Synthesis and in vitro characterization of drug conjugates of l-carnitine as potential prodrugs that target human Octn2

Article abstract of DOI:10.1002/jps.22557

The objective was to evaluate the potential of drug conjugates with l-carnitine as prodrugs that target organic cation/carnitine transporter (OCTN2). Twenty-two l-carnitine analogues were evaluated for human organic cation/carnitine transporter (hOCTN2) inhibition; the 3'-hydroxyl group was found to be the only functional group not contributing to l-carnitine interaction with hOCTN2 among the three functional groups on l-carnitine (i.e., 3'-hydroxyl, amine, and carboxylate). The 3'-hydroxyl group on l-carnitine was therefore chosen as the conjugate site. Three drug-l-carnitine conjugates (i.e., valproyl-l-carnitine, naproxen-l-carnitine, and ketoprofen-l-carnitine) were synthesized along with two ketoprofen analogues that incorporated a linker group (glycolic acid or glycine) between ketoprofen and l-carnitine (i.e., ketoprofen-glycolic acid-l-carnitine and ketoprofen-glycine-l-carnitine). These potential prodrugs were evaluated for their in vitro inhibition, transport, and metabolism properties. All three drug-l-carnitine conjugates and ketoprofen-glycine-l-carnitine were OCTN2 inhibitors, as well as substrates. For valproyl-l-carnitine, K_i = 155± 19μM, K_m = 132± 23μM, and normalized J_max = 0.467± 0.028; for naproxen-l-carnitine, K_i = 5.97± 0.81μM, K_m = 257± 57μM, and normalized J_max = 0.141± 0.012; for ketoprofen-l-carnitine, K_i = 82.2± 5.3μM, K_m = 77.0± 4.0μM, and normalized J_max = 0.412± 0.015; for ketoprofen-glycine-l-carnitine, K_i = 14.4± 1.4μM, K_m = 58.5± 8.7μM, and normalized J_max = 0.0789± 0.0037. Ketoprofen-glycolic acid-l-carnitine was unstable in metabolic buffers and chemical buffers. On the contrary, naproxen-l-carnitine, ketoprofen-l-carnitine, and ketoprofen-glycine-l-carnitine were stable in chemical and metabolic buffers. The results demonstrate the potential of drug-l-carnitine conjugates to serve as prodrugs that target OCTN2.

Full text of DOI:10.1002/jps.22557

Synthesis and in Vitro Characterization of Drug Conjugates of
l-carnitine as Potential Prodrugs That Target Human Octn2
LEI DIAO, JAMES E. POLLI
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201
Received 19 August 2010; revised 21 January 2011; accepted 2 March 2011
Published online 31 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22557
ABSTRACT: The objective was to evaluate the potential of drug conjugates with l-carnitine
as prodrugs that target organic cation/carnitine transporter (OCTN2). Twenty-two l-carnitine
analogues were evaluated for human organic cation/carnitine transporter (hOCTN2) inhibition;
the 3ꢀ-hydroxyl group was found to be the only functional group not contributing to l-carnitine
interaction with hOCTN2 among the three functional groups on l-carnitine (i.e., 3ꢀ-hydroxyl,
amine, and carboxylate). The 3ꢀ-hydroxyl group on l-carnitine was therefore chosen as the con-
jugate site. Three drug–l-carnitine conjugates (i.e., valproyl–l-carnitine, naproxen–l-carnitine,
and ketoprofen–l-carnitine) were synthesized along with two ketoprofen analogues that in-
corporated a linker group (glycolic acid or glycine) between ketoprofen and l-carnitine (i.e.,
ketoprofen–glycolic acid–l-carnitine and ketoprofen–glycine–l-carnitine). These potential pro-
drugs were evaluated for their in vitro inhibition, transport, and metabolism properties. All
three drug–l-carnitine conjugates and ketoprofen–glycine–l-carnitine were OCTN2 inhibitors,
as well as substrates. For valproyl–l-carnitine, Ki = 155 19 :M, Km = 132 23 :M, and nor-
malized Jmax = 0.467 0.028; for naproxen–l-carnitine, Ki = 5.97 0.81 :M, Km = 257 57 :M,
and normalized Jmax = 0.141 0.012; for ketoprofen–l-carnitine, Ki = 82.2 5.3 :M, Km
=
77.0 4.0 :M, and normalized Jmax = 0.412 0.015; for ketoprofen–glycine–l-carnitine, Ki =
14.4 1.4 :M, Km = 58.5 8.7 :M, and normalized Jmax = 0.0789 0.0037. Ketoprofen–gly-
colic acid–l-carnitine was unstable in metabolic buffers and chemical buffers. On the contrary,
naproxen–l-carnitine, ketoprofen–l-carnitine, and ketoprofen–glycine–l-carnitine were stable
in chemical and metabolic buffers. The results demonstrate the potential of drug–l-carnitine
conjugates to serve as prodrugs that target OCTN2. © 2011 Wiley-Liss, Inc. and the American
Pharmacists Association J Pharm Sci 100:3802–3816, 2011
Keywords: prodrugs; transporters; site-specific delivery; renal reabsorption; stability;
substrate; carnitine
placenta.2 l-Carnitine and its endogenous metabolite
INTRODUCTION
acetyl-l-carnitine can also cross the blood–brain bar-
rier (BBB) and are considered to have different phys-
iological roles in the brain, such as helping acetyl-
choline formation.3 Acetyl-l-carnitine may be useful
for the treatment of Alzheimer’s disease.3
The organic cation/carnitine transporter (OCTN2)
is a sodium-dependent high-affinity cation/carnitine
transporter that is widely expressed in hu-
man tissues, including skeletal muscle, kidney,
heart, placenta, and brain.4 OCTN2 is responsi-
ble for l-carnitine tissue distribution and tubu-
lar reabsorption.5–7 Mutations of the OCTN2 gene
in humans cause primary carnitine deficiency.8–10
Patients with primary carnitine deficiency excrete
carnitine in urine due to the defective tubular re-
absorption; plasma and tissue levels of carnitine
drop below 10% of normal values, eliciting clinical
l-Carnitine ($-hydroxy (-trimethylaminobutyrate) is
a hydrophilic endogenous molecule that plays an es-
sential role in the transfer of long- and medium-chain
fatty acids into mitochondria for $-oxidation.1 It plays
a critical role in energy metabolism of peripheral tis-
sues that derive metabolic energy from fatty acid
oxidation such as heart, skeletal muscle, liver, and
Abbreviations used: HBSS, Hanks balanced salt solution;
MDCK, Madin–Darby canine kidney; OCTN2, organic cation/
carnitine transporter; SFB, sodium-free buffer.
