Repeated dosing with NCX1404, a nitric oxide-donating pregabalin, re-establishes normal nociceptive responses in mice with streptozotocin-induced painful diabetic neuropathy

Article abstract of DOI:10.1124/jpet.115.230193

NCX1404 [(3S)-5-methyl-3-(((1-(4-(nitrooxy)butanoyloxy)ethoxy) carbonylamino) methyl)hexanoic acid] is a novel nitric oxide (NO)-donating pregabalin that is readily absorbed and processed in vivo to pregabalin and NO. We determined the antiallodynic response of NCX1404 after acute or after 7, 14, and 21 days of repeated daily oral dosing in mice with streptozotocin (STZ)-induced painful diabetic neuropathy (PDN). Pregabalin and its combination with the NO donor isosorbide mononitrate (ISMN) were used for comparison. The blood levels of pregabalin and nitrites, used as surrogate marker of NO release, after NCX1404 or pregabalin dosing were monitored in parallel experiments using liquid chromatography with tandem mass spectrometry (LC-MS/MS). NCX1404 and pregabalin resulted in similar pregabalin levels as it was their antiallodynic activity after acute dosing in STZ mice. However, NCX1404 resulted in disease-modifying properties when administered daily for 21 days, as indicated by the time- and dose-dependent reversal of STZ-induced mechanical allodynia (paw withdrawal threshold [PWT]_Veh-21d = 1.3 v 0.15 g for vehicle; PWT_NCX1404-21d = 1.4 ± 0.5 g, 2.9 ± 0.2 g^? and 4.1 ± 0.2 g^?, respectively for 19, 63, and 190 μmol/kg, oral gavage [PO] of NCX1404; ^?P, 0.05 versus vehicle). This effect was not shared by pregabalin at equimolar doses (190 μmol/kg, PO, PWT_Pregab-21d = 1.4 ± 0.1 g^?, ^?P < 0.05 versus equimolar NCX1404). In addition, the NO donor ISMN (52.3 μmol/kg, PO) alone or combined with pregabalin (63 μmol/kg) was active at 7 days (PWT_Veh-7d= 1.7 ± 0.16 g; PWT_ISMN-7d = 3.9 ± 0.34 g^?; PWT_Pregab-7d = 1.3 ± 0.07 g; PWT_{ISMN+pregab-7d} = 3.8 ± 0.29 g_?; ^?P, 0.05) but not at later time points. The long-term effect of NCX1404 was independent of residual drug exposure and lasted for several days after the treatment was stopped. In summary, like pregabalin, NCX1404 is an effective antiallodynic agent. Differently from pregabalin, repeated dosing of NCX1404 re-established normal nociceptive responses in STZ-induced PDN in mice.

Full text of DOI:10.1124/jpet.115.230193

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Title page
Repeated dosing with NCX1404, a nitric oxide (NO)-donating pregabalin, re-establishes
normal nociceptive responses in streptozotocin (STZ)-induced painful diabetic neuropathy
in mice
Katia Varani, Fabrizio Vincenzi, Martina Targa, Annalisa Ravani, Elena Bastia, Laura
Storoni, Stefania Brambilla, Nicoletta Almirante and Francesco Impagnatiello
KV, FV, MT and AR, Department of Medical Sciences, Pharmacology Section, University
of Ferrara, Ferrara, Italy.
EB, LS, SB, NA and FI, Nicox Research Institute, Bresso, Italy.

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Running Title Page
Running title: NCX1404 reduces mechanical allodynia development in STZ mice
Corresponding author:
Francesco Impagnatiello
Nicox Research Institute
Via Ariosto 21, 20091, Bresso, Milan, Italy.
Tel. +39 02 610 36 513; Fax +39 02 610 36 232; e-mail: impagnatiello@nicox.it.
N. of text pages: 32
N. of tables: 1
N. of figures: 6 (N.of supplemental figures: 1)
N. of references: 38
N. of abstract words: 246
N. of introduction words: 645
N. of discussion words: 1282
List of abbreviations: NO, nitric oxide; PDN, painful diabetic neuropathy; STZ,
streptozotocin; PWT, paw withdrawal threshold
Recommended section: Behavioral pharmacology

