Prodrugs of pioglitazone for extended-release (XR) injectable formulations

Article abstract of DOI:10.1021/mp500359a

N-Acyloxymethyl derivatives of pioglitazone (PIO) have been prepared and characterized as model candidates for extended-release injectable formulations. All PIO derivatives prepared are crystalline solids as determined by powder X-ray diffraction, and the solubility in aqueous media is below 1 μM at 37 °C. The melting points steadily increase from 55 °C, for the hexanoyloxymethyl derivative, to 85 °C, for the palmitoyloxymethyl derivative; inversely, the solubilities in ethyl oleate decrease as a function of increasing acyl chain length. The butyroyloxymethyl ester has a higher melting point and a lower solubility in ethyl oleate than expected from the trend. The ¹³C solid-state NMR spectra of the PIO homologues between the hexanoyloxymethyl derivative and stearoyloxymethyl derivative suggest a common structural motif with the acyl chains exchanging between two distinct conformations, and the rate of exchange is slower for longer chain derivatives. The butyroyloxymethyl derivative is efficiently converted to PIO in in vitro rat plasma with a half-life of <2 min at 37 ^o C, while the rate of enzymatic cleavage in rat plasma decreases as the ester chain length increases for the longer acyloxymethyl derivatives. The concentration of PIO in plasma increases rapidly, or "spikes," in the hours following intramuscular (IM) injection of either the HCl salt or the butyroyloxymethyl derivative. In contrast, the more lipophilic palmitoyloxymethyl derivative provides slow growth in the PIO concentration over the first day to reach levels that remain steady for 2 weeks. On the basis of its in vivo pharmacokinetic profile, as well as material and solubility properties, the PIO palmitoyloxymethyl derivative has potential as a once-monthly injectable medication to treat diabetes.

Full text of DOI:10.1021/mp500359a

Article
pubs.acs.org/molecularpharmaceutics
Terms of Use
Prodrugs of Pioglitazone for Extended-Release (XR) Injectable
Formulations
Carlos N. Sanrame,‡* Julius F. Remenar, Laura C. Blu†mberg, Julie Waters, Reginald L. Dean, Nan Dong,
̈
Kristi Kriksciukaite, Peixin Cao, and Orn Almarsson
Alkermes plc, 852 Winter Street, Waltham, Massachusetts 02451-1420, United States
S
* Supporting Information
ABSTRACT: N-Acyloxymethyl derivatives of pioglitazone (PIO) have been prepared and characterized as model candidates for
extended-release injectable formulations. All PIO derivatives prepared are crystalline solids as determined by powder X-ray
diffraction, and the solubility in aqueous media is below 1 μM at 37 °C. The melting points steadily increase from 55 °C, for the
hexanoyloxymethyl derivative, to 85 °C, for the palmitoyloxymethyl derivative; inversely, the solubilities in ethyl oleate decrease
as a function of increasing acyl chain length. The butyroyloxymethyl ester has a higher melting point and a lower solubility in
ethyl oleate than expected from the trend. The 13C solid-state NMR spectra of the PIO homologues between the
hexanoyloxymethyl derivative and stearoyloxymethyl derivative suggest a common structural motif with the acyl chains
exchanging between two distinct conformations, and the rate of exchange is slower for longer chain derivatives. The
o
butyroyloxymethyl derivative is efficiently converted to PIO in in vitro rat plasma with a half-life of <2 min at 37 C, while the
rate of enzymatic cleavage in rat plasma decreases as the ester chain length increases for the longer acyloxymethyl derivatives.
The concentration of PIO in plasma increases rapidly, or “spikes,” in the hours following intramuscular (IM) injection of either
the HCl salt or the butyroyloxymethyl derivative. In contrast, the more lipophilic palmitoyloxymethyl derivative provides slow
growth in the PIO concentration over the first day to reach levels that remain steady for 2 weeks. On the basis of its in vivo
pharmacokinetic profile, as well as material and solubility properties, the PIO palmitoyloxymethyl derivative has potential as a
once-monthly injectable medication to treat diabetes.
KEYWORDS: pioglitazone prodrugs, extended-release, prodrugs, N-acyloxy derivatives, solid-state NMR, long acting injectable (LAI),
diabetes
microspheres, alternative extended-release formulations of PIO
are desired. We report herein the synthesis and characterization
of N-acyloxymethyl prodrug derivatives of PIO that have
potential to provide extended release of PIO from an aqueous
suspension of crystalline material.
There are a number of prodrugs utilized in marketed
extended-release injectable formulations of small-molecule
drugs, formulated as oil solutions (haloperidol decanoate,
fluphenazine decanoate, and fluphenazine enanthate) or as a
INTRODUCTION
■
Pioglitazone (PIO) is the leading PPARγ agonist used to treat
Type II Diabetes Mellitus with superior efficacy and tolerability
over other thiazolidinediones;1,2 however, its use is limited by
safety concerns.3,4 An extended-release injectable formulation
of PIO could provide a reduced peak-to-trough ratio in drug
plasma concentrations, which may provide improved safety and
tolerability5 and improved bioavailability by mitigating first-pass
metabolism of the oral therapy.6 Extended-release formulations
of PIO utilizing polymer microspheres to control the rate of
release of the active drug have been reported.7 Since the daily
dose of PIO is considerable (up to 45 mg), the total mass of
injection for a once-monthly injectable formulation is a
concern. As a result of the limited drug-loading achievable in
Received: May 14, 2014
Revised: August 18, 2014
Accepted: August 26, 2014
Published: August 26, 2014
© 2014 American Chemical Society
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nanocrystal aqueous suspension8 (Invega Sustenna (paliper-
idone palmitate)).9 In these examples, a hydroxyl functional
group in the drug molecule has been esterified with a lipophilic
fatty acid to produce a prodrug derivative with desired solubility
properties.