Additional Supporting Information may be found in the online
version of this article. Supporting Information
Correspondence to: James E. Polli (Telephone: +410-706-8292;
Fax: +410-706-5017; E-mail: jpolli@rx.umaryland.edu)
Journal of Pharmaceutical Sciences, Vol. 100, 3802–3816 (2011)
© 2011 Wiley-Liss, Inc. and the American Pharmacists Association
3802
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OCTN2 AS POTENTIAL PRODRUG TARGET
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significant symptoms that include hypoketotic hypo-
glycemia, cardiomyopathy, and skeletal myopathy.10
There is very limited information about the
substrate requirements of OCTN2. Other than
l-carnitine and acylcarnitines, such as acetyl-
l-carnitine, propionyl-l-carnitine, and palmitoyl-l-
carnitine,11,12 few compounds or drugs have been
shown to be OCTN2 substrates. Beta-lactam antibi-
otics are OCTN2 substrates.13 One report demon-
strated the transport of butyryl-l-carnitine by human
organic cation/carnitine transporter (hOCTN2) in a
high-affinity/low-capacity manner using the Xeno-
pus laevis oocyte expression system.14 Another re-
port analyzed the transport efficiency of OCTN2 and
found that OCTN2 did not uptake several drugs, al-
though efficiently translocated mildronate, and con-
cluded that OCTN2 was not a general drug trans-
porter but a highly specific carrier for carnitine and
closely related molecules.15
A drug conjugate of l-carnitine was hypothesized
to be recognized and transported by OCTN2. There-
fore, such conjugates may have potential to serve as
prodrugs that target tissues expressing OCTN2. One
in vivo report demonstrated that the conjugate of
nipecotic acid with l-carnitine showed enhanced pen-
etration of nipecotic acid across the mouse BBB com-
pared with nipecotic acid, and significantly increased
the latency to convulsions induced by pentylenete-
trazole compared with either nipecotic acid or l-
carnitine.16 However, this study did not explore
whether the conjugate was transported by OCTN2,
how parent drug impacted transport properties, or
how linker chemistry between parent drug and l-
carnitine affected conjugate stability and release of
the parent drug.
In this study, 22 l-carnitine analogues were eval-
uated for OCTN2 inhibition in order to find a suit-
able position on l-carnitine for drug conjugation.
Three drug–l-carnitine conjugates (e.g., valproyl–l-
carnitine, naproxen–l-carnitine, and ketoprofen–l-
carnitine) were synthesized by conjugation of the car-
boxylate group on parent drug with the 3ꢀ-hydroxyl
group of l-carnitine. Two analogues of ketoprofen-
l-carnitine that incorporate a linker group (glycolic
acid or glycine) between ketoprofen and l-carnitine
were also synthesized. These potential prodrugs were
evaluated for their in vitro inhibition, transport, and
metabolic properties. Results demonstrated the po-
tential value of drug conjugates with l-carnitine as
prodrugs that target OCTN2.
l-[3H]carnitine was purchased from American Ra-
diolabeled Chemicals (St. Louis, Missouri). l-
Carnitine hydrochloride and ketoprofen were pur-
chased from Spectrum Chemicals & Laboratory
Products (Gardena, California). Naproxen was
purchased from Cayman Chemical (Ann Arbor,
Michigan). N-Ethoxycarbonyl-2-ethoxy-1,2-dihydro-
quinoline (EEDQ) was purchased from Alfa Aesar
(Ward Hill, Massachusetts). 4-Dimethylaminopyri-
dine (DMAP) was purchased from Novabiochem
(Gibbstown, New Jersey). Dicyclohexylcarbodiimide
(DCC) was purchased from Thermo Scientific
(Franklin, Massachusetts). Rat plasma was pur-
chased from Valley Biomedical (Winchester, Virginia).
Rat kidney homogenate was purchased from Pel-
Freez Biologicals (Rogers, Arkansas). Valproic acid,
benzyloxyacetic acid, glycine tert butyl ester hy-
drochloride, l-carnitine analogues in Table 1 (ex-
cept compounds 5–8), mouse liver S9 fraction, mouse
plasma, rat liver microsome, and esterase solution
from porcine liver were purchased from Sigma–
Aldrich (St. Louis, Missouri).
Synthesis of l-Carnitine Analogues
Compounds 5–8 in Table 1 were synthesized by es-
terification of compounds 1–4 (500 mg each) in MeOH
(30 mL) and p-toluenesulfonic acid (100 mg) overnight
at room temperature (RT). To purify compound 5, the
solvent was evaporated under vacuum and the reac-
tion mixture was dissolved in acetone followed by fil-
tration. The filtrate was dried under vacuum to yield
the final compound 5. To purify compounds 6–8, the
crude product was dissolved in 1 M NaOH (20 mL),
extracted into 15 mL EtOAc (3×), and washed with
15 mL of 1 N NaHCO3 (3×), 15 mL of water (3×), and
15 mL of brine (1×). The organic layer was dried over
sodium sulfate and filtered. Removal of EtOAc under
reduced pressure yielded compounds 6–8.
Synthesis of Valproyl–l-Carnitine and
Ketoprofen–l-Carnitine
Schemes 1–4 summarize the synthesis of valproyl–l-
carnitine,
ketoprofen–l-carnitine,
naproxen–l-
carnitine, ketoprofen–glycolic acid–l-carnitine, and
ketoprofen–glycine–l-carnitine, respectively. Synthe-
sis methods for all five compounds were modified from
a previous report and also from applied protection
chemistry.17,18
For
valproyl–l-carnitine
and
ketoprofen–l-
carnitine (Scheme 1), valproic acid or ketoprofen
(1 g) was activated by oxalyl chloride (0.5 mL) for 4 h.
Three drops of dimethylformamide (DMF) was added
as a catalyst. l-Carnitine (1.1 equivalent) dissolved in
trifluoroacetic acid (TFA; 10 mL) was then added
to activated valproic acid or ketoprofen after solvent
evaporation under vacuum. The reaction mixture
was heated to 50◦C for 12 h, after which the solvent
Experimental
Materials
Fetal bovine serum, trypsin–EDTA, and Dulbecco’s
modified Eagle medium (DMEM) were purchased
from Invitrogen Corporation (Carlsbad, California).
DOI 10.1002/jps
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DIAO AND POLLI
Table 1. List of 22 l-Carnitine Analogues Screened as Human Organic Cation/Carnitine Transporter Inhibitors
Number l-Carnitine Analogue Structure
Percent Uptake
Number l-Carnitine Analogue Structure
Percent Uptake
1
2.28 ( 0.09)
12
90.9 ( 4.5)
2
3
4
8.98 ( 0.06)
40.6 ( 1.2)
92.1 ( 3.4)
13
14
15
52.9 ( 1.3)
23.9 ( 0.4)
48.2 ( 1.1)
5
19.5 ( 0.5)
16
83.6 ( 2.1)
6
7
13.9 ( 0.3)
62.8 ( 1.6)
17
18
71.0 ( 2.5)
45.2 ( 0.2)
8
9
91.1 ( 3.4)
94.0 ( 4.9)
19
20
74.1 ( 2.6)
6.05 ( 0.09) using 100 :M
10
11
84.8 ( 2.5)
21
22
15.3 ( 0.3) using 100 :M
73.8 ( 4.2)
16.7 ( 0.6) using 100:M
Cis-inhibition studies of l-carnitine (2.5 :M with spiked l-[3H] carnitine) uptake were carried out using l-carnitine analogue at a single concentration
(500 :M unless indicated) in Hanks balanced salt solution. Results are expressed in terms of percent uptake of l-carnitine, compared with l-carnitine
uptake without l-carnitine analogue present. Lower percent uptake of l-carnitine indicates higher inhibition potency.