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Abstract
NCX1404 is a novel nitric oxide (NO)-donating pregabalin readily absorbed and processed
in vivo to pregabalin and NO. We determined the anti-allodynic response of NCX1404
following acute or after 7, 14 and 21 days of repeated daily oral dosing in streptozotocin
(STZ)-induced painful diabetic neuropathy (PDN) in mice. Pregabalin and its combination
with the NO donor, isosorbide mononitrate (ISMN) were used for comparison. Plasma
levels of pregabalin and nitrites, used as surrogate marker of NO release, after NCX1404
or pregabalin dosing were monitored in parallel experiments using LC-MS/MS. NCX1404
and pregabalin resulted in similar pregabalin levels as it was their anti-allodynic activity
after acute dosing in STZ mice. However, NCX1404 resulted in disease-modifying
properties when administered daily for 21 days as indicated by time-and dose-dependent
reversal of STZ-induced mechanical allodynia (PWTVeh_21d=1.3 ± 0.15g for vehicle,
PWTNCX1404_21d= 1.4±0.5g, 2.9±0.2g*, and 4.1±0.2g*, respectively 19, 63, and
190μmoles/kg, po of NCX1404, * p<0.05 vs. vehicle). This effect was not shared by
pregabalin at equimolar doses (190μmoles/kg, po, PWTPregab_21d=1.4±0.1g, * p<0.05 vs.
equimolar NCX1404). In addition, the NO donor, ISMN (52.3μmoles/kg, po) alone or
combined with pregabalin (63μmoles/kg) was active at 7 days (PWTVeh_7d=1.7±0.16g,
PWTISMN_7d=3.9±0.34g*, PWTPregab_7d=1.3±0.07g and PWTISMN+pregab_7d=3.8±0.29g*) but
not at later time-points. The long-term effect of NCX1404 was independent from residual
drug exposure and lasted for several days after the treatment was stopped. In summary,
like pregabalin, NCX1404 is an effective anti-allodynic agent. Differently from pregabalin,
repeated dosing of NCX1404 re-established normal nociceptive responses in STZ-induced
PDN in mice.

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Introduction
Neuropathic pain is a form of chronic pain that can be classified as peripheral or central.
Peripheral neuropathic pain is caused by injury or infection of peripheral sensory nerves,
whereas central neuropathic pain is caused by damage to the central nervous system
(CNS) or/and the spinal cord. Both peripheral and central neuropathic pain can occur
without obvious initial nerve damage. Various metabolic diseases cause neuropathic
changes which later lead to neuropathic pain. An example is painful diabetic neuropathy
(PDN) which occurs in a large number of patients suffering from diabetes. The
pathophysiology of PDN remains unclear. Nevertheless, neurogenic and vascular
components have been described (Yorek, 2003). For instance, peripheral demyelination,
reduction in nerve conductance, and progressive degeneration of peripheral sensory fibers
(Watkins, 1984) along with micro- and macro-vascular abnormalities dependent on
dysfunctional endothelium, the innermost layer of the blood vessel, have long been
established in experimental animal models and in diabetic patients (Pieper and Gross,
1988; Pitei et al., 1997) as well as in individuals who will later develop diabetes (Cosentino
and Luscher, 1998).
To date, the antidepressant duloxetine (King et al., 2015) and the anticonvulsants
gabapentin and pregabalin (Schreiber et al., 2015) are considered the first-line treatment
strategies for patients affected by various forms of neuropathic pain including PDN. These
drugs are generally safe, but they do not entirely relieve pain symptoms and a substantial
subset of patients is virtually insensitive to treatment. Furthermore, even when they work,
these and other available treatments ameliorate the symptoms but lack effects on the
underlying pathophysiology of the disease, making clinical management difficult as the
disease progresses to its late stages.

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Depending on the circulating concentration and the specific tissue compartment where it is
released, nitric oxide (NO) exerts various functions ultimately leading to either pro-
nociceptive or anti-nociceptive behaviors (Miclescu and Gordh, 2009; Schmidtko et al.,
2009). Low physiologic amounts of NO activate soluble guanylyl cyclase (sGC) causing
vascular relaxation, favor neuronal transmission, and modulate genes transcription
through nuclear factor (NF)-kB signaling (Colasanti and Suzuki, 2000), whilst large
quantities of NO are thought to produce abnormal neuronal activity and central
sensitization (Schmidtko et al., 2009), activate pro-inflammatory cytokines, and promote
tissue-specific oxidative and nitrosative stress, which also are featured in diabetic
conditions (Pacher et al., 2007). Different laboratories (Sasaki et al., 1998; Rodella et al.,
2000) have demonstrated that impaired NO generation in diabetic rats results in
hyperalgesia while the administration of L-arginine, known to enhance the circulating NO
concentration, is antinociceptive in diabetic mice. Pitei and co-workers showed that
decreased NO production contributes to reduced endoneuronal blood flow in diabetic
patients and caused pain (Pitei et al., 1997), while the local application of NO donors, such
as isosorbide dinitrate (Yuen et al., 2002) or nitroglycerin (Francis et al., 1977; Agrawal et
al., 2007), relives pain and burning sensation in patients with PDN.
Early exploratory work from our laboratories documented the ability of NO to enhance the
effect of gabapentin in neuropathic pain models (Wu et al., 2004). Additional evidence of
NO-mediated effects in neuropathic pain models was later obtained with compounds
possessing both antioxidant and NO-releasing properties (Ronchetti et al., 2009). Other
investigators reported the beneficial effect of NO when combined with gabapentin in spite
of the different experimental conditions employed (Curros-Criado and Herrero, 2009).
Similarly, Huang and co-workers, documented the analgesic effects of an NO-donating

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derivative in a rat model of PDN (Huang et al., 2015), while Otari and Upasani reported on
the involvement of NO-cGMP signaling in neuropathic pain processing (Otari and Upasani,
2015).
In the present study, we investigated the anti-allodynic effects of a novel NO-donating
pregabalin derivative, NCX1404, following a single treatment schedule. Furthermore we
addressed whether repeated daily dosing with NCX1404 modulates the development and
maintenance of mechanical allodynia consequent to STZ. In all experiments, the NO donor
isosorbide 5-mononitrate (ISMN) and its combination with pregabalin were used for
comparison.

