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
, ), has been one of the main focuses in this regard [1
]. TRH was the first hypothalamic factor characterized [6
] and it has also served as a lead structure for CNS drug discovery [7
], due to its multitude of central actions [9
]. 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 [10
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 [12
], poor CNS bioavailability [13
] 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
]. Nevertheless, the blood-brain barrier (BBB) has been an obstacle to drug delivery, even with metabolically stable analogs of the peptide [17
]. As it has been well-documented, the BBB prevents the passive transport of the overwhelming majority of chemical entities into the brain from circulation [18
]. 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 [19
Various invasive and non-invasive approaches have been conceived to usher drugs into the brain from the circulation by essentially tricking the BBB [20
]. Among the non-invasive methods, the prodrug approach offers a viable option for CNS-drug delivery of small- and medium-sized neuropeptides [5
]. 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 [23
]. Because lipophilicity is one of the major governing factors for passive transport across the BBB [24
], enhancing this physicochemical property via prodrug creation has been particularly useful for peptides that are 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 [25
]. 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 active agent.
In another prodrug approach that leads to the so-called bioprecursor prodrugs [26
], 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
]. 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
]. 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
]. An additional rationale for replacing the central residue has been to abolish (or at least diminish) the endocrine effects seen with TRH [15
]. Concurrently, the pyridinium moiety of the new central residue can easily be converted via
chemical reduction to a dihydropyridine [2
], and the resultant neutral peptides can serve as bioprecursor prodrugs of these TRH analogs [2
]. 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+
]. 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 [28
], 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.
validation of this design was done by utilizing typical and convenient TRH-associated central activities; specifically, the analeptic [29
] and antidepressant-like effects [31
]. 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 [34
] 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
], 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
, a non-endocrine and metabolically stable TRH-related peptide that possesses numerous central TRH actions [30
], 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 [37
] based on findings that the therapeutically most successful TRH analogs have been derived by pGlu-replacement [38
]. 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 [36
], administered via i.v.
to mice, we recorded a statistically significant antidepressant-like response; yet, this peptide significantly underperformed both TRH and [Glu2
]TRH 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
], in the present study, we explored whether replacement of pGlu with pyridinium-based residues in the TRH sequence itself () 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
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.