7, 15.1. HRMS +ve mode calcd 203.2234 found 203.2229. HRMS –ve
o
mode calcd 260.0469 found 260.0475. Complete melting by 121 C.
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Meclofenamic acid didecyldimethylammonium salt (Mec•NDec Me )
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Sodium carbonate was added to distilled water (200 mL) to adjust the pH of the resulting
solution to ~9. Me sodium salt (2.0 g, 6.30 mmol) was dissolved in 100 mL of this aqueous
basic solution. Didecyldimethylammonium bromide (2.6 g, 6.30 mmol) was also dissolved in
100 mL of aqueous basic solution and 50 mL methanol. The two solutions were mixed and oil
droplets were immediately formed in aqueous medium. The reaction mixture was stirred
further for 30 min and the oil phase was extracted with chloroform (3 x 100 mL). The
combined organic phases were washed with distilled water (3 x 100 mL) until a negative
3 4
AgNO test was obtained. The organic phase was then dried (anhydrous MgSO ), filtered,
evaporated and dried under high vacuum to afford the desired product.
1
Yield 90%. H NMR (CDCl ) δ 8.08 (dd, J = 7.7, 1.6 Hz, 1H), 7.23 (d, J = 8.2 Hz, 1H), 7.07–
3
7.03 (m, 1H), 6.97 (d, J = 8.2, 1H), 6.68–6.64 (m, 1H), 6.23 (dd, J = 8.1, 0.7 Hz, 1H), 3.37–
3
7
1
.33 (m, 4H), 3.31 (s, 6H), 2.37 (s, 3H), 1.58 (br s, 4H), 1.30–1.18 (m, 28H), 0.87 (2 x t, J =
13
.0, 6H). C NMR (CDCl
3
)
δ
173.0, 145.9, 137.6, 136.1, 133.4, 131.9, 130.3, 129.8, 127.6,
26.7, 122.3, 116.7, 112.7, 63.6, 51.1, 31.9, 29.5, 29.4, 29.3, 29.2, 26.3, 22.8, 22.7, 20.7, 14.1.
HRMS +ve calcd 326.3787 found 326.3793 –ve calcd 294.0089 found 294.0087.
Meclofenamic acid 1-hexadecyl-3-methylpyridinium salt
The precursor, 1ꢀhexadecylꢀ3ꢀmethylpyridinium bromide, was initially synthesized from 3ꢀ
1
7
methylpyridine and 1ꢀbromohexadecane according to a literature procedure.
1
Yield 95%. H NMR (CDCl
3
) δ 9.39 (s, 1H), 9.25 (d, J = 6.0 Hz, 1H), 8.24 (d, J = 8.0 Hz,
1H), 7.99 (dd, J = 7.8, 6.2 Hz, 1H), 4.94 (t, J = 7.5 Hz, 2H), 2.64 (s, 3H), 2.09–1.93 (m, 2H),
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.46ꢀ1.12 (m, 26H), 0.86 (t, J = 6.8 Hz, 3H). C NMR (CDCl
3
) δ 146.2, 144.7, 142.4, 139.2,
1
27.8, 61.0, 31.8, 31.1, 29.5 (2C), 29.4, 29.3, 29.2, 28.9, 25.9, 22.6, 18.3, 14.4.
The title compound was synthesized by the procedure used for the synthesis of Mec
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59
60
didecyldimethylammonium salt
.
1
Yield 88%. H NMR (DMSOꢀd ) δ 9.05 (s, 1H), 8.95 (d, J = 6.0 Hz, 1H), 8.43 (d, J = 8.0 Hz,
6
1
H), 8.04 (dd, J = 7.9, 6.1 Hz, 1H), 7.82 (dd, J = 7.6, 1.7 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H),
.17 (dd, J = 8.3, 0.5 Hz, 1H), 6.96–6.92 (m, 1H), 6.55–6.50 (m, 1H), 6.00 (dd, J = 8.1, 0.9
7
Hz, 1H), 4.54 (t, J = 7.6 Hz, 2H), 2.50 (s, 3H), 2.35 (s, 3H), 1.93–1.88 (m, 2H), 1.28–1.23 (m,
1
3
2
1
3
3
6H), 0.85 (t, J = 6.8 Hz, 3H). C NMR (CDCl ) δ 173.3, 146.2, 145.1, 144.6, 142.6, 139.5,
37.4, 136.2, 133.7, 132.0, 130.5, 130.2, 127.9, 127.7, 127.0, 121.6, 116.8, 112.7, 62.0, 32.0,
1.9, 29.8, 29.7 (2C), 29.6, 29.4 (2C), 29.2, 26.2, 22.8, 20.8, 18.6, 14.2. HRMS +ve calcd
318.3161 found 318.3162 –ve calcd 294.0089 found 294.0098.