A series of acyloxymethyl derivatives of PIO with varying
chain lengths of the acyl groups have been synthesized and
evaluated for their thermodynamic and material properties as
well as their extended-release profiles from IM injection in rats.
EXPERIMENTAL SECTION
Recently, LinkeRx technology has been developed to provide
crystalline low-solubility prodrugs from drugs, such as PIO, that
lack hydroxyl functional groups.10,11 These prodrug modifica-
tions allow for engineering the material and physical chemical
properties that are required for extended-release injectable
formulations.12 The desired formulation is a simple aqueous
suspension of crystalline prodrugs. The ideal mechanism of
release is rate-limiting dissolution of the prodrug from the
depot followed by rapid absorption of the prodrug into
systemic circulation and efficient enzymatic cleavage, to provide
sustained, efficacious concentrations of the active drug and
relatively low circulating concentrations of the prodrug
derivative (Scheme 1). This has been accomplished with the
current phase 3 clinical development candidate, aripiprazole
lauroxil, a prodrug of the partial D2 agonist, aripiprazole.10,13
■
Synthesis of the Prodrugs. The N-acyloxymethyl
derivatives of PIO were synthesized in a three-step process
and were isolated as free bases (Scheme 2).
Scheme 2. Synthesis of the PIO N-Acyloxymethyl
a
Derivatives
Scheme 1. Conversion of the LinkeRx Derivatives to PIO
a
Reagents and conditions: (a) ZnCl2, paraformaldehyde, acetonitrile, 0
°C → ambient temperature, 1 h, 90 °C for 15 h, 70% yield; (b) NaI,
acetonitrile, dark, ambient temperature, 15 h, 55% yield; (c) K2CO3,
dimethylformamide, ambient temperature 15−48 h, 21−78% yield.
General Procedure for the Synthesis of Chloromethyl
Esters (Step 1).23 Acid chloride (one equiv) was added
dropwise to an acetonitrile solution of paraformaldehyde (one
equiv) and anhydrous zinc chloride (0.5 equiv) at 0 °C under
argon. After the addition was complete, the reaction mixture
was warmed to 25 °C and stirred for 1 h, then heated to 90 °C
for 15 h. The solid was filtered off and washed with
dichloromethane; the filtrate was concentrated under vacuum
at 37 °C to provide chloromethyl ester, which was used directly
(without purification) in the next step.
General Procedure for the Synthesis of Iodomethyl Ester
(Step 2).24 A solution of chloromethyl ester (one equiv) in
acetonitrile (86 mL) was treated with sodium iodide (three
equiv). The flask was covered in aluminum foil and stirred at 25
°C for at least 15 h. The reaction mixture was partitioned
between dichloromethane and water, and the aqueous layer was
extracted with dichloromethane. The combined organics were
washed with saturated aqueous NaHCO3, 10% aqueous sodium
sulfite solution, and brine, then dried with sodium sulfate and
concentrated to give the iodomethyl ester as a yellow oil, which
was used in the alkylation step without further purification.
General Procedure for the Synthesis of PIO Acylalkoxyl
Derivatives (Step 3). A solution of PIO (one equiv) in
dimethylformamide was treated with dry K2CO3 (three equiv)
at 25 °C. After 40 min, a solution of iodomethyl ester (two
equiv) was added dropwise. The reaction mixture was stirred
for at least 15 h until alkylation was complete, then dumped
The thiazolidinedione ring of PIO has been identified as a
potential site for prodrug modification; this ring system is
considered an imide-type NH acid as defined by Stella.14
Bundgaard and Stella demonstrated the utility of N-acyloxyalkyl
or phosphoryloxyalkyl modifications of weak NH acids to
provide bioreversible derivatives with modified physical
chemical properties.15 One example of a marketed drug that
utilizes this prodrug approach is fosphenytoin, the soluble
phosphoryloxymethyl derivative of phenytoin, a therapy for
epilepsy.16,17 N-Acyloxylmethyl derivatives of imide-containing
drugs are enzymatically cleaved to release N-hydroxymethyl
intermediates that release formaldehyde and the parent
drug.18,19 The rate of dehydroxymethylation of the intermediate
is dependent on the pKa of the parent drug as well as pH; for
example, the pKa of phenytoin is 8.3 and the measured half-life
of phenytoin hydroxymethyl derivative at pH 7.4 and 37 °C is
one second.20,21 On the basis of the reported pKa of PIO
(5.95),22 the predicted half-life of the hydroxymethyl−PIO
derivative at pH 7.4 and 37 °C is less than one second.