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Materials and Methods
In all experiments, animals were cared for and treated in accordance with protocols
approved by the Institutional Animal Care Committees, and all efforts were made to limit
the number of animals and to minimize animal suffering. Male CD1 mice, weighting 20-30
g were kept under standard conditions of temperature (22-24°C) and illumination (12:12-h
light/darkness). They were allowed to adjust to this environment in cages with mesh
bottoms with free access to fresh water and food for at least 7 days before the
experiments began.
NCX1404 ((3S)-5-methyl-3-(((1-(4-(nitrooxy)butanoyloxy)ethoxy)carbonylamino)
methyl)hexanoic acid) is a nitrooxyderivative of pregabalin synthesized in our laboratories
in 8 steps with 12% overall yield. The compound was obtained by coupling pregabalin and
1
-{[(4-nitrophenoxy)carbonyl]oxy}ethyl 4-(nitrooxy)butanoate (9) that was prepared in 7
steps from 4-bromobutanoic acid (Figure 1). To obtain the corresponding 15N- labeled
NCX1404, 15N-AgNO was used instead of AgNO in step b reported in Figure 1.
3
3
1
5N-Isosorbide 5-mononitrate was synthesized in house by adding 15N-Bu
4
NNO , 2,6-di-
3
tert-butyl-4- methyl pyridine and trifluoromethanesulfonic anhydride to a solution of
isosorbide in dichloromethane. 15N-Isosorbide 5-mononitrate was then isolated by flash
chromatography.
Other chemicals and reagents were purchased from Sigma-Aldrich, St Louis, MO unless
otherwise specified.
Streptozotocin (STZ)-induced diabetes mice model

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Diabetes was induced by injecting 200 mg/kg i.p. of streptozotocin (STZ) (Kowaluk
et al., 2000; Vincenzi et al., 2014). Blood glucose levels were assessed from the second
day until the end of the experiment using an Accu-Chek blood glucose monitoring system.
A single administration of STZ induced insulin-dependent diabetes mellitus within 24-48 h
by destruction of pancreatic islet cells (Schenkman, 1991). Plasma glucose levels above
300 mg/dl were considered indicative of diabetes (Migita et al., 2009).
Mechanical allodynia paw withdrawal test
Paw withdrawal thresholds (PWT) were determined using the Dynamic Plantar
Aesthesiometer (Ugo Basile, Italy), an apparatus that generates a mechanical force
linearly increasing with time (Szolcsanyi et al., 2004; Wright et al., 2007). Mice were
placed individually in plastic cages with a wire mesh bottom and allowed to acclimatize for
at least 2 h. Increasing mechanical stimulation (0.25 g/s, cut-off force: 10 g) was applied to
the plantar surface of a hind paw. The nociceptive threshold was defined as the force, in
grams, at which the mouse withdraws its paw. When a withdrawal response occurred, the
stimulus was terminated and the response threshold measured electronically. Data are
reported as the mean ± standard error of the threshold recorded at each trial.
Drug treatment paradigm
Acute schedule. On day 14 post STZ challenge, mice were treated orally with a single
challenge of drugs at the indicated doses or vehicle (Hypromellose 0.5%, DMSO 2%).
Mechanical allodynia, expressed as paw withdrawal threshold (g) was evaluated at 0, 5,
30, 45, 60, 90, 120 and 180 min after treatments.

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Repeated treatment schedule. On day 7 post-STZ challenge, mice were treated orally
each day with the selected drugs at the indicated doses or vehicle (Hypromellose 0.5%,
DMSO 2%) for 21 days. Mechanical allodynia expressed as paw withdrawal threshold (g),
was measured on day 7, 14 and 21, 18-24 h after the last daily treatment. In addition,
changes in the mechanical threshold were continued to be monitored in all groups at 3, 7
and 10 days after dosing was discontinued.
Analytical assessments
Parallel experiments were conducted to monitor the plasma levels of NCX1404 and
pregabalin after acute administration of NCX1404 or pregabalin at 63 μmole/kg, po. For
the acute schedule experiments, blood samples were collected from the left ventricle prior
to (0) and at 5, 15, 30, 60, 120, 240 and 360 min post-dose in anesthetized mice (n=3 for
each time point). The blood samples were later processed for further plasma isolation and
analytical assessments. 15N-Nitrites, used as surrogate marker of NO release, were
monitored at the same time points as above in animals treated with 15N-NCX1404 at the
same dose (63 μmole/kg, po). For the repeated dosing experiments, blood samples were
withdrawn 18-24h after 7, 14, or 21 days of consecutive daily treatment with 63 μmole/kg,
po of pregabalin or NCX1404 and later processed to monitor the levels of pregabalin in
plasma.
NCX1404 and pregabalin quantitation. Aliquots (50 µL) of plasma were added with 10 µL
of 100% DMSO, 10 µL H O, and 500 µL methanol and then centrifuged at 4000 rpm for 10
2
min, 4°C. The supernatants were collected and stored at -80°C and later analyzed by a
sensitive and specific LC/MS/MS method for simultaneous determination of pregabalin and
NCX 1404. The system was an Acquity UPLC (Waters, Milford, MA). The detector was a