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DOI 10.1002/jps

OCTN2 AS POTENTIAL PRODRUG TARGET
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Scheme 3. Synthesis of ketoprofen–glycolic acid–l-
carnitine.
Scheme 1. Synthesis of valproyl–l-carnitine and
ketoprofen–l-carnitine.
DMF. Naproxen (1 equivalent), DCC (1 equivalent),
and DMAP (0.2 equivalent) were then added to con-
jugate naproxen to carboxylate-protected l-carnitine
over 12 h. The reaction mixture was filtered and evap-
orated. Naproxen–l-carnitine was obtained by cat-
alytic hydrogenation to remove the benzyl ester (10%
Pd/charcoal in EtOH at 40–50 psi for 12 h), followed
by purification by preparative thin layer chromatog-
raphy using solvent mixture ethanol and butanone
(1:1). 1H NMR (DMSO, 500 MHz): δ 1.42–1.59 (d, 3H),
2.73–2.88 (s, 2H), 3.04–3.19 (m, 11H), 3.84–4.01 (m,
4H), 5.31–5.51 (m, 1H), 7.11–7.26 (d, 1H), 7.30–7.38
(s, 1H), 7.38–7.51 (t, 1H), 7.68–7.94 (m, 3H).
was evaporated under vacuum. Water was added
and extracted with ether. Aqueous layer was kept
and extracted with butanol saturated with water
(3×). Butanol layer was combined and extracted
with water saturated with butanol (3×). Finally,
butanol fraction was dried with anhydrous sodium
sulfate, filtered, and evaporated under vacuum to
yield valproyl–l-carnitine or ketoprofen–l-carnitine.
1H NMR [dimethyl sulfoxide (DMSO), 500 MHz] for
valproyl–l-carnitine: δ 0.79–0.92 (t, 6H), 1.17–1.31
(m, 4H), 1.33–1.45 (m, 2H), 1.47–1.60 (m, 2H),
2.31–2.39 (m, 1H), 2.49–2.58 (m, 1H), 2.70–2.74 (m,
1H), 3.13 (s, 9H), 3.68–3.74 (d, 1H), 3.83–3.90 (q,
1
1H), 5.43–5.50 (m, 1H). H NMR (DMSO, 500 MHz)
Synthesis of Ketoprofen–Glycolic Acid–l-Carnitine
for ketoprofen–l-carnitine:
3.27–3.69 (s, 9H), 3.68–3.88 (m, 1H), 3.93–4.06 (m,
2H), 5.30–5.40 (m, 1H), 7.52–7.76 (m, 9H).
δ
1.42–1.51 (d, 3H),
Scheme 3 summarizes the synthetic approach for ke-
toprofen–glycolic acid–l-carnitine. Benzyl-l-carnitine
(1 g) and benzyloxyacetic acid (1 equivalent) were cou-
pled by DCC (1 equivalent) and DMAP (0.2 equiv-
alent) in DMF for 12 h. Reaction mixture was then
filtered and dissolved in water after solvent removal
under vacuum. The aqueous solution was then ex-
tracted with butanol saturated with water (3×). Bu-
tanol fraction was combined and dried by anhydrous
sodium sulfate. After catalytic hydrogenation to re-
move the benzyl esters (10% Pd/charcoal in EtOH
at 40–50 psi for 12 h), EtOH was vacuum evaporated
and glycolic acid–l-carnitine conjugate was dissolved
in TFA (10 mL). Oxalyl chloride-activated ketoprofen
(1 equivalent) was added and the reaction mixture
was heated to 50◦C for 12 h, after which the solvent
was evaporated under vacuum. Water was added and
extracted with ether. Aqueous layer was kept and ex-
tracted with butanol saturated with water (3×). Bu-
tanol layer was combined and extracted with water
saturated with butanol (3×). Finally, butanol fraction
was dried with anhydrous sodium sulfate, filtered,
and evaporated under vacuum to yield ketoprofen–g-
Synthesis of Naproxen–l-Carnitine
Scheme 2 summarizes the synthetic approach for
naproxen–l-carnitine. l-Carnitine hydrochloride (1 g)
was dissolved in benzyl alcohol (10 mL). The carboxy-
late group of l-carnitine hydrochloride was protected
by adding DCC (1 equivalent) and DMAP (0.2 equiv-
alent). After stirring for 12 h, the reaction mixture
was filtered and the solvent was evaporated. Acetoni-
trile (10 mL) was added and filtered to give benzyl-
l-carnitine. Benzyl-l-carnitine was then dissolved in
1
lycolic acid–l-carnitine. H NMR (DMSO, 500 MHz):
δ 1.36–1.57 (d, 3H), 2.91–3.18 (m, 9H), 3.63–3.74 (m,
2H), 4.04–4.15 (m, 1H), 5.42–5.58 (m, 1H), 7.38–8.14
(m, 9H).
Scheme 2. Synthesis of naproxen–l-carnitine.
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DIAO AND POLLI
fetal bovine serum, 0.1 mM nonessential amino acid
solution, 50 units/mL penicillin, and 50 :g/mL strep-
tomycin.
Inhibition Study
Inhibition studies of l-carnitine analogues and con-
jugates were conducted as previously described.19
Briefly, after reaching 90% confluence, cells were
seeded in 12-well cluster plates at a density of
1.5 million cells per well and cultured for 4 days. The
culture medium was changed every 48 h. Inhibition
studies were performed on the fourth day and were
conducted in presence of Hank’s balance salts solu-
tion (HBSS), which contains 137 mM sodium chlo-
ride. Cells were exposed to donor solution contain-
ing 2.5 :M l-carnitine (spiked with l-[3H]-carnitine)
in the presence or absence of potential inhibitor (i.e.,
l-carnitine analogue or conjugate) at 37◦C and 50 rpm
orbital shaking for 10 min. The donor solution was
removed and the cells were washed thrice with ice-
cold sodium-free buffer (SFB) wherein sodium chlo-
ride was replaced with 137 mM tetraethylammonium
chloride. Subsequently, cells were lysed using 0.25 mL
of 1 M NaOH for 2 h at RT and neutralized with
0.25 mL of 1 M HCl. Cell lysate was then counted
for the associated radioactivity using a liquid scin-
tillation counter. Jmax of l-carnitine was measured
on each inhibition study occasion. Unless otherwise
noted, data are summarized as mean ( SEM) of three
measurements.