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into water and extracted with ethyl acetate. The combined
organic layers were dried with sodium sulfate and concentrated
under vacuum. The product was purified by flash chromatog-
raphy on silica gel.
Recrystallization. Samples of 4b−h were recrystallized
from isobutyl acetate/heptane or isobutyl acetate/pentane and
dried under vacuum or air-dried. The growth of single crystals
was attempted by slow evaporation from several solvents.
Unfortunately, all attempts at crystallization produced only very
small, fine needles that were unsuitable for single crystal X-ray
diffraction.
PXRD to confirm preservation of the crystalline form during
the experiment.
ClogP. The partition coefficients (logP) were calculated
using ChemBioDraw Ultra version 12.0 from CambridgeSoft
Specific algorithms for calculating logP from fragment-based
methods developed by the Medicinal Chemistry Project and
BioByte.
General Procedure for Aqueous Hydrolysis Measure-
ments. The aqueous hydrolysis of the prodrugs was performed
in the presence of a cosolvent (20% MeCN or DMSO) because
of the very low solubility of the prodrugs in neat aqueous
media. Then, 0.05−0.1 mM solutions of the prodrugs 4a, 4e,
4g, and PIO were prepared in PBS/MeCN 8:2 and/or PBS/
DMSO 8:2 and incubated at 37 °C. Typically, aliquots (0.010
mL) were sampled at different time points and immediately
injected in the HPLC with UV detection and, for some
experiments, with MS detection. Substantial precipitation was
observed for 4e, 4g, and PIO even in the presence of 20%
cosolvent. Therefore, the half-life could only be calculated for
4a.
Microscopy. An Olympus BX51 Reflected Polarized Light
Microscope was used to determine crystal morphology and
characterize the particle size of the suspensions.
Powder X-ray Diffraction (PXRD). PXRD was performed
on a Rigaku Miniflex 2 diffractometer (Rigaku/MSC, Wood-
lands, TX) with a Si zero-background holder with ϕ rotation
and Cu Kα radiation operating at 30 kV/15 mA. Data were
processed using Eva 2 (Bruker).
Solid-State NMR. 13C cross polarized-magic angle spinning
NMR (13C CP/MAS NMR) spectra were obtained on a Varian
General Procedure for Rat Plasma Incubation. Stock
solutions of the prodrugs were prepared in acetonitrile or
acetonitrile/THF. A 0.010 mL aliquot of the stock was spiked
into 1 mL of rat plasma and incubated at 37 °C. The
concentration of the prodrugs in rat plasma was 0.250 mM,
when HPLC−UV was used for sample analysis or 0.003 mM
when HPLC−MS/MS was used. Typically, aliquots (0.050 mL)
were sampled at different time points and immediately
dispensed into Eppendorf tubes containing 0.200 mL of
acetonitrile to precipitate the protein. The tubes were
centrifuged, and the supernatant was isolated and analyzed by
HPLC−UV or HPLC−MS/MS. The peak areas corresponding
to the prodrugs were plotted against time, and the data were
fitted to a first-order monoexponential decay where the rate
constant and the half-life were determined from the slope.
Rat IM PK Dosing. The studies were completed under
protocols approved by the Alkermes Institutional Animal Care
and Use Committee and were conducted in accordance with
the Institute of Laboratory Animal Resources.25 Each
compound was suspended using aseptic techniques at a
concentration of 66.7 mg/mL (PIO equivalents) in a vehicle
containing 2% sodium carboxymethyl cellulose (CMC), 0.2%
polysorbate 20, and PBS buffer, 302 mOsm/kg pH 6.7. The
vehicle was autoclaved before use at 121 °C for 20 min. There
were six rats in each group. A 0.3 mL suspension dose (20 mg
PIO equivalents) was injected immediately into the hind limb
muscles of rats after anesthesia with isoflurane using a single
smooth push with a 1 mL syringe with a 23-gauge, one-inch
needle. Time points: 0.25, 1, and 6 hours and 2, 4, 7, 10, 14, 21,
and 35 days after administration. Approximately 0.250 mL of
whole blood was collected at each sampling time through day
14 and 0.450 mL of whole blood for each time point after day
14. All blood samples were collected via a lateral tail vein after
brief anesthesia with Isoflurane. A 27G half-inch needle and 1
mL syringe without an anticoagulant was used for blood
collection. Once collected, whole blood was transferred to
tubes containing K2-EDTA, inverted 10−15 times, and placed
on ice. The tubes were then centrifuged for 2 min at >14,000g
(11,500 rpm using an Eppendorf centrifuge) at 4−8 °C to
separate plasma. The plasma samples were transferred to chilled
(4−8 °C) plain tubes (96-well plate) and stored frozen at −70
°C.
1
NMR operating at 399.746 MHz for H and 100.527 MHz for
13C. Spectra were collected using a 4 mm T3-HFX probe tuned
to 13C and 1H. All samples were spun at 10 kHz with
temperature set at 10 °C unless otherwise stated. 13C CP/MAS
spectra were collected using the tangentially ramped cross-
polarization pulse sequence (tancpx) included as part of
SolidsPack library of pulse sequences for use with the Vnmrj
3.1a spectrometer operating software by Agilent Technologies.