Diclofenac didecyldimethylammonium salt (Diclof•NDec Me )
2
2
The title compound was synthesized by the procedure used for the synthesis of Tolfꢀ
didecyldimethylammonium salt
.
1
Yield 91%. H NMR (CDCl ) δ 9.22 (br s, 1H), 7.29 (d, J = 8.0 Hz, 2H), 7.22 (dd, J = 7.4, 1.4
3
Hz, 1H), 6.97 (td, J = 7.8, 1.6 Hz, 1H), 6.89 (t, J = 8.0 Hz, 1H), 6.79 (td, J = 7.4, 1.2 Hz, 1H),
6.44 (dd, J = 8.0, 1.0 Hz, 1H), 3.73 (s, 2H), 3.22–3.18 (m, 4H), 3.09 (s, 6H), 1.53 (br s, 4H),
13
3
1.27–1.24 (m, 28H), 0.88 (t, J = 6.9 Hz, 6H). C NMR (CDCl ) δ 176.7, 143.3, 138.4, 130.5,
129.4, 128.8, 128.3, 126.1, 123.0, 120.4, 116.7, 63.4, 50.9, 43.5, 31.8, 29.4, 29.3, 29.2, 29.1,
26.2, 22.6, 14.1. HRMS +ve calcd 326.3787 found 326.3793 –ve calcd 294.0089 found
294.0087.
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Diclofenac benzalkonium salt (Diclof•NAlkBnMe2)
The title compound was synthesised by a slightly modified version of the procedure used for
the synthesis of Mec didecyldimethylammonium salt
in water without methanol.
. In this case the reaction was carried out
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Yield 93% H NMR (CDCl
3
) δ 9.08 (br s, 1H), 7.47–7.37 (m, 5H), 7.28 (d, J = 8.0 Hz, 2H),
7.22 (dd, J = 7.4, 1.3 Hz, 1H), 6.95 (td, J = 7.8, 1.5 Hz, 1H), 6.89 (t, J = 8.0 Hz, 1H), 6.77 (td,
J = 7.4, 1.1 Hz, 1H), 6.44 (dd, J = 7.9, 0.8 Hz, 1H), 4.68 (s, 2H), 3.77 (s, 2H), 3.23–3.19 (m,
13
2H), 3.05 (s, 6H), 1.65 (br s, 2H), 1.32–1.23 (m, 22H), 0.88 (t, J = 6.8 Hz, 3H). C NMR
(
CDCl3)
123.0, 120.5, 116.9, 67.4, 63.1, 49.5, 43.5, 31.9 (2C), 29.7 (2C), 29.6, 29.5, 29.4 (2C), 29.3
2C), 26.3, 22.8, 22.7, 14.2. HRMS +ve calcd 304.3004 found 304.3004 and 332.3317 found
32.3319 –ve calcd 294.0089 found 294.0099.
δ 177.2, 143.4, 138.5, 133.2, 130.6, 130.3, 129.3, 129.0, 128.8, 128.4, 127.6, 126.1,
(
3
Ibuprofen didecyldimethylammonium salt (Ibu•NDec Me )
2
2
The title compound was synthesized by the procedure used for the synthesis of Tolfꢀ
didecyldimethyl ammonium salt.
1
Yield 96%. H NMR (CDCl
3
) δ 7.31 (d, J = 8.0 Hz, 2H), 6.97 (d, J = 8.0 Hz, 2H), 3.56 (q, J =
7.1 Hz, 1H), 3.25–3.20 (m, 4H), 3.13 (s, 6H), 2.38 (d, J = 7.1 Hz, 2H), 1.85–1.75 (m, 1H),
13
1
.52 (br s, 4H), 1.43 (d, J = 7.1 Hz, 3H), 1.28–1.25 (m, 28H), 0.89ꢀ0.85 (m, 12H). C NMR
179.2, 143.0, 138.2, 128.4, 127.5, 63.0, 50.8, 49.2, 45.1, 31.8, 30.2, 29.4, 29.3,
9.2 (2C), 26.2, 22.6 (2C), 22.4 (2C), 19.9, 14.0. HRMS +ve calcd 326.3787 found 326.3795
ve calcd 205.1229 found 205.1225.