Scheme 4. Synthesis of ketoprofen–glycine–l-carnitine.
Synthesis of Ketoprofen–Glycine–l-Carnitine
Scheme 4 summarizes the synthetic approach for
ketoprofen–glycine–l-carnitine. Glycine tert butyl es-
ter hydrochloride (1 g) was dissolved in EtOAc. Tri-
ethylamine (1 equivalent) was added and stirred for
30 min. Ketoprofen (1 equivalent) and EEDQ (1 equiv-
alent) were then added to conjugate ketoprofen to
glycine tert butyl ester. After 12 h, the solvent was
evaporated under vacuum and ketoprofen–glycine
conjugate was obtained by deprotection of tert-butyl
group in dichloromethane (DCM) (10 mL) with TFA
(10 mL) and triisopropylsilane (3 mL) for 4 h. The
crude product was further purified by silica gel col-
umn chromatography using a mobile phase of DCM
and MeOH (90:10). Ketoprofen–glycine was then acti-
vated by oxalyl chloride (0.5 mL) for 4 h. Three drops
of DMF was added as catalyst. l-Carnitine (1.1 equiv-
alent) dissolved in TFA (10 mL) was then added to ac-
tivated ketoprofen–glycine after solvent evaporation
under vacuum. The reaction mixture was heated to
50◦C for 12 h, after which the solvent was evaporated
under vacuum. Water was added and extracted with
ether. Aqueous layer was kept and extracted with
butanol saturated with water (3×). Butanol layer
was combined and extracted with water saturated
with butanol (3×). Finally, butanol fraction was dried
with anhydrous sodium sulfate, filtered, and evap-
orated under vacuum to yield ketoprofen–glycine–l-
carnitine. 1H NMR (DMSO, 500 MHz): δ 1.30–1.42 (d,
3H), 2.61–2.69 (d, 2H), 3.00–3.16 (s, 9H), 3.70–3.92
(m, 4H), 5.43–5.55 (m, 1H), 7.49–7.78 (m, 9H),
8.66–8.75 (m, 1H).
To measure Ki of conjugate, inhibition studies were
performed as described above, where a range of drug
concentrations were applied to inhibit l-carnitine up-
take. The following inhibition model was applied:
Jmax × S
ꢀ
ꢁ
J =
+ Pp × S
(1)
I
Kt 1 +
+ S
/
Ki
where Ki is the inhibition coefficient, I is the con-
centration of inhibitor, and S is the concentration
of l-carnitine (2.5 :M). In applying Eq. 1, only Ki
was estimated using nonlinear regression fitting per-
formed by WinNonlin 4.1 (Pharsight, Mountain View,
California). The other three parameters (i.e., Jmax
,
Kt, and Pp) were estimated from l-carnitine uptake
studies without inhibitor.
Cell Culture
Uptake Study
Stably transfected hOCTN2–Madin–Darby canine
kidney (MDCK) and Calu-3 cells were cultured at
37◦C, 90% relative humidity, and 5% CO2 atmo-
sphere and fed every 2 days, as previously described.19
Media for hOCTN2–MDCK cells was composed of
DMEM supplemented with 10% fetal bovine serum,
50 units/mL penicillin, and 50 :g/mL streptomycin.
Cells were passaged after reaching 80% confluence.
Calu-3 cells were maintained in DMEM with 10%
Human organic cation/carnitine transporter–Madin–
Darby canine kidney and Calu-3 cells were seeded
in 12-well plates (Corning; Corning, New York).
Seeding density was 1.5 million cells/well. On day
4 after seeding, uptake studies were performed for
l-carnitine (spiked with l-[3H]-carnitine) or conju-
gate over a range of concentrations in HBSS and
SFB. Nontransporter-mediated passive uptake was
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OCTN2 AS POTENTIAL PRODRUG TARGET
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assessed by measuring l-carnitine or conjugate up-
take in SFB.
Analytical Methods
l-[3H]-carnitine was quantified by scintillation count-
ing. Conjugate samples were quantified by LC–MS/
MS. The LC–MS/MS instrumental system con-
Uptake was determined to be linear over 10 min. At
the end of the assay (10 min), cells were washed thrice
with chilled SFB. Acetonitrile was then applied and,
after its evaporation, cells were incubated with a mix-
ture of acetonitrile and water (1:1, containing inter-
nal standard compound) for 30 min on a plate shaker
at 50 oscillations per minute.20 Conjugate was quan-
tified by liquid chromatography–tandem mass spec-
trometry (LC–MS/MS). l-Carnitine was quantified by
scintillation counting.
R
sisted of Finnigan Surveyorꢀ Plus Autosampler,
R
Finnigan Surveyorꢀ LC Pump Plus, and Finnigan
R
TSQꢀ Quantum Discovery MAXTM mass spectrome-
ter with an electrospray ionization source and triple-
quadrupole mass analyzer (Thermo Fisher Scien-
tific Inc.; Waltham, MA). The column was a Syner-
giTM Polar-RP column (4 :m, 50 × 2.00 mm; Phe-
nomenex, Torrance, California). A gradient of ace-
tonitrile with 0.1% formic acid and water with 0.1%
formic acid was used as the mobile phase. The gra-
dient was an initial 50% organic phase for 1 min,
a linear increase to 80% organic phase over 3 min,
a plateau of 80% organic phase for 1 min, a linear
decrease to 50% organic phase over 1 min, and a
plateau of 50% organic phase for 1 min, with a flow
rate of 0.4 mL/min. Compounds were monitored by
selective reaction monitoring in positive mode: val-
proic acid–l-carnitine (288.2 → 144.1), naproxen–l-
carnitine (374.0 → 194.1), ketoprofen–l-carnitine
(398.2 → 209.0), ketoprofen–glycolic acid–l-carnitine
Uptake data in SFB were fitted to passive transport
model (Eq. 2):
J = Pp × S
(2)
where J is uptake, Pp is the passive permeability, and
S is conjugate concentration. Uptake data in HBSS
were fitted to the Michaelis–Menten model (Eq. 3):
Jmax × S
J =
+ Pp × S
(3)
Kt + s
(456.2
→
209.0), ketoprofen–glycine–l-carnitine
(455.2 → 209.0), and ketoprofen (255.3 → 209.0).
The assay was linear (r2 > 0.999) over 20–1000 nM
for each compound.
where Jmax and Kt are the Michaelis–Menten coeffi-
cients. Equations 2 and 3 were applied sequentially
to mock and transporter-expressing cell data to es-
timate Pp, Kt, and Jmax. The Pp estimate from mock
cells was applied to Eq. 3. Nonlinear curve fitting was
performed using WinNonlin 4.1 (Pharsight).
Data Analysis
Data were expressed as mean
SEM derived
from three independent wells. Statistical significance
was evaluated using Graphpad Prism (Graphpad
Software, Inc.; La Jolla, California).
