Pharmaceutics 2013, 5, 318-328; doi:10.3390/pharmaceutics5020318
Design and Exploratory Neuropharmacological Evaluation of
Novel Thyrotropin-Releasing Hormone Analogs and Their
Brain-Targeting Bioprecursor Prodrugs
Katalin Prokai-Tatrai 1,2,*, Vien Nguyen 1, Szabolcs Szarka 1, Krisztina Konya 1,† and
Laszlo Prokai 1
1 Department of Molecular Biology and Immunology, University of North Texas Health Science
Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699, USA;
E-Mails: email@example.com (V.N.); firstname.lastname@example.org (S.S.);
2 Department of Pharmaceutical Sciences, UNT System College of Pharmacy,
University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth,
TX 76107-2699, USA
† Current address: Department of Organic Chemistry, University of Debrecen, H-4032 Debrecen,
Egyetem tér 1, Hungary; E-Mail: email@example.com.
* Author to whom correspondence should be addressed; E-Mail: firstname.lastname@example.org;
Tel.: +1-817-735-0617; Fax: +1-817-735-2118.
Received: 21 March 2013; in revised form: 7 May 2013 / Accepted: 10 May 2013 /
Published: 22 May 2013
Abstract: Efforts to take advantage of the beneficial activities of thyrotropin-releasing
hormone (TRH) in the brain are hampered by its poor metabolic stability and lack of
adequate central nervous system bioavailability. We report here novel and metabolically
stable analogs that we derived from TRH by replacing its amino-terminal pyroglutamyl
(pGlu) residue with pyridinium-containing moieties. Exploratory studies have shown that
the resultant compounds were successfully delivered into the mouse brain after systemic
administration via their bioprecursor prodrugs, where they manifested neuropharmacological
responses characteristic of the endogenous parent peptide. On the other hand, the loss of
potency compared to TRH in a model testing antidepressant-like effect with a simultaneous
preservation of analeptic activity has been observed, when pGlu was replaced with
trigonelloyl residue. This finding may indicate an opportunity for designing TRH analogs
with potential selectivity towards cholinergic effects.
Pharmaceutics 2013, 5
Keywords: bioprecursor prodrug; brain-targeting delivery; thyrotropin-releasing hormone;
synthetic peptide analog; analeptic effect; antidepressant-like activity
Our laboratory has been involved in medicinal chemistry-driven research with attention to
facilitating drug delivery of central nervous system (CNS) agents via prodrug approaches.
Thyrotropin-releasing hormone (TRH), a small peptide (pGlu-His-Pro-NH2, Figure 1), has been one of
the main focuses in this regard [1–5]. TRH was the first hypothalamic factor characterized  and it
has also served as a lead structure for CNS drug discovery [7,8], due to its multitude of central
actions [9–11]. These actions are independent of the hypothalamic-pituitary-thyroid axis; thus, the
peptide can also function as a neuromodulator through various neurotransmitters, most prominently
via acetylcholine .
Figure 1. Chemical structure of thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2).
The use of TRH as a neuropharmaceutical agent has been hampered by, e.g., its inadequate
metabolic stability , poor CNS bioavailability  and, therefore, profound endocrine activity due
to high systemic doses needed for establishing therapeutic concentration in the brain. Analogs
preserving the central benefits of TRH and possessing enhanced in vivo metabolic stability may,
however, overcome these limitations [3,11,14–16]. Nevertheless, the blood-brain barrier (BBB) has
been an obstacle to drug delivery, even with metabolically stable analogs of the peptide . As it has
been well-documented, the BBB prevents the passive transport of the overwhelming majority of
chemical entities into the brain from circulation . Only a limited number of small molecules with
suitable physicochemical properties can reach this organ from the blood at adequate concentration in
the absence of specific transporters. Hence, CNS drug delivery has been a challenging endeavor,
especially for peptides .