(CDCl3)
δ
2
–
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Formulation preparation
The formulations were based on long chain (LC) lipids and freshly prepared as homogenous
mixtures of lipids, surfactant and cosolvent. The composition of the LC lipid containing selfꢀ
emulsifying drug delivery systems (LCꢀSEDDS) utilized in the equilibrium solubility, in vitro
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TM
®
and in vivo studies was 60% lipid (soybean oil:Maisine 35ꢀ1, 1:1 w/w), 30% Kolliphor EL
and 10% ethanol. Drugs as the free acid (FA) or IL were subsequently dissolved or suspended
in the formulations to the concentrations specified. Concentrations were confirmed by HPLC.
Equilibrium solubility studies
The equilibrium solubility of the drugs in the different formulations was determined by the
ꢀ
addition of excess drug/drugꢀILs to the LC SEDDS formulation
.
In all cases, 0.5 g of drug or
drugꢀIL was added to 0.5 g of formulation. Dissolution rates in viscous nonꢀaqueous solvents
are typically slow and therefore the formulations were incubated at 37 °C for 3ꢀ7 days in
order to reach equilibrium. Samples were collected at regular intervals and centrifuged
(21,000 x g, 37 °C, 10 min). An aliquot of the resulting particleꢀfree supernatant was
accurately weighed (15ꢀ40 mg) and dissolved in chloroform:methanol (5 mL, 2:1, v/v).
Following further dilution with acetonitrile and mobile phase, samples were analysed for drug
content by HPLC. All solubility tests were performed in triplicate and equilibrium solubility
defined as the value attained when at least three consecutive solubility samples varied by
≤5%. For ILs that were effectively miscible or soluble in the formulations, the equilibrium
solubility was quoted as greater than (
≥
) the measured concentration resulting from mixing a
ꢀ1
1:1 w/w mixture of drugꢀIL and formulation (i.e. 0.5 g plus 0.5 g, nominally 500 mg.g of IL
in the vehicle). To allow better comparison of solubilising capacity, results are quoted as
ꢀ1
ꢀ1
mg.g of free acid equivalents in the vehicle (rather than mg.g of the IL) and are therefore
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lower than the 500 mg.g nominal concentration of IL. Under these circumstances, the
reported ‘solubility’ does not define the maximum solubility per se (since the systems were
miscible) and differs for each IL by virtue of differences in the molecular weight of the
counterions when compared to the free acid.
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Drug solubilisation during in vitro lipid digestion
The methods employed utilised in vitro lipid digestion testing protocols recently published by
2
9
the lipid formulation classification system (LFCS) consortium. The specific methods are
given below. Briefly, formulations were dispersed in simulated intestinal fluid (SIF) in a
temperature controlled vessel (37 °C) for a period of 10ꢀ20 min and then digestion was
initiated by addition of pancreatin. The pH was maintained at intestinal pH (6.5) via the
conduct of experiments in a pH stat titrator. Digestion was allowed to continue for 60 min. At
the end of the initial dispersion period and during the digestion period samples were taken and
centrifuged to separate drug that precipitated out of solution and that which remained
solubilised.
Three comparative formulations based on LCꢀSEDDS and loaded with Tolf•NDec
2 2
Me ,
Tolf•NBuDodecMe , Tolf•NHDec Me, Tolf•NAlkBnMe or Tolf free acid (FA) were freshly
2
2
2
prepared as solutions. Formulations loaded with Tolf•NDec Me , Tolf•NBuDodecMe ,
2
2
2
2 2
Tolf•NHDec Me or Tolf•NAlkBnMe contained the same concentration of Tolf FA
equivalents (~200 mg/g). The concentration of the formulation loaded with less lipidꢀsoluble
Tolf FA was 35 mg/g. Tolf content in formulations was confirmed by HPLC. The SEDDS
formulation (~1 g) containing Tolf ILs or Tolf FA was subsequently dispersed in 39 mL of
SIF (2 mM Trisꢀmaleate, 150 mM NaCl, 1.4 mM CaCl .2H O, 3 mM NaTDC, 0.75 mM PC,
2
2
pH 6.5, 37 °C).