In Vitro Chemical and Metabolic Stability Studies
The following matrices were used to assess the in
vitro chemical and metabolic stability of naproxen–l-
carnitine, ketoprofen–l-carnitine, ketoprofen–glycolic
acid–l-carnitine, or ketoprofen–glycine–l-carnitine:
mouse plasma (lyophilized; reconstituted with 1 mL
water and diluted to 90% with 50 mM Tris–HCl buffer,
pH 7.5), mouse liver S9 fraction (20 mg/mL) in the
presence of 1 mM nicotinamide adenine dinucleotide
phosphate, rat liver microsome (2 mg/mL in phos-
phate buffered saline), 90% rat plasma, rat kidney
homogenate (quickly thawed and centrifuged at 4◦C
for 10 min at 10,000 × g and supernatant was used
for stability study), esterase solution from porcine
liver (50 units/mL in 10 mM borate buffer, pH 8.0),
HBSS (pH 6.8), PBS (pH 7.5), 50 mM Tris–HCl buffer
(pH 7.5), and 0.1 M HCl. Conjugate was incubated
in metabolic and chemical buffers at 37◦C. At a des-
ignated time point, 100 :L aliquot was taken and
quenched immediately with 400 :L acetonitrile, fol-
lowed by centrifugation at 14,000 × g for 10 min at
4◦C. Supernatants were frozen and maintained at
−80◦C prior to LC–MS/MS analysis.
RESULTS AND DISCUSSION
Inhibition Screening of l-Carnitine Analogues
Twenty-two l-carnitine analogues were evaluated
for their inhibition of l-carnitine uptake into
hOCTN2–MDCK cells (Table 1). These studies were
conducted in order to explore the structural require-
ments for compound interacting with OCTN2 and
elucidate a possible suitable position on l-carnitine
to conjugate drug for prodrug design. In Table 1, re-
sults are expressed in terms of percent uptake of l-
carnitine compared with l-carnitine uptake without
l-carnitine analogue present. Lower percent uptake
indicates higher inhibition potency.
Results indicate that the 3ꢀ-hydroxyl group of
l-carnitine is not necessary for OCTN2 inhibition
because (3-carboxylpropyl)trimethylammonium (1)
was a potent inhibitor. The potent inhibition by
4-dimethylaminobutyric acid (2) further indicates
that the 3ꢀ-hydroxyl group is not necessary. Addi-
tionally, d-carnitine (20), acetyl-l-carnitine (21), and
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palmitoyl-l-carnitine (22) each lack 3ꢀ-hydroxyl group
in the R-configuration, yet were potent inhibitors.
Second, results indicate that inhibition potency
depended on the type of amine. Potency showed qua-
ternary amine > tertiary amine > secondary amine
> primary amine, as evident from the results of
compounds (3-carboxylpropyl)trimethylammonium
(1), 4-dimethylaminobutyric acid (2), 4-methyl-
aminobutyric acid (3), and gamma-aminobutyric
acid (4). Results from compounds 5–8, which
are methyl esters of compounds 1–4, showed the
similar trend. The lack of inhibition by S-4-amino-
3-hydroxybutanoic acid (9) also supports the need
for an amine that is not a primary amine. Third, the
carboxylic acid group of l-carnitine contributes to
OCTN2 binding affinity. For example, methyl esteri-
fication reduced inhibitory potency. Compounds 5–8
exhibited reduced inhibition potency compared with
acid counterparts 1–4. N-butyltrimethylammonium
(16) and (3-hydroxypropyl)trimethylammonium (17)
are analogues of compound 1 but lack the carboxylate
and were not potent inhibitors. Tetraethylammonium
(13), tetramethylammonium (14), and tributylmethy-
lammonium (15) are simple quaternary amines that
lack a carboxylate and exhibited less potency than
compound 1. Interestingly, betaine (18) possesses
a carboxylic acid group, but was less potent than
compound 1, suggesting that too close a proximity of
the amine and carboxylate is detrimental to binding
affinity.
cells in HBSS. Each inhibited l-carnitine uptake into
hOCTN2–MDCK monolayers in a concentration-
dependent manner. Figure 2 plots their inhibition
profiles. Kinetic parameters are listed in Table 2.
All three conjugates were hOCTN2 inhibitors, with
Ki of 155
19 :M for valproyl–l-carnitine, 5.97
0.81 :M for naproxen–l-carnitine, and 82.2 5.3 :M
for ketoprofen–l-carnitine.
Uptake of Drug-l-Carnitine Conjugates by OCTN2
Figure 3 represents the concentration-dependent
uptake of the valproyl–l-carnitine, naproxen–l-
carnitine, and ketoprofen–l-carnitine into hOCTN2–
MDCK monolayers in the presence and absence of
sodium. Uptake of each conjugate was greater in the
presence of sodium than in the absence of sodium,
indicating active uptake by the transporter.
Kinetic uptake parameters are listed in Table
2. For valproyl–l-carnitine, Km = 132 23 :M and
Jmax = 0.413 0.023 pmol/(s cm2). For naproxen–l-
carnitine, Km = 257 57 :M and Jmax = 0.159
0.013 pmol/(s cm2). For ketoprofen–l-carnitine, Km
=
77.0 4.0 :M and Jmax = 0.439 0.006 pmol/(s cm2).
In Table 2, the normalized Jmax value is Jmax of
the conjugate normalized for functional hOCTN2 ex-
pression. Specifically, conjugate Jmax is divided by
l-carnitine Jmax from the same study occasion to give
normalized Jmax. The normalized Jmax of valproyl–l-
carnitine, naproxen–l-carnitine, and ketoprofen–L-
carnitine were 0.467 0.028, 0.141 0.012, 0.412
0.015, respectively. Overall, the ketoprofen conjugate
was perhaps the most promising as a substrate, in
that it exhibited the most favorable Km and showed a
Jmax that was about half that of l-carnitine.
These findings of the favorable contributions of the
amine and carboxylate, and the lack of the need for
the 3ꢀ- hydroxyl group, identified the 3ꢀ- hydroxyl
on l-carnitine as a suitable position for conjugation.
Hence, the 3ꢀ- hydroxyl was chosen to conjugate
parent drug (or linker) with l-carnitine. This ap-
proach is consistent with prior results wherein acetyl-
l-carnitine and palmitoyl-l-carnitine were found to be
transported by OCTN2.11,12
To further confirm that the active transport was
mediated by OCTN2, the l-carnitine inhibition of
the uptake of naproxen and ketoprofen conjugates
was investigated. Results are plotted in Figure 4.