Various invasive and non-invasive approaches have been conceived to usher drugs into the brain
from the circulation by essentially tricking the BBB [20–22]. Among the non-invasive methods, the
prodrug approach offers a viable option for CNS-drug delivery of small- and medium-sized
neuropeptides . By definition, a prodrug is an inert precursor of the active agent (parent drug) that
remains inactive until specific enzyme(s) liberates the parent drug in vivo. Prodrugs are synthesized
from the parent agents by transient chemical modification(s), such as esterification . Because
lipophilicity is one of the major governing factors for passive transport across the BBB , enhancing
this physicochemical property via prodrug creation has been particularly useful for peptides that are
Pharmaceutics 2013, 5
generally highly hydrophilic substances. In most cases, the functional group(s) in a peptide sequence
(e.g., -NH2, -COOH or -OH) is derivatized not only to enhance lipophilicity, but also to render the
peptide “neutral” at physiological pH to favor diffusion through the BBB . The chemically
introduced “promoiety(ies)”, whose presence results in the loss of the innate activity of the parent
agent, is metabolically and/or chemically labile. Therefore, removal of the promoieties unmasks the
In another prodrug approach that leads to the so-called bioprecursor prodrugs , no auxiliary
“promoiety” is attached to the parent drug, because a bioreversible chemical manipulation (e.g.,
reduction or oxidation) is carried out within the drug molecule itself [2,3]. We have applied this
particular prodrug methodology with encouraging results to generate centrally active and non-endocrine
TRH analogs upon replacement of the central basic histidyl residue (His) in the TRH sequence with
amino acids having a pyridinium-containing side chain [2,3]. These agents are metabolically stable, as
replacement of His eliminates the TRH-degrading ectoenzyme-sensitive pGlu-His bond mostly
responsible for the very short biological half-life of TRH in the blood [8,12,27]. An additional
rationale for replacing the central residue has been to abolish (or at least diminish) the endocrine
effects seen with TRH [15,27]. Concurrently, the pyridinium moiety of the new central residue can
easily be converted via chemical reduction to a dihydropyridine [2–4,28], and the resultant neutral
peptides can serve as bioprecursor prodrugs of these TRH analogs [2,3]. The preferential activation of
the prodrug to the permanently charged parent agent in the brain occurs via oxidation, analogously to
that of the endogenous NADH → NAD+ reaction . At the same time, the “oxidized prodrugs” (i.e.,
the actual pyridinium-type TRH analogs) should quickly be eliminated from the periphery, due to their
ionic nature , while oxidation of the prodrug in the brain actually prevents efflux from the BBB.
Therefore, this particular prodrug approach is expected to result in brain-enhanced drug delivery.
In vivo validation of this design was done by utilizing typical and convenient TRH-associated
central activities; specifically, the analeptic [29,30] and antidepressant-like effects [31,32]. The latter is
monitored by a swim test introduced by Porsolt et al. . The antagonism of barbiturate-induced
anesthesia (i.e., an analeptic response) has been the most frequently used paradigm to indicate the
extent of the activation of cholinergic neurons  and, thus, a successful central delivery of TRH and
related agents. One of the analogs we created by His replacement with a pyridinium-containing residue
produced TRH-equivalent analeptic and antidepressant-like responses upon intravenous (i.v.)
administration of its brain-targeting dihydropyridine-type of bioprecursor prodrug [2,3], thereby
indicating not only a successful brain delivery of the target TRH analog, but also its ability to retain the
neuropharmacological responses typical of TRH.
Next, we probed whether [Glu2]TRH, pGlu-Glu-Pro-NH2, a non-endocrine and metabolically stable
TRH-related peptide that possesses numerous central TRH actions [30,34–36], could also be utilized
for the same design concept to obtain its pyridinium-containing analogs suitable for convenient
bioprecursor prodrug preparation. In [Glu2]TRH, we replaced the N-terminal pGlu with trigonelloyl
residue  based on findings that the therapeutically most successful TRH analogs have been derived
by pGlu-replacement . Indeed, when the dihydropyridine-based bioprecursor of this agent that was
also “lipidized” as hexyl ester on the central Glu to supply adequate lipophilicity and neutral
character to the prodrug , administered via i.v. to mice, we recorded a statistically significant
antidepressant-like response; yet, this peptide significantly underperformed both TRH and [Glu2]TRH
Pharmaceutics 2013, 5
in the analeptic test. We then reasoned that replacement of pGlu with a pyridinium moiety in TRH may
also allow for improving selectivity towards the antidepressant-like property and, thus, potentially
leading to templates useful for designing a novel class of antidepressants.
Altogether, based on our previous findings [2–5,37], in the present study, we explored whether
replacement of pGlu with pyridinium-based residues in the TRH sequence itself (Figure 2) would also
lead to analogs with improved selectivity towards the antidepressant-like effect over the analeptic
response. In exploratory proof-of-concept studies, we monitored these neuropharmacological measures
upon systemic administration of the bioprecursor prodrugs of the novel TRH analogs having a
permanently charged N-terminus.
Figure 2. Chemical structures of novel TRH analogs (2 and 3) and their brain-targeting
bioprecursor prodrugs (4 and 5, respectively), as well as schematic illustration of prodrug
synthesis from and their bioactivation to the respective TRH analogs.