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Quattro microTM API LC/MS/MS (Waters, Milford, MA). The conditions used to analyze
NCX1404 and pregabalin were: Column, ACQUITY BEH Phenyl 50 x 2.1 mm (5 µm) at
4
0°C; Mobile Phase, A. water + 0.1% formic acid and B. methanol + 0.1% formic acid; flow
rate 0.5 mL/min; Detector Wavelength, 210 nm; MRM transitions (Multiple Reaction
Monitoring) in ESI positive for NCX 1404 was m/z 400.92 325.07; for pregabalin was
m/z 160.2 97.1. The raw data (average peak area at each time point) were interpolated
using Quanlynx v4.1 software to obtain the concentration of compounds at each time
point.
15N-Nitrites. Blood samples were taken from the left ventricle prior to (0) and at 5, 15, 30,
60, 120, 240 and 360 min post-dose, the plasma separated and later extracted with
acetonitrile. The quantification is based on conversion of 15N-Nitrites to 15N-
naphtotriazole using 1.2-diaminonaphtalene in acidic conditions, followed by quantification
by LC-MS/MS.
Briefly, the system used was an Agilent 1200 HPLC (Agilent Technologies, Santaclara,
CA). The detector was an API4000 mass spectrometer (Applied Biosystems). 15N-Nitrites
conversion to naphtotriazole was done mixing 200 L of deproteinized blood sample
spiked with 10µL of 1.2-diaminonaphtalene 1mM in HCl 0.5 M and shaken for 20min at
room temperature. At the end, to stop the conversion, 10 µL of NaOH 1M and 10µL of
DMSO were added to all the samples. The resulting compound was then separated from
the mixture using an ACE C18 AR 50 x 2.0 mm (3 m) column. Mobile phase: A)
water/0.1% formic acid and B) acetonitrile/0.1% formic acid. MRM transition (Multiple
Reaction Monitoring) in ESI positive for 15N-naphtotriazole was m/z 171.1
115.0. Raw
data (average peak area at each time point) were interpolated on a standard curve to
obtain the concentration of 15N-naphtotriazole at each time point.

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Statistics
In all experiments, comparison between treatments groups was performed by two-
way ANOVA followed by a Bonferroni multiple comparison test unless otherwise specified.
P values below 0.05 were considered significant.

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Results
Blood glucose levels and mechanical allodynic threshold following streptozotocin (STZ)-
treatment in CD-1 mice
Physiologic glucose plasma levels in CD-1 mice were 149 ± 4 mg/dl. The
administration of streptozotocin (STZ) resulted in a rapid increase in plasma glucose levels
starting from the following day, and the levels remained elevated throughout the weeks
thereafter reaching 515 ± 17 mg/dl at 4 weeks post-treatment (Table 1).
The injection of STZ also significantly diminished the paw withdrawal threshold (PWT) to
mechanical stimulation starting 14 days after STZ injection (PWT, 4.2 ± 0.31 and 1.3 ±
0.44 g, vehicle and STZ, respectively). The allodynic response remained stable over the
following 2 weeks (Table 1).
NCX1404, pregabalin plasma levels after NCX1404 and pregabalin in mice
The plasma levels of NCX1404 in mice after 63 μmole/kg, po (23,7 mg/kg) were
below the limit of quantitation at all time-points tested. NCX1404 is a nitrooxy ester
derivative of pregabalin, we thus compared the extent of circulating pregabalin as well as
that of nitrites (serving as surrogate marker of NO release) after NCX1404 (63 μmoles/kg,
po) or equimolar pregabalin (63 μmoles/kg, po) single dosing. Parallel experiments were
made using NCX1404 labelled on the nitrate group with 15N to study the levels of nitrites
released into the blood stream after NCX1404 single dosing. Pregabalin levels after
NCX1404 single dosing reached the Cmax of 6.8±0.6 μg/mL 30 min post NCX1404
administration and declined in the next hours to reach levels close to baseline after 360