In Figure 4a, l-carnitine (50 and 100 :M) signifi-
cantly inhibited the uptake of naproxen–l-carnitine
(50 :M) into hOCTN2–MDCK cells in HBSS (p <
0.05). In the presence of l-carnitine, naproxen–l-
carnitine uptake in HBSS was comparable to its up-
take in SFB (99% inhibition), indicating that OCTN2-
mediated active uptake was abolished. In Figure 4b, l-
carnitine (5–20 :M) significantly inhibited the uptake
of ketoprofen–l-carnitine (50–1000 :M) in HBSS (p <
0.05). Ketoprofen–l-carnitine uptake was reduced by
95% in the presence of 1000 :M l-carnitine. Therefore,
the active transport of conjugates by OCTN2 was con-
firmed.
Synthesis of l-Carnitine Prodrugs
Three drug–l-carnitine conjugates were synthesized
and depicted in Figures 1a–1c, and were valproyl–l-
carnitine, naproxen–l-carnitine, and ketoprofen–l-
carnitine. Additionally, two ketoprofen conjugates
that employ glycolic acid and glycine as linkers were
subsequently synthesized in order to promote more
rapid release of the drug compared with ketoprofen–l-
carnitine, as discussed below. These two additional
conjugates were ketoprofen–glycolic acid–l-carnitine
and ketoprofen–glycine–l-carnitine (Figs. 1d and 1e).
Furthermore, ketoprofen–l-carnitine uptake in a
second cell line, Calu-3 cell line, was performed.
Calu-3 cell line is a human bronchial epithelial
cell line and widely used as a transport model to
study drug delivery to the respiratory epithelium.21,22
Calu-3 cells also express OCTN2, but not in an
Inhibition of l-Carnitine Uptake by
Drug–l-Carnitine Conjugates
Valproyl–l-carnitine,
naproxen–l-carnitine,
and
ketoprofen–l-carnitine were evaluated for their
inhibition of l-carnitine uptake into hOCTN2–MDCK
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Figure 1. Structures of the prodrugs. (a) Valproyl–l-carnitine, (b) naproxen–l-carnitine,
(c) ketoprofen–l-carnitine, (d) ketoprofen–glycolic acid–l-carnitine, and (e) ketoprofen–glycine–l-
carnitine.
overexpression fashion as hOCTN2–MDCK cells.23 As
naproxen–l-carnitine (1 :M) was stable in porcine
liver esterase solution (50 units/mL), but degraded
gradually to about 60% in rat plasma (Fig. 5a).
Meanwhile, ketoprofen–l-carnitine (1 :M) was stable
in rat plasma, as well rat kidney homogenate and rat
liver microsomes (2 mg/mL; Fig. 5b). Ketoprofen–l-
carnitine stability is supported by the observation
that no ketoprofen was released in these three
metabolic buffers. On the contrary, ketoprofen–l-
carnitine was susceptible to alkaline hydrolysis.
In 1 h, ketoprofen–l-carnitine fully degraded to
ketoprofen in 0.2 M KOH in methanol incubated at
50◦C.
demonstrated in Supplemental Figure 1, both uptake
of l-carnitine and ketoprofen–l-carnitine into Calu-3
cells were higher in the presence of sodium than in
the absence of sodium, supporting the active uptake
in Calu-3 cells.
In Vitro Stabilities of Naproxen–l-Carnitine
and Ketoprofen–l-Carnitine
Naproxen–l-carnitine and ketoprofen–l-carnitine
were subjected to in vitro metabolic and chemical
stabilities. Results are plotted in Figure 5. Over 5 h,
Table 2. Human Organic Cation Carnitine Transporter Inhibition and Uptake Kinetic Parameters of Prodrugs
Inhibitor Constant Michaelis Constant Michaelis Constant
Jmax [pmol/(s cm2)] Normalized Jmax
Pp × 106 (cm/s)
a
Prodrug
Ki (:M)
Km (:M)
Valproyl–l-carnitine
Naproxen–l-carnitine
Ketoprofen–l-carnitine
Ketoprofen–glycine–l-
carnitine
155 19
5.97 0.81
82.2 5.3
14.4 1.4
132 23
257 57
77.0 4.0
58.5 8.7
0.413 0.023
0.159 0.013
0.439 0.006
0.0485 0.0020
0.467 0.028
0.141 0.012
0.412 0.015
0.0789 0.0037
0.0257 0.033
0.141 0.003
0.0751 0.0082
0.0723 0.0025
Data were summarized as mean ( SEM) of three measurements.
aTo accommodate variation in organic cation/carnitine transporter expression levels across study occasions, Jmax of each prodrug was normalized
against l-carnitine Jmax from the same study occasion, yielding normalized Jmax
.
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Figure 3. Uptake of valproyl–l-carnitine, naproxen–l-
carnitine, and ketoprofen–l-carnitine into human organic
cation/carnitine transporter–Madin–Darby canine kidney
cells in the presence and absence of sodium. (a) Valproyl–l-
carnitine uptake was greater in Hanks balanced salt
solution (HBSS) than in sodium-free buffer (SFB), (b)
naproxen–l-carnitine uptake was greater in HBSS than in
SFB, (c) ketoprofen–l-carnitine uptake was greater in HBSS
than in SFB. Error bars denote SEM.
Figure 2. Inhibition of l-carnitine uptake into human
organic cation/carnitine transporter–Madin–Darby canine
kidney cells by (a) valproyl–l-carnitine, (b) naproxen–l-
carnitine, and (c) ketoprofen–l-carnitine. Error bars denote
SEM.
Design of Ketoprofen–Glycolic Acid–l-Carnitine
and Its in Vitro Stability
well an alcohol that can be conjugated via ketoprofen’s
acid.
Because ketoprofen–l-carnitine exhibited favorable
OCTN2 transport, but insufficient enzymatic activa-
tion by rat plasma, rat kidney homogenate, or rat liver
microsomes, analogues of ketoprofen–l-carnitine that
incorporate a linker group between ketoprofen and
l-carnitine were subsequently synthesized and evalu-
ated. A linker that afforded a less sterically hindered
ester was hypothesized to be more susceptible to en-
zymatic hydrolysis.24 Glycolic acid was selected as a
linker because it possesses a carboxylate that can be
conjugated via the 3ꢀ-hydroxyl group of l-carnitine, as
As shown in Figure 6, the in vitro chemical
and metabolic stability of ketoprofen–glycolic acid–l-
carnitine was evaluated using mouse liver S9 fraction,
mouse plasma, rat plasma, rat kidney homogenate,
and esterase solution from porcine liver, as well as
various chemical buffers including HBSS (pH 6.8),
PBS (pH 7.5), SFB (pH 6.8), Tris–HCl (50 mM, pH
7.5), water, and 0.1 M HCl. Broadly, ketoprofen–gly-
colic acid–l-carnitine was not stable. In mouse liver S9
fraction (Fig. 6a), the conjugate completely degraded
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Figure 4. Inhibition of naproxen–l-carnitine or ketoprofen–l-carnitine uptake into human
organic cation/carnitine transporter–Madin–Darby canine kidney (hOCTN2–MDCK) cells by
l-carnitine. (a) Inhibition of naproxen–l-carnitine uptake (50 :M) into hOCTN2–MDCK cells
by 50 and 100 :M l-carnitine, (b) inhibition of ketoprofen–l-carnitine uptake (50 :M) into
hOCTN2–MDCK cells by 0–1000 :M l-carnitine. ∗, p < 0.05 compared with control in the
absence of l-carnitine. Error bars denote SEM.
in 30 min. In mouse plasma, rat plasma, rat kidney
homogenate, and porcine liver esterase solution (Figs.