The power levels were adjusted to provide π/2 = 2.5 μs for
both channels. Cross-polarization and decoupling parameters
were optimized using glycine, and the spectra were referenced
to the carbonyl peak of glycine at 176.5 ppm. The delay time
between transients was set for at least 1.2 times the value of T1
for 13C CP/MAS spectra, and for >5 times T1 during
1
measurement of H or 13C T1 values. Spectral overlays were
produced using IGOR Pro 6.05 by Wavemetrics, Inc.
Thermal Analysis. Differential scanning calorimetry (DSC)
curves were acquired using a TA Instruments Q1000. Typically,
1−2 mg of sample were weighed into an aluminum pan
(Tzero), sealed with a pinhole lid and heated at 10 °C/min
from 25 to 200 °C. Melting points are reported as the peak
temperature of the melting endotherm. Thermal gravimetric
analysis (TGA) was performed on a TA Instruments TGA
Q500. Samples of 5−10 mg were heated at 10 °C/min from 25
to 250 °C. The data were processed using Universal Analysis
2000 Version 4.3A.
General Procedure for Solubility Measurements in
Aqueous Media. Suspensions of the prodrugs (5 mg/mL) in
PBS were stirred at 37 °C for 3−4 days. Daily, 1 mL aliquots
were filtered; the filtrate diluted with MeCN or MeOH (×2)
and analyzed by HPLC to determine the amount of prodrug
dissolved. After 3−4 days, the remaining suspension was
filtered and the solid analyzed by PXRD to confirm
preservation of the crystal; and after dissolution with MeCN
or MeCN/THF, it was analyzed by HPLC to confirm purity
after the experiment.
General Procedure for Solubility Measurements in
Ethyl Oleate. Suspensions of the prodrugs in ethyl oleate were
stirred at room temperature for 2 days. The samples were
filtered and the filtrate diluted with MeCN/THF (×10 or
×100) and analyzed by HPLC to determine the amount of
prodrug dissolved. The remaining solids were analyzed by
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PIO concentrations in plasma samples were analyzed by
liquid chromatography−mass spectroscopy (HPLC−MS/MS)
using appropriate parameters for each compound. Half-life,
maximal concentration, and AUC were calculated by non-
compartmental analysis using WinNonlin version 5.2 software
(Pharsight, St. Louis, MO) based on measured PIO
concentrations through day 35.
RESULTS AND DISCUSSION
■
Structural Characterization of the Prodrugs by PXRD
and Solid-State NMR. All of the PIO derivatives prepared
were crystalline solids as determined by PXRD (Figure 1). The
Figure 2. 13C CP/MAS NMR spectroscopy on a Varian NMR 400
MHz using a 4 mm probe for 4a−h at 10 °C.
Figure 1. PXRD pattern at room temperature for 4a−h.
structures were characterized with PXRD and 13C CP/MAS
NMR studies that suggest compounds 4a−h pack with similar
structural motifs where the lattice expands for longer chain
lengths. The lowest angle diffraction peak decreased from 3.4 to
2.9° 2θ with increasing chain length, indicating an expansion of
the longest axis of the unit cell. However, many of the peaks, or
clusters, change very little with extending chain length,
suggesting that the overall arrangement of atoms in the lattice
is similar. 13C CP/MAS NMR spectroscopy was used as
another probe, as chemical shifts are sensitive to the local
environment of atoms within the crystal lattice.
Figure 3. Temperature dependence in the alkyl region of the 13C CP/
MAS NMR spectra for 4e and 4g.
The chemical shift values between 50 and 165 ppm in the
13C CP/MAS NMR spectra of the PIO derivatives (Figure
2)26−28 correspond to the linker and the majority of the core
PIO structure. These peaks remain identical throughout the
series with no changes in the number of carbon peaks,
suggesting that crystal packing motif is similar for all of the
derivatives between 4b and 4h. However, there are some
changes in the peaks and chemical shifts that correspond to the
acyl chain and carbonyls, which are suggestive of differences in
the local environment of these groups within the crystal lattices
of the different derivatives.
The changes in the peaks corresponding to the acyl chain,
10−40 ppm, have been explored as a function of temperature,
and they appear to be changes in the acyl chain mobility within
the crystal structure. Figure 3 shows temperature-dependent
changes in the alkyl region (10−40 ppm) of 4e (lauroylox-
ymethyl) and 4g (palmitoyloxymethyl), as representatives of
derivatives containing longer acyl chains (4e, 4f, 4g, and 4h).
The spectra at 0 °C show two pairs of peaks between 14 and 17
ppm, with each pair representing a methyl group. Each of these
pairs coalesces, sharpening to a single peak at higher
temperatures.
The coalescence temperature occurs between 0 and 10 °C
for 4e (lauroyloxymethyl), but in the higher range of 30−50 °C
for 4g (palmitoyloxymethyl). There are no detectable thermal
transitions in the DSC curves at these temperatures, which is
consistent with an increasing rate of motion with temperature
rather than a sudden change from static to free rotation (or a
very low heat capacity transition). Overall, the solid-state data
for 4b−h analogues suggest a common structural motif with the
alkyl chains exchanging between two distinct conformations
and the rate of exchange being slower for longer chains.