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After 5 and 10 min of dispersion, a 1 mL sample was removed, centrifuged (21,000 x g,
37 °C, 10 min) and the concentration of dissolved drug determined by HPLC. After the
dispersion phase, 4 mL pancreatin extract (i.e. 1 mL containing ~10,000 TBU for every ~10
mL of digest) was added to initiate digestion. Digestion was continuously monitored over 60
min using a pHꢀstat titrator, which added 0.2 M NaOH into the reaction vessel in response to
the decrease in pH stimulated by lipid digestion and fatty acid liberation. After 5, 15, 30 and
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60 min of digestion, a 1 mL sample was transferred into an Eppendorf tube containing an
enzyme inhibitor (4ꢀbromophenylboronic acid, 5 µL/mL of a 1.0 M solution in methanol) to
stop digestion and centrifuged (21,000 x g, 37 °C, 15 min). The centrifuged samples separated
to form combinations of a pellet phase at the bottom of the tube containing insoluble
precipitated material, a floating undispersed oily phase and a well dispersed, primarily
micellar, aqueous phase. The aqueous phase, pellet phase and oily phase were separated,
vortexed and diluted. The pellet phase was firstly dissolved in 150 µL 1.0M HCl, 100 µL
chloroform/methanol (2/1:v/v) and 750 µL acetonitrile. The oily phase was first dissolved in 1
mL chloroform/methanol (2/1:v/v). These diluted samples, along with samples of solubilized
drug in the aqueous phase were diluted further with acetonitrile and mobile phase and
analysed by HPLC as below.
Analysis of drug concentrations in formulations and digestion samples
Solubility samples and in vitro digestion samples were assayed for Tolf, Mec, Diclof and Ibu
content via HPLC, using an Alliance 2695 separation module and a 486 tunable UV
absorbance detector (Waters Instruments, Milford, MA) and a Waters Symmetry C18 column
(150 × 3.9 mm, 5 ꢁm). For Tolf, Mec and Diclof assays, mobile phase A was 95:5 (v/v)
water:acetonitrile with 0.01% (v/v) formic acid and mobile phase B was 95:5 (v/v)
acetonitrile:water with 0.01% (v/v) formic acid maintained at a flow rate of 1 mL/min. For
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Tolf, the mobile phase comprised an isocratic mixture of 10% of mobile phase A and 90% of
mobile phase B. For Mec and Diclof, the mobile phase comprised an isocratic mixture of 20%
of mobile phase A and 80% of mobile phase B. The injection volume was 50 ꢁL and UV
absorbance was monitored at 280 nm. The retention times were 2.4 min for Tolf, 2.7 min for
Mec and 2.2 min for Diclof and the concentration range of the calibration standards was 0.8ꢀ
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50 µg/mL. For Ibu, the mobile phase comprised 35:65 (v/v) 5 mM aqueous potassium
dihydrogen phosphate:acetonitrile and was maintained at a flow rate of 1 mL/min. The
injection volume was 25 ꢁL and UV absorbance was monitored at 230 nm. The retention time
was 3.3 min and the concentration range of the calibration standards was 0.8ꢀ50 µg/mL.
Oral bioavailability studies
All experimental procedures were approved by the Monash Institute of Pharmaceutical
Sciences Animal Ethics Committee. Experiments were conducted in fasted male Spragueꢀ
Dawley rats (270ꢀ310 g). A day prior to the study, rats were anaesthetised with isoflurane and
the right carotid artery was surgically cannulated with polyethylene tubing (0.96 x 0.58 mm)
30
to facilitate blood collection as described previously. Animals were allowed to recover
overnight and were fasted up to 12 h prior to and 8 h after dose administration with water
provided ad libitum.