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min (Figure 2A). Pregabalin administered under similar conditions resulted in similar
pregabalin exposure compared to that found after NCX1404 (Figure 2A). The relative
pregabalin bioavailability after NCX1404 compared to pregabalin was 85%.
15N-Nitrites plasma levels after NCX1404 and isosorbide 5-mononitrate in mice.
15N-Nitrites plasma levels after NCX1404 oral treatment (63 μmole/kg, po) were
maximal 5 min after dosing the animals and rapidly declined to basal levels thereafter
(
(
(
Figure 2B). Conversely, 15N-Nitrites after isosorbide 5-mononitrate (ISMN) oral dosing
52.3 μmole/kg, po) in the plasma increased slowly to reach maximum at 60 min
Cmax=1.77±0.4μM) and slowly decayed thereafter to reach basal values during the
following 4 h (Figure 2B).
Anti-allodynic effects of acute dosing of NCX1404 or pregabalin in streptozotocin (STZ)-
induced painful diabetic neuropathy mice model
As shown in figure 3A, acute administration of NCX1404 at 19, 63, and 190
μmoles/kg, po, (7.1, 23.7, and 71 mg/kg) reduced mechanical allodynia in a time- and
dose-dependent fashion (PWTNCX1404_60min=2.6±0.2g, 3.9±0.3g, and 4.6±0.2g for 19, 63,
and 190 μmoles/kg, po of NCX1404, respectively) vs vehicle (PWTVehicle_60min=1.3 ± 0.2g)
in STZ-induced painful diabetic neuropathy (PDN) mice model. The administration of
pregabalin at equimolar doses (3, 10 and 30 mg/kg) also reduced mechanical allodynia in
a time- and dose-dependent fashion (PWTPregab_60min=2.8±0.1g, 3.8±0.3g, 4.3±0.4g after
19, 63 and 190 μmoles/kg, po of pregabalin, respectively) reaching values close to those

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observed in naïve animals (Figure 3B). Consistent with pregabalin exposure after
NCX1404 or pregabalin oral treatment, the anti-allodynic responses of both drugs were
maximal between 45 and 60 min post-dosing and slowly disappeared thereafter to reach
basal values at 120 min (Figure 3A and B).
Reversal of the allodynic response following 21-day repeated dosing with NCX 1404 in
streptozotocin (STZ)-induced painful diabetic neuropathy mice model
These experiments were undertaken to address whether repeated daily
administration of NCX1404 or pregabalin re-establish normal nociceptive responses to
mechanical stimulation in STZ-induced PDN mice model. The compounds were
administered by oral gavage starting 1 week post-STZ treatment whereas testing was
performed at 7, 14 and 21 days, 18-24 h after the respective daily treatment to assure the
absence of circulating pregabalin which was then confirmed in parallel pharmacokinetic
experiments by the absence of detectable levels of pregabalin in plasma samples
collected at the same time-points (data not shown). Furthermore, the duration of the effect
was monitored by assessing the mechanical threshold at 1, 3, 7 and 10 days after the 21
days of repeated daily dosing was ended. Similar treatment paradigm with pregabalin at
equimolar doses was used for comparison.
The repeated oral administration of 19 μmoles/kg (7.1 mg/kg), 63 μmoles/kg (23.7 mg/kg)
and 190 μmoles/kg (71 mg/kg) of NCX1404 resulted in dose- and time-dependent
enhancement of mechanical threshold which was maximal after 21 days (Figure 4A). The
effect of NCX1404 was retained for several days after ending the daily treatment as
indicated by the high mechanical threshold still recorded 3 and 7 days after the treatment

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was completed (Figure 4A). Regardless of the dose used, this effect was not shared by
pregabalin administered at equimolar doses under the same experimental conditions
(Figure 4B) suggesting that these effects likely depend on the release of NO.
Anti-allodynic response following acute or 21-day repeated dosing with the NO donor,
isosorbide 5-mononitrate, pregabalin or their combination in streptozotocin (STZ)-induced
painful diabetic neuropathy mice model
We tested whether isosorbide 5-mononitrate (ISMN) per se or combined with
pregabalin exerts any anti-allodynic effect on STZ-induced PDN mice model. In
exploratory experiments, ISMN was tested at 15.7 μmoles/kg, po (3 mg/kg), 52.3
μmoles/kg, po (10 mg/kg) and 157 μmoles/kg, po (30 mg/kg). In these experiments, 15.7
μmoles/kg did not modify the allodynic threshold to mechanical stimulation in STZ-treated
animals whereas the high dose of 157 μmoles/kg resulted in significant anti-allodynic
activity combined with robust cardiovascular hypotension (data not shown). Conversely,
the administration of ISMN at 52.3 μmoles/kg resulted in significant anti-allodynic effects
with only moderate hypotension, transient in nature and thus, this dose was selected for
further experiments. Animals treated with 52.3 μmoles/kg, po of ISMN showed mild anti-
allodynic response as indicated by the increase in PWT at selected time-points post
dosing (Figure 5). Likewise, 63 μmoles/kg, po (10 mg/kg) administration of pregabalin also
elicited mild to moderate anti-allodynic effects as in previous experiments (Figure 5).
However, the concomitant oral administration of ISMN (52.3 μmoles/kg, po) and
pregabalin (63 μmoles/kg, po) resulted as marginally effective as either drug given alone.