2. Experimental Section
Reagents and solvents used in the study were purchased from Sigma-Aldrich (St. Louis, MO, USA)
and were of reagent grades. Pre-loaded (0.48 mmol/g) Fmoc-Pro-Rink Amide-MBHA resin was from
Anaspec Inc. (Fremont, CA, USA).
Male Swiss-Webster mice (30 ± 2 g body weight) used for monitoring the typical TRH-related
analeptic and antidepressant-like effects were obtained from Charles River Laboratories (Wilmington,
MA, USA). All procedures were reviewed and approved by the institutional animal care and use
committee before initiation of the studies. Animals were housed four per cage with ad libitum access to
water and food in a room where temperature is kept between 21 and 23 °C with a normal day/night
cycle. Each animal was tested only once.
Pharmaceutics 2013, 5
2.3. Synthesis of Test Compounds
Solid-phase peptide synthesis (SPPS) utilizing standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based
chemistry was employed to assemble TRH analogs 2 and 3 (Figure 2), as reported before [2–5,16,37].
Briefly, we used a preloaded Fmoc-Pro-Rink Amide-MBHA resin, 20% (v/v) piperidine in
N,N-dimethylformamide (DMF) for deprotection and DMF and methanol for washing. Coupling was
enforced with (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP)
in the presence of a N,N-diisopropylethylamine (DIPEA) and hydroxybenzotriazole (HOBt) using
amino acid:PyBOP:DIPEA:HOBt, 1:1:2:1 molar ratio. The peptide chain was terminated with
trigonellic acid (1-methylpyridinium-3-carboxylate hydrochloride) for analog 2 or was further
extended with Fmoc-Gly. In the latter case, a solid phase Zincke reaction  was done to obtain 3.
The novel chemical entities (2 and 3) were cleaved from the resin with trifluoroacetic acid/water 98:2
(v/v) and purified by semi-preparative reversed-phase high-performance liquid chromatography
(RP-HPLC). For the preparation of the bioprecursor prodrugs 4 and 5 (Figure 2), the TRH analogs (2
and 3) were reduced to the corresponding neutral dihydropyridines (4 and 5) with a well-established
and straightforward method using sodium dithionite [2,3,28,37,39]. The identity and purity of the test
agents were confirmed by liquid chromatography-mass spectrometry (LC-MS) and analytical
RP-HPLC. Detailed analytical characterizations, including NMR spectral assignments, will be
included in a subsequent publication.
2.4. In Vitro Metabolic Stability
Freshly made mouse brain homogenate and plasma were used for these studies, as reported
before [3–5,16]. Briefly, immediately following removal, the brain was weighed and transferred to a
Potter-Elvehjem tissue grinder (Wheaton, Millville, NJ, USA) on ice bath. Twenty-percent (w/v)
homogenates were prepared in ice-cold, pH 7.4 phosphate-buffered saline. Plasma was prepared by
centrifugation at 1500 rpm for 10 min using a Sorwal (Newtown, CT, USA) GLC-1 general laboratory
centrifuge. The brain homogenate and plasma was used immediately for stability studies. Approximately
100 nmol of test compound was used in 1 mL plasma or brain homogenate, the mixture was incubated
at 37 °C in a temperature-controlled, shaking water bath. Aliquots (100 μL) were removed after 2, 5,
15, 30, 60, 120 and 180 min of incubation and transferred into a 1.5 mL Eppendorf tube containing 200 μL
of ice-cold solution of 5% (v/v) acetic acid in acetonitrile. The samples were centrifuged, and the
supernatant was removed and analyzed by HPLC to monitor the decline in the concentration of the
analyte added. HPLC analyses were performed on an Agilent 1100 HPLC system (Agilent Technologies,
Santa Clara, CA, USA) equipped with a UV-Vis detector and operated at 254 nm. Separation was
carried out on a Phenomenex (Torrance, CA, USA) Aeris PEPTIDE XB-C18 column (150 × 2.10 mm
i.d., 3.6 µm particle size) at ambient temperature with a 0.4 mL/min flow rate. The eluent consisted of
(A) 0.5% trifluoroacetic acid in water (v/v) and (B) 0.05% trifluoroacetic acid in acetonitrile (v/v).