Solubility and Melting Behavior of the Prodrugs. The
solubility of all of the derivatives in aqueous media is very low,
below 1 μm, but a significant solubility difference is observed in
ethyl oleate (EO), a model solvent for assessing solubility in
lipid milieu. The overlay of the melting point and EO solubility
curves as a function of acyl chain length shows an inverse
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correlation (Figure 4). High oil solubility corresponds to low
melting solids and vice versa. This inverse correlation between
lipid solubility and melting points has been reported
previously.16,29
MS, which is consistent with the expected high instability of
this intermediate. On the basis of the reported pKa of PIO
(5.95)22 and hydroxide ion activity (aOH−) at pH 7.4 and 37
°C, which is 6.02 × 10−7 M, the half-life of the hydroxymethyl−
PIO derivative can be predicted to be 0.01 s (= 0.693/kobs),
where kobs = 3883 min−1 (= k1 × aOH−), and the apparent
hydroxide ion catalytic rate constant is k1 = 6.45 × 109 min−1
M−1 (= antilog(14.4−0.77 × 5.95)).20,21
Prodrug Stability in Aqueous Media. The low aqueous
solubility of the long-chain prodrugs (hexanoyloxymethyl
derivative and longer) precludes stability measurements in
aqueous buffer. As a result, the butyroyloxymethyl 4a derivative
is the only derivative for which the stability was determined in
phosphate-buffered saline pH 7.5 (PBS)/DMSO 8:2 at 37 °C.
In Figure 5, the disappearance of 4a and the formation of its
degradants and PIO are plotted against time.
Figure 4. Solubility of 4a−4g in ethyl oleate at room temperature and
melting point vs acyl chain length.
The curves follow a trend of increasing melting point and
decreasing solubility with increasing chain length beginning
with the hexanoyloxymethyl ester. The butyroyloxymethyl
ester, 4a, breaks the trend, melting at higher temperature than
expected and having a lower solubility in ethyl oleate (EO).
The heat of fusion increases linearly with the acyl chain length
from 165 to 200 mJ/mol for 4a−e, and from 195 to 205 mJ/
mol for 4f−h. The palmitoyloxymethyl ester, 4g, one of the
most lipophilic derivatives, has the highest melting point and
lowest solubility in the series.
Prodrug Stability in Rat Plasma. The rate of in vitro
conversion of the PIO derivative to PIO was determined in a
biological matrix known to express carboxylesterase en-
zymes.30−32 The rate of prodrug conversion in rat plasma at
37 °C is represented as the half-life derived from monitoring
the disappearance of prodrugs over time. Data at 37 °C were
fitted to a first-order kinetic equation for prodrug loss and
appearance of PIO. The rate of enzymatic cleavage decreases as
the ester chain increases, with fatty acid chains shorter than six
carbons showing rapid conversion and half-lives less than 2 min
(Table 1).
Figure 5. Area % vs time for 4a and its degradants in PBS/DMSO 8:2
at 37 °C. The mass/charge ratio (m/z) and relative retention times
(RRT) for major peaks in the HPLC−UV−MS chromatograms are
indicated in the legend.
The butyroyloxymethyl prodrug 4a does not convert to PIO
(MW 356.4) in this medium but mainly to a single product
with a molecular weight of 404, as measured by HPLC−MS.
The proposed structure for the breakdown product is shown in
Scheme 3 and corresponds to the hydrolysis of both the ester
Scheme 3. Proposed Structure for the Main Breakdown
Product of 4a in PBS/DMSO 8:2 at 37 °C
Table 1. Half-Life of Prodrug Hydrolysis in Rat Plasma at 37
°C and Calculated logP
half-life (minutes)
ClogP
ab
,
4a (butyroyloxymethyl)
4b (hexanoyloxymethyl)
4e (lauroyloxymethyl)
4f (myristoyloxymethyl)
<2
<2
3.78
4.84
8.01
9.07
10.13
a
a
bc
,
20 , 8
a
∼60
690
bd
,
4g (palmitoyloxymethyl)
group and of the thiazolidine-2,4-dione ring. Further
decomposition of this product to lower molecular weight
byproducts is observed throughout the experiment. PIO has
low solubility in the reaction media, so to confirm that PIO was
not formed in the hydrolysis of 4a, MeOH was added after 18 h
to redissolve any PIO precipitate. Analysis of resulting
methanol solution confirmed the absence of a significant
amount of PIO. A similar result was obtained after incubating at
37 °C 4a in PBS/MeCN 8:2. The peak areas corresponding to
4a were plotted against time, and the data were fitted to a first-
order monoexponential decay where the rate constant and the
half-life, 3.7 h, were calculated from the slope. The half-life for
the 4a degradation in buffer is significantly slower than the
a
b
c
HPLC−UV. HPLC−MS/MS. Deviation from linearity at high
d
conversion (>50%). Extrapolated, calculated from slope.