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To address whether STZ-induced mechanical allodynia was influenced by ISMN, we then
monitored the allodynic response after 7, 14 and 21 days of daily oral treatment with ISMN
(
52.3 μmoles/kg, po) in animals kept treatment-free for 18-24 h prior to testing so to assure
the absence of circulating NO during testing. In these experimental sessions, pregabalin
63 μmoles/kg, po) alone or combined with ISMN (52.3 μmoles/kg, po) administered
(
once/day for 21 consecutive days were used for comparison.
Repeated daily dosing with 52.3 μmoles/kg, po (10 mg/kg) of ISMN reduced the
development of mechanical allodynia in mice treated with STZ one week earlier. In
particular, the effect was evident after 7 days of repeated daily treatment but disappeared
during the following days (Figure 6). Similarly transient was the effect recorded after the
co-administration of pregabalin (63 μmoles/kg, po) and ISMN (52.3 μmoles/kg, po) (Figure
6
).

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Discussion
NCX1404 is a dual acting nitric oxide (NO)-donating derivative of pregabalin featuring a
mononitrate alkyl chain bound to the amino function of pregabalin by an acyloxy-alkyl
carbamate. This novel compound is designed to combine the NO-mediated anti-
inflammatory and anti-oxidant properties with those of pregabalin, which is known to
reduce the symptoms of pain while lacking any measurable effect on the underlining
pathophysiology.
The administration of streptozotocin (STZ) to mice results in sustained diabetes (Kowaluk
et al., 2000) and measurable allodynic response to mechanical stimulation. These effects
are thought to be consequent to the progressive degeneration of peripheral sensory fibers
(
Watkins, 1984) due to dysfunctional vascular endothelium and reduced NO availability
Yorek, 2003). The STZ model is thus particularly interesting to address the acute anti-
(
allodynic effects as well as long-term changes of the nociceptive circuits associated with
the administration of NO-donating drugs like NCX1404.
In our hands, mice receiving a single challenge of STZ rapidly increased their plasma
glucose level and later developed robust allodynic response to mechanical stimulation
which was maximal within 7 days and remained stable over several weeks thereafter. For
this reason, day 14 post STZ treatment was selected to test the acute anti-allodynic
response of NCX1404 versus pregabalin while 7 days post-STZ was used as starting point
for repeated dosing in order to address the effects of NCX1404 on the development of
allodynia in diabetic mice.
NCX1404 is readily absorbed following oral dosing in STZ mice and efficiently cleaved in
vivo to release pregabalin as for other molecules carrying similar approach (Cundy et al.,

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2004). In addition, NCX1404 also releases relevant amounts of NO as determined by the
presence of 15N-nitrites in plasma of animals dosed orally with 15N-labelled NCX1404.
The anti-allodynic effect of NCX1404 after a single oral challenge in STZ-induced diabetic
neuropathic mice model was time- and dose-dependent. Similarly, equimolar doses of
pregabalin also resulted in time- and dose-dependent antiallodynic effects that were
overlapping with that elicited by the respective equimolar dose of NCX1404. Furthermore,
the antiallodynic effects of both NCX1404 and pregabalin after single acute oral dosing
were coincident with the respective time-course of pregabalin plasma exposure. These
data indicate that the acute anti-allodynic effects of NCX1404 are mostly dependent on
pregabalin release with minor, if any, contribution of NO. Alternatively, the absence of
superiority maybe due to partially overlapping mechanisms for pregabalin and NO. In a
different series of experiments, the single oral administration of the NO-donor, isosorbide
5-mononitrate (ISMN) resulted in modest anti-allodynic effect after mechanical stimulation
which was similar to that of a sub-maximal dose (10mg/kg) of pregabalin. Moreover, their
co-administration (concomitant pregabalin and ISMN dosing) was only as effective as the
individual compounds given alone consistent with the concept that the acute anti-allodynic
effect of NO and pregabalin could be due, at least partially, to overlapping mechanisms.
Pregabalin mainly alleviates neuropathic pain symptoms by impairing the trafficking of
2+
2
-1 receptors and, subsequently, reducing Ca influx and spinal sensitization (Sills,
2006). However, additional mechanisms involving the activation of NO/cGMP signaling
pathway have recently been described for another 2 -1 antagonist, gabapentin (Mixcoatl-
Zecuatl et al., 2006; Takasu et al., 2006). Whether similar mechanisms also take place for
pregabalin remain to be established.

