Comparison of N-terminal modifications on neurotensin(8-13) analogues correlates peptide stability but not binding affinity with in vivo efficacy.

Neurotensin(8-13) and two related analogues were used as model systems to directly compare various N-terminal peptide modifications representing both commonly used and novel capping groups. Each N-terminal modification prevented aminopeptidase cleavage but surprisingly differed in its ability to inhibit cleavage at other sites, a phenomenon attributed to long-range conformational effects. None of the capping groups were inherently detrimental to human neurotensin receptor 1 (hNTR1) binding affinity or receptor agonism. Although the most stable peptides exhibited the lowest binding affinities and were the least potent receptor agonists, they produced the largest in vivo effects. Of the parameters studied only stability significantly correlated with in vivo efficacy, demonstrating that a reduction in binding affinity at NTR1 can be countered by increased in vivo stability.

Design, synthesis, and evaluation of the antipsychotic potential of orally bioavailable neurotensin (8-13) analogues containing non-natural arginine and lysine residues.

Neurotensin (NT) and its active fragment NT(8-13) elicit behavioral responses typical of clinically used antipsychotic drugs when administered directly to the brain. However, limited peptide stability and oral bioavailability have prevented these compounds from being developed as relevant pharmaceuticals. Recently, our laboratory designed and studied a first-generation NT(8-13) derivative, KK13, that elicited key pharmacokinetic and behavioral responses typical of clinically used antipsychotic drugs when administered to rats parenterally. This compound was the basis for the rational design of a series of second-generation NT(8-13) analogues (KH1-KH30) studied in this paper. Initial screening of these analogues for CNS activity by monitoring hypothermia induction after peripheral administration defined several compounds (KH11, KH24, KH26, and KH28-KH30) that warranted further investigation. Each compound maintained binding affinity for NTR(1), however, only KH24, KH26, and KH28 (as well as KK13) elicited significant hypothermic responses after oral administration. Of these, KH28 demonstrated an oral activity 3-fold greater than any other analogue; hence it was further characterized in a series of rat behavioral assays. KH28 attenuated d-amphetamine induced hyperlocomotion, a hallmark of current clinically effective antipsychotic drugs, after both IP and oral administration. In addition, tolerance to the compound did not develop after repeated daily dosing, as measured by hypothermic induction as well as attenuation of d-amphetamine induced hyperlocomotion. Finally, KH28 did not produce catalepsy, a deleterious side-effect elicited by classical antipsychotic drugs. KH28 is considered to be an ideal compound for further development as a potential novel antipsychotic.

In vivo behavioral effects of stable, receptor-selective neurotensin[8-13] analogues that cross the blood-brain barrier.

A set of neurotensin[8-13] (NT[8-13]) analogues (KK1-19) has been evaluated in various pre-clinical assays relevant for further development of these compounds as potential antipsychotics. Initial screening of these compounds for induction of hypothermia following systemic (I.V.) injection in rats, an indirect method commonly utilized to measure the central nervous system (CNS) activity of NT[8-13] analogues, identified three peptides, KK1, KK13 and KK14, capable of crossing the blood-brain barrier (BBB). KK1 features 2(S)-azido-7-aminoheptanoic acid (AAHA) in the Arg(8) position and represents the first monosubstituted NT[8-13] analogue that crosses the BBB. KK13 and KK14 both feature AAHA in the Arg(8) position and tert-Leu in the Ile(12) position while KK14 includes a Trp substituted for Tyr(11). When I.P. administered, only the latter two analogues induced a significant hypothermic response. KK13 (1mg/kg) inhibited amphetamine-induced hyperlocomotion after I.P. injection; this assay is highly predictive for potential antipsychotics. Chronic dosing (5mg/kg) of this compound over 5 consecutive days failed to induce hypothermic tolerance while the same dose failed to induce measurable catalepsy. KK13 is thus the first NT[8-13] analogue described to date that demonstrates inhibition of amphetamine-induced hyperlocomotion without inducing catalepsy while maintaining day-to-day hypothermic potency.

In vitro analysis of stable, receptor-selective neurotensin[8-13] analogues.

A set of neurotensin[8-13] (NT[8-13]) analogues featuring substitution of non-natural cationic amino acids in the Arg(8) position have been synthesized and tested for binding potencies against the three cloned human NT receptors (hNTR-1, hNTR-2, hNTR-3), functional agonism of the hNTR1 and for rat serum stability. Three distinct classes of peptides have been identified: Class 1 features alkyl-Arg analogues at Arg(8), Class 2 features alpha-azido-cationic amino acids at Arg(8), and Class 3 feature modified Arg(8) and Tyr(11) residues. Most of the peptides maintain or exceed the binding potency of NT[8-13] to hNTR-1. Class 2 analogues exceed the binding potency of NT[8-13] to hNTR-2 with KK19 binding with higher affinity to hNTR-2 than hNTR-1. Peptides with enhanced binding potencies for hNTR-3 were not found. All analogues are functional agonists of the hNTR1 receptor as indicated by phosphoinositide (PI) determination. Serum stability increased with peptide classification where the half-life of Class 1 < Class 2 < Class 3 which are stable to rat serum for > 24 h.