SEDDS formulations containing Tolf FA or Tolf ILs (Tolf•NBuDodecMe
2 2 2
, Tolf•NDec Me ,
Tolf•NHDec Me) at high drug loadings (150 mg) were dispersed in 0.5 mL of water
2
immediately prior to oral gavage to lightly anaesthetised rats, followed by a further 0.5 mL of
water. Tolf ILs were dissolved in the SEDDS formulation to provide a dose of 100 mg/kg
Tolf FA. To examine whether IL dose had an impact on absorption profiles, the
Tolf•NDec
2
Me
2
formulation was also administered at a lower dose (18 mg/kg). This was
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achieved by dosing a lower quantity of the same formulation (27 mg of formulation dispersed
in 0.5 mL of water, followed by 0.5 mL of water). Tolf FA was dissolved in the SEDDS
2 2
formulation to give a dose of 18 mg/kg to match that of the low dose Tolf•NDec Me
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formulation. Unlike Tolf IL, Tolf FA was not sufficiently soluble in the SEDDS formulation
to provide a dose of 100 mg/kg in solution (to match the high dose ILs) and instead had to be
suspended in the SEDDS formulation to provide a 100 mg/kg dose. Finally Tolf FA was
dosed as an aqueous suspension at 100 mg/kg dose to evaluate the effect of an aqueous versus
lipid suspension formulation. The aqueous suspension comprised 0.5% w/v sodium
carboxymethylcellulose, 0.4% w/v Tween 80 and 0.9% w/v NaCl in water. Blood samples
were collected via the carotid artery cannula at preꢀdose, 0.5, 1, 2, 3, 4, 6, 8, 10, 24, 28, 32
and 48 h postꢀdose. Blood samples were centrifuged at 6,700 x g for 5 min and plasma
collected and stored at ꢀ80 °C until assayed for Tolf content by LCMS.
Quantification of drug concentrations in plasma
Tolf concentrations in rat plasma (except after administration of Tolf•NHDec
2
Me and
Tolf•NDec Me at 18 mg/kg) were assayed using LCMS. Calibration standards for Tolf were
2
2
prepared by spiking blank rat plasma (50 µL) with Tolf standard solutions (5 µL) in
acetonitrile to give plasma concentrations in the range of 50 to 10000 ng/mL. Plasma samples
or calibration standards (50 µL) were spiked with 5 µL of internal standard (Diclof, 50 µg/mL
in acetonitrile) and vortex mixed for 1 min. To precipitate plasma proteins, saturated
ammonium sulphate solution (25 µL) was added and samples vortex mixed for 30 sec,
followed by the addition of acetonitrile (90 µL) (and vortex mixed for a further 1 min). The
samples were then allowed to stand at room temperature for 20 min. After centrifugation at
9,600 x g for 5 min at room temperature, 25 µL of supernatant was transferred into vials for
analysis.
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In most cases, Tolf plasma samples were analysed on a single quadrupole LCMS (Model
2010) with an electrospray ionization (ESI) interface (Shimadzu, Kyoto, Japan). Data
acquisition and processing were performed using LCMS Solutions software (Shimadzu,
Kyoto, Japan). Mobile phase A comprised of water containing 0.1 % (v/v) acetic acid and
mobile phase B was acetonitrile containing 0.1 % (v/v) acetic acid. The mobile phase flow
rate was 0.5 mL/min with the following gradient elution: 35 to 100% mobile phase B from 0ꢀ
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2.5 min; 100% B from 2.5ꢀ3 min; 100 to 35% B from 3ꢀ3.2 min and 35% B from 3.2ꢀ6 min.
Each sample (5 µL) was injected onto a Kinetex C18 100A column (2.6 µm, 50 mm × 2.1
mm, Phenomenex, CA) held at 40 °C. The retention times of Tolf and the internal standard
(Diclof) were 3.1 and 2.7 min, respectively. Tolf and the Diclof were detected in negative ion
mode and selective ion monitoring (SIM) of m/z 260.00 and 294.00, respectively. The heat
block and curved desolvation line (CDL) temperatures were set at 250 and 200 °C,
respectively. Detector and interface voltages were 1.5 kV and 0.5 kV respectively. The
nebulising gas flow rate was 1.2 L/min.