2.5. Analeptic Effect
These studies were conducted according to our established procedure [1–5,16,37]. Briefly, mice
were divided into groups of n = 10. Test compounds were dissolved in saline. The vehicle alone
Pharmaceutics 2013, 5
(30 µL; control group) or equimolar doses of test compounds, including TRH (10 μmol/kg body
weight), were injected through the tail vein of mice. After 10 min, each animal received an
intraperitoneal (i.p.) injection of sodium pentobarbital solution at a dose of 60 mg/kg body weight. The
sleeping time was recorded from the onset of loss of the righting reflex until the reflex was regained.
The two trained observers recorded the sleeping times independently from each other and were
unaware of the treatment regimen.
2.6. Porsolt’s Swim Test (PST) to Assess Antidepressant-Like Activity
Behavioral studies for antidepressant-like activity were conducted with a validated model, as
reported before [16,37,39,40]. Test compounds were administered i.v. (30 µL volume) through the tail
vein at the dose of 3 µmol/kg body weight. The control group received the saline vehicle (30 µL) only.
Briefly, for 6 min, the immobility time (s), the duration of motionless floating after the cessation of
struggling and making only movements necessary to keep the head above the water was recorded
simultaneously by two trained observers that were blinded to the treatment protocol. Immobility is
considered to be “depressed”; therefore, test compounds are deemed to elicit antidepressant-like
activity upon producing significantly shorter immobility time than the vehicle control.
2.7. Data Analysis
Data are expressed as the mean ± SEM, and statistical evaluations were done by one-way ANOVA.
Two-group comparisons employed Dunnett’s or Fisher’s PLSD post hoc tests when a significant
omnibus ANOVA was found (α = 0.05 two-tailed), noting that type-I error correction is not necessary
with orthogonal planned comparisons .
3. Results and Discussion
The synthesis of target compounds with pyridinium-based N-termini (2 and 3, as shown in
Figure 2) via SPPS proceeded smoothly, as our laboratory has been routinely doing such types of
preparative works [1–5,16,25,36,37]. In vitro stability studies in freshly made mouse brain homogenate
and plasma [3–5,16], respectively, showed that 2 and 3 were metabolically stable (data not shown); no
significant degradations of the compounds were detected within 2 h by RP-HPLC. This finding was
expected , since the hydrolysis-sensitive pGlu-His bond  was eliminated when we replaced
pGlu with pyridinium-containing (and also unnatural) residues. Previously, we have also confirmed
that when the central His is replaced in the TRH sequence with pyridinium-based amino acids, the
resultant TRH analogs were metabolically stable [2,3]. In comparison, TRH’s half-life is around
10 min in mouse plasma and 15 min in mouse brain homogenate . Additionally, we did not find
significant differences in the in vitro metabolic oxidation, i.e., bioactivation of the bioprecursor
prodrugs 4 and 5, to the corresponding TRH analogs (2 and 3, respectively, Figure 2) in the selected
biological media. These findings are comparable to those we have reported before for the
dihydropyridine type of prodrugs [2,3,28,37]. Accordingly, the dihydropyridine→pyridine oxidation
(i.e., prodrug to TRH analog conversion) occurred with t½ of about 5 min in mouse brain homogenate,
and around 22 min t½ values were determined in mouse plasma (data not shown). The increased
Pharmaceutics 2013, 5
resilience of 4 and 5 against oxidation in the plasma versus brain should be beneficial for BBB
transport upon their systemic administrations. We also did not find significant differences in these
measures between 4 and 5. It should be noted that the in vitro oxidation of this type of unsubstituted
dihydropyridines in the selected biological matrices apparently does not significantly depend on the
rest of the chemical structure of the prodrugs .
In vivo validation of brain-targeting of 2 and 3 via their bioprecursor prodrugs, 4 and 5, respectively
(Figure 2), was done by utilizing two well-established neuropharmacological paradigms based on
typical TRH-related central actions. Specifically, the analeptic model [29,30] and PST [31,32] were
used for these studies. The analeptic effect is surveyed in a simple paradigm by measuring the reversal
of barbiturate-induced sleeping time in mice [5,7,30]. In PST, after mice give up swimming in a
controlled environment consisting of a water tank, the floating time is recorded within a fixed time
period, as floating is believed to correspond to a “depressive mood” . We used TRH as a positive
control at equimolar concentration (10 μmol/kg body weight) in these studies. The negative control
group received the vehicle only. With exploratory proof-of-concept experiments summarized in
Figure 3, we have shown so far that, for example, analog 2 per se did not elicit an analeptic response,
due to its ionic nature (i.e., having a pyridinium moiety) that prevented its passive transport across the
BBB from the blood after i.v. injection.