6b–6e), ketoprofen–glycolic acid–l-carnitine showed
degradation over 5 h. Qualitatively, ketoprofen re-
lease was observed in all metabolic buffers except
rat plasma, where no generated ketoprofen was ob-
served. Quantitatively, only small amounts of keto-
profen were apparently generated. For example, in
mouse liver S9 fraction (Fig. 6a), at 30 min, when loss
of conjugate was complete, only about 10% of conju-
gate was converted to, or at least present as, ketopro-
fen. By 120 min, and through to 300 min, about 20%
of conjugate was converted to, or at least present as,
ketoprofen.
in water. It was stable in 0.1 M HCl, as perhaps could
be expected, as hydroxide concentration in 0.1 M HCl
is low. Because of its poor stability in HBSS and SFB,
inhibition and uptake studies of ketoprofen–glycolic
acid–l-carnitine were not conducted.
Design of Ketoprofen–Glycine–l-Carnitine and Its
Inhibition, Uptake, and Stability Profiles
Because ketoprofen–l-carnitine was too stable in
various metabolic buffers, whereas ketoprofen–gly-
colic acid–l-carnitine was too unstable in chemical
and metabolic buffers, ketoprofen–glycine–l-carnitine
was devised. Glycine contains an "-amino, rather
than an "-hydroxyl, as is the case for glycolic acid.
Hence, ketoprofen–glycine–l-carnitine possesses an
amide between ketoprofen and the linker, whereas
still affording a less hindered ester compared with
ketoprofen–l-carnitine.
This rapid hydrolysis was also evident even in
chemical buffers (Fig. 6f). In 3 h, ketoprofen–glycolic
acid–l-carnitine degraded to 47% in HBSS, 58% in
PBS, 69% in SFB, 65% in Tris–HCl buffer, and 72%
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Figure 5. In vitro metabolic stabilities of naproxen–l-carnitine and ketoprofen–l-carnitine.
(a) Stability of naproxen–l-carnitine (1 :M) in rat plasma and porcine liver esterase solu-
tion (50 units/mL), (b) stability of ketoprofen–l-carnitine (1 :M) in rat plasma, rat kidney
homogenate, and rat liver microsome (2 mg/mL).
Figure 7 shows inhibition, uptake, and stabil-
ketoprofen–glycine–l-carnitine was actively trans-
ported by hOCTN2. Ketoprofen–glycine–l-carnitine
uptake was reduced by 83% in the presence of
1000 :M l-carnitine.
ity results for ketoprofen–glycine–l-carnitine. In Fig-
ure 7a, the conjugate inhibited l-carnitine uptake
into hOCTN2–MDCK monolayers in a concentration-
dependent manner. Ki = 14.4 1.4 :M (Table 2),
which is lower (i.e., more potent) than ketoprofen–l-
carnitine’s Ki value.
In Figure 7b, ketoprofen–glycine–l-carnitine up-
take into hOCTN2–MDCK cells was greater in the
presence of sodium than in the absence of sodium,
indicating active uptake. Kinetic uptake parame-
ters for uptake of ketoprofen–glycine–l-carnitine are
Ketoprofen–glycine–l-carnitine
was
substan-
tially more stable than ketoprofen–glycolic acid–l-
carnitine. In Figure 7d, over 90% of conjugate
was remaining in mouse plasma and rat kidney
homogenate after 5 h. In HBSS and mouse liver S9
fraction, it degraded to 84% and 79%, respectively,
after 5 h.
listed in Table 2 with Km = 58.5 8.7 :M, Jmax
=
Possible Target Sites for l-Carnitine Prodrugs
0.0485 0.0020 pmol/(s cm2), and normalized Jmax
=
0.0789 0.0037. Hence, its Km was comparable to
ketoprofen–l-carnitine’s Km, but its Jmax is several
folds lower than that of ketoprofen–l-carnitine. In Fig-
ure 7c, l-carnitine (50–1000 :M) significantly inhib-
ited the ketoprofen–glycine–l-carnitine (50 :M) up-
take in HBSS (p < 0.05), further confirming that
Conjugates of naproxen and ketoprofen were evalu-
ated, with naproxen and ketoprofen serving as model
drugs (e.g., medicinal agents with a single carboxy-
late). Naproxen and ketoprofen were also chosen be-
cause they are nonsteroidal anti-inflammatory drugs
(NSAIDs) that may benefit from selective delivery to
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OCTN2 AS POTENTIAL PRODRUG TARGET
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Figure 6. In vitro chemical and metabolic stability of ketoprofen–glycolic acid–l-carnitine
(5 :M), along with the generation of ketoprofen. Filled circles (•) indicate ketoprofen–glycolic
acid–l-carnitine and open circles (◦) indicate ketoprofen. (a) Mouse liver S9, (b) mouse plasma,
(c) rat plasma, (d) rat kidney homogenate, (e) porcine liver esterase solution, and (f) chemical
stability of ketoprofen–glycolic acid–l-carnitine in various buffers.
the kidney. The kidney is a potential target site for an
l-carnitine prodrug strategy.
Additionally, targeting NSAIDs to the proximal
tubular cell may aid in the prevention of tubuloint-
erstitial inflammation, as well as some tubular defect
diseases such as Fanconi and Bartter’s syndrome.26
The study by Haas et al.28 demonstrated that naprox-
en–lysozyme prevented furosemide-induced renal
synthesis of prostaglandin E2, whereas an equimolar
amount of free naproxen was not effective.28 This re-
duction in prostaglandin E2 synthesis indicates that
targeting to the proximal tubular cell of the kidney
could enhance the renal efficacy of NSAIDs, as well
as possibly reduce extrarenal side effects because of
the lower dose.
Another potential site for targeted delivery of l-
carnitine prodrug is the brain, such that a val-
proic acid prodrug was evaluated in this study.