2 2 2
For Tolf•NHDec Me and the bioavailability study for Tolf•NDec Me at the lower dose (18
mg/kg), unavailability of the Shimadzu 2010 instrument necessitated analysis to be
undertaken using a Shimadzu 8030 LCꢀMS/MS system (Shimadzu, Kyoto, Japan) equipped
with an electrospray ionization source. Plasma sample preparation and LC separation
conditions were consistent with the original Shimadzu 2010 LCMS method, however, it was
necessary to reduce the injection volume to 2 μL. The the gradient elution was also modified
as follows: 35 to 70% mobile phase B from 0ꢀ1 min; 70% B from 1ꢀ2 min; 70ꢀ95% from 2ꢀ
2.5 min; 95% B from 2.5ꢀ3 min, 95ꢀ35% B from 3ꢀ3.5 min and 35% B until 4 min. The total
run time was 4 min with Tolf and Diclof eluting at 1.6 and 1.4 min, respectively.
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The MS conditions were optimised as follows: drying gas flow, 15 L/min; nebulizing gas
flow, 2 L/min; desolvation line temperature, 200 °C; heat block temperature, 450 °C; and CID
gas, 230 kPa. Tolf was monitored at m/z 259.90 > 216.10 with the first quadrupole (Q1) and
third quadrupole (Q3) of the mass spectrometer set at 19.0 V and 15.0 V, respectively, and
collision energy at 16.0 V. For the internal standard, Diclof was monitored at m/z 293.90 >
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249.90 with the first quadrupole (Q1) and third quadrupole (Q3) of the mass spectrometer set
at 23.0 V and 17.0 V, respectively, and collision energy at 13.0 V. The chromatographic data
were acquired and analysed using the LabSolutions LCMS Version 5.4 software package for
LCMSꢀ8030 (Shimadzu).
Plasma concentrations in test samples were determined by comparison with a weighted (1/X)
quadratic calibration curve of Tolf:internal standard peak area ratio plotted as a function of
Tolf concentration. The assay was validated by analysis of n=3 spiked plasma quality control
samples at low (50 ng/mL), medium (2000 ng/mL) and high (10000 ng/mL) concentrations.
Intraꢀassay variability was acceptable and accurate to 94.2, 99.8 and 100.7% and precise to ±
2.5, 2.0 and 5.1% of the nominal QC concentrations at 50, 2000 and 10000 ng/mL.
RESULTS AND DISCUSSION
In the current study, a series of weakly acidic drugs were converted to highly lipophilic ILs,
with low melting points and favourable soluteꢀsolvent interactions in lipids. This approach
was taken to increase drug loading in LBFs and to promote the potential utility of LBF for
weak acids with limited lipid solubility. We have previously described a similar approach for
26
weakly basic PWSD, but to this point the in vitro and in vivo behavior of LBF containing IL
of weakly acidic drugs has not been described. Here weakly acidic drugs were converted into
ILs using highly lipophilic basic counterions, many based on quaternary ammonium
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structures similar to the widely used preservative benzalkonium. To address the potential for
differential properties using quaternary versus tertiary ammonium salts, i.e. to assess the
potential impact of a permanent positive charge versus pH dependent ionisation, data was also
obtained for an equivalent tertiary ammonium salt of Tolf.
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Tolf, Mec, Diclof and Ibu were chosen as model drugs (Figure 1). All are nonsteroidal antiꢀ
inflammatory drugs with relatively low water solubility, although in common with most
weakly acidic drugs, solubility under neutral or slightly basic pH in the intestinal tract is
31ꢀ34
significantly enhanced when compared to solubility in the stomach.
All four are relatively
well absorbed after oral administration and as such the intent of the current studies was not to
promote absorption per se, but rather to provide proof of concept data that IL could be
employed to enhance drug loading in LBF, and to evaluate the impact of IL formulation on in
vitro and in vivo behavior.
The selection of lipophilic counterions that are appropriate for use in ionic liquid preparations
of weak acids is complicated since lipophilic cations are, in general, more toxic than the
equivalent anions. We first examined a number of alkyl amines and spermine, a polyamine
35
involved in cellular metabolism, since these are perhaps the simplest and most well defined.