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The main observation made in the present study is, however, the significant time- and
dose-dependent long-term changes of the nociceptive circuits exhibited by NCX1404 after
repeated daily dosing in mice with established neuropathy consequent to the
administration of STZ 1-week earlier. The enhancement of nociceptive threshold following
NCX1404 long-term dosing reflect changes in the nociceptive circuits rather than direct
anti-allodynic effects of the drug and its active metabolites as no detectable levels of
pregabalin and NO were measured at the time (18-24h after the last daily dose) the
behavioral assessments were taken. This effect appears to be unique to NO-donating
drugs like NCX1404, as pregabalin, administered under the same experimental conditions,
did not result in measurable activity. Similar trend, albeit of different intensity, was
observed when the NO-donating derivative of gabapentin (NCX 1236) was administered
under the same conditions (supplemental Figure 1) suggesting the importance of NO
release in mediating this effect. Moreover, ISMN administered alone or combined with
pregabalin was as effective as NCX1404 on day 7 but not on day 14 and 21, time at which
ISMN-mediated tolerance occurs (Daiber and Munzel, 2015). These latter results further
indicate the importance of NO release in the allodynic responses elicited by STZ after
repeated dosing with NCX1404 and, at the same time, suggest potential differences
between NCX1404 and ISMN with respect to their tolerance liability, ability to efficiently
release NO over repeated dosing and/or efficiency to activate the NO/cGMP signaling
pathway (Klemenska and Beresewicz, 2009). NCX1404 releases pregabalin and the
nitrate linker via the action of esterases (data not shown); however, we still lack
information on how the nitrate linker, once cleaved from pregabalin, releases NO. In our
experiments, the kinetics of 15N-nitrites, used as a surrogate marker of NO release,
differed significantly after 15N-NCX1404 and 15N-ISMN dosing. In spite of similar Cmax

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between the two compounds, 15N-nitrites accumulation was rapid (Tmax 5min) and
decayed quickly after 15N-NCX1404. Conversely, 15N-nitrites measured after 15N-ISMN
were largely superior compared to that from 15N-NCX1404 with a Tmax at 60min.
Furthermore, the bioactivation and NO release from ISMN occur via the P450 enzyme that
is down-regulated when repeatedly stimulated (Minamiyama et al., 2004). This
mechanism, along with soluble guanylyl cyclase desensitization and activation of oxidative
stress have been described to mediate ISMN-induced tolerance (Minamiyama et al., 2004;
Daiber and Munzel, 2015) but this mechanism may not be as important for NCX1404 as
similar NO-donating compounds were reported to rely on different bioactivation pathways
compared to ISMN (Govoni et al., 2006).
The effect of NCX1404 is independent from residual drug as pregabalin levels in plasma
samples withdrawn at the time of behavioral assessments were found below the limit of
quantitation (data not shown). Further to that, the reduction of mechanical allodynia in
animals treated with NCX1404 for 21 days was still evident 3 and 7 days after the
treatment was ended making it unlikely that residual compound accounts for the observed
activity. NCX1404 did not affect plasma glycemic levels (data not shown) in these animals
explaining why, soon after the treatment was completed, the allodynic threshold returned
to levels similar to that of vehicle with a time-course resembling that observed after the
initial injection of STZ.
The molecular and cellular mechanisms underlying the long-term effects observed after
NCX1404 repeated dosing are far from clear. Neurogenic and vascular changes are
involved in the pathophysiology of PDN (Yorek, 2003). These include the release of
various inflammatory mediators (Purwata, 2011), vascular endothelial cell dysfunction, and
progressive reduction of NO which is thought to play a pivotal role in the pathophysiology

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JPET #230193
of PDN (Pieper and Gross, 1988). NCX1404 releases biologically relevant amounts of NO
as detected by the plasma increase in 15N-nitrites. Thus, the circulating NO brought about
by NCX1404 might compensate for the reduced endogenous NO production consequent
to endothelial cell dysfunction in STZ-treated animals. Likewise, circulating NO from
NCX1404 might inhibit inflammatory cytokines release. Consistent with this interpretation,
previous studies documented the ability of NO donors to ameliorate endothelial function in
rodent models of diabetes (Pieper and Gross, 1988; Ambrosini et al., 2005; Huang et al.,
2015) and to reduce inflammatory cytokines via modulation of NF-kB translocation
(Colasanti and Suzuki, 2000; Ronchetti et al., 2006).
In summary, acute dosing of NCX1404 is as effective as acute dosing of pregabalin to
reverse mechanical allodynia in STZ-diabetic mice. However, NCX1404, differently from
pregabalin, when administered over 21 days re-establishes normal nociceptive responses
in STZ-induced PDN in mice. This result is likely to be due to a mechanism involving the
release of NO.

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Authorship contributions
Conducted the pharmacological and pharmacokinetic experiments: Fabrizio Vincenzi,
Martina Targa, Annalisa Ravani, Elena Bastia, Stefania Brambilla
Performed data analysis: Elena Bastia, Francesco Impagnatiello, Katia Varani
Designed the synthesis of compounds subject to pharmacological testing: Nicoletta
Almirante and Laura Storoni
Participated to research design: Katia Varani and Francesco Impagnatiello
Contributed to writing the manuscript: Francesco Impagnatiello, Elena Bastia

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Footnotes
Address correspondence and reprint requests to:
Francesco Impagnatiello
Nicox Research Institute
Via Ariosto 21, 20091, Bresso, Milan, Italy.
Tel. +39 02 610 36 513; Fax +39 02 610 36 232
e-mail: impagnatiello@nicox.it.