The half-life for the cleavage of lauroyloxymethyl derivative,
4e, is 20 min. The PIO derivatives with acyl chains longer than
14 carbons (myristoyloxymethyl) appear to be resistant to the
esterase enzymes as the half-lives exceed 400 min for
palmitoyloxymethyl. This dependence of the plasma rate of
hydrolysis on the length of the acyl chain has been previously
observed for lipophilic prodrugs.33
The absence of measurable levels of the intermediate N-
hydroxymethyl−PIO derivative was confirmed by HPLC−MS/
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conversion to PIO in rat plasma (<2 min) at 37 °C; therefore, it
is unlikely that this nonproductive degradation occurs in vivo to
a significant extent.
<30 μM for PIO freebase). The low heat of fusion coupled with
high-lipid solubility of 4a are contributing to its rapid
absorption in rat. The longer-chain 4g derivative shows slower
release of PIO with a delayed tmax of 4 days.
Pharmacokinetics of the Prodrugs in Rats after IM
Injection. After establishing that the PIO derivatives have the
material and solubility properties to allow for formulation as
aqueous suspensions, along with enzymatic activity to act as
productive prodrugs of PIO, we evaluated the prodrugs in vivo
in rat pharmacokinetic studies. One short-chain derivative (4a)
and one long-chain derivative (4g) were dosed as IM injections
of aqueous suspensions of crystalline material. The resulting
plasma concentrations of PIO were measured and compared to
the PIO plasma concentrations achieved when a similar
formulation of PIO HCl is dosed intramuscularly. As shown
in Figure 6, PIO dosed as the hydrochloride salt and the
CONCLUSIONS
■
Use of the methyleneoxy linker group installed on a weakly
acidic thiazolidine-2,4-dione ring creates the opportunity to
make reversible fatty acid ester prodrugs of PIO. Such prodrugs
are crystalline, have low aqueous solubility, and provide
sustained release as aqueous suspensions following intra-
muscular injections. The N-acyloxymethyl prodrugs of PIO
appear to pack in a common motif in the crystal lattice
regardless of acyl chain length, where the long axis of the lattice
expands with increasing the chain length. Longer acyl chains
become simultaneously more resistant to melting and less
soluble in oily solvents, consistent with a stronger packing
efficiency. Likewise, the molecular motions in the lattice slow
with increasing acyl chain length as shown by 13C CP/MAS
NMR. The rate of cleavage in rat plasma at 37 °C is rapid for
derivatives with acyl chain lengths up to lauroyloxymethyl but
steeply decreases for longer-chain derivatives. Rat plasma
concentrations of PIO following an IM injection of the
palmitoyloxymethyl ester of PIO show extended release with
no spike in plasma PIO concentration; while an IM injection of
the shorter-chain butyroyloxymethyl derivative releases PIO
faster than PIO HCl. This study provides a proof-of-concept
that LinkeRx technology can be utilized to tune key
physicochemical properties of a drug that contribute to rate
of absorption from a depot in tissue. The palmitoyloxymethyl
prodrug has the potential to be developed as a once-monthly
injectable formulation based on its reduced aqueous solubility
and absorption rate. On the basis of its IM pharmacokinetic
profile in rats, this novel PIO prodrug has the potential to
provide a very low peak/trough ratio in humans at steady state,
which is a hallmark of well-designed extended-release
injectables.
Figure 6. Mean PIO concentration (linear scale) measured following
IM administration of PIO acyloxymethyl prodrugs 4a (butyroylox-
ymethyl) and 4g (palmitoyloxymethyl) and PIO HCl suspensions to
rats (n = 6). Dose: 20 mg of PIO equivalents (0.3 mL of 66.7 mg/mL
(PIO equivalents) in 2% CMC, 0.2% Tween 20, and PBS buffer, 302
mOsm/kg pH 6.7).
butyroyloxymethyl derivative (4a) show rapid absorption on
day 1, followed by distribution and elimination. The highly
lipophilic palmitoyloxymethyl derivative, 4g, exhibited the
slowest release of PIO with a significantly delayed Tmax
.
The comparison of the AUC0−t achieved with equivalent
doses of 4a and 4g with PIO HCl demonstrated that both
derivatives efficiently converted to PIO in vivo (Table 2). The
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra, HPLC−MS data, and DSC curves
for all compounds are included, as well as the chromatograms
corresponding to the stability in aqueous media of 4a. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Table 2. Calculated Pharmacokinetic Parameters for PIO
HCl, 4a and 4g
PIO AUC
(ng·day/mL)
tmax (d)
0−t
PIO HCl
9270 (1910)
5970 (888)
6760 (1110)
0.3
0.3
4.0
AUTHOR INFORMATION
4a (butyroyloxymethyl)
4g (palmitoyloxymethyl)
■
Corresponding Author
*(C.N.S.) Tel: 781-609-6864. E-mail: carlos.sanrame@
alkermes.com.
absorption profile for PIO after PIO HCl dosing is likely
influenced by the conversion of the PIO HCl salt to the lower
solubility free base form at neutral pH in the muscle. The short-
chain butyloxymethyl modification appears to accelerate
absorption, with 4a providing a higher early Cmax relative to
both PIO and the palmityloxymethyl derivative. Particle size
does not appear to be a key factor in defining the rate of
absorption since microscopy studies show that the particle size
of 4a, which provides a high and early Cmax, is significantly
larger than the particle size of 4g. While the aqueous solubility
of 4a and 4g is lower than PIO free base (3.5 μM in PBS), the
solubility in ethyl oleate, as a model lipid, is much higher for the
prodrugs, especially for the butyroyloxymethyl 4a (80 mM vs
Present Addresses
†(O.A.) Moderna Therapeutics, Inc., 200 Technology Square,
Cambridge, Massachusetts 02139, United States.