Figure 3. Reversal of pentobarbital-induced sleeping times (i.e., analeptic effect) in mice
after administration of TRH, TRH analogue 2 and bioprecursor prodrugs 4 and 5. Sleeping
time is expressed as the mean ± SEM (n = 10 per group). Ten min after i.v. injection of the
test compound at a dose of 10 μmol/kg body weight, pentobarbital (60 mg/kg, i.p.) was
injected. Sleeping time was recorded from the onset of the loss of the righting reflex until
the reflex was regained. * Statistical significance determined using analysis of variance
(ANOVA) followed by post hoc Fisher’s PLSD test for multiple comparisons: p < 0.05
versus vehicle control.
On the other hand, administration of the corresponding bioprecursor prodrug (4) produced a
statistically significant reversal of pentobarbital-induced sleeping time; moreover, this analeptic
response was statistically not different from that of TRH under the experimental conditions applied.
These data imply that (i) the prodrug (4) successfully passed the BBB from the circulation and,
(ii) once in the brain it converted to 2 via oxidation (Figure 2) that, (iii) in turn, elicited the desired
TRH-characteristic neuropharmacological response.
Sleeping time (min)
Pharmaceutics 2013, 5
A slightly longer sleeping time was detected upon administration of 5, which is the bioprecursor
prodrug of 3. In the latter, the pyridinium moiety is attached via a -CH2- linker that may result in more
flexibility compared to 2. Overall, the structural differences between 2 and 3 may indicate that
replacement of pGlu with pyridinium-type moieties could be quite permissive in structural features to
maintain analeptic response.
Similarly to the analeptic studies, analog 2 carrying a permanent positive charge failed to elicit an
antidepressant-like effect in the exploratory PST studies upon systemic administration (Table 1).
However, when it was injected to mice in its bioprecursor prodrug form (4, Figure 2), a statistically
significant decrease in the immobility time (approximately 19%) was recorded compared to the
saline-treated control group (arbitrarily taken as 100%). Nevertheless, analog 2 did still significantly
underperform TRH (approximately a 36% decrease in the immobility time compared to control) in this
paradigm, even though it produced a practically TRH-equivalent analeptic response upon brain-delivery
via its prodrug (Figure 3, Table 1).
Table 1. Porsolt’s swim test (PST) immobility times reflecting the antidepressant-like
effect of the TRH analog 2 with or without utilizing its bioprecursor prodrug 4. TRH was
used as a positive control. Test compounds were administered i.v. at the dose of 3 µmol/kg
body weight. * p < 0.05 versus saline vehicle control (ANOVA followed by Dunnett’s test).
Immobility time (s)
mean ± SEM
288 ± 6
184 ± 5 *
279 ± 8
231 ± 6 *
% Change in immobility
time compared to control a
% Change in sleeping time
compared to control a,b
Prodrug of 2 (4)
a Control arbitrarily taken as 100%; percent decrease is calculated as [1 − (Xtest agent/Xcontrol)] × 100, where X
denotes the given neuropharmacological response; b based on analeptic data shown in Figure 2.
These preliminary findings are in sharp contrast with our original expectation; i.e., we anticipated
that replacement of pGlu with trigonelloyl residue in TRH would also result in enhanced
antidepressant-like activity over analeptic response, as seen with the corresponding [Glu2]TRH
analog . The latter peptide elicited a significantly improved selectivity towards the
antidepressant-like effect, compared to that of TRH and [Glu2]TRH. Apparently, our data presented
here indicate for the first time that the TRH-characteristic analeptic and antidepressant-like activities
may be dissociated upon designing appropriate TRH analogs. This type of agent may also overcome
drug delivery issues in preclinical studies by using the corresponding dihydropyridines, as
brain-targeting bioprecursor prodrugs.
Our exploratory study has shown for the first time that replacement of the N-terminal pGlu of TRH
with pyridinium-type moieties maintains the pharmacological activities of the resultant peptide analogs
in mice. However, the loss of potency in the model testing antidepressant-like effects with a
simultaneous preservation of analeptic potency has been apparent, which may indicate an opportunity
Pharmaceutics 2013, 5
for the discovery of TRH analogs with potential selectivity towards cholinergic effects. In addition, we
have demonstrated, again, the value of a bioprecursor prodrug approach to facilitate brain delivery of
molecules containing pyridinium-type moieties. Collectively, these encouraging data warrant further
investigations, including addressing potential endocrine responses elicited by the TRH analogs
reported in this communication.
This work was supported in part by the National Institutes of Health (MH059360) and the
Robert A. Welch Foundation (endowment BK-0031 to L.P.).
Conflict of Interest
The authors declare no conflict of interest.
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