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Figure legends
Figure 1. Synthesis of NCX 1404.
a) SOCl
N, MeOH, 5 °C, overnight, 84%; d) Ag
chloroethyl chloroformate, Py, CH Cl , r.t., overnight, 97% ; f) NaI, CH
3
2
3 3
, MeOH, r.t, overnight, 85% ; b) AgNO , CH CN, reflux, 4 hours 93 % ; c) LiOH 2
2
O, CH CN / H
3
2
O, r.t., 3 hours, 70% ; e) 1-
CN, 53% ; g) dry
Cl
2
2
toluene, r.t., overnight, 71% ; h) pregabalin, triethylamine, trimethylsilylchloride, CH
r.t., overnight.
2
2
,
Figure 2. A) Pregabalin exposure after single oral administration of NCX 1404 or
pregabalin to mice. NCX 1404 (closed circle, n=3) and pregabalin (open circle, n=3) were
administered at 63 μmole/kg, po, (23.7 and 10 mg/kg for NCX1404 and pregabalin,
respectively) dissolved in vehicle (Hypromellose 0.5%, DMSO 2%). Both drugs were given
by oral gavage at 5ml/kg. B) 15N-Nitrites were monitored following the administration of
NCX1404 (63 μmol/kg, closed circle, n=3) labelled with 15N given at the same dose and
conditions as above of ISMN (52.3 μmol/kg, open square, n=3) labelled with 15N.
Figure 3. Dose-dependent anti-allodynic effect of NCX 1404 (A) and pregabalin (B) in
STZ-diabetic mice. Both drugs were administered dissolved in vehicle (Hypromellose
0.5%, DMSO 2%, grey triangle, n=6). NCX1404 was given by oral gavage (5ml/kg) at 19
μmole/kg (7.1mg/kg, closed diamond, n=6), 63 μmole/kg (23.7mg/kg, closed circle, n=6)
and 190 μmole/kg (71 mg/kg, closed square, n=6). Pregabalin was administered at
equimolar doses as NCX1404 (3 mg/kg, open diamond, n=6; 10 mg/kg, open circle, n=6;
30 mg/kg, open square, n=6). Paw withdrawal threshold following mechanical stimulation
allodynia was monitored at 0 (basal), 15, 30, 45, 60, 90, 120 and 180 min post treatment.

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*
p<0.05 vs vehicle, Two way ANOVA followed by Bonferroni's multiple comparisons test
NCX 1404 19 μmole/kg at 45 min; NCX 1404 63 μmole/kg from 30 to 90 min; NCX 1404
90 μmole/kg from 15 to 90 min; pregabalin 19 μmole/kg from 45 to 60 min; pregabalin 63
(
1
μmole/kg from 30 to 90 min; pregabalin 190 μmole/kg from 30 to 90 min).
Figure 4. Dose-dependent effects of repeated daily oral dosing with of NCX 1404 (A) or
pregabalin (B) in STZ-diabetic mice. Both drugs were administered dissolved in vehicle
(Hypromellose 0.5%, DMSO 2%, grey triangle, n=6). NCX1404 was given by oral gavage
(5ml/kg) at 19 μmole/kg (7.1 mg/kg, closed diamond, n=6), 63 μmole/kg (23.7 mg/kg,
closed circle, n=6) and 190 μmole/kg (71 mg/kg, closed square, n=6). Pregabalin was
administered at equimolar doses as NCX1404 (3 mg/kg, open diamond, n=6; 10 mg/kg,
open circle, n=6; 30 mg/kg, open square, n=6). All treatments were given daily between 9
and 10AM for 21 days starting 7 days after the initial STZ challenge. All measurements of
mechanical allodynia were performed in drug-free conditions. The later aspect was verified
by confirming the absence of a detectable concentration of pregabalin in plasma samples
withdrawn at the time the experiment was performed. *p<0.05 vs vehicle, Two way
ANOVA followed by Bonferroni's multiple comparisons test (NCX 1404 63 μmole/kg from 7
to 21 days; NCX 1404 190 μmole/kg from 7 to 28 days).
Figure 5. Acute anti-allodynic effect of ISMN alone or combined with pregabalin in STZ-
diabetic mice. ISMN (52.3 μmole/kg, po equal to 10mg/kg, cross, n=6), pregabalin (63
μmole/kg, po equal to 10mg/kg, open circle, n=6) or their combination (closed triangle,
n=6) were administered orally (5mL/kg) dissolved in vehicle (Hypromellose 0.5%, DMSO
2
%, grey triangle, n=6). Mechanical allodynia was monitored at 0 (basal), 15, 30, 45, 60,
0 and 120 min post dosing. *p<0.05 vs vehicle, Two way ANOVA followed by Bonferroni's
9