‡(K.K.) Blend Therapeutics, 134 Coolidge Avenue, Watertown,
Massachusetts 02472, United States.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
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Molecular Pharmaceutics
Article
ymethyl prodrugs of phenytoin to structure: potential in vivo
performance implications. J. Pharm. Sci. 1998, 87, 1235−1241.
(17) Gerber, N.; Mays, D. C.; Donn, K. H.; Laddu, A.; Guthrie, R.
M.; Turlapaty, P.; Quon, C. Y.; Rivenburg, W. K. Safety, tolerance and
pharmacokinetics of intravenous doses of the phosphate ester of 3-
hydroxymethyl-5,5-diphenylhydantoin: a new prodrug of phenytoin. J.
Clin. Pharmacol. 1988, 28, 1023−1032.
(18) While formaldehyde is a known carcinogen, prodrugs known to
release formaldehyde at relatively low levels have been shown to be
safe: Griesemer, R.; Ulsamer, A.; Arcos, J.; Beall, J.; Blair, A.; Collins,
T.; de Serres, F.; Everson, R.; Gamble, J.; Gaylor, D.; Groth, D.; Kang,
J.; Keenlyside, R.; Lloyd, J.; Nettesheim, P.; Saffiotti, U.; Weisburger,
E. Report of the federal panel on formaldehyde. Environ. Health
Perspect. 1982, 43, 139−168.
(19) Dhareshwar, S. S.; Stella, V. J. Your prodrug releases
formaldehyde: should you be concerned? No! J. Pharm. Sci. 2008,
97, 4184−4193.
(20) Bundgaard, H.; Johansen, M. Prodrugs as drug delivery systems.
VIII. Bioreversible derivatization of hydantoins by N-hydroxymethy-
lation. Int. J. Pharm. 1980, 5, 67−77.
(21) Johansen, M.; Bundgaard, H. Prodrugs as drug delivery systems.
VI. Kinetics and mechanism of the decomposition of the N-
hydroxymethylated amides and imides in aqueous solutions and
assessment of their suitability as possible prodrugs. Arch. Pharm.
Chemi, Sci. Ed. 1979, 7, 175−192.
(22) Al-Arfaj, N. A.; Al-Abdulkareem, E. A.; Aly, F. A. A validated
adsorptive stripping voltammetric determination of antidiabetic agent
pioglitazone HCl in tablets and biological fluids. Int. J. Biomed. Sci.
2008, 4, 310−318.
(23) Ulich, L. H.; Adams, R. The reaction between acid halides and
aldehydes III. J. Am. Chem. Soc. 1921, 43, 660−667.
ABBREVIATIONS
■
PIO, pioglitazone; PBS, phosphate buffer saline pH 7.5; PXRD,
power X-ray diffraction; HPLC-MS, high-performance liquid
chromatography−mass spectrometry; HPLC−UV, high-per-
formance liquid chromatography/ultraviolet spectrophotome-
try; DSC, differential scanning calorimetry; LC−MS/MS, liquid
chromatography-tandem mass spectrometry; 13C CP/MAS
NMR, 13C cross polarized−magic angle spinning nuclear
magnetic resonance
REFERENCES
■
(1) Scheen, A. J. Hepatotoxicity with thiazolidinediones: is it a class
effect? Drug Saf. 2001, 24, 873−888.
(2) Rasouli, N.; Raue, U.; Miles, L. M.; Lu, T.; Di Gregorio, G. B.;
Elbein, S. C.; Kern, P. A. Pioglitazone improves insulin sensitivity
through reduction in muscle lipid and redistribution of lipid into
adipose tissue. Am. J. Physiol.: Endocrinol. Metab. 2005, 288 (5), E930−
934.
(3) Tobiasova, Z.; Zhang, L.; Yi, T.; Qin, L.; Manes, T. D.; Kulkarni,
S.; Lorber, M. I.; Rodriguez, F. C.; Choi, J.-M.; Tellides, G.; Pober, J.
S.; Kawikova, I.; Bothwell, A. L. M. Peroxisome proliferator-activated
receptor-γ agonists prevent in vivo remodeling of human artery
induced by alloreactive T cells. Circulation 2011, 124, 196−205.
(4) Li, W.; Macdonald, T. M.; Mackenzie, I. S. Pioglitazone and
bladder cancer: A propensity score matched cohort study. Br. J. Clin.
Pharmacol. 2013, 75, 254−259.
(5) Canas, F.; Moller, H. J. Long-acting atypical injectable
̃
̈
antipsychotics in the treatment of schizophrenia: safety and tolerability
review. Expert Opin. Drug Saf. 2010, 9, 683−697.