As described below, these did form ionic complexes with the weak acids studied here, albeit
o
complexes with melting points above 100 C and therefore materials better described as
lipophilic salts than ionic liquids. The advantages in lipid solubility for these materials, above
the free acid, however, were limited. As such, we subsequently turned to bulkier and more
branched lipophilic counterions. This was based on our previous experience with anionic
counterions for basic drugs where bulky branched acids appeared to more effectively disrupt
2
6
packing, reducing melting point and leading to greater increases in lipid solubility. This led
us to the quaternary ammonium compounds employed herein. These are amphiphilic
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surfactants and biocides and are widely used as antiseptic and disinfectants in clinical and
industrial environments. At low concentration benzalkonium (usually as the chloride) is
commonly used as a preservative (e.g. in eye drops). The Registry of Toxic Effects of
Chemical Substances (RTECS) at the Centers for Disease Control and Prevention in Atlanta
GA, suggests that the lowest oral LD50 in rats for benzalkonium is 240 mg/kg
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(https://www.cdc.gov/nioshꢀrtecs/bo3010b0.html). Didecyldimethylammonium chloride is an
antiseptic/disinfectant and has an oral LD50 in rats of 84 mg/kg (GESTIS substance database,
Institute for Occupational Safety and Health of the German Social Accident Insurance)
http://gestisꢀen.itrust.de. Didecylmethyl and butyldodecylammonium chloride are also
disinfectants, however, acute toxicity data after oral administration could not be readily
sourced. These materials are therefore less than ideal as pharmaceutically acceptable
counterions, and as described below, some GI toxicity was evident in our studies. However,
the intent here was to provide initial proof of concept with lipophilic cationic counterions,
rather than to progress potential clinical or commercial candidates.
DrugꢀILs were prepared by metathesis reaction between the sodium salt of the drugs and the
desired quaternary ammonium halide salts, or via acidꢀbase reaction between the drug free
acid and the alkyl amine base. For Tolf, the sodium salt was not readily available and instead
was formed in situ via deprotonation by sodium hydroxide. Metathesis reactions were carried
out under basic conditions (sodium carbonate or sodium bicarbonate) at room temperature in
water or in water/methanol mixtures where the solubility of the ammonium salt in water was
limiting. Acidꢀbase reactions between Tolf free acid and the alkyl amines (butyl, octyl,
didecylmethyl, octadecyl, dodecylamine and spermine) were carried out in methanol.
The resulting waterꢀimmiscible drugꢀILs formed immediately as oil droplets dispersed in the
aqueous reaction medium. DrugꢀILs were extracted with an organic solvent (dichloromethane
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or chloroform) and purified via washing with distilled water. The resultant drugꢀILs were
isolated as liquids or low melting solids. By way of example, Scheme 1 presents the
conversion of the sodium salt of Ibu, a crystalline white powder with a melting point of
0
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190
°C
via metathesis with didecyldimethylammonium bromide to form Ibu
didecyldimethylammonium salt (Ibu•NDec Me ), a low viscosity yellow oil. Synthetic and
2
2
analytical details for all API•ILs are given in the methods and supplementary material.
Once drugꢀILs were isolated, solubility in LBFs was assessed using a model long chain lipidꢀ
containing LBF. The LBF employed was a selfꢀemulsifying drug delivery system (SEDDS)
that disperses in aqueous media under very gentle agitation to form a nanoemulsion with
TM
particle size of 100ꢀ250 nm. The LBF comprised 60% w/w lipid (soybean oil:Maisine 35ꢀ1,
®
1
:1 w/w), 30% w/w surfactant (Kolliphor EL) and 10% cosolvent (ethanol).
2 2
Scheme 1 Preparation of Ibu•NDec Me from crystalline Ibu sodium and didecyldimethyl
ammonium bromide
2 3
Reagents and conditions: (a) didecyldimethyl ammonium bromide, H O/CH OH.
The impact of IL conversion on the melting ranges and solubility of the drugs in the model
LBF is shown in Table 1 and is detailed graphically for some examples in Figure 2.
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Figure 2 Comparison of the solubility of the free acid (FA) of Tolf, Mec and Diclof and
selected drugꢀILs in LCꢀSEDDS. Data shows the measured concentration after mixing a 1:1
w/w blend of formulation and IL. In all cases shown here ILs were effectively miscible at
proportions of 1:1 w/w and as such a true solubility was not determined. The data shown are
lower than the value of 500 mg/g that might be expected in a 1:1 mixture since the data are
expressed as mg/g of the free acid, rather than mg/g IL. The molecular weight of the drug and
the counterion were similar and as such values around 250 mg/g are evident. FA ꢀ free acid;
NDec Me – didecyldimethyl ammonium; NAlkBnMe – benzalkonium; Pyr – hexadecylꢀ3ꢀ
2
2
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methyl pyridinium. The numbers above the bars are the melting point of the FA or IL.
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