The administration of l-carnitine or acetyl-l-carnitine
for the treatment of Alzheimer’s disease3 suggested
that l-carnitine and acetyl-l-carnitine are transported
l-Carnitine and acetyl-l-carnitine are not bound to
plasma proteins and filtered at the glomerulus in the
kidney.25 l-Carnitine and acetyl-l-carnitine are reab-
sorbed by OCTN2 that is expressed in the brush bor-
der membranes of the proximal tubule cells.6 There-
fore, drug conjugate of l-carnitine may be reabsorbed
into proximal tubular cells, achieving targeted deliv-
ery to the kidney. After OCTN2-mediated reabsorp-
tion, drug could be released via prodrug degrada-
tion by various hydrolases because proximal tubular
cells are the most metabolically active cells in the
kidney.26 Naproxen and ketoprofen were chosen be-
cause they are NSAIDs that are used to treat renal
protein excretion via hemodynamic effects.26 How-
ever, these NSAIDs can be toxic in nonrenal tissues,
including causing serious gastrointestinal and central
side effects.27
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DIAO AND POLLI
Figure 7. Inhibition, uptake, and stability profiles of ketoprofen–glycine–l-carnitine. (a) In-
hibition of l-carnitine uptake into organic cation/carnitine transporter–Madin–Darby canine
kidney (MDCK) cells by ketoprofen–glycine–l-carnitine, (b) uptake of ketoprofen–glycine–l-
carnitine into human organic cation/carnitine transporter (hOCTN2)–MDCK cells in the pres-
ence and absence of sodium, (c) inhibition of ketoprofen–glycine–l-carnitine uptake (50 :M) into
hOCTN2–MDCK cells by 0–1000 :M l-carnitine. ∗, p < 0.05 compared with control in the ab-
sence of l-carnitine. (d) The stability of ketoprofen–glycine–l-carnitine (5 :M) in Hanks balanced
salt solution or metabolic buffers.
from the circulating blood into the brain across the
BBB. In vitro and ex vivo studies using brain cap-
illary endothelial cells and microdialysis method in
mouse have demonstrated that l-carnitine and acetyl-
l-carnitine permeate the BBB.29,30 One report demon-
strated the enhanced delivery and successive release
of nipecotic acid in mouse brain after intraperitoneal
injection of nipecotic acid–l-carnitine conjugate com-
pared with nipecotic acid itself.16 Valproic acid was
chosen here because it is used for the treatment of
convulsions, migraines, and bipolar disorder.31
carnitine < valproyl–l-carnitine. The Jmax value
of ketoprofen–glycine–l-carnitine is about one-fifth
of ketoprofen–l-carnitine, indicating that even the
small changes in the substrate structure might cause
significant changes in the transport capacity. The
Jmax values of ketoprofen–l-carnitine and valproyl–l-
carnitine are similar, indicating that OCTN2 can
transport l-carnitine conjugates efficiently as large
as ketoprofen–l-carnitine, not limited to small
l-carnitine conjugates such as valproyl–l-carnitine or
butyryl–l-carnitine.
Prodrug Inhibition and Transport Properties
l-Carnitine Prodrug Considerations
The inhibition Ki value follows the order of
An ideal l-carnitine prodrug would be stable in
plasma, selectively taken up into target tissue by
OCTN2, and then hydrolyzed to release the par-
ent drug in target tissue. All three drug–l-carnitine
conjugates and ketoprofen–glycine–l-carnitine are
OCTN2 substrates. Ketoprofen–l-carnitine, and to
some extent naproxen–l-carnitine, are stable in
metabolic buffers, which perhaps is in agreement
with Brass,32 who suggested the relative in vivo
stability of pivaloyl–l-carnitine. Ketoprofen–glycolic
acid–l-carnitine is unstable in metabolic buffers,
as well as chemical buffers such as HBSS and
SFB; release of ketoprofen from ketoprofen–glycolic
acid–l-carnitine was observed in metabolic buffers.
Ketoprofen–glycine–l-carnitine showed greater sta-
bility than ketoprofen–glycolic acid–l-carnitine in
chemical buffer and metabolic buffers. From in vitro
data, it appears that none of these five conjugates
naproxen–l-carnitine
<
ketoprofen–glycine–l-
carnitine < ketoprofen–l-carnitine < valproyl–l-
carnitine, whereas the Michaelis constant for
uptake Km follows the order of ketoprofen–glycine–l-
carnitine < ketoprofen–l-carnitine < valproyl–l-
carnitine < naproxen–l-carnitine. For valproyl–l-
carnitine and ketoprofen–l-carnitine, Ki and Km
values are essentially identical, indicating that
substrate binding was the rate-limiting step in each
translocation by hOCTN2; for naproxen–l-carntine
and ketoprofen–glycine–l-carnitine, Km values are
apparently larger than corresponding Ki values,
implicating postbinding event(s) as the rate-limiting
step(s) in the uptake of naproxen–l-carnitine and
ketoprofen–glycine–l-carnitine. The normalized Jmax
values follow the order of ketoprofen–glycine–l-
carnitine < naproxen–l-carnitine < ketoprofen–l-
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OCTN2 AS POTENTIAL PRODRUG TARGET
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possess ideal prodrug properties for targeted deliv-
ery. However, in vitro stability results do not nec-
essarily predict in vivo stability. Conjugation of l-
carnitine with nipecotic acid showed enhanced expo-
sure of nipecotic acid within mouse brain compared
with nipecotic acid.16 Therefore, nipecotic acid conju-
gate of l-carnitine definitely degraded and released
nipecotic acid in vivo. Further in vivo studies are be-
ing conducted to investigate the targeting potential
of prodrugs. It is expected that drug conjugates with
l-carnitine might degrade and release drug in vivo,
preferably in target tissue.
and biopharmaceutical implications. Pharm Res 24:1227–
1251.
5. Iwata D, Kato Y, Wakayama T, Sai Y, Kubo Y, Iseki S, Tsuji
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CONCLUSIONS
In this study, drug conjugates with l-carnitine were
investigated for the potential as prodrugs that target
OCTN2. Twenty-two l-carnitine analogues were eval-
uated for OCTN2 inhibition and 3ꢀ-hydroxyl group
was found to be the only functional group not con-
tributing to l-carnitine interaction with hOCTN2
among three functional groups on l-carnitine (i.e., 3ꢀ-
hydroxyl, amine, and carboxylate). The 3ꢀ-hydroxyl
group was subsequently chosen as the conjugate
site on l-carnitine. Three drug–l-carnitine conju-
gates (i.e., valproyl–l-carnitine, naproxen–l-carnitine,
and ketoprofen–l-carnitine), as well as two ana-
logues of ketoprofen–l-carnitine that incorporate a
linker group (glycolic acid or glycine) between ke-
toprofen and l-carnitine, were synthesized. These
five potential prodrugs were evaluated for their in
vitro inhibition, transport, and metabolism prop-
erties. All three drug–l-carnitine conjugates and
ketoprofen–glycine–l-carnitine were OCTN2 sub-
strates. The results demonstrate the potential of drug
conjugates with l-carnitine to serve as prodrugs that
target tissues with OCTN2 expression including the
kidney and brain.
ACKNOWLEDGMENTS
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This work was supported in part by National Insti-
tutes of Health Grant DK67530. The authors kindly
acknowledge Dr. Xin Ming and Dr. Dhiren R. Thakker
(University of North Carolina-Chapel Hill) for provid-
ing the hOCTN2–MDCK cell line used in this study.
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