(24) Finkelstein, H. Ber. Dtsch. Chem. Ges. 1910, 43, 1528−1532.
(25) National Research Council (US) Committee for the Update of
the Guide for the Care and Use of Laboratory Animals. Guide for the
Care and Use of Laboratory Animals, 8th ed.; National Academies Press
(US): Washington, DC, 2011.
(26) Tishmack, P. A.; Bugay, D. E.; Byrn, S. R. Solid-state nuclear
magnetic resonance spectroscopy-pharmaceutical applications. J.
Pharm. Sci. 2003, 92, 441−474.
(6) Baser, O.; Chalk, M.; Rawson, R.; Gastfriend, D. R. Alcohol
dependence treatments: comprehensive healthcare costs, utilization
outcomes, and pharmacotherapy persistence. Am. J. Manag. Care 2011,
Suppl. 8, S222−S234.
(7) Woo, H. J.; Kim, J. S.; Nurunnabi, M.; Huh, K. M.; Cho, K. J.;
Lee, Y. Preparation and characterization of pioglitazone loaded PLGA
nanospheres for the treatment of type 2 diabetes. Polymer 2010, 34,
527−533.
(8) Merisko-Liversidge, E.; Liversidge, G. G. Nanosizing for oral and
parenteral drug delivery: a perspective on formulating poorly-water
soluble compounds using wet media milling technology. Adv. Drug
Delivery Rev. 2011, 63, 427−440.
(9) Remenar, J. F. Making the leap from daily oral dosing to long
acting injectables: lessons from the antipsychotics. Mol. Pharmaceutics.
2014, 11, 1739−1749.
(10) Remenar, J. F.; Blumberg, L. C.; Zeidan, T. A. Heterocyclic
Compounds for the Treatment of Neurological and Psychological
Disorders. U.S. Patent 8,431,576, 30 April 30, 2013.
(11) Blumberg, L. C.; Zeidan, T. A.; Maddaford, A.; Warren, N. C.;
Hutchison, P. Novel N-5-(acyloxyalkoxy)carbonyl prodrugs of
olanzapine with physicochemical properties for extended-release.
RSC Adv. 2013, 3, 16270−16278.
(12) Wright, J. C., Burgess, D. J., Eds. Long Acting Injections and
Implants. In Advances in Delivery Science and Technology; Springer:
New York, 2012.
(27) Geppi, M.; Mollica, G.; Borsacchi, S.; Veracini, C. A. Solid-state
NMR studies of pharmaceutical systems. Appl. Spectrosc. Rev. 2008, 43,
202−302.
(28) Berendt, R. T.; Sperger, D. M.; Munson, E. J.; Isbester, P. K.
Solid-state NMR spectroscopy in pharmaceutical research and analysis.
TrAC, Trends Anal. Chem. 2006, 25, 977−984.
(29) Patton, J. S.; Stone, B.; Papa, C.; Abramowitz, R.; Yalkowsky, S.
H. Solubility of fatty acids and other hydrophobic molecules in liquid
trioleoylglycerol. J. Lipid Res. 1984, 25, 189−97.
(30) Liederer, B. M.; Borchardt, R. T. Enzymes involved in the
bioconversion of ester-based prodrugs. J. Pharm. Sci. 2006, 95, 1177−
1195.
(31) Satoh, T.; Hosokawa, M. The mammalian carboxylesterases:
from molecules to functions. Annu. Rev. Pharmacol. Toxicol. 1998, 38,
257−288.
(32) Ross, M. K.; Crow, J. A. Human carboxylesterases and their role
in xenobiotic and endobiotic metabolism. J. Biochem. Mol. Toxicol.
2007, 21, 187−196.
(33) Yamaoka, Y.; Roberts, R. D.; Stella, V. J. Low-melting phenytoin
prodrugs as alternative oral delivery modes for phenytoin: a model for
other high melting sparingly water-soluble drugs. J. Pharm. Sci. 1983,
72, 400−405.
(13) Turncliff, R.; Potocka, E.; Corey, L.; Lowy, A.; Marandi, M.;
DeSomer, M.; Ehrich, E. ALKS 9070, a novel once monthly prodrug of
aripiprazole, achieves therapeutic levels and is well-tolerated in adults
with schizophrenia. Eur. Neuropsychopharmacol. 2012, 22 (Supplement
3), S330−S331.
(14) Guarino, V. R.; Stella, V. J. Prodrugs of Amides, Imides and
Other NH-Acidic Compounds. In Prodrugs: Challenges and Rewards,
Part 2; Stella, V. J., Borchardt, R. T., Hageman, M. J., Oliyai, R., Maag,
H., Tilley, J. W., Eds.; Springer: New York, 2007; pp 133−187.
(15) Bundgaard, H. Formation of prodrugs of amines, amides,
ureides, and imides. Method Enzymol. 1985, 112, 347−359.
(16) Stella, V. J.; Martodihardjo, S.; Terada, K.; Rao, V. M. Some
relationships between the physical properties of various 3-acylox-
3623
dx.doi.org/10.1021/mp500359a | Mol. Pharmaceutics 2014, 11, 3617−3623